Abstract
Consumption of foods contaminated with aflatoxin B1(AFB1) is a recognized risk factor for developing hepatocellular carcinomas (HCCs). The mutational signature of AFB1 is characterized by high frequency G > T transversions in a limited subset of trinucleotide sequences. The 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B1 (AFB1-FapyGua) has been implicated as the primary DNA lesion responsible for AFB1-induced mutations. This study evaluated the mutagenic potential of AFB1-FapyGua in four contexts, including hot- and cold-spot sequences as apparent in the mutational signature. Vectors containing AFB1-FapyGua were replicated in primate cells and the products of replication were isolated and sequenced. Regardless of the sequence context, AFB1-FapyGua caused base substitutions at frequencies of ~ 80-90%, with G > T transversions being most common. Spectra of mutations were only slightly modulated by the sequence context. These data suggest that mechanism(s) defining sequence context-dependent distribution of AFB1-induced mutations likely operates prior to replication.
INTRODUCTION
Several environmental mutagenic agents, including ultraviolet light, components of tobacco smoke, aristolochic acid, and aflatoxin B1 (AFB1), are established human carcinogens (Report on Carcinogens, National Toxicology Program, U.S. Department of Health and Human Services, Public Health Service, https://doi.org/10.22427/NTP-OTHER-1003). Following exposures of cellular DNA, these agents or their metabolic byproducts induce distinct mutational patterns that differ by the types of mutations, local sequence contexts in which base substitutions preferentially occur, and distribution of mutations throughout genome (Kucab et al. 2019; Alexandrov et al. 2020; Volkova et al. 2020). Such agent-specific mutational signatures are evident in deconstructed profiles of somatic mutations in human cancers (Catalogue Of Somatic Mutations In Cancer (COSMIC), https://cancer.sanger.ac.uk/signatures/) (Alexandrov et al. 2020).
AFB1 is implicated as a significant risk factor for developing hepatocellular carcinoma (HCC) (reviewed in (Kobets et al. 2022)). The most common route of human exposure to AFB1is through consumption of food products contaminated withAspergillus flavus and other related fungi producing this toxin (reviewed in (Kensler et al. 2011)). Although the carcinogenic effect of AFB1 has been recognized for several decades, such exposures remain widespread in many regions of Asia and sub-Saharan Africa (Benkerroum 2020). As an example, recent analyses of somatic mutations in 163 HCCs from Chinese patients demonstrated that ~ 10% of these cancers showed a typical signature of AFB1 (Zhang et al. 2017). The COSMIC data base reports the AFB1 footprint in 19 out of 493 HCCs (single base substitution (SBS) signature 24) (https://cancer.sanger.ac.uk/signatures/sbs/sbs24/).
Following ingestion, AFB1 is metabolically activated by microsomal enzymes to an epoxide, which can intercalate into DNA and covalently bind to it, predominantly at the N7 position of guanine (reviewed in (Kensler et al. 2011; McCullough and Lloyd 2019)). This initial cationictrans -8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) adduct is chemically unstable and can either undergo hydrolytic base loss giving rise to an apurinic (AP) site, or interact with a hydroxyl to produce the imidazole ring-openedtrans -8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B1 (AFB1-FapyGua) (Figure 1 ) (Croy and Wogan 1981). Based on numerous observations summarized below, the AFB1-FapyGua adduct is implicated as a major contributor to AFB1-induced mutagenesis.
In contrast to the initial AFB1-N7-Gua adduct that rapidly disappears from cellular DNA, AFB1-FapyGua persists much longer. Specifically, while it comprised ~80% of the AFB1 adducts detected in rat liver DNA 72 h post intraperitoneal injection (Croy and Wogan 1981), in mice, it represented ~95% of the total AFB1 adducts at 48 h post injection (Vartanian et al. 2017; Coskun et al. 2019). Additionally, prior investigations, using site-specifically modified DNA demonstrated that AFB1-FapyGua was highly mutagenic in Escherichia coli and primate cells (reviewed in (McCullough and Lloyd 2019)). When analyzed in primate (COS7) cells using a SV40-based shuttle vectors, it induced ~84% G to T transversions, 8% G to A transitions, 2.5% G to C transversions, and 2.5% single nucleotide deletions, with only ~3% of progeny vectors containing no mutations at the target site (Lin et al. 2014). Furthermore, there is a correlation between spectrum of AFB1-FapyGua-induced base substitutions (Lin et al. 2014) and the mutagenic properties of AFB1. These have been investigated by a variety of methods, ranging from the assessment of mutations in vector DNA that was pretreated with activated AFB1 and replicated in cells, to sequencing of genomic DNA isolated from exposed animals (reviewed in (McCullough and Lloyd 2019)). Consistently throughout these investigations, the data implicated DNA damage at guanines as a major contributor to mutagenesis, with G > T transversions being the predominant type.
Beyond the overall mutagenic G > T signature, AFB1-induced base substitutions showed non-random, sequence context-dependent distribution (Chawanthayatham et al. 2017; Huang et al. 2017; Zhang et al. 2017; Fedeles and Essigmann 2018; Kucab et al. 2019; Alexandrov et al. 2020; Volkova et al. 2020). When frequencies of AFB1-induced base substitutions were normalized against frequencies of trinucleotide sequences, the dominant G > T transversions concentrated in the CGC and CGG sequence contexts (Fedeles and Essigmann 2018). The molecular mechanisms accounting for difference with respect to sequence contexts are currently unknown. The present study addressed the question whether mutational signature of AFB1 can be explained by differential, sequence-dependent fidelity of replication past AFB1-FapyGua. Site-specifically modified vectors containing this adduct in four different local sequence contexts were constructed, replicated in COS7 cells, and progeny DNA analyzed for mutations.
MATERIAL AND METHODS