NEIL1 protects against aflatoxin-induced hepatocellular carcinoma in mice Vladimir Vartaniana, Irina G. Minkoa, Supawadee Chawanthayathamb, Patricia A. Egnerc, Ying-Chih Lina,d,1, Lauriel F. Earleya,2, Rosemary Makare, Jennifer R. Engf, Matthew T. Campf, Liang Lig, Michael P. Stoneg, Michael R. Lasareva, John D. Groopmanc, Robert G. Croyb, John M. Essigmannb, Amanda K. McCullougha,d,h, and R. Stephen Lloyda,d,h,i,3 aOregon Institute of Occupational Health Sciences, Oregon Health & Science University, Portland, OR 97239; bDepartment of Chemistry, Department Biological Engineering, and Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139; cDepartment of Environmental Health Sciences, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205; dCancer Biology Program, Oregon Health & Science University, Portland, OR 97239; eKnight BioLibrary, Oregon Health & Science University, Portland, OR 97201; fHistopathology Research Core, Oregon Health & Science University, Portland, OR 97201; gDepartment of Chemistry, Department of Biochemistry, Vanderbilt-Ingram Cancer Center, Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN 37235; hDepartment of Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR 97239; and iDepartment of Physiology and Pharmacology, Oregon Health & Science University, Portland, OR 97239 Edited by Michael E. O’Donnell, Rockefeller University, New York, NY, and approved March 7, 2017 (received for review December 20, 2016) Global distribution of hepatocellularcarcinomas(HCCs)isdominated Although there are several different aflatoxin structures, AFB1 by its incidence in developing countries, accounting for >700,000 es- has been demonstrated to be the most potent hepatocarcinogen (5, timated deaths per year, with dietary exposures to aflatoxin (AFB1) 6). Following ingestion and activation by liver microsomal enzymes, and subsequent DNA adduct formation being a significant driver. the intermediate AFB1 epoxide can form a covalently bound ad- duct at N7 guanine in DNA, AFB -N7-dG (Fig. 1A)(7–9). Kinetic Genetic variants that increase individual susceptibility to AFB1-induced 1 analyses of the formation and disappearance of AFB1-DNA ad- HCCs are poorly understood. Herein, it is shown that the DNA base ∼ excision repair (BER) enzyme, DNA glycosylase NEIL1, efficiently rec- ducts revealed that 20% of the initial, quantitatively abundant, ognizes and excises the highly mutagenic imidazole ring-opened cationic AFB1-N7-dG lesions converts to the ring-opened trans-8, 9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)- AFB1-deoxyguanosine adduct (AFB1-Fapy-dG). Consistent with this 9-hydroxy aflatoxin B1 (AFB1-Fapy-dG) adduct (Fig. 1B)within invitroresult,newbornmiceinjectedwithAFB1 show significant −/− 24 h after a single dose. These adducts become the dominant DNA increases in the levels of AFB1-Fapy-dG in Neil1 vs. wild-type liver −/− damage detected in cellular DNA 72 h postexposure, comprising DNA. Further, Neil1 mice are highly susceptible to AFB1-induced up to 80% of all known AFB1-induced lesions (10). The AFB1- HCCs relative to WT controls, with both the frequency and average α −/− Fapy-dG adduct can exist in two interconvertible anomeric forms, size of hepatocellular carcinomas being elevated in Neil1 . The mag- and β, that differ by orientation of the glycosyl bond relative to the −/− nitude of this effect in Neil1 mice is greater than that previously deoxyribose (11). Although the α and β anomers equilibrate at an measured in Xeroderma pigmentosum complementation group A approximately equal ratio in single-stranded environments, the β (XPA) mice that are deficient in nucleotide excision repair (NER). Given form is strongly favored in duplex DNA where it intercalates within that several human polymorphic variants of NEIL1 are catalytically DNA (12) (Fig. 1C). Previously, we demonstrated that in primate inactive for their DNA glycosylase activity, these deficiencies may in- crease susceptibility to AFB1-associated HCCs. Significance aflatoxin | base excision repair | ring-fragmented purines | liver cancer | Human dietary exposures to aflatoxin in some of the most environmental toxicant populated and underdeveloped portions of the world are a major contributing factor to the formation of human hepato- iver cancers pose an international public health concern as cellular carcinomas (HCCs) that account for over 700,000 deaths Lthe second leading cause of cancer-related deaths worldwide, annually. Although the genomic signatures for aflatoxin-driven with >700,000 estimated deaths per year (1–3). This mortality carcinogenesis are G:C to T:A point mutations arising from by- approaches its annual incidence throughout the world, highlight- pass of aflatoxin-induced DNA adducts, maintenance of genome ing the need for development of effective treatments and early stability has been generally attributed to nucleotide excision diagnostic tools. HCCs represent the major histological subtype repair. However, we present three lines of evidence that the among liver cancers. The global distribution of HCCs is domi- DNA base excision repair pathway initiated by the DNA glyco- nated by its incidence in developing countries, especially in eastern sylase NEIL1 is the major contributor in maintaining genomic Asia and Africa, where two major chronic etiological factors drive stability following aflatoxin exposures. These findings suggest this disease: (i) routine dietary exposures to grains and nuts that that inactivating NEIL1 polymorphic variants in the human are contaminated with molds, Aspergillus flavus and Aspergillus population could affect susceptibility to aflatoxin-induced HCC. parasiticus, which produce aflatoxins, and (ii) extremely high rates Author contributions: V.V., I.G.M., J.D.G., R.G.C., J.M.E., A.K.M., and R.S.L. designed re- of hepatitis B (HBV) and C viral infections. In geographical re- search; V.V., I.G.M., S.C., P.A.E., Y.-C.L., L.F.E., J.R.E., M.T.C., L.L., and R.S.L. performed gions of China where aflatoxin contamination of human food research; V.V., I.G.M., S.C., P.A.E., Y.-C.L., L.F.E., R.M., M.P.S., M.R.L., J.D.G., R.G.C., J.M.E., products is highest, there is a large shift in not only the age of A.K.M., and R.S.L. analyzed data; and V.V., I.G.M., M.P.S., M.R.L., J.D.G., R.G.C., J.M.E., A.K.M., and R.S.L. wrote the paper. onset of HCCs, but also in the incidence rate. Within Qidong, a MEDICAL SCIENCES significant number of HCCs occur in males beginning in their early The authors declare no conflict of interest. 20s, with the frequency of HCCs peaking between the ages of This article is a PNAS Direct Submission. 1Present address: Department of Medicine, University of California, San Francisco, CA 40 and 50 (4). These data are in contrast to HCC frequencies in 94143. portions of China, such as Beijing, where aflatoxin exposures are 2Present address: Gene Therapy Center, University of North Carolina, Chapel Hill, NC minimal. The kinetics of HCC formation in aflatoxin-affected 27599. areas are similar to that observed in early onset breast and ovar- 3To whom correspondence should be addressed. Email: [email protected]. ian cancers in women who are carriers (heterozygotic) for inacti- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. vating mutations in BRCA1 or 2. 1073/pnas.1620932114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1620932114 PNAS | April 18, 2017 | vol. 114 | no. 16 | 4207–4212 Downloaded by guest on September 27, 2021 Fig. 1. DNA adduct structures and repair of the AFB1-Fapy-dG adduct by NEIL1. (A) Structure of the AFB1-N7-dG adduct. (B) Structure of the AFB1-Fapy-dG adduct. (C) NMR solution structure of the AFB1-Fapy-dG adduct in a 5′-TXA-3′ sequence context where X is the AFB1-Fapy-dG adduct (12). (D–F) hNEIL1- 32 catalyzed incision of lesion-containing DNA under single-turnover conditions. The P-labeled oligodeoxynucleotides (20 nM) containing AFB1-Fapy-dG either in fully duplex DNA (D) or a bubble structure (E) or Tg in fully duplex DNA (F) were incubated with hNEIL1 (230 nM). The aliquots were removed at the indicated times, and following separation by gel electrophoresis, DNA was visualized using a phosphor screen and Personal Molecular Imager System (Bio- Rad). The representative gel images are shown. The product formation was plotted as a function of time, and the kobs values were obtained from the best fit of the data to a single exponential equation using KaleidaGraph software. cells, the AFB1-Fapy-dG adduct is more mutagenic than AFB1-N7- following a single neonatal challenge with AFB1 (20). A potential dG with mutation frequencies measured at 97% and 32%, respec- role of the base excision repair (BER) pathway in removal of the tively; the mutagenic spectrum for each was dominated by G-to-T AFB -Fapy-dG lesion has previously been investigated. Chetsanga – 1 transversions (13 15). Between the two anomers of AFB1-Fapy-dG, and Frenette (21) presented data suggesting that the E. coli for- the α species was a severe block to replication, whereas the β species mamidopyrimidine DNA glycosylase (Fpg) could incise a portion was implicated as a major contributor to mutagenesis (15). The of the imidazole ring-opened guanines from DNAs that had been spectra of mutations induced by AFB1-dG adducts correlate with previously treated with aflatoxin. This activity could be inhibited the mutation signature observed in genomes of AFB1-associated using an Fpg inhibitor, suggesting an Fpg-catalyzed reaction. HCCs (16, 17), thus underscoring a causal link between these le- However, studies using E. coli cells deficient in Fpg did not sup- sions and genetic alterations associated with these cancers. port a role for this enzyme in aflatoxin adduct repair (18). A role The chemical stability of the AFB1-Fapy-dG adduct is consis- for BER in the repair of AFB -Fapy-dG in human cells has not tent with the long half-lives of many ring-fragmented purines, and 1 been previously investigated.