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  • TC 14012 in br Materials and methods br Results br Discussio


    Materials and methods
    Discussion In rodent species, CYP2E1 and CYP2B have been implicated in CCl4 metabolism. In this study, we used expressed human CYP2E1 to show that this enzyme is a high-affinity catalyst responsible for low-dose CCl4 metabolism. Studies employing an inhibitory CYP2E1 antibody confirmed that the majority of metabolism at 17 μM CCl4 in human microsomes was dependent on CYP2E1. In order to relate this CCl4 concentration with actual exposure conditions, a physiologically based pharmacokinetic model was utilized [15], [16]. This analysis indicated that a maximal liver concentration of 17 μM CCl4 would be achieved on a daily basis in a 200 g rat following one single or repeated 8 h inhalation exposures to approximately 37 ppm CCl4 (Dr Karla Thrall, personal communication). This level of exposure is not hepatotoxic in rats, although it would be toxic in a more susceptible species, such as the mouse. Therefore, it seems likely that human metabolism of CCl4 at doses that are not hepatotoxic is primarily catalyzed by CYP2E1. The importance of CYP2E1 in CCl4 metabolism in humans provides insight into the molecular basis for the association between alcohol consumption and increased susceptibility to CCl4 toxicity observed in previous studies. In one such study, Tomenson et al. [17] examined individuals exposed to occupational levels of CCl4 and demonstrated that alcohol use was associated with alterations in several biochemical and hematological indices of hepatotoxicity. Manno et al. [18] also found that chronic ethanol consumption predisposed humans to CCl4 toxicity. Since ethanol is known to induce CYP2E1 [19], [20], it is likely that the increased response to CCl4 exposure in individuals who consume ethanol is associated with enhanced CCl4 metabolism resulting from elevated CYP2E1 levels. Therefore, other factors such as fasting or diabetes that elevate CYP2E1 levels may also influence individual susceptibility to CCl4 exposure. Clotrimazole decreased CCl4 metabolism by 23% in the human liver microsome sample that exhibited the least (36%) suppression by CYP2E1 TC 14012 in at 530 μM CCl4, suggesting that CYP3A may contribute to CCl4 metabolism in humans at high exposure levels. Since human CYP3A comprises approximately 30 and 70% of hepatic and intestinal total CYP450, respectively, this CYP450 could be important in CCl4 metabolism and systemic uptake following high oral doses. Although CYP2B has been implicated in CCl4 metabolism in rodents [4], [5], studies using expressed human CYP2B6 and inhibitory antibodies against CYP2B6 indicated that this form was unlikely to be important in CCl4 metabolism in humans. It is interesting that the evidence which suggests that rodent CYP2B forms may be involved in CCl4 metabolism and toxicity is primarily based on increased toxicity in animals pre-exposed to phenobarbital, an efficacious CYP2B inducer [5], [6]. However, phenobarbital also induces CYP3A [21], [22], so it is possible that the increased toxicity in phenobarbital-pretreated animals was dependent on increased CYP3A rather than CYP2B expression. Hepatic microsomal metabolic parameters are commonly used to understand and relate interspecies differences in susceptibility to a particular toxicant. In this regard, the CCl4 metabolic rate constants, Km of 56.8 μM and Vmax of 2.26 nmol/min/mg protein, for human liver microsomes varied less than 2-fold from values we measured (unpublished observations) in hepatic microsomes from untreated rats (Km of 59.1 μM and Vmax of 3.10 nmol/min/mg protein), mice (29.3 μM and 2.86 nmol/min/mg protein) and hamsters (30.2 μM and 4.1 nmol/min/mg protein). Therefore, these rodent species appear to make reasonable models for studying CCl4 metabolism and toxicity. In conclusion, these data suggest that CYP2E1 is the major human enzyme responsible for CCl4 bioactivation at lower, environmentally relevant levels. At higher CCl4 levels, CYP3A and possibly other CYP450 forms may contribute to CCl4 metabolism. Individual differences in CYP2E1 expression related to environmental, genetic or pathophysiological factors can therefore be expected to alter CCl4 metabolism and susceptibility to low-dose exposure to this agent.