Low-level sodium arsenite induces apoptosis through inhibiting TrxR activity in pancreatic ß-cells.

In our previous study, we reported that sodium arsenite induced ROS-dependent apoptosis through lysosomal-mitochondrial pathway in pancreatic ß-cells. Since the thioredoxin (Trx) system is the key antioxidant factor in mammalian cells, we investigate whether the inhibition of Trx system contributes to sodium arsenite-induced apoptosis in this study. After treatment with low-level (0.25-1µM) sodium arsenite for 96h, the thioredoxin reductase (TrxR) activity was decreased significantly in pancreatic INS-1 cells. Following with the inactivation of TrxR, ASK1 was released from combining with Trx, which was evidenced by increased levels of ASK1 in sodium arsenite-treated INS-1 cells. Subsequently, activated ASK1 accelerated the expression of proapoptotic protein Bax and reduced the expression of anti-apoptic protein Bcl-2. Finally, low-level sodium arsenite induced apoptosis via caspase-3 in INS-1 cells. Knockdown of ASK1 alleviated sodium arsenite-induced apoptosis. In summary, the precise molecular mechanisms through which arsenic is related to diabetes have not been completely elucidated, inactivation of Trx system might provide insights into the underlying mechanisms at the environmental exposure levels.

Citreoviridin induces ROS-dependent autophagic cell death in human liver HepG2 cells.

Citreoviridin (CIT) is one of toxic mycotoxins derived from fungal species in moldy cereals. Whether CIT exerts hepatotoxicity and the precise molecular mechanisms of CIT hepatotoxicity are not completely elucidated. In this study, the inhibitor of autophagosome formation, 3-methyladenine, protected the cells against CIT cytotoxicity, and the autophagy stimulator rapamycin further decreased the cell viability of CIT-treated HepG2 cells. Knockdown of Atg5 with Atg5 siRNA alleviated CIT-induced cell death. These finding suggested the hypothesis that autophagic cell death contributed to CIT-induced cytotoxicity in HepG2 cells. CIT increased the autophagosome number in HepG2 cells observed under a transmission electron microscope, and this effect was confirmed by the elevated LC3-II levels detected through Western blot. Reduction of P62 protein levels and the result of LC3 turnover assay indicated that the accumulation of autophagosomes in the CIT-treated HepG2 cells was due to increased formation rather than impaired degradation. The pretreatment of HepG2 cells with the ROS inhibitor NAC reduced autophagosome formation and reversed the CIT cytotoxicity, indicating that CIT-induced autophagic cell death was ROS-dependent. In summary, ROS-dependent autophagic cell death of HpeG2 cells described in this study may help to elucidate the underlying mechanism of CIT cytotoxicity.

The role of oxidative stress in citreoviridin-induced DNA damage in human liver-derived HepG2 cells.

We hypothesize that citreoviridin (CIT) induces DNA damage in human liver-derived HepG2 cells through an oxidative stress mechanism and that N-acetyl-l-cysteine (NAC) protects against CIT-induced DNA damage in HepG2 cells. CIT-induced DNA damage in HepG2 cells was evaluated by alkaline single-cell gel electrophoresis assay. To elucidate the genotoxicity mechanisms, the level of oxidative DNA damage was tested by immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG); the intracellular generation of reactive oxygen species (ROS) and reduced glutathione (GSH) were examined; mitochondrial membrane potential and lysosomal membranes' permeability were detected; furthermore, protective effects of NAC on CIT-induced ROS formation and CIT-induced DNA damage were evaluated in HepG2 cells. A significant dose-dependent increment in DNA migration was observed at tested concentrations (2.50-10.00 µM) of CIT. The levels of ROS, 8-OHdG formation were increased by CIT, and significant depletion of GSH in HepG2 cells was induced by CIT. Destabilization of lysosome and mitochondria was also observed in cells treated with CIT. In addition, NAC significantly decreased CIT-induced ROS formation and CIT-induced DNA damage in HepG2 cells. The data indicate that CIT induces DNA damage in HepG2 cells, most likely through oxidative stress mechanisms; that NAC protects against DNA damage induced by CIT in HepG2 cells; and that depolarization of mitochondria and lysosomal protease leakage may play a role in CIT-induced DNA damage in HepG2 cells.

Sodium arsenite induces ROS-dependent autophagic cell death in pancreatic ß-cells.

Inorganic arsenic is a worldwide environmental pollutant. Inorganic arsenic's positive relationship with the incidence of type 2 diabetes mellitus arouses concerns associated with its etiology in diabetes among the general human population. In this study, the inhibitor of autophagosome formation, 3-methyladenine, protected the cells against sodium arsenite cytotoxicity, and the autophagy stimulator rapamycin further decreased the cell viability of sodium arsenite-treated INS-1 cells. These finding suggested the hypothesis that autophagic cell death contributed to sodium arsenite-induced cytotoxicity in INS-1 cells. Sodium arsenite increased the autophagosome-positive puncta in INS-1 cells observed under a fluorescence microscope, and this effect was confirmed by the elevated LC3-II levels detected through Western blot. The LC3 turnover assay indicated that the accumulation of autophagosomes in the arsenite-treated INS-1 cells was due to increased formation rather than impaired degradation. The pretreatment of INS-1 cells with the ROS inhibitor NAC reduced autophagosome formation and reversed the sodium arsenite cytotoxicity, indicating that sodium arsenite-induced autophagic cell death was ROS-dependent. In summary, the precise molecular mechanisms through which arsenic is related to diabetes have not been completely elucidated, but the ROS-dependent autophagic cell death of pancreatic ß-cells described in this study may help to elucidate the underlying mechanism.

Perfluorooctane sulfonate blocked autophagy flux and induced lysosome membrane permeabilization in HepG2 cells.

Perfluorooctane sulfonate (PFOS) is an emerging persistent organic pollutant widely distributed in the environment, wildlife and human. In this study, as observed under the transmission electron microscope, PFOS increased autophagosome numbers in HepG2 cells, and it was confirmed by elevated LC3-II levels in Western blot analysis. PFOS increased P62 level and chloroquine failed to further increase the expression of LC3-II after PFOS treatment, indicating that the accumulation of autophagosome was due to impaired degradation rather than increased formation. With acridine orange staining, we found PFOS caused lysosomal membrane permeabilization (LMP). In this study, autophasome formation inhibitor 3-methyladenine protected cells against PFOS toxicity, autophagy stimulator rapamycin further decreased cell viability and increased LDH release, cathepsin inhibitor did not influence cell viability of PFOS-treated HepG2 cells significantly. These further supported the notion that autophagic cell death contributed to PFOS-induced hepatotoxicity. In summary, the data of the present study revealed that PFOS induced LMP and consequent blockage of autophagy flux, leading to an excessive accumulation of the autophagosomes and turning autophagy into a destructive process eventually. This finding will provide clues for effective prevention and treatment of PFOS-induced hepatic disease.

Genotoxic effects induced by zearalenone in a human embryonic kidney cell line.

Mycotoxins are considered to be significant contaminants of food and animal feed. Zearalenone (ZEA) is a hepatotoxic mycotoxin with estrogenic and anabolic activity found in cereal grains worldwide. ZEA affects hematological and immunological parameters in humans and rodents. The compound can induce cell death, cause lipid peroxidation, inhibit protein and DNA synthesis, and exert genotoxic effects. ZEA may cause increased phagolysosomal fragility in the kidney. Our research showed that exposure of human embryonic kidney (HEK293) cells to ZEA (10 or 20µM) resulted in a concentration-dependent increase in DNA strand breaks measured with the comet assay. Damage was reduced in cells pretreated with NH4Cl, pepstatin A, or desipramine for 1h. Production of reactive oxygen species (ROS) was increased in cells exposed to ZEA, but DNA strand break induction could not be inhibited by the antioxidant hydroxytyrosol (HT). These results suggest that oxidative stress does not play a key role in DNA strand breaks induced by ZEA, that lysosomal injury precedes DNA strand breaks, and that the lysosome may be a primary target for ZEA in HEK293 cells.

[Mechanism of the apoptosis of rat pancreas islet ß cell strain (INS-1 cells) induced by sodium arsenite].

OBJECTIVE: To study mechanism of the apoptosis of rat pancreas islet ß cell strain (INS-1 cells) induced by sodium arsenite. METHODS: INS-1 cells were exposed to sodium arsenite at the different concentrations. MTT assay was used to detect the viability of INS-1 cells. The potentials on mitochondrial membrane and lysosome membrane of INS-1 cells were determined with the fluorescence spectrophotometer. The apoptotic levels of INS-1 cells exposed to sodium arsenite were observed by a fluorescence microscope and flow cytometry. RESULTS: After exposure to sodium arsenite, the viability of INS-1 cells significantly decreased with the doses of sodium arsenite. At 24 h after exposure, the OD values of the mitochondrial membrane potentials declined observably with the doses of sodium arsenite (P < 0.01). At 48 h after exposure, the OD values of the lysosome membrane potentials significantly increased with the doses of sodium arsenite (P < 0.01). At 72 h after exposure, the apoptotic cells were observed under a fluorescence microscope and enhanced with the doses of sodium arsenite. The apoptosis cells with light blue, karyopyknosis, karyorrhexis, apoptotic body and chromatin concentration appeared. The results detected with flow cytometry indicated that after exposure, the apoptotic INS-1E cells significantly increased with the doses of sodium arsenite. CONCLUSIONS: The sodium arsenite can induce the apoptosis of INS-1 cells through the mitochondria-lysosome pathway.

Lysosomal membrane permeabilization contributes to elemene emulsion-induced apoptosis in A549 cells.

Elemene is a broad-spectrum antitumor agent. In the present study, lysosomal membrane permeabilization (LMP) was detected after short elemene emulsion--exposure (12 h) that preceded a decrease of the mitochondrial membrane potential and DNA damage (24 h) in A549 cells. At later time points (36 h) elemene emulsion caused the appearance of A549 cells with apoptotic features, including apoptotic morphology, phosphatidylserine exposure, and caspase-3 activation. A significant increase in protein expression for cathepsin D was also observed utilizing Western blot analysis after exposure to elemene emulsion for 12 h. The present study showed that elemene emulsion induced the increased levels of reactive oxygen species (ROS) and depletion of glutathione (GSH) in A549 cells. Cells treated with pepstatin A, an inhibitor for cathepsin D, showed a significant inhibition in DNA damage, mitochondrial membrane permeabilization, caspase-3 activation, and phosphatidylserine exposure. These results demonstrated that apoptosis induced by elemene emulsion in A549 cells is mediated in part through LMP and lysosomal protease cathepsin D.

ß-Elemene radiosensitizes lung cancer A549 cells by enhancing DNA damage and inhibiting DNA repair.

ß-Elemene is a broad-spectrum antitumor agent. In China, several studies have indicated that ß-elemene enhances the cytotoxic effect of radiation in vitro and in vivo. In this study, the alkaline comet assay and neutral comet assay were used to measure both DNA strand breaks and DNA repair activity in A549 cells exposed to ß-elemene, irradiation or combination treatment. The overall object of the study was to test whether ß-elemene radiosensitization is associated with an enhancement in radiation-induced DNA damage or with a decrease in the repair of radiation-induced damage. The results revealed high levels of DNA single strand breaks (SSB) and double strand breaks (DSB) in A549 cells after exposure to the combination of ß-elemene and irradiation. To assess SSB and DSB repair, alkaline comet assay and neutral comet assay were performed at 24 h postirradiation. The damage induced by the combination of ß-elemene and irradiation was repaired at a slower rate. These findings suggest that ß-elemene can enhance A549 cell radiosensitivity through the enhancement of DNA damage and the suppression of DNA repair.

Patulin-induced oxidative DNA damage and p53 modulation in HepG2 cells.

Patulin (PAT) is a mycotoxin produced by certain species of Penicillium and Aspergillus. The aim of this study was to assess PAT-induced DNA damage and to clarify the mechanisms, using human hepatoma G2 (HepG2) cells. PAT caused significant increase of DNA migration in single cell gel electrophoresis assay. To elucidate the role of glutathione (GSH), the intracellular GSH level was modulated by pre-treatment with buthionine-(S, R)-sulfoximine, a specific GSH synthesis inhibitor. It was observed that PAT significantly induced DNA damage in GSH-depleted HepG2 cells at lower concentrations. PAT induced the increased levels of reactive oxygen species and depletion of GSH in HepG2 cells using 2,7-dichlorofluorescein diacetate and 0-phthalaldehyde, respectively. PAT significantly increased the levels of 8-hydroxydeoxyguanosine and thiobarbituric acid-reactive substances in HepG2 cells. Also, PAT-induced p53 protein accumulation was observed in HepG2 cells, suggesting that the activation of p53 appeared to have been a downstream response to the PAT-induced DNA damage. These results demonstrate that PAT causes DNA strand breaks in HepG2 cells, probably through oxidative stress. Both GSH, as a main intracellular antioxidant, and p53 protein are responsible for cellular defense against PAT-induced DNA damage.

Patulin-induced genotoxicity and modulation of glutathione in HepG2 cells.

Patulin (PAT), a mycotoxin produced by certain species of Penicillium, Aspergillus and Byssochlamys, is mainly found in ripe apple and apple products. In our present study, a significant increase of the micronuclei frequency induced by PAT was found in human hepatoma HepG2 cells. To elucidate the role of glutathione (GSH) in the effect, the intracellular GSH level was modulated by pre-treatment with buthionine-(S, R)-sulfoximine (BSO), a specific GSH synthesis inhibitor, and by pre-treatment with N-acetylcysteine (NAC), a GSH precursor. It was found that depletion of GSH in HepG2 cells with BSO dramatically increased the PAT-induced micronuclei frequencies and that when the intracellular GSH content was elevated by NAC, the chromosome damage induced by PAT was significantly prevented in our test concentrations (0.19-0.75 microM). These results indicate that GSH play an important role in cellular defense against PAT-induced genotoxicity.

Curcumin attenuates acrylamide-induced cytotoxicity and genotoxicity in HepG2 cells by ROS scavenging.

Acrylamide (AA), a proven rodent carcinogen, has recently been discovered in foods heated at high temperatures. This finding raises public health concerns. In our previous study, we found that AA caused DNA fragments and increase of reactive oxygen species (ROS) formation and induced genotoxicity and weak cytotoxicity in HepG2 cells. Presently, curcumin, a natural antioxidant compound present in turmeric was evaluated for its protective effects. The results showed that curcumin at the concentration of 2.5 microg/mL significantly reduced AA-induced ROS production, DNA fragments, micronuclei formation, and cytotoxicity in HepG2 cells. The effect of PEG-catalase on protecting against AA-induced cytotoxicity suggests that AA-induced cytotoxicity is directly dependent on hydrogen peroxide production. These data suggest that curcumin could attenuate the cytotoxicity and genotoxicity induced by AA in HepG2 cells. The protection is probably mediated by an antioxidant protective mechanism. Consumption of curcumin may be a plausible way to prevent AA-mediated genotoxicity.