Mutational spectra of aflatoxin B

Aflatoxin B₁ (AFB₁) and/or hepatitis B and C viruses are risk factors for human hepatocellular carcinoma (HCC). Available evidence supports the interpretation that formation of AFB₁-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 AFB₁ 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 AFB₁ 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 AFB₁ 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 AFB₁, and, as such, is an early detection metric for AFB₁-induced liver cancer in this mouse model that will be a useful tool to reconstruct the molecular etiology of human hepatocarcinogenesis.National Institutes of Health (U.S.) (Grant R01-ES016313)National Institutes of Health (U.S.) (Grant P30-ES002109)National Institutes of Health (U.S.) (Grant T32-ES007020)National Institutes of Health (U.S.) (Grant R01-CA080024

Early Detection of the Aflatoxin B₁ Mutational Fingerprint: A Diagnostic Tool for Liver Cancer

Using duplex-consensus sequencing technology, we recently identified the characteristic high-resolution mutational spectrum of the liver carcinogen aflatoxin B₁ in a mouse model, many months before aflatoxin-induced tumors are detectable. The diagnostic power of this spectrum is then demonstrated by accurately identifying, among the sequenced human liver tumors, the subset of cancers associated with aflatoxin B₁ exposure. Keywords: Duplex sequencing; HCC; hepatocellular carcinoma; mutagenesis; mutational signature; mycotoxin

Mutagenicity of Helicobacter hepaticus infection in the lower bowel mucosa of 129/SvEv Rag2−/−Il10−/−gpt delta mice is influenced by sex

Inflammatory bowel disease and colonic tumors induced by Helicobacter hepaticus (Hh) infection in susceptible mouse strains are utilized to dissect the mechanisms underlying similar human diseases. In our study, infection with genotoxic cytolethal distending toxin-producing Hh in 129/SvEv Rag2−/-Il10−/− gpt delta (RagIl10gpt) mice of both sexes for 21 weeks induced significantly more severe cecal and colonic pathology compared to uninfected controls. The mutation frequencies in the infected RagIl10gpt males were 2.1-fold higher for the cecum and 1.7-fold higher for the colon than male RagIl10gpt controls. In addition, there was a 12.5-fold increase of G:C-to-T:A transversions in the colon of Hh-infected males compared to controls. In contrast, there was no statistical significance in mutation frequencies between infected female Rag2Il10gpt mice and controls. Moreover, Hh infection in RagIl10gpt males significantly up-regulated transcription of Tnfα and iNos, and decreased mRNA levels of cecal Atm compared to the infected females; there was no significant difference in mRNA levels of Il-22, Il-17A, Ifnγ and Atr between the infected males and females. Significantly higher levels of cecal and colonic iNos expression and γH2AX-positive epithelial cells (a biomarker for double-strand DNA breaks [DSB]) in Hh-infected Rag2Il10gpt males vs. Hh-infected females were noted. Finally, Hh infection and associated inflammation increased levels of intestinal mucosa-associated genotoxic colibactin-producing pks+ Escherichia coli. Elevated Tnfα and iNos responses and bacterial genotoxins, in concert with suppression of the DSB repair responses, may have promoted mutagenesis in the lower bowel mucosa of Hh-infected male RagIl10gpt mice.National Institutes of Health (U.S.) (Grant R01–0D01141)National Institutes of Health (U.S.) (Grant T32–0D010978)National Institutes of Health (U.S.) (Grant P30-ES002109)National Institutes of Health (U.S.) (Grant P01-CA26731)National Institutes of Health (U.S.) (Grant R01-CA080024

Prenatal exposure of mice to the human liver carcinogen aflatoxin B

It has become axiomatic that critical windows of susceptibility to genotoxins exist and that genetic damage in utero may be a trigger for later life cancers. Data supporting this critical window hypothesis are remarkably few. This study provides a quantitative bridge between DNA damage by the liver carcinogen aflatoxin B[subscript 1] (AFB[subscript 1]) during prenatal development and the risk of later life genetic disease. AFB[subscript 1] was given to pregnant C57BL/6J mice, carrying F[subscript 1] gestation day 14 (GD14) embryos of the B6C3F1 genotype. Ultra-high performance liquid chromatography and mass spectrometry (UPLC-MS) using aflatoxin-[superscript 15]N[subscript 5]-guanine adduct standards afforded measurement of the AFB[subscript 1]-N[superscript 7]-Gua and AFB[subscript 1]-FAPY adducts 6-hr post dosing in liver DNA of mothers and embryos. A parallel cohort gave birth and the livers of the F[subscript 1] were analyzed for mutations in the gpt gene at 3 and 10 weeks of age. The data revealed mutational spectra dominated by G:C to T:A mutations in both the mother and offspring that are characteristic of AFB[subscript 1] and distinct from background. It was shown that adducts in GD14 embryos were 20-fold more potent inducers of mutagenesis than adducts in parallel-dosed adults. This sensitivity enhancement correlated with Ki67 staining of the liver, reflecting the proliferative potential of the tissue. Taken together, these data provide insight into the relative genetic risks of prenatal and adult exposures to AFB[subscript 1]. Early life exposure, especially during the embryonic period, is strikingly more mutagenic than treatment later in life. Moreover the data provide a baseline against which risk prevention strategies can be evaluated.National Institutes of Health (U.S.) (Grants ES016313, P30-ES002109, P01 ES006052, P30 ES003819, and P30 CA006973)Center of Excellence on Environmental Health, Toxicology and Management of Chemicals, ThailandSchlumberger Foundation. Faculty for the Futur

Editor’s Highlight: Pregnancy Alters Aflatoxin B1 Metabolism and Increases DNA Damage in Mouse Liver

Pregnancy is a complex physiological state, in which the metabolism of endogenous as well as exogenous agents is ostensibly altered. One exogenous agent of concern is the hepatocarcinogen aflatoxin B1(AFB1), a foodborne fungal toxin, that requires phase I metabolic oxidation for conversion to its toxic and carcinogenic form, the AFB1-8,9-exo-epoxide. The epoxide interacts with cellular targets causing toxicity and cell death; these targets include the covalent modification of DNA leading to mutations that can initiate malignant transformation. The main detoxification pathway of the AFB1- epoxide involves phase II metabolic enzymes including the glutathione-S-transferase (GST) family. Pregnancy can modulate both phase I and II metabolism and alter the biological potency of AFB1. The present work investigated the impact of pregnancy on AFB1exposure in mice. A single IP dose of 6 mg/kg AFB1was administered to pregnant C57BL/6 J mice at gestation day 14 and matched non-pregnant controls. Pregnant mice accumulated 2-fold higher AFB1-N7-guanine DNA adducts in the liver when compared with nonpregnant controls 6h post-exposure. Enhanced DNA adduct formation in pregnant animals paralleled elevated hepatic protein expression of mouse CYP1A2 and mouse homologs of human CYP3A4, phase I enzymes capable of bioactivating AFB1. Although phase II enzymes GSTA1/2 showed decreased protein expression, GSTA3, the primary enzymatic protection against the AFB1-epoxide, was unaffected at the protein level. Taken together, our results reveal that pregnancy may constitute a critical window of susceptibility for maternal health, and provide insight into the biochemical factors that could explain the underlying risks.National Institutes of Health (U.S.) (Grant T32-ES007020)National Institutes of Health (U.S.) (Grant P30-ES002109)National Institutes of Health (U.S.) (Grant R01-ES016313)National Institutes of Health (U.S.) (Grant R01-CA080024)National Institutes of Health (U.S.) (Grant P30-CA006973

NEIL1 protects against aflatoxin-induced hepatocellular carcinoma in mice

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[subscript 1]) and subsequent DNA adduct formation being a significant driver. Genetic variants that increase individual susceptibility to AFB[subscript 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[subscript 1]-Fapy-dG). Consistent with this in vitro result, newborn mice injected with AFB[subscript 1] show significant increases in the levels of AFB[subscript 1]-Fapy-dG in Neil1[superscript -/-] vs. wild-type liver DNA. Further, Neil1[superscript -/-] mice are highly susceptible to AFB[subscript 1]-induced HCCs relative to WT controls, with both the frequency and average size of hepatocellular carcinomas being elevated in Neil1[superscript -/-]. The magnitude of this effect in Neil1[superscript -/-] 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[subscript 1]-associated HCCs.National Institutes of Health (U.S.) (Grant R01-ES016313)National Institutes of Health (U.S.) (Grant P30-ES002109)National Institutes of Health (U.S.) (Grant R01-CA080024

NEIL1 protects against aflatoxin-induced hepatocellular carcinoma in mice

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[subscript 1]) and subsequent DNA adduct formation being a significant driver. Genetic variants that increase individual susceptibility to AFB[subscript 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[subscript 1]-Fapy-dG). Consistent with this in vitro result, newborn mice injected with AFB[subscript 1] show significant increases in the levels of AFB[subscript 1]-Fapy-dG in Neil1[superscript -/-] vs. wild-type liver DNA. Further, Neil1[superscript -/-] mice are highly susceptible to AFB[subscript 1]-induced HCCs relative to WT controls, with both the frequency and average size of hepatocellular carcinomas being elevated in Neil1[superscript -/-]. The magnitude of this effect in Neil1[superscript -/-] 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[subscript 1]-associated HCCs.National Institutes of Health (U.S.) (Grant R01-ES016313)National Institutes of Health (U.S.) (Grant P30-ES002109)National Institutes of Health (U.S.) (Grant R01-CA080024