Ewa L. Gregoraszczuk

Effect of bisphenol-A on the expression of selected genes involved in cell cycle and apoptosis in the OVCAR-3 cell line

To support the argument that bisphenol-A (BPA) poses a risk for ovarian cancer, OVCAR-3 cell line was exposed to environmentally relevant concentration of BPA. Expression of selected genes involved in cell cycle and apoptosis were evaluated by real-time PCR. In a dose-dependent manner, BPA increased OVCAR-3 cell proliferation and decreased caspase-3 activity, but it had no effect on DNA fragmentation. We noted 1.2-1.5-fold induction of genes responsible for inducing cell proliferation and 1.2-46-fold suppression of genes responsible for inhibition of proliferation. Moreover, 1.6-8-fold suppression of genes involved in the extrinsic apoptotic pathway was observed. In parallel, 1.3-2.5-fold suppression pro-apoptotic genes and 1.6-51-fold induction of pro-survival genes involved in the intrinsic apoptotic pathway were observed. Additionally, 1.7-fold induction of p53 and 5-fold induction of endonuclease G genes involved in CAD-independent DNA fragmentation were noted under the influence of BPA. In conclusion, we hypothesize that induction of p53 and suppression of caspase-3 and 7 gene expression observed in this study activate the DNA repair process. Therefore, despite the observed induction of endo G gene expression, the action of BPA on DNA fragmentation was not observed.

Bisphenol A induces leptin receptor expression, creating more binding sites for leptin, and activates the JAK/Stat, MAPK/ERK and PI3K/Akt signalling pathways in human ovarian cancer cell

We previously demonstrated that bisphenol A (BPA) promotes proliferation in OVCAR-3 human ovarian cancer cells. This study was designed to investigate the effects of BPA on leptin expression and activity in ovarian cancer. Real-time PCR, Western blot analysis and ELISA assays were used to quantify leptin receptor expression and leptin gene and protein expression after treatment with BPA at doses of 0.2, 2, 8 and 20ng/ml. Our data reveal leptin receptor expression but an absence of leptin gene and protein expression in OVCAR-3 cells. At doses of 8 and 20ng/ml, BPA had stimulatory effects on leptin receptor gene and protein expression. Leptin and BPA alone stimulated cell proliferation but BPA did not potentiate leptin activity. Similarly to leptin, but with different kinetics and duration, BPA induced phosphorylation of Stat3, ERK1/2 and Akt. In co-treatment experiments, the timing of protein phosphorylation represented an additive effect of BPA and leptin treatment. In conclusion, taking into consideration limitation of in vitro study, whether BPA by creating more binding sites for leptin and extending the time of leptin-induced Stat3, ERK1/2 and Akt phosphorylation, can potentiated leptin action in cancer cells, require confirmation by in vivo study.

Valproate inhibits the conversion of testosterone to estradiol and acts as an apoptotic agent in growing porcine ovarian follicular cells.

Summary:  Purpose: Long‐term valproate (VPA) treatment has been associated with hyperandrogenism and polycystic ovaries in women with epilepsy. The exact mechanisms of action of the drug on sex steroid hormone function are still unsettled. The aim of the present study was to investigate the action of VPA on basal and gonadotropin‐stimulated steroid secretion in porcine ovarian follicular cells and to measure the conversion of testosterone to estradiol. Second, the action of VPA on proliferation and apoptosis of follicular cells was investigated.

Valproate-induced alterations in testosterone, estradiol and progesterone secretion from porcine follicular cells isolated from small- and medium-sized ovarian follicles

The aim of the present study was to investigate whether long-term exposure to valproate (VPA) alters follicular steroidogenesis and whether or not this effect is dependent on the degree of follicular development. Small- and medium-sized follicles were obtained from pig ovaries collected, respectively, at days 8-10 and 14-16 of oestrus cycle. Theca interna and granulosa cells were isolated from follicles and placed in the same well in the ratio 1 : 3 with or without the VPA in doses of 100, 300 and 500 micro g ml(-1). The culture medium was changed after 2, 4, 6 and 8 days. In both types of follicles, VPA caused a significant and dose-dependent reduction in both testosterone and estradiol secretion from follicular cells. In small-sized follicles, the testosterone to oestrogen ratio increased at all doses used and after all lengths of time in culture. In medium-sized follicles, a significant increase in the testosterone to oestrogen ratio was only observed at the highest dose level. All doses of VPA caused a marked inhibition of progesterone secretion after 48 hours while during long-term VPA exposure progesterone gradually increased demonstrating luteinization of cells. In conclusion, the present study demonstrates a direct effect of VPA on steroidogenesis. The effect seems to differ to some extent depending on the follicular stage of development. The elevated ratio of testosterone to estradiol suggests that VPA inhibits the conversion of testosterone to estradiol.

Endocrine-Disrupting Chemicals: Some Actions of POPs on Female Reproduction

Persistent organic pollutants (POPs), such as polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and polybrominated ethers (PBDEs), chloronaftalens (PCNs), and bisphenol A (BPA), are stable, lipophilic pollutants that affect fertility and cause serious reproductive problems, including ovotoxic action, lack of ovulation, premature ovarian failure (POF), or polycystic ovarian syndrome (PCOS). Most of the representatives of POPs influence the activation of transcription factors, not only activation of aromatic hydrocarbon receptor (AhR), but also the steroid hormone receptors. This minireview will focus on a variety of PAH activities in oocyte, ovary, placenta, and mammary gland. The complexity and diversity of factors belonging to POPs and disorders of the reproductive function of women indicate that the impact of environmental pollution as an important determinant factor in fertility should not be minimize.

Congener-specific accumulation of polychlorinated biphenyls in ovarian follicular wall follows repeated exposure to PCB 126 and PCB 153. Comparison of tissue levels of PCB and biological changes

OBJECTIVE Small (SF), medium (MF) and large (LF) preovulatory porcine follicles were isolated and incubated in an Erlenmeyer flask containing 5 ml of medium with addition of PCB 126 or PCB 153 to test differences in their accumulation in the follicular wall. METHODS; The follicles were incubated in M199 medium at 37 degrees C with constant shaking at 70 rpm, for 6 days. The media were changed every day and repeated dose 25 pg/ml of PCB 126 or 25 ng/ml of PCB 153 was added each day till 6 days of culture. Media were collected every day and frozen for steroid analysis by RIA. 24 h after the last treatment follicles were frozen for further polychlorinated biphenyls (PCB) content analysis. PCB concentrations in the follicular wall were analysed by mass spectrometry. RESULTS 3.3%; 3.6% and 5.6% of total PCB 126 dose, and 71%; 71.4% and 30.4% of total PCB 153 dose accumulated in SF, MF and LF follicles, respectively. The accumulative effect of PCB was manifested by the disruption of estradiol (E2) secretion. In SF antiestrogenic action of PCB 126 was observed during the whole time of exposure while PCB 153 decreased E2 till 4 days of culture and then estrogenic action was observed. In MF, both these congeners decreased E2 till 5 days of exposure and then estrogenic actions were noted with the highest magnification in the case of PCB 126. In LF both PCB studied increased E2 till 3 days of exposure with the highest magnification of PCB 126, then antiestrogenic action was noted. Testosterone secretion was generally affected in a pattern oposite to that of E2 suggesting action on P450arom activity. CONCLUSION The results of these studies demonstrated that disruption of aromatization process in the follicles following repeated exposure to both congeners is not directly correlated with the bioaccumulation or amount of PCB within the follicular wall.

Dioxin exposure and porcine reproductive hormonal activity

To characterize the action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during both the follicular and luteal phases of the ovarian cycle, the direct effect of TCDD was investigated in vitro using a system of primary monolayer cell culture. Granulosa and theca cells were collected from the preovulatory follicles and cultured as a co-culture, thus resembling follicles in vivo. Luteal cells were isolated from the corpora lutea collected during the midluteal phase. In both cases cells were isolated from the ovaries of animals exhibiting natural estrus cycle. Results of these experiments suggest that TCDD decreases estradiol secretion by follicular cells and progesterone secretion by luteal cells in a dose-dependent manner. It was also shown that TCDD disrupts steroidogenesis through its influence on the activity of enzymes involved in the steroid biosynthesis cascade. In luteal cells, its action is mediated via the aryl hydrocarbon receptor (AhR) and is probably independent of estrogen receptor (ER) stimulation. Endocrine disruptors that interfere with estradiol production in the follicles can act as ovulatory disruptors, and while interfering with progesterone production by luteal cells they can act as abortifacients.

Estrogenic and antiestrogenic effect of in vitro treatment of follicular cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

OBJECTIVE Two types of follicular cells from preovulatory ovary were cultured in vitro separately and in co-culture to test difference in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) action on particular cell types. METHODS The accumulation of TCDD in follicular wall was analysed using coupled capillary gas chromatography mass spectrometry. Whole preovulatory follicles were isolated from ovary and incubated with prolonged exposure to 0.1 nM TCDD or single exposure to 10 nM TCDD for four days. In the second part of experiments direct effects of TCDD on steroidogenesis were investigated in porcine theca cells (Tc) and granulosa cells (Gc) cultured alone and in co-culture (GT). The media were collected after four days for steroid analysis. RESULTS 59.3% and 81.2% of TCDD added to the culture medium was accumulated after 0.1 and 10 nM, respectively. TCDD in a dose-dependent manner increased estradiol secretion with concomitant progesterone secretion by theca interna cells. On the other hand decrease of both progesterone and estradiol secretion by granulosa cells cultured alone and in co-culture with theca cells was noted. CONCLUSION Different cell-specific estrogenic or antiestrogenic effect of TCDD were found in ovarian follicles.

Response of porcine theca and granulosa cells to GH during short-term in vitro culture

In Experiment 1, the influence of exogenous GH on steroid secretion by granulosa and theca interna cells recovered from small (1-3 mm), medium (4-6 mm) and large (8-12 mm) follicles was tested. In the second experiment, theca cells (Tc) and granulosa cells (Gc) obtained from large follicles were cultured separately or in two types, Tc/Gc co-culture, where both types of cells were mixed in one well or Gc and Tc were separated by cell culture membrane inserts. In the third experiment, the influence of GH on the morphology of Gc and Tc cells and activity of Delta(5),3beta-hydroxysteroid dehydrogenase (3beta-HSD) was studied. Cells were grown in the control medium (M199+5% of calf serum) or supplemented with 100 ng/ml GH. Testosterone (10(-7) M) was added as the aromatase substrate to granulosa cells cultures. The media were assayed after 48 h of culture for progesterone and oestradiol by RIA. GH added to the culture media had no effect on oestradiol and progesterone secretion by granulosa cells isolated from small and medium follicles while it stimulated both oestradiol and progesterone secretion by Gc isolated from large preovulatory follicles. A stimulatory effect on oestradiol secretion by Tc isolated from all size follicles was observed. GH did not stimulate progesterone secretion by Tc isolated from small follicles but stimulated progesterone secretion by Tc isolated from medium and large preovulatory follicles. Both co-culture systems exhibited synergistic effect on oestradiol secretion. The stimulatory effect on progesterone secretion under the influence of GH was observed in Gc cultured alone and Tc cultured alone. In contrast, the secretion of progesterone was attenuated in both co-culture systems and the addition of GH further augmented this attenuation. A statistically significant increase in oestradiol secretion was observed in all culture conditions. The addition of GH to the culture medium stimulated the activity of 3beta-HSD compared with the control culture from both types of cells. In conclusion, the present studies indicate that there are direct and follicular development stage dependent actions of GH on steroidogenesis of porcine follicular cells.

The Toxicological Effects of Halogenated Naphthalenes: A Review of Aryl Hydrocarbon Receptor-Mediated (Dioxin-like) Relative Potency Factors

There is no doubt that chloronaphthalenes (PCNs) and their brominated counterparts (PBNs) are dioxin-like compounds, but there is less evidence for mixed bromo/chloronaphthalenes (PXNs). In this article we review information relating to the dioxin-like potency of PCNs and PBNs obtained in vivo, in vitro, and in silico. The aim was to help and improve the quality of data when assessing the contribution of these compounds in the risk analysis of dioxin-like contaminants in foods and other sample types. In vivo and in vitro studies have demonstrated that PCN/PBN congeners are inducers of aryl hydrocarbon hydroxylase, ethoxyresorufin O-deethylase, and luciferase enzymes that are features specifically indicative of planar diaromatic halogenated hydrocarbons such as dioxin and dioxin-like compounds. PCNs in the environment are of multisource origin. The limited data on PBNs in the environment suggest that these also appear to originate from different sources. The toxicological data on these compounds is even scarcer, most of it directed toward explaining the exposure risk from accidental contamination of feed with the commercial PBN containing product, Firemaster BP-6. The occurrence of PBNs and PXNs is possible as ultra-trace environmental and food-chain contaminants produced at least from combustion processes at unknown concentrations. Available toxicological and environmental data enable a focus on PCNs as dioxin analogues to an extent that specific local or regional environmental influences could result in a risk to human health. There is the possibility that they may act synergistically with the better-known classic dioxin and other dioxin-like compounds. PBNs and PXNs are much less studied than the dioxins, but are known to be products of anthropogenic processes that contaminate the environment. A continuously increasing use of bromine for manufacture of brominated flame retardants over the past three decades is anticipated as a stream of “brominated” wastes, that when degraded (combusted), will release PBNs and PXNs. This calls for advanced analytical methods and greater interest toxicologically to understand and control pollution and exposure by PBNs and PXNs. Particular congeners of bromonaphthalene in single studies were found to be much more toxic than their chlorinated counterparts. In addition, brominated/chlorinated naphthalenes also seem to be more potent toxicants than PCNs. About 20% of PCN congeners exhibit a dioxin-like toxicity with relative potencies varying between around 0.003 and 0.000001, but additional and more rigorous data are needed to confirm these figures. Recent food surveys have estimated a small but relevant human exposure to these compounds in foods, giving an additional source of dioxin-like toxicity to those compounds already covered by the World Health Organization–Toxic Equivalency Factors (TEFs) scheme. Given the additivity of response postulated for other dioxin-like compounds, it would seem unwise to ignore this additional contribution. Few data available showed that PBN congeners also exhibit a dioxin-like toxicity and are even more potent than PCN congeners, but the relative potency values were not derived for them until now. There are no toxicological data available for PXNs.