https://aopwiki.org/wiki/api.php?action=feedcontributions&user=Dvillene&feedformat=atomAOP-Wiki - User contributions [en]2024-03-28T18:54:03ZUser contributionsMediaWiki 1.23.2https://aopwiki.org/wiki/index.php/Review:OECD_External_Review_September,_2015_-_Aop:23Review:OECD External Review September, 2015 - Aop:232016-08-11T12:30:27Z<p>Dvillene: /* Reviewer 3: */</p>
<hr />
<div>__NOTOC__<br />
<h2> AOP Information </h2><br />
<br />
<br />
* Snapshot for OECD Review (September 2015):[[File:Aop23-Snapshot-REV-September2015.pdf]]<br />
<br />
* Associated wiki page: [[Aop:23]]<br />
<br />
<br />
<h2> External Review </h2><br />
<br />
The review was performed in September 2015 by a team of 5 reviewers:<br />
<br />
- Jana Blahová (Czech Republic)<br />
- ZhiChao Dang (NL)<br />
- Alex Odermatt (Switzerland)<br />
- Ioanna Katsiadaki (UK)<br />
- R. Balasubramanian (ICAPO)<br />
<br />
<br><br />
----<br />
<br />
<br />
<br />
<h3> Charge question 1:''Check if the AOP incorporates the critical scientific literature and if the scientific content of the AOP reflects the current scientific knowledge on this specific topic''</h3><br />
<br />
<h4> Responses </h4><br />
<br />
====Reviewer 1: ====<br />
<br />
The current AOP is well written with current scientific knowledge. It would be important to have limitation and weight of evidence captured under each key events to understand the regulatory standpoint. The wide applicability of the current AOP to evaluate different taxonomic groups and chemicals would add more clarity.<br />
<br />
'''Response: Weight of evidence and major uncertainties or inconsistencies are covered on the KER pages. The taxonomic domain of applicability of the AOP is described on p.35 of the pdf snapshot.'''<br />
<br />
====Reviewer 2: ====<br />
<br />
====Reviewer 3: ====<br />
The AOP is well described and included the essential literature, which reflects the current understanding in reproductive impairments resulted from the exposure to AR agonists in small model fish. To increase understanding and application of this AOP in the regulatory field, following points need to be clarified and further elucidated. <br />
Two chemicals, 17β-trenbolone and spironolactone, were listed. There are, however, fish data on other AR agonists e.g. 17-methyltestosterone, androstenedione, diazinon, and methyldihydrotestosterone, which should be included too. Explanation is need whether or not these chemicals produce similar effects as described in the AOP. <br />
'''Response: The reviewer raises an interesting point here. There are indeed a number of studies with 17-methyltestosterone (MT). While in vitro MT would be classified as an androgen, in vivo it can be aromatized to an estrogen. As a result, for this compound effects observed may not be consistent with the present AOP. Additional notes regarding this potential limitation of the AOP relative to domain of chemical applicability were added to the "uncertainties and inconsistencies" section of the AOP evaluation. This caveat regarding applicability to non-aromatizable estrogens only was already noted in the "Applicability of the AOP" section of the AOP page. The description for Event:25 also notes that "structures subject to aromatization may behave in vivo as estrogens despite exhibiting potent androgen receptor agonism in vitro."'''<br />
<br />
<br />
Information has been filled in the sections of some, but not all of KEs. In some KEs, fathead minnow and medaka have been listed. But information on the other fish like zebrafish, sheepshead minnow etc has not yet been included. Some KEs may be applicable to vertebrates including fish. It may be important to include some results of e.g. AR and Aromatase knockout mice, and transgenic fish if possible. Information on species similarity and difference in ARs has not yet been included in the AOP. Such information may be of use for the extrapolation among species. <br />
The current description may be only applied to the genomic action. Similar to ERs, both genomic and non-genomic mechanisms are applied to ARs too. It is suggested that this issue should be mentioned too.<br />
Different names for the same assays, e.g. radioimmunoassay and RIA; enzyme immunoassay and ELISA, have been used in KEs. It is important to consistently use the same expressions for these methods in the whole AOP. <br />
In the methods described, no current OECD activities on ARTA were included. In fact, several ARTA assays have been validated and will be developed into the test guideline. This development should be included in this section. <br />
For the in vivo fish tests, secondary sex characteristics in fathead minnow and western mosquitofish were described. How about medaka? Information on medaka should be included too.<br />
Fecundity is an important apical endpoint for fish full/partial life cycle tests, and for TG240 (MEOGT). In addition to TG229, it is important to add these long term toxicity tests. <br />
Interrenal, instead of adrenal, is used in fish. Should the term interrenal be included in the AOP?<br />
<br />
====Reviewer 4: ====<br />
In general the AOP incorporates relevant current knowledge, although some aspects are restricted to one publication only; for example the process of vitellogenesis is solely references as the Tyler and Sumpter paper although it was Wallace and Selman (1981) that first (and in more detail) described the cellular and dynamic aspects of oocyte growth in teleosts. Also I think, that other patterns of spawning, more common amongst fish should be presented as a reference at least. Fish are a highly diverse group encompassing more than 30,000 species of which very few share the spawning pattern observed in continuous spawners such as the fathead minnow and other regulatory species. <br />
As far as the androgen axis goes, the literature doesn't include traits such as the anal fin elongation in the medaka and the spiggin regulation in the stickleback, both of which can provide very insightful information on xenoandrogens. Importantly the AOP doesn't touch at all in the number and type of receptors present in different species; this is very important at binding to the receptor is key for the MIE and fish with only one type of AR are compromised by nature in their ability to detect a wide range of xenobiotics. <br />
The weakest by far link in this AOP is the effect of androgen agonists on gonadotrophins; if we assume there is a negative feedback then the AOP stands strong all the way from the MIE to the KE, the KER and even the population trajectory besides some knowledge gaps there. However, this is not the case; many (if not most) fish species employ different reproductive strategies to the FHM or the medaka and as such they have developed feedback mechanisms that suit them best. During puberty for example many species have a positive feedback mechanism (see review by Trudeau, 1997; Antonopoulou et al, 1999 and many many more!!). I wonder since the AOP relies on this reduction of endogenous androgens leading to reduction of endogenous oestrogens to materialise all downstream KE why there is practically no literature cited here other than a single paper on eels that if I am correct is not relevant as it is an in vitro study. In fact this reference (Huang et al, 1997) is missing from the list but is cited on page 35. <br />
In terms of taxonomic applicability, none of this is relevant to invertebrates as they do not use vertebrate steroids to regulate their reproduction. I suggest we clearly state this rather than say ...not necessarily relevant to invertebrates (i.e. page 3) it's definitely not relevant to them. <br />
A minor point, I am not sure the term lutenising hormone is used in fish; instead we recognize two FSH; trivial perhaps but worth considering<br />
<br />
====Reviewer 5: ====<br />
<br />
<br />
<br />
==== Response from AOP author:==== <br />
<h4> To reviewer 1: </h4> <br />
<h4> To reviewer 2: </h4> <br />
<h4> To reviewer 3: </h4> <br />
<h4> To reviewer 4: </h4> <br />
<h4> To reviewer 5: </h4> <br />
<br />
<br><br />
----<br />
<br />
<h3>Charge question 2:''Verify the weight of evidence judgement/scoring provided by AOP developers for KEs, KERs and the overall AOP''</h3><br />
<br />
<h4> Responses </h4><br />
<br />
====Reviewer 1: ====<br />
<br />
The weight of evidence summary in page 30 of this AOP lists that the WoE for androgen receptor, agonism as 'weak', while the other events are moderate to strong. This is may need further detailing to address the regulatory requirements and ways by which the limitation could be addressed.<br />
<br />
====Reviewer 2: ====<br />
<br />
====Reviewer 3: ====<br />
According to the description of the KE of Gonadotropins, circulating concentrations, Reduction, it seems that there is a problem to measure LH and FSH in small model fish. If so, this may have a conflict with the notion that KEs must be measurable. In addition, KEs have been numbered in the concordance table, in which the KE of Gonadotropin reduction was not included. Explanation is needed why this KE is not included.<br />
Over the relationship: Androgen receptor, Agonism Directly Leads to Gonadotropins, Circulating Concentrations, Reduction, the term “directly leads to” seems confusing because the direct target of AR agonists may not be pituitary and a decrease in gonadotropin levels results from a negative feedback mechanism in which neurotransmitters are involved. This relationship can be understood as indirect process too. <br />
<br />
In the section of Empirical Support for Linkage, results of DEHP/MEHP in mammals have been included. Did they have similar mechanisms to those of fadrozole, prochloraz in fish? <br />
<br />
In the section of Empirical support for linkage, some lines of evidence were based on an increase in 17β-estradiol concentration resulting in an enhancement in VTG in male fish. Such evidence is different from the title of reduction and female of AOPs. <br />
<br />
A weight of evidence table is needed in the annex.<br />
<br />
The table of the KEs was not presented according to the orders of KEs, which may cause some confusing. It is suggested that the table of KEs should be organized according to the order of KEs.<br />
<br />
====Reviewer 4: ====<br />
As expanded above, once we accept that an AR agonist is not aromatisable (species differ enormously on their ability to aromatase or not specific androgens) and has a negative feedback for LH, then the weight of evidence is moderate to strong and the AOP stands. However, my concerns are entirely on how relevant is this for ecologically important species that ultimately we are trying to protect via this exercise. If we are interested in the adverse outcome for a laboratory strain of FHM, then by all means the evidence is there in most relationships including the adversity. However, I do not believe this is how most species regulate their reproduction and more information should be made available to regulators on this critical assumptions. <br />
<br />
====Reviewer 5: ====<br />
<br />
<br />
==== Response from AOP author:==== <br />
<h4> To reviewer 1: </h4> <br />
<h4> To reviewer 2: </h4> <br />
<h4> To reviewer 3: </h4> <br />
<h4> To reviewer 4: </h4> <br />
<h4> To reviewer 5: </h4> <br />
<br />
<br><br />
----<br />
<br />
<h3> Charge question 3:''What would be the regulatory applicability of this AOP in your opinion?''</h3><br />
<br />
<h4> Responses </h4><br />
<br />
====Reviewer 1: ====<br />
<br />
Globally across different geographies identification and evaluation of EDCs are recommended by various regulatory agencies. This AOP covers the existing and additional events of evaluation. The set of regulatory specification/ data requirement for the AOP could help broader usage for different chemicals types<br />
<br />
====Reviewer 2: ====<br />
<br />
====Reviewer 3: ====<br />
Identification of Endocrine Disrupting Chemicals (EDCs) is needed under several pieces of European Union (EU) legislation, including the Regulation on industrial chemicals (Registration, Evaluation, Authorization and restriction of Chemicals, EC 1907/2006, REACH), the Plant Protection Products Regulation (EC 1107/2009, PPPR), and the Biocides Products Regulation (528/2012, BPR). Currently, the regulatory identification of EDCs is mainly based on the general consensus on the WHO definition, which consists of three essential elements, i.e. chemical-induced adverse effects (adversity), chemical specific endocrine modes/mechanisms of action (MOAs) and the causal relationship (causality) between adverse effects and endocrine MOAs. AOPs cover all essential elements for identification of EDCs and show the complex biology of adversity and MOAs. These will help regulators understand the complexity of identification of EDCs. Besides, current regulatory tests focus on EATS pathways. In contrast, AOPs include not only EATS pathways but also other pathways, e.g. PPARs, RXR, that are essential to development, growth, and reproduction. Within each AOP, different targets at molecular, cellular, organ/tissue and individual levels could be identified and the adverse outcome would be predicated. Such information would be of help for prioritizing chemicals, for grouping chemicals and for developing an integrated testing strategy. It is important to indicate that the AOP needs extensive amount of data which might be possible for a few chemicals but will not be possible for a majority of chemicals. Current data requirements under REACH, PPPR, BPR, etc. do not cover all key events of the AOP.<br />
<br />
====Reviewer 4: ====<br />
This is restricted clearly to female fish only as adversity is linked to reduced oestrogen synthesis (via reduced androgen synthesis); it is also limited to fully reproductive mature fish (not fish entering puberty or juvenile fish) and importantly is limited to fish that once they reach sexual maturity they spawn constantly. The latter is a reproductive strategy employed by fish that tend to occupy tropical areas (around the equator). Unfortunately most fish species have different reproductive strategies (annual life cycle) hence the level of gonadotropin expression (and consequently steroid production) is regulated by photoperiodic and temperature changes throughout the year. Even if a negative feedback mechanism operates in all of these species and in all life stages (which is certainly not the case) we still need to establish what is the relative strength of the AR agonist induced negative feedback to the environment-induced stimulation of gonadotropins! This link has never been studied and is critical if we really mean to protect wildlife. <br />
<br />
====Reviewer 5: ====<br />
<br />
<br />
==== Response from AOP author:==== <br />
<h4> To reviewer 1: </h4> <br />
<h4> To reviewer 2: </h4> <br />
<h4> To reviewer 3: </h4> <br />
<h4> To reviewer 4: </h4> <br />
<h4> To reviewer 5: </h4> <br />
<br />
<br><br />
----<br />
<br />
<h3> Charge question 4:''Overall Assessment of the AOP - Would you recommend this AOP to be submitted to the Working group of the National Coordinators for the Test Guidelines Programme (WNT) and the Task Force on Hazard Assessment (TFHA) for endorsement?''</h3><br />
<br />
<h4> Responses </h4><br />
<br />
====Reviewer 1: ====<br />
<br />
YES, recommend this to be submitted to the WNT. This will set the right direction for regulatory application sooner. Additionally, the strengths and weakness of this AOP should be clearly stated for continuous usage and updation<br />
<br />
====Reviewer 2: ====<br />
<br />
====Reviewer 3: ====<br />
Yes. It is important to get the official stamp for publishing the AOP. To increase the regulatory applications of the AOP, following points are suggested: <br />
The current AOP focuses only on female fish. As the majority of test guidelines include both males and females, it is important to include certain information/statement over male fish so that regulators can get an overall picture. It is also important to indicate that the major androgen in male teleost fish is 11-ketotestosterone. In addition, it is important to specify the uncertainties or inconsistencies that are related to chemicals. This would be of great help for non-experts and risk assessors to understand confounding factors.<br />
<br />
====Reviewer 4: ====<br />
Yes as long as the limitations based on feedback mechanisms and reproductive strategies are made even clearer than currently are. This is relevant ONLY for small repeat-spawning fish species but this is mentioned only once in the title throughout the AOP. Assuming this is presented as a warning on the first paragraph, then my recommendation would be to submit to WNT.<br />
<br />
====Reviewer 5: ====<br />
<br />
<br />
==== Response from AOP author:==== <br />
<h4> To reviewer 1: </h4> <br />
<h4> To reviewer 2: </h4> <br />
<h4> To reviewer 3: </h4> <br />
<h4> To reviewer 4: </h4> <br />
<h4> To reviewer 5: </h4> <br />
<br />
<br><br />
----<br />
<br />
<h3> Other comments </h3><br />
<br />
====Reviewer 1: ====<br />
<br />
====Reviewer 2: ====<br />
<br />
====Reviewer 3: ====<br />
<br />
====Reviewer 4: ====<br />
<br />
====Reviewer 5: ====</div>Dvillenehttps://aopwiki.org/wiki/index.php/Review:OECD_External_Review_September,_2015_-_Aop:23Review:OECD External Review September, 2015 - Aop:232016-08-11T12:27:26Z<p>Dvillene: /* Reviewer 3: */</p>
<hr />
<div>__NOTOC__<br />
<h2> AOP Information </h2><br />
<br />
<br />
* Snapshot for OECD Review (September 2015):[[File:Aop23-Snapshot-REV-September2015.pdf]]<br />
<br />
* Associated wiki page: [[Aop:23]]<br />
<br />
<br />
<h2> External Review </h2><br />
<br />
The review was performed in September 2015 by a team of 5 reviewers:<br />
<br />
- Jana Blahová (Czech Republic)<br />
- ZhiChao Dang (NL)<br />
- Alex Odermatt (Switzerland)<br />
- Ioanna Katsiadaki (UK)<br />
- R. Balasubramanian (ICAPO)<br />
<br />
<br><br />
----<br />
<br />
<br />
<br />
<h3> Charge question 1:''Check if the AOP incorporates the critical scientific literature and if the scientific content of the AOP reflects the current scientific knowledge on this specific topic''</h3><br />
<br />
<h4> Responses </h4><br />
<br />
====Reviewer 1: ====<br />
<br />
The current AOP is well written with current scientific knowledge. It would be important to have limitation and weight of evidence captured under each key events to understand the regulatory standpoint. The wide applicability of the current AOP to evaluate different taxonomic groups and chemicals would add more clarity.<br />
<br />
'''Response: Weight of evidence and major uncertainties or inconsistencies are covered on the KER pages. The taxonomic domain of applicability of the AOP is described on p.35 of the pdf snapshot.'''<br />
<br />
====Reviewer 2: ====<br />
<br />
====Reviewer 3: ====<br />
The AOP is well described and included the essential literature, which reflects the current understanding in reproductive impairments resulted from the exposure to AR agonists in small model fish. To increase understanding and application of this AOP in the regulatory field, following points need to be clarified and further elucidated. <br />
Two chemicals, 17β-trenbolone and spironolactone, were listed. There are, however, fish data on other AR agonists e.g. 17-methyltestosterone, androstenedione, diazinon, and methyldihydrotestosterone, which should be included too. Explanation is need whether or not these chemicals produce similar effects as described in the AOP. <br />
'''Response: The reviewer raises an interesting point here. There are indeed a number of studies with 17-methyltestosterone (MT). While in vitro MT would be classified as an androgen, in vivo it can be aromatized to an estrogen. As a result, for this compound effects observed may not be consistent with the present AOP. Additional notes regarding this potential limitation of the AOP relative to domain of chemical applicability were added to the "uncertainties and inconsistencies" section of the AOP evaluation. This caveat regarding applicability to non-aromatizable estrogens only was already noted in the "Applicability of the AOP" section of the AOP page.'''<br />
<br />
<br />
Information has been filled in the sections of some, but not all of KEs. In some KEs, fathead minnow and medaka have been listed. But information on the other fish like zebrafish, sheepshead minnow etc has not yet been included. Some KEs may be applicable to vertebrates including fish. It may be important to include some results of e.g. AR and Aromatase knockout mice, and transgenic fish if possible. Information on species similarity and difference in ARs has not yet been included in the AOP. Such information may be of use for the extrapolation among species. <br />
The current description may be only applied to the genomic action. Similar to ERs, both genomic and non-genomic mechanisms are applied to ARs too. It is suggested that this issue should be mentioned too.<br />
Different names for the same assays, e.g. radioimmunoassay and RIA; enzyme immunoassay and ELISA, have been used in KEs. It is important to consistently use the same expressions for these methods in the whole AOP. <br />
In the methods described, no current OECD activities on ARTA were included. In fact, several ARTA assays have been validated and will be developed into the test guideline. This development should be included in this section. <br />
For the in vivo fish tests, secondary sex characteristics in fathead minnow and western mosquitofish were described. How about medaka? Information on medaka should be included too.<br />
Fecundity is an important apical endpoint for fish full/partial life cycle tests, and for TG240 (MEOGT). In addition to TG229, it is important to add these long term toxicity tests. <br />
Interrenal, instead of adrenal, is used in fish. Should the term interrenal be included in the AOP?<br />
<br />
====Reviewer 4: ====<br />
In general the AOP incorporates relevant current knowledge, although some aspects are restricted to one publication only; for example the process of vitellogenesis is solely references as the Tyler and Sumpter paper although it was Wallace and Selman (1981) that first (and in more detail) described the cellular and dynamic aspects of oocyte growth in teleosts. Also I think, that other patterns of spawning, more common amongst fish should be presented as a reference at least. Fish are a highly diverse group encompassing more than 30,000 species of which very few share the spawning pattern observed in continuous spawners such as the fathead minnow and other regulatory species. <br />
As far as the androgen axis goes, the literature doesn't include traits such as the anal fin elongation in the medaka and the spiggin regulation in the stickleback, both of which can provide very insightful information on xenoandrogens. Importantly the AOP doesn't touch at all in the number and type of receptors present in different species; this is very important at binding to the receptor is key for the MIE and fish with only one type of AR are compromised by nature in their ability to detect a wide range of xenobiotics. <br />
The weakest by far link in this AOP is the effect of androgen agonists on gonadotrophins; if we assume there is a negative feedback then the AOP stands strong all the way from the MIE to the KE, the KER and even the population trajectory besides some knowledge gaps there. However, this is not the case; many (if not most) fish species employ different reproductive strategies to the FHM or the medaka and as such they have developed feedback mechanisms that suit them best. During puberty for example many species have a positive feedback mechanism (see review by Trudeau, 1997; Antonopoulou et al, 1999 and many many more!!). I wonder since the AOP relies on this reduction of endogenous androgens leading to reduction of endogenous oestrogens to materialise all downstream KE why there is practically no literature cited here other than a single paper on eels that if I am correct is not relevant as it is an in vitro study. In fact this reference (Huang et al, 1997) is missing from the list but is cited on page 35. <br />
In terms of taxonomic applicability, none of this is relevant to invertebrates as they do not use vertebrate steroids to regulate their reproduction. I suggest we clearly state this rather than say ...not necessarily relevant to invertebrates (i.e. page 3) it's definitely not relevant to them. <br />
A minor point, I am not sure the term lutenising hormone is used in fish; instead we recognize two FSH; trivial perhaps but worth considering<br />
<br />
====Reviewer 5: ====<br />
<br />
<br />
<br />
==== Response from AOP author:==== <br />
<h4> To reviewer 1: </h4> <br />
<h4> To reviewer 2: </h4> <br />
<h4> To reviewer 3: </h4> <br />
<h4> To reviewer 4: </h4> <br />
<h4> To reviewer 5: </h4> <br />
<br />
<br><br />
----<br />
<br />
<h3>Charge question 2:''Verify the weight of evidence judgement/scoring provided by AOP developers for KEs, KERs and the overall AOP''</h3><br />
<br />
<h4> Responses </h4><br />
<br />
====Reviewer 1: ====<br />
<br />
The weight of evidence summary in page 30 of this AOP lists that the WoE for androgen receptor, agonism as 'weak', while the other events are moderate to strong. This is may need further detailing to address the regulatory requirements and ways by which the limitation could be addressed.<br />
<br />
====Reviewer 2: ====<br />
<br />
====Reviewer 3: ====<br />
According to the description of the KE of Gonadotropins, circulating concentrations, Reduction, it seems that there is a problem to measure LH and FSH in small model fish. If so, this may have a conflict with the notion that KEs must be measurable. In addition, KEs have been numbered in the concordance table, in which the KE of Gonadotropin reduction was not included. Explanation is needed why this KE is not included.<br />
Over the relationship: Androgen receptor, Agonism Directly Leads to Gonadotropins, Circulating Concentrations, Reduction, the term “directly leads to” seems confusing because the direct target of AR agonists may not be pituitary and a decrease in gonadotropin levels results from a negative feedback mechanism in which neurotransmitters are involved. This relationship can be understood as indirect process too. <br />
<br />
In the section of Empirical Support for Linkage, results of DEHP/MEHP in mammals have been included. Did they have similar mechanisms to those of fadrozole, prochloraz in fish? <br />
<br />
In the section of Empirical support for linkage, some lines of evidence were based on an increase in 17β-estradiol concentration resulting in an enhancement in VTG in male fish. Such evidence is different from the title of reduction and female of AOPs. <br />
<br />
A weight of evidence table is needed in the annex.<br />
<br />
The table of the KEs was not presented according to the orders of KEs, which may cause some confusing. It is suggested that the table of KEs should be organized according to the order of KEs.<br />
<br />
====Reviewer 4: ====<br />
As expanded above, once we accept that an AR agonist is not aromatisable (species differ enormously on their ability to aromatase or not specific androgens) and has a negative feedback for LH, then the weight of evidence is moderate to strong and the AOP stands. However, my concerns are entirely on how relevant is this for ecologically important species that ultimately we are trying to protect via this exercise. If we are interested in the adverse outcome for a laboratory strain of FHM, then by all means the evidence is there in most relationships including the adversity. However, I do not believe this is how most species regulate their reproduction and more information should be made available to regulators on this critical assumptions. <br />
<br />
====Reviewer 5: ====<br />
<br />
<br />
==== Response from AOP author:==== <br />
<h4> To reviewer 1: </h4> <br />
<h4> To reviewer 2: </h4> <br />
<h4> To reviewer 3: </h4> <br />
<h4> To reviewer 4: </h4> <br />
<h4> To reviewer 5: </h4> <br />
<br />
<br><br />
----<br />
<br />
<h3> Charge question 3:''What would be the regulatory applicability of this AOP in your opinion?''</h3><br />
<br />
<h4> Responses </h4><br />
<br />
====Reviewer 1: ====<br />
<br />
Globally across different geographies identification and evaluation of EDCs are recommended by various regulatory agencies. This AOP covers the existing and additional events of evaluation. The set of regulatory specification/ data requirement for the AOP could help broader usage for different chemicals types<br />
<br />
====Reviewer 2: ====<br />
<br />
====Reviewer 3: ====<br />
Identification of Endocrine Disrupting Chemicals (EDCs) is needed under several pieces of European Union (EU) legislation, including the Regulation on industrial chemicals (Registration, Evaluation, Authorization and restriction of Chemicals, EC 1907/2006, REACH), the Plant Protection Products Regulation (EC 1107/2009, PPPR), and the Biocides Products Regulation (528/2012, BPR). Currently, the regulatory identification of EDCs is mainly based on the general consensus on the WHO definition, which consists of three essential elements, i.e. chemical-induced adverse effects (adversity), chemical specific endocrine modes/mechanisms of action (MOAs) and the causal relationship (causality) between adverse effects and endocrine MOAs. AOPs cover all essential elements for identification of EDCs and show the complex biology of adversity and MOAs. These will help regulators understand the complexity of identification of EDCs. Besides, current regulatory tests focus on EATS pathways. In contrast, AOPs include not only EATS pathways but also other pathways, e.g. PPARs, RXR, that are essential to development, growth, and reproduction. Within each AOP, different targets at molecular, cellular, organ/tissue and individual levels could be identified and the adverse outcome would be predicated. Such information would be of help for prioritizing chemicals, for grouping chemicals and for developing an integrated testing strategy. It is important to indicate that the AOP needs extensive amount of data which might be possible for a few chemicals but will not be possible for a majority of chemicals. Current data requirements under REACH, PPPR, BPR, etc. do not cover all key events of the AOP.<br />
<br />
====Reviewer 4: ====<br />
This is restricted clearly to female fish only as adversity is linked to reduced oestrogen synthesis (via reduced androgen synthesis); it is also limited to fully reproductive mature fish (not fish entering puberty or juvenile fish) and importantly is limited to fish that once they reach sexual maturity they spawn constantly. The latter is a reproductive strategy employed by fish that tend to occupy tropical areas (around the equator). Unfortunately most fish species have different reproductive strategies (annual life cycle) hence the level of gonadotropin expression (and consequently steroid production) is regulated by photoperiodic and temperature changes throughout the year. Even if a negative feedback mechanism operates in all of these species and in all life stages (which is certainly not the case) we still need to establish what is the relative strength of the AR agonist induced negative feedback to the environment-induced stimulation of gonadotropins! This link has never been studied and is critical if we really mean to protect wildlife. <br />
<br />
====Reviewer 5: ====<br />
<br />
<br />
==== Response from AOP author:==== <br />
<h4> To reviewer 1: </h4> <br />
<h4> To reviewer 2: </h4> <br />
<h4> To reviewer 3: </h4> <br />
<h4> To reviewer 4: </h4> <br />
<h4> To reviewer 5: </h4> <br />
<br />
<br><br />
----<br />
<br />
<h3> Charge question 4:''Overall Assessment of the AOP - Would you recommend this AOP to be submitted to the Working group of the National Coordinators for the Test Guidelines Programme (WNT) and the Task Force on Hazard Assessment (TFHA) for endorsement?''</h3><br />
<br />
<h4> Responses </h4><br />
<br />
====Reviewer 1: ====<br />
<br />
YES, recommend this to be submitted to the WNT. This will set the right direction for regulatory application sooner. Additionally, the strengths and weakness of this AOP should be clearly stated for continuous usage and updation<br />
<br />
====Reviewer 2: ====<br />
<br />
====Reviewer 3: ====<br />
Yes. It is important to get the official stamp for publishing the AOP. To increase the regulatory applications of the AOP, following points are suggested: <br />
The current AOP focuses only on female fish. As the majority of test guidelines include both males and females, it is important to include certain information/statement over male fish so that regulators can get an overall picture. It is also important to indicate that the major androgen in male teleost fish is 11-ketotestosterone. In addition, it is important to specify the uncertainties or inconsistencies that are related to chemicals. This would be of great help for non-experts and risk assessors to understand confounding factors.<br />
<br />
====Reviewer 4: ====<br />
Yes as long as the limitations based on feedback mechanisms and reproductive strategies are made even clearer than currently are. This is relevant ONLY for small repeat-spawning fish species but this is mentioned only once in the title throughout the AOP. Assuming this is presented as a warning on the first paragraph, then my recommendation would be to submit to WNT.<br />
<br />
====Reviewer 5: ====<br />
<br />
<br />
==== Response from AOP author:==== <br />
<h4> To reviewer 1: </h4> <br />
<h4> To reviewer 2: </h4> <br />
<h4> To reviewer 3: </h4> <br />
<h4> To reviewer 4: </h4> <br />
<h4> To reviewer 5: </h4> <br />
<br />
<br><br />
----<br />
<br />
<h3> Other comments </h3><br />
<br />
====Reviewer 1: ====<br />
<br />
====Reviewer 2: ====<br />
<br />
====Reviewer 3: ====<br />
<br />
====Reviewer 4: ====<br />
<br />
====Reviewer 5: ====</div>Dvillenehttps://aopwiki.org/wiki/index.php/Aop:23Aop:232016-08-11T12:17:31Z<p>Dvillene: /* Weight of Evidence Summary */</p>
<hr />
<div>__NOTOC__<br />
{{#widget:Revisions}}<br />
{{#widget:Network}}<br />
== AOP Title ==<br />
<br />
<div class='Title'> Androgen receptor agonism leading to reproductive dysfunction </div><br />
<br />
<div id='shortTitle' class='Title2'>Short name: Androgen receptor agonism leading to reproductive dysfunction </div><br />
<br />
== Authors ==<br />
Dan Villeneuve, US EPA Mid-Continent Ecology Division (villeneuve.dan@epa.gov)<br />
<br />
== Status ==<br />
<br />
'''Please follow the link to [//{{SERVERNAME}}/aops/{{PAGENAMEE}}/snapshots snapshots page] to view and create Snapshots of this AOP.'''<br />
<br />
'''Open for comment'''<br />
<br />
[http://www.oecd.org/env/ehs/testing/listsofprojectsontheaopdevelopmentprogrammeworkplan.htm OECD Project 1.12: The Adverse outcome pathways linking aromatase inhibition, androgen receptor agonism, estrogen receptor antagonism, or steroidogenesis inhibition, to impaired reproduction in small repeat-spawning fish species]<br />
<br />
This AOP page was last modified on {{REVISIONMONTH1}}/{{REVISIONDAY}}/{{REVISIONYEAR}}.<br />
<br />
<span id='ToggleRevision' style="text-decoration: underline;">Click here to show/hide revision dates for related pages</span><br />
{|class="wikitable sortable" id="Revtable"<br />
<br />
!Page<br />
!Revision Date/Time<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Abstract ==<br />
This adverse outcome pathway details the linkage between binding and activation of androgen receptor as a nuclear transcription factor in females and reproductive dysfunction as evidenced through reductions cumulative fecundity and spawning in repeat-spawning fish species. Cumulative fecundity is the most apical endpoint considered in the OECD 229 Fish Short Term Reproduction Assay. The OECD 229 assay serves as screening assay for endocrine disruption and associated reproductive impairment (OECD 2012). Cumulative fecundity is one of several variables known to be of demographic significance in forecasting fish population trends. Therefore, this AOP has utility in supporting the application of measures of androgen receptor binding and activation as a nuclear transcription factor as a means to identify chemicals with known potential to adversely affect fish populations.<br />
<br />
== Summary of the AOP ==<br />
[[Category:Adverse Outcome Pathway]]<br />
Please follow link to [//{{SERVERNAME}}/aops/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== Molecular Initiating Event ===<br />
<br />
{|class="wikitable sortable" id="MIEproof"<br />
<br />
!Molecular Initiating Event<br />
!Support for Essentiality<br />
<br />
|-<br />
<br />
|[[Event:25|Androgen receptor, Agonism]]||[[Aop:23#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Key Events ===<br />
<br />
{|class="wikitable sortable" id="KEproof"<br />
<br />
!Event<br />
!Support for Essentiality<br />
<br />
|-<br />
<br />
|[[Event:274|Testosterone synthesis by ovarian theca cells, Reduction]]||[[Aop:23#Essentiality of the Key Events|Moderate]]<br />
<br />
|-<br />
<br />
|[[Event:3|17beta-estradiol synthesis by ovarian granulosa cells, Reduction]]||[[Aop:23#Essentiality of the Key Events|Moderate]]<br />
<br />
|-<br />
<br />
|[[Event:219|Plasma 17beta-estradiol concentrations, Reduction]]||[[Aop:23#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|[[Event:285|Vitellogenin synthesis in liver, Reduction]]||[[Aop:23#Essentiality of the Key Events|Moderate]]<br />
<br />
|-<br />
<br />
|[[Event:78|Cumulative fecundity and spawning, Reduction]]||[[Aop:23#Essentiality of the Key Events|Moderate]]<br />
<br />
|-<br />
<br />
|[[Event:221|Plasma vitellogenin concentrations, Reduction]]||[[Aop:23#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|[[Event:309|Vitellogenin accumulation into oocytes and oocyte growth/development, Reduction]]||[[Aop:23#Essentiality of the Key Events|Weak]]<br />
<br />
|-<br />
<br />
|[[Event:129|Gonadotropins, circulating concentrations, Reduction]]||[[Aop:23#Essentiality of the Key Events|Moderate]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Adverse Outcome ===<br />
<br />
{|class="wikitable sortable" id="AOproof"<br />
<br />
!Adverse Outcome<br />
<br />
|-<br />
<br />
|[[Event:360|Population trajectory, Decrease]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Relationships Among Key Events and the Adverse Outcome ===<br />
<br />
{| class="wikitable sortable" id="relation"<br />
<br />
!Event<br />
!Description<br />
!Triggers<br />
!Weight of Evidence<br />
!Quantitative Understanding<br />
<br />
|-<br />
<br />
|[[Event:25|Androgen receptor, Agonism]]||[[Relationship:31|Directly Leads to]]||[[Event:129|Gonadotropins, circulating concentrations, Reduction]]||[[Relationship:31#Weight of Evidence|Weak]]||[[Relationship:31#Quantitative Understanding of the Linkage|Weak]]<br />
<br />
|-<br />
<br />
|[[Event:221|Plasma vitellogenin concentrations, Reduction]]||[[Relationship:255|Directly Leads to]]||[[Event:309|Vitellogenin accumulation into oocytes and oocyte growth/development, Reduction]]||[[Relationship:255#Weight of Evidence|Moderate]]||[[Relationship:255#Quantitative Understanding of the Linkage|Weak]]<br />
<br />
|-<br />
<br />
|[[Event:274|Testosterone synthesis by ovarian theca cells, Reduction]]||[[Relationship:302|Directly Leads to]]||[[Event:3|17beta-estradiol synthesis by ovarian granulosa cells, Reduction]]||[[Relationship:302#Weight of Evidence|Strong]]||[[Relationship:302#Quantitative Understanding of the Linkage|Weak]]<br />
<br />
|-<br />
<br />
|[[Event:3|17beta-estradiol synthesis by ovarian granulosa cells, Reduction]]||[[Relationship:5|Directly Leads to]]||[[Event:219|Plasma 17beta-estradiol concentrations, Reduction]]||[[Relationship:5#Weight of Evidence|Strong]]||[[Relationship:5#Quantitative Understanding of the Linkage|Weak]]<br />
<br />
|-<br />
<br />
|[[Event:219|Plasma 17beta-estradiol concentrations, Reduction]]||[[Relationship:252|Directly Leads to]]||[[Event:285|Vitellogenin synthesis in liver, Reduction]]||[[Relationship:252#Weight of Evidence|Strong]]||[[Relationship:252#Quantitative Understanding of the Linkage|Moderate]]<br />
<br />
|-<br />
<br />
|[[Event:285|Vitellogenin synthesis in liver, Reduction]]||[[Relationship:315|Directly Leads to]]||[[Event:221|Plasma vitellogenin concentrations, Reduction]]||[[Relationship:315#Weight of Evidence|Strong]]||[[Relationship:315#Quantitative Understanding of the Linkage|Moderate]]<br />
<br />
|-<br />
<br />
|[[Event:309|Vitellogenin accumulation into oocytes and oocyte growth/development, Reduction]]||[[Relationship:337|Directly Leads to]]||[[Event:78|Cumulative fecundity and spawning, Reduction]]||[[Relationship:337#Weight of Evidence|Moderate]]||[[Relationship:337#Quantitative Understanding of the Linkage|Moderate]]<br />
<br />
|-<br />
<br />
|[[Event:78|Cumulative fecundity and spawning, Reduction]]||[[Relationship:94|Directly Leads to]]||[[Event:360|Population trajectory, Decrease]]||[[Relationship:94#Weight of Evidence|Moderate]]||[[Relationship:94#Quantitative Understanding of the Linkage|Moderate]]<br />
<br />
|-<br />
<br />
|[[Event:129|Gonadotropins, circulating concentrations, Reduction]]||[[Relationship:143|Directly Leads to]]||[[Event:274|Testosterone synthesis by ovarian theca cells, Reduction]]||[[Relationship:143#Weight of Evidence|Strong]]||[[Relationship:143#Quantitative Understanding of the Linkage|Weak]]<br />
<br />
|-<br />
<br />
|[[Event:25|Androgen receptor, Agonism]]||[[Relationship:32|Indirectly Leads to]]||[[Event:274|Testosterone synthesis by ovarian theca cells, Reduction]]||[[Relationship:32#Weight of Evidence|Strong]]||[[Relationship:32#Quantitative Understanding of the Linkage|Weak]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Network View ===<br />
<div id="cytoscapeweb"><br />
Cytoscape Web will replace the contents of this div with your graph.<br />
</div><br />
<div id="note"><br />
<p>Click nodes or edges.</p><br />
</div><br />
<br />
=== Life Stage Applicability ===<br />
<br />
{|class="wikitable sortable" id="LSproof"<br />
<br />
!Life Stage<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|Adult, reproductively mature||||<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|Pimephales promelas||Pimephales promelas||||[http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=90988 NCBI]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Sex Applicability ===<br />
<br />
{|class="wikitable sortable" id="Sexproof"<br />
<br />
!Sex<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|Female||||<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Graphical Representation ===<br />
<br />
*[https://aopkb.org/common/AOP_Graphic_Template2.pptx Click to download template for graphical representation]<br />
<br />
[[File:Androgen receptor agonism leading to reproductive dysfunction.jpg | 900px | Click to upload graphical representation]]<br />
<br />
== Overall Assessment of the AOP ==<br />
<br />
===Weight of Evidence Summary=== <br />
[[#Relationships Among Key Events and the Adverse Outcome | Summary Table]]<br><br />
<em><br />
Provide an overall summary of the weight of evidence based on the evaluations of the individual linkages from the Key Event Relationship pages.<br />
</em><br />
<br />
::'''Biological Plausibility''' <br />
::*The biochemistry of steroidogenesis and the predominant role of the gonad in synthesis of the sex steroids are well established. <br />
::*Similarly, the role of E2 as the major regulator of hepatic vitellogenin production is widely documented in the literature. <br />
::*The direct link between reduced VTG concentrations in the plasma and reduced uptake into oocytes is highly plausible, as the plasma is the primary source of the VTG. <br />
::*The direct connection between reduced VTG uptake and impaired spawning/reduced cumulative fecundity is more tentative. It is not clear, for instance whether impaired VTG uptake limits oocyte growth and failure to reach a critical size in turn impairs physical or inter-cellular signaling processes that promote release of the oocyte from the surrounding follicles. In at least one experiment, oocytes with similar size to vitellogenic oocytes, but lacking histological staining characteristic of vitellogenic oocytes was observed (R. Johnson, personal communication). At present, the link between reductions in circulating VTG concentrations and reduced cumulative fecundity are best supported by the correlation between those endpoints across multiple experiments, including those that impact VTG via other molecular initiating events (Miller et al. 2007). <br />
::* At present, negative feedback is the most biologically plausible explanation for the reductions in ex vivo T and E2 production following exposure to 17β-trenbolone. In vitro exposure of fathead minnow ovary tissue to 17β-trenbolone or spironolactone does not cause reductions in T or E2 synthesis at concentrations comparable to those that produce significant responses in vivo (i.e., at non-cytotoxic concentrations; D.L. Villeneuve, unpublished data; C.A. LaLone unpublished data), nor are there any known reports of 17β-trenbolone directly inhibiting steroid biosynthesis. When tested in an in vitro steroidogenesis assay using H295R adrenal carcinoma cells, trenbolone caused a concentration-dependent increase in estradiol production, as opposed to any reductions in steroid hormone concentrations, an effect that was concurrent with increased transcription of CYP19 (aromatase) in the cell line (Gracia et al. 2007). Given the lack of any established direct effect on steroidogenic enzyme activity, negative feedback is currently the most likely explanation for the consistent effects observed in vivo. That said, many uncertainties regarding the exact mechanisms through which an exogenous, non-aromatizable, AR agonist elicits negative feedback remain. <br />
<br />
'''Concordance of dose-response relationships:''' <br />
There are a limited number of studies in which multiple key events were considered in the same study following exposure to known, non-aromatizable, AR agonists. These were considered the most useful for evaluating the concordance of dose-response relationships. In general, effects on downstream key events occurred at concentrations equal to or greater than those at which upstream events occurred [https://aopkb.org/aopwiki/images/d/dc/AR_agonism_concordance_table.pdf Concordance Table]. For exposures to 17b-trenbolone, key events related to steroid production and circulating estradiol and vitellogenin concentrations were impacted at the same dose at which effects on cumulative fecundity were observed. Effects on vitellogenin transcription were only observed at greater concentrations, but data for comparable species and dose ranges were unavailable at present. For two other AR agonists tested in fish, available studies examined a single time-point only. Consequently, it was unclear whether lower effect concentrations for certain downstream KEs, relative to upstream were due to a lack of dose-response concordance, or due to decreased sensitivity of the upstream later in the exposure time-course.<br />
<br />
While not directly addressing dose-response concordance, the dependence of the key events on the concentration of the androgen agonist has been established for all key events starting at and down-stream of reduced T synthesis. However, to date we are not aware of any studies that have established a concentration-response relationship between exposure to non-endogenous AR agonists (e.g., xenobiotics, pharmaceuticals) and circulating gonadotropin concentrations in fish or other vertebrates.<br />
::* Exposure of female fathead minnows to the AR agonist 17β-trenbolone for 21 d caused concentration-dependent reductions in circulating T, E2, and VTG concentrations over a range from 0.005 to 0.5 μg/L. The concentration response for all three variables had a “U”-shaped concentration response curve which may indicate concentration-dependent differences in the feedback response and/or compensatory processes. Histological evidence of reduced VTG uptake and reduced gonad stage were evident, although the concentration-response of histological effects was not determined. Despite the “U”-shaped concentration-response at the biochemical level, concentration-dependent reductions in cumulative fecundity were observed (Ankley et al. 2003). Effective concentrations were consistent with those causing phenotypic masculinization in female fish. <br />
::* Jensen et al. (2006) also demonstrated concentration-dependent reductions in circulating T, E2, and VTG following 21 d of in vivo exposure to 17α-trenbolone (Jensen et al. 2006).<br />
::* In a time-course experiment in which female fathead minnows were exposed to to 33 or 472 ng 17β-trenbolone/L ex vivo T, ex vivo E2, plasma E2, and plasma VTG all showed concentration-dependent reductions that were consistent with the AOP (Ekman et al. 2011).<br />
::* Exposure of female fathead minnows to spironolactone, a pharmaceutical that binds the fathead minnow AR, for 21 d caused concentration-dependent reductions in cumulative fecundity, plasma VTG and VTG mRNA expression, and plasma E2 concentrations. The frequency and severity of females with decreased yolk accumulation, and increased oocyte atresia was concentration-dependent. The chemical also induced phenotypic masculinization in female fish. (Lalone et al. 2013).<br />
::* Exposure of female medaka to spironolactone caused concentration-dependent reductions in cumulative fecundity and VTG mRNA expression (impacts on steroid hormone concentrations were not measured). Spironolactone also caused phenotypic masculinization of female medaka (Lalone et al. 2013).<br />
<br />
: '''Temporal concordance among the key events and adverse effect''': Temporal concordance between activation of the AR as a nuclear transcription factor and onset of a negative feedback response resulting in decreased gonadotropin secretion has not been established. Temporal concordance of the key events starting with reduced T biosynthesis and proceeding through reductions in plasma vitellogenin has been established [https://aopkb.org/aopwiki/images/d/dc/AR_agonism_concordance_table.pdf Concordance table]. Temporal concordance beyond the key event of reductions in plasma vitellogenin has not been established, in large part due to disconnect in the time-scales over which the events can be measured. For example, most small fish used in reproductive toxicity testing can spawn anywhere from once daily to several days per week. Given the variability in daily spawning rates, it is neither practical nor effective to evaluate cumulative fecundity at a time scale shorter than roughly a week. Since the impacts at lower levels of biological organization can be detected within hours of exposure, lack of impact on cumulative fecundity before the other key events are impacted cannot be effectively measured. Overall, among those key events whose temporal concordance can reasonably be evaluated based on currently available data, the temporal profile observed is consistent with the AOP.<br />
<br />
: '''Consistency''': We are aware of no cases where the pattern of key events described was observed without also observing a significant impact on cumulative fecundity. Due to variability in the cumulative fecundity endpoint and potential compensatory responses ((Villeneuve et al. 2009; Villeneuve et al. 2013; Ankley et al. 2009b; Zhang et al. 2008; Ekman et al. 2012), the cumulative fecundity endpoint can be less sensitive than key events measured at lower levels of biological organization. Nonetheless, the occurrence of the final adverse outcome when the other key events are observed is very consistent. The final adverse effect is not specific to this AOP. Many of the key events included in this AOP overlap with AOPs linking other molecular initiating events to reproductive dysfunction in small fish.<br />
::* In general, there is a consistent body of evidence linking exposure to an AR agonist to decreased T synthesis, E2 synthesis, circulating E2 and VTG concentrations, and cumulative fecundity in female fish. For example, the association between 17β-trenbolone exposure and reduced vitellogenin concentrations in females has been replicated in over a dozen independent experiments (Ekman et al. 2011; Ankley et al. 2003; Jensen et al. 2006; Ankley et al. 2010; Hemmer et al. 2008; Seki et al. 2006; Brockmeier et al. 2013). However, relatively few exogenous, non-aromatizable, AR agonists have been tested. Other than recent work with spironolactone (Lalone et al. 2013), we are not aware of the profile of responses being demonstrated for other AR agonists. <br />
<br />
: '''Uncertainties, inconsistencies, and data gaps''': There are three major areas of uncertainty and data gaps in the current AOP. *First, there remains considerable uncertainty as to the specific mechanism(s) through which AR agonism elicits a negative feedback response at the level of the hypothalamus and/or pituitary. There is also a substantial data gap relative to establishing that exposure to an AR agonist like 17β-trenbolone causes concentration-dependent reductions in circulating gonadotropins. <br />
*The second major uncertainty in this AOP relates to whether there is a direct biological linkage between impaired VTG uptake into oocytes and impaired spawning/reduced cumulative fecundity. Plausible biological connections have been hypothesized, but have not yet been tested experimentally.<br />
*A third uncertainty pertains to the chemical domain of applicability. In vivo, a number of chemicals that are detected as androgens in in vitro screening assays such as receptor binding assays or ligand-activated transcriptional assay can be aromatized to functional estrogens. Thus, in vivo such compounds may produce a profile of effects more consistent with estrogen receptor activation than AR activation or may produced mixed effects characteristic of either estrogen or androgen exposures. Examples of such aromatizable androgens include, testosterone, methyltestosterone, and androstenedione. Consequently, caution is warranted in applying this AOP based on in vitro screening data alone, without consideration for possible conversion to estrogens.<br />
<br />
===Essentiality of the Key Events===<br />
[[#Molecular Initiating Event | Molecular Initiating Event Summary]], <br />
[[#Key Events | Key Event Summary]]<br><br />
<br />
*In general, few studies have directly addressed the essentiality of the proposed sequence of key events.<br />
*Ekman et al. 2011 provide evidence that in fathead minnow, cessation of trenbolone exposure resulted in recovery of plasma E2 and VTG concentrations which were depressed by continuous exposure to 17beta trenbolone. This provides some support for the essentiality of these two key events.<br />
*Essentiality of the proposed negative feedback key event is supported by experimental work that evaluated the ability of AR agonists to reduce T or E2 production in vitro. In vitro exposure of fathead minnow ovary tissue to 17β-trenbolone or spironolactone does not cause reductions in T or E2 synthesis at concentrations comparable to those that produce significant responses in vivo (i.e., at non-cytotoxic concentrations; D.L. Villeneuve, unpublished data; C.A. LaLone unpublished data), nor are there any known reports of 17β-trenbolone directly inhibiting steroid biosynthesis. When tested in an in vitro steroidogenesis assay using H295R adrenal carcinoma cells, trenbolone caused a concentration-dependent increase in estradiol production, as opposed to any reductions in steroid hormone concentrations, an effect that was concurrent with increased transcription of CYP19 (aromatase) in the cell line (Gracia et al. 2007).<br />
<br />
===Quantitative Considerations===<br />
[[#Relationships Among Key Events and the Adverse Outcome | Summary Table]]<br><br />
: '''Assessment of quantitative understanding of the AOP''': At present, the quantitative understanding of the AOP is insufficient to directly link a measure of chemical potency as an AR agonist (e.g., as measured in a transcriptional activation assay) to a predicted effect concentration at the level of cumulative fecundity. However, a number of mechanistic and statistical models are sufficiently developed to facilitate predictions of cumulative outcomes based on intermediate key event measurements such as circulating vitellogenin concentrations. Because the current models were developed based on a fairly limited range of model compounds and species, the general applicability and degree of accuracy and precision in the model-derived predictions remains uncertain.<br />
<br />
===Applicability of the AOP===<br />
[[#Life Stage Applicability | Life Stage Applicability]], <br />
[[#Taxonomic Applicability | Taxonomic Applicability]], <br />
[[#Sex Applicability | Sex Applicability]]<br><br />
<br />
'''Domain(s) of Applicability'''<br />
: '''Chemical''': This AOP applies to non-aromatizable androgens. Compounds which can bind the AR in vitro, but are converted to high potency estrogens in vivo through aromatization do not produce the profile of effects described in the present AOP (e.g., methyltestosterone [Ankley et al. 2001; Pawlowski et al. 2004]; androstenedione [OECD 2007]).<br />
: '''Sex''': The AOP applies to females only.<br />
: '''Life stages''': The relevant life stages for this AOP are reproductively mature adults. This AOP does not apply to adult stages that lack a sexually mature ovary, for example as a result of seasonal or environmentally-induced gonadal senescence (i.e., through control of temperature, photo-period, etc. in a laboratory setting). <br />
: '''Taxonomic''': At present, the assumed taxonomic applicability domain of this AOP is iteroparous teleost fish species. However, to date the majority of toxicological data on which this AOP is based has been limited to two small fish species, fathead minnow (Pimephales promelas) and Japanese medaka (Oryzias latipes). Given that species dependent differences in endocrine feedback responses, likely associated with different reproductive strategies, have been reported the applicability domain may prove more restricted than currently assumed.<br />
::* Reductions in plasma VTG concentrations and/or hepatic VTG mRNA abundance in females following exposure to 17β-trenbolone has been observed in Pimephales promelas, Oryzias latipes, Danio rerio, (Seki et al. 2006), Cyprinodon variegatus (Hemmer et al. 2008), Gambusia holbrooki and Gambusia affinis (Brockmeier et al. 2013)<br />
::* European eel may be an exception to the generalizability of the negative feedback response to a non-aromatizable xenoandrogen (Huang et al. 1997).<br />
<br />
== Considerations for Potential Applications of the AOP (optional) ==<br />
<br />
== References ==<br />
<br />
<references /><br />
*1. Amano M, Iigo M, Ikuta K, Kitamura S, Yamada H, Yamamori K. 2000. Roles of melatonin in gonadal maturation of underyearling precocious male masu salmon. General and comparative endocrinology 120(2): 190-197.<br />
*2. Ankley GT, Bencic D, Cavallin JE, Jensen KM, Kahl MD, Makynen EA, et al. 2009b. Dynamic nature of alterations in the endocrine system of fathead minnows exposed to the fungicide prochloraz. Toxicol Sci 112(2): 344-353.<br />
*3. Ankley GT, Bencic DC, Cavallin JE, Jensen KM, Kahl MD, Makynen EA, et al. 2009a. Dynamic nature of alterations in the endocrine system of fathead minnows exposed to the fungicide prochloraz. Toxicological sciences : an official journal of the Society of Toxicology 112(2): 344-353.<br />
*4. Ankley GT, Cavallin JE, Durhan EJ, Jensen KM, Kahl MD, Makynen EA, et al. 2012. A time-course analysis of effects of the steroidogenesis inhibitor ketoconazole on components of the hypothalamic-pituitary-gonadal axis of fathead minnows. Aquatic toxicology 114-115: 88-95.<br />
*5. Ankley GT, Jensen KM, Durhan EJ, Makynen EA, Butterworth BC, Kahl MD, et al. 2005. Effects of two fungicides with multiple modes of action on reproductive endocrine function in the fathead minnow (Pimephales promelas). Toxicol Sci 86(2): 300-308.<br />
*6. Ankley GT, Jensen KM, Kahl MD, Durhan EJ, Makynen EA, Cavallin JE, et al. 2010. Use of chemical mixtures to differentiate mechanisms of endocrine action in a small fish model. Aquatic toxicology 99(3): 389-396.<br />
*7. Ankley GT, Jensen KM, Kahl MD, Makynen EA, Blake LS, Greene KJ, et al. 2007. Ketoconazole in the fathead minnow (Pimephales promelas): reproductive toxicity and biological compensation. Environ Toxicol Chem 26(6): 1214-1223.<br />
*8. Ankley GT, Jensen KM, Makynen EA, Kahl MD, Korte JJ, Hornung MW, et al. 2003. Effects of the androgenic growth promoter 17--trenbolone on fecundity and reproductive endocrinology of the fathead minnow. Environmental Toxicology and Chemistry 22(6): 1350-1360.<br />
*9. Ankley GT, Kahl MD, Jensen KM, Hornung MW, Korte JJ, Makynen EA, et al. 2002. Evaluation of the aromatase inhibitor fadrozole in a short-term reproduction assay with the fathead minnow (Pimephales promelas). Toxicological Sciences 67: 121-130.<br />
*10. Ankley GT, Miller DH, Jensen KM, Villeneuve DL, Martinovic D. 2008. Relationship of plasma sex steroid concentrations in female fathead minnows to reproductive success and population status. Aquatic toxicology 88(1): 69-74.<br />
*11. Araki N, Ohno K, Nakai M, Takeyoshi M, Iida M. 2005. Screening for androgen receptor activities in 253 industrial chemicals by in vitro reporter gene assays using AR-EcoScreen cells. Toxicology in vitro : an international journal published in association with BIBRA 19(6): 831-842.<br />
*12. Arukwe A, Goksøyr A. 2003. Eggshell and egg yolk proteins in fish: hepatic proteins for the next generation: oogenetic, population, and evolutionary implications of endocrine disruption. Comparative Hepatology 2(4): 1-21.<br />
*13. Baker ME. 1997. Steroid receptor phylogeny and vertebrate origins. Molecular and cellular endocrinology 135(2): 101-107.<br />
*14. Baker ME. 2011. Origin and diversification of steroids: co-evolution of enzymes and nuclear receptors. Molecular and cellular endocrinology 334(1-2): 14-20.<br />
*15. Benninghoff AD, Thomas P. 2006. Gonadotropin regulation of testosterone production by primary cultured theca and granulosa cells of Atlantic croaker: I. Novel role of CaMKs and interactions between calcium- and adenylyl cyclase-dependent pathways. General and comparative endocrinology 147(3): 276-287.<br />
*16. Biales AD, Bencic DC, Lazorchak JL, Lattier DL. 2007. A quantitative real-time polymerase chain reaction method for the analysis of vitellogenin transcripts in model and nonmodel fish species. Environ Toxicol Chem 26(12): 2679-2686.<br />
*17. Bohl CE, Chang C, Mohler ML, Chen J, Miller DD, Swaan PW, et al. 2004. A ligand-based approach to identify quantitative structure-activity relationships for the androgen receptor. Journal of medicinal chemistry 47(15): 3765-3776.<br />
*18. Bowman CJ, Kroll KJ, Hemmer MJ, Folmar LC, Denslow ND. 2000. Estrogen-induced vitellogenin mRNA and protein in sheepshead minnow (Cyprinodon variegatus). General and comparative endocrinology 120(3): 300-313.<br />
*19. Breen MS, Villeneuve DL, Breen M, Ankley GT, Conolly RB. 2007. Mechanistic computational model of ovarian steroidogenesis to predict biochemical responses to endocrine active compounds. Annals of biomedical engineering 35(6): 970-981.<br />
*20. Brockmeier EK, Ogino Y, Iguchi T, Barber DS, Denslow ND. 2013. Effects of 17beta-trenbolone on Eastern and Western mosquitofish (Gambusia holbrooki and G. affinis) anal fin growth and gene expression patterns. Aquatic toxicology 128-129: 163-170.<br />
*21. Campbell BK, Baird DT, Webb R. 1998. Effects of dose of LH on androgen production and luteinization of ovine theca cells cultured in a serum-free system. Journal of reproduction and fertility 112(1): 69-77.<br />
*22. Centenera MM, Harris JM, Tilley WD, Butler LM. 2008. The contribution of different androgen receptor domains to receptor dimerization and signaling. Molecular endocrinology 22(11): 2373-2382.<br />
*23. Cheng GF, Yuen CW, Ge W. 2007. Evidence for the existence of a local activin follistatin negative feedback loop in the goldfish pituitary and its regulation by activin and gonadal steroids. The Journal of endocrinology 195(3): 373-384.<br />
*24. Claessens F, Denayer S, Van Tilborgh N, Kerkhofs S, Helsen C, Haelens A. 2008. Diverse roles of androgen receptor (AR) domains in AR-mediated signaling. Nuclear receptor signaling 6: e008.<br />
*25. Cutress ML, Whitaker HC, Mills IG, Stewart M, Neal DE. 2008. Structural basis for the nuclear import of the human androgen receptor. Journal of cell science 121(Pt 7): 957-968.<br />
*26. Eick GN, Thornton JW. 2011. Evolution of steroid receptors from an estrogen-sensitive ancestral receptor. Molecular and cellular endocrinology 334(1-2): 31-38.<br />
*27. Ekman DR, Hartig PC, Cardon M, Skelton DM, Teng Q, Durhan EJ, et al. 2012. Metabolite profiling and a transcriptional activation assay provide direct evidence of androgen receptor antagonism by bisphenol A in fish. Environmental science & technology 46(17): 9673-9680.<br />
*28. Ekman DR, Villeneuve DL, Teng Q, Ralston-Hooper KJ, Martinovic-Weigelt D, Kahl MD, et al. 2011. Use of gene expression, biochemical and metabolite profiles to enhance exposure and effects assessment of the model androgen 17beta-trenbolone in fish. Environmental toxicology and chemistry / SETAC 30(2): 319-329.<br />
*29. Eppig JJ. 1994. Further reflections on culture systems for the growth of oocytes in vitro. Human reproduction 9(6): 974-976.<br />
*30. Genovese G, Regueira M, Piazza Y, Towle DW, Maggese MC, Lo Nostro F. 2012. Time-course recovery of estrogen-responsive genes of a cichlid fish exposed to waterborne octylphenol. Aquatic toxicology 114-115: 1-13.<br />
*31. Gopurappilly R, Ogawa S, Parhar IS. 2013. Functional significance of GnRH and kisspeptin, and their cognate receptors in teleost reproduction. Frontiers in endocrinology 4: 24.<br />
*32. Govoroun M, Chyb J, Breton B. 1998. Immunological cross-reactivity between rainbow trout GTH I and GTH II and their alpha and beta subunits: application to the development of specific radioimmunoassays. General and comparative endocrinology 111(1): 28-37.<br />
*33. Gracia T, Hilscherova K, Jones PD, Newsted JL, Higley EB, Zhang X, et al. 2007. Modulation of steroidogenic gene expression and hormone production of H295R cells by pharmaceuticals and other environmentally active compounds. Toxicology and applied pharmacology 225(2): 142-153.<br />
*34. Habibi HR, Huggard DL. 1998. Testosterone regulation of gonadotropin production in goldfish. Comparative biochemistry and physiology Part C, Pharmacology, toxicology & endocrinology 119(3): 339-344.<br />
*35. Havelock JC, Rainey WE, Carr BR. 2004. Ovarian granulosa cell lines. Molecular and cellular endocrinology 228(1-2): 67-78.<br />
*36. Heemers HV, Tindall DJ. 2007. Androgen receptor (AR) coregulators: a diversity of functions converging on and regulating the AR transcriptional complex. Endocrine reviews 28(7): 778-808.<br />
*37. Hemmer MJ, Cripe GM, Hemmer BL, Goodman LR, Salinas KA, Fournie JW, et al. 2008. Comparison of estrogen-responsive plasma protein biomarkers and reproductive endpoints in sheepshead minnows exposed to 17beta-trenbolone. Aquatic toxicology 88(2): 128-136.<br />
*38. Hong H, Fang H, Xie Q, Perkins R, Sheehan DM, Tong W. 2003. Comparative molecular field analysis (CoMFA) model using a large diverse set of natural, synthetic and environmental chemicals for binding to the androgen receptor. SAR and QSAR in environmental research 14(5-6): 373-388.<br />
*39. Iguchi T, Irie F, Urushitani H, Tooi O, Kawashima Y, Roberts M, et al. 2006. Availability of in vitro vitellogenin assay for screening of estrogenic and anti-estrogenic activities of environmental chemicals. Environ Sci 13(3): 161-183.<br />
*40. Jamnongjit M, Hammes SR. 2005. Oocyte maturation: the coming of age of a germ cell. Seminars in reproductive medicine 23(3): 234-241.<br />
*41. Jensen K, Korte J, Kahl M, Pasha M, Ankley G. 2001. Aspects of basic reproductive biology and endocrinology in the fathead minnow (Pimephales promelas). Comparative Biochemistry and Physiology Part C 128: 127-141.<br />
*42. Jensen KM, Makynen EA, Kahl MD, Ankley GT. 2006. Effects of the feedlot contaminant 17alpha-trenbolone on reproductive endocrinology of the fathead minnow. Environmental science & technology 40(9): 3112-3117.<br />
*43. Jolly C, Katsiadaki I, Le Belle N, Mayer I, Dufour S. 2006. Development of a stickleback kidney cell culture assay for the screening of androgenic and anti-androgenic endocrine disrupters. Aquatic toxicology 79(2): 158-166.<br />
*44. Kah O, Pontet A, Nunez Rodriguez J, Calas A, Breton B. 1989. Development of an enzyme-linked immunosorbent assay for goldfish gonadotropin. Biology of reproduction 41(1): 68-73.<br />
*45. Kim TS, Yoon CY, Jung KK, Kim SS, Kang IH, Baek JH, et al. 2010. In vitro study of Organization for Economic Co-operation and Development (OECD) endocrine disruptor screening and testing methods- establishment of a recombinant rat androgen receptor (rrAR) binding assay. The Journal of toxicological sciences 35(2): 239-243.<br />
*46. Korte JJ, Kahl MD, Jensen KM, Mumtaz SP, Parks LG, LeBlanc GA, et al. 2000. Fathead minnow vitellogenin: complementary DNA sequence and messenger RNA and protein expression after 17B-estradiol treatment. Environmental Toxicology and Chemistry 19(4): 972-981.<br />
*47. Lalone CA, Villeneuve DL, Cavallin JE, Kahl MD, Durhan EJ, Makynen EA, et al. 2013. Cross species sensitivity to a novel androgen receptor agonist of environmental concern, spironolactone. Environmental toxicology and chemistry / SETAC (in press).<br />
*48. Leino R, Jensen K, Ankley G. 2005. Gonadal histology and characteristic histopathology associated with endocrine disruption in the adult fathead minnow. Environmental Toxicology and Pharmacology 19: 85-98.<br />
*49. Levavi-Sivan B, Bogerd J, Mananos EL, Gomez A, Lareyre JJ. 2010. Perspectives on fish gonadotropins and their receptors. General and comparative endocrinology 165(3): 412-437.<br />
*50. Li Z, Kroll KJ, Jensen KM, Villeneuve DL, Ankley GT, Brian JV, et al. 2011a. A computational model of the hypothalamic: pituitary: gonadal axis in female fathead minnows (Pimephales promelas) exposed to 17alpha-ethynylestradiol and 17beta-trenbolone. BMC systems biology 5: 63.<br />
*51. Li Z, Villeneuve DL, Jensen KM, Ankley GT, Watanabe KH. 2011b. A computational model for asynchronous oocyte growth dynamics in a batch-spawning fish. Can J Fish Aquat Sci 68: 1528-1538.<br />
*52. Magoffin DA. 2005. Ovarian theca cell. The international journal of biochemistry & cell biology 37(7): 1344-1349.<br />
*53. Mak P, Cruz FD, Chen S. 1999. A yeast screen system for aromatase inhibitors and ligands for androgen receptor: yeast cells transformed with aromatase and androgen receptor. Environmental health perspectives 107(11): 855-860.<br />
*54. Markov GV, Laudet V. 2011. Origin and evolution of the ligand-binding ability of nuclear receptors. Molecular and cellular endocrinology 334(1-2): 21-30.<br />
*55. McMaster ME MK, Jardine JJ, Robinson RD, Van Der Kraak GJ. 1995. Protocol for measuring in vitro steroid production by fish gonadal tissue. Canadian Technical Report of Fisheries and Aquatic Sciences 1961 1961: 1-78.<br />
*56. Miller DH, Ankley GT. 2004. Modeling impacts on populations: fathead minnow (Pimephales promelas) exposure to the endocrine disruptor 17trenbolone as a case study. Ecotoxicology and Environmental Safety 59: 1-9.<br />
*57. Miller DH, Jensen KM, Villeneuve DL, Kahl MD, Makynen EA, Durhan EJ, et al. 2007. Linkage of biochemical responses to population-level effects: a case study with vitellogenin in the fathead minnow (Pimephales promelas). Environ Toxicol Chem 26(3): 521-527.<br />
*58. Miller DH, Tietge JE, McMaster ME, Munkittrick KR, Xia X, Ankley GT. 2013. Assessment of Status of White Sucker (Catostomus Commersoni) Populations Exposed to Bleached Kraft Pulp Mill Effluent. Environmental toxicology and chemistry / SETAC.<br />
*59. Miller WL, Strauss JF, 3rd. 1999. Molecular pathology and mechanism of action of the steroidogenic acute regulatory protein, StAR. The Journal of steroid biochemistry and molecular biology 69(1-6): 131-141.<br />
*60. Miller WL. 1988. Molecular biology of steroid hormone synthesis. Endocrine reviews 9(3): 295-318.<br />
*61. Murphy CA, Rose KA, Rahman MS, Thomas P. 2009. Testing and applying a fish vitellogenesis model to evaluate laboratory and field biomarkers of endocrine disruption in Atlantic croaker (Micropogonias undulatus) exposed to hypoxia. Environmental toxicology and chemistry / SETAC 28(6): 1288-1303.<br />
*62. Murphy CA, Rose KA, Thomas P. 2005. Modeling vitellogenesis in female fish exposed to environmental stressors: predicting the effects of endocrine disturbance due to exposure to a PCB mixture and cadmium. Reproductive toxicology 19(3): 395-409.<br />
*63. Nagahama Y, Yoshikumi M, Yamashita M, Sakai N, Tanaka M. 1993. Molecular endocrinology of oocyte growth and maturation in fish. Fish Physiology and Biochemistry 11: 3-14.<br />
*64. Navas JM, Segner H. 2006. Vitellogenin synthesis in primary cultures of fish liver cells as endpoint for in vitro screening of the (anti)estrogenic activity of chemical substances. Aquat Toxicol 80(1): 1-22.<br />
*65. Norris DO. 2007. Vertebrate Endocrinology. Fourth ed. New York: Academic Press.<br />
*66. Norris JD, Joseph JD, Sherk AB, Juzumiene D, Turnbull PS, Rafferty SW, et al. 2009. Differential presentation of protein interaction surfaces on the androgen receptor defines the pharmacological actions of bound ligands. Chemistry & biology 16(4): 452-460.<br />
*67. Oakley AE, Clifton DK, Steiner RA. 2009. Kisspeptin signaling in the brain. Endocrine reviews 30(6): 713-743.<br />
*68. OECD. 2012. Test No. 229: Fish Short Term Reproduction Assay. Paris, France:Organization for Economic Cooperation and Development.<br />
*69. Olsson P-E, Berg A, von Hofsten J, Grahn B, Hellqvist A, Larsson A, et al. 2005. Molecular cloning and characterization of a nuclear androgen receptor activated by 11-ketotestosterone. Reproductive Biology and Endocrinology 3: 1-17.<br />
*70. Payne AH, Hales DB. 2004. Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocrine reviews 25(6): 947-970.<br />
*71. Prat F, Sumpter JP, Tyler CR. 1996. Validation of radioimmunoassays for two salmon gonadotropins (GTH I and GTH II) and their plasma concentrations throughout the reproductivecycle in male and female rainbow trout (Oncorhynchus mykiss). Biology of reproduction 54(6): 1375-1382.<br />
*72. Prescott J, Coetzee GA. 2006. Molecular chaperones throughout the life cycle of the androgen receptor. Cancer letters 231(1): 12-19.<br />
*73. Quignot N, Bois FY. 2013. A computational model to predict rat ovarian steroid secretion from in vitro experiments with endocrine disruptors. PloS one 8(1): e53891.<br />
*74. Schmid T, Gonzalez-Valero J, Rufli H, Dietrich DR. 2002. Determination of vitellogenin kinetics in male fathead minnows (Pimephales promelas). Toxicol Lett 131(1-2): 65-74.<br />
*75. Schmieder P, Tapper M, Linnum A, Denny J, Kolanczyk R, Johnson R. 2000. Optimization of a precision-cut trout liver tissue slice assay as a screen for vitellogenin induction: comparison of slice incubation techniques. Aquat Toxicol 49(4): 251-268.<br />
*76. Schultz IR, Orner G, Merdink JL, Skillman A. 2001. Dose-response relationships and pharmacokinetics of vitellogenin in rainbow trout after intravascular administration of 17alpha-ethynylestradiol. Aquatic toxicology 51(3): 305-318.<br />
*77. Seki M, Fujishima S, Nozaka T, Maeda M, Kobayashi K. 2006. Comparison of response to 17 beta-estradiol and 17 beta-trenbolone among three small fish species. Environmental toxicology and chemistry / SETAC 25(10): 2742-2752.<br />
*78. Serafimova R, Walker J, Mekenyan O. 2002. Androgen receptor binding affinity of pesticide "active" formulation ingredients. QSAR evaluation by COREPA method. SAR and QSAR in environmental research 13(1): 127-134.<br />
*79. Shoemaker JE, Gayen K, Garcia-Reyero N, Perkins EJ, Villeneuve DL, Liu L, et al. 2010. Fathead minnow steroidogenesis: in silico analyses reveals tradeoffs between nominal target efficacy and robustness to cross-talk. BMC systems biology 4: 89.<br />
*80. Skolness SY, Blanksma CA, Cavallin JE, Churchill JJ, Durhan EJ, Jensen KM, et al. 2013. Propiconazole Inhibits Steroidogenesis and Reproduction in the Fathead Minnow (Pimephales promelas). Toxicological sciences : an official journal of the Society of Toxicology 132(2): 284-297.<br />
*81. Skolness SY, Durhan EJ, Garcia-Reyero N, Jensen KM, Kahl MD, Makynen EA, et al. 2011. Effects of a short-term exposure to the fungicide prochloraz on endocrine function and gene expression in female fathead minnows (Pimephales promelas). Aquat Toxicol 103(3-4): 170-178.<br />
*82. Sower SA, Freamat M, Kavanaugh SI. 2009. The origins of the vertebrate hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-thyroid (HPT) endocrine systems: new insights from lampreys. General and comparative endocrinology 161(1): 20-29.<br />
*83. Sperry TS, Thomas P. 1999. Identification of two nuclear androgen receptors in kelp bass (Paralabrax clathratus) and their binding affinities for xenobiotics: comparison with Atlantic croaker (Micropogonias undulatus) androgen receptors. Biology of reproduction 61(4): 1152-1161.<br />
*84. Sun L, Wen L, Shao X, Qian H, Jin Y, Liu W, et al. 2010. Screening of chemicals with anti-estrogenic activity using in vitro and in vivo vitellogenin induction responses in zebrafish (Danio rerio). Chemosphere 78(7): 793-799.<br />
*85. Sun L, Zha J, Spear PA, Wang Z. 2007. Toxicity of the aromatase inhibitor letrozole to Japanese medaka (Oryzias latipes) eggs, larvae and breeding adults. Comp Biochem Physiol C Toxicol Pharmacol 145(4): 533-541.<br />
*86. Thornton JW. 2001. Evolution of vertebrate steroid receptors from an ancestral estrogen receptor by ligand exploitation and serial genome expansions. Proceedings of the National Academy of Sciences of the United States of America 98(10): 5671-5676.<br />
*87. Tilley WD, Marcelli M, Wilson JD, McPhaul MJ. 1989. Characterization and expression of a cDNA encoding the human androgen receptor. Proceedings of the National Academy of Sciences of the United States of America 86(1): 327-331.<br />
*88. Todorov M, Mombelli E, Ait-Aissa S, Mekenyan O. 2011. Androgen receptor binding affinity: a QSAR evaluation. SAR and QSAR in environmental research 22(3): 265-291.<br />
*89. Trudeau VL, Spanswick D, Fraser EJ, Lariviére K, Crump D, Chiu S, et al. 2000. The role of amino acid neurotransmitters in the regulation of pituitary gonadotropin release in fish. Biochemistry and Cell Biology 78: 241-259.<br />
*90. Trudeau VL. 1997. Neuroendocrine regulation of gonadotropin II release and gonadal growth in the goldfish, Carassius auratus. Reviews of Reproduction 2: 55-68.<br />
*91. Tyler C, Sumpter J. 1996. Oocyte growth and development in teleosts. Reviews in Fish Biology and Fisheries 6: 287-318.<br />
*92. Tyler C, van der Eerden B, Jobling S, Panter G, Sumpter J. 1996. Measurement of vitellogenin, a biomarker for exposure to oestrogenic chemicals, in a wide variety of cyprinid fish. Journal of Comparative Physiology and Biology 166: 418-426.<br />
*93. van der Burg B, Winter R, Man HY, Vangenechten C, Berckmans P, Weimer M, et al. 2010. Optimization and prevalidation of the in vitro AR CALUX method to test androgenic and antiandrogenic activity of compounds. Reproductive toxicology 30(1): 18-24.<br />
*94. Villeneuve DL, Ankley GT, Makynen EA, Blake LS, Greene KJ, Higley EB, et al. 2007. Comparison of fathead minnow ovary explant and H295R cell-based steroidogenesis assays for identifying endocrine-active chemicals. Ecotoxicol Environ Saf 68(1): 20-32.<br />
*95. Villeneuve DL, Breen M, Bencic DC, Cavallin JE, Jensen KM, Makynen EA, et al. 2013. Developing Predictive Approaches to Characterize Adaptive Responses of the Reproductive Endocrine Axis to Aromatase Inhibition: I. Data Generation in a Small Fish Model. Toxicological sciences : an official journal of the Society of Toxicology.<br />
*96. Villeneuve DL, Mueller ND, Martinovic D, Makynen EA, Kahl MD, Jensen KM, et al. 2009. Direct effects, compensation, and recovery in female fathead minnows exposed to a model aromatase inhibitor. Environ Health Perspect 117(4): 624-631.<br />
*97. Waller CL, Juma BW, Gray EJ, Kelce WR. 1996. Three-dimensional quantitative structure-activity relationships for androgen receptor ligands. Toxicology and Applied Pharmacolgy 137: 219-227.<br />
*98. Wilson VS, Bobseine K, Lambright CR, Gray LE. 2002. A novel cell line, MDA-kb2, that stably expresses an androgen- and glucocorticoid-responsive reporter for the detection of hormone receptor agonists and antagonists. Toxicological Sciences 66: 69-81.<br />
*99. Wilson VS, Cardon MC, Gray LE, Jr., Hartig PC. 2007. Competitive binding comparison of endocrine-disrupting compounds to recombinant androgen receptor from fathead minnow, rainbow trout, and human. Environmental toxicology and chemistry / SETAC 26(9): 1793-1802.<br />
*100. Wolf JC, Dietrich DR, Friederich U, Caunter J, Brown AR. 2004. Qualitative and quantitative histomorphologic assessment of fathead minnow Pimephales promelas gonads as an endpoint for evaluating endocrine-active compounds: a pilot methodology study. Toxicol Pathol 32(5): 600-612.<br />
*101. Yaron Z. 1995. Endocrine control of gametogenesis and spawning induction in the carp. Aquaculture 129: 49-73.<br />
*102. Yin D, He Y, Perera MA, Hong SS, Marhefka C, Stourman N, et al. 2003. Key structural features of nonsteroidal ligands for binding and activation of the androgen receptor. Molecular pharmacology 63(1): 211-223.<br />
*103. Young JM, McNeilly AS. 2010. Theca: the forgotten cell of the ovarian follicle. Reproduction 140(4): 489-504.<br />
*104. Zhang X, Hecker M, Tompsett AR, Park JW, Jones PD, Newsted J, et al. 2008. Responses of the medaka HPG axis PCR array and reproduction to prochloraz and ketoconazole. Environ Sci Technol 42(17): 6762-6769.<br />
*105. Organization for Economic Cooperation and Development. 2007. Report of Eight 21-day Fish Endocrine Screening Assays With Additional Test Substances for Phase-3 of the OECD Validation Program: Studies with Octylphenol in the Fathead Minnow (Pimephales promelas) and Zebrafish (Danio rerio) and with Sodium Pentachlorophenol and Androstenedione in the Fathead Minnow (Pimephales promelas). Phase-3 OECD 21-day Fish Screening Assay Validation Report Additional Test Substances Studies. Paris, France.<br />
*106. Pawlowski S, Sauer A, Shears JA, Tyler CR, Braunbeck T. 2004. Androgenic and estrogenic effects of the synthetic androgen 17α-methyltestosterone on sexual development and reproductive performance in the fathead minnow (Pimephales promelas) determined using the gonadal recrudescence assay. Aquat Toxicol 68:277-291.<br />
*107. Ankley GT, Jensen KM, Kahl MD, Korte JJ, Makynen EA. 2001. Description and evaluation of a short-term reproduction test with the fathead minnow (Pimephales promelas). Environ Toxicol Chem 20:1276-1290.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:423Event:4232016-07-06T17:16:24Z<p>Dvillene: /* Evidence Supporting Taxonomic Applicability */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Clonal Expansion / Cell Proliferation to Form Pre-Neoplastic Altered Hepatic Foci </div><br />
<div id='shortTitle' class='Title2'> Pre-neoplastic AHF Lesion Formation </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Level of Biological Organization : Organ ==<br />
Altered hepatic foci (AHF) are formed in the liver; they are clonal in origin, and are identified with immunohistochemical techniques, typically due to their synthesis/expression of GST-P.<br />
<br />
== How this Key Event works ==<br />
The occurrence of altered hepatic foci (AHF) as precursors to liver tumors in AFB1-treated rats has been recognized for decades. Originally, these foci were observed as histologically different from the surrounding parenchyma. (Harada et al., 1989, 1990; Gil et al., 1988; Bannasch et al., 1985). In addition, enzyme alterations were used to identify AHF foci, most notably, the occurrence of a placental form of glutathione-S-transferase (GSTP+). (Godlewski et al., 1985; Dragan et al., 1994a, 1995; Kirby et al., 1990) The growth and occurrence of foci are expressed as the number of AHF in a volume of liver, possibly the entire liver, and the volume fraction of the liver occupied by AHF. (Dragan et al., 1997) Both of these reflect focal growth because single cell foci are not detectable with the immunohistochemical staining technique. The assumption is that single transformed cells in which apoptosis is blocked by tumor-critical mutations will grow into AHF. (Grassl-Kraupp et al., 1997). A number of agents regarded as tumor promoters appear to enhance the growth of foci, acting further to inhibit apoptosis and also creating an overall proliferative stimulus. (Angsubhakorn et al., 2002; Wyde et al., 2002). <br />
<br />
AFB1 appears to be a “complete” carcinogen in that the toxin acts as an initiator through the formation of pro-mutagenic DNA adducts (the MIE) and as a promoter through increasing oxidative stress and inflammation. (Ohnishi et al., 2013; Caballero et al., 2004).<br />
<br />
== Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
Chemoprevention studies, reviewed in another section of this AOP, suggest a strong relationship between altered hepatic foci (AHF) and HCC tumor formation (Olden and Vulimiri, 2014; Liby et al., 2008; Yates et al., 2007; Yates and Kensler, 2007; Kensler et al., 2004). For example, Johnson et al. (2014) observed background levels of AHF along with a complete absence of tumors in rats treated with a triterpenoid chemoprotectant CDDO-Im, despite maintaining a significant burden of AFB1-induced adducts. (Johnson et al., 2014) Cell proliferation appears to be six- to seven-fold greater in AHF than in surrounding liver parenchyma. (Dragan et al., 1994) However, the measurements were made from liver biopsies, and whether the increased expression was associated with foci is not known.<br />
<br />
== How it is Measured or Detected ==<br />
Quantitative stereology has been used to quantify the growth of AHF (Pitot et al., 1996; Dragan et al., 1995; Xu et al., 1990). Growth of foci appears to follow the Moolgavkar-Venzon-Knudson model of initiation and promotion. (Dewanji et al., 1991; Dragan et al., 1995) Most recently, Johnson et al. (2014) have shown that a chemoprotective agent reduces the occurrence of AHF to background levels and completely protects against tumors, although pro-mutagenic adducts are still present at easily quantifiable levels.<br />
<br />
== Evidence Supporting Taxonomic Applicability ==<br />
The occurrence of AHF appears to be universal and has been observed in mammals, including humans, as well as in birds and in fish. (Ribback et al., 2013; Thoolen et al., 2012; Kirby et al., 1990).<br />
<br />
== References ==<br />
Angsubhakorn S, Pradermwong A, Phanwichien K, Nguansangiam S (2002) Promotion of aflatoxin B1-induced hepatocarcinogenesis by dichlorodiphenyl trichloroethane (DDT). Southeast Asian J Trop Med Public Health 33: 613-623.<br />
<br />
Bannasch P, Benner U, Enzmann H, Hacker HJ (1985) Tigroid cell foci and neoplastic nodules in the liver of rats treated with a single dose of aflatoxin B1. Carcinogenesis 6: 1641-1648.<br />
<br />
Caballero F, Meiss R, Gimenez A, Batlle A, Vazquez E (2004) Immunohistochemical analysis of heme oxygenase-1 in preneoplastic and neoplastic lesions during chemical hepatocarcinogenesis. Int J Exp Pathol 85: 213-222.<br />
<br />
Dewanji A, Moolgavkar SH, Luebeck EG (1991) Two-mutation model for carcinogenesis: joint analysis of premalignant and malignant lesions. Math Biosci 104: 97-109.<br />
<br />
Dragan Y, Teeguarden J, Campbell H, Hsia S, Pitot H (1995a) The quantitation of altered hepatic foci during multistage hepatocarcinogenesis in the rat: transforming growth factor alpha expression as a marker for the stage of progression. Cancer Lett 93: 73-83.<br />
<br />
Dragan YP, Campbell HA, Baker K, Vaughan J, Mass M, Pitot HC (1994) Focal and non-focal hepatic expression of placental glutathione S-transferase in carcinogen-treated rats. Carcinogenesis 15: 2587-2591.<br />
<br />
Dragan YP, Campbell HA, Xu XH, Pitot HC (1997) Quantitative stereological studies of a 'selection' protocol of hepatocarcinogenesis following initiation in neonatal male and female rats. Carcinogenesis 18: 149-158.<br />
<br />
Dragan YP, Hully J, Baker K, Crow R, Mass MJ, Pitot HC (1995b) Comparison of experimental and theoretical parameters of the Moolgavkar-Venzon-Knudson incidence function for the stages of initiation and promotion in rat hepatocarcinogenesis. Toxicology 102: 161-175.<br />
<br />
Gil R, Callaghan R, Boix J, Pellin A, Llombart-Bosch A (1988) Morphometric and cytophotometric nuclear analysis of altered hepatocyte foci induced by N-nitrosomorpholine (NNM) and aflatoxin B1 (AFB1) in liver of Wistar rats. Virchows Arch B Cell Pathol Incl Mol Pathol 54: 341-349.<br />
<br />
Godlewski CE, Boyd JN, Sherman WK, Anderson JL, Stoewsand GS (1985) Hepatic glutathione S-transferase activity and aflatoxin B1-induced enzyme altered foci in rats fed fractions of brussels sprouts. Cancer Lett 28: 151-157.<br />
<br />
Grasl-Kraupp B, Ruttkay-Nedecky B, Müllauer L, Taper H, Huber W, et al (1997) Inherent increase of apoptosis in liver tumors: implications for carcinogenesis and tumor regression. Hepatology 25: 906-912.<br />
<br />
Harada T, Maronpot RR, Morris RW, Boorman GA (1990) Effects of mononuclear cell leukemia on altered hepatocellular foci in Fischer 344 rats. Vet Pathol 27: 110-116.<br />
<br />
Harada T, Maronpot RR, Morris RW, Stitzel KA, Boorman GA (1989) Morphological and stereological characterization of hepatic foci of cellular alteration in control Fischer 344 rats. Toxicol Pathol 17: 579-593.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, et al (2014) Complete protection against aflatoxin B1-induced liver cancer with triterpenoid: DNA adduct dosimetry, molecular signature and genotoxicity threshold. Cancer Prev Res (Phila) .<br />
<br />
Kirby GM, Stalker M, Metcalfe C, Kocal T, Ferguson H, Hayes MA (1990) Expression of immunoreactive glutathione S-transferases in hepatic neoplasms induced by aflatoxin B1 or 1,2-dimethylbenzanthracene in rainbow trout (Oncorhynchus mykiss). Carcinogenesis 11: 2255-2257.<br />
<br />
Ohnishi S, Ma N, Thanan R, Pinlaor S, Hammam O, et al (2013) DNA damage in inflammation-related carcinogenesis and cancer stem cells. Oxid Med Cell Longev 2013: 387014.<br />
<br />
Pitot HC, Dragan YP, Teeguarden J, Hsia S, Campbell H (1996) Quantitation of multistage carcinogenesis in rat liver. Toxicol Pathol 24: 119-128<br />
<br />
Ribback S, Calvisi DF, Cigliano A, Sailer V, Peters M, et al (2013) Molecular and metabolic changes in human liver clear cell foci resemble the alterations occurring in rat hepatocarcinogenesis. J Hepatol 58: 1147-1156.<br />
<br />
Thoolen B, Ten Kate FJ, van Diest PJ, Malarkey DE, Elmore SA, Maronpot RR (2012) Comparative histomorphological review of rat and human hepatocellular proliferative lesions. J Toxicol Pathol 25: 189-199.<br />
<br />
Wyde ME, Cambre T, Lebetkin M, Eldridge SR, Walker NJ (2002) Promotion of altered hepatic foci by 2,3,7,8-tetrachlorodibenzo-p-dioxin and 17beta-estradiol in male Sprague-Dawley rats. Toxicol Sci 68: 295-303.<br />
<br />
Xu YH, Maronpot R, Pitot HC (1990) Quantitative stereologic study of the effects of varying the time between initiation and promotion on four histochemical markers in rat liver during hepatocarcinogenesis. Carcinogenesis 11: 267-272.<br />
<br />
Xu YH, Campbell HA, Sattler GL, Hendrich S, Maronpot R, et al (1990) Quantitative stereological analysis of the effects of age and sex on multistage hepatocarcinogenesis in the rat by use of four cytochemical markers. Cancer Res 50: 472-479.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:423Event:4232016-07-06T17:16:08Z<p>Dvillene: /* How it is Measured or Detected */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Clonal Expansion / Cell Proliferation to Form Pre-Neoplastic Altered Hepatic Foci </div><br />
<div id='shortTitle' class='Title2'> Pre-neoplastic AHF Lesion Formation </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Level of Biological Organization : Organ ==<br />
Altered hepatic foci (AHF) are formed in the liver; they are clonal in origin, and are identified with immunohistochemical techniques, typically due to their synthesis/expression of GST-P.<br />
<br />
== How this Key Event works ==<br />
The occurrence of altered hepatic foci (AHF) as precursors to liver tumors in AFB1-treated rats has been recognized for decades. Originally, these foci were observed as histologically different from the surrounding parenchyma. (Harada et al., 1989, 1990; Gil et al., 1988; Bannasch et al., 1985). In addition, enzyme alterations were used to identify AHF foci, most notably, the occurrence of a placental form of glutathione-S-transferase (GSTP+). (Godlewski et al., 1985; Dragan et al., 1994a, 1995; Kirby et al., 1990) The growth and occurrence of foci are expressed as the number of AHF in a volume of liver, possibly the entire liver, and the volume fraction of the liver occupied by AHF. (Dragan et al., 1997) Both of these reflect focal growth because single cell foci are not detectable with the immunohistochemical staining technique. The assumption is that single transformed cells in which apoptosis is blocked by tumor-critical mutations will grow into AHF. (Grassl-Kraupp et al., 1997). A number of agents regarded as tumor promoters appear to enhance the growth of foci, acting further to inhibit apoptosis and also creating an overall proliferative stimulus. (Angsubhakorn et al., 2002; Wyde et al., 2002). <br />
<br />
AFB1 appears to be a “complete” carcinogen in that the toxin acts as an initiator through the formation of pro-mutagenic DNA adducts (the MIE) and as a promoter through increasing oxidative stress and inflammation. (Ohnishi et al., 2013; Caballero et al., 2004).<br />
<br />
== Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
Chemoprevention studies, reviewed in another section of this AOP, suggest a strong relationship between altered hepatic foci (AHF) and HCC tumor formation (Olden and Vulimiri, 2014; Liby et al., 2008; Yates et al., 2007; Yates and Kensler, 2007; Kensler et al., 2004). For example, Johnson et al. (2014) observed background levels of AHF along with a complete absence of tumors in rats treated with a triterpenoid chemoprotectant CDDO-Im, despite maintaining a significant burden of AFB1-induced adducts. (Johnson et al., 2014) Cell proliferation appears to be six- to seven-fold greater in AHF than in surrounding liver parenchyma. (Dragan et al., 1994) However, the measurements were made from liver biopsies, and whether the increased expression was associated with foci is not known.<br />
<br />
== How it is Measured or Detected ==<br />
Quantitative stereology has been used to quantify the growth of AHF (Pitot et al., 1996; Dragan et al., 1995; Xu et al., 1990). Growth of foci appears to follow the Moolgavkar-Venzon-Knudson model of initiation and promotion. (Dewanji et al., 1991; Dragan et al., 1995) Most recently, Johnson et al. (2014) have shown that a chemoprotective agent reduces the occurrence of AHF to background levels and completely protects against tumors, although pro-mutagenic adducts are still present at easily quantifiable levels.<br />
<br />
== KE#3: Evidence Supporting Taxonomic Applicability ==<br />
The occurrence of AHF appears to be universal and has been observed in mammals, including humans, as well as in birds and in fish. (Ribback et al., 2013; Thoolen et al., 2012; Kirby et al., 1990).<br />
<br />
== References ==<br />
Angsubhakorn S, Pradermwong A, Phanwichien K, Nguansangiam S (2002) Promotion of aflatoxin B1-induced hepatocarcinogenesis by dichlorodiphenyl trichloroethane (DDT). Southeast Asian J Trop Med Public Health 33: 613-623.<br />
<br />
Bannasch P, Benner U, Enzmann H, Hacker HJ (1985) Tigroid cell foci and neoplastic nodules in the liver of rats treated with a single dose of aflatoxin B1. Carcinogenesis 6: 1641-1648.<br />
<br />
Caballero F, Meiss R, Gimenez A, Batlle A, Vazquez E (2004) Immunohistochemical analysis of heme oxygenase-1 in preneoplastic and neoplastic lesions during chemical hepatocarcinogenesis. Int J Exp Pathol 85: 213-222.<br />
<br />
Dewanji A, Moolgavkar SH, Luebeck EG (1991) Two-mutation model for carcinogenesis: joint analysis of premalignant and malignant lesions. Math Biosci 104: 97-109.<br />
<br />
Dragan Y, Teeguarden J, Campbell H, Hsia S, Pitot H (1995a) The quantitation of altered hepatic foci during multistage hepatocarcinogenesis in the rat: transforming growth factor alpha expression as a marker for the stage of progression. Cancer Lett 93: 73-83.<br />
<br />
Dragan YP, Campbell HA, Baker K, Vaughan J, Mass M, Pitot HC (1994) Focal and non-focal hepatic expression of placental glutathione S-transferase in carcinogen-treated rats. Carcinogenesis 15: 2587-2591.<br />
<br />
Dragan YP, Campbell HA, Xu XH, Pitot HC (1997) Quantitative stereological studies of a 'selection' protocol of hepatocarcinogenesis following initiation in neonatal male and female rats. Carcinogenesis 18: 149-158.<br />
<br />
Dragan YP, Hully J, Baker K, Crow R, Mass MJ, Pitot HC (1995b) Comparison of experimental and theoretical parameters of the Moolgavkar-Venzon-Knudson incidence function for the stages of initiation and promotion in rat hepatocarcinogenesis. Toxicology 102: 161-175.<br />
<br />
Gil R, Callaghan R, Boix J, Pellin A, Llombart-Bosch A (1988) Morphometric and cytophotometric nuclear analysis of altered hepatocyte foci induced by N-nitrosomorpholine (NNM) and aflatoxin B1 (AFB1) in liver of Wistar rats. Virchows Arch B Cell Pathol Incl Mol Pathol 54: 341-349.<br />
<br />
Godlewski CE, Boyd JN, Sherman WK, Anderson JL, Stoewsand GS (1985) Hepatic glutathione S-transferase activity and aflatoxin B1-induced enzyme altered foci in rats fed fractions of brussels sprouts. Cancer Lett 28: 151-157.<br />
<br />
Grasl-Kraupp B, Ruttkay-Nedecky B, Müllauer L, Taper H, Huber W, et al (1997) Inherent increase of apoptosis in liver tumors: implications for carcinogenesis and tumor regression. Hepatology 25: 906-912.<br />
<br />
Harada T, Maronpot RR, Morris RW, Boorman GA (1990) Effects of mononuclear cell leukemia on altered hepatocellular foci in Fischer 344 rats. Vet Pathol 27: 110-116.<br />
<br />
Harada T, Maronpot RR, Morris RW, Stitzel KA, Boorman GA (1989) Morphological and stereological characterization of hepatic foci of cellular alteration in control Fischer 344 rats. Toxicol Pathol 17: 579-593.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, et al (2014) Complete protection against aflatoxin B1-induced liver cancer with triterpenoid: DNA adduct dosimetry, molecular signature and genotoxicity threshold. Cancer Prev Res (Phila) .<br />
<br />
Kirby GM, Stalker M, Metcalfe C, Kocal T, Ferguson H, Hayes MA (1990) Expression of immunoreactive glutathione S-transferases in hepatic neoplasms induced by aflatoxin B1 or 1,2-dimethylbenzanthracene in rainbow trout (Oncorhynchus mykiss). Carcinogenesis 11: 2255-2257.<br />
<br />
Ohnishi S, Ma N, Thanan R, Pinlaor S, Hammam O, et al (2013) DNA damage in inflammation-related carcinogenesis and cancer stem cells. Oxid Med Cell Longev 2013: 387014.<br />
<br />
Pitot HC, Dragan YP, Teeguarden J, Hsia S, Campbell H (1996) Quantitation of multistage carcinogenesis in rat liver. Toxicol Pathol 24: 119-128<br />
<br />
Ribback S, Calvisi DF, Cigliano A, Sailer V, Peters M, et al (2013) Molecular and metabolic changes in human liver clear cell foci resemble the alterations occurring in rat hepatocarcinogenesis. J Hepatol 58: 1147-1156.<br />
<br />
Thoolen B, Ten Kate FJ, van Diest PJ, Malarkey DE, Elmore SA, Maronpot RR (2012) Comparative histomorphological review of rat and human hepatocellular proliferative lesions. J Toxicol Pathol 25: 189-199.<br />
<br />
Wyde ME, Cambre T, Lebetkin M, Eldridge SR, Walker NJ (2002) Promotion of altered hepatic foci by 2,3,7,8-tetrachlorodibenzo-p-dioxin and 17beta-estradiol in male Sprague-Dawley rats. Toxicol Sci 68: 295-303.<br />
<br />
Xu YH, Maronpot R, Pitot HC (1990) Quantitative stereologic study of the effects of varying the time between initiation and promotion on four histochemical markers in rat liver during hepatocarcinogenesis. Carcinogenesis 11: 267-272.<br />
<br />
Xu YH, Campbell HA, Sattler GL, Hendrich S, Maronpot R, et al (1990) Quantitative stereological analysis of the effects of age and sex on multistage hepatocarcinogenesis in the rat by use of four cytochemical markers. Cancer Res 50: 472-479.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:423Event:4232016-07-06T17:15:52Z<p>Dvillene: /* Evidence Supporting Essentiality */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Clonal Expansion / Cell Proliferation to Form Pre-Neoplastic Altered Hepatic Foci </div><br />
<div id='shortTitle' class='Title2'> Pre-neoplastic AHF Lesion Formation </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Level of Biological Organization : Organ ==<br />
Altered hepatic foci (AHF) are formed in the liver; they are clonal in origin, and are identified with immunohistochemical techniques, typically due to their synthesis/expression of GST-P.<br />
<br />
== How this Key Event works ==<br />
The occurrence of altered hepatic foci (AHF) as precursors to liver tumors in AFB1-treated rats has been recognized for decades. Originally, these foci were observed as histologically different from the surrounding parenchyma. (Harada et al., 1989, 1990; Gil et al., 1988; Bannasch et al., 1985). In addition, enzyme alterations were used to identify AHF foci, most notably, the occurrence of a placental form of glutathione-S-transferase (GSTP+). (Godlewski et al., 1985; Dragan et al., 1994a, 1995; Kirby et al., 1990) The growth and occurrence of foci are expressed as the number of AHF in a volume of liver, possibly the entire liver, and the volume fraction of the liver occupied by AHF. (Dragan et al., 1997) Both of these reflect focal growth because single cell foci are not detectable with the immunohistochemical staining technique. The assumption is that single transformed cells in which apoptosis is blocked by tumor-critical mutations will grow into AHF. (Grassl-Kraupp et al., 1997). A number of agents regarded as tumor promoters appear to enhance the growth of foci, acting further to inhibit apoptosis and also creating an overall proliferative stimulus. (Angsubhakorn et al., 2002; Wyde et al., 2002). <br />
<br />
AFB1 appears to be a “complete” carcinogen in that the toxin acts as an initiator through the formation of pro-mutagenic DNA adducts (the MIE) and as a promoter through increasing oxidative stress and inflammation. (Ohnishi et al., 2013; Caballero et al., 2004).<br />
<br />
== Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
Chemoprevention studies, reviewed in another section of this AOP, suggest a strong relationship between altered hepatic foci (AHF) and HCC tumor formation (Olden and Vulimiri, 2014; Liby et al., 2008; Yates et al., 2007; Yates and Kensler, 2007; Kensler et al., 2004). For example, Johnson et al. (2014) observed background levels of AHF along with a complete absence of tumors in rats treated with a triterpenoid chemoprotectant CDDO-Im, despite maintaining a significant burden of AFB1-induced adducts. (Johnson et al., 2014) Cell proliferation appears to be six- to seven-fold greater in AHF than in surrounding liver parenchyma. (Dragan et al., 1994) However, the measurements were made from liver biopsies, and whether the increased expression was associated with foci is not known.<br />
<br />
== KE#3: How it is Measured or Detected ==<br />
Quantitative stereology has been used to quantify the growth of AHF (Pitot et al., 1996; Dragan et al., 1995; Xu et al., 1990). Growth of foci appears to follow the Moolgavkar-Venzon-Knudson model of initiation and promotion. (Dewanji et al., 1991; Dragan et al., 1995) Most recently, Johnson et al. (2014) have shown that a chemoprotective agent reduces the occurrence of AHF to background levels and completely protects against tumors, although pro-mutagenic adducts are still present at easily quantifiable levels.<br />
<br />
== KE#3: Evidence Supporting Taxonomic Applicability ==<br />
The occurrence of AHF appears to be universal and has been observed in mammals, including humans, as well as in birds and in fish. (Ribback et al., 2013; Thoolen et al., 2012; Kirby et al., 1990).<br />
<br />
== References ==<br />
Angsubhakorn S, Pradermwong A, Phanwichien K, Nguansangiam S (2002) Promotion of aflatoxin B1-induced hepatocarcinogenesis by dichlorodiphenyl trichloroethane (DDT). Southeast Asian J Trop Med Public Health 33: 613-623.<br />
<br />
Bannasch P, Benner U, Enzmann H, Hacker HJ (1985) Tigroid cell foci and neoplastic nodules in the liver of rats treated with a single dose of aflatoxin B1. Carcinogenesis 6: 1641-1648.<br />
<br />
Caballero F, Meiss R, Gimenez A, Batlle A, Vazquez E (2004) Immunohistochemical analysis of heme oxygenase-1 in preneoplastic and neoplastic lesions during chemical hepatocarcinogenesis. Int J Exp Pathol 85: 213-222.<br />
<br />
Dewanji A, Moolgavkar SH, Luebeck EG (1991) Two-mutation model for carcinogenesis: joint analysis of premalignant and malignant lesions. Math Biosci 104: 97-109.<br />
<br />
Dragan Y, Teeguarden J, Campbell H, Hsia S, Pitot H (1995a) The quantitation of altered hepatic foci during multistage hepatocarcinogenesis in the rat: transforming growth factor alpha expression as a marker for the stage of progression. Cancer Lett 93: 73-83.<br />
<br />
Dragan YP, Campbell HA, Baker K, Vaughan J, Mass M, Pitot HC (1994) Focal and non-focal hepatic expression of placental glutathione S-transferase in carcinogen-treated rats. Carcinogenesis 15: 2587-2591.<br />
<br />
Dragan YP, Campbell HA, Xu XH, Pitot HC (1997) Quantitative stereological studies of a 'selection' protocol of hepatocarcinogenesis following initiation in neonatal male and female rats. Carcinogenesis 18: 149-158.<br />
<br />
Dragan YP, Hully J, Baker K, Crow R, Mass MJ, Pitot HC (1995b) Comparison of experimental and theoretical parameters of the Moolgavkar-Venzon-Knudson incidence function for the stages of initiation and promotion in rat hepatocarcinogenesis. Toxicology 102: 161-175.<br />
<br />
Gil R, Callaghan R, Boix J, Pellin A, Llombart-Bosch A (1988) Morphometric and cytophotometric nuclear analysis of altered hepatocyte foci induced by N-nitrosomorpholine (NNM) and aflatoxin B1 (AFB1) in liver of Wistar rats. Virchows Arch B Cell Pathol Incl Mol Pathol 54: 341-349.<br />
<br />
Godlewski CE, Boyd JN, Sherman WK, Anderson JL, Stoewsand GS (1985) Hepatic glutathione S-transferase activity and aflatoxin B1-induced enzyme altered foci in rats fed fractions of brussels sprouts. Cancer Lett 28: 151-157.<br />
<br />
Grasl-Kraupp B, Ruttkay-Nedecky B, Müllauer L, Taper H, Huber W, et al (1997) Inherent increase of apoptosis in liver tumors: implications for carcinogenesis and tumor regression. Hepatology 25: 906-912.<br />
<br />
Harada T, Maronpot RR, Morris RW, Boorman GA (1990) Effects of mononuclear cell leukemia on altered hepatocellular foci in Fischer 344 rats. Vet Pathol 27: 110-116.<br />
<br />
Harada T, Maronpot RR, Morris RW, Stitzel KA, Boorman GA (1989) Morphological and stereological characterization of hepatic foci of cellular alteration in control Fischer 344 rats. Toxicol Pathol 17: 579-593.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, et al (2014) Complete protection against aflatoxin B1-induced liver cancer with triterpenoid: DNA adduct dosimetry, molecular signature and genotoxicity threshold. Cancer Prev Res (Phila) .<br />
<br />
Kirby GM, Stalker M, Metcalfe C, Kocal T, Ferguson H, Hayes MA (1990) Expression of immunoreactive glutathione S-transferases in hepatic neoplasms induced by aflatoxin B1 or 1,2-dimethylbenzanthracene in rainbow trout (Oncorhynchus mykiss). Carcinogenesis 11: 2255-2257.<br />
<br />
Ohnishi S, Ma N, Thanan R, Pinlaor S, Hammam O, et al (2013) DNA damage in inflammation-related carcinogenesis and cancer stem cells. Oxid Med Cell Longev 2013: 387014.<br />
<br />
Pitot HC, Dragan YP, Teeguarden J, Hsia S, Campbell H (1996) Quantitation of multistage carcinogenesis in rat liver. Toxicol Pathol 24: 119-128<br />
<br />
Ribback S, Calvisi DF, Cigliano A, Sailer V, Peters M, et al (2013) Molecular and metabolic changes in human liver clear cell foci resemble the alterations occurring in rat hepatocarcinogenesis. J Hepatol 58: 1147-1156.<br />
<br />
Thoolen B, Ten Kate FJ, van Diest PJ, Malarkey DE, Elmore SA, Maronpot RR (2012) Comparative histomorphological review of rat and human hepatocellular proliferative lesions. J Toxicol Pathol 25: 189-199.<br />
<br />
Wyde ME, Cambre T, Lebetkin M, Eldridge SR, Walker NJ (2002) Promotion of altered hepatic foci by 2,3,7,8-tetrachlorodibenzo-p-dioxin and 17beta-estradiol in male Sprague-Dawley rats. Toxicol Sci 68: 295-303.<br />
<br />
Xu YH, Maronpot R, Pitot HC (1990) Quantitative stereologic study of the effects of varying the time between initiation and promotion on four histochemical markers in rat liver during hepatocarcinogenesis. Carcinogenesis 11: 267-272.<br />
<br />
Xu YH, Campbell HA, Sattler GL, Hendrich S, Maronpot R, et al (1990) Quantitative stereological analysis of the effects of age and sex on multistage hepatocarcinogenesis in the rat by use of four cytochemical markers. Cancer Res 50: 472-479.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:423Event:4232016-07-06T17:15:32Z<p>Dvillene: /* How this Key Event works */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Clonal Expansion / Cell Proliferation to Form Pre-Neoplastic Altered Hepatic Foci </div><br />
<div id='shortTitle' class='Title2'> Pre-neoplastic AHF Lesion Formation </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Level of Biological Organization : Organ ==<br />
Altered hepatic foci (AHF) are formed in the liver; they are clonal in origin, and are identified with immunohistochemical techniques, typically due to their synthesis/expression of GST-P.<br />
<br />
== How this Key Event works ==<br />
The occurrence of altered hepatic foci (AHF) as precursors to liver tumors in AFB1-treated rats has been recognized for decades. Originally, these foci were observed as histologically different from the surrounding parenchyma. (Harada et al., 1989, 1990; Gil et al., 1988; Bannasch et al., 1985). In addition, enzyme alterations were used to identify AHF foci, most notably, the occurrence of a placental form of glutathione-S-transferase (GSTP+). (Godlewski et al., 1985; Dragan et al., 1994a, 1995; Kirby et al., 1990) The growth and occurrence of foci are expressed as the number of AHF in a volume of liver, possibly the entire liver, and the volume fraction of the liver occupied by AHF. (Dragan et al., 1997) Both of these reflect focal growth because single cell foci are not detectable with the immunohistochemical staining technique. The assumption is that single transformed cells in which apoptosis is blocked by tumor-critical mutations will grow into AHF. (Grassl-Kraupp et al., 1997). A number of agents regarded as tumor promoters appear to enhance the growth of foci, acting further to inhibit apoptosis and also creating an overall proliferative stimulus. (Angsubhakorn et al., 2002; Wyde et al., 2002). <br />
<br />
AFB1 appears to be a “complete” carcinogen in that the toxin acts as an initiator through the formation of pro-mutagenic DNA adducts (the MIE) and as a promoter through increasing oxidative stress and inflammation. (Ohnishi et al., 2013; Caballero et al., 2004).<br />
<br />
== KE#3: Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
Chemoprevention studies, reviewed in another section of this AOP, suggest a strong relationship between altered hepatic foci (AHF) and HCC tumor formation (Olden and Vulimiri, 2014; Liby et al., 2008; Yates et al., 2007; Yates and Kensler, 2007; Kensler et al., 2004). For example, Johnson et al. (2014) observed background levels of AHF along with a complete absence of tumors in rats treated with a triterpenoid chemoprotectant CDDO-Im, despite maintaining a significant burden of AFB1-induced adducts. (Johnson et al., 2014) Cell proliferation appears to be six- to seven-fold greater in AHF than in surrounding liver parenchyma. (Dragan et al., 1994) However, the measurements were made from liver biopsies, and whether the increased expression was associated with foci is not known.<br />
<br />
== KE#3: How it is Measured or Detected ==<br />
Quantitative stereology has been used to quantify the growth of AHF (Pitot et al., 1996; Dragan et al., 1995; Xu et al., 1990). Growth of foci appears to follow the Moolgavkar-Venzon-Knudson model of initiation and promotion. (Dewanji et al., 1991; Dragan et al., 1995) Most recently, Johnson et al. (2014) have shown that a chemoprotective agent reduces the occurrence of AHF to background levels and completely protects against tumors, although pro-mutagenic adducts are still present at easily quantifiable levels.<br />
<br />
== KE#3: Evidence Supporting Taxonomic Applicability ==<br />
The occurrence of AHF appears to be universal and has been observed in mammals, including humans, as well as in birds and in fish. (Ribback et al., 2013; Thoolen et al., 2012; Kirby et al., 1990).<br />
<br />
== References ==<br />
Angsubhakorn S, Pradermwong A, Phanwichien K, Nguansangiam S (2002) Promotion of aflatoxin B1-induced hepatocarcinogenesis by dichlorodiphenyl trichloroethane (DDT). Southeast Asian J Trop Med Public Health 33: 613-623.<br />
<br />
Bannasch P, Benner U, Enzmann H, Hacker HJ (1985) Tigroid cell foci and neoplastic nodules in the liver of rats treated with a single dose of aflatoxin B1. Carcinogenesis 6: 1641-1648.<br />
<br />
Caballero F, Meiss R, Gimenez A, Batlle A, Vazquez E (2004) Immunohistochemical analysis of heme oxygenase-1 in preneoplastic and neoplastic lesions during chemical hepatocarcinogenesis. Int J Exp Pathol 85: 213-222.<br />
<br />
Dewanji A, Moolgavkar SH, Luebeck EG (1991) Two-mutation model for carcinogenesis: joint analysis of premalignant and malignant lesions. Math Biosci 104: 97-109.<br />
<br />
Dragan Y, Teeguarden J, Campbell H, Hsia S, Pitot H (1995a) The quantitation of altered hepatic foci during multistage hepatocarcinogenesis in the rat: transforming growth factor alpha expression as a marker for the stage of progression. Cancer Lett 93: 73-83.<br />
<br />
Dragan YP, Campbell HA, Baker K, Vaughan J, Mass M, Pitot HC (1994) Focal and non-focal hepatic expression of placental glutathione S-transferase in carcinogen-treated rats. Carcinogenesis 15: 2587-2591.<br />
<br />
Dragan YP, Campbell HA, Xu XH, Pitot HC (1997) Quantitative stereological studies of a 'selection' protocol of hepatocarcinogenesis following initiation in neonatal male and female rats. Carcinogenesis 18: 149-158.<br />
<br />
Dragan YP, Hully J, Baker K, Crow R, Mass MJ, Pitot HC (1995b) Comparison of experimental and theoretical parameters of the Moolgavkar-Venzon-Knudson incidence function for the stages of initiation and promotion in rat hepatocarcinogenesis. Toxicology 102: 161-175.<br />
<br />
Gil R, Callaghan R, Boix J, Pellin A, Llombart-Bosch A (1988) Morphometric and cytophotometric nuclear analysis of altered hepatocyte foci induced by N-nitrosomorpholine (NNM) and aflatoxin B1 (AFB1) in liver of Wistar rats. Virchows Arch B Cell Pathol Incl Mol Pathol 54: 341-349.<br />
<br />
Godlewski CE, Boyd JN, Sherman WK, Anderson JL, Stoewsand GS (1985) Hepatic glutathione S-transferase activity and aflatoxin B1-induced enzyme altered foci in rats fed fractions of brussels sprouts. Cancer Lett 28: 151-157.<br />
<br />
Grasl-Kraupp B, Ruttkay-Nedecky B, Müllauer L, Taper H, Huber W, et al (1997) Inherent increase of apoptosis in liver tumors: implications for carcinogenesis and tumor regression. Hepatology 25: 906-912.<br />
<br />
Harada T, Maronpot RR, Morris RW, Boorman GA (1990) Effects of mononuclear cell leukemia on altered hepatocellular foci in Fischer 344 rats. Vet Pathol 27: 110-116.<br />
<br />
Harada T, Maronpot RR, Morris RW, Stitzel KA, Boorman GA (1989) Morphological and stereological characterization of hepatic foci of cellular alteration in control Fischer 344 rats. Toxicol Pathol 17: 579-593.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, et al (2014) Complete protection against aflatoxin B1-induced liver cancer with triterpenoid: DNA adduct dosimetry, molecular signature and genotoxicity threshold. Cancer Prev Res (Phila) .<br />
<br />
Kirby GM, Stalker M, Metcalfe C, Kocal T, Ferguson H, Hayes MA (1990) Expression of immunoreactive glutathione S-transferases in hepatic neoplasms induced by aflatoxin B1 or 1,2-dimethylbenzanthracene in rainbow trout (Oncorhynchus mykiss). Carcinogenesis 11: 2255-2257.<br />
<br />
Ohnishi S, Ma N, Thanan R, Pinlaor S, Hammam O, et al (2013) DNA damage in inflammation-related carcinogenesis and cancer stem cells. Oxid Med Cell Longev 2013: 387014.<br />
<br />
Pitot HC, Dragan YP, Teeguarden J, Hsia S, Campbell H (1996) Quantitation of multistage carcinogenesis in rat liver. Toxicol Pathol 24: 119-128<br />
<br />
Ribback S, Calvisi DF, Cigliano A, Sailer V, Peters M, et al (2013) Molecular and metabolic changes in human liver clear cell foci resemble the alterations occurring in rat hepatocarcinogenesis. J Hepatol 58: 1147-1156.<br />
<br />
Thoolen B, Ten Kate FJ, van Diest PJ, Malarkey DE, Elmore SA, Maronpot RR (2012) Comparative histomorphological review of rat and human hepatocellular proliferative lesions. J Toxicol Pathol 25: 189-199.<br />
<br />
Wyde ME, Cambre T, Lebetkin M, Eldridge SR, Walker NJ (2002) Promotion of altered hepatic foci by 2,3,7,8-tetrachlorodibenzo-p-dioxin and 17beta-estradiol in male Sprague-Dawley rats. Toxicol Sci 68: 295-303.<br />
<br />
Xu YH, Maronpot R, Pitot HC (1990) Quantitative stereologic study of the effects of varying the time between initiation and promotion on four histochemical markers in rat liver during hepatocarcinogenesis. Carcinogenesis 11: 267-272.<br />
<br />
Xu YH, Campbell HA, Sattler GL, Hendrich S, Maronpot R, et al (1990) Quantitative stereological analysis of the effects of age and sex on multistage hepatocarcinogenesis in the rat by use of four cytochemical markers. Cancer Res 50: 472-479.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:423Event:4232016-07-06T17:15:16Z<p>Dvillene: /* Level of Biological Organization : Organ */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Clonal Expansion / Cell Proliferation to Form Pre-Neoplastic Altered Hepatic Foci </div><br />
<div id='shortTitle' class='Title2'> Pre-neoplastic AHF Lesion Formation </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Level of Biological Organization : Organ ==<br />
Altered hepatic foci (AHF) are formed in the liver; they are clonal in origin, and are identified with immunohistochemical techniques, typically due to their synthesis/expression of GST-P.<br />
<br />
== KE#3: How this Key Event works ==<br />
The occurrence of altered hepatic foci (AHF) as precursors to liver tumors in AFB1-treated rats has been recognized for decades. Originally, these foci were observed as histologically different from the surrounding parenchyma. (Harada et al., 1989, 1990; Gil et al., 1988; Bannasch et al., 1985). In addition, enzyme alterations were used to identify AHF foci, most notably, the occurrence of a placental form of glutathione-S-transferase (GSTP+). (Godlewski et al., 1985; Dragan et al., 1994a, 1995; Kirby et al., 1990) The growth and occurrence of foci are expressed as the number of AHF in a volume of liver, possibly the entire liver, and the volume fraction of the liver occupied by AHF. (Dragan et al., 1997) Both of these reflect focal growth because single cell foci are not detectable with the immunohistochemical staining technique. The assumption is that single transformed cells in which apoptosis is blocked by tumor-critical mutations will grow into AHF. (Grassl-Kraupp et al., 1997). A number of agents regarded as tumor promoters appear to enhance the growth of foci, acting further to inhibit apoptosis and also creating an overall proliferative stimulus. (Angsubhakorn et al., 2002; Wyde et al., 2002). <br />
<br />
AFB1 appears to be a “complete” carcinogen in that the toxin acts as an initiator through the formation of pro-mutagenic DNA adducts (the MIE) and as a promoter through increasing oxidative stress and inflammation. (Ohnishi et al., 2013; Caballero et al., 2004).<br />
<br />
== KE#3: Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
Chemoprevention studies, reviewed in another section of this AOP, suggest a strong relationship between altered hepatic foci (AHF) and HCC tumor formation (Olden and Vulimiri, 2014; Liby et al., 2008; Yates et al., 2007; Yates and Kensler, 2007; Kensler et al., 2004). For example, Johnson et al. (2014) observed background levels of AHF along with a complete absence of tumors in rats treated with a triterpenoid chemoprotectant CDDO-Im, despite maintaining a significant burden of AFB1-induced adducts. (Johnson et al., 2014) Cell proliferation appears to be six- to seven-fold greater in AHF than in surrounding liver parenchyma. (Dragan et al., 1994) However, the measurements were made from liver biopsies, and whether the increased expression was associated with foci is not known.<br />
<br />
== KE#3: How it is Measured or Detected ==<br />
Quantitative stereology has been used to quantify the growth of AHF (Pitot et al., 1996; Dragan et al., 1995; Xu et al., 1990). Growth of foci appears to follow the Moolgavkar-Venzon-Knudson model of initiation and promotion. (Dewanji et al., 1991; Dragan et al., 1995) Most recently, Johnson et al. (2014) have shown that a chemoprotective agent reduces the occurrence of AHF to background levels and completely protects against tumors, although pro-mutagenic adducts are still present at easily quantifiable levels.<br />
<br />
== KE#3: Evidence Supporting Taxonomic Applicability ==<br />
The occurrence of AHF appears to be universal and has been observed in mammals, including humans, as well as in birds and in fish. (Ribback et al., 2013; Thoolen et al., 2012; Kirby et al., 1990).<br />
<br />
== References ==<br />
Angsubhakorn S, Pradermwong A, Phanwichien K, Nguansangiam S (2002) Promotion of aflatoxin B1-induced hepatocarcinogenesis by dichlorodiphenyl trichloroethane (DDT). Southeast Asian J Trop Med Public Health 33: 613-623.<br />
<br />
Bannasch P, Benner U, Enzmann H, Hacker HJ (1985) Tigroid cell foci and neoplastic nodules in the liver of rats treated with a single dose of aflatoxin B1. Carcinogenesis 6: 1641-1648.<br />
<br />
Caballero F, Meiss R, Gimenez A, Batlle A, Vazquez E (2004) Immunohistochemical analysis of heme oxygenase-1 in preneoplastic and neoplastic lesions during chemical hepatocarcinogenesis. Int J Exp Pathol 85: 213-222.<br />
<br />
Dewanji A, Moolgavkar SH, Luebeck EG (1991) Two-mutation model for carcinogenesis: joint analysis of premalignant and malignant lesions. Math Biosci 104: 97-109.<br />
<br />
Dragan Y, Teeguarden J, Campbell H, Hsia S, Pitot H (1995a) The quantitation of altered hepatic foci during multistage hepatocarcinogenesis in the rat: transforming growth factor alpha expression as a marker for the stage of progression. Cancer Lett 93: 73-83.<br />
<br />
Dragan YP, Campbell HA, Baker K, Vaughan J, Mass M, Pitot HC (1994) Focal and non-focal hepatic expression of placental glutathione S-transferase in carcinogen-treated rats. Carcinogenesis 15: 2587-2591.<br />
<br />
Dragan YP, Campbell HA, Xu XH, Pitot HC (1997) Quantitative stereological studies of a 'selection' protocol of hepatocarcinogenesis following initiation in neonatal male and female rats. Carcinogenesis 18: 149-158.<br />
<br />
Dragan YP, Hully J, Baker K, Crow R, Mass MJ, Pitot HC (1995b) Comparison of experimental and theoretical parameters of the Moolgavkar-Venzon-Knudson incidence function for the stages of initiation and promotion in rat hepatocarcinogenesis. Toxicology 102: 161-175.<br />
<br />
Gil R, Callaghan R, Boix J, Pellin A, Llombart-Bosch A (1988) Morphometric and cytophotometric nuclear analysis of altered hepatocyte foci induced by N-nitrosomorpholine (NNM) and aflatoxin B1 (AFB1) in liver of Wistar rats. Virchows Arch B Cell Pathol Incl Mol Pathol 54: 341-349.<br />
<br />
Godlewski CE, Boyd JN, Sherman WK, Anderson JL, Stoewsand GS (1985) Hepatic glutathione S-transferase activity and aflatoxin B1-induced enzyme altered foci in rats fed fractions of brussels sprouts. Cancer Lett 28: 151-157.<br />
<br />
Grasl-Kraupp B, Ruttkay-Nedecky B, Müllauer L, Taper H, Huber W, et al (1997) Inherent increase of apoptosis in liver tumors: implications for carcinogenesis and tumor regression. Hepatology 25: 906-912.<br />
<br />
Harada T, Maronpot RR, Morris RW, Boorman GA (1990) Effects of mononuclear cell leukemia on altered hepatocellular foci in Fischer 344 rats. Vet Pathol 27: 110-116.<br />
<br />
Harada T, Maronpot RR, Morris RW, Stitzel KA, Boorman GA (1989) Morphological and stereological characterization of hepatic foci of cellular alteration in control Fischer 344 rats. Toxicol Pathol 17: 579-593.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, et al (2014) Complete protection against aflatoxin B1-induced liver cancer with triterpenoid: DNA adduct dosimetry, molecular signature and genotoxicity threshold. Cancer Prev Res (Phila) .<br />
<br />
Kirby GM, Stalker M, Metcalfe C, Kocal T, Ferguson H, Hayes MA (1990) Expression of immunoreactive glutathione S-transferases in hepatic neoplasms induced by aflatoxin B1 or 1,2-dimethylbenzanthracene in rainbow trout (Oncorhynchus mykiss). Carcinogenesis 11: 2255-2257.<br />
<br />
Ohnishi S, Ma N, Thanan R, Pinlaor S, Hammam O, et al (2013) DNA damage in inflammation-related carcinogenesis and cancer stem cells. Oxid Med Cell Longev 2013: 387014.<br />
<br />
Pitot HC, Dragan YP, Teeguarden J, Hsia S, Campbell H (1996) Quantitation of multistage carcinogenesis in rat liver. Toxicol Pathol 24: 119-128<br />
<br />
Ribback S, Calvisi DF, Cigliano A, Sailer V, Peters M, et al (2013) Molecular and metabolic changes in human liver clear cell foci resemble the alterations occurring in rat hepatocarcinogenesis. J Hepatol 58: 1147-1156.<br />
<br />
Thoolen B, Ten Kate FJ, van Diest PJ, Malarkey DE, Elmore SA, Maronpot RR (2012) Comparative histomorphological review of rat and human hepatocellular proliferative lesions. J Toxicol Pathol 25: 189-199.<br />
<br />
Wyde ME, Cambre T, Lebetkin M, Eldridge SR, Walker NJ (2002) Promotion of altered hepatic foci by 2,3,7,8-tetrachlorodibenzo-p-dioxin and 17beta-estradiol in male Sprague-Dawley rats. Toxicol Sci 68: 295-303.<br />
<br />
Xu YH, Maronpot R, Pitot HC (1990) Quantitative stereologic study of the effects of varying the time between initiation and promotion on four histochemical markers in rat liver during hepatocarcinogenesis. Carcinogenesis 11: 267-272.<br />
<br />
Xu YH, Campbell HA, Sattler GL, Hendrich S, Maronpot R, et al (1990) Quantitative stereological analysis of the effects of age and sex on multistage hepatocarcinogenesis in the rat by use of four cytochemical markers. Cancer Res 50: 472-479.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:425Event:4252016-07-06T17:13:47Z<p>Dvillene: </p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Thyroidal iodide uptake, Decreased </div><br />
<div id='shortTitle' class='Title2'> </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:54|Inhibition of Na+/I- symporter (NIS) decreases TH synthesis leading to learning and memory deficits in children ]]||KE||[[Aop:54#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|[[Aop:134|Sodium Iodide Symporter (NIS) Inhibition and Subsequent Adverse Neurodevelopmental Outcomes in Mammals]]||KE||[[Aop:134#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Cellular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== How this Key Event works ==<br />
Iodine (I2) is a non-metallic chemical element which is required for the normal cellular metabolism. It is one of the essential components of the TH, comprising 65% and 58% of T4's and T3's weight, respectively and therefore it is crucial for the normal thyroid function. It is a trace element and a healthy human body contains 15-20 mg of iodine, most of which is concentrated in the thyroid gland (Dunn, 1998). Iodide (I-) that enters the thyroid gland remains in the free state only briefly and subsequently it bounds to the tyrosine residues of thyroglobulin to form the precursors of the thyroid hormones mono-iodinated tyrosine (MIT) or di-iodinated tyrosine (DIT) (Berson and Yalow, 1955). The bounding rate of iodide is 50-100% of the intrathyroidal iodide pool, meaning that only a very small proportion of this element is free in the thyroid and this comes mainly by the deiodination of MIT and DIT. <br />
<br />
The body is not able to produce or make iodine, thus the diet is the only source of this element. Iodine is found in nature in various forms, such as inorganic sodium and potassium salts (iodides and iodates), inorganic diatomic iodine and organic monoatomic iodine (Patrick, 2008). Thus, it is widely distributed in the earth's environment but in many regions of the world the soil's iodine has been depleted due to different environmental phenomena. In these regions, the incidence of iodine deficiency is greatly increased (Ahad and Ganie, 2010). <br />
<br />
The daily iodine intake of adult humans varies greatly due to the different dietary habits between the different regions on earth (Dunn, 1993). In any case, the ingested iodine is absorbed through the intestine and transported into the plasma to reach the thyroid gland. However, thyroid is not the only organ of the body that concentrates iodide. It has been shown that other tissues have also the ability of iodide concentration, such as the salivary glands, the gastric mucosa, the mammary glands and the choroid plexus, all of which express NIS, the well-known iodine transporter protein (Jhiang et al., 1998; Cho et al., 2000). The thyroid, salivary glands and the gastric mucosa have a common embryologic origin, from the primitive alimentary tract, which may explain the reason of the NIS expression in these tissues. Furthermore, in regards to the gastric mucosa and the breast, there is an obvious value of concentrating iodide, as it is the route for its derivation to the bloodstream and to the breast milk, respectively. The iodide from the circulation will eventually reach the thyroid in order to participate in its most important function, namely the production of thyroid hormones. In contrast, the biological role of iodide in the salivary glands and the choroid plexus is not yet specified, but it is a research area of high interest, as it is believed that it may be involved in important pathways but yet undiscovered. <br />
<br />
The most important role of iodine is the formation of the thyroid hormones (T4 and T3). The thyroid actively concentrates the circulating iodide through the basolateral membrane of the thyrocytes by the sodium/iodide symporter protein (NIS). The concentrated thyroid-iodine is oxidized in the follicular cells of the gland and consequently binds to tyrosines to form mono- or di-iodotyrosines (MIT and DIT respectively), being incorporated into thyroglobulin. This newly formed iodothyroglobulin forms one of the most important constituents of the colloid material, present in the follicle of the thyroid unit. If two di-iodotyrosine molecules couple together, the result is the formation of thyroxin (T4). If a di-iodotyrosine and a mono-iodotyrosine are coupled together, the result is the formation of tri-iodothyronine (T3). From the perspective of the formation of thyroid hormone, the major coupling reaction is the di-iodotyrosine coupling to produce T4. Although T3 is more biologically active than T4, the major production of T3 actually occurs outside of the thyroid gland. The majority of T3 is produced by peripheral conversion from T4 in a deiodination reaction involving a specific enzyme which removes one iodine from the outer ring of T4.<br />
<br />
A sodium-iodide (Na/I) symporter pumps iodide (I−) actively into the cell, which previously has crossed the endothelium by largely unknown mechanisms. This iodide enters the follicular lumen from the cytoplasm by the transporter pendrin, in a purportedly passive manner. In the colloid, iodide (I−) is oxidized to iodine (I0) by an enzyme called thyroid peroxidase (TPO). Iodine (I0) is very reactive and iodinates the thyroglobulin at tyrosyl residues in its protein chain. In conjugation, adjacent tyrosyl residues are paired together. Thyroglobulin binds the megalin receptor for endocytosis back into the follicular cell. Proteolysis by various proteases liberates thyroxine (T4) and triiodothyronine molecules (T3), which enter the bloodstream where they are bound to thyroid hormone binding proteins. The major thyroid hormone binding protein is thyroxin binding globulin (TBG) which accounts for about 75% of the bound hormone.<br />
In order to attain normal levels of thyroid hormone synthesis, an adequate supply of iodine is essential. In iodine sufficient areas, the adult thyroid absorbs 60-80 μg of iodide per day to maintain the thyroid homeostasis (Degroot, 1966). Inadequate amount of iodide results to deficient production of thyroid hormones, which consequently leads to an increase of TSH secretion and goiter, as compensating effect (Delange, 2000). On the other hand, excess iodide could also inhibit TH synthesis (Wolff and Chaikoff, 1948). The proposed mechanism for this latter effect is the possible formation of 2-iodohexadecanal that inhibits the generation of H2O2 and the subsequent oxidation of iodide in the thyroid follicular cells. The lack of oxidized free radicals of iodide affects the reaction with the tyrosine residues of Thyroglobulin (Tg) and the subsequent formation of MIT and DIT (Panneels et al., 1994). <br />
During pregnancy, the organism of the mother is also supporting the needs of the foetus and therefore the iodide requirements are greatly increased (Glinoer, 1997). Additionally, small iodine concentrations have been found to have significant antioxidant effects that resembles to ascorbic acid (Smyth, 2003).<br />
<br />
== How it is Measured or Detected ==<br />
The radioactive iodine uptake test, or RAIU test, is a type of scan used in the diagnosis of thyroid gland dysfunction. The patient swallows radioactive iodine in the form of capsule or fluid, and its absorption by the thyroid is studied after 4–6 hours and after 24 hours with the aid of a gamma scintillation counter. The percentage of RAIU 24 hours after the administration of radioiodide is the most useful, since this is the time when the thyroid gland has reached the plateau of isotope accumulation, and because it has been shown that at this time, the best separation between high, normal, and low uptake is obtained. <br />
The test does not measure hormone production and release but merely the avidity of the thyroid gland for iodide and its rate of clearance relative to the kidney.<br />
<br />
== Evidence Supporting Taxonomic Applicability ==<br />
Animal studies have proven that iodine normalizes elevated adrenal corticosteroid hormone secretion and has the ability to reverse the effects of hypothyroidism in the ovaries, testicles and thymus in thyroidectomized rats (Nolan et al., 2000).<br />
<br />
== References ==<br />
<br />
Ahad F, Ganie SA. (2010). Iodine, iodine metabolism and iodine deficiency disorders revisited. Indian J Endocrinol Metab. 14: 13-17.<br />
<br />
Berson SA, Yalow RS. (1955). The iodide trapping and binding functions of the thyroid. J Clin Invest. 34: 186-204.<br />
<br />
Cho JY, Leveille R, Kao R, Rousset B, Parlow AF, Burak WE Jr, Mazzaferri EL, Jhiang SM.(2000). Hormonal regulation of radioiodide uptake activity and Na+/I- symporter expression in mammary glands. J Clin Endocrinol Metab. 85:2936-2943.<br />
<br />
Degroot LJ.(1966). Kinetic analysis of iodine metabolism. J Clin Endocrinol Metab. 26: 149-173.<br />
<br />
Delange F. (2000). Iodine deficiency. In: Braverman L, Utiger R, editors. Werner and Ingbar's the thyroid: a fundamental and clinical text. Philadelphia: JD Lippincott. pp 295-316.<br />
<br />
Dunn JT. (1993). Sources of dietary iodine in industrialized countries. In: Delange F, Dunn JT, Glinoer D, editors. Iodine deficiency in Europe. A continuing concern. New York: Plenum press. pp 17-21. <br />
<br />
Dunn JT. (1998). What's happening to our iodine? J Clin Endocrinol Metab. 83: 3398-3400.<br />
Glinoer D. (1997). The regulation of thyroid function in pregnancy: pathways of endocrine adaptation from physiology to pathology. Endocr Rev. 18: 404-433.<br />
<br />
Jhiang SM, Cho JY, Ryu KY, DeYoung BR, Smanik PA, McGaughy VR, Fischer AH, Mazzaferri EL.(1998). An immunohistochemical study of Na+/I- symporter in human thyroid tissues and salivary gland tissues. Endocrinology. 139:4416-4419.<br />
<br />
Nolan LA, Windle RJ, Wood SA, Kershaw YM, Lunness HR, Lightman SL, Ingram CD, Levy A. (2000). Chronic iodine deprivation attenuates stress-induced and diurnal variation in corticosterone secretion in female Wistar rats. J Neuroendocrinol. 12:1149-1159.<br />
<br />
Panneels V, Van den Bergen H, Jacoby C, Braekman JC, Van Sande J, Dumont JE, Boeynaems JM. (1994). Inhibition of H2O2 production by iodoaldehydes in cultured dog thyroid cells. Mol Cell Endocrinol. 102:167-176.<br />
<br />
Patrick L. (2008).Iodine:Deficiency and therapeutic considerations. Altern MedRev. 13:166-127.<br />
<br />
Smyth PA. (2003). Role of iodine in antioxidant defense in thyroid and breast disease. Biofactors. 19:121-130.<br />
<br />
Wolff J, Chaikoff IL. (1948). Plasma inorganic iodide as a homeostatic regulator of thyroid function. J Biol Chem. 174: 555-564.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:378Event:3782016-07-06T17:13:37Z<p>Dvillene: /* AO: References */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Hepatocellular carcinoma, Tumorigenesis </div><br />
<div id='shortTitle' class='Title2'> Hepatocellular Carcinoma (HCC) </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]][[Category:Adverse Outcome]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||AO||<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Affected Organs ===<br />
<br />
{|class="wikitable sortable" id="Organproof"<br />
<br />
!Synonym<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|---||liver||[http://www.ontobee.org/browser/rdf.php?o=UBERON&iri=http://purl.obolibrary.org/obo/UBERON_0002107 Ontobee]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Individual<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Level of Biological Organization : Organism ==<br />
<br />
== How this Key Event works ==<br />
The AO final key event results from the processes that occur in the earlier series of key events, which for AFB1 is a mutagenic MOA—the AFB1 induction of mutations in critical cancer genes that alter the phenotype of the mutant cell and set the stage for that cell to progress to a pre-neoplastic lesion and ultimately an HCC. The biological processes described in this AO, however, are not specific to a mutagenic MOA—nor necessarily demonstrated for AFB1 exposure, but occur in development of HCC from all MOAs for HCC. Thus the final key events (AHF and HCC) represent the final stages of the pathway that leads to HCC from a mutagenic MOA or other MOAs. <br />
<br />
Hepatocellular carcinoma (HCC) is a cancer of hepatocytes, and this disease is almost always lethal in the absence of extreme intervention measures (e.g., surgery, liver transplant). A number of factors are associated with HCC including AFB1 exposure, infection with hepatitis virus (HBV), and alcohol use. A common etiologic feature of HCC, whether produced by AFB1 intoxication, HBV, cirrhosis or something else, is the presence of oxidative damage in the liver. (Ravinayagam et al., 2012 Int J Hepatol; Kim et al., 2011 J Ginseng Res).<br />
<br />
AFB1 produces specific pro-mutagenic adducts that are believed to lead to a mutation in the p53 gene, which affects its functioning. P53 is generally considered to be a tumor suppressor gene involved in cell cycle regulation and initiation of apoptosis. When applied in vitro to hepatocytes, AFB1 produced cellular swelling, bleb formation, and lysis. These effects may be due to lipid peroxidation affecting the cell membrane from the downstream dialdehyde metabolite of the AFB1 epoxide metabolites. (Mathijs et al., 2009, 2010) This damage is reflective of oxidative stress, a known contributor to HCC (Ravinayagam et al., 2012 Int J Hepatol; Kim et al., 2011 J Ginseng Res). As discussed elsewhere in this AOP, the Nrf2-Keap1 anti-oxidant response induced by a number of chemoprotective agents can be quite effective in preventing HCC [3-8], even in the presence of a significant burden of N7- AFB1-G adducts. <br />
<br />
The cellular damage produced by exposure to AFB1 likely leads to chronic inflammation, also a contributor to tumor progression. (Ellinger-Ziegelbauer et al., 2004) Heme oxygenase-1 (HO-1) breaks down heme to bilirubin and biliverdin that have anti-oxidant and anti-inflammatory activities (Keum et al., 2006; Caballero et al 2004) , thus countering the inflammatory response. The induction of HO-1 is part of the Nrf2-Keap1 anti-oxidant response. <br />
<br />
From a systems biology and biochemistry perspective, the presence of oxidative stress and inflammation, although not specific only to AFB1 exposure, are strong contributors to cancer progression.(Ohnishi et al., 2013; Zheng et al., 2013; Higgs et al., 2014).<br />
<br />
== How it is Measured or Detected ==<br />
Hepatocellular carcinoma is detected in humans by clinical examination confirmed by pathological examination, and in laboratory test species by pathological examination.<br />
<br />
== Evidence Supporting Taxonomic Applicability ==<br />
Hepatocellular carcinoma occurs in many vertebrate species including birds, fish, and mammals such as humans.<br />
<br />
== Regulatory Examples Using This Adverse Outcome ==<br />
Although not specifically used EPA for regulatory determinations vis-à-vis AFB1, HCC has been used as an adverse endpoint in many hazard assessments that can be used as input to risk management decisions. The U.S. EPA Integrated Risk Information System (IRIS database) contains 111 instances wherein HCC has been considered in hazard assessment of environmental contaminants. For example, HCC in rats formed part of the weight of evidence in categorizing polychlorinated biphenyls as probable human carcinogens. These tumors, combined with other liver tumors, also formed the basis for quantitative dose-response assessment for cancer induced by polychlorinated biphenyls by the oral route.(USEPA, 2014).<br />
<br />
Given that AFB1 can be a contaminant in both human food and animal feed, FDA has established allowable limits. http://www.fda.gov/downloads/advisorycommittees/committeesmeetingmaterials/foodadvisorycommittee/ucm428947.pdf<br />
<br />
== References ==<br />
Caballero F, Meiss R, Gimenez A, Batlle A, Vazquez E (2004) Immunohistochemical analysis of heme oxygenase-1 in preneoplastic and neoplastic lesions during chemical hepatocarcinogenesis. Int J Exp Pathol 85: 213-222.<br />
<br />
Higgs MR, Chouteau P, Lerat H (2014) 'Liver let die': oxidative DNA damage and hepatotropic viruses. J Gen Virol 95: 991-1004.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, et al (2014) Complete protection against aflatoxin B1-induced liver cancer with triterpenoid: DNA adduct dosimetry, molecular signature and genotoxicity threshold. Cancer Prev Res (Phila) .<br />
<br />
Liby KT, Sporn MB (2012) Synthetic oleanane triterpenoids: multifunctional drugs with a broad range of applications for prevention and treatment of chronic disease. Pharmacol Rev 64: 972-1003.<br />
<br />
Liby K, Yore MM, Roebuck BD, Baumgartner KJ, Honda T, et al (2008) A novel acetylenic tricyclic bis-(cyano enone) potently induces phase 2 cytoprotective pathways and blocks liver carcinogenesis induced by aflatoxin. Cancer Res 68: 6727-6733.<br />
<br />
Moudgil V, Redhu D, Dhanda S, Singh J (2013) A review of molecular mechanisms in the development of hepatocellular carcinoma by aflatoxin and hepatitis B and C viruses. J Environ Pathol Toxicol Oncol 32: 165-175.<br />
<br />
Ohnishi S, Ma N, Thanan R, Pinlaor S, Hammam O, et al (2013) DNA damage in inflammation-related carcinogenesis and cancer stem cells. Oxid Med Cell Longev 2013: 387014.<br />
<br />
Roebuck BD (2004) Hyperplasia, partial hepatectomy, and the carcinogenicity of aflatoxin B1. J Cell Biochem 91: 243-249.<br />
<br />
Shelton P, Jaiswal AK (2013) The transcription factor NF-E2-related factor 2 (Nrf2): a protooncogene? FASEB J 27: 414-423.<br />
<br />
U.S. EPA IRIS, (2014) available at http://www.epa.gov/iris/subst/0294.htm#woe<br />
<br />
Yates MS, Tauchi M, Katsuoka F, Flanders KC, Liby KT, et al (2007) Pharmacodynamic characterization of chemopreventive triterpenoids as exceptionally potent inducers of Nrf2-regulated genes. Mol Cancer Ther 6: 154-162.<br />
<br />
Yates MS, Kensler TW (2007) Keap1 eye on the target: chemoprevention of liver cancer. Acta Pharmacol Sin 28: 1331-1342.<br />
<br />
Yates MS, Kwak MK, Egner PA, Groopman JD, Bodreddigari S, et al (2006) Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole. Cancer Res 66: 2488-2494.<br />
<br />
Zhang Y, Guan L, Wang X, Wen T, Xing J, Zhao J (2008) Protection of chlorophyllin against oxidative damage by inducing HO-1 and NQO1 expression mediated by PI3K/Akt and Nrf2. Free Radic Res 42: 362-371.<br />
<br />
Zheng YW, Nie YZ, Taniguchi H (2013) Cellular reprogramming and hepatocellular carcinoma development. World J Gastroenterol 19: 8850-8860.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:378Event:3782016-07-06T17:13:21Z<p>Dvillene: /* AO: Regulatory Examples Using This Adverse Outcome */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Hepatocellular carcinoma, Tumorigenesis </div><br />
<div id='shortTitle' class='Title2'> Hepatocellular Carcinoma (HCC) </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]][[Category:Adverse Outcome]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||AO||<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Affected Organs ===<br />
<br />
{|class="wikitable sortable" id="Organproof"<br />
<br />
!Synonym<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|---||liver||[http://www.ontobee.org/browser/rdf.php?o=UBERON&iri=http://purl.obolibrary.org/obo/UBERON_0002107 Ontobee]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Individual<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Level of Biological Organization : Organism ==<br />
<br />
== How this Key Event works ==<br />
The AO final key event results from the processes that occur in the earlier series of key events, which for AFB1 is a mutagenic MOA—the AFB1 induction of mutations in critical cancer genes that alter the phenotype of the mutant cell and set the stage for that cell to progress to a pre-neoplastic lesion and ultimately an HCC. The biological processes described in this AO, however, are not specific to a mutagenic MOA—nor necessarily demonstrated for AFB1 exposure, but occur in development of HCC from all MOAs for HCC. Thus the final key events (AHF and HCC) represent the final stages of the pathway that leads to HCC from a mutagenic MOA or other MOAs. <br />
<br />
Hepatocellular carcinoma (HCC) is a cancer of hepatocytes, and this disease is almost always lethal in the absence of extreme intervention measures (e.g., surgery, liver transplant). A number of factors are associated with HCC including AFB1 exposure, infection with hepatitis virus (HBV), and alcohol use. A common etiologic feature of HCC, whether produced by AFB1 intoxication, HBV, cirrhosis or something else, is the presence of oxidative damage in the liver. (Ravinayagam et al., 2012 Int J Hepatol; Kim et al., 2011 J Ginseng Res).<br />
<br />
AFB1 produces specific pro-mutagenic adducts that are believed to lead to a mutation in the p53 gene, which affects its functioning. P53 is generally considered to be a tumor suppressor gene involved in cell cycle regulation and initiation of apoptosis. When applied in vitro to hepatocytes, AFB1 produced cellular swelling, bleb formation, and lysis. These effects may be due to lipid peroxidation affecting the cell membrane from the downstream dialdehyde metabolite of the AFB1 epoxide metabolites. (Mathijs et al., 2009, 2010) This damage is reflective of oxidative stress, a known contributor to HCC (Ravinayagam et al., 2012 Int J Hepatol; Kim et al., 2011 J Ginseng Res). As discussed elsewhere in this AOP, the Nrf2-Keap1 anti-oxidant response induced by a number of chemoprotective agents can be quite effective in preventing HCC [3-8], even in the presence of a significant burden of N7- AFB1-G adducts. <br />
<br />
The cellular damage produced by exposure to AFB1 likely leads to chronic inflammation, also a contributor to tumor progression. (Ellinger-Ziegelbauer et al., 2004) Heme oxygenase-1 (HO-1) breaks down heme to bilirubin and biliverdin that have anti-oxidant and anti-inflammatory activities (Keum et al., 2006; Caballero et al 2004) , thus countering the inflammatory response. The induction of HO-1 is part of the Nrf2-Keap1 anti-oxidant response. <br />
<br />
From a systems biology and biochemistry perspective, the presence of oxidative stress and inflammation, although not specific only to AFB1 exposure, are strong contributors to cancer progression.(Ohnishi et al., 2013; Zheng et al., 2013; Higgs et al., 2014).<br />
<br />
== How it is Measured or Detected ==<br />
Hepatocellular carcinoma is detected in humans by clinical examination confirmed by pathological examination, and in laboratory test species by pathological examination.<br />
<br />
== Evidence Supporting Taxonomic Applicability ==<br />
Hepatocellular carcinoma occurs in many vertebrate species including birds, fish, and mammals such as humans.<br />
<br />
== Regulatory Examples Using This Adverse Outcome ==<br />
Although not specifically used EPA for regulatory determinations vis-à-vis AFB1, HCC has been used as an adverse endpoint in many hazard assessments that can be used as input to risk management decisions. The U.S. EPA Integrated Risk Information System (IRIS database) contains 111 instances wherein HCC has been considered in hazard assessment of environmental contaminants. For example, HCC in rats formed part of the weight of evidence in categorizing polychlorinated biphenyls as probable human carcinogens. These tumors, combined with other liver tumors, also formed the basis for quantitative dose-response assessment for cancer induced by polychlorinated biphenyls by the oral route.(USEPA, 2014).<br />
<br />
Given that AFB1 can be a contaminant in both human food and animal feed, FDA has established allowable limits. http://www.fda.gov/downloads/advisorycommittees/committeesmeetingmaterials/foodadvisorycommittee/ucm428947.pdf<br />
<br />
== AO: References ==<br />
Caballero F, Meiss R, Gimenez A, Batlle A, Vazquez E (2004) Immunohistochemical analysis of heme oxygenase-1 in preneoplastic and neoplastic lesions during chemical hepatocarcinogenesis. Int J Exp Pathol 85: 213-222.<br />
<br />
Higgs MR, Chouteau P, Lerat H (2014) 'Liver let die': oxidative DNA damage and hepatotropic viruses. J Gen Virol 95: 991-1004.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, et al (2014) Complete protection against aflatoxin B1-induced liver cancer with triterpenoid: DNA adduct dosimetry, molecular signature and genotoxicity threshold. Cancer Prev Res (Phila) .<br />
<br />
Liby KT, Sporn MB (2012) Synthetic oleanane triterpenoids: multifunctional drugs with a broad range of applications for prevention and treatment of chronic disease. Pharmacol Rev 64: 972-1003.<br />
<br />
Liby K, Yore MM, Roebuck BD, Baumgartner KJ, Honda T, et al (2008) A novel acetylenic tricyclic bis-(cyano enone) potently induces phase 2 cytoprotective pathways and blocks liver carcinogenesis induced by aflatoxin. Cancer Res 68: 6727-6733.<br />
<br />
Moudgil V, Redhu D, Dhanda S, Singh J (2013) A review of molecular mechanisms in the development of hepatocellular carcinoma by aflatoxin and hepatitis B and C viruses. J Environ Pathol Toxicol Oncol 32: 165-175.<br />
<br />
Ohnishi S, Ma N, Thanan R, Pinlaor S, Hammam O, et al (2013) DNA damage in inflammation-related carcinogenesis and cancer stem cells. Oxid Med Cell Longev 2013: 387014.<br />
<br />
Roebuck BD (2004) Hyperplasia, partial hepatectomy, and the carcinogenicity of aflatoxin B1. J Cell Biochem 91: 243-249.<br />
<br />
Shelton P, Jaiswal AK (2013) The transcription factor NF-E2-related factor 2 (Nrf2): a protooncogene? FASEB J 27: 414-423.<br />
<br />
U.S. EPA IRIS, (2014) available at http://www.epa.gov/iris/subst/0294.htm#woe<br />
<br />
Yates MS, Tauchi M, Katsuoka F, Flanders KC, Liby KT, et al (2007) Pharmacodynamic characterization of chemopreventive triterpenoids as exceptionally potent inducers of Nrf2-regulated genes. Mol Cancer Ther 6: 154-162.<br />
<br />
Yates MS, Kensler TW (2007) Keap1 eye on the target: chemoprevention of liver cancer. Acta Pharmacol Sin 28: 1331-1342.<br />
<br />
Yates MS, Kwak MK, Egner PA, Groopman JD, Bodreddigari S, et al (2006) Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole. Cancer Res 66: 2488-2494.<br />
<br />
Zhang Y, Guan L, Wang X, Wen T, Xing J, Zhao J (2008) Protection of chlorophyllin against oxidative damage by inducing HO-1 and NQO1 expression mediated by PI3K/Akt and Nrf2. Free Radic Res 42: 362-371.<br />
<br />
Zheng YW, Nie YZ, Taniguchi H (2013) Cellular reprogramming and hepatocellular carcinoma development. World J Gastroenterol 19: 8850-8860.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:378Event:3782016-07-06T17:13:03Z<p>Dvillene: /* AO: Evidence Supporting Taxonomic Applicability */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Hepatocellular carcinoma, Tumorigenesis </div><br />
<div id='shortTitle' class='Title2'> Hepatocellular Carcinoma (HCC) </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]][[Category:Adverse Outcome]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||AO||<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Affected Organs ===<br />
<br />
{|class="wikitable sortable" id="Organproof"<br />
<br />
!Synonym<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|---||liver||[http://www.ontobee.org/browser/rdf.php?o=UBERON&iri=http://purl.obolibrary.org/obo/UBERON_0002107 Ontobee]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Individual<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Level of Biological Organization : Organism ==<br />
<br />
== How this Key Event works ==<br />
The AO final key event results from the processes that occur in the earlier series of key events, which for AFB1 is a mutagenic MOA—the AFB1 induction of mutations in critical cancer genes that alter the phenotype of the mutant cell and set the stage for that cell to progress to a pre-neoplastic lesion and ultimately an HCC. The biological processes described in this AO, however, are not specific to a mutagenic MOA—nor necessarily demonstrated for AFB1 exposure, but occur in development of HCC from all MOAs for HCC. Thus the final key events (AHF and HCC) represent the final stages of the pathway that leads to HCC from a mutagenic MOA or other MOAs. <br />
<br />
Hepatocellular carcinoma (HCC) is a cancer of hepatocytes, and this disease is almost always lethal in the absence of extreme intervention measures (e.g., surgery, liver transplant). A number of factors are associated with HCC including AFB1 exposure, infection with hepatitis virus (HBV), and alcohol use. A common etiologic feature of HCC, whether produced by AFB1 intoxication, HBV, cirrhosis or something else, is the presence of oxidative damage in the liver. (Ravinayagam et al., 2012 Int J Hepatol; Kim et al., 2011 J Ginseng Res).<br />
<br />
AFB1 produces specific pro-mutagenic adducts that are believed to lead to a mutation in the p53 gene, which affects its functioning. P53 is generally considered to be a tumor suppressor gene involved in cell cycle regulation and initiation of apoptosis. When applied in vitro to hepatocytes, AFB1 produced cellular swelling, bleb formation, and lysis. These effects may be due to lipid peroxidation affecting the cell membrane from the downstream dialdehyde metabolite of the AFB1 epoxide metabolites. (Mathijs et al., 2009, 2010) This damage is reflective of oxidative stress, a known contributor to HCC (Ravinayagam et al., 2012 Int J Hepatol; Kim et al., 2011 J Ginseng Res). As discussed elsewhere in this AOP, the Nrf2-Keap1 anti-oxidant response induced by a number of chemoprotective agents can be quite effective in preventing HCC [3-8], even in the presence of a significant burden of N7- AFB1-G adducts. <br />
<br />
The cellular damage produced by exposure to AFB1 likely leads to chronic inflammation, also a contributor to tumor progression. (Ellinger-Ziegelbauer et al., 2004) Heme oxygenase-1 (HO-1) breaks down heme to bilirubin and biliverdin that have anti-oxidant and anti-inflammatory activities (Keum et al., 2006; Caballero et al 2004) , thus countering the inflammatory response. The induction of HO-1 is part of the Nrf2-Keap1 anti-oxidant response. <br />
<br />
From a systems biology and biochemistry perspective, the presence of oxidative stress and inflammation, although not specific only to AFB1 exposure, are strong contributors to cancer progression.(Ohnishi et al., 2013; Zheng et al., 2013; Higgs et al., 2014).<br />
<br />
== How it is Measured or Detected ==<br />
Hepatocellular carcinoma is detected in humans by clinical examination confirmed by pathological examination, and in laboratory test species by pathological examination.<br />
<br />
== Evidence Supporting Taxonomic Applicability ==<br />
Hepatocellular carcinoma occurs in many vertebrate species including birds, fish, and mammals such as humans.<br />
<br />
== AO: Regulatory Examples Using This Adverse Outcome ==<br />
Although not specifically used EPA for regulatory determinations vis-à-vis AFB1, HCC has been used as an adverse endpoint in many hazard assessments that can be used as input to risk management decisions. The U.S. EPA Integrated Risk Information System (IRIS database) contains 111 instances wherein HCC has been considered in hazard assessment of environmental contaminants. For example, HCC in rats formed part of the weight of evidence in categorizing polychlorinated biphenyls as probable human carcinogens. These tumors, combined with other liver tumors, also formed the basis for quantitative dose-response assessment for cancer induced by polychlorinated biphenyls by the oral route.(USEPA, 2014).<br />
<br />
Given that AFB1 can be a contaminant in both human food and animal feed, FDA has established allowable limits. http://www.fda.gov/downloads/advisorycommittees/committeesmeetingmaterials/foodadvisorycommittee/ucm428947.pdf<br />
<br />
== AO: References ==<br />
Caballero F, Meiss R, Gimenez A, Batlle A, Vazquez E (2004) Immunohistochemical analysis of heme oxygenase-1 in preneoplastic and neoplastic lesions during chemical hepatocarcinogenesis. Int J Exp Pathol 85: 213-222.<br />
<br />
Higgs MR, Chouteau P, Lerat H (2014) 'Liver let die': oxidative DNA damage and hepatotropic viruses. J Gen Virol 95: 991-1004.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, et al (2014) Complete protection against aflatoxin B1-induced liver cancer with triterpenoid: DNA adduct dosimetry, molecular signature and genotoxicity threshold. Cancer Prev Res (Phila) .<br />
<br />
Liby KT, Sporn MB (2012) Synthetic oleanane triterpenoids: multifunctional drugs with a broad range of applications for prevention and treatment of chronic disease. Pharmacol Rev 64: 972-1003.<br />
<br />
Liby K, Yore MM, Roebuck BD, Baumgartner KJ, Honda T, et al (2008) A novel acetylenic tricyclic bis-(cyano enone) potently induces phase 2 cytoprotective pathways and blocks liver carcinogenesis induced by aflatoxin. Cancer Res 68: 6727-6733.<br />
<br />
Moudgil V, Redhu D, Dhanda S, Singh J (2013) A review of molecular mechanisms in the development of hepatocellular carcinoma by aflatoxin and hepatitis B and C viruses. J Environ Pathol Toxicol Oncol 32: 165-175.<br />
<br />
Ohnishi S, Ma N, Thanan R, Pinlaor S, Hammam O, et al (2013) DNA damage in inflammation-related carcinogenesis and cancer stem cells. Oxid Med Cell Longev 2013: 387014.<br />
<br />
Roebuck BD (2004) Hyperplasia, partial hepatectomy, and the carcinogenicity of aflatoxin B1. J Cell Biochem 91: 243-249.<br />
<br />
Shelton P, Jaiswal AK (2013) The transcription factor NF-E2-related factor 2 (Nrf2): a protooncogene? FASEB J 27: 414-423.<br />
<br />
U.S. EPA IRIS, (2014) available at http://www.epa.gov/iris/subst/0294.htm#woe<br />
<br />
Yates MS, Tauchi M, Katsuoka F, Flanders KC, Liby KT, et al (2007) Pharmacodynamic characterization of chemopreventive triterpenoids as exceptionally potent inducers of Nrf2-regulated genes. Mol Cancer Ther 6: 154-162.<br />
<br />
Yates MS, Kensler TW (2007) Keap1 eye on the target: chemoprevention of liver cancer. Acta Pharmacol Sin 28: 1331-1342.<br />
<br />
Yates MS, Kwak MK, Egner PA, Groopman JD, Bodreddigari S, et al (2006) Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole. Cancer Res 66: 2488-2494.<br />
<br />
Zhang Y, Guan L, Wang X, Wen T, Xing J, Zhao J (2008) Protection of chlorophyllin against oxidative damage by inducing HO-1 and NQO1 expression mediated by PI3K/Akt and Nrf2. Free Radic Res 42: 362-371.<br />
<br />
Zheng YW, Nie YZ, Taniguchi H (2013) Cellular reprogramming and hepatocellular carcinoma development. World J Gastroenterol 19: 8850-8860.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:378Event:3782016-07-06T17:12:46Z<p>Dvillene: /* AO: How it is Measured or Detected */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Hepatocellular carcinoma, Tumorigenesis </div><br />
<div id='shortTitle' class='Title2'> Hepatocellular Carcinoma (HCC) </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]][[Category:Adverse Outcome]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||AO||<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Affected Organs ===<br />
<br />
{|class="wikitable sortable" id="Organproof"<br />
<br />
!Synonym<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|---||liver||[http://www.ontobee.org/browser/rdf.php?o=UBERON&iri=http://purl.obolibrary.org/obo/UBERON_0002107 Ontobee]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Individual<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Level of Biological Organization : Organism ==<br />
<br />
== How this Key Event works ==<br />
The AO final key event results from the processes that occur in the earlier series of key events, which for AFB1 is a mutagenic MOA—the AFB1 induction of mutations in critical cancer genes that alter the phenotype of the mutant cell and set the stage for that cell to progress to a pre-neoplastic lesion and ultimately an HCC. The biological processes described in this AO, however, are not specific to a mutagenic MOA—nor necessarily demonstrated for AFB1 exposure, but occur in development of HCC from all MOAs for HCC. Thus the final key events (AHF and HCC) represent the final stages of the pathway that leads to HCC from a mutagenic MOA or other MOAs. <br />
<br />
Hepatocellular carcinoma (HCC) is a cancer of hepatocytes, and this disease is almost always lethal in the absence of extreme intervention measures (e.g., surgery, liver transplant). A number of factors are associated with HCC including AFB1 exposure, infection with hepatitis virus (HBV), and alcohol use. A common etiologic feature of HCC, whether produced by AFB1 intoxication, HBV, cirrhosis or something else, is the presence of oxidative damage in the liver. (Ravinayagam et al., 2012 Int J Hepatol; Kim et al., 2011 J Ginseng Res).<br />
<br />
AFB1 produces specific pro-mutagenic adducts that are believed to lead to a mutation in the p53 gene, which affects its functioning. P53 is generally considered to be a tumor suppressor gene involved in cell cycle regulation and initiation of apoptosis. When applied in vitro to hepatocytes, AFB1 produced cellular swelling, bleb formation, and lysis. These effects may be due to lipid peroxidation affecting the cell membrane from the downstream dialdehyde metabolite of the AFB1 epoxide metabolites. (Mathijs et al., 2009, 2010) This damage is reflective of oxidative stress, a known contributor to HCC (Ravinayagam et al., 2012 Int J Hepatol; Kim et al., 2011 J Ginseng Res). As discussed elsewhere in this AOP, the Nrf2-Keap1 anti-oxidant response induced by a number of chemoprotective agents can be quite effective in preventing HCC [3-8], even in the presence of a significant burden of N7- AFB1-G adducts. <br />
<br />
The cellular damage produced by exposure to AFB1 likely leads to chronic inflammation, also a contributor to tumor progression. (Ellinger-Ziegelbauer et al., 2004) Heme oxygenase-1 (HO-1) breaks down heme to bilirubin and biliverdin that have anti-oxidant and anti-inflammatory activities (Keum et al., 2006; Caballero et al 2004) , thus countering the inflammatory response. The induction of HO-1 is part of the Nrf2-Keap1 anti-oxidant response. <br />
<br />
From a systems biology and biochemistry perspective, the presence of oxidative stress and inflammation, although not specific only to AFB1 exposure, are strong contributors to cancer progression.(Ohnishi et al., 2013; Zheng et al., 2013; Higgs et al., 2014).<br />
<br />
== How it is Measured or Detected ==<br />
Hepatocellular carcinoma is detected in humans by clinical examination confirmed by pathological examination, and in laboratory test species by pathological examination.<br />
<br />
== AO: Evidence Supporting Taxonomic Applicability ==<br />
Hepatocellular carcinoma occurs in many vertebrate species including birds, fish, and mammals such as humans.<br />
<br />
== AO: Regulatory Examples Using This Adverse Outcome ==<br />
Although not specifically used EPA for regulatory determinations vis-à-vis AFB1, HCC has been used as an adverse endpoint in many hazard assessments that can be used as input to risk management decisions. The U.S. EPA Integrated Risk Information System (IRIS database) contains 111 instances wherein HCC has been considered in hazard assessment of environmental contaminants. For example, HCC in rats formed part of the weight of evidence in categorizing polychlorinated biphenyls as probable human carcinogens. These tumors, combined with other liver tumors, also formed the basis for quantitative dose-response assessment for cancer induced by polychlorinated biphenyls by the oral route.(USEPA, 2014).<br />
<br />
Given that AFB1 can be a contaminant in both human food and animal feed, FDA has established allowable limits. http://www.fda.gov/downloads/advisorycommittees/committeesmeetingmaterials/foodadvisorycommittee/ucm428947.pdf<br />
<br />
== AO: References ==<br />
Caballero F, Meiss R, Gimenez A, Batlle A, Vazquez E (2004) Immunohistochemical analysis of heme oxygenase-1 in preneoplastic and neoplastic lesions during chemical hepatocarcinogenesis. Int J Exp Pathol 85: 213-222.<br />
<br />
Higgs MR, Chouteau P, Lerat H (2014) 'Liver let die': oxidative DNA damage and hepatotropic viruses. J Gen Virol 95: 991-1004.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, et al (2014) Complete protection against aflatoxin B1-induced liver cancer with triterpenoid: DNA adduct dosimetry, molecular signature and genotoxicity threshold. Cancer Prev Res (Phila) .<br />
<br />
Liby KT, Sporn MB (2012) Synthetic oleanane triterpenoids: multifunctional drugs with a broad range of applications for prevention and treatment of chronic disease. Pharmacol Rev 64: 972-1003.<br />
<br />
Liby K, Yore MM, Roebuck BD, Baumgartner KJ, Honda T, et al (2008) A novel acetylenic tricyclic bis-(cyano enone) potently induces phase 2 cytoprotective pathways and blocks liver carcinogenesis induced by aflatoxin. Cancer Res 68: 6727-6733.<br />
<br />
Moudgil V, Redhu D, Dhanda S, Singh J (2013) A review of molecular mechanisms in the development of hepatocellular carcinoma by aflatoxin and hepatitis B and C viruses. J Environ Pathol Toxicol Oncol 32: 165-175.<br />
<br />
Ohnishi S, Ma N, Thanan R, Pinlaor S, Hammam O, et al (2013) DNA damage in inflammation-related carcinogenesis and cancer stem cells. Oxid Med Cell Longev 2013: 387014.<br />
<br />
Roebuck BD (2004) Hyperplasia, partial hepatectomy, and the carcinogenicity of aflatoxin B1. J Cell Biochem 91: 243-249.<br />
<br />
Shelton P, Jaiswal AK (2013) The transcription factor NF-E2-related factor 2 (Nrf2): a protooncogene? FASEB J 27: 414-423.<br />
<br />
U.S. EPA IRIS, (2014) available at http://www.epa.gov/iris/subst/0294.htm#woe<br />
<br />
Yates MS, Tauchi M, Katsuoka F, Flanders KC, Liby KT, et al (2007) Pharmacodynamic characterization of chemopreventive triterpenoids as exceptionally potent inducers of Nrf2-regulated genes. Mol Cancer Ther 6: 154-162.<br />
<br />
Yates MS, Kensler TW (2007) Keap1 eye on the target: chemoprevention of liver cancer. Acta Pharmacol Sin 28: 1331-1342.<br />
<br />
Yates MS, Kwak MK, Egner PA, Groopman JD, Bodreddigari S, et al (2006) Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole. Cancer Res 66: 2488-2494.<br />
<br />
Zhang Y, Guan L, Wang X, Wen T, Xing J, Zhao J (2008) Protection of chlorophyllin against oxidative damage by inducing HO-1 and NQO1 expression mediated by PI3K/Akt and Nrf2. Free Radic Res 42: 362-371.<br />
<br />
Zheng YW, Nie YZ, Taniguchi H (2013) Cellular reprogramming and hepatocellular carcinoma development. World J Gastroenterol 19: 8850-8860.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:378Event:3782016-07-06T17:12:27Z<p>Dvillene: /* AO: How this Key Event works */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Hepatocellular carcinoma, Tumorigenesis </div><br />
<div id='shortTitle' class='Title2'> Hepatocellular Carcinoma (HCC) </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]][[Category:Adverse Outcome]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||AO||<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Affected Organs ===<br />
<br />
{|class="wikitable sortable" id="Organproof"<br />
<br />
!Synonym<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|---||liver||[http://www.ontobee.org/browser/rdf.php?o=UBERON&iri=http://purl.obolibrary.org/obo/UBERON_0002107 Ontobee]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Individual<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Level of Biological Organization : Organism ==<br />
<br />
== How this Key Event works ==<br />
The AO final key event results from the processes that occur in the earlier series of key events, which for AFB1 is a mutagenic MOA—the AFB1 induction of mutations in critical cancer genes that alter the phenotype of the mutant cell and set the stage for that cell to progress to a pre-neoplastic lesion and ultimately an HCC. The biological processes described in this AO, however, are not specific to a mutagenic MOA—nor necessarily demonstrated for AFB1 exposure, but occur in development of HCC from all MOAs for HCC. Thus the final key events (AHF and HCC) represent the final stages of the pathway that leads to HCC from a mutagenic MOA or other MOAs. <br />
<br />
Hepatocellular carcinoma (HCC) is a cancer of hepatocytes, and this disease is almost always lethal in the absence of extreme intervention measures (e.g., surgery, liver transplant). A number of factors are associated with HCC including AFB1 exposure, infection with hepatitis virus (HBV), and alcohol use. A common etiologic feature of HCC, whether produced by AFB1 intoxication, HBV, cirrhosis or something else, is the presence of oxidative damage in the liver. (Ravinayagam et al., 2012 Int J Hepatol; Kim et al., 2011 J Ginseng Res).<br />
<br />
AFB1 produces specific pro-mutagenic adducts that are believed to lead to a mutation in the p53 gene, which affects its functioning. P53 is generally considered to be a tumor suppressor gene involved in cell cycle regulation and initiation of apoptosis. When applied in vitro to hepatocytes, AFB1 produced cellular swelling, bleb formation, and lysis. These effects may be due to lipid peroxidation affecting the cell membrane from the downstream dialdehyde metabolite of the AFB1 epoxide metabolites. (Mathijs et al., 2009, 2010) This damage is reflective of oxidative stress, a known contributor to HCC (Ravinayagam et al., 2012 Int J Hepatol; Kim et al., 2011 J Ginseng Res). As discussed elsewhere in this AOP, the Nrf2-Keap1 anti-oxidant response induced by a number of chemoprotective agents can be quite effective in preventing HCC [3-8], even in the presence of a significant burden of N7- AFB1-G adducts. <br />
<br />
The cellular damage produced by exposure to AFB1 likely leads to chronic inflammation, also a contributor to tumor progression. (Ellinger-Ziegelbauer et al., 2004) Heme oxygenase-1 (HO-1) breaks down heme to bilirubin and biliverdin that have anti-oxidant and anti-inflammatory activities (Keum et al., 2006; Caballero et al 2004) , thus countering the inflammatory response. The induction of HO-1 is part of the Nrf2-Keap1 anti-oxidant response. <br />
<br />
From a systems biology and biochemistry perspective, the presence of oxidative stress and inflammation, although not specific only to AFB1 exposure, are strong contributors to cancer progression.(Ohnishi et al., 2013; Zheng et al., 2013; Higgs et al., 2014).<br />
<br />
== AO: How it is Measured or Detected ==<br />
Hepatocellular carcinoma is detected in humans by clinical examination confirmed by pathological examination, and in laboratory test species by pathological examination.<br />
<br />
== AO: Evidence Supporting Taxonomic Applicability ==<br />
Hepatocellular carcinoma occurs in many vertebrate species including birds, fish, and mammals such as humans.<br />
<br />
== AO: Regulatory Examples Using This Adverse Outcome ==<br />
Although not specifically used EPA for regulatory determinations vis-à-vis AFB1, HCC has been used as an adverse endpoint in many hazard assessments that can be used as input to risk management decisions. The U.S. EPA Integrated Risk Information System (IRIS database) contains 111 instances wherein HCC has been considered in hazard assessment of environmental contaminants. For example, HCC in rats formed part of the weight of evidence in categorizing polychlorinated biphenyls as probable human carcinogens. These tumors, combined with other liver tumors, also formed the basis for quantitative dose-response assessment for cancer induced by polychlorinated biphenyls by the oral route.(USEPA, 2014).<br />
<br />
Given that AFB1 can be a contaminant in both human food and animal feed, FDA has established allowable limits. http://www.fda.gov/downloads/advisorycommittees/committeesmeetingmaterials/foodadvisorycommittee/ucm428947.pdf<br />
<br />
== AO: References ==<br />
Caballero F, Meiss R, Gimenez A, Batlle A, Vazquez E (2004) Immunohistochemical analysis of heme oxygenase-1 in preneoplastic and neoplastic lesions during chemical hepatocarcinogenesis. Int J Exp Pathol 85: 213-222.<br />
<br />
Higgs MR, Chouteau P, Lerat H (2014) 'Liver let die': oxidative DNA damage and hepatotropic viruses. J Gen Virol 95: 991-1004.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, et al (2014) Complete protection against aflatoxin B1-induced liver cancer with triterpenoid: DNA adduct dosimetry, molecular signature and genotoxicity threshold. Cancer Prev Res (Phila) .<br />
<br />
Liby KT, Sporn MB (2012) Synthetic oleanane triterpenoids: multifunctional drugs with a broad range of applications for prevention and treatment of chronic disease. Pharmacol Rev 64: 972-1003.<br />
<br />
Liby K, Yore MM, Roebuck BD, Baumgartner KJ, Honda T, et al (2008) A novel acetylenic tricyclic bis-(cyano enone) potently induces phase 2 cytoprotective pathways and blocks liver carcinogenesis induced by aflatoxin. Cancer Res 68: 6727-6733.<br />
<br />
Moudgil V, Redhu D, Dhanda S, Singh J (2013) A review of molecular mechanisms in the development of hepatocellular carcinoma by aflatoxin and hepatitis B and C viruses. J Environ Pathol Toxicol Oncol 32: 165-175.<br />
<br />
Ohnishi S, Ma N, Thanan R, Pinlaor S, Hammam O, et al (2013) DNA damage in inflammation-related carcinogenesis and cancer stem cells. Oxid Med Cell Longev 2013: 387014.<br />
<br />
Roebuck BD (2004) Hyperplasia, partial hepatectomy, and the carcinogenicity of aflatoxin B1. J Cell Biochem 91: 243-249.<br />
<br />
Shelton P, Jaiswal AK (2013) The transcription factor NF-E2-related factor 2 (Nrf2): a protooncogene? FASEB J 27: 414-423.<br />
<br />
U.S. EPA IRIS, (2014) available at http://www.epa.gov/iris/subst/0294.htm#woe<br />
<br />
Yates MS, Tauchi M, Katsuoka F, Flanders KC, Liby KT, et al (2007) Pharmacodynamic characterization of chemopreventive triterpenoids as exceptionally potent inducers of Nrf2-regulated genes. Mol Cancer Ther 6: 154-162.<br />
<br />
Yates MS, Kensler TW (2007) Keap1 eye on the target: chemoprevention of liver cancer. Acta Pharmacol Sin 28: 1331-1342.<br />
<br />
Yates MS, Kwak MK, Egner PA, Groopman JD, Bodreddigari S, et al (2006) Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole. Cancer Res 66: 2488-2494.<br />
<br />
Zhang Y, Guan L, Wang X, Wen T, Xing J, Zhao J (2008) Protection of chlorophyllin against oxidative damage by inducing HO-1 and NQO1 expression mediated by PI3K/Akt and Nrf2. Free Radic Res 42: 362-371.<br />
<br />
Zheng YW, Nie YZ, Taniguchi H (2013) Cellular reprogramming and hepatocellular carcinoma development. World J Gastroenterol 19: 8850-8860.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:378Event:3782016-07-06T17:12:02Z<p>Dvillene: /* AO: Level of Biological Organization : Organism */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Hepatocellular carcinoma, Tumorigenesis </div><br />
<div id='shortTitle' class='Title2'> Hepatocellular Carcinoma (HCC) </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]][[Category:Adverse Outcome]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||AO||<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Affected Organs ===<br />
<br />
{|class="wikitable sortable" id="Organproof"<br />
<br />
!Synonym<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|---||liver||[http://www.ontobee.org/browser/rdf.php?o=UBERON&iri=http://purl.obolibrary.org/obo/UBERON_0002107 Ontobee]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Individual<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Level of Biological Organization : Organism ==<br />
<br />
== AO: How this Key Event works ==<br />
The AO final key event results from the processes that occur in the earlier series of key events, which for AFB1 is a mutagenic MOA—the AFB1 induction of mutations in critical cancer genes that alter the phenotype of the mutant cell and set the stage for that cell to progress to a pre-neoplastic lesion and ultimately an HCC. The biological processes described in this AO, however, are not specific to a mutagenic MOA—nor necessarily demonstrated for AFB1 exposure, but occur in development of HCC from all MOAs for HCC. Thus the final key events (AHF and HCC) represent the final stages of the pathway that leads to HCC from a mutagenic MOA or other MOAs. <br />
<br />
Hepatocellular carcinoma (HCC) is a cancer of hepatocytes, and this disease is almost always lethal in the absence of extreme intervention measures (e.g., surgery, liver transplant). A number of factors are associated with HCC including AFB1 exposure, infection with hepatitis virus (HBV), and alcohol use. A common etiologic feature of HCC, whether produced by AFB1 intoxication, HBV, cirrhosis or something else, is the presence of oxidative damage in the liver. (Ravinayagam et al., 2012 Int J Hepatol; Kim et al., 2011 J Ginseng Res).<br />
<br />
AFB1 produces specific pro-mutagenic adducts that are believed to lead to a mutation in the p53 gene, which affects its functioning. P53 is generally considered to be a tumor suppressor gene involved in cell cycle regulation and initiation of apoptosis. When applied in vitro to hepatocytes, AFB1 produced cellular swelling, bleb formation, and lysis. These effects may be due to lipid peroxidation affecting the cell membrane from the downstream dialdehyde metabolite of the AFB1 epoxide metabolites. (Mathijs et al., 2009, 2010) This damage is reflective of oxidative stress, a known contributor to HCC (Ravinayagam et al., 2012 Int J Hepatol; Kim et al., 2011 J Ginseng Res). As discussed elsewhere in this AOP, the Nrf2-Keap1 anti-oxidant response induced by a number of chemoprotective agents can be quite effective in preventing HCC [3-8], even in the presence of a significant burden of N7- AFB1-G adducts. <br />
<br />
The cellular damage produced by exposure to AFB1 likely leads to chronic inflammation, also a contributor to tumor progression. (Ellinger-Ziegelbauer et al., 2004) Heme oxygenase-1 (HO-1) breaks down heme to bilirubin and biliverdin that have anti-oxidant and anti-inflammatory activities (Keum et al., 2006; Caballero et al 2004) , thus countering the inflammatory response. The induction of HO-1 is part of the Nrf2-Keap1 anti-oxidant response. <br />
<br />
From a systems biology and biochemistry perspective, the presence of oxidative stress and inflammation, although not specific only to AFB1 exposure, are strong contributors to cancer progression.(Ohnishi et al., 2013; Zheng et al., 2013; Higgs et al., 2014).<br />
<br />
== AO: How it is Measured or Detected ==<br />
Hepatocellular carcinoma is detected in humans by clinical examination confirmed by pathological examination, and in laboratory test species by pathological examination.<br />
<br />
== AO: Evidence Supporting Taxonomic Applicability ==<br />
Hepatocellular carcinoma occurs in many vertebrate species including birds, fish, and mammals such as humans.<br />
<br />
== AO: Regulatory Examples Using This Adverse Outcome ==<br />
Although not specifically used EPA for regulatory determinations vis-à-vis AFB1, HCC has been used as an adverse endpoint in many hazard assessments that can be used as input to risk management decisions. The U.S. EPA Integrated Risk Information System (IRIS database) contains 111 instances wherein HCC has been considered in hazard assessment of environmental contaminants. For example, HCC in rats formed part of the weight of evidence in categorizing polychlorinated biphenyls as probable human carcinogens. These tumors, combined with other liver tumors, also formed the basis for quantitative dose-response assessment for cancer induced by polychlorinated biphenyls by the oral route.(USEPA, 2014).<br />
<br />
Given that AFB1 can be a contaminant in both human food and animal feed, FDA has established allowable limits. http://www.fda.gov/downloads/advisorycommittees/committeesmeetingmaterials/foodadvisorycommittee/ucm428947.pdf<br />
<br />
== AO: References ==<br />
Caballero F, Meiss R, Gimenez A, Batlle A, Vazquez E (2004) Immunohistochemical analysis of heme oxygenase-1 in preneoplastic and neoplastic lesions during chemical hepatocarcinogenesis. Int J Exp Pathol 85: 213-222.<br />
<br />
Higgs MR, Chouteau P, Lerat H (2014) 'Liver let die': oxidative DNA damage and hepatotropic viruses. J Gen Virol 95: 991-1004.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, et al (2014) Complete protection against aflatoxin B1-induced liver cancer with triterpenoid: DNA adduct dosimetry, molecular signature and genotoxicity threshold. Cancer Prev Res (Phila) .<br />
<br />
Liby KT, Sporn MB (2012) Synthetic oleanane triterpenoids: multifunctional drugs with a broad range of applications for prevention and treatment of chronic disease. Pharmacol Rev 64: 972-1003.<br />
<br />
Liby K, Yore MM, Roebuck BD, Baumgartner KJ, Honda T, et al (2008) A novel acetylenic tricyclic bis-(cyano enone) potently induces phase 2 cytoprotective pathways and blocks liver carcinogenesis induced by aflatoxin. Cancer Res 68: 6727-6733.<br />
<br />
Moudgil V, Redhu D, Dhanda S, Singh J (2013) A review of molecular mechanisms in the development of hepatocellular carcinoma by aflatoxin and hepatitis B and C viruses. J Environ Pathol Toxicol Oncol 32: 165-175.<br />
<br />
Ohnishi S, Ma N, Thanan R, Pinlaor S, Hammam O, et al (2013) DNA damage in inflammation-related carcinogenesis and cancer stem cells. Oxid Med Cell Longev 2013: 387014.<br />
<br />
Roebuck BD (2004) Hyperplasia, partial hepatectomy, and the carcinogenicity of aflatoxin B1. J Cell Biochem 91: 243-249.<br />
<br />
Shelton P, Jaiswal AK (2013) The transcription factor NF-E2-related factor 2 (Nrf2): a protooncogene? FASEB J 27: 414-423.<br />
<br />
U.S. EPA IRIS, (2014) available at http://www.epa.gov/iris/subst/0294.htm#woe<br />
<br />
Yates MS, Tauchi M, Katsuoka F, Flanders KC, Liby KT, et al (2007) Pharmacodynamic characterization of chemopreventive triterpenoids as exceptionally potent inducers of Nrf2-regulated genes. Mol Cancer Ther 6: 154-162.<br />
<br />
Yates MS, Kensler TW (2007) Keap1 eye on the target: chemoprevention of liver cancer. Acta Pharmacol Sin 28: 1331-1342.<br />
<br />
Yates MS, Kwak MK, Egner PA, Groopman JD, Bodreddigari S, et al (2006) Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole. Cancer Res 66: 2488-2494.<br />
<br />
Zhang Y, Guan L, Wang X, Wen T, Xing J, Zhao J (2008) Protection of chlorophyllin against oxidative damage by inducing HO-1 and NQO1 expression mediated by PI3K/Akt and Nrf2. Free Radic Res 42: 362-371.<br />
<br />
Zheng YW, Nie YZ, Taniguchi H (2013) Cellular reprogramming and hepatocellular carcinoma development. World J Gastroenterol 19: 8850-8860.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:417Event:4172016-07-06T17:09:50Z<p>Dvillene: </p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> sex ratio, skewed </div><br />
<div id='shortTitle' class='Title2'> </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]][[Category:Adverse Outcome]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:52|ER agonism leading to skewed sex ratios due to altered sexual differentiation in males]]||AO||<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Affected Organs ===<br />
<br />
{|class="wikitable sortable" id="Organproof"<br />
<br />
!Synonym<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|}<br />
<br />
== How this Key Event works ==<br />
<br />
== How it is Measured or Detected ==<br />
<em><br />
Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above.<br />
All other methods, including those well established in the published literature, should be described here. <br />
Consider the following criteria when describing each method:<br />
1. Is the assay fit for purpose?<br />
2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final<br />
adverse effect in question?<br />
3. Is the assay repeatable?<br />
4. Is the assay reproducible?<br />
</em><br />
<br />
== Evidence Supporting Taxonomic Applicability ==<br />
<br />
== References ==<br />
<br />
<references /></div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:409Event:4092016-07-06T17:08:26Z<p>Dvillene: /* Evidence Supporting Essentiality */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id ='longTitle' class='Title'> Step: #0: pre-MIE </div><br />
<div id ='longTitle' class='Title'> Action: Metabolism of AFB1 </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||KE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Molecular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Biological Process: Production of Reactive Electrophiles ==<br />
Aflatoxin B1 (AFB1) can be oxidized to a variety of metabolites, many of which can be further metabolized, for example by conjugation with glutathione (GSH) via Glutathione-S-transferase (GST). In order for DNA binding and formation of a pro-mutagenic DNA adduct to occur, AFB1 must be metabolized via Cytochrome P450 metabolism to a specific highly reactive form of AFB1, the exo-epoxide AFB1-8,9-epoxide. CYP3A4 forms the exo-form of this reactive epoxide only. CYP1A2, inducible in liver, forms both the exo- and the endo-epoxides; in human liver, CYP1A2 metabolism occurs with a lower Vmax (enzymatic rate) and higher Km (half-maximal concentrations) than CYP3A4 (Degen and Neumann,1981; Groopman and Kensler, 2005; Guengerich et al., 1996; Ueng et al., 1995). Figure 1, from Pottenger et al., 2014, depicts the metabolism of AFB1.<br />
<br />
Metabolic activation of AFB1 occurs mainly in the liver, where the highest levels of the CYP isozymes are located. Competitive metabolism with other hepatic and extra-hepatic P450 isozymes may decrease the proportion of the specific metabolite, AFB1-8,9-epoxide; this can reduce the effectiveness of hepatic activation of AFB1. Alternatively, induction of either hepatic or extra-hepatic GST activity can reduce the levels of AFB1-epoxide available for reaction with DNA by increasing conjugation of the epoxide with GSH. This conjugation renders the epoxide inactive and directs it towards eventual elimination (Guengerich et al., 1996).[[File:Figure1met.jpg|thumb|alt=Figure 1 alt text|Figure 1. Metabolism of AFB1,]] Figure 1 was obtained from Pottenger et al., 2014.<br />
<br />
The AFB1 pro-mutagenic DNA adduct is most likely the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct, or N7-AFB1-G (Croy et al., 1978). Once the exo-epoxide is bound to the N7-guanine, it is subject to ring-opening to form the more highly pro-mutagenic 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-pyrimid-9-yl-foramido)-9-hydroxyaflatoxin B1; this is known as the formamidopyrimidine adduct or AFB1-FAPy adduct (Brown et al., 2006).<br />
<br />
== Taxonomic Applicability ==<br />
The metabolic information above is applicable to all mammalian systems evaluated; it is also applicable to certain birds such as turkeys (Gregory et al., 1983; IARC, 1993). Humans, non-human primates, rats, mice, poultry and fish have all demonstrated susceptibility to AFB1-induced liver tumors (Asplin and Canaghan, 1961; Eaton and Gallagher, 1994). Species that preferentially metabolize AFB1 to the exo-8,9-epoxide are more susceptible to AFB1 carcinogenicity. Sulfation by GST enzymes or other enzymatic detoxification of AFB1 metabolites can reduce available levels of the exo-epoxide, and subsequent steps or even the progression of Key Events may not occur in these species. Mice are less susceptible to AFB1-induced cancer than rats, likely due to increased detoxification (Monroe and Eaton, 1987). This increased detoxication capability can also be instigated by dietary exposures to compounds that modulate CYP450 expression or modify detoxication activities (Elegbede and Gould, 2002; Primiano et al., 1995; Roebuck et al. 1991, 2003; Kensler et al., 1998; Wang et al., 1999; Yates et al., 2006).<br />
<br />
== Level of Biological Organization : Molecular ==<br />
Metabolic activation of AFB1 occurs at the intracellular level, with sub-cellular organelles involved due to the localization of CYP450 in the endoplasmic reticulum, coupled with the epoxide hydratase; GST is localized in the cytosol (Guengerich et al., 1996). Organ architecture also plays a role, as the structure of the liver is orientated around the O2 gradient set by the portal artery and hepatic vein; hepatocytes nearest the portal artery demonstrate increased tissue oxygen while those around the portal vein have decreased tissue O2 and correspondingly higher levels of CYP450 activity.<br />
<br />
== How this Key Event works ==<br />
The reactive exo-epoxide is formed in hepatocytes (or extra-hepatically) by metabolism of the parent AFB1 by CYP450 (Larsson et al., 1990; Larsson and Tjalve, 1993). The reactive metabolite then escapes the endoplasmic reticulum where the CYP450 is located. The reactive metabolite must evade conjugation with GSH in the cytoplasm or binding with other cytoplasmic nucleophiles. It then traverses the nuclear membrane in order to reach the cell nucleus and the nuclear DNA. Once the reactive metabolite is in the cell nucleus, binding to nuclear DNA and the formation of DNA adducts can occur.<br />
<br />
== How it is Measured or Detected ==<br />
Formation of the exo-epoxide can be produced with in vitro systems and detected using techniques for structural quantitation of AFB1 metabolites (Himmelstein et al., 2009), including liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). By using subcellular fractions (e.g., microsomes), cellular homogenates, or cells in culture, it is possible to measure formation of AFB1 exo-epoxide. Such data can also be collected from in vivo systems; samples of plasma or blood from AFB1-treated animals can be analyzed for the AFB1 exo-epoxide with similar mass spectrometric based detection systems (e.g., LC-MS/MS). Samples of blood from humans in AFB1-endemic regions have demonstrated presence of AFB1-albumin adducts, which are formed from the AFB1 exo-epoxide. AFB1-treated animals may also provide tissue samples for analysis of AFB1 exo-epoxide. Special trapping techniques may be required as the reactive AFB1 exo-epoxide metabolite has a short half-life in biological matrices.<br />
<br />
== Evidence Supporting Taxonomic Applicability ==<br />
Ample data across phyla demonstrate metabolic activation of AFB1 to the exo-epoxide via CYP450. These taxons include several mammalian species (humans, non-human primates, rats, mice) in addition to birds (turkeys) and fish (Eaton and Gallagher, 1994; IARC, 1993).<br />
<br />
== Evidence for Chemical Initiation of this Initial Molecular Event ==<br />
Pre-MIE:<br />
There is an extensive database on AFB1 in many different systems demonstrating formation of the AFB1 exo-epoxide. This database includes several mammalian species (humans, non-human primates, rats, mice) in addition to birds (turkeys) and fish (Eaton and Gallagher, 1994; IARC, 1993).<br />
<br />
== Evidence Supporting Essentiality ==<br />
Pre-MIE<br />
<br />
Strong<br />
<br />
Many studies show that in the absence of AFB1 metabolic activation, tumors do not occur. Treatment of rats with the CYP450-inhibitor and GST-inducer oltipraz reduces the levels of AFB1 DNA adducts by 65-70%, and the later-forming altered hepatic foci (AHF) and liver tumors are also reduced by 97 and 100%, respectively; thus, no hepatocellular carcinomas (HCC) form in the oltipraz-treated rats dosed with AFB1 (Roebuck et al., 1991). More recent data show even more effective prevention of tumors and of hepatic foci in rats pre-treated with a triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) (Johnson et al., 2014). The pre-treated rats were reported with a 70% reduction of AFB1-induced adducts in urine but had 100% inhibition of tumors, and only 1 rat out of 23 pre-treated had any altered hepatic foci (AHF). There are similar data in humans treated with oltipraz. Kensler et al. (1998) report a significant reduction in a biomarker of exposure AFB1-induced albumin adducts, supporting the human relevance of this metabolic activation <br />
<br />
Another line of evidence for essentiality of the pre-MIE is the recognized difference in sensitivity to AFB1-induced liver tumors between mice and rats (Degen and Neumann, 1981). Mice, have considerably increased metabolic activation of AFB1 to the exo-epoxide compared to rats; mice are nonetheless much less sensitive to AFB1-induced liver tumors. It is thought that this difference is due to the constitutive presence of GST-alpha activity in mice vs rats, where this activity is not found.<br />
<br />
== References ==<br />
F.D. Asplin, R.B.A. Carnaghan, (1961). The toxicity of certain groundnut meals for poultry with special reference to their effect on ducklings and chickens. Vet. Rec. 73:1215– 1219. <br />
<br />
Brown KL, Deng JZ, Iyer RS, Iyer LG, Voehler MW, Stone MP, Harris CM, Harris TM (2006). Unraveling the aflatoxin-FAPY conundrum: Structural basis of the formamidopyrimidine-type DNA adduct of aflatoxin B1. J Am Chem Soc 128:15188-15199.<br />
<br />
Croy RG, Essigman JM, Reinhold VN, Wogan GN (1978). Identification of the principal aflatoxin N1-DNA adduct formed in vivo in rat liver. Proc Natl Acad Sci USA 75:1745-1749.<br />
<br />
Degen GH, Neumann HG (1981). Differences in aflatoxin B1-susceptibility of rat and mouse are correlated with the capability in vitro to inactivate aflatoxin B1-epoxide. Carcinogenesis 2:299–306.<br />
<br />
Eaton DL, and Gallagher EP (1994). Mechanisms of aflatoxin carcinogenesis. Annu Rev Pharmacol Toxicol 34:135-172.<br />
<br />
Elegbede JA, and Gould MN. (2002). Monoterpenes reduced adducts formation in rats exposed to aflatoxin B1. African J Biotech, 1, 46–49.<br />
<br />
Gregory 3rd JF, Goldstein SL, Edds GT. (1983). Metabolite distribution and rate of residue clearance in turkeys fed a diet containing aflatoxin B1. Food Chem Toxicol, 21, 463–7.<br />
<br />
Groopman JD, Kensler TW (2005). Role of metabolism and viruses in aflatoxin-induced liver cancer. Toxicol Appl Pharmacol 206:131-137.<br />
<br />
Guengerich FP, Johnson WW, Ueng Y-F, Yamazaki H, Shimada T (1996). Involvement of Cytochrome P450, glutathione S-transferase, and epoxide hydrolase in the metabolism of aflatoxin B1 and relevance to risk of human liver cancer. Environ Health Perspect 104(Suppl 3):557-562.<br />
<br />
Himmelstein MW, Boogaard PJ, Cadet J, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: II.Overview of methods of identification and quantitation of DNA damage. Crit Rev Toxicol, 39, 679–694.<br />
<br />
IARC (1993). Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. IARC Monographs on the Evaluation of Carcinogenic Risk to Humans. Vol. 56, 245-395.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, Sutter TR, Groopman JD, Kensler TW, Roebuck BD. (2014). Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold. Cancer Prev Res. 7(7):658-665.<br />
<br />
Kensler TW, He X, Otieno M, et al. (1998). Oltipraz chemoprevention trial in Qidong, People’s Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol Biomarkers Prev, 7, 127–34.<br />
<br />
Larsson P, Hoedaya, WI, Tjalve H. (1990). Disposition of 3H-aflatoxin H in mice: formation and retention of tissue bound metabolites in nasal glands. Pharmacol Toxicol, 67, 162–71.<br />
<br />
Larsson P, and Tjalve H. (1993). Distribution and metabolism of aflatoxin B1 in the marmoset monkey (Callithrix jacchus). Carcinogenesis, 14, 1–6.<br />
<br />
Monroe DH, Eaton DL. (1987). Comparative effects of butylated hydroxyanisole on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B1 in the rat and the mouse. Toxicol Appl Pharmacol, 90, 401–409.<br />
<br />
Pottenger, L.H., Andrews LS, Bachman AN, Boogaard PJ, Cadet J, Embry MR, Farmer PB, Himmelstein MW, Jarabek AM, Martin EA, Mauthe RJ, Persaud R, Preston RJ, Schoeny R, Skare J, Swenberg JA, Williams GM, Zeiger E, Zhang F, Kim JH. (2014). An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride. Crit. Rev. Toxicol. 44(4):348-391.<br />
<br />
Primiano T, Egner PA, Sutter TR, et al. (1995). Intermittent dosing with oltipraz: relationship between chemoprevention of aflatoxin-induced tumorigenesis and induction of glutathione-S-transferases. Cancer Res, 55, 4319–4324.<br />
<br />
Roebuck BD, Liu Y-L, Rogers AE, et al. (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short term molecular dosimetry. Cancer Res, 51, 5501–5506.<br />
<br />
Ueng Y-F, Shimada T, Yamazaki H, Guengerich FP (1995). Oxidation of aflatoxin B1 by bacterial recombinant human cytochrome P450 enzymes. Chem Res Toxiol 8:218-225.<br />
<br />
Wang J-S, Shen X, He X, et al. (1999). Protective alerations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qidong, People’s Republic of China. J Natl Cancer Inst, 91, 347–354.<br />
<br />
Yates MS, Kwak M-K, Egner PA, et al. (2006). Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl] imidazole. Cancer Res, 66, 2488–2494.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:409Event:4092016-07-06T17:07:19Z<p>Dvillene: /* References */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id ='longTitle' class='Title'> Step: #0: pre-MIE </div><br />
<div id ='longTitle' class='Title'> Action: Metabolism of AFB1 </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||KE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Molecular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Biological Process: Production of Reactive Electrophiles ==<br />
Aflatoxin B1 (AFB1) can be oxidized to a variety of metabolites, many of which can be further metabolized, for example by conjugation with glutathione (GSH) via Glutathione-S-transferase (GST). In order for DNA binding and formation of a pro-mutagenic DNA adduct to occur, AFB1 must be metabolized via Cytochrome P450 metabolism to a specific highly reactive form of AFB1, the exo-epoxide AFB1-8,9-epoxide. CYP3A4 forms the exo-form of this reactive epoxide only. CYP1A2, inducible in liver, forms both the exo- and the endo-epoxides; in human liver, CYP1A2 metabolism occurs with a lower Vmax (enzymatic rate) and higher Km (half-maximal concentrations) than CYP3A4 (Degen and Neumann,1981; Groopman and Kensler, 2005; Guengerich et al., 1996; Ueng et al., 1995). Figure 1, from Pottenger et al., 2014, depicts the metabolism of AFB1.<br />
<br />
Metabolic activation of AFB1 occurs mainly in the liver, where the highest levels of the CYP isozymes are located. Competitive metabolism with other hepatic and extra-hepatic P450 isozymes may decrease the proportion of the specific metabolite, AFB1-8,9-epoxide; this can reduce the effectiveness of hepatic activation of AFB1. Alternatively, induction of either hepatic or extra-hepatic GST activity can reduce the levels of AFB1-epoxide available for reaction with DNA by increasing conjugation of the epoxide with GSH. This conjugation renders the epoxide inactive and directs it towards eventual elimination (Guengerich et al., 1996).[[File:Figure1met.jpg|thumb|alt=Figure 1 alt text|Figure 1. Metabolism of AFB1,]] Figure 1 was obtained from Pottenger et al., 2014.<br />
<br />
The AFB1 pro-mutagenic DNA adduct is most likely the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct, or N7-AFB1-G (Croy et al., 1978). Once the exo-epoxide is bound to the N7-guanine, it is subject to ring-opening to form the more highly pro-mutagenic 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-pyrimid-9-yl-foramido)-9-hydroxyaflatoxin B1; this is known as the formamidopyrimidine adduct or AFB1-FAPy adduct (Brown et al., 2006).<br />
<br />
== Taxonomic Applicability ==<br />
The metabolic information above is applicable to all mammalian systems evaluated; it is also applicable to certain birds such as turkeys (Gregory et al., 1983; IARC, 1993). Humans, non-human primates, rats, mice, poultry and fish have all demonstrated susceptibility to AFB1-induced liver tumors (Asplin and Canaghan, 1961; Eaton and Gallagher, 1994). Species that preferentially metabolize AFB1 to the exo-8,9-epoxide are more susceptible to AFB1 carcinogenicity. Sulfation by GST enzymes or other enzymatic detoxification of AFB1 metabolites can reduce available levels of the exo-epoxide, and subsequent steps or even the progression of Key Events may not occur in these species. Mice are less susceptible to AFB1-induced cancer than rats, likely due to increased detoxification (Monroe and Eaton, 1987). This increased detoxication capability can also be instigated by dietary exposures to compounds that modulate CYP450 expression or modify detoxication activities (Elegbede and Gould, 2002; Primiano et al., 1995; Roebuck et al. 1991, 2003; Kensler et al., 1998; Wang et al., 1999; Yates et al., 2006).<br />
<br />
== Level of Biological Organization : Molecular ==<br />
Metabolic activation of AFB1 occurs at the intracellular level, with sub-cellular organelles involved due to the localization of CYP450 in the endoplasmic reticulum, coupled with the epoxide hydratase; GST is localized in the cytosol (Guengerich et al., 1996). Organ architecture also plays a role, as the structure of the liver is orientated around the O2 gradient set by the portal artery and hepatic vein; hepatocytes nearest the portal artery demonstrate increased tissue oxygen while those around the portal vein have decreased tissue O2 and correspondingly higher levels of CYP450 activity.<br />
<br />
== How this Key Event works ==<br />
The reactive exo-epoxide is formed in hepatocytes (or extra-hepatically) by metabolism of the parent AFB1 by CYP450 (Larsson et al., 1990; Larsson and Tjalve, 1993). The reactive metabolite then escapes the endoplasmic reticulum where the CYP450 is located. The reactive metabolite must evade conjugation with GSH in the cytoplasm or binding with other cytoplasmic nucleophiles. It then traverses the nuclear membrane in order to reach the cell nucleus and the nuclear DNA. Once the reactive metabolite is in the cell nucleus, binding to nuclear DNA and the formation of DNA adducts can occur.<br />
<br />
== How it is Measured or Detected ==<br />
Formation of the exo-epoxide can be produced with in vitro systems and detected using techniques for structural quantitation of AFB1 metabolites (Himmelstein et al., 2009), including liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). By using subcellular fractions (e.g., microsomes), cellular homogenates, or cells in culture, it is possible to measure formation of AFB1 exo-epoxide. Such data can also be collected from in vivo systems; samples of plasma or blood from AFB1-treated animals can be analyzed for the AFB1 exo-epoxide with similar mass spectrometric based detection systems (e.g., LC-MS/MS). Samples of blood from humans in AFB1-endemic regions have demonstrated presence of AFB1-albumin adducts, which are formed from the AFB1 exo-epoxide. AFB1-treated animals may also provide tissue samples for analysis of AFB1 exo-epoxide. Special trapping techniques may be required as the reactive AFB1 exo-epoxide metabolite has a short half-life in biological matrices.<br />
<br />
== Evidence Supporting Taxonomic Applicability ==<br />
Ample data across phyla demonstrate metabolic activation of AFB1 to the exo-epoxide via CYP450. These taxons include several mammalian species (humans, non-human primates, rats, mice) in addition to birds (turkeys) and fish (Eaton and Gallagher, 1994; IARC, 1993).<br />
<br />
== Evidence for Chemical Initiation of this Initial Molecular Event ==<br />
Pre-MIE:<br />
There is an extensive database on AFB1 in many different systems demonstrating formation of the AFB1 exo-epoxide. This database includes several mammalian species (humans, non-human primates, rats, mice) in addition to birds (turkeys) and fish (Eaton and Gallagher, 1994; IARC, 1993).<br />
<br />
== Evidence Supporting Essentiality of Pre-MIE (Step #0) ==<br />
Strong<br />
<br />
Many studies show that in the absence of AFB1 metabolic activation, tumors do not occur. Treatment of rats with the CYP450-inhibitor and GST-inducer oltipraz reduces the levels of AFB1 DNA adducts by 65-70%, and the later-forming altered hepatic foci (AHF) and liver tumors are also reduced by 97 and 100%, respectively; thus, no hepatocellular carcinomas (HCC) form in the oltipraz-treated rats dosed with AFB1 (Roebuck et al., 1991). More recent data show even more effective prevention of tumors and of hepatic foci in rats pre-treated with a triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) (Johnson et al., 2014). The pre-treated rats were reported with a 70% reduction of AFB1-induced adducts in urine but had 100% inhibition of tumors, and only 1 rat out of 23 pre-treated had any altered hepatic foci (AHF). There are similar data in humans treated with oltipraz. Kensler et al. (1998) report a significant reduction in a biomarker of exposure AFB1-induced albumin adducts, supporting the human relevance of this metabolic activation <br />
<br />
Another line of evidence for essentiality of the pre-MIE is the recognized difference in sensitivity to AFB1-induced liver tumors between mice and rats (Degen and Neumann, 1981). Mice, have considerably increased metabolic activation of AFB1 to the exo-epoxide compared to rats; mice are nonetheless much less sensitive to AFB1-induced liver tumors. It is thought that this difference is due to the constitutive presence of GST-alpha activity in mice vs rats, where this activity is not found.<br />
<br />
== References ==<br />
F.D. Asplin, R.B.A. Carnaghan, (1961). The toxicity of certain groundnut meals for poultry with special reference to their effect on ducklings and chickens. Vet. Rec. 73:1215– 1219. <br />
<br />
Brown KL, Deng JZ, Iyer RS, Iyer LG, Voehler MW, Stone MP, Harris CM, Harris TM (2006). Unraveling the aflatoxin-FAPY conundrum: Structural basis of the formamidopyrimidine-type DNA adduct of aflatoxin B1. J Am Chem Soc 128:15188-15199.<br />
<br />
Croy RG, Essigman JM, Reinhold VN, Wogan GN (1978). Identification of the principal aflatoxin N1-DNA adduct formed in vivo in rat liver. Proc Natl Acad Sci USA 75:1745-1749.<br />
<br />
Degen GH, Neumann HG (1981). Differences in aflatoxin B1-susceptibility of rat and mouse are correlated with the capability in vitro to inactivate aflatoxin B1-epoxide. Carcinogenesis 2:299–306.<br />
<br />
Eaton DL, and Gallagher EP (1994). Mechanisms of aflatoxin carcinogenesis. Annu Rev Pharmacol Toxicol 34:135-172.<br />
<br />
Elegbede JA, and Gould MN. (2002). Monoterpenes reduced adducts formation in rats exposed to aflatoxin B1. African J Biotech, 1, 46–49.<br />
<br />
Gregory 3rd JF, Goldstein SL, Edds GT. (1983). Metabolite distribution and rate of residue clearance in turkeys fed a diet containing aflatoxin B1. Food Chem Toxicol, 21, 463–7.<br />
<br />
Groopman JD, Kensler TW (2005). Role of metabolism and viruses in aflatoxin-induced liver cancer. Toxicol Appl Pharmacol 206:131-137.<br />
<br />
Guengerich FP, Johnson WW, Ueng Y-F, Yamazaki H, Shimada T (1996). Involvement of Cytochrome P450, glutathione S-transferase, and epoxide hydrolase in the metabolism of aflatoxin B1 and relevance to risk of human liver cancer. Environ Health Perspect 104(Suppl 3):557-562.<br />
<br />
Himmelstein MW, Boogaard PJ, Cadet J, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: II.Overview of methods of identification and quantitation of DNA damage. Crit Rev Toxicol, 39, 679–694.<br />
<br />
IARC (1993). Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. IARC Monographs on the Evaluation of Carcinogenic Risk to Humans. Vol. 56, 245-395.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, Sutter TR, Groopman JD, Kensler TW, Roebuck BD. (2014). Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold. Cancer Prev Res. 7(7):658-665.<br />
<br />
Kensler TW, He X, Otieno M, et al. (1998). Oltipraz chemoprevention trial in Qidong, People’s Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol Biomarkers Prev, 7, 127–34.<br />
<br />
Larsson P, Hoedaya, WI, Tjalve H. (1990). Disposition of 3H-aflatoxin H in mice: formation and retention of tissue bound metabolites in nasal glands. Pharmacol Toxicol, 67, 162–71.<br />
<br />
Larsson P, and Tjalve H. (1993). Distribution and metabolism of aflatoxin B1 in the marmoset monkey (Callithrix jacchus). Carcinogenesis, 14, 1–6.<br />
<br />
Monroe DH, Eaton DL. (1987). Comparative effects of butylated hydroxyanisole on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B1 in the rat and the mouse. Toxicol Appl Pharmacol, 90, 401–409.<br />
<br />
Pottenger, L.H., Andrews LS, Bachman AN, Boogaard PJ, Cadet J, Embry MR, Farmer PB, Himmelstein MW, Jarabek AM, Martin EA, Mauthe RJ, Persaud R, Preston RJ, Schoeny R, Skare J, Swenberg JA, Williams GM, Zeiger E, Zhang F, Kim JH. (2014). An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride. Crit. Rev. Toxicol. 44(4):348-391.<br />
<br />
Primiano T, Egner PA, Sutter TR, et al. (1995). Intermittent dosing with oltipraz: relationship between chemoprevention of aflatoxin-induced tumorigenesis and induction of glutathione-S-transferases. Cancer Res, 55, 4319–4324.<br />
<br />
Roebuck BD, Liu Y-L, Rogers AE, et al. (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short term molecular dosimetry. Cancer Res, 51, 5501–5506.<br />
<br />
Ueng Y-F, Shimada T, Yamazaki H, Guengerich FP (1995). Oxidation of aflatoxin B1 by bacterial recombinant human cytochrome P450 enzymes. Chem Res Toxiol 8:218-225.<br />
<br />
Wang J-S, Shen X, He X, et al. (1999). Protective alerations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qidong, People’s Republic of China. J Natl Cancer Inst, 91, 347–354.<br />
<br />
Yates MS, Kwak M-K, Egner PA, et al. (2006). Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl] imidazole. Cancer Res, 66, 2488–2494.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:409Event:4092016-07-06T17:06:27Z<p>Dvillene: /* Evidence for Chemical Initiation of this Initial Molecular Event */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id ='longTitle' class='Title'> Step: #0: pre-MIE </div><br />
<div id ='longTitle' class='Title'> Action: Metabolism of AFB1 </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||KE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Molecular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Biological Process: Production of Reactive Electrophiles ==<br />
Aflatoxin B1 (AFB1) can be oxidized to a variety of metabolites, many of which can be further metabolized, for example by conjugation with glutathione (GSH) via Glutathione-S-transferase (GST). In order for DNA binding and formation of a pro-mutagenic DNA adduct to occur, AFB1 must be metabolized via Cytochrome P450 metabolism to a specific highly reactive form of AFB1, the exo-epoxide AFB1-8,9-epoxide. CYP3A4 forms the exo-form of this reactive epoxide only. CYP1A2, inducible in liver, forms both the exo- and the endo-epoxides; in human liver, CYP1A2 metabolism occurs with a lower Vmax (enzymatic rate) and higher Km (half-maximal concentrations) than CYP3A4 (Degen and Neumann,1981; Groopman and Kensler, 2005; Guengerich et al., 1996; Ueng et al., 1995). Figure 1, from Pottenger et al., 2014, depicts the metabolism of AFB1.<br />
<br />
Metabolic activation of AFB1 occurs mainly in the liver, where the highest levels of the CYP isozymes are located. Competitive metabolism with other hepatic and extra-hepatic P450 isozymes may decrease the proportion of the specific metabolite, AFB1-8,9-epoxide; this can reduce the effectiveness of hepatic activation of AFB1. Alternatively, induction of either hepatic or extra-hepatic GST activity can reduce the levels of AFB1-epoxide available for reaction with DNA by increasing conjugation of the epoxide with GSH. This conjugation renders the epoxide inactive and directs it towards eventual elimination (Guengerich et al., 1996).[[File:Figure1met.jpg|thumb|alt=Figure 1 alt text|Figure 1. Metabolism of AFB1,]] Figure 1 was obtained from Pottenger et al., 2014.<br />
<br />
The AFB1 pro-mutagenic DNA adduct is most likely the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct, or N7-AFB1-G (Croy et al., 1978). Once the exo-epoxide is bound to the N7-guanine, it is subject to ring-opening to form the more highly pro-mutagenic 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-pyrimid-9-yl-foramido)-9-hydroxyaflatoxin B1; this is known as the formamidopyrimidine adduct or AFB1-FAPy adduct (Brown et al., 2006).<br />
<br />
== Taxonomic Applicability ==<br />
The metabolic information above is applicable to all mammalian systems evaluated; it is also applicable to certain birds such as turkeys (Gregory et al., 1983; IARC, 1993). Humans, non-human primates, rats, mice, poultry and fish have all demonstrated susceptibility to AFB1-induced liver tumors (Asplin and Canaghan, 1961; Eaton and Gallagher, 1994). Species that preferentially metabolize AFB1 to the exo-8,9-epoxide are more susceptible to AFB1 carcinogenicity. Sulfation by GST enzymes or other enzymatic detoxification of AFB1 metabolites can reduce available levels of the exo-epoxide, and subsequent steps or even the progression of Key Events may not occur in these species. Mice are less susceptible to AFB1-induced cancer than rats, likely due to increased detoxification (Monroe and Eaton, 1987). This increased detoxication capability can also be instigated by dietary exposures to compounds that modulate CYP450 expression or modify detoxication activities (Elegbede and Gould, 2002; Primiano et al., 1995; Roebuck et al. 1991, 2003; Kensler et al., 1998; Wang et al., 1999; Yates et al., 2006).<br />
<br />
== Level of Biological Organization : Molecular ==<br />
Metabolic activation of AFB1 occurs at the intracellular level, with sub-cellular organelles involved due to the localization of CYP450 in the endoplasmic reticulum, coupled with the epoxide hydratase; GST is localized in the cytosol (Guengerich et al., 1996). Organ architecture also plays a role, as the structure of the liver is orientated around the O2 gradient set by the portal artery and hepatic vein; hepatocytes nearest the portal artery demonstrate increased tissue oxygen while those around the portal vein have decreased tissue O2 and correspondingly higher levels of CYP450 activity.<br />
<br />
== How this Key Event works ==<br />
The reactive exo-epoxide is formed in hepatocytes (or extra-hepatically) by metabolism of the parent AFB1 by CYP450 (Larsson et al., 1990; Larsson and Tjalve, 1993). The reactive metabolite then escapes the endoplasmic reticulum where the CYP450 is located. The reactive metabolite must evade conjugation with GSH in the cytoplasm or binding with other cytoplasmic nucleophiles. It then traverses the nuclear membrane in order to reach the cell nucleus and the nuclear DNA. Once the reactive metabolite is in the cell nucleus, binding to nuclear DNA and the formation of DNA adducts can occur.<br />
<br />
== How it is Measured or Detected ==<br />
Formation of the exo-epoxide can be produced with in vitro systems and detected using techniques for structural quantitation of AFB1 metabolites (Himmelstein et al., 2009), including liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). By using subcellular fractions (e.g., microsomes), cellular homogenates, or cells in culture, it is possible to measure formation of AFB1 exo-epoxide. Such data can also be collected from in vivo systems; samples of plasma or blood from AFB1-treated animals can be analyzed for the AFB1 exo-epoxide with similar mass spectrometric based detection systems (e.g., LC-MS/MS). Samples of blood from humans in AFB1-endemic regions have demonstrated presence of AFB1-albumin adducts, which are formed from the AFB1 exo-epoxide. AFB1-treated animals may also provide tissue samples for analysis of AFB1 exo-epoxide. Special trapping techniques may be required as the reactive AFB1 exo-epoxide metabolite has a short half-life in biological matrices.<br />
<br />
== Evidence Supporting Taxonomic Applicability ==<br />
Ample data across phyla demonstrate metabolic activation of AFB1 to the exo-epoxide via CYP450. These taxons include several mammalian species (humans, non-human primates, rats, mice) in addition to birds (turkeys) and fish (Eaton and Gallagher, 1994; IARC, 1993).<br />
<br />
== Evidence for Chemical Initiation of this Initial Molecular Event ==<br />
Pre-MIE:<br />
There is an extensive database on AFB1 in many different systems demonstrating formation of the AFB1 exo-epoxide. This database includes several mammalian species (humans, non-human primates, rats, mice) in addition to birds (turkeys) and fish (Eaton and Gallagher, 1994; IARC, 1993).<br />
<br />
== Evidence Supporting Essentiality of Pre-MIE (Step #0) ==<br />
Strong<br />
<br />
Many studies show that in the absence of AFB1 metabolic activation, tumors do not occur. Treatment of rats with the CYP450-inhibitor and GST-inducer oltipraz reduces the levels of AFB1 DNA adducts by 65-70%, and the later-forming altered hepatic foci (AHF) and liver tumors are also reduced by 97 and 100%, respectively; thus, no hepatocellular carcinomas (HCC) form in the oltipraz-treated rats dosed with AFB1 (Roebuck et al., 1991). More recent data show even more effective prevention of tumors and of hepatic foci in rats pre-treated with a triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) (Johnson et al., 2014). The pre-treated rats were reported with a 70% reduction of AFB1-induced adducts in urine but had 100% inhibition of tumors, and only 1 rat out of 23 pre-treated had any altered hepatic foci (AHF). There are similar data in humans treated with oltipraz. Kensler et al. (1998) report a significant reduction in a biomarker of exposure AFB1-induced albumin adducts, supporting the human relevance of this metabolic activation <br />
<br />
Another line of evidence for essentiality of the pre-MIE is the recognized difference in sensitivity to AFB1-induced liver tumors between mice and rats (Degen and Neumann, 1981). Mice, have considerably increased metabolic activation of AFB1 to the exo-epoxide compared to rats; mice are nonetheless much less sensitive to AFB1-induced liver tumors. It is thought that this difference is due to the constitutive presence of GST-alpha activity in mice vs rats, where this activity is not found.<br />
<br />
== Pre-MIE: References ==<br />
F.D. Asplin, R.B.A. Carnaghan, (1961). The toxicity of certain groundnut meals for poultry with special reference to their effect on ducklings and chickens. Vet. Rec. 73:1215– 1219. <br />
<br />
Brown KL, Deng JZ, Iyer RS, Iyer LG, Voehler MW, Stone MP, Harris CM, Harris TM (2006). Unraveling the aflatoxin-FAPY conundrum: Structural basis of the formamidopyrimidine-type DNA adduct of aflatoxin B1. J Am Chem Soc 128:15188-15199.<br />
<br />
Croy RG, Essigman JM, Reinhold VN, Wogan GN (1978). Identification of the principal aflatoxin N1-DNA adduct formed in vivo in rat liver. Proc Natl Acad Sci USA 75:1745-1749.<br />
<br />
Degen GH, Neumann HG (1981). Differences in aflatoxin B1-susceptibility of rat and mouse are correlated with the capability in vitro to inactivate aflatoxin B1-epoxide. Carcinogenesis 2:299–306.<br />
<br />
Eaton DL, and Gallagher EP (1994). Mechanisms of aflatoxin carcinogenesis. Annu Rev Pharmacol Toxicol 34:135-172.<br />
<br />
Elegbede JA, and Gould MN. (2002). Monoterpenes reduced adducts formation in rats exposed to aflatoxin B1. African J Biotech, 1, 46–49.<br />
<br />
Gregory 3rd JF, Goldstein SL, Edds GT. (1983). Metabolite distribution and rate of residue clearance in turkeys fed a diet containing aflatoxin B1. Food Chem Toxicol, 21, 463–7.<br />
<br />
Groopman JD, Kensler TW (2005). Role of metabolism and viruses in aflatoxin-induced liver cancer. Toxicol Appl Pharmacol 206:131-137.<br />
<br />
Guengerich FP, Johnson WW, Ueng Y-F, Yamazaki H, Shimada T (1996). Involvement of Cytochrome P450, glutathione S-transferase, and epoxide hydrolase in the metabolism of aflatoxin B1 and relevance to risk of human liver cancer. Environ Health Perspect 104(Suppl 3):557-562.<br />
<br />
Himmelstein MW, Boogaard PJ, Cadet J, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: II.Overview of methods of identification and quantitation of DNA damage. Crit Rev Toxicol, 39, 679–694.<br />
<br />
IARC (1993). Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. IARC Monographs on the Evaluation of Carcinogenic Risk to Humans. Vol. 56, 245-395.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, Sutter TR, Groopman JD, Kensler TW, Roebuck BD. (2014). Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold. Cancer Prev Res. 7(7):658-665.<br />
<br />
Kensler TW, He X, Otieno M, et al. (1998). Oltipraz chemoprevention trial in Qidong, People’s Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol Biomarkers Prev, 7, 127–34.<br />
<br />
Larsson P, Hoedaya, WI, Tjalve H. (1990). Disposition of 3H-aflatoxin H in mice: formation and retention of tissue bound metabolites in nasal glands. Pharmacol Toxicol, 67, 162–71.<br />
<br />
Larsson P, and Tjalve H. (1993). Distribution and metabolism of aflatoxin B1 in the marmoset monkey (Callithrix jacchus). Carcinogenesis, 14, 1–6.<br />
<br />
Monroe DH, Eaton DL. (1987). Comparative effects of butylated hydroxyanisole on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B1 in the rat and the mouse. Toxicol Appl Pharmacol, 90, 401–409.<br />
<br />
Pottenger, L.H., Andrews LS, Bachman AN, Boogaard PJ, Cadet J, Embry MR, Farmer PB, Himmelstein MW, Jarabek AM, Martin EA, Mauthe RJ, Persaud R, Preston RJ, Schoeny R, Skare J, Swenberg JA, Williams GM, Zeiger E, Zhang F, Kim JH. (2014). An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride. Crit. Rev. Toxicol. 44(4):348-391.<br />
<br />
Primiano T, Egner PA, Sutter TR, et al. (1995). Intermittent dosing with oltipraz: relationship between chemoprevention of aflatoxin-induced tumorigenesis and induction of glutathione-S-transferases. Cancer Res, 55, 4319–4324.<br />
<br />
Roebuck BD, Liu Y-L, Rogers AE, et al. (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short term molecular dosimetry. Cancer Res, 51, 5501–5506.<br />
<br />
Ueng Y-F, Shimada T, Yamazaki H, Guengerich FP (1995). Oxidation of aflatoxin B1 by bacterial recombinant human cytochrome P450 enzymes. Chem Res Toxiol 8:218-225.<br />
<br />
Wang J-S, Shen X, He X, et al. (1999). Protective alerations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qidong, People’s Republic of China. J Natl Cancer Inst, 91, 347–354.<br />
<br />
Yates MS, Kwak M-K, Egner PA, et al. (2006). Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl] imidazole. Cancer Res, 66, 2488–2494.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:409Event:4092016-07-06T17:06:09Z<p>Dvillene: /* Evidence for Chemical Initiation of this Initial Molecular Event */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id ='longTitle' class='Title'> Step: #0: pre-MIE </div><br />
<div id ='longTitle' class='Title'> Action: Metabolism of AFB1 </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||KE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Molecular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Biological Process: Production of Reactive Electrophiles ==<br />
Aflatoxin B1 (AFB1) can be oxidized to a variety of metabolites, many of which can be further metabolized, for example by conjugation with glutathione (GSH) via Glutathione-S-transferase (GST). In order for DNA binding and formation of a pro-mutagenic DNA adduct to occur, AFB1 must be metabolized via Cytochrome P450 metabolism to a specific highly reactive form of AFB1, the exo-epoxide AFB1-8,9-epoxide. CYP3A4 forms the exo-form of this reactive epoxide only. CYP1A2, inducible in liver, forms both the exo- and the endo-epoxides; in human liver, CYP1A2 metabolism occurs with a lower Vmax (enzymatic rate) and higher Km (half-maximal concentrations) than CYP3A4 (Degen and Neumann,1981; Groopman and Kensler, 2005; Guengerich et al., 1996; Ueng et al., 1995). Figure 1, from Pottenger et al., 2014, depicts the metabolism of AFB1.<br />
<br />
Metabolic activation of AFB1 occurs mainly in the liver, where the highest levels of the CYP isozymes are located. Competitive metabolism with other hepatic and extra-hepatic P450 isozymes may decrease the proportion of the specific metabolite, AFB1-8,9-epoxide; this can reduce the effectiveness of hepatic activation of AFB1. Alternatively, induction of either hepatic or extra-hepatic GST activity can reduce the levels of AFB1-epoxide available for reaction with DNA by increasing conjugation of the epoxide with GSH. This conjugation renders the epoxide inactive and directs it towards eventual elimination (Guengerich et al., 1996).[[File:Figure1met.jpg|thumb|alt=Figure 1 alt text|Figure 1. Metabolism of AFB1,]] Figure 1 was obtained from Pottenger et al., 2014.<br />
<br />
The AFB1 pro-mutagenic DNA adduct is most likely the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct, or N7-AFB1-G (Croy et al., 1978). Once the exo-epoxide is bound to the N7-guanine, it is subject to ring-opening to form the more highly pro-mutagenic 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-pyrimid-9-yl-foramido)-9-hydroxyaflatoxin B1; this is known as the formamidopyrimidine adduct or AFB1-FAPy adduct (Brown et al., 2006).<br />
<br />
== Taxonomic Applicability ==<br />
The metabolic information above is applicable to all mammalian systems evaluated; it is also applicable to certain birds such as turkeys (Gregory et al., 1983; IARC, 1993). Humans, non-human primates, rats, mice, poultry and fish have all demonstrated susceptibility to AFB1-induced liver tumors (Asplin and Canaghan, 1961; Eaton and Gallagher, 1994). Species that preferentially metabolize AFB1 to the exo-8,9-epoxide are more susceptible to AFB1 carcinogenicity. Sulfation by GST enzymes or other enzymatic detoxification of AFB1 metabolites can reduce available levels of the exo-epoxide, and subsequent steps or even the progression of Key Events may not occur in these species. Mice are less susceptible to AFB1-induced cancer than rats, likely due to increased detoxification (Monroe and Eaton, 1987). This increased detoxication capability can also be instigated by dietary exposures to compounds that modulate CYP450 expression or modify detoxication activities (Elegbede and Gould, 2002; Primiano et al., 1995; Roebuck et al. 1991, 2003; Kensler et al., 1998; Wang et al., 1999; Yates et al., 2006).<br />
<br />
== Level of Biological Organization : Molecular ==<br />
Metabolic activation of AFB1 occurs at the intracellular level, with sub-cellular organelles involved due to the localization of CYP450 in the endoplasmic reticulum, coupled with the epoxide hydratase; GST is localized in the cytosol (Guengerich et al., 1996). Organ architecture also plays a role, as the structure of the liver is orientated around the O2 gradient set by the portal artery and hepatic vein; hepatocytes nearest the portal artery demonstrate increased tissue oxygen while those around the portal vein have decreased tissue O2 and correspondingly higher levels of CYP450 activity.<br />
<br />
== How this Key Event works ==<br />
The reactive exo-epoxide is formed in hepatocytes (or extra-hepatically) by metabolism of the parent AFB1 by CYP450 (Larsson et al., 1990; Larsson and Tjalve, 1993). The reactive metabolite then escapes the endoplasmic reticulum where the CYP450 is located. The reactive metabolite must evade conjugation with GSH in the cytoplasm or binding with other cytoplasmic nucleophiles. It then traverses the nuclear membrane in order to reach the cell nucleus and the nuclear DNA. Once the reactive metabolite is in the cell nucleus, binding to nuclear DNA and the formation of DNA adducts can occur.<br />
<br />
== How it is Measured or Detected ==<br />
Formation of the exo-epoxide can be produced with in vitro systems and detected using techniques for structural quantitation of AFB1 metabolites (Himmelstein et al., 2009), including liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). By using subcellular fractions (e.g., microsomes), cellular homogenates, or cells in culture, it is possible to measure formation of AFB1 exo-epoxide. Such data can also be collected from in vivo systems; samples of plasma or blood from AFB1-treated animals can be analyzed for the AFB1 exo-epoxide with similar mass spectrometric based detection systems (e.g., LC-MS/MS). Samples of blood from humans in AFB1-endemic regions have demonstrated presence of AFB1-albumin adducts, which are formed from the AFB1 exo-epoxide. AFB1-treated animals may also provide tissue samples for analysis of AFB1 exo-epoxide. Special trapping techniques may be required as the reactive AFB1 exo-epoxide metabolite has a short half-life in biological matrices.<br />
<br />
== Evidence Supporting Taxonomic Applicability ==<br />
Ample data across phyla demonstrate metabolic activation of AFB1 to the exo-epoxide via CYP450. These taxons include several mammalian species (humans, non-human primates, rats, mice) in addition to birds (turkeys) and fish (Eaton and Gallagher, 1994; IARC, 1993).<br />
<br />
== Evidence for Chemical Initiation of this Initial Molecular Event ==<br />
Pre-MIE<br />
There is an extensive database on AFB1 in many different systems demonstrating formation of the AFB1 exo-epoxide. This database includes several mammalian species (humans, non-human primates, rats, mice) in addition to birds (turkeys) and fish (Eaton and Gallagher, 1994; IARC, 1993).<br />
<br />
== Evidence Supporting Essentiality of Pre-MIE (Step #0) ==<br />
Strong<br />
<br />
Many studies show that in the absence of AFB1 metabolic activation, tumors do not occur. Treatment of rats with the CYP450-inhibitor and GST-inducer oltipraz reduces the levels of AFB1 DNA adducts by 65-70%, and the later-forming altered hepatic foci (AHF) and liver tumors are also reduced by 97 and 100%, respectively; thus, no hepatocellular carcinomas (HCC) form in the oltipraz-treated rats dosed with AFB1 (Roebuck et al., 1991). More recent data show even more effective prevention of tumors and of hepatic foci in rats pre-treated with a triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) (Johnson et al., 2014). The pre-treated rats were reported with a 70% reduction of AFB1-induced adducts in urine but had 100% inhibition of tumors, and only 1 rat out of 23 pre-treated had any altered hepatic foci (AHF). There are similar data in humans treated with oltipraz. Kensler et al. (1998) report a significant reduction in a biomarker of exposure AFB1-induced albumin adducts, supporting the human relevance of this metabolic activation <br />
<br />
Another line of evidence for essentiality of the pre-MIE is the recognized difference in sensitivity to AFB1-induced liver tumors between mice and rats (Degen and Neumann, 1981). Mice, have considerably increased metabolic activation of AFB1 to the exo-epoxide compared to rats; mice are nonetheless much less sensitive to AFB1-induced liver tumors. It is thought that this difference is due to the constitutive presence of GST-alpha activity in mice vs rats, where this activity is not found.<br />
<br />
== Pre-MIE: References ==<br />
F.D. Asplin, R.B.A. Carnaghan, (1961). The toxicity of certain groundnut meals for poultry with special reference to their effect on ducklings and chickens. Vet. Rec. 73:1215– 1219. <br />
<br />
Brown KL, Deng JZ, Iyer RS, Iyer LG, Voehler MW, Stone MP, Harris CM, Harris TM (2006). Unraveling the aflatoxin-FAPY conundrum: Structural basis of the formamidopyrimidine-type DNA adduct of aflatoxin B1. J Am Chem Soc 128:15188-15199.<br />
<br />
Croy RG, Essigman JM, Reinhold VN, Wogan GN (1978). Identification of the principal aflatoxin N1-DNA adduct formed in vivo in rat liver. Proc Natl Acad Sci USA 75:1745-1749.<br />
<br />
Degen GH, Neumann HG (1981). Differences in aflatoxin B1-susceptibility of rat and mouse are correlated with the capability in vitro to inactivate aflatoxin B1-epoxide. Carcinogenesis 2:299–306.<br />
<br />
Eaton DL, and Gallagher EP (1994). Mechanisms of aflatoxin carcinogenesis. Annu Rev Pharmacol Toxicol 34:135-172.<br />
<br />
Elegbede JA, and Gould MN. (2002). Monoterpenes reduced adducts formation in rats exposed to aflatoxin B1. African J Biotech, 1, 46–49.<br />
<br />
Gregory 3rd JF, Goldstein SL, Edds GT. (1983). Metabolite distribution and rate of residue clearance in turkeys fed a diet containing aflatoxin B1. Food Chem Toxicol, 21, 463–7.<br />
<br />
Groopman JD, Kensler TW (2005). Role of metabolism and viruses in aflatoxin-induced liver cancer. Toxicol Appl Pharmacol 206:131-137.<br />
<br />
Guengerich FP, Johnson WW, Ueng Y-F, Yamazaki H, Shimada T (1996). Involvement of Cytochrome P450, glutathione S-transferase, and epoxide hydrolase in the metabolism of aflatoxin B1 and relevance to risk of human liver cancer. Environ Health Perspect 104(Suppl 3):557-562.<br />
<br />
Himmelstein MW, Boogaard PJ, Cadet J, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: II.Overview of methods of identification and quantitation of DNA damage. Crit Rev Toxicol, 39, 679–694.<br />
<br />
IARC (1993). Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. IARC Monographs on the Evaluation of Carcinogenic Risk to Humans. Vol. 56, 245-395.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, Sutter TR, Groopman JD, Kensler TW, Roebuck BD. (2014). Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold. Cancer Prev Res. 7(7):658-665.<br />
<br />
Kensler TW, He X, Otieno M, et al. (1998). Oltipraz chemoprevention trial in Qidong, People’s Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol Biomarkers Prev, 7, 127–34.<br />
<br />
Larsson P, Hoedaya, WI, Tjalve H. (1990). Disposition of 3H-aflatoxin H in mice: formation and retention of tissue bound metabolites in nasal glands. Pharmacol Toxicol, 67, 162–71.<br />
<br />
Larsson P, and Tjalve H. (1993). Distribution and metabolism of aflatoxin B1 in the marmoset monkey (Callithrix jacchus). Carcinogenesis, 14, 1–6.<br />
<br />
Monroe DH, Eaton DL. (1987). Comparative effects of butylated hydroxyanisole on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B1 in the rat and the mouse. Toxicol Appl Pharmacol, 90, 401–409.<br />
<br />
Pottenger, L.H., Andrews LS, Bachman AN, Boogaard PJ, Cadet J, Embry MR, Farmer PB, Himmelstein MW, Jarabek AM, Martin EA, Mauthe RJ, Persaud R, Preston RJ, Schoeny R, Skare J, Swenberg JA, Williams GM, Zeiger E, Zhang F, Kim JH. (2014). An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride. Crit. Rev. Toxicol. 44(4):348-391.<br />
<br />
Primiano T, Egner PA, Sutter TR, et al. (1995). Intermittent dosing with oltipraz: relationship between chemoprevention of aflatoxin-induced tumorigenesis and induction of glutathione-S-transferases. Cancer Res, 55, 4319–4324.<br />
<br />
Roebuck BD, Liu Y-L, Rogers AE, et al. (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short term molecular dosimetry. Cancer Res, 51, 5501–5506.<br />
<br />
Ueng Y-F, Shimada T, Yamazaki H, Guengerich FP (1995). Oxidation of aflatoxin B1 by bacterial recombinant human cytochrome P450 enzymes. Chem Res Toxiol 8:218-225.<br />
<br />
Wang J-S, Shen X, He X, et al. (1999). Protective alerations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qidong, People’s Republic of China. J Natl Cancer Inst, 91, 347–354.<br />
<br />
Yates MS, Kwak M-K, Egner PA, et al. (2006). Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl] imidazole. Cancer Res, 66, 2488–2494.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:376Event:3762016-07-06T17:04:53Z<p>Dvillene: /* KE#2: References */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Induced Mutations in Critical Genes, Increased </div><br />
<div id='shortTitle' class='Title'> Induced in Critical Genes, Mutation </div><br />
<br />
== Action ==<br />
Induced mutation<br />
<br />
== Biological Process/Object ==<br />
Critical genes for carcinogenesis<br />
<br />
== Induced Mutation in Critical Genes ==<br />
The AFB1-induced pro-mutagenic DNA adduct is either not repaired or is mis-repaired resulting in a mutation in one or more critical genes. In bacteria and mammalian cells (both in vitro and in vivo) the primary mutation associated with AFB1 is a guanine to thymine transversion (Foster et al., 1983; Dycaico et al., 1996). A G:T transversion is expected for the pro-mutagenic DNA adduct AFB1-FAPy. <br />
<br />
A specific critical mutation in codon 249 of the p53 gene has been identified in human hepatocellular carcinoma (HCC) (See section below on essentiality).<br />
<br />
== Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
A specific critical mutation in the p53 gene has been identified in human hepatocellular carcinoma (HCC). Demonstrating that AFB1 can induce this specific p53 gene mutation would be the highest level of evidence that AFB1-induced HCC involves mutation as a KE. Absent such information, the next best level of evidence is the induction of the specific type of mutation (G:C to T:A transversion) in a variety of gene mutation assays measuring a range of target genes. <br />
<br />
The codon 249 mutation is present in a significant proportion of human HCCs. In fact codon 249 mutation is detected in up to 50% of liver cancers in Qidong, China (Hsu et al., 1991) and in Mozambique, both areas with high likelihood of AFB1 exposure. The codon 249 G:C to T:A mutation in the third base is seen in up to 75% of HCC in high-incidence areas of China and East Africa (Gouas et al., 2009). In contrast, this specific mutation is very rare in HCC from areas with no or low exposure to AFB1 (Hsu et al. 1991 and Bressac et al. Nature, 350:429-431, 1991). This mutation is also very rare in other types of tumors (Gouas et al., 2009). According to Gouas et al. (2009), populations with AFB1 exposure are likely to be exposed to hepatitis B virus (HBV) as well and the effects of each are difficult to separate.This mutation is very rare in HCC from non/low -aflatoxin areas (Hsu et al. 1991 and Bressac et al., 1991) and also very rare in other types of tumors.<br />
<br />
Some indirect evidence of the essentiality of mutation in tumor development is provided by the clear species difference between adult mice and adult rats both in the induction of surrogate gene mutations and in the induction of tumors. Adult mice exposed to AFB1 do not get tumors and there is no increase in mutant frequency (MF) for Big Blue mice exposed as adults. That is, Lac I mutants from the AFB1- exposed adult mice showed a spontaneous mutational spectrum. Rats, however, showed a large increase in MF and, more specifically a large increase in G:C to T:A transversions (Dycaico et al., 1996). In addition, for mice there is an difference between neonata and adult mice. Neonatal mice treated with AFB1 (6 mg/kg—a dose that does result in tumors) showed an increase in cII mutation with G:C to T:A transversion as the major mutation. Adult mice treated at 6 and 60 mg/kg (doses that do not produce tumors) did not have a significant increase in cII mutation (but did give a different mutational spectrum than controls) (Chen et al. 2010).<br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||KE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Cellular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Taxonomic Applicability ==<br />
The induction of G:C to T:A transversions following AFB1 exposure is seen in a wide variety of species from bacteria to mammals. Assays that measure mutation in surrogate genes (that is, genes unrelated to the critical cancer genes) can be used to evaluate the full spectrum of mutational events that can be induced by a chemical. One such system is the AS52 assay, an in vitro mammalian transgenic mutation assay that measures mutation in the gpt gene. After exposure to AFB1 in culture the predominant mutation is G:C to T:A transversion, although a number of other types of mutations were also seen (Wattanawaraporn et al., 2012). There is a species difference between adult mice and rats. No increase in MF is seen in the in vivo Big Blue™ mutation assay for mice exposed to AFB1. That is, Lac I mutations from the mice showed a spontaneous mutational spectrum. By contrast rats showed a large increase in MF with a large increase in G:C to T:A transversions (Dycaico et al., 1996). However, neonatal mice treated with AFB1 (6 mg/kg—a dose that induces tumors in neonates) do show an increase in cII mutation with G:C to T:A transversion as the major mutation. Adult mice treated at 6 and 60 mg/kg (doses that do not induce tumors) did not show a significant increase in cII mutations but did produce a different mutational spectrum than controls (Chen et al., 2010.)<br />
<br />
== Level of Biological Organization : Cellular ==<br />
The induction of mutation occurs within the nucleus of cells and involves permanent alterations in the primary DNA sequence that is passed to subsequent cell generations and, thus, is heritable.<br />
<br />
== How this Key Event works ==<br />
Following the formation of DNA adducts, which are either mis-repaired or not repaired, the sequence of base pairs in the DNA is changed due to insertion of an incorrect base opposite the DNA adduct during DNA replication, so that a G is permanently replaced by a T. This type of mutation is observed in the mutants seen in surrogate gene mutation assays using AFB1 exposureA high frequency of codon 249 p53 mutations occurs in human tumors in high AFB1 exposure regions.Hence, codon 249 of the p53 gene contains a base pair susceptible to insufficient or misrepair of DNA adducts.<br />
<br />
Puisieux et al. (1991) provide evidence that the AFB1 epoxide adduct binds preferentially to codon 249 of the p53 gene. Using a plasmid with containing full-length human p53 DNA, adduct formation was observed in exons 5, 6, 7 and 8 (a total of 1086 bases) and 20% of the bases were targeted by AFB1 with a preference for guanine residues. Binding of AFB1 to p53 sequences was restricted to fewer residues and was more specific for guanine than was the binding of B[a]P (Puisieux et al.,1991). Binding of AFB1 in the region around codon 249 of p53 AFB1 was reported to be “stronger” than that of B[a]P. The last nucleotide of codon 249 is a guanine and was targeted by AFB1 but not by B[a]P. This guanine residue is the mutational hotspot in human liver cancers from patients in high AFB1 exposure regions. <br />
<br />
While mutations at codon 249 of the p53 gene have been observed in association with HCC in humans, the question remains whether this mutation occurs as a direct result of adduct formation at this site or by some different mechanism..In human HepG2 hepatocytes exposed to microsomally activated AFB1, a dose-dependent increase in G:C to T:A transversions were observed at 10 additional locations using ligation-mediated PCR, and at 4 additional locations using terminal-transferred dependent PCR.(Denissenko et al.,1998). These authors suggest that codon 249 may not present a key adduct site. However, other more recently published data identified codon 249 of the P53 gene as an unusually mutagenic adduct conformation based on the local DNA sequence and concluded that a higher mutation rate may occur there rather than at other locations because of increased DNA polymerase bypass (Pussieux et al., 1991; Lin et al., 2014a,b).<br />
<br />
== How it is Measured or Detected ==<br />
Historically the detection of critical cancer gene specific mutations has not been technically feasible. A newly developed method, allele specific competitive blocker-polymerase chain reaction (ACB-PCR) has proven useful in providing such information, and data on specific chemical-induced mutations are available for a very small number of chemicals (Parsons et al., 2010). Unfortunately, there are no data for AFB1.<br />
<br />
There are, however, a number of gene mutation assays that have been widely used for determining the general ability of chemicals, including AFB1 to induce mutations.These assays use selection methods that allow only mutant cells to survive and grow. The AS52 in vitro gene mutation assay using the gpt gene, and 6-thioguanine selection has been used to demonstrate that AFB1 exposure increases the MF at the gpt gene. In vivo transgenic assays use molecular methods to recover the transgene from isolated DNA and to evaluate the MF in the transgene. Molecular methods can detect the presence (above a certain sensitivity level) of mutant cells. DNA from tumors can be sequenced to determine the presence of mutations in specific genes. DNA sequencing has been used on human tumors to detect the presence of the Codon 249 p53 mutation.<br />
<br />
== Evidence Supporting Taxonomic Applicability ==<br />
There are data across phyla demonstrating the induction of mutations, specifically the induction to G:C to T:A transversions following AFB1 exposure.<br />
<br />
== References ==<br />
Bressac, B., Kew, M., Wands, J., & Ozturk, M. (1991). Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern africa. Nature, 350(6317), 429-31. doi:10.1038/350429a<br />
<br />
Chen, T., Heflich, R. H., Moore, M. M., & Mei, N. (2010). Differential mutagenicity of aflatoxin B1 in the liver of neonatal and adult mice. Environ Mol Mutagen, 51(2), 156-63. doi:10.1002/em.2051<br />
<br />
Denissenko MF, Koudriakova TB, Smith L, O'Connor TR, Riggs AD, and Pfeifer GP). The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts. (Oncogene. 1998, Dec 10;17(23):3007-14.<br />
<br />
Dycaico, M. J., Stuart, G. R., Tobal, G. M., de Boer, J. G., Glickman, B. W., & Provost, G. S. (1996). Species-specific differences in hepatic mutant frequency and mutational spectrum among lambda/laci transgenic rats and mice following exposure to aflatoxin B1. Carcinogenesis, 17(11), 2347-56<br />
<br />
Foster, P. L., Eisenstadt, E., & Miller, J. H. (1983). Base substitution mutations induced by metabolically activated aflatoxin B1. Proceedings of the National Academy of Sciences of the United States of America, 80(9), 2695-8.<br />
<br />
Gouas, D., Shi, H., & Hainaut, P. (2009). The aflatoxin-induced TP53 mutation at codon 249 (R249S): Biomarker of exposure, early detection and target for therapy. Cancer Lett, 286(1), 29-37. doi:10.1016/j.canlet.2009.02.057<br />
<br />
Hsu, I. C., Metcalf, R. A., Sun, T., Welsh, J. A., Wang, N. J., & Harris, C. C. (1991). Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature, 350(6317), 427-8. doi:10.1038/350427a<br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014a). Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua. J Biol Chem. 289:18497-18506. <br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014b). Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis 35(7):1461-1468<br />
<br />
Parsons BL, Myers MB, Meng F, Wang Y, McKinzie PB. 2010. Oncomutations as biomarkers of cancer risk. Environ Mol Mutagen. 51(8-9):836-850.<br />
<br />
Puisieux, A., Lim, S., Groopman, J., & Ozturk, M. (1991). Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res, 51(22), 6185-9.<br />
<br />
Wattanawaraporn, R., Kim, M. Y., Adams, J., Trudel, L. J., Woo, L. L., Croy, R. G., . . . Wogan, G. N. (2012). AFB(1) -induced mutagenesis of the gpt gene in AS52 cells. Environ Mol Mutagen, 53(7), 567-73. doi:10.1002/em.2171</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:376Event:3762016-07-06T17:04:36Z<p>Dvillene: /* KE#2: Evidence Supporting Taxonomic Applicability */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Induced Mutations in Critical Genes, Increased </div><br />
<div id='shortTitle' class='Title'> Induced in Critical Genes, Mutation </div><br />
<br />
== Action ==<br />
Induced mutation<br />
<br />
== Biological Process/Object ==<br />
Critical genes for carcinogenesis<br />
<br />
== Induced Mutation in Critical Genes ==<br />
The AFB1-induced pro-mutagenic DNA adduct is either not repaired or is mis-repaired resulting in a mutation in one or more critical genes. In bacteria and mammalian cells (both in vitro and in vivo) the primary mutation associated with AFB1 is a guanine to thymine transversion (Foster et al., 1983; Dycaico et al., 1996). A G:T transversion is expected for the pro-mutagenic DNA adduct AFB1-FAPy. <br />
<br />
A specific critical mutation in codon 249 of the p53 gene has been identified in human hepatocellular carcinoma (HCC) (See section below on essentiality).<br />
<br />
== Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
A specific critical mutation in the p53 gene has been identified in human hepatocellular carcinoma (HCC). Demonstrating that AFB1 can induce this specific p53 gene mutation would be the highest level of evidence that AFB1-induced HCC involves mutation as a KE. Absent such information, the next best level of evidence is the induction of the specific type of mutation (G:C to T:A transversion) in a variety of gene mutation assays measuring a range of target genes. <br />
<br />
The codon 249 mutation is present in a significant proportion of human HCCs. In fact codon 249 mutation is detected in up to 50% of liver cancers in Qidong, China (Hsu et al., 1991) and in Mozambique, both areas with high likelihood of AFB1 exposure. The codon 249 G:C to T:A mutation in the third base is seen in up to 75% of HCC in high-incidence areas of China and East Africa (Gouas et al., 2009). In contrast, this specific mutation is very rare in HCC from areas with no or low exposure to AFB1 (Hsu et al. 1991 and Bressac et al. Nature, 350:429-431, 1991). This mutation is also very rare in other types of tumors (Gouas et al., 2009). According to Gouas et al. (2009), populations with AFB1 exposure are likely to be exposed to hepatitis B virus (HBV) as well and the effects of each are difficult to separate.This mutation is very rare in HCC from non/low -aflatoxin areas (Hsu et al. 1991 and Bressac et al., 1991) and also very rare in other types of tumors.<br />
<br />
Some indirect evidence of the essentiality of mutation in tumor development is provided by the clear species difference between adult mice and adult rats both in the induction of surrogate gene mutations and in the induction of tumors. Adult mice exposed to AFB1 do not get tumors and there is no increase in mutant frequency (MF) for Big Blue mice exposed as adults. That is, Lac I mutants from the AFB1- exposed adult mice showed a spontaneous mutational spectrum. Rats, however, showed a large increase in MF and, more specifically a large increase in G:C to T:A transversions (Dycaico et al., 1996). In addition, for mice there is an difference between neonata and adult mice. Neonatal mice treated with AFB1 (6 mg/kg—a dose that does result in tumors) showed an increase in cII mutation with G:C to T:A transversion as the major mutation. Adult mice treated at 6 and 60 mg/kg (doses that do not produce tumors) did not have a significant increase in cII mutation (but did give a different mutational spectrum than controls) (Chen et al. 2010).<br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||KE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Cellular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Taxonomic Applicability ==<br />
The induction of G:C to T:A transversions following AFB1 exposure is seen in a wide variety of species from bacteria to mammals. Assays that measure mutation in surrogate genes (that is, genes unrelated to the critical cancer genes) can be used to evaluate the full spectrum of mutational events that can be induced by a chemical. One such system is the AS52 assay, an in vitro mammalian transgenic mutation assay that measures mutation in the gpt gene. After exposure to AFB1 in culture the predominant mutation is G:C to T:A transversion, although a number of other types of mutations were also seen (Wattanawaraporn et al., 2012). There is a species difference between adult mice and rats. No increase in MF is seen in the in vivo Big Blue™ mutation assay for mice exposed to AFB1. That is, Lac I mutations from the mice showed a spontaneous mutational spectrum. By contrast rats showed a large increase in MF with a large increase in G:C to T:A transversions (Dycaico et al., 1996). However, neonatal mice treated with AFB1 (6 mg/kg—a dose that induces tumors in neonates) do show an increase in cII mutation with G:C to T:A transversion as the major mutation. Adult mice treated at 6 and 60 mg/kg (doses that do not induce tumors) did not show a significant increase in cII mutations but did produce a different mutational spectrum than controls (Chen et al., 2010.)<br />
<br />
== Level of Biological Organization : Cellular ==<br />
The induction of mutation occurs within the nucleus of cells and involves permanent alterations in the primary DNA sequence that is passed to subsequent cell generations and, thus, is heritable.<br />
<br />
== How this Key Event works ==<br />
Following the formation of DNA adducts, which are either mis-repaired or not repaired, the sequence of base pairs in the DNA is changed due to insertion of an incorrect base opposite the DNA adduct during DNA replication, so that a G is permanently replaced by a T. This type of mutation is observed in the mutants seen in surrogate gene mutation assays using AFB1 exposureA high frequency of codon 249 p53 mutations occurs in human tumors in high AFB1 exposure regions.Hence, codon 249 of the p53 gene contains a base pair susceptible to insufficient or misrepair of DNA adducts.<br />
<br />
Puisieux et al. (1991) provide evidence that the AFB1 epoxide adduct binds preferentially to codon 249 of the p53 gene. Using a plasmid with containing full-length human p53 DNA, adduct formation was observed in exons 5, 6, 7 and 8 (a total of 1086 bases) and 20% of the bases were targeted by AFB1 with a preference for guanine residues. Binding of AFB1 to p53 sequences was restricted to fewer residues and was more specific for guanine than was the binding of B[a]P (Puisieux et al.,1991). Binding of AFB1 in the region around codon 249 of p53 AFB1 was reported to be “stronger” than that of B[a]P. The last nucleotide of codon 249 is a guanine and was targeted by AFB1 but not by B[a]P. This guanine residue is the mutational hotspot in human liver cancers from patients in high AFB1 exposure regions. <br />
<br />
While mutations at codon 249 of the p53 gene have been observed in association with HCC in humans, the question remains whether this mutation occurs as a direct result of adduct formation at this site or by some different mechanism..In human HepG2 hepatocytes exposed to microsomally activated AFB1, a dose-dependent increase in G:C to T:A transversions were observed at 10 additional locations using ligation-mediated PCR, and at 4 additional locations using terminal-transferred dependent PCR.(Denissenko et al.,1998). These authors suggest that codon 249 may not present a key adduct site. However, other more recently published data identified codon 249 of the P53 gene as an unusually mutagenic adduct conformation based on the local DNA sequence and concluded that a higher mutation rate may occur there rather than at other locations because of increased DNA polymerase bypass (Pussieux et al., 1991; Lin et al., 2014a,b).<br />
<br />
== How it is Measured or Detected ==<br />
Historically the detection of critical cancer gene specific mutations has not been technically feasible. A newly developed method, allele specific competitive blocker-polymerase chain reaction (ACB-PCR) has proven useful in providing such information, and data on specific chemical-induced mutations are available for a very small number of chemicals (Parsons et al., 2010). Unfortunately, there are no data for AFB1.<br />
<br />
There are, however, a number of gene mutation assays that have been widely used for determining the general ability of chemicals, including AFB1 to induce mutations.These assays use selection methods that allow only mutant cells to survive and grow. The AS52 in vitro gene mutation assay using the gpt gene, and 6-thioguanine selection has been used to demonstrate that AFB1 exposure increases the MF at the gpt gene. In vivo transgenic assays use molecular methods to recover the transgene from isolated DNA and to evaluate the MF in the transgene. Molecular methods can detect the presence (above a certain sensitivity level) of mutant cells. DNA from tumors can be sequenced to determine the presence of mutations in specific genes. DNA sequencing has been used on human tumors to detect the presence of the Codon 249 p53 mutation.<br />
<br />
== Evidence Supporting Taxonomic Applicability ==<br />
There are data across phyla demonstrating the induction of mutations, specifically the induction to G:C to T:A transversions following AFB1 exposure.<br />
<br />
== KE#2: References ==<br />
Bressac, B., Kew, M., Wands, J., & Ozturk, M. (1991). Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern africa. Nature, 350(6317), 429-31. doi:10.1038/350429a<br />
<br />
Chen, T., Heflich, R. H., Moore, M. M., & Mei, N. (2010). Differential mutagenicity of aflatoxin B1 in the liver of neonatal and adult mice. Environ Mol Mutagen, 51(2), 156-63. doi:10.1002/em.2051<br />
<br />
Denissenko MF, Koudriakova TB, Smith L, O'Connor TR, Riggs AD, and Pfeifer GP). The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts. (Oncogene. 1998, Dec 10;17(23):3007-14.<br />
<br />
Dycaico, M. J., Stuart, G. R., Tobal, G. M., de Boer, J. G., Glickman, B. W., & Provost, G. S. (1996). Species-specific differences in hepatic mutant frequency and mutational spectrum among lambda/laci transgenic rats and mice following exposure to aflatoxin B1. Carcinogenesis, 17(11), 2347-56<br />
<br />
Foster, P. L., Eisenstadt, E., & Miller, J. H. (1983). Base substitution mutations induced by metabolically activated aflatoxin B1. Proceedings of the National Academy of Sciences of the United States of America, 80(9), 2695-8.<br />
<br />
Gouas, D., Shi, H., & Hainaut, P. (2009). The aflatoxin-induced TP53 mutation at codon 249 (R249S): Biomarker of exposure, early detection and target for therapy. Cancer Lett, 286(1), 29-37. doi:10.1016/j.canlet.2009.02.057<br />
<br />
Hsu, I. C., Metcalf, R. A., Sun, T., Welsh, J. A., Wang, N. J., & Harris, C. C. (1991). Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature, 350(6317), 427-8. doi:10.1038/350427a<br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014a). Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua. J Biol Chem. 289:18497-18506. <br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014b). Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis 35(7):1461-1468<br />
<br />
Parsons BL, Myers MB, Meng F, Wang Y, McKinzie PB. 2010. Oncomutations as biomarkers of cancer risk. Environ Mol Mutagen. 51(8-9):836-850.<br />
<br />
Puisieux, A., Lim, S., Groopman, J., & Ozturk, M. (1991). Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res, 51(22), 6185-9.<br />
<br />
Wattanawaraporn, R., Kim, M. Y., Adams, J., Trudel, L. J., Woo, L. L., Croy, R. G., . . . Wogan, G. N. (2012). AFB(1) -induced mutagenesis of the gpt gene in AS52 cells. Environ Mol Mutagen, 53(7), 567-73. doi:10.1002/em.2171</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:409Event:4092016-07-06T17:04:28Z<p>Dvillene: /* Evidence Supporting Taxonomic Applicability */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id ='longTitle' class='Title'> Step: #0: pre-MIE </div><br />
<div id ='longTitle' class='Title'> Action: Metabolism of AFB1 </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||KE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Molecular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Biological Process: Production of Reactive Electrophiles ==<br />
Aflatoxin B1 (AFB1) can be oxidized to a variety of metabolites, many of which can be further metabolized, for example by conjugation with glutathione (GSH) via Glutathione-S-transferase (GST). In order for DNA binding and formation of a pro-mutagenic DNA adduct to occur, AFB1 must be metabolized via Cytochrome P450 metabolism to a specific highly reactive form of AFB1, the exo-epoxide AFB1-8,9-epoxide. CYP3A4 forms the exo-form of this reactive epoxide only. CYP1A2, inducible in liver, forms both the exo- and the endo-epoxides; in human liver, CYP1A2 metabolism occurs with a lower Vmax (enzymatic rate) and higher Km (half-maximal concentrations) than CYP3A4 (Degen and Neumann,1981; Groopman and Kensler, 2005; Guengerich et al., 1996; Ueng et al., 1995). Figure 1, from Pottenger et al., 2014, depicts the metabolism of AFB1.<br />
<br />
Metabolic activation of AFB1 occurs mainly in the liver, where the highest levels of the CYP isozymes are located. Competitive metabolism with other hepatic and extra-hepatic P450 isozymes may decrease the proportion of the specific metabolite, AFB1-8,9-epoxide; this can reduce the effectiveness of hepatic activation of AFB1. Alternatively, induction of either hepatic or extra-hepatic GST activity can reduce the levels of AFB1-epoxide available for reaction with DNA by increasing conjugation of the epoxide with GSH. This conjugation renders the epoxide inactive and directs it towards eventual elimination (Guengerich et al., 1996).[[File:Figure1met.jpg|thumb|alt=Figure 1 alt text|Figure 1. Metabolism of AFB1,]] Figure 1 was obtained from Pottenger et al., 2014.<br />
<br />
The AFB1 pro-mutagenic DNA adduct is most likely the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct, or N7-AFB1-G (Croy et al., 1978). Once the exo-epoxide is bound to the N7-guanine, it is subject to ring-opening to form the more highly pro-mutagenic 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-pyrimid-9-yl-foramido)-9-hydroxyaflatoxin B1; this is known as the formamidopyrimidine adduct or AFB1-FAPy adduct (Brown et al., 2006).<br />
<br />
== Taxonomic Applicability ==<br />
The metabolic information above is applicable to all mammalian systems evaluated; it is also applicable to certain birds such as turkeys (Gregory et al., 1983; IARC, 1993). Humans, non-human primates, rats, mice, poultry and fish have all demonstrated susceptibility to AFB1-induced liver tumors (Asplin and Canaghan, 1961; Eaton and Gallagher, 1994). Species that preferentially metabolize AFB1 to the exo-8,9-epoxide are more susceptible to AFB1 carcinogenicity. Sulfation by GST enzymes or other enzymatic detoxification of AFB1 metabolites can reduce available levels of the exo-epoxide, and subsequent steps or even the progression of Key Events may not occur in these species. Mice are less susceptible to AFB1-induced cancer than rats, likely due to increased detoxification (Monroe and Eaton, 1987). This increased detoxication capability can also be instigated by dietary exposures to compounds that modulate CYP450 expression or modify detoxication activities (Elegbede and Gould, 2002; Primiano et al., 1995; Roebuck et al. 1991, 2003; Kensler et al., 1998; Wang et al., 1999; Yates et al., 2006).<br />
<br />
== Level of Biological Organization : Molecular ==<br />
Metabolic activation of AFB1 occurs at the intracellular level, with sub-cellular organelles involved due to the localization of CYP450 in the endoplasmic reticulum, coupled with the epoxide hydratase; GST is localized in the cytosol (Guengerich et al., 1996). Organ architecture also plays a role, as the structure of the liver is orientated around the O2 gradient set by the portal artery and hepatic vein; hepatocytes nearest the portal artery demonstrate increased tissue oxygen while those around the portal vein have decreased tissue O2 and correspondingly higher levels of CYP450 activity.<br />
<br />
== How this Key Event works ==<br />
The reactive exo-epoxide is formed in hepatocytes (or extra-hepatically) by metabolism of the parent AFB1 by CYP450 (Larsson et al., 1990; Larsson and Tjalve, 1993). The reactive metabolite then escapes the endoplasmic reticulum where the CYP450 is located. The reactive metabolite must evade conjugation with GSH in the cytoplasm or binding with other cytoplasmic nucleophiles. It then traverses the nuclear membrane in order to reach the cell nucleus and the nuclear DNA. Once the reactive metabolite is in the cell nucleus, binding to nuclear DNA and the formation of DNA adducts can occur.<br />
<br />
== How it is Measured or Detected ==<br />
Formation of the exo-epoxide can be produced with in vitro systems and detected using techniques for structural quantitation of AFB1 metabolites (Himmelstein et al., 2009), including liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). By using subcellular fractions (e.g., microsomes), cellular homogenates, or cells in culture, it is possible to measure formation of AFB1 exo-epoxide. Such data can also be collected from in vivo systems; samples of plasma or blood from AFB1-treated animals can be analyzed for the AFB1 exo-epoxide with similar mass spectrometric based detection systems (e.g., LC-MS/MS). Samples of blood from humans in AFB1-endemic regions have demonstrated presence of AFB1-albumin adducts, which are formed from the AFB1 exo-epoxide. AFB1-treated animals may also provide tissue samples for analysis of AFB1 exo-epoxide. Special trapping techniques may be required as the reactive AFB1 exo-epoxide metabolite has a short half-life in biological matrices.<br />
<br />
== Evidence Supporting Taxonomic Applicability ==<br />
Ample data across phyla demonstrate metabolic activation of AFB1 to the exo-epoxide via CYP450. These taxons include several mammalian species (humans, non-human primates, rats, mice) in addition to birds (turkeys) and fish (Eaton and Gallagher, 1994; IARC, 1993).<br />
<br />
== Pre-MIE: Evidence for Chemical Initiation of this Initial Molecular Event/Pre-MIE ==<br />
There is an extensive database on AFB1 in many different systems demonstrating formation of the AFB1 exo-epoxide. This database includes several mammalian species (humans, non-human primates, rats, mice) in addition to birds (turkeys) and fish (Eaton and Gallagher, 1994; IARC, 1993).<br />
<br />
== Evidence Supporting Essentiality of Pre-MIE (Step #0) ==<br />
Strong<br />
<br />
Many studies show that in the absence of AFB1 metabolic activation, tumors do not occur. Treatment of rats with the CYP450-inhibitor and GST-inducer oltipraz reduces the levels of AFB1 DNA adducts by 65-70%, and the later-forming altered hepatic foci (AHF) and liver tumors are also reduced by 97 and 100%, respectively; thus, no hepatocellular carcinomas (HCC) form in the oltipraz-treated rats dosed with AFB1 (Roebuck et al., 1991). More recent data show even more effective prevention of tumors and of hepatic foci in rats pre-treated with a triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) (Johnson et al., 2014). The pre-treated rats were reported with a 70% reduction of AFB1-induced adducts in urine but had 100% inhibition of tumors, and only 1 rat out of 23 pre-treated had any altered hepatic foci (AHF). There are similar data in humans treated with oltipraz. Kensler et al. (1998) report a significant reduction in a biomarker of exposure AFB1-induced albumin adducts, supporting the human relevance of this metabolic activation <br />
<br />
Another line of evidence for essentiality of the pre-MIE is the recognized difference in sensitivity to AFB1-induced liver tumors between mice and rats (Degen and Neumann, 1981). Mice, have considerably increased metabolic activation of AFB1 to the exo-epoxide compared to rats; mice are nonetheless much less sensitive to AFB1-induced liver tumors. It is thought that this difference is due to the constitutive presence of GST-alpha activity in mice vs rats, where this activity is not found.<br />
<br />
== Pre-MIE: References ==<br />
F.D. Asplin, R.B.A. Carnaghan, (1961). The toxicity of certain groundnut meals for poultry with special reference to their effect on ducklings and chickens. Vet. Rec. 73:1215– 1219. <br />
<br />
Brown KL, Deng JZ, Iyer RS, Iyer LG, Voehler MW, Stone MP, Harris CM, Harris TM (2006). Unraveling the aflatoxin-FAPY conundrum: Structural basis of the formamidopyrimidine-type DNA adduct of aflatoxin B1. J Am Chem Soc 128:15188-15199.<br />
<br />
Croy RG, Essigman JM, Reinhold VN, Wogan GN (1978). Identification of the principal aflatoxin N1-DNA adduct formed in vivo in rat liver. Proc Natl Acad Sci USA 75:1745-1749.<br />
<br />
Degen GH, Neumann HG (1981). Differences in aflatoxin B1-susceptibility of rat and mouse are correlated with the capability in vitro to inactivate aflatoxin B1-epoxide. Carcinogenesis 2:299–306.<br />
<br />
Eaton DL, and Gallagher EP (1994). Mechanisms of aflatoxin carcinogenesis. Annu Rev Pharmacol Toxicol 34:135-172.<br />
<br />
Elegbede JA, and Gould MN. (2002). Monoterpenes reduced adducts formation in rats exposed to aflatoxin B1. African J Biotech, 1, 46–49.<br />
<br />
Gregory 3rd JF, Goldstein SL, Edds GT. (1983). Metabolite distribution and rate of residue clearance in turkeys fed a diet containing aflatoxin B1. Food Chem Toxicol, 21, 463–7.<br />
<br />
Groopman JD, Kensler TW (2005). Role of metabolism and viruses in aflatoxin-induced liver cancer. Toxicol Appl Pharmacol 206:131-137.<br />
<br />
Guengerich FP, Johnson WW, Ueng Y-F, Yamazaki H, Shimada T (1996). Involvement of Cytochrome P450, glutathione S-transferase, and epoxide hydrolase in the metabolism of aflatoxin B1 and relevance to risk of human liver cancer. Environ Health Perspect 104(Suppl 3):557-562.<br />
<br />
Himmelstein MW, Boogaard PJ, Cadet J, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: II.Overview of methods of identification and quantitation of DNA damage. Crit Rev Toxicol, 39, 679–694.<br />
<br />
IARC (1993). Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. IARC Monographs on the Evaluation of Carcinogenic Risk to Humans. Vol. 56, 245-395.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, Sutter TR, Groopman JD, Kensler TW, Roebuck BD. (2014). Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold. Cancer Prev Res. 7(7):658-665.<br />
<br />
Kensler TW, He X, Otieno M, et al. (1998). Oltipraz chemoprevention trial in Qidong, People’s Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol Biomarkers Prev, 7, 127–34.<br />
<br />
Larsson P, Hoedaya, WI, Tjalve H. (1990). Disposition of 3H-aflatoxin H in mice: formation and retention of tissue bound metabolites in nasal glands. Pharmacol Toxicol, 67, 162–71.<br />
<br />
Larsson P, and Tjalve H. (1993). Distribution and metabolism of aflatoxin B1 in the marmoset monkey (Callithrix jacchus). Carcinogenesis, 14, 1–6.<br />
<br />
Monroe DH, Eaton DL. (1987). Comparative effects of butylated hydroxyanisole on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B1 in the rat and the mouse. Toxicol Appl Pharmacol, 90, 401–409.<br />
<br />
Pottenger, L.H., Andrews LS, Bachman AN, Boogaard PJ, Cadet J, Embry MR, Farmer PB, Himmelstein MW, Jarabek AM, Martin EA, Mauthe RJ, Persaud R, Preston RJ, Schoeny R, Skare J, Swenberg JA, Williams GM, Zeiger E, Zhang F, Kim JH. (2014). An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride. Crit. Rev. Toxicol. 44(4):348-391.<br />
<br />
Primiano T, Egner PA, Sutter TR, et al. (1995). Intermittent dosing with oltipraz: relationship between chemoprevention of aflatoxin-induced tumorigenesis and induction of glutathione-S-transferases. Cancer Res, 55, 4319–4324.<br />
<br />
Roebuck BD, Liu Y-L, Rogers AE, et al. (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short term molecular dosimetry. Cancer Res, 51, 5501–5506.<br />
<br />
Ueng Y-F, Shimada T, Yamazaki H, Guengerich FP (1995). Oxidation of aflatoxin B1 by bacterial recombinant human cytochrome P450 enzymes. Chem Res Toxiol 8:218-225.<br />
<br />
Wang J-S, Shen X, He X, et al. (1999). Protective alerations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qidong, People’s Republic of China. J Natl Cancer Inst, 91, 347–354.<br />
<br />
Yates MS, Kwak M-K, Egner PA, et al. (2006). Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl] imidazole. Cancer Res, 66, 2488–2494.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:376Event:3762016-07-06T17:04:18Z<p>Dvillene: /* KE#2: How it is Measured or Detected */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Induced Mutations in Critical Genes, Increased </div><br />
<div id='shortTitle' class='Title'> Induced in Critical Genes, Mutation </div><br />
<br />
== Action ==<br />
Induced mutation<br />
<br />
== Biological Process/Object ==<br />
Critical genes for carcinogenesis<br />
<br />
== Induced Mutation in Critical Genes ==<br />
The AFB1-induced pro-mutagenic DNA adduct is either not repaired or is mis-repaired resulting in a mutation in one or more critical genes. In bacteria and mammalian cells (both in vitro and in vivo) the primary mutation associated with AFB1 is a guanine to thymine transversion (Foster et al., 1983; Dycaico et al., 1996). A G:T transversion is expected for the pro-mutagenic DNA adduct AFB1-FAPy. <br />
<br />
A specific critical mutation in codon 249 of the p53 gene has been identified in human hepatocellular carcinoma (HCC) (See section below on essentiality).<br />
<br />
== Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
A specific critical mutation in the p53 gene has been identified in human hepatocellular carcinoma (HCC). Demonstrating that AFB1 can induce this specific p53 gene mutation would be the highest level of evidence that AFB1-induced HCC involves mutation as a KE. Absent such information, the next best level of evidence is the induction of the specific type of mutation (G:C to T:A transversion) in a variety of gene mutation assays measuring a range of target genes. <br />
<br />
The codon 249 mutation is present in a significant proportion of human HCCs. In fact codon 249 mutation is detected in up to 50% of liver cancers in Qidong, China (Hsu et al., 1991) and in Mozambique, both areas with high likelihood of AFB1 exposure. The codon 249 G:C to T:A mutation in the third base is seen in up to 75% of HCC in high-incidence areas of China and East Africa (Gouas et al., 2009). In contrast, this specific mutation is very rare in HCC from areas with no or low exposure to AFB1 (Hsu et al. 1991 and Bressac et al. Nature, 350:429-431, 1991). This mutation is also very rare in other types of tumors (Gouas et al., 2009). According to Gouas et al. (2009), populations with AFB1 exposure are likely to be exposed to hepatitis B virus (HBV) as well and the effects of each are difficult to separate.This mutation is very rare in HCC from non/low -aflatoxin areas (Hsu et al. 1991 and Bressac et al., 1991) and also very rare in other types of tumors.<br />
<br />
Some indirect evidence of the essentiality of mutation in tumor development is provided by the clear species difference between adult mice and adult rats both in the induction of surrogate gene mutations and in the induction of tumors. Adult mice exposed to AFB1 do not get tumors and there is no increase in mutant frequency (MF) for Big Blue mice exposed as adults. That is, Lac I mutants from the AFB1- exposed adult mice showed a spontaneous mutational spectrum. Rats, however, showed a large increase in MF and, more specifically a large increase in G:C to T:A transversions (Dycaico et al., 1996). In addition, for mice there is an difference between neonata and adult mice. Neonatal mice treated with AFB1 (6 mg/kg—a dose that does result in tumors) showed an increase in cII mutation with G:C to T:A transversion as the major mutation. Adult mice treated at 6 and 60 mg/kg (doses that do not produce tumors) did not have a significant increase in cII mutation (but did give a different mutational spectrum than controls) (Chen et al. 2010).<br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||KE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Cellular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Taxonomic Applicability ==<br />
The induction of G:C to T:A transversions following AFB1 exposure is seen in a wide variety of species from bacteria to mammals. Assays that measure mutation in surrogate genes (that is, genes unrelated to the critical cancer genes) can be used to evaluate the full spectrum of mutational events that can be induced by a chemical. One such system is the AS52 assay, an in vitro mammalian transgenic mutation assay that measures mutation in the gpt gene. After exposure to AFB1 in culture the predominant mutation is G:C to T:A transversion, although a number of other types of mutations were also seen (Wattanawaraporn et al., 2012). There is a species difference between adult mice and rats. No increase in MF is seen in the in vivo Big Blue™ mutation assay for mice exposed to AFB1. That is, Lac I mutations from the mice showed a spontaneous mutational spectrum. By contrast rats showed a large increase in MF with a large increase in G:C to T:A transversions (Dycaico et al., 1996). However, neonatal mice treated with AFB1 (6 mg/kg—a dose that induces tumors in neonates) do show an increase in cII mutation with G:C to T:A transversion as the major mutation. Adult mice treated at 6 and 60 mg/kg (doses that do not induce tumors) did not show a significant increase in cII mutations but did produce a different mutational spectrum than controls (Chen et al., 2010.)<br />
<br />
== Level of Biological Organization : Cellular ==<br />
The induction of mutation occurs within the nucleus of cells and involves permanent alterations in the primary DNA sequence that is passed to subsequent cell generations and, thus, is heritable.<br />
<br />
== How this Key Event works ==<br />
Following the formation of DNA adducts, which are either mis-repaired or not repaired, the sequence of base pairs in the DNA is changed due to insertion of an incorrect base opposite the DNA adduct during DNA replication, so that a G is permanently replaced by a T. This type of mutation is observed in the mutants seen in surrogate gene mutation assays using AFB1 exposureA high frequency of codon 249 p53 mutations occurs in human tumors in high AFB1 exposure regions.Hence, codon 249 of the p53 gene contains a base pair susceptible to insufficient or misrepair of DNA adducts.<br />
<br />
Puisieux et al. (1991) provide evidence that the AFB1 epoxide adduct binds preferentially to codon 249 of the p53 gene. Using a plasmid with containing full-length human p53 DNA, adduct formation was observed in exons 5, 6, 7 and 8 (a total of 1086 bases) and 20% of the bases were targeted by AFB1 with a preference for guanine residues. Binding of AFB1 to p53 sequences was restricted to fewer residues and was more specific for guanine than was the binding of B[a]P (Puisieux et al.,1991). Binding of AFB1 in the region around codon 249 of p53 AFB1 was reported to be “stronger” than that of B[a]P. The last nucleotide of codon 249 is a guanine and was targeted by AFB1 but not by B[a]P. This guanine residue is the mutational hotspot in human liver cancers from patients in high AFB1 exposure regions. <br />
<br />
While mutations at codon 249 of the p53 gene have been observed in association with HCC in humans, the question remains whether this mutation occurs as a direct result of adduct formation at this site or by some different mechanism..In human HepG2 hepatocytes exposed to microsomally activated AFB1, a dose-dependent increase in G:C to T:A transversions were observed at 10 additional locations using ligation-mediated PCR, and at 4 additional locations using terminal-transferred dependent PCR.(Denissenko et al.,1998). These authors suggest that codon 249 may not present a key adduct site. However, other more recently published data identified codon 249 of the P53 gene as an unusually mutagenic adduct conformation based on the local DNA sequence and concluded that a higher mutation rate may occur there rather than at other locations because of increased DNA polymerase bypass (Pussieux et al., 1991; Lin et al., 2014a,b).<br />
<br />
== How it is Measured or Detected ==<br />
Historically the detection of critical cancer gene specific mutations has not been technically feasible. A newly developed method, allele specific competitive blocker-polymerase chain reaction (ACB-PCR) has proven useful in providing such information, and data on specific chemical-induced mutations are available for a very small number of chemicals (Parsons et al., 2010). Unfortunately, there are no data for AFB1.<br />
<br />
There are, however, a number of gene mutation assays that have been widely used for determining the general ability of chemicals, including AFB1 to induce mutations.These assays use selection methods that allow only mutant cells to survive and grow. The AS52 in vitro gene mutation assay using the gpt gene, and 6-thioguanine selection has been used to demonstrate that AFB1 exposure increases the MF at the gpt gene. In vivo transgenic assays use molecular methods to recover the transgene from isolated DNA and to evaluate the MF in the transgene. Molecular methods can detect the presence (above a certain sensitivity level) of mutant cells. DNA from tumors can be sequenced to determine the presence of mutations in specific genes. DNA sequencing has been used on human tumors to detect the presence of the Codon 249 p53 mutation.<br />
<br />
== KE#2: Evidence Supporting Taxonomic Applicability ==<br />
There are data across phyla demonstrating the induction of mutations, specifically the induction to G:C to T:A transversions following AFB1 exposure.<br />
<br />
== KE#2: References ==<br />
Bressac, B., Kew, M., Wands, J., & Ozturk, M. (1991). Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern africa. Nature, 350(6317), 429-31. doi:10.1038/350429a<br />
<br />
Chen, T., Heflich, R. H., Moore, M. M., & Mei, N. (2010). Differential mutagenicity of aflatoxin B1 in the liver of neonatal and adult mice. Environ Mol Mutagen, 51(2), 156-63. doi:10.1002/em.2051<br />
<br />
Denissenko MF, Koudriakova TB, Smith L, O'Connor TR, Riggs AD, and Pfeifer GP). The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts. (Oncogene. 1998, Dec 10;17(23):3007-14.<br />
<br />
Dycaico, M. J., Stuart, G. R., Tobal, G. M., de Boer, J. G., Glickman, B. W., & Provost, G. S. (1996). Species-specific differences in hepatic mutant frequency and mutational spectrum among lambda/laci transgenic rats and mice following exposure to aflatoxin B1. Carcinogenesis, 17(11), 2347-56<br />
<br />
Foster, P. L., Eisenstadt, E., & Miller, J. H. (1983). Base substitution mutations induced by metabolically activated aflatoxin B1. Proceedings of the National Academy of Sciences of the United States of America, 80(9), 2695-8.<br />
<br />
Gouas, D., Shi, H., & Hainaut, P. (2009). The aflatoxin-induced TP53 mutation at codon 249 (R249S): Biomarker of exposure, early detection and target for therapy. Cancer Lett, 286(1), 29-37. doi:10.1016/j.canlet.2009.02.057<br />
<br />
Hsu, I. C., Metcalf, R. A., Sun, T., Welsh, J. A., Wang, N. J., & Harris, C. C. (1991). Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature, 350(6317), 427-8. doi:10.1038/350427a<br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014a). Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua. J Biol Chem. 289:18497-18506. <br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014b). Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis 35(7):1461-1468<br />
<br />
Parsons BL, Myers MB, Meng F, Wang Y, McKinzie PB. 2010. Oncomutations as biomarkers of cancer risk. Environ Mol Mutagen. 51(8-9):836-850.<br />
<br />
Puisieux, A., Lim, S., Groopman, J., & Ozturk, M. (1991). Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res, 51(22), 6185-9.<br />
<br />
Wattanawaraporn, R., Kim, M. Y., Adams, J., Trudel, L. J., Woo, L. L., Croy, R. G., . . . Wogan, G. N. (2012). AFB(1) -induced mutagenesis of the gpt gene in AS52 cells. Environ Mol Mutagen, 53(7), 567-73. doi:10.1002/em.2171</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:409Event:4092016-07-06T17:04:12Z<p>Dvillene: /* How it is Measured or Detected */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id ='longTitle' class='Title'> Step: #0: pre-MIE </div><br />
<div id ='longTitle' class='Title'> Action: Metabolism of AFB1 </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||KE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Molecular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Biological Process: Production of Reactive Electrophiles ==<br />
Aflatoxin B1 (AFB1) can be oxidized to a variety of metabolites, many of which can be further metabolized, for example by conjugation with glutathione (GSH) via Glutathione-S-transferase (GST). In order for DNA binding and formation of a pro-mutagenic DNA adduct to occur, AFB1 must be metabolized via Cytochrome P450 metabolism to a specific highly reactive form of AFB1, the exo-epoxide AFB1-8,9-epoxide. CYP3A4 forms the exo-form of this reactive epoxide only. CYP1A2, inducible in liver, forms both the exo- and the endo-epoxides; in human liver, CYP1A2 metabolism occurs with a lower Vmax (enzymatic rate) and higher Km (half-maximal concentrations) than CYP3A4 (Degen and Neumann,1981; Groopman and Kensler, 2005; Guengerich et al., 1996; Ueng et al., 1995). Figure 1, from Pottenger et al., 2014, depicts the metabolism of AFB1.<br />
<br />
Metabolic activation of AFB1 occurs mainly in the liver, where the highest levels of the CYP isozymes are located. Competitive metabolism with other hepatic and extra-hepatic P450 isozymes may decrease the proportion of the specific metabolite, AFB1-8,9-epoxide; this can reduce the effectiveness of hepatic activation of AFB1. Alternatively, induction of either hepatic or extra-hepatic GST activity can reduce the levels of AFB1-epoxide available for reaction with DNA by increasing conjugation of the epoxide with GSH. This conjugation renders the epoxide inactive and directs it towards eventual elimination (Guengerich et al., 1996).[[File:Figure1met.jpg|thumb|alt=Figure 1 alt text|Figure 1. Metabolism of AFB1,]] Figure 1 was obtained from Pottenger et al., 2014.<br />
<br />
The AFB1 pro-mutagenic DNA adduct is most likely the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct, or N7-AFB1-G (Croy et al., 1978). Once the exo-epoxide is bound to the N7-guanine, it is subject to ring-opening to form the more highly pro-mutagenic 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-pyrimid-9-yl-foramido)-9-hydroxyaflatoxin B1; this is known as the formamidopyrimidine adduct or AFB1-FAPy adduct (Brown et al., 2006).<br />
<br />
== Taxonomic Applicability ==<br />
The metabolic information above is applicable to all mammalian systems evaluated; it is also applicable to certain birds such as turkeys (Gregory et al., 1983; IARC, 1993). Humans, non-human primates, rats, mice, poultry and fish have all demonstrated susceptibility to AFB1-induced liver tumors (Asplin and Canaghan, 1961; Eaton and Gallagher, 1994). Species that preferentially metabolize AFB1 to the exo-8,9-epoxide are more susceptible to AFB1 carcinogenicity. Sulfation by GST enzymes or other enzymatic detoxification of AFB1 metabolites can reduce available levels of the exo-epoxide, and subsequent steps or even the progression of Key Events may not occur in these species. Mice are less susceptible to AFB1-induced cancer than rats, likely due to increased detoxification (Monroe and Eaton, 1987). This increased detoxication capability can also be instigated by dietary exposures to compounds that modulate CYP450 expression or modify detoxication activities (Elegbede and Gould, 2002; Primiano et al., 1995; Roebuck et al. 1991, 2003; Kensler et al., 1998; Wang et al., 1999; Yates et al., 2006).<br />
<br />
== Level of Biological Organization : Molecular ==<br />
Metabolic activation of AFB1 occurs at the intracellular level, with sub-cellular organelles involved due to the localization of CYP450 in the endoplasmic reticulum, coupled with the epoxide hydratase; GST is localized in the cytosol (Guengerich et al., 1996). Organ architecture also plays a role, as the structure of the liver is orientated around the O2 gradient set by the portal artery and hepatic vein; hepatocytes nearest the portal artery demonstrate increased tissue oxygen while those around the portal vein have decreased tissue O2 and correspondingly higher levels of CYP450 activity.<br />
<br />
== How this Key Event works ==<br />
The reactive exo-epoxide is formed in hepatocytes (or extra-hepatically) by metabolism of the parent AFB1 by CYP450 (Larsson et al., 1990; Larsson and Tjalve, 1993). The reactive metabolite then escapes the endoplasmic reticulum where the CYP450 is located. The reactive metabolite must evade conjugation with GSH in the cytoplasm or binding with other cytoplasmic nucleophiles. It then traverses the nuclear membrane in order to reach the cell nucleus and the nuclear DNA. Once the reactive metabolite is in the cell nucleus, binding to nuclear DNA and the formation of DNA adducts can occur.<br />
<br />
== How it is Measured or Detected ==<br />
Formation of the exo-epoxide can be produced with in vitro systems and detected using techniques for structural quantitation of AFB1 metabolites (Himmelstein et al., 2009), including liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). By using subcellular fractions (e.g., microsomes), cellular homogenates, or cells in culture, it is possible to measure formation of AFB1 exo-epoxide. Such data can also be collected from in vivo systems; samples of plasma or blood from AFB1-treated animals can be analyzed for the AFB1 exo-epoxide with similar mass spectrometric based detection systems (e.g., LC-MS/MS). Samples of blood from humans in AFB1-endemic regions have demonstrated presence of AFB1-albumin adducts, which are formed from the AFB1 exo-epoxide. AFB1-treated animals may also provide tissue samples for analysis of AFB1 exo-epoxide. Special trapping techniques may be required as the reactive AFB1 exo-epoxide metabolite has a short half-life in biological matrices.<br />
<br />
== Pre-MIE: Evidence Supporting Taxonomic Applicability ==<br />
Ample data across phyla demonstrate metabolic activation of AFB1 to the exo-epoxide via CYP450. These taxons include several mammalian species (humans, non-human primates, rats, mice) in addition to birds (turkeys) and fish (Eaton and Gallagher, 1994; IARC, 1993).<br />
<br />
== Pre-MIE: Evidence for Chemical Initiation of this Initial Molecular Event/Pre-MIE ==<br />
There is an extensive database on AFB1 in many different systems demonstrating formation of the AFB1 exo-epoxide. This database includes several mammalian species (humans, non-human primates, rats, mice) in addition to birds (turkeys) and fish (Eaton and Gallagher, 1994; IARC, 1993).<br />
<br />
== Evidence Supporting Essentiality of Pre-MIE (Step #0) ==<br />
Strong<br />
<br />
Many studies show that in the absence of AFB1 metabolic activation, tumors do not occur. Treatment of rats with the CYP450-inhibitor and GST-inducer oltipraz reduces the levels of AFB1 DNA adducts by 65-70%, and the later-forming altered hepatic foci (AHF) and liver tumors are also reduced by 97 and 100%, respectively; thus, no hepatocellular carcinomas (HCC) form in the oltipraz-treated rats dosed with AFB1 (Roebuck et al., 1991). More recent data show even more effective prevention of tumors and of hepatic foci in rats pre-treated with a triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) (Johnson et al., 2014). The pre-treated rats were reported with a 70% reduction of AFB1-induced adducts in urine but had 100% inhibition of tumors, and only 1 rat out of 23 pre-treated had any altered hepatic foci (AHF). There are similar data in humans treated with oltipraz. Kensler et al. (1998) report a significant reduction in a biomarker of exposure AFB1-induced albumin adducts, supporting the human relevance of this metabolic activation <br />
<br />
Another line of evidence for essentiality of the pre-MIE is the recognized difference in sensitivity to AFB1-induced liver tumors between mice and rats (Degen and Neumann, 1981). Mice, have considerably increased metabolic activation of AFB1 to the exo-epoxide compared to rats; mice are nonetheless much less sensitive to AFB1-induced liver tumors. It is thought that this difference is due to the constitutive presence of GST-alpha activity in mice vs rats, where this activity is not found.<br />
<br />
== Pre-MIE: References ==<br />
F.D. Asplin, R.B.A. Carnaghan, (1961). The toxicity of certain groundnut meals for poultry with special reference to their effect on ducklings and chickens. Vet. Rec. 73:1215– 1219. <br />
<br />
Brown KL, Deng JZ, Iyer RS, Iyer LG, Voehler MW, Stone MP, Harris CM, Harris TM (2006). Unraveling the aflatoxin-FAPY conundrum: Structural basis of the formamidopyrimidine-type DNA adduct of aflatoxin B1. J Am Chem Soc 128:15188-15199.<br />
<br />
Croy RG, Essigman JM, Reinhold VN, Wogan GN (1978). Identification of the principal aflatoxin N1-DNA adduct formed in vivo in rat liver. Proc Natl Acad Sci USA 75:1745-1749.<br />
<br />
Degen GH, Neumann HG (1981). Differences in aflatoxin B1-susceptibility of rat and mouse are correlated with the capability in vitro to inactivate aflatoxin B1-epoxide. Carcinogenesis 2:299–306.<br />
<br />
Eaton DL, and Gallagher EP (1994). Mechanisms of aflatoxin carcinogenesis. Annu Rev Pharmacol Toxicol 34:135-172.<br />
<br />
Elegbede JA, and Gould MN. (2002). Monoterpenes reduced adducts formation in rats exposed to aflatoxin B1. African J Biotech, 1, 46–49.<br />
<br />
Gregory 3rd JF, Goldstein SL, Edds GT. (1983). Metabolite distribution and rate of residue clearance in turkeys fed a diet containing aflatoxin B1. Food Chem Toxicol, 21, 463–7.<br />
<br />
Groopman JD, Kensler TW (2005). Role of metabolism and viruses in aflatoxin-induced liver cancer. Toxicol Appl Pharmacol 206:131-137.<br />
<br />
Guengerich FP, Johnson WW, Ueng Y-F, Yamazaki H, Shimada T (1996). Involvement of Cytochrome P450, glutathione S-transferase, and epoxide hydrolase in the metabolism of aflatoxin B1 and relevance to risk of human liver cancer. Environ Health Perspect 104(Suppl 3):557-562.<br />
<br />
Himmelstein MW, Boogaard PJ, Cadet J, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: II.Overview of methods of identification and quantitation of DNA damage. Crit Rev Toxicol, 39, 679–694.<br />
<br />
IARC (1993). Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. IARC Monographs on the Evaluation of Carcinogenic Risk to Humans. Vol. 56, 245-395.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, Sutter TR, Groopman JD, Kensler TW, Roebuck BD. (2014). Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold. Cancer Prev Res. 7(7):658-665.<br />
<br />
Kensler TW, He X, Otieno M, et al. (1998). Oltipraz chemoprevention trial in Qidong, People’s Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol Biomarkers Prev, 7, 127–34.<br />
<br />
Larsson P, Hoedaya, WI, Tjalve H. (1990). Disposition of 3H-aflatoxin H in mice: formation and retention of tissue bound metabolites in nasal glands. Pharmacol Toxicol, 67, 162–71.<br />
<br />
Larsson P, and Tjalve H. (1993). Distribution and metabolism of aflatoxin B1 in the marmoset monkey (Callithrix jacchus). Carcinogenesis, 14, 1–6.<br />
<br />
Monroe DH, Eaton DL. (1987). Comparative effects of butylated hydroxyanisole on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B1 in the rat and the mouse. Toxicol Appl Pharmacol, 90, 401–409.<br />
<br />
Pottenger, L.H., Andrews LS, Bachman AN, Boogaard PJ, Cadet J, Embry MR, Farmer PB, Himmelstein MW, Jarabek AM, Martin EA, Mauthe RJ, Persaud R, Preston RJ, Schoeny R, Skare J, Swenberg JA, Williams GM, Zeiger E, Zhang F, Kim JH. (2014). An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride. Crit. Rev. Toxicol. 44(4):348-391.<br />
<br />
Primiano T, Egner PA, Sutter TR, et al. (1995). Intermittent dosing with oltipraz: relationship between chemoprevention of aflatoxin-induced tumorigenesis and induction of glutathione-S-transferases. Cancer Res, 55, 4319–4324.<br />
<br />
Roebuck BD, Liu Y-L, Rogers AE, et al. (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short term molecular dosimetry. Cancer Res, 51, 5501–5506.<br />
<br />
Ueng Y-F, Shimada T, Yamazaki H, Guengerich FP (1995). Oxidation of aflatoxin B1 by bacterial recombinant human cytochrome P450 enzymes. Chem Res Toxiol 8:218-225.<br />
<br />
Wang J-S, Shen X, He X, et al. (1999). Protective alerations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qidong, People’s Republic of China. J Natl Cancer Inst, 91, 347–354.<br />
<br />
Yates MS, Kwak M-K, Egner PA, et al. (2006). Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl] imidazole. Cancer Res, 66, 2488–2494.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:409Event:4092016-07-06T17:03:54Z<p>Dvillene: /* How this Key Event works */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id ='longTitle' class='Title'> Step: #0: pre-MIE </div><br />
<div id ='longTitle' class='Title'> Action: Metabolism of AFB1 </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||KE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Molecular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Biological Process: Production of Reactive Electrophiles ==<br />
Aflatoxin B1 (AFB1) can be oxidized to a variety of metabolites, many of which can be further metabolized, for example by conjugation with glutathione (GSH) via Glutathione-S-transferase (GST). In order for DNA binding and formation of a pro-mutagenic DNA adduct to occur, AFB1 must be metabolized via Cytochrome P450 metabolism to a specific highly reactive form of AFB1, the exo-epoxide AFB1-8,9-epoxide. CYP3A4 forms the exo-form of this reactive epoxide only. CYP1A2, inducible in liver, forms both the exo- and the endo-epoxides; in human liver, CYP1A2 metabolism occurs with a lower Vmax (enzymatic rate) and higher Km (half-maximal concentrations) than CYP3A4 (Degen and Neumann,1981; Groopman and Kensler, 2005; Guengerich et al., 1996; Ueng et al., 1995). Figure 1, from Pottenger et al., 2014, depicts the metabolism of AFB1.<br />
<br />
Metabolic activation of AFB1 occurs mainly in the liver, where the highest levels of the CYP isozymes are located. Competitive metabolism with other hepatic and extra-hepatic P450 isozymes may decrease the proportion of the specific metabolite, AFB1-8,9-epoxide; this can reduce the effectiveness of hepatic activation of AFB1. Alternatively, induction of either hepatic or extra-hepatic GST activity can reduce the levels of AFB1-epoxide available for reaction with DNA by increasing conjugation of the epoxide with GSH. This conjugation renders the epoxide inactive and directs it towards eventual elimination (Guengerich et al., 1996).[[File:Figure1met.jpg|thumb|alt=Figure 1 alt text|Figure 1. Metabolism of AFB1,]] Figure 1 was obtained from Pottenger et al., 2014.<br />
<br />
The AFB1 pro-mutagenic DNA adduct is most likely the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct, or N7-AFB1-G (Croy et al., 1978). Once the exo-epoxide is bound to the N7-guanine, it is subject to ring-opening to form the more highly pro-mutagenic 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-pyrimid-9-yl-foramido)-9-hydroxyaflatoxin B1; this is known as the formamidopyrimidine adduct or AFB1-FAPy adduct (Brown et al., 2006).<br />
<br />
== Taxonomic Applicability ==<br />
The metabolic information above is applicable to all mammalian systems evaluated; it is also applicable to certain birds such as turkeys (Gregory et al., 1983; IARC, 1993). Humans, non-human primates, rats, mice, poultry and fish have all demonstrated susceptibility to AFB1-induced liver tumors (Asplin and Canaghan, 1961; Eaton and Gallagher, 1994). Species that preferentially metabolize AFB1 to the exo-8,9-epoxide are more susceptible to AFB1 carcinogenicity. Sulfation by GST enzymes or other enzymatic detoxification of AFB1 metabolites can reduce available levels of the exo-epoxide, and subsequent steps or even the progression of Key Events may not occur in these species. Mice are less susceptible to AFB1-induced cancer than rats, likely due to increased detoxification (Monroe and Eaton, 1987). This increased detoxication capability can also be instigated by dietary exposures to compounds that modulate CYP450 expression or modify detoxication activities (Elegbede and Gould, 2002; Primiano et al., 1995; Roebuck et al. 1991, 2003; Kensler et al., 1998; Wang et al., 1999; Yates et al., 2006).<br />
<br />
== Level of Biological Organization : Molecular ==<br />
Metabolic activation of AFB1 occurs at the intracellular level, with sub-cellular organelles involved due to the localization of CYP450 in the endoplasmic reticulum, coupled with the epoxide hydratase; GST is localized in the cytosol (Guengerich et al., 1996). Organ architecture also plays a role, as the structure of the liver is orientated around the O2 gradient set by the portal artery and hepatic vein; hepatocytes nearest the portal artery demonstrate increased tissue oxygen while those around the portal vein have decreased tissue O2 and correspondingly higher levels of CYP450 activity.<br />
<br />
== How this Key Event works ==<br />
The reactive exo-epoxide is formed in hepatocytes (or extra-hepatically) by metabolism of the parent AFB1 by CYP450 (Larsson et al., 1990; Larsson and Tjalve, 1993). The reactive metabolite then escapes the endoplasmic reticulum where the CYP450 is located. The reactive metabolite must evade conjugation with GSH in the cytoplasm or binding with other cytoplasmic nucleophiles. It then traverses the nuclear membrane in order to reach the cell nucleus and the nuclear DNA. Once the reactive metabolite is in the cell nucleus, binding to nuclear DNA and the formation of DNA adducts can occur.<br />
<br />
== Pre-MIE: How it is Measured or Detected ==<br />
Formation of the exo-epoxide can be produced with in vitro systems and detected using techniques for structural quantitation of AFB1 metabolites (Himmelstein et al., 2009), including liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). By using subcellular fractions (e.g., microsomes), cellular homogenates, or cells in culture, it is possible to measure formation of AFB1 exo-epoxide. Such data can also be collected from in vivo systems; samples of plasma or blood from AFB1-treated animals can be analyzed for the AFB1 exo-epoxide with similar mass spectrometric based detection systems (e.g., LC-MS/MS). Samples of blood from humans in AFB1-endemic regions have demonstrated presence of AFB1-albumin adducts, which are formed from the AFB1 exo-epoxide. AFB1-treated animals may also provide tissue samples for analysis of AFB1 exo-epoxide. Special trapping techniques may be required as the reactive AFB1 exo-epoxide metabolite has a short half-life in biological matrices.<br />
<br />
== Pre-MIE: Evidence Supporting Taxonomic Applicability ==<br />
Ample data across phyla demonstrate metabolic activation of AFB1 to the exo-epoxide via CYP450. These taxons include several mammalian species (humans, non-human primates, rats, mice) in addition to birds (turkeys) and fish (Eaton and Gallagher, 1994; IARC, 1993).<br />
<br />
== Pre-MIE: Evidence for Chemical Initiation of this Initial Molecular Event/Pre-MIE ==<br />
There is an extensive database on AFB1 in many different systems demonstrating formation of the AFB1 exo-epoxide. This database includes several mammalian species (humans, non-human primates, rats, mice) in addition to birds (turkeys) and fish (Eaton and Gallagher, 1994; IARC, 1993).<br />
<br />
== Evidence Supporting Essentiality of Pre-MIE (Step #0) ==<br />
Strong<br />
<br />
Many studies show that in the absence of AFB1 metabolic activation, tumors do not occur. Treatment of rats with the CYP450-inhibitor and GST-inducer oltipraz reduces the levels of AFB1 DNA adducts by 65-70%, and the later-forming altered hepatic foci (AHF) and liver tumors are also reduced by 97 and 100%, respectively; thus, no hepatocellular carcinomas (HCC) form in the oltipraz-treated rats dosed with AFB1 (Roebuck et al., 1991). More recent data show even more effective prevention of tumors and of hepatic foci in rats pre-treated with a triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) (Johnson et al., 2014). The pre-treated rats were reported with a 70% reduction of AFB1-induced adducts in urine but had 100% inhibition of tumors, and only 1 rat out of 23 pre-treated had any altered hepatic foci (AHF). There are similar data in humans treated with oltipraz. Kensler et al. (1998) report a significant reduction in a biomarker of exposure AFB1-induced albumin adducts, supporting the human relevance of this metabolic activation <br />
<br />
Another line of evidence for essentiality of the pre-MIE is the recognized difference in sensitivity to AFB1-induced liver tumors between mice and rats (Degen and Neumann, 1981). Mice, have considerably increased metabolic activation of AFB1 to the exo-epoxide compared to rats; mice are nonetheless much less sensitive to AFB1-induced liver tumors. It is thought that this difference is due to the constitutive presence of GST-alpha activity in mice vs rats, where this activity is not found.<br />
<br />
== Pre-MIE: References ==<br />
F.D. Asplin, R.B.A. Carnaghan, (1961). The toxicity of certain groundnut meals for poultry with special reference to their effect on ducklings and chickens. Vet. Rec. 73:1215– 1219. <br />
<br />
Brown KL, Deng JZ, Iyer RS, Iyer LG, Voehler MW, Stone MP, Harris CM, Harris TM (2006). Unraveling the aflatoxin-FAPY conundrum: Structural basis of the formamidopyrimidine-type DNA adduct of aflatoxin B1. J Am Chem Soc 128:15188-15199.<br />
<br />
Croy RG, Essigman JM, Reinhold VN, Wogan GN (1978). Identification of the principal aflatoxin N1-DNA adduct formed in vivo in rat liver. Proc Natl Acad Sci USA 75:1745-1749.<br />
<br />
Degen GH, Neumann HG (1981). Differences in aflatoxin B1-susceptibility of rat and mouse are correlated with the capability in vitro to inactivate aflatoxin B1-epoxide. Carcinogenesis 2:299–306.<br />
<br />
Eaton DL, and Gallagher EP (1994). Mechanisms of aflatoxin carcinogenesis. Annu Rev Pharmacol Toxicol 34:135-172.<br />
<br />
Elegbede JA, and Gould MN. (2002). Monoterpenes reduced adducts formation in rats exposed to aflatoxin B1. African J Biotech, 1, 46–49.<br />
<br />
Gregory 3rd JF, Goldstein SL, Edds GT. (1983). Metabolite distribution and rate of residue clearance in turkeys fed a diet containing aflatoxin B1. Food Chem Toxicol, 21, 463–7.<br />
<br />
Groopman JD, Kensler TW (2005). Role of metabolism and viruses in aflatoxin-induced liver cancer. Toxicol Appl Pharmacol 206:131-137.<br />
<br />
Guengerich FP, Johnson WW, Ueng Y-F, Yamazaki H, Shimada T (1996). Involvement of Cytochrome P450, glutathione S-transferase, and epoxide hydrolase in the metabolism of aflatoxin B1 and relevance to risk of human liver cancer. Environ Health Perspect 104(Suppl 3):557-562.<br />
<br />
Himmelstein MW, Boogaard PJ, Cadet J, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: II.Overview of methods of identification and quantitation of DNA damage. Crit Rev Toxicol, 39, 679–694.<br />
<br />
IARC (1993). Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. IARC Monographs on the Evaluation of Carcinogenic Risk to Humans. Vol. 56, 245-395.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, Sutter TR, Groopman JD, Kensler TW, Roebuck BD. (2014). Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold. Cancer Prev Res. 7(7):658-665.<br />
<br />
Kensler TW, He X, Otieno M, et al. (1998). Oltipraz chemoprevention trial in Qidong, People’s Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol Biomarkers Prev, 7, 127–34.<br />
<br />
Larsson P, Hoedaya, WI, Tjalve H. (1990). Disposition of 3H-aflatoxin H in mice: formation and retention of tissue bound metabolites in nasal glands. Pharmacol Toxicol, 67, 162–71.<br />
<br />
Larsson P, and Tjalve H. (1993). Distribution and metabolism of aflatoxin B1 in the marmoset monkey (Callithrix jacchus). Carcinogenesis, 14, 1–6.<br />
<br />
Monroe DH, Eaton DL. (1987). Comparative effects of butylated hydroxyanisole on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B1 in the rat and the mouse. Toxicol Appl Pharmacol, 90, 401–409.<br />
<br />
Pottenger, L.H., Andrews LS, Bachman AN, Boogaard PJ, Cadet J, Embry MR, Farmer PB, Himmelstein MW, Jarabek AM, Martin EA, Mauthe RJ, Persaud R, Preston RJ, Schoeny R, Skare J, Swenberg JA, Williams GM, Zeiger E, Zhang F, Kim JH. (2014). An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride. Crit. Rev. Toxicol. 44(4):348-391.<br />
<br />
Primiano T, Egner PA, Sutter TR, et al. (1995). Intermittent dosing with oltipraz: relationship between chemoprevention of aflatoxin-induced tumorigenesis and induction of glutathione-S-transferases. Cancer Res, 55, 4319–4324.<br />
<br />
Roebuck BD, Liu Y-L, Rogers AE, et al. (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short term molecular dosimetry. Cancer Res, 51, 5501–5506.<br />
<br />
Ueng Y-F, Shimada T, Yamazaki H, Guengerich FP (1995). Oxidation of aflatoxin B1 by bacterial recombinant human cytochrome P450 enzymes. Chem Res Toxiol 8:218-225.<br />
<br />
Wang J-S, Shen X, He X, et al. (1999). Protective alerations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qidong, People’s Republic of China. J Natl Cancer Inst, 91, 347–354.<br />
<br />
Yates MS, Kwak M-K, Egner PA, et al. (2006). Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl] imidazole. Cancer Res, 66, 2488–2494.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:376Event:3762016-07-06T17:03:53Z<p>Dvillene: /* KE#2: How this Key Event works */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Induced Mutations in Critical Genes, Increased </div><br />
<div id='shortTitle' class='Title'> Induced in Critical Genes, Mutation </div><br />
<br />
== Action ==<br />
Induced mutation<br />
<br />
== Biological Process/Object ==<br />
Critical genes for carcinogenesis<br />
<br />
== Induced Mutation in Critical Genes ==<br />
The AFB1-induced pro-mutagenic DNA adduct is either not repaired or is mis-repaired resulting in a mutation in one or more critical genes. In bacteria and mammalian cells (both in vitro and in vivo) the primary mutation associated with AFB1 is a guanine to thymine transversion (Foster et al., 1983; Dycaico et al., 1996). A G:T transversion is expected for the pro-mutagenic DNA adduct AFB1-FAPy. <br />
<br />
A specific critical mutation in codon 249 of the p53 gene has been identified in human hepatocellular carcinoma (HCC) (See section below on essentiality).<br />
<br />
== Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
A specific critical mutation in the p53 gene has been identified in human hepatocellular carcinoma (HCC). Demonstrating that AFB1 can induce this specific p53 gene mutation would be the highest level of evidence that AFB1-induced HCC involves mutation as a KE. Absent such information, the next best level of evidence is the induction of the specific type of mutation (G:C to T:A transversion) in a variety of gene mutation assays measuring a range of target genes. <br />
<br />
The codon 249 mutation is present in a significant proportion of human HCCs. In fact codon 249 mutation is detected in up to 50% of liver cancers in Qidong, China (Hsu et al., 1991) and in Mozambique, both areas with high likelihood of AFB1 exposure. The codon 249 G:C to T:A mutation in the third base is seen in up to 75% of HCC in high-incidence areas of China and East Africa (Gouas et al., 2009). In contrast, this specific mutation is very rare in HCC from areas with no or low exposure to AFB1 (Hsu et al. 1991 and Bressac et al. Nature, 350:429-431, 1991). This mutation is also very rare in other types of tumors (Gouas et al., 2009). According to Gouas et al. (2009), populations with AFB1 exposure are likely to be exposed to hepatitis B virus (HBV) as well and the effects of each are difficult to separate.This mutation is very rare in HCC from non/low -aflatoxin areas (Hsu et al. 1991 and Bressac et al., 1991) and also very rare in other types of tumors.<br />
<br />
Some indirect evidence of the essentiality of mutation in tumor development is provided by the clear species difference between adult mice and adult rats both in the induction of surrogate gene mutations and in the induction of tumors. Adult mice exposed to AFB1 do not get tumors and there is no increase in mutant frequency (MF) for Big Blue mice exposed as adults. That is, Lac I mutants from the AFB1- exposed adult mice showed a spontaneous mutational spectrum. Rats, however, showed a large increase in MF and, more specifically a large increase in G:C to T:A transversions (Dycaico et al., 1996). In addition, for mice there is an difference between neonata and adult mice. Neonatal mice treated with AFB1 (6 mg/kg—a dose that does result in tumors) showed an increase in cII mutation with G:C to T:A transversion as the major mutation. Adult mice treated at 6 and 60 mg/kg (doses that do not produce tumors) did not have a significant increase in cII mutation (but did give a different mutational spectrum than controls) (Chen et al. 2010).<br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||KE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Cellular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Taxonomic Applicability ==<br />
The induction of G:C to T:A transversions following AFB1 exposure is seen in a wide variety of species from bacteria to mammals. Assays that measure mutation in surrogate genes (that is, genes unrelated to the critical cancer genes) can be used to evaluate the full spectrum of mutational events that can be induced by a chemical. One such system is the AS52 assay, an in vitro mammalian transgenic mutation assay that measures mutation in the gpt gene. After exposure to AFB1 in culture the predominant mutation is G:C to T:A transversion, although a number of other types of mutations were also seen (Wattanawaraporn et al., 2012). There is a species difference between adult mice and rats. No increase in MF is seen in the in vivo Big Blue™ mutation assay for mice exposed to AFB1. That is, Lac I mutations from the mice showed a spontaneous mutational spectrum. By contrast rats showed a large increase in MF with a large increase in G:C to T:A transversions (Dycaico et al., 1996). However, neonatal mice treated with AFB1 (6 mg/kg—a dose that induces tumors in neonates) do show an increase in cII mutation with G:C to T:A transversion as the major mutation. Adult mice treated at 6 and 60 mg/kg (doses that do not induce tumors) did not show a significant increase in cII mutations but did produce a different mutational spectrum than controls (Chen et al., 2010.)<br />
<br />
== Level of Biological Organization : Cellular ==<br />
The induction of mutation occurs within the nucleus of cells and involves permanent alterations in the primary DNA sequence that is passed to subsequent cell generations and, thus, is heritable.<br />
<br />
== How this Key Event works ==<br />
Following the formation of DNA adducts, which are either mis-repaired or not repaired, the sequence of base pairs in the DNA is changed due to insertion of an incorrect base opposite the DNA adduct during DNA replication, so that a G is permanently replaced by a T. This type of mutation is observed in the mutants seen in surrogate gene mutation assays using AFB1 exposureA high frequency of codon 249 p53 mutations occurs in human tumors in high AFB1 exposure regions.Hence, codon 249 of the p53 gene contains a base pair susceptible to insufficient or misrepair of DNA adducts.<br />
<br />
Puisieux et al. (1991) provide evidence that the AFB1 epoxide adduct binds preferentially to codon 249 of the p53 gene. Using a plasmid with containing full-length human p53 DNA, adduct formation was observed in exons 5, 6, 7 and 8 (a total of 1086 bases) and 20% of the bases were targeted by AFB1 with a preference for guanine residues. Binding of AFB1 to p53 sequences was restricted to fewer residues and was more specific for guanine than was the binding of B[a]P (Puisieux et al.,1991). Binding of AFB1 in the region around codon 249 of p53 AFB1 was reported to be “stronger” than that of B[a]P. The last nucleotide of codon 249 is a guanine and was targeted by AFB1 but not by B[a]P. This guanine residue is the mutational hotspot in human liver cancers from patients in high AFB1 exposure regions. <br />
<br />
While mutations at codon 249 of the p53 gene have been observed in association with HCC in humans, the question remains whether this mutation occurs as a direct result of adduct formation at this site or by some different mechanism..In human HepG2 hepatocytes exposed to microsomally activated AFB1, a dose-dependent increase in G:C to T:A transversions were observed at 10 additional locations using ligation-mediated PCR, and at 4 additional locations using terminal-transferred dependent PCR.(Denissenko et al.,1998). These authors suggest that codon 249 may not present a key adduct site. However, other more recently published data identified codon 249 of the P53 gene as an unusually mutagenic adduct conformation based on the local DNA sequence and concluded that a higher mutation rate may occur there rather than at other locations because of increased DNA polymerase bypass (Pussieux et al., 1991; Lin et al., 2014a,b).<br />
<br />
== KE#2: How it is Measured or Detected ==<br />
Historically the detection of critical cancer gene specific mutations has not been technically feasible. A newly developed method, allele specific competitive blocker-polymerase chain reaction (ACB-PCR) has proven useful in providing such information, and data on specific chemical-induced mutations are available for a very small number of chemicals (Parsons et al., 2010). Unfortunately, there are no data for AFB1.<br />
<br />
There are, however, a number of gene mutation assays that have been widely used for determining the general ability of chemicals, including AFB1 to induce mutations.These assays use selection methods that allow only mutant cells to survive and grow. The AS52 in vitro gene mutation assay using the gpt gene, and 6-thioguanine selection has been used to demonstrate that AFB1 exposure increases the MF at the gpt gene. In vivo transgenic assays use molecular methods to recover the transgene from isolated DNA and to evaluate the MF in the transgene. Molecular methods can detect the presence (above a certain sensitivity level) of mutant cells. DNA from tumors can be sequenced to determine the presence of mutations in specific genes. DNA sequencing has been used on human tumors to detect the presence of the Codon 249 p53 mutation.<br />
<br />
== KE#2: Evidence Supporting Taxonomic Applicability ==<br />
There are data across phyla demonstrating the induction of mutations, specifically the induction to G:C to T:A transversions following AFB1 exposure.<br />
<br />
== KE#2: References ==<br />
Bressac, B., Kew, M., Wands, J., & Ozturk, M. (1991). Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern africa. Nature, 350(6317), 429-31. doi:10.1038/350429a<br />
<br />
Chen, T., Heflich, R. H., Moore, M. M., & Mei, N. (2010). Differential mutagenicity of aflatoxin B1 in the liver of neonatal and adult mice. Environ Mol Mutagen, 51(2), 156-63. doi:10.1002/em.2051<br />
<br />
Denissenko MF, Koudriakova TB, Smith L, O'Connor TR, Riggs AD, and Pfeifer GP). The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts. (Oncogene. 1998, Dec 10;17(23):3007-14.<br />
<br />
Dycaico, M. J., Stuart, G. R., Tobal, G. M., de Boer, J. G., Glickman, B. W., & Provost, G. S. (1996). Species-specific differences in hepatic mutant frequency and mutational spectrum among lambda/laci transgenic rats and mice following exposure to aflatoxin B1. Carcinogenesis, 17(11), 2347-56<br />
<br />
Foster, P. L., Eisenstadt, E., & Miller, J. H. (1983). Base substitution mutations induced by metabolically activated aflatoxin B1. Proceedings of the National Academy of Sciences of the United States of America, 80(9), 2695-8.<br />
<br />
Gouas, D., Shi, H., & Hainaut, P. (2009). The aflatoxin-induced TP53 mutation at codon 249 (R249S): Biomarker of exposure, early detection and target for therapy. Cancer Lett, 286(1), 29-37. doi:10.1016/j.canlet.2009.02.057<br />
<br />
Hsu, I. C., Metcalf, R. A., Sun, T., Welsh, J. A., Wang, N. J., & Harris, C. C. (1991). Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature, 350(6317), 427-8. doi:10.1038/350427a<br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014a). Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua. J Biol Chem. 289:18497-18506. <br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014b). Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis 35(7):1461-1468<br />
<br />
Parsons BL, Myers MB, Meng F, Wang Y, McKinzie PB. 2010. Oncomutations as biomarkers of cancer risk. Environ Mol Mutagen. 51(8-9):836-850.<br />
<br />
Puisieux, A., Lim, S., Groopman, J., & Ozturk, M. (1991). Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res, 51(22), 6185-9.<br />
<br />
Wattanawaraporn, R., Kim, M. Y., Adams, J., Trudel, L. J., Woo, L. L., Croy, R. G., . . . Wogan, G. N. (2012). AFB(1) -induced mutagenesis of the gpt gene in AS52 cells. Environ Mol Mutagen, 53(7), 567-73. doi:10.1002/em.2171</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:376Event:3762016-07-06T17:03:36Z<p>Dvillene: /* KE#2: Level of Biological Organization : Cellular */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Induced Mutations in Critical Genes, Increased </div><br />
<div id='shortTitle' class='Title'> Induced in Critical Genes, Mutation </div><br />
<br />
== Action ==<br />
Induced mutation<br />
<br />
== Biological Process/Object ==<br />
Critical genes for carcinogenesis<br />
<br />
== Induced Mutation in Critical Genes ==<br />
The AFB1-induced pro-mutagenic DNA adduct is either not repaired or is mis-repaired resulting in a mutation in one or more critical genes. In bacteria and mammalian cells (both in vitro and in vivo) the primary mutation associated with AFB1 is a guanine to thymine transversion (Foster et al., 1983; Dycaico et al., 1996). A G:T transversion is expected for the pro-mutagenic DNA adduct AFB1-FAPy. <br />
<br />
A specific critical mutation in codon 249 of the p53 gene has been identified in human hepatocellular carcinoma (HCC) (See section below on essentiality).<br />
<br />
== Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
A specific critical mutation in the p53 gene has been identified in human hepatocellular carcinoma (HCC). Demonstrating that AFB1 can induce this specific p53 gene mutation would be the highest level of evidence that AFB1-induced HCC involves mutation as a KE. Absent such information, the next best level of evidence is the induction of the specific type of mutation (G:C to T:A transversion) in a variety of gene mutation assays measuring a range of target genes. <br />
<br />
The codon 249 mutation is present in a significant proportion of human HCCs. In fact codon 249 mutation is detected in up to 50% of liver cancers in Qidong, China (Hsu et al., 1991) and in Mozambique, both areas with high likelihood of AFB1 exposure. The codon 249 G:C to T:A mutation in the third base is seen in up to 75% of HCC in high-incidence areas of China and East Africa (Gouas et al., 2009). In contrast, this specific mutation is very rare in HCC from areas with no or low exposure to AFB1 (Hsu et al. 1991 and Bressac et al. Nature, 350:429-431, 1991). This mutation is also very rare in other types of tumors (Gouas et al., 2009). According to Gouas et al. (2009), populations with AFB1 exposure are likely to be exposed to hepatitis B virus (HBV) as well and the effects of each are difficult to separate.This mutation is very rare in HCC from non/low -aflatoxin areas (Hsu et al. 1991 and Bressac et al., 1991) and also very rare in other types of tumors.<br />
<br />
Some indirect evidence of the essentiality of mutation in tumor development is provided by the clear species difference between adult mice and adult rats both in the induction of surrogate gene mutations and in the induction of tumors. Adult mice exposed to AFB1 do not get tumors and there is no increase in mutant frequency (MF) for Big Blue mice exposed as adults. That is, Lac I mutants from the AFB1- exposed adult mice showed a spontaneous mutational spectrum. Rats, however, showed a large increase in MF and, more specifically a large increase in G:C to T:A transversions (Dycaico et al., 1996). In addition, for mice there is an difference between neonata and adult mice. Neonatal mice treated with AFB1 (6 mg/kg—a dose that does result in tumors) showed an increase in cII mutation with G:C to T:A transversion as the major mutation. Adult mice treated at 6 and 60 mg/kg (doses that do not produce tumors) did not have a significant increase in cII mutation (but did give a different mutational spectrum than controls) (Chen et al. 2010).<br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||KE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Cellular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Taxonomic Applicability ==<br />
The induction of G:C to T:A transversions following AFB1 exposure is seen in a wide variety of species from bacteria to mammals. Assays that measure mutation in surrogate genes (that is, genes unrelated to the critical cancer genes) can be used to evaluate the full spectrum of mutational events that can be induced by a chemical. One such system is the AS52 assay, an in vitro mammalian transgenic mutation assay that measures mutation in the gpt gene. After exposure to AFB1 in culture the predominant mutation is G:C to T:A transversion, although a number of other types of mutations were also seen (Wattanawaraporn et al., 2012). There is a species difference between adult mice and rats. No increase in MF is seen in the in vivo Big Blue™ mutation assay for mice exposed to AFB1. That is, Lac I mutations from the mice showed a spontaneous mutational spectrum. By contrast rats showed a large increase in MF with a large increase in G:C to T:A transversions (Dycaico et al., 1996). However, neonatal mice treated with AFB1 (6 mg/kg—a dose that induces tumors in neonates) do show an increase in cII mutation with G:C to T:A transversion as the major mutation. Adult mice treated at 6 and 60 mg/kg (doses that do not induce tumors) did not show a significant increase in cII mutations but did produce a different mutational spectrum than controls (Chen et al., 2010.)<br />
<br />
== Level of Biological Organization : Cellular ==<br />
The induction of mutation occurs within the nucleus of cells and involves permanent alterations in the primary DNA sequence that is passed to subsequent cell generations and, thus, is heritable.<br />
<br />
== KE#2: How this Key Event works ==<br />
Following the formation of DNA adducts, which are either mis-repaired or not repaired, the sequence of base pairs in the DNA is changed due to insertion of an incorrect base opposite the DNA adduct during DNA replication, so that a G is permanently replaced by a T. This type of mutation is observed in the mutants seen in surrogate gene mutation assays using AFB1 exposureA high frequency of codon 249 p53 mutations occurs in human tumors in high AFB1 exposure regions.Hence, codon 249 of the p53 gene contains a base pair susceptible to insufficient or misrepair of DNA adducts.<br />
<br />
Puisieux et al. (1991) provide evidence that the AFB1 epoxide adduct binds preferentially to codon 249 of the p53 gene. Using a plasmid with containing full-length human p53 DNA, adduct formation was observed in exons 5, 6, 7 and 8 (a total of 1086 bases) and 20% of the bases were targeted by AFB1 with a preference for guanine residues. Binding of AFB1 to p53 sequences was restricted to fewer residues and was more specific for guanine than was the binding of B[a]P (Puisieux et al.,1991). Binding of AFB1 in the region around codon 249 of p53 AFB1 was reported to be “stronger” than that of B[a]P. The last nucleotide of codon 249 is a guanine and was targeted by AFB1 but not by B[a]P. This guanine residue is the mutational hotspot in human liver cancers from patients in high AFB1 exposure regions. <br />
<br />
While mutations at codon 249 of the p53 gene have been observed in association with HCC in humans, the question remains whether this mutation occurs as a direct result of adduct formation at this site or by some different mechanism..In human HepG2 hepatocytes exposed to microsomally activated AFB1, a dose-dependent increase in G:C to T:A transversions were observed at 10 additional locations using ligation-mediated PCR, and at 4 additional locations using terminal-transferred dependent PCR.(Denissenko et al.,1998). These authors suggest that codon 249 may not present a key adduct site. However, other more recently published data identified codon 249 of the P53 gene as an unusually mutagenic adduct conformation based on the local DNA sequence and concluded that a higher mutation rate may occur there rather than at other locations because of increased DNA polymerase bypass (Pussieux et al., 1991; Lin et al., 2014a,b).<br />
<br />
== KE#2: How it is Measured or Detected ==<br />
Historically the detection of critical cancer gene specific mutations has not been technically feasible. A newly developed method, allele specific competitive blocker-polymerase chain reaction (ACB-PCR) has proven useful in providing such information, and data on specific chemical-induced mutations are available for a very small number of chemicals (Parsons et al., 2010). Unfortunately, there are no data for AFB1.<br />
<br />
There are, however, a number of gene mutation assays that have been widely used for determining the general ability of chemicals, including AFB1 to induce mutations.These assays use selection methods that allow only mutant cells to survive and grow. The AS52 in vitro gene mutation assay using the gpt gene, and 6-thioguanine selection has been used to demonstrate that AFB1 exposure increases the MF at the gpt gene. In vivo transgenic assays use molecular methods to recover the transgene from isolated DNA and to evaluate the MF in the transgene. Molecular methods can detect the presence (above a certain sensitivity level) of mutant cells. DNA from tumors can be sequenced to determine the presence of mutations in specific genes. DNA sequencing has been used on human tumors to detect the presence of the Codon 249 p53 mutation.<br />
<br />
== KE#2: Evidence Supporting Taxonomic Applicability ==<br />
There are data across phyla demonstrating the induction of mutations, specifically the induction to G:C to T:A transversions following AFB1 exposure.<br />
<br />
== KE#2: References ==<br />
Bressac, B., Kew, M., Wands, J., & Ozturk, M. (1991). Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern africa. Nature, 350(6317), 429-31. doi:10.1038/350429a<br />
<br />
Chen, T., Heflich, R. H., Moore, M. M., & Mei, N. (2010). Differential mutagenicity of aflatoxin B1 in the liver of neonatal and adult mice. Environ Mol Mutagen, 51(2), 156-63. doi:10.1002/em.2051<br />
<br />
Denissenko MF, Koudriakova TB, Smith L, O'Connor TR, Riggs AD, and Pfeifer GP). The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts. (Oncogene. 1998, Dec 10;17(23):3007-14.<br />
<br />
Dycaico, M. J., Stuart, G. R., Tobal, G. M., de Boer, J. G., Glickman, B. W., & Provost, G. S. (1996). Species-specific differences in hepatic mutant frequency and mutational spectrum among lambda/laci transgenic rats and mice following exposure to aflatoxin B1. Carcinogenesis, 17(11), 2347-56<br />
<br />
Foster, P. L., Eisenstadt, E., & Miller, J. H. (1983). Base substitution mutations induced by metabolically activated aflatoxin B1. Proceedings of the National Academy of Sciences of the United States of America, 80(9), 2695-8.<br />
<br />
Gouas, D., Shi, H., & Hainaut, P. (2009). The aflatoxin-induced TP53 mutation at codon 249 (R249S): Biomarker of exposure, early detection and target for therapy. Cancer Lett, 286(1), 29-37. doi:10.1016/j.canlet.2009.02.057<br />
<br />
Hsu, I. C., Metcalf, R. A., Sun, T., Welsh, J. A., Wang, N. J., & Harris, C. C. (1991). Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature, 350(6317), 427-8. doi:10.1038/350427a<br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014a). Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua. J Biol Chem. 289:18497-18506. <br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014b). Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis 35(7):1461-1468<br />
<br />
Parsons BL, Myers MB, Meng F, Wang Y, McKinzie PB. 2010. Oncomutations as biomarkers of cancer risk. Environ Mol Mutagen. 51(8-9):836-850.<br />
<br />
Puisieux, A., Lim, S., Groopman, J., & Ozturk, M. (1991). Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res, 51(22), 6185-9.<br />
<br />
Wattanawaraporn, R., Kim, M. Y., Adams, J., Trudel, L. J., Woo, L. L., Croy, R. G., . . . Wogan, G. N. (2012). AFB(1) -induced mutagenesis of the gpt gene in AS52 cells. Environ Mol Mutagen, 53(7), 567-73. doi:10.1002/em.2171</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:409Event:4092016-07-06T17:03:34Z<p>Dvillene: /* Level of Biological Organization : Molecular */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id ='longTitle' class='Title'> Step: #0: pre-MIE </div><br />
<div id ='longTitle' class='Title'> Action: Metabolism of AFB1 </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||KE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Molecular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Biological Process: Production of Reactive Electrophiles ==<br />
Aflatoxin B1 (AFB1) can be oxidized to a variety of metabolites, many of which can be further metabolized, for example by conjugation with glutathione (GSH) via Glutathione-S-transferase (GST). In order for DNA binding and formation of a pro-mutagenic DNA adduct to occur, AFB1 must be metabolized via Cytochrome P450 metabolism to a specific highly reactive form of AFB1, the exo-epoxide AFB1-8,9-epoxide. CYP3A4 forms the exo-form of this reactive epoxide only. CYP1A2, inducible in liver, forms both the exo- and the endo-epoxides; in human liver, CYP1A2 metabolism occurs with a lower Vmax (enzymatic rate) and higher Km (half-maximal concentrations) than CYP3A4 (Degen and Neumann,1981; Groopman and Kensler, 2005; Guengerich et al., 1996; Ueng et al., 1995). Figure 1, from Pottenger et al., 2014, depicts the metabolism of AFB1.<br />
<br />
Metabolic activation of AFB1 occurs mainly in the liver, where the highest levels of the CYP isozymes are located. Competitive metabolism with other hepatic and extra-hepatic P450 isozymes may decrease the proportion of the specific metabolite, AFB1-8,9-epoxide; this can reduce the effectiveness of hepatic activation of AFB1. Alternatively, induction of either hepatic or extra-hepatic GST activity can reduce the levels of AFB1-epoxide available for reaction with DNA by increasing conjugation of the epoxide with GSH. This conjugation renders the epoxide inactive and directs it towards eventual elimination (Guengerich et al., 1996).[[File:Figure1met.jpg|thumb|alt=Figure 1 alt text|Figure 1. Metabolism of AFB1,]] Figure 1 was obtained from Pottenger et al., 2014.<br />
<br />
The AFB1 pro-mutagenic DNA adduct is most likely the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct, or N7-AFB1-G (Croy et al., 1978). Once the exo-epoxide is bound to the N7-guanine, it is subject to ring-opening to form the more highly pro-mutagenic 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-pyrimid-9-yl-foramido)-9-hydroxyaflatoxin B1; this is known as the formamidopyrimidine adduct or AFB1-FAPy adduct (Brown et al., 2006).<br />
<br />
== Taxonomic Applicability ==<br />
The metabolic information above is applicable to all mammalian systems evaluated; it is also applicable to certain birds such as turkeys (Gregory et al., 1983; IARC, 1993). Humans, non-human primates, rats, mice, poultry and fish have all demonstrated susceptibility to AFB1-induced liver tumors (Asplin and Canaghan, 1961; Eaton and Gallagher, 1994). Species that preferentially metabolize AFB1 to the exo-8,9-epoxide are more susceptible to AFB1 carcinogenicity. Sulfation by GST enzymes or other enzymatic detoxification of AFB1 metabolites can reduce available levels of the exo-epoxide, and subsequent steps or even the progression of Key Events may not occur in these species. Mice are less susceptible to AFB1-induced cancer than rats, likely due to increased detoxification (Monroe and Eaton, 1987). This increased detoxication capability can also be instigated by dietary exposures to compounds that modulate CYP450 expression or modify detoxication activities (Elegbede and Gould, 2002; Primiano et al., 1995; Roebuck et al. 1991, 2003; Kensler et al., 1998; Wang et al., 1999; Yates et al., 2006).<br />
<br />
== Level of Biological Organization : Molecular ==<br />
Metabolic activation of AFB1 occurs at the intracellular level, with sub-cellular organelles involved due to the localization of CYP450 in the endoplasmic reticulum, coupled with the epoxide hydratase; GST is localized in the cytosol (Guengerich et al., 1996). Organ architecture also plays a role, as the structure of the liver is orientated around the O2 gradient set by the portal artery and hepatic vein; hepatocytes nearest the portal artery demonstrate increased tissue oxygen while those around the portal vein have decreased tissue O2 and correspondingly higher levels of CYP450 activity.<br />
<br />
== Pre-MIE: How this Key Event works ==<br />
The reactive exo-epoxide is formed in hepatocytes (or extra-hepatically) by metabolism of the parent AFB1 by CYP450 (Larsson et al., 1990; Larsson and Tjalve, 1993). The reactive metabolite then escapes the endoplasmic reticulum where the CYP450 is located. The reactive metabolite must evade conjugation with GSH in the cytoplasm or binding with other cytoplasmic nucleophiles. It then traverses the nuclear membrane in order to reach the cell nucleus and the nuclear DNA. Once the reactive metabolite is in the cell nucleus, binding to nuclear DNA and the formation of DNA adducts can occur.<br />
<br />
== Pre-MIE: How it is Measured or Detected ==<br />
Formation of the exo-epoxide can be produced with in vitro systems and detected using techniques for structural quantitation of AFB1 metabolites (Himmelstein et al., 2009), including liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). By using subcellular fractions (e.g., microsomes), cellular homogenates, or cells in culture, it is possible to measure formation of AFB1 exo-epoxide. Such data can also be collected from in vivo systems; samples of plasma or blood from AFB1-treated animals can be analyzed for the AFB1 exo-epoxide with similar mass spectrometric based detection systems (e.g., LC-MS/MS). Samples of blood from humans in AFB1-endemic regions have demonstrated presence of AFB1-albumin adducts, which are formed from the AFB1 exo-epoxide. AFB1-treated animals may also provide tissue samples for analysis of AFB1 exo-epoxide. Special trapping techniques may be required as the reactive AFB1 exo-epoxide metabolite has a short half-life in biological matrices.<br />
<br />
== Pre-MIE: Evidence Supporting Taxonomic Applicability ==<br />
Ample data across phyla demonstrate metabolic activation of AFB1 to the exo-epoxide via CYP450. These taxons include several mammalian species (humans, non-human primates, rats, mice) in addition to birds (turkeys) and fish (Eaton and Gallagher, 1994; IARC, 1993).<br />
<br />
== Pre-MIE: Evidence for Chemical Initiation of this Initial Molecular Event/Pre-MIE ==<br />
There is an extensive database on AFB1 in many different systems demonstrating formation of the AFB1 exo-epoxide. This database includes several mammalian species (humans, non-human primates, rats, mice) in addition to birds (turkeys) and fish (Eaton and Gallagher, 1994; IARC, 1993).<br />
<br />
== Evidence Supporting Essentiality of Pre-MIE (Step #0) ==<br />
Strong<br />
<br />
Many studies show that in the absence of AFB1 metabolic activation, tumors do not occur. Treatment of rats with the CYP450-inhibitor and GST-inducer oltipraz reduces the levels of AFB1 DNA adducts by 65-70%, and the later-forming altered hepatic foci (AHF) and liver tumors are also reduced by 97 and 100%, respectively; thus, no hepatocellular carcinomas (HCC) form in the oltipraz-treated rats dosed with AFB1 (Roebuck et al., 1991). More recent data show even more effective prevention of tumors and of hepatic foci in rats pre-treated with a triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) (Johnson et al., 2014). The pre-treated rats were reported with a 70% reduction of AFB1-induced adducts in urine but had 100% inhibition of tumors, and only 1 rat out of 23 pre-treated had any altered hepatic foci (AHF). There are similar data in humans treated with oltipraz. Kensler et al. (1998) report a significant reduction in a biomarker of exposure AFB1-induced albumin adducts, supporting the human relevance of this metabolic activation <br />
<br />
Another line of evidence for essentiality of the pre-MIE is the recognized difference in sensitivity to AFB1-induced liver tumors between mice and rats (Degen and Neumann, 1981). Mice, have considerably increased metabolic activation of AFB1 to the exo-epoxide compared to rats; mice are nonetheless much less sensitive to AFB1-induced liver tumors. It is thought that this difference is due to the constitutive presence of GST-alpha activity in mice vs rats, where this activity is not found.<br />
<br />
== Pre-MIE: References ==<br />
F.D. Asplin, R.B.A. Carnaghan, (1961). The toxicity of certain groundnut meals for poultry with special reference to their effect on ducklings and chickens. Vet. Rec. 73:1215– 1219. <br />
<br />
Brown KL, Deng JZ, Iyer RS, Iyer LG, Voehler MW, Stone MP, Harris CM, Harris TM (2006). Unraveling the aflatoxin-FAPY conundrum: Structural basis of the formamidopyrimidine-type DNA adduct of aflatoxin B1. J Am Chem Soc 128:15188-15199.<br />
<br />
Croy RG, Essigman JM, Reinhold VN, Wogan GN (1978). Identification of the principal aflatoxin N1-DNA adduct formed in vivo in rat liver. Proc Natl Acad Sci USA 75:1745-1749.<br />
<br />
Degen GH, Neumann HG (1981). Differences in aflatoxin B1-susceptibility of rat and mouse are correlated with the capability in vitro to inactivate aflatoxin B1-epoxide. Carcinogenesis 2:299–306.<br />
<br />
Eaton DL, and Gallagher EP (1994). Mechanisms of aflatoxin carcinogenesis. Annu Rev Pharmacol Toxicol 34:135-172.<br />
<br />
Elegbede JA, and Gould MN. (2002). Monoterpenes reduced adducts formation in rats exposed to aflatoxin B1. African J Biotech, 1, 46–49.<br />
<br />
Gregory 3rd JF, Goldstein SL, Edds GT. (1983). Metabolite distribution and rate of residue clearance in turkeys fed a diet containing aflatoxin B1. Food Chem Toxicol, 21, 463–7.<br />
<br />
Groopman JD, Kensler TW (2005). Role of metabolism and viruses in aflatoxin-induced liver cancer. Toxicol Appl Pharmacol 206:131-137.<br />
<br />
Guengerich FP, Johnson WW, Ueng Y-F, Yamazaki H, Shimada T (1996). Involvement of Cytochrome P450, glutathione S-transferase, and epoxide hydrolase in the metabolism of aflatoxin B1 and relevance to risk of human liver cancer. Environ Health Perspect 104(Suppl 3):557-562.<br />
<br />
Himmelstein MW, Boogaard PJ, Cadet J, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: II.Overview of methods of identification and quantitation of DNA damage. Crit Rev Toxicol, 39, 679–694.<br />
<br />
IARC (1993). Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. IARC Monographs on the Evaluation of Carcinogenic Risk to Humans. Vol. 56, 245-395.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, Sutter TR, Groopman JD, Kensler TW, Roebuck BD. (2014). Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold. Cancer Prev Res. 7(7):658-665.<br />
<br />
Kensler TW, He X, Otieno M, et al. (1998). Oltipraz chemoprevention trial in Qidong, People’s Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol Biomarkers Prev, 7, 127–34.<br />
<br />
Larsson P, Hoedaya, WI, Tjalve H. (1990). Disposition of 3H-aflatoxin H in mice: formation and retention of tissue bound metabolites in nasal glands. Pharmacol Toxicol, 67, 162–71.<br />
<br />
Larsson P, and Tjalve H. (1993). Distribution and metabolism of aflatoxin B1 in the marmoset monkey (Callithrix jacchus). Carcinogenesis, 14, 1–6.<br />
<br />
Monroe DH, Eaton DL. (1987). Comparative effects of butylated hydroxyanisole on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B1 in the rat and the mouse. Toxicol Appl Pharmacol, 90, 401–409.<br />
<br />
Pottenger, L.H., Andrews LS, Bachman AN, Boogaard PJ, Cadet J, Embry MR, Farmer PB, Himmelstein MW, Jarabek AM, Martin EA, Mauthe RJ, Persaud R, Preston RJ, Schoeny R, Skare J, Swenberg JA, Williams GM, Zeiger E, Zhang F, Kim JH. (2014). An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride. Crit. Rev. Toxicol. 44(4):348-391.<br />
<br />
Primiano T, Egner PA, Sutter TR, et al. (1995). Intermittent dosing with oltipraz: relationship between chemoprevention of aflatoxin-induced tumorigenesis and induction of glutathione-S-transferases. Cancer Res, 55, 4319–4324.<br />
<br />
Roebuck BD, Liu Y-L, Rogers AE, et al. (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short term molecular dosimetry. Cancer Res, 51, 5501–5506.<br />
<br />
Ueng Y-F, Shimada T, Yamazaki H, Guengerich FP (1995). Oxidation of aflatoxin B1 by bacterial recombinant human cytochrome P450 enzymes. Chem Res Toxiol 8:218-225.<br />
<br />
Wang J-S, Shen X, He X, et al. (1999). Protective alerations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qidong, People’s Republic of China. J Natl Cancer Inst, 91, 347–354.<br />
<br />
Yates MS, Kwak M-K, Egner PA, et al. (2006). Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl] imidazole. Cancer Res, 66, 2488–2494.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:376Event:3762016-07-06T17:03:12Z<p>Dvillene: /* KE#2: Taxonomic Applicability */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Induced Mutations in Critical Genes, Increased </div><br />
<div id='shortTitle' class='Title'> Induced in Critical Genes, Mutation </div><br />
<br />
== Action ==<br />
Induced mutation<br />
<br />
== Biological Process/Object ==<br />
Critical genes for carcinogenesis<br />
<br />
== Induced Mutation in Critical Genes ==<br />
The AFB1-induced pro-mutagenic DNA adduct is either not repaired or is mis-repaired resulting in a mutation in one or more critical genes. In bacteria and mammalian cells (both in vitro and in vivo) the primary mutation associated with AFB1 is a guanine to thymine transversion (Foster et al., 1983; Dycaico et al., 1996). A G:T transversion is expected for the pro-mutagenic DNA adduct AFB1-FAPy. <br />
<br />
A specific critical mutation in codon 249 of the p53 gene has been identified in human hepatocellular carcinoma (HCC) (See section below on essentiality).<br />
<br />
== Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
A specific critical mutation in the p53 gene has been identified in human hepatocellular carcinoma (HCC). Demonstrating that AFB1 can induce this specific p53 gene mutation would be the highest level of evidence that AFB1-induced HCC involves mutation as a KE. Absent such information, the next best level of evidence is the induction of the specific type of mutation (G:C to T:A transversion) in a variety of gene mutation assays measuring a range of target genes. <br />
<br />
The codon 249 mutation is present in a significant proportion of human HCCs. In fact codon 249 mutation is detected in up to 50% of liver cancers in Qidong, China (Hsu et al., 1991) and in Mozambique, both areas with high likelihood of AFB1 exposure. The codon 249 G:C to T:A mutation in the third base is seen in up to 75% of HCC in high-incidence areas of China and East Africa (Gouas et al., 2009). In contrast, this specific mutation is very rare in HCC from areas with no or low exposure to AFB1 (Hsu et al. 1991 and Bressac et al. Nature, 350:429-431, 1991). This mutation is also very rare in other types of tumors (Gouas et al., 2009). According to Gouas et al. (2009), populations with AFB1 exposure are likely to be exposed to hepatitis B virus (HBV) as well and the effects of each are difficult to separate.This mutation is very rare in HCC from non/low -aflatoxin areas (Hsu et al. 1991 and Bressac et al., 1991) and also very rare in other types of tumors.<br />
<br />
Some indirect evidence of the essentiality of mutation in tumor development is provided by the clear species difference between adult mice and adult rats both in the induction of surrogate gene mutations and in the induction of tumors. Adult mice exposed to AFB1 do not get tumors and there is no increase in mutant frequency (MF) for Big Blue mice exposed as adults. That is, Lac I mutants from the AFB1- exposed adult mice showed a spontaneous mutational spectrum. Rats, however, showed a large increase in MF and, more specifically a large increase in G:C to T:A transversions (Dycaico et al., 1996). In addition, for mice there is an difference between neonata and adult mice. Neonatal mice treated with AFB1 (6 mg/kg—a dose that does result in tumors) showed an increase in cII mutation with G:C to T:A transversion as the major mutation. Adult mice treated at 6 and 60 mg/kg (doses that do not produce tumors) did not have a significant increase in cII mutation (but did give a different mutational spectrum than controls) (Chen et al. 2010).<br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||KE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Cellular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Taxonomic Applicability ==<br />
The induction of G:C to T:A transversions following AFB1 exposure is seen in a wide variety of species from bacteria to mammals. Assays that measure mutation in surrogate genes (that is, genes unrelated to the critical cancer genes) can be used to evaluate the full spectrum of mutational events that can be induced by a chemical. One such system is the AS52 assay, an in vitro mammalian transgenic mutation assay that measures mutation in the gpt gene. After exposure to AFB1 in culture the predominant mutation is G:C to T:A transversion, although a number of other types of mutations were also seen (Wattanawaraporn et al., 2012). There is a species difference between adult mice and rats. No increase in MF is seen in the in vivo Big Blue™ mutation assay for mice exposed to AFB1. That is, Lac I mutations from the mice showed a spontaneous mutational spectrum. By contrast rats showed a large increase in MF with a large increase in G:C to T:A transversions (Dycaico et al., 1996). However, neonatal mice treated with AFB1 (6 mg/kg—a dose that induces tumors in neonates) do show an increase in cII mutation with G:C to T:A transversion as the major mutation. Adult mice treated at 6 and 60 mg/kg (doses that do not induce tumors) did not show a significant increase in cII mutations but did produce a different mutational spectrum than controls (Chen et al., 2010.)<br />
<br />
== KE#2: Level of Biological Organization : Cellular ==<br />
The induction of mutation occurs within the nucleus of cells and involves permanent alterations in the primary DNA sequence that is passed to subsequent cell generations and, thus, is heritable.<br />
<br />
== KE#2: How this Key Event works ==<br />
Following the formation of DNA adducts, which are either mis-repaired or not repaired, the sequence of base pairs in the DNA is changed due to insertion of an incorrect base opposite the DNA adduct during DNA replication, so that a G is permanently replaced by a T. This type of mutation is observed in the mutants seen in surrogate gene mutation assays using AFB1 exposureA high frequency of codon 249 p53 mutations occurs in human tumors in high AFB1 exposure regions.Hence, codon 249 of the p53 gene contains a base pair susceptible to insufficient or misrepair of DNA adducts.<br />
<br />
Puisieux et al. (1991) provide evidence that the AFB1 epoxide adduct binds preferentially to codon 249 of the p53 gene. Using a plasmid with containing full-length human p53 DNA, adduct formation was observed in exons 5, 6, 7 and 8 (a total of 1086 bases) and 20% of the bases were targeted by AFB1 with a preference for guanine residues. Binding of AFB1 to p53 sequences was restricted to fewer residues and was more specific for guanine than was the binding of B[a]P (Puisieux et al.,1991). Binding of AFB1 in the region around codon 249 of p53 AFB1 was reported to be “stronger” than that of B[a]P. The last nucleotide of codon 249 is a guanine and was targeted by AFB1 but not by B[a]P. This guanine residue is the mutational hotspot in human liver cancers from patients in high AFB1 exposure regions. <br />
<br />
While mutations at codon 249 of the p53 gene have been observed in association with HCC in humans, the question remains whether this mutation occurs as a direct result of adduct formation at this site or by some different mechanism..In human HepG2 hepatocytes exposed to microsomally activated AFB1, a dose-dependent increase in G:C to T:A transversions were observed at 10 additional locations using ligation-mediated PCR, and at 4 additional locations using terminal-transferred dependent PCR.(Denissenko et al.,1998). These authors suggest that codon 249 may not present a key adduct site. However, other more recently published data identified codon 249 of the P53 gene as an unusually mutagenic adduct conformation based on the local DNA sequence and concluded that a higher mutation rate may occur there rather than at other locations because of increased DNA polymerase bypass (Pussieux et al., 1991; Lin et al., 2014a,b).<br />
<br />
== KE#2: How it is Measured or Detected ==<br />
Historically the detection of critical cancer gene specific mutations has not been technically feasible. A newly developed method, allele specific competitive blocker-polymerase chain reaction (ACB-PCR) has proven useful in providing such information, and data on specific chemical-induced mutations are available for a very small number of chemicals (Parsons et al., 2010). Unfortunately, there are no data for AFB1.<br />
<br />
There are, however, a number of gene mutation assays that have been widely used for determining the general ability of chemicals, including AFB1 to induce mutations.These assays use selection methods that allow only mutant cells to survive and grow. The AS52 in vitro gene mutation assay using the gpt gene, and 6-thioguanine selection has been used to demonstrate that AFB1 exposure increases the MF at the gpt gene. In vivo transgenic assays use molecular methods to recover the transgene from isolated DNA and to evaluate the MF in the transgene. Molecular methods can detect the presence (above a certain sensitivity level) of mutant cells. DNA from tumors can be sequenced to determine the presence of mutations in specific genes. DNA sequencing has been used on human tumors to detect the presence of the Codon 249 p53 mutation.<br />
<br />
== KE#2: Evidence Supporting Taxonomic Applicability ==<br />
There are data across phyla demonstrating the induction of mutations, specifically the induction to G:C to T:A transversions following AFB1 exposure.<br />
<br />
== KE#2: References ==<br />
Bressac, B., Kew, M., Wands, J., & Ozturk, M. (1991). Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern africa. Nature, 350(6317), 429-31. doi:10.1038/350429a<br />
<br />
Chen, T., Heflich, R. H., Moore, M. M., & Mei, N. (2010). Differential mutagenicity of aflatoxin B1 in the liver of neonatal and adult mice. Environ Mol Mutagen, 51(2), 156-63. doi:10.1002/em.2051<br />
<br />
Denissenko MF, Koudriakova TB, Smith L, O'Connor TR, Riggs AD, and Pfeifer GP). The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts. (Oncogene. 1998, Dec 10;17(23):3007-14.<br />
<br />
Dycaico, M. J., Stuart, G. R., Tobal, G. M., de Boer, J. G., Glickman, B. W., & Provost, G. S. (1996). Species-specific differences in hepatic mutant frequency and mutational spectrum among lambda/laci transgenic rats and mice following exposure to aflatoxin B1. Carcinogenesis, 17(11), 2347-56<br />
<br />
Foster, P. L., Eisenstadt, E., & Miller, J. H. (1983). Base substitution mutations induced by metabolically activated aflatoxin B1. Proceedings of the National Academy of Sciences of the United States of America, 80(9), 2695-8.<br />
<br />
Gouas, D., Shi, H., & Hainaut, P. (2009). The aflatoxin-induced TP53 mutation at codon 249 (R249S): Biomarker of exposure, early detection and target for therapy. Cancer Lett, 286(1), 29-37. doi:10.1016/j.canlet.2009.02.057<br />
<br />
Hsu, I. C., Metcalf, R. A., Sun, T., Welsh, J. A., Wang, N. J., & Harris, C. C. (1991). Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature, 350(6317), 427-8. doi:10.1038/350427a<br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014a). Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua. J Biol Chem. 289:18497-18506. <br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014b). Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis 35(7):1461-1468<br />
<br />
Parsons BL, Myers MB, Meng F, Wang Y, McKinzie PB. 2010. Oncomutations as biomarkers of cancer risk. Environ Mol Mutagen. 51(8-9):836-850.<br />
<br />
Puisieux, A., Lim, S., Groopman, J., & Ozturk, M. (1991). Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res, 51(22), 6185-9.<br />
<br />
Wattanawaraporn, R., Kim, M. Y., Adams, J., Trudel, L. J., Woo, L. L., Croy, R. G., . . . Wogan, G. N. (2012). AFB(1) -induced mutagenesis of the gpt gene in AS52 cells. Environ Mol Mutagen, 53(7), 567-73. doi:10.1002/em.2171</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:409Event:4092016-07-06T17:03:05Z<p>Dvillene: /* Taxonomic Applicability */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id ='longTitle' class='Title'> Step: #0: pre-MIE </div><br />
<div id ='longTitle' class='Title'> Action: Metabolism of AFB1 </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||KE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Molecular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Biological Process: Production of Reactive Electrophiles ==<br />
Aflatoxin B1 (AFB1) can be oxidized to a variety of metabolites, many of which can be further metabolized, for example by conjugation with glutathione (GSH) via Glutathione-S-transferase (GST). In order for DNA binding and formation of a pro-mutagenic DNA adduct to occur, AFB1 must be metabolized via Cytochrome P450 metabolism to a specific highly reactive form of AFB1, the exo-epoxide AFB1-8,9-epoxide. CYP3A4 forms the exo-form of this reactive epoxide only. CYP1A2, inducible in liver, forms both the exo- and the endo-epoxides; in human liver, CYP1A2 metabolism occurs with a lower Vmax (enzymatic rate) and higher Km (half-maximal concentrations) than CYP3A4 (Degen and Neumann,1981; Groopman and Kensler, 2005; Guengerich et al., 1996; Ueng et al., 1995). Figure 1, from Pottenger et al., 2014, depicts the metabolism of AFB1.<br />
<br />
Metabolic activation of AFB1 occurs mainly in the liver, where the highest levels of the CYP isozymes are located. Competitive metabolism with other hepatic and extra-hepatic P450 isozymes may decrease the proportion of the specific metabolite, AFB1-8,9-epoxide; this can reduce the effectiveness of hepatic activation of AFB1. Alternatively, induction of either hepatic or extra-hepatic GST activity can reduce the levels of AFB1-epoxide available for reaction with DNA by increasing conjugation of the epoxide with GSH. This conjugation renders the epoxide inactive and directs it towards eventual elimination (Guengerich et al., 1996).[[File:Figure1met.jpg|thumb|alt=Figure 1 alt text|Figure 1. Metabolism of AFB1,]] Figure 1 was obtained from Pottenger et al., 2014.<br />
<br />
The AFB1 pro-mutagenic DNA adduct is most likely the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct, or N7-AFB1-G (Croy et al., 1978). Once the exo-epoxide is bound to the N7-guanine, it is subject to ring-opening to form the more highly pro-mutagenic 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-pyrimid-9-yl-foramido)-9-hydroxyaflatoxin B1; this is known as the formamidopyrimidine adduct or AFB1-FAPy adduct (Brown et al., 2006).<br />
<br />
== Taxonomic Applicability ==<br />
The metabolic information above is applicable to all mammalian systems evaluated; it is also applicable to certain birds such as turkeys (Gregory et al., 1983; IARC, 1993). Humans, non-human primates, rats, mice, poultry and fish have all demonstrated susceptibility to AFB1-induced liver tumors (Asplin and Canaghan, 1961; Eaton and Gallagher, 1994). Species that preferentially metabolize AFB1 to the exo-8,9-epoxide are more susceptible to AFB1 carcinogenicity. Sulfation by GST enzymes or other enzymatic detoxification of AFB1 metabolites can reduce available levels of the exo-epoxide, and subsequent steps or even the progression of Key Events may not occur in these species. Mice are less susceptible to AFB1-induced cancer than rats, likely due to increased detoxification (Monroe and Eaton, 1987). This increased detoxication capability can also be instigated by dietary exposures to compounds that modulate CYP450 expression or modify detoxication activities (Elegbede and Gould, 2002; Primiano et al., 1995; Roebuck et al. 1991, 2003; Kensler et al., 1998; Wang et al., 1999; Yates et al., 2006).<br />
<br />
== Pre-MIE: Level of Biological Organization : Molecular ==<br />
Metabolic activation of AFB1 occurs at the intracellular level, with sub-cellular organelles involved due to the localization of CYP450 in the endoplasmic reticulum, coupled with the epoxide hydratase; GST is localized in the cytosol (Guengerich et al., 1996). Organ architecture also plays a role, as the structure of the liver is orientated around the O2 gradient set by the portal artery and hepatic vein; hepatocytes nearest the portal artery demonstrate increased tissue oxygen while those around the portal vein have decreased tissue O2 and correspondingly higher levels of CYP450 activity.<br />
<br />
== Pre-MIE: How this Key Event works ==<br />
The reactive exo-epoxide is formed in hepatocytes (or extra-hepatically) by metabolism of the parent AFB1 by CYP450 (Larsson et al., 1990; Larsson and Tjalve, 1993). The reactive metabolite then escapes the endoplasmic reticulum where the CYP450 is located. The reactive metabolite must evade conjugation with GSH in the cytoplasm or binding with other cytoplasmic nucleophiles. It then traverses the nuclear membrane in order to reach the cell nucleus and the nuclear DNA. Once the reactive metabolite is in the cell nucleus, binding to nuclear DNA and the formation of DNA adducts can occur.<br />
<br />
== Pre-MIE: How it is Measured or Detected ==<br />
Formation of the exo-epoxide can be produced with in vitro systems and detected using techniques for structural quantitation of AFB1 metabolites (Himmelstein et al., 2009), including liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). By using subcellular fractions (e.g., microsomes), cellular homogenates, or cells in culture, it is possible to measure formation of AFB1 exo-epoxide. Such data can also be collected from in vivo systems; samples of plasma or blood from AFB1-treated animals can be analyzed for the AFB1 exo-epoxide with similar mass spectrometric based detection systems (e.g., LC-MS/MS). Samples of blood from humans in AFB1-endemic regions have demonstrated presence of AFB1-albumin adducts, which are formed from the AFB1 exo-epoxide. AFB1-treated animals may also provide tissue samples for analysis of AFB1 exo-epoxide. Special trapping techniques may be required as the reactive AFB1 exo-epoxide metabolite has a short half-life in biological matrices.<br />
<br />
== Pre-MIE: Evidence Supporting Taxonomic Applicability ==<br />
Ample data across phyla demonstrate metabolic activation of AFB1 to the exo-epoxide via CYP450. These taxons include several mammalian species (humans, non-human primates, rats, mice) in addition to birds (turkeys) and fish (Eaton and Gallagher, 1994; IARC, 1993).<br />
<br />
== Pre-MIE: Evidence for Chemical Initiation of this Initial Molecular Event/Pre-MIE ==<br />
There is an extensive database on AFB1 in many different systems demonstrating formation of the AFB1 exo-epoxide. This database includes several mammalian species (humans, non-human primates, rats, mice) in addition to birds (turkeys) and fish (Eaton and Gallagher, 1994; IARC, 1993).<br />
<br />
== Evidence Supporting Essentiality of Pre-MIE (Step #0) ==<br />
Strong<br />
<br />
Many studies show that in the absence of AFB1 metabolic activation, tumors do not occur. Treatment of rats with the CYP450-inhibitor and GST-inducer oltipraz reduces the levels of AFB1 DNA adducts by 65-70%, and the later-forming altered hepatic foci (AHF) and liver tumors are also reduced by 97 and 100%, respectively; thus, no hepatocellular carcinomas (HCC) form in the oltipraz-treated rats dosed with AFB1 (Roebuck et al., 1991). More recent data show even more effective prevention of tumors and of hepatic foci in rats pre-treated with a triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) (Johnson et al., 2014). The pre-treated rats were reported with a 70% reduction of AFB1-induced adducts in urine but had 100% inhibition of tumors, and only 1 rat out of 23 pre-treated had any altered hepatic foci (AHF). There are similar data in humans treated with oltipraz. Kensler et al. (1998) report a significant reduction in a biomarker of exposure AFB1-induced albumin adducts, supporting the human relevance of this metabolic activation <br />
<br />
Another line of evidence for essentiality of the pre-MIE is the recognized difference in sensitivity to AFB1-induced liver tumors between mice and rats (Degen and Neumann, 1981). Mice, have considerably increased metabolic activation of AFB1 to the exo-epoxide compared to rats; mice are nonetheless much less sensitive to AFB1-induced liver tumors. It is thought that this difference is due to the constitutive presence of GST-alpha activity in mice vs rats, where this activity is not found.<br />
<br />
== Pre-MIE: References ==<br />
F.D. Asplin, R.B.A. Carnaghan, (1961). The toxicity of certain groundnut meals for poultry with special reference to their effect on ducklings and chickens. Vet. Rec. 73:1215– 1219. <br />
<br />
Brown KL, Deng JZ, Iyer RS, Iyer LG, Voehler MW, Stone MP, Harris CM, Harris TM (2006). Unraveling the aflatoxin-FAPY conundrum: Structural basis of the formamidopyrimidine-type DNA adduct of aflatoxin B1. J Am Chem Soc 128:15188-15199.<br />
<br />
Croy RG, Essigman JM, Reinhold VN, Wogan GN (1978). Identification of the principal aflatoxin N1-DNA adduct formed in vivo in rat liver. Proc Natl Acad Sci USA 75:1745-1749.<br />
<br />
Degen GH, Neumann HG (1981). Differences in aflatoxin B1-susceptibility of rat and mouse are correlated with the capability in vitro to inactivate aflatoxin B1-epoxide. Carcinogenesis 2:299–306.<br />
<br />
Eaton DL, and Gallagher EP (1994). Mechanisms of aflatoxin carcinogenesis. Annu Rev Pharmacol Toxicol 34:135-172.<br />
<br />
Elegbede JA, and Gould MN. (2002). Monoterpenes reduced adducts formation in rats exposed to aflatoxin B1. African J Biotech, 1, 46–49.<br />
<br />
Gregory 3rd JF, Goldstein SL, Edds GT. (1983). Metabolite distribution and rate of residue clearance in turkeys fed a diet containing aflatoxin B1. Food Chem Toxicol, 21, 463–7.<br />
<br />
Groopman JD, Kensler TW (2005). Role of metabolism and viruses in aflatoxin-induced liver cancer. Toxicol Appl Pharmacol 206:131-137.<br />
<br />
Guengerich FP, Johnson WW, Ueng Y-F, Yamazaki H, Shimada T (1996). Involvement of Cytochrome P450, glutathione S-transferase, and epoxide hydrolase in the metabolism of aflatoxin B1 and relevance to risk of human liver cancer. Environ Health Perspect 104(Suppl 3):557-562.<br />
<br />
Himmelstein MW, Boogaard PJ, Cadet J, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: II.Overview of methods of identification and quantitation of DNA damage. Crit Rev Toxicol, 39, 679–694.<br />
<br />
IARC (1993). Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. IARC Monographs on the Evaluation of Carcinogenic Risk to Humans. Vol. 56, 245-395.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, Sutter TR, Groopman JD, Kensler TW, Roebuck BD. (2014). Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold. Cancer Prev Res. 7(7):658-665.<br />
<br />
Kensler TW, He X, Otieno M, et al. (1998). Oltipraz chemoprevention trial in Qidong, People’s Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol Biomarkers Prev, 7, 127–34.<br />
<br />
Larsson P, Hoedaya, WI, Tjalve H. (1990). Disposition of 3H-aflatoxin H in mice: formation and retention of tissue bound metabolites in nasal glands. Pharmacol Toxicol, 67, 162–71.<br />
<br />
Larsson P, and Tjalve H. (1993). Distribution and metabolism of aflatoxin B1 in the marmoset monkey (Callithrix jacchus). Carcinogenesis, 14, 1–6.<br />
<br />
Monroe DH, Eaton DL. (1987). Comparative effects of butylated hydroxyanisole on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B1 in the rat and the mouse. Toxicol Appl Pharmacol, 90, 401–409.<br />
<br />
Pottenger, L.H., Andrews LS, Bachman AN, Boogaard PJ, Cadet J, Embry MR, Farmer PB, Himmelstein MW, Jarabek AM, Martin EA, Mauthe RJ, Persaud R, Preston RJ, Schoeny R, Skare J, Swenberg JA, Williams GM, Zeiger E, Zhang F, Kim JH. (2014). An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride. Crit. Rev. Toxicol. 44(4):348-391.<br />
<br />
Primiano T, Egner PA, Sutter TR, et al. (1995). Intermittent dosing with oltipraz: relationship between chemoprevention of aflatoxin-induced tumorigenesis and induction of glutathione-S-transferases. Cancer Res, 55, 4319–4324.<br />
<br />
Roebuck BD, Liu Y-L, Rogers AE, et al. (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short term molecular dosimetry. Cancer Res, 51, 5501–5506.<br />
<br />
Ueng Y-F, Shimada T, Yamazaki H, Guengerich FP (1995). Oxidation of aflatoxin B1 by bacterial recombinant human cytochrome P450 enzymes. Chem Res Toxiol 8:218-225.<br />
<br />
Wang J-S, Shen X, He X, et al. (1999). Protective alerations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qidong, People’s Republic of China. J Natl Cancer Inst, 91, 347–354.<br />
<br />
Yates MS, Kwak M-K, Egner PA, et al. (2006). Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl] imidazole. Cancer Res, 66, 2488–2494.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:409Event:4092016-07-06T17:02:47Z<p>Dvillene: /* Biological Process: Production of Reactive Electrophiles */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id ='longTitle' class='Title'> Step: #0: pre-MIE </div><br />
<div id ='longTitle' class='Title'> Action: Metabolism of AFB1 </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||KE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Molecular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Biological Process: Production of Reactive Electrophiles ==<br />
Aflatoxin B1 (AFB1) can be oxidized to a variety of metabolites, many of which can be further metabolized, for example by conjugation with glutathione (GSH) via Glutathione-S-transferase (GST). In order for DNA binding and formation of a pro-mutagenic DNA adduct to occur, AFB1 must be metabolized via Cytochrome P450 metabolism to a specific highly reactive form of AFB1, the exo-epoxide AFB1-8,9-epoxide. CYP3A4 forms the exo-form of this reactive epoxide only. CYP1A2, inducible in liver, forms both the exo- and the endo-epoxides; in human liver, CYP1A2 metabolism occurs with a lower Vmax (enzymatic rate) and higher Km (half-maximal concentrations) than CYP3A4 (Degen and Neumann,1981; Groopman and Kensler, 2005; Guengerich et al., 1996; Ueng et al., 1995). Figure 1, from Pottenger et al., 2014, depicts the metabolism of AFB1.<br />
<br />
Metabolic activation of AFB1 occurs mainly in the liver, where the highest levels of the CYP isozymes are located. Competitive metabolism with other hepatic and extra-hepatic P450 isozymes may decrease the proportion of the specific metabolite, AFB1-8,9-epoxide; this can reduce the effectiveness of hepatic activation of AFB1. Alternatively, induction of either hepatic or extra-hepatic GST activity can reduce the levels of AFB1-epoxide available for reaction with DNA by increasing conjugation of the epoxide with GSH. This conjugation renders the epoxide inactive and directs it towards eventual elimination (Guengerich et al., 1996).[[File:Figure1met.jpg|thumb|alt=Figure 1 alt text|Figure 1. Metabolism of AFB1,]] Figure 1 was obtained from Pottenger et al., 2014.<br />
<br />
The AFB1 pro-mutagenic DNA adduct is most likely the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct, or N7-AFB1-G (Croy et al., 1978). Once the exo-epoxide is bound to the N7-guanine, it is subject to ring-opening to form the more highly pro-mutagenic 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-pyrimid-9-yl-foramido)-9-hydroxyaflatoxin B1; this is known as the formamidopyrimidine adduct or AFB1-FAPy adduct (Brown et al., 2006).<br />
<br />
== Pre-MIE: Taxonomic Applicability ==<br />
The metabolic information above is applicable to all mammalian systems evaluated; it is also applicable to certain birds such as turkeys (Gregory et al., 1983; IARC, 1993). Humans, non-human primates, rats, mice, poultry and fish have all demonstrated susceptibility to AFB1-induced liver tumors (Asplin and Canaghan, 1961; Eaton and Gallagher, 1994). Species that preferentially metabolize AFB1 to the exo-8,9-epoxide are more susceptible to AFB1 carcinogenicity. Sulfation by GST enzymes or other enzymatic detoxification of AFB1 metabolites can reduce available levels of the exo-epoxide, and subsequent steps or even the progression of Key Events may not occur in these species. Mice are less susceptible to AFB1-induced cancer than rats, likely due to increased detoxification (Monroe and Eaton, 1987). This increased detoxication capability can also be instigated by dietary exposures to compounds that modulate CYP450 expression or modify detoxication activities (Elegbede and Gould, 2002; Primiano et al., 1995; Roebuck et al. 1991, 2003; Kensler et al., 1998; Wang et al., 1999; Yates et al., 2006).<br />
<br />
== Pre-MIE: Level of Biological Organization : Molecular ==<br />
Metabolic activation of AFB1 occurs at the intracellular level, with sub-cellular organelles involved due to the localization of CYP450 in the endoplasmic reticulum, coupled with the epoxide hydratase; GST is localized in the cytosol (Guengerich et al., 1996). Organ architecture also plays a role, as the structure of the liver is orientated around the O2 gradient set by the portal artery and hepatic vein; hepatocytes nearest the portal artery demonstrate increased tissue oxygen while those around the portal vein have decreased tissue O2 and correspondingly higher levels of CYP450 activity.<br />
<br />
== Pre-MIE: How this Key Event works ==<br />
The reactive exo-epoxide is formed in hepatocytes (or extra-hepatically) by metabolism of the parent AFB1 by CYP450 (Larsson et al., 1990; Larsson and Tjalve, 1993). The reactive metabolite then escapes the endoplasmic reticulum where the CYP450 is located. The reactive metabolite must evade conjugation with GSH in the cytoplasm or binding with other cytoplasmic nucleophiles. It then traverses the nuclear membrane in order to reach the cell nucleus and the nuclear DNA. Once the reactive metabolite is in the cell nucleus, binding to nuclear DNA and the formation of DNA adducts can occur.<br />
<br />
== Pre-MIE: How it is Measured or Detected ==<br />
Formation of the exo-epoxide can be produced with in vitro systems and detected using techniques for structural quantitation of AFB1 metabolites (Himmelstein et al., 2009), including liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). By using subcellular fractions (e.g., microsomes), cellular homogenates, or cells in culture, it is possible to measure formation of AFB1 exo-epoxide. Such data can also be collected from in vivo systems; samples of plasma or blood from AFB1-treated animals can be analyzed for the AFB1 exo-epoxide with similar mass spectrometric based detection systems (e.g., LC-MS/MS). Samples of blood from humans in AFB1-endemic regions have demonstrated presence of AFB1-albumin adducts, which are formed from the AFB1 exo-epoxide. AFB1-treated animals may also provide tissue samples for analysis of AFB1 exo-epoxide. Special trapping techniques may be required as the reactive AFB1 exo-epoxide metabolite has a short half-life in biological matrices.<br />
<br />
== Pre-MIE: Evidence Supporting Taxonomic Applicability ==<br />
Ample data across phyla demonstrate metabolic activation of AFB1 to the exo-epoxide via CYP450. These taxons include several mammalian species (humans, non-human primates, rats, mice) in addition to birds (turkeys) and fish (Eaton and Gallagher, 1994; IARC, 1993).<br />
<br />
== Pre-MIE: Evidence for Chemical Initiation of this Initial Molecular Event/Pre-MIE ==<br />
There is an extensive database on AFB1 in many different systems demonstrating formation of the AFB1 exo-epoxide. This database includes several mammalian species (humans, non-human primates, rats, mice) in addition to birds (turkeys) and fish (Eaton and Gallagher, 1994; IARC, 1993).<br />
<br />
== Evidence Supporting Essentiality of Pre-MIE (Step #0) ==<br />
Strong<br />
<br />
Many studies show that in the absence of AFB1 metabolic activation, tumors do not occur. Treatment of rats with the CYP450-inhibitor and GST-inducer oltipraz reduces the levels of AFB1 DNA adducts by 65-70%, and the later-forming altered hepatic foci (AHF) and liver tumors are also reduced by 97 and 100%, respectively; thus, no hepatocellular carcinomas (HCC) form in the oltipraz-treated rats dosed with AFB1 (Roebuck et al., 1991). More recent data show even more effective prevention of tumors and of hepatic foci in rats pre-treated with a triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) (Johnson et al., 2014). The pre-treated rats were reported with a 70% reduction of AFB1-induced adducts in urine but had 100% inhibition of tumors, and only 1 rat out of 23 pre-treated had any altered hepatic foci (AHF). There are similar data in humans treated with oltipraz. Kensler et al. (1998) report a significant reduction in a biomarker of exposure AFB1-induced albumin adducts, supporting the human relevance of this metabolic activation <br />
<br />
Another line of evidence for essentiality of the pre-MIE is the recognized difference in sensitivity to AFB1-induced liver tumors between mice and rats (Degen and Neumann, 1981). Mice, have considerably increased metabolic activation of AFB1 to the exo-epoxide compared to rats; mice are nonetheless much less sensitive to AFB1-induced liver tumors. It is thought that this difference is due to the constitutive presence of GST-alpha activity in mice vs rats, where this activity is not found.<br />
<br />
== Pre-MIE: References ==<br />
F.D. Asplin, R.B.A. Carnaghan, (1961). The toxicity of certain groundnut meals for poultry with special reference to their effect on ducklings and chickens. Vet. Rec. 73:1215– 1219. <br />
<br />
Brown KL, Deng JZ, Iyer RS, Iyer LG, Voehler MW, Stone MP, Harris CM, Harris TM (2006). Unraveling the aflatoxin-FAPY conundrum: Structural basis of the formamidopyrimidine-type DNA adduct of aflatoxin B1. J Am Chem Soc 128:15188-15199.<br />
<br />
Croy RG, Essigman JM, Reinhold VN, Wogan GN (1978). Identification of the principal aflatoxin N1-DNA adduct formed in vivo in rat liver. Proc Natl Acad Sci USA 75:1745-1749.<br />
<br />
Degen GH, Neumann HG (1981). Differences in aflatoxin B1-susceptibility of rat and mouse are correlated with the capability in vitro to inactivate aflatoxin B1-epoxide. Carcinogenesis 2:299–306.<br />
<br />
Eaton DL, and Gallagher EP (1994). Mechanisms of aflatoxin carcinogenesis. Annu Rev Pharmacol Toxicol 34:135-172.<br />
<br />
Elegbede JA, and Gould MN. (2002). Monoterpenes reduced adducts formation in rats exposed to aflatoxin B1. African J Biotech, 1, 46–49.<br />
<br />
Gregory 3rd JF, Goldstein SL, Edds GT. (1983). Metabolite distribution and rate of residue clearance in turkeys fed a diet containing aflatoxin B1. Food Chem Toxicol, 21, 463–7.<br />
<br />
Groopman JD, Kensler TW (2005). Role of metabolism and viruses in aflatoxin-induced liver cancer. Toxicol Appl Pharmacol 206:131-137.<br />
<br />
Guengerich FP, Johnson WW, Ueng Y-F, Yamazaki H, Shimada T (1996). Involvement of Cytochrome P450, glutathione S-transferase, and epoxide hydrolase in the metabolism of aflatoxin B1 and relevance to risk of human liver cancer. Environ Health Perspect 104(Suppl 3):557-562.<br />
<br />
Himmelstein MW, Boogaard PJ, Cadet J, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: II.Overview of methods of identification and quantitation of DNA damage. Crit Rev Toxicol, 39, 679–694.<br />
<br />
IARC (1993). Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. IARC Monographs on the Evaluation of Carcinogenic Risk to Humans. Vol. 56, 245-395.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, Sutter TR, Groopman JD, Kensler TW, Roebuck BD. (2014). Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold. Cancer Prev Res. 7(7):658-665.<br />
<br />
Kensler TW, He X, Otieno M, et al. (1998). Oltipraz chemoprevention trial in Qidong, People’s Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol Biomarkers Prev, 7, 127–34.<br />
<br />
Larsson P, Hoedaya, WI, Tjalve H. (1990). Disposition of 3H-aflatoxin H in mice: formation and retention of tissue bound metabolites in nasal glands. Pharmacol Toxicol, 67, 162–71.<br />
<br />
Larsson P, and Tjalve H. (1993). Distribution and metabolism of aflatoxin B1 in the marmoset monkey (Callithrix jacchus). Carcinogenesis, 14, 1–6.<br />
<br />
Monroe DH, Eaton DL. (1987). Comparative effects of butylated hydroxyanisole on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B1 in the rat and the mouse. Toxicol Appl Pharmacol, 90, 401–409.<br />
<br />
Pottenger, L.H., Andrews LS, Bachman AN, Boogaard PJ, Cadet J, Embry MR, Farmer PB, Himmelstein MW, Jarabek AM, Martin EA, Mauthe RJ, Persaud R, Preston RJ, Schoeny R, Skare J, Swenberg JA, Williams GM, Zeiger E, Zhang F, Kim JH. (2014). An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride. Crit. Rev. Toxicol. 44(4):348-391.<br />
<br />
Primiano T, Egner PA, Sutter TR, et al. (1995). Intermittent dosing with oltipraz: relationship between chemoprevention of aflatoxin-induced tumorigenesis and induction of glutathione-S-transferases. Cancer Res, 55, 4319–4324.<br />
<br />
Roebuck BD, Liu Y-L, Rogers AE, et al. (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short term molecular dosimetry. Cancer Res, 51, 5501–5506.<br />
<br />
Ueng Y-F, Shimada T, Yamazaki H, Guengerich FP (1995). Oxidation of aflatoxin B1 by bacterial recombinant human cytochrome P450 enzymes. Chem Res Toxiol 8:218-225.<br />
<br />
Wang J-S, Shen X, He X, et al. (1999). Protective alerations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qidong, People’s Republic of China. J Natl Cancer Inst, 91, 347–354.<br />
<br />
Yates MS, Kwak M-K, Egner PA, et al. (2006). Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl] imidazole. Cancer Res, 66, 2488–2494.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:376Event:3762016-07-06T17:02:43Z<p>Dvillene: /* KE#2: Evidence Supporting Essentiality */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Induced Mutations in Critical Genes, Increased </div><br />
<div id='shortTitle' class='Title'> Induced in Critical Genes, Mutation </div><br />
<br />
== Action ==<br />
Induced mutation<br />
<br />
== Biological Process/Object ==<br />
Critical genes for carcinogenesis<br />
<br />
== Induced Mutation in Critical Genes ==<br />
The AFB1-induced pro-mutagenic DNA adduct is either not repaired or is mis-repaired resulting in a mutation in one or more critical genes. In bacteria and mammalian cells (both in vitro and in vivo) the primary mutation associated with AFB1 is a guanine to thymine transversion (Foster et al., 1983; Dycaico et al., 1996). A G:T transversion is expected for the pro-mutagenic DNA adduct AFB1-FAPy. <br />
<br />
A specific critical mutation in codon 249 of the p53 gene has been identified in human hepatocellular carcinoma (HCC) (See section below on essentiality).<br />
<br />
== Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
A specific critical mutation in the p53 gene has been identified in human hepatocellular carcinoma (HCC). Demonstrating that AFB1 can induce this specific p53 gene mutation would be the highest level of evidence that AFB1-induced HCC involves mutation as a KE. Absent such information, the next best level of evidence is the induction of the specific type of mutation (G:C to T:A transversion) in a variety of gene mutation assays measuring a range of target genes. <br />
<br />
The codon 249 mutation is present in a significant proportion of human HCCs. In fact codon 249 mutation is detected in up to 50% of liver cancers in Qidong, China (Hsu et al., 1991) and in Mozambique, both areas with high likelihood of AFB1 exposure. The codon 249 G:C to T:A mutation in the third base is seen in up to 75% of HCC in high-incidence areas of China and East Africa (Gouas et al., 2009). In contrast, this specific mutation is very rare in HCC from areas with no or low exposure to AFB1 (Hsu et al. 1991 and Bressac et al. Nature, 350:429-431, 1991). This mutation is also very rare in other types of tumors (Gouas et al., 2009). According to Gouas et al. (2009), populations with AFB1 exposure are likely to be exposed to hepatitis B virus (HBV) as well and the effects of each are difficult to separate.This mutation is very rare in HCC from non/low -aflatoxin areas (Hsu et al. 1991 and Bressac et al., 1991) and also very rare in other types of tumors.<br />
<br />
Some indirect evidence of the essentiality of mutation in tumor development is provided by the clear species difference between adult mice and adult rats both in the induction of surrogate gene mutations and in the induction of tumors. Adult mice exposed to AFB1 do not get tumors and there is no increase in mutant frequency (MF) for Big Blue mice exposed as adults. That is, Lac I mutants from the AFB1- exposed adult mice showed a spontaneous mutational spectrum. Rats, however, showed a large increase in MF and, more specifically a large increase in G:C to T:A transversions (Dycaico et al., 1996). In addition, for mice there is an difference between neonata and adult mice. Neonatal mice treated with AFB1 (6 mg/kg—a dose that does result in tumors) showed an increase in cII mutation with G:C to T:A transversion as the major mutation. Adult mice treated at 6 and 60 mg/kg (doses that do not produce tumors) did not have a significant increase in cII mutation (but did give a different mutational spectrum than controls) (Chen et al. 2010).<br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||KE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Cellular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== KE#2: Taxonomic Applicability ==<br />
The induction of G:C to T:A transversions following AFB1 exposure is seen in a wide variety of species from bacteria to mammals. Assays that measure mutation in surrogate genes (that is, genes unrelated to the critical cancer genes) can be used to evaluate the full spectrum of mutational events that can be induced by a chemical. One such system is the AS52 assay, an in vitro mammalian transgenic mutation assay that measures mutation in the gpt gene. After exposure to AFB1 in culture the predominant mutation is G:C to T:A transversion, although a number of other types of mutations were also seen (Wattanawaraporn et al., 2012). There is a species difference between adult mice and rats. No increase in MF is seen in the in vivo Big Blue™ mutation assay for mice exposed to AFB1. That is, Lac I mutations from the mice showed a spontaneous mutational spectrum. By contrast rats showed a large increase in MF with a large increase in G:C to T:A transversions (Dycaico et al., 1996). However, neonatal mice treated with AFB1 (6 mg/kg—a dose that induces tumors in neonates) do show an increase in cII mutation with G:C to T:A transversion as the major mutation. Adult mice treated at 6 and 60 mg/kg (doses that do not induce tumors) did not show a significant increase in cII mutations but did produce a different mutational spectrum than controls (Chen et al., 2010.)<br />
<br />
== KE#2: Level of Biological Organization : Cellular ==<br />
The induction of mutation occurs within the nucleus of cells and involves permanent alterations in the primary DNA sequence that is passed to subsequent cell generations and, thus, is heritable.<br />
<br />
== KE#2: How this Key Event works ==<br />
Following the formation of DNA adducts, which are either mis-repaired or not repaired, the sequence of base pairs in the DNA is changed due to insertion of an incorrect base opposite the DNA adduct during DNA replication, so that a G is permanently replaced by a T. This type of mutation is observed in the mutants seen in surrogate gene mutation assays using AFB1 exposureA high frequency of codon 249 p53 mutations occurs in human tumors in high AFB1 exposure regions.Hence, codon 249 of the p53 gene contains a base pair susceptible to insufficient or misrepair of DNA adducts.<br />
<br />
Puisieux et al. (1991) provide evidence that the AFB1 epoxide adduct binds preferentially to codon 249 of the p53 gene. Using a plasmid with containing full-length human p53 DNA, adduct formation was observed in exons 5, 6, 7 and 8 (a total of 1086 bases) and 20% of the bases were targeted by AFB1 with a preference for guanine residues. Binding of AFB1 to p53 sequences was restricted to fewer residues and was more specific for guanine than was the binding of B[a]P (Puisieux et al.,1991). Binding of AFB1 in the region around codon 249 of p53 AFB1 was reported to be “stronger” than that of B[a]P. The last nucleotide of codon 249 is a guanine and was targeted by AFB1 but not by B[a]P. This guanine residue is the mutational hotspot in human liver cancers from patients in high AFB1 exposure regions. <br />
<br />
While mutations at codon 249 of the p53 gene have been observed in association with HCC in humans, the question remains whether this mutation occurs as a direct result of adduct formation at this site or by some different mechanism..In human HepG2 hepatocytes exposed to microsomally activated AFB1, a dose-dependent increase in G:C to T:A transversions were observed at 10 additional locations using ligation-mediated PCR, and at 4 additional locations using terminal-transferred dependent PCR.(Denissenko et al.,1998). These authors suggest that codon 249 may not present a key adduct site. However, other more recently published data identified codon 249 of the P53 gene as an unusually mutagenic adduct conformation based on the local DNA sequence and concluded that a higher mutation rate may occur there rather than at other locations because of increased DNA polymerase bypass (Pussieux et al., 1991; Lin et al., 2014a,b).<br />
<br />
== KE#2: How it is Measured or Detected ==<br />
Historically the detection of critical cancer gene specific mutations has not been technically feasible. A newly developed method, allele specific competitive blocker-polymerase chain reaction (ACB-PCR) has proven useful in providing such information, and data on specific chemical-induced mutations are available for a very small number of chemicals (Parsons et al., 2010). Unfortunately, there are no data for AFB1.<br />
<br />
There are, however, a number of gene mutation assays that have been widely used for determining the general ability of chemicals, including AFB1 to induce mutations.These assays use selection methods that allow only mutant cells to survive and grow. The AS52 in vitro gene mutation assay using the gpt gene, and 6-thioguanine selection has been used to demonstrate that AFB1 exposure increases the MF at the gpt gene. In vivo transgenic assays use molecular methods to recover the transgene from isolated DNA and to evaluate the MF in the transgene. Molecular methods can detect the presence (above a certain sensitivity level) of mutant cells. DNA from tumors can be sequenced to determine the presence of mutations in specific genes. DNA sequencing has been used on human tumors to detect the presence of the Codon 249 p53 mutation.<br />
<br />
== KE#2: Evidence Supporting Taxonomic Applicability ==<br />
There are data across phyla demonstrating the induction of mutations, specifically the induction to G:C to T:A transversions following AFB1 exposure.<br />
<br />
== KE#2: References ==<br />
Bressac, B., Kew, M., Wands, J., & Ozturk, M. (1991). Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern africa. Nature, 350(6317), 429-31. doi:10.1038/350429a<br />
<br />
Chen, T., Heflich, R. H., Moore, M. M., & Mei, N. (2010). Differential mutagenicity of aflatoxin B1 in the liver of neonatal and adult mice. Environ Mol Mutagen, 51(2), 156-63. doi:10.1002/em.2051<br />
<br />
Denissenko MF, Koudriakova TB, Smith L, O'Connor TR, Riggs AD, and Pfeifer GP). The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts. (Oncogene. 1998, Dec 10;17(23):3007-14.<br />
<br />
Dycaico, M. J., Stuart, G. R., Tobal, G. M., de Boer, J. G., Glickman, B. W., & Provost, G. S. (1996). Species-specific differences in hepatic mutant frequency and mutational spectrum among lambda/laci transgenic rats and mice following exposure to aflatoxin B1. Carcinogenesis, 17(11), 2347-56<br />
<br />
Foster, P. L., Eisenstadt, E., & Miller, J. H. (1983). Base substitution mutations induced by metabolically activated aflatoxin B1. Proceedings of the National Academy of Sciences of the United States of America, 80(9), 2695-8.<br />
<br />
Gouas, D., Shi, H., & Hainaut, P. (2009). The aflatoxin-induced TP53 mutation at codon 249 (R249S): Biomarker of exposure, early detection and target for therapy. Cancer Lett, 286(1), 29-37. doi:10.1016/j.canlet.2009.02.057<br />
<br />
Hsu, I. C., Metcalf, R. A., Sun, T., Welsh, J. A., Wang, N. J., & Harris, C. C. (1991). Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature, 350(6317), 427-8. doi:10.1038/350427a<br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014a). Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua. J Biol Chem. 289:18497-18506. <br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014b). Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis 35(7):1461-1468<br />
<br />
Parsons BL, Myers MB, Meng F, Wang Y, McKinzie PB. 2010. Oncomutations as biomarkers of cancer risk. Environ Mol Mutagen. 51(8-9):836-850.<br />
<br />
Puisieux, A., Lim, S., Groopman, J., & Ozturk, M. (1991). Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res, 51(22), 6185-9.<br />
<br />
Wattanawaraporn, R., Kim, M. Y., Adams, J., Trudel, L. J., Woo, L. L., Croy, R. G., . . . Wogan, G. N. (2012). AFB(1) -induced mutagenesis of the gpt gene in AS52 cells. Environ Mol Mutagen, 53(7), 567-73. doi:10.1002/em.2171</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:376Event:3762016-07-06T17:02:26Z<p>Dvillene: /* Step: KE#2: Induced Mutation in Critical Genes */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Induced Mutations in Critical Genes, Increased </div><br />
<div id='shortTitle' class='Title'> Induced in Critical Genes, Mutation </div><br />
<br />
== Action ==<br />
Induced mutation<br />
<br />
== Biological Process/Object ==<br />
Critical genes for carcinogenesis<br />
<br />
== Induced Mutation in Critical Genes ==<br />
The AFB1-induced pro-mutagenic DNA adduct is either not repaired or is mis-repaired resulting in a mutation in one or more critical genes. In bacteria and mammalian cells (both in vitro and in vivo) the primary mutation associated with AFB1 is a guanine to thymine transversion (Foster et al., 1983; Dycaico et al., 1996). A G:T transversion is expected for the pro-mutagenic DNA adduct AFB1-FAPy. <br />
<br />
A specific critical mutation in codon 249 of the p53 gene has been identified in human hepatocellular carcinoma (HCC) (See section below on essentiality).<br />
<br />
== KE#2: Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
A specific critical mutation in the p53 gene has been identified in human hepatocellular carcinoma (HCC). Demonstrating that AFB1 can induce this specific p53 gene mutation would be the highest level of evidence that AFB1-induced HCC involves mutation as a KE. Absent such information, the next best level of evidence is the induction of the specific type of mutation (G:C to T:A transversion) in a variety of gene mutation assays measuring a range of target genes. <br />
<br />
The codon 249 mutation is present in a significant proportion of human HCCs. In fact codon 249 mutation is detected in up to 50% of liver cancers in Qidong, China (Hsu et al., 1991) and in Mozambique, both areas with high likelihood of AFB1 exposure. The codon 249 G:C to T:A mutation in the third base is seen in up to 75% of HCC in high-incidence areas of China and East Africa (Gouas et al., 2009). In contrast, this specific mutation is very rare in HCC from areas with no or low exposure to AFB1 (Hsu et al. 1991 and Bressac et al. Nature, 350:429-431, 1991). This mutation is also very rare in other types of tumors (Gouas et al., 2009). According to Gouas et al. (2009), populations with AFB1 exposure are likely to be exposed to hepatitis B virus (HBV) as well and the effects of each are difficult to separate.This mutation is very rare in HCC from non/low -aflatoxin areas (Hsu et al. 1991 and Bressac et al., 1991) and also very rare in other types of tumors.<br />
<br />
Some indirect evidence of the essentiality of mutation in tumor development is provided by the clear species difference between adult mice and adult rats both in the induction of surrogate gene mutations and in the induction of tumors. Adult mice exposed to AFB1 do not get tumors and there is no increase in mutant frequency (MF) for Big Blue mice exposed as adults. That is, Lac I mutants from the AFB1- exposed adult mice showed a spontaneous mutational spectrum. Rats, however, showed a large increase in MF and, more specifically a large increase in G:C to T:A transversions (Dycaico et al., 1996). In addition, for mice there is an difference between neonata and adult mice. Neonatal mice treated with AFB1 (6 mg/kg—a dose that does result in tumors) showed an increase in cII mutation with G:C to T:A transversion as the major mutation. Adult mice treated at 6 and 60 mg/kg (doses that do not produce tumors) did not have a significant increase in cII mutation (but did give a different mutational spectrum than controls) (Chen et al. 2010).<br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||KE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Cellular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== KE#2: Taxonomic Applicability ==<br />
The induction of G:C to T:A transversions following AFB1 exposure is seen in a wide variety of species from bacteria to mammals. Assays that measure mutation in surrogate genes (that is, genes unrelated to the critical cancer genes) can be used to evaluate the full spectrum of mutational events that can be induced by a chemical. One such system is the AS52 assay, an in vitro mammalian transgenic mutation assay that measures mutation in the gpt gene. After exposure to AFB1 in culture the predominant mutation is G:C to T:A transversion, although a number of other types of mutations were also seen (Wattanawaraporn et al., 2012). There is a species difference between adult mice and rats. No increase in MF is seen in the in vivo Big Blue™ mutation assay for mice exposed to AFB1. That is, Lac I mutations from the mice showed a spontaneous mutational spectrum. By contrast rats showed a large increase in MF with a large increase in G:C to T:A transversions (Dycaico et al., 1996). However, neonatal mice treated with AFB1 (6 mg/kg—a dose that induces tumors in neonates) do show an increase in cII mutation with G:C to T:A transversion as the major mutation. Adult mice treated at 6 and 60 mg/kg (doses that do not induce tumors) did not show a significant increase in cII mutations but did produce a different mutational spectrum than controls (Chen et al., 2010.)<br />
<br />
== KE#2: Level of Biological Organization : Cellular ==<br />
The induction of mutation occurs within the nucleus of cells and involves permanent alterations in the primary DNA sequence that is passed to subsequent cell generations and, thus, is heritable.<br />
<br />
== KE#2: How this Key Event works ==<br />
Following the formation of DNA adducts, which are either mis-repaired or not repaired, the sequence of base pairs in the DNA is changed due to insertion of an incorrect base opposite the DNA adduct during DNA replication, so that a G is permanently replaced by a T. This type of mutation is observed in the mutants seen in surrogate gene mutation assays using AFB1 exposureA high frequency of codon 249 p53 mutations occurs in human tumors in high AFB1 exposure regions.Hence, codon 249 of the p53 gene contains a base pair susceptible to insufficient or misrepair of DNA adducts.<br />
<br />
Puisieux et al. (1991) provide evidence that the AFB1 epoxide adduct binds preferentially to codon 249 of the p53 gene. Using a plasmid with containing full-length human p53 DNA, adduct formation was observed in exons 5, 6, 7 and 8 (a total of 1086 bases) and 20% of the bases were targeted by AFB1 with a preference for guanine residues. Binding of AFB1 to p53 sequences was restricted to fewer residues and was more specific for guanine than was the binding of B[a]P (Puisieux et al.,1991). Binding of AFB1 in the region around codon 249 of p53 AFB1 was reported to be “stronger” than that of B[a]P. The last nucleotide of codon 249 is a guanine and was targeted by AFB1 but not by B[a]P. This guanine residue is the mutational hotspot in human liver cancers from patients in high AFB1 exposure regions. <br />
<br />
While mutations at codon 249 of the p53 gene have been observed in association with HCC in humans, the question remains whether this mutation occurs as a direct result of adduct formation at this site or by some different mechanism..In human HepG2 hepatocytes exposed to microsomally activated AFB1, a dose-dependent increase in G:C to T:A transversions were observed at 10 additional locations using ligation-mediated PCR, and at 4 additional locations using terminal-transferred dependent PCR.(Denissenko et al.,1998). These authors suggest that codon 249 may not present a key adduct site. However, other more recently published data identified codon 249 of the P53 gene as an unusually mutagenic adduct conformation based on the local DNA sequence and concluded that a higher mutation rate may occur there rather than at other locations because of increased DNA polymerase bypass (Pussieux et al., 1991; Lin et al., 2014a,b).<br />
<br />
== KE#2: How it is Measured or Detected ==<br />
Historically the detection of critical cancer gene specific mutations has not been technically feasible. A newly developed method, allele specific competitive blocker-polymerase chain reaction (ACB-PCR) has proven useful in providing such information, and data on specific chemical-induced mutations are available for a very small number of chemicals (Parsons et al., 2010). Unfortunately, there are no data for AFB1.<br />
<br />
There are, however, a number of gene mutation assays that have been widely used for determining the general ability of chemicals, including AFB1 to induce mutations.These assays use selection methods that allow only mutant cells to survive and grow. The AS52 in vitro gene mutation assay using the gpt gene, and 6-thioguanine selection has been used to demonstrate that AFB1 exposure increases the MF at the gpt gene. In vivo transgenic assays use molecular methods to recover the transgene from isolated DNA and to evaluate the MF in the transgene. Molecular methods can detect the presence (above a certain sensitivity level) of mutant cells. DNA from tumors can be sequenced to determine the presence of mutations in specific genes. DNA sequencing has been used on human tumors to detect the presence of the Codon 249 p53 mutation.<br />
<br />
== KE#2: Evidence Supporting Taxonomic Applicability ==<br />
There are data across phyla demonstrating the induction of mutations, specifically the induction to G:C to T:A transversions following AFB1 exposure.<br />
<br />
== KE#2: References ==<br />
Bressac, B., Kew, M., Wands, J., & Ozturk, M. (1991). Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern africa. Nature, 350(6317), 429-31. doi:10.1038/350429a<br />
<br />
Chen, T., Heflich, R. H., Moore, M. M., & Mei, N. (2010). Differential mutagenicity of aflatoxin B1 in the liver of neonatal and adult mice. Environ Mol Mutagen, 51(2), 156-63. doi:10.1002/em.2051<br />
<br />
Denissenko MF, Koudriakova TB, Smith L, O'Connor TR, Riggs AD, and Pfeifer GP). The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts. (Oncogene. 1998, Dec 10;17(23):3007-14.<br />
<br />
Dycaico, M. J., Stuart, G. R., Tobal, G. M., de Boer, J. G., Glickman, B. W., & Provost, G. S. (1996). Species-specific differences in hepatic mutant frequency and mutational spectrum among lambda/laci transgenic rats and mice following exposure to aflatoxin B1. Carcinogenesis, 17(11), 2347-56<br />
<br />
Foster, P. L., Eisenstadt, E., & Miller, J. H. (1983). Base substitution mutations induced by metabolically activated aflatoxin B1. Proceedings of the National Academy of Sciences of the United States of America, 80(9), 2695-8.<br />
<br />
Gouas, D., Shi, H., & Hainaut, P. (2009). The aflatoxin-induced TP53 mutation at codon 249 (R249S): Biomarker of exposure, early detection and target for therapy. Cancer Lett, 286(1), 29-37. doi:10.1016/j.canlet.2009.02.057<br />
<br />
Hsu, I. C., Metcalf, R. A., Sun, T., Welsh, J. A., Wang, N. J., & Harris, C. C. (1991). Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature, 350(6317), 427-8. doi:10.1038/350427a<br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014a). Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua. J Biol Chem. 289:18497-18506. <br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014b). Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis 35(7):1461-1468<br />
<br />
Parsons BL, Myers MB, Meng F, Wang Y, McKinzie PB. 2010. Oncomutations as biomarkers of cancer risk. Environ Mol Mutagen. 51(8-9):836-850.<br />
<br />
Puisieux, A., Lim, S., Groopman, J., & Ozturk, M. (1991). Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res, 51(22), 6185-9.<br />
<br />
Wattanawaraporn, R., Kim, M. Y., Adams, J., Trudel, L. J., Woo, L. L., Croy, R. G., . . . Wogan, G. N. (2012). AFB(1) -induced mutagenesis of the gpt gene in AS52 cells. Environ Mol Mutagen, 53(7), 567-73. doi:10.1002/em.2171</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:376Event:3762016-07-06T17:02:03Z<p>Dvillene: /* KE#2: Biological Process/Object */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Induced Mutations in Critical Genes, Increased </div><br />
<div id='shortTitle' class='Title'> Induced in Critical Genes, Mutation </div><br />
<br />
== Action ==<br />
Induced mutation<br />
<br />
== Biological Process/Object ==<br />
Critical genes for carcinogenesis<br />
<br />
== Step: KE#2: Induced Mutation in Critical Genes ==<br />
The AFB1-induced pro-mutagenic DNA adduct is either not repaired or is mis-repaired resulting in a mutation in one or more critical genes. In bacteria and mammalian cells (both in vitro and in vivo) the primary mutation associated with AFB1 is a guanine to thymine transversion (Foster et al., 1983; Dycaico et al., 1996). A G:T transversion is expected for the pro-mutagenic DNA adduct AFB1-FAPy. <br />
<br />
A specific critical mutation in codon 249 of the p53 gene has been identified in human hepatocellular carcinoma (HCC) (See section below on essentiality).<br />
<br />
== KE#2: Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
A specific critical mutation in the p53 gene has been identified in human hepatocellular carcinoma (HCC). Demonstrating that AFB1 can induce this specific p53 gene mutation would be the highest level of evidence that AFB1-induced HCC involves mutation as a KE. Absent such information, the next best level of evidence is the induction of the specific type of mutation (G:C to T:A transversion) in a variety of gene mutation assays measuring a range of target genes. <br />
<br />
The codon 249 mutation is present in a significant proportion of human HCCs. In fact codon 249 mutation is detected in up to 50% of liver cancers in Qidong, China (Hsu et al., 1991) and in Mozambique, both areas with high likelihood of AFB1 exposure. The codon 249 G:C to T:A mutation in the third base is seen in up to 75% of HCC in high-incidence areas of China and East Africa (Gouas et al., 2009). In contrast, this specific mutation is very rare in HCC from areas with no or low exposure to AFB1 (Hsu et al. 1991 and Bressac et al. Nature, 350:429-431, 1991). This mutation is also very rare in other types of tumors (Gouas et al., 2009). According to Gouas et al. (2009), populations with AFB1 exposure are likely to be exposed to hepatitis B virus (HBV) as well and the effects of each are difficult to separate.This mutation is very rare in HCC from non/low -aflatoxin areas (Hsu et al. 1991 and Bressac et al., 1991) and also very rare in other types of tumors.<br />
<br />
Some indirect evidence of the essentiality of mutation in tumor development is provided by the clear species difference between adult mice and adult rats both in the induction of surrogate gene mutations and in the induction of tumors. Adult mice exposed to AFB1 do not get tumors and there is no increase in mutant frequency (MF) for Big Blue mice exposed as adults. That is, Lac I mutants from the AFB1- exposed adult mice showed a spontaneous mutational spectrum. Rats, however, showed a large increase in MF and, more specifically a large increase in G:C to T:A transversions (Dycaico et al., 1996). In addition, for mice there is an difference between neonata and adult mice. Neonatal mice treated with AFB1 (6 mg/kg—a dose that does result in tumors) showed an increase in cII mutation with G:C to T:A transversion as the major mutation. Adult mice treated at 6 and 60 mg/kg (doses that do not produce tumors) did not have a significant increase in cII mutation (but did give a different mutational spectrum than controls) (Chen et al. 2010).<br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||KE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Cellular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== KE#2: Taxonomic Applicability ==<br />
The induction of G:C to T:A transversions following AFB1 exposure is seen in a wide variety of species from bacteria to mammals. Assays that measure mutation in surrogate genes (that is, genes unrelated to the critical cancer genes) can be used to evaluate the full spectrum of mutational events that can be induced by a chemical. One such system is the AS52 assay, an in vitro mammalian transgenic mutation assay that measures mutation in the gpt gene. After exposure to AFB1 in culture the predominant mutation is G:C to T:A transversion, although a number of other types of mutations were also seen (Wattanawaraporn et al., 2012). There is a species difference between adult mice and rats. No increase in MF is seen in the in vivo Big Blue™ mutation assay for mice exposed to AFB1. That is, Lac I mutations from the mice showed a spontaneous mutational spectrum. By contrast rats showed a large increase in MF with a large increase in G:C to T:A transversions (Dycaico et al., 1996). However, neonatal mice treated with AFB1 (6 mg/kg—a dose that induces tumors in neonates) do show an increase in cII mutation with G:C to T:A transversion as the major mutation. Adult mice treated at 6 and 60 mg/kg (doses that do not induce tumors) did not show a significant increase in cII mutations but did produce a different mutational spectrum than controls (Chen et al., 2010.)<br />
<br />
== KE#2: Level of Biological Organization : Cellular ==<br />
The induction of mutation occurs within the nucleus of cells and involves permanent alterations in the primary DNA sequence that is passed to subsequent cell generations and, thus, is heritable.<br />
<br />
== KE#2: How this Key Event works ==<br />
Following the formation of DNA adducts, which are either mis-repaired or not repaired, the sequence of base pairs in the DNA is changed due to insertion of an incorrect base opposite the DNA adduct during DNA replication, so that a G is permanently replaced by a T. This type of mutation is observed in the mutants seen in surrogate gene mutation assays using AFB1 exposureA high frequency of codon 249 p53 mutations occurs in human tumors in high AFB1 exposure regions.Hence, codon 249 of the p53 gene contains a base pair susceptible to insufficient or misrepair of DNA adducts.<br />
<br />
Puisieux et al. (1991) provide evidence that the AFB1 epoxide adduct binds preferentially to codon 249 of the p53 gene. Using a plasmid with containing full-length human p53 DNA, adduct formation was observed in exons 5, 6, 7 and 8 (a total of 1086 bases) and 20% of the bases were targeted by AFB1 with a preference for guanine residues. Binding of AFB1 to p53 sequences was restricted to fewer residues and was more specific for guanine than was the binding of B[a]P (Puisieux et al.,1991). Binding of AFB1 in the region around codon 249 of p53 AFB1 was reported to be “stronger” than that of B[a]P. The last nucleotide of codon 249 is a guanine and was targeted by AFB1 but not by B[a]P. This guanine residue is the mutational hotspot in human liver cancers from patients in high AFB1 exposure regions. <br />
<br />
While mutations at codon 249 of the p53 gene have been observed in association with HCC in humans, the question remains whether this mutation occurs as a direct result of adduct formation at this site or by some different mechanism..In human HepG2 hepatocytes exposed to microsomally activated AFB1, a dose-dependent increase in G:C to T:A transversions were observed at 10 additional locations using ligation-mediated PCR, and at 4 additional locations using terminal-transferred dependent PCR.(Denissenko et al.,1998). These authors suggest that codon 249 may not present a key adduct site. However, other more recently published data identified codon 249 of the P53 gene as an unusually mutagenic adduct conformation based on the local DNA sequence and concluded that a higher mutation rate may occur there rather than at other locations because of increased DNA polymerase bypass (Pussieux et al., 1991; Lin et al., 2014a,b).<br />
<br />
== KE#2: How it is Measured or Detected ==<br />
Historically the detection of critical cancer gene specific mutations has not been technically feasible. A newly developed method, allele specific competitive blocker-polymerase chain reaction (ACB-PCR) has proven useful in providing such information, and data on specific chemical-induced mutations are available for a very small number of chemicals (Parsons et al., 2010). Unfortunately, there are no data for AFB1.<br />
<br />
There are, however, a number of gene mutation assays that have been widely used for determining the general ability of chemicals, including AFB1 to induce mutations.These assays use selection methods that allow only mutant cells to survive and grow. The AS52 in vitro gene mutation assay using the gpt gene, and 6-thioguanine selection has been used to demonstrate that AFB1 exposure increases the MF at the gpt gene. In vivo transgenic assays use molecular methods to recover the transgene from isolated DNA and to evaluate the MF in the transgene. Molecular methods can detect the presence (above a certain sensitivity level) of mutant cells. DNA from tumors can be sequenced to determine the presence of mutations in specific genes. DNA sequencing has been used on human tumors to detect the presence of the Codon 249 p53 mutation.<br />
<br />
== KE#2: Evidence Supporting Taxonomic Applicability ==<br />
There are data across phyla demonstrating the induction of mutations, specifically the induction to G:C to T:A transversions following AFB1 exposure.<br />
<br />
== KE#2: References ==<br />
Bressac, B., Kew, M., Wands, J., & Ozturk, M. (1991). Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern africa. Nature, 350(6317), 429-31. doi:10.1038/350429a<br />
<br />
Chen, T., Heflich, R. H., Moore, M. M., & Mei, N. (2010). Differential mutagenicity of aflatoxin B1 in the liver of neonatal and adult mice. Environ Mol Mutagen, 51(2), 156-63. doi:10.1002/em.2051<br />
<br />
Denissenko MF, Koudriakova TB, Smith L, O'Connor TR, Riggs AD, and Pfeifer GP). The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts. (Oncogene. 1998, Dec 10;17(23):3007-14.<br />
<br />
Dycaico, M. J., Stuart, G. R., Tobal, G. M., de Boer, J. G., Glickman, B. W., & Provost, G. S. (1996). Species-specific differences in hepatic mutant frequency and mutational spectrum among lambda/laci transgenic rats and mice following exposure to aflatoxin B1. Carcinogenesis, 17(11), 2347-56<br />
<br />
Foster, P. L., Eisenstadt, E., & Miller, J. H. (1983). Base substitution mutations induced by metabolically activated aflatoxin B1. Proceedings of the National Academy of Sciences of the United States of America, 80(9), 2695-8.<br />
<br />
Gouas, D., Shi, H., & Hainaut, P. (2009). The aflatoxin-induced TP53 mutation at codon 249 (R249S): Biomarker of exposure, early detection and target for therapy. Cancer Lett, 286(1), 29-37. doi:10.1016/j.canlet.2009.02.057<br />
<br />
Hsu, I. C., Metcalf, R. A., Sun, T., Welsh, J. A., Wang, N. J., & Harris, C. C. (1991). Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature, 350(6317), 427-8. doi:10.1038/350427a<br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014a). Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua. J Biol Chem. 289:18497-18506. <br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014b). Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis 35(7):1461-1468<br />
<br />
Parsons BL, Myers MB, Meng F, Wang Y, McKinzie PB. 2010. Oncomutations as biomarkers of cancer risk. Environ Mol Mutagen. 51(8-9):836-850.<br />
<br />
Puisieux, A., Lim, S., Groopman, J., & Ozturk, M. (1991). Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res, 51(22), 6185-9.<br />
<br />
Wattanawaraporn, R., Kim, M. Y., Adams, J., Trudel, L. J., Woo, L. L., Croy, R. G., . . . Wogan, G. N. (2012). AFB(1) -induced mutagenesis of the gpt gene in AS52 cells. Environ Mol Mutagen, 53(7), 567-73. doi:10.1002/em.2171</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:376Event:3762016-07-06T17:01:45Z<p>Dvillene: /* KE#2: Action */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Induced Mutations in Critical Genes, Increased </div><br />
<div id='shortTitle' class='Title'> Induced in Critical Genes, Mutation </div><br />
<br />
== Action ==<br />
Induced mutation<br />
<br />
== KE#2: Biological Process/Object ==<br />
Critical genes for carcinogenesis<br />
<br />
== Step: KE#2: Induced Mutation in Critical Genes ==<br />
The AFB1-induced pro-mutagenic DNA adduct is either not repaired or is mis-repaired resulting in a mutation in one or more critical genes. In bacteria and mammalian cells (both in vitro and in vivo) the primary mutation associated with AFB1 is a guanine to thymine transversion (Foster et al., 1983; Dycaico et al., 1996). A G:T transversion is expected for the pro-mutagenic DNA adduct AFB1-FAPy. <br />
<br />
A specific critical mutation in codon 249 of the p53 gene has been identified in human hepatocellular carcinoma (HCC) (See section below on essentiality).<br />
<br />
== KE#2: Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
A specific critical mutation in the p53 gene has been identified in human hepatocellular carcinoma (HCC). Demonstrating that AFB1 can induce this specific p53 gene mutation would be the highest level of evidence that AFB1-induced HCC involves mutation as a KE. Absent such information, the next best level of evidence is the induction of the specific type of mutation (G:C to T:A transversion) in a variety of gene mutation assays measuring a range of target genes. <br />
<br />
The codon 249 mutation is present in a significant proportion of human HCCs. In fact codon 249 mutation is detected in up to 50% of liver cancers in Qidong, China (Hsu et al., 1991) and in Mozambique, both areas with high likelihood of AFB1 exposure. The codon 249 G:C to T:A mutation in the third base is seen in up to 75% of HCC in high-incidence areas of China and East Africa (Gouas et al., 2009). In contrast, this specific mutation is very rare in HCC from areas with no or low exposure to AFB1 (Hsu et al. 1991 and Bressac et al. Nature, 350:429-431, 1991). This mutation is also very rare in other types of tumors (Gouas et al., 2009). According to Gouas et al. (2009), populations with AFB1 exposure are likely to be exposed to hepatitis B virus (HBV) as well and the effects of each are difficult to separate.This mutation is very rare in HCC from non/low -aflatoxin areas (Hsu et al. 1991 and Bressac et al., 1991) and also very rare in other types of tumors.<br />
<br />
Some indirect evidence of the essentiality of mutation in tumor development is provided by the clear species difference between adult mice and adult rats both in the induction of surrogate gene mutations and in the induction of tumors. Adult mice exposed to AFB1 do not get tumors and there is no increase in mutant frequency (MF) for Big Blue mice exposed as adults. That is, Lac I mutants from the AFB1- exposed adult mice showed a spontaneous mutational spectrum. Rats, however, showed a large increase in MF and, more specifically a large increase in G:C to T:A transversions (Dycaico et al., 1996). In addition, for mice there is an difference between neonata and adult mice. Neonatal mice treated with AFB1 (6 mg/kg—a dose that does result in tumors) showed an increase in cII mutation with G:C to T:A transversion as the major mutation. Adult mice treated at 6 and 60 mg/kg (doses that do not produce tumors) did not have a significant increase in cII mutation (but did give a different mutational spectrum than controls) (Chen et al. 2010).<br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||KE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Cellular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== KE#2: Taxonomic Applicability ==<br />
The induction of G:C to T:A transversions following AFB1 exposure is seen in a wide variety of species from bacteria to mammals. Assays that measure mutation in surrogate genes (that is, genes unrelated to the critical cancer genes) can be used to evaluate the full spectrum of mutational events that can be induced by a chemical. One such system is the AS52 assay, an in vitro mammalian transgenic mutation assay that measures mutation in the gpt gene. After exposure to AFB1 in culture the predominant mutation is G:C to T:A transversion, although a number of other types of mutations were also seen (Wattanawaraporn et al., 2012). There is a species difference between adult mice and rats. No increase in MF is seen in the in vivo Big Blue™ mutation assay for mice exposed to AFB1. That is, Lac I mutations from the mice showed a spontaneous mutational spectrum. By contrast rats showed a large increase in MF with a large increase in G:C to T:A transversions (Dycaico et al., 1996). However, neonatal mice treated with AFB1 (6 mg/kg—a dose that induces tumors in neonates) do show an increase in cII mutation with G:C to T:A transversion as the major mutation. Adult mice treated at 6 and 60 mg/kg (doses that do not induce tumors) did not show a significant increase in cII mutations but did produce a different mutational spectrum than controls (Chen et al., 2010.)<br />
<br />
== KE#2: Level of Biological Organization : Cellular ==<br />
The induction of mutation occurs within the nucleus of cells and involves permanent alterations in the primary DNA sequence that is passed to subsequent cell generations and, thus, is heritable.<br />
<br />
== KE#2: How this Key Event works ==<br />
Following the formation of DNA adducts, which are either mis-repaired or not repaired, the sequence of base pairs in the DNA is changed due to insertion of an incorrect base opposite the DNA adduct during DNA replication, so that a G is permanently replaced by a T. This type of mutation is observed in the mutants seen in surrogate gene mutation assays using AFB1 exposureA high frequency of codon 249 p53 mutations occurs in human tumors in high AFB1 exposure regions.Hence, codon 249 of the p53 gene contains a base pair susceptible to insufficient or misrepair of DNA adducts.<br />
<br />
Puisieux et al. (1991) provide evidence that the AFB1 epoxide adduct binds preferentially to codon 249 of the p53 gene. Using a plasmid with containing full-length human p53 DNA, adduct formation was observed in exons 5, 6, 7 and 8 (a total of 1086 bases) and 20% of the bases were targeted by AFB1 with a preference for guanine residues. Binding of AFB1 to p53 sequences was restricted to fewer residues and was more specific for guanine than was the binding of B[a]P (Puisieux et al.,1991). Binding of AFB1 in the region around codon 249 of p53 AFB1 was reported to be “stronger” than that of B[a]P. The last nucleotide of codon 249 is a guanine and was targeted by AFB1 but not by B[a]P. This guanine residue is the mutational hotspot in human liver cancers from patients in high AFB1 exposure regions. <br />
<br />
While mutations at codon 249 of the p53 gene have been observed in association with HCC in humans, the question remains whether this mutation occurs as a direct result of adduct formation at this site or by some different mechanism..In human HepG2 hepatocytes exposed to microsomally activated AFB1, a dose-dependent increase in G:C to T:A transversions were observed at 10 additional locations using ligation-mediated PCR, and at 4 additional locations using terminal-transferred dependent PCR.(Denissenko et al.,1998). These authors suggest that codon 249 may not present a key adduct site. However, other more recently published data identified codon 249 of the P53 gene as an unusually mutagenic adduct conformation based on the local DNA sequence and concluded that a higher mutation rate may occur there rather than at other locations because of increased DNA polymerase bypass (Pussieux et al., 1991; Lin et al., 2014a,b).<br />
<br />
== KE#2: How it is Measured or Detected ==<br />
Historically the detection of critical cancer gene specific mutations has not been technically feasible. A newly developed method, allele specific competitive blocker-polymerase chain reaction (ACB-PCR) has proven useful in providing such information, and data on specific chemical-induced mutations are available for a very small number of chemicals (Parsons et al., 2010). Unfortunately, there are no data for AFB1.<br />
<br />
There are, however, a number of gene mutation assays that have been widely used for determining the general ability of chemicals, including AFB1 to induce mutations.These assays use selection methods that allow only mutant cells to survive and grow. The AS52 in vitro gene mutation assay using the gpt gene, and 6-thioguanine selection has been used to demonstrate that AFB1 exposure increases the MF at the gpt gene. In vivo transgenic assays use molecular methods to recover the transgene from isolated DNA and to evaluate the MF in the transgene. Molecular methods can detect the presence (above a certain sensitivity level) of mutant cells. DNA from tumors can be sequenced to determine the presence of mutations in specific genes. DNA sequencing has been used on human tumors to detect the presence of the Codon 249 p53 mutation.<br />
<br />
== KE#2: Evidence Supporting Taxonomic Applicability ==<br />
There are data across phyla demonstrating the induction of mutations, specifically the induction to G:C to T:A transversions following AFB1 exposure.<br />
<br />
== KE#2: References ==<br />
Bressac, B., Kew, M., Wands, J., & Ozturk, M. (1991). Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern africa. Nature, 350(6317), 429-31. doi:10.1038/350429a<br />
<br />
Chen, T., Heflich, R. H., Moore, M. M., & Mei, N. (2010). Differential mutagenicity of aflatoxin B1 in the liver of neonatal and adult mice. Environ Mol Mutagen, 51(2), 156-63. doi:10.1002/em.2051<br />
<br />
Denissenko MF, Koudriakova TB, Smith L, O'Connor TR, Riggs AD, and Pfeifer GP). The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts. (Oncogene. 1998, Dec 10;17(23):3007-14.<br />
<br />
Dycaico, M. J., Stuart, G. R., Tobal, G. M., de Boer, J. G., Glickman, B. W., & Provost, G. S. (1996). Species-specific differences in hepatic mutant frequency and mutational spectrum among lambda/laci transgenic rats and mice following exposure to aflatoxin B1. Carcinogenesis, 17(11), 2347-56<br />
<br />
Foster, P. L., Eisenstadt, E., & Miller, J. H. (1983). Base substitution mutations induced by metabolically activated aflatoxin B1. Proceedings of the National Academy of Sciences of the United States of America, 80(9), 2695-8.<br />
<br />
Gouas, D., Shi, H., & Hainaut, P. (2009). The aflatoxin-induced TP53 mutation at codon 249 (R249S): Biomarker of exposure, early detection and target for therapy. Cancer Lett, 286(1), 29-37. doi:10.1016/j.canlet.2009.02.057<br />
<br />
Hsu, I. C., Metcalf, R. A., Sun, T., Welsh, J. A., Wang, N. J., & Harris, C. C. (1991). Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature, 350(6317), 427-8. doi:10.1038/350427a<br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014a). Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua. J Biol Chem. 289:18497-18506. <br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014b). Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis 35(7):1461-1468<br />
<br />
Parsons BL, Myers MB, Meng F, Wang Y, McKinzie PB. 2010. Oncomutations as biomarkers of cancer risk. Environ Mol Mutagen. 51(8-9):836-850.<br />
<br />
Puisieux, A., Lim, S., Groopman, J., & Ozturk, M. (1991). Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res, 51(22), 6185-9.<br />
<br />
Wattanawaraporn, R., Kim, M. Y., Adams, J., Trudel, L. J., Woo, L. L., Croy, R. G., . . . Wogan, G. N. (2012). AFB(1) -induced mutagenesis of the gpt gene in AS52 cells. Environ Mol Mutagen, 53(7), 567-73. doi:10.1002/em.2171</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:375Event:3752016-07-06T16:58:36Z<p>Dvillene: /* KE#1: Evidence Supporting Taxonomic Applicability */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Replaced by Event:493 </div><br />
<br />
== Action ==<br />
Insufficient repair or mis-repair of DNA<br />
<br />
== Biological Process/Object ==<br />
pro-mutagenic DNA adducts, a KE for this AOP<br />
<br />
== Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
Evidence supporting the insufficient repair or mis-repair of pro-mutagenic DNA adducts is strong, but mainly indirect. This evidence comes from different biological systems and datasets, mostly mammalian, and is based in large part on the biological understanding of a DNA-reactive mode of action requiring a change in primary DNA sequence (mutation). Such a change occurs when DNA lesions are not repaired prior to cell replication (Preston and Williams, 2005; Jarabek et al, 2009; Pottenger et al., 2014). AFB1 forms two types of pro-mutagenic adducts: N7-AFB1-G and AFB1-FAPy DNA adducts, and the resulting mutations are predominantly G:C to T:A transversions. These changes are the expected result of such adducts not being repaired or undergoing mis-repair (Bedard and Massey, 2006; Lin et al., 2014a,b).<br />
<br />
Data from diverse cell types and systems demonstrate that AFB1-induced DNA adducts are repaired by a variety of DNA repair processes, including SOS repair, nucleotide excision repair (NER), homologous recombination (HR), and post-replication repair (Bedard and Massey, 2006). The work by Guo et al. (2005), conducted in transgenic yeast modified to express human CYP1A2, is particularly useful as several DNA repair systems were evaluated for efficacy towards the AFB1-induced DNA adducts. Mutations were more likely to be induced in strains deficient in certain repair systems. Mutations were also induced in strains with active secondary repair pathways; these include pathways for error-prone post-replication repair and those relying on apurinic endonucleases. However, NER appears to be most important for repair of AFB1-induced adducts in mammalian systems (Bedard and Massey, 2006).<br />
<br />
The codon 249 of the p53 gene has been identified as a particular target of AFB1-induced adduction and subsequent mutation. Using DNA polymerase fingerprint analysis, Puiseux et al. (1991) showed a specificity of AFB1 epoxide binding for this codon. This specificity was not found with benzo[a]pyrene (B[a]P), although B[a]P also forms bulky, intercalating epoxides, similar to AFB1 exo-epoxide, which result in bulky N7-B[a]P-G adducts. <br />
<br />
Essentiality of this key event of insufficient or mis-repair is supported in part by datasets demonstrating that a reduction in AFB1 adduct burden (e.g., 65-70% reduction) results in a significantly reduced or even eliminated altered hepatic foci (AHF) and liver tumor burden in experimental animals (Roebuck et al., 1991; Elegbede and Gould, 2002; Yates et al., 2006; Johnson et al., 2014). Data from studies in human populations show that treatment with known modulators of AFB1 metabolism (e.g., with oltipraz or chlorophyllin) resulted in reduced urinary levels of N7-AFB1-G, derived from AFB1-induced DNA adducts (Egner et al., 2006). These treatments are also observed to decrease levels of a human biomarker of exposure, AFB1-induced albumin adducts (Kensler et al., 1998). A similar dataset in rats pre-treated with the triterpenoid, ODDC-Im, provides an even more convincing picture, wherein a chronic bioassay demonstrated a ~60% reduction in AFB1 DNA adducts, complete elimination of liver tumors, and nearly complete elimination of altered hepatic foci (Johnson et al., 2014).<br />
Some studies suggest that eukaryotic NER expression is induced by exposure to phytochemicals (Gross-Steinmeyer et al. 2010), and alteration of DNA repair has been suggested as a pathway of chemoprevention for AFB1 carcinogenesis (Gross-Steinmeyer and Eaton, 2012).<br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Taxonomic Applicability ==<br />
Measurements of repair of AFB1-induced DNA adduct have focused mainly on in vitro systems with bacteria, yeast, and mammalian cells, including cell lines derived from rats and non-human primates (Oleykowski et al., 1993; Leadon et al., 1981; Levy et al.,1992; Sarasin et al., 1977; Guo et al., 2005; Gross-Steinmeyer and Eaton, 2012). Relevant recent studies evaluated mutation induction by AFB1 in Caenorhabditis elegans (Leung et al., 2010; Meir et al., 2014). It is likely that all species which are capable of DNA repair following binding of AFB1 exo-epoxide to DNA—are subject to insufficient or mis-repair.<br />
<br />
== Level of Biological Organization : Cellular ==<br />
Repair of DNA adducts occurs at the sub-cellular level, as the target is nuclear DNA. For AFB1, the cross-species critical target is repair of hepatocyte nuclear DNA, as the eventual AFB1-induced tumor is hepatocellular carcinoma (HCC), which is initiated in hepatocytes.<br />
<br />
== How this Key Event works ==<br />
The AFB1-induced pro-mutagenic DNA adducts, N7-AFB1-G and /or AFB1-FAPy, are recognized by DNA repair proteins or systems, which then initiate their repair processes (Bedard and Massey, 2006). <br />
<br />
Although there are no data specific to AFB1 for this key event of insufficient/mis-repair of DNA, it is a process known to occur and known to result in mutations. The system that recognizes the damage depends in part upon which stage of the cell cycle is in progress when the pro-mutagenic DNA adducts are recognized. Initial steps can include recruitment of a series of proteins specific to that repair system and can also include blocking of progress of the DNA replication fork. This latter step ensures that DNA replication waits for repair to occur before proceeding, preventing replication of the damaged DNA, thus avoiding a mutation when the repair might otherwise faithfully copy the error.<br />
<br />
[[Mis-repair:]] For many of the DNA repair systems the repair process is quite faithful; that is, damage is correctly repaired with the correct or original base substituted for the adducted base. This is not always the case, however, and erroneous repair is one source of mutations, when an incorrect base inserted opposite a correct or an adducted base (mis-repair). <br />
<br />
[[Insufficient repair:]] An alternative route to mutation can occur when on-going DNA replication is not stopped, and DNA replication occurs across an adducted (typically non-informational lesion) base that has not undergone any repair (insufficient repair). This situation can result in the incorporation of an incorrect base into the nascent DNA strand. <br />
<br />
Insufficient repair or mis-repair results in an incorrect base incorporated into the nascent DNA strand, an error which is then eventually replicated, resulting in a so-called “fixed” change in the primary DNA sequence—a mutation. This mutation may be silent (no impact on protein product structure and/or function) or expressed (non-functional or differently functional protein product). Depending on the location or gene of the expressed mutation, the biological impact may none, slight, or substantial. For example, if the mutation is located in codon 249 of the p53 gene and is expressed, there is potential for substantial biological impact, including progression toward carcinogenesis. A substantial biological impact is less likely when the mutation results in no change in the coded amino acid due to degenerate nature of the genetic code for amino acid sequence. Likewise a change in amino acid sequence may not result in any change in the activity or function of the protein product.<br />
<br />
== How it is Measured or Detected ==<br />
One approach would be quantitation of chemical-specific DNA adducts, such as the N7-AFB1-G and AFB1-FAPy DNA adducts, before and after DNA repair occurs combined with DNA sequence data to determine whether the adducts were correctly repaired or not. Such is not a very practical approach. Measurement of mutations following AFB1 treatment of yeast with a variety of DNA repair deficiencies has been used to elucidate the role of DNA repair (Guo et al., 2005) by comparing mutant frequency (MF) in wild type vs. DNA repair deficient strains. In this instance MF serves as a marker for the insufficient or mis-repair of the AFB1 DNA adducts. The exquisitely sensitive analytical techniques available for structural quantification of these chemical-specific DNA adducts require specialized analytical chemistry techniques conducted on DNA isolated from tissues or cells and subjected to neutral thermal or enzyme or acid hydrolysis to release the adducted bases, which are then further analyzed (Himmelstein et al., 2009). Demonstration of adduct dose-responses and temporal relationships are possible with administration of a variety of dose regimens, including repeated doses.<br />
<br />
== Evidence Supporting Taxonomic Applicability ==<br />
Measurements of repair of AFB1-induced DNA adduct have focused mainly on in vitro systems with bacteria, yeast, and mammalian cells, including cell lines derived from rats and non-human primates (Oleykowski et al., 1993; Leadon et al., 1981; Levy et al.,1992; Sarasin et al., 1977; Guo et al., 2005; Gross-Steinmeyer and Eaton, 2012). Relevant recent studies evaluated mutation induction of AFB1 in Caenorhabditis elegans (Leung et al., 2010; Meir et al., 2014), Likely, all species capable of DNA repair following binding of AFB1 exo-epoxide to DNA are subject to insufficient or mis-repair These species include bacteria, yeast, birds, mammals, and fish.<br />
<br />
== References ==<br />
Bedard, L.L., and Massey, T.T. (2006). Aflatoxin B1-induced DNA damage and its repair. Cancer Lett. 241(2):174-183.<br />
<br />
Egner PA, Groopman JD, Wang J-S, Kensler TW, Friesen MD (2006). Quantification of aflatoxin-B1-N7-Guanine in human urine by high-performance liquid chromatography and isotope dilution tandem mass spectrometry. Chem Res Toxicol 19:1191-1195.<br />
<br />
Elegbede JA, and Gould MN. (2002). Monoterpenes reduced adducts formation in rats exposed to aflatoxin B1. African J Biotech, 1, 46–49.<br />
<br />
Gross-Steinmeyer K, and Eaton DL. (2012). Dietary modulation of the biotransformation and genetoxicity of aflatoxin B1. Toxicology, 299: 69–79.<br />
<br />
Guo Y, Breeden LL, Zarbl H, et al. (2005). Expression of a human cytochrome p450 in yeast permits analysis of pathways for response to and repair of aflatoxin-induced DNA damage. Mol Cell Biol, 25, 5823–5833.<br />
<br />
Himmelstein MW, Boogaard PJ, Cadet J, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: II.Overview of methods of identification and quantitation of DNA damage. Crit Rev Toxicol, 39, 679–694.<br />
<br />
Jarabek, AM, Pottenger, LH, Andrews, LS, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: I. Data organization. Crit. Rev. Toxicol. 39: 659-678.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, Sutter TR, Groopman JD, Kensler TW, Roebuck BD. (2014). Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold. Cancer Prev Res. 7(7):658-665.<br />
<br />
Kensler TW, He X, Otieno M, et al. (1998). Oltipraz chemoprevention trial in Qidong, People’s Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol Biomarkers Prev, 7, 127–134.<br />
<br />
Leadon SA, Tyrrell RM, Cerutti PA. (1981). Excision repair of aflatoxin B1-DNA adducts in human fibroblasts. Cancer Res. 41: 5125-5129.<br />
<br />
Leung MC, Goldstone JV, Boyd WA, Freedman JH, and Meyer JN. (2010). Caenorhabditis elegans generates biologically relevant levels of genotoxic metabolites from aflatoxin B1 but not benzo[a]pyrene in vivo. Toxicol Sci.118(2):444-453.<br />
<br />
Levy DD, Groopman JD, Lim SE, et al. (1992). Sequence specificity of aflatoxin B1-induced mutations in a plasmid replicated in xeroderma pigmentosum and DNA repair proficient human cells. Cancer Res. 52: 5668–5673.<br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014a). Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua. J Biol Chem. 289:18497-18506. <br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014b). Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis 35(7):1461-1468<br />
<br />
Meier B, Cooke SL, Weiss J, Bailly AP, Alexandrov LB, Marshall J, et al. (2014). C. elegans whole genome sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency. Genome Res. 24(10):1624-1636.<br />
<br />
Oleykowski CA, Mayernick JA, Lim SE, et al. (1993). Repair of aflatoxin B1 DNA adducts by the UvrABC endonuclease of Escherichia coli. J Biol.Chem. 268: 7900–8002.<br />
<br />
Pottenger, L.H., Andrews LS, Bachman AN, Boogaard PJ, Cadet J, Embry MR, Farmer PB, Himmelstein MW, Jarabek AM, Martin EA, Mauthe RJ, Persaud R, Preston RJ, Schoeny R, Skare J, Swenberg JA, Williams GM, Zeiger E, Zhang F, Kim JH. (2014). An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride. Crit. Rev. Toxicol. 44(4):348-391.<br />
<br />
Preston RJ, Williams GM. (2005). DNA-reactive carcinogens: mode of action and human cancer hazard. Crit Rev Toxicol, 35, 673–83<br />
<br />
Puisieux A, Lim S, Groopman J, Ozturk M. (1991). Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res. 51(22):6185-6189.<br />
<br />
Roebuck BD, Liu Y-L, Rogers AE, et al. (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short term molecular dosimetry. Cancer Res, 51, 5501–5506.<br />
<br />
Sarasin AR, Smith CA Hanawalt PC. (1977). Repair of DNA in human cells after treatment with activated aflatoxin B1. Cancer Res. 37: 1786–1793.<br />
<br />
Yates MS, Kwak M-K, Egner PA, et al. (2006). Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl] imidazole. Cancer Res, 66, 2488–2494.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:375Event:3752016-07-06T16:58:19Z<p>Dvillene: /* KE#1: How it is Measured or Detected */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Replaced by Event:493 </div><br />
<br />
== Action ==<br />
Insufficient repair or mis-repair of DNA<br />
<br />
== Biological Process/Object ==<br />
pro-mutagenic DNA adducts, a KE for this AOP<br />
<br />
== Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
Evidence supporting the insufficient repair or mis-repair of pro-mutagenic DNA adducts is strong, but mainly indirect. This evidence comes from different biological systems and datasets, mostly mammalian, and is based in large part on the biological understanding of a DNA-reactive mode of action requiring a change in primary DNA sequence (mutation). Such a change occurs when DNA lesions are not repaired prior to cell replication (Preston and Williams, 2005; Jarabek et al, 2009; Pottenger et al., 2014). AFB1 forms two types of pro-mutagenic adducts: N7-AFB1-G and AFB1-FAPy DNA adducts, and the resulting mutations are predominantly G:C to T:A transversions. These changes are the expected result of such adducts not being repaired or undergoing mis-repair (Bedard and Massey, 2006; Lin et al., 2014a,b).<br />
<br />
Data from diverse cell types and systems demonstrate that AFB1-induced DNA adducts are repaired by a variety of DNA repair processes, including SOS repair, nucleotide excision repair (NER), homologous recombination (HR), and post-replication repair (Bedard and Massey, 2006). The work by Guo et al. (2005), conducted in transgenic yeast modified to express human CYP1A2, is particularly useful as several DNA repair systems were evaluated for efficacy towards the AFB1-induced DNA adducts. Mutations were more likely to be induced in strains deficient in certain repair systems. Mutations were also induced in strains with active secondary repair pathways; these include pathways for error-prone post-replication repair and those relying on apurinic endonucleases. However, NER appears to be most important for repair of AFB1-induced adducts in mammalian systems (Bedard and Massey, 2006).<br />
<br />
The codon 249 of the p53 gene has been identified as a particular target of AFB1-induced adduction and subsequent mutation. Using DNA polymerase fingerprint analysis, Puiseux et al. (1991) showed a specificity of AFB1 epoxide binding for this codon. This specificity was not found with benzo[a]pyrene (B[a]P), although B[a]P also forms bulky, intercalating epoxides, similar to AFB1 exo-epoxide, which result in bulky N7-B[a]P-G adducts. <br />
<br />
Essentiality of this key event of insufficient or mis-repair is supported in part by datasets demonstrating that a reduction in AFB1 adduct burden (e.g., 65-70% reduction) results in a significantly reduced or even eliminated altered hepatic foci (AHF) and liver tumor burden in experimental animals (Roebuck et al., 1991; Elegbede and Gould, 2002; Yates et al., 2006; Johnson et al., 2014). Data from studies in human populations show that treatment with known modulators of AFB1 metabolism (e.g., with oltipraz or chlorophyllin) resulted in reduced urinary levels of N7-AFB1-G, derived from AFB1-induced DNA adducts (Egner et al., 2006). These treatments are also observed to decrease levels of a human biomarker of exposure, AFB1-induced albumin adducts (Kensler et al., 1998). A similar dataset in rats pre-treated with the triterpenoid, ODDC-Im, provides an even more convincing picture, wherein a chronic bioassay demonstrated a ~60% reduction in AFB1 DNA adducts, complete elimination of liver tumors, and nearly complete elimination of altered hepatic foci (Johnson et al., 2014).<br />
Some studies suggest that eukaryotic NER expression is induced by exposure to phytochemicals (Gross-Steinmeyer et al. 2010), and alteration of DNA repair has been suggested as a pathway of chemoprevention for AFB1 carcinogenesis (Gross-Steinmeyer and Eaton, 2012).<br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Taxonomic Applicability ==<br />
Measurements of repair of AFB1-induced DNA adduct have focused mainly on in vitro systems with bacteria, yeast, and mammalian cells, including cell lines derived from rats and non-human primates (Oleykowski et al., 1993; Leadon et al., 1981; Levy et al.,1992; Sarasin et al., 1977; Guo et al., 2005; Gross-Steinmeyer and Eaton, 2012). Relevant recent studies evaluated mutation induction by AFB1 in Caenorhabditis elegans (Leung et al., 2010; Meir et al., 2014). It is likely that all species which are capable of DNA repair following binding of AFB1 exo-epoxide to DNA—are subject to insufficient or mis-repair.<br />
<br />
== Level of Biological Organization : Cellular ==<br />
Repair of DNA adducts occurs at the sub-cellular level, as the target is nuclear DNA. For AFB1, the cross-species critical target is repair of hepatocyte nuclear DNA, as the eventual AFB1-induced tumor is hepatocellular carcinoma (HCC), which is initiated in hepatocytes.<br />
<br />
== How this Key Event works ==<br />
The AFB1-induced pro-mutagenic DNA adducts, N7-AFB1-G and /or AFB1-FAPy, are recognized by DNA repair proteins or systems, which then initiate their repair processes (Bedard and Massey, 2006). <br />
<br />
Although there are no data specific to AFB1 for this key event of insufficient/mis-repair of DNA, it is a process known to occur and known to result in mutations. The system that recognizes the damage depends in part upon which stage of the cell cycle is in progress when the pro-mutagenic DNA adducts are recognized. Initial steps can include recruitment of a series of proteins specific to that repair system and can also include blocking of progress of the DNA replication fork. This latter step ensures that DNA replication waits for repair to occur before proceeding, preventing replication of the damaged DNA, thus avoiding a mutation when the repair might otherwise faithfully copy the error.<br />
<br />
[[Mis-repair:]] For many of the DNA repair systems the repair process is quite faithful; that is, damage is correctly repaired with the correct or original base substituted for the adducted base. This is not always the case, however, and erroneous repair is one source of mutations, when an incorrect base inserted opposite a correct or an adducted base (mis-repair). <br />
<br />
[[Insufficient repair:]] An alternative route to mutation can occur when on-going DNA replication is not stopped, and DNA replication occurs across an adducted (typically non-informational lesion) base that has not undergone any repair (insufficient repair). This situation can result in the incorporation of an incorrect base into the nascent DNA strand. <br />
<br />
Insufficient repair or mis-repair results in an incorrect base incorporated into the nascent DNA strand, an error which is then eventually replicated, resulting in a so-called “fixed” change in the primary DNA sequence—a mutation. This mutation may be silent (no impact on protein product structure and/or function) or expressed (non-functional or differently functional protein product). Depending on the location or gene of the expressed mutation, the biological impact may none, slight, or substantial. For example, if the mutation is located in codon 249 of the p53 gene and is expressed, there is potential for substantial biological impact, including progression toward carcinogenesis. A substantial biological impact is less likely when the mutation results in no change in the coded amino acid due to degenerate nature of the genetic code for amino acid sequence. Likewise a change in amino acid sequence may not result in any change in the activity or function of the protein product.<br />
<br />
== How it is Measured or Detected ==<br />
One approach would be quantitation of chemical-specific DNA adducts, such as the N7-AFB1-G and AFB1-FAPy DNA adducts, before and after DNA repair occurs combined with DNA sequence data to determine whether the adducts were correctly repaired or not. Such is not a very practical approach. Measurement of mutations following AFB1 treatment of yeast with a variety of DNA repair deficiencies has been used to elucidate the role of DNA repair (Guo et al., 2005) by comparing mutant frequency (MF) in wild type vs. DNA repair deficient strains. In this instance MF serves as a marker for the insufficient or mis-repair of the AFB1 DNA adducts. The exquisitely sensitive analytical techniques available for structural quantification of these chemical-specific DNA adducts require specialized analytical chemistry techniques conducted on DNA isolated from tissues or cells and subjected to neutral thermal or enzyme or acid hydrolysis to release the adducted bases, which are then further analyzed (Himmelstein et al., 2009). Demonstration of adduct dose-responses and temporal relationships are possible with administration of a variety of dose regimens, including repeated doses.<br />
<br />
== KE#1: Evidence Supporting Taxonomic Applicability ==<br />
Measurements of repair of AFB1-induced DNA adduct have focused mainly on in vitro systems with bacteria, yeast, and mammalian cells, including cell lines derived from rats and non-human primates (Oleykowski et al., 1993; Leadon et al., 1981; Levy et al.,1992; Sarasin et al., 1977; Guo et al., 2005; Gross-Steinmeyer and Eaton, 2012). Relevant recent studies evaluated mutation induction of AFB1 in Caenorhabditis elegans (Leung et al., 2010; Meir et al., 2014), Likely, all species capable of DNA repair following binding of AFB1 exo-epoxide to DNA are subject to insufficient or mis-repair These species include bacteria, yeast, birds, mammals, and fish.<br />
<br />
== References ==<br />
Bedard, L.L., and Massey, T.T. (2006). Aflatoxin B1-induced DNA damage and its repair. Cancer Lett. 241(2):174-183.<br />
<br />
Egner PA, Groopman JD, Wang J-S, Kensler TW, Friesen MD (2006). Quantification of aflatoxin-B1-N7-Guanine in human urine by high-performance liquid chromatography and isotope dilution tandem mass spectrometry. Chem Res Toxicol 19:1191-1195.<br />
<br />
Elegbede JA, and Gould MN. (2002). Monoterpenes reduced adducts formation in rats exposed to aflatoxin B1. African J Biotech, 1, 46–49.<br />
<br />
Gross-Steinmeyer K, and Eaton DL. (2012). Dietary modulation of the biotransformation and genetoxicity of aflatoxin B1. Toxicology, 299: 69–79.<br />
<br />
Guo Y, Breeden LL, Zarbl H, et al. (2005). Expression of a human cytochrome p450 in yeast permits analysis of pathways for response to and repair of aflatoxin-induced DNA damage. Mol Cell Biol, 25, 5823–5833.<br />
<br />
Himmelstein MW, Boogaard PJ, Cadet J, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: II.Overview of methods of identification and quantitation of DNA damage. Crit Rev Toxicol, 39, 679–694.<br />
<br />
Jarabek, AM, Pottenger, LH, Andrews, LS, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: I. Data organization. Crit. Rev. Toxicol. 39: 659-678.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, Sutter TR, Groopman JD, Kensler TW, Roebuck BD. (2014). Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold. Cancer Prev Res. 7(7):658-665.<br />
<br />
Kensler TW, He X, Otieno M, et al. (1998). Oltipraz chemoprevention trial in Qidong, People’s Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol Biomarkers Prev, 7, 127–134.<br />
<br />
Leadon SA, Tyrrell RM, Cerutti PA. (1981). Excision repair of aflatoxin B1-DNA adducts in human fibroblasts. Cancer Res. 41: 5125-5129.<br />
<br />
Leung MC, Goldstone JV, Boyd WA, Freedman JH, and Meyer JN. (2010). Caenorhabditis elegans generates biologically relevant levels of genotoxic metabolites from aflatoxin B1 but not benzo[a]pyrene in vivo. Toxicol Sci.118(2):444-453.<br />
<br />
Levy DD, Groopman JD, Lim SE, et al. (1992). Sequence specificity of aflatoxin B1-induced mutations in a plasmid replicated in xeroderma pigmentosum and DNA repair proficient human cells. Cancer Res. 52: 5668–5673.<br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014a). Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua. J Biol Chem. 289:18497-18506. <br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014b). Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis 35(7):1461-1468<br />
<br />
Meier B, Cooke SL, Weiss J, Bailly AP, Alexandrov LB, Marshall J, et al. (2014). C. elegans whole genome sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency. Genome Res. 24(10):1624-1636.<br />
<br />
Oleykowski CA, Mayernick JA, Lim SE, et al. (1993). Repair of aflatoxin B1 DNA adducts by the UvrABC endonuclease of Escherichia coli. J Biol.Chem. 268: 7900–8002.<br />
<br />
Pottenger, L.H., Andrews LS, Bachman AN, Boogaard PJ, Cadet J, Embry MR, Farmer PB, Himmelstein MW, Jarabek AM, Martin EA, Mauthe RJ, Persaud R, Preston RJ, Schoeny R, Skare J, Swenberg JA, Williams GM, Zeiger E, Zhang F, Kim JH. (2014). An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride. Crit. Rev. Toxicol. 44(4):348-391.<br />
<br />
Preston RJ, Williams GM. (2005). DNA-reactive carcinogens: mode of action and human cancer hazard. Crit Rev Toxicol, 35, 673–83<br />
<br />
Puisieux A, Lim S, Groopman J, Ozturk M. (1991). Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res. 51(22):6185-6189.<br />
<br />
Roebuck BD, Liu Y-L, Rogers AE, et al. (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short term molecular dosimetry. Cancer Res, 51, 5501–5506.<br />
<br />
Sarasin AR, Smith CA Hanawalt PC. (1977). Repair of DNA in human cells after treatment with activated aflatoxin B1. Cancer Res. 37: 1786–1793.<br />
<br />
Yates MS, Kwak M-K, Egner PA, et al. (2006). Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl] imidazole. Cancer Res, 66, 2488–2494.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:375Event:3752016-07-06T16:57:59Z<p>Dvillene: /* KE#1: How this Key Event works */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Replaced by Event:493 </div><br />
<br />
== Action ==<br />
Insufficient repair or mis-repair of DNA<br />
<br />
== Biological Process/Object ==<br />
pro-mutagenic DNA adducts, a KE for this AOP<br />
<br />
== Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
Evidence supporting the insufficient repair or mis-repair of pro-mutagenic DNA adducts is strong, but mainly indirect. This evidence comes from different biological systems and datasets, mostly mammalian, and is based in large part on the biological understanding of a DNA-reactive mode of action requiring a change in primary DNA sequence (mutation). Such a change occurs when DNA lesions are not repaired prior to cell replication (Preston and Williams, 2005; Jarabek et al, 2009; Pottenger et al., 2014). AFB1 forms two types of pro-mutagenic adducts: N7-AFB1-G and AFB1-FAPy DNA adducts, and the resulting mutations are predominantly G:C to T:A transversions. These changes are the expected result of such adducts not being repaired or undergoing mis-repair (Bedard and Massey, 2006; Lin et al., 2014a,b).<br />
<br />
Data from diverse cell types and systems demonstrate that AFB1-induced DNA adducts are repaired by a variety of DNA repair processes, including SOS repair, nucleotide excision repair (NER), homologous recombination (HR), and post-replication repair (Bedard and Massey, 2006). The work by Guo et al. (2005), conducted in transgenic yeast modified to express human CYP1A2, is particularly useful as several DNA repair systems were evaluated for efficacy towards the AFB1-induced DNA adducts. Mutations were more likely to be induced in strains deficient in certain repair systems. Mutations were also induced in strains with active secondary repair pathways; these include pathways for error-prone post-replication repair and those relying on apurinic endonucleases. However, NER appears to be most important for repair of AFB1-induced adducts in mammalian systems (Bedard and Massey, 2006).<br />
<br />
The codon 249 of the p53 gene has been identified as a particular target of AFB1-induced adduction and subsequent mutation. Using DNA polymerase fingerprint analysis, Puiseux et al. (1991) showed a specificity of AFB1 epoxide binding for this codon. This specificity was not found with benzo[a]pyrene (B[a]P), although B[a]P also forms bulky, intercalating epoxides, similar to AFB1 exo-epoxide, which result in bulky N7-B[a]P-G adducts. <br />
<br />
Essentiality of this key event of insufficient or mis-repair is supported in part by datasets demonstrating that a reduction in AFB1 adduct burden (e.g., 65-70% reduction) results in a significantly reduced or even eliminated altered hepatic foci (AHF) and liver tumor burden in experimental animals (Roebuck et al., 1991; Elegbede and Gould, 2002; Yates et al., 2006; Johnson et al., 2014). Data from studies in human populations show that treatment with known modulators of AFB1 metabolism (e.g., with oltipraz or chlorophyllin) resulted in reduced urinary levels of N7-AFB1-G, derived from AFB1-induced DNA adducts (Egner et al., 2006). These treatments are also observed to decrease levels of a human biomarker of exposure, AFB1-induced albumin adducts (Kensler et al., 1998). A similar dataset in rats pre-treated with the triterpenoid, ODDC-Im, provides an even more convincing picture, wherein a chronic bioassay demonstrated a ~60% reduction in AFB1 DNA adducts, complete elimination of liver tumors, and nearly complete elimination of altered hepatic foci (Johnson et al., 2014).<br />
Some studies suggest that eukaryotic NER expression is induced by exposure to phytochemicals (Gross-Steinmeyer et al. 2010), and alteration of DNA repair has been suggested as a pathway of chemoprevention for AFB1 carcinogenesis (Gross-Steinmeyer and Eaton, 2012).<br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Taxonomic Applicability ==<br />
Measurements of repair of AFB1-induced DNA adduct have focused mainly on in vitro systems with bacteria, yeast, and mammalian cells, including cell lines derived from rats and non-human primates (Oleykowski et al., 1993; Leadon et al., 1981; Levy et al.,1992; Sarasin et al., 1977; Guo et al., 2005; Gross-Steinmeyer and Eaton, 2012). Relevant recent studies evaluated mutation induction by AFB1 in Caenorhabditis elegans (Leung et al., 2010; Meir et al., 2014). It is likely that all species which are capable of DNA repair following binding of AFB1 exo-epoxide to DNA—are subject to insufficient or mis-repair.<br />
<br />
== Level of Biological Organization : Cellular ==<br />
Repair of DNA adducts occurs at the sub-cellular level, as the target is nuclear DNA. For AFB1, the cross-species critical target is repair of hepatocyte nuclear DNA, as the eventual AFB1-induced tumor is hepatocellular carcinoma (HCC), which is initiated in hepatocytes.<br />
<br />
== How this Key Event works ==<br />
The AFB1-induced pro-mutagenic DNA adducts, N7-AFB1-G and /or AFB1-FAPy, are recognized by DNA repair proteins or systems, which then initiate their repair processes (Bedard and Massey, 2006). <br />
<br />
Although there are no data specific to AFB1 for this key event of insufficient/mis-repair of DNA, it is a process known to occur and known to result in mutations. The system that recognizes the damage depends in part upon which stage of the cell cycle is in progress when the pro-mutagenic DNA adducts are recognized. Initial steps can include recruitment of a series of proteins specific to that repair system and can also include blocking of progress of the DNA replication fork. This latter step ensures that DNA replication waits for repair to occur before proceeding, preventing replication of the damaged DNA, thus avoiding a mutation when the repair might otherwise faithfully copy the error.<br />
<br />
[[Mis-repair:]] For many of the DNA repair systems the repair process is quite faithful; that is, damage is correctly repaired with the correct or original base substituted for the adducted base. This is not always the case, however, and erroneous repair is one source of mutations, when an incorrect base inserted opposite a correct or an adducted base (mis-repair). <br />
<br />
[[Insufficient repair:]] An alternative route to mutation can occur when on-going DNA replication is not stopped, and DNA replication occurs across an adducted (typically non-informational lesion) base that has not undergone any repair (insufficient repair). This situation can result in the incorporation of an incorrect base into the nascent DNA strand. <br />
<br />
Insufficient repair or mis-repair results in an incorrect base incorporated into the nascent DNA strand, an error which is then eventually replicated, resulting in a so-called “fixed” change in the primary DNA sequence—a mutation. This mutation may be silent (no impact on protein product structure and/or function) or expressed (non-functional or differently functional protein product). Depending on the location or gene of the expressed mutation, the biological impact may none, slight, or substantial. For example, if the mutation is located in codon 249 of the p53 gene and is expressed, there is potential for substantial biological impact, including progression toward carcinogenesis. A substantial biological impact is less likely when the mutation results in no change in the coded amino acid due to degenerate nature of the genetic code for amino acid sequence. Likewise a change in amino acid sequence may not result in any change in the activity or function of the protein product.<br />
<br />
== KE#1: How it is Measured or Detected ==<br />
One approach would be quantitation of chemical-specific DNA adducts, such as the N7-AFB1-G and AFB1-FAPy DNA adducts, before and after DNA repair occurs combined with DNA sequence data to determine whether the adducts were correctly repaired or not. Such is not a very practical approach. Measurement of mutations following AFB1 treatment of yeast with a variety of DNA repair deficiencies has been used to elucidate the role of DNA repair (Guo et al., 2005) by comparing mutant frequency (MF) in wild type vs. DNA repair deficient strains. In this instance MF serves as a marker for the insufficient or mis-repair of the AFB1 DNA adducts. The exquisitely sensitive analytical techniques available for structural quantification of these chemical-specific DNA adducts require specialized analytical chemistry techniques conducted on DNA isolated from tissues or cells and subjected to neutral thermal or enzyme or acid hydrolysis to release the adducted bases, which are then further analyzed (Himmelstein et al., 2009). Demonstration of adduct dose-responses and temporal relationships are possible with administration of a variety of dose regimens, including repeated doses.<br />
<br />
== KE#1: Evidence Supporting Taxonomic Applicability ==<br />
Measurements of repair of AFB1-induced DNA adduct have focused mainly on in vitro systems with bacteria, yeast, and mammalian cells, including cell lines derived from rats and non-human primates (Oleykowski et al., 1993; Leadon et al., 1981; Levy et al.,1992; Sarasin et al., 1977; Guo et al., 2005; Gross-Steinmeyer and Eaton, 2012). Relevant recent studies evaluated mutation induction of AFB1 in Caenorhabditis elegans (Leung et al., 2010; Meir et al., 2014), Likely, all species capable of DNA repair following binding of AFB1 exo-epoxide to DNA are subject to insufficient or mis-repair These species include bacteria, yeast, birds, mammals, and fish.<br />
<br />
== References ==<br />
Bedard, L.L., and Massey, T.T. (2006). Aflatoxin B1-induced DNA damage and its repair. Cancer Lett. 241(2):174-183.<br />
<br />
Egner PA, Groopman JD, Wang J-S, Kensler TW, Friesen MD (2006). Quantification of aflatoxin-B1-N7-Guanine in human urine by high-performance liquid chromatography and isotope dilution tandem mass spectrometry. Chem Res Toxicol 19:1191-1195.<br />
<br />
Elegbede JA, and Gould MN. (2002). Monoterpenes reduced adducts formation in rats exposed to aflatoxin B1. African J Biotech, 1, 46–49.<br />
<br />
Gross-Steinmeyer K, and Eaton DL. (2012). Dietary modulation of the biotransformation and genetoxicity of aflatoxin B1. Toxicology, 299: 69–79.<br />
<br />
Guo Y, Breeden LL, Zarbl H, et al. (2005). Expression of a human cytochrome p450 in yeast permits analysis of pathways for response to and repair of aflatoxin-induced DNA damage. Mol Cell Biol, 25, 5823–5833.<br />
<br />
Himmelstein MW, Boogaard PJ, Cadet J, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: II.Overview of methods of identification and quantitation of DNA damage. Crit Rev Toxicol, 39, 679–694.<br />
<br />
Jarabek, AM, Pottenger, LH, Andrews, LS, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: I. Data organization. Crit. Rev. Toxicol. 39: 659-678.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, Sutter TR, Groopman JD, Kensler TW, Roebuck BD. (2014). Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold. Cancer Prev Res. 7(7):658-665.<br />
<br />
Kensler TW, He X, Otieno M, et al. (1998). Oltipraz chemoprevention trial in Qidong, People’s Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol Biomarkers Prev, 7, 127–134.<br />
<br />
Leadon SA, Tyrrell RM, Cerutti PA. (1981). Excision repair of aflatoxin B1-DNA adducts in human fibroblasts. Cancer Res. 41: 5125-5129.<br />
<br />
Leung MC, Goldstone JV, Boyd WA, Freedman JH, and Meyer JN. (2010). Caenorhabditis elegans generates biologically relevant levels of genotoxic metabolites from aflatoxin B1 but not benzo[a]pyrene in vivo. Toxicol Sci.118(2):444-453.<br />
<br />
Levy DD, Groopman JD, Lim SE, et al. (1992). Sequence specificity of aflatoxin B1-induced mutations in a plasmid replicated in xeroderma pigmentosum and DNA repair proficient human cells. Cancer Res. 52: 5668–5673.<br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014a). Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua. J Biol Chem. 289:18497-18506. <br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014b). Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis 35(7):1461-1468<br />
<br />
Meier B, Cooke SL, Weiss J, Bailly AP, Alexandrov LB, Marshall J, et al. (2014). C. elegans whole genome sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency. Genome Res. 24(10):1624-1636.<br />
<br />
Oleykowski CA, Mayernick JA, Lim SE, et al. (1993). Repair of aflatoxin B1 DNA adducts by the UvrABC endonuclease of Escherichia coli. J Biol.Chem. 268: 7900–8002.<br />
<br />
Pottenger, L.H., Andrews LS, Bachman AN, Boogaard PJ, Cadet J, Embry MR, Farmer PB, Himmelstein MW, Jarabek AM, Martin EA, Mauthe RJ, Persaud R, Preston RJ, Schoeny R, Skare J, Swenberg JA, Williams GM, Zeiger E, Zhang F, Kim JH. (2014). An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride. Crit. Rev. Toxicol. 44(4):348-391.<br />
<br />
Preston RJ, Williams GM. (2005). DNA-reactive carcinogens: mode of action and human cancer hazard. Crit Rev Toxicol, 35, 673–83<br />
<br />
Puisieux A, Lim S, Groopman J, Ozturk M. (1991). Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res. 51(22):6185-6189.<br />
<br />
Roebuck BD, Liu Y-L, Rogers AE, et al. (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short term molecular dosimetry. Cancer Res, 51, 5501–5506.<br />
<br />
Sarasin AR, Smith CA Hanawalt PC. (1977). Repair of DNA in human cells after treatment with activated aflatoxin B1. Cancer Res. 37: 1786–1793.<br />
<br />
Yates MS, Kwak M-K, Egner PA, et al. (2006). Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl] imidazole. Cancer Res, 66, 2488–2494.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:375Event:3752016-07-06T16:57:42Z<p>Dvillene: /* KE#1: Level of Biological Organization : Cellular */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Replaced by Event:493 </div><br />
<br />
== Action ==<br />
Insufficient repair or mis-repair of DNA<br />
<br />
== Biological Process/Object ==<br />
pro-mutagenic DNA adducts, a KE for this AOP<br />
<br />
== Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
Evidence supporting the insufficient repair or mis-repair of pro-mutagenic DNA adducts is strong, but mainly indirect. This evidence comes from different biological systems and datasets, mostly mammalian, and is based in large part on the biological understanding of a DNA-reactive mode of action requiring a change in primary DNA sequence (mutation). Such a change occurs when DNA lesions are not repaired prior to cell replication (Preston and Williams, 2005; Jarabek et al, 2009; Pottenger et al., 2014). AFB1 forms two types of pro-mutagenic adducts: N7-AFB1-G and AFB1-FAPy DNA adducts, and the resulting mutations are predominantly G:C to T:A transversions. These changes are the expected result of such adducts not being repaired or undergoing mis-repair (Bedard and Massey, 2006; Lin et al., 2014a,b).<br />
<br />
Data from diverse cell types and systems demonstrate that AFB1-induced DNA adducts are repaired by a variety of DNA repair processes, including SOS repair, nucleotide excision repair (NER), homologous recombination (HR), and post-replication repair (Bedard and Massey, 2006). The work by Guo et al. (2005), conducted in transgenic yeast modified to express human CYP1A2, is particularly useful as several DNA repair systems were evaluated for efficacy towards the AFB1-induced DNA adducts. Mutations were more likely to be induced in strains deficient in certain repair systems. Mutations were also induced in strains with active secondary repair pathways; these include pathways for error-prone post-replication repair and those relying on apurinic endonucleases. However, NER appears to be most important for repair of AFB1-induced adducts in mammalian systems (Bedard and Massey, 2006).<br />
<br />
The codon 249 of the p53 gene has been identified as a particular target of AFB1-induced adduction and subsequent mutation. Using DNA polymerase fingerprint analysis, Puiseux et al. (1991) showed a specificity of AFB1 epoxide binding for this codon. This specificity was not found with benzo[a]pyrene (B[a]P), although B[a]P also forms bulky, intercalating epoxides, similar to AFB1 exo-epoxide, which result in bulky N7-B[a]P-G adducts. <br />
<br />
Essentiality of this key event of insufficient or mis-repair is supported in part by datasets demonstrating that a reduction in AFB1 adduct burden (e.g., 65-70% reduction) results in a significantly reduced or even eliminated altered hepatic foci (AHF) and liver tumor burden in experimental animals (Roebuck et al., 1991; Elegbede and Gould, 2002; Yates et al., 2006; Johnson et al., 2014). Data from studies in human populations show that treatment with known modulators of AFB1 metabolism (e.g., with oltipraz or chlorophyllin) resulted in reduced urinary levels of N7-AFB1-G, derived from AFB1-induced DNA adducts (Egner et al., 2006). These treatments are also observed to decrease levels of a human biomarker of exposure, AFB1-induced albumin adducts (Kensler et al., 1998). A similar dataset in rats pre-treated with the triterpenoid, ODDC-Im, provides an even more convincing picture, wherein a chronic bioassay demonstrated a ~60% reduction in AFB1 DNA adducts, complete elimination of liver tumors, and nearly complete elimination of altered hepatic foci (Johnson et al., 2014).<br />
Some studies suggest that eukaryotic NER expression is induced by exposure to phytochemicals (Gross-Steinmeyer et al. 2010), and alteration of DNA repair has been suggested as a pathway of chemoprevention for AFB1 carcinogenesis (Gross-Steinmeyer and Eaton, 2012).<br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Taxonomic Applicability ==<br />
Measurements of repair of AFB1-induced DNA adduct have focused mainly on in vitro systems with bacteria, yeast, and mammalian cells, including cell lines derived from rats and non-human primates (Oleykowski et al., 1993; Leadon et al., 1981; Levy et al.,1992; Sarasin et al., 1977; Guo et al., 2005; Gross-Steinmeyer and Eaton, 2012). Relevant recent studies evaluated mutation induction by AFB1 in Caenorhabditis elegans (Leung et al., 2010; Meir et al., 2014). It is likely that all species which are capable of DNA repair following binding of AFB1 exo-epoxide to DNA—are subject to insufficient or mis-repair.<br />
<br />
== Level of Biological Organization : Cellular ==<br />
Repair of DNA adducts occurs at the sub-cellular level, as the target is nuclear DNA. For AFB1, the cross-species critical target is repair of hepatocyte nuclear DNA, as the eventual AFB1-induced tumor is hepatocellular carcinoma (HCC), which is initiated in hepatocytes.<br />
<br />
== KE#1: How this Key Event works ==<br />
The AFB1-induced pro-mutagenic DNA adducts, N7-AFB1-G and /or AFB1-FAPy, are recognized by DNA repair proteins or systems, which then initiate their repair processes (Bedard and Massey, 2006). <br />
<br />
Although there are no data specific to AFB1 for this key event of insufficient/mis-repair of DNA, it is a process known to occur and known to result in mutations. The system that recognizes the damage depends in part upon which stage of the cell cycle is in progress when the pro-mutagenic DNA adducts are recognized. Initial steps can include recruitment of a series of proteins specific to that repair system and can also include blocking of progress of the DNA replication fork. This latter step ensures that DNA replication waits for repair to occur before proceeding, preventing replication of the damaged DNA, thus avoiding a mutation when the repair might otherwise faithfully copy the error.<br />
<br />
[[Mis-repair:]] For many of the DNA repair systems the repair process is quite faithful; that is, damage is correctly repaired with the correct or original base substituted for the adducted base. This is not always the case, however, and erroneous repair is one source of mutations, when an incorrect base inserted opposite a correct or an adducted base (mis-repair). <br />
<br />
[[Insufficient repair:]] An alternative route to mutation can occur when on-going DNA replication is not stopped, and DNA replication occurs across an adducted (typically non-informational lesion) base that has not undergone any repair (insufficient repair). This situation can result in the incorporation of an incorrect base into the nascent DNA strand. <br />
<br />
Insufficient repair or mis-repair results in an incorrect base incorporated into the nascent DNA strand, an error which is then eventually replicated, resulting in a so-called “fixed” change in the primary DNA sequence—a mutation. This mutation may be silent (no impact on protein product structure and/or function) or expressed (non-functional or differently functional protein product). Depending on the location or gene of the expressed mutation, the biological impact may none, slight, or substantial. For example, if the mutation is located in codon 249 of the p53 gene and is expressed, there is potential for substantial biological impact, including progression toward carcinogenesis. A substantial biological impact is less likely when the mutation results in no change in the coded amino acid due to degenerate nature of the genetic code for amino acid sequence. Likewise a change in amino acid sequence may not result in any change in the activity or function of the protein product.<br />
<br />
== KE#1: How it is Measured or Detected ==<br />
One approach would be quantitation of chemical-specific DNA adducts, such as the N7-AFB1-G and AFB1-FAPy DNA adducts, before and after DNA repair occurs combined with DNA sequence data to determine whether the adducts were correctly repaired or not. Such is not a very practical approach. Measurement of mutations following AFB1 treatment of yeast with a variety of DNA repair deficiencies has been used to elucidate the role of DNA repair (Guo et al., 2005) by comparing mutant frequency (MF) in wild type vs. DNA repair deficient strains. In this instance MF serves as a marker for the insufficient or mis-repair of the AFB1 DNA adducts. The exquisitely sensitive analytical techniques available for structural quantification of these chemical-specific DNA adducts require specialized analytical chemistry techniques conducted on DNA isolated from tissues or cells and subjected to neutral thermal or enzyme or acid hydrolysis to release the adducted bases, which are then further analyzed (Himmelstein et al., 2009). Demonstration of adduct dose-responses and temporal relationships are possible with administration of a variety of dose regimens, including repeated doses.<br />
<br />
== KE#1: Evidence Supporting Taxonomic Applicability ==<br />
Measurements of repair of AFB1-induced DNA adduct have focused mainly on in vitro systems with bacteria, yeast, and mammalian cells, including cell lines derived from rats and non-human primates (Oleykowski et al., 1993; Leadon et al., 1981; Levy et al.,1992; Sarasin et al., 1977; Guo et al., 2005; Gross-Steinmeyer and Eaton, 2012). Relevant recent studies evaluated mutation induction of AFB1 in Caenorhabditis elegans (Leung et al., 2010; Meir et al., 2014), Likely, all species capable of DNA repair following binding of AFB1 exo-epoxide to DNA are subject to insufficient or mis-repair These species include bacteria, yeast, birds, mammals, and fish.<br />
<br />
== References ==<br />
Bedard, L.L., and Massey, T.T. (2006). Aflatoxin B1-induced DNA damage and its repair. Cancer Lett. 241(2):174-183.<br />
<br />
Egner PA, Groopman JD, Wang J-S, Kensler TW, Friesen MD (2006). Quantification of aflatoxin-B1-N7-Guanine in human urine by high-performance liquid chromatography and isotope dilution tandem mass spectrometry. Chem Res Toxicol 19:1191-1195.<br />
<br />
Elegbede JA, and Gould MN. (2002). Monoterpenes reduced adducts formation in rats exposed to aflatoxin B1. African J Biotech, 1, 46–49.<br />
<br />
Gross-Steinmeyer K, and Eaton DL. (2012). Dietary modulation of the biotransformation and genetoxicity of aflatoxin B1. Toxicology, 299: 69–79.<br />
<br />
Guo Y, Breeden LL, Zarbl H, et al. (2005). Expression of a human cytochrome p450 in yeast permits analysis of pathways for response to and repair of aflatoxin-induced DNA damage. Mol Cell Biol, 25, 5823–5833.<br />
<br />
Himmelstein MW, Boogaard PJ, Cadet J, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: II.Overview of methods of identification and quantitation of DNA damage. Crit Rev Toxicol, 39, 679–694.<br />
<br />
Jarabek, AM, Pottenger, LH, Andrews, LS, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: I. Data organization. Crit. Rev. Toxicol. 39: 659-678.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, Sutter TR, Groopman JD, Kensler TW, Roebuck BD. (2014). Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold. Cancer Prev Res. 7(7):658-665.<br />
<br />
Kensler TW, He X, Otieno M, et al. (1998). Oltipraz chemoprevention trial in Qidong, People’s Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol Biomarkers Prev, 7, 127–134.<br />
<br />
Leadon SA, Tyrrell RM, Cerutti PA. (1981). Excision repair of aflatoxin B1-DNA adducts in human fibroblasts. Cancer Res. 41: 5125-5129.<br />
<br />
Leung MC, Goldstone JV, Boyd WA, Freedman JH, and Meyer JN. (2010). Caenorhabditis elegans generates biologically relevant levels of genotoxic metabolites from aflatoxin B1 but not benzo[a]pyrene in vivo. Toxicol Sci.118(2):444-453.<br />
<br />
Levy DD, Groopman JD, Lim SE, et al. (1992). Sequence specificity of aflatoxin B1-induced mutations in a plasmid replicated in xeroderma pigmentosum and DNA repair proficient human cells. Cancer Res. 52: 5668–5673.<br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014a). Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua. J Biol Chem. 289:18497-18506. <br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014b). Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis 35(7):1461-1468<br />
<br />
Meier B, Cooke SL, Weiss J, Bailly AP, Alexandrov LB, Marshall J, et al. (2014). C. elegans whole genome sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency. Genome Res. 24(10):1624-1636.<br />
<br />
Oleykowski CA, Mayernick JA, Lim SE, et al. (1993). Repair of aflatoxin B1 DNA adducts by the UvrABC endonuclease of Escherichia coli. J Biol.Chem. 268: 7900–8002.<br />
<br />
Pottenger, L.H., Andrews LS, Bachman AN, Boogaard PJ, Cadet J, Embry MR, Farmer PB, Himmelstein MW, Jarabek AM, Martin EA, Mauthe RJ, Persaud R, Preston RJ, Schoeny R, Skare J, Swenberg JA, Williams GM, Zeiger E, Zhang F, Kim JH. (2014). An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride. Crit. Rev. Toxicol. 44(4):348-391.<br />
<br />
Preston RJ, Williams GM. (2005). DNA-reactive carcinogens: mode of action and human cancer hazard. Crit Rev Toxicol, 35, 673–83<br />
<br />
Puisieux A, Lim S, Groopman J, Ozturk M. (1991). Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res. 51(22):6185-6189.<br />
<br />
Roebuck BD, Liu Y-L, Rogers AE, et al. (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short term molecular dosimetry. Cancer Res, 51, 5501–5506.<br />
<br />
Sarasin AR, Smith CA Hanawalt PC. (1977). Repair of DNA in human cells after treatment with activated aflatoxin B1. Cancer Res. 37: 1786–1793.<br />
<br />
Yates MS, Kwak M-K, Egner PA, et al. (2006). Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl] imidazole. Cancer Res, 66, 2488–2494.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:375Event:3752016-07-06T16:57:25Z<p>Dvillene: /* KE#1: Taxonomic Applicability */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Replaced by Event:493 </div><br />
<br />
== Action ==<br />
Insufficient repair or mis-repair of DNA<br />
<br />
== Biological Process/Object ==<br />
pro-mutagenic DNA adducts, a KE for this AOP<br />
<br />
== Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
Evidence supporting the insufficient repair or mis-repair of pro-mutagenic DNA adducts is strong, but mainly indirect. This evidence comes from different biological systems and datasets, mostly mammalian, and is based in large part on the biological understanding of a DNA-reactive mode of action requiring a change in primary DNA sequence (mutation). Such a change occurs when DNA lesions are not repaired prior to cell replication (Preston and Williams, 2005; Jarabek et al, 2009; Pottenger et al., 2014). AFB1 forms two types of pro-mutagenic adducts: N7-AFB1-G and AFB1-FAPy DNA adducts, and the resulting mutations are predominantly G:C to T:A transversions. These changes are the expected result of such adducts not being repaired or undergoing mis-repair (Bedard and Massey, 2006; Lin et al., 2014a,b).<br />
<br />
Data from diverse cell types and systems demonstrate that AFB1-induced DNA adducts are repaired by a variety of DNA repair processes, including SOS repair, nucleotide excision repair (NER), homologous recombination (HR), and post-replication repair (Bedard and Massey, 2006). The work by Guo et al. (2005), conducted in transgenic yeast modified to express human CYP1A2, is particularly useful as several DNA repair systems were evaluated for efficacy towards the AFB1-induced DNA adducts. Mutations were more likely to be induced in strains deficient in certain repair systems. Mutations were also induced in strains with active secondary repair pathways; these include pathways for error-prone post-replication repair and those relying on apurinic endonucleases. However, NER appears to be most important for repair of AFB1-induced adducts in mammalian systems (Bedard and Massey, 2006).<br />
<br />
The codon 249 of the p53 gene has been identified as a particular target of AFB1-induced adduction and subsequent mutation. Using DNA polymerase fingerprint analysis, Puiseux et al. (1991) showed a specificity of AFB1 epoxide binding for this codon. This specificity was not found with benzo[a]pyrene (B[a]P), although B[a]P also forms bulky, intercalating epoxides, similar to AFB1 exo-epoxide, which result in bulky N7-B[a]P-G adducts. <br />
<br />
Essentiality of this key event of insufficient or mis-repair is supported in part by datasets demonstrating that a reduction in AFB1 adduct burden (e.g., 65-70% reduction) results in a significantly reduced or even eliminated altered hepatic foci (AHF) and liver tumor burden in experimental animals (Roebuck et al., 1991; Elegbede and Gould, 2002; Yates et al., 2006; Johnson et al., 2014). Data from studies in human populations show that treatment with known modulators of AFB1 metabolism (e.g., with oltipraz or chlorophyllin) resulted in reduced urinary levels of N7-AFB1-G, derived from AFB1-induced DNA adducts (Egner et al., 2006). These treatments are also observed to decrease levels of a human biomarker of exposure, AFB1-induced albumin adducts (Kensler et al., 1998). A similar dataset in rats pre-treated with the triterpenoid, ODDC-Im, provides an even more convincing picture, wherein a chronic bioassay demonstrated a ~60% reduction in AFB1 DNA adducts, complete elimination of liver tumors, and nearly complete elimination of altered hepatic foci (Johnson et al., 2014).<br />
Some studies suggest that eukaryotic NER expression is induced by exposure to phytochemicals (Gross-Steinmeyer et al. 2010), and alteration of DNA repair has been suggested as a pathway of chemoprevention for AFB1 carcinogenesis (Gross-Steinmeyer and Eaton, 2012).<br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Taxonomic Applicability ==<br />
Measurements of repair of AFB1-induced DNA adduct have focused mainly on in vitro systems with bacteria, yeast, and mammalian cells, including cell lines derived from rats and non-human primates (Oleykowski et al., 1993; Leadon et al., 1981; Levy et al.,1992; Sarasin et al., 1977; Guo et al., 2005; Gross-Steinmeyer and Eaton, 2012). Relevant recent studies evaluated mutation induction by AFB1 in Caenorhabditis elegans (Leung et al., 2010; Meir et al., 2014). It is likely that all species which are capable of DNA repair following binding of AFB1 exo-epoxide to DNA—are subject to insufficient or mis-repair.<br />
<br />
== KE#1: Level of Biological Organization : Cellular ==<br />
Repair of DNA adducts occurs at the sub-cellular level, as the target is nuclear DNA. For AFB1, the cross-species critical target is repair of hepatocyte nuclear DNA, as the eventual AFB1-induced tumor is hepatocellular carcinoma (HCC), which is initiated in hepatocytes.<br />
<br />
== KE#1: How this Key Event works ==<br />
The AFB1-induced pro-mutagenic DNA adducts, N7-AFB1-G and /or AFB1-FAPy, are recognized by DNA repair proteins or systems, which then initiate their repair processes (Bedard and Massey, 2006). <br />
<br />
Although there are no data specific to AFB1 for this key event of insufficient/mis-repair of DNA, it is a process known to occur and known to result in mutations. The system that recognizes the damage depends in part upon which stage of the cell cycle is in progress when the pro-mutagenic DNA adducts are recognized. Initial steps can include recruitment of a series of proteins specific to that repair system and can also include blocking of progress of the DNA replication fork. This latter step ensures that DNA replication waits for repair to occur before proceeding, preventing replication of the damaged DNA, thus avoiding a mutation when the repair might otherwise faithfully copy the error.<br />
<br />
[[Mis-repair:]] For many of the DNA repair systems the repair process is quite faithful; that is, damage is correctly repaired with the correct or original base substituted for the adducted base. This is not always the case, however, and erroneous repair is one source of mutations, when an incorrect base inserted opposite a correct or an adducted base (mis-repair). <br />
<br />
[[Insufficient repair:]] An alternative route to mutation can occur when on-going DNA replication is not stopped, and DNA replication occurs across an adducted (typically non-informational lesion) base that has not undergone any repair (insufficient repair). This situation can result in the incorporation of an incorrect base into the nascent DNA strand. <br />
<br />
Insufficient repair or mis-repair results in an incorrect base incorporated into the nascent DNA strand, an error which is then eventually replicated, resulting in a so-called “fixed” change in the primary DNA sequence—a mutation. This mutation may be silent (no impact on protein product structure and/or function) or expressed (non-functional or differently functional protein product). Depending on the location or gene of the expressed mutation, the biological impact may none, slight, or substantial. For example, if the mutation is located in codon 249 of the p53 gene and is expressed, there is potential for substantial biological impact, including progression toward carcinogenesis. A substantial biological impact is less likely when the mutation results in no change in the coded amino acid due to degenerate nature of the genetic code for amino acid sequence. Likewise a change in amino acid sequence may not result in any change in the activity or function of the protein product.<br />
<br />
== KE#1: How it is Measured or Detected ==<br />
One approach would be quantitation of chemical-specific DNA adducts, such as the N7-AFB1-G and AFB1-FAPy DNA adducts, before and after DNA repair occurs combined with DNA sequence data to determine whether the adducts were correctly repaired or not. Such is not a very practical approach. Measurement of mutations following AFB1 treatment of yeast with a variety of DNA repair deficiencies has been used to elucidate the role of DNA repair (Guo et al., 2005) by comparing mutant frequency (MF) in wild type vs. DNA repair deficient strains. In this instance MF serves as a marker for the insufficient or mis-repair of the AFB1 DNA adducts. The exquisitely sensitive analytical techniques available for structural quantification of these chemical-specific DNA adducts require specialized analytical chemistry techniques conducted on DNA isolated from tissues or cells and subjected to neutral thermal or enzyme or acid hydrolysis to release the adducted bases, which are then further analyzed (Himmelstein et al., 2009). Demonstration of adduct dose-responses and temporal relationships are possible with administration of a variety of dose regimens, including repeated doses.<br />
<br />
== KE#1: Evidence Supporting Taxonomic Applicability ==<br />
Measurements of repair of AFB1-induced DNA adduct have focused mainly on in vitro systems with bacteria, yeast, and mammalian cells, including cell lines derived from rats and non-human primates (Oleykowski et al., 1993; Leadon et al., 1981; Levy et al.,1992; Sarasin et al., 1977; Guo et al., 2005; Gross-Steinmeyer and Eaton, 2012). Relevant recent studies evaluated mutation induction of AFB1 in Caenorhabditis elegans (Leung et al., 2010; Meir et al., 2014), Likely, all species capable of DNA repair following binding of AFB1 exo-epoxide to DNA are subject to insufficient or mis-repair These species include bacteria, yeast, birds, mammals, and fish.<br />
<br />
== References ==<br />
Bedard, L.L., and Massey, T.T. (2006). Aflatoxin B1-induced DNA damage and its repair. Cancer Lett. 241(2):174-183.<br />
<br />
Egner PA, Groopman JD, Wang J-S, Kensler TW, Friesen MD (2006). Quantification of aflatoxin-B1-N7-Guanine in human urine by high-performance liquid chromatography and isotope dilution tandem mass spectrometry. Chem Res Toxicol 19:1191-1195.<br />
<br />
Elegbede JA, and Gould MN. (2002). Monoterpenes reduced adducts formation in rats exposed to aflatoxin B1. African J Biotech, 1, 46–49.<br />
<br />
Gross-Steinmeyer K, and Eaton DL. (2012). Dietary modulation of the biotransformation and genetoxicity of aflatoxin B1. Toxicology, 299: 69–79.<br />
<br />
Guo Y, Breeden LL, Zarbl H, et al. (2005). Expression of a human cytochrome p450 in yeast permits analysis of pathways for response to and repair of aflatoxin-induced DNA damage. Mol Cell Biol, 25, 5823–5833.<br />
<br />
Himmelstein MW, Boogaard PJ, Cadet J, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: II.Overview of methods of identification and quantitation of DNA damage. Crit Rev Toxicol, 39, 679–694.<br />
<br />
Jarabek, AM, Pottenger, LH, Andrews, LS, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: I. Data organization. Crit. Rev. Toxicol. 39: 659-678.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, Sutter TR, Groopman JD, Kensler TW, Roebuck BD. (2014). Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold. Cancer Prev Res. 7(7):658-665.<br />
<br />
Kensler TW, He X, Otieno M, et al. (1998). Oltipraz chemoprevention trial in Qidong, People’s Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol Biomarkers Prev, 7, 127–134.<br />
<br />
Leadon SA, Tyrrell RM, Cerutti PA. (1981). Excision repair of aflatoxin B1-DNA adducts in human fibroblasts. Cancer Res. 41: 5125-5129.<br />
<br />
Leung MC, Goldstone JV, Boyd WA, Freedman JH, and Meyer JN. (2010). Caenorhabditis elegans generates biologically relevant levels of genotoxic metabolites from aflatoxin B1 but not benzo[a]pyrene in vivo. Toxicol Sci.118(2):444-453.<br />
<br />
Levy DD, Groopman JD, Lim SE, et al. (1992). Sequence specificity of aflatoxin B1-induced mutations in a plasmid replicated in xeroderma pigmentosum and DNA repair proficient human cells. Cancer Res. 52: 5668–5673.<br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014a). Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua. J Biol Chem. 289:18497-18506. <br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014b). Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis 35(7):1461-1468<br />
<br />
Meier B, Cooke SL, Weiss J, Bailly AP, Alexandrov LB, Marshall J, et al. (2014). C. elegans whole genome sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency. Genome Res. 24(10):1624-1636.<br />
<br />
Oleykowski CA, Mayernick JA, Lim SE, et al. (1993). Repair of aflatoxin B1 DNA adducts by the UvrABC endonuclease of Escherichia coli. J Biol.Chem. 268: 7900–8002.<br />
<br />
Pottenger, L.H., Andrews LS, Bachman AN, Boogaard PJ, Cadet J, Embry MR, Farmer PB, Himmelstein MW, Jarabek AM, Martin EA, Mauthe RJ, Persaud R, Preston RJ, Schoeny R, Skare J, Swenberg JA, Williams GM, Zeiger E, Zhang F, Kim JH. (2014). An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride. Crit. Rev. Toxicol. 44(4):348-391.<br />
<br />
Preston RJ, Williams GM. (2005). DNA-reactive carcinogens: mode of action and human cancer hazard. Crit Rev Toxicol, 35, 673–83<br />
<br />
Puisieux A, Lim S, Groopman J, Ozturk M. (1991). Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res. 51(22):6185-6189.<br />
<br />
Roebuck BD, Liu Y-L, Rogers AE, et al. (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short term molecular dosimetry. Cancer Res, 51, 5501–5506.<br />
<br />
Sarasin AR, Smith CA Hanawalt PC. (1977). Repair of DNA in human cells after treatment with activated aflatoxin B1. Cancer Res. 37: 1786–1793.<br />
<br />
Yates MS, Kwak M-K, Egner PA, et al. (2006). Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl] imidazole. Cancer Res, 66, 2488–2494.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:375Event:3752016-07-06T16:56:55Z<p>Dvillene: /* KE #1: Evidence Supporting Essentiality */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Replaced by Event:493 </div><br />
<br />
== Action ==<br />
Insufficient repair or mis-repair of DNA<br />
<br />
== Biological Process/Object ==<br />
pro-mutagenic DNA adducts, a KE for this AOP<br />
<br />
== Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
Evidence supporting the insufficient repair or mis-repair of pro-mutagenic DNA adducts is strong, but mainly indirect. This evidence comes from different biological systems and datasets, mostly mammalian, and is based in large part on the biological understanding of a DNA-reactive mode of action requiring a change in primary DNA sequence (mutation). Such a change occurs when DNA lesions are not repaired prior to cell replication (Preston and Williams, 2005; Jarabek et al, 2009; Pottenger et al., 2014). AFB1 forms two types of pro-mutagenic adducts: N7-AFB1-G and AFB1-FAPy DNA adducts, and the resulting mutations are predominantly G:C to T:A transversions. These changes are the expected result of such adducts not being repaired or undergoing mis-repair (Bedard and Massey, 2006; Lin et al., 2014a,b).<br />
<br />
Data from diverse cell types and systems demonstrate that AFB1-induced DNA adducts are repaired by a variety of DNA repair processes, including SOS repair, nucleotide excision repair (NER), homologous recombination (HR), and post-replication repair (Bedard and Massey, 2006). The work by Guo et al. (2005), conducted in transgenic yeast modified to express human CYP1A2, is particularly useful as several DNA repair systems were evaluated for efficacy towards the AFB1-induced DNA adducts. Mutations were more likely to be induced in strains deficient in certain repair systems. Mutations were also induced in strains with active secondary repair pathways; these include pathways for error-prone post-replication repair and those relying on apurinic endonucleases. However, NER appears to be most important for repair of AFB1-induced adducts in mammalian systems (Bedard and Massey, 2006).<br />
<br />
The codon 249 of the p53 gene has been identified as a particular target of AFB1-induced adduction and subsequent mutation. Using DNA polymerase fingerprint analysis, Puiseux et al. (1991) showed a specificity of AFB1 epoxide binding for this codon. This specificity was not found with benzo[a]pyrene (B[a]P), although B[a]P also forms bulky, intercalating epoxides, similar to AFB1 exo-epoxide, which result in bulky N7-B[a]P-G adducts. <br />
<br />
Essentiality of this key event of insufficient or mis-repair is supported in part by datasets demonstrating that a reduction in AFB1 adduct burden (e.g., 65-70% reduction) results in a significantly reduced or even eliminated altered hepatic foci (AHF) and liver tumor burden in experimental animals (Roebuck et al., 1991; Elegbede and Gould, 2002; Yates et al., 2006; Johnson et al., 2014). Data from studies in human populations show that treatment with known modulators of AFB1 metabolism (e.g., with oltipraz or chlorophyllin) resulted in reduced urinary levels of N7-AFB1-G, derived from AFB1-induced DNA adducts (Egner et al., 2006). These treatments are also observed to decrease levels of a human biomarker of exposure, AFB1-induced albumin adducts (Kensler et al., 1998). A similar dataset in rats pre-treated with the triterpenoid, ODDC-Im, provides an even more convincing picture, wherein a chronic bioassay demonstrated a ~60% reduction in AFB1 DNA adducts, complete elimination of liver tumors, and nearly complete elimination of altered hepatic foci (Johnson et al., 2014).<br />
Some studies suggest that eukaryotic NER expression is induced by exposure to phytochemicals (Gross-Steinmeyer et al. 2010), and alteration of DNA repair has been suggested as a pathway of chemoprevention for AFB1 carcinogenesis (Gross-Steinmeyer and Eaton, 2012).<br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|}<br />
<br />
== KE#1: Taxonomic Applicability ==<br />
Measurements of repair of AFB1-induced DNA adduct have focused mainly on in vitro systems with bacteria, yeast, and mammalian cells, including cell lines derived from rats and non-human primates (Oleykowski et al., 1993; Leadon et al., 1981; Levy et al.,1992; Sarasin et al., 1977; Guo et al., 2005; Gross-Steinmeyer and Eaton, 2012). Relevant recent studies evaluated mutation induction by AFB1 in Caenorhabditis elegans (Leung et al., 2010; Meir et al., 2014). It is likely that all species which are capable of DNA repair following binding of AFB1 exo-epoxide to DNA—are subject to insufficient or mis-repair. <br />
<br />
== KE#1: Level of Biological Organization : Cellular ==<br />
Repair of DNA adducts occurs at the sub-cellular level, as the target is nuclear DNA. For AFB1, the cross-species critical target is repair of hepatocyte nuclear DNA, as the eventual AFB1-induced tumor is hepatocellular carcinoma (HCC), which is initiated in hepatocytes.<br />
<br />
== KE#1: How this Key Event works ==<br />
The AFB1-induced pro-mutagenic DNA adducts, N7-AFB1-G and /or AFB1-FAPy, are recognized by DNA repair proteins or systems, which then initiate their repair processes (Bedard and Massey, 2006). <br />
<br />
Although there are no data specific to AFB1 for this key event of insufficient/mis-repair of DNA, it is a process known to occur and known to result in mutations. The system that recognizes the damage depends in part upon which stage of the cell cycle is in progress when the pro-mutagenic DNA adducts are recognized. Initial steps can include recruitment of a series of proteins specific to that repair system and can also include blocking of progress of the DNA replication fork. This latter step ensures that DNA replication waits for repair to occur before proceeding, preventing replication of the damaged DNA, thus avoiding a mutation when the repair might otherwise faithfully copy the error.<br />
<br />
[[Mis-repair:]] For many of the DNA repair systems the repair process is quite faithful; that is, damage is correctly repaired with the correct or original base substituted for the adducted base. This is not always the case, however, and erroneous repair is one source of mutations, when an incorrect base inserted opposite a correct or an adducted base (mis-repair). <br />
<br />
[[Insufficient repair:]] An alternative route to mutation can occur when on-going DNA replication is not stopped, and DNA replication occurs across an adducted (typically non-informational lesion) base that has not undergone any repair (insufficient repair). This situation can result in the incorporation of an incorrect base into the nascent DNA strand. <br />
<br />
Insufficient repair or mis-repair results in an incorrect base incorporated into the nascent DNA strand, an error which is then eventually replicated, resulting in a so-called “fixed” change in the primary DNA sequence—a mutation. This mutation may be silent (no impact on protein product structure and/or function) or expressed (non-functional or differently functional protein product). Depending on the location or gene of the expressed mutation, the biological impact may none, slight, or substantial. For example, if the mutation is located in codon 249 of the p53 gene and is expressed, there is potential for substantial biological impact, including progression toward carcinogenesis. A substantial biological impact is less likely when the mutation results in no change in the coded amino acid due to degenerate nature of the genetic code for amino acid sequence. Likewise a change in amino acid sequence may not result in any change in the activity or function of the protein product.<br />
<br />
== KE#1: How it is Measured or Detected ==<br />
One approach would be quantitation of chemical-specific DNA adducts, such as the N7-AFB1-G and AFB1-FAPy DNA adducts, before and after DNA repair occurs combined with DNA sequence data to determine whether the adducts were correctly repaired or not. Such is not a very practical approach. Measurement of mutations following AFB1 treatment of yeast with a variety of DNA repair deficiencies has been used to elucidate the role of DNA repair (Guo et al., 2005) by comparing mutant frequency (MF) in wild type vs. DNA repair deficient strains. In this instance MF serves as a marker for the insufficient or mis-repair of the AFB1 DNA adducts. The exquisitely sensitive analytical techniques available for structural quantification of these chemical-specific DNA adducts require specialized analytical chemistry techniques conducted on DNA isolated from tissues or cells and subjected to neutral thermal or enzyme or acid hydrolysis to release the adducted bases, which are then further analyzed (Himmelstein et al., 2009). Demonstration of adduct dose-responses and temporal relationships are possible with administration of a variety of dose regimens, including repeated doses.<br />
<br />
== KE#1: Evidence Supporting Taxonomic Applicability ==<br />
Measurements of repair of AFB1-induced DNA adduct have focused mainly on in vitro systems with bacteria, yeast, and mammalian cells, including cell lines derived from rats and non-human primates (Oleykowski et al., 1993; Leadon et al., 1981; Levy et al.,1992; Sarasin et al., 1977; Guo et al., 2005; Gross-Steinmeyer and Eaton, 2012). Relevant recent studies evaluated mutation induction of AFB1 in Caenorhabditis elegans (Leung et al., 2010; Meir et al., 2014), Likely, all species capable of DNA repair following binding of AFB1 exo-epoxide to DNA are subject to insufficient or mis-repair These species include bacteria, yeast, birds, mammals, and fish.<br />
<br />
== References ==<br />
Bedard, L.L., and Massey, T.T. (2006). Aflatoxin B1-induced DNA damage and its repair. Cancer Lett. 241(2):174-183.<br />
<br />
Egner PA, Groopman JD, Wang J-S, Kensler TW, Friesen MD (2006). Quantification of aflatoxin-B1-N7-Guanine in human urine by high-performance liquid chromatography and isotope dilution tandem mass spectrometry. Chem Res Toxicol 19:1191-1195.<br />
<br />
Elegbede JA, and Gould MN. (2002). Monoterpenes reduced adducts formation in rats exposed to aflatoxin B1. African J Biotech, 1, 46–49.<br />
<br />
Gross-Steinmeyer K, and Eaton DL. (2012). Dietary modulation of the biotransformation and genetoxicity of aflatoxin B1. Toxicology, 299: 69–79.<br />
<br />
Guo Y, Breeden LL, Zarbl H, et al. (2005). Expression of a human cytochrome p450 in yeast permits analysis of pathways for response to and repair of aflatoxin-induced DNA damage. Mol Cell Biol, 25, 5823–5833.<br />
<br />
Himmelstein MW, Boogaard PJ, Cadet J, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: II.Overview of methods of identification and quantitation of DNA damage. Crit Rev Toxicol, 39, 679–694.<br />
<br />
Jarabek, AM, Pottenger, LH, Andrews, LS, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: I. Data organization. Crit. Rev. Toxicol. 39: 659-678.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, Sutter TR, Groopman JD, Kensler TW, Roebuck BD. (2014). Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold. Cancer Prev Res. 7(7):658-665.<br />
<br />
Kensler TW, He X, Otieno M, et al. (1998). Oltipraz chemoprevention trial in Qidong, People’s Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol Biomarkers Prev, 7, 127–134.<br />
<br />
Leadon SA, Tyrrell RM, Cerutti PA. (1981). Excision repair of aflatoxin B1-DNA adducts in human fibroblasts. Cancer Res. 41: 5125-5129.<br />
<br />
Leung MC, Goldstone JV, Boyd WA, Freedman JH, and Meyer JN. (2010). Caenorhabditis elegans generates biologically relevant levels of genotoxic metabolites from aflatoxin B1 but not benzo[a]pyrene in vivo. Toxicol Sci.118(2):444-453.<br />
<br />
Levy DD, Groopman JD, Lim SE, et al. (1992). Sequence specificity of aflatoxin B1-induced mutations in a plasmid replicated in xeroderma pigmentosum and DNA repair proficient human cells. Cancer Res. 52: 5668–5673.<br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014a). Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua. J Biol Chem. 289:18497-18506. <br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014b). Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis 35(7):1461-1468<br />
<br />
Meier B, Cooke SL, Weiss J, Bailly AP, Alexandrov LB, Marshall J, et al. (2014). C. elegans whole genome sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency. Genome Res. 24(10):1624-1636.<br />
<br />
Oleykowski CA, Mayernick JA, Lim SE, et al. (1993). Repair of aflatoxin B1 DNA adducts by the UvrABC endonuclease of Escherichia coli. J Biol.Chem. 268: 7900–8002.<br />
<br />
Pottenger, L.H., Andrews LS, Bachman AN, Boogaard PJ, Cadet J, Embry MR, Farmer PB, Himmelstein MW, Jarabek AM, Martin EA, Mauthe RJ, Persaud R, Preston RJ, Schoeny R, Skare J, Swenberg JA, Williams GM, Zeiger E, Zhang F, Kim JH. (2014). An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride. Crit. Rev. Toxicol. 44(4):348-391.<br />
<br />
Preston RJ, Williams GM. (2005). DNA-reactive carcinogens: mode of action and human cancer hazard. Crit Rev Toxicol, 35, 673–83<br />
<br />
Puisieux A, Lim S, Groopman J, Ozturk M. (1991). Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res. 51(22):6185-6189.<br />
<br />
Roebuck BD, Liu Y-L, Rogers AE, et al. (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short term molecular dosimetry. Cancer Res, 51, 5501–5506.<br />
<br />
Sarasin AR, Smith CA Hanawalt PC. (1977). Repair of DNA in human cells after treatment with activated aflatoxin B1. Cancer Res. 37: 1786–1793.<br />
<br />
Yates MS, Kwak M-K, Egner PA, et al. (2006). Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl] imidazole. Cancer Res, 66, 2488–2494.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:375Event:3752016-07-06T16:56:38Z<p>Dvillene: /* KE#1: Biological Process/Object */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Replaced by Event:493 </div><br />
<br />
== Action ==<br />
Insufficient repair or mis-repair of DNA<br />
<br />
== Biological Process/Object ==<br />
pro-mutagenic DNA adducts, a KE for this AOP<br />
<br />
== KE #1: Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
Evidence supporting the insufficient repair or mis-repair of pro-mutagenic DNA adducts is strong, but mainly indirect. This evidence comes from different biological systems and datasets, mostly mammalian, and is based in large part on the biological understanding of a DNA-reactive mode of action requiring a change in primary DNA sequence (mutation). Such a change occurs when DNA lesions are not repaired prior to cell replication (Preston and Williams, 2005; Jarabek et al, 2009; Pottenger et al., 2014). AFB1 forms two types of pro-mutagenic adducts: N7-AFB1-G and AFB1-FAPy DNA adducts, and the resulting mutations are predominantly G:C to T:A transversions. These changes are the expected result of such adducts not being repaired or undergoing mis-repair (Bedard and Massey, 2006; Lin et al., 2014a,b).<br />
<br />
Data from diverse cell types and systems demonstrate that AFB1-induced DNA adducts are repaired by a variety of DNA repair processes, including SOS repair, nucleotide excision repair (NER), homologous recombination (HR), and post-replication repair (Bedard and Massey, 2006). The work by Guo et al. (2005), conducted in transgenic yeast modified to express human CYP1A2, is particularly useful as several DNA repair systems were evaluated for efficacy towards the AFB1-induced DNA adducts. Mutations were more likely to be induced in strains deficient in certain repair systems. Mutations were also induced in strains with active secondary repair pathways; these include pathways for error-prone post-replication repair and those relying on apurinic endonucleases. However, NER appears to be most important for repair of AFB1-induced adducts in mammalian systems (Bedard and Massey, 2006).<br />
<br />
The codon 249 of the p53 gene has been identified as a particular target of AFB1-induced adduction and subsequent mutation. Using DNA polymerase fingerprint analysis, Puiseux et al. (1991) showed a specificity of AFB1 epoxide binding for this codon. This specificity was not found with benzo[a]pyrene (B[a]P), although B[a]P also forms bulky, intercalating epoxides, similar to AFB1 exo-epoxide, which result in bulky N7-B[a]P-G adducts. <br />
<br />
Essentiality of this key event of insufficient or mis-repair is supported in part by datasets demonstrating that a reduction in AFB1 adduct burden (e.g., 65-70% reduction) results in a significantly reduced or even eliminated altered hepatic foci (AHF) and liver tumor burden in experimental animals (Roebuck et al., 1991; Elegbede and Gould, 2002; Yates et al., 2006; Johnson et al., 2014). Data from studies in human populations show that treatment with known modulators of AFB1 metabolism (e.g., with oltipraz or chlorophyllin) resulted in reduced urinary levels of N7-AFB1-G, derived from AFB1-induced DNA adducts (Egner et al., 2006). These treatments are also observed to decrease levels of a human biomarker of exposure, AFB1-induced albumin adducts (Kensler et al., 1998). A similar dataset in rats pre-treated with the triterpenoid, ODDC-Im, provides an even more convincing picture, wherein a chronic bioassay demonstrated a ~60% reduction in AFB1 DNA adducts, complete elimination of liver tumors, and nearly complete elimination of altered hepatic foci (Johnson et al., 2014).<br />
Some studies suggest that eukaryotic NER expression is induced by exposure to phytochemicals (Gross-Steinmeyer et al. 2010), and alteration of DNA repair has been suggested as a pathway of chemoprevention for AFB1 carcinogenesis (Gross-Steinmeyer and Eaton, 2012).<br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|}<br />
<br />
== KE#1: Taxonomic Applicability ==<br />
Measurements of repair of AFB1-induced DNA adduct have focused mainly on in vitro systems with bacteria, yeast, and mammalian cells, including cell lines derived from rats and non-human primates (Oleykowski et al., 1993; Leadon et al., 1981; Levy et al.,1992; Sarasin et al., 1977; Guo et al., 2005; Gross-Steinmeyer and Eaton, 2012). Relevant recent studies evaluated mutation induction by AFB1 in Caenorhabditis elegans (Leung et al., 2010; Meir et al., 2014). It is likely that all species which are capable of DNA repair following binding of AFB1 exo-epoxide to DNA—are subject to insufficient or mis-repair. <br />
<br />
== KE#1: Level of Biological Organization : Cellular ==<br />
Repair of DNA adducts occurs at the sub-cellular level, as the target is nuclear DNA. For AFB1, the cross-species critical target is repair of hepatocyte nuclear DNA, as the eventual AFB1-induced tumor is hepatocellular carcinoma (HCC), which is initiated in hepatocytes.<br />
<br />
== KE#1: How this Key Event works ==<br />
The AFB1-induced pro-mutagenic DNA adducts, N7-AFB1-G and /or AFB1-FAPy, are recognized by DNA repair proteins or systems, which then initiate their repair processes (Bedard and Massey, 2006). <br />
<br />
Although there are no data specific to AFB1 for this key event of insufficient/mis-repair of DNA, it is a process known to occur and known to result in mutations. The system that recognizes the damage depends in part upon which stage of the cell cycle is in progress when the pro-mutagenic DNA adducts are recognized. Initial steps can include recruitment of a series of proteins specific to that repair system and can also include blocking of progress of the DNA replication fork. This latter step ensures that DNA replication waits for repair to occur before proceeding, preventing replication of the damaged DNA, thus avoiding a mutation when the repair might otherwise faithfully copy the error.<br />
<br />
[[Mis-repair:]] For many of the DNA repair systems the repair process is quite faithful; that is, damage is correctly repaired with the correct or original base substituted for the adducted base. This is not always the case, however, and erroneous repair is one source of mutations, when an incorrect base inserted opposite a correct or an adducted base (mis-repair). <br />
<br />
[[Insufficient repair:]] An alternative route to mutation can occur when on-going DNA replication is not stopped, and DNA replication occurs across an adducted (typically non-informational lesion) base that has not undergone any repair (insufficient repair). This situation can result in the incorporation of an incorrect base into the nascent DNA strand. <br />
<br />
Insufficient repair or mis-repair results in an incorrect base incorporated into the nascent DNA strand, an error which is then eventually replicated, resulting in a so-called “fixed” change in the primary DNA sequence—a mutation. This mutation may be silent (no impact on protein product structure and/or function) or expressed (non-functional or differently functional protein product). Depending on the location or gene of the expressed mutation, the biological impact may none, slight, or substantial. For example, if the mutation is located in codon 249 of the p53 gene and is expressed, there is potential for substantial biological impact, including progression toward carcinogenesis. A substantial biological impact is less likely when the mutation results in no change in the coded amino acid due to degenerate nature of the genetic code for amino acid sequence. Likewise a change in amino acid sequence may not result in any change in the activity or function of the protein product.<br />
<br />
== KE#1: How it is Measured or Detected ==<br />
One approach would be quantitation of chemical-specific DNA adducts, such as the N7-AFB1-G and AFB1-FAPy DNA adducts, before and after DNA repair occurs combined with DNA sequence data to determine whether the adducts were correctly repaired or not. Such is not a very practical approach. Measurement of mutations following AFB1 treatment of yeast with a variety of DNA repair deficiencies has been used to elucidate the role of DNA repair (Guo et al., 2005) by comparing mutant frequency (MF) in wild type vs. DNA repair deficient strains. In this instance MF serves as a marker for the insufficient or mis-repair of the AFB1 DNA adducts. The exquisitely sensitive analytical techniques available for structural quantification of these chemical-specific DNA adducts require specialized analytical chemistry techniques conducted on DNA isolated from tissues or cells and subjected to neutral thermal or enzyme or acid hydrolysis to release the adducted bases, which are then further analyzed (Himmelstein et al., 2009). Demonstration of adduct dose-responses and temporal relationships are possible with administration of a variety of dose regimens, including repeated doses.<br />
<br />
== KE#1: Evidence Supporting Taxonomic Applicability ==<br />
Measurements of repair of AFB1-induced DNA adduct have focused mainly on in vitro systems with bacteria, yeast, and mammalian cells, including cell lines derived from rats and non-human primates (Oleykowski et al., 1993; Leadon et al., 1981; Levy et al.,1992; Sarasin et al., 1977; Guo et al., 2005; Gross-Steinmeyer and Eaton, 2012). Relevant recent studies evaluated mutation induction of AFB1 in Caenorhabditis elegans (Leung et al., 2010; Meir et al., 2014), Likely, all species capable of DNA repair following binding of AFB1 exo-epoxide to DNA are subject to insufficient or mis-repair These species include bacteria, yeast, birds, mammals, and fish.<br />
<br />
== References ==<br />
Bedard, L.L., and Massey, T.T. (2006). Aflatoxin B1-induced DNA damage and its repair. Cancer Lett. 241(2):174-183.<br />
<br />
Egner PA, Groopman JD, Wang J-S, Kensler TW, Friesen MD (2006). Quantification of aflatoxin-B1-N7-Guanine in human urine by high-performance liquid chromatography and isotope dilution tandem mass spectrometry. Chem Res Toxicol 19:1191-1195.<br />
<br />
Elegbede JA, and Gould MN. (2002). Monoterpenes reduced adducts formation in rats exposed to aflatoxin B1. African J Biotech, 1, 46–49.<br />
<br />
Gross-Steinmeyer K, and Eaton DL. (2012). Dietary modulation of the biotransformation and genetoxicity of aflatoxin B1. Toxicology, 299: 69–79.<br />
<br />
Guo Y, Breeden LL, Zarbl H, et al. (2005). Expression of a human cytochrome p450 in yeast permits analysis of pathways for response to and repair of aflatoxin-induced DNA damage. Mol Cell Biol, 25, 5823–5833.<br />
<br />
Himmelstein MW, Boogaard PJ, Cadet J, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: II.Overview of methods of identification and quantitation of DNA damage. Crit Rev Toxicol, 39, 679–694.<br />
<br />
Jarabek, AM, Pottenger, LH, Andrews, LS, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: I. Data organization. Crit. Rev. Toxicol. 39: 659-678.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, Sutter TR, Groopman JD, Kensler TW, Roebuck BD. (2014). Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold. Cancer Prev Res. 7(7):658-665.<br />
<br />
Kensler TW, He X, Otieno M, et al. (1998). Oltipraz chemoprevention trial in Qidong, People’s Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol Biomarkers Prev, 7, 127–134.<br />
<br />
Leadon SA, Tyrrell RM, Cerutti PA. (1981). Excision repair of aflatoxin B1-DNA adducts in human fibroblasts. Cancer Res. 41: 5125-5129.<br />
<br />
Leung MC, Goldstone JV, Boyd WA, Freedman JH, and Meyer JN. (2010). Caenorhabditis elegans generates biologically relevant levels of genotoxic metabolites from aflatoxin B1 but not benzo[a]pyrene in vivo. Toxicol Sci.118(2):444-453.<br />
<br />
Levy DD, Groopman JD, Lim SE, et al. (1992). Sequence specificity of aflatoxin B1-induced mutations in a plasmid replicated in xeroderma pigmentosum and DNA repair proficient human cells. Cancer Res. 52: 5668–5673.<br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014a). Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua. J Biol Chem. 289:18497-18506. <br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014b). Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis 35(7):1461-1468<br />
<br />
Meier B, Cooke SL, Weiss J, Bailly AP, Alexandrov LB, Marshall J, et al. (2014). C. elegans whole genome sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency. Genome Res. 24(10):1624-1636.<br />
<br />
Oleykowski CA, Mayernick JA, Lim SE, et al. (1993). Repair of aflatoxin B1 DNA adducts by the UvrABC endonuclease of Escherichia coli. J Biol.Chem. 268: 7900–8002.<br />
<br />
Pottenger, L.H., Andrews LS, Bachman AN, Boogaard PJ, Cadet J, Embry MR, Farmer PB, Himmelstein MW, Jarabek AM, Martin EA, Mauthe RJ, Persaud R, Preston RJ, Schoeny R, Skare J, Swenberg JA, Williams GM, Zeiger E, Zhang F, Kim JH. (2014). An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride. Crit. Rev. Toxicol. 44(4):348-391.<br />
<br />
Preston RJ, Williams GM. (2005). DNA-reactive carcinogens: mode of action and human cancer hazard. Crit Rev Toxicol, 35, 673–83<br />
<br />
Puisieux A, Lim S, Groopman J, Ozturk M. (1991). Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res. 51(22):6185-6189.<br />
<br />
Roebuck BD, Liu Y-L, Rogers AE, et al. (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short term molecular dosimetry. Cancer Res, 51, 5501–5506.<br />
<br />
Sarasin AR, Smith CA Hanawalt PC. (1977). Repair of DNA in human cells after treatment with activated aflatoxin B1. Cancer Res. 37: 1786–1793.<br />
<br />
Yates MS, Kwak M-K, Egner PA, et al. (2006). Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl] imidazole. Cancer Res, 66, 2488–2494.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:375Event:3752016-07-06T16:56:21Z<p>Dvillene: /* KE#1: Action */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Replaced by Event:493 </div><br />
<br />
== Action ==<br />
Insufficient repair or mis-repair of DNA<br />
<br />
== KE#1: Biological Process/Object ==<br />
pro-mutagenic DNA adducts, a KE for this AOP<br />
<br />
== KE #1: Evidence Supporting Essentiality ==<br />
Strong<br />
<br />
Evidence supporting the insufficient repair or mis-repair of pro-mutagenic DNA adducts is strong, but mainly indirect. This evidence comes from different biological systems and datasets, mostly mammalian, and is based in large part on the biological understanding of a DNA-reactive mode of action requiring a change in primary DNA sequence (mutation). Such a change occurs when DNA lesions are not repaired prior to cell replication (Preston and Williams, 2005; Jarabek et al, 2009; Pottenger et al., 2014). AFB1 forms two types of pro-mutagenic adducts: N7-AFB1-G and AFB1-FAPy DNA adducts, and the resulting mutations are predominantly G:C to T:A transversions. These changes are the expected result of such adducts not being repaired or undergoing mis-repair (Bedard and Massey, 2006; Lin et al., 2014a,b).<br />
<br />
Data from diverse cell types and systems demonstrate that AFB1-induced DNA adducts are repaired by a variety of DNA repair processes, including SOS repair, nucleotide excision repair (NER), homologous recombination (HR), and post-replication repair (Bedard and Massey, 2006). The work by Guo et al. (2005), conducted in transgenic yeast modified to express human CYP1A2, is particularly useful as several DNA repair systems were evaluated for efficacy towards the AFB1-induced DNA adducts. Mutations were more likely to be induced in strains deficient in certain repair systems. Mutations were also induced in strains with active secondary repair pathways; these include pathways for error-prone post-replication repair and those relying on apurinic endonucleases. However, NER appears to be most important for repair of AFB1-induced adducts in mammalian systems (Bedard and Massey, 2006).<br />
<br />
The codon 249 of the p53 gene has been identified as a particular target of AFB1-induced adduction and subsequent mutation. Using DNA polymerase fingerprint analysis, Puiseux et al. (1991) showed a specificity of AFB1 epoxide binding for this codon. This specificity was not found with benzo[a]pyrene (B[a]P), although B[a]P also forms bulky, intercalating epoxides, similar to AFB1 exo-epoxide, which result in bulky N7-B[a]P-G adducts. <br />
<br />
Essentiality of this key event of insufficient or mis-repair is supported in part by datasets demonstrating that a reduction in AFB1 adduct burden (e.g., 65-70% reduction) results in a significantly reduced or even eliminated altered hepatic foci (AHF) and liver tumor burden in experimental animals (Roebuck et al., 1991; Elegbede and Gould, 2002; Yates et al., 2006; Johnson et al., 2014). Data from studies in human populations show that treatment with known modulators of AFB1 metabolism (e.g., with oltipraz or chlorophyllin) resulted in reduced urinary levels of N7-AFB1-G, derived from AFB1-induced DNA adducts (Egner et al., 2006). These treatments are also observed to decrease levels of a human biomarker of exposure, AFB1-induced albumin adducts (Kensler et al., 1998). A similar dataset in rats pre-treated with the triterpenoid, ODDC-Im, provides an even more convincing picture, wherein a chronic bioassay demonstrated a ~60% reduction in AFB1 DNA adducts, complete elimination of liver tumors, and nearly complete elimination of altered hepatic foci (Johnson et al., 2014).<br />
Some studies suggest that eukaryotic NER expression is induced by exposure to phytochemicals (Gross-Steinmeyer et al. 2010), and alteration of DNA repair has been suggested as a pathway of chemoprevention for AFB1 carcinogenesis (Gross-Steinmeyer and Eaton, 2012).<br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|}<br />
<br />
== KE#1: Taxonomic Applicability ==<br />
Measurements of repair of AFB1-induced DNA adduct have focused mainly on in vitro systems with bacteria, yeast, and mammalian cells, including cell lines derived from rats and non-human primates (Oleykowski et al., 1993; Leadon et al., 1981; Levy et al.,1992; Sarasin et al., 1977; Guo et al., 2005; Gross-Steinmeyer and Eaton, 2012). Relevant recent studies evaluated mutation induction by AFB1 in Caenorhabditis elegans (Leung et al., 2010; Meir et al., 2014). It is likely that all species which are capable of DNA repair following binding of AFB1 exo-epoxide to DNA—are subject to insufficient or mis-repair. <br />
<br />
== KE#1: Level of Biological Organization : Cellular ==<br />
Repair of DNA adducts occurs at the sub-cellular level, as the target is nuclear DNA. For AFB1, the cross-species critical target is repair of hepatocyte nuclear DNA, as the eventual AFB1-induced tumor is hepatocellular carcinoma (HCC), which is initiated in hepatocytes.<br />
<br />
== KE#1: How this Key Event works ==<br />
The AFB1-induced pro-mutagenic DNA adducts, N7-AFB1-G and /or AFB1-FAPy, are recognized by DNA repair proteins or systems, which then initiate their repair processes (Bedard and Massey, 2006). <br />
<br />
Although there are no data specific to AFB1 for this key event of insufficient/mis-repair of DNA, it is a process known to occur and known to result in mutations. The system that recognizes the damage depends in part upon which stage of the cell cycle is in progress when the pro-mutagenic DNA adducts are recognized. Initial steps can include recruitment of a series of proteins specific to that repair system and can also include blocking of progress of the DNA replication fork. This latter step ensures that DNA replication waits for repair to occur before proceeding, preventing replication of the damaged DNA, thus avoiding a mutation when the repair might otherwise faithfully copy the error.<br />
<br />
[[Mis-repair:]] For many of the DNA repair systems the repair process is quite faithful; that is, damage is correctly repaired with the correct or original base substituted for the adducted base. This is not always the case, however, and erroneous repair is one source of mutations, when an incorrect base inserted opposite a correct or an adducted base (mis-repair). <br />
<br />
[[Insufficient repair:]] An alternative route to mutation can occur when on-going DNA replication is not stopped, and DNA replication occurs across an adducted (typically non-informational lesion) base that has not undergone any repair (insufficient repair). This situation can result in the incorporation of an incorrect base into the nascent DNA strand. <br />
<br />
Insufficient repair or mis-repair results in an incorrect base incorporated into the nascent DNA strand, an error which is then eventually replicated, resulting in a so-called “fixed” change in the primary DNA sequence—a mutation. This mutation may be silent (no impact on protein product structure and/or function) or expressed (non-functional or differently functional protein product). Depending on the location or gene of the expressed mutation, the biological impact may none, slight, or substantial. For example, if the mutation is located in codon 249 of the p53 gene and is expressed, there is potential for substantial biological impact, including progression toward carcinogenesis. A substantial biological impact is less likely when the mutation results in no change in the coded amino acid due to degenerate nature of the genetic code for amino acid sequence. Likewise a change in amino acid sequence may not result in any change in the activity or function of the protein product.<br />
<br />
== KE#1: How it is Measured or Detected ==<br />
One approach would be quantitation of chemical-specific DNA adducts, such as the N7-AFB1-G and AFB1-FAPy DNA adducts, before and after DNA repair occurs combined with DNA sequence data to determine whether the adducts were correctly repaired or not. Such is not a very practical approach. Measurement of mutations following AFB1 treatment of yeast with a variety of DNA repair deficiencies has been used to elucidate the role of DNA repair (Guo et al., 2005) by comparing mutant frequency (MF) in wild type vs. DNA repair deficient strains. In this instance MF serves as a marker for the insufficient or mis-repair of the AFB1 DNA adducts. The exquisitely sensitive analytical techniques available for structural quantification of these chemical-specific DNA adducts require specialized analytical chemistry techniques conducted on DNA isolated from tissues or cells and subjected to neutral thermal or enzyme or acid hydrolysis to release the adducted bases, which are then further analyzed (Himmelstein et al., 2009). Demonstration of adduct dose-responses and temporal relationships are possible with administration of a variety of dose regimens, including repeated doses.<br />
<br />
== KE#1: Evidence Supporting Taxonomic Applicability ==<br />
Measurements of repair of AFB1-induced DNA adduct have focused mainly on in vitro systems with bacteria, yeast, and mammalian cells, including cell lines derived from rats and non-human primates (Oleykowski et al., 1993; Leadon et al., 1981; Levy et al.,1992; Sarasin et al., 1977; Guo et al., 2005; Gross-Steinmeyer and Eaton, 2012). Relevant recent studies evaluated mutation induction of AFB1 in Caenorhabditis elegans (Leung et al., 2010; Meir et al., 2014), Likely, all species capable of DNA repair following binding of AFB1 exo-epoxide to DNA are subject to insufficient or mis-repair These species include bacteria, yeast, birds, mammals, and fish.<br />
<br />
== References ==<br />
Bedard, L.L., and Massey, T.T. (2006). Aflatoxin B1-induced DNA damage and its repair. Cancer Lett. 241(2):174-183.<br />
<br />
Egner PA, Groopman JD, Wang J-S, Kensler TW, Friesen MD (2006). Quantification of aflatoxin-B1-N7-Guanine in human urine by high-performance liquid chromatography and isotope dilution tandem mass spectrometry. Chem Res Toxicol 19:1191-1195.<br />
<br />
Elegbede JA, and Gould MN. (2002). Monoterpenes reduced adducts formation in rats exposed to aflatoxin B1. African J Biotech, 1, 46–49.<br />
<br />
Gross-Steinmeyer K, and Eaton DL. (2012). Dietary modulation of the biotransformation and genetoxicity of aflatoxin B1. Toxicology, 299: 69–79.<br />
<br />
Guo Y, Breeden LL, Zarbl H, et al. (2005). Expression of a human cytochrome p450 in yeast permits analysis of pathways for response to and repair of aflatoxin-induced DNA damage. Mol Cell Biol, 25, 5823–5833.<br />
<br />
Himmelstein MW, Boogaard PJ, Cadet J, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: II.Overview of methods of identification and quantitation of DNA damage. Crit Rev Toxicol, 39, 679–694.<br />
<br />
Jarabek, AM, Pottenger, LH, Andrews, LS, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: I. Data organization. Crit. Rev. Toxicol. 39: 659-678.<br />
<br />
Johnson NM, Egner PA, Baxter VK, Sporn MB, Wible RS, Sutter TR, Groopman JD, Kensler TW, Roebuck BD. (2014). Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold. Cancer Prev Res. 7(7):658-665.<br />
<br />
Kensler TW, He X, Otieno M, et al. (1998). Oltipraz chemoprevention trial in Qidong, People’s Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol Biomarkers Prev, 7, 127–134.<br />
<br />
Leadon SA, Tyrrell RM, Cerutti PA. (1981). Excision repair of aflatoxin B1-DNA adducts in human fibroblasts. Cancer Res. 41: 5125-5129.<br />
<br />
Leung MC, Goldstone JV, Boyd WA, Freedman JH, and Meyer JN. (2010). Caenorhabditis elegans generates biologically relevant levels of genotoxic metabolites from aflatoxin B1 but not benzo[a]pyrene in vivo. Toxicol Sci.118(2):444-453.<br />
<br />
Levy DD, Groopman JD, Lim SE, et al. (1992). Sequence specificity of aflatoxin B1-induced mutations in a plasmid replicated in xeroderma pigmentosum and DNA repair proficient human cells. Cancer Res. 52: 5668–5673.<br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014a). Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua. J Biol Chem. 289:18497-18506. <br />
<br />
Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014b). Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis 35(7):1461-1468<br />
<br />
Meier B, Cooke SL, Weiss J, Bailly AP, Alexandrov LB, Marshall J, et al. (2014). C. elegans whole genome sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency. Genome Res. 24(10):1624-1636.<br />
<br />
Oleykowski CA, Mayernick JA, Lim SE, et al. (1993). Repair of aflatoxin B1 DNA adducts by the UvrABC endonuclease of Escherichia coli. J Biol.Chem. 268: 7900–8002.<br />
<br />
Pottenger, L.H., Andrews LS, Bachman AN, Boogaard PJ, Cadet J, Embry MR, Farmer PB, Himmelstein MW, Jarabek AM, Martin EA, Mauthe RJ, Persaud R, Preston RJ, Schoeny R, Skare J, Swenberg JA, Williams GM, Zeiger E, Zhang F, Kim JH. (2014). An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride. Crit. Rev. Toxicol. 44(4):348-391.<br />
<br />
Preston RJ, Williams GM. (2005). DNA-reactive carcinogens: mode of action and human cancer hazard. Crit Rev Toxicol, 35, 673–83<br />
<br />
Puisieux A, Lim S, Groopman J, Ozturk M. (1991). Selective targeting of p53 gene mutational hotspots in human cancers by etiologically defined carcinogens. Cancer Res. 51(22):6185-6189.<br />
<br />
Roebuck BD, Liu Y-L, Rogers AE, et al. (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short term molecular dosimetry. Cancer Res, 51, 5501–5506.<br />
<br />
Sarasin AR, Smith CA Hanawalt PC. (1977). Repair of DNA in human cells after treatment with activated aflatoxin B1. Cancer Res. 37: 1786–1793.<br />
<br />
Yates MS, Kwak M-K, Egner PA, et al. (2006). Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl] imidazole. Cancer Res, 66, 2488–2494.</div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:742Event:7422016-07-06T16:52:28Z<p>Dvillene: </p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Androgen receptor activity, Decreased </div><br />
<div id='shortTitle' class='Title2'> </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]][[Category:Molecular Initiating Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:111|Decrease in androgen receptor activity leading to Leydig cell tumors (in rat)]]||MIE||<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Chemical Initiators ===<br />
The following are chemical initiators that operate directly through this Event:<br />
<br />
#[[Chem_Init:136|Vinclozalin]]<br />
#[[Chem_Init:137|Flutamide]]<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|}<br />
<br />
== How this Key Event works ==<br />
<br />
== How it is Measured or Detected ==<br />
<em><br />
Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above.<br />
All other methods, including those well established in the published literature, should be described here. <br />
Consider the following criteria when describing each method:<br />
1. Is the assay fit for purpose?<br />
2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final<br />
adverse effect in question?<br />
3. Is the assay repeatable?<br />
4. Is the assay reproducible?<br />
</em><br />
<br />
== Evidence Supporting Taxonomic Applicability ==<br />
<br />
== References ==<br />
<br />
<references /></div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:741Event:7412016-07-06T16:51:27Z<p>Dvillene: </p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Adenomas/carcinomas (follicular cell), Increase </div><br />
<div id='shortTitle' class='Title2'> </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]][[Category:Adverse Outcome]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:110|Inhibition of iodide pump activity leading to follicular cell adenomas and carcinomas (in rat and mouse)]]||AO||<br />
<br />
|-<br />
<br />
|[[Aop:119|Inhibition of thyroid peroxidase leading to follicular cell adenomas and carcinomas (in rat and mouse)]]||AO||<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Affected Organs ===<br />
<br />
{|class="wikitable sortable" id="Organproof"<br />
<br />
!Synonym<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|}<br />
<br />
== How this Key Event works ==<br />
<br />
== How it is Measured or Detected ==<br />
<em><br />
Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above.<br />
All other methods, including those well established in the published literature, should be described here. <br />
Consider the following criteria when describing each method:<br />
1. Is the assay fit for purpose?<br />
2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final<br />
adverse effect in question?<br />
3. Is the assay repeatable?<br />
4. Is the assay reproducible?<br />
</em><br />
<br />
== Evidence Supporting Taxonomic Applicability ==<br />
<br />
== References ==<br />
<br />
<references /></div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:737Event:7372016-07-06T16:50:36Z<p>Dvillene: </p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Uptake of inorganic iodide, Decreased </div><br />
<div id='shortTitle' class='Title2'> </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]][[Category:Molecular Initiating Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:110|Inhibition of iodide pump activity leading to follicular cell adenomas and carcinomas (in rat and mouse)]]||MIE||<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Chemical Initiators ===<br />
The following are chemical initiators that operate directly through this Event:<br />
<br />
#[[Chem_Init:66|Perchlorate]]<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|}<br />
<br />
== How this Key Event works ==<br />
<br />
== How it is Measured or Detected ==<br />
<em><br />
Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above.<br />
All other methods, including those well established in the published literature, should be described here. <br />
Consider the following criteria when describing each method:<br />
1. Is the assay fit for purpose?<br />
2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final<br />
adverse effect in question?<br />
3. Is the assay repeatable?<br />
4. Is the assay reproducible?<br />
</em><br />
<br />
== Evidence Supporting Taxonomic Applicability ==<br />
<br />
== References ==<br />
<br />
<references /></div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:736Event:7362016-07-06T16:49:42Z<p>Dvillene: </p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Adenomas/carcinomas (bronchioloalveolar), Increase </div><br />
<div id='shortTitle' class='Title2'> </div><br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]][[Category:Adverse Outcome]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:109|Cytotoxicity leading to bronchioloalveolar adenomas and carcinomas (in mouse)]]||AO||<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Affected Organs ===<br />
<br />
{|class="wikitable sortable" id="Organproof"<br />
<br />
!Synonym<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|}<br />
<br />
== How this Key Event works ==<br />
<br />
== How it is Measured or Detected ==<br />
<em><br />
Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above.<br />
All other methods, including those well established in the published literature, should be described here. <br />
Consider the following criteria when describing each method:<br />
1. Is the assay fit for purpose?<br />
2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final<br />
adverse effect in question?<br />
3. Is the assay repeatable?<br />
4. Is the assay reproducible?<br />
</em><br />
<br />
== Evidence Supporting Taxonomic Applicability ==<br />
<br />
== References ==<br />
<br />
<references /></div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:373Event:3732016-07-06T16:43:44Z<p>Dvillene: /* MIE: References */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Pro-mutagenic DNA Adducts, Formation </div><br />
<div id='shortTitle' class='Title2'> </div><br />
<br />
== Molecular Initiating Event ==<br />
<br />
== Action - how the event is affected ==<br />
Alkylation, binding<br />
<br />
== Biological Process/Object ==<br />
The Molecular Initiating Event for this AOP<br />
Formation of AFB1-induced pro-mutagenic DNA adducts: N7-AFB1-G and/or AFB1-FAPy<br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]][[Category:Molecular Initiating Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||MIE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Chemical Initiators ===<br />
The following are chemical initiators that operate directly through this Event:<br />
<br />
#[[Chem_Init:61|Aflatoxin B1]]<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|rainbow trout||Oncorhynchus mykiss||[[Event:373#Evidence Supporting Taxonomic Applicability|Strong]]||[http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8022 NCBI]<br />
<br />
|-<br />
<br />
|Human, rat, mouse||||[[Event:373#Evidence Supporting Taxonomic Applicability|Strong]]||<br />
<br />
|-<br />
<br />
|chickens, ducks, turkeys||||[[Event:373#Evidence Supporting Taxonomic Applicability|Strong]]||<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Molecular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Evidence Supporting Essentiality ==<br />
Strong <br />
<br />
Evidence supporting the formation of an AFB1-induced pro-mutagenic DNA adduct as the molecular initiating event (MIE) is strong and stems from many datasets in different biological systems. The formation of N7-AFB1-G DNA adducts after AFB1 exposure has been demonstrated across phyla, from bacteria through yeast, fish, birds, and including many mammalian systems up through non-human primates and humans (Croy et al., 1978; IARC, 1993; Cupid et al., 2004). <br />
<br />
The reactive metabolite AFB1 exo-epoxide intercalates into DNA and then binds to the nucleophilic N7-G residue via an SN2 reaction. This N7-G DNA adduct can then spontaneously ring-open to form the more highly pro-mutagenic 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-pyrimid-9-yl-foramido-9-hydroxyaflatoxin B1, or formamidopyrimidine adduct, AFB1-FAPy (Brown et al., 2006).<br />
<br />
The essentiality of this MIE is demonstrated by the effects of modulation of metabolism to reactive forms. Inhibition of activation results in reduced formation of the critical exo-epoxide. Likewise, increased GST activity results in increased metabolism of the exo-epoxide to less toxic forms. In both cases, less reactive metabolite is available to form DNA adducts, resulting in fewer adducts (Guengerich et al., 1996). Pre-treatment of rats with oltipraz provides a specific example, wherein a 65-70% reduction in AFB1-induced DNA adducts was demonstrated due to increased GST activity; this corresponds with a subsequent 100% reduction in liver tumors (Roebuck et al., 1991; Kensler et al., 1998). <br />
<br />
<br />
Another line of evidence for essentiality of the MIE is the recognized species difference in sensitivity to AFB1-induced liver tumors between mice and rats. Mice, with considerably increased metabolic activation of AFB1 to the exo-epoxide compared with rats, are nonetheless much less sensitive to AFB1-induced liver tumors (Degen and Neumann, 1981). This difference is believed to be due to the constitutive presence of GST-alpha activity in mice vs. rats, where this activity is not found (Monroe and Eaton, 1987).<br />
<br />
== Taxonomic Applicabilty ==<br />
AFB1-induced DNA adduct measurements have focused mainly on mammalian species, including rats, mice, non-human primates, and humans; however, all species capable of metabolic activation of AFB1 to the exo-epoxide—including yeast, birds, and fish--will form the pro-mutagenic N7-AFB1-G DNA and AFB1-FAPy adducts described above (IARC, 1993).<br />
<br />
== Level of Biological Organization : Molecular ==<br />
Formation of DNA adducts is at the sub-cellular level, as the target is nuclear DNA, leading to a potential outcome of heritable mutation. For AFB1, the cross-species critical target is hepatocyte nuclear DNA, as the apical outcome is hepatocellular carcinoma (HCC).<br />
<br />
== How this Key Event works ==<br />
The initial AFB1-induced pro-mutagenic DNA adduct is the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct, or N7-AFB1-G (Croy et al., 1971). Once the exo-epoxide is bound to the N7-guanine, it can then ring-open to form the more highly pro-mutagenic 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-pyrimid-9-yl-foramido)-9-hydroxyaflatoxin B1, or formamidopyrimidine adduct, AFB1-FAPy (Brown et al., 2006).<br />
<br />
The N7-AFB1-G adduct has a short half-life; it can spontaneously depurinate, leaving an apurinic (AP) site, a DNA lesion that typically is rapidly repaired (Denissenko et al., 1998). AP sites are the predominant background or endogenous lesion identified to date in DNA from control rats, with about 30,000 AP sites/cell present ubiquitously and continually (Swenberg et al., 2011). Thus, although the N7-AFB1-G is considered to be a pro-mutagenic lesion due to its capability to intercalate in DNA and its bulkiness (Bailey et al., 1996), it may not be the most important DNA adduct in the process of AFB1-induced tumorigenesis.<br />
<br />
The AFB1-FAPy adduct has a longer half-life and demonstrates higher mutagenic efficiency or potency than the N7-AFB1-G (Brown et al., 2006). Data indicate that about 20% of the N7-AFB1-G adducts undergo opening of the ring to become AFB1-FAPy adducts (Bedard et al., 2005; Croy and Wogan, 1981a); others report that by about 24 post-exposure, AFB1-FAPy adducts predominate (Boysen et al., 2009; Croy and Wogan, 1981a). These adducts do not spontaneously depurinate, thus can accumulate over time, which likely contributes to their increased mutagenic efficacy (Smela et al., 2002).<br />
<br />
The pro-mutagenicity of these two adducts was demonstrated by assessing their mutant frequencies (MF) in non-human primate-derived cell line COS-7; these cells employ an error-prone replication bypass repair system. The N7-AFB1-G adducts demonstrated a MF of 45% in COS-7 cells (Lin et al., 2014a), while the N7-AFB1-FAPy adduct MF was 97% (Lin et al., 2014b).<br />
<br />
== How it is Measured or Detected ==<br />
Sensitive analytical techniques are available for structural quantification of the AFB1-specific DNA adducts, including the N7-AFB1-G and AFB1-FAPy adducts (Himmelstein et al., 2009). DNA is isolated from tissues or cells and the isolated DNA subjected to neutral thermal or enzyme or acid hydrolysis. This releases the adducted bases, which are then further analyzed with specialized approaches. Techniques include high pressure liquid chromatography (HPLC) or liquid chromatography (LC) separation coupled with tandem mass spectrometry (HPLC-MS/MS or LC-MS/MS). These techniques allow for definitive identification of the AFB1-related adducts using authentic standards. These capabilities can be further enhanced by the use of stable isotope-labelled test materials, e.g., with 13C, 15N, or D3. More sensitivity is reported with accelerated mass spectrometry (AMS) approaches; these require the use of radiolabelled (14C) test material but can detect adducts down into the attomolar range. Demonstration of dose-responses of adduct formation and temporal-response relationships are possible with administration of a variety of dose regimens, including repeated doses<br />
<br />
== Evidence Supporting Taxonomic Applicability ==<br />
AFB1-induced DNA adduct measurements have focused mainly on mammalian species, including rats, mice, non-human primates, and humans; however, all species seem capable of metabolic activation of AFB1 to the exo-epoxide, including yeast, birds, and fish. These will form the pro-mutagenic N7-AFB1-G DNA and AFB1-FAPy adducts described above (Croy et al., 1978; IARC, 1993; Cupid et al., 2004; Lin et al., 2014b; Smela et al., 2002).<br />
<br />
== Evidence for Chemical Initiation of this Molecular Initiating Event==<br />
An extensive database demonstrates the formation of AFB1-specific DNA adducts in many different systems and from several laboratories. In particular, Groopman’s lab and Essigman’s group, among others, have provided pivotal data to demonstrate the formation of these pro-mutagenic AFB1-induced DNA adducts (Croy and Wogan, 1981a,b; Croy et al., 1978; Groopman et al., 1992; Smela et al., 2002; Egner et al., 2006). Lutz (1987) summarized data from a thesis that measured tritiated DNA in liver following p.o. administration of tritiated AFB1 to male F344 rats over a range of doses, from 1 ng AFB1/kg bw to 104 ng AFB1/kg bw and the dose-response was reported to be linear; only limited experimental details are available for this dataset, which relied on less sophisticated and less specific analytical methods than are currently available.<br />
<br />
== References ==<br />
<p>Bailey EA, Iyer RS, Stone MP, et al. (1996). Mutational properties of the primary aflatoxin B1-DNA adduct. Proc Natl Acad Sci USA, 93, 1535–1539.</p><br />
<p>Bedard L, Alessi, M, Davey SK, Massey TE (2005). Susceptibility to aflatoxin B1-induced carcinogenesis correlates with tissue-specific differences in DNA repair activity in mouse and in rat. Cancer Res 65:1265-1270. </p><br />
<p>Boysen G, Pachkowski BF, Nakamura J, Swenberg JA. (2009). The formation and biological significance of N7-guanine adducts. Mutat Res, 678, 76–94. </p><br />
<p>Brown KL, Deng JZ, Iyer RS, Iyer LG, Voehler MW, Stone MP, Harris CM, Harris TM (2006). Unraveling the aflatoxin-FAPY conundrum: Structural basis of the formamidopyrimidine-type DNA adduct of aflatoxin B1. J Am Chem Soc 128:15188-15199. </p><br />
<p>Croy RG, Wogan GN (1981a). Temporal patterns of covalent DNA adducts in rat liver after single and multiple doses of aflatoxin B1. Cancer Res 41:197-203. </p><br />
<p>Croy RG, Wogan GN (1981b). Quantitative comparison of covalent aflatoxin-DNA adducts formed in rat and mouse livers and kidneys. J Natl Cancer Inst 66:761-768. </p><br />
<p>Croy RG, Essigman JM, Reinhold VN, Wogan GN (1978). Identification of the principal aflatoxin N1-DNA adduct formed in vivo in rat liver. Proc Natl Acad Sci USA 75:1745-1749. </p><br />
<p>Cupid BC, Lightfoot TJ, Russell D, Grant SJ, Turner PC, Dingley KH, Curtis KD, Leveson SH, Turteltaub KW, Garner RC (2004). The formation of AFB1-macromolecular adducts in rats and humans at dietary levels of exposure. Food Chem Toxicol 42:559-569. </p><br />
<p>Degen GH, Neumann HG (1981). Differences in aflatoxin B1-susceptibility of rat and mouse are correlated with the capability in vitro to inactivate aflatoxin B1-epoxide. Carcinogenesis 2:299–306. </p><br />
<p>Denissenko MF, Koudriakova TB, Smith L, O'Connor TR, Riggs AD, and Pfeifer GP. The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts. Oncogene. 1998, Dec 10;17(23):3007-14. </p><br />
<p>Egner PA, Groopman JD, Wang J-S, Kensler TW, Friesen MD (2006). Quantification of aflatoxin-B1-N7-Guanine in human urine by high-performance liquid chromatography and isotope dilution tandem mass spectrometry. Chem Res Toxicol 19:1191-1195. </p><br />
<p>Groopman JD, Roebuck BD, Kensler TW. (1992). Molecular dosimetry of aflatoxin DNA adducts in humans and experimental rat models. Prog Clin Biol Res. 374:139-155. </p><br />
<p>Guengerich FP, Johnson WW, Ueng Y-F, Yamazaki H, Shimada T (1996). Involvement of Cytochrome P450, glutathione S-transferase, and epoxide hydrolase in the metabolism of aflatoxin B1 and relevance to risk of human liver cancer. Environ Health Perspect. 104(Suppl 3):557-562. </p><br />
<p>Himmelstein MW, Boogaard PJ, Cadet J, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: II.Overview of methods of identification and quantitation of DNA damage. Crit Rev Toxicol, 39, 679–694. </p><br />
<p>IARC (1993). Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. IARC Monographs on the Evaluation of Carcinogenic Risk to Humans. Vol. 56, 245-395. </p><br />
<p>Kensler TW, He X, Otieno M, et al. (1998). Oltipraz chemoprevention trial in Qidong, People’s Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol Biomarkers Prev, 7, 127–34. </p><br />
<p>Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014a). Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua. J Biol Chem. 289:18497-18506. </p><br />
<p>Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014b). Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis 35(7):1461-1468. </p><br />
<p>Lutz, W. (1987). Quantitative evaluation of DNA-binding data in vivo for low-dose extrapolations. Arch.Toxicol, Suppl. 11: 66-74. </p><br />
<p>Monroe DH, Eaton DL. (1987). Comparative effects of butylated hydroxyanisole on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B1 in the rat and the mouse. Toxicol Appl Pharmacol, 90, 401–409. </p><br />
<p>Roebuck BD, Liu Y-L, Rogers AE, et al. (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short term molecular dosimetry. Cancer Res, 51, 5501–5506. </p><br />
<p>Smela ME, Hamm ML, Henderson PT, Harris CM, Harris TM, Essigmann JM (2002). The aflatoxin B(1) formamidopyrimidine adduct plays a major role in causing the types of mutations observed in human hepatocellular carcinoma. Proc Natl Acad Sci USA 99:6655-6660. </p><br />
<p>Swenberg JA, Lu K, Moeller BC, et al. (2011). Endogenous versus exogenous DNA adducts: their role in carcinogenesis, epidemiology, and risk assessment. Tox Sci, 120, S130–45. </p></div>Dvillenehttps://aopwiki.org/wiki/index.php/Event:373Event:3732016-07-06T16:43:16Z<p>Dvillene: /* MIE: Evidence for Chemical Initiation of this Molecular Initiating Event */</p>
<hr />
<div>__ForceTOC__<br />
<br />
== Event Title ==<br />
<div id='longTitle' class='Title'> Pro-mutagenic DNA Adducts, Formation </div><br />
<div id='shortTitle' class='Title2'> </div><br />
<br />
== Molecular Initiating Event ==<br />
<br />
== Action - how the event is affected ==<br />
Alkylation, binding<br />
<br />
== Biological Process/Object ==<br />
The Molecular Initiating Event for this AOP<br />
Formation of AFB1-induced pro-mutagenic DNA adducts: N7-AFB1-G and/or AFB1-FAPy<br />
<br />
== Key Event Overview ==<br />
Please follow link to [//{{SERVERNAME}}/events/{{PAGENAMEE}} widget page] to edit this section.<br />
<br />
<span style="color:#FF0000">'''If you manually enter text in this section, it will get automatically altered or deleted in subsequent edits using the widgets.'''</span><br />
<br />
=== AOPs Including This Key Event ===<br />
<br />
[[Category:Key Event]][[Category:Molecular Initiating Event]]<br />
<br />
{|class="wikitable sortable" id="Table1"<br />
<br />
!AOP Name<br />
!Event Type<br />
!Essentiality<br />
<br />
|-<br />
<br />
|[[Aop:46|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)]]||MIE||[[Aop:46#Essentiality of the Key Events|Strong]]<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Chemical Initiators ===<br />
The following are chemical initiators that operate directly through this Event:<br />
<br />
#[[Chem_Init:61|Aflatoxin B1]]<br />
<br />
=== Taxonomic Applicability ===<br />
<br />
{|class="wikitable sortable" id="Specproof"<br />
<br />
!Name<br />
!Scientific Name<br />
!Evidence<br />
!Links<br />
<br />
|-<br />
<br />
|rainbow trout||Oncorhynchus mykiss||[[Event:373#Evidence Supporting Taxonomic Applicability|Strong]]||[http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8022 NCBI]<br />
<br />
|-<br />
<br />
|Human, rat, mouse||||[[Event:373#Evidence Supporting Taxonomic Applicability|Strong]]||<br />
<br />
|-<br />
<br />
|chickens, ducks, turkeys||||[[Event:373#Evidence Supporting Taxonomic Applicability|Strong]]||<br />
<br />
|-<br />
<br />
|}<br />
<br />
=== Level of Biological Organization ===<br />
<br />
{|class="wikitable sortable" id="BioProof"<br />
<br />
!Biological Organization<br />
<br />
|-<br />
<br />
|Molecular<br />
<br />
|-<br />
<br />
|}<br />
<br />
== Evidence Supporting Essentiality ==<br />
Strong <br />
<br />
Evidence supporting the formation of an AFB1-induced pro-mutagenic DNA adduct as the molecular initiating event (MIE) is strong and stems from many datasets in different biological systems. The formation of N7-AFB1-G DNA adducts after AFB1 exposure has been demonstrated across phyla, from bacteria through yeast, fish, birds, and including many mammalian systems up through non-human primates and humans (Croy et al., 1978; IARC, 1993; Cupid et al., 2004). <br />
<br />
The reactive metabolite AFB1 exo-epoxide intercalates into DNA and then binds to the nucleophilic N7-G residue via an SN2 reaction. This N7-G DNA adduct can then spontaneously ring-open to form the more highly pro-mutagenic 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-pyrimid-9-yl-foramido-9-hydroxyaflatoxin B1, or formamidopyrimidine adduct, AFB1-FAPy (Brown et al., 2006).<br />
<br />
The essentiality of this MIE is demonstrated by the effects of modulation of metabolism to reactive forms. Inhibition of activation results in reduced formation of the critical exo-epoxide. Likewise, increased GST activity results in increased metabolism of the exo-epoxide to less toxic forms. In both cases, less reactive metabolite is available to form DNA adducts, resulting in fewer adducts (Guengerich et al., 1996). Pre-treatment of rats with oltipraz provides a specific example, wherein a 65-70% reduction in AFB1-induced DNA adducts was demonstrated due to increased GST activity; this corresponds with a subsequent 100% reduction in liver tumors (Roebuck et al., 1991; Kensler et al., 1998). <br />
<br />
<br />
Another line of evidence for essentiality of the MIE is the recognized species difference in sensitivity to AFB1-induced liver tumors between mice and rats. Mice, with considerably increased metabolic activation of AFB1 to the exo-epoxide compared with rats, are nonetheless much less sensitive to AFB1-induced liver tumors (Degen and Neumann, 1981). This difference is believed to be due to the constitutive presence of GST-alpha activity in mice vs. rats, where this activity is not found (Monroe and Eaton, 1987).<br />
<br />
== Taxonomic Applicabilty ==<br />
AFB1-induced DNA adduct measurements have focused mainly on mammalian species, including rats, mice, non-human primates, and humans; however, all species capable of metabolic activation of AFB1 to the exo-epoxide—including yeast, birds, and fish--will form the pro-mutagenic N7-AFB1-G DNA and AFB1-FAPy adducts described above (IARC, 1993).<br />
<br />
== Level of Biological Organization : Molecular ==<br />
Formation of DNA adducts is at the sub-cellular level, as the target is nuclear DNA, leading to a potential outcome of heritable mutation. For AFB1, the cross-species critical target is hepatocyte nuclear DNA, as the apical outcome is hepatocellular carcinoma (HCC).<br />
<br />
== How this Key Event works ==<br />
The initial AFB1-induced pro-mutagenic DNA adduct is the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct, or N7-AFB1-G (Croy et al., 1971). Once the exo-epoxide is bound to the N7-guanine, it can then ring-open to form the more highly pro-mutagenic 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-pyrimid-9-yl-foramido)-9-hydroxyaflatoxin B1, or formamidopyrimidine adduct, AFB1-FAPy (Brown et al., 2006).<br />
<br />
The N7-AFB1-G adduct has a short half-life; it can spontaneously depurinate, leaving an apurinic (AP) site, a DNA lesion that typically is rapidly repaired (Denissenko et al., 1998). AP sites are the predominant background or endogenous lesion identified to date in DNA from control rats, with about 30,000 AP sites/cell present ubiquitously and continually (Swenberg et al., 2011). Thus, although the N7-AFB1-G is considered to be a pro-mutagenic lesion due to its capability to intercalate in DNA and its bulkiness (Bailey et al., 1996), it may not be the most important DNA adduct in the process of AFB1-induced tumorigenesis.<br />
<br />
The AFB1-FAPy adduct has a longer half-life and demonstrates higher mutagenic efficiency or potency than the N7-AFB1-G (Brown et al., 2006). Data indicate that about 20% of the N7-AFB1-G adducts undergo opening of the ring to become AFB1-FAPy adducts (Bedard et al., 2005; Croy and Wogan, 1981a); others report that by about 24 post-exposure, AFB1-FAPy adducts predominate (Boysen et al., 2009; Croy and Wogan, 1981a). These adducts do not spontaneously depurinate, thus can accumulate over time, which likely contributes to their increased mutagenic efficacy (Smela et al., 2002).<br />
<br />
The pro-mutagenicity of these two adducts was demonstrated by assessing their mutant frequencies (MF) in non-human primate-derived cell line COS-7; these cells employ an error-prone replication bypass repair system. The N7-AFB1-G adducts demonstrated a MF of 45% in COS-7 cells (Lin et al., 2014a), while the N7-AFB1-FAPy adduct MF was 97% (Lin et al., 2014b).<br />
<br />
== How it is Measured or Detected ==<br />
Sensitive analytical techniques are available for structural quantification of the AFB1-specific DNA adducts, including the N7-AFB1-G and AFB1-FAPy adducts (Himmelstein et al., 2009). DNA is isolated from tissues or cells and the isolated DNA subjected to neutral thermal or enzyme or acid hydrolysis. This releases the adducted bases, which are then further analyzed with specialized approaches. Techniques include high pressure liquid chromatography (HPLC) or liquid chromatography (LC) separation coupled with tandem mass spectrometry (HPLC-MS/MS or LC-MS/MS). These techniques allow for definitive identification of the AFB1-related adducts using authentic standards. These capabilities can be further enhanced by the use of stable isotope-labelled test materials, e.g., with 13C, 15N, or D3. More sensitivity is reported with accelerated mass spectrometry (AMS) approaches; these require the use of radiolabelled (14C) test material but can detect adducts down into the attomolar range. Demonstration of dose-responses of adduct formation and temporal-response relationships are possible with administration of a variety of dose regimens, including repeated doses<br />
<br />
== Evidence Supporting Taxonomic Applicability ==<br />
AFB1-induced DNA adduct measurements have focused mainly on mammalian species, including rats, mice, non-human primates, and humans; however, all species seem capable of metabolic activation of AFB1 to the exo-epoxide, including yeast, birds, and fish. These will form the pro-mutagenic N7-AFB1-G DNA and AFB1-FAPy adducts described above (Croy et al., 1978; IARC, 1993; Cupid et al., 2004; Lin et al., 2014b; Smela et al., 2002).<br />
<br />
== Evidence for Chemical Initiation of this Molecular Initiating Event==<br />
An extensive database demonstrates the formation of AFB1-specific DNA adducts in many different systems and from several laboratories. In particular, Groopman’s lab and Essigman’s group, among others, have provided pivotal data to demonstrate the formation of these pro-mutagenic AFB1-induced DNA adducts (Croy and Wogan, 1981a,b; Croy et al., 1978; Groopman et al., 1992; Smela et al., 2002; Egner et al., 2006). Lutz (1987) summarized data from a thesis that measured tritiated DNA in liver following p.o. administration of tritiated AFB1 to male F344 rats over a range of doses, from 1 ng AFB1/kg bw to 104 ng AFB1/kg bw and the dose-response was reported to be linear; only limited experimental details are available for this dataset, which relied on less sophisticated and less specific analytical methods than are currently available.<br />
<br />
== MIE: References ==<br />
<p>Bailey EA, Iyer RS, Stone MP, et al. (1996). Mutational properties of the primary aflatoxin B1-DNA adduct. Proc Natl Acad Sci USA, 93, 1535–1539.</p><br />
<p>Bedard L, Alessi, M, Davey SK, Massey TE (2005). Susceptibility to aflatoxin B1-induced carcinogenesis correlates with tissue-specific differences in DNA repair activity in mouse and in rat. Cancer Res 65:1265-1270. </p><br />
<p>Boysen G, Pachkowski BF, Nakamura J, Swenberg JA. (2009). The formation and biological significance of N7-guanine adducts. Mutat Res, 678, 76–94. </p><br />
<p>Brown KL, Deng JZ, Iyer RS, Iyer LG, Voehler MW, Stone MP, Harris CM, Harris TM (2006). Unraveling the aflatoxin-FAPY conundrum: Structural basis of the formamidopyrimidine-type DNA adduct of aflatoxin B1. J Am Chem Soc 128:15188-15199. </p><br />
<p>Croy RG, Wogan GN (1981a). Temporal patterns of covalent DNA adducts in rat liver after single and multiple doses of aflatoxin B1. Cancer Res 41:197-203. </p><br />
<p>Croy RG, Wogan GN (1981b). Quantitative comparison of covalent aflatoxin-DNA adducts formed in rat and mouse livers and kidneys. J Natl Cancer Inst 66:761-768. </p><br />
<p>Croy RG, Essigman JM, Reinhold VN, Wogan GN (1978). Identification of the principal aflatoxin N1-DNA adduct formed in vivo in rat liver. Proc Natl Acad Sci USA 75:1745-1749. </p><br />
<p>Cupid BC, Lightfoot TJ, Russell D, Grant SJ, Turner PC, Dingley KH, Curtis KD, Leveson SH, Turteltaub KW, Garner RC (2004). The formation of AFB1-macromolecular adducts in rats and humans at dietary levels of exposure. Food Chem Toxicol 42:559-569. </p><br />
<p>Degen GH, Neumann HG (1981). Differences in aflatoxin B1-susceptibility of rat and mouse are correlated with the capability in vitro to inactivate aflatoxin B1-epoxide. Carcinogenesis 2:299–306. </p><br />
<p>Denissenko MF, Koudriakova TB, Smith L, O'Connor TR, Riggs AD, and Pfeifer GP. The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts. Oncogene. 1998, Dec 10;17(23):3007-14. </p><br />
<p>Egner PA, Groopman JD, Wang J-S, Kensler TW, Friesen MD (2006). Quantification of aflatoxin-B1-N7-Guanine in human urine by high-performance liquid chromatography and isotope dilution tandem mass spectrometry. Chem Res Toxicol 19:1191-1195. </p><br />
<p>Groopman JD, Roebuck BD, Kensler TW. (1992). Molecular dosimetry of aflatoxin DNA adducts in humans and experimental rat models. Prog Clin Biol Res. 374:139-155. </p><br />
<p>Guengerich FP, Johnson WW, Ueng Y-F, Yamazaki H, Shimada T (1996). Involvement of Cytochrome P450, glutathione S-transferase, and epoxide hydrolase in the metabolism of aflatoxin B1 and relevance to risk of human liver cancer. Environ Health Perspect. 104(Suppl 3):557-562. </p><br />
<p>Himmelstein MW, Boogaard PJ, Cadet J, et al. (2009). Creating context for the use of DNA adduct data in cancer risk assessment: II.Overview of methods of identification and quantitation of DNA damage. Crit Rev Toxicol, 39, 679–694. </p><br />
<p>IARC (1993). Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. IARC Monographs on the Evaluation of Carcinogenic Risk to Humans. Vol. 56, 245-395. </p><br />
<p>Kensler TW, He X, Otieno M, et al. (1998). Oltipraz chemoprevention trial in Qidong, People’s Republic of China: Modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiol Biomarkers Prev, 7, 127–34. </p><br />
<p>Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014a). Error-prone replication bypass of the primary aflatoxin B1 DNA adduct, AFB1-N7-Gua. J Biol Chem. 289:18497-18506. </p><br />
<p>Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, Lloyd RS. (2014b). Molecular basis of aflatoxin-induced mutagenesis-role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis 35(7):1461-1468. </p><br />
<p>Lutz, W. (1987). Quantitative evaluation of DNA-binding data in vivo for low-dose extrapolations. Arch.Toxicol, Suppl. 11: 66-74. </p><br />
<p>Monroe DH, Eaton DL. (1987). Comparative effects of butylated hydroxyanisole on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B1 in the rat and the mouse. Toxicol Appl Pharmacol, 90, 401–409. </p><br />
<p>Roebuck BD, Liu Y-L, Rogers AE, et al. (1991). Protection against aflatoxin B1-induced hepatocarcinogenesis in F344 rats by 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz): predictive role for short term molecular dosimetry. Cancer Res, 51, 5501–5506. </p><br />
<p>Smela ME, Hamm ML, Henderson PT, Harris CM, Harris TM, Essigmann JM (2002). The aflatoxin B(1) formamidopyrimidine adduct plays a major role in causing the types of mutations observed in human hepatocellular carcinoma. Proc Natl Acad Sci USA 99:6655-6660. </p><br />
<p>Swenberg JA, Lu K, Moeller BC, et al. (2011). Endogenous versus exogenous DNA adducts: their role in carcinogenesis, epidemiology, and risk assessment. Tox Sci, 120, S130–45. </p></div>Dvillene