Error-prone Replication Bypass of the Primary Aflatoxin B1 DNA Adduct, AFB1-N7-Gua

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Routine dietary consumption of foods that contain aflatoxins is the second leading cause of environmental carcinogenesis worldwide. Aflatoxin-driven mutagenesis is initiated through metabolic activation of aflatoxin B1(AFB1) to its epoxide form that reacts with N7 guanine in DNA. The resulting AFB1-N7-dG adduct undergoes either spontaneous depurination or imidazole-ring opening yielding formamidopyrimidine AFB1(AFB1-Fapy-dG). Because this latter adduct is known to persist in human tissues and contributes to the high frequency G-to-T mutation signature associated with many hepatocellular carcinomas, we sought to establish the identity of the polymerase(s) involved in processing this lesion. Although our previous biochemical analyses demonstrated the ability of polymerase ζ (pol ζ) to incorporate an A opposite AFB1-Fapy-dG and extend from this mismatch, biological evidence supporting a unique role for this polymerase in cellular tolerance following aflatoxin exposure has not been established. Following challenge with AFB1, survival of mouse cells deficient in pol ζ (Rev3L−/−) was significantly reduced relative toRev3L+/−cells orRev3L−/−cells complemented through expression of the wild-type human REV3L. Furthermore, cell-cycle progression ofRev3L−/−mouse embryo fibroblasts was arrested in late S/G2 following AFB1exposure. TheseRev3L−/−cells showed an increase in replication-dependent formation of γ-H2AX foci, micronuclei, and chromosomal aberrations (chromatid breaks and radials) relative toRev3L+/−cells. These data suggest that pol ζ is essential for processing AFB1-induced DNA adducts and that, in its absence, cells do not have an efficient backup polymerase or a repair/tolerance mechanism facilitating survival.

Section: Resultsmentioning

confidence: 99%

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confidence: 89%

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confidence: 99%

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DNA polymerase ζ limits chromosomal damage and promotes cell survival following aflatoxin exposure

Lin

Owen

Minko

et al. 2016

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“…The G:C→T:A dominance in the spectra was expected due to the mutational properties of individual AFB 1 -DNA adducts (all of which occur at guanines) (12,13,29,30), the mutational spectra in vitro and in vivo of AFB 1 in cells and tissues (12)(13)(14)(15)(16)(17)(18), and observed patterns of mutations in human tumors (27,28). Unexpected, however, was the high G→T mutation yield at selected guanyl residues in the 16 possible three-base contexts (Fig.…”

Section: Discussionmentioning

confidence: 99%

Mutational spectra of aflatoxin B ₁ in vivo establish biomarkers of exposure for human hepatocellular carcinoma

Chawanthayatham

Valentine

Fedeles

et al. 2017

108

113

Aflatoxin B 1 (AFB 1 ) and/or hepatitis B and C viruses are risk factors for human hepatocellular carcinoma (HCC). Available evidence supports the interpretation that formation of AFB 1 -DNA adducts in hepatocytes seeds a population of mutations, mainly G:C→T:A, and viral processes synergize to accelerate tumorigenesis, perhaps via inflammation. Responding to a need for early-onset evidence predicting disease development, highly accurate duplex sequencing was used to monitor acquisition of high-resolution mutational spectra (HRMS) during the process of hepatocarcinogenesis. Four-day-old male mice were treated with AFB 1 using a regimen that induced HCC within 72 wk. For analysis, livers were separated into tumor and adjacent cellular fractions. HRMS of cells surrounding the tumors revealed predominantly G:C→T:A mutations characteristic of AFB 1 exposure. Importantly, 25% of all mutations were G→T in one trinucleotide context (CGC; the underlined G is the position of the mutation), which is also a hotspot mutation in human liver tumors whose incidence correlates with AFB 1 exposure. The technology proved sufficiently sensitive that the same distinctive spectrum was detected as early as 10 wk after dosing, well before evidence of neoplasia. Additionally, analysis of tumor tissue revealed a more complex pattern than observed in surrounding hepatocytes; tumor HRMS were a composite of the 10-wk spectrum and a more heterogeneous set of mutations that emerged during tumor outgrowth. We propose that the 10-wk HRMS reflects a short-term mutational response to AFB 1 , and, as such, is an early detection metric for AFB 1 -induced liver cancer in this mouse model that will be a useful tool to reconstruct the molecular etiology of human hepatocarcinogenesis. duplex sequencing | mycotoxins | cancer | mutagenesis | mouse model

“…cells, the AFB 1 -Fapy-dG adduct is more mutagenic than AFB 1 -N7-dG with mutation frequencies measured at 97% and 32%, respectively; the mutagenic spectrum for each was dominated by G-to-T transversions (13)(14)(15). Between the two anomers of AFB 1 -Fapy-dG, the α species was a severe block to replication, whereas the β species was implicated as a major contributor to mutagenesis (15).…”

Section: Significancementioning

confidence: 99%

NEIL1 protects against aflatoxin-induced hepatocellular carcinoma in mice

Vartanian

Minko

Chawanthayatham

et al. 2017

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109

Global distribution of hepatocellular carcinomas (HCCs) is dominated by its incidence in developing countries, accounting for >700,000 estimated deaths per year, with dietary exposures to aflatoxin (AFB 1 ) and subsequent DNA adduct formation being a significant driver. Genetic variants that increase individual susceptibility to AFB 1 -induced HCCs are poorly understood. Herein, it is shown that the DNA base excision repair (BER) enzyme, DNA glycosylase NEIL1, efficiently recognizes and excises the highly mutagenic imidazole ring-opened AFB 1 -deoxyguanosine adduct (AFB 1 -Fapy-dG). Consistent with this in vitro result, newborn mice injected with AFB 1 show significant increases in the levels of AFB 1 -Fapy-dG in Neil1 −/− vs. wild-type liver DNA. Further, Neil1 −/− mice are highly susceptible to AFB 1 -induced HCCs relative to WT controls, with both the frequency and average size of hepatocellular carcinomas being elevated in Neil1 −/− . The magnitude of this effect in Neil1 −/− mice is greater than that previously measured in Xeroderma pigmentosum complementation group A (XPA) mice that are deficient in nucleotide excision repair (NER). Given that several human polymorphic variants of NEIL1 are catalytically inactive for their DNA glycosylase activity, these deficiencies may increase susceptibility to AFB 1 -associated HCCs.aflatoxin | base excision repair | ring-fragmented purines | liver cancer | environmental toxicant L iver cancers pose an international public health concern as the second leading cause of cancer-related deaths worldwide, with >700,000 estimated deaths per year (1-3). This mortality approaches its annual incidence throughout the world, highlighting the need for development of effective treatments and early diagnostic tools. HCCs represent the major histological subtype among liver cancers. The global distribution of HCCs is dominated by its incidence in developing countries, especially in eastern Asia and Africa, where two major chronic etiological factors drive this disease: (i) routine dietary exposures to grains and nuts that are contaminated with molds, Aspergillus flavus and Aspergillus parasiticus, which produce aflatoxins, and (ii) extremely high rates of hepatitis B (HBV) and C viral infections. In geographical regions of China where aflatoxin contamination of human food products is highest, there is a large shift in not only the age of onset of HCCs, but also in the incidence rate. Within Qidong, a significant number of HCCs occur in males beginning in their early 20s, with the frequency of HCCs peaking between the ages of 40 and 50 (4). These data are in contrast to HCC frequencies in portions of China, such as Beijing, where aflatoxin exposures are minimal. The kinetics of HCC formation in aflatoxin-affected areas are similar to that observed in early onset breast and ovarian cancers in women who are carriers (heterozygotic) for inactivating mutations in BRCA1 or 2.Although there are several different aflatoxin structures, AFB 1 has been demonstrated to be the most potent hepatoc...