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  • In principle in vitro techniques for assessing enzyme induct


    In principle, in vitro techniques for assessing enzyme induction might include the use of cultured hepatocytes, precision-cut liver slices, immortalized Nalidixic acid lines (such as those derived from hepatomas) and reporter gene constructs (where appropriate cells are transfected with recombinant DNA encoding a conveniently measured enzyme, such as luciferase, under the control of the regulatory element [cis-acting factor] that normally controls the induction of a selected CYP450 gene). In this context, appropriate cells are those that express the appropriate receptors and transcription factors [trans-acting factors] that mediate cytochrome P450 induction (e.g., PXR). Each of these in vitro systems has its advantages and disadvantages. Over the last decade, there has been a growing consensus that cultures of primary human hepatocytes are the ‘gold standard’ for determining the enzyme induction potential of NCEs in vitro. More recently, however, cell lines transiently transfected with expression vectors containing sequences for various nuclear receptors and reporter constructs driven by promoter sequences from specific CYP450 enzymes are making a rapid surge into the forefront of screening technology. The impact of these systems on the future of drug discovery and development has yet to be realized. 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 cells 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 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 Nalidixic acid 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.