The overall objective of this research proposal is to enhance the safety of currently used volatile anesthetic agents, and certain agents presently under development, by diminishing their toxicity. This will be accomplished by elucidating the enzymatic mechanisms of anesthetic bioactivation and clinical pharmacology of anesthetic toxification. Halothane, the most widely used volatile anesthetic agent in the world, is known to cause a rare but fatal fulminant hepatic necrosis. Moreover, it commonly causes altered postoperative mixed function oxidase activity. Fluorinated ether anesthetics such as methoxyflurane, enflurane and sevoflurane are hepatically defluorinated, releasing free fluoride ion which is nephrotoxic. The toxicity of all these anesthetics is intrinsically linked to their metabolism by hepatic cytochrome P450. For example, the immunologic cascade culminating in the fulminant hepatic necrosis known as halothane hepatitis is thought to be initiated by cytochrome P450-mediated halothane oxidation to products which bind covalently to liver proteins, rendering them antigenic. Hepatic cytochrome P450 also activates halothane to free radicals which initiate lipid peroxidation, and is responsible for releasing free fluoride ion from fluorinated ethers. The central hypothesis to be tested in this proposal is that specific isozymes of human cytochrome P450 are responsible for anesthetic toxification, and that selective inactivation of these isozymes can be accomplished clinically to diminish anesthetic metabolism and toxicity. Human liver microsomal tissue will be used to establish an in vitro model for human volatile anesthetic metabolism, which will then be used to: 1) Identify the predominant human hepatic P450 isozyme(s) catalyzing oxidative and reductive halothane metabolism and formation of halothane-altered liver neoantigens, 2) Identify the principal human P450 isozyme catalyzing the oxidation of fluorinated ether anesthetics to fluoride ion and toxic acetylating species, and 3) Evaluate the activity of these specific isozymes towards individual stereoisomers of the chiral anesthetics, in order to assess the potential for therapeutic advantage of individual enantiomers. Once defined, selective inhibitors of these specific P450 isozymes will be used as in vivo clinical probes, to render them catalytically inactive in patients undergoing anesthesia and surgery. If the tested hypothesis is correct, then volatile anesthetic metabolism will be diminished and the in vivo role of specific P450 isozymes confirmed. Elucidation of the P450 isozymes catalyzing anesthetic toxification, coupled with known influences of disease and induction states on P450 activity, may be used to identify patient populations and individuals potentially at risk for anesthetic toxicity. Clinical strategies may then be devised for avoiding specific anesthetic agents or administering protective adjuvants to diminish anesthetic toxicity.
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