Activity of Benzo[a]pyrene and Its Hydroxylated Metabolites in an Estrogen Receptor-alpha Reporter Gene Assay

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Abstract
A human breast cancer cell line, MCF-7, transiently transfected with a chimeric estrogen receptor (Gal4-HEG0) and a luciferase reporter plasmid (17m5-G-Luc), was used to investigate the estrogenic activity of benzo[a]pyrene (B[a]P), a prototypical polyaromatic hydrocarbon (PAH). B[a]P at concentrations ≥ 1 μM produced responses comparable to that of 0.1 nM 17β-estradiol (E2). The ER antagonist ICI 182,780 (ICI) completely inhibited the response to both E2 and B[a]P, indicating that the responses were ER-mediated. However, 2 μM α-napthoflavone (α-NF), an Ah receptor antagonist and P450 inhibitor, also decreased the response to B[a]P but not to E2. Analysis of the profile of B[a]P metabolites in the transfected MCF-7 cultures indicated that α-NF inhibited the production of the 3- and 9-hydroxy (3-OH and 9-OH), as well as the 7,8- and 9,10-dihydroxy (7,8-OH and 9,10-OH) B[a]P species. In the ER-α reporter assay, the 3-OH and 9-OH metabolites produced maximal responses comparable to E2, with EC50 values of 1.2 μM and 0.7 μM, respectively. The 9,10-OH metabolite exhibited minimal activity in the assay. These responses were inhibited by ICI for both the 3-OH and the 9-OH species; however, α-NF inhibited only the response to the 9-OH metabolite. The 7,8-OH metabolite did not exhibit significant estrogenic activity. Furthermore, 7,8-OH B[a]P displayed observable cytotoxicity at concentrations ≥ 10–7 M. This cytotoxic response was completely inhibited by α-NF, suggesting that 7,8-OH B[a]P was being further metabolized to one or more cytotoxic metabolites.

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