[CANCER RESEARCH 61, 3942–3948, May 15, 2001] Differential Regulation of Cytochrome P450 1A1 and 1B1 by a Combination of Dioxin and Pesticides in the Breast Tumor Cell Line MCF-7 1 Xavier Coumoul, Monique Diry, Cedric Robillot, and Robert Barouki 2 Institut National de la Sante ´ et de la Recherche Me ´dicale U490, Universite ´ Rene ´ Descartes, 75270 Paris Cedex 06 [X. C., M. D., R. B.], and Laboratoire Environnement et Chimie Analytique CNRS ERS 657, Ecole Supe ´rieure de Physique et de Chimie Industrielles de la Ville de Paris, 75231 Paris Cedex 05 [C. R.], France ABSTRACT Dioxin and pesticides with xenoestrogenic activity are environmental contaminants that are suspected of promoting human diseases such as cancers. However, few studies have addressed the molecular consequences of a combination of these contaminants, a situation that is likely to occur in the environment. We investigated the effects of natural and xenoestro- gens on basal and 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cyto- chrome P450 (CYP) 1A1 and 1B1. The CYP1B1/1A1 ratio is a critical determinant of the metabolism and toxicity of estradiol in mammary cells. Here we show that in MCF-7 cells, 17-estradiol and -endosulfan can repress whole cell ethoxyresorufin-O-deethylase activity, lowering CYP1A1 mRNA levels as well as promoter activity as assessed by tran- sient transfection assays. These negative effects are observed at both the basal and tetrachlorodibenzo-p-dioxin-induced levels. Under the same conditions, CYP1B1 mRNA levels and promoter activity are not affected. The effects on mRNA-induced levels are also observed in another mam- mary cell line, T47D, but not in mammary cell lines that do not express aryl hydrocarbon receptor and estrogen receptor (ER). Moreover, the use of ER antagonists shows that these effects are ER dependent in MCF-7 cells. In human hepatoma HepG2 cells, which lack functional ER, - endosulfan, but not 17-estradiol, displays a repressive effect on CYP1A1 through a different mechanism. These results show that xenoestrogens, by altering the ratio of CYP1B1/CYP1A1, could redirect estradiol metabo- lism in a more toxic pathway in the breast cell line MCF-7. INTRODUCTION Several synthetic compounds, such as pesticides, herbicides, or industrial byproducts, display xenoestrogenic activity. These com- pounds tend to accumulate in the environment and can be found in products consumed by human, particularly food; they are thus con- sidered major environmental contaminants in addition to dioxins (known as polychlorinated dibenzodioxin), furans (known as poly- chlorinated dibenzofuran), polyaromatic hydrocarbons, and heavy metals. It is assumed that these compounds, alone or in combination, may promote human diseases (1). Xenoestrogens bind to and activate the ER, 3 thus increasing the estrogen-dependent transcription of target genes and promoting un- desirable estrogenic effects. They are suspected to play a role in decreasing the quantity and quality of human and animal semen during the last 50 years and in the increasing incidence of testicular cancer in men and breast cancer in women in industrialized countries (2– 4). Breast carcinogenesis depends on several parameters, such as the absence or presence of ER, exposure to xenobiotics, and/or genetic factors (5–7). Genotoxic molecules can be exogenous or endogenous. Estrogen catechols produced by the breast epithelium are an example of genotoxic molecules derived from the hydroxylation of the natural estrogens estrone, E2, and estriol, respectively. The predominant forms are 2OH-E2 and 4OH-E2. Another hydroxylated metabolite is 16-hydroxy-estrone (8). The catechols 2OH-E2 and 4OH-E2 can be oxidized to quinones, which are putative tumor initiators (9, 10). Quinones derived from 2OH-E2 react with DNA to form stable adducts but do not seem to generate mutations. In contrast, the 3,4-catechol estradiol quinone, derived from 4OH-E2, forms depuri- nating adducts and readily leads to mutation events (10 –12). Thus, this 4-hydroxylated form of E2 appears to be one of the most geno- toxic metabolites of E2 in the breast epithelium. In contrast, the influence of the 2OH-E2 is controversial; whereas some studies have established a protective effect of this form (13), others have suggested that it could be to be carcinogenic (14). Moreover, 2OH-E2 could inhibit the genotoxicity of 4OH-E2 via its 2-methoxy form (15, 16). Thus, the ratio 2OH-E2/4OH-E2 could be a critical parameter of the carcinogenicity of E2 (17–20). In breast epithelium, the production of estrogen catechols depends on the activity of monooxygenases, particularly cytochromes CYP1A1 and -1B1 (21, 22). CYPs are drug-metabolizing enzymes that play a major role in the metabolism of hydrophobic xenobiotics and endogenous compounds (23). CYP1A1 displays hydroxylase ac- tivity at the C2 position, but also at the C6 and C15 positions of E2 (24), whereas CYP1B1 displays its primary activity on the C4 position (25). CYP1A1 produces primarily 2OH-E2, whereas CYP1B1 pro- duces 4OH-E2 (9). As in the case of the catechol ratio, the relative expression of these genes and the CYP1A1/1B1 ratio could then be critical in terms of carcinogenesis. The expression of CYP1A1, -1A2, and -1B1 depends on the activ- ity of the AhR. The AhR belongs to the basic helix loop helix/ per-ARNT-sim protein family (26). It is expressed in many tissues, including breast epithelium (27). Activation of the cytoplasmic com- plex containing the AhR depends on the binding of a ligand. As a consequence, the AhR enters the nucleus and forms an active het- erodimer with a nuclear protein called ARNT, which also belongs to the basic helix loop helix/per-ARNT-sim family (28). The AhR- ARNT complex binds to specific responsive elements, xenobiotic responsive elements, located in the promoters and enhancers of target genes and activates their transcription (26). Polyaromatic hydrocar- bons or halogenated aromatic hydrocarbons can be ligands of the AhR. Among these molecules, the best characterized activator is TCDD, which belongs to the family of molecules called dioxins (29). Studies using animal models have revealed that exposure to TCDD leads to many toxicological symptoms, including increased frequency of cancers (30). Epidemiological studies on highly exposed human populations revealed a more moderate toxicity, but recent evidence confirmed a significant increase in cancer and other pathologies in these populations (30). A confusing factor of population studies is the influence of other contaminating compounds. In this report, we address this question by Received 8/11/00; accepted 3/16/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by INSERM, Universite ´ Paris V-Rene ´ Descartes, Fondation pour la Recherche Me ´dicale (Grant 1000031401), Ministe `re de l’environnement, and Re ´gion Ile de France. 2 To whom requests for reprints should be addressed, at INSERM U490, Universite ´ Rene ´ Descartes, 45 rue des Saints-Pe `res 75270 Paris cedex 06, France. Phone: 33- 142862075; E-mail: robert.barouki@biomedicale.univ-paris5.fr. 3 The abbreviations used are: ER, estrogen receptor; E2, 17-estradiol; 2OH-E2 and 4OH-E2, 2-hydroxy- and 4-hydroxy-17-estradiol; CYP, cytochrome P450; AhR, aryl hydrocarbon receptor; ARNT, AhR nuclear translocator; TCDD: 2,3,7,8-tetrachlorod- ibenzo-p-dioxin; INSERM, Institut National de la Sante ´ et de la Recherche Me ´dicale; FL, firefly luciferase; RL, Renilla luciferase; EROD, ethoxyresorufin-O-deethylase; PXR, pregnane X receptor. 3942 Research. on October 6, 2021. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from