CONTROL OF DRUG AND ETHANOL METABOLISM

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DESCRIPTION: (Adapted from the Investigator's Abstract) During the recent funding period, we have collected evidence supporting the hypothesis that early alcohol-induced liver injury involves activation of Kupffer cells by gut-derived endotoxin, oxidants and the toxic cytokine TNFa. Moreover, using the enteral feeding model of Tsukamoto and French, the investigators discovered that female rats, like humans, exhibit more injury than males and have higher blood endotoxin levels. This finding led to our discovery that estrogen increases Kupffer cell expression of the endotoxin receptor (CD14) and production of TNF(. Both parameters were decreased when the gut was sterilized with non-absorbable antibiotics. Moreover the investigators showed that increased sensitivity of Kupffer cells to endotoxin caused by acute ethanol is associated with an increase in CD14 expression. To date, reports on mechanisms of early alcohol-induced liver disease relied on work with inhibitors, which may not be specific. Accordingly, to fill critical gaps in our knowledge, the investigators will use knockout technology where specific receptors or proteins are genetically deleted to obtain clear information on causal events in mechanisms of early alcohol-induced liver injury. The unifying hypothesis we will test here using knockout technology is that endotoxin activates Kupffer cells to produce oxidants which increase TNFa production leading to early alcohol-induced liver injury. The investigators also proposed that gender differences could be explained by this hypothesis. Initially, in Aim 1 the PI will optimize, characterize and validate a mouse model of enteral alcohol delivery by determining pathology in wild-type mice, which are background strains for selected knockouts. In aim 2, the PI will determine if the primary source of oxidants in early alcohol-induced liver disease is the ethanol-inducible cytochrome P450 (CYP2E1) which the investigators expect will come predominantly from parenchymal cells or NADPH oxidase from Kupffer cells. The p47phox knockout mouse, which is NADPH oxidase deficient, will be compared wit h the CYP2E1 knockout to test the hypothesis that radical generation by Kupffer cells is a primary event in early alcohol-induced liver injury, using the enteral model characterized in Aim 1. Using the endotoxin receptor (CD14) knockout and the TNFa receptor 1 (TNFR1) knockout in enteral studies with alcohol, the investigators will test the hypothesis that endotoxin and TNFa are causally involved in early alcohol-induced liver injury in Aim 3. In Aim 4, estrogen receptor knockout mice will be used to determine if gender differences in liver injury due to alcohol can be explained by the involvement of estrogens. This work is timely and exciting since it will provide a new enteral feeding model in the mouse that will allow the investigators and others to investigate the roles of specific proteins and enzymes in alcohol-induced liver disease using the power of knockout technology. This will uniquely position us to provide unequivocal new information and fill critical gaps in our knowledge on mechanisms of early alcohol-induced liver injury.