Differential effects of ambient PAH mixtures on cellular and steroidogenic properties of placental JEG-3 and BeWo cells.

We determined the action of a mixture of 16 priority PAHs present in high concentrations in maternal blood (Mix I) and the same but in low concentrations detected in placental tissue (Mix II) on AhR, ERα, NFκB, CYP1A1, CYP1B1, and COMT protein expression and cell proliferation of JEG-3 and BeWo cell lines. Both mixtures induced AhR expression in JEG-3 and BeWo; however, in JEG-3 cells expression of ERα, CYP1A1, CYP1B1, and COMT was upregulated, while in BeWo cells downregulated. The opposite effect of mixtures on NFκB protein expression (inhibitory in JEG-3, stimulatory in BeWo) was noted. We suggest AhR-mediated activation of PAHs metabolism in JEG-3 cells, and AhR interaction with NFκB and AhR cross-talk with ERα causing inhibition of detoxification in BeWo cells. As a result there was a stimulatory effect on JEG-3 and an inhibitory one on BeWo cell proliferation.

Leptin Receptor Antagonists' Action on HDAC Expression Eliminating the Negative Effects of Leptin in Ovarian Cancer.

BACKGROUND/AIM: A common finding in cancer cells is the overexpression of histone deacetylases (HDACs), leading to altered expression and activity of numerous proteins involved in carcinogenesis. Considering that leptin can modulate the levels of HDACs, we hypothesised that leptin receptor antagonists can alter HDAC expression. MATERIALS AND METHODS: HDAC expression in cells exposed to leptin and leptin receptor antagonists (SHLA and Lan2) were evaluated in ovarian epithelial (OVCAR-3, CaOV3) and folliculoma (COV434, KGN) cells. RESULTS: Higher HDAC expression was found in epithelial compared to folliculoma cells. Leptin increased class I and II HDACs only in OVCAR-3 cells, and SHLA was more potent then Lan-2. In folliculoma cells, leptin only increased class II HDAC expression, Lan-2 was more potent than SHLA in the COV434 and neither antagonist affected the KGN cells. CONCLUSION: SHLA and Lan2 eliminate the negative effects of leptin on HDAC expression in a cell-type-dependent manner. This is the first report testing leptin receptor blockers as HDAC inhibitors in ovarian cancer cells.

Effects of human blood levels of two PAH mixtures on the AHR signalling activation pathway and CYP1A1 and COMT target genes in granulosa non-tumor and granulosa tumor cell lines.

Epidemiological studies have shown a link between problems with offspring of couples living in a contaminated environment in comparison to those who live in an uncontaminated environment. We measured the concentrations of 16 priority polycyclic aromatic hydrocarbons (PAHs) in maternal and cord blood. To explore the mechanism of the effects of PAH mixtures on nonluteinized granulosa cells (HGrC1) and granulosa tumor cells (COV434), as well as cell proliferation and apoptosis, we investigated the effect of PAH mixtures on the expression of the aryl hydrocarbon receptor (AHR), aryl hydrocarbon receptor nuclear translocator (ARNT) and aryl hydrocarbon receptor repressor (AHRR) genes, as well as the expression and activity of target genes cytochrome P450 1A1 (CYP1A1) and catechol-O-methyltransferase (COMT). The cells were exposed to mixture 1 (M1), composed of all 16 priority PAHs, and mixture 2 (M2), composed of five PAHs which are not classified as human carcinogens, and which are observed in the highest amounts both in maternal and cord blood. All 16 priority PAHs were bioavailable in maternal and cord plasma, suggesting that perinatal exposure should be considered. In HGrC1 cells, M1 increased AHR and ARNT, but decreased AHRR expression, in parallel with increased CYP1A1 and COMT expression and activity. M2 decreased AHR and AHRR, and increased ARNT, with no effect on CYP1A1 expression and activity; however, it did increase COMT expression and activity. In tumor cells, M1 lowered AHR and up-regulated AHRR and ARNT expression, consequently decreasing CYP1A1 expression and COMT activity. M2 up-regulated AHR and ARNT, down-regulated AHRR, and had no effect on CYP1A1 and COMT expression, but decreased COMT activity. We hypothesise that, dependent on composition, mixtures of PAHs activate the AHR differently through varying transcription responses: in HGrC1, a canonical AHR mechanism of M1, with activation of CYP1A1 important for detoxication, while in COV434, a noncanonical AHR mechanism, probably by activation the nuclear factor NFkB.

Differences in the action of lower and higher chlorinated polychlorinated naphthalene (PCN) congeners on estrogen dependent breast cancer cell line viability and apoptosis, and its correlation with Ahr and CYP1A1 expression.

There are data showing that exposition to PCNs mixture increased incidence of gastrointestinal and respiratory neoplasms, but data regarding incidence of hormone-dependent cancer so far not shown. The objective was to determine if exposure to single lower and higher chlorinated PCN congeners is associated with altered proliferation and apoptosis of estrogen dependent breast cancer cells, and whether such effects are related to induction of AhR and CYP1A1 protein expression. MCF-7 cells were exposed to PCN 34, 39, 42, 46, 48, 52, 53, 54, 66, 67, 70, 71, 73 and 74 at concentrations of 100-10,000pg/ml. We evaluated the action of these PCN congeners on cell proliferation, DNA fragmentation and caspase-8,-9 activity. AhR and CYP1A1 protein expression and CYP1A1 activity was evaluated at a concentration of 1000pg/ml. An opposite action of tri- to tetraCNs than of penta-to heptaCNs on cell proliferation and apoptosis was evident. Tetra PCNs increased cell proliferation, but had no effect on DNA fragmentation nor caspase activity. Fast induction of CYP1A1 protein expression under the influence of lower chlorinated PCNs suggests faster metabolism and a possible stimulatory action of locally formed metabolites on cell proliferation. None of the higher chlorinated PCNs affected cell proliferation but all higher chlorinated PCNs increased caspase-8 activity, and hexa PCNs also increased caspase-9 activity. The rapid activation of the Ah receptor and CYP1A1 protein expression by higher chlorinated PCNs point to their toxicity; however, it is not sufficient for potential carcinogenicity. Action of lower chlorinated naphthalenes metabolites should be explored.

Effect of Chemotherapeutic Drugs on Caspase-3 Activity, as a Key Biomarker for Apoptosis in Ovarian Tumor Cell Cultured as Monolayer. A Pilot Study.

We aimed to develop a cost-effective and robust method to predict drug resistance in individual patients. Representative tissue fragments were obtained from tumors removed from female patients, aged 24-74 years old. The tumor tissue was taken by a histopathology's or a surgeon under sterile conditions. Cells obtained by enzymatic dissociation from tumor after surgery, were cultured as a monolayer for 6 days. Paclitaxel, doxorubicin, carboplatin and endoxan alone or in combination were added at the beginning of culture and after 6 days, Alamar blue test was used for showing action on cell proliferation why caspase- 3 activity assays for verifying action on apoptosis. Inhibitory action on cell proliferation was noted in 2 of 12 patients tumor treated with both single and combined drugs. Using caspase-3 assay we showed that 50% of tumor cells was resistant to single chemotherapeutic drugs and 40% for combined. In 2 of 12 tumors, which did not reacted on single drugs, positive synergistic action on cell proliferation was observed in combination of D + E and C + E. This pilot study suggests: 1) monolayer culture of tumor cells, derived from individual patients, before chemotherapy could provide a suitable model for studying resistance for drugs; 2) caspase-3 activity is cheap and useful methods; 3) Alamar blue test should be taken into consideration for measuring cell proliferation.

Effects of bisphenol A and 17ß-estradiol on vascular endothelial growth factor A and its receptor expression in the non-cancer and cancer ovarian cell lines.

Tumours secrete several pro-angiogenic factors, among which vascular endothelial growth factor (VEGF) and its receptor (VEGF-R) are the most extensively studied but not in ovarian cancer cells. The study was designed to investigate the effect of bisphenol A (BPA) (environmental oestrogen) and of 17ß-estradiol (E2) (endogenous estrogen) on the gene (real-time PCR) and protein (Western blotting) expression of VEGF-R2 and VEGF-A in human non-cancer (HOSEpiC) and ovarian cancer cell lines (SKOV-3 and OVCAR-3). In addition, VEGF-A levels were measured in culture supernatants using a colorimetric assay. Cells were exposed to BPA (1, 40 and 100 nM) or 17ß-estradiol (0.1, 10 and 40 nM) for 3 to 48 h. Since differential expression levels of basal oestrogen receptor (ERα and ERß) between non-cancer and cancer cell lines may affect the response to oestrogens, receptor expression was measured both at the gene and protein levels. Basal ERß expression was similar in all cell lines, and ERα expression was significantly higher in the SKOV-3 cell line. Basal VEGF-R2 expression was higher in cancer than non-cancer cell lines, and in contrast, VEGF-A expression was significantly lower in both SKOV-3 and OVCAR-3 cancer cell lines. Exposure of non-cancer cells to BPA and E2 was associated with a significant increase in VEGF-R2 expression but had no effect on VEGF-A expression or secretion. In contrast, exposure of cancer cells to BPA, but not E2, increased VEGF-R2 and VEGF-A expression and secretion. In conclusion, (1) BPA and E2 regulated VEGF-R2 and VEGF-A expression differently in non-cancer and cancer cells, and (2) BPA has a direct stimulatory effect on VEGF-R2 and VEGF-A expression in both, while E2 appears to be uninvolved in the regulation of VEGF-R2 and VEGF-A expression in cancer cells. Graphical Abstract A schematic representation showing BPA and E2 action on VEGF-R2 and VEGF-A expression in non-cancer (HOSEpiC) and cancer cells (SKOV-3, OVCAR-3).

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

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.

Ghrelin negatively affects the function of ovarian follicles in mature pigs by direct action on basal and gonadotropin-stimulated steroidogenesis.

We previously showed that expression of ghrelin messenger RNA is significantly increased in the ovaries of cycling pigs but not in prepubertal animals and that ghrelin stimulates estradiol (E2) secretion by ovarian follicles in prepubertal animals. The present study investigated in vitro the role of ghrelin in regulating the ovarian steroidogenesis during estrus cycle in mature pigs. Small (SFs), medium (MFs), and large (LFs) ovarian follicles were collected on days 4 to 6, 10 to 12, and 16 to 18 of the estrous cycle from cycling pigs and exposed to 20, 100, and 500 pg/mL ghrelin for 24 hours. In additional experiments, MFs were exposed to ghrelin plus 100 ng/mL follicle-stimulating hormone (FSH) or luteinizing hormone (LH). Levels of progesterone (P4), testosterone (T), and E2 in culture medium were determined by enzyme-linked immunosorbent assay, and the expression of the steroid pathway enzymes 3ß hydroxysteroid dehydrogenase (HSD), 17ß-HSD, and cytochrome P450 aromatase (CYP19) was evaluated by Western blotting. Ghrelin had no effect on steroid secretion when present at 20 pg/mL, its concentration in follicular fluid, whereas at 100 pg/mL and 500 pg/mL, its concentration in serum, ghrelin significantly decreased secretion of P4, T, and E2. Moreover, all concentrations of ghrelin decreased steroid secretion in FSH- and LH-stimulated follicles. Western blot analysis showed that ghrelin inhibited expression of 3ß-HSD, 17ß-HSD, and CYP19 proteins. These results suggest that ghrelin, by direct inhibition of 3ß-HSD, 17ß-HSD, and CYP19 protein expression, inhibits LH- and FSH-stimulated steroid secretion by ovarian follicles, thus negatively affecting ovarian steroidogenesis in mature pigs.

Cooperation of bisphenol A and leptin in inhibition of caspase-3 expression and activity in OVCAR-3 ovarian cancer cells.

This study was designed to investigate the effect of bisphenol A and leptin on caspase-3 expression and activity in OVCAR-3 ovarian cancer cells. Caspase-3 and survivin expression was measured at the transcript level by real-time PCR and at the protein level by Western blotting. In addition, caspase-3 activity was measured, using a fluorometric assay, upon exposure to bisphenol A (40 nM) alone, leptin (2.5 nM) alone, and the combination of both agents. 17ß-estradiol (40 nM) was used as a positive control for estrogenic properties of bisphenol A. Results showed that the interaction between bisphenol A and leptin, which was similar to that observed between 17ß-estradiol and leptin, led to the inhibition of caspase-3 expression and activity in OVCAR-3 cells. Surprisingly, survivin was found to not be involved in the anti-apoptotic activity of either agent. Also, results showed that leptin inhibits caspase-3 activity by acting on the signal transducers and activators of transcription 3 (STAT3) pathway, but bisphenol A and 17ß-estradiol by the extracellular-signal-regulated kinases 1/2 (ERK1/2) pathway. In conclusion, the study reveals that bisphenol A and leptin interact to inhibit caspase-3 expression and activity by modulating STAT3 and ERK1/2 signaling pathways in OVCAR-3 cells.

Effects of single and repeated in vitro exposure of three forms of parabens, methyl-, butyl- and propylparabens on the proliferation and estradiol secretion in MCF-7 and MCF-10A cells.

BACKGROUND: Here, we analyzed the dose- (0.2, 2, 20, 200 nM or 2 µM) and time- (48, 96, 144 and 196 h) dependent activity of a single or repeated exposure of methyl-, butyl- and propylparaben on the proliferation of MCF-7 human breast cancer cells and MCF-10A human breast epithelial cells. Additionally, the effect on estradiol secretion, gene and protein expression of aromatase (CYP19A1) was investigated. METHODS: Cell proliferation was determined by AlamarBlue assay, and estradiol secretion by ELISA kits. Gene and protein expression of CYP19A1 was measurement using real time PCR and western blot, respectively. RESULTS: Stimulatory effect of a single exposure of all doses of tested parabens and time dependent effect of repeated exposure to methylparaben, propylparaben and butylparaben, the same as that of 17ß-estradiol, on proliferation of MCF-7 cells was observed. Only at low doses methyl- and butylparabens increased MCF-10A cells proliferation after single exposure, but no effect of repeated exposure was noted. Exposure at low doses of all of the parabens significantly increased 17ß-estradiol (E2) secretion in MCF-7 cells but had the opposite effect on MCF-10A cells. It was correlated with gene and protein expression of CYP19A1 in MCF-7 and MCF-10 cells. CONCLUSIONS: In summary, present study indicates a different mechanism of proliferative action of parabens in investigated cell lines. In MCF-7 breast cancer cell line it is probably due to stimulatory action on estradiol secretion and aromatase activity. In MCF-10A by an unknown mechanism, independent on stimulatory action on estradiol section, which requires further investigation.

Leptin stimulation of cell cycle and inhibition of apoptosis gene and protein expression in OVCAR-3 ovarian cancer cells.

The OVCAR-3 cell line expressing the long (ObRb) and short (ObRt) isoforms of leptin receptor mRNA was used to analyze the effect of leptin on the expression of selected genes and proteins involved in the cell cycle and apoptosis. OVCAR-3 cells were exposed to 2, 20, 40, and 100 ng/ml of leptin. Cell proliferation was determined using the alamarBlue cell viability test and flow cytometry. Apoptosis was measured using a cellular DNA fragmentation ELISA kit. The expression of selected cell cycle and apoptosis genes was evaluated by real-time PCR and confirmed by western blot. The stimulatory action of leptin on cell proliferation was observed as an increase in cells in the S and G2/M phases. Up-regulation of genes responsible for inducing cell proliferation and suppression of genes responsible for inhibition of proliferation were noted. Western blots revealed increased expression of cyclins D and A and inhibition of p21WAF1/CIP1 protein expression by leptin. Inhibition of DNA fragmentation was observed under all leptin doses. Suppression of genes involved in the extrinsic and intrinsic apoptotic pathway was observed. Western blots illustrated decreased Bad, TNFR1, and caspase 6 protein expression in response to leptin treatment. Leptin promotes ovarian cancer cell line growth by up-regulating genes and proteins responsible for inducing cell proliferation as well as down-regulating pro-apoptotic genes and proteins in apoptotic pathways. Results of this study warrant examining the relationship between the risk of ovarian cancer and elevated leptin levels in obese women.

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.

Effects of valproic acid and levetiracetam on viability and cell cycle regulatory genes expression in the OVCAR-3 cell line.

Concentration- and time-dependent effects of two antiepileptic drugs (AEDs), levetiracetam (LEV) and valproic acid (VPA), on proliferation, cytotoxicity and expression of cell cycle regulatory genes were investigated in a human ovarian cancer cell line, OVCAR-3. Cells were cultured with VPA or LEV, at concentrations between 100 µM and 10 mM. Cell proliferation was determined by alamarBlue and BrdU incorporation assays; cytotoxic effects by tetrazolium hydroxide (XTT), acid phosphatase (AP) and lactate dehydrogenase (LDH) assays. Expression of cell cycle regulatory genes was determined by real-time PCR. Exposure to VPA caused a concentration- and time-dependent decrease in cell proliferation (alamarBlue and BrdU incorporation assays), cytotoxic effects above 2.5 mM (XTT and AP assays) and modulated expression of genes primarily responsible for cell cycle arrest in G(1) phase. Cell proliferation was unaffected by exposure to LEV for 24 h and 120 h (alamarBlue assay), but increased when exposed to LEV for 72 h and 168 h, at concentrations from 250 µM to 1 mM. The BrdU incorporation assay showed no effect of LEV on cell proliferation. LEV was cytotoxic at higher concentrations (AP assay), but modulation in expression of cell cycle regulatory genes was not observed. Changes in LDH release were not observed with either AED. In summary, VPA apparently decreased cell proliferation by down-regulating genes responsible for transition from G(1) to S phase and up-regulating genes responsible for G(1) phase arrest, which suggest its potential as an anticancer drug. LEV does not exhibit such action.

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.

Effects of valproic acid (VPA) and levetiracetam (LEV) on proliferation, apoptosis and hormone secretion of the human choriocarcinoma BeWo cell line.

Epilepsy has been associated with poor obstetric outcomes that may be the result of the epilepsy or a direct effect of anti-epileptic drugs on placentation. To investigate any direct effect of anti-epileptic drugs on cell proliferation, apoptosis and hormone secretion with focus on human chorionic gonadotropin (ß-hCG), progesterone (P4) and 17ß-estradiol (E2), BeWo cell line was cultured in the presence of different concentrations of sodium valproate (0.45, 0.6, 1.5 or 2 mM) or levetiracetam (0.07, 0.12, 0.3 or 0.5 mM) with appropriate solvent controls. Cell proliferation was measured using BrdU incorporation. Caspase-3 activity was used as a marker of cell apoptosis and was evaluated by a fluorometric assay. Additionally, hormone secretion was evaluated by ELISA kits. Dose-dependent action of VPA on cell proliferation occurred in parallel to stimulation of caspase-3 activity. LEV had no effect on cell proliferation, and after long term exposure to the drug, a decrease in caspase-3 activity was observed. A significant decrease in ß-hCG, P4 and E2 production was observed when the cells were treated with VPA. LEV decreased ß-hCG and E2 secretion but had no effect on P4 level. Direct inhibition of cell proliferation and hormone secretion along with apoptotic action suggest that exposure to VPA at therapeutic doses during early pregnancy should be approached with caution. Trophoblast cells appear to be less sensitive to LEV; however, further studies involving placental tissue are necessary to determine the safety of the drug.

Combinatory effects of PBDEs and 17ß-estradiol on MCF-7 cell proliferation and apoptosis.

In the present work, we analyzed whether polybrominated diphenyl ethers (PBDEs) (47, 99, 100 and 209) interfere with the effect of 17ß-estradiol on the proliferation and apoptosis of the MCF-7 cell line. MCF-7 cells were cultured in DMEM without phenol red; upplemented with 5% charcoal-treated fetal bovine serum for 3 days with 10 nM 17ß-estradiol; with 0.1 µM, 0.5 µM or 1 µM of the tested PBDE congeners; or with both 17ß-estradiol and a congener. Cell proliferation was determined by measuring BrdU incorporation, and cell apoptosis was measured by caspase-9 activity. No PBDE congener had an effect on basal cell proliferation, but they all significantly decreased basal caspase-9 activity. An additive anti-apoptotic activity and ability to induce cell proliferation was observed in the presence of 17ß-estradiol.

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.

Differential accumulation of HCBz and PeCBz in porcine ovarian follicles and their opposing actions on steroid secretion and CYP11, CYP17, 17ß-HSD and CYP19 protein expression. A tissue culture approach.

In the current study, we determined in vitro accumulation of hexachlorobenzene (HCBz) and pentachlorobenzene (PeCBz) in porcine ovarian follicles, the effect on steroidogenesis and the expression of enzymes responsible for steroid synthesis. Sixty percent of the HCBz and almost 100% of the PeCBz that was added to the culture medium accumulated in ovarian tissue, and only 1% of each was found in the medium. An inhibitory HCBz effect and stimulatory PeCBz effect on testosterone and estradiol secretion were noted. Immunoblot analyses showed an inhibitory effect of HCBz on CYP17, 17ß-HSD and CYP19, a stimulatory effect of PeCBz on CYP17 and CYP19 and no effect on 17ß-HSD protein expression. In conclusion, the greater exposure to an estrogenic action of PeCBz than anti-estrogenic HCBz would be a consequence of the preferential accumulation of PeCBz in the ovarian follicles. As one of the mechanisms of action, we propose modulation of steroidogenic enzymes expression.

Comparison of combinatory effects of PCBs (118, 138, 153 and 180) with 17 beta-estradiol on proliferation and apoptosis in MCF-7 breast cancer cells.

We analyzed whether polychlorinated biphenyls (PCBs) interfere with the activity of 17 ß-estradiol in the proliferation and apoptosis of the MCF-7 cell line. MCF-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) without phenol red supplemented with 5% charcoal-treated fetal bovine serum (CD-FBS) for 3 days with 10 nM 17 ß-estradiol or 0.1 µM, 0.5 µM and 1 µM of the tested PCB congeners (118, 138, 153 and 180), or both. Cell proliferation was determined by measuring 5-bromo-2'-deoxyuridine (BrdU) incorporation, and cell apoptosis was measured by caspase-9 activity. From the PCB congeners tested, PCB138 and 153 had the highest stimulatory effects on basal cell proliferation as well as the highest inhibitory actions on basal caspase-9 activity. The proliferative and anti-apoptotic actions of PCB138 and 153 were still observed in the presence of 17 ß-estradiol, while the actions of PCB118 and 180 were reversed. In conclusion, the results of this study suggest the possibility that PCB138 and 153 contribute to the action of endogenous 17 ß-estradiol on cell proliferation and apoptosis in the breast cancer cell line MCF-7.

Induction of cytochrome P450 1A1 in MCF-7 human breast cancer cells by 4-chlorobiphenyl (PCB3) and the effects of its hydroxylated metabolites on cellular apoptosis.

Several studies suggest an involvement of PCBs in breast cancer formation, but the results are ambiguous and the mechanisms not clear. We propose that local activation of cytochrome P450 enzymes, CYP1A1 and CYP1B1 by PCB3, may generate active metabolites which affect apoptosis and thereby promote mammary carcinogenesis. To test this hypothesis MCF-7 human breast cancer cells were exposed to 300 nM PCB3 and its hydroxylated metabolites, 4OH-PCB and 3,4diOH-PCB3. The enzyme activity for CYP1A1 was assayed using the EROD assay, and CYP1A1 and CYP1B1 protein expression by western blotting. PCB3 increased CYP1A1 activity (~1.5fold) and protein levels within 6h after exposure. No effect on CYP1B1 protein expression was observed. The effects of PCB3 and both its metabolites on staurosporine-induced apoptosis were determined by measuring DNA fragmentation using ELISA and TUNEL assays, and by measuring caspase-8 and caspase-9 activity. We found that PCB3 and both of its hydroxylated metabolites had no effect on caspase-8 and caspase-9 activity when cells were grown in medium deprived of estrogen, but reduced caspase-9 activity when cells were grown in medium supplemented with serum containing estradiol. Interestingly, a decrease of DNA fragmentation was observed upon treatment with 3,4diOH-PCB3 in both culture conditions, suggesting that 3,4diOH-PCB3 affects a caspase-independent pathway of cell death. In summary, interactions of PCB3 and its metabolites with estradiol by yet unknown mechanisms inhibit caspase 9-related apoptosis and additional, other death pathways are affected by the catechol metabolite 3,4diOH-PCB3. These anti-apoptotic effects and the change in metabolic activity may contribute to the carcinogenic effect of PCBs.