Cytotoxicity and mitochondrial dysfunction of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in isolated rat hepatocytes

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant that induces hepatic and extrahepatic oxidative stress and the mechanisms of TCDD-induced reactive oxygen species are not fully investigated. Moreover, the potential toxicity of TCDD in isolated rat hepatocytes is not fully explored. The aim of the current study was to explore the possible cytotoxic effect of TCDD on primary rat hepatocytes and to explore the impact of mitochondria in TCDD-induced toxicity. Hepatocytes were isolated from adult rat liver and incubated with 0, 5, 10 or 15 nM of TCDD for 24, 48 and 72 h. Cell viability, lactate dehydrogenase (LDH) leakage into media along with reactive oxygen species (ROS) generation and hydrogen peroxide (H₂O₂) production, mitochondrial membrane potential (Δψ_m), superoxide dismutase (SOD), catalse (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), total thiol contents, hepatic aryl hydrocarbon hydroxylase (AHH), and ethoxyresorufin O-deethylase (EROD) were performed in hepatocytes. In addition, superoxide anion generation, lipid peroxidation (LPO), mitochondrial protein carbonyl content and respiratory chain complexes II and IV were assayed in hepatocyte mitochondria. Cell viability was significantly decreased while LDH leakage into media was significantly increased in a dose and time related manner. ROS generation and H₂O₂ production along with EROD and AHH activities were significantly increased in hepatocytes in the same pattern. The antioxidant enzymes SOD, CAT, GPx and GR and the non-enzymatic protein thiols, in addition to Δψ_m were significantly decreased in hepatocytes in a concentration and time dependent pattern. On the other side, mitochondrial superoxide anion along with LPO and mitochondrial protein carbonyl content were significantly increased while the respiratory chain complexes II and IV activities were significantly decreased in hepatocyte mitochondria. This effect may lead to disruption in the functional integrity of hepatocytes and hepatocyte mitochondria. In conclusion, our data clearly show that TCDD induces hepatocyte toxicity and mitochondrial dysfunction by a mechanism involving generation of ROS. Mitochondria might be the primary source of (or at least contribute to) the oxidative stress response and resulting toxicological outcomes elicited by TCDD.     

Vitamin E protects against the mitochondrial damage caused by cyclosporin A in LLC-PK1 cells

Cyclosporin A (CsA) has nephrotoxic effects known to involve reactive oxygen species (ROS), since antioxidants prevent the kidney damage induced by this drug. Given that mitochondria are among the main sources of intracellular ROS, the aims of our study were to examine the mitochondrial effects of CsA in the porcine renal endothelial cell line LLC-PK1 and the influence of the antioxidant Vitamin E (Vit E).Following the treatment of LLC-PK1 cells with CsA, we assessed the mitochondrial synthesis of superoxide anion, permeability transition pore opening, mitochondrial membrane potential, cardiolipin peroxidation, cytochrome c release and cellular apoptosis, using flow cytometry and confocal microscopy procedures. Similar experiments were done after Vit E preincubation of cells.CsA treatment increased superoxide anion in a dose-dependent way. CsA opened the permeability transition pores, caused Bax migration to mitochondria, and decreased mitochondrial membrane potential and cardiolipin content. Also CsA released cytochrome c into cytosol and provoked cellular apoptosis. Vit E pretreatment inhibited the effects that CsA induced on mitochondrial structure and function in LLC-PK1 cells and avoided apoptosis.CsA modifies mitochondrial LLC-PK1 cell physiology with loss of negative electrochemical gradient across the inner mitochondrial membrane and increased lipid peroxidation. These features are related to apoptosis and can explain the cellular damage that CsA induces. As Vit E inhibited these effects, our results suggest that they were mediated by an increase in ROS production by mitochondria.     

Mitochondrial Dysfunction and Ca2+ Overload in Injured Sertoli Cells Exposed to Bisphenol A

Bisphenol‐A (BPA) is well known as one of endocrine‐disrupting chemicals and testicular toxicant. In this present study, we determined whether BPA caused cell injury through mitochondria impairment and ROS overproduction. The cellular ROS production, mitochondrial ATP synthetase activity and Ca²⁺ concentration were examined. We have found BPA caused the cellular mitochondria dysfunction and followed by cell death in Sertoli cells. Moreover cytoplasm Ca²⁺ overload was also involved. Furthermore, pretreatment with N‐acetyl‐L‐cysteine (NAC) could alleviate the damage by causing a remarkable decrease in ROS production and mitochondrial dysfunction. Collectively, our results showed that BPA exposure induced Sertoli cell apoptosis because of excessive ROS generation and mitochondrial dysfunction. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 823–831, 2017.     

Silver nanoparticles induce oxidative cell damage in human liver cells through inhibition of reduced glutathione and induction of mitochondria-involved apoptosis

Silver nanoparticles (AgNPs), which have well-known antimicrobial properties, are extensively used in various medical and general applications. Despite the widespread use of AgNPs, relatively few studies have been undertaken to determine the cytotoxic effects of AgNPs exposure. This study investigates possible molecular mechanisms underlying the cytotoxic effects of AgNPs. Here, we show that AgNPs-induced cytotoxicity was higher compared than that observed when AgNO₃ was used as a silver ion source. AgNPs induced reactive oxygen species (ROS) generation and suppression of reduced glutathione (GSH) in human Chang liver cells. ROS generated by AgNPs resulted in damage to various cellular components, DNA breaks, lipid membrane peroxidation, and protein carbonylation. Upon AgNPs exposure, cell viability decreased due to apoptosis, as demonstrated by the formation of apoptotic bodies, sub-G₁ hypodiploid cells, and DNA fragmentation. AgNPs induced a mitochondria-dependent apoptotic pathway via modulation of Bax and Bcl-2 expressions, resulting in the disruption of mitochondrial membrane potential (Δψ_m). Loss of Δψ_m was followed by cytochrome c release from the mitochondria, resulting in the activation of caspases 9 and 3. The apoptotic effect of AgNPs was exerted via the activation of c-Jun NH₂-terminal kinase (JNK) and was abrogated by the JNK-specific inhibitor, SP600125 and siRNA targeting JNK. In summary, the results suggest that AgNPs cause cytotoxicity by oxidative stress-induced apoptosis and damage to cellular components.     

Cytotoxicity and oxidative stress study in cultured rat Sertoli cells with Methyl tert-butyl ether (MTBE) exposure

Cultured Sertoli cells were tested for their cytotoxicity and oxidative stress induced by Methyl tert-butyl ether (MTBE) which has been extensively used as a gasoline additive. In cytotoxic experiments, Sertoli cells were cultured with medium alone (control), 5, 500, or 50,000 μM MTBE. Lactate dehydrogcnase (LDH) leakage assay, staining with fluorescein diacetate (FDA) and propidium iodide (PI), and flow cytometric analyses were used. In oxidative stress experiments, Sertoli cells were cultured with medium alone (control), 0.5, 50, or 5000 μM MTBE. The production of reactive oxygen species (ROS), maleic dialdehyde (MDA) content and the level of superoxide dismutase (SOD) activity in cell supernatants were measured. Meanwhile, the expression level of 8-oxoguanine DNA glycosidase (OGG1) and extracellular form of superoxide dismutase (SOD_EX) in Sertoli cells were determined by RT-PCR. We also compared the current findings with the previous findings in rat spermatogenic cells exposed to MTBE. The present data indicate that high dose MTBE may exert a direct toxic effect on Sertoli cells. Oxidative stress induced by MTBE is a possible mechanism of cytotoxicity.     

Taurine reverses endosulfan-induced oxidative stress and apoptosis in adult rat testis

The present study was aimed to investigate the mechanistic aspect of endosulfan toxicity and its protection by taurine in rat testes. Pre-treatment with taurine (100 mg/kg/day) significantly reversed the decrease in testes weight, and the reduction in sperm count, motility, viability and daily sperm production in endosulfan (5 mg/kg/day)-treated rats. Sperm chromatin integrity and epididymal L-carnitine were markedly decreased by endosulfan treatment. Endosulfan significantly decreased the level of serum testosterone and testicular 3β-HSD, 17β-HSD, G6PDH and LDH-X. Sperm Δψm and mitochondrial cytochrome c content were significantly decreased after endosulfan. Testicular caspases-3, -8 and -9 activities were significantly increased but taurine showed significant protection from endosulfan-induced apoptosis. Oxidative stress was induced by endosulfan treatment as evidenced by increased H₂O₂ level and LPO and decreased the antioxidant enzymes SOD, CAT and GPx activities and GSH content. These alterations were effectively prevented by taurine pre-treatment.In conclusion, endosulfan decreases rat testes weight, and inhibits spermatogenesis and steroidogenesis. It induces oxidative stress and apoptosis by possible mechanisms of both mitochondria and non-mitochondria pathways. These data provide insight into the mode of action of endosulfan-induced toxicity and the beneficial role provided by taurine to counteract endosulfan-induced oxidative stress and apoptosis in rat testis.     

Olaquindox induces apoptosis through the mitochondrial pathway in HepG2 cells

Olaquindox is used in China as feed additive for growth promotion in pigs. Recently, we have demonstrated that olaquindox induced genome DNA damage and oxidative stress in HepG2 cells. The aim of this study was to explore the molecular mechanism of cell cycle arrest and apoptosis induced by olaquindox in HepG2 cells. In the present study olaquindox induced cell cycle arrest to the S phase and dose-dependent apoptotic cell death in HepG2 cells, indicated by accumulation of sub-G1 cell population, nuclear condenstion, DNA fragmentation, caspases activation and PARP cleavage. Meanwhile, the data showed that olaquindox triggered ROS-mediated apoptosis in HepG2 cells correlated with both the mitochondrial DNA damage and nuclear DNA damage, collapse of Δψ_m, opening of mPTP, down-regulation of Bcl-2 and up-regulation of Bax. Furthermore, we also found that olaquindox increased the expression of p53 protein and induced the release of cytochrome C from mitochondria to cytosol. In conclusion, olaquindox induced apoptosis of HepG2 cells through a caspase-9 and -3 dependent mitochondrial pathway, involving p53, Bcl-2 family protein expression, Δψ_m disruption and mPTP opening.     

Involvement of the mitochondrion-dependent pathway and oxidative stress in the apoptosis of murine splenocytes induced by areca nut extract

Areca quid chewing is a major risk factor for oral submucous fibrosis and oral cancer. Clinical evidence suggests that the pathophysiology of the oral diseases is closely associated with immune deterioration. The objective of the present studies was to investigate the pro-apoptotic effect of areca nut extract (ANE) in lymphocytes. Exposure of naïve splenic lymphocytes to ANE significantly enhanced apoptosis in a time- and concentration-dependent manner. Results from Hoechst staining confirmed the morphological features characteristic of apoptosis in ANE-treated cells. ANE treatment induced the depolarization of mitochondrial membrane potential (Δψ_m), which preceded the occurrence of apoptosis. In parallel with the disruption of Δψ_m, ANE induced the release of cytochrome c, and the activation of caspase-9, indicating the activation of the mitochondrion-dependent pathway. Moreover, an increased level in the intracellular reactive oxygen species was detected in ANE-treated lymphocytes undergoing apoptosis. ANE-mediated apoptosis, caspase-9 activation and ROS production, but not Δψ_m depolarization, were partially but significantly attenuated in the presence of the antioxidant N-acetyl-l-cysteine (NAC). Collectively, these results demonstrated the pro-apoptotic effect of ANE in primary lymphocytes, which was mediated, at least in part, by the activation of the mitochondrion-dependent pathway and oxidative stress.     

Nonylphenol-induced oxidative stress and cytotoxicity in testicular Sertoli cells

Nonylphenol (NP) as an environment contaminant has been demonstrated to adversely affect male reproduction. The main objective of this study was to evaluate NP-induced oxidative stress and toxicity in testicular Sertoli cells. Sertoli cells were exposed to 10–40 μM NP for 24 h. Cell death and growth inhibition were observed by flow cytometric analysis and 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) assay, while fluorescein diacetate (FDA) and propidium iodide (PI) staining was used to examine the morphological changes following NP exposure. Subsequently, we found that short-term treatment (2 h) of NP caused intracellular accumulation of reactive oxygen species (ROS), which was evaluated by loading of 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) without visible morphological changes. Loss of mitochondrial membrane potential (MMP) was detected following 12 and 24 h treatment of NP and assessment by Rhodamine 123 (Rh123) staining. In addition, incubation with NP for 12 h also increased lipid peroxidation of Sertoli cells. These results indicated that low micromolar concentrations of NP induce an adverse oxidative stress in rat Sertoli cells.     

In vitro toxicity evaluation of melamine on mouse TM4 Sertoli cells

The male reproductive toxicity of melamine (MA) has been recognized in recent years excepted for its renal toxicity. Our previous in vivo studies revealed that the damages of Sertoli cell barrier played a critical role in MA-induced testicular toxicity in mice. Herein, we performed an in vitro study to comprehensively evaluate the toxicity of MA on Sertoli cell by examining the influences of MA on the viability, morphology, mortality and intercellular junctions of mouse TM4 Sertoli cells (TM4 cells). The results showed that MA suppressed cell viability, induced obvious ultrastructural changes and cell apoptosis in concentration-dependent manner. Moreover, MA down-regulated the expressions of junction-associated proteins including occludin, N-cadherin, and vimentin, suggesting that MA disrupted the integrity of Sertoli cell barrier. Thus, these results indicated that Sertoli cell might be an important cellular target for MA-induced male reproductive toxicity.     

Emodin induces apoptosis in human lung adenocarcinoma cells through a reactive oxygen species-dependent mitochondrial signaling pathway

Emodin, a natural anthraquinone derivative isolated from Rheum palmatum L., has been reported to exhibit anti-cancer effect on several human cancers such as liver cancers and lung cancers. However, the molecular mechanisms of emodin-mediated tumor regression have not been fully defined. In this study, we show that treatment with 50 μM emodin resulted in a pronounced release of cytochrome c, activation of caspase-2, -3, and -9, and apoptosis in human lung adenocarcinoma A549 cells. These events were accompanied by the inactivation of ERK and AKT, generation of reactive oxygen species (ROS), disruption of mitochondrial membrane potential (Δψ_m), decrease of mitochondrial Bcl-2, and increase of mitochondrial Bax content. Ectopic expression of Bcl-2, or treatment with aurintricarboxylic acid, furosemide or caspase inhibitors markedly blocked emodin-induced apoptosis. Conversely, pharmacologic ERK and AKT inhibition promoted emodin-induced apoptosis. Furthermore, the free radical scavenger ascorbic acid and N-acetylcysteine attenuated emodin-mediated ROS production, ERK and AKT inactivation, mitochondrial dysfunction, Bcl-2/Bax modulation, and apoptosis. Take together, these findings suggest that in A549 cells, emodin-mediated oxidative injury acts as an early and upstream change in the cell death cascade to antagonize cytoprotective ERK and AKT signaling, triggers mitochondrial dysfunction, Bcl-2 and Bax modulation, mitochondrial cytochrome c release, caspase activation, and consequent leading to apoptosis.     

The effects of antimycin A on endothelial cells in cell death,reactive oxygen species and GSH levels

Antimycin A (AMA) inhibits mitochondrial electron transport chain between cytochrome b and c. Here, we evaluated the effects of AMA on the growth and death of endothelial cells (ECs) in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. AMA inhibited the growth of calf pulmonary artery endothelial cells (CPAEC) and human umbilical vein endothelial cells (HUVEC). AMA also induced apoptosis in both ECs which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m). HUVEC were more sensitive to AMA than CPAEC. AMA increased ROS level in CPAEC but decreased the levels in HUVEC. Z-VAD (pan-caspase inhibitor) mildly prevented apoptosis in AMA-treated ECs without the significant changes of ROS. N-acetyl-cysteine (NAC; a well-known antioxidant) decreased ROS levels in AMA-treated ECs. NAC reduced CPAEC death by AMA but enhanced HUVEC death by it. Furthermore, AMA increased GSH depleted cell numbers in ECs. Buthionine sulfoximine (BSO; an inhibitor of GSH synthesis), showing a pro-apoptotic effect on AMA-treated HUVEC, significantly increased GSH depleted cell number but it did not affect cell death and GSH depletion in AMA-treated CPAEC. In conclusion, AMA inhibited the growth of ECs via caspase-dependent apoptosis. ROS level change by AMA was partially related to CPAEC death, but did not affect HUVEC death. The change of GSH contents by AMA influenced the death of ECs.     

Actein alleviates 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin‐mediated cellular dysfunction in osteoblastic MC3T3‐E1 cells

The environmental pollutant 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is known to affect bone metabolism. We evaluated the protective effects of the triterpene glycoside actein from the herb black cohosh against TCDD‐induced toxicity in MC3T3‐E1 osteoblastic cells. We found that TCDD significantly reduced cell viability and increased apoptosis and autophagy in MC3T3‐E1 osteoblastic cells (P < .05). In addition, TCDD treatment resulted in a significant increase in intracellular calcium concentration, mitochondrial membrane potential collapse, reactive oxygen species (ROS) production, and cardiolipin peroxidation, whereas pretreatment with actein significantly mitigated these effects (P < .05). The effects of TCDD on extracellular signal‐related kinase (ERK), aryl hydrocarbon receptor, aryl hydrocarbon receptor repressor, and cytochrome P450 1A1 levels in MC3T3‐E1 cells were significantly inhibited by actein. The levels of superoxide dismutase, ERK1, and nuclear factor kappa B mRNA were also effectively restored by pretreatment with actein. Furthermore, actein treatment resulted in a significant increase in alkaline phosphatase (ALP) activity and collagen content, as well as in the expression of genes associated with osteoblastic differentiation (ALP, type I collagen, osteoprotegerin, bone sialoprotein, and osterix). This study demonstrates the underlying molecular mechanisms of cytoprotection exerted by actein against TCDD‐induced oxidative stress and osteoblast damage.     

Tributyltin chloride induced testicular toxicity by JNK and p38 activation,redox imbalance and cell death in sertoli-germ cell co-culture

The widespread use of tributyltin (TBT) as biocides in antifouling paints and agricultural chemicals has led to environmental and marine pollution. Human exposure occurs mainly through TBT contaminated seafood and drinking water. It is a well known endocrine disruptor in mammals, but its molecular mechanism in testicular damage is largely unexplored. This study was therefore, designed to ascertain effects of tributyltin chloride (TBTC) on sertoli-germ cell co-culture in ex-vivo and in the testicular tissue in-vivo conditions. An initial Ca²⁺ rise followed by ROS generation and glutathione depletion resulted in oxidative damage and cell death. We observed p38 and JNK phosphorylation, stress proteins (Nrf2, MT and GST) induction and mitochondrial depolarization leading to caspase-3 activation. Prevention of TBTC reduced cell survival and cell death by Ca²⁺ inhibitors and free radical scavengers specify definitive role of Ca²⁺ and ROS. Sertoli cells were found to be more severely affected which in turn can hamper germ cells functionality. TBTC exposure in-vivo resulted in increased tin content in the testis with enhanced Evans blue leakage into the testicular tissue indicating blood–testis barrier disruption. Tesmin levels were significantly diminished and histopathological studies revealed marked tissue damage. Our data collectively indicates the toxic manifestations of TBTC on the male reproductive system and the mechanisms involved.     

Microcystin-LR causes cytotoxicity effects in rat testicular Sertoli cells

Microcystins (MCs) are produced by cyanobacteria. The most toxic and widely distributed MC is microcystin-LR (MC-LR). The aim of this study was to investigate whether exposure to MC-LR could induce oxidative stress, leading the further toxicity effects on Sertoli cells in vitro. Sertoli cells obtained from rats were cultured with a medium containing 0, 0.5, 5, 50 or 500 nM/l MC-LR. We examined the decrease of mitochondrial membrane potential (MMP), the increase of reactive oxygen species (ROS) production, the increase of lipid peroxidation and decrease of superoxide dismutase (SOD) activity in Sertoli cells after treatment with MC-LR in vitro, and higher expression of caspase-9 and caspase-3, the increase of apoptosis rate. Therefore, we deduced that direct exposure to microcystin-LR could induce oxidative stress generation in Sertoli cells, and subsequently depressed cellular viability and caused cells to undergo apoptosis, resulting in the reproductive toxicity in male rats.     

Xanthohumol ameliorates 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin–induced cellular toxicity in cultured MC3T3‐E1 osteoblastic cells

2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is an environmental contaminant. Xanthohumol is a prenylated flavonoid found in hops (Humulus lupulus) and beer. The aim of the current study was to explore the role of xanthohumol in modulating the toxicity of TCDD in MC3T3‐E1 osteoblastic cells. In cells treated with TCDD alone, intracellular Ca²⁺ concentrations, mitochondrial membrane potential disruption, reactive oxygen species production, cardiolipin peroxidation, nitric oxide release and cytochrome P450 1A1 expression were significantly increased. TCDD treatment increased the mRNA levels of extracellular signal‐regulated kinase 1 and nuclear factor kappa B, and significantly decreased the level of protein kinase B (AKT) in MC3T3‐E1 osteoblastic cells. However, the presence of xanthohumol alleviated the pathological effects of TCDD. In addition, xanthohumol treatment significantly increased the expression of genes associated with osteoblast differentiation (alkaline phosphatase, osteocalcin, osteoprotegerin and osterix). We conclude that xanthohumol has a beneficial influence and may antagonize TCDD toxicity in osteoblastic cells.     

Nonylphenol induces apoptosis in rat testicular Sertoli cells via endoplasmic reticulum stress

Nonylphenol (NP) is a widely distributed environment contaminant and has been documented to disrupt testicular development and decrease male fertility. Amongst possible targets of this compound are testicular Sertoli cells, which play a crucial role in supporting and nourishing sperm cells. In the present study, we found that NP treatment could cause dramatic morphological changes as well as decreased cell viability of Sertoli cells, while the following Annexin V–PI staining demonstrated that NP treatment led to increased proportion of cell apoptosis, which was evidenced again by the detection of condensation and marginal changes of chromatins using Hoechst staining and transmission microscopy observation. In addition, increased intracellular Ca²⁺ levels and changes of endoplasmic reticulum (ER) ultrastructure were also observed in NP-treated groups, indicating the action of NP on ER. The subsequent data showed that the expressions of ER-stress signaling targeted genes GRP78 and gadd153 were elevated, suggesting the activation of ER-stress signal pathway. Furthermore, the detection of ER-stress related proteins by western blotting revealed that the expression of gadd153 was upregulated by NP, whereas the expressions of GRP78 and ERp57 were both first upregulated and then inhibited. Taken together, it is suggested that NP can induce ER stress in Sertoli cells, which may plays an important role in the induction of apoptosis.     

Gallic acid inhibits the growth of HeLa cervical cancer cells via apoptosis and/or necrosis

Gallic acid (GA) is widely distributed in various plants and foods, and its various biological effects have been reported. Here, we evaluated the effects of GA on HeLa cells in relation to cell growth inhibition and death. HeLa cell growth was diminished with an IC₅₀ of approximately 80 μM GA at 24 h whereas an IC₅₀ of GA in human umbilical vein endothelial cells (HUVEC) was approximately 400 μM. GA-induced apoptosis and/or necrosis in HeLa cells and HUVEC, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m). The percents of MMP (ΔΨ_m) loss cells and death cells were lower in HUVEC than HeLa cells. All the tested caspase inhibitors (pan-caspase, caspase-3, -8 or -9 inhibitor) significantly rescued HeLa cells from GA-induced cell death. GA increased reactive oxygen species (ROS) level and GSH (glutathione) depleted cell number in HeLa cells. Caspase inhibitors reduced GSH depleted cell number but not ROS level in GA-treated HeLa cells. In conclusion, GA inhibited the growth of HeLa cells and HUVEC via apoptosis and/or necrosis. The susceptibility of HeLa cells to GA was higher than that of HUVEC. GA-induced HeLa cell death was accompanied by ROS increase and GSH depletion.     

Nonylphenol regulates cyclooxygenase‐2 expression via Ros‐activated NF‐κB pathway in sertoli TM4 cells

The aim of this study was to investigate the signaling pathways involved in the cyclooxygenase (COX)‐2 regulation induced by nonylphenol (NP) in mouse testis Sertoli TM4 cells. Our results showed that treatment of TM4 cells with NP increased COX‐2 protein expression and interleukin‐6 (IL)‐6 and prostaglandin E2 (PGE2) secretion in a dose‐dependent manner. Pretreatment with reactive oxygen species (ROS) scavenger, N‐acetylcysteine (NAC), attenuated NP‐induced ROS production, COX‐2 expression, and IL‐6 and PGE2 release in TM4 cells. Exposure to NP stimulated activation of NF‐κB, whereas the NF‐κB inhibitor, pyrrolidine dithiocarbamate, attenuated NP‐enhanced COX‐2 expression and IL‐6 and PGE2 release in TM4 cells in a dose‐dependent manner. Furthermore, NAC blocked NP‐induced activation of NF‐κB. In addition, inhibition of COX‐2 mitigated NP‐induced IL‐6 release. In conclusion, NP induced ROS generation, activation of NF‐κB pathway, COX‐2 upregulation, and IL‐6 and PGE2 secretion in TM4 cells. NP may regulate COX‐2 expression via ROS‐activated NF‐κB pathway in Sertoli TM4 cells. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1144–1152, 2015.     

Pyrogallol-induced calf pulmonary arterial endothelial cell death via caspase-dependent apoptosis and GSH depletion

Pyrogallol (PG) as a polyphenol induces apoptosis in cells. Here, we evaluated the effects of PG on the growth and death of endothelial cells (ECs). PG dose-dependently inhibited the growth of calf pulmonary artery endothelial cells (CPAEC) and human umbilical vein endothelial cells (HUVEC). PG also induced apoptosis in both cells accompanied by the loss of mitochondrial membrane potential (ΔΨ_m). CPAEC were more sensitive to PG than HUVEC concerning cell growth and death. Caspase inhibitors (pan-caspase, caspase-3, -8 or -9 inhibitor) did not affect the growth inhibition of CPAEC by PG. However, pan-caspase inhibitor (Z-VAD) significantly reduced apoptosis and the loss of ΔΨ_m in PG-treated CPAEC. PG reduced ROS level and increased GSH depleted cell numbers in CPAEC. While Z-VAD increased ROS levels in PG-treated CPAEC, it decreased GSH depleted cell numbers. In conclusion, PG inhibited the growth of ECs, especially CPAEC via caspase-dependent apoptosis and GSH depletion.