Zinc inhibition of endotoxemia in alcoholic liver injury Abstract Alcoholic liver disease (ALD) is an important health problem, but an FDA-approved effective therapy is not available. A better understanding of the mechanisms involved in the pathogenesis of ALD will certainly pave the way to develop effective therapies for treating this disease. Several hypotheses regarding the mechanisms by which alcohol induces liver injury have been postulated, including ethanol metabolism-generated oxidative stress, endotoxemia, and proinflammatory cytokine production. Each of the three factors has been shown to contribute significantly to ALD. However, the critical trigger for these detrimental events has not been defined. Although previous studies have largely focused on the direct effects of ethanol on the liver, clinical evidence indicates that excessive ethanol consumption is necessary but not sufficient to induce liver injury. Gut-derived endotoxin has been suggested as one of the most important cofactors in development of ALD. Endotoxin is known to trigger proinflammatory cytokine production in the liver, leading to inflammation, oxidative stress and eventually cell death. While endotoxemia is well documented in patients with ALD, the mechanisms involved in the development of endotoxemia have not been fully understood. Given the fact that only alcoholics with gut leakiness develop ALD, intestinal barrier dysfunction is likely the cause of endotoxemia. Therefore, suppression of endotoxemia by sealing the leaky gut could be very an attractive approach for treating ALD. Our previous studies have shown that inhibition of endotoxemia is associated with zinc prevention of acute ethanol-induced liver injury. Importantly, our preliminary data identify a novel potential mechanism by which ethanol and zinc regulate the intestinal barrier. We found that zinc deficiency and dysfunction of zinc finger transcription factors disrupt the barrier function of Caco-2 cells through depressing junction proteins. In this context, we hypothesize that zinc homeostasis plays a critical role in regulation of intestinal barrier function. We will carry out the following specific aims and research approaches in this project. Aim 1 will determine the role of zinc deficiency in the pathogenesis of ethanol-induced intestinal barrier dysfunction and endotoxemia. A chronic ethanol exposure rat model will be utilized to induce alcoholic endotoxemia. First, we will determine disturbance of intestinal zinc homeostasis in association with ethanol-induced intestinal barrier dysfunction and endotoxemia. Second, the impact of zinc deficiency on intestinal barrier function and the possible synergistic action with ethanol will be determined by using a dietary zinc deficiency model. We expect the results of Aim 1 to demonstrate that zinc deprivation is a causal factor in the pathogenesis of ethanol-induced intestinal barrier disruption. Aim 2 will investigate the molecular mechanisms by which zinc deficiency interferes with intestinal barrier function. Caco-2 cell culture model will be utilized to determine whether ethanol-induced zinc deficiency lead to dysfunction of zinc finger transcription factors, and whether zinc finger transcription factors critically regulate epithelial barrier structure and function. We will first define the links between ethanol-induced ROS generation and zinc mobilization and depression of zinc finger transcription factors. We will then define the role of zinc finger transcription factors in regulation of tight junction proteins and barrier function by gene silencing and expression. We expect Aim 2 to demonstrate that zinc mobilization from zinc finger transcription factors is a critical molecular event in ethanol-induced epithelial barrier disruption. Aim 3 will evaluate the beneficial effect of dietary zinc supplementation on ethanol-induced gut leakiness and endotoxemia. A chronic ethanol exposure rat model will be utilized to evaluate the beneficial effects of dietary zinc supplementation on ethanol-induced intestinal leakiness and the consequent endotoxemia and hepatitis. We expect that dietary zinc supplementation through preserving zinc finger transcription factors will seal the leaky gut and attenuate endotoxemia and the consequent hepatitis. These studies will provide novel insights into the role of zinc in modulation of ethanol-induced gut leakiness and the consequent endotoxemia and hepatitis. The results would have high potential leading to a simplistic approach to prevention/treatment of ALD.