Caigao Zhong

Hexavalent chromium induces energy metabolism disturbance and p53-dependent cell cycle arrest via reactive oxygen species in L-02 hepatocytes

Hexavalent chromium [Cr(VI)] has become a non-negligible pollutant in the world. Cr(VI) exposure leads to severe damage to the liver, but the mechanisms involved in Cr(VI)-mediated toxicity in the liver are unclear. The present study aimed to explore whether Cr(VI) induces energy metabolism disturbance and cell cycle arrest in human L-02 hepatocytes. We showed that Cr(VI) inhibited state 3 respiration, respiratory control rate (RCR), and subsequently induced energy metabolism disturbance with decreased ATP production. Interestingly, cell cycle analysis by flow cytometry and protein expression analysis by western blotting revealed that low dose of Cr(VI) (4 uM) exposure induced S phase cell cycle arrest with decreased mediator of replication checkpoint 1 (Mrc1) and cyclin-dependent kinase 2 (CDK2), while higher doses of Cr(VI) (16, 32 uM) exposure resulted in G2/M phase arrest with decreased budding uninhibited by benzimidazoles-related 1 (BubR1) and cell division cycle 25 (CDC25). Mechanism study revealed that Cr(VI) decreased the activities of mitochondrial respiratory chain complex (MRCC) I and II, thus leading to ROS accumulation. Moreover, inhibiting ROS production by antioxidant N-acetyl-l-cysteine (NAC) rescued Cr(VI)-induced ATP depletion and cell cycle arrest. ROS-mediated p53 activation was found to involve in Cr(VI)-induced cell cycle arrest, and p53 inhibitor Pifithrin-α (PFT-α) rescued Cr(VI)-induced reduction of check point proteins Mrc1 and BubR1, thus inhibiting cell cycle arrest. In summary, the present study provides experimental evidence that Cr(VI) leads to energy metabolism disturbance and p53-dependent cell cycle arrest via ROS in L-02 hepatocytes.

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 Ca2+ 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.

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.

Reactive Oxygen Species Play a Central Role in Hexavalent Chromium-Induced Apoptosis in Hep3B Cells without the Functional Roles of p53 and Caspase-3

Background/Aims: Hexavalent chromium [Cr(VI)] and its compounds, which have the extensive application in diverse industries including metallurgy, textile and electroplating, are known to be genotoxic and mutagenic to humans. Although it is supported by a large body of literatures that p53 and caspase-3 played key roles in Cr(VI)-induced cytotoxicity, it is clear that Cr(VI) could induce apoptosis either without activating caspase, or in a p53-independent manner. Methods: In the present study, by using Z-VAD-fmk to inhibit caspase-3 in p53-deficient Hep3B cells, we explored the effect of Cr(VI) on apoptosis induction and the related mechanisms when the functions of p53 and caspase were simultaneously blocked. Results: We found that Cr(VI) still induced DNA damage, mitochondrial injury, oxidative stress and apoptosis in Hep3B cells without the functional roles of p53 and caspase-3, and the mechanism study revealed that this was in a ROS-dependent manner since NAC co-treatment showed the protective effect against Cr(VI)-induced apoptosis. Conclusion: Our research has disclosed the mechanism involved in Cr(VI)-induced cytotoxicity following the loss of p53 and caspase-3 functions and shed light on the importance of using antioxidants for primary and secondary prevention in Cr(VI) occupational exposure populations.

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.

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.

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

ABSTRACT 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.

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.

Effect of hexavalent chromium on electron leakage of respiratory chain in mitochondria isolated from rat liver.

Background/Aims: In the present study, we explored reactive axygen species (ROS) production in mitochondria, the mechanism of hexavalent chromium (Cr(VI)) hepatotoxicity, and the role of protection by GSH. Methods: Intact mitochondria were isolated from rat liver tissues and mitochondrial basal respiratory rates of NADH and FADH2 respiratory chains were determined. Mitochondria were treated with Cr(VI), GSH and several complex inhibitors. Mitochondria energized by glutamate/malate were separately or jointly treated with Rotenone (Rot), diphenyleneiodonium (DPI) and antimycinA (Ant), while mitochondria energized by succinate were separately or jointly treated with Rot, DPI ‚ thenoyltrifluoroacetone (TTFA) and Ant. Results: Cr(VI) concentration-dependently induced ROS production in the NADH and FADH2 respiratory chain in liver mitochondria. Basal respiratory rate of the mitochondrial FADH2 respiratory chain was significantly higher than that of NADH respiratory chain. Hepatic mitochondrial electron leakage induced by Cr(VI) from NADH respiratory chain were mainly from ubiquinone binding sites of complex I and complex III. Conclusion: Treatment with 50µM Cr(VI) enhances forward movement of electrons through FADH2 respiratory chain and leaking through the ubiquinone binding site of complex III. Moreover, the protective effect of GSH on liver mitochondria electron leakage is through removing excess H2O2 and reducing total ROS.

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

BackgroudDrinking 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.MethodsIn 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.ResultsWe found Cr (VI) caused hepatotoxicity characterized by the alterations of several enzymatic and cytokine markers including aspartate aminotransferase (AST), alanine aminotransferase (ALT), interleukine-1β (IL-1β), 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.ConclusionsWe 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.