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