Disruption of spermatogenesis and differential regulation of testicular estrogen receptor expression in mice after polychlorinated biphenyl exposure

The testicular toxicity of polychlorinated biphenyls (PCBs) has been extensively studied. However, the detailed mechanism is still obscure. In the present study, male C57 mice were treated with different doses of Aroclor 1254 (a commercial PCB mixture) once every 3 days by oral gavage. After exposure to Aroclor 1254 for 50 days, the sperm count decreased in a dose-dependent manner. Cell proliferation and apoptosis are key processes regulating development of the testis, and alterations in these processes may underlie testicular dysgenesis. Our results showed that the germ cell proliferation was inhibited and the apoptosis of the germ cell was induced in a dose-dependent manner after treatment with Aroclor 1254. Although there was no significant change in serum testosterone levels and androgen receptor expression levels after treatment with different dosages of Aroclor 1254, the estradiol levels decreased and the expression of estrogen receptor (ER) β increased in a dose-dependent manner, whereas an elevation of the expression of ERα was only observed in the 50μg/kg group. The data as a whole suggested that inhibited proliferation and induced apoptosis in germ cells, and a differential regulation of ER, may be involved in the testicular toxicity of PCBs.     

The toxic effects of Aroclor 1254 exposure on the osteoblastic cell line MC3T3-E1 and its molecular mechanism

Polychlorinated biphenyls (PCBs) are still prevalent in the environment despite the fact that they have been banned in many countries for several decades. Recent epidemiologic studies have demonstrated a link between PCBs exposure and pathological alterations of bone tissues. The aim of this study was to investigate the toxic effects of the PCBs mixture Aroclor 1254 on MC3T3-E1 preosteoblasts and explore the underlying molecular mechanism. Different doses of Aroclor 1254 were used to treat MC3T3-E1 and the cell viability, apoptosis, ALP activity, intracellular calcium (Ca2+) level and oxidative stress response were measured. The expression level of related proteins TRPV6, Apaf-1 and Bax was evaluated with Western blot assay. The subcellular distribution of TRPV6 protein was detected with immunofluorescence assay. The results indicated that the higher dose of Aroclor 1254 (>10 mg/L) could inhibit the cell proliferation and induce apoptosis in MC3T3-E1. The ROS level following Aroclor 1254 exposure was elevated with the concentration, while the ALP activity and intracellular calcium (Ca2+) level decreased. After Aroclor 1254 exposure, the expression level of calcium transport related protein TRPV6 was down-regulated, while the expression level of apoptosis related proteins Apaf-1 and Bax up-regulated in a dose dependant manner. The immunofluorescence assay results showed that the expression of TRPV6 in the cytoplasm was greatly suppressed after Aroclor 1254 exposure. In conclusion, Aroclor 1254 exposure could induce toxic effects in MC3T3-E1 as evidenced by inhibition of proliferation, induction of apoptosis and suppression of ALP activity. The ROS production and alteration of intracellular Ca2+ level induced by down-regulation of TRPV6 might involve the toxic effects, and cell apoptosis induced by Aroclor 1254 exposure is associated with the pro-apoptotic Apaf-1 pathway as well as alteration of Bcl-2/Bax ratio.     

Aroclor 1254 causes atrophy of exocrine pancreas in mice and the mechanism involved

Polychlorinated biphenyls (PCBs) are a class of organic pollutants that have been linked to pancreatic disease. However, their role in affecting the exocrine function of pancreas and the underlying mechanism remains elusive. In the present study, male C57 mice were treated with Aroclor 1254, a commercially available PCBs mixture, at a dosage of 0.5, 5, 50, or 500 μg kg^?1 every 3 days by oral gavage. Decrease in pancreas/soma index and acinar atrophy were observed in the mice after exposure for 50 days. Aroclor 1254 exposure significantly decreased the PCNA‐positive cells in the pancreatic acini in a dose‐dependent manner. In addition, western blot analysis showed that PCNA expression was decreased in pancreas in the presence of Aroclor 1254, which suggests that Aroclor 1254 suppresses cell proliferation. TUNEL‐positive apoptotic cells as well as the expression of Bcl2, BclXL, BAX, and Bad of exocrine pancreas did not show significant changes in the treated mice, indicating that Aroclor 1254 has no effect on apoptosis. We also found that phosphorylation of ERK1/2, P90RSK1 and Bad was increased in the treated groups; this compensatory activation of phosphorylation in ERK1/2‐P90RSK1‐Bad signaling cascade could protect cell from apoptosis to maintain the cell numbers and function of exocrine pancreas. Moreover, we found that the expression of Kras and TNFα was increased in the pancreas, indicating that Aroclor 1254 exposure could result in increased risk of inflammation and carcinoma. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 671–678, 2016.     

Propolis induces cell cycle arrest and apoptosis in human leukemic U937 cells through Bcl-2/Bax regulation

We investigated mechanism(s) where propolis induces apoptosis in human leukemic U937 cells. Propolis inhibited the proliferation of U937 cells in a dose-dependent manner by inducing apoptosis and blocking cell cycle progression in the G2/M phase. Western blot analysis showed that propolis increases the expression of p21 and p27 proteins, and decreases the levels of cyclin B1, cyclin A, Cdk2 and Cdc2, thereby contributing to cell cycle arrest. DAPI staining assay revealed typical morphology features of apoptotic cells. Propolis-induced apoptosis was also confirmed by assays with annexin V-FITC, PI-labeling and DNA fragmentation assay. The increase in apoptosis level induced by propolis was associated with down-regulation of Bcl-2 and activation of caspase-3, but not with Bax. These results suggests that propolis-induced apoptosis is related to the selective activation of caspase-3 and induction of Bcl-2/Bax regulation.     

Polychlorinated biphenyl mixtures (Aroclors) induce apoptosis via Bcl-2, Bax and caspase-3 proteins in neuronal cell cultures

Polychlorinated biphenyls (PCBs) are a group of persistent and widely dispersed environmental pollutants, some of which may be neurotoxic. In the present study, we have investigated the effect of PCB commercial mixtures (Aroclors) on neuronal cell cultures by assessing cell viability and apoptotic cell death. We have combined morphological and biochemical techniques to establish the relevance of apoptosis in neuronal cell death induced by Aroclors. Treatment with both Aroclor 1248 and Aroclor 1260 caused the loss of cell viability and accelerated apoptosis both in a concentration- and time-dependent manner. However, the extent of apoptosis resulted greater for Aroclor 1248 than for Aroclor 1260. This is correlated with the loss of cell viability since Aroclor 1248 is more cytotoxic. The apoptosis induced by Aroclors involves the increase of caspase-3 activity. To correlate the caspase-3 activity with respect to changes in protein processing, caspase-3 precursor protein (procaspase-3) was evaluated by Western blot analysis. Also, Bcl-2 and Bax protein were assessed in order to elucidate the cell death machinery induced in cortical neuronal cell cultures by Aroclor 1248. The results indicate that the increase in Aroclor-induced apoptosis correlates with a reduction in the expression of antiapoptotic Bcl-2 and an increase in the expression of proapoptotic Bax. These results suggest that, with our experimental conditions, Aroclors induce apoptosis in primary cultures of cortical neurons via proteins of the Bcl-2 and caspase families.     

The inhibition of LPS-induced splenocyte proliferation by ortho-substituted and microbially dechlorinated polychlorinated biphenyls is associated with a decreased expression of cyclin D2

Immunological effects of polychlorinated biphenyls (PCBs) have been demonstrated in our laboratories with the peferential inhibition of lipopolysaccharide (LPS)-induced splenocyte proliferation by ortho-substituted PCB congeners. An investigation of the mechanism behind this immunotoxicity revealed an interruption in the progression of murine lymphocytes from G0/G1 into S phase by Aroclor 1242 and the di-ortho-substituted congener, 2,2'-chlorobiphenyl (CB), whereas, a non-ortho-substituted congener, 4,4'-CB, did not affect cell cycle progression. This interruption of cell cycle progression by 2,2'-CB and Aroclor 1242 was associated with a decreased expression of the cell cycle regulatory protein, cyclin D2, while expression was not affected by exposure to the non-ortho-substituted 4,4'-CB. These results suggest the preferential inhibition of LPS-induced splenocyte proliferation by ortho-substituted congeners is a result of a decreased expression of cyclin D2, which leads to an interruption in cell cycle progression. In addition, PCB mixtures with an increased percentage of chlorines in the ortho position following an environmentally occurring degradation process inhibited LPS-induced proliferation, interrupted cell cycle progression, and decreased cyclin D2 expression. This study provides evidence for a mechanism of action of the immunological effects of ortho-substituted individual congeners as well as environmentally relevant mixtures enriched in congeners with this substitution pattern.     

Fenofibrate induced PPAR alpha expression was attenuated by oestrogen receptor alpha overexpression in Hep3B cells

The physiological regulation of Oestrogen receptor α (ERα) and peroxisome proliferator‐activated receptor alpha (PPARα) in Hepatocellular carcinoma (HCC) remains unknown. The present study we first treat the cells with fenofibrate and further investigated the possible mechanisms of 17β‐estradiol (E₂) and/or ERα on regulating PPARα expression. We also found higher PPARα expression in the tumor area than adjacent areas and subsequently compared PPARα expression in four different hepatic cancer cell lines. Hep3B cells were found to express more PPARα than the other cell lines. Using the PPARα agonist fenofibrate, we found that fenofibrate increased Hep3B cell proliferation efficiency by increasing cell cycle proteins, such as cyclin D1 and PCNA, and inhibiting p27 and caspase 3 expressions. Next, we performed transient transfections and immuno‐precipitation studies using the pTRE2/ERα plasmid to evaluate the interaction between ERα and PPARα. ERα interacted directly with PPARα and negatively regulated its function. Moreover, in Tet‐on ERα over‐expressed Hep3B cells, E₂ treatment inhibited PPARα, its downstream gene acyl‐CoA oxidase (ACO), cyclin D1 and PCNA expression and further increased p27 and caspase 3 expressions. However, over‐expressed ERα plus 17‐β‐estradiol (10⁻⁸ M) reversed the fenofibrate effect and induced apoptosis, which was blocked in ICI/melatonin/fenofibrate‐treated cells. This study illustrates that PPARα expression and function were negatively regulated by ERα expression in Hep3B cells.     

Berberine induces G1 arrest and apoptosis in human glioblastoma T98G cells through mitochondrial/caspases pathway

Berberine is an isoquinoline plant alkaloid with a long history of being used for the treatment of many diseases in Chinese herbal medicine. Berberine has a wide range of biochemical and pharmacological effects, including antitumor activities, but its mechanism of action is not clearly understood. In this study, we investigated that the relationship between the antiproliferative activities of berberine and the apoptotic pathway associated with its molecular mechanism of action in human glioblastoma T98G cells. Berberine treatment of T98G cell lines inhibited cell proliferation and induced cell death in a dose (50-200 microg/ml) dependent manner with an IC50 value of 134 microg/ml, which was associated with an increase in G1 arrest. Western blot analysis showed that the berberine-induced G1 arrest was mediated through the increased expression of P27 and the decreased expression of cyclin-dependent kinase (CDK) 2, CDK4, cyclin D, and cyclin E proteins. Berberine treatment also markedly enhanced apoptosis in T98G cells through the induction of a higher ratio of the Bax/Bcl-2 proteins, the disruption of mitochondrial membrane potential, and the activation of procaspase-9, caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP). Berberine can inhibit T98G cell proliferation by inducing G1 arrest and apoptosis. These results demonstrate that the berberine-induced apoptosis of T98G cells is primarily mediated through the mitochondrial/caspases-dependent pathway.     

(-)-Syringaresinol inhibits proliferation of human promyelocytic HL-60 leukemia cells via G1 arrest and apoptosis

We examined the effect of (-)-syringaresinol, a furofuran-type lignan isolated from Daphne genkwa, on cell cycle regulation in HL-60 human promyelocytic leukemia cells in vitro. (-)-Syringaresinol decreased the viability of HL-60 cells by inducing G(1) arrest followed by apoptosis in a dose- and time-dependent manner. The G(0)/G(1) phase of the cell cycle is regulated by cyclin-dependent kinases (Cdk), cyclins and cyclin-dependent kinase inhibitors (Cdki). We show by western blot analysis, that the (-)-syringaresinol-induced G(1) arrest was mediated through the increased expression of Cdki proteins (p21(cip1/waf1) and p27(kip1)) with a simultaneous decrease in cdk2, cdk4, cdk6, cyclin D(1), cyclin D(2), and cyclin E expression. The induction of apoptosis after treatment with (-)-syringaresinol for 24 h was demonstrated by morphological changes, DNA fragmentation, altered ratio of Bax/Bcl-2, cleavage of poly(ADP-ribose) polymerase and flow cytometry analysis. (-)-Syringaresinol also induced cytochrome c release and activation of caspase-3 and caspase-9. To our knowledge, this is the first time that (-)-syringaresinol has been reported to potently inhibit the proliferation of human promyelocytic HL-60 cells through G(1) arrest and induction of apoptosis. These findings suggest that (-)-syringaresinol may be a potential chemotherapeutic agent for the treatment of cancer.     

Differential effects of PCBs on the induction of apoptosis machinery and PKCalpha translocation in rat renal tubular cell cultures

We have demonstrated previously [Pérez-Reyes, P.L., Sánchez-Alonso, J.A., López-Aparicio, P., Recio, M.N., Pérez-Albarsanz, M.A., 2001. Different molecular capacity in the induction of apoptosis by polychlorinated biphenyl congeners in rat renal tubular cell cultures. Biosci. Rep. 6, 765-778] that the polychlorinated biphenyls (PCBs) cause loss of cell viability and accelerate apoptosis in cell kidney cultures. Further investigations are necessary to elucidate the mechanism of apoptosis induction. In this way, we have analyzed in the present work the effects of PCBs on protein kinase C (PKC, a protein family intimately involved in the regulation of cell survival) and the expression of two proapoptotic (caspase-3 and Bax) and one antiapoptotic (Bcl-2) proteins. Aroclor 1248 (a commercial PCB mixture with 48% chlorine by weight), PCB 153 (2,2',4,4',5,5'-hexachlorobiphenyl, a di-ortho-substituted nonplanar congener) and PCB 77 (3,3',4,4'-tetrachlorobiphenyl, a non-ortho-substituted planar congener), significantly increased PKCalpha activity compared to control cells in the cytosolic and particulate cell fractions, and increased the PKCalpha protein content in the particulate fraction. The nonplanar PCB 153 showed stronger effects than the coplanar congener PCB 77. In addition, Aroclor 1248 decreased both, procaspase-3 levels and the Bcl-2/Bax protein ratio. These findings indicate that PCBs, particularly nonplanar congeners, can induce apoptosis in primary renal tubular cells through the PKCalpha, caspase-3 and Bcl-2/Bax pathway.     

The selective estrogen receptor modulator bazedoxifene inhibits hormone-independent breast cancer cell growth and down-regulates estrogen receptor α and cyclin D1

Bazedoxifene (BZA) is a third-generation selective estrogen receptor modulator (SERM) that has been approved for the prevention and treatment of postmenopausal osteoporosis. It has antitumor activity; however, its mechanism of action remains unclear. In the present study, we characterized the effects of BZA and several other SERMs on the proliferation of hormone-dependent MCF-7 and T47D breast cancer cells and hormone-independent MCF-7:5C and MCF-7:2A cells and examined its mechanism of action in these cells. We found that all of the SERMs inhibited the growth of MCF-7, T47D, and MCF-7:2A cells; however, only BZA and fulvestrant (FUL) inhibited the growth of hormone-independent MCF-7:5C cells. Cell cycle analysis revealed that BZA and FUL induced G(1) blockade in MCF-7:5C cells; however, BZA down-regulated cyclin D1, which was constitutively overexpressed in these cells, whereas FUL suppressed cyclin A. Further analysis revealed that small interfering RNA knockdown of cyclin D1 reduced the basal growth of MCF-7:5C cells, and it blocked the ability of BZA to induce G(1) arrest in these cells. BZA also down-regulated estrogen receptor-α (ERα) protein by increasing its degradation and suppressing cyclin D1 promoter activity in MCF-7:5C cells. Finally, molecular modeling studies demonstrated that BZA bound to ERα in an orientation similar to raloxifene; however, a number of residues adopted different conformations in the induced-fit docking poses compared with the experimental structure of ERα-raloxifene. Together, these findings indicate that BZA is distinct from other SERMs in its ability to inhibit hormone-independent breast cancer cell growth and to regulate ERα and cyclin D1 expression in resistant cells.     

Isokotomolide A, a new butanolide extracted from the leaves of Cinnamomum kotoense, arrests cell cycle progression and induces apoptosis through the induction of p53/p21 and the initiation of mitochondrial system in human non-small cell lung cancer A549 cells

This study is the first to investigate isokotomolide A (IKA), a butanolide compound isolated from the leaves of Cinnamomum kotoense Kanehira & Sasaki (Lauraceaee), which exhibits an anti-proliferative activity in human non-small cell lung cancer A549 cells. The results show that IKA inhibits the proliferation of A549 by blocking cell cycle progression in the G0/G1 phase and inducing apoptosis. Blockade of cell cycle was associated with increased p21/WAF1 levels and reduced amounts of cyclin D1, cyclin E, Cdk2, Cdk4, and Cdk6 in a p53-mediated manner. IKA treatment also increased p53 phosphorylation (Ser15) and decreased the interaction of p53-MDM2. IKA treatment triggered the mitochondrial apoptotic pathway, indicated by changing Bax/Bcl-2 ratios, cytochrome c release and caspase-9 activation. In addition, pre-treatment of cells with caspase-9 inhibitor inhibited IKA-induced apoptosis, indicating that caspase-9 activation was involved in A549 cells' apoptosis induced by IKA. Our study reports here for the first time that the induction of p53/p21 and the initiation of the mitochondrial apoptotic system may participate in the anti-proliferative activity of IKA in human non-small cell lung cancer cells.     

丹参酮ⅡA抗乳腺癌细胞增殖作用研究

目的利用雌激素受体(estrogenic receptor,ER)阳性乳腺癌T47D细胞和ER阴性乳腺癌MDA-MB-231细胞观察丹参酮ⅡA(TanshinoneⅡA)对细胞增殖活性的影响及其对雌激素受体亚型的调节功能。方法以ER拮抗剂ICI182,780为工具药,采用MTT细胞增殖实验观察1×10-6mol.L-1和1×10-7mol.L-1丹参酮ⅡA对T47D和MDA-MB-231细胞增殖的影响。利用实时荧光定量PCR法及流式细胞术检测其对T47D细胞ERα和ERβmRNA及蛋白表达情况的影响。结果丹参酮ⅡA能够抑制T47D细胞增殖,且该作用可被ICI182,780部分拮抗;对MDA-MB-231细胞增殖的抑制作用较其对T47D细胞的作用更为明显。丹参酮ⅡA可使T47D细胞ERα和ERβmRNA和蛋白表达明显增加,并使ERα/ERβ比值有所上升。结论丹参酮ⅡA具有抑制乳腺癌细胞增殖的作用,抑制强度与对其ER亚型的调节作用相关。     

Anti-tumor potential of 15,16-dihydrotanshinone I against breast adenocarcinoma through inducing G1 arrest and apoptosis

Chemotherapeutic drugs are usually designed to induce cancer cell death via cell cycle arrest and/or apoptosis pathways. In this study, we used the chemical drug 15,16-dihydrotanshinone I (DHTS) to inhibit breast cancer cell proliferation and tumor growth, and investigate the underlying molecular mechanisms. Human breast cancer cell lines MCF-7 and MDA-MB-231 were both used in this study, and DHTS was found to significantly decrease cell proliferation by a dose-dependent manner in both cells. Flow cytometry indicated that DHTS induced G1 phase arrest in synchronous MCF-7 and MDA-MB-231 cells. When analyzing the expression of cell cycle-related proteins, we found that DHTS reduced cyclin D1, cyclin D3, cyclin E, and CDK4 expression, and increased CDK inhibitor p27 expression in a dose-dependent manner. In addition, DHTS inhibited the kinase activities of CDK2 and CDK4 by an immunocomplex kinase assay. In addition, DHTS also induced apoptosis in both cells through mainly mitochondrial apoptosis pathways. We found that DHTS decreased the anti-apoptotic protein Bcl-xL level and increased the loss of mitochondria membrane potential and the amount of cytochrome c released. Moreover, DHTS activated caspase-9, caspase-3, and caspase-7 and caused cell apoptosis. The fact that DHTS-induced apoptosis could be blocked by pretreating cells with pan-caspase inhibitor confirmed that it is mediated through activation of the caspase-3-dependent pathway. In a nude mice xenograft experiment, DHTS significantly inhibited the tumor growth of MDA-MB-231 cells. Taken together, these results suggest that DHTS can inhibit human breast cancer cell proliferation and tumor growth, and might have potential chemotherapeutic applications.     

一种选择性环氧化酶2—抑制剂JTE—522抑制RL95—2细胞的增殖及诱导其凋亡

目的:探讨环氧化酶-2抑制剂JTE-522是否对人子宫内膜癌细胞株RL95-2细胞有抑制增殖和诱导凋亡的作用及其分子机理.方法:应用体外噻唑兰法、琼脂糖凝胶电泳、酶联免疫试验、流式细胞术、RT-PCR及Western blot等方法研究JTE-522对RL952细胞增殖和凋亡的作用及其分子机理.结果:JTE522抑制RL95-2细胞的增殖、诱导其凋亡、引起G_0/G_1期阻滞和 S期抑制,并伴有COX-2 mRNA、磷酸化 Rb、CDK4蛋白表达的抑制及 p53,p21和cyclin D_1蛋白表达水平的上调.另外,细胞经 JTE-522处理后,还可见caspase-3活性的增加.结论:JTE-522抑制RL95-2细胞的增殖及诱导其凋亡,可能与COX-2mRNA,磷酸化Rb、CDK4蛋白水平的下降及 p53,p21和 cyclin D_1蛋白表达的上调有关,还可能与caspase-3的激活有关.     

Down-regulation of the HGF/MET autocrine loop induced by celecoxib and mediated by P-gp in MDR-positive human hepatocellular carcinoma cell line

Many tumors are resistant to drug-induced cell-cycle arrest and apoptosis. We have reported that apoptosis can be restored in human multidrug-resistant (MDR) hepatocellular carcinoma cell lines by celecoxib. Here we show that P-glycoprotein (P-gp) mediates cell-cycle arrest and autophagy induced by celecoxib in human MDR overexpressing hepatocellular carcinoma cell line by down-regulation of the HGF/MET autocrine loop and Bcl-2 expression. Exposure of cells to a low concentration of celecoxib down-regulated the expression of mTOR and caused G1 arrest and autophagy, while higher concentration triggered apoptosis. Cell growth inhibition and autophagy were associated with up-regulation of the expression of TGFβ1, p16^INK4b, p21^Cip1 and p27^Kip1 and down-regulation of cyclin D1, cyclin E, pRb and E2F. The role of P-glycoprotein expression in resistance of MDR cell clone to cell-cycle arrest, autophagy and apoptosis was shown in cells transfected with MDR1 small interfering RNA. These findings demonstrate that the constitutive expression of P-gp is involved in the HGF/MET autocrine loop that leads to increased expression of Bcl-2 and mTor, inhibition of eIF2α expression, resistance to autophagy/apoptosis and progression in the cell-cycle. Since mTor inhibitors have been proposed in treatment of “drug resistant” cancer, these data may help explain the reversing effect of mTor inhibitors.     

Role of N-methyl-D-aspartate receptors in polychlorinated biphenyl mediated neurotoxicity

Polychlorinated biphenyls (PCBs) are widespread persistent environmental pollutants. Chronic human and animal exposure to PCBs results in various harmful effects including neurotoxicity. This study investigates the effects of the PCB mixture Aroclor 1254 (A1254) and two PCB congeners (coplanar, non-ortho PCB 126, and non coplanar PCB 99) on the expression of N-methyl-D-aspartate receptors (NMDARs) and the subsequent toxic effects using a human SHS5-SY neuroblastoma cell line. NMDAR was measured using a radiolabeled phencyclidine receptor ligand [³H]-MK801, apoptosis was quantified using fluorogenic substrates specific for caspase-3 (DEVD-AFC) and cell death using lactate dehydrogenase (LDH) release. After treatment, a positive dose–response relationship of increasing NMDARS, increasing caspase-3 activity and cell death was observed in all PCB compounds. The non-coplanar PCB compounds were found to be significantly more toxic than the coplanar congener and the PCB mixture A1254. PCB-mediated cell death was attenuated with 10 μM NMDAR antagonists: 1-amino-3,5-dimethyladamantane hydrochloride (memantine) and (+)-5-methyl-10,11-dihydro-5H-debenzocyclhepten-5,10-imine maleate ((+)-MK-801), thus demonstrating the importance of NMDAR in PCB neurotoxicity. Intracellular calcium [Ca²⁺]_i chelator BAPTA-AM (1 μM) partially attenuated the neurotoxic effect of the PCBs suggesting a role of calcium homeostasis disruption in the neurotoxicity of PCBs. These results suggest that the neurotoxicity of PCBs can be mediated through activation of NMDARs.     

Neuroprotective effects of oestrogen against oxidative toxicity through activation of G-protein-coupled receptor 30 receptor

1. 17-β-oestradiol (E2) plays a critical role in neuroprotection through both genomic and non-genomic mechanisms. The aim of the present study was to investigate the role of G-protein-coupled receptor 30 (GPR30), a new kind of oestrogen receptor, in the neuroprotection against oxidative insult. 2. The neuroprotection evoked by GPR30 stimulation was examined in cultured cortical neurons. Hoechst 33258/propidium iodide double staining, flow cytometric analysis and western blotting were applied to assess neuronal apoptosis induced by H(2)O(2) . 3. We found that the GPR30 agonist, G1, and E2 attenuated apoptosis induced by H(2)O(2) exposure. Furthermore, G1 (1 nmol/L) or E2 (1 nmol/L) significantly increased the levels of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), Bcl-2 and pro-caspase-3. Pretreatment with ICI182780, a highly selective nuclear oestrogen receptor antagonist that is used to block the classical ERα and ERβ receptors, did not totally block the neuroprotective effects of E2 (1 nmol/L) and had no effect on the neuroprotective effects of G1 (1 nmol/L). 4. Our data suggest that GPR30 is involved in the neuroprotection against oxidative insult. The neuroprotection evoked by GPR30 stimulation was associated with the signalling through the ERK1/2 kinase pathway. In addition, the anti-apoptotic activity of GPR30 was dependent on the expression of Bcl-2 and pro-caspase-3. GPR30 might be a potential therapeutic target for neuroprotection and oxidative stress.     

维生素D对过氧化氢诱导MIN6细胞凋亡的保护作用

目的探讨维生素D(vitamin D,VitD)对过氧化氢(hydrogen peroxide,H 2O 2)诱导小鼠胰岛β细胞株MIN6细胞凋亡的保护作用及机制。方法VitD预处理后,用H 2O 2处理MIN6细胞,分别运用CCK-8法、Hoechst 33258荧光染色法、流式细胞术检测MIN6细胞增殖、形态及凋亡百分率。Western blot检测增殖与凋亡相关基因Bax、Bcl-2、Caspase-3以及Cleaved caspase-3的表达。结果H 2O 2呈时间和剂量依赖性抑制MIN6细胞增殖,诱导细胞凋亡,降低Bcl-2表达、增加Bax及Cleaved caspase-3表达,Bcl-2/Bax比值降低,Cleaved caspase-3/caspase-3比值增加;当用VitD预处理后,Bcl-2表达增加,Bax、Cleaved caspase-3表达降低,Bcl-2/Bax比值增加,Cleaved caspase-3/caspase-3比值降低,MIN6细胞活力增加。结论VitD预处理后,通过增加抗凋亡Bcl-2表达,降低促凋亡Bax和Cleaved caspase-3表达,减少由H 2O 2诱导的小鼠胰岛β细胞株MIN6细胞凋亡。     

Aryl hydrocarbon receptor-activating polychlorinated biphenyls and their hydroxylated metabolites induce cell proliferation in contact-inhibited rat liver epithelial cells.

Polychlorinated biphenyls (PCBs) exhibit tumor-promoting effects in experimental animals. We investigated effects of six model PCB congeners and hydroxylated PCB metabolites on proliferation of contact-inhibited rat liver epithelial WB-F344 cells. The 'dioxin-like' PCB congeners, PCB 126, PCB 105, and 4'-OH-PCB 79, a metabolite of the planar PCB 77 congener, induced cell proliferation in a concentration-dependent manner. In contrast, the 'non-dioxin-like' compounds that are not aryl hydrocarbon receptor (AhR) agonists, PCB 47, PCB 153, and 4-OH-PCB 187, an abundant noncoplanar PCB metabolite, had no effect on cell proliferation at concentrations up to 10 muM. The concentrations of dioxin-like PCBs leading to cell proliferation corresponded with the levels inducing the expression of cytochrome P450 1A1 mRNA, suggesting that the release from contact inhibition was associated with AhR activation. The effects of PCB 126 and PCB 153 on expression of proteins controlling G0/G1-S-phase transition and S-phase progression were compared. Only PCB 126 was found to upregulate cyclin A and D2 protein levels, and to increase both total cyclin-dependent kinase 2 (cdk2) and cyclin A/cdk2 complex activities. Despite the observed upregulation of cyclin D2, no increase in cdk4 activity was observed. The expression of cdk inhibitor p27Kip1 was not affected by either PCB 126 or PCB 153. These results suggest that dioxin-like PCBs can induce cell proliferation of contact-inhibited rat liver epithelial cells by increasing cyclin A protein levels, a process that then leads to upregulation of cyclin A/cdk2 activity and initiation of DNA replication. This mechanism could be involved in tumor-promoting effects of dioxin-like PCBs.