The initial granting period focused on the role of the blood neutrophil (PMN) in liver injury. The toxic actions of Alpha-naphthylisothiocyanate (ANIT) provide a model for the study of chemically induced liver injury that involves periportal PMN accumulation, edema, cholestasis and necrosis of parenchymal cells and biliary epithelial cells. We have found that ANIT stimulates rat PMNs in vitro to release toxic oxygen metabolites and lysosomal enzymes that could injure tissue. Manipulations that modify ANIT toxicity in vivo also altered the capacity of ANIT to stimulate PMNs in vitro. Importantly, depletion of blood PMNs protected against ANIT hepatotoxicity in vivo, indicating that PMNs are involved in ANIT-induced liver injury. A focus of this proposal will be to determine mechanisms by which PMNs participate in liver injury. Depletion of hepatic glutathione (GSH) was also associated with protection from ANIT-induced liver injury, suggesting that GSH plays a causal or permissive role in ANIT toxicity. Thiolether leukotrienes (e.g., LTC4, LTD4) are formed by conjunction of GSH with LTA4 and are potent biological agents capable of causing tissue injury, whereas other LTs (e.g., LTB4) are potent chemotaxins for PMNs. Accordingly, we will test the unifying hypothesis that LTs mediate ANIT hepatotoxicity. Our recent finding that a selective inhibitor of LT biosynthesis ameliorates ANIT hepatotoxicity in vivo renders this hypothesis compelling. We will determine whether biliary, blood or liver LT concentrations are elevated after ANIT treatment, whether administration of LTs into the portal circulation of rats causes injury similar to that caused by ANIT, and whether blockade of LT synthesis or LT receptors attenuates hepatic insult from ANIT. Injury caused by LTs may relate to PMNs, since these cells respond to LTs and participate in their production. We will determine in vitro whether the presence of PMNs renders liver target cells (i.e., hepatocytes and biliary epithelial cells) more sensitive to injury from ANIT and whether nonparenchymal cells (esp. Kupffer cells) promote injury to liver target cells. Cell culture and coculture studies will uncover specific cellular sources of LTs in ANIT toxicity, elucidate important interactions among cells and determine actions of LTs on specific cell types that may be critical in ANIT-induced liver injury. Agents that destroy toxic oxygen metabolites or inhibit lysosomal proteases released by PMNs will be evaluated for their abilities to afford protection in vitro and in vivo. Animals (e.g., beige mice) having a deficiency in the ability of their PMNs to release lysosomal proteases will be evaluated to determine if they are less sensitive to ANIT hepatotoxicity. These studies will uncover mechanisms by which PMNs and LTs participate in ANIT hepatotoxicity. Importantly, they will elucidate novel mechanisms by which xenobiotic agents injure liver and will uncover interactions among liver target cells (i.e., parenchymal cells biliary epithelium) and effector cells (i.e., PMNs, Kupffer cells) that contribute to hepatotoxicity.
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