PROJECT SUMMARY: Inorganic Arsenic (iAs) is one of the largest toxic exposures to impact humanity worldwide, with over 140 million people exposed to iAs via contaminated drinking water. Exposure to iAs during pregnancy disrupts normal DNA methylation patterns in developing offspring and leads to the onset of adult diseases such as type II diabetes (T2D), cardiovascular disease, and cancers. More specifically, disruption during pregnancy attenuates the proper remodeling of the epigenome of the F1 developing offspring and potentially its F2 grand-offspring via disruption of fetal primordial germ cells (PGCs). However, there is a limited understanding between the correlation between the disease phenotype and methylation profile within the F1 offspring. Furthermore, we do not know whether iAs exposure alters epigenetic remodeling in F2 grand- offspring and if the F2 generation is also at risk for developing iAs associated diseases even without direct iAs exposure. Mechanistically, epigenetic reprogramming could be disrupted within the primordial germ cells, resulting in differential DNA methylation and detrimental metabolic health in F2 offspring. The mechanism is as follows: iAs is metabolized enzymatically by methylation, using S-adenosylmethionine (SAM) as a methyl group donor. SAM is also normally used in DNA methylation to maintain the epigenome via the enzymatic addition of a methyl group to unmodified cytosines. Thus, the competition of SAM for both methylation pathways results in methyl group deficiency leading to less iAs conversion (increased toxicity) and less DNA methylation (mis-regulation of gene expression). The central hypothesis is that intergenerational effects of in utero iAs exposure will impact the epigenetic profile and onset of disease phenotypes within F1 and F2 adult offspring, despite the life-long absence of direct arsenic exposure within these generations. Aim 1 will establish the effects of iAs on F1 epigenetic remodeling during embryogenesis, focusing on genes associated with development of obesity and T2D during adulthood. To confirm the maternal effect of iAs exposure on epigenetic remodeling, the validated Agouti Avy mouse model epigenetic biosensor will be used to indicate loss or gain of DNA methylation by shifting coat color. Aim 2 will characterize the F2 (adult grand-offspring) effects of arsenic exposure on epigenetic remodeling in F1 primordial germ cells. For both aims, global DNA methylation and hydroxymethylation will be quantified in somatic and germ tissues by liquid chromatography mass-spectrometry and site specific methylation by pyrosequencing. Glucose tolerance and body composition will be measured in adult F1 and F2 offspring as indications of disease present in adulthood. Collectively, these experiments will provide a better understanding of the effects of iAs heavy metal exposure on intergenerational epigenetics, the onset of adult disease in subsequent generations, and targets of multigenerational ...