1. 1 Robert D. Mitchell III, 2 Andrew Wallace, 2 Ernest Hodgson, 1 Anirudh Dhammi and 1 R. Michael Roe 1 Department of Entomology, North Carolina State University, Raleigh, NC, USA, 2 Department of Toxicology, North Carolina State University, Raleigh, NC, USA Global Molecular Impact of DEET and Fipronil on Human Health The authors gratefully acknowledge Elizabeth Scholl (NCSU), Jeff Roach (UNC-CH), Sam Suarez (NCSU), and the North Carolina State University Genome Sciences Laboratory (GSL) for their assistance with the bioinformatics analyses. This research was supported in part by the U.S. Central Appalachian Regional Education and Research Center (CARERC) Pilot Study. 1.While both DEET and fipronil have different structures and modes of action, there is a common pathway by which these vastly different toxicants are metabolized. 2.The combination of the two compounds together displayed a more than additive relationship as 1,939 genes were dysregulated that were unique to the combinatorial treatment of DEET plus fipronil. 3.Important components involved in endocrine regulation were disrupted by DEET, fipronil, and the combination of the two, suggesting that DEET may not be as benign as previously expected, especially when in combination with another environmental chemical, which is the more common occurrence. 1.Utilize next-gen sequencing technology to analyze the impact of DEET and Fipronil, singly or in combination, on global gene expression in human hepatocytes. 2.Identity specific systems and pathways where genes are up- or down-regulated after chemical exposure. 3.Determine how DEET and Fipronil alter gene expression and whether or not these changes are prognostic or diagnostic for disease. 4.Establish a method for risk assessment in human hepatocytes that can be used to analyze the genetic and epigenetic effects of environmental chemicals. New paradigms for human health risk assessment of environmental chemicals emphasize the use of molecular methods and human-derived cell lines; thus it seemed imperative to examine the impact of such chemicals on a human cell type. The examples chosen were the effects of the insect repellent DEET (N, N-diethyl-m-toluamide) and the phenylpyrozole insecticide fipronil (fluocyanobenpyrazole) on primary human hepatocytes. These chemicals were tested individually or, since interactions are of critical importance, together. RNA-Seq showed that although DEET is a potent inducer (transcript levels higher for 108 genes and lower for 64), fipronil (2,246 transcripts up-regulated and 1,428 repressed) is considerably more effective than DEET. The combined treatment of DEET and fipronil showed interactive and greater than additive effects (3,017 transcripts up-regulated and 2,087 repressed). The genes affected were primarily involved in catalytic activity and binding while the principal pathway affected was the steroid hormone biosynthesis pathway. When primary human hepatocytes were treated with 100 µM DEET, 172 genes were differentially expressed (α = 0.05), of which 108 transcripts were up-regulated and 64 were down-regulated. When treated with 10 µM fipronil, 3,674 genes were differentially expressed where 2,246 transcripts were up-regulated and 1,428 were down-regulated. When hepatocytes were treated with a combination of 100 µM DEET and 10 µM fipronil together, 5,104 transcripts were dysregulated, 3,017 transcripts were up-regulated and 2,087 were down-regulated. Several elements of the steroid hormone biosynthesis pathway were disrupted when primary human hepatocytes were exposed to DEET, fipronil, and DEET plus fipronil. Steroid hormones, derived from cholesterol in mammals, act as intermediaries between the brain and specific tissues or organs and participate in a wide variety of functions in the human body including inflammatory events, pregnancy, and mineral and sugar metabolism. Disruptions in the production of steroid hormones can contribute to defects in development, metabolism, and physiology. Chromosomal mapping shows how the 172 genes that were dysregulated by 100 µM DEET after 72 hours are situated on the chromosomes of a female donor, age 62, with no history of smoking or alcohol. 149 of the 172 genes that were up- or down- regulated by DEET were also differentially regulated by fipronil, along with 3,525 other genes. If the effect on hepatocyte transcript levels by DEET and fipronil were independent, the additive response expected would have been 3,846 dysregulated genes. What was observed from the mixture was 5,104 transcripts that were affected, 1.3 fold higher than the expected or an additional 1,258 transcripts. 1.National Research Council (2007) Toxicity testing in the 21st century: a vision and a strategy. National Academy Press. Washington DC. 2.Tang, J., Usmani, K. A., Hodgson, E. and Rose, R. L. (2004) In vitro metabolism of fipronil by human and rat cytochrome P450 and its interaction with testosterone and diazepam. Chemico-Biol. Interact. 147: 319-329. 3.Das, P. C., Cao, Y., Rose, R. L., Cherrington, N. and Hodgson, E. (2008) Enzyme induction and cytotoxicity in human hepatocytes by chlorpyrifos and N,N-diethyl-m-toluamide (DEET). Drug Metabol. Drug Interact. 23: 237-260. DEETFipronil Introduction Objectives Results Conclusions Acknowledgements References Methods There are 57 known human cytochrome P450 oxidase (CYP) genes and 59 pseudogenes currently whose products serve many roles including the metabolism of endogenous substrates. They perform the bulk of the activities in phase I (enzymatic transformation) of xenobiotic metabolism where lipid-soluble compounds are chemically converted into more readily excreted water-soluble compounds.