4-hydroxy-2-nonenal and 1-2-dithiol-3-thione

3H-1,2-dithiole-3-thione (D3T), a cruciferous organosulfur compound, induces cytoprotective enzymes in animal cardiovascular cells. However, it remains unknown if D3T also upregulates antioxidants and phase 2 enzymes in human cardiomyocytes, and protects against cell injury induced by oxidative/electrophilic species as well as doxorubicin. In this study, we found that D3T (10-50 muM) potently induced a series of antioxidants and phase 2 enzymes in primary cultured human cardiomyocytes, including superoxide dismutase (SOD), glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx) glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase 1 (NQO1), aldose reductase (AR), and heme oxygenase (HO). D3T treatment also caused elevation of SOD, GSH, GR, GPx and GST in the isolated mitochondria. We also observed a time-dependent induction by D3T of mRNA expression for Cu,ZnSOD, MnSOD, gamma-glutamylcysteine ligase, GR, GSTA1, GSTM1, NQO1, AR, and HO-1. Pretreatment with D3T conferred concentration-dependent protection against cell injury induced by xanthine oxidase (XO)/xanthine, H(2)O(2), 3-morpholinosydnonimine, 4-hydroxy-2-nonenal, and doxorubicin. Pretreatment with D3T also reduced the formation of intracellular reactive oxygen species by XO/xanthine, H(2)O(2), and doxorubicin. In conclusion, this study demonstrated that D3T potently upregulated many antioxidants and phase 2 enzymes in human cardiomyocytes, which was accompanied by increased resistance to oxidative/electrophilic stress and doxorubicin toxicity.

Topics: Aldehydes; Antineoplastic Agents; Antioxidants; Cell Line; Cytoprotection; Doxorubicin; Humans; Metabolic Detoxication, Phase II; Mitochondria; Molsidomine; Myocytes, Cardiac; Oxidative Stress; Reactive Oxygen Species; Thiones; Thiophenes; Up-Regulation; Xanthine Oxidase

Astrocytes possess important roles in maintaining normal brain function and providing trophic support to the neurons. They also suffer a range of toxic insults, being a chief target of prooxidants such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridinium (MPP(+)), 6-hydroxydopamine (6-OHDA), 4-hydroxy-2-nonenal (HNE), and acrolein. Recently, we have observed that the cellular antioxidants and phase 2 enzymes can be upregulated by 3H-1,2-dithiole-3-thione (D3T), a nutraceutical found in cruciferous vegetables, against many prooxidants in human neuroblastoma cell lines (SH-SY5Y). However, the regulation of the above cellular factors by D3T in astrocytes and their role in ameliorating the neurotoxic effects of the above neurotoxins have not been investigated. In this study, we show that incubation of human primary astrocytes with micromolar concentrations (5-100 microM) of D3T for 24 h resulted in significant increases in the levels of reduced glutathione (GSH), glutathione reductase (GR), and the phase 2 enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1). D3T treatment also caused time-dependent increases in mRNA expression of the gamma-glutamylcysteine ligase catalytic subunit (GCLC), GR, and of NQO1 in these cells. Pretreatment of astrocytes with D3T was found to afford remarkable protection against the neurocytotoxicity elicited by MPTP, MPP(+), 6-OHDA, HNE and acrolein. Taken together, this study demonstrates for the first time that in human astrocytes, the cruciferous nutraceutical D3T potently induces the cellular GSH system and the phase 2 enzyme NQO1, which is accompanied by dramatically increased resistance of these cells to the damage induced by various neurotoxicants. The results of this study may have important implications for the development of novel neuroprotective strategies.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; 1-Methyl-4-phenylpyridinium; Acrolein; Aldehydes; Astrocytes; Brassicaceae; Cells, Cultured; Glutathione; Glutathione Reductase; Humans; NAD(P)H Dehydrogenase (Quinone); Oxidants; Oxidopamine; RNA, Messenger; Thiones; Thiophenes

Evidence suggests oxidative and electrophilic stress as a major factor contributing to the neuronal cell death in neurodegenerative disorders, especially Parkinson's disease. Consistent with this concept, administration of exogenous antioxidants has been shown to be protective against oxidative/electrophilic neurodegeneration. However, whether induction of endogenous antioxidants and phase 2 enzymes by the unique chemoprotectant, 3H-1,2-dithiole-3-thione (D3T) in neuronal cells also affords protection against oxidative and electrophilic neurocytotoxicity has not been carefully investigated. In this study, we showed that incubation of SH-SY5Y neuroblastoma cells or primary human neurons with micromolar concentrations (10-100 microM) of D3T for 24 h resulted in significant increases in the levels of reduced glutathione (GSH) and NAD(P)H:quinone oxidoreductase 1 (NQO1), two crucial cellular defenses against oxidative and electrophilic stress. D3T treatment also caused increases in mRNA expression of gamma-glutamylcysteine ligase catalytic subunit and NQO1 in SH-SY5Y cells. In addition, D3T treatment of the neuronal cells also resulted in a marked elevation of GSH content in the mitochondrial compartment. To determine the protective effects of the D3T-induced cellular defenses on neurotoxicant-elicited cell injury, SH-SY5Y cells were pretreated with D3T for 24 h and then exposed to dopamine, 6-hydroxydopamine (6-OHDA), 4-hydroxy-2-nonenal (HNE), or H2O2, agents that are known to be involved in neuron degeneration. We observed that D3T-pretreatment of SH-SY5Y cells led to significant protection against the cytotoxicity elicited by the above neurotoxicants. Similar neurocytoprotective effects of D3T-pretreatment were also observed in primary human neurons exposed to 6-OHDA or HNE. Taken together, this study demonstrates that D3T potently induces neuronal cellular GSH and NQO1 as well as mitochondrial GSH, and that such upregulated endogenous defenses are accompanied by increased resistance to oxidative and electrophilic neurocytotoxicity.

Topics: Aldehydes; Antioxidants; Cell Line, Tumor; Cells, Cultured; Dopamine; Glutathione; Humans; Hydrogen Peroxide; Mitochondria; NAD(P)H Dehydrogenase (Quinone); Nerve Degeneration; Neurons; Neuroprotective Agents; Oxidative Stress; Oxidopamine; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiones; Thiophenes

4-Hydroxy-2-nonenal (HNE) has been suggested to contribute to the pathogenesis of atherosclerosis. One of the major metabolic transformation pathways of HNE involves conjugation with glutathione (GSH) catalyzed by GSH S-transferase (GST). In this study, we have characterized the induction of GSH and GST by 3H-1,2-dithiole-3-thione (D3T) and the protective effects of the D3T-elevated cellular defenses on HNE-mediated toxicity in rat aortic smooth muscle A10 cells. Incubation of A10 cells with D3T resulted in a marked concentration- dependent induction of both GSH and GST. The induction of cellular GST by D3T also exhibited a time-dependent response. Pretreatment of A10 cells with D3T led to a dramatic decrease of HNE-induced cytotoxicity, as assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay and scanning electron microscopy. Incubation of A10 cells with HNE for 0.5 h and 1 h resulted in a significant depletion of cellular GSH, which preceded the decrease of cell viability. To further demonstrate the involvement of GSH and GST in protecting against HNE-induced cytotoxicity, buthionine sulfoximine (BSO) and sulfasalazine were used to inhibit cellular GSH biosynthesis and GST activity, respectively. Either depletion of GSH by BSO or inhibition of GST by sulfasalazine caused great potentiation of HNE-mediated cytotoxicity. Moreover, cotreatment of A10 cells with BSO was found to completely block the D3T-mediated GSH induction and to largely reverse the cytoprotective effects of D3T on HNE-induced toxicity. Taken together, this study demonstrates that D3T can induce both GSH and GST in aortic smooth muscle cells, and that the D3T-augmented cellular defenses afford a marked protection against HNE-induced vascular cell injury.

Topics: Aldehydes; Animals; Antineoplastic Agents; Aorta; Buthionine Sulfoximine; Cell Survival; Cells, Cultured; Chemoprevention; Dose-Response Relationship, Drug; Enzyme Induction; Glutathione; Glutathione Transferase; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Rats; Sulfasalazine; Thiones; Thiophenes

Extensive evidence suggests that reactive oxygen species are critically involved in the pathogenesis of cardiovascular diseases, such as atherosclerosis and myocardial ischemia-reperfusion injury. Consistent with this concept, administration of exogenous antioxidants has been shown to be protective against oxidative cardiovascular injury. However, whether induction of endogenous antioxidants by chemical inducers in vasculature also affords protection against oxidative vascular cell injury has not been extensively investigated. In this study, using rat aortic smooth muscle A10 cells as an in vitro system, we have studied the induction of cellular antioxidants by the unique chemoprotector, 3H-1,2-dithiole-3-thione [corrected] (D3T) and the protective effects of the D3T-induced cellular antioxidants against oxidative cell injury. Incubation of A10 cells with micromolar concentrations of D3T for 24 h resulted in a significant induction of a battery of cellular antioxidants in a concentration-dependent manner. These included reduced glutathione (GSH), GSH peroxidase, GSSG reductase, GSH S-transferase, superoxide dismutase, and catalase. To further examine the protective effects of the induced endogenous antioxidants against oxidative cell injury, A10 cells were pretreated with D3T and then exposed to either xanthine oxidase (XO)/xanthine, 4-hydroxynonenal, or cadmium. We observed that D3T pretreatment of A10 cells led to significant protection against the cytotoxicity induced by XO/xanthine, 4-hydroxynonenal or cadmium, as determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium reduction assay. Taken together, this study demonstrates for the first time that a number of endogenous antioxidants in vascular smooth muscle cells can be induced by exposure to D3T, and that this chemical induction of cellular antioxidants is accompanied by markedly increased resistance to oxidative vascular cell injury.

Topics: Aldehydes; Animals; Antioxidants; Cadmium; Catalase; Cell Line; Cell Survival; Cytoplasm; Cytoprotection; Dose-Response Relationship, Drug; Glutathione; Muscle, Smooth, Vascular; Oxidative Stress; Rats; Superoxide Dismutase; Thiones; Thiophenes; Xanthine Oxidase