Anti-apoptotic defense of bcl-2 gene against hydroperoxide-induced cytotoxicity together with suppressed lipid peroxidation,enhanced ascorbate uptake,and upregulated Bcl-2 protein

Although it is well known that Bcl-2 can prevent apoptosis, the Bcl-2's anti-apoptotic mechanism is not fully understood. Here, we investigate the mechanism of oxidant-induced cell death and to investigate the role of Bcl-2 in the tert-butyl hydroperoxide (t-BuOOH)-induced oxidant injury in Rat-1 fibroblasts and their bcl-2 transfected counterparts, b5 cells. Treatment with t-BuOOH causes mitochondrial disfunction and induced morphological features consistent with apoptosis more markedly in Rat-1 cells than in b5 cells. The hydroperoxide t-BuOOH at concentrations less than 100 nM for as long as 48 h or with higher concentrations (up to 100 microM) for only 3 h induces death in Rat-1 cells, whereas their bcl-2 transfectants were significantly resistant to cytotoxicity by both time and all concentration other than 100 microM. The similar results were obtained also for DNA strand cleavages as detected by TUNEL stain. The bcl-2 transfectants significantly suppressed t-BuOOH-induced increases in both lipid peroxidation and caspase-3 activation 3 and 1 h after t-BuOOH exposure, respectively, but failed to suppress either caspase-1 activation or an enhanced production of the intracellular reactive oxygen species (ROS). Intracellular uptake of [1-(14)C] ascorbic acid (Asc) into the bcl-2 transfectants was superior to that into the non-transfectants always under examined conditions regardless of serum addition to culture medium and cell density. Upregulation of Bcl-2 proteins was rapidly induced after t-BuOOH exposure in the transfectants, but not in non-transfectants, and restored till 24 h to the normal Bcl-2 level. Thus suppressions of both lipid peroxidation and the subsequent cell death events such as caspase-3 activation and DNA cleavage were concerned with the inhibitory effects of Bcl-2 on the t-BuOOH-induced cytotoxicity. And some of these events may correlate with Bcl-2 expression-induced partial enhanced anti-oxidant cellular ability including enrichment of intracellular Asc and oxidative stress-induced upregulation of Bcl-2 protein. On the other hand, ROS production and caspase-1 activation were not related to cytoprotection by Bcl-2.     

t-Butyl hydroperoxide and oxidized low density lipoprotein enhance phospholipid hydrolysis in lipopolysaccharide-stimulated retinal pericytes

Free radicals induced by organic peroxides or oxidized low density lipoprotein (oxLDL) play a critical role in the development of atherosclerosis. In investigating this process, and the concomitant inflammatory response, the role of pericytes, cells supporting the endothelial ones in blood vessels, has received little attention. In this study we tested the hypothesis that tert-butyl hydroperoxide (t-BuOOH) and oxLDL, administered in sublethal doses to the culture medium of retinal pericytes, function as prooxidant signals to increase the stimulation of the peroxidation process induced by lipopolysaccharide (LPS). Confluent cell monolayers were exposed to t-BuOOH (25-400 microM), native LDL or oxLDL (3.4-340 nmol hydroperoxides/mg protein, 1-100 micro). LPS (1 microg/ml), t-BuOOH (200 microM), and oxLDL (100 microM), but not native LDL, incubated for 24 h with cells, markedly increased lipid peroxidation, cytosolic phospholipase A2 (cPLA2) activity and arachidonic acid (AA) release in a time- and dose-dependent manner. AACOCF(3), a potent cPLA2 inhibitor, and the antioxidant alpha-tocopherol strongly inhibited the prooxidant-stimulated AA release. Long-term exposure to maximal concentrations of t-BuOOH (400 microM) or oxLDL (100 microM) had a sharp cytotoxic effect on the cells, described by morphological and biochemical indices. The presence of t-BuOOH or oxLDL at the same time, synergistically increased phospholipid hydrolysis induced by LPS alone. 400 microM t-BuOOH or 100 microM oxLDL had no significant effect on the stimulation of an apoptosis process estimated by DNA laddering and light and electron microscopy. The results indicate that (i) pericytes may be the target of extensive oxidative damage; (ii) activation of cPLA2 mediates AA liberation; (iii) as long-term regulatory signals, organic peroxide and specific constituents of oxLDL increase the pericyte ability to degrade membrane phospholipids mediated by LPS which was used, in the present study, to simulate in vitro an inflammatory burst in the retinal capillaries.     

Estrogenic activity of procymidone in rainbow trout (Oncorhynchus mykiss) hepatocytes: a possible mechanism of action

It is known that procymidone modifies sexual differentiation in vivo and in vitro, and that it induces vitellogenin (Vtg) synthesis in primary cultured rainbow trout hepatocytes. The aim of this study was to evaluate the mechanism underlying this latter in vitro estrogenic action. The cells were treated for 24 h with procymidone 150 microM (with 17beta-estradiol [E2] 20 microM as a positive control) combined with an estrogen receptor (ER) antagonist (tamoxifen 20 microM or ICI 182,780 1 microM) or, given the drug toxic action on the production of reactive oxygen species (ROS), a free radical scavenger (alpha-tocopherol 30 microM). The results from ELISA experiments provided evidence that procymidone Vtg-induction is inhibited by ER antagonists and by alpha-tocopherol suggesting that both ER and ROS are involved in this effect. The ROS detection revealed that the treatment with alpha-tocopherol and tamoxifen completely prevented ROS induction by procymidone, that was not inhibited by ICI 182,780. In exploring the mechanism mediating these events and its timing, we found that procymidone induced mitogen-activated protein kinase (MAPK) at 30 and 60 min, and that this effect was blocked by co-treatment with alpha-tocopherol. In summary, the results of the study clearly support the idea that the estrogenic activity of procymidone in primary cultured trout hepatocytes is mediated by ROS production, and that this activity is similar to that of the ligand-independent ER activation involving MAPK.     

The protective effect of ascorbic acid derivative on PC12 cells: involvement of its ROS scavenging ability

L-ascorbic acid 2-phosphate-6-palmitate (Asc2P6P) was synthesized and its effect on the damage of PC12 cells induced by H2O2 was investigated. 200 microM H2O2 in a treatment period of 4 hours in our experiment resulted in substantial cell loss. With the increasing concentration of antioxidants, such H2O2-induced cytotoxicity was significantly prevented and the corresponding intracellular and extracellular ROS levels decreased concurrently by pre-treatment with Asc2P6P and Asc. It was found that Asc2P6P was superior to L-ascorbic acid in its protective role and showed a dose-dependent manner during a 24-hour treatment. The higher potency of Asc2P6P's protective role on PC12 cells was correlated with its more effective ROS scavenging ability. HPLC assay demonstrated that Asc2P6P could easily enter the cells and be converted into Asc persistently, which contributed to its distinguished role in protecting PC12 cells against H2O2-induced cytotoxicity.     

Slow-down of age-dependent telomere shortening is executed in human skin keratinocytes by hormesis-like-effects of trace hydrogen peroxide or by anti-oxidative effects of pro-vitamin C in common concurrently with reduction of intracellular oxidative stress

The cellular life-span of cultivated human skin epidermis keratinocytes NHEK-F was shown to be extended up to 150% of population doubling levels (PDLs) by repetitive addition with two autooxidation-resistant derivatives of ascorbic acid (Asc), Asc-2-O-phosphate (Asc2P), and Asc-2-O-alpha-glucoside (Asc2G), respectively, but to be not extended with Asc itself. In contrast, hydrogen peroxide (H(2)O(2)) as dilute as 20 microM which was non-cytotoxic to the keratinocytes, or at 60 microM being marginally cytotoxic achieved the cellular longevity, unexpectedly, up to 160 and 120% of PDLs, respectively, being regarded as a hormesis-like stimulatory effect. The lifespan-extended cells that were administered with Asc2P, Asc2G, or 20 microM H(2)O(2) were prevented from senescence-induced symptoms such as PDL-dependent enlargement of a cell size of 14.7 microm finally up to 17.4 microm upon Hayflick's limit-called loss of proliferation ability as estimated with a channelizer, and retained young cell morphological aspects such as thick and compact shape and intense attachment to the culture substratum even upon advanced PDLs, whereas other non-extended cells looked like thin or fibrous shape and large size upon lower PDLs. The PDL-dependent shortening of telomeric DNA of 11.5 kb finally down to 9.12-8.10 kb upon Hayflick's limit was observed in common for each additive-given cells, but was decelerated in the following order: 20 microM H(2)O(2) > Asc2P = Asc2G > 60 microM H(2)O(2) > Asc = no additive, being in accord with the order of cell longevity. Intracellular reactive oxygen species (ROS) was diminished by Asc2P, Asc2G or 20 microM H(2)O(2), but not significantly by Asc or 60 microM H(2)O(2) as estimated by fluorometry using the redox indicator dye CDCFH. There was no appreciable difference among NHEK keratinocytes that were administered with or without diverse additives in terms of telomerase activity per cell, which was 1.40 x 10(4)-4.48 x 10(4) times lower for the keratinocytes than for HeLa cells which were examined as the typical tumor cells. Thus longevity of the keratinocytes was suggested to be achieved by slowdown of age-dependent shortening of telomeric DNA rather than by telomerase; telomeres may suffer from less DNA lesions due to the continuous and thorough repression of intracellular ROS, which was realized either by pro-vitamin C such as Asc2P or Asc2G that exerted an antioxidant ability more persistent than Asc itself or by 20 microM H(2)O(2) which diminished intracellular ROS assumedly through a hormesis-like effect.     

Overexpression of aldehyde dehydrogenase-2 (ALDH2) transgene prevents acetaldehyde-induced cell injury in human umbilical vein endothelial cells: role of ERK and p38 mitogen-activated protein kinase

Acetaldehyde, the major ethanol metabolite that is far more toxic and reactive than ethanol, has been postulated to be responsible for alcohol-induced tissue and cell injury. This study was to examine whether facilitated acetaldehyde metabolism affects acetaldehyde-induced oxidative stress and apoptosis. Transgene-encoding human aldehyde dehydrogenase-2 (ALDH2), which converts acetaldehyde into acetate, was constructed under chicken beta-actin promoter and transfected into human umbilical vein endothelial cells (HUVECs). Efficacy of ALDH2 transfection was verified using green fluorescent protein and ALDH2 enzymatic assay. Generation of reactive oxygen species (ROS) was measured using chloromethyl-2',7'-dichlorodihydrofluorescein diacetate. Apoptosis was evaluated by 4',6'-diamidino-2'-phenylindoladihydrochloride fluorescence microscopy, quantitative DNA fragmentation, and caspase-3 assay. Acetaldehyde (0-200 microm) elicited ROS generation and apoptosis in HUVECs in a time- and concentration-dependent manner, associated with activation of the stress signal molecules ERK1/2 and p38 mitogen-activated protein (MAP) kinase. A close liner correlation was observed between the acetaldehyde-induced ROS generation and apoptosis. Interestingly, the acetaldehyde-induced ROS generation, apoptosis, activation of ERK1/2, and p38 MAP kinase were prevented by the ALDH2 transgene or antioxidant alpha-tocopherol. The involvement of ERK1/2 and p38 MAP kinase in acetaldehyde-induced apoptosis was confirmed by selective kinase inhibitors U0126, SB203580, and SB202190. Collectively, our data revealed that facilitation of acetaldehyde metabolism by ALDH2 transgene overexpression may prevent acetaldehyde-induced cell injury and activation of stress signals. These results indicated therapeutic potential of ALDH2 enzyme in the prevention and detoxification of acetaldehyde or alcohol-induced cell injury.     

On the cytotoxicity and status of oxidative stress of two novel synthesized tri-aza macrocyclic diamides as studied in the V79 cell lines

Two tri-aza macrocycles as diamide derivatives of macrocyclic compounds possess a hydrophilic cavity surrounded by hydrophobic ring, which enables them to diffuse cell membrane and interfere with different living systems. In this study, we comparatively evaluated cytotoxicity effects of tri-aza dibenzo sulfoxide (TSD) and dibenzo sulfide (TTS) macrocyclic diamides in a range of doses (0.5-8mM) and the role of oxidative stress in V79 cell culture. We assessed the effects of these substances on ROS level, cellular viability, apoptosis events, activity of antioxidant enzymes including superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT), and on some macromolecules' oxidative damage end-products: malondialdehyde (MDA), dityrosine, and 8-hydroxy-deoxyguanosine (8-OH-dG) that were assessed by spectrometry and HPLC methods. Both compounds revealed cytotoxicity effects on cell culture particularly at doses >1mM after 24-h incubation. They decreased cellular viability and significantly promoted ROS generation, increased enzyme activities, and enhanced oxidative damages in which TSD was more effective. Treatment of cells with each compound alone increased significantly the percent of apoptotic events at 2 and then 4mM. Co-treatment with alpha-tocopherol (alpha-TCP) drastically reduced these events. Cells' exposure with mixture of 30 microM alpha-tocopherol and 8mM of each compound exerted significant decrease in the levels of ROS, enzyme activities, and oxidative damage biomarkers. As conclusion, our study documented the oxidative radical forming ability of the studied compounds and further strengthened the documentation of their cytotoxicity effects through lipids, proteins and DNA oxidation damages.     

Involvement of NADPH oxidase 1 in UVB-induced cell signaling and cytotoxicity in human keratinocytes

Members of NADPH oxidase (Nox) enzyme family are important sources of reactive oxygen species (ROS) and are known to be involved in several physiological functions in response to various stimuli including UV irradiation. UVB-induced ROS have been associated with inflammation, cytotoxicity, cell death, or DNA damage in human keratinocytes. However, the source and the role of UVB-induced ROS remain undefined.Here, we show that Nox1 is involved in UVB-induced p38/MAPK activation and cytotoxicity via ROS generation in keratinocytes. Nox1 knockdown or inhibitor decreased UVB-induced ROS production in human keratinocytes. Nox1 knockdown impaired UVB-induced p38 activation, accompanied by reduced IL-6 levels and attenuated cell toxicity. Treatment of cells with N-acetyl-L-cysteine (NAC), a potent ROS scavenger, suppressed p38 activation as well as consequent IL-6 production and cytotoxicity in response to UVB exposure. p38 inhibitor also suppressed UVB-induced IL-6 production and cytotoxicity. Furthermore, the blockade of IL-6 production by IL-6 neutralizing antibody reduced UVB-induced cell toxicity.In vivo assay using wild-type mice, the intradermal injection of lysates from UVB-irradiated control cells, but not from UVB-irradiated Nox1 knockdown cells, induced inflammatory swelling and IL-6 production in the skin of ears. Moreover, administration of Nox1 inhibitor suppressed UVB-induced increase in IL-6 mRNA expression in mice skin.Collectively, these data suggest that Nox1-mediated ROS production is required for UVB-induced cytotoxicity and inflammation through p38 activation and inflammatory cytokine production, such as IL-6. Thus, our findings suggest Nox1 as a therapeutic target for cytotoxicity and inflammation in response to UVB exposure.     

Oxidative damage to DNA and replicative lifespan in cultured adrenocortical cells

Oxidative damage to DNA in cultured bovine adrenocortical cells was investigated by exposing cells to a sublethal concentration (10 microM) of cumene hydroperoxide under conditions previously shown to be deficient in the biological antioxidants selenium and alpha-tocopherol (vitamin E). DNA prepared from cells incubated for 4 h with 10 microM cumene hydroperoxide had a greater fraction showing resistance to S1 nuclease after denaturation and reassociation to a log C0t of -3. Cross-linking by cumene hydroperoxide was abolished in cells that had been grown in the presence of 20 nM selenite or 1 microM alpha-tocopherol for 96 h prior to peroxide addition, whereas such cells remained susceptible to cross-linking by nitrogen mustard. Extensive strand breaks in DNA from peroxide-treated cells as assessed by alkaline sucrose gradient centrifugation were greatly reduced in cells grown in selenite or alpha-tocopherol. Despite the evidence of damage to DNA, cumene hydroperoxide was not detectably mutagenic, in contrast to 5 microM methylnitronitrosoguanidine (MNNG), when assessed as the incidence of resistance to 25 microM ouabain. We confirmed that cumene hydroperoxide at greater than 10 microM lowers cloning efficiency and that this is largely prevented by selenite or alpha-tocopherol. Additionally, selenite or alpha-tocopherol produced increased clonogenicity in cells not incubated with peroxide. To examine effects of the biological antioxidants on replicative lifespan, cells were grown continuously in fetal bovine serum (FBS), fibroblast growth factor (FGF), and selenite or alpha-tocopherol. Selenium increased replicative lifespan by 10-20% and alpha-tocopherol by 22-30%. Levels of DNA cross-links and strand breaks did not differ under any circumstances between early (second) passage and late (30th) passage cells. The experiments on replicative potential were all performed in the presence of FGF. When FGF was omitted from the culture medium, replicative lifespan was reduced by 85%. We conclude that types of damage to DNA resulting from peroxide exposure are not present in cells under standard culture conditions at early or late stages of the lifespan. Other work has noted a relationship between clonogenicity and replicative lifespan; thus, the increase in cloning efficiency seen with selenium and alpha-tocopherol may cause the observed slight increase in replicative lifespan. Oxidative damage does not appear to be a major determinant of cellular senescence in adrenocortical cells.     

Coenzyme Q cytoprotective mechanisms for mitochondrial complex I cytopathies involves NAD(P)H: quinone oxidoreductase 1(NQO1)

The commonest mitochondrial diseases are probably those impairing the function of complex I of the respiratory electron transport chain. Such complex I impairment may contribute to various neurodegenerative disorders e.g. Parkinson's disease. In the following, using hepatocytes as a model cell, we have shown for the first time that the cytotoxicity caused by complex I inhibition by rotenone but not that caused by complex III inhibition by antimycin can be prevented by coenzyme Q (CoQ1) or menadione. Furthermore, complex I inhibitor cytotoxicity was associated with the collapse of the mitochondrial membrane potential and reactive oxygen species (ROS) formation. ROS scavengers or inhibitors of the mitochondrial permeability transition prevented cytotoxicity. The CoQ1 cytoprotective mechanism required CoQ1 reduction by DT-diaphorase (NQO1). Furthermore, the mitochondrial membrane potential and ATP levels were restored at low CoQ1 concentrations (5 microM). This suggests that the CoQ1H2 formed by NQO1 reduced complex III and acted as an electron bypass of the rotenone block. However cytoprotection still occurred at higher CoQ1 concentrations (>10 microM), which were less effective at restoring ATP levels but readily restored the cellular cytosolic redox potential (i.e. lactate: pyruvate ratio) and prevented ROS formation. This suggests that CoQ1 or menadione cytoprotection also involves the NQO1 catalysed reoxidation of NADH that accumulates as a result of complex I inhibition. The CoQ1H2 formed would then also act as a ROS scavenger.     

Bradykinin induces mitochondrial ROS generation via NO,cGMP, PKG,and mitoKATP channel opening and leads to cardioprotection

Bradykinin (BK) mimics ischemic preconditioning by generating reactive oxygen species (ROS). To identify intermediate steps that lead to ROS generation, rabbit cardiomyocytes were incubated in reduced MitoTracker Red stain, which becomes fluorescent after exposure to ROS. Fluorescence intensity in treated cells was expressed as a percentage of that in paired, untreated cells. BK (500 nM) caused a 51 +/- 16% increase in ROS generation (P < 0.001). Coincubation with either the BK B2-receptor blocker HOE-140 (5 microM) or the free radical scavenger N-(2-mercaptopropionyl)glycine (1 mM) prevented this increase, which confirms that the response was receptor mediated and ROS were actually being measured. Closing mitochondrial ATP-sensitive K+ (mitoKATP) channels with 5-hydroxydecanoate (5-HD, 1 mM) prevented increased ROS generation. BK-induced ROS generation was blocked by Nomega-nitro-m-arginine methyl ester (m-NAME, 200 microM), which implicates nitric oxide as an intermediate. Blockade of guanylyl cyclase with 1-H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 10 microM) aborted BK-induced ROS generation but not that from diazoxide, a direct opener of mitoKATP channels. The protein kinase G (PKG) blocker 8-bromoguanosine-3',5'-cyclic monophosphorothioate (25 microM) eliminated the effects of BK. Conversely, direct activation of PKG with 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (100 microM) increased ROS generation (39 +/- 15%; P < 0.004) similar to BK. This increase was blocked by 5-HD. Finally, the nitric oxide donor S-nitroso-N-acetylpenicillamine (1 microM) increased ROS by 34 +/- 6%. This increase was also blocked by 5-HD. In intact rabbit hearts, BK (400 nM) decreased infarction from 30.5 +/- 3.0 of the risk zone in control hearts to 11.9 +/- 1.4% (P < 0.01). This protection was aborted by either 200 microM m-NAME or 2 microM ODQ (35.4 +/- 5.7 and 30.4 +/- 3.0% infarction, respectively; P = not significant vs. control). Hence, BK preconditions through receptor-mediated production of nitric oxide, which activates guanylyl cyclase. The resulting cGMP activates PKG, which opens mitoKATP. Subsequent release of ROS triggers cardioprotection.     

Cytoprotection of vascular endotheliocytes by phosphorylated ascorbate through suppression of oxidative stress that is generated immediately after post-anoxic reoxygenation or with alkylhydroperoxides

Vascular endotheliocytes BAE-2 underwent the gradually proceeding cell death until 48 h after reoxygenation (Reox) following 3 h anoxia (Anox), but protected by pre-Anox administration with L-ascorbic acid (Asc)-2-O-phosphate (Asc2P), an autooxidation-resistant Asc derivative, but not by Asc itself. This cytoprotection with Asc2P was achieved in a glucose (Glc)-lacking buffer more advantageously than in a Glc-containing buffer where less efficiency had been demonstrated for Asc entry into BAE-2 cells than in a Glc-lacking buffer. Superoxide anion radicals were detected explosively in the extracellular space at 2-5 min after Reox following the Anox treatment of HUVE endotheliocytes, and were thereafter retained at levels as high as approximately one-half of the maximum level until 60 min after Reox, as shown by cytochrome c reduction assay. Superoxide anions at 3 and 60 min after Reox were suppressed by pre-Anox administration with Asc2P, but not with Asc or dehydro-Asc, and were not suppressed by post-Anox administration with Asc2P; the cytoprotection may need the intracellular accumulation of the ROS-scavenging effector Asc that is converted from Asc2P until 3 min after Reox. The ROS-generator tert-butylhydroperoxide (t-BuOOH) also induced both the diminished cell viability and nuclear DNA strand cleavages of BAE-2 endotheliocytes, which were also protected dose-dependently with Asc2P. The cytoprotection was attributed to reduction of intracellular ROS including hydroperoxide and hydrogen peroxide with Asc2P as shown by fluorometry with the redox indicator CDCFH-DA. Thus Anox/Reox-induced cell death can be prevented by Asc2P that suppresses ROS-generation immediately after Reox following Anox more efficiently in the intracellular sphere rather than in the extracellular space.     

Endosulfan decreases cell growth and apoptosis in human HaCaT keratinocytes: partial ROS-dependent ERK1/2 mechanism

Endosulfan is an organochlorine insecticide described as a potential carcinogen in humans. This insecticide was recently reported to alter the mitogen-activated protein (MAP) kinase signaling pathways and is suspected to affect cell growth and differentiation in human keratinocytes. This study was designed to assess the mitogenic, apoptogenic, and genotoxic effects of endosulfan on the HaCaT cell line. We first found that 25 microM endosulfan led to persistent extracellular signal-regulated kinase (ERK)1/2 phosphorylation with an accumulation of the phosphorylated form in the nucleus, probably caused by MAP kinase phosphatase (MKP) inhibition. As previously described under sustained ERK1/2 activation, cell growth was decreased: delayed confluency and 35% decrease of BrdU incorporation was demonstrated in endosulfan-treated keratinocytes. In addition, endosulfan has been shown to generate transient reactive oxygen species (ROS), and blocking this oxidative stress by N-acetyl cysteine (NAC) strongly prevented both persistent nuclear ERK1/2 phosphorylation and cell growth decrease. Additional experiments demonstrated that unchanged endosulfan rather than its metabolites has mutagenic effects (Ames positive without S9) and increased DNA strand breaks (Comet assay) in HaCaT cells, via a ROS-dependent mechanism. Therefore, to assess the putative pro-apoptotic response of damaged cells, caspases 3/7 activity and poly(ADP-ribose)-polymerase (PARP) cleavage were measured. The results clearly indicated that endosulfan inhibited both spontaneous and staurosporine-induced apoptosis. Taken together, these findings strongly support that endosulfan induces ROS generation leading to sustained ERK1/2 phosphorylation and decrease in cell growth. Moreover, endosulfan was found to inhibit apoptosis and this could contribute to mutant cell survival and therefore have possible carcinogenic effects.     

Cadmium-dependent generation of reactive oxygen species and mitochondrial DNA breaks in photosynthetic and non-photosynthetic strains of Euglena gracilis

The photosynthetic strain Z of Euglena gracilis is more susceptible to cadmium chloride (Cd) than the non-photosynthetic strain SMZ. We investigated the correlation of intracellular reactive oxygen species (ROS) levels with Cd-induced cellular damage. Flow cytometry with dihydrorhodamine 123 showed that strain Z generated higher levels of ROS, probably H(2)O(2) and/or ONOO(-), than strain SMZ, and that this difference between the two strains became more pronounced with increasing Cd dose. The levels of ROS increased at cytotoxic concentrations of Cd, at over 10 microM Cd for Z and 50 microM Cd for SMZ. These results show an association of Cd cytotoxicity with ROS generation. Considering that strain SMZ is non-photosynthetic, the higher levels of ROS in strain Z might be due to blockage of photosynthetic electron flow by Cd. Using terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labeling analysis in combination with 4',6-diamidino-2-phenylindole, dihydrochloride staining, we observed DNA breaks in the mitochondria of both strains after Cd exposure. The results suggest that the mitochondrion is the primary target organelle of Cd in E. gracilis cells.     

Altered mitochondrial function and overgeneration of reactive oxygen species precede the induction of apoptosis by 1-O-octadecyl-2-methyl-rac-glycero-3-phosphocholine in p53-defective hepatocytes.

The mechanism of induction of apoptosis by the novel anti-cancer drug 1-O-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) was investigated in p53-defective SV40 immortalized rat hepatocytes (CWSV1). Exposure to 12 microM ET-18-OCH3 for 36 h induced apoptosis as determined using classical morphological features and agarose gel electrophoresis of genomic DNA. Increased levels of reactive oxygen species (ROS) were detected spectrophotometrically using a nitroblue tetrazolium (NBT) assay in cells treated with ET-18-OCH3. Both the increased generation of ROS and the induction of apoptosis were inhibited when cells were treated concurrently with ET-18-OCH3 in the presence of the antioxidant alpha-tocopherol. Similar results were achieved when cells were switched acutely to choline-deficient (CD) medium in the presence of the antioxidant. The possible role of mitochondria in the generation of ROS was investigated. Both ET-18-OCH3 and CD decreased the phosphatidylcholine (PC) content of mitochondrial and associated membranes, which correlated with depolarization of the mitochondrial membrane as analyzed using 5,5',6,6'-tetramethylbenzimidazolcarbocyanine iodide (JC-1), a sensitive probe of mitochondrial membrane potential. Rotenone, an inhibitor of the mitochondrial electron transport chain, significantly reduced the intracellular level of ROS and prevented mitochondrial membrane depolarization, correlating with a reduction of apoptosis in response to either ET-18-OCH3 or CD. Taken together, these results suggest that the form of p53-independent apoptosis induced by ET-18-OCH3 is mediated by alterations in mitochondrial membrane PC, a loss of mitochondrial membrane potential, and the release of ROS, resulting in completion of apoptosis.     

Differential effects of glutathione depletion and metallothionein induction on the induction of DNA single-strand breaks and cytotoxicity by tert-butyl hydroperoxide in cultured mammalian cells

Induction of DNA single-strand breaks (ssb), their resealing and cytotoxicity by tert-butyl hydroperoxide (t-BuOOH) were investigated in cultured Chinese hamster V79 cells. The effect of the depletion of cellular glutathione (GSH), iron chelation and induction of metallothionein (MT) on these parameters was studied. t-BuOOH in a concentration range of 0.02-0.5 mM induced DNA ssb in a dose-dependent fashion. Strand breakage increased as a function of time up to 1 h. Divalent iron chelator o-phenanthroline suppressed markedly the induction of DNA ssb while the trivalent iron chelator desferrioxamine had no effect. GSH-depletion increased cytotoxicity of t-BuOOH. In contrast, the depletion of GSH did not affect the efficiency of formation of DNA ssb by t-BuOOH and the rate of resealing of the DNA damage. The induction of MT did not influence the efficiency of formation of DNA ssb by t-BuOOH. In summary, while GSH depletion and MT induction affected the formation of DNA ssb and cytotoxicity differently divalent iron was required for both. Therefore, appears likely that DNA breakage and cytotoxicity by t-BuOOH are caused by independent mechanisms.     

Flow cytometric measurement of lipid peroxidation in vital cells using parinaric acid.

A method for measuring lipid peroxidation using time resolved flow cytometry is described. Because of its chemical nature, the naturally fluorescent fatty acid cis-parinaric acid is readily consumed in lipid peroxidation reactions. It could be loaded into Chinese hamster ovary cells in a time and concentration dependent manner at 37 degrees C, with 5 microM for 60' giving consistent, bright fluorescence without evidence of cytotoxicity. Examination of cells by fluorescence microscopy showed diffuse staining of surface and internal membranes. Cells were maintained at 37 degrees C while being examined in an Epics Elite flow cytometer equipped with a 325 nm HeCd laser, and parinaric acid fluorescence at 405 nm was measured over time. Addition of the oxidant tert-butyl hydroperoxide resulted in a burst of intracellular oxidation, shown by simultaneously loading the cells with dichlorofluorescein, and loss of parinaric fluorescence over time. This was followed by cell death, indicated by loss of forward light scatter and uptake of propidium iodide. Pretreatment of the cells with the antioxidant alpha-tocopherol, 200 microM, reduced the rate of loss of parinaric acid fluorescence and delayed the onset of cell death. Simultaneous biochemical determination of the lipid peroxidation breakdown product malondialdehyde confirmed a close temporal relationship with loss of parinaric acid fluorescence, both with and without alpha-tocopherol pretreatment and suggested that the flow cytometric assay for lipid peroxidation is of comparable sensitivity. The mitochondrial stain dodecyl acridine orange and the cyanine dye DiOC(6)3 were combined with cis-parinaric acid staining and could be excited by the latter using resonance energy transfer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of albumin dimers induced by exposure to peroxides in human plasma: a possible biomarker for oxidative stress

Human serum albumin (HSA) has one free thiol residue at Cys-34 that is likely oxidized by various reactive oxygen species (ROS). We attempted to identify the oxidation product of Cys-34 of HSA following exposure of plasma to ROS. Oxidation induced by tert-butyl hydroperoxide (t-BuOOH) of this free cysteine residue in HSA was observed in detail. Analysis of oxidized albumin in a partially purified fraction obtained by affinity column chromatography clearly revealed the formation of albumin disulfide dimers following t-BuOOH exposure. Albumin disulfide dimer formation was observed in normal plasma following treatment with various peroxides, as well as in untreated plasma from patients on hemodialysis using SDS-PAGE and Western blot analysis. The present results indicate that albumin dimers are oxidative products derived from peroxides, and that their presence in plasma might be a marker of oxidative stress as secondary metabolites of peroxidation.