Increased, Insufficient repair or mis-repair of pro-mutagenic DNA adducts leads to Increased, Induced Mutations in Critical Genes

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.

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.