Switching off HSP70 and i-NOS to study their role in normal and H2O2-stressed human fibroblasts

i-NOS and HSP70 antisense oligonucleotides were used to study the role of the two well known stress-regulated molecules on cell survival of both untreated control, and H2O2-stressed human fibroblasts. Cell survival was assessed either by LDH release or by MTT assay. The levels of cytosolic i-NOS and HSP70 were tested by using immunoblotting analysis, and reactive oxygen species (ROS) production was quantified. Compared to the values observed in untreated control cells, anti HSP70-transfected human fibroblasts showed an increase in ROS production, i-NOS level and LDH release. The addition of 0.12 mM H2O2 for 20 min. to the HSP70-deprived fibroblasts did not modify the percentage of LDH release observed in H2O2 stressed cells, but reduced cell viability increasing both ROS production and i-NOS level. Anti i-NOS-transfected fibroblasts, compared to the control untreated cells, showed no modification in ROS production, while cell survival was improved. When treated with H2O2 the i-NOS depleted cells counteracted ROS formation as well as LDH release but negatively affected cell viability and HSP70 levels, compared to the results obtained with H2O2 alone-treated fibroblasts. The data indicates that the induced decrease in HSP70 level in oxidative stress conditions makes fibroblasts more prone to oxidative injury and also increases i-NOS level. Whereas in one way the forced decrease in i-NOS expression seems to counteract ROS production stimulated by the oxidative insult in the cells, in another way, since it causes a decrease in HSP70 expression as well as in cell viability, it seems to activate some unidentified pathways affecting cell demise.     

双酚A 干扰大鼠生精过程时TJ 渗透性屏障的体外研究

目的:研究体外大鼠睾丸支持细胞紧密连接蛋白(SCJP)在类雌激素-双酚A(BPA)干扰下的损伤机制。方法:对Wistar大鼠睾丸支持细胞(Sertoli细胞)离体原代培养4-5d,通过双室培养模型建立体外紧密连接(TJ)渗透性屏障,并测量其跨上皮电阻值(TER)反应紧密连接结构的形成及BPA对紧密连接的损害程度。设溶剂(DMSO)做阴性对照,以终浓度为25μM、100μM的BPA作用于支持细胞24h,MTT法测不同浓度BPA作用的Sertoli细胞增殖活性。Western bloting观察occludin、ZO-1、Cx43表达的变化。结果:成功分离并培养Wistar大鼠睾丸支持细胞,并建立良好的体外TJ屏障模型。双室培养支持细胞上皮TER值在培养的d4达到顶峰,然后在d4-9维持相对较稳定的状态,d4以200μM,100μM,25μM BPA染毒,分别于染毒后24,48,72,96和120h测TER:与DMSO溶剂对照组相比,200μM,100μM的BPA组TER值明显下降(P<0.05),而25μM的BPA组在染毒后TER值无明显变化(P>0.05)。MTT结果显示:经不同浓度BPA作用24h后,Sertoli细胞的吸光度(OD值)随着染毒剂量的增加而逐渐降低。102、103μM浓度组与溶剂对照组有显著性差异(P<0.05),而10-2、10-1、100、101μM组和溶剂对照组无显著性差异(P>0.05)。Western blot结果显示:occludin、ZO-1、Cx43在各剂量组均有表达,与溶剂对照组相比,occludin、ZO-1表达均分别随作用剂量的增加而降低:25μM组、100μM组与溶剂对照组相比,差异均存在显著性(P<0.05);100μM组与25μM组相比,差异亦存在显著性(P<0.05)。Cx43的表达却随染毒剂量的增加而增加,与溶剂对照组相比,25μM组表达无明显增加(P>0.05),而100μM组则明显增加(P<0.05);与25μM组相比,100μM组表达明显增加(P<0.05)。结论:双酚A可通过损伤支持细胞连接蛋白正常表达,破坏了TJ屏障渗透性,从而影响正常的精子形成过程。     

Electrochemical cell-based chip for the detection of toxic effects of bisphenol-A on neuroblastoma cells

A cell-based chip was fabricated for the electrochemical detection of the dose-dependent effects of bisphenol-A (BPA) on neuroblastoma cells (SH-SY5Y), which showed dual-mode correlation as a standard curve. Toxicity assessment of BPA became very important in environmental toxicants detection since BPA can be reached out easily from various common plastic-based product and give negative cellular effects on living organism. Cell chip was fabricated by immobilizing cells on C(RGD)(4) peptide coated electrode to detect the cytotoxicity of BPA electrochemically. Redox properties in living cells were determined by cyclic voltammetry using a home-made three-electrode system, and the cathodic peak current (I(pc)) was used as a parameter for measurement of the effect of BPA on cell viability. The peak current, I(pc) value increased with the concentration of BPA up to 300 nM and then decreased because of the stimulation of cancer cell activity at the concentration of BPA below 300nM and cytotoxicity at the concentration of BPA above 300 nM, respectively. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and optical microscopy-based morphological analysis confirmed the results of electrochemical study. This dual-mode correlation between the concentration of BPA and voltammetric signal intensity should be firstly considered to analyze its dose-dependent stimulus and cytotoxic effects on neuroblastoma cells by cell chip.     

双酚A暴露对东亚三角涡虫急性毒性及神经酶的影响

旨在探讨双酚A (BPA)对东亚三角涡虫的急性毒性及神经系统相关酶活性的影响。采用不同浓度的BPA处理涡虫24 h、48 h、72 h,求出半致死浓度,以此为基础,采用不同浓度的BPA处理涡虫24 h、72 h、144 h,测定AchE、ChAT及Na+~K+^-ATP酶活性。BPA对三角涡虫的24 h、48 h、72 h,LC50分别为12.18 mg/L、8.49 mg/L、6.43 mg/L。ChE、ChAT、Na+~K+^-ATP酶活性对BPA反应敏感,具有较好的规律性。24 h处理组,AChE酶活力随BPA浓度的升高而升高,72 h和144 h处理组则呈现先上升后下降的趋势,除0.643 mgBPA/L以外,其它处理组均表现出了时间-效应关系;在BPA胁迫下,ChAT酶活力均呈现降低趋势,在BPA浓度为0.643 mg/L、1.286 mg/L时,表现出了时间-效应关系;24 h、72 h处理组Na+~K+^-ATP酶活力随BPA浓度升高呈现先上升后下降的趋势,144 h处理组则呈现下降趋势,Na+~K+^-ATP酶活力随BPA胁迫时间的延长呈现先升高后下降的趋势。BPA对涡虫具有较强的生态毒性,AchE、ChAT、Na+~K+^-ATP酶活性可与其他敏感指标一起作为水体BPA污染的早期监测指标。     

Micronucleus assay in bovine lymphocytes after exposure to bisphenol A in vitro

Bisphenol A (BPA) [2,2-bis-(4-hydroxyphenyl) propane] is an important industrial agent, made by combining acetone and phenol, that is used extensively as a monomer in the production of polycarbonate plastics and as a precursor of epoxy resins. Micronucleus assays have served as an index of cytogenetic damage in in vivo and in vitro studies. We studied the genotoxic and cytotoxic effects of BPA on bovine peripheral lymphocytes in vitro. Lymphocyte cultures from two donors were exposed to four different concentrations of BPA (1?×?10^?4, 1?×?10^?5, 1?×?10^?6, and 1?×?10^?7 mol.L^?1) for 48 h. The highest concentration of BPA (1?×?10^?4 mol.L^?1) resulted in a significant increase in the number of micronuclei in comparison with the negative control (67.50?±?2.121/1,000 binucleated cells versus 36.0?±?5.657/1,000 binucleated cells in the DMSO control, P??=??0.018). BPA did not affect the nuclear division index at any treatment concentrations. The present results thus demonstrate a significant genotoxic effect by BPA on bovine peripheral lymphocytes in vitro, only at the highest concentration.     

Bisphenol A triggers proliferation and migration of laryngeal squamous cell carcinoma via GPER mediated upregulation of IL‐6

Bisphenol A (BPA) can be accumulated into the human body via food intake and inhalation. Numerous studies indicated that BPA can trigger the tumorigenesis and progression of cancer cells. Laryngeal cancer cells can be exposed to BPA directly via food digestion, while there were very limited data concerning the effect of BPA on the development of laryngeal squamous cell carcinoma (LSCC). Our present study revealed that nanomolar BPA can trigger the proliferation of LSCC cells. Bisphenol A also increased the in vitro migration and invasion of LSCC cells and upregulated the expression of matrix metallopeptidase 2. Among various chemokines tested, the expression of IL‐6 was significantly increased in LSCC cells treated with BPA for 24 hours. Neutralization antibody of IL‐6 or si–IL‐6 can attenuate BPA‐induced proliferation and migration of LSCC cells. Targeted inhibition of G protein–coupled estrogen receptor, while not estrogen receptor (ERα), abolished BPA‐induced IL‐6 expression, proliferation, and migration of LSCC cells. The increased IL‐6 can further activate its downstream signal molecule STAT3, which was evidenced by the results of increased phosphorylation and nuclear translocation of STAT3, while si–IL‐6 and si‐GPER can both reverse BPA‐induced activation of STAT3. Collectively, our present study revealed that BPA can trigger the progression of LSCC via GPER‐mediated upregulation of IL‐6. Therefore, more attention should be paid for the BPA exposure on the development of laryngeal cancer.     

Contribution of environmental pollutants to male infertily: a working model of germ cell apoptosis induced by plasticizers

Bisphenol A [2,2-bis(4-hydroxyphenyl)propane] (BPA), 4-nonylphenol (NP) and di(2-ethylhexyl)phthalate (DEHP), and its metabolite mono-2-ethylhexyl phthalate (MEHP) are chemicals found in plastics, which act as endocrine disruptors (EDs) in animals, including human. EDs act like hormones in the endocrine system, and disrupt the physiologic function of endogenous hormones. Most people are exposed to different endocrine disruptors and concern has been raised about their true effect on reproductive organs. In the testis, they seem to preferentially attack developing testis during puberty rather than adult organs. However, the lack of information about the molecular mechanism, and the apparently controversial effect observed in different models has hampered the understanding of their effects on mammalian spermatogenesis. In this review, we critically discuss the available information regarding the effect of BPA, NP and DEHP/ MEHP upon mammalian spermatogenesis, a major target of EDs. Germ cell sloughing, disruption of the blood-testis-barrier and germ cell apoptosis are the most common effects reported in the available literature. We propose a model at the molecular level to explain the effects at the cellular level, mainly focused on germ cell apoptosis.     

Mechanisms of transport of p-borono-phenylalanine through the cell membrane in vitro

The mechanisms of transport of p-(dihydroxyboryl)-phenylalanine (BPA) through the cell membrane were investigated in vitro, evaluating especially the systems responsible for the transport of neutral amino acids as potential carriers for BPA. Rat 9L gliosarcoma cells and Chinese hamster V79 cells were exposed to BPA under controlled conditions and in a defined medium that was free of amino acids. The time course of (10)B (delivered by BPA) uptake and efflux was measured under different conditions. To analyze the intracellular boron content, direct-current plasma atomic emission spectroscopy (DCP-AES) was used after separating the cells from extracellular boron in the cell medium using an oil filtration technique. The dependence of factors such as cell type, temperature, composition and concentration of amino acids and specific substrates for amino acid transport systems in the culture medium or in intracellular compartments on BPA uptake and efflux were studied. The results of this study support the hypothesis that BPA is transported by the L system and that transport can be further stimulated by amino acids preaccumulated in the cell by either the L or A amino acid transport system. Copyright [bj54] by Radiation Research Society     

Bisphenol A exposure modifies DNA methylation of imprint genes in mouse fetal germ cells

Bisphenol A (BPA) is an estrogenic environmental toxin widely used for the production of plastics. Human frequent exposure to this chemical has been proposed to be a potential public health risk. The objective of this study was to assess the effects of BPA on DNA methylation of imprinting genes in fetal mouse germ cell. Pregnant mice were treated with BPA at doses of 0, 40, 80 and 160 μg BPA/kg body weight/day from 0.5 day post coitum. DNA methylation of imprinting genes, Igf2r, Peg3 and H19, was decreased with the increase of BPA concentration in fetal mouse germ cells (p < 0.01).The relative mRNA levels of Nobox were lower in BPA-treated group compared to control (BPA free) in female fetal germ cells, but in male fetal germ cells, a significant higher in Nobox expression was observed in BPA-treated group compared to control. Decreased mRNA expression of specific meiotic genes including Stimulated by Stra8 and Dazl were obtained in the female fetal germ cells. In conclusion, BPA exposure can affect the DNA methylation of imprinting genes in fetal mouse germ cells.     

Endocrine disruptors (EDs) and hormone-dependent cancers: Correlation or causal relationship?

The selective increase in the incidence of hormone-dependent cancers (breast, prostate, testicular) in industrialized countries is associated with the increasing number of endocrine disruptors (EDs) in the environment and raises questions about the role of EDs in mammary carcinogenesis. Answering these questions is difficult because the number of EDs is large and varies with time. Moreover hormonal carcinogenesis is multifactorial and progresses slowly and in stages. This discussion will be limited to breast cancer and three EDs: distilbene, bisphenol A (BPA), and dichlorodiphenyltrichloroethane (DDT). All these three EDs bind estrogen receptors, albeit with widely different affinities. Several complementary approaches have been used: French cancer records, epidemiological studies on cohorts followed over several decades, numerous in vitro experimental studies using cell cultures and in vivo animal studies. These approaches all converge to the same result, strongly suggesting a causal relationship between EDs and precancerous lesions. Except for distilbene, the mechanisms and molecular targets involved are still unclear, which makes it difficult to look for substitute products that are just as efficient, but less toxic.     

Effects of bisphenol A and 4-nonylphenol on cellular responses through the different induction of LPA receptors in liver epithelial WB-F344 cells

Abstract

Lysophosphatidic acid (LPA) signaling via G protein-coupled transmembrane LPA receptors (LPA₁ to LPA₆) mediates a variety of cellular functions, including cell proliferation, migration, morphogenesis, and differentiation. Recently, we demonstrated that the different induction of LPA receptors by estrogens regulates cell motile activity of rat liver epithelial WB-F344 cells. In the present study, to assess whether endocrine disruptors (EDs) are involved in cellular functions through LPA signaling, we measured cell motile activity and LPA receptor expressions in WB-F344 cells treated with bisphenol A (BPA) and 4-nonylphenol (4-NP). Using quantitative real time RT-PCR analysis, the Lpar1 expression was elevated in BPA-treated cells, whereas the Lpar3 expression was decreased. In contrast, 4-NP increased the Lpar3 expression, but not the Lpar1 and Lpar2. For cell motility assay with a Cell Culture Insert, cell motile activity of BPA-treated cells was significantly lower than that of untreated cells. In contrast, 4-NP markedly enhanced cell motile activity. The effects of BPA and 4-NP on cell motility were inhibited by the Lpar1 or Lpar3 knockdown. These results suggest that BPA and 4-NP may regulate cell motile activity through the different induction of LPA receptors in WB-F344 cells.     

Bisphenol-A exposure during adolescence leads to enduring alterations in cognition and dendritic spine density in adult male and female rats

We have previously demonstrated that adolescent exposure of rats to bisphenol-A (BPA), an environmental endocrine disrupter, increases anxiety, impairs spatial memory, and decreases dendritic spine density in the CA1 region of the hippocampus (CA1) and medial prefrontal cortex (mPFC) when measured in adolescents in both sexes. The present study examined whether the behavioral and morphological alterations following BPA exposure during adolescent development are maintained into adulthood. Male and female, adolescent rats received BPA, 40 μg/kg/bodyweight, or control treatments for one week. In adulthood, subjects were tested for anxiety and locomotor activity, spatial memory, non-spatial visual memory, and sucrose preference. Additionally, stress-induced serum corticosterone levels and dendritic spine density in the mPFC and CA1 were measured. BPA-treated males, but not females, had decreased arm visits on the elevated plus maze, but there was no effect on anxiety. Non-spatial memory, object recognition, was also decreased in BPA treated males, but not in females. BPA exposure did not alter spatial memory, object placement, but decreased exploration during the tasks in both sexes. No significant group differences in sucrose preference or serum corticosterone levels in response to a stress challenge were found. However, BPA exposure, regardless of sex, significantly decreased spine density of both apical and basal dendrites on pyramidal cells in CA1 but had no effect in the mPFC. Current data are discussed in relation to BPA dependent changes, which were present during adolescence and did, or did not, endure into adulthood. Overall, adolescent BPA exposure, below the current reference safe daily limit set by the U.S.E.P.A., leads to alterations in some behaviors and neuronal morphology that endure into adulthood.     

Simple and accurate determination of bisphenol A in red blood cells prepared with basic glycine buffer using liquid chromatography-electrochemical detection

For an accurate determination of bisphenol A (BPA) in red blood cells (RBC), the effect of pH on the concentration of BPA was investigated. Also, BPA recovery using ferric heme, methemoglobin (metHb) and hematin, were investigated to confirm whether BPA binds to ferric heme. BPA recovery in hemolysate was high at alkaline pH and was very low at acidic pH where oxyHb changed to metHb. BPA recovery decreased dose-dependently in metHb and hematin, but inorganic iron ions did not influence the recovery. These results suggested that BPA could be bound to ferric heme in RBC. The use of glycine-NaOH buffer (pH 11) as well as plasma had the highest recovery (97%). BPA was not detected in red blood cells of healthy adult volunteers (n=6). In sheep blood contaminated with BPA, BPA was detected in both plasma and RBC (10 times lower than in plasma), indicating that BPA could have migrated from plasma into RBC.     

Bisphenol A induces apoptosis and G2-to-M arrest of ovarian granulosa cells

We investigated the impact of bisphenol A (BPA) on murine ovarian granulosa cells. Ovarian granulosa cells were cultured with 100 fM to 100 microM BPA for 24 h to 72 h. BPA decreased granulosa cell viability in a dose- and time-dependent manner. The lowest concentration that induced a significant decrease was 100 pM (89.2 +/- 4.0% of the control). TUNEL analysis demonstrated that treatment with BPA increased apoptosis of granulosa cells in a dose- and time-dependent manner. In addition, flow cytometry analyses revealed that treatment with BPA resulted in G2-to-M arrest, which was most prominent at 48 h. BPA increased the expression of Bax and concomitantly decreased the expression of Bcl2 at both protein and mRNA levels of granulosa cells. These findings suggest that low, presumably environmentally relevant doses of BPA, decrease the viability of granulosa cells by inducing apoptosis and G2-to-M arrest. Up-regulation of Bax and down-regulation of Bcl2 were suggested to be involved in this apoptotic effect.     

Estrogen and bisphenol A disrupt spontaneous [Ca(2+)](i) oscillations in mouse oocytes

The present work aims to study the effects of estrogen or endocrine disrupters (EDs) on the dynamic changes in intracellular Ca(2+) concentration of mouse immature oocytes (IOs) loaded with Ca(2+)-sensitive dye Fura-2 using an image analyzer. The majority of IOs isolated from the ovary exhibited spontaneous Ca(2+) oscillations at regular intervals. Entry of external Ca(2+), probably through gap junctions, contributes to Ca(2+) oscillations since they were reversibly inhibited by removing Ca(2+) from the bathing medium or by the application of a gap-junction inhibitor carbenoxolone (CBX, 30 microM). Both 17beta-estradiol (E2) and E2-BSA, a membrane impermeable estrogen, shortened the duration of Ca(2+) oscillations in a dose-dependent manner (1-1000 nM), and produced an irregular pattern of the oscillations, strongly suggesting that E2 acts on the plasma membrane of the oocyte. For bisphenol A (BPA), one of the estrogen-mimicking EDs, a 10,000-fold higher concentration (100 microM) was necessary to exert similar inhibitory action to that of E2.