t-BOOH Treatment Research Articles

The bZIP-type transcription factors NapA and RsmA modulate the volumetric ratio and the relative superoxide ratio of mitochondria in Aspergillus nidulans.

Basic leucine zipper (bZIP) transcription factors are crucial components of differentiation, cellular homeostasis and the environmental stress defense of eukaryotes. In this work, we further studied the consequence of gene deletion and overexpression of two bZIP transcription factors, NapA and RsmA, on superoxide production, mitochondrial morphology and hyphal diameter of Aspergillus nidulans. We have found that reactive oxygen species production was influenced by both gene deletion and overexpression of napA under tert-butylhydroperoxide (tBOOH) elicited oxidative stress. Furthermore, gene expression of napA negatively correlated with mitochondrial volumetric ratio as well as sterigmatocystin production of A. nidulans. High rsmA expression was accompanied with elevated relative superoxide ratio in the second hyphal compartment. A negative correlation between the expression of rsmA and catalase enzyme activity or mitochondrial volumetric ratio was also confirmed by statistical analysis. Hyphal diameter was independent on either rsmA and napA expression as well as 0.2mM tBOOH treatment.

Rapid Hypoxia‐Induced Upregulation of Glutathione‐Related Genes May Protect Elephant Seal Endothelial Cells Against Oxidative Stress

Northern elephant seals (Mirounga angustirostris) experience extreme fluctuations in blood oxygen levels during repeated bouts of deep diving. Such fluctuations characterize human cardiovascular pathologies including myocardial infarction and ischemic stroke, though seals do not incur vascular injury during the repetitive hypoxia/reoxygenation cycles associated with diving. Marine mammals diving at sea remain inaccessible for most real‐time in vivo biochemical investigations, thus we developed a proliferative arterial endothelial cell culture system from expelled placentae from elephant seals and humans to assess the molecular and biochemical response to oxidative stress. Seal endothelial cells generate oxidants when pharmacologically stimulated but remain protected against lipid peroxidation when treated with tert‐Butyl hydroperoxide (t‐BOOH, 300 uM), while human cells exposed to the same conditions display a doubling in lipid peroxidation levels (p=0.0002). Seal endothelial cells maintain viability at moderate (100 uM) doses of t‐BOOH while human cells display significantly reduced viability at the same dose. Interestingly, protein expression of the only two enzymes capable of repairing lipid peroxidation – glutathione peroxidase 4 (Gpx4) and peroxiredoxin 6 (Prdx6) – decreases in both seal and human endothelial cells after t‐BOOH treatment (Human: Gpx4 17% decrease, p=0.08; Prdx6 37% decrease, p=0.007; Seal: Gpx4 53% decrease, p=0.036; Prdx6 35% decrease, p=0.007). Additionally, seal endothelial cells display lower baseline glutathione peroxidase activity than human cells (p<0.0001) and activity in human (p=0.0002) but not seal cells declines after t‐BOOH treatment. Seal endothelial cells exposed to 1% O2 for up to 6 h upregulate expression of genes in the KEGG glutathione metabolism pathway within 30 minutes of hypoxia onset, while expression of these genes returns to baseline levels beyond 1 h in hypoxia. Among those genes contributing to the overrepresentation of the glutathione pathway are several glutathione S‐transferases (GSTM3, GSTK1, GSTO1, MGST2), suggesting that glutathione S‐transferases may limit oxidative damage in seal endothelial cells during hypoxia/reoxygenation bouts. That this upregulation occurs absent the reintroduction of oxygen into the system may suggest that the seal glutathione system is “primed” for rapid response to protect against oxidative injury during the repeated hypoxia/reoxygenation cycles associated with diving.

The Zebrafish Embryo as a Model to Test Protective Effects of Food Antioxidant Compounds.

The antioxidant activity of food compounds is one of the properties generating the most interest, due to its health benefits and correlation with the prevention of chronic disease. This activity is usually measured using in vitro assays, which cannot predict in vivo effects or mechanisms of action. The objective of this study was to evaluate the in vivo protective effects of six phenolic compounds (naringenin, apigenin, rutin, oleuropein, chlorogenic acid, and curcumin) and three carotenoids (lycopene B, β-carotene, and astaxanthin) naturally present in foods using a zebrafish embryo model. The zebrafish embryo was pretreated with each of the nine antioxidant compounds and then exposed to tert-butyl hydroperoxide (tBOOH), a known inducer of oxidative stress in zebrafish. Significant differences were determined by comparing the concentration-response of the tBOOH induced lethality and dysmorphogenesis against the pretreated embryos with the antioxidant compounds. A protective effect of each compound, except β-carotene, against oxidative-stress-induced lethality was found. Furthermore, apigenin, rutin, and curcumin also showed protective effects against dysmorphogenesis. On the other hand, β-carotene exhibited increased lethality and dysmorphogenesis compared to the tBOOH treatment alone.

The Roles of Superoxide on At-Level Spinal Cord Injury Pain in Rats.

Background: In the present study, we examined superoxide-mediated excitatory nociceptive transmission on at-level neuropathic pain following spinal thoracic 10 contusion injury (SCI) in male Sprague Dawley rats. Methods: Mechanical sensitivity at body trunk, neuronal firing activity, and expression of superoxide marker/ionotropic glutamate receptors (iGluRs)/CamKII were measured in the T7/8 dorsal horn, respectively. Results: Topical treatment of superoxide donor t-BOOH (0.4 mg/kg) increased neuronal firing rates and pCamKII expression in the naïve group, whereas superoxide scavenger Tempol (1 mg/kg) and non-specific ROS scavenger PBN (3 mg/kg) decreased firing rates in the SCI group (* p < 0.05). SCI showed increases of iGluRs-mediated neuronal firing rates and pCamKII expression (* p < 0.05); however, t-BOOH treatment did not show significant changes in the naïve group. The mechanical sensitivity at the body trunk in the SCI group (6.2 ± 0.5) was attenuated by CamKII inhibitor KN-93 (50 μg, 3.9 ± 0.4) or Tempol (1 mg, 4 ± 0.4) treatment (* p < 0.05). In addition, the level of superoxide marker Dhet showed significant increase in SCI rats compared to the sham group (11.7 ± 1.7 vs. 6.6 ± 1.5, * p < 0.05). Conclusions: Superoxide and the pCamKII pathway contribute to chronic at-level neuropathic pain without involvement of iGluRs following SCI.

Characterization of Suicidal Erythrocyte Death (Eryptosis) in Dogs.

Suicidal erythrocyte death (eryptosis) is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface following a Ca2+ entry in the cell. Eryptosis is stimulated by increased cytosolic Ca2+ ([Ca2+]i), oxidative stress, energy depletion, or high osmotic shock. Eryptosis signaling includes p38 mitogen-activated protein kinase (MAPK), caspases, casein kinase 1 (CK1), janus kinase 3 (JAK3), and protein kinase C (PKC). Dog and human erythrocytes have different characteristics, for example, dog erythrocytes lack Na+/K+- ATPase activity. Whether eryptosis occurs in dog erythrocytes in an analogous way as that in humans remains unclear. Eryptosis in dogs has not been investigated. This study aimed to explore which stimulator and signaling molecules are involved in eryptosis in healthy dog erythrocytes. Erythrocytes were isolated from 10 dogs, and eryptosis was stimulated by oxidative stress with tert-butyl hydroperoxide (tBOOH), high osmotic shock with excessive sucrose condition, energy depletion with minus glucose condition, and high [Ca2+]i with ionomycin. Phosphatidylserine exposure was estimated using annexin V binding. Erythrocyte volume and [Ca2+]i were measured by forward scatter and Fluo3-fluorescence, respectively. In addition, the role of certain mediators was assessed using the following inhibitors to determine the detailed mechanisms of eryptosis in dog erythrocytes: p38MAPK, caspase family, CK1, JAK3, and PKC inhibitors. All eryptosis-inducing factors resulted in phosphatidylserine exposures, except for ionomycin. In addition, the erythrocyte volume increased with ionomycin and tBOOH but decreased with excessive sucrose and minus glucose condition. All treatments increased [Ca2+]i. Furthermore, WH1-P154 and chelerythrine significantly blunted the increase of annexin V binding erythrocytes following the tBOOH treatment. Eryptosis in dogs is triggered by oxidative stress, hyperosmotic shock, and energy depletion. It is suggested that JAK3 and PKC play an important role in eryptosis following an oxidative stress in dog erythrocytes.

Toxicity of Aqueous L-Selenomethionine and Tert-Butyl Hydroperoxide Exposure to Zebrafish (Danio rerio) Embryos Following Tert-Butyl Hydroquinone Treatment.

Aqueous L-selenomethionine (SeMet) embryo exposures represent a rapid and simplified method for investigating the embryotoxic effects of SeMet. Using zebrafish (Danio rerio) as a model organism, the objective of the present study was to characterize the effects of waterborne exposure to both SeMet and tert-butyl hydroperoxide (tBOOH) to early life stages of zebrafish pre-treated with the antioxidant tert-butyl hydroquinone (tBHQ) in an attempt to investigate the mechanism of Se toxicity as it relates to oxidative stress. During the initial concentration range finding experiment, recently fertilized embryos were exposed for five days to 5, 25, 125, and 625 µg Se/L (as SeMet). These exposures informed the second experiment in which embryos were exposed to two concentrations of SeMet (25 and 125 µg Se/L) and 75 mg/L tBOOH either with (tBOOH-t, 25-t, 125-t) or without (tBOOH, 25, 125) a 4 h 100 µg/L tBHQ pre-treatment. Survival, hatchability, time to hatch, the frequency and severity of deformities (total and type), and changes in the expression of seven antioxidant-associated genes were determined. Exposures to SeMet and tBOOH reduced hatchability, increased time to hatch, decreased survival, increased the incidence and severity of deformities, and increased glutathione-disulfide reductase (gsr) expression in the pre-treated tBOOH treatment group.

Antigenotoxic and antimutagenic effects of diphenyl ditelluride against several known mutagens in Chinese hamster lung fibroblasts.

The present study evaluates antigenotoxic and antimutagenic properties of diphenyl ditelluride (DPDT) against several known mutagens in Chinese hamster lung fibroblasts (V79 cells). DPDT was not cytotoxic and genotoxic at concentrations ranging from 0.01 to 0.1 μM. The pre-treatment for 2h with this organotellurium compound at non-cytotoxic dose range (0.01, 0.05 and 0.1 μM) increased cell survival after challenge with hydrogen peroxide (H2O2), t-butyl hydroperoxide (t-BOOH), methylmethanesulphonate (MMS) or ultraviolet (UV)C radiation. In addition, the pre-treatment with DPDT decreased the DNA damage and Formamidopyrimidine DNA-glycosylase (Fpg)- and Endonuclease III (Endo III) sensitive sites induction by the studied genotoxic agents, as verified by comet assay and modified comet assay, respectively. The pre-treatment also reduced micronucleus frequency, revealing the protector effect of DPDT against MMS and UVC-induced mutagenesis. Our results demonstrate that DPDT-treated cells at concentration range of 0.01-0.1 μM do not change thiobarbituric acid reactive species (TBARS) levels and ROS generation. Moreover, DPDT pre-treatment at this concentration range decreases the ROS induction by H2O2 and t-BOOH treatment indicating antioxidant potential. On the other hand, concentrations higher than 0.1 μM increase TBARS formation and inhibited superoxide dismutase (SOD) activity, suggesting pro-oxidative effect of this compound at high concentrations. Our results suggest that DPDT presents antigenotoxic and antimutagenic properties at concentration range of 0.01-0.1 μM. The protection effect could be attributed to antioxidant capacity of DPDT at this concentration range in V79 cells.

Impact of Cyclin-Dependent Kinase CDK4 Inhibition on Eryptosis

Background/Aims: The cyclin-dependent kinase 4 (CDK4) participates in the regulation of apoptosis of nucleated cells by altering transcriptional regulation of genes governing cell proliferation and cell death. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure at the cell surface. As mature erythrocytes lack nuclei, acute stimulation of eryptosis cannot result from altered gene expression. Eryptosis is triggered by isotonic cell shrinkage following Cl^- removal (replacement with the impermeant organic anion gluconate) or by oxidative stress (exposure to 0.3 mM tertbutyl-hydroperoxide [tBOOH]). The present study explored whether CDK4 is expressed in erythrocytes and whether the CDK4 inhibitors II (NSC625987) and III (ryuvidine) influence eryptosis. Methods: Western blotting was utilized for determination of the presence of CDK4 protein in human erythrocytes, and FACS analysis to determine Fluo3 fluorescence (reflecting cytosolic Ca²⁺), annexin-V-binding (reflecting PS-exposure) and forward scatter (reflecting cell volume). Results: CDK4 protein was present in human erythrocytes. Cl^- removal was followed by decrease of forward scatter and increase of both annexin-V-binding and Fluo3 fluorescence, an effect significantly curtailed by CDK4 inhibitors II and III. Furthermore, CDK4 inhibition blunted enhanced PS-exposure elicited by tBOOH treatment. Conclusions: The present observations disclose the presence of CDK4 protein in human erythrocytes and the suppression of suicidal erythrocyte death by pharmacological inhibition of CDK4.

Determination of in vitro and in vivo protective effects of Ghrelin against oxidative stress: Experimental Study

Antioxidant properties of ghrelin have been recently reported in animal models and cell culture experiments. This study was set to examine the possible in vitro and in vivo anti-oxidative effects of ghrelin in rat and HepG2 cell culture. In this study, thirty six male Wistar Rats were randomly allocated into six experimental groups of six; Intraperitoneally, group 1 (Control) received 1 ml PBS, group 2 received 0.1 mM/kg tert-Butylhydroperoxide (tBOOH), groups 3 and 4 received 0.1 mM/kg t-BOOH and then received 10 and 50 µg/kg ghrelin, respectively. Groups 5 and 6 received 10 and 50 µg/kg ghrelin, respectively. Rats were anesthetized 24 h after last injection and blood samples were taken by cardiac puncture. Carbonylated proteins, nitric oxide (NO) and total antioxidant capacity (TAC) levels were measured in sera. HepG2 cells were plateaued at a density of 1.5 x 10 5 per well in eight plates. After treatment with ghrelin (0, 10, 25 and 50 nM) for 30 min, cells were treated with TBOOH (100 or 200 µM) for 24 h to analyze cell proliferation by MTT assay at 570 nm. Evidence of oxidative stress including increased carbonylated proteins and NO levels and decreased TAC level were observed after tBOOH injection. In rats with oxidative stress, subsequent treatment with ghrelin decreased NO and carbonylated proteins level and increased TAC level. The cell viability was decreased after t-BOOH treatment in dose dependent manner; in contrast, ghrelin in all used concentrations caused an elevation in cell viability after 24 h incubation time. These data taken together indicate that ghrelin reduces oxidative stress, but its exact mechanism is yet to be investigated.

Reactive oxygen species contribute to neuropathic pain and locomotor dysfunction via activation of CamKII in remote segments following spinal cord contusion injury in rats

In this study, we examined whether blocking spinal cord injury (SCI)-induced increases in reactive oxygen species (ROS) by a ROS scavenger would attenuate below-level central neuropathic pain and promote recovery of locomotion. Rats with T10 SCI developed mechanical allodynia in both hind paws and overproduction of ROS, as assayed by Dhet intensity, in neurons in the lumbar 4/5 dorsal horn (∗P<0.05). To scavenge ROS, phenyl-N-tert-butylnitrone (PBN, a ROS scavenger) was administered immediately after SCI and for 7 consecutive days (early treatment) by either intrathecal (it; 1 and 3mg) or systemic (ip; 10, 50 and 100mg) injections. In addition, the high doses of it (3mg) or ip (100mg) injections were performed at 35days (delayed treatment) after SCI. High doses of PBN (ip, 100mg, and it, 3mg) significantly attenuated mechanical allodynia in both hind paws at both early and delayed treatments, respectively (∗P<0.05). The abnormal hyperexcitability of wide dynamic range neurons after SCI was significantly attenuated by both early and delayed PBN treatment (∗P<0.05). Early PBN treatment (100mg, ip, and 3mg, it) attenuated overproduction of ROS in neurons in the lumbar 4/5 dorsal horn. In addition, it and ip t-BOOH (ROS donor) treatment dose-dependently produced mechanical allodynia in both hind paws (∗P<0.05). Both SCI and t-BOOH treatment groups showed significantly increased phospho-CamKII (pCamKII) expression in neurons and KN-93 (an inhibitor of pCamKII) significantly attenuated mechanical allodynia (∗P<0.05). In addition, high doses of PBN significantly promoted the recovery of locomotion (∗P<0.05). In conclusion, the present data suggest that overproduction of ROS contribute to sensory and motor abnormalities in remote segments below the lesion after thoracic SCI.

Mechanism of tert-butylhydroperoxide induced cytotoxicity in U-937 macrophages by alteration of mitochondrial function and generation of ROS

tert-Butylhydroperoxide has been reported to inhibit growth and induce apoptosis in number of cell types, but little is known about the molecular mechanism mediating these effects. In the present study, we determined the molecular pathways that lead to apoptosis after treatment of cells with t-BOOH. The cells were exposed to different concentrations of t-BOOH (100–750 μM) for 1–4 h and various parameters such as cytotoxicity, ROS (reactive oxygen species) generation, MMP (mitochondrial membrane potential), intracellular Ca ++ levels and expression of various proteins involved in apoptosis were determined. Exposure of U-937 cells to t-BOOH induced cytotoxicity in a time dependent manner with about 50% toxicity at 400 μM t-BOOH in 4 h. t-BOOH treatment resulted in a time dependent increase in reactive oxygen species levels, Ca ++ influx and annexin V positive cells. There was a significant fall in MMP following exposure to t-BOOH with time. t-BOOH treatment of U-937 cells leads to apoptosis, which is accompanied by activation of caspase-3. The caspase-3 inhibitor (Ac-DEVD-CHO) inhibits the cytotoxicity induced by t-BOOH, indicating a direct link between caspase-3 activation and cell death. This activation of apoptosis is accompanied by release of cytochrome c, down regulation of anti-apoptotic protein Bcl-2 levels with concurrent increase in pro-apoptotic proteins Bax and Bad levels. These observations indicate that t-BOOH induces cell death in U-937 macrophages by apoptosis, which is mediated through mitochondrial pathway.

Complex regulation of the organic hydroperoxide resistance gene (ohr) from Xanthomonas involves OhrR, a novel organic peroxide-inducible negative regulator, and posttranscriptional modifications.

Analysis of the sequence immediate upstream of ohr revealed an open reading frame, designated ohrR, with the potential to encode a 17-kDa peptide with moderate amino acid sequence homology to the MarR family of negative regulators of gene expression. ohrR was transcribed as bicistronic mRNA with ohr, while ohr mRNA was found to be 95% monocistronic and 5% bicistronic with ohrR. Expression of both genes was induced by tert-butyl hydroperoxide (tBOOH) treatment. High-level expression of ohrR negatively regulated ohr expression. This repression could be overcome by tBOOH treatment. In vivo promoter analysis showed that the ohrR promoter (P1) has organic peroxide-inducible, strong activity, while the ohr promoter (P2) has constitutive, weak activity. Only P1 is autoregulated by OhrR. ohr primer extension results revealed three major primer extension products corresponding to the 5' ends of ohr mRNA, and their levels were strongly induced by tBOOH treatment. Sequence analysis of regions upstream of these sites showed no typical Xanthomonas promoter. Instead, the regions can form a stem-loop secondary structure with the 5' ends of ohr mRNA located in the loop section. The secondary structure resembles the structure recognized and processed by RNase III enzyme. These findings suggest that the P1 promoter is responsible for tBOOH-induced expression of the ohrR-ohr operon. The bicistronic mRNA is then processed by RNase III-like enzymes to give high levels of ohr mRNA, while ohrR mRNA is rapidly degraded.

Effect of caffeic acid on tert-butyl hydroperoxide-induced oxidative stress in U937

Nonvitamin phenolic compounds are ubiquitous in food plants and therefore potentially present in human plasma in a diet-dependent concentration. The aim of this study was to evaluate the ability of caffeic acid, a phenolic acid with antioxidant activity, to affect cellular response in U937 human monocytic cells to t-butyl hydroperoxide-induced oxidative stress. In our experimental conditions caffeic acid was incorporated into cells without any cytotoxic effect. Caffeic acid-treated cells showed an increased resistance to oxidative challenge, as revealed by an higher percent of survival and the maintenance of an higher proliferative capacity in respect to control cells. This effect seems to be due to the ability of caffeic acid to reduce glutathione depletion and to inhibit lipid peroxidation during tBOOH treatment. It can be concluded that caffeic acid exerts an antioxidant action inside the cell, responsible for the observed modulation of the cellular response to oxidative challenge. Due to its presence in the diet, therefore, caffeic acid may play a role in the modulation of oxidative processes in vivo.

Nitric Oxide Inhibited Peroxyl and Alkoxyl Radical Formation with Concomitant Protection against Oxidant Injury in Intestinal Epithelial Cells

A model compound of lipid peroxidation,tert-butyl hydroperoxide (tBOOH), was usedin vitroto investigate (i). the generation of tBOOH-derived peroxyl and alkoxyl radicals by rat intestinal epithelial cells or enterocytes and (ii) the role of nitric oxide (NO) on cell-generated free radical formation and cellular cytotoxicity. Peroxyl, alkoxyl, and methyl radicals were detected and characterized by direct and spin-trapping electron paramagnetic resonance spectroscopy in incubations containing tBOOH and hematin, enterocytes, or intestinal epithelial cell line-6 cells. The direct interactions of tBOOH-derived radicals and NO from nitrosoglutathione (GSNO), nitrosoacetyl penicillamine (SNAP), or 1-{b3-aminopropy-4-(3-aminopropylammonio)} butylamino-diazeniumdiolate (SpNONOate) were demonstrated as their levels were depleted in these incubations. SNAP, not GSNO or SpNONOate, was capable of trapping methyl radical produced during hematin-catalyzed decomposition of tBOOH. Cellular cytotoxicity expressed by percentage of dead cells and lactate dehydrogenase was increased with tBOOH treatment. Addition of GSNO, SNAP, or SpNONOate suppressed tBOOH-induced elevation of cell cytotoxicity. The NO donor precursor glutathione, acetylpenicillamine, or spermine did not have any effects on tBOOH-derived radical generation or cell cytotoxicity. These findings demonstrated free radical–free radical reactions between NO- and tBOOH-derived alkoxyl and peroxyl radicals generated by enterocytes. These reactions, at least in part, describe the protective role of NO from hydroperoxide-induced injury in intestinal epithelial cells.

Bcl-2 Overexpression in the HaCaT Cell Line is Associated With A Different Membrane Fatty Acid Composition and Sensitivity to Oxidative Stress

Different mechanisms have been proposed for the activity of the Bcl-2 proto-oncogene product. A bona fide antioxidant activity and a pro-oxidant setting up of the cell have been suggested using different experimental models, yet many uncertainties exist about the biochemical mechanism of Bcl-2 action. In the present paper, we report the characterization of the cellular response to mild oxidative stress of a cultured cell line of immortalized keratinocytes (HaCaT), overexpressing the Bcl-2 oncogene product. A sublethal oxidative stress was induced by 1 h treatment with 200 μM tert-butyl-hydroperoxide (t-BOOH). Following peroxide treatment, the formation of reactive oxygen species was lower in Bcl-2 expressing cells, suggesting a better capacity to counter oxidative stress. Total Superoxide Dismutase activity was induced by oxidative t-BOOH treatment in bcl-2 transfected cells, which also accumulated less damage to membrane lipids and proteins, as assessed by TBA-RS and carbonyl formation respectively. On the other hand, the formation of 4-hydroxy-nonenal, a more specific marker of peroxidative damage to polyunsaturated fatty acids, was higher in bcl-2 transfected cells than in control cells. Bcl-2 over-expression was also associated with significant changes in the fatty acid composition of cell membranes. Transfected cells presented a higher proportion of mono-unsaturated fatty acids and ω6 poly unsaturated fatty acids and a lower proportion of penta-enoic PUFA, thus resulting in a higher unsaturation index with respect to control cells. Changes in Protein kinase C activity were also associated to bcl-2 expression, possibly resulting from the differences in membrane fatty acid composition. These data may be an important background for the understanding of Bcl-2 involvement in the control of apoptotic response as well as in the induction of antioxidant cell defenses against oxidative stress.

The reduction of glutathione disulfide produced by t-butyl hydroperoxide in respiring mitochondria

Factors affecting the reduction of GSSG by rat liver mitochondria after a t-butyl hydroperoxide-induced (t-BOOH) oxidative stress were studied. The amounts of ADP and mitochondrial protein were adjusted to consume less than 50% of the available oxygen during the 8-min experimental period. A 4-min treatment of mitochondria with 24 nmol t-BOOH/mg protein (60 μM) oxidized 91% of total glutathione. In the presence of glutamate/malate, succinate or ascorbate/ N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD) (state 4 respiration), 84, 84, and 28% of the GSSG formed during t-BOOH treatment was reduced after 4 min, respectively. A similar extent of reduction was seen during state 3 respiration (1.5 mM ADP) with glutamate/malate, but no reduction occurred during state 3 respiration with either succinate or ascorbate/TMPD. The succinate-supported reduction of GSSG was completely blocked by rotenone, antimycin A, carbonyl cyanide p-trifluoromethoxyphe-nylhydrazone (FCCP), or 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). In contrast, oligomycin potentiated GSSG reduction using either glutamate/malate or succinate as substrates. Rotenone partially blocked glutamate/ malate-supported GSSG reduction. NADPH levels were altered in direct proportion to the effects on GSSG reduction. The current data indicate that the reduction of GSSG in oxidatively- stressed isolated rat liver mitochondria occurs most efficiently during state 4 respiration and is independent of ATP synthesis. Both transhydrogenation and the transmembrane proton gradient appear to be important in NADPH regeneration and consequent GSSG reduction.

Inward potassium transport systems in skeletal muscle derived cells are highly sensitive to oxidant exposure

Strenuous physical exercise causes a remarkable perturbation of K+ homeostasis in skeletal muscle tissue. Potassium efflux is crucial for a number of physiological control processes; however, exercise-induced perturbation of K+ homeostasis in skeletal muscle is suggested to be implicated in the generation of muscle fatigue. Physical exercise is also known to induce oxidative stress; a possible contribution of oxygen free radicals to the development of muscle fatigue has been hypothesized. To reveal the dose-dependent effect of oxidant exposure on inward and outward K+ (86RbCl) transporting systems, skeletal muscle derived L6 cells were treated with different concentrations of tert-butylhydroperoxide (TBOOH). We document the responses of (1) the ouabain-sensitive component of K+ influx (Na+, K+ pump), (2) bumetanide-sensitive ouabain-insensitive component of K+ influx (Na+, K+, 2Cl− cotransporter), (3) ouabain- and bumetanide-insensitive component of K+ influx (passive permeability of the cell membrane to inward K+), (4) ouabain-insensitive component of K+ efflux, and (5) passive leakage component of K+ efflux following exposure of L6 cells to oxidant treatment. Even very low doses of TBOOH (25 μmol) caused powerful activation of the Na+, K+ pump. Following TBOOH treatment, activity of the Na+, K+, 2Cl− cotransporter was remarkably inhibited. Such a treatment also significantly decreased the permeability of the cell membrane to inward flux of K+ (passive influx). Thus, we observed that even very low doses of oxidant had remarkable specific effects on the different components of K+ influx in the skeletal muscle derived cells. However, K+ efflux mechanisms appeared to be rather insensitive to the extracellular oxidant challenge.