This pathway to independence grant application describes the training and career development plan for Dr. John Clarke. Dr. Clarke's immediate career goal is to complete the necessary postdoctoral training in order establish a research project in toxicology that is independent from the work of his post-doctoral advisor, Dr. Nathan Cherrington. This goal is directly tied to the research proposed in the application, since this project takes the liver disease phenotype studied in Dr. Cherrington's group and applies it to exposure and toxicity of an environmental stressor. Dr. Clarke's long-term career goals are to 1) improve human health by advancing our understanding of the interaction between liver disease and environmental toxicant exposure, and 2) become a mentor to subsequent generations of scientists. These immediate and long-term career goals will be accomplished through specific aspects of the training environment and the research project. Dr. Clarke's training plan includes expanding his knowledge and recognition in the field of toxicology, developing new technical expertise, improving his mentoring and communication skills, and improving grant writing skills. In order to increase knowledge and recognition in the field of toxicology Dr. Clarke will participate in educational and training activities available through the NIEHS training grant, the Society of Toxicology, the Pharmacology and Toxicology Department, and the Arizona Health Sciences Center. These include seminars, journal clubs, colloquia, and conferences. Dr. Clarke has assembled a dream team of mentors and collaborators that will be an incredible asset for his career development and research plan. Dr. Clarke will develop new technical expertise in xenobiotic transport kinetics, in vivo toxicokinetics analyses, organ specific toxicity, and physiologically based toxicokinetics modeling (PBTK). An important aspect of being an independent scientist is the ability to mentor and communicate effectively. As part of the training environment Dr. Clarke will continue to present data at conferences and publish in peer-reviewed journals. He will also continue to mentor high school, graduate, and undergraduate students in Dr. Cherrington's lab. Finally, he will improve his grant writing skills by participatig in a series of grant writing workshops offered at the University of Arizona and through the NIH. By implementing each aspect of this training plan, Dr. Clarke will receive the additional training he needs to successfully transition into an independent faculty position in toxicology. This research project seeks to understand how the liver disease nonalcoholic steatohepatitis (NASH) alters the toxicokinetics and toxicity of the waterborne toxin microcystin-LR (MCLR). The prevalence of NASH, which is the most severe form of nonalcoholic fatty liver disease, continues to increase worldwide and is estimated to be present in 5% to 17% of adults in the United States. In addition to the liver problems this population faces, there is also a higher incidence of chronic kidney disease among these patients, which may be associated with MCLR exposure. MCLR-producing cyanobacteria are ubiquitous in sources of drinking water around the world, and the occurrence of blooms and MCLR contaminated drinking water are expected to increase as anthropomorphic eutrophication of water systems and global climate change continue to worsen. Unfortunately, there is a dearth of information regarding MCLR toxicokinetics in humans and how NASH-associated changes in xenobiotic transporter expression influence MCLR toxicokinetics and MCLR-induced toxicity. The objectives of this research are to 1) provide a complete picture of the xenobiotic transporters responsible for the disposition of MCLR and its main metabolites, MCLR-glutathione and MCLR-cysteine, 2) determine how altered expression of these transporters in NASH affects MCLR disposition and toxicity, and 3) move these findings towards risk assessment by constructing a PBTK model for human MCLR exposure in healthy and NASH populations. Dr. Clarke's previous work has demonstrated that NASH causes decreased expression of the liver uptake transporters important for MCLR-induced liver toxicity: organic anion transporting polypeptide-1b2 (Oatp1b2) in rodents and OATP1B1 and OATP1B3 in humans. He has also shown that this NASH-induced decrease in rodent Oatp1b2 shifted the disposition of several drugs that are substrates for OATPs away from liver exposure towards kidney exposure. In the case of MCLR, it has been shown that genetic knockout of Oatp1b2 completely protected the mice from MCLR-induced liver toxicity. In a recently completed preliminary study Dr. Clarke has shown that MCLR exposure in NASH dramatically increase kidney toxicity compared to healthy controls exposed to MCLR. These data lead to the central hypothesis that people with NASH are at increased risk of altered MCLR exposure and MCLR-induced renal toxicity due to changes in hepatic OATP transporters. To test this hypothesis and achieve the objectives of this project several methods will be employed. First, in vitro transporter kinetics experiments will be performed to identify transporters, beyond the Oatps, that contribute to MCLR disposition. Second, the effect of NASH on MCLR disposition will be determined by measuring toxicokinetics profiles after oral and intravenous MCLR administration in healthy and NASH rats. Third, acute and sub-chronic, sub-lethal toxicity studies will be performed to determine if MCLR exposure in NASH increases renal toxicity. Lastly, a PBTK model for MCLR exposure in humans will be constructed using the Simcyp software to help translate this work into exposure information for human populations. The research project outlined here is important to the development of Dr. Clarke's career but, more importantly, it is important to millions of people worldwide who have NASH and are exposed to MCLR.