Kaminsky, LS; Fraser, JM; Seaman, M; Dunbar, D
2,2,2-Trifluoroethanol (TFE) is a metabolite of anesthetic agents and chlorofluorocarbon alternatives. Its toxicity in rats is a consequence of its metabolism to 2,2,2-trifluoroacetaldehyde (TFAld) and then to trifluoroacetic acid (TFAA). The enzymes involved in the toxic metabolic pathway have been investigated in this study. For the reaction of TFE to TFAld, the major hepatic metabolism associated with toxicity (as assessed by pyrazole-inhibitability) was NADPH dependent and occurred in the microsomes, whereas for TFAld conversion to TFAA, NADPH-dependent microsomal metabolism was significant, but mitochondrial and cytosolic metabolism in the presence of NADPH were also major contributors. NADPH-dependent hepatic microsomal metabolism of TFE to TFAld and TFAld to TFAA was inhibited by carbon monoxide, 2-allyl-2-isopropylacetamide, SKF-525A, metyrapone, imidazole, and pyrazole, and both reactions were oxygen dependent. The metabolism of TFE to TFAld was inhibited by diethyldithiocarbamate, a specific inhibitor of cytochrome P450E1, and by a monoclonal antibody to P4502E1, whereas the metabolism of TFAld was inhibited by neither. Ethanol pretreatment of rats enhanced the Vmax for hepatic microsomal metabolism of TFE to TFAld from 5.3 to 9.7 nmol/mg protein/min, while for TFAld to TFAA the Vmax was increased from 4.3 to 6.5 and the Km was unaffected for both reactions. Phenobarbital pretreatment of the rats did not affect any of these kinetic parameters. Coadministration of ethanol and a lethal dose of TFE very markedly decreased the lethality. Both the lethality (LD50 0.21 to 0.44 g/kg) and the metabolic kinetic parameters [(Vmax/Km)H(Vmax/Km)D = 4.2] were affected markedly when deuterated TFE replaced TFE. In contrast, deuteration of TFAld did not affect its lethality or rates of metabolism, but did affect its Km. Taken together these results indicate that P4502E1 catalyzed toxicity-associated hepatic metabolism of TFE to TFAld, while TFAld metabolism was catalyzed by a P450 which was not P4502E1. The hepatic metabolism of TFAld was not associated with its toxicity, which has been determined previously to be associated with its intestinal metabolism.
