Expression of Clusterin suppresses Cr(VI)-induced premature senescence through activation of PI3K/AKT pathway.

Our group found that long-term low-dose exposure to hexavalent chromium [Cr(VI)] in L-02 hepatocytes resulted in premature senescence, which accompanied by the increased expression of Clusterin (CLU), but the functional role of CLU in premature senescence has never been explored. In the present study, the CLU overexpressed or silenced L-02 hepatocytes were established by lentiviral vector transfection. Cell viability assay, cell cycle analysis, western blotting, plate clone formation assay, and confocal microcopy were performed. The results indicated that Cr(VI)-induced premature senescence was associated with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway inhibition, and high expression of CLU in the senescent cells exerted its functional role of promoting cell proliferation. CLU could complex with eukaryotic translation initiation factor 3 subunit I (EIF3I) and prevent its degradation, leading to the increase of AKT activity in Cr(VI)-exposed senescent hepatocytes. Blockage of the PI3K/AKT pathway with its inhibitor LY294002 eliminated the inhibitory effect of CLU on Cr(VI)-induced premature senescence. We concluded that high expression of CLU suppressed Cr(VI)-induced premature senescence through activation of PI3K/AKT pathway, which will provide the experimental basis for the study of Cr(VI)-induced liver cancer, especially for the elucidation of the mechanism of liver cancer cells escaping from senescence.

Cr(VI)-Induced Autophagy Protects L-02 Hepatocytes from Apoptosis Through the ROS-AKT-mTOR Pathway.

BACKGROUND/AIMS: Hexavalent chromium [Cr(VI)] pollution has become a global concern for both ecosystems and human health. Our previous study revealed Cr(VI) could induce both apoptosis and autophagy in L-02 hepatocytes. Here, we sought to explore the underlying mechanism of Cr(VI)-induced autophagy and its exact role in cell death. METHODS: Autophagy ultrastructure was observed under transmission electron microscope (TEM), autophagy flux was measured with double-tagged mCherry-green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) assay, long-lived protein degradation assay, and LC3II expression assay in the presence of lysosomal inhibitor, bafilomycin A1 (BafA1). Reactive oxygen species (ROS) level was determined using fluorescent probe dichloro-dihydrofluorescein diacetate (DCFH-DA). The expression levels of Beclin-1, LC3, p62/ SQSTM1, and AKT-mammalian target of rapamycin (mTOR) pathway-related molecules including phosphorylation (p)-AKT, AKT, p-mTOR, and mTOR were examined using real-time polymerase chain reaction (RT-PCR) and western blotting. Apoptosis was determined using Annexin V- fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. RESULTS: Our results demonstrated Cr(VI) exposure activated autophagy in L-02 hepatocytes, as evidenced by the accumulation of autophagosomes, the increase of LC3-II and degradation of p62/ SQSTM1, and the enhanced overall degradation of proteins. We also confirmed Cr(VI)-induced LC3-II elevation mainly came from autophagy induction rather than lysosomal degradation impairment. ROS-AKT-mTOR pathway was associated with Cr(VI)-induced autophagy, and ROS scavenger N-acetylcysteine (NAC) pretreatment inhibited Cr(VI)-induced autophagy by alleviating the inhibition of the AKT-mTOR pathway. Autophagy inhibitors 3-methyladenine (3-MA) and chloroquine diphosphate (CDP) promoted Cr(VI)-induced apoptotic death. CONCLUSION: These findings indicated Cr(VI)-induced autophagy protected L-02 hepatocytes from apoptosis through the ROS-AKT-mTOR pathway.

Retraction: Cd induces G2/M cell cycle arrest by up-regulating miR-133b via directly targeting PPP2R2D in L02 hepatocytes.

Retraction of 'Cd induces G2/M cell cycle arrest by up-regulating miR-133b via directly targeting PPP2R2D in L02 hepatocytes' by Yujing Zhang et al., Metallomics, 2018, 10, 1510-1523.

Cd induces G2/M cell cycle arrest by up-regulating miR-133b via directly targeting PPP2R2D in L02 hepatocytes.

Previous research has shown that cadmium (Cd), which accumulates primarily in the liver and kidneys, could cause cell cycle arrest in various cell types. In the present study, the effect of Cd on the cell cycle progression of hepatocytes and the underlying mechanisms were investigated. Our results showed that Cd inhibited the growth and proliferation of L02 hepatocytes. Further study revealed that Cd significantly induced G2-phase cell cycle arrest accompanied by an overproduction of reactive oxygen species (ROS). Cd-induced G2-phase arrest could be prevented by inhibiting ROS with the use of antioxidant Trolox. Additionally, treatment with Cd induced an increase in the expression of miR-133b, which could be reversed with Trolox pretreatment. Moreover, miR-133b was shown to regulate PPP2R2D expression by binding to the 3'-untranslated region (UTR) of PPP2R2D mRNA. We also found that treatment with Cd decreased the expression of PPP2R2D, and inhibition of PPP2R2D expression by siRNA partially aggravated Cd-induced cell cycle arrest. By artificially increasing the expression of the protein phosphatase 2A-B55δ subunit (PP2A-B55δ, encoded by the PPP2R2D gene), we confirmed that PPP2R2D can be regulated by miR-133b in Cd-treated cells, and the G2-phase arrest effect could be suppressed by PP2A-B55δ. Collectively, these findings indicated that Cd increased ROS and induced the expression of miR-133b, which consequently inhibited the expression of PPP2R2D and led to G2-phase arrest in L02 hepatocytes. This study provides novel insights into the molecular mechanisms of Cd-induced cell cycle arrest.

Mitochondrial Biogenesis in Response to Chromium (VI) Toxicity in Human Liver Cells.

Hexavalent chromium (Cr(VI)) is a ubiquitous environmental pollutant, which poses a threat to human public health. Recent studies have shown that mitochondrial biogenesis can be activated by inflammatory and oxidative stress. However, whether mitochondrial biogenesis is involved in Cr(VI)-induced hepatotoxicity is unclear. Here, we demonstrated the induction of inflammatory response and oxidative stress, as indicated by upregulation of inflammatory factors and reactive oxygen species (ROS). Subsequently, we demonstrated that mitochondrial biogenesis, comprising the mitochondrial DNA copy number and mitochondrial mass, was significantly increased in HepG2 cells exposed to low concentrations of Cr(VI). Expression of genes related to mitochondrial function complex I and complex V was upregulated at low concentrations of Cr(VI). mRNA levels of antioxidant enzymes, including superoxide dismutase 1 and 2 (SOD1 and SOD2, respectively), kech like ECH associate protein 1 (KEAP1) and nuclear respiratory factor 2 (NRF-2), were also upregulated. Consistent with the above results, mRNA and protein levels of key transcriptional regulators of mitochondrial biogenesis such as the peroxisome-proliferator-activated receptor γ coactivator-1α (PGC-1α), NRF-1 and mitochondrial transcription factor A (TFAM) were increased by low concentrations of Cr(VI) in HepG2 cells. Moreover, we found that PGC-1α and NRF-1 tended to translocate into the nucleus. The expression of genes potentially involved in mitochondrial biogenesis pathways, including mRNA level of silent information regulator-1 (SIRT1), forkhead box class-O (FOXO1), threonine kinase 1 (AKT1), and cAMP response element-binding protein (CREB1), was also upregulated. In contrast, mitochondrial biogenesis was inhibited and the expression of its regulatory factors and antioxidants was downregulated at high and cytotoxic concentrations of Cr(VI) in HepG2 cells. It is believed that pretreatment with α-tocopherol could be acting against the mitochondrial biogenesis imbalance induced by Cr(VI). In conclusion, our study suggests that the homeostasis of mitochondrial biogenesis may be an important cellular compensatory mechanism against Cr(VI)-induced toxicity and a promising detoxification target.

An evaluation of the protective role of vitamin C in reactive oxygen species-induced hepatotoxicity due to hexavalent chromium in vitro and in vivo.

BACKGROUD: Drinking water contamination with hexavalent chromium [Cr (VI)] has become one of the most serious public health problems, thus the investigation of Cr (VI)-induced hepatotoxicity has attracted much attention in recent years. METHODS: In the present study, by determining the indices of hepatotoxicity induced by Cr (VI), the source of accumulated reactive oxygen species (ROS), and the protective effect of the antioxidant Vitamin C (Vit C), we explored the mechanisms involved in Cr (VI)-induced hepatotoxicity in vitro and in vivo. RESULTS: We found Cr (VI) caused hepatotoxicity characterized by the alterations of several enzymatic and cytokine markers including aspartate aminotransferase (AST), alanine aminotransferase (ALT), interleukine-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α), etc. ROS production after Cr (VI) exposure was origins from the inhibition of electron transfer chain (ETC) and antioxidant system. Vit C inhibited ROS accumulation thus protected against Cr (VI)-induced hepatotoxicity in L-02 hepatocytes and in the rat model. CONCLUSIONS: We concluded that ROS played a role in Cr (VI)-induced hepatotoxicity and Vit C exhibited protective effect. Our current data provides important clues for studying the mechanisms involved in Cr (VI)-induced liver injury, and may be of great help to develop therapeutic strategies for prevention and treatment of liver diseases involving ROS accumulation for occupational exposure population.

CoQ10 Deficiency May Indicate Mitochondrial Dysfunction in Cr(VI) Toxicity.

To investigate the toxic mechanism of hexavalent chromium Cr(VI) and search for an antidote for Cr(VI)-induced cytotoxicity, a study of mitochondrial dysfunction induced by Cr(VI) and cell survival by recovering mitochondrial function was performed. In the present study, we found that the gene expression of electron transfer flavoprotein dehydrogenase (ETFDH) was strongly downregulated by Cr(VI) exposure. The levels of coenzyme 10 (CoQ10) and mitochondrial biogenesis presented by mitochondrial mass and mitochondrial DNA copy number were also significantly reduced after Cr(VI) exposure. The subsequent, Cr(VI)-induced mitochondrial damage and apoptosis were characterized by reactive oxygen species (ROS) accumulation, caspase-3 and caspase-9 activation, decreased superoxide dismutase (SOD) and ATP production, increased methane dicarboxylic aldehyde (MDA) content, mitochondrial membrane depolarization and mitochondrial permeability transition pore (MPTP) opening, increased Ca2+ levels, Cyt c release, decreased Bcl-2 expression, and significantly elevated Bax expression. The Cr(VI)-induced deleterious changes were attenuated by pretreatment with CoQ10 in L-02 hepatocytes. These data suggest that Cr(VI) induces CoQ10 deficiency in L-02 hepatocytes, indicating that this deficiency may be a biomarker of mitochondrial dysfunction in Cr(VI) poisoning and that exogenous administration of CoQ10 may restore mitochondrial function and protect the liver from Cr(VI) exposure.

Cr(VI) induces cytotoxicity in vitro through activation of ROS-mediated endoplasmic reticulum stress and mitochondrial dysfunction via the PI3K/Akt signaling pathway.

The occupational and environmental toxicant hexavalent chromium [Cr(VI)] can cause severe damage to the liver; however, the exact mechanisms associated with its toxicity have not been thoroughly demonstrated. In the present study, the underlying mechanisms of Cr(VI)-induced hepatotoxicity were investigated. Our results showed that Cr(VI) inhibited the growth and proliferation of L-02 hepatocytes. Further study revealed that Cr(VI) significantly induced S-phase cell cycle arrest and apoptosis accompanying with the overproduction of reactive oxygen species (ROS). Cr(VI)-induced apoptosis could be prevented by inhibiting ROS with N-acetyl-l-cysteine (NAC). Additionally, our data showed that Cr(VI)-induced endoplasmic reticulum (ER) stress and mitochondrial dysfunction were concentration- and time-dependent. Moreover, inhibition of C/EBA homologous protein (CHOP) expression by siRNA partially prevented Cr(VI)-induced cell apoptosis, mitochondrial dysfunction and ROS generation. We also found that Cr(VI) treatment inhibited the PI3K/Akt pathway in a concentration- and time-dependent manner. After using IGF-1 (50ng/mL), the specific agonist of the PI3K/AKT signaling pathway, the facilitating effects of Cr(VI) were depressed. This finding demonstrated the relationship between the PI3K/Akt pathway, ER stress and mitochondrial dysfunction. Collectively, these findings indicated that Cr(VI) increased ROS production. Increased ROS production may account for inhibition of the PI3K/Akt pathway and lead to ER stress and mitochondrial dysfunction, which consequently induces apoptosis in L-02 hepatocytes. This study provides novel insights into the molecular mechanisms of Cr(VI)-induced cytotoxicity.

In vitro cytotoxicity assessment of roundup (glyphosate) in L-02 hepatocytes.

The goal of the present study was to elucidate the in vitro cytotoxicity of Roundup and to reveal the possible related mechanisms in L-02 hepatocytes. By detecting reactive oxygen species (ROS) production, glutathione (GSH)/superoxide dismutase (SOD) levels, mitochondrial permeability transition pore (PTP) open rate, apoptosis-inducing factor (AIF) release, intracellular Ca2+ concentration, and alanine aminotransferease (ALT)/aspartate aminotransferase (AST) leakage, we determined that Roundup induced anti-oxidant system inhibition, mitochondria damage, DNA damage, membrane integrity and permeability changes, and apoptosis in L-02 hepatocytes. By revealing the mechanistic insights of Roundup-induced cytotoxicity, our results are valuable for the design of preventive and therapeutic strategies for the occupational population exposed to Roundup and other pesticides.

Overexpression of TNKS1BP1 in lung cancers and its involvement in homologous recombination pathway of DNA double-strand breaks.

TNKS1BP1 is a member of the poly(ADP-ribose) polymerase (PARP) superfamily. Our previous studies have demonstrated that TNKS1BP1 plays an important role in DNA damage response. But whether and how TNKS1BP1 associates with cancer is still not clear. Here, we found that TNKS1BP1 was upregulated in human lung adenocarcinoma (LAC) tissues, and was associated with poor overall survival (OS) in LAC patients. Dysregulation of TNKS1BP1 affected the sensitivity of A549 cells to several DNA damage agents including cisplatin, bleomycin, and ionizing radiation. Mechanically, overexpression of TNKS1BP1 increased the accumulation of S phase cells, which was accompanied by a decrease in M phase cells. More importantly, we found TNKS1BP1 regulated genome stability, mainly through affecting the homologous recombination pathway of DNA double-strand breaks by inhibiting the RAD51 foci formation. Overall, our study indicates that, in LAC, aberrant expressions of TNKS1BP1 are common events, and overexpression of TNKS1BP1 might affect outcomes of cancer patients to chemotherapy and radiotherapy.

A Ca2+ chelator ameliorates chromium (VI)-induced hepatocyte L-02 injury via down-regulation of voltage-Dependent anion channel 1 (VDAC1) expression.

Hexavalent chromium could result in cell malfunctions. Intracellular Ca2+ ([Ca2+]i) content and VDAC1 expression are both important features related to cell survial. This study aimed to explore the mechanism of cell injury induced by Cr(VI) and tentatively offer clues to repairing this cell damage using [Ca2+]i and VDAC1. L-02 hepatocytes were treated with Cr(VI)/BAPTA, and the levels of [Ca2+]i and cell injury associated with Cr(VI) were determined in addition to the effect of BAPTA. The expression of VDAC1 in Cr(VI)-induced cells was evaluated. The results showed a dose-dependent elevation of the level of VDAC1 and the mRNA level of the VDAC1 biogenesis-related gene Sam50. BAPTA could ameliorate less severe damage induced by 4µM Cr(VI) via reducing VDAC1 and Sam50. Additionally, cell injury caused by less than 4µM Cr(VI) could be ameliorated by VDAC1 knockdown. Taken together, the findings of this study suggest that inhibition of intracellular Ca2± overload could protect cells from damage and that VDAC1 plays a considerable role in Cr(VI)-induced liver injury.

The role of STIM1 in the Cr(vi)-induced [Ca2+]i increase and cell injury in L-02 hepatocytes.

Hexavalent chromium [Cr(vi)] is a potent cytotoxin and carcinogen. In recent years, drinking water contamination with Cr(vi) has become a worldwide problem of significant public health importance, thus much attention has been paid to the investigation of Cr(vi)-induced hepatotoxicity. The concentration of intracellular calcium ions ([Ca2+]i) was found to be increased after Cr(vi) exposure, but the exact underlying mechanisms involved in the Ca2+ homeostasis imbalance remain poorly characterized. In the present study, by utilizing the antagonist of store-operated calcium channels (SOCCs) 2-aminoethoxydiphenyl borate (2-APB), small interfering RNA against stromal interaction molecule 1 (si-STIM1) and antioxidant N-acetylcysteine (NAC), we found that Cr(vi) induces [Ca2+]i increase, cell viability loss and transaminase (AST/ALT) leakage, and that these could be suppressed by both 2-APB and si-STIM1. NAC significantly alleviated Cr(vi)-induced up-regulation of STIM1, phosphorylated-extracellular-signal-regulated kinases 1 and 2 (p-ERK1/2), ERK1/2 and nuclear factor κB (NF-κB). By utilizing the ERK inhibitor U0126 and the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC), we confirmed that STIM1 can be regulated by ERK and NF-κB. Thus we concluded that STIM1 plays a role in the Cr(vi)-induced [Ca2+]i increase and cell injury. Our current data provide new insights into the mechanisms of STIM1 function in Cr(vi)-induced hepatotoxicity, and may provide experimental clues for the prevention and treatment of liver diseases in the occupational population exposed to Cr(vi).

Cr(VI) induces premature senescence through ROS-mediated p53 pathway in L-02 hepatocytes.

Hexavalent Chromium [Cr(VI)], which can be found of various uses in industries such as metallurgy and textile dying, can cause a number of human disease including inflammation and cancer. Unlike previous research that focused on Cr(VI)-induced oxidative damage and apoptosis, this study placed emphasis on premature senescence that can be induced by low-dose and long-term Cr(VI) exposure. We found Cr(VI) induced premature senescence in L-02 hepatocytes, as confirmed by increase in senescence associated-ß-galactosidase (SA-ß-Gal) activity. Cr(VI) stabilized p53 through phosphorylation at Ser15 and increased expression of p53-transcriptional target p21. Mechanism study revealed Cr(VI) targeted and inhibited mitochondrial respiratory chain complex (MRCC) I and II to enhance reactive oxygen species (ROS) production. By applying antioxidant Trolox, we also confirmed that ROS mediated p53 activation. A tetracycline-inducible lentiviral expression system containing shRNA to p53 was used to knockout p53. We found p53 could inhibit pro-survival genes B-cell lymphoma-2 (Bcl-2), myeloid leukemia-1 (Mcl-1) and S phase related cell cycle proteins cyclin-dependent kinase 2 (CDK2), Cyclin E to induce premature senescence, and the functional role of ROS in Cr(VI)-induced premature senescence is depend on p53. The results suggest that Cr(VI) has a role in premature senescence by promoting ROS-dependent p53 activation in L-02 hepatocytes.

Association of NOS2 and NOS3 gene polymorphisms with susceptibility to type 2 diabetes mellitus and diabetic nephropathy in the Chinese Han population.

Inducible nitric oxide synthase (NOS2) and endothelial nitric oxide synthase (NOS3) gene play important roles in the susceptibility to type 2 diabetes mellitus (T2DM). The present study aims to detect the potential association of NOS2 and NOS3 gene polymorphisms with the susceptibility toT2DM and diabetic nephropathy (DN) in the Chinese Han population. Four hundred and ninety T2DM patients and 485 healthy controls were enrolled in this case-control study. The genotypes of NOS2 and NOS3 gene polymorphisms were analyzed by the polymerase chain reaction (PCR)-ligase detection reaction (LDR) method. Our data demonstrated that the NOS2 rs2779248 and NOS2 rs1137933 genetic polymorphisms were significantly associated with the increased susceptibility to T2DM in the heterozygote comparison, dominant model, and allele contrast; and NOS3 rs3918188 genetic polymorphism was significantly associated with the increased susceptibility to T2DM in the homozygote comparison and recessive model. The allele-C and genotype-TC of NOS2 rs2779248, allele-A and genotype-GA of NOS2 rs1137933 and genotype-AA of NOS3 rs3918188 genetic polymorphisms might be the risk factors for increasing the susceptibility to T2DM. And a significant haplotype effect of NOS2 rs10459953/C- rs1137933/G- rs2779248/T was found between T2DM cases and controls. Moreover, NOS3 rs1800783 polymorphism was significantly associated with the increased susceptibility to DN in the heterozygote comparison, recessive model and allele contrast. At last, a positive correlation of family history of diabetes with NOS3 rs11771443 polymorphism was found in DN. These preliminary findings indicate that the NOS2 rs2779248, NOS2 rs1137933, and NOS3 rs3918188 genetic polymorphisms are potentially related to the susceptibility to T2DM, and the rs1800783 polymorphism might be considered as genetic risk factors for diabetic nephropathy, and family history of diabetes was closely associated with rs11771443 polymorphism in DN, and the genetic variants might be used as molecular markers for evaluating the risk of T2DM and diabetic nephropathy. © 2016 IUBMB Life, 68(7):516-525, 2016.

DNA-PKcs Negatively Regulates Cyclin B1 Protein Stability through Facilitating Its Ubiquitination Mediated by Cdh1-APC/C Pathway.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a critical component of the non-homologous end-joining pathway of DNA double-stranded break repair. DNA-PKcs has also been shown recently functioning in mitotic regulation. Here, we report that DNA-PKcs negatively regulates the stability of Cyclin B1 protein through facilitating its ubiquitination mediated by Cdh1 / E 3 ubiquitin ligase APC/C pathway. Loss of DNA-PKcs causes abnormal accumulation of Cyclin B1 protein. Cyclin B1 degradation is delayed in DNA-PKcs-deficient cells as result of attenuated ubiquitination. The impact of DNA-PKcs on Cyclin B1 stability relies on its kinase activity. Our study further reveals that DNA-PKcs interacts with APC/C core component APC2 and its co-activator Cdh1. The destruction of Cdh1 is accelerated in the absence of DNA-PKcs. Moreover, overexpression of exogenous Cdh1 can reverse the increase of Cyclin B1 protein in DNA-PKcs-deficient cells. Thus, DNA-PKcs, in addition to its direct role in DNA damage repair, functions in mitotic progression at least partially through regulating the stability of Cyclin B1 protein.

TNKS1BP1 functions in DNA double-strand break repair though facilitating DNA-PKcs autophosphorylation dependent on PARP-1.

TNKS1BP1 was originally identified as an interaction protein of tankyrase 1, which belongs to the poly(ADP-ribose) polymerase (PARP) superfamily. PARP members play important roles for example in DNA repair, telomere stability and mitosis regulation. Although the TNKS1BP1 protein was considered to be a poly(ADP-ribosyl)ation acceptor of tankyrase 1, its function is still unknown. Here we firstly identified that TNKS1BP1 was up-regulated by ionizing radiation (IR) and the depletion of TNKS1BP1 significantly sensitized cancer cells to IR. Neutral comet assay, pulsed-field gel electrophoresis, and γH2AX foci analysis indicated that TNKS1BP1 is required for the efficient repair of DNA double-strand breaks (DSB). The TNKS1BP1 protein was demonstrated to interact with DNA-dependent protein kinase (DNA-PKcs) and poly(ADP-ribose) polymerase 1 (PARP-1), by co-immunoprecipitation analysis. Moreover, TNKS1BP1 was shown to promote the association of PARP-1 and DNA-PKcs. Overexpression of TNKS1BP1 induced the autophosphorylation of DNA-PKcs/Ser2056 in a PARP-1 dependent manner, which contributed to an increased capability of DNA DSB repair. Inhibition of PARP-1 blocked the TNKS1BP1-mediated DNA-PKcs autophosphorylation and attenuated the PARylation of DNA-PKcs. TNKS1BP1 is a newly described component of the DNA DSB repair machinery, which provides much more mechanistic evidence for the rationale of developing effective anticancer measures by targeting PARP-1 and DNA-PKcs.

Role of mitochondrial electron transport chain dysfunction in Cr(VI)-induced cytotoxicity in L-02 hepatocytes.

BACKGROUND: Hexavalent chromium [Cr(VI)] and its compounds (e.g. chromates), which are extensively used in numerous industrial processes including leather tanning and steel manufacturing, are considered as priority pollutants. There is growing evidence supporting that Cr(VI) could be a human carcinogen that induces primary liver cancer after oral exposure, and this sheds light on the importance of the investigation of Cr(VI)-induced hepatotoxicity. Although it is known that mitochondria are major targets for heavy metals, the mechanisms of electron transfer chain (ETC) dysfunction involved in Cr(VI)-induced cytotoxicity are unclear. METHODS: In the present study, by using mitochondrial respiratory chain complex (MRCC) I inhibitor rotenone (ROT) and its substrates glutamate/malate (Glu/Mal), MRCC III inhibitor antimycin A (AA) and its substrate coenzyme Q (CoQ), and the antioxidant Vitamin C (Vit C), we aimed to elucidate the role of mitochondrial ETC dysfunction in Cr(VI)-induced cytotoxicity. RESULTS: We found that Cr(VI) targeted and inhibited MRCC I and III to induce ETC dysfunction, which played an important role in Cr(VI)-induced cytotoxicity. CONCLUSION: Our current data provides novel important insight into the mechanisms of mitochondrial ETC dysfunction in Cr(VI)-induced cytotoxicity in the hepatocytes, and we will be poised to develop new methods in the prevention and treatment of liver diseases involving mitochondrial ETC dysfunction for the occupational exposure population.

Activation of autophagy protects against ROS-mediated mitochondria-dependent apoptosis in L-02 hepatocytes induced by Cr(VI).

BACKGROUND: Hexavalent chromium (Cr(VI)) overdose causes hepatocellular injuries by inducing mitochondrial damage and subsequent apoptosis in animals and humans. Autophagy can selectively remove damaged organelles, especially impaired mitochondria, and in turn, protects against mitochondria-dependent cell death. The present study was designed to explore the effects of autophagy on the Cr(VI)-induced hepatotoxicity. METHODS: L-02 hepatocytes were incubated with different concentrations of Cr(VI) for 24h and several indicators for evaluating mitochondrial damage and hepatocellular apoptosis were measured. Then effects of ROS scavenger NAC on ROS production and calcium overload during Cr(VI)-induced hepatotoxicity were examined. Finally, the study further investigated the role of autophagy played in repairing mitochondrial damage and subsequent hepatocyte injuries. RESULTS: After exposed to different concentrations of Cr(VI) for 24h, cell viability, mitochondria membrane potential, ATP content were significantly decreased and caspase-3 activities and apoptosis rates increased in L-02 hepatocytes. The treatment of NAC reduced ROS formation and Ca(2+) content, restored CRAC channel activities and further diminished mitochondrial injuries. Furthermore, autophagy inducer, rapamycin is beneficial for repairing mitochondrial function and limiting hepatocytes damage, and pharmacological inhibition of autophagy by 3-methyladenine further exacerbated Cr(VI)-induced hepatotoxicity. CONCLUSIONS: ROS production is a critical reason for Cr(VI)-induced mitochondria-dependent apoptosis. And activation of autophagy could repair mitochondria function to protect hepatocytes potentially by removing damaged mitochondria.

Desipramine ameliorates Cr(VI)-induced hepatocellular apoptosis via the inhibition of ceramide channel formation and mitochondrial PTP opening.

BACKGROUND: Hexavalent chromium (Cr(VI)) is a common environmental pollutant. Cr(VI) exposure can lead to severe damage in the liver, but the preventive measures to diminish Cr(VI)-induced hepatotoxicity need further study. Acid sphingomyelinase (ASMase) is responsible for the production of ceramide via the hydrolysis of sphingomyelin. The present study was designed to investigate effects of desipramine (DES), as an ASMase inhibitor, on Cr(VI)-induced hepatotoxicity. METHODS: L-02 hepatocytes were incubated with different concentrations of Cr(VI) for 24h, and ASMase activities and ceramide levels were measured. Moreover, the study investigated the role of DES played in ASMase activities and ceramide levels. Finally, effects of DES on mRNA and protein expressions of the components of mitochondrial permeability transition pore (PTP) and PTP opening were detected. RESULTS: The ASMase activities and ceramide contents increased in L-02 hepatocytes treated with Cr(VI). The results demonstrated that apoptosis rates, ASMase activities and ceramide content decreased in groups treated with the combination of DES and Cr(VI) compared to Cr(VI) groups. Furthermore, DES inhibited Cr(VI)-induced mitochondrial PTP opening by intervening the mRNA and protein expressions of the components of mitochondrial PTP. CONCLUSIONS: DES may exert protective effects on Cr(VI)-induced hepatocellular apoptosis probably by inhibiting ceramide channel formation and mitochondrial PTP opening.

[Effects of 10% Liuyangmycin emulsifiable on mitochondrial function in L-02 hepatocytes].

OBJECTIVE: To investigate the effects of 10% Liuyangmycin emulsifiable (LY-EC) on mitochondrial function in L-02 hepatocytes. METHODS: To detect the effect of different concentration of LY-EC (0 -200 mg/L) on L-02 hepatocytes survival rate with MTT method, and choose proper LY-EC treated concentrations (survival rate > 70%) for the following experiments. After hepatocytes were treated with LY-EC at 0.39, 1.56, 6.25 and 25.00 mg/L respectively for 12 h, MDA and GSH contents, SOD, SDH, ATPase activities were respectively analyzed by their assay kits using colorimetry. Then mitochondrial PTP open percentage and the contents of ATP, ADP and AMP were measured with HPLC assays, and ATP/ADP ratio and energy charge (EC) values were calculated. The effects of LY-EC on mitochondrial ultra-structures were observed by transmission electron microscopy. RESULTS: LY-EC induced the decrease of cell survival rates in a concentrate- dependent manner (r = 0.939, P < 0.05). Compared with control group, GSH contents and SOD, SDH and ATPase activities in groups treated with LY-EC decreased significantly, MDA contents and mitochondrial PTP open percentages increased (P < 0.05), the levels of cellular ATP, TAN and ATP/ADP ratio in L-02 hepatocytes decreased, while energy charge (EC) increased significantly (P < 0.05). Hepatocytes treated with LY-EC were observed cytoplasmic edema, mitochondria swelling and the deformation of mitochondrial cristae with transmission electron microscope. CONCLUSION: LY-EC induced oxidative stress, mitochondrial function damage and energy metabolism disorder in L-02 hepatocytes.