This research project is designed to provide new knowledge concerning the nature of the intermediate pathological processes which occur subsequent to the initial metabolism of hepatotoxic xenobiotics such as carbon tetrachloride (CC1-4). Recent work in this laboratory has shown that exposure of isolated hepatocytes to CC1-4 activates phospholipase A2 (PLA2). This work has provided the necessary methodology to pursue studies to determine whether PLA2 activation occurs during the course of action of a number of primary hepatotoxins. Most of the work will be carried out with suspensions of hepatocytes in which PLA2 activity is determined by measuring either the release of 3H-arachidonic acid from cellular phospholipids prelabeled with 3H-arachidonate, or by measuring the formation of 14C-lysophospholipids from cellular phospholipids prelabeled with 14C-ethanolamine. Toxic agents to be studied in addition to CC14 will be those known or suspected to act through a lipoperoxidative mechanism and/or through disturbance of hepatocellular calcium homeostasis, such as bromotrichloromethane, 1,2-dibromoethane, bromobenzene, and vinylidene chloride. We also intend to determine the extent of hepatic PLA2 activation in vivo after administration of various hepatotoxins including CC1-4. Because of the connection between elevated levels of cytosolic free calcium and PLA2 activation, a continuation of ongoing studies of toxigenic disturbances of hepatocyte calcium homeostasis by the calcium probe Fura-2 is planned. The hepatotoxic action of CC1-4 is potentiated by chlordecone (Kepone) and isopropanol. An examination of whether this potentiation involves potentiation of PLA2 activity is projected. The prostaglandins, 16,16-dimethyl PGE2 and PGI2, and other agents inhibit CC1-4-induced liver injury even when given several hours after CC1-4. Projected studies will reveal if the cytoprotective action involved an inhibition of PLA2. Further characterization of the CC1-4-induced activation of PLA2 will include a determination of the subcellular location of the activated phospholipase and a temporal analysis of the relationship between phospholipase activation and indices of cell injury. To understand the fundamental biochemical mechanisms involved in the activation of PLA2 as they relate to toxic cell injury, a search for conditions in vitro which promote phospholipase activity is projected. In particular, a study of the effects of lipid peroxidation and covalent binding of CC1-4 cleavage products on PLA2 activity is planned.
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