Induction of Apoptosis by Pyrrolidinedithiocarbamate and N-Acetylcysteine in Vascular Smooth Muscle Cells (original) (raw)
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Basic Research in Cardiology, 2001
Oxidation of lipids is considered a key feature of atherogenesis. Lipid peroxidation products such as oxidized LDL or the bioactive aldehyde 4-hydroxynonenal (HNE) exert mitogenic effects on vascular smooth muscle cells (VSMC). These effects appear to be concentration-dependent since in addition to our previous reports on growth promotion at lower concentrations we here indicate induction of apoptosis in VSMC by 4-hydroxynonenal (HNE) at higher concentrations (100 µmol/L). In a line with HNE's previously documented effects on key mitogenic signaling elements, we also report on activation by this aldehyde of the redox-sensitive transcription factor NF-B, a key regulator of apoptosis: HNE (1.0 µmol/L) induced DNA-binding of NF-B in VSMC. The effect was inhibited by antioxidants, N-acetylcysteine and pyrrolidine dithio-carbamate. HNE caused phosphorylation but not degradation of the inhibitory subunit IB-␣. HNE itself acts as an oxidant as was investigated with measurements of 8-isoprostane which ranks among the most valuable available biomarkers of lipid peroxidation: HNE (1.0 µmol/L) increased 8-isoprostane levels in VSMC by 4.5-fold (p < 0.05). Compared to the controls, plasma samples from apoE null mice exhibited elevated levels of 8-isoprostane (40 pg/mL, 3.2-fold increase) and the combined aldehydes HNE and malonaldehyde (1.5 µmol/L, 2.5-fold increase), (p < 0.05, resp). In addition, immunohistochemistry indicated the presence of HNE-protein adducts in atheroscerlotic lesions of apoE null mice. Thus HNE is present in atherosclerotic tissue at concentrations that are bioactive in vitro. The data further indicate the involvement of the lipid peroxidation product HNE in atherogenesis.
Journal of Applied Toxicology, 2008
N-nitrosopiperidine (NPIP) and N-nitrosodibutylamine (NDBA) belong to a group of N-nitrosamines that are widely distributed in foodstuffs and the occupational environment. In the present study, the human promyelocytic leukemia cell line HL-60, was used to characterize the apoptotic effects of N-nitrosamines, and to examine the production of reactive oxygen species (ROS). Apoptotic cells were identified by (i) chromatin condensation (ii) flow cytometry analysis and (iii) poly(ADP-ribose) polymerase (PARP) cleavage. NPIP and NDBA induced morphological changes consistent with apoptotic events in HL-60 cells. Flow cytometry analysis showed that both N-nitrosamines induced apoptotic cell death in a concentration and time dependent-manner. It was observed that NDBA was stronger than NPIP, since it induced a significant apoptotic cell death after 18 h starting from a concentration of 2 mm, whereas NPIP was effective at 10 mm. Furthermore, PARP was markedly cleaved with 0.5 mm of NDBA and 5 mm of NPIP after treatments for 3 and 18 h, respectively. Finally, the ROS level was found to be elevated after 0.5 h of treatment with both N-nitrosamines. Antioxidant N-acetylcysteine (NAC) completely inhibited the ROS production induced by NPIP and NDBA. However, this action seems not to be associated with the apoptosis because NAC did not block N-nitrosamines-induced apoptosis. The data demonstrate that NPIP and NDBA induce apoptosis and ROS production in HL-60 cells. Copyright © 2007 John Wiley & Sons, Ltd.
NeuroToxicology, 2007
In order to specifically elucidate the involvement of oxidative stress, the effects of various types of stressors and antioxidants on PC12 cells were examined. In this study, the following four stressors were studied in detail: free radicals generated from 2,2 0 -azobis(2-amidinopropane) dihydrochloride (AAPH), 4-hydroxynonenal (HNE), 7-ketocholesterol (KC), and arsenic trioxide (As 2 O 3 ). Undifferentiated PC12 cells were treated with 50% lethal concentration (LC 50 ) of these stressors, and subsequently the viability, apoptosis/necrosis ratio, reactive oxygen species (ROS) production, caspase-3 activity, and protection by antioxidants were measured to elucidate the underlying mechanisms that determine the action of these stressors on PC12 cells. The cytotoxicity did not correlate directly with the intracellular formation of ROS. For example, as compared to AAPH, As 2 O 3 produced considerably smaller amounts of ROS at LC 50 . As observed in the cells incubated with As 2 O 3 , KC and HNE exerted cell toxicity, but with a moderate production of ROS. With the exception of HNE, the apoptosis/necrosis ratio of all the stressors evaluated by annexin Vand propidium iodide assays increased with an increase in the incubation time at the LC 50 values of these stressors. In accordance with apoptosis ratio, caspase activity was detected in the cells incubated with AAPH, As 2 O 3 , and KC, but not HNE at LC 50 for 24 h. The protective effect of a-tocopherol, 17b-estradiol, 2,3-dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butylbenzofuran (BO653), glutathione, and N-acetylcysteine (NAC) against cytotoxicity depended on the type of stressors. These antioxidants were found to be effective against the abovementioned stressors, except As 2 O 3 against which only NAC was effective. These results suggest that the involvement of ROS and the protective effect of antioxidants depend on the type of stressors. #
Journal of Biological Chemistry, 2001
We have investigated the ability of intracellular vitamin C to protect human umbilical vein endothelial cells from exposure to hypochlorous acid (HOCl) and a range of derived chloramines. Ascorbate provided minimal protection against the cytotoxicity induced by these oxidants, as measured by propidium iodide uptake. In contrast, there was a marked effect on apoptosis, monitored by caspase-3 activation and phosphatidylserine exposure. Extended incubation of the cells with glycine chloramine or histamine chloramine completely blocked apoptosis initiated in the cells by serum withdrawal. This effect was significantly abrogated by ascorbate. Inhibition of apoptosis required the oxidant to be present for an extended period after serum withdrawal and occurred prior to caspase-3 activation. General protection of thiols by ascorbate was not responsible for the protection of apoptosis, because intracellular oxidation by HOCl or chloramines was not prevented in supplemented cells. The results...
Oxidative stress–induced apoptosis of endothelial cells
Free Radical Biology and Medicine, 2000
Endothelial cells (ECs) are subjected to oxidative stress during many pathological processes, including ischemia/reperfusion and general inflammation. In the present study, we examined the effects of oxidative stress on rates of apoptosis in EC cultures. We treated large and microvessel ECs with menadione for 1 h in vitro to simulate the most common physiological form of oxidative stress, exposure to O 2 •Ϫ. Capillary ECs were resistant to menadione-induced apoptosis when compared with large-vessel ECs. Treatment with 35 M menadione resulted in an apoptotic rate of ϳ5% in capillary EC cultures compared with ϳ45% in large-vessel EC cultures. At higher concentrations of menadione (35-75 M), both types of ECs exhibited a concentration-related increase in apoptosis. Necrotic cell death only became evident at menadione concentrations ranging from 75-100 M for both cell types. The timing of the apoptotic response to a 1 h menadione exposure was very specific. For both EC types, peaks of apoptosis occurred in two distinct waves, at 6-8 and 18-22 h after treatment. Analysis of the events leading up to the first peak of apoptosis indicated that specific matrix metalloproteinases (MMPs) were activated, suggesting that MMPs may be involved in initiating the apoptotic process.
Inhibition of Acrolein-Induced Apoptosis by the Antioxidant N-Acetylcysteine
Journal of Pharmacology and Experimental Therapeutics, 2007
Acrolein is a highly electrophilic alpha,beta-unsaturated aldehyde to which humans are exposed in many situations. It is an environmental pollutant that is responsible for multiple respiratory diseases and has been implicated in neurodegenerative diseases such as Alzheimer&amp;amp;amp;amp;amp;#39;s disease. The hypothesis of the study is that the antioxidant N-acetylcysteine (NAC), a precursor of glutathione, could protect cells against acrolein-induced apoptosis. Exposure of Chinese hamster ovary cells to a noncytotoxic dose of acrolein (4 fmol/cell) depleted intracellular glutathione to 45% of initial levels. NAC, which increased intracellular glutathione levels by 30%, afforded protection against acrolein-induced cytotoxicity (loss of cell proliferation) and apoptosis. NAC protected against apoptosis by diminishing acrolein-induced activation of the mitochondrial death pathway. NAC inhibited acrolein-induced Bad translocation from the cytosol to the mitochondria, as well as Bcl-2 translocation from mitochondria to the cytosol, as evaluated by Western blot analysis. However, NAC had no effect on acrolein-induced Bax translocation to mitochondria and cytochrome c liberation into the cytosol. Meanwhile, NAC inhibited depolarization of mitochondrial membrane potential, as evaluated by rhodamine fluorescence using flow cytometry. NAC also inhibited procaspase-9 processing, activation of enzymatic activity of caspase-9, -7, and -8, and poly(ADP-ribose) polymerase cleavage induced by acrolein. Inhibition of acrolein-induced apoptosis using NAC was confirmed morphologically by diminished condensation of nuclear chromatin, as evaluated by fluorescence microscopy. These findings suggest that NAC could be potentially useful as a protective agent for people exposed to acrolein.
Arteriosclerosis, Thrombosis, and Vascular Biology, 1999
Vascular cell death is a key feature of atherosclerotic lesions and may contribute to the plaque "necrotic" core, cap rupture, and thrombosis. Oxidatively modified low-density lipoproteins (LDLs) are implicated in the pathogenesis of atherosclerosis, and dietary antioxidants are thought to protect the vasculature against LDL-induced cytotoxicity. Because LDL oxidative modification may vary within atherosclerotic lesions, we examined the effects of defined, oxidatively modified LDL species on human arterial smooth muscle cell apoptosis and the cytoprotective effects of vitamin C. Moderately oxidized LDL (0 to 300 g protein/mL), which has the highest content of lipid hydroperoxides, induced smooth muscle cell apoptosis within 6 hours, whereas native LDL and mildly and highly oxidized LDL had no effect. Moderately oxidized LDL increased cellular DNA fragmentation, release of fragmented DNA into the culture medium, and annexin V binding and decreased mitochondrial dehydrogenase activity and expression of the antiapoptotic mediator Bcl-x L . Treatment of cells with native LDL together with the lipid hydroperoxide 13(S)-hydroperoxyoctadeca-9Z,11E-dienoic acid (HPODE, 200 mol/L, 6 to 24 hours) also induced apoptotic cell death. Pretreatment of smooth muscle cells with vitamin C (0 to 100 mol/L, 24 hours) attenuated the cytotoxicity and apoptosis induced by both moderately oxidized LDL and HPODE. Our findings suggest that moderately oxidized LDL, with its high lipid hydroperoxide content, rather than mildly or highly oxidized LDL, causes apoptosis of human smooth muscle cells and that vitamin C supplementation may provide protection against plaque instability in advanced atherosclerosis.