Hydrogen Peroxide-induced Cytotoxicity Research Articles

Effects of naringin on hydrogen peroxide-induced cytotoxicity and apoptosis in P388 cells.

Flavonoids are widely recognized as naturally occurring antioxidants. Naringin (NG) is one of the flavonoid components in citrus fruits such as grapefruit. Hydrogen peroxide (H2O2) causes cytotoxicity through oxidative stress and apoptosis. In this paper, we examined the effects of NG on H2O2-induced cytotoxicity and apoptosis in mouse leukemia P388 cells. Cytotoxicity was determined by mitochondrial activity (MTT assay). Apoptosis and DNA damage were analyzed by measuring chromatin condensation and Comet assay (alkaline single cell gel electrophoresis), respectively. H2O2-induced cytotoxicity was significantly attenuated by NG or the reduced form of glutathione (GSH), a typical intracellular antioxidant. NG suppressed chromatin condensation and DNA damage induced by H2O2. These results indicate that NG from natural products is a useful drug having antioxidant and anti-apoptopic properties.

Effects of Naringin on Hydrogen Peroxide-Induced Cytotoxicity and Apoptosis in P388 Cells

Effects of Naringin on Hydrogen Peroxide-Induced Cytotoxicity and Apoptosis in P388 Cells

Protective effects of resveratrol on hydrogen peroxide-induced apoptosis in rat pheochromocytoma (PC12) cells

Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities. One of the plausible ways to prevent the reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical augmentation of endogenous antioxidant defense capacity. Resveratrol (3,5,4′-trihydroxy- trans-stilbene), one of the major antioxidative constituents found in the skin of grapes, has been considered to be responsible in part for the protective effects of red wine consumption against coronary heart disease (‘French Pardox’). In this study, we have investigated the effects of resveratrol on hydrogen peroxide-induced oxidative stress and apoptotic death in cultured rat pheochromocytoma (PC12) cells. PC12 cells treated with hydrogen peroxide underwent apoptotic death as determined by characteristic morphological features, internucleosomal DNA fragmentation and positive in situ end-labeling by terminal transferase (TUNEL staining). Resveratrol pretreatment attenuated hydrogen peroxide-induced cytotoxicity, DNA fragmentation, and intracellular accumulation of ROS. Hydrogen peroxide transiently induced activation of NF-κB in PC12 cells, which was mitigated by resveratrol pretreatment. These results suggest that resveratrol has the potential to prevent oxidative stress-induced cell death.

Indian medicinal plants as antiradicals and DNA cleavage protectors

Celastrus paniculatus L. (Celastraceae) (CP), Picrorhiza kurroa L. (Scrophulariaceae) (PK) and Withania somnifera L. (Solanaceae) (WS) are Indian medicinal plants having a remarkable reputation, as a factor of health care, among the indigenous medical practitioners. The plants exhibit varying degrees of therapeutic value some of which useful in the treatment of cognitive dysfunction, epilepsy, insomnia, rheumatism, gout, dyspepsia. In this work, we have investigated the free radical scavenging capacity of methanolic extracts from CP, PK, WS and the effect on DNA cleavage induced by H2O2 UV-photholysis. In addition, we investigated whether these plant extracts are capable of reducing the hydrogen peroxide-induced cytotoxicity and DNA damage in human non-immortalized fibroblasts. These extracts showed a dose-dependent free radical scavenging capacity and a protective effect on DNA cleavage; methanolic extracts from PK was more active than extracts from CP and WS. These results were confirmed by a significant protective effect on H2O2-induced cytoxicity and DNA damage in human non-immortalized fibroblasts. These antioxidant effects of active principle of CP, PK and WS may explain, at least in part, the reported anti-stress, immunomodulatory, cognition-facilitating, anti-inflammatory and antiaging effects produced by them in experimental animal and in clinical situations and may justify the further investigation of their other beneficial biological properties.

Neuroprotection by pramipexole against dopamine- and levodopa-induced cytotoxicity

Pramipexole, a novel non-ergoline dopamine (DA) agonist, has been applied successfully for treatment of Parkinson's disease (PD). We report here that pramipexole can protect dopaminergic cell line Mes23.5 against dopamine- and levodopa-induced cytotoxicity possibly through a mechanism related to antioxidant activity. In the MES 23.5 cultures, DA and L-DOPA induce a dose- and time-dependent cytotoxicity, as determined by tetrazolium salt and trypan blue assays. Furthermore, an in situ terminal deoxynucleotidyl transferase assay demonstrates that DA-induced cell death is apoptotic. Pretreatment with pramipexole in a concentration range (4–100 μM) significantly attenuates DA- or L-DOPA-induced cytotoxicity and apoptosis, an action which is not blocked by D 3 antagonist U-99194 A or D 2 antagonist raclopride. Pramipexole also protects MES 23.5 cells from hydrogen peroxide-induced cytotoxicity in a dose-dependent manner. In cell-free system, pramipexole can effectively inhibit the formation of melanin, an end product resulting from DA or L-DOPA oxidation. These results indicate that pramipexole exerts its neuroprotective effect possibly through a mechanism, which is independent of DA receptors but related to antioxidation or scavenging of free radicals (e.g. hydrogen peroxide). As a direct DA agonist and potentially neuroprotective agent, pramipexole remains attractive in the treatment of PD.

Protective effect of ebselen against hydrogen peroxide-induced cytotoxicity and DNA damage in HepG 2 cells

The protective effect of ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a selenoorganic compound, against hydrogen peroxide (H 2O 2)-induced cytotoxicity and DNA damage was investigated in a human hepatoma cell line, HepG 2. The inhibitory effect of H 2O 2 on cell growth was determined using the tetrazolium dye colorimetric test (MTT test), and the cytotoxicity and lipid peroxidation were estimated by lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) formation, respectively. DNA damage was detected using single cell gel electrophoresis (comet assay), and intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA). The results showed that H 2O 2 suppressed the growth of HepG 2 cells and the addition of ebselen significantly reduced the suppression. Furthermore, ebselen also displayed a dose-dependent reduction of LDH leakage and MDA formation in H 2O 2-treated cells. The results also demonstrate that ebselen was able to reduce the ROS formation and DNA damaging effect caused by H 2O 2 in a dose-dependent manner. These findings suggest that ebselen has a strong protective ability against the cytotoxicity and DNA damaging effect caused by reactive oxygen species.

Protective effect of neurotrophic factors, neuropoietic cytokines and dibutyryl cyclic amp on hydrogen peroxide-induced cytotoxicity on PC12 cells: a possible link with the state of differentiation

We present evidence that the survival of PC12 cells exposed to hydroxyl radicals generated by hydrogen peroxide applied for 30 min at 1 mM was effective when they were differentiated in response to Nerve Growth Factor (NGF) and/or other inducers of neurite outgrowth such as basic-fibroblast growth factor and dibutyryl cyclic AMP. The time- and dose-dependent differentiation triggered by NGF was (1) markedly increased by basic fibroblast growth factor, interleukin-6 or dibutyryl cyclic AMP; (2) diminished by leukemia inhibitory factor or ciliary neurotrophic factor; (3) not potentiated by insulin-like growth factor I or progesterone. The influence of these various factors and agents on PC12 cells was evaluated by the estimation of neurite outgrowth, whereas their possible protective effects were assessed by the measurement of cell survival. Our results would indicate that the factors and agents that induced differentiation were also able to protect the cells against an oxidative stress.

Suppression of Hydrogen Peroxide-Induced Cytotoxicity toward Chinese Hamster Lung Fibroblasts by Chemically Modified Curcumin.

We assessed the inhibitory effects of curcumin and its chemically modified homologues against H2O2-induced cytotoxicity toward Chinese hamster lung fibroblasts V79 cells with a colony formation assay. Among 4 curcuminoids, dihydroxycurcumin and dihydroxytetrahydrocurcumin suppressed H2O2-induced cytotoxicity. On the contrary, neither curcumin nor tetrahydrocurcumin showed any suppressive effects. These results support our empirical rule that the o-dihydroxy (catechol) moiety is essential for the inhibitory effects.

Role of cytochrome P-450 in hydrogen peroxide-induced cytotoxicity to LLC-PK1 cells

The current study was designed to test the role of cytochrome P-450 in hydrogen peroxide-induced cytotoxicity, and to determine whether it may serve as a source of catalytic iron. Hydrogen peroxide led to iron release (as measured by iron/bathophenanthroline complex) from the microsomes prepared from LLC-PK1 cells. Cimetidine, which inhibits cytochrome P-450 by interacting with the heme iron, significantly blocked iron release, whereas ranitidine, which has a similar structure as cimetidine but is a weak inhibitor of P-450, did not have an effect (H2O2, 0.42 +/- 0.04; H2O2 + cimetidine, 0.23 +/- 0.02 nmol/mg protein; N = 4, P < 0.01). Exposure of LLC-PK1 cells to hydrogen peroxide (2.5 mM) resulted in a significant increase in the bleomycin-detectable iron (iron capable of catalyzing free radical reactions) content that was prevented by cimetidine, but not ranitidine. We then examined the effect of the inhibitors of cytochrome P-450 on cell death (as measured by Trypan blue exclusion) after exposure of LLC-PK1 cells to 2.5 mM hydrogen peroxide for 120 minutes. Inhibition of cytochrome P-450 by cimetidine significantly reduced the cell death; the effect was observed with 0.05 mM and was concentration dependent with 1 mM affording almost complete protection (H2O2, 59 +/- 1.3% vs. H2O2 + cimetidine, 11 +/- 0.7%; N = 5, P < 0.01). In contrast, ranitidine did not show any protection. We confirmed that the protective effect of cimetidine was not related to scavenging hydrogen peroxide or hydroxyl radicals or chelating iron. A second inhibitor of cytochrome P-450, piperonyl butoxide, had a similar dose-dependent beneficial effect against hydrogen peroxide-induced cell injury. Our data thus indicate an important role of cytochrome P-450 in hydrogen peroxide-induced cytotoxicity to LLC-PK1 cells and suggest that cytochrome P-450 may serve as a source of catalytic iron.

Long-term protection against oxidative stress by linsidomine: Ferritin as a possible mediator

Using cultured endothelial cells, the present study investigates the effect of the molsidomine metabolite and nitric oxide donor linsidomine (SIN-1) on hydrogen peroxide-induced cytotoxicity and expression of ferritin heavy-chain mRNA. In porcine aortic endothelial cells, a 20-h incubation with hydrogen peroxide (0.5 mM) reduced the number of viable cells to 44% of control. A 6-h preincubation with SIN-1 (0.5 mM) protected endothelial cells from hydrogen peroxide-dependent cytotoxicity and increased viability to 81% of control. This protection was associated with a 2-fold induction of ferritin heavy-chain mRNA by SIN-1. A protective effect comparable with SIN-1 was observed when preincubating the cells with iron-free apoferritin (1 mg mL−1). These findings suggest that ferritin induction may be a mechanism underlying long-term cytoprotection by SIN-1 against oxidative stress.

ROLE OF CYTOCHROME P-450 IN HYDROGEN PEROXIDE-INDUCED CYTOTOXICITY IN LLC-PK1 CELLS. † 2131

The current study was designed to test the role of cytochrome P-450 in hydrogen peroxide-induced cytotoxicity to LLC-PK1 cells, and to determine whether it may serve as a source of catalytic iron. Hydrogen peroxide led to iron release as measured by Fe2+-(BPS)3 from the microsomes prepared from LLC-PK1 cells. Cimetidine, which inhibits cytochrome P-450 by interacting with the heme iron, significantly blocked iron release, whereas ranitidine which has a similar structure as cimetidine but is a weak inhibitor of P-450, did not have an effect (H2O2: 0.42± 0.04, H2O2 + cimetidine: 0.23 ± 0.02 nmol/mg protein, n=4, p < 0.01). Exposure of LLC-PK1 cells to hydrogen peroxide (2.5 mM) resulted in a significant increase in the bleomycin-detectable iron (iron capable of catalyzing free radical reactions) content which was prevented by cimetidine, but not ranitidine. We then examined the effect of the inhibitors of cytochrome P-450 on cell death (as measured by trypan blue exclusion) after exposure of LLC-PK1 cells to 2.5 mM hydrogen peroxide for 120 min. Inhibition of cytochrome P-450 by cimetidine significantly reduced the cell death. The effect was observed with 0.05 mM and was concentration dependent with 1 mM affording almost complete protection (H2O2: 58.5 ± 1.3 vs H2O2 + cimetidine: 10.9 ± 0.7, n=5, p < 0.01). In contrast, ranitidine did not show any protection. We confirmed that the protective effect of cimetidine was not related to scavenging hydrogen peroxide or hydroxyl radicals or chelating iron. A second inhibitor of cytochrome P-450, piperonyl butoxide, had a similar dose-dependent beneficial effect against hydrogen peroxide-induced cell injury. Our data thus indicate an important role of cytochrome P-450 in hydrogen peroxide-induced cytotoxicity to LLC-PK1 cells suggesting that cytochrome P-450 may serve as a source of catalytic iron.

The L-histidine-mediated enhancement of hydrogen peroxide-induced DNA double strand breakage and cytotoxicity does not involve metabolic processes

The cytotoxic response of Chinese hamster ovary (CHO) cells to challenge with hydrogen peroxide was highly dependent upon the temperature of exposure, being markedly higher at 37 ° than at 4 °C. Increasing intracellular levels of L-histidine prior to challenge with hydrogen peroxide increased the toxicity elicited by the oxidant at both physiologic and ice-bath temperatures. The effect of the amino acid, however, was more pronounced under conditions at 4 °C, as compared to 37 °C. Indeed, at 4 °C the oxidant was nontoxic at submillimolar levels and pre-exposure to L-histidine restored cytotoxicity to levels slightly higher than those observed after treatment at 37 °C (in the micromolar range). Pre-exposure to the amino acid increased the production of DNA double-strand breaks (DSBs) elicited by treatment with the oxidant both at 37 ° and 4 °C. A remarkable correlation was found when the level of this lesion was plotted against the cytotoxic response observed using different concentrations of L-histidine or hydrogen peroxide, or treating the cells with the oxidant either at 37 ° or 4 °C, thus suggesting the existence of a cause-effect relationship. The overlapping correlation curves obtained with cells challenged with the oxidant at 4 ° or 37 °C also suggest that similar molecular mechanisms mediate the formation of DNA DSBs under both experimental conditions. Two lines of evidence provide experimental support for this inference: (1) the kinetics of repair of DNA DSBs generated at 37 ° or 4 °C were virtually superimposable; this would suggest that the same repair pathway(s) is/are responsible for the removal of DNA DSBs generated at the two temperatures; and (2) the size distribution of double-stranded DNA fragments produced under the two treatment conditions, resulting in a similar cytotoxic response, was basically identical. This is indicative of remarkable similarities in the topology of chromosomal domains where DSBs are generated. Overall, the results presented in this paper provide further experimental evidence supporting the notion that DNA DSBs are responsible for the L-histidine-mediated enhancement of hydrogen peroxide-induced cytotoxicity, and demonstrate that the mechanism whereby the amino acid enhances the ability of hydrogen peroxide to produce DNA double strand breakage and cell killing does not depend on cellular metabolism and/or energy-dependent reactions.

The L-histidine-mediated enhancement of hydrogen peroxide-induced DNA double strand breakage and cytotoxicity does not involve metabolic processes

The L-histidine-mediated enhancement of hydrogen peroxide-induced DNA double strand breakage and cytotoxicity does not involve metabolic processes

The l-histidine-mediated enhancement of hydrogen peroxide-induced cytotoxicity is a general response in cultured mammalian cell lines and is always associated with the formation of DNA double strand breaks

Micromolar concentrations of l-histidine increase the cytotoxicity of hydrogen peroxide in a number of cell lines including CHO(hamster), EAHY, McCoy's, U937 and CCRF-CEM (human), Vero (monkey) and SC-1 (mouse). Importantly, these cell lines displayed differentt degrees of sensitivity to H 2O 2 alone and the extent of enhancement elicited by the amino acid was more pronounced in resistant cell lines. The increased cytotoxicity was invariably associated with the formation of DNA DSBs and a remarkable correlation was found by plotting the level of DNA DSBs against the cytotoxic response. These results strongly support the hypothesis that the mechanism whereby l-histidine increases the toxicity elicited by H 2O 2 involves the formation of DNA DSBs and are consistent with the possibility that the amino acid might participate in the regulation of the physio-pathological response to oxidative stress in mammals.

L-histidine-mediated enhancement of hydrogen peroxide-induced cytotoxicity: relationships between dna single/double strand breakage and cell killing

Results presented in this study demonstrate an association between the L-Histidine-mediated enhancement of H 2O 2-induced cytotoxicity and the formation of DNA double strand breakage (DSB), whereas no relationship exists between the increased cytotoxic response and DNA single strand breakage (SSB). Indeed, the higher lethality and the production of DNA DSB occurred in oxidatively-injured cells regardless of whether the exposure to L-Histidine was performed before or during challenge with the oxidant. In fact, the increased level of DNA SSB detected in cells simultaneously exposed to the oxidant and the amino acid was not observed in cells pre-treated with L-Histidine and then challenged with hydrogen peroxide. Further experiments have demonstrated an association between the kinetics of DNA DSB formation and the enhancement of the cytotoxic response. In conclusion, intracellular L-Histidine seems to mediate the formation of DNA DSB and the increased growth-inhibitory response elicited by the oxidant. In addition, these results suggest that the enhancement of DNA SSB is produced by the extracellular/plasma membrane fraction of the amino acid and not causally related to the L-Histidine-mediated increase of the growth-inhibitory response to H 2O 2-treated cells.

Suppression of Hydrogen Peroxide-induced Cytotoxicity toward Chinese Hamster Lung Fibroblasts by Decylubiquinone, a Coenzyme Q Homolog

The cytotoxicity of hydrogen peroxide (H2O2) toward Chinese hamster V79 cells was assessed with a colony-formation assay. Decylubiquinone, a coenzyme Q homolog, suppressed H2O2-induced cytotoxicity. Almost all the incorporated decylubiquinone was detected as decylubiquinol after incubating the cells for 4h. These results suggest that decylubiquinol reduced in the cells acted as an antioxidant against H2O2-induced oxidative cell injury.

Cytotoxicity and dentin permeability of carbamide peroxide and hydrogen peroxide vital bleaching materials, in vitro.

There has been recent concern about the inadvertent exposure of dentin with patent tubules as well as gingiva to bleaching systems containing 10-15% carbamide peroxide or 2-10% hydrogen peroxide for more than a few minutes. The aims of the present study were: (1) to determine the cytotoxicity of dilutions of hydrogen peroxide in cell culture; (2) to measure hydrogen peroxide diffusion from bleaching agents through dentin in vitro; and (3) to determine the risk of hydrogen peroxide-induced cytotoxicity from exposure of dentin to these vital bleaching agents. The 50% inhibitory dose (ID50) of hydrogen peroxide to succinyl dehydrogenase activity in cultured cells was found to be 0.58 mmol/L after 1 h. All bleaching materials demonstrated diffusion of hydrogen peroxide through dentin in an "in vitro pulp chamber" device. The one- and six-hour diffusates of all bleaching agents through 0.5-mm dentin exceeded the ID50 in monolayer cultures. Inhibition of succinyl dehydrogenase activity corresponded to the amount of hydrogen peroxide that can rapidly diffuse through dentin in vitro and reach concentrations which are toxic to cultured cells in less than 1 h.

L-Glutamine prevents the l-histidine-mediated enhancement of hydrogen peroxide-induced cytotoxicity

Results presented in this study demonstrate that l-glutamine, a competitive inhibitor of l-histidine uptake, inhibits in a concentration-dependent fashion the l-histidine-mediated enhancement of H 2O 2-induced cytotoxicity. l-Glutamine also prevents the induction of DNA double strand breaks (DSB) but does not affect the enhancing effect of l-histidine on DNA single strand break induction by H 2O 2. Taken together, these data demonstrate that l-histidine, in order to allow the formation of DNA double strand breakage and increase the toxicity elicited by the oxidant, has to enter the cell. In addition, these results indicate that the enhancement of DNA single strand breakage is a consequence of the action of the amino acid at the extracellular level and/or outer surface of the plasma membrane and does not appear related to the mechanism whereby l-histidine increases the cytotoxic response to H 2O 2. The latter mechanism very likely involves the formation of DNA DSB.

Hydrogen peroxide-induced cytotoxicity in cultured epithelial cells (WISH): A functional and morphological study

The effect of hydrogen peroxide on cultured epithelial cells (WISH) was investigated with particular emphasis on cell functions: cell morphology and cytoskeletal components were also studied. The presence of low concentrations of H 2O 2 (0.1–0.4 mM) in the culture medium markedly inhibited cell growth, although WISH contained catalase and glutathione-peroxidase activities. After 1 hr of incubation with H 2O 2 up to 5 mM, the majority of the cells were still alive, but reincubation in normal medium for 24 hr clearly reduced cell viability. Cell adhesion was dose-dependently reduced by H 2O 2 treatment (0.1–0.5 mM) for 4 hr. Incubation with 1.5 mM-H 2O 2 gave rise to a bleb appearance on the cell surface and to mitochondrial swelling, as shown, respectively, by scanning and transmission electron microscopy. Immunofluorescence studies revealed changes in microtubules and microfilaments, which are two of the main cytoskeletal components. The modification of microtubules was also confirmed by Western blot analysis of WISH protein homogenates submitted to SDS-PAGE. WISH treated with 1.5 mM-H 2O 2 showed decreased levels of GSH compared with control cells: glutathione transferase activity was reduced, whereas other enzymes of the glutathione cycle were unchanged.

Pyruvate and related α-ketoacids protect mammalian cells in culture against hydrogen peroxide-induced cytotoxicity

Pyruvate efficiently protected V79 Chinese hamster cells against the lethal effects of hydrogen peroxide. Protection was also provided by other α-ketoacids, such as α-ketobutyrate, α-ketoglutarate and α-ketoadipate, although higher concentrations were required. The corresponding β-ketoacids had no effect. The results indicate that pyruvate and other α-ketoacids possess antioxidant activity in vitro and, probably, in vivo.