Upstream eventIncreased, Insufficient repair or mis-repair of pro-mutagenic DNA adducts
Increased, Induced Mutations in Critical Genes
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|AFB1: Mutagenic Mode-of-Action leading to Hepatocellular Carcinoma (HCC)||adjacent||Moderate|
Life Stage Applicability
Key Event Relationship Description
There is no direct information concerning insufficient or mis-repair of AFB1 promutagenic adducts leading directly to mutations in critical genes. It is well known, however, that in general when the repair of DNA adducts is either done incorrectly or is insufficient to remove the DNA adduct and correct the DNA sequence prior to DNA replication, a mutation at the site of the DNA adduct will result in the daughter cells upon DNA replication.
Evidence Supporting this KER
When DNA adducts are not repaired, mutations result if cell replication (and DNA synthesis) takes place.
Evidence supporting the insufficient repair or mis-repair of pro-mutagenic DNA adducts is strong, but mainly indirect although there is some direct evidence from Archaea. The mechanism of DNA repair in Archaea is evolutionarily conserved and similar to that in eukaryotes. Banerjee et al. (2011) showed the P2 DNA polymerase in S. solfataricus bypasses the AFB1-N7-G adducts in an error-free manner but conducts error-prone replication past the AFB1-FAPy adduct and misinserts dATP, consistent with G:C to T:A transversion. (Banerjee et al., 2011).
Guo et al. (2005) used a transgenic yeast model expressing human CYP1A2 with defects in a range of DNA repair and cell cycle checkpoint pathways. This study demonstrated that nucleotide excision repair (NER), homologous recombination repair, post-replication repair, and several cell cycle checkpoint proteins all function in the repair of AFB1 adducts in yeast. Error-prone post-replication repair and apurinic endonucleases are secondary repair pathways that are used by cells when the major pathways are not functional or are overwhelmed. These secondary pathways tend to produce mutations.
The majority of both AFB1-N7-G and AFB1-N7-FAPy adducts are repaired by NER. (Alekseyev et al., 2004; Kew, 2013; Mulder et al., 2014). The evidence supporting the role of NER in humans is fairly strong. In populations in the Gambia and in China with high AFB1 exposure studies show that polymorphisms in the DNA repair gene x-ray repair cross complementary group (XRCC) are a risk factor for hepatocellular carcinoma (HCC). (Kirk et al., 2005; Long et al., 2006, 2008). In addition, these polymorphisms are associated with both increased levels of AFB1-DNA adducts and mutations at codon 249 of p53. (Long et al., 2008a; 2009). The correlation between AFB1 exposure, codon 249 mutations, and HCC has been established in humans. (Besaratinia et al., 2009) The secondary repair mechanisms play an important role in genesis of mutations from AFB1 adducts; this is particularly true for individuals with NER deficiencies such as those associated with XRCC polymorphisms. One possibility is that mutations occur through mis-repair by NER; alternatively, if NER is insufficient, the mutations may result through another pathway such as post-replication repair.
In the nematode, Caenorhabditis elegans, AFB1 is metabolized by CYP1, CYP2 and CYP3 family enzymes, and exposure to AFB1 results in significant DNA damage. (Leung et al., 2010). In C. elegans, whole genome sequencing was used to determine the mutational signature of AFB1 in worms deficient in DNA repair. A dose-response relationship was observed between AFB1 dose and G:C to T:A transversions and G:C to A:T transitions. (Meier et al., 2014). NER-deficient mutants showed an increase in these mutations compared to wild-type. In addition, the G:C to T:A transversions and G:C to A:T transitions were observed with a strand bias, indicative of transcription-coupled NER mechanisms similar to those observed in AFB1-associated p53 mutations in humans (Besaratinia et al., 2009; Carvalho et al., 2013; Gursoy-Yuzugullu et al., 2011; Symanska et al., 2009; Villar et al., 2011) This work with C. elegans is the first to link AFB1 adducts with mutations in a eukaryotic organism.
Other biological systems and datasets provide additional evidence from primarily mammalian species. The support provided by this evidence is based on the biological knowledge that a change in primary DNA sequence—in other words, a mutation—requires a mode of action that includes DNA damage. Mutations occur when DNA lesions are not repaired prior to cell replication (Preston and Williams, 2005; Pottenger et al., 2014). AFB1 forms two types of pro-mutagenic adducts: N7-AFB1-G and AFB1-FAPy DNA adducts, and resulting mutations are predominantly G:C to T:A transversions, which is the expected result of such adducts not being repaired or undergoing mis-repair (Bedard and Massey, 2006; Lin et al., 2014a,b).
These AFB1-induced DNA adducts undergo repair by SOS repair, NER, homologous recombination (HR), and post-replication repair (Bedard and Massey, 2006). NER appears to be most important for repair of AFB1-induced adducts in mammalian systems (Bedard and Massey, 2006). However, experiments in transgenic yeast expressing human CYP1A2 revealed different efficacy of the various DNA repair systems for AFB1-induced DNA adducts (Guo et al., 2005). 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, such as the pathways for error-prone post-replication repair or those relying on apurinic endonucleases.
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, Puisieux et al. (1991) showed specificity of the 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, intercalating N7-B[a]P-G adducts.
Uncertainties and Inconsistencies
Quantitative Understanding of the Linkage
In mammals, AFB1-N7-G and AFB1-N7-FAPy adducts are repaired by nucleotide excision repair (NER). AFB1-N7-G adducts are more readily repaired, or removed by spontaneous depurination, likely because these distort the structure of DNA to a greater extent than do AFB1-FAPy adducts. (Bedard and Massey, 2006). Species and tissue differences in repair capacities occur. Nuclear extracts from the livers of mice administered AFB1 in vivo seemed to repair AFB1 N7-G adducts at a greater rate than AFB1-N7-FAPy adducts, whereas repair of both types of adducts was similar in rat liver extracts. (Bedard et al., 2005). Given the heterogeneity in the human population with regard to DNA repair, the ability to repair AFB1 adducts could vary greatly.
There are no available data in either rodents or humans, which directly quantify the relationship between the number of AFB1 adducts and the frequency of mutation, either in surrogate genes or in the critical cancer gene. However, there are examples from other direct and indirect-acting alkylating agents (Doak et al., 2007; Gocke and Muller, 2009; Pottenger et al., 2009; Bryce et al., 2010; Dobo et al., 2011) that demonstrate non-linear dose-responses. Additional data on AFB1 could clarify the relationship between AFB1 pro-mutagenic adduct formation and induced mutations. This is a complex issue that requires further carefully designed experimental work.
There have been several studies that provide some quantitative information. Bailey et al. (1996) inserted an oligonucleotide containing a single AFB1-N7-G adduct into the genome of phage M13. In E. coli, the SOS response consists of the activation of over 20 unlinked genes involved in DNA damage tolerance and repair. (Smith and Walker, 1998). Replication of the oligonucleotide-bearing M13 phage in E. coli undergoing an induced SOS response yielded a mutation frequency of 4% for the AFB1-N7-G adduct (Bailey et al., 1996). The conditions in this study were extremely stringent, including single stranded DNA target (M13), and thus there was no ability to use the other DNA strand as an alternate template for bypass repair. These very stringent experimental conditions likely represent a significantly worst-case scenario for mutation induction, conditions with extremely limited repair opportunities and unlike normal conditions in eukaryotes.
While mutations at codon 249 of the p53 gene have been observed in association with HCC in humans, it is not known whether this mutation occurs as a direct result of adduct formation at this site early in the process or by some other mechanism. Human HepG2 hepatocytes were exposed to AFB1 and microsomes for metabolism. A dose-dependent increase in G:C to T:A transversions was observed at 10 additional locations as measured by ligation-mediated PCR, and at 4 additional locations using terminal-transferred dependent PCR. (Denissenko et al., 1998) These authors suggest that codon 249 may present an unusually mutagenic adduct conformation based on the local DNA sequence, and that a higher mutation rate may occur there rather than at other locations because of increased DNA polymerase bypass.
Leung et al. (2010) provide dose-response data for AFB1 resulting in DNA lesions in C. elegans, and Meier et al. (2014) provide dose-response data for AFB1-induced DNA base changes in C. elegans. However, the extent to which these quantitative relationships are applicable to humans or rodents is not yet clear.
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
1. Bedard LL, and Massey TE. Aflatoxin B1-induced DNA damage and its repair. Cancer Lett. 2006, Sep 28;241(2):174-83.
2. Bedard LL, Alessi M, Davey S, and Massey TE. Susceptibility to aflatoxin B1-induced carcinogenesis correlates with tissue-specific differences in DNA repair activity in mouse and in rat. Cancer Res. 2005, Feb 15;65(4):1265-70.
3. Smith BT, and Walker GC. Mutagenesis and more: umuDC and the Escherichia coli SOS response. Genetics. 1998, Apr;148(4):1599-610.
4. Bailey EA, Iyer RS, Stone MP, Harris TM, and Essigmann JM. Mutational properties of the primary aflatoxin B1-DNA adduct. Proc Natl Acad Sci U S A. 1996, Feb 20;93(4):1535-9.
5. 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.
6. Banerjee S, Brown KL, Egli M, and Stone MP. Bypass of aflatoxin B1 adducts by the Sulfolobus solfataricus DNA polymerase IV. J Am Chem Soc. 2011, Aug 17;133(32):12556-68.
7. Guo Y, Breeden LL, Zarbl H, Preston BD, and Eaton DL. 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. 2005, Jul;25(14):5823-33.
8. Alekseyev YO, Hamm ML, and Essigmann JM. Aflatoxin B1 formamidopyrimidine adducts are preferentially repaired by the nucleotide excision repair pathway in vivo. Carcinogenesis. 2004, Jun;25(6):1045-51. 9. Kew MC. Aflatoxins as a cause of hepatocellular carcinoma. J Gastrointestin Liver Dis. 2013, Sep;22(3):305-10.
10. Mulder JE, Bondy GS, Mehta R, and Massey TE. Up-regulation of nucleotide excision repair in mouse lung and liver following chronic exposure to aflatoxin B₁ and its dependence on p53 genotype. Toxicol Appl Pharmacol. 2014, Mar 1;275(2):96-103.
11. Kirk GD, Turner PC, Gong Y, Lesi OA, Mendy M, Goedert JJ, et al. Hepatocellular carcinoma and polymorphisms in carcinogen-metabolizing and DNA repair enzymes in a population with aflatoxin exposure and hepatitis B virus endemicity. Cancer Epidemiol Biomarkers Prev. 2005, Feb;14(2):373-9.
12. Long XD, Ma Y, Wei YP, and Deng ZL. The polymorphisms of GSTM1, GSTT1, HYL1*2, and XRCC1, and aflatoxin B1-related hepatocellular carcinoma in Guangxi population, China. Hepatol Res. 2006, Sep;36(1):48-55.
13. Long XD, Ma Y, Qu de Y, Liu YG, Huang ZQ, Huang YZ, et al. The polymorphism of XRCC3 codon 241 and AFB1-related hepatocellular carcinoma in Guangxi population, China. Ann Epidemiol. 2008, Jul;18(7):572-8.
14. Long XD, Ma Y, Huang HD, Yao JG, Qu de Y, and Lu YL. Polymorphism of XRCC1 and the frequency of mutation in codon 249 of the p53 gene in hepatocellular carcinoma among Guangxi population, China. Mol Carcinog. 2008, Apr;47(4):295-300.
15. Long X-D, Ma Y, and Deng Z-L. GSTM1 and XRCC3 polymorphisms: Effects on levels of aflatoxin B1-DNA adducts. Chinese Journal of Cancer Research. 2009, Sep;21(3):177-184.
16. Besaratinia A, Kim SI, Hainaut P, and Pfeifer GP. In vitro recapitulating of TP53 mutagenesis in hepatocellular carcinoma associated with dietary aflatoxin B1 exposure. Gastroenterology. 2009, Sep;137(3):1127-37, 1137.e1-5.
17. Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, and Lloyd RS. Error-prone Replication Bypass of the Primary Aflatoxin B1 DNA Adduct, AFB1-N7-Gua. J Biol Chem. 2014, May 16;
18. Lin YC, Li L, Makarova AV, Burgers PM, Stone MP, and Lloyd RS. Molecular basis of aflatoxin-induced mutagenesis--role of the aflatoxin B1-formamidopyrimidine adduct. Carcinogenesis. 2014, Feb 7;
19. Leung MC, Goldstone JV, Boyd WA, Freedman JH, and Meyer JN. Caenorhabditis elegans generates biologically relevant levels of genotoxic metabolites from aflatoxin B1 but not benzo[a]pyrene in vivo. Toxicol Sci. 2010, Dec;118(2):444-53.
20. Meier B, Cooke SL, Weiss J, Bailly AP, Alexandrov LB, Marshall J, et al. C. elegans whole genome sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency. Genome Res. 2014, Jul 16;
21. de Carvalho FM, de Almeida Pereira T, Gonçalves PL, Jarske RD, Pereira FE, and Louro ID. Hepatocellular carcinoma and liver cirrhosis TP53 mutation analysis reflects a moderate dietary exposure to aflatoxins in Espírito Santo State, Brazil. Mol Biol Rep. 2013, Aug;40(8):4883-7.
22. Gursoy-Yuzugullu O, Yuzugullu H, Yilmaz M, and Ozturk M. Aflatoxin genotoxicity is associated with a defective DNA damage response bypassing p53 activation. Liver Int. 2011, Apr;31(4):561-71.
23. Szymañska K, Chen JG, Cui Y, Gong YY, Turner PC, Villar S, et al. TP53 R249S mutations, exposure to aflatoxin, and occurrence of hepatocellular carcinoma in a cohort of chronic hepatitis B virus carriers from Qidong, China. Cancer Epidemiol Biomarkers Prev. 2009, May;18(5):1638-43.
24. Villar S, Le Roux-Goglin E, Gouas DA, Plymoth A, Ferro G, Boniol M, et al. Seasonal variation in TP53 R249S-mutated serum DNA with aflatoxin exposure and hepatitis B virus infection. Environ Health Perspect. 2011, Nov;119(11):1635-40.
25. Macé K, Aguilar F, Wang JS, Vautravers P, Gómez-Lechón M, Gonzalez FJ, et al. Aflatoxin B1-induced DNA adduct formation and p53 mutations in CYP450-expressing human liver cell lines. Carcinogenesis. 1997, Jul;18(7):1291-7.
26. Aguilar F, Hussain SP, and Cerutti P. Aflatoxin B1 induces the transversion of G-->T in codon 249 of the p53 tumor suppressor gene in human hepatocytes. Proc Natl Acad Sci U S A. 1993, Sep 15;90(18):8586-90.
27. Preston RJ, Williams GM. (2005). DNA-reactive carcinogens: mode of action and human cancer hazard. Crit Rev Toxicol, 35, 673–83
28. 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.
29. 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.