Filling in data gaps: studies of epigenetic alterations in complex disease

Three specific aims were created to investigate the involvement of epigenomic and epigenetic alterations in complex disease phenotypes induced by environmental exposure to toxic substances, and to address the central hypothesis that epigenetic alterations occur in a tissue- and/or disease state-specific manner as a result of exposure to known genotoxic chemical carcinogens.

Aim 1:To investigate the role of epigenetic alterations, as well as genotoxic damage, in hepatocellular carcinoma (HCC) using a mouse model designed specifically to emulate the most common human HCC phenotype. The molecular mechanisms leading to the development of HCC are complex and include both genetic and epigenetic events. To determine the relative contribution of these alterations in liver tumorigenesis, we evaluated epigenetic modifications at both the global and the gene specific level, as well as the mutational profile of genes commonly altered in liver tumors using a mouse model of fibrosis-associated liver cancer designed to emulate cirrhotic liver (Uehara et al., 2013), a prevailing disease state

observed in most humans with HCC (Fattovich et al., 2004; Farazi and DePinho, 2006). We characterized underlying molecular signatures associated with the greater-than-additive increase in tumor incidence that was previously observed in this mouse model of co-morbidity and HCC. We hypothesized that a correlation exists between the increased tumor incidence in mice treated with genotoxic and pro-fibrogenic agents and epigenetic alterations, relative to mice treated with only one of the chemicals.

Aim 2: To characterize the tissue specificity of epigeneticalterations and DNA damage that result from exposure to the genotoxic chemical 1,3-butadiene (1,3-butadiene). 1,3- butadiene, a known human carcinogen, poses an occupational health hazard to humans due to its use in the synthesis of plastics and rubber (White, 2007). 1,3-butadiene is also an environmental pollutant because it is present in automobile exhaust and cigarette smoke (Hecht, 1999). Although genotoxicity is an established mechanism of the carcinogenicity of 1,3-butadiene, epigenetic alterations have also been observed in the liver of mice exposed to 1,3-butadiene by inhalation (Koturbash et al., 2011b; Koturbash et al., 2011c). Further, epigenetic reprogramming has been proposed as an integral part of the “genome instability” enabling characteristic of cancer cells. Based on these findings, we aim to characterize the diverse molecular mechanisms of 1,3- butadiene-induced tumorigenicity by evaluating both genotoxic and “epigenotoxic” effects of 1,3- butadiene exposure in mouse tissues that are targets (lung and liver) and non-targets (kidney) of 1,3-butadiene-induced tumorigenesis. While extensive DNA damage has been observed in the liver, lungs, and kidneys of mice chronically exposed to 1,3-butadiene (Goggin et al., 2009; Goggin et al., 2011; Swenberg et al., 2011), only the lung and liver develop tumors. We hypothesized that tissue-specific epigenetic alterations may explain, at least in part, the differential tissue susceptibility to 1,3-butadiene-induced tumorigenesis in mice.

Aim 3: To understand the relationship between alterations in epigenetic regulatory elements and transcriptional responses as a consequence of exposure to the genotoxic agent 1,3-butadiene. Aim 3 expanded on the findings of Aim 2 by examining mouse strain-and

tissue-specific transcriptional responses to 1,3-butadiene, as well as alterations to epigenetic regulators of gene expression. In this aim, a strain known to be susceptible to DNA damage and global changes to the epigenome (C57BL/6J), and a strain that has been shown to be resistant to the same 1,3-butadiene-responses (CAST/EiJ) (Koturbash et al., 2011b) were used to address the hypothesis that 1,3-butadiene-induced alterations in mRNA and miRNA vary across strains and tissues, and that differentially expressed miRNAs modulate the 1,3-butadiene-induced changes in gene expression. Measurement of differentially expressed genes and functional annotation enabled the identification and evaluation of genetic pathways involved in the toxicity of the classic genotoxic agent 1,3-butadiene. We also evaluated changes in the expression of microRNAs and their potential association with differential gene expression in response to 1,3- butadiene. Alterations in miRNA expression have been implicated in many human cancers, and have also been shown to be influenced by exposure to toxic substances (Lema and Cunningham, 2010; Berindan-Neagoe et al., 2014).

REFERENCES

Abdel-Rahman SZ, Ammenheuser MM and Ward JB, Jr. (2001) Human sensitivity to 1,3- butadiene: role of microsomal epoxide hydrolase polymorphisms. Carcinogenesis 22:415-423.

Abdel-Rahman SZ, El-Zein RA, Ammenheuser MM, Yang Z, Stock TH, Morandi M and Ward JB, Jr. (2003) Variability in human sensitivity to 1,3-butadiene: Influence of the allelic variants of the microsomal epoxide hydrolase gene. Environmental and molecular mutagenesis41:140-146.

Alyea RA, Moore NP, LeBaron MJ, Gollapudi BB and Rasoulpour RJ (2012) Is the current product safety assessment paradigm protective for epigenetic mechanisms? Journal of pharmacological and toxicological methods66:207-214.

Bai W, Chen Y, Yang J, Niu P, Tian L and Gao A (2014a) Aberrant miRNA profiles associated with chronic benzene poisoning. Exp Mol Pathol96:426-430.

Bai W, Yang J, Yang G, Niu P, Tian L and Gao A (2014b) Long non-coding RNA NR_045623 and NR_028291 involved in benzene hematotoxicity in occupationally benzene-exposed workers. Experimental and molecular pathology96:354-360.

Baik JH, Griffiths S, Giuili G, Manson M, Siegrist S and Guellaen G (1991) DNA methylation patterns of the rat gamma-glutamyl transpeptidase gene in embryonic, adult and neoplastic liver. Carcinogenesis12:1035-1039.

Barkley LR and Santocanale C (2013) MicroRNA-29a regulates the benzo[a]pyrene dihydrodiol epoxide-induced DNA damage response through Cdc7 kinase in lung cancer cells. Oncogenesis2:e57.

Barrett JC (1993) Mechanisms of multistep carcinogenesis and carcinogen risk assessment. Environ Health Perspect100:9-20.

Baylin SB (2005) DNA methylation and gene silencing in cancer. Nat Clin Pract Oncol2 Suppl 1:S4-11.

Beland FA, Beranek DT, Dooley KL, Heflich RH and Kadlubar FF (1983) Arylamine-DNA adducts in vitro and in vivo: their role in bacterial mutagenesis and urinary bladder carcinogenesis. Environ Health Perspect49:125-134.

Berindan-Neagoe I, Monroig Pdel C, Pasculli B and Calin GA (2014) MicroRNAome genome: a treasure for cancer diagnosis and therapy. CA: a cancer journal for clinicians64:311- 336.

Bollati V, Baccarelli A, Hou L, Bonzini M, Fustinoni S, Cavallo D, Byun HM, Jiang J, Marinelli B, Pesatori AC, Bertazzi PA and Yang AS (2007) Changes in DNA methylation patterns in subjects exposed to low-dose benzene. Cancer Res67:876-880.

Bradley C, van der Meer R, Roodi N, Yan H, Chandrasekharan MB, Sun ZW, Mernaugh RL and Parl FF (2007) Carcinogen-induced histone alteration in normal human mammary

Brauze D, Wielgosz SM, Pawlak AL and Baer-Dubowska W (1997) Effect of the route of benzo[a]pyrene administration on sister chromatid exchange and DNA binding in bone marrow of mice differing with respect to cytochrome P450 1A1 induction. Toxicology letters91:211-217.

Caiment F, Gaj S, Claessen S and Kleinjans J (2015) High-throughput data integration of RNA- miRNA-circRNA reveals novel insights into mechanisms of benzo[a]pyrene-induced carcinogenicity. Nucleic Acids Res43:2525-2534.

Chan KT, Hsieh DP and Lung ML (2003) In vitro aflatoxin B1-induced p53 mutations. Cancer letters199:1-7.

Chao MR, Wang CJ, Wu MT, Pan CH, Kuo CY, Yang HJ, Chang LW and Hu CW (2008) Repeated measurements of urinary methylated/oxidative DNA lesions, acute toxicity, and mutagenicity in coke oven workers. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology17:3381-3389.

Chappell G, Kobets T, O'Brien B, Tretyakova N, Sangaraju D, Kosyk O, Sexton KG, Bodnar W, Pogribny IP and Rusyn I (2014a) Epigenetic events determine tissue-specific toxicity of inhalational exposure to the genotoxic chemical 1,3-butadiene in male C57BL/6J mice. Toxicol Sci142:375-384.

Chappell G, Kutanzi K, Uehara T, Tryndyak V, Hong HH, Hoenerhoff M, Beland FA, Rusyn I and Pogribny IP (2014b) Genetic and epigenetic changes in fibrosis-associated hepatocarcinogenesis in mice. Int J Cancer134:2778-2788.

Chen JX, Zheng Y, West M and Tang MS (1998) Carcinogens preferentially bind at methylated CpG in the p53 mutational hot spots. Cancer Res58:2070-2075.

Cheng KC, Preston BD, Cahill DS, Dosanjh MK, Singer B and Loeb LA (1991) The vinyl chloride DNA derivative N2,3-ethenoguanine produces G----A transitions in Escherichia coli. Proc Natl Acad Sci U S A88:9974-9978.

Choi YM, An S, Lee EM, Kim K, Choi SJ, Kim JS, Jang HH, An IS and Bae S (2012) CYP1A1 is a target of miR-892a-mediated post-transcriptional repression. Int J Oncol41:331-336. Costa S, Coelho P, Costa C, Silva S, Mayan O, Santos LS, Gaspar J and Teixeira JP (2008)

Genotoxic damage in pathology anatomy laboratory workers exposed to formaldehyde. Toxicology252:40-48.

Coulter JB, O'Driscoll CM and Bressler JP (2013) Hydroquinone increases 5-

hydroxymethylcytosine formation through ten eleven translocation 1 (TET1) 5- methylcytosine dioxygenase. J Biol Chem288:28792-28800.

Damiani LA, Yingling CM, Leng S, Romo PE, Nakamura J and Belinsky SA (2008) Carcinogen- induced gene promoter hypermethylation is mediated by DNMT1 and causal for

transformation of immortalized bronchial epithelial cells. Cancer research68:9005-9014. Davie JR, Delcuve GP, Nickel BE, Moirier R and Bailey G (1987) Reduced levels of histones

H1o and H1b, and unaltered content of methylated DNA in rainbow trout hepatocellular carcinoma chromatin. Cancer Res47:5407-5410.

Deaton AM and Bird A (2011) CpG islands and the regulation of transcription. Genes Dev 25:1010-1022.

DeBord DG, Cheever KL, Booth-Jones AD, Swearengin TF and Savage RE, Jr. (1995) Alterations of histone phosphorylation in rat spleen cells after treatment with the aromatic amine, 4,4'-methylene-bis(2-chloroaniline). J Biochem Toxicol10:19-23. DeMarini DM, Landi S, Tian D, Hanley NM, Li X, Hu F, Roop BC, Mass MJ, Keohavong P, Gao

W, Olivier M, Hainaut P and Mumford JL (2001) Lung tumor KRAS and TP53 mutations in nonsmokers reflect exposure to PAH-rich coal combustion emissions. Cancer

research61:6679-6681.

Deng Q, Huang S, Zhang X, Zhang W, Feng J, Wang T, Hu D, Guan L, Li J, Dai X, Deng H, Zhang X and Wu T (2014) Plasma microRNA expression and micronuclei frequency in workers exposed to polycyclic aromatic hydrocarbons. Environ Health Perspect122:719- 725.

Denissenko MF, Chen JX, Tang MS and Pfeifer GP (1997) Cytosine methylation determines hot spots of DNA damage in the human P53 gene. Proc Natl Acad Sci U S A94:3893-3898. Deppe J, Steinritz D, Santovito D, Egea V, Schmidt A, Weber C and Ries C (2015) Upregulation

of miR-203 and miR-210 affect growth and differentiation of keratinocytes after exposure to sulfur mustard in normoxia and hypoxia. Toxicol Lett.

Diala ES and Hoffman RM (1982) DNA methylation levels in normal and chemically-transformed mouse 3T3 cells. Biochemical and biophysical research communications104:1489- 1494.

Dong H, Bonala RR, Suzuki N, Johnson F, Grollman AP and Shibutani S (2004) Mutagenic potential of benzo[a]pyrene-derived DNA adducts positioned in codon 273 of the human P53 gene. Biochemistry43:15922-15928.

Duan H, He Z, Ma J, Zhang B, Sheng Z, Bin P, Cheng J, Niu Y, Dong H, Lin H, Dai Y, Zhu B, Chen W, Xiao Y and Zheng Y (2013) Global and MGMT promoter hypomethylation independently associated with genomic instability of lymphocytes in subjects exposed to high-dose polycyclic aromatic hydrocarbon. Arch Toxicol87:2013-2022.

Farazi PA and DePinho RA (2006) Hepatocellular carcinoma pathogenesis: from genes to environment. Nature reviews Cancer6:674-687.

Fattovich G, Stroffolini T, Zagni I and Donato F (2004) Hepatocellular carcinoma in cirrhosis: incidence and risk factors. Gastroenterology127:S35-50.

Feng Y, Xue WJ, Li P, Sha ZY, Huang H, Rui L, Li HX and Mao QS (2012) RASSF1A

hypermethylation is associated with aflatoxin B1 and polycyclic aromatic hydrocarbon exposure in hepatocellular carcinoma. Hepatogastroenterology59:1883-1888.

Fustinoni S, Rossella F, Polledri E, Bollati V, Campo L, Byun HM, Agnello L, Consonni D, Pesatori AC, Baccarelli A and Bertazzi PA (2012) Global DNA methylation and low-level exposure to benzene. Med Lav103:84-95.

Gao A, Zuo X, Liu Q, Lu X, Guo W and Tian L (2010) Methylation of PARP-1 promoter involved in the regulation of benzene-induced decrease of PARP-1 mRNA expression. Toxicol Lett195:114-118.

Gao A, Zuo X, Song S, Guo W and Tian L (2011) Epigenetic modification involved in benzene- induced apoptosis through regulating apoptosis-related genes expression. Cell Biol Int 35:391-396.

Gao L, Mai A, Li X, Lai Y, Zheng J, Yang Q, Wu J, Nan A, Ye S and Jiang Y (2013) LncRNA- DQ786227-mediated cell malignant transformation induced by benzo(a)pyrene. Toxicology letters223:205-210.

Garzon R, Fabbri M, Cimmino A, Calin GA and Croce CM (2006) MicroRNA expression and function in cancer. Trends in molecular medicine12:580-587.

Goggin M, Sangaraju D, Walker VE, Wickliffe J, Swenberg JA and Tretyakova N (2011) Persistence and repair of bifunctional DNA adducts in tissues of laboratory animals exposed to 1,3-butadiene by inhalation. Chem Res Toxicol24:809-817.

Goggin M, Swenberg JA, Walker VE and Tretyakova N (2009) Molecular dosimetry of 1,2,3,4- diepoxybutane-induced DNA-DNA cross-links in B6C3F1 mice and F344 rats exposed to 1,3-butadiene by inhalation. Cancer Res69:2479-2486.

Gomaa AI, Khan SA, Toledano MB, Waked I and Taylor-Robinson SD (2008) Hepatocellular carcinoma: epidemiology, risk factors and pathogenesis. World J Gastroenterol14:4300- 4308.

Gordon MW, Yan F, Zhong X, Mazumder PB, Xu-Monette ZY, Zou D, Young KH, Ramos KS and Li Y (2015) Regulation of p53-targeting microRNAs by polycyclic aromatic

hydrocarbons: Implications in the etiology of multiple myeloma. Mol Carcinog54:1060- 1069.

Guengerich FP (1992) Roles of the vinyl chloride oxidation products 1-chlorooxirane and 2- chloroacetaldehyde in the in vitro formation of etheno adducts of nucleic acid bases [corrected]. Chemical research in toxicology5:2-5.

Guengerich FP, Mason PS, Stott WT, Fox TR and Watanabe PG (1981) Roles of 2-

haloethylene oxides and 2-haloacetaldehydes derived from vinyl bromide and vinyl chloride in irreversible binding to protein and DNA. Cancer research41:4391-4398. Han Z, Yang Q, Liu B, Wu J, Li Y, Yang C and Jiang Y (2012) MicroRNA-622 functions as a

tumor suppressor by targeting K-Ras and enhancing the anticarcinogenic effect of resveratrol. Carcinogenesis33:131-139.

Han Z, Zhang Y, Xu Y, Ji J, Xu W, Zhao Y, Luo F, Wang B, Bian Q and Liu Q (2014) Cell cycle changes mediated by the p53/miR-34c axis are involved in the malignant transformation of human bronchial epithelial cells by benzo[a]pyrene. Toxicol Lett225:275-284.

Hanahan D and Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell144:646- 674.

Hecht SS (1999) Tobacco smoke carcinogens and lung cancer. J Natl Cancer Inst91:1194- 1210.

Herberth G, Bauer M, Gasch M, Hinz D, Roder S, Olek S, Kohajda T, Rolle-Kampczyk U, von Bergen M, Sack U, Borte M, Lehmann I, Lifestyle, Environmental F and Their Influence on Newborns Allergy Risk study g (2014) Maternal and cord blood miR-223 expression associates with prenatal tobacco smoke exposure and low regulatory T-cell numbers. The Journal of allergy and clinical immunology133:543-550.

Herbstman JB, Tang D, Zhu D, Qu L, Sjodin A, Li Z, Camann D and Perera FP (2012) Prenatal exposure to polycyclic aromatic hydrocarbons, benzo[a]pyrene-DNA adducts, and genomic DNA methylation in cord blood. Environ Health Perspect120:733-738.

Herceg Z, Lambert MP, van Veldhoven K, Demetriou C, Vineis P, Smith MT, Straif K and Wild CP (2013) Towards incorporating epigenetic mechanisms into carcinogen identification and evaluation. Carcinogenesis34:1955-1967.

Hernandez-Vargas H, Castelino J, Silver MJ, Dominguez-Salas P, Cros MP, Durand G, Calvez- Kelm FL, Prentice AM, Wild CP, Moore SE, Hennig BJ, Herceg Z, Gong YY and

Routledge MN (2015) Exposure to aflatoxin B1 in utero is associated with DNA

methylation in white blood cells of infants in The Gambia. Int J Epidemiol44:1238-1248. Heston WE (1953a) Occurrence of tumors in mice injected subcutaneously with sulfur mustard

and nitrogen mustard. J Natl Cancer Inst14:131-140.

Heston WE (1953b) Pulmonary tumors in strain A mice exposed to mustard gas. Proc Soc Exp Biol Med82:457-460.

Hoyos-Giraldo LS, Escobar-Hoyos LF, Saavedra-Trujillo D, Reyes-Carvajal I, Munoz A, Londono-Velasco E, Tello A, Cajas-Salazar N, Ruiz M, Carvajal S and Santella RM (2015) Gene-specific promoter methylation is associated with micronuclei frequency in urothelial cells from individuals exposed to organic solvents and paints. J Expo Sci Environ Epidemiol.

Hu G, Yang T, Zheng J, Dai J, Nan A, Lai Y, Zhang Y, Yang C and Jiang Y (2015) Functional role and mechanism of lncRNA LOC728228 in malignant 16HBE cells transformed by anti-benzopyrene-trans-7,8-dihydrodiol-9,10-epoxide. Mol Carcinog54 Suppl 1:E192- 204.

Hu J, Ma H, Zhang W, Yu Z, Sheng G and Fu J (2014) Effects of benzene and its metabolites on global DNA methylation in human normal hepatic L02 cells. Environmental toxicology 29:108-116.

Hu W, Feng Z and Tang MS (2003) Preferential carcinogen-DNA adduct formation at codons 12 and 14 in the human K-ras gene and their possible mechanisms. Biochemistry

42:10012-10023.

Huan LC, Wu JC, Chiou BH, Chen CH, Ma N, Chang CY, Tsen YK and Chen SC (2014) MicroRNA regulation of DNA repair gene expression in 4-aminobiphenyl-treated HepG2 cells. Toxicology322:69-77.

Huang H, Hu G, Cai J, Xia B, Liu J, Li X, Gao W, Zhang J, Liu Y and Zhuang Z (2014) Role of poly(ADP-ribose) glycohydrolase silencing in DNA hypomethylation induced by

Huang XY, Yao JG, Huang HD, Wang C, Ma Y, Xia Q and Long XD (2013) MicroRNA-429 Modulates Hepatocellular Carcinoma Prognosis and Tumorigenesis. Gastroenterol Res Pract2013:804128.

Huff JE, Melnick RL, Solleveld HA, Haseman JK, Powers M and Miller RA (1985) Multiple organ carcinogenicity of 1,3-butadiene in B6C3F1 mice after 60 weeks of inhalation exposure. Science227:548-549.

IARC (1975) IARC monographs on the evaluation of the carcinogenic risk of chemicals to man: some aziridines, N-, S- & O-mustards and selenium. IARC Monogr Eval Carcinog Risk Chem Man9:1-268.

IARC (1983) Polynuclear aromatic compounds, Part 1, Chemical, environmental and experimental data. IARC Monogr Eval Carcinog Risk Chem Hum32:1-453.

IARC (2006) Formaldehyde, 2-butoxyethanol and 1-tert-butoxypropan-2-ol. IARC Monogr Eval Carcinog Risks Hum88:1-478.

IARC (2008) IARC monographs on the evaluation of carcinogenic risks to humans. Volume 97. 1,3-butadiene, ethylene oxide and vinyl halides (vinyl fluoride, vinyl chloride and vinyl bromide). IARC monographs on the evaluation of carcinogenic risks to humans / World Health Organization, International Agency for Research on Cancer97:3-471.

IARC (2010a) Painting, firefighting, and shiftwork. IARC Monogr Eval Carcinog Risks Hum98:9- 764.

IARC (2010b) Some non-heterocyclic polycyclic aromatic hydrocarbons and some related exposures. IARC Monogr Eval Carcinog Risks Hum92:1-853.

IARC (2012) Chemical Agents and Related Occupations - A Review of Human Carcinogens. IARC monographs on the evaluation of carcinogenic risks to humans / World Health Organization, International Agency for Research on Cancer100F:1-567.

Ibuki Y, Toyooka T, Zhao X and Yoshida I (2014) Cigarette sidestream smoke induces histone H3 phosphorylation via JNK and PI3K/Akt pathways, leading to the expression of proto- oncogenes. Carcinogenesis35:1228-1237.

Infante PF (2006) Benzene exposure and multiple myeloma: a detailed meta-analysis of benzene cohort studies. Ann N Y Acad Sci1076:90-109.

Infante PF, Rinsky RA, Wagoner JK and Young RJ (1977) Leukaemia in benzene workers. Lancet2:76-78.

Jackson TE, Lilly PD, Recio L, Schlosser PM and Medinsky MA (2000) Inhibition of cytochrome P450 2E1 decreases, but does not eliminate, genotoxicity mediated by 1,3-butadiene. Toxicological sciences : an official journal of the Society of Toxicology55:266-273. Ji Z, Zhang L, Peng V, Ren X, McHale CM and Smith MT (2010) A comparison of the

cytogenetic alterations and global DNA hypomethylation induced by the benzene