Lack of zonal uptake of estrone sulfate in enriched periportal and perivenous isolated rat hepatocytes.
The zonal uptake of estrone sulfate (E1S; 1 to 400 microM) was investigated in periportal and perivenous rat hepatocytes and cells isolated from whole liver (regular hepatocytes). Transport of E1S by periportal, perivenous, and regular hepatocytes was described by saturable (Kms of 24 to 26 microM and Vmaxs of 1.8 nmol/min/mg protein) and nonsaturable components (2.5 to 3.2 microl/min/mg protein) that were not different among the zonal regions (p >.05, ANOVA). These kinetic constants represented pooled values for the entire complement of transporters for E1S, including two known transporters of E1S: Ntcp, Na+-taurocholate cotransporting polypeptide, and oatp1, the organic anion transporting polypeptide cloned from rat liver. Uptake of E1S was significantly reduced by estradiol 17beta-glucuronide (50 microM) and bumetanide (200 microM), and was inhibited strongly and competitively by pregnenolone sulfate with an inhibition constant of 6.7 microM. Further segregation of the kinetic constants as the sodium-dependent and -independent systems was achieved through simultaneous fitting of data obtained in the presence and absence of sodium from parallel hepatocytic uptake studies. For the periportal, perivenous, and regular hepatocytes, two saturable systems: a sodium-dependent transport system, characterized by similar Vmaxs (1.1 to 1.4 nmol/min/mg protein) and Kms (49 to 55 microM), a sodium-independent transport system of comparable Vmaxs (0.70 to 0.84 nmol/min/mg protein) and Kms (16 to 22 microM), and a linear clearance of 1.7 to 2.7 microl/min/mg protein (ANOVA, p >.05) were obtained. The data suggest that hepatic uptake of E1S involved sodium-dependent and -independent transporter systems. No heterogeneity in transport was observed. (+info)
Laboratory assay reproducibility of serum estrogens in umbilical cord blood samples.
We evaluated the reproducibility of laboratory assays for umbilical cord blood estrogen levels and its implications on sample size estimation. Specifically, we examined correlation between duplicate measurements of the same blood samples and estimated the relative contribution of variability due to study subject and assay batch to the overall variation in measured hormone levels. Cord blood was collected from a total of 25 female babies (15 Caucasian and 10 Chinese-American) from full-term deliveries at two study sites between March and December 1997. Two serum aliquots per blood sample were assayed, either at the same time or 4 months apart, for estrone, total estradiol, weakly bound estradiol, and sex hormone-binding globulin (SHBG). Correlation coefficients (Pearson's r) between duplicate measurements were calculated. We also estimated the components of variance for each hormone or protein associated with variation among subjects and variation between assay batches. Pearson's correlation coefficients were >0.90 for all of the compounds except for total estradiol when all of the subjects were included. The intraclass correlation coefficient, defined as a proportion of the total variance due to between-subject variation, for estrone, total estradiol, weakly bound estradiol, and SHBG were 92, 80, 85, and 97%, respectively. The magnitude of measurement error found in this study would increase the sample size required for detecting a difference between two populations for total estradiol and SHBG by 25 and 3%, respectively. (+info)
Estrogens induce apoptosis in mouse peritoneal macrophages.
AIM: To study whether estrogen might induce apoptosis in mouse peritoneal macrophages (MPM). METHOD: The MPM were isolated and incubated in culture medium containing 17-beta-estradiol, estrone, or equal volume of 100% ethanol as control. DNA fragmentation was visualized by agarose gel electrophoresis. RESULTS: 17-beta-Estradiol 0.01-1 mumol.L-1 or estrone 10-20 mumol.L-1 elicited typical morphological apoptosis and DNA fragmentation in a concentration-dependent manner in MPM. Staurosporine (Sta) 0.01 mumol.L-1, cycloheximide (Cyc) 1 mg.L-1, and tamoxifen (Tam) 10 mumol.L-1 inhibited the DNA fragmentation induced by 17-beta-estradiol 1 mumol.L-1 or estrone 20 mumol.L-1. CONCLUSION: Estradiol and estrone induced apoptosis in MPM. (+info)
Caffeine consumption and menstrual function.
The relation between caffeine intake and menstrual function was examined in 403 healthy premenopausal women who belonged to Kaiser Permanente Medical Care Program in 1990-1991. A telephone interview collected information about caffeinated beverage intake as well as other lifestyle, demographic, occupational, and environmental factors. Subjects collected daily urine samples and completed a daily diary for an average of five menstrual cycles. Metabolites of estrogen and progesterone were measured in the urine, each cycle was characterized as anovulatory or ovulatory, and a probable day of ovulation was selected when appropriate. Logistic regression and repeated measures analyses were performed on menstrual parameters. Women whose caffeine consumption was heavy (>300 mg of caffeine per day) had less than a third of the risk for long menses (> or =8 days) compared with women who did not consume caffeine (adjusted odds ratio = 0.30, 95% confidence interval 0.14-0.66). Those whose caffeine consumption was heavy also had a doubled risk for short cycle length (< or =24 days) (adjusted odds ratio = 2.00, 95% confidence interval 0.98-4.06); this association was also evident in those whose caffeine consumption was heavy who did not smoke (adjusted odds ratio = 2.11, 95% confidence interval 1.03-4.33). Caffeine intake was not strongly related to an increased risk for anovulation, short luteal phase (< or =10 days), long follicular phase (> or =24 days), long cycle (> or =36 days), or measures of within-woman cycle variability. (+info)
Polyspecific substrate uptake by the hepatic organic anion transporter Oatp1 in stably transfected CHO cells.
The rat liver organic anion transporting polypeptide (Oatp1) has been extensively characterized mainly in the Xenopus laevis expression system as a polyspecific carrier transporting organic anions (bile salts), neutral compounds, and even organic cations. In this study, we extended this characterization using a mammalian expression system and confirm the basolateral hepatic expression of Oatp1 with a new antibody. Besides sulfobromophthalein [Michaelis-Menten constant (Km) of approximately 3 microM], taurocholate (Km of approximately 32 microM), and estradiol- 17beta-glucuronide (Km of approximately 4 microM), substrates previously shown to be transported by Oatp1 in transfected HeLa cells, we determined the kinetic parameters for cholate (Km of approximately 54 microM), glycocholate (Km of approximately 54 microM), estrone-3-sulfate (Km of approximately 11 microM), CRC-220 (Km of approximately 57 microM), ouabain (Km of approximately 3,000 microM), and ochratoxin A (Km of approximately 29 microM) in stably transfected Chinese hamster ovary (CHO) cells. In addition, three new substrates, taurochenodeoxycholate (Km of approximately 7 microM), tauroursodeoxycholate (Km of approximately 13 microM), and dehydroepiandrosterone sulfate (Km of approximately 5 microM), were also investigated. The results establish the polyspecific nature of Oatp1 in a mammalian expression system and definitely identify conjugated dihydroxy bile salts and steroid conjugates as high-affinity endogenous substrates of Oatp1. (+info)
Molecular cloning and characterization of a new multispecific organic anion transporter from rat brain.
A cDNA encoding the new member of the multispecific organic anion transporter family, OAT3, was isolated by the reverse transcription-polymerase chain reaction cloning method. Degenerate primers were designed based on the sequences conserved among OAT1, OAT2, and organic cation transporter 1 (OCT1), and reverse transcription-polymerase chain reaction was performed using rat brain poly(A)+ RNA. The 536-amino acid protein sequence encoded by OAT3 showed 49, 39, and 36% identity to those of OAT1, OAT2, and OCT1, respectively. Northern blot analysis revealed that rat OAT3 mRNA is expressed in the liver, brain, kidney, and eye. When expressed in Xenopus laevis oocytes, OAT3 mediated the uptake of organic anions, such as p-aminohippurate (Km = 65 microM), ochratoxin A (Km = 0.74 microM), and estrone sulfate (Km = 2.3 microM) and a cationic compound, cimetidine. OAT3-mediated uptake of [3H]estrone sulfate was sodium-independent. para-Aminohippuric acid, estrone sulfate or ochratoxin A did not show any trans-stimulatory effect on either influx or efflux of [3H]estrone sulfate via OAT3. Organic anions such as sulfobromophthalein, probenecid, indocyanine green, bumetanide, piroxicam, furosemide, azidodeoxythymidine, 4, 4'-diisothiocyanostilbene-3,3'-disulfonic acid, and benzylpenicillin inhibited OAT3-mediated estrone sulfate uptake, while ouabain and digoxin did not. Organic cations such as tetraethylammonium, guanidine, verapamil, and quinidine did not interact with OAT3. Acidic metabolites of neurotransmitters derived from dopamine, epinephrine, norepinephrine, and serotonin inhibited the uptake of estrone sulfate via OAT3. These results suggest an important role of OAT3 in the excretion/detoxification of endogenous and exogenous organic anions, especially from the brain. (+info)
Reproducibility and validity of radioimmunoassays for urinary hormones and metabolites in pre- and postmenopausal women.
The reproducibility of RIAs of circulating sex hormones has been evaluated as part of recent epidemiological investigations, but none seem to have addressed the reproducibility or validity of RIAs for urinary hormones or their metabolites. As part of a case-control study of breast cancer in Asian-American women, 12-h overnight urine samples were obtained, and a methodological study was conducted to identify laboratories capable of assaying urinary hormones. For the reproducibility component of this study, two laboratories with extensive experience in hormone assays measured urinary estrone, estradiol, estriol, pregnanediol glucuronide, and estrone glucuronide using samples from 15 women (5 midfollicular, 5 midluteal, and 5 postmenopausal). Variance estimates from these measurements were used to calculate the laboratory variability (coefficient of variation) and to assess the magnitude of the biological variability among the women in relation to the total variability (intraclass correlation coefficient). For the validity component, urinary estrone, estradiol, and estriol levels were measured in the same samples by gas chromatography-mass spectroscopy in the laboratory of Dr. Herman Adlercreutz (University of Helsinki, Helsinki, Finland). We found that the degree of assay reproducibility differed between the laboratories, but that laboratory variability was usually low compared with the range of hormone values among women, particularly for the estrogens. Values for estrone and estradiol were well correlated among all of the laboratories. For estriol, the RIAs tended to overestimate levels compared with gas chromatography-mass spectroscopy. In one laboratory, assays for pregnanediol glucuronide and estrone glucuronide were consistently reproduced; in the other, the reproducibility of the RIA for pregnanediol glucuronide was problematic, and estrone glucuronide was not measured. Despite some limitations, urinary hormones and their metabolites can be reliably measured by current RIAs in large investigations attempting to link hormone level to disease risk and may be particularly advantageous for studies of postmenopausal women, where serum concentrations of estrone and estradiol are low and assay measurements are not as dependable. (+info)
Fecal estrone sulfate profile in sows during gestation.
The aim of this study was to establish radioimmunoassay (RIA) for fecal estrone sulfate (E1S) and to elucidate changes in fecal E1S during pregnancy in the sow. Fecal E1S was extracted on a commercially available solid phase column, and the E1S fraction obtained was subjected to RIA. The sensitivity of the RIA was 8.5 pg/tube. The intra- and inter-assay coefficients of variation were 8.8-8.9% and 10.7-14.2%, respectively. Mean recovery for E1S added to fecal samples was as high as 95.0%. A significant positive correlation (r = 0.904, n = 147 p < 0.0001) existed between fecal and plasma E1S concentrations. Mean E1S concentration in feces and plasma fluctuated exhibiting two peaks. The first peak of E1S concentration was evident on day 28-32 in feces and on days 26-30 in plasma. The E1S concentration in both feces and plasma remained at baseline levels during mid-pregnancy, but began to rise gradually around days 72-82 and 70-80, in feces and plasma respectively, and reached a peak concentration on days 110-114. Following parturition, the concentration of E1S in plasma declined rapidly, but there was a two-day delay before a decline in fecal E1S. Apart from this two-day delay, changes in fecal E1S were similar to those in plasma E1S. The study indicates that the measurement of E1S in feces could be a useful tool for early pregnancy diagnosis and for monitoring fetal development in sows and gilts. (+info)