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  • Inasmuch as hepatocytes are initially refractory to the enzy


    Inasmuch as hepatocytes are initially refractory to the enzyme inducing effects of CYP450 inducers, cultures must be maintained for at least 36–48 h before treatment with test articles and/or known CYP450 inducers, at which time the hepatocytes are expressing liver-specific genes and a near-normal morphology, i.e., the doxercalciferol are bipolar with newly formed cell–cell communications and bile canaliculi (see Section 2) (LeCluyse et al., 2000a, LeCluyse et al., 2001, Maurel, 1996a). It is important to appreciate that hepatocellular morphology and function are restored, not maintained, when hepatocytes are isolated and placed in culture. Hepatocytes can be cultured for up to 2 weeks with little or no loss of responsiveness to CYP450 enzyme inducers (Section 2.1; Maurel, 1996a). If enzyme activities are measured, the induction regimen normally involves a 2- to 5-day exposure to various concentrations of the test articles and/or known CYP450 inducers dissolved in buffer or organic solvent, such as dimethylsulfoxide. If mRNA is determined by quantitative PCR, then much shorter exposure periods are required (<6 h for potent inducers of CYP3A4) (Coon et al., 1999, Silva, 2000). Like all in vitro techniques, the use of cultured hepatocytes for screening CYP450 enzyme inducers is prone to artifacts. In terms of evaluating drugs and NCEs for their potential to induce cytochrome P450, false negatives might arise for any one of the following reasons: (1) The induction of cytochrome P450 by a chemical is dependent on the formation of a metabolite that is not formed in vitro. This is probably the case with musk xylene, which must undergo nitro-reduction by intestinal bacteria in order to induce CYP2B enzymes in rats in vivo (Lehmann doxercalciferol et al., 1998). (2) The induction of cytochrome P450 is not the result of a direct effect of the chemical on the liver. For example, the ability of streptozotocin to induce CYP2E1 is secondary to the ability of this drug to destroy the insulin-secreting cells of the pancreas and thereby induce a diabetic state, during which various ketone bodies induce the synthesis of CYP2E1. (3) The induction of cytochrome P450 is not primarily dependent on transcriptional activation but involves increased translational efficiency of pre-existing mRNA and/or stabilization of the pre-existing enzyme, as is often the case with CYP2E1. (4) The concentration of inducer is too low or too high, especially as it relates to clinical or toxicological concentrations. Conversely, false positives might arise because: (1) The chemical is rapidly metabolized or otherwise degraded in vivo but slowly metabolized/degraded in vitro, and (2) the concentration of inducer is too high, especially as it related to clinical or toxicological concentrations. A factor that complicates the use of human liver cells is one of variability, which arises in part because many of the human liver samples available for research show various stages of a variety of disease states (steatosis (fatty liver), cirrhosis, carcinogenesis in adjacent tissue), although care is taken to avoid liver infected with hepatitis, HIV or other potentially infectious agents. However, even among human livers that would be considered normal, there is tremendous inter-individual variability in the expression of CYP450 enzymes. Some of this variation is genetically based, inasmuch as some of the CYP450 enzymes, such as CYP2D6 and CYP2C19, are highly polymorphic (Parkinson, 1996a, Parkinson, 1996b). For other CYP450 enzymes, such as CYP1A2 and CYP3A4, much of the inter-subject variability likely arises from individual differences in exposure to inducers, such as cigarette smoke, diet and medications, or suppressors, such as active systemic infections (Parkinson, 1996a, Parkinson, 1996b, Maurel, 1996b). Despite the variability, species differences in CYP450 enzyme regulation suggest that it is important to utilize human hepatocytes to evaluate the ability of drugs and NCEs to induce cytochrome P450. For example, omeprazole and rifampin, which are prototypical inducers of human CYP1A2 and CYP3A4, respectively, cause little or no induction of the corresponding CYP450 enzymes in rats or mice (Diaz et al., 1990, Pichard et al., 1990). In addition, the variability observed in primary cultures of human hepatocytes better reflects the variability in drug-induced enzyme regulation observed in the general population (Flockhart, D., personal communication).