Difference in receptor-binding contributes to difference in biological activity between the unique guinea pig GnRH and mammalian GnRH.

In the current study, we compared the in vitro potency of a unique form of gonadotropin-releasing hormone (GnRH) present in the brain of the guinea pig (gpGnRH) with that of mammalian GnRH (mGnRH) as well as their binding affinities to the GnRH receptor. In gpGnRH, the highly conserved histidine in position 2 (His(2)) and leucine in position 7 (Leu(7)) are substituted by tyrosine and valine, respectively. In vivo, gpGnRH was shown to be less potent than mGnRH, possibly in part because of higher susceptibility to enzymatic degradation. In the present in vitro experiments, we observed that gpGnRH was less potent than mGnRH in stimulating the release of luteinizing hormone (LH) from primary pituitary cell cultures of the rat, and at lower concentrations from primary pituitary cell culture of the guinea pig, too. These results were confirmed by radioligand-binding studies. It is concluded that the lower biological activity of gpGnRH in both rat and guinea pig may be explained by the difference in binding to the target cells, although additional factors such as proteolytic degradation may also contribute to the observed phenomenon.

Further evaluation of the biological activity of the unique gonadotropin-releasing hormone peptide in the guinea pig brain.

In this study we compared the biological activity of a unique form of gonadotropin-releasing hormone (GnRH) in the brain of the guinea pig (gpGnRH) with mammalian GnRH (mGnRH). In gpGnRH, the highly conserved histidine in position 2 (His(2)) and leucine in position 7 (Leu(7)) are substituted by tyrosine and valine, respectively. The gpGnRH was less potent than mGnRH in stimulating the release of luteinizing hormone (LH) in vivo in the guinea pig and displayed only low activity in the rat. The gpGnRH was more rapidly degraded by serum proteolytic enzymes than mGnRH. It is concluded that gpGnRH displays lower biological activity than mGnRH in both rat and guinea pig, which may be due in part to its greater susceptibility to proteolytic degradation besides differences in receptor affinity and/or activation.

Internal exposure to pollutants and sexual maturation in Flemish adolescents.

Flanders is densely populated with much industry and intensive farming. Sexual maturation of adolescents (aged 14-15 years) was studied in relation to internal exposure to pollutants. Serum levels of pollutants and sex hormones were measured in 1679 participants selected as a random sample of the adolescents residing in the study areas. Data on sexual development were obtained from the medical school examination files. Self-assessment questionnaires provided information on health, use of medication and lifestyle factors. In boys, serum levels of hexachlorobenzene (HCB), p,p'-DDE and polychlorinated biphenyls (sum of marker PCB138, 153 and 180) were significantly and positively associated with pubertal staging (pubic hair and genital development). Higher levels of serum HCB and blood lead were associated with, respectively, a lower and a higher risk of gynecomastia. In girls, significant and negative associations were detected between blood lead and pubic hair development; higher exposure to PCBs was significantly associated with a delay in timing of menarche. Environmental exposures to pollutants at levels actually present in the Flemish population are associated with measurable effects on pubertal development. However, further understanding of toxic mode of action and sensitive windows of exposure is needed to explain the current findings.

Internal exposure to pollutants and sex hormone levels in Flemish male adolescents in a cross-sectional study: associations and dose-response relationships.

Flanders is densely populated with much industry and intensive farming. Hormonal status of 14- to 15-year-old male adolescents was studied in relation to internal exposure to pollutants. A total of 887 participants were selected as a random sample of the adolescents residing in the study areas. Confounding factors and significant covariates were taken into account. Serum levels of testosterone, free testosterone and estradiol, and the aromatase index showed significant positive associations with serum levels of marker polychlorobiphenyls (sum of PCBs 138, 153, and 180) and of hexachlorobenzene (HCB) and a negative association with urinary cadmium concentration. Serum levels of estradiol also showed a positive association with serum levels of dichlorodiphenyldichloroethylene (DDE). A doubling of serum concentrations of marker PCBs and HCB and of urinary concentration of cadmium were, respectively, associated with an increase of 16.4% (P<0.00001) and 16.6% (P<0.001) and a decrease of 9.6% (P<0.001) in serum testosterone concentration. Similar findings were made after additional adjustment for concurrent exposures. Associations between biological effects and internal exposures were, in terms of the regression coefficient, often stronger at exposures below the median. Environmental exposures to pollutants resulting in "normal" levels of internal exposure were associated with quite substantial differences in hormone concentrations.

Internal exposure to pollutants and body size in Flemish adolescents and adults: associations and dose-response relationships.

Flanders is densely populated with much industry and intensive farming. Body size of 14- to 15-year old adolescents and of adults aged 50-65 was studied in relation to internal exposure to pollutants. 1679 adolescents (887 boys and 792 girls), 775 men and 808 women were selected as a random sample of the population. Concentrations of pollutants in blood or urine were measured in accordance with quality control/quality assurance procedures. Self-assessment questionnaires provided information on personal and life-style factors. Height and weight of subjects were measured. Confounding factors and significant covariates were taken into account. For boys and girls, height and body mass index (BMI) showed a negative association with urinary concentration of cadmium and BMI also with serum concentration of hexachlorobenzene (HCB) and with the sum of serum concentrations of polychlorinated biphenyls (PCBs) 138, 153, and 180 (marker PCBs), whereas BMI showed a positive association with serum concentration of PCB 118. For boys, height showed a negative association with urinary concentration of 1-hydroxypyrene (1-OHP) and positive associations with serum concentrations of HCB and PCB 118. For adults no significant associations between internal exposure and height were observed. For men, BMI showed negative associations with urinary cadmium concentration and with serum levels of marker PCBs and positive associations with serum levels of HCB, p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE), PCB 118 and the dioxin fraction of dioxin-like activity. For women, BMI showed a negative association with urinary cadmium concentration, with blood lead concentration and with the concentration of marker PCBs in serum, and a positive association with serum concentrations of HCB, p,p'-DDE and PCB 118. Associations between biological effects and internal exposures were, in terms of the regression coefficient, often stronger at exposures below the median. Environmental exposures to pollutants resulting in "normal" levels of internal exposure were associated with quite substantial differences in body mass index.

Endocrine disruptors and abnormalities of pubertal development.

Onset and development of puberty is regulated by the neuroendocrine system. Population-based studies worldwide have observed secular trends towards earlier puberty development. These changes are apparently caused by environmental factors such as improved socio-economic status, improved health care and nutrition. However, they may also partly result from endocrine-disrupting chemicals in the environment. Epidemiological studies have investigated the relationship between pubertal development and exposure to endocrine-disrupting chemicals (polychlorinated biphenyls, polybrominated biphenyls, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane, phthalate esters, furans and the pesticide endosulfan). Associations with both perinatal and postnatal exposure have been reported. Studies in experimental animals support some of these findings and point to differential endocrine regulatory mechanisms linked to pubertal development acting in the perinatal and the pre-pubertal period. Pubertal development is naturally associated with growth and body composition. There is increasing evidence for a link between prenatal development and pubertal onset. In girls born small for gestational age (SGA), pubertal onset and age at menarche often are advanced, especially if there has been an extensive catch-up growth during the first months of life. In utero growth retardation may have multiple causes including exposure to xenobiotic substances as was suggested for some endocrine-disrupting chemicals. An abnormal perinatal environment of children born SGA may alter the endocrine status and the sensitivity of the receptors for endocrine and metabolic signalling that may have effects on maturation of brain and gonads. However, the causal pathways and the molecular mechanisms that may link the pubertal growth pattern of children born SGA, pubertal development and endocrine-disrupting chemicals need further study.

Reproductive parameters of community-dwelling men from 2 regions in flanders are associated with the consumption of self-grown vegetables.

Although regional differences in semen parameters have been described, little is known about the etiologic factors underlying these variations in male fertility status. We previously reported people from a rural area (Peer) in Flanders to have lower sperm parameters and free testosterone than men from the city of Antwerp. In the present study, our objectives were to investigate to what extent these differences were associated with lifestyle or environmental factors. People in Peer were slightly older and had a higher body mass index, factors known to affect testosterone concentrations but not sperm parameters. People consuming locally produced vegetables (n = 37 of 94) but not fruit had significantly lower serum free testosterone and luteinizing hormone (LH) (both P = .04) and nonsignificantly lower follicle-stimulating hormone (FSH) (P = .05). Per unit increase of monthly intake of locally produced vegetables, free testosterone declined by 0.7% (P = .01) and sperm concentration by 2.3% (P = .04) over the whole range of the explanatory variable, whereas LH declined by 3.6% (P = .02), FSH declined by 3.5% (P = .08), and sperm morphology by 7% (P = .002) in the range of 0-10 consumptions per month. No relationship was found with lifelong exposure to cadmium. These results support a hypothesis of impaired gonadotropic signaling causing the regional difference in reproductive parameters. The surprising strong impact of self-grown vegetable consumption did not seem to be related to soil contamination by cadmium. We could not exclude pesticide exposure by inappropriate application or other factors such as nutritional deficiency, physical activity, or stress as contributors to the observed regional differences.

Estrogen receptor signaling is an unstable feature of the gonadotropic LbetaT2 cell line.

The murine, gonadotropic LbetaT2 cell line was assessed as a potential in vitro model to analyze estrogen receptor (ER)-mediated regulation of luteinizing hormone (LH) synthesis and secretion. In agreement with limited literature data, repeated exposure to (sub) physiological concentrations of gonadotropin-releasing hormone enhanced LHbeta-subunit gene expression, being the rate-limiting step of LH synthesis, and the corresponding LH secretory response. However, in the same subclone of the LbetaT2 cell line, we observed that LH production was not affected following exposure to E(2), which is in contrast to previously reported weak or modest effects. One explanation may be the absence of measurable ERalpha protein expression on the one hand and impaired ER signal transduction on the other. Furthermore, an alternative ERalpha mRNA splicing variant was detected in the LbetaT2 cell line, which (theoretically) encodes for a protein that may alter ERalpha transcriptional activity, depending on the cellular context. The studied LbetaT2 subclone did not show a generalized impairment of nuclear receptor function, as we observed androgen- and glucocorticoid-induced gene transcription, together with enhanced LH secretory response following dexamethasone treatment. Since its development, the gonadotropic LbetaT2 cell line served as a reference model to study gonadotroph-specific effects because of its mature properties. Nevertheless, this cell line does not seem to be a suitable in vitro model for the study of estrogenic regulatory effects at the level of the pituitary gonadotrophs in view of the unstable nature of ER signaling in LbetaT2 cells.

Estrogen receptor alpha (ERalpha) and insulin-like growth factor I receptor (IGF-IR) cross-talk in the gonadotropic alphaT3-1 cell line.

In reproductive tissues such as the breast and the uterus, cell proliferation and differentiation is strongly regulated by complex interactions between estrogen receptor alpha (ERalpha) and growth factor receptors. In the present study, we investigated the potential occurrence of such cross-talk in the murine, gonadotropic alphaT3-1 cell line, which expresses ERalpha and the IGF-I receptor (IGF-IR). Under estrogen-free conditions, basal cell proliferation and ER-mediated gene transcription was strongly inhibited by the selective estrogen receptor modulator (SERM) 4-hydroxy-tamoxifen (4-OH-Tam) and by the pure anti-estrogen ICI 182,780 (ICI). These effects can be reversed by either 17-beta-estradiol (E(2)) or insulin-like growth factor I (IGF-I), both exerting modest mitogenic effects in the alphaT3-1 cell line. Furthermore, IGF-I enhanced both basal and E(2)-induced ER-driven gene transcription. This may be explained, at least in part, by enhanced phosphorylation of ERalpha at serine 118, a prerequisite for the transactivation capacity of the receptor. Finally, the IGF-I-induced response on cell growth and ER-mediated transactivation can be inhibited with either ICI or 4-OH-Tam. In conclusion, our data indicate IGF-IR and ER interactions in the alphaT3-1 cell line, an in vitro model for the pituitary gonadotrophs, hereby suggesting a role of IGF-I in the regulation of gonadotropin synthesis and secretion.

Regional variations in semen quality of community-dwelling young men from Flanders are not paralleled by hormonal indices of testicular function.

Epidemiological studies of sperm quality are hampered by problems such as low participation rates and poor comparability of results due to methodological differences in semen analysis. More objective sperm quality-related serum markers would facilitate worldwide comparisons of male reproductive status. Our objectives were to investigate to what extent a set of hormonal indices of testicular function, previously established in a clinical setting, could predict regional variations in seminal parameters in men from the general population. We recruited 101 men aged 20-40 years from two regions in Flanders, and assessed their sperm parameters and levels of serum reproductive hormones. In one region compared to another, the participants had lower sperm counts (by 34%; P=.06), lower total sperm counts (by 41%; P=.02) and poorer sperm morphology (by 32%; P<.001), which were paralleled by significantly lower levels of free testosterone (by 11%; P=.03), while for total testosterone (T) and follicle-stimulating hormone (FSH), the differences were not significant (both P=.09) at 10% and 17%, respectively. There were no differences in inhibin B and the T to luteinizing hormone (LH) ratio, which are markers of testicular function. Receiver operating characteristic curve analysis revealed that T:LH, inhibin B, and the inhibin B/FSH ratio had significant discriminatory power between men with sperm concentrations below or above 13.5x10(6)/mL. Regional variations in the semen quality of community-dwelling individuals are not necessarily reflected in altered hormonal indices of testicular function and thus, these markers, validated in clinical settings, are not valid substitutes for the traditional semen quality assessment used in epidemiological population studies.

Subtle side-chain modifications of the hop phytoestrogen 8-prenylnaringenin result in distinct agonist/antagonist activity profiles for estrogen receptors alpha and beta.

In search of therapeutic agents for estrogen-related pathologies, phytoestrogens are being extensively explored. In contrast to naringenin, 8-prenylnaringenin is a potent hop-derived estrogenic compound, highlighting the importance of the prenyl group for hormonal activity. We investigated the effects of substituting the prenyl group at C(8) with alkyl chains of varying lengths and branching patterns on estrogen receptor (ER) subtype ERalpha- and ERbeta-binding affinities and transcriptional activities. In addition, features of the ligand-induced receptor conformations were explored using a set of specific ER-binding peptides. The new 8-alkylnaringenins were found to span an activity spectrum ranging from full agonism to partial agonism to antagonism. Most strikingly, 8-(2,2-dimethylpropyl)naringenin exhibited full agonist character on ERalpha, but pronounced antagonist character on ERbeta. Knowledge on how ER-subtype-selective activities can be designed provides valuable information for future drug or tool compound discovery.

Serum dioxin-like activity is associated with reproductive parameters in young men from the general Flemish population.

BACKGROUND: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and some related environmental contaminants are aryl hydrocarbon receptor (AhR) ligands that exert reproductive and developmental toxicity in laboratory animals. In humans, fertility-related effects are less documented. OBJECTIVE: The aim of this study was to investigate the relationship between dioxin-like biological activity in serum and parameters of reproductive status in men from the general population 5 months after a polychlorinated biphenyl and dioxin food-contamination episode in Belgium. DESIGN: In the framework of the cross-sectional Flemish Environment and Health Study (FLEHS), we recruited 101 men 20-40 years of age and evaluated sperm parameters, measured sex hormones, and gathered information on a number of lifestyle factors. In addition, we determined the AhR-mediated enzymatic response elicited by individual serum samples and expressed it as TCDD equivalent concentrations (CALUX-TEQs) using an established transactivation assay. RESULTS: Age (p = 0.04) and the frequency of fish (p = 0.02) and egg (p = 0.001) consumption were independent positive determinants of serum dioxin-like activity. After correcting for possible confounders, we found that a 2-fold increase in CALUX-TEQ > 16 pg/L was associated with a 7.1% and 6.8% (both p = 0.04) decrease in total and free testosterone, respectively. We also observed a more pronounced drop in semen volume of 16.0% (p = 0.03), whereas sperm concentration rose by 25.2% (p = 0.07). No relationship was found with total sperm count or sperm morphology. CONCLUSIONS: These data suggest an interaction of dioxin-like compounds with the secretory function of the seminal vesicles or prostate, possibly indirectly through an effect on testosterone secretion, at levels not affecting spermatogenesis as such.

Experimental parameters affecting sensitivity and specificity of a yeast assay for estrogenic compounds: results of an interlaboratory validation exercise.

In vitro assays are considered as the first step in a tiered approach to compound screening for hormonal activity. Although many new assays have been developed in recent years, little attention has been paid towards assay validation. Our objective was to identify critical experimental parameters in a yeast estrogen screen (YES) that affect its sensitivity and specificity. We investigated the role of incubation time, solvent type, yeast inoculum growth stage and concentration on the outcome of the YES. Compounds tested included new and established agonists, antagonists and negative controls, and results were evaluated according to prefixed statistical criteria. In addition, we assessed the assay's performance in a blind interlaboratory validation exercise (IVE). An incubation time of five days was necessary to positively identify the estrogenic properties of all agonists tested, when dissolved in DMSO. Longer incubation times were required when using an ethanol protocol. Similar estrogenic activity was reported for benzyl butyl phthalate, bisphenol-A, methoxychlor, permethrin and genistein in the IVE. One out of the three laboratories did not classify alpha,beta-endosulfan, dissolved in DMSO, as an estrogen. The same was true for 4,4'-DDE and lindane, dissolved in ethanol, a result that might be attributable to an inappropriate yeast start concentration and/or growth stage. These validation experiments show that under appropriate experimental conditions the YES yields sensitive, specific and reliable results. Therefore it fulfills the requirements as a first step screening assay to evaluate the capacity of chemicals to interact with the estrogen receptor.

The prenylflavonoid isoxanthohumol from hops (Humulus lupulus L.) is activated into the potent phytoestrogen 8-prenylnaringenin in vitro and in the human intestine.

Hops, an essential beer ingredient, are a source of prenylflavonoids, including 8-prenylnaringenin (8-PN), one of the most potent phytoestrogens. Because 8-PN concentrations in beers are generally low, its health effects after moderate beer consumption were considered negligible. However, human intestinal microbiota may activate up to 4 mg/L isoxanthohumol (IX) in beer into 8-PN. Depending on interindividual differences in the intestinal transformation potential, this conversion could easily increase the 8-PN exposure 10-fold upon beer consumption. Here, we present a further investigation of the process both in vitro and in vivo. In vitro experiments with the dynamic SHIME model showed that hop prenylflavonoids pass unaltered through the stomach and small intestine and that activation of IX into 8-PN (up to 80% conversion) occurs only in the distal colon. In vitro incubations of 51 fecal samples from female volunteers with IX enabled us to separate the fecal microbiota into high (8 of 51), moderate (11 of 51) and slow (32 of 51) 8-PN producers, clearly illustrating an interindividual variability. Three women, selected from the respective groups, received a daily dose of 5.59 mg IX for 4 d. Intestinal IX activation and urinary 8-PN excretion were correlated (R(2) = 0.6417, P < 0.01). These data show that intestinal conversion of IX upon moderate beer consumption can lead to 8-PN exposure values that might fall within the range of human biological activity.

Regioselective synthesis and estrogenicity of (+/-)-8-alkyl-5,7-dihydroxy-4-(4-hydroxyphenyl)-3,4-dihydrocoumarins.

Nine new (+/-)-8-alkyl-5,7-dihydroxy-4-(4-hydroxyphenyl)-3,4-dihydrocoumarins have been synthesized from 2,4,6-trimethoxybenzaldehyde via a short, efficient, and regioselective pathway, together with the unsubstituted analogue (+/-)-5,7-dihydroxy-4-(4-hydroxyphenyl)-3,4-dihydrocoumarin. The compounds were tested for estrogenic activity using a yeast-based estrogen screen. Weak estrogenicity was determined for seven members of the series.

Advanced water treatment with manganese oxide for the removal of 17alpha-ethynylestradiol (EE2).

Municipal wastewater is supposed to be one of the most important sources of endocrine-disrupting compounds (EDCs) in water. Therefore, advanced treatments and cost-efficient techniques should be developed to prevent the spread of this type of pollution into the environment. In this view, experiments were conducted in which the removal of 17alpha-ethynylestradiol (EE2), a synthetic and persistent estrogen, from water was monitored in three upstream bioreactors (UBRs), filled with, respectively, sand, granulated activated carbon (GAC) and MnO(2) granules. Tap water, spiked with 15,000ngEE2/L was filtered through the reactors with a hydraulic retention time of approximately 1h. The removal of EE2 in the sand, GAC and MnO(2) reactors was, respectively, 17.3%,>99.8% and 81.7%. The removal in the GAC reactor was mainly due to adsorption. The MnO(2) reactor, however, removed significantly more EE2 than could be predicted from its adsorption capacity, probably thanks to its catalytic properties. These catalytic properties could make it a cost-efficient technique for the removal of EE2, but further research at more environmentally relevant concentrations is needed.