Innovative Research for a Sustainable Future www.epa.gov/research Yue Ge l ge.yue@epa.gov l 919-541-2202 Investigating Steatosis Susceptibility in Pancreas Tissue of Mouse in Response to Vinyl Chloride Exposure aYue Ge, aMaribel Bruno, aBrian N. Chorley, bMatthew C. Cave, cJuliane I. Beier a US Environmental Protection Agency; RTP, NC 27711; b University of Louisville; c University of Pittsburgh Objective Methods Results Conclusions Alterations in physiological processes in pancreas have been associated with various metabolic problems and fatty disease syndromes, such as inflammation, fibrosis, necrosis, abdominal obesity, and steatosis. The alterations of normal pancreatic functions can result from environmental exposures, such as chemicals and diets, which can impact susceptibility to metabolic problems and fatty diseases. It has been reported that environmental vinyl chloride (VC) exposure significantly increased steatosis in mice fed a high‐fat diet (HFD) but not low-fat diet (LFD). However, little is known about molecular basis such as toxicity pathways and biomarkers and toxic mechanisms underlying the VC- increased environmental susceptibility, steatosis and other fatty disease induced by HFD. The present study was undertaken to examine the protein responses to VC exposure in pancreas tissues of C57BL/6J mice fed LFD or HFD, with focus on the investigation of the changes at expression and/or phosphorylation levels of some protein biomarkers of fat metabolisms, and key regulators of steatotic pathways such as cell proliferation and differentiation, fatty acid oxidation , energy metabolism, cell adhesion and migration, oxidative stress, and inflammation. Our hypothesis is that the protein alterations may modify the environmental susceptibility of mouse to the existing steatotic pathways and adverse outcomes induced by HFD after the exposure to VC. Six-week-old C57BL/6J mice (Jackson Laboratory) fed LFD or HFD (Envigo Teklad Diets, Madison) were exposed to VC (targeted concentration: 0.1 ppm) by air inhalation in chambers for 6 hours per day, 5 days per week for 12 weeks. The pancreas tissue samples were homogenized using polytron for 20sends in the buffer containing 150 mM NaCl, 20 mM Tris pH 7.7, 1mM EDTA, 1 mM EGTA and 1% Triton X-100. After incubation in ice for one hour, the homogenized tissue samples were then centrifuged at 10,000 g for 10 minutes. The supernatants containing total tissue proteins of pancreas were analyzed for identification of changes at expression and/or phosphorylation levels of protein biomarkers using a combination of 200 Mouse cytokines array (QAM-CAA- 4000, Raybiotech, Norcross GA) and Western blot. Pancreas tissues from four groups of mice treated with LFD, HFD, LFD+VC, LFD+VC were used for the study. The accession numbers, relative fold changes, and IDs of the differentially altered proteins were tabulated and imported into ingenuity pathway analysis (IPA) for identification of top canonical pathways, toxlists, molecular and biological functions, biomarkers, and protein interaction networks that were regulated by the identified proteins. Figure 1: Hierarchical clustering of differentially expressed and phosphorylated proteins in in pancreas mice treated with LFD and HFD with or without VC. Blue areas represented downregulation and red ones represented upregulation of proteins. As shown in Fig 1, different treatment groups (LFD, LFD+VC, HFD, or HFD+VC) were distinctively separated (left side of the Figure), which suggested that protein expression and phosphorylation patterns in pancreas proteome of mice treated with LFD, LFD+VC, HFD, or HFD+VC are different, and HFD-induced protein change is similar as the one induced by HFD+VC, as compared to those induced by LFD and LFD+VC. The lower portion of the dendogram depicted seven main clusters and each cluster usually consisted of two proteins. These clusters included the cluster of TCK-1and PIGF2, Eotaxin 2 and CD48, GSTµ and pAKT, cystatin C an