The long term goal of this grant is to understand the mechanism of toxicity of both acute hepatic necrosis and chronic sub-clinical impairment of liver function that result from anesthetic metabolism in humans. We will carry out experiments in two complementary experimental systems: Monolayers of hepatocytes in primary culture will allow study of the effects of metabolism on the integrated systems of the cell as well as attack of hepatocytes by macrophages activated by metabolically-produced chemo attractants or antigens. A synthetic model of a liver cell formed by reconstituting human cytochrome p-450 and NADPH cytochrome P-450 reductase into phospholipid vesicles will allow study of individual steps of anesthetic metabolism, formation of chemo-attractant leukotrienes, and antigenic metabolites on the cell surface. The following experiments with halothane will also contribute to the understanding of how other halogenated hydrocarbons cause liver disease and cancer. 1) We wil continue our studies of the toxicity of halothane metabolism in monolayers of hepatocytes under conditions that mimic those that exacerbate halothane toxicity in vivo, especially the effect of hypoxia. 2) We will use reconstituted human cytochromes P-450 to continue our studies of production of arachidonic acid hydroperoxides during metabolism of halothane and their conversion by specific isozymes of human cytochrome P-450 to the potent chemoattractant leukotriene B-4 (LTB-4) 3) We will measure production and release of LTB-4 from monolayers of hepatocytes in response to injury by halothane metabolism as a function of oxygen concentration. 4) We will isolate both leukocytes and the resident liver macrophages, Kupffer cells, and measure their lysis of hepatocytes as a consequence of activation by leukotrienes produced during halothane metabolism. 5) We will use reconstituted human cytochrome P-450 to study production of N-trifluoroacetyl-phosphatidylethanolamide, a known metabolite of halothane that we suggest may act as an antigen if exposed on the hepatocyte plasma membrane. We will prepare antibodies to this antigen. 6) We will allow monolayers of hepatocytes to metabolize halothane to produce the proposed antigen on the plasma membrane surface, add the specific antibodies, and measure immune complex mediated lysis of hepatocytes by leukocytes or Kupffer cells.
| Costa, A K; Baker, M A; Brown, J M et al. (1989) In vitro hepatotoxicity of SR 4233 (3-amino-1,2,4-benzotriazine-1,4-dioxide), a hypoxic cytotoxin and potential antitumor agent. Cancer Res 49:925-9 |
