Elsevier

Toxicology in Vitro

Volume 19, Issue 2, March 2005, Pages 275-287
Toxicology in Vitro

Cell-based high-throughput bioassays to assess induction and inhibition of CYP1A enzymes

https://doi.org/10.1016/j.tiv.2004.10.003Get rights and content

Abstract

CYP1A is a subfamily of cytochrome P450 enzymes involved in the metabolism of numerous therapeutic drugs and in the bioactivation of procarcinogens to mutagens. Because of their diverse metabolic capacities, differences in expression of CYP1A enzymes may profoundly influence drug–drug interactions and drug or carcinogen activation and detoxification. Here, we demonstrate that cell-based bioassays are capable of identifying xenobiotics that either alter aryl hydrocarbon receptor (AhR)-mediated CYP1A levels or produce inhibition of enzyme activity. To assess induction, a stable cell line harboring a luciferase reporter driven by multiple dioxin response elements (DREs) was developed. Using this cell line, AhR agonists and antagonists were identified among drugs, dietary agents, and environmental compounds. Of the chemicals examined, the therapeutic agent omeprazole induced reporter gene activity 12.5 ± 0.41 fold above control, whereas the phytochemical, chrysin and environmental pollutant, benzanthracene enhanced luciferase activity 3.3 ± 0.03 and 28.7 ± 1.7 fold above control, respectively. Several natural products, polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) prevented TCDD-mediated increases in luciferase expression. For example, the botanical kava inhibited TCDD-mediated induction by 88%. Northern blot analyses of CYP1A1 in HepG2 cells treated with similar agents validated results generated in the stable cell line. The stable cells were further used to identify inhibitors of CYP1A-mediated metabolism. Resveratrol and furafylline exhibited dose-dependent decreases in CYP1A1 and CYP1A2 enzyme activities with IC50 values of 1.89 and 0.79 μM, respectively. In summary, chemicals that possess the ability to alter CYP1A expression or inhibit CYP1A enzyme activities can be rapidly identified with the cell-based bioassays described here.

Introduction

The dioxin or aryl hydrocarbon receptor (AhR) functions as a ligand activated transcription factor that belongs to the basic helix-loop-helix family of DNA binding proteins (Burbach et al., 1992, Safe, 2001). The receptor is a ubiquitous intra-cellular protein that regulates the expression of a battery of genes including drug-metabolizing enzymes in the CYP1A subfamily (Postlind et al., 1993). The molecular mechanisms involved in CYP1A induction have been extensively studied. The transcriptional activation of CYP1A includes a sequence of events that begins with binding of an inducer to the AhR followed by ligand-dependent heterodimerization between the AhR and an AhR nuclear translocator. The next step is the interaction of the heterodimer with a consensus DNA sequence known as the dioxin response element (DRE). This interaction is followed by transmission of the induction signal from the enhancer to the CYP1A promoter, alteration of chromatin structure, and eventually enhanced expression of CYP1A proteins (Whitlock, 1999).

In addition to enhanced expression of P450 enzymes, dioxin and structurally related compounds elicit a broad range of biological effects through activation of AhR. Many of these events are toxic and in the case of TCDD, include lethality, tumor promotion, teratogenicity, immune suppression, and alteration in endocrine homeostasis (DeVito et al., 1994). In addition to TCDD, other environmental contaminants, including the polycyclic aromatic hydrocarbons (PAHs), their halogenated derivatives and polychlorinated biphenyls (PCBs) increase expression of the CYP1 family (Jones and Anderson, 1999, Safe, 1989). Several non-PAH compounds are also inducers of CYP1As including indole-3,2-β-carbazole, an acid derivative of a chemical found in cruciferous vegetables (Bradfield and Bjeldanes, 1987), the therapeutic, omeprazole (Dzeletovic et al., 1997, Shih et al., 1999, Quattrochi and Tukey, 1993), curcumin, a polyphenolic compound found in the spice turmeric (Ciolino et al., 1998a), trytophan metabolites (Heath-Pagliuso et al., 1998) and bilirubin (Phelan et al., 1998).

In humans, the CYP1A subfamily is comprised of two members, CYP1A1 and CYP1A2. Accordingly, constitutive CYP1A1 expression appears to be low and largely extrahepatic, whereas CYP1A2 is primarily expressed in the human liver. Metabolism of certain chemicals by CYP1As, such as the PAHs results in activation and the generation of genotoxic metabolites that bind to DNA (Pelkonen and Nebert, 1982). Enhanced expression of CYP1A enzymes has been associated with carcinogenesis resulting from substrate bioactivation (Kuljukka et al., 1996). In addition to the PAHs, CYP1As metabolize a variety of therapeutic agents including caffeine, clozapine, imipramine and theophylline (Brosen, 1995). Exposure to an inducer of these enzymes prior to administration of a drug substrate can result in a lack of therapeutic efficacy. For example, enhanced expression of CYP1A2 resulting from exposure to cruciferous vegetables has been associated with a decreased efficacy of warfarin, theophylline and clozapine (Abernethy and Flockhart, 2000). Similarly, charcoal-grilled meats that contain components that induce CYP1A, increase the metabolism of theophylline and warfarin and thereby decrease their effectiveness (Heck et al., 2000).

In addition to enzyme induction, inhibition of P450-mediated metabolism is probably the most frequently encountered form of metabolism-based drug interactions observed clinically. Unlike enzyme induction, which is a slow regulatory process involving activation of transcription machinery and protein translation, enzyme inhibition is an almost immediate response. Because many agents are metabolized by the CYP1A enzymes including simultaneously administered drugs, these P450s represent a major site for drug interactions. Inhibitors of CYP1A enzymes can cause marked increases in the plasma concentrations of drugs that are substrates for these enzymes. For drugs with a narrow therapeutic index, this can lead to over-dosage and/or toxicity (Lin and Lu, 1998). Exposure to dietary supplements or environmental pollutants, combined with prescribed medicines also poses risks for xenochemical–drug interactions. For example, naturally occurring flavonoids that are present in many herbs exhibit inhibitory effects on caffeine N-demethylation, catalyzed by human CYP1A2 (Lee et al., 1998). In order to circumvent altered drug responses or prevent toxicity from exposure to environmental contaminants, it is important to identify chemical modulators of CYP1A activity.

Determination of gene expression profiles of drug-metabolizing enzymes in response to xenobiotic exposure almost exclusively depended upon the use of human tissues and primary hepatocytes. These assays are limited by the availability and the inter-individual differences in response of the donors. Here, we demonstrated that cell-based bioassays are capable of identifying xenobiotics that induce CYP1A1 and CYP1A2 through AhR-mediated transactivation. Furthermore, hepatoma cells over-expressing CYP1A were utilized for characterization of inhibitors of CYP1A enzymes. With these cell-based bioassays, several therapeutics, botanicals, and environmental compounds that possess the ability to alter expression of CYP1A proteins were identified in the present investigation. An attraction of this high-throughput system allows easy identification of new chemical entities that possess the capacity to enhance or repress expression of AhR-mediated genes or inhibit metabolism by CYP1A1 and CYP1A2 at the early stages of drug discovery.

Section snippets

Preparation of a construct containing the dioxin response element (DRE)

The core sequence of AhR binding motif, DRE, (5′T/GNGCGTGAA/CG/CAA3)′ identified in the regulatory region of many TCDD-inducible genes including CYP1A2, CYP1B1, UDPglucuronosyltransferase 1A1 and glutathione S-transferase Ya C (Lusska et al., 1993, Whitlock, 1999, Yueh et al., 2003) was utilized to construct an oligonucleotide. A pair of complimentary oligonucleotides, containing three copies of the DRE in tandem with the restriction enzymes KpnI and XhoI as linkers, were constructed that had

Results

A stable cell line was developed to identify xenobiotics that alter CYP1A gene expression through the AhR. Selection of antibiotic resistant colonies produced the stable cell line, DRE12-6, that harbored three copies of the dioxin response element (DRE) in a luciferase reporter gene construct. Because the stably integrated DRE12-6 cells exhibited enhanced luciferase activity upon exposure to TCDD, they were further characterized. To determine optimal xenobiotic exposure, DRE12-6 stable cells

Discussion

In the present investigation, stably integrated cell lines harboring the dioxin response element were developed to identify inducers and inhibitors of CYP1A P450 enzymes. The cell-based bioassays were used to monitor alterations in CYP1A gene expression either by AhR-mediated transactivation or by assessing enzyme activity with isoform-specific substrates. We also found that by producing over-expression of CYP1A1 or CYP1A2 in the stable cell lines, we could identify inhibitors of activities

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    This work was supported by NIH grants GM58287 (M-FY), GM49511 (JR), and AA08990 (JR). Portions of this work were presented at the International Society for the Study of Xenobiotics (ISSX) meetings in Providence, RI, October, 2003.

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