Leptin induces CYP1B1 expression in MCF-7 cells through ligand-independent activation of the ERα pathway.

Leptin, a hormone with multiple biological actions, is produced predominantly by adipose tissue. Among its functions, leptin can stimulate tumour cell growth. Oestrogen receptor α (ERα), which plays an essential role in breast cancer development, can be transcriptionally activated in a ligand-independent manner. In this study, we investigated the effect of leptin on CYP1B1 expression and its mechanism in breast cancer cells. Leptin induced CYP1B1 protein, messenger RNA expression and promoter activity in ERα-positive MCF-7 cells but not in ERα-negative MDA-MB-231 cells. Additionally, leptin increased 4-hydroxyoestradiol in MCF-7 cells. Also, ERα knockdown by siRNA significantly blocked the induction of CYP1B1 expression by leptin, indicating that leptin induced CYP1B1 expression via an ERα-dependent mechanism. Transient transfection with CYP1B1 deletion promoter constructs revealed that the oestrogen response element (ERE) plays important role in the up-regulation of CYP1B1 by leptin. Furthermore, leptin stimulated phosphorylation of ERα at serine residues 118 and 167 and increased ERE-luciferase activity, indicating that leptin induced CYP1B1 expression by ERα activation. Finally, we found that leptin activated ERK and Akt signalling pathways, which are upstream kinases related to ERα phosphorylation induced by leptin. Taken together, our results indicate that leptin-induced CYP1B1 expression is mediated by ligand-independent activation of the ERα pathway as a result of the activation of ERK and Akt in MCF-7 cells.

Early changes in pulmonary gene expression following tobacco exposure shed light on the role of estrogen metabolism in lung carcinogenesis.

This perspective on Meireles et al. (beginning on p. 707 in this issue of the journal) discusses the increasing evidence for the role of female steroid hormones in lung cancer development and progression. The novel work of Meireles et al. is the first evidence for the rapid upregulation by tobacco smoke of a key cytochrome P450 gene that can metabolize estrogens such as beta-estradiol to potentially carcinogenic catechol and quinine forms, as well as the first evidence for the colocalization of beta-estradiol and estrogen receptors in murine airway epithelium. Actions of estrogens that contribute to lung carcinogenesis, especially in the presence of tobacco smoke, may involve both reactive intermediates that damage DNA and steroid hormone receptor signaling that promotes growth.

Ovarian cancer risk and polymorphisms involved in estrogen catabolism.

Polymorphisms within genes responsible for estrogen catabolism could alter cellular levels of genotoxic 4-hydroxylated catechol estrogens and antiangiogenic 2-methoxyestradiol, thus influencing risk of developing ovarian cancer. We carried out a population-based case-control study of 310 epithelial ovarian cancer cases and 585 controls in African-American and Caucasian women ages 35 to 54 years from Seattle, Atlanta, and Detroit metropolitan areas. Subjects were interviewed and genotyped for CYP1A1 m1, m2, m3, and m4; CYP1B1 Arg(48)Gly, Ala(119)Ser, Val(432)Leu, and Asn(453)Ser; COMT Val(158)Met; UGT1A1 A(TA)nTAA; and SULT1A1 Arg(213)His polymorphisms. Unconditional logistic regression was used to calculate odds ratios (OR). Haplotypes were inferred and analyzed using models based on expectation-maximization with progressive ligation and Bayesian coalescence theory. CYP1B1 Leu(432) carriers were at increased risk of ovarian cancer, with an adjusted OR of 1.5 (95% confidence interval, 1.1-2.3) compared with Val(432) homozygotes. The most common CYP1B1 haplotype was Arg(48)-Ala(119)-Val(432)-Asn(453). All other haplotypes with frequencies >5% contained the Leu(432) allele. In diplotype analyses, relative to women homozygous for Arg(48)-Ala(119)-Val(432)-Asn(453), women with diplotypes containing at least one Leu(432) allele had adjusted ORs ranging from 1.3 to 2.2. Among women homozygous for COMT Met(158), carriers of CYP1B1 Leu(432) had a 2.6-fold increase in risk relative to CYP1B1 Val(432) homozygotes (95% confidence interval, 1.1-5.9). This latter result is opposite in direction from a similar analysis conducted by other investigators in a different study population. No association of ovarian cancer risk was observed with any of the other polymorphisms examined, either alone or in combination.

Spectral characterization of catechol estrogen quinone (CEQ)-derived DNA adducts and their identification in human breast tissue extract.

Estrogens, including the natural hormones estrone (E(1)) and estradiol (E(2)), are thought to be involved in tumor induction. Catechol estrogen quinones (CEQ) derived from 4-hydroxyestrone (4-OHE(1)) and 4-hydroxyestradiol (4-OHE(2)) react with DNA and form depurinating N7Gua and N3Ade adducts that might be responsible for tumor initiation (Cavalieri, E. L., et al. (2000) J. Natl. Cancer Inst. Monogr. 27, 75). Current detection limits for the CEQ-derived DNA adducts by high-performance liquid chromatography with multichannel electrochemical detection are in the picomole range. To improve the limit of detection (LOD) for CEQ-derived DNA adducts, spectrophotometric monitoring was investigated. Spectroscopic studies of 4-OHE(1)-1-N3Ade, 4-OHE(1)-1-N7Gua, 4-OHE(2)-1-N3Ade, and 4-OHE(2)-1-N7Gua adduct standards were performed at 77 and 300 K. Upon laser excitation at 257 nm, the 4-OHE(1)- and 4-OHE(2)-derived N7Gua and N3Ade adducts are strongly phosphorescent at T = 77 K. No phosphorescence was observed at 300 K. Both N3Ade and N7Gua adduct types have weak phosphorescence origin bands near 383 and 385 nm, respectively. The corresponding phosphorescence lifetimes are 1.11 +/- 0.05 and 0.37 +/- 0.05 s. The LOD, based on phosphorescence measurements, is in the low femtomole range. The concentration LOD is approximately 10(-9) M, i.e., similar to that recently obtained for CEQ-derived N-acetylcysteine conjugates (Jankowiak, R., et al. (2003) Chem. Res. Toxicol. 16, 304). The LOD in capillary electrophoresis (CE) with field-amplified sample stacking and absorbance detection is about 3 x 10(-8) M. To verify whether CEQ-derived DNA adducts are formed in humans or not, tissue extracts from two breast cancer patients were analyzed by CE interfaced with room temperature absorption and low temperature (laser-excited) phosphorescence spectroscopies. For the first time, formation of CEQ-derived DNA adducts is shown in humans. For example, the level of 4-OHE(1)-1-N3Ade in the breast tissue extract from a patient with breast carcinoma (8.40 +/- 0.05 pmol/g of tissue) is larger by a factor of about 30 than that in the breast tissue sample from a woman without breast cancer (0.25 +/- 0.05 pmol/g of tissue). In contrast, similar amounts of 4-OHE(2)-1-N3Ade were observed in both types of tissue. Although more breast tissue samples from women with and without breast cancer need to be studied, these results suggest that the N3Ade adducts could serve as biomarkers to predict the risk of breast cancer.

Catechol estrogen formation in liver microsomes from female ACI and Sprague-Dawley rats: comparison of 2- and 4-hydroxylation revisited.

Estradiol (E(2))-hydroxylation was studied in liver microsomes from ACI and Sprague-Dawley female rats, which differ markedly in their susceptibility to E(2)-induced formation of mammary tumors. NADPH-dependent oxidation of E(2) by liver microsomes from ACI and Sprague-Dawley rats produced several metabolites of which 2-hydroxyestradiol (2-OH-E(2)), estrone (E(1)), and 2-hydroxyestrone (2-OH-E(1)) were predominant. Incubations with either low (9 nM) or high (50 microM) concentrations of radiolabeled E(2) and with varying amounts of microsomal protein indicated the formation of only small amounts of 4-hydroxyestradiol (4-OH-E(2)). The ratio of 2-OH-E(2) to 4-OH-E(2) formed with the low concentration of E(2) was about 10:1 regardless of the amount of microsomal protein used, and about 20:1 using a high concentration of E(2). Thus, oxidation of E(2) by liver microsomes from female ACI and Sprague-Dawley rats occurs primarily via 2-hydroxylation, and 4-hydroxylation is only a minor pathway. These results are in disagreement with a recent report indicating substantial 4-hydroxylation of E(2) by liver microsomes from female ACI rats.

Case-control study of ovarian cancer and polymorphisms in genes involved in catecholestrogen formation and metabolism.

Steroid hormones, such as estrogens, appear to be associated with ovarian carcinogenesis, but the precise biological mechanisms are unclear. Polymorphisms in genes that regulate the concentration of estrogens and their metabolites may contribute directly to the individual variation in ovarian cancer risk through a mechanism involving oxidative stress or indirectly by influencing ovarian cancer susceptibility associated with ovulation and reproduction. We conducted a population-based, case-control study of primary ovarian cancer between 1993 and 1999 in Hawaii to test several genetic and related hypotheses. A personal interview and blood specimen were obtained in the subjects' homes. In a sample of 129 epithelial ovarian cancer cases and 144 controls, we compared the frequencies of several polymorphisms in genes that regulate steroid hormone metabolism and catecholestrogen formation. Multivariate unconditional logistic regression was used to model the association of each genetic polymorphism separately after adjusting for age, ethnicity, and other covariates. The high-activity Val432 allele of the CYP1B1 gene, which may be linked to oxidative stress through elevated 4-hydroxylated catecholestrogen formation, was associated with an increased risk of ovarian cancer. The Val/Leu genotype for CYP1B1 was associated with an odds ratio of 1.8 (95% confidence interval, 1.0-3.3) and the Val/Val genotype with an odds ratio of 3.8 (95% confidence interval, 1.2-11.4) compared with the Leu/Leu genotype (P = 0.005). Tobacco smokers with at least one CYP1A1 (MspI) m2 allele, one CYP1B1 Val allele, one COMT Met allele, or two CYP1A2 A alleles were at significantly increased risk of ovarian cancer compared to never-smokers with CYP1A1 (MspI) ml/ml, CYP1B1 Leu/Leu, COMT Val/Val, or CYP1A2 A/A genotypes, respectively. We found a positive statistical interaction (P = 0.03) between tobacco smoking and the CYP1A1 (MspI) polymorphism on the risk of ovarian cancer. None of the other gene-environment (pregnancy, oral contraceptive pill use) or gene-gene interactions were statistically significant. Although not significant, there was a suggestion that the effect of the CYP1B1 Val allele was reduced substantially in the presence of the high-activity COMT Met allele. These findings suggest that the CYP1B1-Val allele and perhaps other genetic polymorphisms in combination with environmental or hormonal exposures are susceptibility factors for ovarian cancer.

Effect of chlorinated hydrocarbons on expression of cytochrome P450 1A1, 1A2 and 1B1 and 2- and 4-hydroxylation of 17beta-estradiol in female Sprague-Dawley rats.

Chlorinated hydrocarbons (CHCs) are environmental contaminants that bioaccumulate and hence are detected in human tissues. Epidemiological evidence suggests that the increased incidence of a variety of human cancers, such as lymphoma, leukemia and liver and breast cancers, might be attributed to exposure to these agents. The ability of CHCs to disrupt estrogen homeostasis is hypothesized to be responsible for their biological effects. The present study examined the effect of CHCs on the expression of cytochrome P450 (CYP)1A1, CYP1A2 and CYP1B1 mRNAs and the consequent 2- and 4-hydroxylation of 17beta-estradiol (E(2)) in female Sprague-Dawley rats. Animals were administered a single dose of the LD(50) of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) (25 microg/kg), 2, 4-dichlorophenoxyacetic acid (2,4-D) (375 mg/kg) and dieldrin (DED) (38 mg/kg) by gavage. Seventy-two hours after treatment, increased expression of CYP1A1, CYP1A2 and CYP1B1 was observed in the liver, kidney and mammary tissue. Since CYP1A and CYP1B1 are the major enzymes catalyzing 2- and 4-hydroxylation of E(2), respectively, the effect of these CHCs on the metabolism of E(2) was investigated in rat tissues. Formation of 2- and 4-catechol estrogens was increased in a tissue-specific manner in response to treatment. TCDD was the most potent inducer for CYP1 enzyme mRNA and for the 2- and 4-hydroxylation of E(2). 2,4-D and DED induced similar responses, but less than that of TCDD. These results suggest that induction of CYP1 family enzymes and consequent increases in estrogen metabolism by CHCs in target tissues may be factors contributing to the biological effects associated with exposure to these agents.

Quantitative analysis of constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cytochrome P450 1B1 expression in human lymphocytes.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) results in a broad spectrum of biological responses, including altered metabolism, disruption of normal hormone signaling pathways, reproductive and developmental effects, and cancer. Cytochrome P450 1B1 (CYP1B1) is a dioxin-inducible gene that is active in the formation of 4-hydroxyestradiol, a potentially genotoxic catechol estrogen. Therefore, the analysis of CYP1B1 in humans may be useful in establishing relationships between dioxin exposure and adverse health effects. In this study, we examined the expression of CYP1B1 in human peripheral blood lymphocytes of unexposed individuals using a quantitative reverse transcription-PCR method. Absolute CYP1B1 RNA levels varied more than 30-fold in uncultured mononuclear cells obtained from 10 individuals. In vitro treatment of mitogen-stimulated lymphocytes with TCDD for 1-5 days of culture resulted in a peak induction of CYP1B1 after 3 days. The induction of CYP1B1 RNA levels after 3 days of culture was dose-dependent, exhibited a maximum response above 10 nM TCDD, and varied greatly among different individuals. However, the half maximal dose required for this induction was similar between individuals and comparable to that observed in the MCF-7 and HepG2 human cell lines. These observations indicate that CYP1B1 exhibits variable constitutive expression and is inducible in vitro by TCDD in human lymphocytes and that the magnitude of induction varies within the population. These data define the suitability of CYP1B1 for use as a mechanistically based biomarker in ongoing molecular epidemiological studies of human populations exposed to dioxins and related chemicals that bind the aromatic hydrocarbon receptor.

Coordination of differential effects of primary estrogen and catecholestrogen on two distinct targets mediates embryo implantation in the mouse.

In the mouse, estrogen is essential for blastocyst implantation in the progesterone (P4)-primed uterus. The mechanism(s) by which estrogen initiates this response still remains elusive. The present investigation, using delayed implantation in the mouse, examined the differential role of estradiol-17beta (E2) and its catechol metabolite 4-hydroxy-E2 (4-OH-E2) in uterine and blastocyst activation for implantation. The conditions of delayed implantation were induced by ovariectomizing mice on day 4 (day 1 = vaginal plug) of pregnancy or pseudopregnancy and maintaining them with P4 from days 5-7. The binding of EGF to blastocysts was used as a marker for blastocyst activation. Our results show that whereas E2 fails to activate dormant blastocysts (with respect to EGF binding in vitro), 4-OH-E2, cAMP, or prostaglandin E2, is effective in this response. Further, whereas 4-OH-E2 induced-activation is not blocked by an antiestrogen, an inhibitor of PG synthesis, adenylyl cyclase or protein kinase A effectively blocks this activation. These results suggest that 4-OH-E2 effects on blastocysts are mediated by PGs, which, in turn, stimulate cAMP production and thus activation of protein kinase A. Two-fluoro-E2 is a poor substrate and an inhibitor of catecholestrogen synthesis, but it is estrogenic, with respect to uterine growth and gene expression. Using blastocyst transfer experiments, we observed that dormant blastocysts incubated with 4-OH-E2 in vitro, but not with E2, are capable of implanting in P4-treated delayed implanting mice receiving two-fluoro-E2. The results suggest that whereas E2 is necessary for preparation of the uterus, uterine-derived catecholestrogen is important for blastocyst activation for implantation. Indeed, the receptive uterus has the capacity to synthesize 4-OH-E2. Collectively, we demonstrate that the primary ovarian estrogen E2, via its interaction with nuclear estrogen receptors, participates in the preparation of the P4-primed uterus to the receptive state in an endocrine manner, whereas its metabolite 4-OH-E2, produced from E2 in the uterus, mediates blastocyst activation for implantation in a paracrine manner. Our results also establish that these target-specific effects of primary estrogen and catecholestrogen are both essential for implantation and that successful implantation occurs only when the activated stage of the blastocyst coincides with the receptive state of the uterus.

Formation of catechol estrogen glutathione conjugates and gamma-glutamyl transpeptidase-dependent nephrotoxicity of 17beta-estradiol in the golden Syrian hamster.

In an animal model of hormone-mediated carcinogenesis, male golden Syrian hamsters develop renal carcinoma following prolonged exposure to 17beta-estradiol. The basis for the species and tissue specificity is unclear. Detailed information on the disposition of 17beta-estradiol in this model is lacking. Because catechol estrogens have been implicated in this model of carcinogenesis, we investigated the metabolism and nephrotoxicity of 17beta-estradiol in golden Syrian hamsters, with emphasis on the formation of catechol estrogen thioethers. 17beta-Estradiol (50 micromol/kg, i.p.) is a mild nephrotoxicant, causing significant elevations in the urinary excretion of gamma-glutamyl transpeptidase (gamma-GT), alkaline phosphatase, glutathione S-transferase (GST) and glucose. Increases in renal protein carbonyls and lipid hydroperoxides, which are markers of oxidative damage, also occur after administration of 17beta-estradiol (50 micromol/kg, i.p.). 17beta-Estradiol-mediated nephrotoxicity is reduced by treating animals with acivicin, an inhibitor of gamma-GT, implying that toxicity is mediated by metabolites requiring metabolism by this enzyme. Following administration of 17beta-[14C]estradiol (100 micromol/kg) to hamsters, 9.7% of the dose is recovered in bile after 5 h, the majority (7.9%) representing aqueous metabolites. Seven catechol estrogen GSH conjugates were identified, 2-hydroxy-1,4-bis-(glutathion-S-yl)-17beta-estradiol, 2-hydroxy-4-(glutathion-S-yl)-17beta-estradiol, 2-hydroxy-4-(glutathion-S-yl)-estrone, 4-hydroxy-1-(glutathion-S-yl)-estrone, 2-hydroxy-1-(glutathion-S-yl)-estrone, 4-hydroxy-1-(glutathion-S-yl)-17beta-estradiol, and 2-hydroxy-1-(glutathion-S-yl)-17beta-estradiol. At 5.4 micromol/kg of 17beta-estradiol, a dose-reflective of daily exposure levels in the hamster model of nephrocarcinogenicity, 12% of the dose is recovered within 5 h as a combination of GSH conjugates of 2- and 4-hydroxy-17beta-estradiol and 2- and 4-hydroxyestrone. In summary, oxidation of catechol estrogens, followed by GSH conjugation, occurs in vivo and 17beta-estradiol is a mild nephrotoxicant in a manner dependent on the activity of gamma-GT.

Inhibition of catechol O-methyltransferase-catalyzed O-methylation of 2- and 4-hydroxyestradiol by quercetin. Possible role in estradiol-induced tumorigenesis.

Catecholestrogens have been postulated to mediate the induction of kidney tumors by estradiol in male Syrian hamsters. In this study, we examined the mechanism of inhibition by quercetin of the catechol O-methyltransferase-catalyzed O-methylation of catecholestrogens as a basis for the previously reported enhancement of estradiol-induced tumorigenesis by this flavonoid. In hamsters treated with 50 micrograms of [6,7-3H]estradiol, quercetin increased concentrations of 2- and 4-hydroxyestradiol in kidney by 80 and 59%, respectively. In animals treated with two 10-mg estradiol implants, quercetin also decreased by 63-65% the urinary excretion of 2- and 4-hydroxyestradiol monomethyl ethers. Taken together, these results demonstrate the in vivo inhibition of the O-methylation of catecholestrogens by quercetin. S-Adenosyl-L-homocysteine, produced by the methylation of catecholestrogens, noncompetitively inhibited the O-methylation of 2- and 4-hydroxyestradiol by hamster kidney cytosolic catechol O-methyltransferase (IC50 approximately 10-14 microM). Due to the rapid O-methylation of quercetin itself, quercetin decreased renal concentrations of S-adenosyl-L-methionine by approximately 25% in control or estradiol-treated hamsters and increased concentrations of S-adenosyl-L-homocysteine by 5-15 nmol/g of wet tissue, which was estimated to cause a 30-70% inhibition of the enzymatic O-methylation of catecholestrogens. Quercetin or fisetin (a structural analog) inhibited the O-methylation of 2- and 4-hydroxyestradiol by a competitive plus noncompetitive mechanism (IC50 approximately 2-5 microM). These results suggest that the in vivo O-methylation of catecholestrogens is inhibited more by S-adenosyl-L-homocysteine than by quercetin. The accumulation of 2- and 4-hydroxyestradiol during co-administration of estradiol and quercetin may enhance metabolic redox cycling of catecholestrogens and thus estradiol-induced kidney tumorigenesis.

Catechol estrogen production in rat microsomes after treatment with indole-3-carbinol, ascorbigen, or beta-naphthaflavone: a comparison of stable isotope dilution gas chromatography-mass spectrometry and radiometric methods.

Compounds like indole-3-carbinol (I3C) have been shown to increase catechol estrogen formation and reduce mammary tumor incidence in mice. These compounds may exert a protective effect for breast cancer development by decreasing the overall estrogen pool available for the formation of 16 alpha-hydroxyestrone (16 alpha-OHE1), a metabolite that retains significant estrogenic activity, may be mutagenic and could represent a potential carcinogenic intermediate of estradiol degradation. I3C and ascorbigen originate from the breakdown of glucobrassicin. We have compared the inductive effects of I3C with ascorbigen and beta-naphthaflavone (Bnf) in microsomes from rats pretreated with these compounds using isotope dilution GC-MS and a radiometric method. Incubated microsomes from rats pretreated with I3C and ascorbigen yielded high levels of 2-hydroxyestradiol (2-OHE2) that were comparable to levels induced by Bnf and were significantly above control group levels (p < 0.005). Absolute values determined by the radiometric method were approximately 40% lower than 2-OHE2 concentrations determined by GC-MS, although the relative changes in each group were the same. These differences may be attributed to the radiolabel becoming trapped in microsomal intermediates in the sequence leading to tritium entering the aqueous compartment. Both ascorbigen- and Bnf-treated animals exhibited significant increases in 2-hydroxyestrone (2-OHE1) (p < 0.05). The ability of ascorbigen to induce estradiol C-2 hydroxylation has not been previously reported. Based on these data, we speculate that ascorbigen will act as an anticarcinogenic agent and will inhibit or reduce the incidence of mammary tumor formation.

The sexually differentiated metabolism of [6,7-3H]17 beta-oestradiol in rats: male-specific 15 alpha- and male-selective 16 alpha-hydroxylation and female-selective catechol formation.

The oxygenated-metabolite profiles of exogenous 17 beta-oestradiol (E2) in adult male and female Wistar rats have been characterized and major sex-dependent biotransformations observed which correlate with the regioselectivities of known sexually differentiated hepatic P450. [6,7-3H]E2 (27 micrograms/kg) was given i.v. The metabolites of E2 were rapidly and extensively excreted in bile (46 and 78% of the dose over 1 and 6 h, respectively). Female rats metabolized E2 by one major pathway: oxidation to oestrone (E1) followed by C-2 hydroxylation and O-methylation; the principal aglycones (0-1 h bile collections) were E1 (14%), 2-hydroxyE1 (2-OHE1) (42%) and 2-methoxyE1 (24%). Male rats metabolized E2 principally by two parallel composite pathways of E1 hydroxylation which yielded a complex mixture of mono- and di-oxygenated compounds: 15 alpha-OHE1 (33%), 2,15 alpha-diOHE1 (7%), and 2-methoxy-15 alpha OHE1 (14%); 16 alpha-OHE1 (13%), 2,16 alpha-diOHE1 (4%) and 2-methoxy-16 alpha-OHE1 (2%). 15 alpha-Hydroxylation was unique to males. The balance of aromatic and alkyl hydroxylation in males was dose-dependent: at 3 mg/kg, 15 alpha-hydroxylation was decreased approx. 50% in favour of 2-hydroxylation whilst 16 alpha-hydroxylation was largely unaffected. The male-specific 15 alpha-hydroxylation and male-predominant 16 alpha-hydroxylation of E1 derived from E2 in vivo may be ascribable to the male-specific isoforms P450IIC13 and P450IIC11, respectively.

The metabolism of 2,4-dibromo [6,7-3H]17 beta-oestradiol in the rat: ring-A dibromination blocks male-specific 15 alpha-hydroxylation and catechol formation.

The metabolism in the rat of 2,4-dibromo-17 beta-oestradiol (2,4-DBE2), a compound of potential use for tumour imaging and assessment, has been studied. 2,4-DB[6,7-3H]E2 was synthesized by bromination of [6,7-3H]E2 with N-bromosuccinimide, and administered (40 micrograms/kg, i.v.) to anaesthetized male and female rats. Metabolites were rapidly and extensively excreted in bile (60 and 82% of the dose over 1 and 6 h, respectively). No unchanged compound was excreted. 2,4-DBE2 was almost entirely oxidized to 2,4-DB-oestrone; which was largely eliminated as its glucuronide but partly (approx. 30%) metabolized to 2,4-DB-16 alpha-hhydroxyoesterone and, to a minor extent, 2,4-DB-oestriol. No products of either oxidative or reductive debromination were detected. Neither of the two oxidative transformations of 2,4-DBE2 in the rat, in contrast with those of exogenous E2, was sex-selective, and 2,4-DB-oestrone underwent less extensive hydroxylation than oestrone formed from E2. In female rats, the substituents selectively redirected the principal site of hydroxylation from C-2 to C-16, whereas in males they had no significant effect on the existing 16 alpha-hydroxylation but did block the major pathway, 15 alpha-hydroxylation. Thus the sexual differentiation of E2 oxidative metabolism was abolished by direct blockage causing metabolic switching to a latent reaction in the female rat and long-range inhibition of the vicinal hydroxylation in the male rat.

Influence of indole-3-carbinol on the hepatic microsomal formation of catechol estrogens.

The oral administration of indole-3-carbinol (IC), present in cabbage and other members of the Cruciferae family, to female rats almost doubled their ability to convert estradiol to catechol estrogens in the liver. This was determined by the release of 3H from C-2 of the estrogen and also by isolation of the 14C-labeled catechol derivative after incubation with hepatic microsomal fractions. The yield of 4-hydroxyestradiol was also elevated and these effects were similar to those produced by 3-methylcholanthrene (MC), a well-characterized cytochrome P450 inducer. Further evidence for the involvement of a mixed-function oxidase was provided by a 70% to 80% decrease in the yield of 3H2O and water-soluble radioactivity by SKF-525A (0.1 mM) when added to the microsomal fractions isolated from the livers of control or IC-treated rats. In addition, NADPH could not be replaced by NADH in these experiments. Pretreatment with ethionine prevented the increase in estradiol metabolism brought about by oral administration of IC. Both IC and MC inhibited catechol estrogen formation when added directly to the liver microsomal system, confirming earlier findings that in vivo inducers can act as in vitro inhibitors. However, IC was less inhibitory than MC, supporting the theory that IC is converted to a more active product in the stomach. Thus, IC may be conferring protection against estrogen-dependent neoplasia by increasing the hepatic oxidation of estradiol, thereby lowering the amount of available active estrogen.

Hepatic catecholestrogen synthases: differential effect of sex, inducers of cytochromes P-450 and of antibody to the glucocorticoid inducible cytochrome P-450 on NADPH-dependent estrogen-2-hydroxylase and on organic hydroperoxide-dependent estrogen-2/4-hydroxylase activity of rat hepatic microsomes.

Formation of catecholestrogens (CE) by rat hepatic microsomes was re-examined because as recently shown; (1) CE formation can be catalyzed by an NADPH-dependent estrogen-4-hydroxylase (E-4-H(NADPH)) and by a peroxidatic, organic hydroperoxide-dependent estrogen-2/4-hydroxylase (E-2/4-H(OHP)), in addition to the established NADPH-dependent estrogen 2-hydroxylase (E-2-H(NADPH)); and (2) the indirect radiometric and the COMT-coupled radioenzymatic assays, used in many previous studies, may fail to provide an accurate measure, in particular, of 4-OH-CE. Using a direct product isolation assay, hepatic microsomes of both male and female rats were shown to express E-2/4-H(OHP) activity with properties similar to those of peroxidatic activity in other tissues. The activities of E-2/4-H(OHP) and E-2-H(NADPH) were affected differently by 5 out of 7 inducers of cytochromes P-450 administered in vivo. Phenobarbital and dexamethasone caused a 4- and 2-3-fold increase in E-2-H(NADPH) activity, respectively, but only a 38 and 20% increase in E-2/4-H(OHP) activity. Ketoconazol and beta-naphtoflavone caused a modest increase in E-2-H(NADPH) activity but a decrease in OHP-dependent activity. Clofibrate decreased peroxidatic activity by 50% and NADPH-dependent activity by approximately 20%. Both activities were increased by ethanol but decreased by isoniazide, an agent which induces the same form of cytochromes P-450 as ethanol. Polyclonal antibody against P-450p, a form of P-450 induced by glucocorticoids, inhibited E-2-H(NADPH) but not E-2/4-H(OHP) activity of untreated and of dexamethasone- and phenobarbital-treated rats. This study establishes that CE formation may occur in liver via the peroxidatic pathway and indicates that this pathway depends on forms of P-450 different from those mediating E-2-H(NADPH) activity. It also confirms and extends previous observations of the involvement of multiple, constitutive and induced forms of cytochrome P-450 in NADPH-dependent 2-hydroxylation in liver.

Effect of chronic estrogen treatment of Syrian hamsters on microsomal enzymes mediating formation of catecholestrogens and their redox cycling: implications for carcinogenesis.

Estrogens have previously been shown to induce DNA damage in Syrian hamster kidney, a target organ of estrogen-induced cancer. The biochemical mechanism of DNA adduction has been postulated to involve free radicals generated by redox cycling of estrogens. As part of an examination of this postulate, we measured the effect of chronic estrogen treatment of hamsters on renal microsomal enzymes mediating catechol estrogen formation and free radical generation by redox cycling of catechol estrogens. In addition, the activities of the same enzymes were assayed in liver in which tumors do not develop under these conditions. At saturating substrate concentration, 2- and 4-hydroxyestradiol were formed in approximately equal amounts (26 and 28 pmol/mg protein/min, respectively), which is 1-2 orders of magnitude higher than reported previously. Estradiol treatment for 2 months decreased 2-hydroxylase activity per mg protein by 75% and 4-hydroxylase activity by 25%. Hepatic 2- and 4-hydroxylase activities were 1256 and 250 pmol/mg protein/min, respectively. Estrogen treatment decreased both activities by 40-60%. Basal peroxidatic activity of cytochrome P-450, the enzyme which oxidizes estrogen hydroquinones to quinones in the redox cycle, was 2.5-fold higher in liver than in kidney and did not change with estrogen treatment. However, when normalized for specific content of cytochrome P-450 the enzyme activity in kidney was 2.5-fold higher than in liver and increased further by 2-3-fold with chronic estrogen treatment. The activity of cytochrome P-450 reductase, which reduces quinones to hydroquinones in the estrogen redox cycle, was 6-fold higher in liver than in kidney of both control and estrogen-treated animals. When normalized for cytochrome P-450, the activity of this enzyme was similar in liver and kidney, but over 4-fold higher in kidney than liver after estrogen treatment. Basal concentrations of superoxide, a product of redox cycling, were 2-fold higher in liver than in kidney. Estrogen treatment did not affect this parameter in liver, but increased it in kidney by 40%. These data provide evidence for a preferential preservation of enzymes involved in estrogen activation.

Catechol estrogen formation in the mouse uterus and its role in implantation.

Estradiol-2/4-hydroxylase (E-2/4-H) activity was determined in the mouse uterus during early pregnancy as well as in ovarian steroid hormone-treated ovariectomized uterus. Under the assay conditions used, E-4-H was the predominant catechol estrogen-forming monooxygenase enzyme. The inhibition of E-4-H activity by SKF-525A, metyrapone and alpha-naphthoflavone suggested involvement of cytochrome P450-dependent monooxygenases. A haloestrogen, 2-fluoroestradiol (2-FL-E2), also inhibited this activity. During the peri-implantation period, no change in uterine E-4-H activity was noted on the morning of days 2 through 5, but the activity significantly (P less than 0.01) increased in the afternoon of day 4 of pregnancy. A single injection of estradiol-17 beta (E2, 100 ng/mouse) to ovariectomized mice significantly (P less than 0.01) elevated the level of E-4-H activity at 24 h as did injections of progesterone (P4, 2 mg/mouse) for 2 days. When 2 days of P4 (2 mg/mouse) treatment was combined with a single injection of E2 (20 ng/mouse), E-4-H activity increased 1.3-fold (P less than 0.05) by 24 h above that of P4 treatment alone. Dexamethasone (200 micrograms/mouse) and cholesterol (2 mg/mouse) treatment for 2 days had no effect on E-4-H activity. Thus, the stimulatory effect of P4 and E2 on E-4-H activity appeared to be specific. The increased activity of uterine E-4-H prior to implantation on day 4 evening and the modulation of its activity by P4 and/or E2 suggest an involvement of 4-hydroxyestradiol in embryo implantation.(ABSTRACT TRUNCATED AT 250 WORDS)