Highly Chlorinated Pcbs Inhibit the Human Xenobiotic Response Mediated by the Steroid and Xenobiotic Receptor (SXR) Michelle M
Total Page：16
File Type：pdf, Size：1020Kb
Research | Article Highly Chlorinated PCBs Inhibit the Human Xenobiotic Response Mediated by the Steroid and Xenobiotic Receptor (SXR) Michelle M. Tabb,1 Vladyslav Kholodovych,2 Felix Grün1, Changcheng Zhou,1 William J. Welsh,2 and Bruce Blumberg1 1Department of Developmental and Cell Biology, University of California, Irvine, California, USA; 2University of Medicine & Dentistry of New Jersey–Robert Wood Johnson Medical School and The UMDNJ Informatics Institute, Piscataway, New Jersey USA (Blumberg et al. 1998; Jones et al. 2000; Polychlorinated biphenyls (PCBs) are a family of persistent organic contaminants suspected to cause Maglich et al. 2002), we infer that the metab- adverse effects in wildlife and humans. In rodents, PCBs bind to the aryl hydrocarbon (AhR) and olism of some compounds will be correspond- pregnane X receptors (PXR) inducing the expression of catabolic cytochrome p450 enzymes of the ingly different between humans and model CYP1A and 3A families. We found that certain highly chlorinated PCBs are potent activators of organisms. To test this possibility, we tested a rodent PXR but antagonize its human ortholog, the steroid and xenobiotic receptor (SXR), inhibit- variety of PCBs to determine their ability to ing target gene induction. Thus, exposure to PCBs may blunt the human xenobiotic response, activate human and rodent SXR/PXR. We inhibiting the detoxification of steroids, bioactive dietary compounds, and xenobiotics normally found that more highly chlorinated PCB con- mediated by SXR. The antagonistic PCBs are among the most stable and abundant in human tis- geners were able to activate rodent PXR but sues. These findings have important implications for understanding the biologic effects of PCB not human SXR. These same PCBs were able exposure and the use of animal models to predict the attendant risk. Key words: metabolism, poly- to directly bind to human SXR and antago- chlorinated biphenyls, PXR, SXR, xenobiotic. Environ Health Perspect 112:163–169 (2004). nize its activation and target gene induction in doi:10.1289/ehp.6560 available via http://dx.doi.org/ [Online 24 October 2003] primary human hepatocytes and LS180 human colon carcinoma cells. Using SXR transcriptional activation and antagonism Polychlorinated biphenyls (PCBs) are a major (Borlakoglu and Wilkins 1993). Thus, highly data, a predictive molecular model for PCB health concern. They exhibit many negative chlorinated PCBs linger in human tissues, binding to SXR was formulated, tested, and biologic effects, they are ubiquitously distrib- increasing their potential to disrupt normal used to identify additional antagonistic PCBs. uted, and they persist in the environment. signaling pathways. Consequently, the ability The PCBs we identified are among the most Although the manufacture and use of PCBs of a PCB to induce carcinogenesis or mutage- persistent and abundant congeners in human was banned in the United States in the late nesis or to alter endocrine signaling is strongly tissues and show striking differences in their 1970s, PCBs were still made and used for associated with its metabolic fate. Dogs and potential to be metabolized in rodents and many years in other countries. Certain PCBs rats metabolize PCBs more readily than mon- humans. PCBs strongly induce their own or PCB mixtures elicit biochemical and toxi- keys (Matthews and Dedrick 1984), suggest- metabolism in rodents; however, our findings cologic responses in humans and laboratory ing that the metabolism and biologic activity suggest that they antagonize their own metab- animals that mimic those caused by 2,3,7,8- of individual PCBs may be species specific. olism—and that of other xenobiotics, dietary tetrachlorodibenzo-p-dioxin (TCDD; The mammalian xenobiotic response is compounds, and endogenous steroids—in dioxin); however, many PCBs, especially mediated primarily by two broad-specificity humans. Thus, the use of rats to predict the more highly chlorinated congeners, do not sensors: the orphan nuclear receptors risk of human exposure to these PCBs or mix- and many of their nondioxin-like effects SXR/PXR (human steroid and xenobiotic tures that contain them will likely lead to erro- remain unknown (Carpenter et al. 2002). receptor/rodent pregnane X receptor) neous conclusions. These findings suggest PCBs and their metabolites can exert estro- (Blumberg et al. 1998; Kliewer et al. 1998) more broadly that the literature concerning genic effects by binding to the estrogen recep- and the constitutive androstane receptor the effects of xenobiotic chemicals and the tor (ER) (Bonefeld-Jorgensen et al. 2001) or (CAR) (Forman et al. 1998; Xie et al. 2000b; attendant risks for human and wildlife popu- by inhibiting estrogen catabolism (Kester et al. reviewed by Dussault and Forman 2002; lations will need to be reevaluated where the 2000). PCB metabolites have been reported to Willson and Kliewer 2002). SXR/PXR plays interfere with transcriptional activation medi- a critical role in the regulation of phase I Address correspondence to B. Blumberg, Department ated by thyroid hormone (Iwasaki et al. 2002) (cytochrome P450), phase II (conjugating), of Developmental and Cell Biology, University of and androgens (Portigal et al. 2002). and phase III (ABC family transporters) California, 5205 McGaugh Hall, Irvine, CA 92697- PCBs are lipophilic and accumulate in detoxifying enzymes, coordinately regulating 2300 USA. Telephone: (949) 824-8573. Fax: (949) 824-4709. E-mail: [email protected] liver and adipose tissue (Guvenius et al. steroid, drug, and xenobiotic clearance in the We thank J. Kanno, T. Iguchi, L. Guillette, 2002). Coplanar PCBs that are structurally liver and intestine (Dussault and Forman C. Zilinski, and members of the Blumberg labora- similar to dioxin and mono-ortho-substituted 2002; Staudinger et al. 2001; Xie et al. tory for critically reading the manuscript, and PCBs induce their own metabolism by bind- 2000a). SXR/PXR is activated by a diverse B. Forman and I. Dussault for plasmids. ing to and activating the aryl hydrocarbon group of steroid hormones, dietary com- Normal human hepatocytes were obtained through receptor (AhR) (Shimada et al. 2002), which pounds (e.g., phytoestrogens), prescription the Liver Tissue Procurement and Distribution System, (Pittsburgh, PA) funded by National induces CYP1A and CYP1B isozymes drugs (e.g., taxol, rifampicin), medicinal Institutes of Health (NIH) Contract N01-DK-9- (Rendic and Di Carlo 1997). The resulting herbs (e.g., St. John’s Wort), and xenobiotics 2310. This study was supported by grants from the hydroxylated and methylsulfonyl PCB (e.g., organochlorine pesticides) that are all U.S. Environmental Protection Agency (STAR G1T1 metabolites covalently bind to DNA, produc- substrates for the SXR-induced enzymes 0704) and the National Cancer Institute (CA-87222) ing double-stranded breaks (Srinivasan et al. (reviewed by Dussault and Forman 2002; to B.B. M.M.T was supported by a training grant 2001). Highly chlorinated PCB congeners are Willson and Kliewer 2002). from the NIH (Ruth L. Kirschstein NRSA T32 HD- 07029). metabolized slowly, if at all, because rapid Because SXR/PXR exhibits species-specific The authors declare they have no competing financial metabolism requires two adjacent, unsubsti- differences in its response to phytoestrogens, interests. tuted carbon atoms on the biphenyl ring clinically important drugs, and xenobiotics Received 2 July 2003; accepted 23 October 2003. Environmental Health Perspectives • VOLUME 112 | NUMBER 2 | February 2004 163 Article | Tabb et al. behavior of xenobiotic sensors differs across formaldehyde gels then transferred to (Packard, Meriden, CT). Counts remaining species. The differential metabolism of drugs, Hybond N membrane (Amersham Biosciences, after the addition of 10 µM clotrimazole were xenobiotics, and dietary compounds mediated Piscataway, NJ). Probes specific for full-length taken as nonspecific background and sub- by SXR/PXR provides both an explanation human CYP3A4 or rat CYP3A1 were generated tracted from all wells (Jones et al. 2000). All and a molecular tool with which to address by PCR and labeled with α-(32P) dCTP using assays were performed in triplicate and repro- the often contradictory and controversial liter- the NEBlot kit (New England Biolabs, Beverly, duced in independent experiments. ature on the effects of dietary and environ- MA). Blots were probed overnight in QuikHyb Molecular modeling. All molecular model- mental chemicals on human health. (Stratagene, Cedar Creek, TX) at 68°C fol- ing operations were performed on a Silicon lowed by manufacturer’s recommended washes. Graphics Octane workstation running under Materials and Methods Quantitative real-time RT-PCR. Primary the IRIX 6.5 operating system (Silicon Rat PXR cloning. Human Gal-SXR and human hepatocytes or LS180 cells were treated Graphics, Inc., Mountain View, CA). The mouse Gal-PXR plasmids were described pre- with various ligands for 48 hr and total RNA PCB structures in this series were constructed viously (Blumberg et al. 1998); these plasmids was prepared as described above. For RT-PCR using the Sybyl 6.8 molecular modeling pro- contain the yeast Gal4 DNA binding domain analysis, 1 µg total RNA was reverse tran- gram (Tripos, Inc., St. Louis, MO), followed fused to the ligand binding domain of SXR or scribed using Superscript II (Invitrogen). by initial geometry optimization using the PXR. Reverse-transcriptase polymerase chain Quantitative real-time RT-PCR (QRT-PCR) MMFF force field and Gasteiger-Marsili