U.S. Environmental Protection Agency Office of Research and Development Senthilkumar P. Kuppusamy 1, J. Phillip Kaiser 2, and Scott C. Wesselkamper 2 1 ORISE Postdoctoral Fellow at the U.S. EPA, Cincinnati, OH; 2 U.S. EPA, ORD, NCEA, Cincinnati, OH Background Epigenetics refers to changes in phenotype and gene expression that occur without alterations in DNA sequence. Three distinct and interrelated epigenetic mechanisms are DNA methylation, histone modification, and regulation by non-coding RNAs. DNA methylation, which is an integral epigenetic component, occurs at the 5’-carbon position of cytosine residues in CpG islands by DNA methyltransferase using S-adenosylmethionine as a methyl donor. Hypomethylation and Hypermethylation of a gene can lead to gene overexpression and silencing, respectively. Although human and animal studies have shown a strong involvement of epigenetic dysregulation in the etiology of several toxicological conditions, applicability of epigenetic data across the current human health assessment paradigm is unclear. The objective of this study is to compare the sensitivity of epigenetic alterations with the development of tumors in animal toxicological studies and to explore the possibility of incorporating epigenetic information into the hazard identification process for human health risk assessments. Methods Summary and Conclusions  This analysis shows that DNA methylation changes are more sensitive than the corresponding tumor incidences; thus, DNA methylation could potentially be considered as a precursor event in human health risk assessment of suspected carcinogens.  In addition, the exposure duration for all the epigenetic studies (except for hydrazine) were shorter than the carcinogenicity studies, suggesting that epigenetic studies may be more time- and cost-efficient compared to a 2-year carcinogenicity bioassay.  A larger database of chemicals focusing on epigenetic dose-response analysis including evaluation of additional epigenetic endpoints is needed to continue to ascertain how epigenetic data can be applied in human health risk assessment. Senthilkumar P. Kuppusamy, PHOTO PHOOPHOTO PHOPHOTO Figure 1. Mechanisms of Epigenetic Regulation [Kim et al (2011). Pul Circulation. 1(3), ] Figure 2. DNA Methylation and Gene Expression [Verma et al (2002). Lancet Oncol. 3(12), ] Recent investigations have identified a number of environmental toxicants that cause epigenetic alterations in genes. These heritable changes in gene expression may be involved in chemically-mediated adverse health outcomes, such as cancer. Figure 3. Trends in Biomedical Epigenetic Research Publications Adapted from Kim et al (2011). Pul Circulation. 1(3), Step 1: Probable and known environmental human carcinogens were identified from the U.S. EPA's Integrated Risk Information System (IRIS) and the National Toxicology Program (NTP) databases. Step 2: Using PubMed, literature searches were performed on chemicals identified in Step 1 using the chemical name and the search term “DNA methylation”. Chemicals with suitable DNA methylation data in laboratory animal studies were identified. Step 3: Identified chemicals from Step 2 were then mined for appropriate tumor incidence data in the same species, sex and organ as evaluated in the DNA methylation studies. Eight chemicals were selected for this study: di(2-ethylhexyl) phthalate (DEHP), bromodichloromethane, dibromochloromethane, chloroform, hydrazine, trichloroethylene, benzidine, and trichloroacetic acid. Step 4: In all studies, animal doses were converted to corresponding human equivalent doses (HEDs) [1]. A No-observed-adverse-effect level (NOAEL) was identified for DNA methylation. In the absence of a NOAEL, a 10-fold uncertainty factor was applied to the lowest-observed-adverse- effect level (LOAEL) to approximate a NOAEL [2]. Tumor incidence data were analyzed using U.S. EPA’s Benchmark Dose (BMD) modeling (version 2.2.1) software. Step 5: The resulting BMD values were compared to the NOAELs for changes in DNA methylation. Results Table 1. Selected DNA methylation and carcinogenicity studies Chemical Sex, strain, species, and oral exposure type Duration of DNA methylation studies Duration of carcinogenicity studies BromodichloromethaneMale, B6C3Fl, mouse, gavage5 and 28 days [3]2 years [4] DibromochloromethaneFemale, B6C3F1, mouse, gavage11 days [5]105 weeks [6] ChloroformFemale, B6C3F1, mouse, gavage11 days [7]526 days [8] HydrazineMale, Syrian, hamster, drinking water21 months [9]2 years [10] TrichloroethyleneFemale, B6C3F1, mouse, gavage5, 12, and 33 days [11]90 weeks [12] BenzidineMale, B6C3F1, mouse, drinking water1 year [13]33 months [14] Trichloroacetic acidFemale, B6C3F1, mouse, gavage5 days [15]576 days [16] DEHPMale, Sprague Dawley, rats, gavageTreated for GD14-19; examined on PND60. [17] 159 weeks [18] * GD - Gestation Day; PND - Postnatal Day Figure 4. Illustration of BMD models of tumor incidence data Note: The Multistage ‑ cancer model in the EPA benchmark dose software (BMDS) was fit to the tumor incidence data using the extra risk option. An adequate model fit was judged by three criteria: goodness ‑ of ‑ fit p ‑ value (p > 0.1), visual inspection of the dose ‑ response curve, and scaled residual at the data point in the vicinity of the BMR. Among all the models providing adequate fit to the data, the BMD from the best fitting Multistage-cancer model as judged by the goodness ‑ of ‑ fit p ‑ value, is selected as the point of departure. Liver tumor in F B6C3Fl mouse administered with trichloroacetic acidTesticular tumor in M Sprague Dawley rats administered with DEHP Chemical Target organ DNA methylation (Approximated NOAEL HED ) Cancer (BMD HED ) Ratio of BMD HED / NOAEL HED Sensitivity of DNA methylation greater than tumor incidence? BromodichloromethaneKidney Yes DibromochloromethaneLiver Yes ChloroformLiver Yes HydrazineLiver Yes TrichloroethyleneLiver Yes BenzidineLiver Yes Trichloroacetic acidLiver Similar DEHPTestes Yes Table 2. Point of departures for DNA methylation (NOAEL) and tumor incidence (BMD) Table 3. Specific DNA methylation alterations ChemicalPromoter methylationGene methylation BromodichloromethaneNDHypomethylation of whole DNA DibromochloromethaneHypomethylation of c-Myc promoterND ChloroformHypomethylation of c-Myc promoterHypomethylation of whole DNA HydrazineND Hypomethylation of c-Jun and p53; Hypermethylation of c-Ha-Ras and DNA methyltransferase Trichloroethylene Hypomethylation of c-Myc and c-Jun promoter Hypomethylation of whole DNA BenzidineNDHypomethylation of Ha-Ras and Ki-Ras Trichloroacetic acid Hypomethylation of c-Myc and c-Jun promoter ND DEHPHypomethylation of mineralocorticoid receptor (MR) promoter Hypermethylation of MR gene * ND - No Data available Table 4. Average costs of toxicity tests Study typeApproximate cost in dollars 14 day exposure (rat)50, day exposure (rat)125,000 1-year (oral gavage, rat)300,000 2-year (oral gavage, rat)700,000 1)U.S. EPA. (2005). Guidelines for Carcinogen Risk Assessment. Washington, DC 2)Dourson and Stara (1983). Regul Toxicol Pharmacol. 3, ) Tao et al. (2005). Toxicol. Sci. 87(2), 344–352 4) NTP (1987). National Toxicology Program Tech Rep Ser No & 7 ) Coffin et al. (2000). Toxicol Sci. 58 (2), )NTP (1985). Natl Toxicol Program Tech Rep Ser. 282, )NCI (1976). Natl Tech Inform Service No. PB264018/AS 9 ) Zheng and Shank (1996). Carcinogenesis. 17(12), ) Bosan et al. (1987). Carcinogenesis. 8(3), ) Tao et al. (1999). J Biochem Mol Toxicol. 13(5), )NCI (1976). CAS No , NCI-CG-TR-2 13) Vorce et al. (1989). Toxicol Appl Pharmacol. 100 (3), ) Littlefield et al. (1984). Fundam Appl Toxicol. 4 (1), ) Tao et al. (2000). Toxicol Sci. 54 (2), ) Pereira et al. (1996). Fundam Appl Toxicol. 31, ) Marinez-Arguellas et al. (2009). Endocrinology. 150 (12), ) Voss et al. (2005). Toxicology. 206 (3), The views expressed in this poster are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency Environmental Epigenetics: Potential Application in Human Health Risk Assessment