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Glucocorticoids (GCs) are known to play an important role in maintaining basal and stress-related homeostasis by interacting with endocrine mediators and prostaglandins (PGs). Although a growing body of evidence shows that GCs exert their regulatory action at a multitude of sites in the reproductive axis through corticosteroid receptors, little is known about the direct role of cortisol, an active form of GCs, in the equine endometrium. Thus, the study aimed to determine the effect of cortisol on PGF2α synthesis in the endometrial tissue and cells in vitro. In Exp.1, the immunolocalization and the expression of the glucocorticoid receptor (GCR) in the endometrium throughout the estrous cycle were established. In Exp. 2 and 3, the effects of cortisol on PGF2α secretion and transcripts associated with the arachidonic acid (AA) cascade in endometrial tissues, and cells were defined. Endometrial tissues obtained from the early, mid, and late luteal phases and the follicular phase of the estrous cycle were exposed to cortisol (100, 200, and 400 nM) for 24 h. Endometrial epithelial and stromal cells (early phase of estrous cycle) were exposed to cortisol (100 nM) for 24 h. Then, PGF2α secretion and transcripts associated with the AA cascade (PLA2G2A, PLA2G4A, PTGS2, and PGFS) were assessed. GCR was expressed in the cytoplasm and the nucleus in the luminal and glandular epithelium as well as in the stroma. Endometrial GCR protein abundance was up-regulated at the late luteal phase compared to the mid-luteal phase of the estrous cycle. Cortisol dose-dependently decreased PGF2α secretion, PLA2G2A and PLA2G4A transcripts in endometrial tissues. Additionally, cortisol treatment decreased PGF2α secretion from endometrial epithelial and stromal cells. Moreover, it affected PLA2G2A, PLA2G4A, and PTGS2 transcripts in endometrial stromal cells. These findings suggest that cortisol suppresses the synthesis of PGF2α by affecting the AA cascade in the equine endometrium during the estrous cycle.

Triclosan (TCS) is a well-known compound that can be found in disinfectants, personal care products. There is one publication concerning the involvement of PPARγ in the mechanism of action of TCS. It is known that activation of PPARγ regulates the expression of the NF-κB mediated inflammation by acting on nitric oxide synthase (NOS) genes. However, there are no studies demonstrating a relationship between the effects of TCS on the PPARγ signaling pathway, changes in NF-κB expression, and NOS isoform synthesis. Therefore, the aim of this study was to evaluate the effect of TCS on the expression of PPARγ, NF-κB, nNOS, iNOS, and eNOS in mouse neocortical neurons. In addition, the effects of co-administration of synthetic alpha-naphthoflavone (αNF) or beta-naphthoflavone (βNF) flavonoids and triclosan were investigated. Our results show that TCS alters PPARγ, NF-κB, iNOS, and eNOS expression in mouse neurons in vitro. After 48 h of exposure, TCS increased PPARγ expression and decreased NF-κB expression. Moreover, under the influence of TCS, the expression of iNOS was increased and at the same time the expression of nNOS was decreased, which was probably caused by high levels of ROS. The experiments have shown that both αNF and βNF are able to modulate the effects of TCS in primary cultures of mouse cortical neurons.

DDE (dichlorodiphenyldichloroethylene) is an environmental metabolite of the pesticide DDT, which is still present in the environment, and its insecticidal properties are used to fight malaria and the Zika virus disease. We showed for the first time that the neurotoxic effects of DDE involve autophagy, as demonstrated by elevated levels of Becn1, Map1lc3a/MAP1LC3A, Map1lc3b, and Nup62/NUP62 and an increase in autophagosome formation. The suggestion that the aryl hydrocarbon receptor (AHR) and the constitutive androstane receptor (CAR) are involved in the neurotoxic effect of DDE was supported by increases in the mRNA and protein expression of these receptors, as detected by qPCR, ELISA, immunofluorescence labeling and confocal microscopy. Selective antagonists of the receptors, including alpha-naphthoflavone, CH223191, and CINPA 1, inhibited p,p'-DDE- and o,p'-DDE-induced LDH release and caspase-3 activity, while specific siRNAs (Ahr and Car siRNA) reduced the levels of p,p'-DDE- and o,p'-DDE-induced autophagosome formation. Although the neurotoxic effects of DDE were isomer independent, the mechanisms of p,p'- and o,p'-DDE were isomer specific. Therefore, we identified previously unknown mechanisms of the neurotoxic actions of DDE that, in addition to inducing apoptosis, stimulate autophagy in mouse neocortical cultures and induce AHR and CAR signaling.

Triclocarban is a phenyl ether that has recently been classified as a contaminant of emerging concern. Evidence shows that triclocarban is present in human tissues, but little is known about the impact of triclocarban on the nervous system, particularly at early developmental stages. This study demonstrated that triclocarban that was used at environmentally relevant concentrations induced apoptosis in mouse embryonic neurons, inhibited sumoylation, and changed the epigenetic status, as evidenced by impaired activities of HDAC, sirtuins, and DNMT, global DNA hypomethylation, and alterations of methylation levels of bax, bcl2, Ahr, and Car genes. The use of selective antagonists and specific siRNAs, which was followed by the co-localization of aryl hydrocarbon receptor (AHR) and constitutive androstane receptor (CAR) in mouse neurons, points to the involvement of AHR and CAR in triclocarban-induced neurotoxicity. A 24-h treatment with triclocarban enhanced protein levels of the receptors which was paralleled by Car hypomethylation and Ahr hypermethylation. Car hypomethylation is in line with global DNA hypomethylation and explains the increased mRNA and protein levels of CAR in response to triclocarban. Ahr hypermethylation could reflect reduced Ahr mRNA expression and corresponds to lowered protein levels after 3- and 6-h exposures to triclocarban that is likely related to proteasomal degradation of activated AHR. We hypothesize that the triclocarban-induced apoptosis in mouse neurons and the disruption of epigenetic status involve both AHR- and CAR-mediated effects, which may substantiate a fetal basis of the adult onset of neurological diseases; however, the expression of the receptors is regulated in different ways.

Several lines of evidence suggest that exposures to Endocrine Disrupting Chemicals (EDCs) such as pesticides increase the risks of neuropsychiatric disorders. Despite extended residual persistence of dichlorodiphenyltrichloroethane (DDT) in the environment, the mechanisms of perinatal actions of DDT that could account for adult-onset of depression are largely unknown. This study demonstrated the isomer-specific induction of depressive-like behavior and impairment of Htr1a/serotonin signaling in one-month-old mice that were prenatally exposed to DDT. The effects were reversed by the antidepressant citalopram as evidenced in the forced swimming (FST) and tail suspension (TST) tests in the male and female mice. Prenatally administered DDT accumulated in mouse brain as determined with gas chromatography and tandem mass spectrometry, led to global DNA hypomethylation, and altered the levels of methylated DNA in specific genes. The induction of depressive-like behavior and impairment of Htr1a/serotonin signaling were accompanied by p,p'-DDT-specific decrease in the levels of estrogen receptors i.e. ESR1 and/or GPER1 depending on sex. In contrast, o,p'-DDT did not induce depressive-like effects and exhibited quite distinct pattern of biochemical alterations that was related to aryl hydrocarbon receptor (AHR), its nuclear translocator ARNT, and ESR2. Exposure to o,p'-DDT increased AHR expression in male and female brains, and reduced expression levels of ARNT and ESR2 in the female brains. The evolution of p,p'-DDT-induced depressive-like behavior was preceded by attenuation of Htr1a and Gper1/GPER1 expression as observed in the 7-day-old mouse pups. Because p,p'-DDT caused sex- and age-independent attenuation of GPER1, we suggest that impairment of GPER1 signaling plays a key role in the propagation of DDT-induced depressive-like symptoms.

Dibutyl phthalate (di-n-butyl phthalate, DBP) is one of the most commonly used phthalate esters. DBP is widely used as a plasticizer in a variety of household industries and consumer products. Because phthalates are not chemically bound to products, they can easily leak out to enter the environment. DBP can pass through the placental and blood–brain barriers due to its chemical structure, but little is known about its mechanism of action in neuronal cells. This study demonstrated the toxic and apoptotic effects of DBP in mouse neocortical neurons in primary cultures. DBP stimulated caspase-3 and LDH activities as well as ROS formation in a concentration (10 nM–100 µM) and time-dependent (3–48 h) manner. DBP induced ROS formation at nanomolar concentrations, while it activated caspase-3 and LDH activities at micromolar concentrations. The biochemical effects of DBP were accompanied by decreased cell viability and induction of apoptotic bodies. Exposure to DBP reduced Erα and Pparγ mRNA expression levels, which were inversely correlated with protein expression of the receptors. Treatment with DBP enhanced Ahr mRNA expression, which was reflected by the increased AhR protein level observed at 3 h after exposure. ERα, ERβ, and PPARγ antagonists stimulated DBP-induced caspase-3 and LDH activities. AhR silencing demonstrated that DBP-induced apoptosis and neurotoxicity are mediated by AhR, which is consistent with the results from DBP-induced enhancement of AhR mRNA and protein expression. Our study showed that AhR is involved in DBP-induced apoptosis and neurotoxicity, while the ERs and PPARγ signaling pathways are impaired by the phthalate.

Background Tetrabromobisphenol A (TBBPA) is a brominated flame retardant widely used in a variety of commercial and household products. TBBPA can become bioaccumulated in human body fluids, and also in different brain regions. The aim of the present study was to determine the viability and apoptosis of cultured mouse hippocampal neurons in vitro after exposure to TBBPA. Additionally, we examined the involvement of ROS generation in the effect of TBBPA. Methods Primary hippocampal neuron cultures were prepared from Swiss mouse embryos on day 17/18 of gestation. The cultures were treated with TBBPA at concentrations ranging from 1 nM to 100 μM for 30 min or 3, 6 or 24 h. To study apoptosis, the activity of caspase-3 was measured, and apoptotic body formation was evaluated. To investigate the cytotoxic effect of TBBPA, the level of lactate dehydrogenase (LDH) was measured in the culture medium. Results Our results demonstrated that TBBPA concentrations ranging from 100 nM to 100 μM caused caspase-3 activation and apoptotic body formation. The cytotoxic effects of TBBPA were observed at concentrations ranging from 50 nM to 100 μM. To detect intracellular ROS, the fluorogenic dye H2DCFDA was used. We did not observe any significant increase in the level of cellular ROS in cultured cells after TBBPA treatment. However, in a cell-free model, TBBPA at concentrations ranging from 10 to 100 μM interacted with H2DCFDA and enhanced the fluorescence signal. Conclusion We suggest that the H2DCFDA assay cannot be used to measure TBBPA-stimulated cell-mediated ROS production.

Triclosan (TCS) is commonly used worldwide in a range of personal care and sanitizing products. A number of studies have revealed the presence of TCS in human tissues. It has recently been shown that TCS can interact with AhR in mouse neurons and the one of its effects is the stimulation of reactive oxygen species (ROS) production. Reactive oxygen species perform a wide spectrum of functions in neuronal cells, where they are generated as by-products of cellular metabolism. Therefore the aim of the study was to investigate effects of two synthetic naphthoflavones, the beta-naphthoflavone (βNF) and alpha-naphthoflavone (αNF), well known agonist and antagonist of AhR on TCS-stimulated cytotoxicity, apoptosis and ROS production in mouse primary cortical neurons in vitro cultures. The results showed that both agonist (βNF) and antagonist (αNF) of AhR enhanced the LDH release and caspase-3 activity stimulated by TCS. Interestingly, both naphthoflavones decreased the TCS-stimulated ROS production, however, they showed no scavenging properties as revealed by ABTS•+ and DPPH• methods. What's more, both βNF as well as αNF inhibited the activity of xanthine oxidase (XO) stimulated by TCS. Thus, we can assume that αNF or βNF act in a competitive way over TCS and inhibit its effect on antioxidant enzyme activity.

Degradation products of elastin, i.e. elastin-derived peptides (EDPs), are involved in various physiological and pathological processes. EDPs are detectable in cerebrospinal fluid in healthy people and in patients after ischemic stroke. However, to date, no studies concerning the role of EDP in the nervous system were conducted. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play important roles during the repair phases of cerebral ischemia, particularly during angiogenesis and reestablishment of cerebral blood flow. Therefore, the aim of this study was to investigate the impact of the specific elastin-derived peptide VGVAPG on Mmp-2, -9 and Timp-1, -2, -3 and -4 mRNA expression in mouse cortical glial cells in vitro. Primary glial cells were maintained in DMEM/F12 without phenol red supplemented with 10% fetal bovine serum and the cells were exposed to 50 nM, 1 and 50 μM of the VGVAPG peptide. After 3 and 6 h of exposition to the peptide, expression of Mmp-2, -9 and Timp-1, -2, -3 and -4 mRNA was measured. Moreover, siRNA gene knockdown, cytotoxicity and apoptosis measurement were included in our experiments, which showed that VGVAPG in a wide range of concentrations exhibited neither proapoptotic nor cytotoxic properties in mouse glial cells in vitro. The peptides enhanced mRNA expression of Timp-2 and Timp-3 genes in an elastin-binding protein (EBP)-dependent manner. However, changes in mRNA expression of Mmp-2, Mmp-9 and Timp-4 were partially EBP-dependent. The decrease in mRNA expression of Timp-1 was EBP-independent. However, further studies underlying the VGVAPG peptide’s mechanism of action in the nervous system are necessary. Electronic supplementary material The online version of this article (10.1007/s12640-018-9935-x) contains supplementary material, which is available to authorized users.

Triclosan (TCS) is a commonly used antimicrobial agent in personal care and sanitizing products, as well as in household items. Numerous studies have demonstrated the presence of TCS in various human tissues. Several studies have reported the accumulation of TCS in fish and human brain tissue. The aim of the present study was to investigate the effect of TCS on apoptosis in mouse neocortical neurons after 7 days of culture in vitro following 3, 6 and 24 h of exposure. To explore the mechanism underlying the effects of TCS in neurons, we studied the activation and protein expression of the Fas receptor (FasR) and caspase- 8, caspase-9 and caspase-3, as well as DNA fragmentation in TCS-treated cells. Cultures of neocortical neurons were prepared from Swiss mouse embryos on day 15/16 of gestation. The cells were cultured in phenol red-free Neurobasal medium with B27 and glutamine. The cultures were treated with concentrations of TCS ranging from 1 nM to 100 lM for 3, 6 and 24 h. The level of lactate dehydrogenase (LDH) was measured in the culture medium to exclude the cytotoxic concentrations. The cytotoxic effects were only observed when the highest concentrations of TCS were used (50 and 100 lM). To study apoptosis, the activities of caspase- 8, caspase-9 and caspase-3 were measured, and DNA fragmentation was evaluated. Our results are the first time to demonstrate that TCS can induce an apoptotic process in neocortical neurons in vitro. The data demonstrated that TCS caused caspase-3 activation, DNA fragmentation and apoptotic body formation. Non-cytotoxic concentrations of TCS activated the extrinsic apoptotic signaling pathway, which is dependent on FasR and caspase-8 activation. However, it is also possible that TCS may activate the intrinsic apoptotic pathway after long-term exposure. Therefore, further studies on the mechanism underlying the effects of TCS on the nervous system are needed.

Even though high doses of organic pollutants are toxic, relatively low concentrations have been reported to cause long-term alterations in functioning of individual organisms, populations and even next generations. Among these pollutants are dioxins, polychlorinated biphenyls, pesticides, brominated flame retardants, plasticizers (bisphenol A, nonylphenol, and phthalates) as well as personal care products and drugs. In addition to toxic effects, they are able to interfere with hormone receptors, hormone synthesis or hormone conversion. Because these chemicals alter hormone-dependent processes and disrupt functioning of the endocrine glands, they have been classified as endocrine-disrupting chemicals (EDCs). Because certain EDCs are able to alter neural transmission and the formation of neural networks, the term neural-disrupting chemicals has been introduced, thus implicating EDCs in the etiology of neurological disorders. Recently, public concern has been focused on the effects of EDCs on brain function, concomitantly with an increase in neuropsychiatric disorders, including autism, attention deficit and hyperactivity disorder as well as learning disabilities and aggressiveness. Several lines of evidence suggest that exposure to EDCs is associated with depression and could result in neural degeneration. EDCs act via several classes of receptors with the best documented mechanisms being reported for nuclear steroid and xenobiotic receptors. Low doses of EDCs have been postulated to cause incomplete methylation of specific gene regions in the young brain and to impair neural development and brain functions across generations. Efforts are needed to develop systematic epidemiological studies and to investigate the mechanisms of action of EDCs in order to fully understand their effects on wildlife and humans.

Triclosan (TCS) is an antimicrobial agent that is used extensively in personal care and in sanitizing products, such as soaps, toothpastes, and hair products. A number of studies have revealed the presence of TCS in human tissues, such as fat, liver and brain, in addition to blood and breast milk. The aim of the present study was to investigate the impact of TCS on AhR and Cyp1a1/Cyp1b1 signaling in mouse neocortical neurons in primary cultures. In addition to the use of selective ligands and siRNAs, expression levels of mRNA and proteins as well as caspase-3 activity, reactive oxygen species (ROS) formation, and lactate dehydrogenase (LDH) release have been measured. We also studied the involvement of the AhR in TCS-induced LDH release and caspase-3 activation as well as the effect of TCS on ROS generation. Cultures of neocortical neurons were prepared from Swiss mouse embryos on day 15/16 of gestation. The cells were cultured in phenol red-free Neurobasal medium with B27 and glutamine, and the neurons were exposed to 1 and 10 mM TCS. Our experiments showed that the expression of AhR and Cyp1a1 mRNA decreased in cells exposed to 10 mM TCS for 3 or 6 h. In the case of Cyp1b1, mRNA expression remained unchanged compared with the control group following 3 h of exposure to TCS, but after 6 h, the mRNA expression of Cyp1b1 was decreased. Our results confirmed that the AhR is involved in the TCS mechanism of action, and our data demonstrated that after the cells were transfected with AhR siRNA, the cytotoxic and pro-apoptotic properties of TCS were decreased. The decrease in Cyp1a1 mRNA and protein expression levels accompanied by a decrease in its activity. The stimulation of Cyp1a1 activity produced by the application of an AhR agonist (βNF) was attenuated by TCS, whereas the addition of AhR antagonist (αNF) reversed the inhibitory effects of TCS. In our experiments, TCS diminished Cyp1b1 mRNA and enhanced its protein expression. In case of Cyp1a1 we observed paradoxical effect of TCS action, which caused the decrease in activity and protein expression of Cyp1a1 and the increase in protein level of AhR. Therefore, we determined the effects of TCS on the production of ROS. Our results revealed that TCS increased the production of ROS and that this effect of TCS was reversed by 10 mM Nacetyl-L-cysteine (NAC), the ROS scavenger. To confirm an involvement of ROS in TCS-induced neurotoxicity we measured AhR, Cyp1a1, and Cyp1b1 mRNA expression levels in cells co-treated with TCS and NAC. In the presence of NAC, TCS enhanced mRNA expression of the cytochromes and AhR at 3 and 6 h, respectively. We postulate that TCS exhibits primary and secondary effects. The primary effects such as impairment of Cyp1a1 signaling are mediated by TCS-induced ROS production, whereas secondary effects of TCS are due to transcriptional activity of AhR and estrogenic properties of TCS.

This study investigated in vitro endocrine disrupting effects of three mixtures of POPs: 'Marine mix' extracted from Atlantic cod liver, and two mixtures extracted from burbot liver, 'Mjosa mix' and 'Losna mix'. The POP mixtures were chemically characterized. Co-culture of theca and granulosa cells, were exposed for 48h with different doses of 'Marine mix', 'Mjosa mix' or 'Losna mix'. As an end point cell viability was determinated by LDH test, steroid analysis by EIA and caspase-3 by colorimetric substrate. Chemical characterization of the mixtures demonstrated that the 'Marine mix' contained high levels of DDTs and PCBs. In the 'Mjosa mix', the dominant pollutants were BDEs and HBCD. The concentrations of POPs measured in the 'Losna mix' were considerably lower. All mixtures used in the present study had a stimulatory effect on testosterone and estradiol secretion with 'Marine mix'>'Mjosa mix'>'Losna mix'. These results show that even a mixture containing background concentrations of POPs significantly affected steroid secretion. A higher steroidogenic response in low dose ranges, compared with high dose ranges indicated xenobiotic-conditioning hormesis. This could complicate predictions of effects in risk assessments.

Leptin (L) is recognised as an important regulator of puberty and a factor which controls reproduction. Whole pig ovarian follicles were incubated with different doses of leptin (2, 20 and 200 ng/ml) added alone or in combination with 100 ng/ml of GH or 50 ng/ml of IGF-I. The expression of the functional long form leptin receptor (Ob-Rb) mRNA was examined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) in follicular cells cultured with GH or IGF-I. Both GH and IGF-I increased leptin receptor expression in prepubertal pig ovaries. In separate experiments, the action of leptin on ovarian follicular steroidogenesis and cell apoptosis was examined. After 24 h of incubation with leptin alone or in combination with GH or IGF-I, oestradiol (E2) levels were determined in the culture medium while follicular tissue was used for the estimation of caspase-3 activity. Leptin increased E2 secretion and significantly diminished caspase-3 activity at all doses used. Both GH and IGF-I stimulated oestradiol secretion and decreased caspase-3 activity. No differences were demonstrable in oestradiol secretion and caspase-3 activity between cells treated with GH plus leptin and GH alone or cells treated with IGF-I plus leptin as compared to cultures treated with GH or IGF-I alone. However, GH diminished leptin-stimulated oestradiol secretion while IGF-I was without effect on it. Both GH and IGF-I reversed the anti-apoptotic action of leptin. In conclusion, we infer that (1) leptin directly affects ovarian function in prepubertal animals by its action on oestradiol secretion and cell apoptosis, (2) GH and IGF-I modulate the action of leptin, and (3) at least in part, the direct effect of GH/IGF-I on leptin production is due to an action on leptin receptor expression.

Summary: Purpose: Long-term valproate (VPA) treatment has been associated with reproductive endocrine disorders characterized by hyperandrogenism and polycystic changes in the ovaries in women with epilepsy. Levetiracetam (LEV) is a promising, new antiepileptic drug that may represent an alternative to VPA for many patients. Here the effect of LEV and VPA on basal and gonadotropin-stimulated steroid secretion from prepubertal porcine ovarian follicular cells was compared and the conversion of testosterone to estradiol is measured. Methods: Ovarian follicles were obtained from prepubertal pigs. Follicular theca and granulosa cells were cocultured and different concentrations of LEV or VPA added to the control or gonadotropin-stimulated cultures. Results: VPA, but not LEV, caused a significant increase of LH-stimulated testosterone secretion and decreased FSHstimulated estradiol secretion. VPA decreased conversion of testosterone to estradiol in both basal and FSH-stimulated cultures, while LEV only decreased testosterone to estradiol conversion after FSH stimulation and only at the highest, nontherapeutic drug concentration. Both drugs increased basal testosterone secretion at therapeutic drug levels. VPA also reduced basal estradiol secretion, while LEV decreased basal estradiol secretion only at nontherapeutic drug levels. Conclusion: Both LEV and VPA affect endocrine function in the prepubertal ovary. But while VPA alters both basal and gonadotropin-stimulated testosterone and estradiol secretion at therapeutic drug concentrations, LEV only affects basal hormone secretion at this concentration level. The possibility that LEV could be an alternative treatment to VPA if reproductive endocrine problems emerge in adult women, is discussed. However, extrapolation to the clinical situation is problematic and particular emphasis is placed on the need for further studies. Key Words: Levetiracetam—Valproate—Steroidogenesis—Ovary.

Theca interna and granulosa cells from small, medium and large preovulatory porcine follicles were cultured as a monolayer to compare the effects of 4-chlorobiphenyl (PCB3) and its metabolites on estradiol secretion. Cells were treated with PCB3 or its mono- and di-hydroxylated metabolites 4-OH-PCB3 and 3,4-di-OH-PCB3 at concentrations of 0.06, 0.6, 6 and 60 ng/ml medium for 24 h. Each compound significantly increased estradiol levels detected in the medium of small, medium and large follicle cultures. This effect was evident at even the lowest concentration tested (0.06 ng/ml). A time-series with 6.0 ng/ml PCB3 or metabolites showed estrogenic action at each test interval (24, 48, 72, 96 h) and established a rankorder potency of 3,4-OH-PCB > 4-OH-PCB3 ≥ PCB3. In small and medium follicles treated with PCB3 metabolites the temporal increase led to estradiol accumulations >20,000-fold over control culture media. Large preovulatory follicles had the highest estrogenic action at 72 h of exposure, decreasing somewhat at 96 h. These findings indicate that PCB3 and its hydroxylated metabolites induce a surge in estradiol secretion in ovarian follicle cells. Such a response would in vivo be expected to disrupt reproductive processes, through enhanced aging of the follicle and negative feedback to the hypothalamus, and perhaps increase the risk for ovarian and breast cancer.

Apoptosis causes the elimination of ovarian germ cells and the atretic degeneration of ovarian follicles. Here we have used cultured rat preovulatory follicles to examine the regulation of effector caspase-3 and -7 in follicles undergoing apoptosis in the presence or absence of gonadotropins or IGF-I. Culturing follicles in the presence or absence of serum resulted in the induction of apoptosis of granulosa cells (GC), which was accompanied by effector caspase activation. Surprisingly, the addition of the survival factors LH or FSH, but not IGF-I, further increased caspase-3 and -7 activity. Immunohistochemistry studies of the LH- and FSH-treated follicles indicated that cleaved caspase-3 was predominantly localized to the peripheral theca-interstitial cells (TIC). Western blot analysis and caspase-3 and -7 activity assays of the separated follicular compartments confirmed that both LH and FSH treatments significantly enhance caspase-3 and -7 activity in TIC. The elevation in caspase-3 and -7 activity in TIC was accompanied by an increase in apoptosis. Interestingly, LH and FSH also induced an increase in caspase-3 and -7 activity in GC; however, this increase was accompanied by a decrease in apoptosis. Finally, we demonstrate that in freshly isolated preovulatory follicles and in antral follicles in intact ovaries, the expression level of procaspase-3 is significantly higher in TIC than in GC. Thus, LH and FSH have a dual effect on the cultured rat preovulatory follicle: an antiapoptotic effect on GC and a proapoptotic effect on TIC.

The aim of the presented study was to evaluate the effects of PCB 126 and PCB 153 on granulosa and theca cell apoptosis. Granulosa and theca cells were collected from small, medium, and large preovulatory porcine follicles and cultured as monolayers. Cells were initially cultured for 24 h to allow attachment to the plates. Media were changed and 100 pg/ml PCB 126 or 100 ng/ml PCB 153 were added. After 48 h, granulosa and theca cells were fixed for assessment of the number of apoptotic cells utilizing a Hoechst staining technique or frozen for measurement of caspase-3 activity. Media were collected for testosterone concentration analysis from theca cell cultures or estradiol from granulosa cell cultures. Neither PCB 153 nor PCB 126 had an effect on testosterone secretion by theca cells collected from small and medium size follicles, while both PCBs decreased testosterone secretion by large follicles. The decrease in testosterone secretion by large follicles under the influence of both PCBs was paralleled by a suppression of caspase-3 activity and a decreased incidence of apoptotic bodies. Neither of the PCBs had an effect on estradiol secretion by granulosa cells collected from small and medium size follicles, while both PCBs increased estradiol in granulosa cells collected from large follicles. PCB-associated increased estradiol secretion by granulosa cells collected from large follicles was accompanied by suppression of caspase-3 activity and a decreased incidence of apoptotic bodies. In conclusion, we have presented evidence that in preovulatory follicles PCBs inhibit both theca and granulosa cells apoptosis. Therefore, an exposure to PCBs may cause alterations in the pattern of terminal differentiation of follicles and attenuate spontaneous elimination of atretic follicles.

In our previous paper[1], we demonstrated that porcine follicles collected during the early stage of development are the most sensitive to the toxic action of polychlorinated biphenyl 153 (PCB 153). Follicles of this type were collected to test the effect of PCB 153 on cell steroidogenesis and viability. Cocultures of granulosa and theca cells were grown in M199 medium at 37ºC. Control cultures were maintained in that medium alone, while experimental ones were supplemented with PCB 153 at doses of 5, 10, 50, and 100 ng/ml. After 48, 96, and 144 h, media were collected for steroid analysis and cell viability was measured using an LDH (lactate dehydrogenase activity) cytotoxicity test. A 2-day exposure of follicular cells to all the investigated doses of PCB 153 caused a statistically significant decrease in progesterone (P4) secretion, while in doses of 50 and 100 ng/ml there was also a decrease in testosterone (T) secretion. No effect on estradiol (E2) secretion was observed. The observed decrease in P4 and T secretion, and lack of any statistically significant effect on E2 secretion by cells from small follicles exposed for 48 h to PCB, suggests that PCB 153 acts before P4 formation. Longer exposures caused an increase in P4 secretion, with a concomitant drastic decrease in T secretion and a tendency to decrease the E2 secretion, suggesting inhibition of P450 17αhydroxysteroid dehydrogenase, an enzyme that converts P4 to T. The observed PCB 153–induced increase in P4 secretion by cells collected from small antral follicles, with a concomitant decrease in E2 secretion, accounts for the induction of luteinization and, in this case, inhibition of aromatization process in the follicles. However, in all doses tested and at all times of exposure, PCB 153 had no effect on cell viability. These findings suggest different time of exposure–dependent action of PCB 153 on particular steps of steroidogenesis but not action on cell viability. These results should be considered preliminary, pending confirmation by other studies.

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 μ 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.

Phytoestrogens have received considerable attention because they provide an array of beneficial effects, such as neuroprotection. To better understand the molecular and functional link between phytoestrogens and classical as well as membrane estrogen receptors (ERs), we investigated the effect of daidzein on the glutamate-mediated apoptotic pathway. Our study demonstrated that daidzein (0.1–10 μM) inhibited the pro-apoptotic and neurotoxic effects caused by glutamate treatment. Hippocampal, neocortical and cerebellar tissues responded to the inhibitory action of daidzein on glutamate-activated caspase-3 and lactate dehydrogenase (LDH) release in a similar manner. Biochemical data were supported at the cellular level by Hoechst 33342 and calcein AM staining. The sensitivity of neuronal cells to daidzein-mediated protection was most prominent in hippocampal cultures at an early stage of development 7th day in vitro. A selective estrogen receptor β (ERβ) antagonist, 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5,-a]pyrimidin-3-yl]phenol (PHTPP), and a selective G-protein-coupled receptor 30 (GPR30) antagonist, 3aS∗,4R∗,9bR∗)-4-(6-Bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinoline (G15), reversed the daidzein-mediated inhibition of glutamate-induced loss of membrane mitochondrial potential, caspase-3 activity, and LDH release. A selective ERα antagonist, methyl-piperidino-pyrazole (MPP), did not influence any anti-apoptotic effect of daidzein. However, a high-affinity estrogen receptor antagonist, 7α,17β-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI) 182,780, and a selective GPR30 agonist, (±)-1-[(3aR∗,4S∗,9bS∗)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone (G1), intensified the protective action of daidzein against glutamate-induced loss of membrane mitochondrial potential and LDH release. In siRNA ERβ- and siRNA GPR30-transfected cells, daidzein did not inhibit the glutamate-induced effects. Twenty-four hour exposure to glutamate did not affect the cellular distribution of ERβ and GPR30, but caused greater than 100% increase in the levels of the receptors. Co-treatment with daidzein decreased the level of ERβ without significant changing of the GPR30 protein level. Here, we elucidated neuroprotective effects of daidzein at low micromolar concentrations and demonstrated that the phytoestrogens may exert their effects through novel extranuclear GPR30 and the classical transcriptionally acting ERβ. These studies uncover key roles of the ERβ and GPR30 intracellular signaling pathways in mediating the anti-apoptotic action of daidzein and may provide insight into new strategies to treat or prevent neural degeneration.

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.

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.

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.

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.

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.