The 4-hydroxy metabolite of 178-estradiol (E2) has been implicated in the carcinogenicity of this hormone. Previous studies showed that aryl hydrocarbonreceptor agonists induced a cytochrome P450 that catalyzed the 4-hydroxylation of E2. This activity was associated with human P450 lBi. To determine the relationship of the human P450 lBl gene product and E2 4-hydroxylation, the protein was expressed in Saccharomyces cerevisiae. Microsomes from the transformed yeast catalyzed the 4-and 2-hydroxylation of E2 with Km values of 0.71 and 0.78 ,uM and turnover numbers of 1.39 and 0.27 nmol product min'l nmol P450-1, respectively. Treatment of MCF-7 human breast cancer cells with the aryl hydrocarbon-receptor ligand indolo[3,2-b]carbazole resulted in a concentration-dependent increase in P450 lBl and P450 lAl mRNA levels, and caused increased rates of 2-, 4-, 6c-, and 15a-hydroxylation of E2. At an E2 concentration of 10 nM, the increased rates of 2-and 4-hydroxylation were approximately equal, emphasizing the significance of the low Km P450 lBl-component of E2 metabolism. These studies demonstrate that human P450 lB1 is a catalytically efficient E2 4-hydroxylase that is likely to participate in endocrine regulation and the toxicity of estrogens.The importance of estrogens in the etiology of breast and uterine cancer is widely recognized (1-3). The carcinogenicity of estrogens has been primarily attributed to their action as agonists of the estrogen receptor, through which concerted gene regulation controls cellular growth and differentiation in estrogen responsive tissues. Increasing evidence of another mechanism of carcinogenicity has focused attention on the catechol estrogen metabolites, which are less potent estrogens than 17f3-estradiol (E2). The 2-and 4-hydroxylated metabolites of both E2 and estrone (E1) can directly or indirectly damage DNA, proteins, and lipids through the generation of reactive free radicals by the reductive-oxidative cycling of these catechol estrogens between their semiquinone and quinone forms (4-6).4-Hydroxylated metabolites represent only a small percentage of the total urinary catechol-estrogen content, and 4-hydroxylation was previously thought to be only a minor metabolic route (7). However, tissue-specific 4-hydroxylation of E2 may be significant in the metabolic control of estrogen homeostasis. In human (8) and mouse uteri (9), rat pituitary (10), and hamster kidney (11) the rate of E2 4-hydroxylation approaches or exceeds that of 2-hydroxylation. Interestingly, these organs are targets of estrogen-induced tumorigenesis (2, 12-14), and higher E2 4-hydroxylase activity has been measured in tumors of the human breast (15, 16) and uterus (8), each compared with normal tissue. Furthermore, in the male hamster kidney, the carcinogenic and DNA-damaging activity of 4-hydroxyestradiol (4-OHE2), and lack of activity of 2-hydroxyestradiol (2-OHE2), (17)(18)(19), implicate the 4-hydroxylated metabolites in estrogen-induced carcinogenesis. Pertinent to elucidating the contribution of 4...

Human cytochromes P450 1A1 (CYP1A1) and P450 1B1 (CYP1B1) catalyze the metabolic activation of a number of procarcinogens and the hydroxylation of 17beta-estradiol (E2) at the C-2 and C-4 positions, respectively. The aromatic hydrocarbon receptor (AhR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has a marked effect on estrogen metabolism in MCF-7 breast-tumor cells by induction of these two enzymes. To investigate whether induction of CYP1A1 and CYP1B1 by AhR agonists and the associated increase in E2 metabolism are common to all breast epithelial cells and breast-tumor cells, we determined the effects of TCDD on E2 metabolism, and CYP1A1 and CYP1B1 mRNA levels in a series of non-tumor-derived breast epithelial (184A1 and MCF-10A) and breast-tumor (MCF-7, T-47D, ZR-75-1, BT-20, MDA-MB-157, MDA-MB-231 and MDA-MB-436) cell lines. In 184A1 cells, which did not express detectable estrogen receptor (ER) alpha mRNA, CYP1A1 mRNA and activity were induced by TCDD, and enhanced E2 metabolism in TCDD-treated cells was predominantly E2 2-hydroxylation. In MCF-10A, MCF-7, T-47D, ZR-75-1 and BT-20 cells, which expressed varying levels of ER alpha mRNA, both CYP1A1 and CYP1B1 mRNA levels and rates of both E2 2- and 4-hydroxylation were highly elevated following exposure to TCDD. In MDA-MB-157, MDA-MB-231 and MDA-MB-436 cells, which did not express detectable ER alpha mRNA and generally displayed fibroblastic or mesenchymal rather than epithelial morphology, CYP1B1 induction was favored, and the rate of E2 4-hydroxylation exceeded that of 2-hydroxylation in TCDD-treated cells. These results show that breast epithelial cells and tumor cells vary widely with regard to AhR-mediated CYP1A1 and CYP1B1 induction, suggesting that factors in addition to the AhR regulate CYP1A1 and CYP1B1 gene expression. In these cell lines, significant CYP1A1 inducibility was restricted to cultures displaying epithelial morphology, whereas CYP1B1 inducibility was observed in cells of both epithelial and mesenchymal morphology.

Cytochrome P450 (CYP)1A1 and CYP1B1, which are under the regulatory control of the aryl hydrocarbon (Ah) receptor (AhR), catalyze the metabolic activation of numerous procarcinogens and the hydroxylation of 17beta-estradiol (E2) at the C-2 and C-4 positions, respectively. There is evidence of cross-talk between estrogen receptor alpha (ERalpha)- and AhR-mediated signaling in breast and endometrial cells. To further examine these interactions, we investigated the short- and long-term effects of E2 exposure on Ah responsiveness in MCF-7 human breast cancer cells. Short-term exposure to 1 nM E2 elevated the ratio of the 4- to 2-hydroxylation pathways of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced E2 metabolism and the ratio of the induced CYP1B1 to CYP1A1 mRNA levels, as determined by real-time PCR. Cells maintained long-term (9-12 months) in low-E2 medium progressively lost Ah responsiveness, as indicated by diminished rates of TCDD-induced E2 metabolism and ethoxyresorufin O-deethylase activity, and the reduced expression of the CYP1A1 and CYP1B1 mRNAs and proteins levels. These E2-deprived cells showed elevated levels of ERalpha mRNA, depressed levels of AhR mRNA, and unchanged levels of the AhR nuclear translocator mRNA. Transient transfection studies using a CYP1B1-promoter-luciferase reporter construct showed that reduced CYP1B1 promoter activity in E2-deprived cells could be restored by co-transfection with an AhR expression construct, indicating that AhR expression was limiting in these cells. The reduced Ah responsiveness of E2-deprived cells was reversed by culture for four passages in medium supplemented with 1 nM E2; ERalpha and AhR mRNAs returned to near-normal levels and the inducibility of the CYP1A1 and CYP1B1 mRNAs, proteins, and E2 metabolic activities by TCDD was restored. These studies indicate that the continued presence of estrogen is required to maintain high levels of AhR expression and inducibility of the procarcinogen-bioactivating enzymes, CYP1A1 and CYP1B1, in MCF-7 cells.

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ABSTRACT:Polycyclic aromatic hydrocarbons (PAHs) and metals are often environmental cocontaminants, yet there have been relatively few studies of combined effects of PAHs and metals on cytochrome P450 (P450)-catalyzed metabolism. We examined the effects of NaAsO 2 in combination with benzo[a]pyrene (BAP) on CYP1A1 and CYP1B1 in T-47D human breast cancer cells by using estrogen metabolism as a probe of their activities. Exposure to BAP caused elevated rates of the 2-and 4-hydroxylation pathways of estrogen metabolism, indicating induction of both CYP1A1, an estradiol 2-hydroxylase, and CYP1B1, an estradiol 4-hydroxylase. BAP-induced metabolism peaked 9 to 16 h after exposure and returned to near-basal levels by 48 h. Concentration-response studies showed maximal induction of the 2-and 4-hydroxylation pathways at 3 M BAP; higher levels caused reduced rates of metabolism due to inhibition of CYP1A1 and CYP1B1. NaAsO 2 caused pronounced decreases in the induction of CYP1A1 and CYP1B1 by 3 M BAP because cotreatment with 10 M NaAsO 2 inhibited the rates of the 2-and 4-hydroxylation pathways by 86 and 92%, respectively. Western immunoblots showed diminished levels of BAP-induced CYP1A1 by coexposure to NaAsO 2 . The levels of the CYP1A1 and CYP1B1 mRNAs induced by BAP were not significantly affected by coexposure to NaAsO 2 ; however, heme oxygenase 1 mRNA levels were markedly induced by coexposure to BAP and NaAsO 2 . These results indicate a post-transcriptional inhibitory effect of arsenite on the expression of CYP1A1 and CYP1B1 in T-47D cells, possibly resulting from reduced heme availability.Among the environmental contaminants of most concern due to their toxicity, including carcinogenicity, are heavy metals, such as arsenic, lead, and mercury, and polycyclic aromatic hydrocarbons (PAHs 1 ) typified by benzo[a]pyrene (BAP). PAHs and heavy metals are often cocontaminants in the environment, yet there have been relatively few studies of the combined toxic and particularly the carcinogenic effects elicited by PAHs and heavy metals. The carcinogenicity of BAP and other PAHs is a consequence of their metabolic activation catalyzed by cytochromes P450 (P450) and epoxide hydrolase (Wood et al., 1976;Kapitulnik et al., 1978). Covalent adducts formed by the reaction of PAH diol epoxide metabolites with guanine in mutational hotspots of critical genes such as that of the p53 tumor suppressor (Denissenko et al., 1996), if not efficiently repaired, may initiate tumorigenesis.Several members of the P450 superfamily in conjunction with epoxide hydrolase have been shown to catalyze the metabolism of BAP to carcinogenic intermediates. In extrahepatic tissues, CYP1A1 and CYP1B1 are thought to be the most important enzymes in catalyzing the formation of mutagenic intermediates from BAP and a number of other PAHs, including several that are potent mammary gland carcinogens in rodents. CYP1B1 appears to be more active than CYP1A1 in the conversion of a number of PAHs to genotoxic intermediates (Shimada et al., 1996). In the presence...

In a previous study of nine human breast-derived cell lines, rates of metabolism of 17beta-estradiol (E(2)) were greatly enhanced when cultures were exposed to the aromatic hydrocarbon receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Elevated rates of E(2) hydroxylation at the C-2, -4, -6alpha and -15alpha positions were observed concomitant with the induction of cytochromes P450 1A1 and 1B1. In each cell line, 2- and 4-hydroxyestradiol (2- and 4-OHE(2)) were converted to 2- and 4-methoxyestradiol (2- and 4-MeOE(2)) by the action of catechol O:-methyltransferase. In this study, conjugation of these estrogen metabolites was investigated. A comparison of the levels of metabolites determined with and without prior treatment of the media with a crude beta-glucuronidase/sulfatase preparation showed that most of the 2-MeOE(2) present was in conjugated form, whereas 4-MeOE(2), 6alpha-OHE(2) and 15alpha-OHE(2) were minimally conjugated. Inhibitor studies suggested that it was the sulfatase activity of the preparation that hydrolyzed the 2-MeOE(2) conjugates in MCF-7 cell media; the presence of 2-MeOE(2)-3-sulfate in MCF-7 culture media was confirmed by electrospray ion-trap mass spectrometry. To identify the enzyme catalyzing this conjugation, the expression of mRNAs encoding five sulfotransferases (SULT1A1, SULT1A2, SULT1A3, SULT1E1 and SULT2A1) was evaluated in the nine cell lines by use of the reverse transcription-polymerase chain reaction. Only expression of SULT1A1 mRNA correlated with the observed conjugation of nanomolar levels of 2-MeOE(2) in these cell lines. Cloning and sequencing of SULT1A1 cDNA from MCF-7 cells revealed that mRNAs encoding two previously identified allelic variants, SULT1A1*1 ((213)Arg) and SULT1A1*2 ((213)His), were expressed in these cells. Heterologous cDNA-directed expression of either variant in MDA-MB-231 cells, which do not normally express SULT1A1, conferred 2-MeOE(2) sulfonation activity. The SULT1A1 allelic variants were also expressed in SF:9 insect cells, from which post-microsomal supernatants were used to determine K:(m) values of 0.90 +/- 0.12 and 0.81 +/- 0.06 microM for SULT1A1*1 and SULT1A1*2, respectively, with 2-MeOE(2) as substrate. These results show that SULT1A1 is an efficient and selective catalyst of 2-MeOE(2) sulfonation and, as such, may be important in modulating the anticarcinogenic effects of 2-MeOE(2) that have been described recently.

The cumulative exposure to estrogens is an important determinant in the risk of breast cancer, yet the full range of mechanisms involving estrogens in the genesis and progression of breast cancer remains a subject of debate. Interactions of estrogens and environmental toxicants have received attention as putative factors contributing to carcinogenesis. Mechanistic studies have demonstrated interactions between estrogen receptor α (ERα) and the aryl hydrocarbon receptor (AhR), with consequences on the genes that they regulate. Many studies of ERα and AhR-mediated effects and crosstalk between them have focused on the initial molecular events. In this study, we investigated ERα- and AhR-mediated effects in long-term estrogen exposed (LTEE) MCF-7 human breast cancer cells, which were obtained by continuous culturing for at least 12 weeks in medium supplemented with 1 nM of 17β-estradiol (E2). With these LTEE cells and with parallel control cells cultured without E2 supplementation, we performed an extensive study of cytochrome P450 (CYP) induction, carcinogen bioactivation, global gene expression, and tumorigenicity in immunocompromised mice. We found that LTEE cells, in comparison with control cells, had higher levels of AhR mRNA and protein, greater responsiveness for AhR-regulated CYP1A1 and CYP1B1 induction, a 6-fold higher initial level of benzo(a)pyrene-DNA adducts as determined by liquid chromatography tandem mass spectrometry, marked differences in the expression of numerous genes, and a higher rate of E2-dependent tumor growth as xenografts. These studies indicate that LTEE causes adaptive responses in MCF-7 cells, which may reflect processes that contribute to the overall carcinogenic effect of E2.

The interactions of polycyclic aromatic hydrocarbons (PAH) and cytochromes P450 (CYP) are complex; PAHs are enzyme inducers, substrates, and inhibitors. In T-47D breast cancer cells, exposure to 0.1 to 1 μM benzo(k)fluoranthene (BKF) induced CYP1A1/1B1-catalyzed 17β-estradiol (E 2 ) metabolism, whereas BKF levels greater than 1 μM inhibited E 2 metabolism. Time-course studies showed that induction of CYP1-catalyzed E 2 metabolism persisted after the disappearance of BKF or co-exposed benzo(a)pyrene, suggesting that BKF metabolites retaining Ah receptor agonist activity were responsible for prolonged CYP1 induction. BKF metabolites were shown, through the use of ethoxyresorufin O-deethylase and CYP1A1-promoter-luciferase reporter assays, to induce CYP1A1/1B1 in T-47D cells. Metabolites formed by oxidation at the C-2/C-3 region of BKF had potencies for CYP1 induction exceeding those of BKF, whereas C-8/C-9 oxidative metabolites were somewhat less potent than BKF. The activities of expressed human CYP1A1 and 1B1 with BKF as substrate were investigated by use of HPLC with fluorescence detection, and by GC/MS. The results showed that both enzymes efficiently catalyzed the formation of 3-, 8-, and 9-OHBKF from BKF. These studies indicate that the inductive effects of PAH metabolites as potent CYP1 inducers are likely to be additional important factors in PAH-CYP interactions that affect metabolism and bioactivation of other PAHs, ultimately modulating PAH toxicity and carcinogenicity.