A TRANSGENIC MOUSE MODEL WITH A LUCIFERASE REPORTER FOR STUDYING IN VIVO TRANSCRIPTIONAL REGULATION OF THE HUMAN CYP 3 A 4 GENE

Cytochrome P450 3A4 (CYP3A4) plays an important role in drug metabolism, and the enzymatic activity of CYP3A4 contributes to many adverse drug-drug interactions. Here we describe a transgenic mouse model that is useful in monitoring the in vivo transcriptional regulation of the human CYP3A4 gene. A reporter construct consisting of 13 kilobases of the human CYP3A4 promoter controlling the firefly luciferase gene was used to generate a transgenic mouse line [FVB/N-Tg(CYP3A4-luc)Xen]. Reporter gene expression was assessed using an in vivo imaging system (IVIS) in anesthetized mice. Basal expression of the reporter was highest in liver and kidney, and moderate in the duodenum in male transgenic mice, whereas the basal luciferase activity was highest in the duodenum and lower in kidney and liver in females. Injections of pregnenolone, phenobarbital, rifampicin, nifedipine, dexamethasone, 5-pregnen-3 -ol-20-one-16 -carbonitrile (PCN), and clotrimazole resulted in a time-dependent induction of luciferase expression, primarily in liver, that peaked at 6 h post injection. The greatest induction was found with clotrimazole, dexamethasone, and PCN, whereas the lowest induction followed pregnenolone, phenobarbital, and rifampicin injection. In general, male mice responded to these drugs more strongly than did females. Our results suggest that the human CYP3A4 promoter functions in transgenic mice and that this in vivo model can be used to study transcriptional regulation of the CYP3A4 gene. Cytochrome P450 (P450) enzymes are a superfamily of hemecontaining proteins encoded by at least 57 P450 genes in humans (Nelson, 2002). P450s are involved in the metabolism of a variety of structurally diverse substances including endogenous chemicals and xenobiotics such as drugs, carcinogens, and environmental pollutants (Shimada et al., 1994; Li et al., 1995). The cytochrome P450 3A4 (CYP3A4) is the P450 enzyme responsible for oxidation of 50 to 60% of clinical drugs and is expressed primarily in liver and small intestine (Li et al., 1995). Regulation of enzymatic activity and gene expression of CYP3A4 contributes to the unexpected pharmacological effects of many drugs and may contribute to adverse drug-drug interactions (Maurel, 1996). Regulation of the CYP3A4 promoter has been extensively studied. A xenobiotic response element, an everted repeat separated by six nucleotides in the proximal promoter region, and a rifampicin distal enhancer module approximately 8 kilobases from the transcriptional start site have been identified (Hashimoto et al., 1993; Goodwin et al., 1999). CYP3A4 induction by xenobiotics occurs when a compound binds to the xenobiotic receptor PXR and the complex then dimerizes with the retinoid X receptor. The heterodimer subsequently binds to xenobiotic response elements of the CYP3A4 promoter to modulate transcription (Bertilsson et al., 1998; Blumberg et al., 1998). Transcriptional regulation of CYP3A genes in response to xenobiotics appears to be species-specific (Barwick et al., 1996) and is partly determined by divergence in ligand binding domains of PXR (Jones et al., 2000). Induction of P450 gene expression may be as important as inhibition of enzymatic activity in determining drug efficacy. Induction of P450 enzymes by a drug increases the capacity for drug metabolism, and this may attenuate the pharmacological effect of the primary drug or other subsequently administered drugs. Currently, human and animal primary hepatocytes are widely used to evaluate P450 transcriptional regulation (Schuetz et al., 1988; Kocarek et al., 1995), because most hepatoma cell lines lose the ability to express the major P450 genes. However, even hepatocytes tend to decrease expression of major P450 enzymes during culture (Schuetz et al., 1988; Silva et al., 1998), despite many improvements of culture conditions (Decad et al., 1977; Baffet et al., 1982). Furthermore, the response to xenobiotics of human primary hepatocytes from different donors varies dramatically, producing significant assay variability (Kostrubsky et al., 1999; LeCluyse et al., 2000). Recently, activation of the PXR using in vitro reporter assays was suggested as an approach to predict the potency of CYP3A4 inducers (Jones et al., 2000). These in vitro assays do not always agree with in vivo data. For example, dexamethasone, a potent inducer of the human CYP3A4 (Watkins et al., 1989), showed little activation of human PXR (hPXR) in in vitro receptor assays (Bertilsson et al., 1998; Blumberg et al., 1998; Lehmann et al., 1998). Similarly, rifampicin moderately induced mouse Cyp3a11 mRNA and 1 Abbreviations used are: P450, cytochrome P450; PXR, pregnane X receptor; h, human; m, murine; kb, kilobase(s); PCR, polymerase chain reaction; DMSO, dimethyl sulfoxide; PCN, 5-pregnen-3 -ol-20-one-16 -carbonitrile; PBS, phosphate-buffered saline; MANOVA, multivariate analysis of variance; ARE, androgen