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Triclosan is an antimicrobial agent frequently used in pharmaceuticals and personal care products. We analyzed triclosan for its action on placental secretion of progesterone, estradiol and human chorionic gonadotropin in vitro in the JEG-3 cells. We also investigated its action on cell viability, proliferation and apoptosis. The JEG-3 cells were cultured with increasing doses of triclosan (1×10(-9)-1×10(-4) M) for 24, 48 and 72 h. Triclosan was found to increase estradiol and progesterone secretion after short- and long-term exposure. The stimulatory effect was observed up to 10 μM after short- and long-term exposure to triclosan. In addition, triclosan caused an adverse effect on β-hCG secretion. The highest doses of triclosan (50 and 100 μM) showed a strong cytotoxic effect. Anti proliferative and pro-apoptotic effects were also observed. Overall, this study demonstrates that triclosan may indirectly disrupt steroidogenesis which may, in turn, affect placental development and consequently fetal growth.

The aim of the study was to evaluate the pentoxifylline administration on the foetal-placental circulation and neonatal outcome in women with threatened preterm labour. Pentoxifylline was given as a supplement to standard tocolytic therapy in a group of 43 patients (pentoxifylline group) as an intravenous infusion and oral supplementation in a total dosage of 800 mg/day. The drug was administered within 3 weeks after admission. No pentoxifylline was given in the control group (53 patients). Doppler velocimetry of pulsatility indices (PI) of the umbilical (UA) and middle cerebral (MCA) arteries as well as cerebro-placental ratio (CPR) were calculated. Also, the neonatal outcome was estimated in both groups. From the second week of therapy with pentoxifylline, the PI decreased in umbilical artery and increased in the MCA, whereas in the control group, there were no changes. The value of PIUA, evaluated after the third week of pentoxifylline administration, was statistically significantly lower when compared to data obtained on admission (mean: 0.99 ± 0.22 versus 0.82 ± 0.12; p =0.016). Pentoxifylline significantly increased CPR values calculated after third week of drug administration, which were statistically significantly higher in the pentoxifylline group when compared with respective data in the control group (mean: 2.30 versus 1.61; p = 0.001). The risk of severe neonatal complications was significantly lower in the pentoxifylline group (p = 0.026). Pentoxifylline changed foetal-placental blood circulation in patients with threatened preterm labour and improved neonatal outcome.

Insulin-like growth factor-I (IGF-I) in concert with insulin-like binding protein 3 (IGFBP-3), insulin-like binding protein 2 (IGFBP-2), human growth hormone (GH) and P53 protein is involved in autocrine/paracrine growth signaling pathways as an adaptive response to environmental stimuli.The study evaluated the local secretion of PRL, hGH, IGF-I, IGFBP-2 and IGFBP-3 by breast cancer tissue explants in relation to the overexpression of P53 protein in breast cancer tissue.Breast cancer explants were obtained during radical mastectomies. The overexpression of P53 protein was assessed immunohistochemically using monoclonal antibody (DAKO, Anti-Human P53 protein, clone DO-7); the results of the reaction were stratified into 5 groups. The lack of P53 protein overexpression was defined as 0% of cells that overexpressed P53 protein. IGF-I, IGFBP-3, IGFBP-2, and hGH levels were measured with RIA kits, and prolactin was measured with the MEIA kit.The local secretion of hGH by tumour explants - presenting a positive immunohistochemical reaction (IHCR) to the product of P53 gene - was twice as high as those with no IHCR to product of P53 gene; the opposite was noted in the case of IGF-I, IGFBP-2 and IGFBP-3 secretion. In both cases, the level of hGH, IGF-I and IGFBP-3 secretion did not correlate with the ratio of cells overexpressing P53 protein. There was a significant decrease in local, basic IGFBP-2 secretion along with an increased ratio of cells with positive IHCR to product of P53 gene. Furthermore, local PRL secretion was not correlated with the ratio of cells overexpressing P53 protein in breast cancer tissue. Prolactin also exerts no influence on IGF-I secretion.Our results may suggest the presence of local hGH/IGF-I feedback in breast tissue as well as the possibility of P53/hGH/IGF-I/IGFBP-3 but not P53/PRL/IGF-I axis.

In recent years, the major concern has been focused on persistent organic pollutants (POPs), which are present in ecosphere in increasing concentrations, especially since 1950s. Among of these pollutants are dioxins and polychlorinated biphenyls (PCBs) released during vast burning and plastics processing, as well as pesticides which were industrial chemicals intensively produced for many years.In last decade, dioxins together with PCBs and pesticides have been classified as endocrine disrupting chemicals, because they are able to alter hormone-dependent processes and disrupt functioning of endocrine glands, e.g. thyroid and gonads. Furthermore, these pollutants have been included in neural disrupting chemicals due to their ability of altering neural transmission and formation of neural networks. Since POPs may persist in the environment for dozens of years, an exposure to these organic pollutants creates a serious issue for environmental toxicologists. POP intoxication creates severe clinical problems, which became evident in dramatic circumstances, e.g. Yusho incident in Japan, Yu-Cheng incident in Tajwan, Michigan Lake poisoning. Clinical problems have been recognized as disruption of thyroid and gonadal functions, immunodeficiency as well as psychomotor deficits and increased occurrence of hormone-dependent cancers. Thus, knowledge on POP effects on human nervous system has been related mainly to toxic effects of these organic pollutants. Little is known, however, about the action of very low, so called background, doses of POPs and their effects on hormonal homeostasis in developing brain. It is of particular importance, because doses which are low for adults might become toxic for fetuses, infants or children. Recently, the public concern has been focused on POP effects on brain function, concomitantly with the increase in neuropsychiatric disorders, including autism, attention deficit and hyperactivity disorder (ADHD) as well as learning disabilities. Although some of epidemiological data are controversial, most of them point to adverse effects of prenatal exposure to polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAH) on cognitive function and, in general, mental development of infants and children. Studies on prenatal exposure to pesticides demonstrated increased incidence of autism and ADHD as well as deficits in psychomotor and visio-spatial skills, which were observed in infants and 8 years old children, respectively. Psychomotor deficits were also indicated in 6 months old infants exposed prenatally to polychlorinated dibenzo-p-dioxin (PCDD) and in 1-6 year old children affected prenatally by polybrominated difenyl ethers (PBDE). Recent data also demonstrate the strong correlation between exposure to bisphenol A at early pregnancy and increased locomotor activity and aggressiveness of children.Our knowledge on POP effects on human nervous system seems rather extensive, but is related mainly to toxic effects of these organic pollutants. Little is known, however, about the action of very low doses of POPs and their effects on hormonal homeostasis in developing brain. Therefore, the role of scientists and clinicians is to recognize the mechanisms of POP action, especially in respect to prenatal and early postnatal period when the nervous system develops and is particularly sensitive to hormonally active chemicals present in the environment.

Verify that resistance to aspirin may have an impact on pregnancy and neonatal outcome.We enrolled 43 pregnant women, aged 30.7 ± 4.0 years regularly taking 75 mg of aspirin daily and 32 (aged 30.8 ± 4 years) pregnant women not receiving aspirin who served as control group. Laboratory tests were performed at 18 to 22 weeks of gestation, 28 to 32 weeks of gestation and 16 to 32 weeks after delivery. Resistance to aspirin was defined as urinary 11-dehydrothromboxane B2 (u11-dTXB2) concentrations in the highest quartile and additionally, as the resistance index (RI) calculated for each woman, defined as the difference between u11-dTXB2 concentration of each woman treated with aspirin and the median value at the same time point measured in the control group.Women taking aspirin in the highest quartile of u11-dTXB2 delivered prematurely (35.8±3.4 vs 38.1±1.7 weeks, p=0.02). Delivery of small for gestational age (SGA) newborns (p=0.003) as well as fetal distress (p=0.014) and preeclampsia (p=0.003) occured more frequently in aspirin-resistant women. Resistance to aspirin based on the RI value was also associated with higher prevalence of preeclampsia (p=0.02) and SGA newborns delivery (p=0.01). The two groups resistant to ASA designed on the basis of both (RI and u11-dTXB2 urine levels) methods compared with ASA sensitive group differed in frequency of SLE prevalence.Aspirin resistance may be associated with increased risk of adverse pregnancy outcomes including preeclampsia, premature delivery and delivery of SGA newborns.

The present work investigated the effects of two different natural mixtures on aryl hydrocarbon receptor (AhR) and oestrogen receptor (ER)beta protein levels, as well as on the activity of cytochrome P450 (CYP) 1A1 and CYP2B. Consequently, the authors observed the effects of these mixtures on gonadotropine-stimulated steroid secretion by ovarian follicles. The natural mixtures that were studied were 'Mjosa' extracted from burbot liver, which contains a high level of PBDEs, and 'Marine mix', extracted from Atlantic cod liver, which contains a high level of polychlorinated biphenyls (PCBs). Follicular cells were exposed in vitro to 'Marine mix' and 'Mjosa mix' at doses of 3.6 and 1.4 microg ml(-1), respectively. Media were collected and used for steroid analysis and cell viability assays. Cells were used to estimate aromatase activity (CYP19), AhR and ER protein levels, and CYP1A1 and CYP2B1 activity. Western blot analysis indicated down-regulation of AhR by 'Marine mix' and down-regulation of ERbeta by Mjosa mix. Up-regulation of CYP1A1 expression and activity were seen following treatment with Marine mix, but not Mjosa mix. Increased CYP2B1 activity was noted after treatment with both 'Marine mix' and Mjosa mix. Both mixtures increased luteinizing hormone (LH)-stimulated progesterone and testosterone secretion, follicle-stimulating hormone (FSH)-stimulated oestradiol secretion, and CYP19 activity. These results suggest that: (1) 'Marine mix' is a mixed-type CYP inducer; (2) 'Mjosa mix' is an inducer of ERbeta and CYP2B; and (3) both 'Marine mix' and 'Mjosa mix' stimulate aromatase activity as a consequence of oestradiol secretion through activation of CYP19.

Activation of aryl hydrocarbon receptors (AhRs) induces neuronal damage, but the mechanism by which this occurs is largely unknown. This study evaluated the effects of an AhR agonist, beta-naphthoflavone, on apoptotic pathways in mouse primary neuronal cell cultures. beta-Naphthoflavone (0.1-100 micronhanced caspase-3 activity and lactate dehydrogenase (LDH) release in neocortical and hippocampal cells. These data were supported at the cellular level with Hoechst 33342 and calcein AM staining. alpha-Naphthoflavone inhibited the action of beta-naphthoflavone, thus confirming specific activation of AhRs. A high-affinity estrogen receptor (ER) antagonist, ICI 182,780, and a selective estrogen receptor modulator (SERM), tamoxifen, enhanced beta-naphthoflavone-mediated apoptosis. Another SERM, raloxifene, and an ERalpha antagonist, methyl-piperidino-pyrazole, did not affect beta-naphthoflavone-induced caspase-3 activity. However, they inhibited beta-naphthoflavone-induced LDH release at a late hour of treatment, thus suggesting delayed control of AhR-mediated neuronal cell death. The apoptotic effects of beta-naphthoflavone were accompanied by increased levels of AhRs, and these receptors colocalized with ERbeta as demonstrated by confocal microscopy. These data strongly support apoptotic effects of AhR activation in neocortical and hippocampal tissues. Moreover, this study provides evidence for direct interaction of the AhR-mediated apoptotic pathway with estrogen receptor signaling, which provides insight into new strategies to treat or prevent AhR-mediated neurotoxicity.

Explants of human placenta were used to study the effects of two isomers of DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) [p,p′-DDT and o,p′-DDT] and their DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene) metabolites [p,p′-DDE and o,p′-DDE] on the secretion of progesterone and human chorionic gonadotropin (hCG). Explants were treated with 1, 10, 100 or 1000 ng/ml of each compound for 24 h or 72 h. We found opposite effects (stimulatory after short-term and inhibitory after long-term exposure) of all compounds on progesterone secretion. However, both short- and long-term exposure to all investigated compounds caused decreased hCG secretion. In conclusion, we suggest the existence of a local axis between steroid hormones and hCG in placenta. DDT (which has estrogenic properties) increases progesterone secretion and consequently decreases hCG secretion. After long-term exposure, the low level of hCG is insufficient to stimulate progesterone.

Placental explants were used to compare the effects of two isomers of DDT (1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane), p,p'-DDT and o,p'-DDT and their metabolites p,p'-DDE and o,p'-DDE (1,1,-dichloro-2,2-bis(p-chlorophenyl)ethylene) on steroid hormone secretion (estradiol (E2) and progesterone (P4)). Explants were treated with 1, 10, 100ng/ml or 1microg/ml of each compound for 24h. We found that all investigated compounds at all doses caused reductions of estradiol secretion. Moreover, it was shown that the inhibition of estradiol secretion was due to direct action on aromatase activity. Twenty-four-hour exposure to p,p'-DDE, o,p'-DDT or o,p'-DDE at doses of 100ng/ml or 1microg/ml increased P4 secretion, suggesting that these compounds act on P450scc. The fluorometric assay confirmed that all investigated compounds inhibited aromatase activity at a concentration of 100ng/ml. Our findings suggest that by decreasing estradiol secretion with concomitant stimulation of progesterone secretion, DDT could be a factor that influences the outcome of pregnancy.

The paper summarizes results of a series of studies concerning luteolysis and early pregnancy in pigs. The involvement of the oxytocin (OT)/OT receptor system in the mechanism of corpus luteum (CL) protection during early pregnancy as well as the implication of luteinizing hormone (LH) in the endometrial prostaglandin (PG) release and synthesis are described. In addition, the role of leptin in the regulation of ovarian steroidogenesis and the expression of leptin and its receptor (OB-Rb) genes in hypothalamus, pituitary and reproductive tissues are reported. Moreover, a strong emphasis was placed on the mechanism of PGE2 participation in the local endocrine regulations of reproductive processes occurring in the utero-ovarian area as well as on the vascular endothelial growth factor (VEGF) ligand-receptor system in the ovary and uterus.

Since 01.07.1993 to 30.09.2004, 675 children with ALL-SR were diagnosed and treated according to the modified ALL-BFM 90 protocol. Subject to statistical analysis (Kaplan-Meier method) were thus 197 children with ALL-SR treated with HD-MTX in a dose 5.0g/ m2. Among them, 21 patients failed to respond to therapy: 2 (1.0%) early deaths, 2 (1.0%) deaths during I complete remission, 16 (8.2%) relapses, 1 (0.5%) second neoplasm. Relapses occured: 12 (6.2%) in bone marrow, 2 (1.0%) in central nervous system, 1 (0.5%) in testicle and in 1 (0.5%) child combined relapse was observed. Probability rates for 11-year event free survival (EFS) was 0.80 (0.03). Application of high dose of methotrexate is safety and effective in prevention of relapses, especially meningeal and testicular involvement.

Since 01.07.1993 to 30.09.2002, 640 children (48.2% girls and 51.8% boys) with ALL-SR were diagnosed and treated according to the modified ALL-BFM 90 protocol. In 29 children the treatment was intensified because of poor corticosteroid response. Subject to statistical analysis (Kaplan-Meier method) were thus 611 children with ALL-SR. Among them, 89 patients failed to respond to therapy: 10 (1.6%) early deaths, 15 (2.5%) deaths during I complete remission, 64 (10.5%) relapses. Relapses occurred: 45 (7.4%) in bone marrow, 11 (1.8%) in central nervous system, 4 (0.7%) in testicular and in 4 (0.7%) children combined relapses were observed. Probability rates for 9-year event free survival (EFS) and relapse free survival (RFS) for all patients were 0.77 (0.02) and 0.79 (0.02), respectively. Application of high dose of methotrexate is effective in prevention of relapses, especially meningeal and testicular involvement.

The aim of the present study was to investigate whether exposure of porcine ovarian follicular cells to clinically relevant concentrations of valproate affected steroid production in vitro and to which extent these effects were reversed by removal of the drug from the culture medium. Small and medium follicles were obtained from ovaries collected, respectively, at days 8-10 and 14-16 of the estrous cycle. Theca and granulosa cells were collected from follicles and co-cultured in one well. To show whether the effect of valproate was reversible, cells were cultured for 24, 48, or 72 h with valproate 100 or 250 microg/ml. The medium was then changed to fresh medium without drugs for an additional 24, 48, or 72 h. Valproate added to the culture medium caused a significant reduction of estradiol secretion with concomitant increase in both testosterone and progesterone secretion by small follicles. In medium-sized follicles, 100 microg/ml valproate was without effect on estradiol secretion while 250 microg/ml caused a small, but statistically significant decrease. The effects of valproate on steroid secretion patterns were irreversibly independent of concentration, exposure time, and time of restoration after drug exposure up to 72 h in both small and medium follicles. In conclusion, the present study demonstrated that even short-term valproate treatment disrupted follicular steroidogenesis in isolated ovarian follicular cells resulting in increased testosterone and progesterone and decreased estradiol secretion. It was not possible to reverse the steroidogenic effects of valproate by removing the drug from the cell cultures.

Abstract —Vascular endothelial growth factor (VEGF) is known to induce the release of nitric oxide (NO) from endothelial cells. However, the effect of NO on VEGF synthesis is not clear. Accordingly, the effect of endogenous and exogenous NO on VEGF synthesis by rat vascular smooth muscle cells (VSMCs) was investigated. Two in vitro models were used: (1) VSMCs stimulated to produce NO by treatment with interleukin (IL)-1β (10 ng/mL) and (2) VSMCs lipotransfected with pKecNOS plasmid, containing the endothelial constitutive NO synthase (ecNOS) cDNA. The synthesis of NO was inhibited by N ω -nitro- l -arginine methyl ester (L-NAME, 2 to 5 mmol/L) or diaminohydroxypyrimidine (DAHP, 2.5 to 5 mmol/L), inhibitors of NOS and GTP cyclohydrolase I, respectively. Some cells treated with L-NAME or DAHP were supplemented with l -arginine (10 mmol/L) or tetrahydrobiopterin (BH 4 ; 100 μmol/L), respectively. In addition, we studied the effect of sodium nitroprusside (SNP; 10 and 100 μmol/L) and chemically related compounds, potassium ferrocyanide and ferricyanide, on VEGF generation. IL-1β induced iNOS expression and NO generation and significantly upregulated VEGF mRNA expression and protein synthesis. L-NAME and DAHP totally inhibited NO generation and decreased the IL-1β–upregulated VEGF synthesis by 30% to 40%. Supplementation with l -arginine or BH 4 increased NO generation by L-NAME– or DAHP-treated cells, and VEGF synthesis was augmented by addition of BH 4 . The cells generating NO after pKecNOS transfection released significantly higher amounts of VEGF than cells transfected with control plasmids. Inhibition of NO generation by L-NAME decreased VEGF synthesis. In contrast to the effect of endogenous NO, we observed the inhibition of VEGF synthesis in the presence of high (10 or 100 μmol/L) concentrations of SNP. This effect was mimicked by chemically related ferricyanide and ferrocyanide compounds, suggesting that the inhibitory effect of sodium nitroprusside may be mediated by an NO-independent mechanism. The results indicate that endogenous NO enhances VEGF synthesis. The positive interaction between endogenous NO and VEGF may have implications for endothelial regeneration after balloon angioplasty and for angiogenesis.

Lanthanide-saturated peptides analogous to calcium-binding loops of EF-hand proteins can be used to stabilize the alpha-helical structure of peptide or protein segments attached to their C-termini. To study conformational properties of such loop-containing hybrids it is necessary to produce them in bacteria. In peptides obtained in this way the helix will be destabilized by the negatively charged C-terminal alpha-carboxyl groups. We propose to block them by the homoserine lactone. The results presented in this paper indicate that the presence of the lactone even at the C-terminus of the loop does not have any negative effect on the loop helix-nucleation ability. On the other hand, the presence of the alpha-NH3+ at the loop N-terminus leads to a drop of metal-binding constant and loss of the rigid structure of the alpha-helical segment of the loop. The alpha-amino group separated by one glycine residue from the loop N-terminus should also be avoided because it perturbs the conformation of the N-terminal part of the loop and may reduce the loop affinity to lanthanide ions.

The goal of the present study was to investigate the action of TBBPA on PPARγ protein expression in vitro in human choriocarcinoma-derived placental JEG-3 cells. We also analyzed TBBPA for its action on placental secretion of progesterone and β-hCG, cell viability, and apoptosis. Our results showed that after TBBPA treatment at 10 nM and 10 µM, PPARγ protein expression increased in a time-dependent manner until 48 h and then slightly decreased at 72 h but was still above the control level. This alteration in PPARγ protein expression was accompanied by a decreased β-hCG level. Interestingly, co-treatment with the PPARγ antagonist GW9662 reversed the TBBPA-mediated changes in PPARγ protein expression but, according to β-hCG secretion, potentiated an inhibitory effect of TBBPA. Additionally, in our study, we assessed the ability of TBBPA to increase progesterone levels in JEG-3 cells compared with those of controls. Finally, in the present study, we demonstrated that TBBPA at all of the tested doses significantly increased caspase-3 activity compared with that of the vehicle control. The apoptotic action of TBBPA was also confirmed by Hoechst 33342 staining. These results showed the up-regulation of PPARγ protein expression after TBBPA exposure in human placental cells. Although co-treatment with antagonist of PPARγ reversed the TBBPA-mediated increase in this protein expression and restored it to the control level, it did not reverse the effect on β-hCG secretion. This indicated that the mechanism of TBBPA-induced changes in β-hCG secretion is PPARγ-independent.

Dichlorodiphenyldichloroethylene (DDE) is a primary environmental and metabolic degradation product of the pesticide dichlorodiphenyltrichloroethane (DDT). It is one of the most toxic compounds belonging to organochlorines. DDE has never been commercially produced; however, the parent pesticide DDT is still used in some developing countries for disease-vector control of malaria. DDT and DDE remain in the environment because these chemicals are resistant to degradation and bioaccumulate in the food chain. Little is known, however, about DDE toxicity during the early stages of neural development. The results of the present study demonstrate that DDE induced a caspase-3-dependent apoptosis and caused the global DNA hypomethylation in mouse embryonic neuronal cells. This study also provided evidence for DDE-isomer-non-specific alterations of retinoid X receptor α (RXRα)- and retinoid X receptor β (RXRβ)-mediated intracellular signaling, including changes in the levels of the receptor mRNAs and changes in the protein levels of the receptors. DDE-induced stimulation of RXRα and RXRβ was verified using selective antagonist and specific siRNAs. Co-localization of RXRα and RXRβ was demonstrated using confocal microscopy. The apoptotic action of DDE was supported at the cellular level through Hoechst 33342 and calcein AM staining experiments. In conclusion, the results of the present study demonstrated that the stimulation of RXRα- and RXRβ-mediated intracellular signaling plays an important role in the propagation of DDE-induced apoptosis during early stages of neural development.