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Event: 376

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. More help

Increased, Induced Mutations in Critical Genes

Short name
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Increased, Induced Mutations in Critical Genes
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Biological Context

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Level of Biological Organization
Cellular

Cell term

The location/biological environment in which the event takes place.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.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Cell term
eukaryotic cell

Organ term

The location/biological environment in which the event takes place.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.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; https://aopwiki.org/info_pages/2/info_linked_pages/7#List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling).  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 signaling 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.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Process Object Action
mutation cellular tumor antigen p53 increased

Key Event Overview

AOPs Including This Key Event

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AOP Name Role of event in AOP Point of Contact Author Status OECD Status
Mutagenic MOA for Cancer 2 KeyEvent Agnes Aggy (send email) Open for citation & comment EAGMST Under Review

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KE.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

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Sex Applicability

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Key Event Description

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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

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.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). Do not provide detailed protocols. More help

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

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided).  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. 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