Short Communication DIFFERENTIAL REGULATION OF THE HUMAN CYP3A4 PROMOTER IN TRANSGENIC MICE AND RATS

Previously we described a transgenic mouse model [FVB/NTg(CYP3A4-luc)Xen] using a reporter construct consisting of 13 kilobases of the human CYP3A4 promoter driving the firefly luciferase gene in the inbred FVB/N mouse strain. Here we report regulation of the same CYP3A4-luc reporter gene in a transgenic outbred mouse strain (CD-1) and in a transgenic rat (SpragueDawley). Basal reporter expression and responses to several xenobiotics in the transgenic CD-1 mice [CD-1/Crl-Tg(CYP3A4luc)Xen] were similar to those in the transgenic FVB/N mice. In both mouse backgrounds, the basal levels of the reporter were higher in male compared with female, and in the FVB/N strain there was greater induction for all drugs in male compared with female; however, in the CD-1 background this gender difference for induction was not obvious. In contrast with transgenic mice, transgenic rats [SD/Tac-Tg(CYP3A4-luc)Xen] expressed the luciferase reporter at higher basal levels in female compared with male rats. Responses to some compounds were much greater in rats than in mice, and the kinetics of induction was different with peak induction occurring later in the rat compared with the mouse. Our results suggest that the human CYP3A4 promoter is regulated differently in transgenic mice and rats in some aspects. Modulation of cytochrome P450 (P450) enzyme activities and induction of P450 gene expression in humans lead to changes in metabolism of therapeutic drugs, which can potentially result in a variety of adverse effects (Lin and Lu, 1998). Inhibition or activation of P450 enzymatic activity is usually a rapid process, whereas gene induction, which increases the total amount of enzyme and metabolic capacity, is relatively slower. Cytochrome P450 3A4 (CYP3A4) is one of the most important enzymes that metabolizes drugs. This gene is primarily expressed in liver and small intestine and is up-regulated by structurally diverse chemical compounds, thus increasing the capacity for metabolism of xenobiotics (Li et al., 1995). Increased expression of CYP3A4 is one factor that may produce adverse drugdrug interactions (Maurel, 1996). Current models for evaluating the regulation of P450 genes by xenobiotics include in vitro assays using primary hepatocytes, hepatoma cell lines, human pregnane X receptor (hPXR) activation of reporters, and in vivo animal models. An in vivo reporter assay in transiently transduced mouse liver for induction of CYP3A4-luc has also been reported (Schuetz et al., 2002; Zhang et al., 2003a). More recently, we described an in vivo transgenic CYP3A4luc reporter in an inbred FVB/N mouse strain, allowing us to noninvasively monitor CYP3A4 induction kinetics (Zhang et al., 2003b). The conventional rodent models used in toxicology and pharmacology are outbred rat strains that are genetically heterogeneous to capture the full genetic complement influencing the response. These outbred rat strains are useful for firstand second-level screening in biomedical research (Gill, 1980). Some investigators have suggested that it would be useful to complete toxicology testing in several different inbred strains since the results might be less variable due to less genetic heterogeneity (Festing, 1986). Since the rat is the most commonly used rodent model in toxicology and pharmacology (Kacew, 2001), we have developed a CYP3A4-luc transgenic reporter in the Sprague-Dawley outbred rat strain. In addition, to compare the response of the CYP3A4-luc transgene in both an inbred and an outbred mouse strain, we have further created a CYP3A4-luc mouse in the outbred CD-1 mouse strain to complement the existing model in the FVB/N inbred strain that we have characterized (Zhang et al., 2003b). Having the same reporter in outbred mouse and outbred rat strains also allows us to compare species differences in the regulation of the human CYP3A4 promoter. Our results demonstrated that the human CYP3A4 promoter was regulated differently in mice and rats in basal and induced level of expression and kinetics of induction; only slight differences between outbred and inbred mouse strains were observed. Materials and Methods Generation of the CYP3A4-luc Transgenic Mouse and Rat Lines. The CYP3A4-luc reporter construct previously described by Zhang et al. (2003a) was used to make transgenic mice in outbred (CD-1) mice and SpragueDawley rats. This 15-kb DNA fragment containing a 13-kb CYP3A4 promoter, a chimeric intron, and the firefly luciferase cDNA was microinjected into single-cell stage Crl:CD-1 (ICR)BR (Charles River Laboratories, Inc., Wilmington, MA) mouse embryos and Tac:N(SD) Sprague-Dawley rat (Taconic Farms, Germantown, NY) embryos (Xenogen Biosciences, Cranbury, NJ). The injected embryos were implanted into pseudopregnant mice or rats. Transgenic founders and progeny were genotyped by PCR (Zhang et al., 2003b). Primary screening of transgenic lines was completed as previously described (Zhang et al., 2003b). Twelve independent CYP3A4-luc CD-1 mouse lines [CD-1/CrlTg(CYP3A4-luc)Xen] and 17 independent CYP3A4-luc SD rat lines [SD/TacTg(CYP3A4-luc)Xen] were screened for their response to several known CYP3A4 inducers. One line from each species was selected for further char1 Abbreviations used are: P450, cytochrome P450; PXR, pregnane X receptor; SD, Sprague-Dawley; PCN, 5-pregnen-3 -ol-20-one-16 -carbonitrile; DMSO, dimethyl sulfoxide; kb, kilobase. Address correspondence to: Weisheng Zhang, Xenogen Corporation, 860 Atlantic Avenue, Alameda, CA 94501. E-mail: [email protected] 0090-9556/04/3202-163–167$20.00 DRUG METABOLISM AND DISPOSITION Vol. 32, No. 2 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 1189/1116828 DMD 32:163–167, 2004 Printed in U.S.A. 163 at A PE T Jornals on July 6, 2017 dm d.aspurnals.org D ow nladed from