Covalent Binding of Oxidative Biotransformation Reactive Intermediates to Protein Influences Halothane-Associated Hepatotoxicity in Guinea Pigs
Halothane (CF3CBrClH; H) biotransformation by cyt P-450 produces reactive intermediates along both oxidative (acyl chloride) and reductive (free radical) pathways that ultimately generate the metabolites trifluoroacetic acid and F−, respectively. Inhibiting oxidative metabolism with deuterated halothane (d-H) reduces resultant injury in our guinea pig model of acute H hepatotoxicity. To elucidate whether covalent binding of rective intermediates to proteins (oxidative pathway) or lipids (reductive pathway) is a mechanism of necrosis, male outbred Hartley guinea pigs (600–725 g), N=8, were exposed to either 1% (v/v) H or d-H at either 40% or 10% O2 for 4 hr. One-half of the animals were killed immediately after exposure for binding studies; the remainder at 96 hr post for evaluation of hepatotoxicity. Covalent binding of halothane intermediates to liver protein or lipid was determined by measuring the fluoride content of the bound moieties. The use of d-H and/or 10% O2 during exposure led to 63–88% reductions (p < 0.01) in plasma trifluoroacetic acid concentrations (H-40% O2 = 546; 73 mM, N=8) which were accompanied by 33–60% decreases (p <0.01) in binding to liver proteins (H-40% O2 = 1.36; 0.26 nmoles bound F−/mg protein, N=4), 78–84% decreases (p < 0.05) in 48 hr plasma ALT levels (H-40% O2 = 308; 219, control = 23 + 3, N=4) and a total amelioration of centilobular necrosis. Deuteration of H did not alter plasma F−concentrations or binding to lipid (H-40% O2 = 6.7; 1.9 M F−, N=8 and 5.6;1.8 nmoles bound F−/mg lipid phosphate, N=4). Exposure to H or d-H under 10% O2 increased (p < 0.05) plasma F− by almost 2 × and covalent binding to lipid by 5–6 × (p < 0.01) but ameliorated the centrilobular lesion. Covalent binding of oxidative pathway generated H reactive intermediates to protein is thus indicated as the mechanism of H-necrosis in guinea pigs.
KeywordsCovalent Binding Liver Protein Reductive Pathway Lipid Phosphate Centrilobular Necrosis
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