Effects of 2,2',4,4'-tetrabromodiphenyl ether (BDE47) on the enzymes of phase I (CYP2B1/2) and phase II (SULT1A and COMT) metabolism, and differences in the action of parent BDE-47 and its hydroxylated metabolites, 5-OH-BDE-47 and 6-OH-BDE47, on steroid secretion by luteal cells.

UNLABELLED: In this study we determined the effects of BDE-47 on the expression and activity of phase I (CYP2B1/2) and phase II (SULT1A and COMT) enzymes, and assessed the actions of BDE-47 and its metabolites on luteal steroidogenesis. Luteal cells collected during early (ELP), middle (MLP) and late (LLP) luteal phase were exposed to BDE-47 (0.5, 25, and 50ng/ml) or metabolites (2.5, 5 and 25ng/ml). BDE-47 decreased CYP2B1/2 activity and expression but had no effect on SULT1A or COMT. BDE-47 exerted a stimulatory action on estrogen secretion in MLP and an inhibitory in LLP, but had no effect on progesterone secretion. 5-OH-BDE-47 and 6-OH-BDE-47 decreased progesterone, but had no effect on estrogen secretion. CONCLUSIONS: The inhibitory effect of BDE-47 on CYP2B1/2 suggests the possibility of BDE-47 accumulation in the corpus luteum; by affecting steroid secretion and steroidogenesis enzymes, BDE-47 and its metabolites can be responsible for shortening luteal phase.

Hexachlorobenzene and pentachlorobenzene accumulation, metabolism and effect on steroid secretion and on CYP11A1 and CYP19 expression in cultured human placental tissue.

Hexachlorobenzene and pentachlorobenzene accumulation and the effect on CYP1A1, SULT1A, COMT and steroid secretion in term placental tissue were determined. Explants of placental tissue were exposed to between 0.02 and 2 ng/ml HCBz or PeCBz for 6-72 h. Accumulation was measured by capillary gas chromatography and quadrupole mass spectrometry. CYP1A1, SULT1A, COMT activity and progesterone secretion were analysed by EIA. Protein expression was quantified by Western blot; 6% HCBz and 7% PeCBz were detected in the tissue. Fast induction of CYP1A1 activity and protein expression in the presence of HCBz were observed. HCBz increased, while PeCBz decreased COMT protein expression. The stimulatory effect of HCBz, and the inhibitory of PeCBz on progesterone secretion and CYP11A1 protein expression were noted. Later activation of CYP1A1, inhibition of COMT protein expression and progesterone secretion by PeCBz suggest greater exposure to PeCBz and pointing at PeCBz as the main factor responsible for the disruption of placental function.

Activation of the enzymes of phase I (CYP2B1/2) and phase II (SULT1A and COMT) metabolism by 2,2',4,4'-tetrabromodiphenyl ether (BDE47) in the pig ovary.

The aim of the current study was to determine metabolism of polybrominated diphenyl ether (BDE-47) in the porcine ovary. We analyzed the activity and expression of enzymes involved in phase I (CYP1A1 and CYP2B1/2) and phase II (SULT1A and COMT) of BDE-47 metabolism. Basal CYP1A1 and CYP2B1/2 activity increased during culture. BDE-47 had no effect on CYP1A1, however increased CYP2B1/2 activity after exposure for 6h. Basal SULT1A activity was 2.5 fold lower than that of COMT, and both proteins were stable during culture. BDE-47 increased SULT1A after exposure for 6 h, and COMT activity after exposure for 24 and 48 h. BDE-47 had no effect on the expression of all investigated enzymes. In conclusion, fast activation of CYP2B1/2 and late activation of COMT (with a very low basal SULT1A activity) indicates a possible action of locally produced hydroxylated metabolites prior to their detoxification.

Expression of ghrelin and the ghrelin receptor in different stages of porcine corpus luteum development and the inhibitory effects of ghrelin on progesterone secretion, 3ß-hydroxysteroid dehydrogenase (3ß-honestly significant difference (HSD)) activity and protein expression.

Recent studies have suggested that ghrelin plays a direct role in controlling female reproduction. The aim of the present study was to investigate the mRNA and protein expression of ghrelin and its receptor (via real time PCR, Western blot and immunohistochemistry analysis, respectively) in porcine corpora lutea (CL) collected during early (CL1: 1-2 days after ovulation), middle (CL2: 7-10 after ovulation), and late luteal phase (CL3: 13-15 after ovulation). Ghrelin expression and concentration of both acylated and unacylated forms of ghrelin significantly increased during CL development. Immunohistochemistry analysis shown localization of ghrelin protein in the cytoplasm of large luteal cells. No changes in the expression of the ghrelin receptor were observed. Direct in vitro effects of ghrelin on progesterone (P4) secretion and 3-beta-hydroxysteroid dehydrogenase (3ß-honestly significant difference (HSD)) activity, which were measured by the conversion of pregnenolone (P5) to P4, and 3ß-HSD protein expression were then analyzed. To assess 3ß-HSD activities, mature luteal cells were first cultured for 24 h with ghrelin at 100, 250, 500 and 1000 pg/mL with P5, or with aminoglutethimide (AMG). AMG is an inhibitor of CYP11A1-mediated hydroxylation; an addition of AMG and P5 enabled P4 production to serve as an index of 3ß-HSD activity. Inhibitory effects of ghrelin on P4 secretion, 3ß-HSD activity and protein expression were observed. In conclusion, the presence of ghrelin and its receptor in porcine corpora lutea and the direct inhibitory effects of ghrelin on luteal P4 secretion and 3ß-HSD suggest potential auto/paracrine regulation by ghrelin in the luteal phase of ovary function.

Congener-specific action of PBDEs on steroid secretion, CYP17, 17ß-HSD and CYP19 activity and protein expression in porcine ovarian follicles.

The available data on reproductive toxicity of PBDEs are limited. In the present study we evaluated the direct effects of BDE-47, -99 and -100 on porcine ovarian follicular steroid secretions and the activity and expression of enzymes involved in its synthesis. Follicles were exposed to BDE-47 (0.5, 25 and 50 ng/ml), BDE-99 (0.25, 10 and 17.5 ng/ml), or BDE-100 (0.1, 4 and 12.5 ng/ml) for 24h. Progesterone (P4), androstenedione (A4), testosterone (T) and estradiol (E2) levels in the media were determined by EIA. CYP17, 17ß-hydroxysteroid dehydrogenase (17ß-HSD) and CYP19 activity was measured by conversion of P4>A4, A4>T and T to E2, respectively. Protein expression of CYP17, 17ß-HSD and CYP19 was measured by western blot. All of the congeners explored in this study increase testosterone secretion. However, in the case of BDE-47 due to activation of 17 ß-HSD and BDE-100 due to activation of CYP17, a corresponding failure to activate CYP19 expression and inhibition of CYP19 activity was seen. The lack of an effect of BDE-99 on the expression and activity of all of the investigated enzymes indicates action on enzymes before progesterone secretion, i.e., STAR or 3ß-HSD activity.

Mixture of dominant PBDE congeners (BDE-47, -99, -100 and -209) at levels noted in human blood dramatically enhances progesterone secretion by ovarian follicles.

OBJECTIVE: Polybrominated diphenyl ethers (PBDEs) are used as flame retardants in a wide variety of products. They show an accumulation pattern similar to that of well-known persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs), DDT and others. So far, most of information showing adverse effects of various POPs was based on the exposure to single chemicals. However, in this study we intended to evaluate in vitro effect of the mixture of four most abundant PBDE congeners (PBDE-47, -99, -100 and -209) as usually found in wildlife and humans on the secretion of gonad hormones by ovarian follicles. METHODS: Cocultures of theca and granulosa cells collected from medium size follicles of cycling pigs were exposed for 24 or 48 h to three different levels of respective PBDE congeners mixture: 1. low level (0.5; 0.25; 0.1; 0.05 ng/ml); 2. medium level (25; 10; 4.0; 0.25 ng/ml) and high level (50; 17.5; 12.5; 0.5 ng/ml). Additionally, to show if the effect on steroid secretion by ovarian follicles is reversible, the cells were cultured for 24 h with the reagents and, after the medium was exchanged, the cells were cultured without reagents for additional 48 h. At the end of each culture procedure, progesterone (P4), testosterone (T) and estradiol (E2) secretion in the culture medium were determined by ELISA. RESULTS: After short term exposure increased secretion of all investigated steroids was noted only under the influence of small dose of mixture. However, after longer time of exposure the stimulation of estradiol and testosterone secretion was observed under the influence of both the low and medium dose of mixture, while 3.0-, 6.8- and 7.2-fold increase in progesterone secretion was noted under the influence of low, medium and high doses of mixture, respectively. CONCLUSION: Taken together, our findings from this and previous studies allow several interrelated conclusions to be drawn. First, the increase of P4/T ratio with a parallel decrease of T/E2 ratio was found suggesting preterm luteinization in antral follicles followed by the disruption of ovulation and, second, it was not possible to reverse the steroidogenic effects of PBDEs mixture by removing reagents from the cell cultures.