Suppression of aromatase activity in vitro by PCBs 28 and 105 and Aroclor 1221.
The effects of polychlorinated biphenyls (PCBs) on human cytochrome P450 aromatase activity in vitro were investigated using a commercially available microsomal fraction obtained from baculovirus infected insects that had been transfected with the human CYP19 gene and cytochrome P450 reductase. The assay measured the conversion of tritiated testosterone to estradiol in Tris buffer at pH 7.4. When aroclors, commercial preparations of PCBs, were added to aromatase assays at a 10 microM concentration, Aroclor 1221 caused a reduction in the aromatase activity, whereas other aroclors (1016, 1232, 1242, 1248, 1254, 1260, 5432, 5442 and 5460) were without effect. Further investigation of the effect of Aroclor 1221 on aromatase activity showed that the inhibition was dose dependent. When a reconstituted mixture (RM) of PCBs that represented the congeneric content of human milk was investigated, no inhibition of aromatase activity at the maximum treatment of 15.0 microM was observed. None of the congeners present in the reconstituted mixture, except PCB 28 and 105, affected P450 arom activity. PCB 28 showed a statistically significant inhibition of aromatase activity (P<0.05) at 1.5 and 15 microM and a significant inhibition of aromatase activity by PCB 105 was also observed, but only at 15 microM. In three separate kinetic analyses the Km(app) for aromatase was 64, 89 and 69 nM (mean 74 nM). In addition, PCB 28 resulted in an increase in the Km(app) without a significant effect on Vmax(app), suggesting competitive inhibition by this congener. This conclusion was supported by slope (Km(app)/Vmax(app) versus [inhibitor]) and intercept (1/Vmax(app) versus [inhibitor]) replots. The slope replots gave Ki(app) values for PCB 28 of 0.9, 1.3 and 2.0 microM (mean 1.4 microM), whereas intercept replots were almost horizontal. Thus, PCB 28 is a competitive inhibitor of aromatase with a Ki(app) value approximately 20-fold the Km(app) value. Based on these studies, we conclude that most PCBs are not inhibitors of aromatase activity in vitro. However, as being inhibitors of aromatase activity, Aroclor 1221, PCB 28 and PCB 105 would remain a priority for further study as possible endocrine disrupters.
Woodhouse
Amanda J
AJ
Toxicology Research Division, Health Products and Foods Branch, Health Canada, Department of Cellular and Molecular Medicine, University of Ottawa, Sir Frederick G. Banting Research Centre, Tunney's Pasture, Ottawa, ON, Canada.
Cooke
Gerard M
GM
D000818:Animals; D001140:Aroclors / Q000633:toxicity*; D001141:Aromatase / Q000494:pharmacology*; D047072:Aromatase Inhibitors; D016367:Baculoviridae / Q000235:genetics; D004347:Drug Interactions; D004703:Endocrine System / Q000187:drug effects; D004785:Environmental Pollutants / Q000633:toxicity*; D004958:Estradiol / Q000032:analysis; D006801:Humans; D007313:Insecta; D007700:Kinetics; D008861:Microsomes; D011078:Polychlorinated Biphenyls / Q000633:toxicity*; D013739:Testosterone / Q000378:metabolism; D014162:Transfection
D001140:Aroclors; D047072:Aromatase Inhibitors; D004785:Environmental Pollutants; C067940:2,3,3',4,4'-pentachlorobiphenyl; C032739:aroclor 1221; D013739:Testosterone; D004958:Estradiol; C081766:2,4,4'-trichlorobiphenyl; D011078:Polychlorinated Biphenyls; D001141:Aromatase