Duplex sequencing of liver genomes in Neil1-deficient and WT mice following aflatoxin B1 exposure

Increased risk for the development of hepatocellular carcinoma is driven by a number of etiological factors including hepatitis viral infection and dietary exposures to foods contaminated with aflatoxin-producing molds. Once ingested, aflatoxin is primarily bioactivated in the liver to a DNA-reactive epoxide to form base adducts, one of which is the stable trans-8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy aflatoxin B1 (AFB1-Fapy-dG). In the absence of DNA repair, replication bypass of this lesion is highly mutagenic, yielding predominantly G to T transversions. These mutations, along with minor accumulations of G to A transitions and G to C transversions constitute the aflatoxin-specific mutational signature, COSMIC single base substitution (SBS) signature 24, in which there are distinctive trinucleotide sequence-context dependent variation. Previously, we demonstrated that in addition to nucleotide excision repair, the DNA glycosylase, NEIL1 efficiently initiated the base excision repair pathway for AFB1-Fapy-dG, with Neil1-/- mice significantly more susceptible to aflatoxin-induced HCC relative to wild-type or XPA-deficient mice. To test whether Neil1 deficiency would result in greater frequencies of these mutations, and possibly modulate the overall trinucleotide-dependent mutation signature, wild-type and Neil1-/- mice were challenged with a single, 4 mg/kg dose of aflatoxin. Total nuclear and mitochondrial liver DNAs were harvested 2.5 months post-injection and the mutation frequencies and spectra analyzed using duplex sequencing technologies. Relative to control liver DNAs from untreated animals, sequence analyses of genomic DNAs revealed a ~25-fold increase in SBS mutations in aflatoxin-challenged mice. Although the specificity of the trinucleotide sequence context of the mutagenic signature was not changed in the Neil1-/- mice, the overall mutation frequency was significantly elevated. In contrast to the very large increases in genomic DNA mutagenesis, analyses of the integrity of the mitochondrial DNA pool revealed an overall decrease in mutations in aflatoxin-treated mice, suggesting an ongoing cleansing of damaged mitochondrial DNAs. Consistent with our previous carcinogenesis data, concomitant carcinogenesis studies confirmed elevated HCCs in Neil1-/- mice.