TOXICOLOGY AND APPLIED PHARMACOLOGY 142, 160–168 (1997) ARTICLE NO. TO968022 Antiestrogenic Activity of Hydroxylated Polychlorinated Biphenyl Congeners Identified in Human Serum M. MOORE,* M. MUSTAIN,* K. DANIEL,* I. CHEN,* S. SAFE,* T. ZACHAREWSKI,² B. G ILLESBY,² A. JOYEUX,‡ AND P. BALAGUER‡ *Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843-4466; ² Department of Pharmacology and Toxicology, University of Western Ontario, London, Ontario, Canada; and ‡INSERM U58, 60 rue de Navacelles, 34090 Montpellier, France Received January 29, 1996; accepted August 26, 1996 1989; McFarland and Clarke, 1989). The biochemical and Antiestrogenic Activity of Hydroxylated Polychlorinated Biphe- toxic effects of PCBs have been extensively studied in labo- nyl Congeners Identified in Human Serum. MOORE, M., MUSTAIN, ratory animals and a broad spectrum of biochemical and M., DANIEL, K., CHEN, I., SAFE, S., ZACHAREWSKI, T., GILLESBY, toxic effects have been reported and these studies have been B., JOYEUX, A., AND BALAGUER, P. (1997). Toxicol. Appl. Pharma- extensively reviewed (Safe, 1984, 1990, 1994). PCBs are col. 142, 160–168. complex mixtures of congeners, and up to 132 individual Several hydroxylated polychlorinated biphenyls (PCBs) identi- compounds have been identified in industrial products fied in human serum have been synthesized and these include (Schulz et al., 1989). Since the effects of PCBs are due to 2,2,3,4,5,5-hexachloro-4-biphenylol; 2,3,3,4,5-pentachloro-4- activities of individual compounds present in these mixtures, biphenylol; 2,3,3,4,5-pentachloro-4-biphenylol; 2,2,3,3,4,5- the structure-dependent activities have been extensively in- hexachloro-4-biphenylol; 2,2,3,3,4,5,5-heptachloro-4-bipheny- vestigated (Safe, 1994). The ‘‘nonortho’’ or coplanar PCBs lol; 2,2,3,4,5,5,6-heptachloro-4-biphenylol; and 2,2,3,4,4,5,5- typified by 3,3,4,4-tetraCB, 3,4,4,5-tetraCB, 3,3,4,4,5- heptachloro-3-biphenylol. The hydroxy-PCBs exhibited minimal pentaCB, and 3,3,4,4,5,5-hexaCB bind to the aryl hydro- binding to the rat uterine cytosolic estrogen receptor (ER) and carbon (Ah) receptor and exhibit activity similar to that de- did not induce proliferation of estrogen-responsive MCF-7 human scribed for 2,3,7,8-tetrachlorodibenzo-p-dioxin and related breast cancercells at concentrations ranging from 10 05 to 10 08 M. compounds. The monoortho-substituted analogs of the co- The estrogenic activity of these compounds was further investi- gated utilizing two estrogen-responsive in vitro bioassays, namely, planar PCBs also exhibit Ah-receptor agonist activity; how- (i) HeLa cells stably transfected with a Gal4:human ER chimera ever, these compounds also elicit Ah-receptor-independent and a 17-mer-regulated luciferase reporter gene, and (ii) MCF-7 responses including induction of CYP2B gene expression cells transiently transfected with a full-length human ER expres- (Parkinson et al., 1981a,b, 1983). The structure-dependent sion plasmid and a plasmid containing an estrogen-responsive effects of other PCB congeners have not been defined; how- vitellogenin A2 promoter linked to a chloramphenicol acetyl trans- ever, several studies report that other structural classes of ferase (CAT) reporter gene. None of the hydroxy-PCBs signifi- PCB congeners induce Ah-receptor-independent responses. cantly induced luciferase activity in the stably transfected HeLa For example, (i) various structural classes of ortho-substi- cells or CAT activity in MCF-7 cells at concentrations as high as tuted PCBs resemble phenobarbital (PB) as inducers of 10 05 M. The antiestrogenic effects of the hydroxy-PCBs were also CYP2B gene expression (Denomme et al., 1983; Parkinson investigated using the same bioassays in which the cells were co- et al., 1983; Connor et al., 1996); (ii) some of these same treated with 17b-estradiol plus the hydroxy-PCBs. All of the hy- compounds also resemble PB as liver tumor promoters in droxy-PCB congeners inhibited one or more estrogenic response, and one congener, 2,2,3,4,5,5,6-heptachloro-4-biphenylol, inhib- rodents (Buchmann et al., 1986, 1991; Laib et al., 1991; ited 17b-estradiol-induced cell proliferation and CAT activity in Sargent et al., 1991); and (iii) ortho-substituted PCB conge- MCF-7 cells and luciferase activity in HeLa cells.  1997 Academic ners have also been characterized as neurotoxins (Seegal et Press al., 1990, 1991; Kodavanti et al., 1993, 1995; Maier et al., 1994) and modulators of neutrophil functions (Brown and Ganey, 1995; Tithof et al., 1996). Polychlorinated biphenyls (PCBs) are halogenated aro- The metabolism of PCBs results in formation of various matic industrial compounds which have been widely identi- metabolites including hydroxylated PCBs, dihydrodiols and fied in almost every component of the global ecosystem catechols, phenolic conjugates, glutathione conjugates, and including fish, wildlife, and human adipose tissue, breast methylsulfonyl metabolites (Sipes and Schnellmann, 1987; Safe, 1989). Metabolite formation is a major pathway for milk, and serum (Ballschmiter et al., 1989; Kannan et al., 160 0041-008X/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.