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Event: 373

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Formation, Pro-mutagenic DNA Adducts

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
Formation, Pro-mutagenic DNA Adducts

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help
Level of Biological Organization
Molecular

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help
Cell term
hepatocyte

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help
Process Object Action
DNA alkylation Nuclear deoxyribonucleic acid abnormal

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
Mutagenic MOA for Cancer 2 MolecularInitiatingEvent Ted Simon (send email) Open for citation & comment EAGMST Under Review

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
rainbow trout Oncorhynchus mykiss High NCBI
Human, rat, mouse Human, rat, mouse High NCBI
chickens, ducks, turkeys chickens, ducks, turkeys High NCBI

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help

Sex Applicability

No help message More help

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

Evidence Supporting Essentiality

Strong

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).

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).

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).

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).

Taxonomic Applicabilty

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).

How this Key Event works

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).

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.

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).

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).

How It Is Measured or Detected

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

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

Domain of Applicability

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

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).

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

When a specific MIE can be defined (i.e., the molecular target and nature of interaction is known), in addition to describing the biological state associated with the MIE, how it can be measured, and its taxonomic, life stage, and sex applicability, it is useful to list stressors known to trigger the MIE and provide evidence supporting that initiation. This will often be a list of prototypical compounds demonstrated to interact with the target molecule in the manner detailed in the MIE description to initiate a given pathway (e.g., 2,3,7,8-TCDD as a prototypical AhR agonist; 17α-ethynyl estradiol as a prototypical ER agonist). Depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). Known stressors should be included in the MIE description, but it is not expected to include a comprehensive list. Rather initially, stressors identified will be exemplary and the stressor list will be expanded over time. For more information on MIE, please see pages 32-33 in the User Handbook.

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.

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help

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.

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.

Boysen G, Pachkowski BF, Nakamura J, Swenberg JA. (2009). The formation and biological significance of N7-guanine adducts. Mutat Res, 678, 76–94.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Lutz, W. (1987). Quantitative evaluation of DNA-binding data in vivo for low-dose extrapolations. Arch.Toxicol, Suppl. 11: 66-74.

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.

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.

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.

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.