A pharmacokinetic model of anaerobic in vitro carbon tetrachloride metabolism

Abstract

Carbon tetrachloride (CCl 4) is a potent hepatotoxic agent whose toxicity is mediated through cytochome P450-dependent metabolism. Results from anaerobic in vitro experiments with hepatic microsomes isolated from male F-344 rats indicate that chlorofom (CHCl 3) formation from CCl 4 is nonlinear with dose. Dose is traditionally expressed as the amount of CCl 4 added to the vial. In this study, a pharmacokinetic model has been developed to calculate the concentration of CCl 4 in the microsomal suspension. Hepatic microsomes prepared from fed and fasted animals were incubated with CCl 4 under anaerobic conditions and formation of CHCl 3 over a 5-min incubation period was monitored by headspace gas chromatography. Dose-response curves, based on total amount of CCl 4 added to the microsomes, revealed a nonlinear, biphasic appearance of CHCl 3, with fasting slightly increasing CHCl 3 production in microsomes prepared from fasted rats. Microsomes were also pretreated with the CYP2E1 inhibitor, diallyl sulfone (DAS), before addition of CCl 4. In uninhibited microsomes, there appeared to be a high-affinity saturable phase of metabolism occurring at lower concentrations followed by a linear phase at higher CCl 4 concentrations. Following DAS pretreatment, the saturable portion of the dose-response curve was inhibited more than the linear phase with the biphasic CHCl 3 production becoming more linear. DAS inhibition eliminated the effect of fasting on CHCl 3 formation. The best fit kinetic constants for the saturable phase resulted in an estimate of V max of 0.017 mg/h/mg protein ( V maxc = 7.61 mg/h/kg) and K rmm of 2.3 mg/l (15 μM). The linear phase rate constant ( k f) was determined to be 0.046 h −1 ( k fc = 0.03 h −1). In conclusion, a pharmacokinetic model has been developed for anaerobic in vitro metabolism of CCl 4 to CHCl 3 that estimates metabolic rates based on CHCl 3 formation and actual CCl 4 concentration in the microsomal suspension.