A Repressive Cross-Regulation between Catalytic and Promoter Activities of the CYP1A1 and CYP2E1 Genes: Role of H2O2 YANNICK MOREL, ISABELLE DE WAZIERS, and ROBERT BAROUKI

Cytochrome P450 enzymes catalyze the first step of the metabolism and subsequent elimination of hydrophobic xenobiotics. However, the activity of some isoforms, among them CYP1A1 and CYP2E1, may result in cellular insults such as oxidative stress and activation of procarcinogen compounds into reactive metabolites. The regulation of the expression of these enzymes is therefore important. We have previously shown that the CYP1A1 gene promoter was repressed by oxidative stress. We show here that the CYP2E1 gene promoter is down-regulated by exogenous H2O2 addition and glutathione depletion. It is also repressed by the transfection of a CYP2E1 expression vector, which elicits an intracellular H2O2 generation. This autoregulation is limited by catalase (which catalyzes the catabolism of H2O2), thus implying H2O2 as a mediator of the negative feedback mechanism. Furthermore, we observed that the activity of CYP1A1 resulting either from the stimulation of the endogenous gene by benzo[a]pyrene treatment or from the transfection of an expression vector, repressed the activity of the CYP2E1 gene promoter. Conversely, CYP2E1 overexpression repressed the activity of the CYP1A1 gene promoter. In both cases, catalase and a specific inhibitor of one enzyme prevented the repression of the other. This suggests that the generation of H2O2 during the catalytic cycle of these enzymes is a mediator of the cross-regulatory mechanisms. These novel repressive mechanisms of autoregulation and cross-regulation using H2O2 as a common mediator may limit the potential toxicity resulting from high cytochrome P450 activity within the cell. Cytochrome P450 monooxygenases constitute a multigenic superfamily of enzymes that metabolize both endogenous and exogenous compounds. They play an important role in the metabolism of hydrophobic xenobiotics by initiating a process that leads to their solubilization and elimination. In some cases, however, they activate their substrates into reactive metabolites that can form adducts with proteins or DNA. The activity of cytochromes P450 can thus be implicated in cytotoxic processes. In this respect, it is important to control their expression. Most isoforms involved in the metabolism of xenobiotics are inducible, often by their own substrates. Conversely, several pathophysiological conditions, such as infection and inflammation, repress the expression of several isoforms (Morgan et al., 1998). Extensive studies have been published on the transcriptional regulation of CYP1A1 (Whitlock, 1999), CYP3A4 (reviewed in Guengerich, 1999), and other isoforms including CYP2B and CYP4A (Waxman, 1999). The expression of the human CYP1A1 gene is mainly controlled by the regulation of its promoter. Its induction by polycyclic aromatic compounds such as 2,3,7,8 tetrachlorodibenzo-p-dioxin or benzo[a]pyrene (BP) is particularly potent. In HepG2 cells, a 100-fold increase was observed (Kress et al., 1998). The mechanism involves the stimulation of the Ah receptor (for a recent review, see Whitlock, 1999). On the contrary, CYP1A1 mRNA levels are depressed by inflammatory cytokines and growth factors (Barker et al., 1992; Muntane-Relat et al., 1995). A common mechanism for these repressions could involve reactive oxygen species (ROS). Indeed, the CYP1A1 gene promoter is inhibited by oxidative stress (Morel and Barouki, 1998). The human CYP2E1 gene is induced by ethanol ingestion (Wrighton et al., 1986) and has therefore been widely studied. Contrary to other major isoforms, this induction mainly involves post-transcriptional mechanisms. Several potent CYP2E1 inducers, including ethanol, do not induce mRNA synthesis (Carroccio et al., 1994); rather, they increase the translation efficiency (Kim et al., 1990) and the protein halflife. Compared with other isoforms, CYP2E1 normally has a shorter half-life of about 6 h (Yang and Cederbaum, 1997) because it is degraded after ubiquitination by a specific rapid pathway involving the proteasome complex (Tierney et al., ABBREVIATIONS: BP, benzo[a]pyrene; ROS, reactive oxygen species; UTR, untranslated region; DCF, 29,79-dichlorofluorescein; BSO, Lbuthionine-S,R-sulfoximine; 4-MP, 4-methlpyrrazole; NFI, nuclear factor I. 0026-895X/00/061158-07$3.00/0 MOLECULAR PHARMACOLOGY Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics MOL 57:1158–1164, 2000 /13013/824865 1158 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from 1992; Yang and Cederbaum, 1997). On binding to CYP2E1, some of its substrates stabilize the protein, which then displays a half-life of about 37 h, similar to that of other P450 isoforms (Roberts et al., 1995). For these reasons, the transcriptional regulation of the CYP2E1 gene was less studied than that of CYP1A1. However, because inflammatory cytokines (Abdel-Razzak et al., 1993), cause a decrease in CYP2E1 mRNA, we asked whether oxidative stress could regulate the CYP2E1 gene promoter. ROS are important regulators of cellular functions. For example, they can either induce (Dalton et al., 1999) or repress (Morel and Barouki, 1999) gene expression. ROS generation can be triggered by several cellular stresses, such as mitochondrial dysfunction, inflammation, or UV radiations (Morel and Barouki, 1999). The metabolism of endogenous or xenobiotic compounds is also an important generator of ROS. Indeed, several microsomal cytochromes P450 have been shown to produce ROS in vitro, especially when metabolizing uncoupled substrates. This phenomenon was first observed in intact hepatoma cells with CYP2E1 (Dai et al., 1993). In lymphocytes, the overexpression of several isoforms was also shown to increase ROS production (Puntarulo and Cederbaum, 1998). Moreover, 2,3,7,8 tetrachlorodibenzo-p-dioxin (a potent CYP1A1 inducer) generates an oxidative stress in vivo (Shertzer et al., 1998). We have shown recently that CYP1A1 activity triggers an intracellular H2O2 production (Morel et al., 1999). This ROS release by CYP1A1 is involved in negative feedback that limits CYP1A1 induction and the related toxicity. In this study, we asked whether the catalytic activity of one cytochrome P450 isoform could regulate the gene expression of another isoform. We first showed that both exogenous H2O2 and the catalytic activity of either CYP2E1 or CYP1A1 repress the CYP2E1 gene promoter. We also showed that the activity of CYP2E1 could inhibit the CYP1A1 gene promoter. These regulations constitute a novel cross-regulatory mechanism. Materials and Methods Chemicals. H2O2 was used from a 30% stock obtained from Merck (Darmstadt, Germany), Other chemicals were obtained from Sigma (Saint-Quentin Fallavier, France) and oligonucleotides from Genset (Paris, France). Cell Culture. The human hepatoma cell line HepG2 was used because the CYP1A1 gene promoter is regulated by oxidative stress (Morel and Barouki, 1998) and because of its good transfection efficiency (Morel et al., 1999). It was maintained at 37°C in an incubator under an atmosphere containing 5% CO2. The medium used in cell cultures was half Dulbecco’s modified essential medium and half Ham’s F12 (Life Technologies, Paisley, UK), supplemented with 10% fetal calf serum (Life Technologies), 0.5 mg/ml fungizone (BristolMyers Squibb, Paris La Défense, France), 100 U/ml penicillin G (Diamant, Puteaux, France), and 100 U/ml streptomycin (Life Tech-