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Dietary exposure to aflatoxin B₁ (AFB₁) is a risk factor for the development of hepatocellular carcinomas (HCCs). Following metabolic activation, AFB₁ reacts with guanines to form covalent DNA adducts, which induce high-frequency G > T transversions. The molecular signature associated with these mutational events aligns with the single base substitution signature 24 (SBS24) in the Catalog of Somatic Mutations in Cancer (COSMIC) database. Deficiencies in either base excision repair (BER) due to the absence of Nei-like DNA glycosylase 1 (NEIL1) or nucleotide excision repair (NER) due to the absence of xeroderma complementation group A protein (XPA) contribute to HCCs in murine models. In the current study, ultra-low error duplex sequencing was used to characterize mutational profiles in liver DNAs of NEIL1-deficient, XPA-deficient, and DNA repair-proficient mice following neonatal injection of 1 mg/kg AFB₁. Analyses of AFB₁-induced mutations showed high cosine similarity to SBS24, regardless of repair proficiency status. The absence of NEIL1 resulted in an approximately 30% increase in the frequency of mutations, with distribution suggesting preferential NEIL1-dependent repair of AFB₁ lesions in open chromatin regions. A trend of increased mutagenesis was also observed in the absence of XPA. Consistent with the role of XPA in transcription-coupled repair, mutational profiles in XPA-deficient mice showed disruption of the transcriptional bias in mutations associated with SBS24. Implications: Our findings define the roles of DNA repair pathways in AFB₁-induced mutagenesis and carcinogenesis in murine models, with these findings having implications in human health for those with BER and NER deficiencies.

Increased risk for the development of hepatocellular carcinoma (HCC) is driven by a number of etiological factors including hepatitis viral infection and dietary exposures to foods contaminated with aflatoxin-producing molds. Intracellular metabolic activation of aflatoxin B 1 (AFB 1 ) to a reactive epoxide generates highly mutagenic AFB 1 -Fapy-dG adducts. Previously, we demonstrated that repair of AFB 1 -Fapy-dG adducts can be initiated by the DNA glycosylase NEIL1 and that male Neil1 -/- mice were significantly more susceptible to AFB 1 -induced HCC relative to wild-type mice. To investigate the mechanisms underlying this enhanced carcinogenesis, WT and Neil1 -/- mice were challenged with a single, 4 mg/kg dose of AFB 1 and frequencies and spectra of mutations were analyzed in liver DNAs 2.5 months post-injection using duplex sequencing. The analyses of DNAs from AFB 1 -challenged mice revealed highly elevated mutation frequencies in the nuclear genomes of both males and females, but not the mitochondrial genomes. In both WT and Neil1 -/- mice, mutation spectra were highly similar to the AFB 1 -specific COSMIC signature SBS24. Relative to wild-type, the NEIL1 deficiency increased AFB 1 -induced mutagenesis with concomitant elevated HCCs in male Neil1 -/- mice. Our data establish a critical role of NEIL1 in limiting AFB 1 -induced mutagenesis and ultimately carcinogenesis., (© The Author(s) 2024. Published by Oxford University Press on behalf of NAR Molecular Medicine.)