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Event: 376

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

Increased, Induced Mutations in Critical Genes

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
Increased, Induced Mutations in Critical Genes

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
Cellular

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
eukaryotic cell

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
mutation cellular tumor antigen p53 increased

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 KeyEvent 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

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

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. 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

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.

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.

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).

Induced Mutation in Critical Genes

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.

A specific critical mutation in codon 249 of the p53 gene has been identified in human hepatocellular carcinoma (HCC) (See section below on essentiality).

Level of Biological Organization : Cellular

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.

Evidence Supporting Essentiality

Strong

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.

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.

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).

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). ?

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.

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.

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

There are data across phyla demonstrating the induction of mutations, specifically the induction to G:C to T:A transversions following AFB1 exposure.

Taxonomic Applicability

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.)

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

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

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

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.

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

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.

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

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

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

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.

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.

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