Quantitative analysis of constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cytochrome P450 1B1 expression in human lymphocytes. (1/114)

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.  (+info)

Serum 2-hydroxyestradiol 17-sulphate in pregnancy. (2/114)

2-Hydroxyestradiol 17-sulphate (2-OH E2-17-S) is a catecholized form of sulphated estrogen. In vitro studies showed that its antioxidative effect is almost equal to that of free catecholestrogens, such as 2-OH E2 or 4-OH E2 and alpha-tocoferol, but the existance of 2-OH E2-17-S in human serum has not yet been made clear. 2-OH E2-17-S strongly antagonizes lipid peroxidation, and so it may play an important role in pregnancy, for example as an anti-oxidant in pregnancy-induced hypertension (PIH). The serum level of 2-OH E2-17-S was measured during mid to late pregnancy by a direct radioimmunoassay (RIA) without hydrolysis. The serum levels at 28-31 weeks, 32-35 weeks and 36-40 weeks of gestation were 4.68+/-0.93 (mean+/-SE), 8.38+/-1.21 and 18.31+/-3.41 nmol/l, respectively. The serum level in PIH cases at 36-40 weeks (4.64+/-1.29 nmol/l) was significantly lower than that in normal pregnancy. The 2-OH E2-17-S level in umbilical arteries was significantly higher than that in maternal peripheral vein. These results suggest that the feto-placental unit plays an important role in catecholizing E2-17-S to 2-OH E2-17-S, which may act as an antioxidant in pregnancy.  (+info)

A new ELISA kit for measuring urinary 2-hydroxyestrone, 16alpha-hydroxyestrone, and their ratio: reproducibility, validity, and assay performance after freeze-thaw cycling and preservation by boric acid. (3/114)

There is considerable controversy regarding the role of estrogen metabolites in breast cancer risk, fueled in part by the development of a rapid ELISA that is suitable for large scale investigations. An earlier version of the ELISA could detect values of the 2-hydroxyestrone (2-OHE1) and 16alpha-hydroxyestrone (16alpha-OHE1) metabolites as low as 2 ng/ml and produce consistent results in premenopausal urines. However, reproducibility was problematic in postmenopausal urines where concentrations of these compounds are much lower. In response to our concern, a new ELISA was developed with a sensitivity of 0.625 ng/ml, which we evaluated using the same pre- and postmenopausal urine samples analyzed in the earlier ELISA. In this report, we present findings on the new kit with regard to reproducibility of the 2-OHE1 and 16alpha-OHE1 measurements, comparability of results with gas chromatography-mass spectroscopy values, and with regard to the stability of the metabolites after repeated freeze-thaw cycles and after preservation by boric acid. For the most part, we found the new ELISA to be reproducible, with assay coefficients of variation ranging from 10 to 20%, and intraclass correlation coefficients (ICCs) ranging from 80 to 95% in both the pre- and postmenopausal urines. ELISA results for 16alpha-OHE1 differed from 1 day (i.e., batch) to the next, and the absolute values of the metabolites obtained by the ELISA were consistently lower than but well correlated with those obtained by gas chromatography-mass spectroscopy. Values of the 2-OHE1:16alpha-OHE1 ratio also differed between the methods, but because the range of values was not large, the magnitude of these differences was not as great. For the ratio, the correlation between methods was excellent, and the ICCs were high for both groups of women. After preservation by boric acid, values of the ratio varied according to acid concentration but not in a linear fashion. Ratio values were similar in urine samples exposed to four different freeze-thaw cycle treatments, although values for all treatments were consistently lower in one batch. Because batch-to-batch variability was not negligible, it is advisable that matched cases and controls be analyzed in the same batch. Provided this is done, the relatively low assay coefficient of variation and high ICC demonstrate that the new ELISA kit can reliably measure the 2-OHE1:16alpha-OHE1 ratio and detect small case-control differences in large population-based studies, where rapid and relatively easy laboratory methods are critical.  (+info)

Induction of uterine adenocarcinoma in CD-1 mice by catechol estrogens. (4/114)

Catechol estrogens may mediate estrogen-induced carcinogenesis because 4-hydroxyestradiol induces DNA damage and renal tumors in hamsters, and this metabolite is formed in the kidney and estrogen target tissues by a specific estrogen 4-hydroxylase. We examined the carcinogenic potential of catechol estrogen in an experimental model previously reported to result in a high incidence of uterine adenocarcinoma after neonatal exposure to diethylstilbestrol. Outbred female CD-1 mice were treated with 2- or 4-hydroxyestradiol, 17beta-estradiol, or 17alpha-ethinyl estradiol on days 1-5 of neonatal life (2 microg/pup/day) and sacrificed at 12 or 18 months of age. Mice treated with 17beta-estradiol or 17a-ethinyl estradiol had a total uterine tumor incidence of 7% or 43%, respectively. 2-Hydroxyestradiol induced tumors in 12% of the mice, but 4-hydroxyestradiol was the most carcinogenic estrogen, with a 66% incidence of uterine adenocarcinoma. Both 2- and 4-hydroxylated catechols were estrogenic and increased uterine wet weights in these neonates. These data demonstrate that both 2- and 4-hydroxyestradiol are carcinogenic metabolites. The high tumor incidence induced by 4-hydroxyestradiol supports the postulated role of this metabolite in hormone-associated cancers.  (+info)

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. (5/114)

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.  (+info)

Periovulatory changes in catfish ovarian oestradiol-17beta, oestrogen-2-hydroxylase and catechol-O-methyltransferase during GnRH analogue-induced ovulation and in vitro induction of oocyte maturation by catecholoestrogens. (6/114)

In the catfish Heteropneustes fossilis and Clarias batrachus, ovarian oestrogen-2-hydroxylase (OE-2-H) activity increased significantly at 8 h after the injection of an ovulatory dose (0.15 microg/g body weight) of a mammalian GnRH analogue ([d -Ala(6)-Pro(9)]-LHRH ethylamide) and was restored to the 0 h (control) level after egg-stripping at 16 h. On the other hand, ovarian oestradiol-17beta (OE2) level and catechol-O-methyltransferase (COMT) activity decreased significantly at 8 h. While the OE2 level was restored to the 0 h level, COMT activity increased significantly at 16 h. Changes in ovarian OE2 level and enzymes indicate higher synthesis of 2-hydroxylated catecholoestrogens and their degradation during the periovulatory period. Under in vitro conditions, the synthetic catecholoestrogens (CEs, 2- and 4-hydroxylated oestradiol17beta and oestrone (OE1)) induced germinal vesicle break down (GVBD) in a dose- (0.01-10 microg/ml) and duration- (1-36 h) dependent manner, the mean values of the responses being in the order 2-OH OE2>4-OH OE2> 2-OH OE1>4-OH OE1. The CE-induced GVBD response (8 h induction) was not blocked by prior and subsequent incubations with steroid synthesis inhibitors (cyanoketone, epostane and aminoglutethimide) up to 36 h, suggesting that de novo steroidogenesis is not essential for the response. The percentage of GVBD response to 2-h induction by CEs was significantly inhibited by actinomycin D (a transcriptional inhibitor) and cycloheximide (a translational inhibitor), indicating the involvement of both RNA and protein synthesis. The CE-induced 8-h stimulation of GVBD was mildly blocked by propranolol, the beta-adrenergic inhibitor, suggesting the response was partly mediated through a beta-adrenergic receptor mechanism. Incubations with phentolamine, an alpha-adrenergic inhibitor, did not interfere with the CE-induced GVBD response. The results demonstrate CE-related enzymatic changes in teleost (catfish) ovaries and maturation-inducing substance activity of CEs.  (+info)

Catechol estrogen conjugates and DNA adducts in the kidney of male Syrian golden hamsters treated with 4-hydroxyestradiol: potential biomarkers for estrogen-initiated cancer. (7/114)

Formation of depurinating adducts by reaction of catechol estrogen-3,4-quinones with DNA was proposed to be a tumor initiating event by estrogens [E.L. Cavalieri et al. (1997) Proc. Natl Acad. Sci. USA, 94, 10937-10942]. Under estrogenic imbalance, oxidation of catechol estrogens to quinones may compete with their detoxification by protective enzymes. The quinones formed can be detoxified by reaction with glutathione (GSH) or can covalently bind to DNA. To provide more support for this hypothesis, we developed a method to identify and quantify GSH, cysteine (Cys) and N-acetylCys conjugates of 4-hydroxyestrogens (4-OHE) in the kidneys of male Syrian hamsters treated with 4-hydroxyestradiol (4-OHE2) by intraperitoneal injection. The highest level of conjugates was observed 1 h after treatment, and almost none was detected after 24 h. Dose-response studies indicated conjugate formation after treatment with 0.5 micromol of 4-OHE2/100 g body weight, and formation increased up to a treatment level of 12 micromol/100 g body weight. GSH, Cys and N-acetylCys conjugates of 4-OHE were identified in the picomole range by high-performance liquid chromatography (HPLC) with multichannel electrochemical detection and confirmed by HPLC/tandem mass spectrometry. Treatment of tissue homogenates with beta-glucuronidase/sulfatase at 37 degrees C for 6 h before extraction resulted in a 12- to 20-fold increase in Cys conjugates from picomole to nanomole levels. Similar enhancement was observed by just incubating the tissue at 37 degrees C for 6 h. Evidence for the 4-OHE-1-N7Gua depurinating adducts was obtained by mass spectrometry. We conclude that GSH and Cys conjugates of the 4-OHE and the 4-OHE-N7Gua adducts can be utilized as biomarkers to detect estrogenic imbalance and potential susceptibility to tumor initiation.  (+info)

Case-control study of ovarian cancer and polymorphisms in genes involved in catecholestrogen formation and metabolism. (8/114)

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.  (+info)