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Your Environment. Your Health.

Final Progress Reports: University of Albany - SUNY: Studies of the Alterations in Estrogen Metabolism Caused by Exposure to PCBs

Superfund Research Program

Studies of the Alterations in Estrogen Metabolism Caused by Exposure to PCBs

Project Leader: David C. Spink
Grant Number: P42ES004913
Funding Period: 1995 - 2000

Project-Specific Links

Final Progress Reports

Year:   1999 

Human cytochrome P450 1B1 (CYP1B1), which is induced by chlorinated dioxins and PCBs, catalyzes the hydroxylation of 17b-estradiol (E2) at C-4, with a lesser activity at C-2. Project investigators recently cloned and expressed a variant form of human CYP1B1 from MCF-7 cells with leucine substituted for valine at position 432 and serine substituted for asparagine at position 453. To investigate the NADPH- and organic hydroperoxide-supported E2 hydroxylase activities of the 432L, 453S form of human CYP1B1, the enzyme was expressed in Sf9 insect cells. In microsomal assays supplemented with human NADPH:cytochrome P450 oxidoreductase, the expressed 432L, 453S form catalyzed NADPH-supported E2 hydroxylation with a similar preference for 4-hydroxylation as the 432V, 453N form, with maximal rates of 1.97 and 0.37 nmol(min)-1(nmol cytochrome P450)-1 for 4- and 2-hydroxylation, respectively. Cumeme hydroperoxide efficiently supported E2 hydroxylation by both the 432V, 453N and 432L, 453S forms at several-fold higher rates than the NADPH-supported activities, and with a lesser preference for E2 4- versus 2-hydroxylation (2:1). The hydroperoxide-supported activities of both forms were potently inhibited by the CYP1B1 inhibitor, 3,3',4,4',5,5'-hexachlorobiphenyl (PCB 169). These results indicate that the 432V, 453N and 432L, 453S forms of CYP1B1 have similar catalytic properties for E2 hydroxylation, and that human CYP1B1 is very efficient in catalyzing the hydroperoxide-dependent formation of catecholestrogens.

The activities of CYP1A1 and CYP1B1 with E2 as substrate have been well characterized, whereas their activities with estrone (E1) have only been evaluated in a few limited studies. An assay technique employing gas chromatography-mass spectrometry for the analysis of metabolites of E1 as methyl oxime-trimethylsilyl derivatives was developed and used to evaluate the activities of cDNA-expressed CYP1A1 and CYP1B1 with E1 as substrate. The results indicated that CYP1A1 is primarily an E1 2-hydroxylase, while CYP1B1 is primarily an E1 4-hydroxylase.

Significant progress was also made in the studies of the phase II component of PCB-induced estrogen metabolism. An assay for the determination of the terminal metabolite, 2-methoxyestradiol-3-sulfate (2-MeOE2-3-sulfate), was developed employing electrospray ion trap mass spectrometry with quantitation by stable isotope dilution. Since expression of thermostable phenol sulfotransferase (SULT1A1 by the new nomenclature) correlated with the observed conjugation of nanomolar levels of 2-MeOE2 formed in dioxin- and PCB-exposed cultures of breast cancer cells, this enzyme was targeted for characterization. Cloning and sequencing of SULT1A1 cDNA from MCF-7 cells revealed that mRNAs encoding two previously identified alleles, SULT1A1*1 (213Arg) and SULT1A1*2 (213His), were expressed in these cells. Heterologous cDNA-directed expression the two alleles in Sf9 insect cells was achieved, and post-microsomal supernatants from the transfected Sf9 cells were used to determine Km values of 0.90 + 0.12 and 0.81 + 0.06 mM for SULT1A1*1 and SULT1A1*2, respectively, with 2-MeOE2 as substrate. These results show that SULT1A1 is an efficient and selective catalyst of 2-MeOE2 sulfation, and as such may be important in attenuating the physiologic effects of 2-MeOE2 that have recently been described.

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