Activity of benzo[a]pyrene and its hydroxylated metabolites in an estrogen receptor-alpha reporter gene assay.
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 > or = 1 microM produced responses comparable to that of 0.1 nM 17beta-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 microM alpha-napthoflavone (alpha-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 alpha-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-alpha reporter assay, the 3-OH and 9-OH metabolites produced maximal responses comparable to E2, with EC50 values of 1.2 microM and 0.7 microM, 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, alpha-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 > or = 10(-7) M. This cytotoxic response was completely inhibited by alpha-NF, suggesting that 7,8-OH B[a]P was being further metabolized to one or more cytotoxic metabolites.
Charles
G D
GD
Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan 48674, USA. gdcharles@dow.com
Bartels
M J
MJ
Zacharewski
T R
TR
Gollapudi
B B
BB
Freshour
N L
NL
Carney
E W
EW
D001564:Benzo(a)pyrene / Q000378:metabolism / Q000494:pharmacology*; D001571:Benzoflavones / Q000494:pharmacology; D001943:Breast Neoplasms / Q000201:enzymology / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D005260:Female; D000077267:Fulvestrant; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter / Q000187:drug effects*; D005821:Genetic Techniques; D006801:Humans; D006900:Hydroxylation; D008156:Luciferases / Q000378:metabolism; D011960:Receptors, Estrogen / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000378:metabolism
D001571:Benzoflavones; D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D011960:Receptors, Estrogen; D000077267:Fulvestrant; D001564:Benzo(a)pyrene; D004958:Estradiol; C011512:alpha-naphthoflavone; D008156:Luciferases; D001616:beta-Galactosidase