In vivo and in vitro effects of PCB126 and PCB153 on rat testicular androgenesis

In this study we compared the effects of PCB126 and PCB153 on adult rat testicular androgenesis and the status of antioxidant enzymes in the interstitial cell compartment 96h after local intratesticular application. Obtained results indicated PCB126-induced inhibition of conversion of progesterone (P) and Δ(4)-androstenedione (A(4)) to testosterone (T), and stimulation of conversion of P to T induced by PCB153, while combined application had no effect. Activities of antioxidant enzymes were unchanged, except of decreased activity of SOD in PCB126-treated group. In parallel experiments, adult purified Leydig cells challenged with PCB congeners were incubated for 2h in the presence of corresponding steroid substrates. Results demonstrated that in the presence of subsaturating substrate concentrations PCB126 induced inhibition of conversion of P and A(4) to T at nM to μM doses, while PCB153 caused stimulation at nM concentrations. Further studies should indicate possible mechanism(s) of modulation of androgenesis by tested PCB congeners.     

Quercetin attenuates cadmium-induced oxidative damage and apoptosis in granulosa cells from chicken ovarian follicles

The attenuating effect of quercetin on cadmium-induced oxidative damage and apoptosis was investigated in cultured granulosa cells from chicken ovarian follicles. Results showed that exposure to 5 μM CdCl(2) induced a decrease in granulosa cell number and viability, caused chromatin condensation and DNA fragmentation. Moreover, cadmium treatment markedly increased malondialdehyde level and decreased glutathione peroxidase and superoxide dismutase activities. Furthermore, cadmium provoked higher BAX expression, inhibited expression of BCL2 and X-linked inhibitor of apoptosis protein (XIAP) and activated caspase-3. However, simultaneous supplementation with 1 μg/ml quercetin protected granulosa cells against cadmium-induced cytotoxicity through attenuating lipid peroxidation, renewing antioxidant enzymes activities and alleviating apoptosis by modulating XIAP, BAX and BCL2 expression, and inhibiting caspase-3 activity. Therefore, these results suggested that quercetin, as a widely distributed dietary antioxidant, contributes potentially to prevent cadmium-induced cytotoxicity in granulosa cells through attenuating lipid peroxidation, elevating intracellular antioxidant status and inhibiting apoptosis to ensure reproductive health.     

Effects of lipopolysaccharide (LPS) and peptidoglycan (PGN) on estradiol production in bovine granulosa cells from small and large follicles

In cows, postpartum uterine infection due to bacteria that produce lipopolysaccharide (LPS) or peptidoglycan (PGN) leads to ovarian dysfunction. The aim of this study was to determine the effect of LPS and/or PGN on estradiol production from granulosa cells from small and large follicles in the bovine ovary. Granulosa cells from small and large ovarian follicles were exposed to LPS and/or PGN in vitro. LPS inhibited the expression of TLR4, CD14, MD2 and NOD1 genes in FSH-treated granulosa cells from small follicles. LPS suppressed estradiol (E2) production in granulosa cells from small and large follicles, while PGN inhibited E2 production in granulosa cells from large follicles. LPS or PGN did not affect granulosa cell survival. Although LPS alone suppressed E2 production in granulosa cells from small and large follicles, E2 production was not further suppressed when PGN was added to culture medium with LPS alone. Our data demonstrated that susceptibility to LPS or PGN in granulosa cells depends on the follicle developmental stage. The results of the present study suggest that ovarian dysfunction in cows with postpartum uterine infection may be caused by inhibitory effects of LPS and PGN on E2 production in granulosa cells.     

Effect of triptolide on progesterone production from cultured rat granulosa cells

Triptolide(CAS 38748-32-2), a major active component of Tripterygium wilfordii Hook F (TWHF), is known to have multiple pharmacological activities. However, studies have also shown that triptolide is highly disrupt to the reproductive system by disrupting normal steroid hormone signaling. In the present study, we investigated the effect of triptolide (5, 10, or 20 nM for 24 h) on progesterone production by rat granulosa cells. Triptolide inhibited both basal and human chorionic gonadotropin (HCG)- and 8-bromo-cAMP-stimulated progesterone production as revealed by RIA assay. Furthermore, the HCG-evoked increase in cellular cAMP content was also inhibited by triptolide, indicating that disruption of the cAMP/PKA signaling pathway may mediate the deleterious effects of triptolide on progesterone regulation. In addition, triptolide inhibited 25-OH-cholesterol-stimulated progesterone production, suggesting that activity of the P450 side chain cleavage (P450scc) enzyme was also be inhibited by triptolide. Western blot and quantitative real-time PCR (qRT-PCR) assays further revealed that triptolide decreased mRNA and protein expression of P450scc and the steroidogenic regulatory (StAR) protein in granulosa cells. In contrast, cell viability tests using 3-(4,5-dimethyl-thiazol-2-yl)-2,5- diphenyl-tetrazolium bromide (MTT) indicated that triptolide did not cause measurable cell death at doses that suppressed steroidogenesis. The reproductive toxicity of triptolide may be caused by disruption of cAMP/PKA-mediated expression of a number of progesterone synthesis enzymes or regulatory proteins, leading to reduced progesterone synthesis and reproductive dysfunction.     

雄激素诱导小鼠颗粒细胞凋亡及其机制探讨

目的 通过睾酮诱导小鼠颗粒细胞凋亡,观察热休克蛋白(HSP) 10在高雄激素下的表达,探讨HSP10在高雄激素诱导颗粒细胞凋亡中的作用.方法 原代培养小鼠颗粒细胞,用0、10-8M、10-6M、10-5M睾酮处理24 h,检测HSP10基因表达;处理48 h,检测细胞活力及活性Caspase-3的表达.用AdCMV-H1-siRNA/HSP10腺病毒在小鼠颗粒细胞中干涉HSP10的表达,MTT检测细胞活力,流式细胞术检测细胞周期,并检测Ki67、Bcl-2、Bax基因水平变化.结果 与0M睾酮处理组比较,10-6 M、10-5M睾酮处理组颗粒细胞的细胞活力降低,且Caspase-3表达增加,HSP10表达降低(P＜0.05);感染AdCMV-H1-siRNA/HSP10腺病毒后,颗粒细胞的细胞活力降低,且细胞增殖阻滞在G2期,Bcl-2、Ki67表达降低而Bax表达增加(P＜0.05).结论 高雄激素可诱导小鼠颗粒细胞凋亡.其可能的机制是睾酮能降低HSP10表达,经由降低Bcl-2和增加Bax表达的通路.     

Effects of perinatal exposure to low doses of PCB 153 and PCB 126 on lymphocyte proliferation and hematology in goat kids

Pregnant does (10 goats/group) were dosed orally with either PCB 153 or PCB 126 dissolved in corn oil or only corn oil (control group) from day 60 of gestation until delivery. Effects on in vitro mitogen-induced lymphocyte proliferation and blood cell counts in their goat kids exposed to low levels of PCB 153 and PCB 126 during gestation and lactation were assessed. The concentrations of PCB 153 and PCB 126 in adipose tissue in the goat kids 9 mo postpartum were 5800 ng/g (fat weight) and 0.49 ng/g (fat weight), respectively. Kids exposed to PCB 153 had a significantly higher number of white blood cells, neutrophils, and lymphocytes at 2 wk of age compared to controls. In the kids exposed to PCB 126 there was a significantly lower concentration of monocytes at 2, 4, and 8 wk of age. The mean lymphocyte response to phytohemagglutinin (PHA) and to concanavalin A (Con A) was significant lower in the PCB 153 compared to the control group at wk 2, 4, and 8 postnatally. The results of the present study support previous reports on immunotoxic effects of PCB exposure in animals. However, this is the first report to demonstrate immunotoxicity in animals by using low doses of PCB 153. The difference in results between PCB 126 and PCB 153 treatment groups may strengthen the hypothesis that PCBs mediate immunotoxic effects through both AhR-dependent and -independent mechanisms.     

Perinatal exposure to low doses of PCB 153 and PCB 126 affects maternal and neonatal immunity in goat kids

Pregnant does (10 goats/group) were dosed orally either with polychlorinated biphenyl (PCB) 153 (98 microg/kg body weight/d) or PCB 126 (ng/kg body weight/d) dissolved in corn oil or with corn oil only (control group) from gestation day (GD) 60 until delivery. An additional group (n = 5) of pregnant does received the synthetic estrogen diethylstilbestrol (DES; 0.4 microg/kg body weight/d) by intramuscular injection using the same treatment schedule as for the PCB groups. Blood samples for immune analysis were collected at wk 0, 1, 2, 4, 6, and 8 of age. The effects of perinatal PCB exposure on postnatal humoral immune responses were examined by assessing the levels of total immunoglobulin G (IgG) and immunoglobulins to specific microbes at wk 0, 1, 2, 4, 6, and 8 of age, and immune responses following immunization of kids at 2 wk of age. PCB 153 exposure suppressed maternal and neonatal immunity, as demonstrated by reduced transfer of maternal IgG and specific antibodies to the environmental microbes Arcanobacterium pyogenes, Mannheimia haemolytica, and reovirus (REO-1). Furthermore, PCB 153 reduced the level of maternal antibodies to Mycobacterium avium paratuberculosis and equine influenza virus (EIV-1) in the newborn kids. The antibody response against EIV-1 was significantly higher in PCB 153-exposed kids 2 wk following immunization. PCB 126 exposure reduced the levels of maternal antibodies to REO-1. In contrast, gestational exposure to PCB 126 increased the concentrations of maternal antibodies to tetanus toxoid. No differences from controls in plasma total IgG levels at birth or colostrum IgG concentrations were observed in the PCB 126-treated does. However, a significant reduction in IgG levels from GD 60 until delivery was found in this group. Gestational exposure to DES reduced the concentrations of maternal antibodies against A. pyogenes, M. haemolytica, M. avium Paratuberculosis, and REO-1. These results suggest that perinatal exposure to low doses of PCB 126 and PCB 153 affects the maternal immunity in kids. The difference in responses between PCB 126 and PCB 153 treatment groups may strengthen the hypothesis that PCBs mediate immunotoxic effects through both AhR-dependent and -independent mechanisms. The observation that the effects produced by PCB 153 resembled those produced by DES raises the question of whether this congener may modulate immunity by estrogenic mechanisms.     

Effects of nicotine and cotinine on bovine theca interna and granulosa cells

The purpose of this study was to determine if nicotine or cotinine inhibits steroidogenesis in the ovarian follicle. Theca interna and granulosa cells were isolated from bovine follicles, cultured with nicotine or cotinine for 24 h, and culture media were assayed for androstenedione or estradiol. Treatment of theca interna with 6, 60, and 600 μM nicotine decreased (P≤0.002) production of androstenedione to 55, 53, and 24% of control levels, respectively. Levels of androstenedione in theca interna treated with cotinine were not different from control values. In granulosa cells, nicotine inhibited production of estradiol at the highest dose tested. Treatment with 600 μM nicotine decreased (P≤0.001) estradiol concentration to 12% of control values, attributable to a general cytotoxic effect. Cotinine had no effect on estradiol production by granulosa cells. These results provide novel evidence for inhibitory effects of nicotine on androgen production by theca interna.     

Different effects of PCB101, PCB118, PCB138 and PCB153 alone or mixed in MCF-7 breast cancer cells

Polychlorinated biphenyls (PCBs) are ubiquitous, persistent environmental contaminants that can be a potential health hazard. In the present study we analyzed the potential estrogenic effect in MCF-7 cells of four biologically relevant PCB congeners, alone or in mixtures, present in dairy products, vegetable oil and fish: PCB101, PCB118, PCB138 and PCB153. The mixture of four PCB was tested at seven different concentrations. We investigated the ability of these PCBs, alone or mixed, to induce cell proliferation, and the level of estrogen-regulated protein pS2, in human MCF-7 breast cancer cells. PCB153 (35 microM) stimulated cell proliferation from 48 h up to day 6, PCB118 (40 microM) only at 48 h, but PCB101 (45 microM) and PCB138 (15 microM) applied to the cells for 6 days had no effect. In contrast, the various concentrations of mixtures significantly reduced cell proliferation at different times. No change in pS2 levels was seen after treatment with the PCBs alone or mixed. In exploring the mechanism of these events, we found that PCB153 induced mitogen-activated protein kinase (MAPK) ERK1/2 at 4, 8 and 12 h, while the antiproliferative effect seemed to be related to an apoptotic action beginning at 12 h and ending at 48 h. These findings indicate that these PCBs alone or mixed have no estrogenic effect in MCF-7 cells, although PCB153 induce an ERK1/2-mediated mitogenic effect. On the contrary the mixture of PCBs induces an antiproliferative effect, ascribable to an apoptotic action.     

Comparison of pharmacokinetic interactions and physiologically based pharmacokinetic modeling of PCB 153 and PCB 126 in nonpregnant mice, lactating mice, and suckling pups.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that can induce neurological defects in infants and children via placental and lactational transfer. To investigate the lactational transfer of PCBs and compare pharmacokinetic interactions among nonpregnant, lactating mice and suckling pups, quantitative time-course measurements of PCB accumulation in tissues were performed. On postnatal day 1, nonpregnant and lactating C57BL/6 mice were exposed to PCB 153 (2,2',4,4',5,5'-hexachlorobiphenyl, 20 mg/kg) alone or a mixture of PCB 153 (20 mg/kg) and PCB 126 (3,3',4,4',5-pentachlorobiphenyl, 0.2 mg/kg) by oral gavage. At 1, 3, 6, and 13 days after treatment, PCB 153 and PCB 126 were determined in nonpregnant and maternal tissues as well as in neonatal tissues by gas chromatography (GC). Coadministration of PCB 153 and PCB 126 increased PCB 153 retention in the liver and decreased PCB 153 accumulation in the fat of nonpregnant mice. Lactational transfer was confirmed to be an efficient elimination mechanism for the lactating mice but a major source of exposure in the pups. However, little or no significant pharmacokinetic interactions were observed in lactating mice and suckling pups. To describe pharmacokinetic interactions between PCB 153 and PCB 126, a physiologically based pharmacokinetic model for PCB 153 disposition was developed. The effects of PCB 126 on the fat content in liver and a diffusion permeation constant in fat were incorporated into the physiologically based pharmacokinetic (PBPK) model. This model successfully describes PCB 153 disposition altered by PCB 126 in nonpregnant mice.     

Effects of PCB 126 on primary immune organs and thymocyte apoptosis in chicken embryos

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyl (PCB) 126 produce thymic atrophy and immunosuppression. This study explored the hypothesis that the thymic atrophy produced by developmental exposure to PCB 126 is associated with an increase in apoptotic thymocytes at the end of incubation in chicken embryos. Eggs were injected via the air cell with PCB 126 (0.05, 0.13, 0.32, 0.64, and 0.80 ng/g egg) on d 0 of incubation, and tissues were collected on d 20. Controls included noninjected and vehicle-injected (sunflower oil) eggs. Thymocytes were cultured for 6 h and analyzed by flow cytometry for decreased DNA content (propidium iodide staining) and cell size (forward scatter), which indicate apoptosis. PCB 126 induced dose-dependent mortality with an LD50 of 1.01 ng/g and lowest-observed-effect concentration (LOEC) of 0.32 ng/g. Teratogenic effects commonly associated with TCDD and planar PCBs, including cranial and foot deformities and subcutaneous edema, tended to increase with dose of PCB 126. PCB 126 reduced thymus mass by approximately 20% at 0.64 and 0.8 ng/g, the number of viable thymocytes by approximately 20-24% at and above 0.13 ng/g, and the number of bursal lymphoid cells by 57% at 0.64 ng/g. The percentage of apoptotic thymocytes increased with dose, reaching levels 2 times greater than controls at 0.8 ng/g. Electrophoresis of low-molecular-weight DNA from thymocytes of all doses demonstrated fragments in multiples of 180 bp. This DNA laddering is a hallmark of apoptosis. At all doses, thymocytes exhibited caspase-3 activation, another indicator of apoptosis. The results of this experiment supported the hypothesis that the thymic atrophy produced by developmental exposure to PCB 126 in chicken embryos is associated with an increase in apoptotic thymocytes on embryonic d 20.     

Cell death mechanisms in GT1-7 GnRH cells exposed to polychlorinated biphenyls PCB74, PCB118, and PCB153

Exposure to endocrine disrupting chemicals (EDCs) such as polychlorinated biphenyls (PCBs) causes functional deficits in neuroendocrine systems. We used an immortalized hypothalamic GT1-7 cell line, which synthesizes the neuroendocrine peptide gonadotropin-releasing hormone (GnRH), to examine the neurotoxic and endocrine disrupting effects of PCBs and their mechanisms of action. Cells were treated for 1, 4, 8, or 24 h with a range of doses of a representative PCB from each of three classes: coplanar (2,4,4′,5-tetrachlorobiphenyl: PCB74), dioxin-like coplanar (2′,3,4,4′,5′ pentachlorobiphenyl: PCB118), non-coplanar (2,2′,4,4′,5,5′-hexachlorobiphenyl: PCB153), or their combination. GnRH peptide concentrations, cell viability, apoptotic and necrotic cell death, and caspase activation were quantified. In general, GnRH peptide levels were suppressed by high doses and longer durations of PCBs, and elevated at low doses and shorter timepoints. The suppression of GnRH peptide levels was partially reversed in cultures co-treated with the estrogen receptor antagonist ICI 182,780. All PCBs reduced viability and increased both apoptotic and necrotic cell death. Although the effects for the three classes of PCBs were often similar, subtle differences in responses, together with evidence that the combination of PCBs acted slightly different from individual PCBs, suggest that the three tested PCB compounds may act via slightly different or more than one mechanism. These results provide evidence that PCB congeners have endocrine disrupting and/or neurotoxic effects on the hypothalamic GnRH cell line, a finding that has implications for environmental endocrine disruption in animals.     

Action of defined mixtures of PCBs, p,p'-DDT and its metabolite p,p'-DDE, on co-culture of porcine theca and granulosa cells: steroid secretion, cell proliferation and apoptosis

We recently reported on a stimulatory effect of a mixture of persistent organic pollutants (POPs) extracted from the liver oil of Atlantic cod (Gadus morhua) on estradiol (E2) secretion by porcine ovarian follicular cells [Gregoraszczuk EL, Milczarek K, Wójtowicz A, Berg V, Skaare JU, Ropstad E. Steroid secretion following exposure of ovarian follicular cells to three different natural mixtures of persistent organic pollutants (POPs). Reproductive Toxicol 2007;25:58-66]. The objective of the present study was to identify what compound or compounds within this natural mixture could be responsible for the stimulatory action on E2 secretion. Co-cultures of porcine ovarian theca and granulosa cells were exposed for 48h to individual congeners of polychlorinated biphenyls (PCBs) alone or in combination, at environmentally relevant concentrations (PCB153 at 8microg/ml; PCB118 at 3microg/ml; PCB138 at 8microg/ml; PCB180 at 3microg/ml; PCB126 at 8ng/ml; DDT at 5microg/ml; p,p'-DDE at 10microg/ml). At the end of culture media samples were collected and used for cell viability and steroid determination, and cells were used for the measurement of caspase-3 activity. A threefold decrease in E2 secretion was noted when PCB126 was added in combination with PCB153. Only DDT and DDE could reverse the action of PCB126. Stimulatory secretion of E2 occurred in parallel with a decrease in testosterone (T) secretion. Among the single congeners used in the present study, DDE had the strongest stimulatory action on E2 secretion. In conclusion, the present findings indicate that the overall effect on steroid secretion by ovarian follicular cells could be dependent on the concentration of DDE in environmental POPs and that the predicted effect of a mixture based on a sum of independent effects of individual congeners is higher than that noted under the influence of defined mixture.     

Transcriptomics identifies differences between ultrapure non-dioxin-like polychlorinated biphenyls (PCBs) and dioxin-like PCB126 in cultured peripheral blood mononuclear cells

Polychlorinated biphenyls (PCBs) remain ubiquitously present in human lipids despite the ban on their production and use. Their presence can be chemically monitored in peripheral blood samples of the general population. We tested whether in vitro exposure to different PCB congeners induced different gene expression profiles in peripheral blood cells. We have isolated peripheral blood mononuclear cells (PBMC) from whole blood of 8 healthy individuals and exposed these cells in vitro to individual non-dioxin-like (NDL)-PCB congeners (PCB52, 138 or 180; 10μM) or dioxin-like (DL)-PCB congener PCB126 (1μM) during 18h. Differential gene expression response was measured using Agilent whole-human genome microarrays. Two-way ANOVA analysis of the data showed that both gender and PCB exposure are important factors influencing gene expression responses in blood cells. Hierarchical cluster analysis of genes influenced by PCB exposure, revealed that DL-PCB126 induced a different gene expression response compared to the NDL-PCBs. Biological interpretation of the results revealed that exposure to PCB126 induced the AhR signaling pathway, whereas the induction of nuclear receptor pathways by the NDL-PCBs was limited in blood cells. Nevertheless, molecular responses of blood cells to individual PCB congeners revealed significantly expressed genes that play a role in biological functions and processes known to be affected by PCB exposure in vivo. Observed gene expression changes in this in vitro model were found to be related to hepatotoxicity, immune and inflammatory response and disturbance of lipid and cholesterol homeostasis.     

人毛乳头细胞的生物学特性研究

目的观察毛乳头细胞在体外培养条件下的生长特性,为毛囊重建提供参考资料。方法采用二步酶消化法培养正常人头皮毛囊的毛乳头细胞,在体外进行传代培养,用免疫组化方法观察不同传代毛乳头细胞生长因子表达的差异,同时收集低代培养毛乳头细胞的条件培养基,观察其对高代毛乳头细胞的影响。结果高代培养毛乳头细胞逐渐丧失其凝集性生长的特性,且生长因子的表达逐渐消失,低代毛乳头条件培养基可恢复高代毛乳头细胞的某些特性。结论诸多因素影响毛乳头细胞生物学特性,体外培养的毛乳头细胞与体内有差异。低代毛乳头条件培养基可恢复毛乳头细胞的部分特性。     

Effects of fenvalerate on progesterone production in cultured rat granulosa cells

In this study, primary serum-free cultured rat granulosa cells (rGCs) were used as a cellular model to investigate the effects of fenvalerate on progesterone production. Various concentrations (0, 1, 5, 25, 125 and 625 microM) of fenvalerate were added to the cell cultures for 24 h. rGCs were stimulated by compounds such as follicle-stimulating hormone (FSH), 8-bromo-cAMP or 22(R)-hydroxycholesterol (22R-HC). Progesterone production and intracellular cAMP content were measured in control and treated groups. Expression of P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory protein (StAR) were monitored by real-time PCR and Western blotting. Results showed that fenvalerate inhibited basal progesterone production in rGCs in the absence of stimulators. This inhibition was stronger in the presence of FSH and was not fully reversed by 8-bromo-cAMP or 22R-HC. The increase of cAMP content, stimulated by FSH, was inhibited by fenvalerate implicating that the intracellular cAMP-dependent signal pathway was involved. Fenvalerate reduced mRNA and protein expression of P450scc. These results suggested that multi-site inhibition of progesterone production by fenvalerate including a cAMP-dependent protein kinase pathway and reduction on P450scc gene expression and/or its enzymatic activity in rGCs.     

Study on PCB 101, PCB 153, mercury and methyl mercury content in blue crab Portunus Pelagicus from Khuzestan shore (Persian Gulf)

Distribution of polychlorinated biphenyl (PCB 101, PCB 153), Mercury (Hg) and methyl mercury (MMHg) in muscle, gill and hepatopancreas of blue swimming crab Portunus segnis from Persian Gulf were investigated. In addition, the relationships between crab size (carapace width) and PCBs, Hg, MMHg levels in tissues were investigated by linear regression analysis. There were significant differences in crab PCB 101, PCB 153, Hg and MMHg concentrations were detected between different tissues (ANOVA, P<0.05). The pollutants concentrations were highest in hepatopancreas whereas lowest in the muscle of all crab species. The recorded mean concentrations were 0.485 μg g^?1 PCB 101, 1.89 μg g^?1 PCB 0.463, 0.815 μg g^?1 Hg and 0.471 μg g^?1 MMHg. The results showed that significant relationships between PCB and Hg-MMHg levels and crab size were positive. Comparison between male and female indicated that the average PCBs and Hg-MMHg concentrations in tissues of male crab were found to be significantly higher than those found in the female crab.     

Fas抗体对体外培养的气道上皮细胞凋亡增殖的影响

目的观察Fas抗体、白细胞介素-1β(IL-1β)对体外培养的气道上皮细胞(AEC)凋亡、增殖的影响。方法分离培养兔的AEC,加入IL-1β与不同浓度的Fas抗体共同孵育,以流式细胞术和末端脱氧核苷酸转移酶介导脱氧三磷酸核苷缺口标记技术(TUNEL)法检测AEC凋亡的变化,以免疫细胞化学法检测AEC增殖细胞核抗原(PCNA)的表达程度。结果随Fas抗体浓度(500,1000ng/ml)增加,PCNA表达阳性率逐渐增高,分别为(49.7±6.7)%和(57.0±1.9)%,IL-1β组和IL-1β+Fas抗体(500ng/ml)组PCNA表达阳性率为(72.2±5.9)%和(44.6±8.0)%,以上各实验组与对照组表达阳性率[(36.5±2.1)%]比较,差异均有统计学意义(P<0.05),各加Fas抗体组与单加IL-1β组比较,差异均有统计学意义(P<0.05)。结论Fas抗体能够有效地诱导正常和炎症状态下原代AEC发生凋亡,IL-1β能有效地增强AEC的增殖作用,Fas抗体可以有效抑制IL-1β所致AEC的增殖作用,可能对抑制气道炎症和气道重塑有积极作用。     

Exposure to the Three Structurally Different PCB Congeners (PCB 118, 153, and 126) Results in Decreased Protein Expression and Altered Steroidogenesis in the Human Adrenocortical Carcinoma Cell Line H295R

Polychlorinated biphenyls (PCB), synthetic, persistent organic pollutants (POP), are detected ubiquitously, in water, soil, air, and sediments, as well as in animals and humans. PCB are associated with range of adverse health effects, such as interference with the immune system and nervous system, reproductive abnormalities, fetotoxicity, carcinogenicity, and endocrine disruption. Our objective was to determine the effects of three structurally different PCB congeners, PCB118, PCB 126, and PCB 153, each at two concentrations, on the steroidogenic capacity and proteome of human adrenocortical carcinoma cell line cultures (H295R) . After 48 h of exposure, cell viability was monitored and estradiol, testosterone, cortisol and progesterone secretion measured to quantify steroidogenic capacity of the cells. Two-dimensional (2D) gel-based proteomics was used to screen for proteome alterations in H295R cells in response to the PCB. Exposure to PCB 118 increased estradiol and cortisol secretion, while exposure to PCB 153 elevated estradiol secretion. PCB 126 was the most potent congener, increasing estradiol, cortisol, and progesterone secretion in exposed H295R cells. Seventy-three of the 711 spots analyzed showed a significant difference in normalized spot volumes between controls (vehicle only) and at least one exposure group. Fourteen of these protein spots were identified by liquid chromatography with mass spectroscopy (LC-MS/MS). Exposure to three PCB congeners with different chemical structure perturbed steroidogenesis and protein expression in the H295R in vitro model. This study represents an initial analysis of the effects on proteins and hormones in the H295R cell model, and additional studies are required in order to obtain a more complete understanding of the pathways disturbed by PCB congeners in H295R cells. Overall, alterations in protein regulation and steroid hormone synthesis suggest that exposure to PCB disturbs several cellular processes, including protein synthesis, stress response, and apoptosis.     

Inhibitory effect of nomegestrol acetate on steroidogenesis of cultured granulosa cells from rat ovary in vitro.

AIM: To study the effect of nomegestrol acetate, a new synthetic progesterone on granulosa cells' viability and steroidogenesis function. METHODS: Granulosa cells were cultured in McCoy's 5A medium. Trypan blue stain was used to measure viable cells. FSH and testosterone were added to stimulate the steroid secretion. Specific RIA assay was used to evaluate the estrogen and progesterone secretion respectively. RESULTS: IC50 of nomegestrol acetate to damage cells is 6.85 mg/L (95 % confidence limits 5.36-8.75 mg/L). Nomegestrol acetate 0.45, 0.9, and 1.8 mg/L greatly inhibited the estrogen secretion from granulosa cells by 7.6 %, 12.5 %, 28.3 % in the presence of testosterone 0.5 micromol/L and FSH 10 U/L without affecting the number of viable cells. The secretion of progesteron were markedly decreased by 44.5 %, 53.3 %, and 62.0 % concurrently. CONCLUSION: Nomegestrol acetate directly inhibited the steroidogenesis of granulosa cells.