Anna Karpeta

Congener-specific action of PBDEs on steroid secretion, CYP17, 17β-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, 17β-hydroxysteroid dehydrogenase (17β-HSD) and CYP19 activity was measured by conversion of P4>A4, A4>T and T to E2, respectively. Protein expression of CYP17, 17β-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 β-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 3β-HSD activity.

Expression of ghrelin and the ghrelin receptor in different stages of porcine corpus luteum development and the inhibitory effects of ghrelin on progesterone secretion, 3β-hydroxysteroid dehydrogenase (3β-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 (3β-honestly significant difference (HSD)) activity, which were measured by the conversion of pregnenolone (P5) to P4, and 3β-HSD protein expression were then analyzed. To assess 3β-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 3β-HSD activity. Inhibitory effects of ghrelin on P4 secretion, 3β-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 3β-HSD suggest potential auto/paracrine regulation by ghrelin in the luteal phase of ovary function.

The 2,2 ,4,4 -tetrabromodiphenyl ether hydroxylated metabolites 5-OH-BDE-47 and 6-OH-BDE-47 stimulate estradiol secretion in the ovary by activating aromatase expression

The aim of the present study was to assess the effect of two hydroxylated BDE-47 metabolites, 5-OH-BDE-47 and 6-OH-BDE-47, on steroidogenesis in the ovary. Both metabolites failed to affect the production of androstenedione and testosterone but increased the secretion of estradiol at all concentrations tested. The increased secretion of estradiol was due to the stimulation of aromatase gene and protein expression. Direct assessment of aromatase activity by dibenzylfluorescein assay and indirect assessment of aromatase activity by measurement of the conversion of testosterone to estradiol confirmed that 5-OH-BDE-47 and 6-OH-BDE-47 stimulate aromatase activity. The aromatase inhibitor CGS 16949A abolished this stimulatory activity and reduced estradiol levels in the control and treatment groups.

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.

Action of Halowax 1051 on Enzymes of Phase I (CYP1A1) and Phase II (SULT1A and COMT) Metabolism in the Pig Ovary

Polychlorinated naphthalenes (PCNs) are a group of organochlorinated compounds exhibiting dioxin-like properties. Previously published data showed the direct action of PCN-rich Halowax 1051 on ovarian follicular steroidogenesis. Taking into consideration that the observed biological effects of PCNs may be frequently side effects of metabolites generated by their detoxification, the aim of this study was to determine the activity and expression of enzymes involved in phase I (cytochrome P450, family 1 (CYP1A1)) and phase II (sulfotransferase (SULT1A) and catechol-O-methyltransferase (COMT)) detoxification metabolism. Cocultures of granulosa and theca interna cells collected from sexually mature pigs were exposed to 1 pg/mL to 10 ng/mL of Halowax 1051 for 1 to 48 hours, after which levels and activities of CYP1A1, SULT1A, and COMT were measured. Dose-dependent increases of CYP1A1 activity and expression were observed. High doses of Halowax 1051 were inhibitory to COMT and SULT1A activity and reduced their protein levels. In conclusion, fast activation of phase I enzymes with simultaneous inhibition of phase II enzymes indicates that the previously observed effect of Halowax 1051 on follicular steroidogenesis may partially result from metabolite action occurring locally in ovarian follicles.

Different action of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and its hydroxylated metabolites on ERα and ERβ gene and protein expression.

In our previously published data we showed that PBDEs act as endocrine disruptors in ovarian follicles by altering steroid secretion. In this study we try to answer a question if BDE-47 and its hydroxylated metabolites (5-OH-BDE-47 and 6-OH-BDE-47) can act as endocrine disruptors in the ovary by changing the expression of the steroid nuclear receptors, estrogen receptor alpha (ERα) and beta (ERβ), androgen receptor (AR), and receptors associated with the metabolism of xenobiotics and steroid hormones, constitutive androstane receptor (CAR) and pregnane X-receptor (PXR), in porcine ovarian follicles. Expression of mRNA was evaluated by real-time PCR, whereas protein level by western blotting. CAR and PXR mRNAs were not expressed in porcine ovarian follicular cells. BDE-47 and its hydroxylated metabolites had no effect on the expression of AR mRNA and protein. Decreased expression of ERβ mRNA and protein under BDE-47 influence and increase both ERα and ERβ gene and protein expression in cells exposed to hydroxylated metabolites was noted. These findings indicate that BDE-47, by altering the ratio of ERα to ERβ toward ERα, and the hydroxylated metabolites of BDE-47, by increase estrogen receptors expression, may result in excessive ovarian exposure to estrogens.

Regulatory Role of Gonadotropins and Local Factors Produced by Ovarian Follicles on In Vitro Resistin Expression and Action on Porcine Follicular Steroidogenesis

ABSTRACT Resistin, a hormone secreted by adipocytes, is thought to be important in reproduction. Our previous study demonstrated resistin expression in porcine ovarian follicles and its direct effect on steroidogenesis. The aim of the current study was to evaluate the effect of gonadotropins and the local ovarian factors, such as insulin-like growth factor type 1 (IGF1) and steroids (progesterone, testosterone, and 17 beta-estradiol), on the expression and secretion of resistin, as well as its steroidogenic action. Porcine ovarian follicles were exposed to follicle-stimulating hormone (FSH) and luteinizing hormone (LH) at 50–150 ng/ml, IGF1 (10–100 ng/ml), and steroids at 10−8 to 10−6 M for 24 h. Then, mRNA, protein expression, and medium concentration of resistin were determined using real-time PCR, Western blot analysis, and ELISA, respectively. In the subsequent experiments, ovarian follicles were exposed to resistin and/or FSH, LH, IGF1, and steroids, and ovarian steroidogenesis was analyzed. Additionally, we examined the direct effect of resistin on the protein expression of receptors for gonadotropins and investigated local factors. The results showed that gonadotropins and steroids have stimulatory effects but that IGF1 has an inhibitory effect on resistin expression and secretion. Resistin decreased gonadotropins and local hormone-induced steroid secretion and inhibited 3beta-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase, and cytochrome P450 aromatase protein expression. Additionally, we demonstrated that resistin increased the expression of receptors for progesterone and testosterone. These findings all show that the expression and function of resistin are regulated by gonadotropins and local factors produced by ovarian follicles.

Different mechanisms of action of 2, 2’, 4, 4’‐tetrabromodiphenyl ether (BDE‐47) and its metabolites (5‐OH‐BDE‐47 and 6‐OH‐BDE‐47) on cell proliferation in OVCAR‐3 ovarian cancer cells and MCF‐7 breast cancer cells

Data concerning the possible action of polybrominated diphenyl ethers (PBDEs) in hormone‐dependent cancer are scarce. Some data showed that PBDEs may directly affect breast cancer cells formation and only one research showed increased proliferation of the OVCAR‐3 cells, but the results are ambiguous and the mechanisms are not clear. There is growing evidence that not only parent compounds but also its metabolites may be involved in cancer development. The present study was, therefore, designed to determine the effect of BDE‐47 and its metabolites (2.5 to 50 ng ml–1) on proliferation (BrdU), cell‐cycle genes (real‐time PCR) and protein expression (Western blot), protein expression of oestrogen receptors (α β), extracellular signal‐regulated kinases 1 and 2 (ERK1/2) and protein kinase Cα (PKCα) in OVCAR‐3 ovarian and MCF‐7 breast cancer cells. In OVCAR‐3 cells, the parent compound stimulated cell proliferation by activating CDK1, CDK7, E2F1 and E2F2. Independent of time of exposure, BDE‐47 had no effect on ERα and ERβ protein expression and ERK1/2 and PKCα phosphorylation. Metabolites had no effect on cell proliferation but increased both ERs protein expression and ERK1/2 and PKCα phosphorylation. In MCF‐7 cells, the parent compound displayed no effect on cell proliferation but decreased ERα and increased ERβ protein expression with concomitant induction of PKCα phosphorylation. Both metabolites increased MCF‐7 cell proliferation, ERK1/2 and PKCα phosphorylation and decreased ERα and ERβ protein expression.We suggest that studies concerning PBDEs with fewer bromine atoms should be continued to understand environmental links to different hormone‐dependent cancers. Copyright

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.

Differences in the mechanisms of action of BDE-47 and its metabolites on OVCAR-3 and MCF-7 cell apoptosis.

Data concerning possible carcinogenic action of polybrominated diphenyl ethers (PBDEs) in hormone‐dependent tissues are limited. Our earlier studies showed that 2,2′,4,4′‐tetrabromodiphenyl ether (BDE‐47) stimulated OVCAR‐3 and MCF‐7 cell proliferation, while its hydroxylated metabolites (5‐OH‐BDE‐47 and 6‐OH‐BDE‐47) increased estrogen receptors protein expression and extracellular signal‐regulated kinase 1/2 and protein kinase Cα phosphorylation in these cell lines. In addition to cell proliferative disorder, a failure in the regulation of apoptosis can also lead to the formation and development of tumors. Therefore, in the present study, we investigated the effect of BDE‐47 and its metabolites (2.5–50 ng ml–1) on the expression of apoptosis regulatory genes and proteins, caspase‐8 and ‐9 activity and DNA fragmentation induced by extracellular signal‐regulated kinase inhibitor (PD098059) and protein kinase Cα inhibitor (Gӧ 6976) in ovarian (OVCAR‐3) and breast (MCF‐7) cancer cells. In OVCAR‐3 cells, BDE‐47 upregulated expression of most of the investigated genes and increased protein expression of tumor necrosis factor (TNF)‐α, TNF receptor 1, caspase‐6, Bcl‐xl and caspase‐8 activity. Whereas in MCF‐7 cells, BDE‐47 resulted in the downregulation of most of the investigated genes, and decreased caspase‐8 and ‐9 activity. In both OVCAR‐3 and MCF‐7 cells, the expression of most of the investigated genes were downregulated by metabolites. Exposure of OVCAR‐3 cells to 5‐OH‐BDE‐47 corresponded with a decrease in the protein expression of caspase‐6, caspase‐9 and Bcl‐xl and treatment with 6‐OH‐BDE‐47 decreased Bcl‐xl and TNF receptor 1 expression in OVCAR‐3 cells and caspase‐9 expression in MCF‐7 cells. Hydroxylated metabolites of BDE‐47 have strong inhibitory effects on apoptosis in ovarian and breast tumor cells and thus should be considered potential carcinogens in hormone‐dependent cancers. Copyright