Pharmacological inhibition of TLR4/NF‐κB with TLR4‐IN‐C34 attenuated microcystin‐leucine arginine toxicity in bovine Sertoli cells

This study investigated the pharmacological inhibition of the toll‐like receptor 4 (TLR4) genes as a measure to attenuate microcystin‐LR (MC‐LR) reproductive toxicity. Bovine Sertoli cells were pretreated with TLR4‐IN‐C34 (C34) for 1 hour. Thereafter the pretreated and non‐pretreated Sertoli cells were cultured in medium containing 10% heat‐activated fetal bovine serum + 80 μg/L MC‐LR for 24 hours to assess the ability of TLR4‐IN‐C34 to attenuate the toxic effects of MC‐LR. The results showed that TLR4‐IN‐C34 inhibited MC‐LR‐induced mitochondria membrane damage, mitophagy and downregulation of blood‐testis barrier constituent proteins via TLR4/nuclear factor‐kappaB and mitochondria‐mediated apoptosis signaling pathway blockage. The downregulation of the mitochondria electron transport chain, energy production and DNA replication related genes (mt‐ND2, COX‐1, COX‐2, ACAT, mtTFA) and upregulation of inflammatory cytokines (interleukin [IL]‐6, tumor necrosis factor‐α, IL‐1β, interferon‐γ, IL‐4, IL‐10, IL‐13 and transforming growth factor β1) were modulated by TLR4‐IN‐C34. Taken together, we conclude that TLR4‐IN‐C34 inhibits MC‐LR‐related disruption of mitochondria membrane, mitophagy and downregulation of blood‐testis barrier proteins of the bovine Sertoli cell via cytochrome c release and TLR4 signaling blockage.     

Epigenetic modification of H3K4 and oxidative stress are involved in MC‐LR‐induced apoptosis in testicular cells of SD rats

Microcystin‐leucine arginine (MC‐LR) is a cyclic heptapeptide, produced by aquatic cyanobacteria such as microcystis, with strong reproductive toxicity which poses greater threat to the reproductive abilities of humans and animals. By exploring the role of trimethylation of histone H3 at lysine 4 (H3K4me3) and the role of oxidative stress in MC‐LR‐induced apoptosis in testicular Sertoli cells in Sprague‐Dawley (SD) rats, this study indicated that MC‐LR increased the expression levels of apoptosis‐related genes by raising the levels of H3K4me3. 5′‐Deoxy‐5′‐methylthioadenosine (MTA), the inhibitor of H3K4me3, reduced apoptosis, indicating for the first time that epigenetic modification is closely related to the testicular reproductive toxicity induced by MC‐LR. MC‐LR also induced oxidative stress by stimulating the generation of reactive oxygen species (ROS), and subsequently triggering mitochondria‐mediated apoptotic pathway by decreasing mitochondrial membrane potential and increasing the levels of Bax, Bcl‐2, Caspase‐3, and so on. MC‐LR‐induced apoptosis of testicular cells could be decreased after pretreatment with oxidative stress inhibitor N‐acetyl‐cysteine (NAC). Furthermore, the pathological damage to mitochondria and testes were observed in SD rats. These results show that MC‐LR can induce apoptosis by raising the levels of H3K4me3, and pretreatment with MTA can ameliorate the MC‐LR‐induced apoptosis of cocultured cells by lowering the levels of H3K4me3. Furthermore, NAC has a protective effect on MC‐LR‐induced apoptosis of testicular cells in SD rats by inhibiting the oxidative stress.     

Toxic effects of microcystin‐LR on the HepG2 cell line under hypoxic and normoxic conditions

Microcystins (MCs) are highly liver‐specific and evidenced as a liver tumor promoter. Oxidative stress is one of the most important toxicity mechanisms of MCs, which is tightly related to oxygen concentration. The effects of MCs on animals and cell lines in normoxia and the mechanisms have been well studied, but such effects in different oxygen conditions were still unclear. The aim of the present study was to explore the cellular response of the human hepatocellular carcinoma cell line (HepG2) to MC‐LR exposure under hypoxic (1% O₂) and normoxic (21% O₂) conditions. We examined cell viability, mitochondrial membrane potential (MMP), superoxide dismutase (SOD) activity and gene expression posttoxin exposure. Cell viability was increased by MC‐LR in normoxia although decreased in hypoxia. MC‐LR markedly induced MMP loss under hypoxic condition but only slightly MMP loss under normoxic condition. SOD activity was significantly induced by MC‐LR in hypoxia, indicating prolonged oxidative stress. Inhibitory apoptosis protein (c‐IAP2) was significantly up‐regulated by MC‐LR under normoxic condition, suggesting that c‐IAP2 played an important role in the promotion of cell proliferation by MC‐LR. These results indicate that MC‐LR promotes cell proliferation under normoxic condition whereas induces cell apoptosis under hypoxic condition. Copyright © 2012 John Wiley & Sons, Ltd.     

The role of GSH in microcystin‐induced apoptosis in rat liver: Involvement of oxidative stress and NF‐κB

Microcystins (MCs) are potent and specific hepatotoxins produced by cyanobacteria in eutrophic waters, representing a health hazard to animals and humans. The objectives of this study are to determine the relationship between oxidative stress and NF‐κB activity in MC‐induced apoptosis in rat liver and the role of glutathione (GSH). Sprague‐Dawley rats were intraperitoneally (i.p.) injected with microcystin‐LR (MC‐LR) at 0.25 and 0.5 LD₅₀ with or without pretreatment of buthionine‐(S,R)‐sulfoximine (BSO), a specific GSH synthesis inhibitor. MC‐LR induced time‐dependent alterations of GSH levels in rat liver. Increased malondialdehyde (MDA) and significant changes of antioxidant enzymes including GSH peroxidase (GPX) and GSH reductase (GR) were also observed, particularly at 24 h post‐exposure. The results indicated that acute exposure to MC‐LR induced oxidative stress, and GSH depletion (BSO pretreatment) enhanced the level of oxidative stress. Furthermore, the modulation of pro‐apoptotic gene p53 and Bax and anti‐apoptotic gene Bcl‐2 was observed in 0.5 LD₅₀ group at 24 h, and the alteration was more pronounced by BSO injection before MC‐LR treatment, suggesting that GSH played a protective role against MC‐induced toxicity. Additionally, electrophoretic mobility shift assay (EMSA) showed that NF‐κB was induced at 0.25 LD₅₀ but inhibited at 0.5 LD₅₀. The above results indicated that the possible crosstalk of oxidative stress and NF‐κB activity was associated with MC‐LR‐induced hepatocytes apoptosis in vivo. Our data will provide a new perspective for understanding the mechanisms of MC‐induced liver injury. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 552–560, 2016.     

NF‐κB/p53‐activated inflammatory response involves in diquat‐induced mitochondrial dysfunction and apoptosis

Inflammation generated by environmental toxicants including pesticides could be one of the factors underlying neuronal cell damage in neurodegenerative diseases. In this study, we investigated the mechanisms by which inflammatory responses contribute to apoptosis in PC12 cells treated with diquat. We found that diquat induced apoptosis, as demonstrated by the activation of caspases and nuclear condensation, inhibition of mitochondrial complex I activity, and decreased ATP level in PC12 cells. Diquat also reduced the dopamine level, indicating that cell death induced by diquat is due to cytotoxicity of dopaminergic neuronal components in these cells. Exposure of PC12 cells to diquat led to the production of reactive oxygen species (ROS), and the antioxidant N‐acetyl‐cystein attenuated the cytotoxicity of caspase‐3 pathways. These results demonstrate that diquat‐induced apoptosis is involved in mitochondrial dysfunction through production of ROS. Furthermore, diquat increased expression of cyclooxygenase‐2 (COX‐2) and tumor necrosis factor‐α (TNF‐α) via inflammatory stimulation. Diquat induced nuclear accumulation of NF‐κB and p53 proteins. Importantly, an inhibitor of NF‐κB nuclear translocation blocked the increase of p53. Both NF‐κB and p53 inhibitors also blocked the diquat‐induced inflammatory response. Pretreatment of cells with meloxicam, a COX‐2 inhibitor, also blocked apoptosis and mitochondrial dysfunction. These results represent a unique molecular characterization of diquat‐induced cytotoxicity in PC12 cells. Our results demonstrate that diquat induces cell damage in part through inflammatory responses via NF‐κB‐mediated p53 signaling. This suggests the potential to generate mitochondrial damage via inflammatory responses and inflammatory stimulation‐related neurodegenerative disease.     

67‐kDa Laminin receptor contributes to hypoxia‐induced migration and invasion of trophoblast‐like cells by mediating matrix metalloproteinase‐9

Insufficient trophoblast invasion often occurs in patients experiencing preeclampsia. The 67‐kDa laminin receptor (LR1) is a multifunctional protein that binds to laminin and interacts with the extracellular matrix. We recently demonstrated that LR1 is implicated in trophoblast migration and invasion. However, whether LR1 is involved in hypoxia‐mediated trophoblastic invasion remains unclear and requires further investigation. This study demonstrates that two trophoblast‐like cell lines (JEG3 and BeWo cells) cultured at 3% oxygen exerted enhanced migratory and invasive capabilities as compared with their counterparts exposed to 20% oxygen. LR1 expression was increased in hypoxic JEG3 cells but decreased after transfection with hypoxia‐inducible factor 1 alpha (HIF‐1α) specific siRNA. Moreover, shRNA targeting LR1 mRNA significantly inhibited hypoxia‐induced increase in matrix metalloproteinase (MMP)‐9 activity in JEG3 cells. Forced overexpression of LR1 augmented JEG3 cell migration and invasion, and enhanced MMP‐9 expression and activity. Additionally, the blockade of the MMP‐9 effect with its neutralizing antibody reduced LR1 elevation‐promoted trophoblastic invasion. In summary, this study demonstrates that LR1 contributes to hypoxia‐induced migration and invasion of trophoblast cells at least partly by mediating MMP‐9 in vitro.     

Microcystin‐LR induces a wide variety of biochemical changes in the A549 human non‐small cell lung cancer cell line: Roles for protein phosphatase 2A and its substrates

Our previous studies have described the toxic effects of microcystin‐LR (MC‐LR) in various normal cell lines and human hepatoma SMMC‐7721 cells, but the specific effects of MC‐LR in other types of cancer cells with respect to protein phosphatase 2A (PP2A) have not been fully elaborated. A549 human lung adenocarcinoma cells have been identified to express organic anion‐transporting polypeptides (OATP) involved in cellular uptake of MC‐LR, and thus probably make an appropriate in vitro model to assess MC‐LR's cytotoxicity. Hence, in our present study, A549 cells were treated with various concentrations of MC‐LR for 24 h. The presence of MC‐LR in A549 cells was confirmed, and PP2A activity, PP2A substrates, cytoskeleton, apoptosis, and proliferation were subsequently explored. The results showed that 5–10 μM MC‐LR inhibited PP2A activity significantly but 0.5–1 μM MC‐LR did not change PP2A activity dramatically. The inhibition could result from the hyperphosphorylation of PP2A/C at Tyr307, an elevation in the total PP2A/C expression and the dissociation of α4/PP2A/C complexes. Moreover, MC‐LR led to rearrangements of filamentous actin and microtubules, which might be correlated with the hyperphosphorylation of Ezrin, VASP and HSP27 due to PP2A inhibition and mitogen‐activated protein kinase (MAPK) activation. However, exposure to MC‐LR for 24 h failed to trigger either apoptosis or proliferation, which might be related to PP2A‐inhibition‐induced hyperphosphorylation of Bcl‐2 and Bad and the activation status of Akt. In conclusion, our data indicated that MC‐LR induced extensive molecular and cellular alterations in A549 cells through a PP2A‐centered pathway, which differed in some respects from our previous study in SMMC‐7721 cells. To our knowledge, this is the first report comprehensively demonstrating the effects of MC‐LR in A549 cells, and our findings provide insights into the mechanism of MC‐LR toxicity in cancer cells. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1065–1078, 2017.     

Silica nanoparticles induced intrinsic apoptosis in neuroblastoma SH-SY5Y cells via CytC/Apaf-1 pathway

The present study was to investigate effects of Silica nanoparticles (SiNPs) on nervous system and explore potential mechanisms in human neuroblastoma cells (SH-SY5Y). Cytotoxicity was detected by cell viability and Lactate dehydrogenase (LDH) release. Flow cytometry analysis was applied to assess mitochondrial membrane potential (MMP) loss, intracellular Ca²⁺ and apoptosis. To clarify the mechanism of SiNPs-induced apoptosis, intrinsic apoptosis-related proteins were detected. Our results showed that SiNPs caused cytotoxicity, cell membrane damage and Ca²⁺ increase in a dose-dependent manner in SH-SY5Y cells. Both the mitochondrial membrane potential (MMP) loss and potential mitochondria damage resulted in Cyt C release to the cytoplasm. The elevated Cyt C and Apaf1 further triggered intrinsic apoptosis via executive molecular caspase-9 and caspase-3. The present study confirmed that SiNPs induced intrinsic apoptosis in neuroblastoma SH-SY5Y cells via CytC/Apaf-1 pathway and provided a better understanding of the potential toxicity induced by SiNPs on human neurocyte.     

Microcystin‐LR induces protein phosphatase 2A alteration in a human liver cell line

Microcystin‐LR (MC‐LR) is a potent inhibitor of protein phosphatases 1 and 2A, and has potent hepatotoxicity and tumor promotion activity. Numerous studies on MC‐LR toxicity have been conducted in rat hepatocytes, but few studies of the effects of microcystins on human hepatocytes have been done. In this study, HL7702 cells (a human normal liver cell line) were incubated in MC‐LR for 24 h. The existence of MC‐LR in HL7702 cells was confirmed. Furthermore, PP2A activity and the alteration of PP2A subunits were assessed. The results show that PP2A activity decreased from the concentration of 1 μM MC‐LR, showing a concentration‐dependent decline, to about 34% at 10 μM MC‐LR. This activity undergone opposite change with alternations of phosphorylated Y307‐PP2A/C and PP2A/C subunit but showed same change with the alteration of the ratio of methylated L309‐PP2A/C to PP2A/C. B55α, a regulatory subunit of PP2A, was slightly increases in cells treated with the highest concentration of MC‐LR (10 μM), and colocalized increasedly with rearranged‐microtubules after 1 μM MC‐LR exposure. However, the proportion of early apoptotic cells did not show any change at various concentration of MC‐LR for 24 h. To our knowledge, this is the first report showing MC‐LR‐induced alteration of PP2A phosphatase in human cultured hepatocytes, and the mechanism of action seems to be similar as described before in vitro. The alteration of PP2A and microtubule seems to be the early event induced by MC‐LR exposure. © 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 1236–1244, 2014.     

Aldosterone induces p21‐regulated apoptosis via increased synthesis and secretion of tumour necrosis factor‐α in human proximal tubular cells

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Fluoxetine Induces Apoptosis in Ovarian Carcinoma Cell Line OVCAR‐3 Through Reactive Oxygen Species‐Dependent Activation of Nuclear Factor‐κB

Abstract: The apoptotic effect of fluoxetine (FLX), an antidepressant, against human epithelial ovarian cancer cell lines OVCAR‐3 and SK‐OV‐3 was investigated in relation to the mitochondria‐mediated cell death process and nuclear factor (NF)‐κB activation. FLX‐induced mitochondrial membrane permeability change and formation of reactive oxygen species, leading to cell death. FLX‐induced increase in mitochondrial Bax levels, decrease in cytosolic Bid and Bcl‐2 levels, loss of the mitochondrial transmembrane potential, cytochrome c release, caspase‐3 activation and up‐regulation of p53. Oxidant scavengers and Bay 11‐7085 [an inhibitor of nuclear factor kappaB (NF‐κB) activation] prevented the FLX‐induced cell death, increase in phosphorylated inhibitory κB‐α and NF‐κB p65 levels, and binding of NF‐κB p65 to DNA. Results from this study suggest that FLX may exhibit apoptotic effect against ovarian cancer cell lines by inducing the mitochondrial membrane permeability change, which leads to cytochrome c release and subsequent caspase‐3 activation, through reactive oxygen species‐dependent activation of NF‐κB.     

Microcystin‐LR induces cytotoxicity and affects carp immune cells by impairment of their phagocytosis and the organization of the cytoskeleton

Microcystin‐LR (MC‐LR) is the main isoform of hepatotoxin produced by cyanobacteria, which occur worldwide in the aquatic environment. The present study investigated the in vitro toxic MC‐LR effects on immune cells isolated from the blood of carp. Cells were exposed to different MC‐LR concentrations ranging from 0.01 to 1 µg ml^–1 for 2, 6 and 24 h. In addition, the effect of the toxin on the phagocytic activity of leukocytes and on actin and tubulin re‐organization in phagocytic cells was studied. We observed that MC‐LR induces apoptosis in lymphocytes 2 h after incubation, whereas high toxin concentrations induced necrosis in lymphocytes in a time‐ and concentration‐dependent manner. Incubation of the cells for 2 h with 0.1 and 1 µg ml^–1 MC‐LR inhibited phagocytosis without affecting apoptosis or glutathione (GSH) levels. Moreover, at this time point and with these concentrations, the toxin also induced a significant re‐organization of the actin cytoskeleton in phagocytes, which subsequently collapsed around the nucleus leading to cell shrinkage and the disappearance of filopodia. These results suggest that both phagocytes and lymphocytes are targets for MC‐LR and the disturbances of phagocytosis may impair the balance of the immune system. Copyright © 2012 John Wiley & Sons, Ltd.     

Apoptosis,Dysbiosis and Expression of Inflammatory Cytokines are Sequential Events in the Development of 5‐Fluorouracil‐Induced Intestinal Mucositis in Mice

The chemotherapeutic agent 5‐fluorouracil (5‐FU) causes intestinal mucositis with severe diarrhoea, but the pathogenesis is not fully understood. In this study, we investigated the pathogenic effects of 5‐FU in mice, focusing on apoptosis, enterobacteria and inflammatory cytokines. Repeated administration of 5‐FU caused severe intestinal mucositis on day 6, accompanied by diarrhoea and body‐weight loss. TNF‐α expression increased 1 day after exposure to the drug, and spiked a second time on day 4, at which point myeloperoxidase activity and IL‐1β expression also increased. Apoptotic cells were observed in intestinal crypts only on day 1. 5‐FU also induced dysbiosis, notably decreasing the abundance of intestinal Firmicutes while increasing the abundance of Bacteroidetes and Verrucomicrobia. Twice‐daily co‐administration of oral antibiotics significantly reduced the severity of intestinal mucositis and dysbiosis, and blocked the increase in myeloperoxidase activity and cytokine expression on day 6, without affecting apoptosis and TNF‐α up‐regulation on day 1. In cultured colonic epithelial cells, exposure to 5‐FU also up‐regulated TNF‐α expression. Collectively, the data suggest that crypt apoptosis, dysbiosis and expression of inflammatory cytokines are sequential events in the development of intestinal mucositis after exposure to 5‐FU. In particular, 5‐FU appears to directly induce apoptosis via TNF‐α and to suppress intestinal cell proliferation, thereby resulting in degradation of the epithelial barrier, as well as in secondary inflammation mediated by inflammatory cytokines.     

Microcystin‐LR‐Caused ROS generation involved in p38 activation and tau hyperphosphorylation in neuroendocrine (PC12) cells

Microcystin‐LR (MC‐LR), a potent specific hepatotoxin produced by cyanobacteria, has recently been reported to show neurotoxicity. Our previous study demonstrated that MC‐LR caused the reorganization of cytoskeleton architectures and hyperphosphorylation of the cytoskeletal‐associated proteins tau and HSP27 in neuroendocrine PC12 cell line by direct PP2A inhibition and indirect p38 mitogen‐activated protein kinase (MAPK) activation. It has been shown that oxidative stress is extensively associated with MC‐LR toxicity, mainly resulting from an excessive production of reactive oxygen species (ROS). However, the mechanisms by which ROS mediates the cytotoxic action of MC‐LR are unclear. In the present study, we investigated whether ROS might play a critical role in MC‐LR‐induced hyperphosphorylation of microtubule‐associated protein tau and the activation of the MAPKs in PC12 cell line. The results showed that MC‐LR had time‐ and concentration‐dependent effects on ROS generation, p38‐MAPK activation and tau phosphorylation. The time‐course studies indicated similar biphasic changes in ROS generation and tau hyperphosphorylation, which started to increase within 1 h and reached the maximum level at 3 h followed by a decrease after prolonged treatment. Furthermore, pretreatment with the antioxidants, N‐acetylcysteine and vitamin C, significantly decreased MC‐LR‐induced ROS generation and effectively attenuated p38‐MAPK activation as well as tau hyperphosphorylation. Taken together, these findings suggest that ROS generation triggered by MC‐LR is a key intracellular event that contributes to an induction of p38‐MAPK activation and tau phosphorylation, and that blockade of this ROS‐mediated redox‐sensitive signal cascades may attenuate the toxic effects of MC‐LR. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 366–374, 2015.     

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.     

α‐Lipoic acid protects against microcystin‐LR induced hepatotoxicity through regeneration of glutathione via activation of Nrf2

Microcystins (MCs), as the most dominant bloom‐forming strains in eutrophic surface water, can induce hepatotoxicity by oxidative stress. Alpha‐lipoic acid (α‐LA) is a super antioxidant that can induce the synthesis of antioxidants, such as glutathione (GSH), by nuclear factor erythroid 2‐related factor 2 (Nrf2). However, the potential molecular mechanism of α‐LA regeneration of GSH remains unclear. The present study aimed to investigate whether α‐LA could reduce the toxicity of MCs induced in human hepatoma (HepG2), Bel7420 cells, and BALB/c mice by activating Nrf2 to regenerate GSH. Results showed that exposure to 10 μM microcystin‐leucine arginine (MC‐LR) reduced viability of HepG2 and Bel7402 cells and promoted the formation of reactive oxygen species (ROS) compared with untreated cells. Moreover, the protection of α‐LA included reducing the level of ROS, increasing superoxide dismutase activity, and decreasing malondialdehyde. Levels of reduced glutathione (rGSH) and rGSH/oxidized glutathione were significantly increased in cells cotreated with α‐LA and MC‐LR compared to those treated with MC‐LR alone, indicating an ability of α‐LA to attenuate oxidative stress and MC‐LR‐induced cytotoxicity by increasing the amount of rGSH. α‐LA can mediate GSH regeneration through the Nrf2 pathway under the action of glutathione reductase in MC‐LR cell lines. Furthermore, the data also showed that α‐LA‐induced cytoprotection against MC‐LR is associated with Nrf2 mediate pathway in vivo. These findings demonstrated the potential of α‐LA to resist MC‐LR‐induced oxidative damage of liver.     

AlCl3 induces lymphocyte apoptosis in rats through the mitochondria–caspase dependent pathway

To investigate apoptosis mechanisms in lymphocytes induced by aluminum trichloride (AlCl₃) through the mitochondria–caspase dependent pathway, the spleen lymphocytes of rats were cultured with RPMI‐1640 medium and exposed to AlCl₃·6H₂O in the final concentrations of 0 (control group, CG), 0.3 (low‐dose group, LG), 0.6 (mid‐dose group, MG), and 1.2 (high‐dose group, HG) mmol·L^?1 for 24 h, respectively. Mitochondrial transmembrane potential (ΔΨm), cytochrome C (Cyt C) protein expression in cytoplasm, Caspase‐3 and Caspase‐9 activity, Bcl‐2, Bax, Caspase‐3 and Caspase‐9 mRNA expressions, DNA ladder and lymphocytes apoptosis index were detected. The results showed that Cyt C protein expression in cytoplasm, Caspase‐3 and Caspase‐9 activity, Bcl‐2, Bax, Caspase‐3 and Caspase‐9 mRNA expressions, the ratio of Bcl‐2 and Bax mRNA expression, lymphocytes apoptosis index increased, while ΔΨm decreased in the AlCl₃‐treated groups compared with those in CG. The results indicate that AlCl₃ induces lymphocyte apoptosis in rats through the mitochondria–caspase dependent pathway. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 385–394, 2016.     

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.