Effects Of Tert-butyl Hydroperoxide Research Articles

Tris(2-metoxy-5-chlorophenyl)antimony Dicarboxylates (5-Cl-2-MeO-C6H3)3Sb[OC(O)R]2, Where R = CH2Br, CH2C6H4F-3, and C6H3F2-2,3: Synthesis and Structure

In a crystal of tris(2-methoxy-5-chlorophenyl)antimony benzene solvate (5-Cl-2-MeO-C6H3)3Sb (I) ⋅ 1/2С6Н6 synthesized from SbCl3 and 2-methoxy-5-chlorophenyllithium with recrystallization from benzene, the antimony atoms are in a trigonal environment of ligands with the oxygen atoms of MeO groups coordinated to the metal atom (intramolecular Sb∙∙∙OМе distances are 3.053(1), 3.054(1), and 3.011(1) A, CN = 3 + 3). The effect of tert-butyl hydroperoxide on a mixture of tris(2-methoxy-5-chlorophenyl)antimony and bromoacetic, 3-fluorophenylacetic, and 2,3-difluorobenzoic acids in ether leads to the formation of trigonal bipyramidal triarylantimony dicarboxylates (5-Cl-2-MeO-C6H3)3Sb[OC(O)R]2, where R = CH2Br (II), CH2C6H4F-3 (III), C6H3F2-2,3 (IV) with carboxylate ligands in apical positions. In crystals of compounds II–IV, the metal atom is additionally coordinated to the oxygen atoms of О=С and МеО groups.

The in vitro effect of the diabetes-associated markers insulin, leptin and oxidative stress on cellular characteristics promoting breast cancer progression is GLUT1-dependent

Obesity and type 2 diabetes mellitus (T2DM) associate with increased incidence and mortality from many cancers, including breast cancer. The mechanisms involved in this relation remain poorly understood. Our study aimed to investigate the in vitro effect of high levels of glucose, insulin, leptin, TNF-α, INF-γ and oxidative stress (induced with tert-butylhydroperoxide (TBH)), which are associated with T2DM, upon glucose uptake by breast cancer (MCF-7 and MDA-MB-231) and non-cancer (MCF-12A) cells and to correlate this effect with their effects upon cellular characteristics associated with cancer progression (cell proliferation, viability, migration, angiogenesis and apoptosis).3H-DG uptake was markedly inhibited by a selective GLUT1 inhibitor (BAY-876) in all cell lines, proving that 3H-DG uptake is mainly GLUT1-mediated. TBH (2.5 μM), insulin (50 nM), leptin (500 ng/ml) and INF-y (100 ng/ml) stimulate GLUT1-mediated 3H-DG (1 mM) uptake by both ER-positive and triple-negative breast cancer cell lines. TBH and leptin, but not insulin and INF-γ, increase GLUT1 mRNA levels. Insulin and leptin (in both ER-positive and triple-negative breast cancer cell lines) and TBH (in the triple-negative cell line) have a proproliferative effect and leptin possesses a cytoprotective effect in both breast cancer cell lines that can contribute to cancer progression. The effects of TBH, insulin, leptin and INF-γ upon breast cancer cell proliferation and viability are GLUT1-dependent.In conclusion, T2DM-associated characteristics induce changes in GLUT1-mediated glucose uptake that can contribute to cancer progression. Moreover, we conclude that BAY-876 can be a strong candidate for development of a new effective anticancer agent against breast cancer.

O33 Oxidative stress induced both apoptosis and necroptosis in endothelial cells

Oxidative stress plays an essential role in mediating endothelial cell death while the underlying mechanisms remain elusive. Previous reports suggested that ROS overproduction induced both apoptosis and necrosis. Herein, the pro-death effect of tert-butyl hydroperoxide (t-BHP), an alternative for H2O2, on human umbilical vein endothelial cells was investigated with a low concentration (50 μM) of t-BHP (t-BHPL) and a high concentration (500 μM) of t-BHP (t-BHPH). T-BHPL induced caspase-dependent apoptosis and ROS generation, which was inhibited by N-acetyl- l -cysteine (NAC). Furthermore, NADPH oxidase inhibitor diphenyleneiodonium (DPI), NOX4 siRNA, and NOX4 inhibitor GKT137831 reduced t-BHPL-induced ROS generation while mitochondrial respiratory chain inhibitors rotenone (Rot), 2-thenoyltrifluoroacetone (TTFA), and antimycin A (AA) failed to do so. NOX4 overexpression resulted in increased ROS generation and Akt expression but decreased sensitivity to t-BHPL. In contrast, RIP1 inhibitor necrostatin-1, MLKL inhibitor necrosulfonamide and silencing RIP1, RIP3, and MLKL inhibited t-BHPH-induced cell death while pan-caspase inhibitor Z-VAD-FMK showed no effect. t-BHPH-induced ROS production was inhibited by TTFA, AA and Rot while DPI showed no effect. T-BHPH induced RIP1/RIP3 interaction, which was decreased by Rot, TTFA, and AA. Silencing RIP1 and RIP3, but not MLKL, inhibited t-BHPH-induced mitochondrial membrane potential (MMP) decrease and ROS production. In summary, t-BHP induced both apoptosis and necroptosis in endothelial cells which was mediated by ROS. ROS derived from NADPH oxidase and mitochondria contributed to t-BHPL and t-BHPH-induced apoptosis and necroptosis, respectively.

Stimulation of dopamine oxidation in liver mitochondria by palmitic acid in the presence of ATP and tert-butylhydroperoxide

The effect of palmitic acid on the oxidation of dopamine, i.e., on the monoamine oxidase (MA-oxidase) activity, was investigated on deenergized liver mitochondria, upon energization by ATP and also in the presence of an oxidizing agent tert-butylhydroperoxide (TBH). It was found that palmitic acid reduces the value of the apparent Km for dopamine without alteration of the apparent Vmax. This points to stimulation of the mitochondrial MA-oxidase activity by palmitic acid at low concentrations of dopamine. Stimulatory effect of palmitic acid may be related to the ability of amphiphilic compounds to increase the negative charge density on the outer mitochondrial membrane. This leads to an increase in the local concentration of positively charged ions of dopamine in the layer adjacent to the membrane near the active site of monoamine oxidase. ATP eliminates the ability of palmitic acid to stimulate the MA-oxidase activity of mitochondria. This effect of ATP is not observed in the presence of the FOF1-ATP-synthase inhibitor oligomycin. Apparently, in the case of vector transport of H+ from the matrix induced by ATP-hydrolysis, protonation of palmitic acid anions occurs on the outer mitochondrial membrane, followed by the movement of the neutral molecules to the outer and then to the inner monolayer of the inner membrane. It was found that TBH at a concentration of 300 μM has no significant effect on the ATPase activity of mitochondria and in the presence of ATP and palmitic acid reduces the value of the apparent Km for dopamine without alteration of the apparent Vmax. Antioxidant thiourea eliminates this effect of TBH. We propose that the TBH-induced oxidative stress in the case of ATP-energized mitochondria results in the movement of palmitic acid molecules from the inner to the outer membrane. This leads to an increase in the density of negative charges on the surface of this membrane and, therefore, to the stimulation of the dopamine oxidation.

Features of inhibition of glycerol-3-phosphate oxidase activity of liver mitochondria by palmitic acid in the presence of ATP and tert-butylhydroperoxide

The effect of palmitic acid on glycerol-3-phosphate oxidation, i.e., on the glycerol-3-phosphate oxidase (GP oxidase) activity of liver mitochondria was investigated in the presence and absence of ATP, as well as under conditions of tert-butylhydroperoxide (TBH)-induced oxidative stress. It was found that palmitic and lauric acids inhibit GP oxidase activity of deenergized liver mitochondria formally in the competitive manner. In this case lauric acid, which is less hydrophobic than palmitic acid, inhibits GP oxidase activity of mitochondria significantly weaker. It is noted that the competitive inhibition of mitochondrial GP oxidase activity by these saturated fatty acids may be related to their ability, as amphiphilic compounds, to increase the negative charge density on the outer surface of the inner mitochondrial membrane. It is shown that ATP at a concentration of 2 mM eliminates the ability of palmitic acid to inhibit GP oxidase activity of mitochondria. This effect of ATP is not observed in the presence of F O F 1-ATP synthase inhibitor oligomycin. Apparently, in the case of vector transport of H+ from the matrix to the intermembrane space of mitochondria induced by ATP hydrolysis there is occurs protonation of palmitic acid anions on the outer surface of the inner membrane and subsequent movement of its neutral molecules to the inner monolayer of the inner membrane. It was found that TBH at a concentration of 300 µM in the presence of ATP and palmitic acid inhibits their GP oxidase activity formally in the competitive manner without significant effect on ATPase activity of liver mitochondria. Thiourea antioxidant eliminates this effect of TBH. It is assumed that TBH-induced oxidative stress in the case of ATP-energized mitochondria results in an increase in transport rate of palmitic acid anions from the inner monolayer of the inner membrane to its outer monolayer. This, in turn, is accompanied by an increase in the density of negative charges on the outer surface of the inner mitochondrial membrane and inhibition of GP oxidase activity of liver mitochondrial formally in the competitive manner.

The water extract of Angelica Sinensis Radix protects cultured PC12 cells against oxidative stress: suppression of reactive oxygen species and activation of anti-oxidant response elements

Objective: Angelica Sinensis Radix (ASR; Dang Gui; the root of Angelica sinensis) is a herbal supplement that has been used in invigorating blood circulation. Here, we provided different lines of evidence to support the beneficial role of ASR against oxidative stress in cultured PC12 cell, a rat pheochromocytoma cell line. Methods: The water extract of ASR inhibited the activity of xanthine oxidase in vitro. In cultures, the pre-treatment of ASR water extract reduced the cytotoxic effect of tert-butyl hydroperoxide (tBHP), an oxidative stress inducer. Results: The protecting mechanisms of ASR were shown to be mediated by: (i) suppression of tBHP-induced reactive oxygen species (ROS) formation; (ii) induction of caspase-3 and PARP activities; and (iii) stimulation of mRNAs encoding antioxidative genes, glutathione S-transferase A2 (GSTA2) and NAD(P)H dehydrogenase quinone oxidoreductase 1 (NQO1), via the transcriptional activation of antioxidant response element (ARE). The outcome was the prevention of tBHP-induced cell apoptosis. Conclusion: Interestingly, the protective effect of ASR extract in PC12 cells were insignificant when challenging by insults of -amlyoid (for Alzheimer’s disease) and MPP+ (for Parkinson’s disease). Taken together, we revealed a neurobeneficial role of ASR in protecting neuronal cells against oxidative damage, which might be useful in developing health food supplements for disease prevention in future.

Oxidative stress decreases uptake of neutral amino acids in a human placental cell line (BeWo cells)

Increased oxidative stress (OS) is implicated in the pathophysiology of several pregnancy disorders. We aimed to investigate the effect of tert-butylhydroperoxide (TBHP)-induced OS upon the placental transport of the neutral amino acids l-methionine (l-Met) and l-alanine (l-Ala), by using a human trophoblast cell model (BeWo cells). TBHP reduced both total and Na+-independent 14C-l-Met intracellular steady-state accumulation over time (Amax), by reducing non-system l-mediated uptake – most probably system y+ – while having no effect on system L. Moreover, TBHP reduced total 14C-l-Ala Amax through an inhibition of system A. The effect of TBHP upon total, but not system A-mediated, 14C-l-Ala uptake was dependent upon phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) activation, and was completely prevented by the polyphenol quercetin. In conclusion, a reduction in placental uptake of neutral amino acids may contribute to the deleterious effects of pregnancy disorders associated with OS.

Mechanism of induction of oxidative stress in liver mitochondria by low concentrations of tert-butyl hydroperoxide

The mechanism of the effect of tert-butyl hydroperoxide (tBHP) on the kinetics of decrease in liver mitochondrial ΔΨ (transmembrane electric potential) in response to successive additions of tBHP in low concentrations has been studied. FeSO(4) was found to increase significantly the damaging effect of tBHP; this effect was shown to increase in the presence of low concentrations of Ca2+ starting from 2 µM CaCl(2). Cyclosporin A prevents these effects. The data show that the damaging effect of low concentrations of tBHP in the course of pyruvate oxidation in isolated liver mitochondria is caused by the opening of the nonspecific Ca2+-dependent cyclosporin A-sensitive pore in the inner mitochondrial membrane. Application of a method of studying oxidative stress regulators, developed in this work, is illustrated by an example of the prooxidant action of ascorbate. This method is proposed for studying mitochondria in hemochromatosis, a pathology caused by excessive accumulation of iron.

The effect of tert-butyl hydroperoxide on hepatic transcriptome expression patterns in the striped sea bream (Lithognathus mormyrus; Teleostei)

The study was aimed at examining the effects of tert-butyl hydroperoxide (tBHP) on hepatic transcriptome expression patterns of the teleost fish Lithognathus mormyrus. tBHP is an organic hydro-peroxide, widely used as a model pro-oxidant. It generates the reactive oxygen species (ROS) tert-butoxyl and tert-butylperoxyl. Complementary DNAs of tBHP-treated vs control fish were applied onto a previously produced cDNA microarray of ∼ 1500 unique sequences. The effects of the tBHP application were demonstrated by leukocyte infiltration into the liver and by differential expression of various genes, some already known to be involved in ROS-related responses. Indicator genes of putative ROS effects were: aldehyde dehydrogenase 3A2, Heme oxygenase and the hemopexin-like protein. Putative indicators of transendothelial leukocyte migration and function were: p22phox, Rac1 and CD63-like genes. Interestingly, 7-dehydrocholesterol reductase was significantly down-regulated in response to all treatments. Several non-annotated genes revealed uniform directions of differential expression in response to all treatments.

Protective role of ortho-substituted Mn(III) N-alkylpyridylporphyrins against the oxidative injury induced by tert-butylhydroperoxide

The present work addresses the role of two ortho-substituted Mn(III) N-alkylpyridylporphyrins, alkyl being ethyl in MnTE-2-PyP5+ and n-hexyl in MnTnHex-2-PyP5+, on the protection against the oxidant tert-butylhydroperoxide (TBHP). Their protective role was studied in V79 cells using endpoints of cell viability (MTT and crystal violet assays), intracellular O2•– generation (dihydroethidium assay) and glutathione status (DTNB and monochlorobimane assays). MnPs per se did not show cytotoxicity (up to 25 μM, 24 h). The exposure to TBHP resulted in a significant decrease in cell viability and in an increase in the intracellular O2•– levels. Also, TBHP depleted total and reduced glutathione and increased GSSG. The two MnPs counteracted remarkably the effects of TBHP. Even at low concentrations, both MnPs were protective in terms of cell viability and abrogated the intracellular O2•– increase in a significant way. Also, they augmented markedly the total and reduced glutathione contents in TBHP-treated cells, highlighting the multiple mechanisms of protection of these SOD mimics, which at least in part may be ascribed to their electron-donating ability.

Hypochlorous acid-induced oxidative damage of human red blood cells: effects of tert-butyl hydroperoxide and nitrite on the HOCl reaction with erythrocytes

Hypochlorous acid, one of the most powerful biological oxidants, is believed to be important in the pathogenesis of some diseases. The purpose of this study was to further characterise the membrane and intracellular events which resulted in HOCl-induced oxidative impairments and haemolysis of human erythrocytes and interaction of different oxidative agents, which accumulated during respiratory burst, in the process of RBS oxidation. The sequence of cellular events after red blood cell exposure to HOCl: cell morphological transformations, oxidation of cellular constituents, enzyme modifications, and haemolysis have been evaluated. It was shown that HOCl-treated cells underwent colloid-osmotic haemolysis, preceded by rapid morphological transformations and membrane structural transitions. The activation energy of the process of haemolysis (after removal of the excess of oxidative agent) was estimated to be 146±22 kJ/mol at temperatures above the break point of Arrhenius plot (31–32 °C). This value corresponds to the activation energy of the process of protein denaturation. Modification of erythrocytes by HOCl inhibited membrane acetylcholinesterase (uncompetitive type of inhibition), depleted intracellular glutathione, activated intracellular glutathione peroxidase, but did not induce membrane lipid peroxidation. The presence of other oxidants, nitrite or tert-butyl hydroperoxide ( t-BHP), promoted the oxidative damage induced by HOCl and led to new oxidative reactions.

Lipids up-regulate uncoupling protein 2 expression in rat hepatocytes

Background & Aims: Hepatic steatosis reflects the accumulation of triglycerides and free fatty acids in hepatocytes. Although lipids and their metabolites are potentially hepatotoxic, the absence of overt injury in fatty livers suggests that adaptive responses to lipid accumulation occur. Fatty acids induce mitochondrial uncoupling proteins (UCP) 2 and 3 in muscle and fat, providing a mechanism to dispose of excessive fatty acids. Although hepatocytes do not normally express uncoupling proteins, UCP-2 is expressed in hepatocytes of genetically obese mice with fatty livers, suggesting that lipids also induce UCP-2 in hepatocytes. Methods: To test whether lipids up-regulate hepatocyte UCP-2, cultures of rat hepatocytes were treated with lipid emulsions, linoleic or oleic acid, and UCP-2 expression was evaluated by Northern blotting and immunocytochemistry. Because increased reactive oxygen species (ROS) production may contribute to lipid-related UCP-2 induction, the DNA-binding activity of the ROS-activated transcription factor, NF-κB, was measured, and the effects of tert-butyl hydroperoxide (TBHP) and glutathione (GSH) on UCP-2 induction were also assessed. Results: Lipid emulsions increased the DNA-binding activity of NF-κB and resulted in a dose- and time-dependent induction of UCP-2 transcripts in cultured hepatocytes; after 24 hours, UCP-2 messenger RNA levels were increased 4.5-fold, and increased UCP-2 protein was shown by immunocytochemistry. Consistent with the possibility that ROS generated intracellularly during lipid metabolism participates in UCP-2 induction, addition of the cell-impermeable antioxidant GSH did not alter lipid-related induction of UCP-2. Furthermore, TBHP, which is known to increase hepatocyte mitochondrial ROS production, also increased UCP-2 messenger RNA levels. Conclusions: Lipids increase ROS and induce UCP-2 in hepatocytes. Thus, the liver may adapt to an excessive supply of lipid substrates by inducing UCP-2 to facilitate substrate disposal while constraining ROS production. GASTROENTEROLOGY 1999;116:1184-1193

The Inositol 1,4,5-Trisphosphate-Generating Agonist ATP Enhances DNA Cleavage Induced bytert-Butylhydroperoxide

In this paper we present experimental evidence indicating that DNA cleavage induced bytert-butylhydroperoxide in U937 cells can be enhanced via ATP-mediated activation of membrane receptors coupled with hydrolysis of phosphatidylinositol 4,5-bisphosphate. The mechanism whereby ATP exerts this effect involves release of Ca2+from the inositol 1,4,5-trisphosphate (IP3)-sensitive stores, further release of the cation from the ryanodine receptor, mitochondrial clearance of the fraction of Ca2+derived from the ryanodine receptor, and Ca2+-dependent mitochondrial formation of DNA-damaging species. IP3-generating agonists must therefore be considered as potential modulators of the genotoxic effects oftert-butylhydroperoxide.

The effect of tert-butyl hydroperoxide on peritoneal macrophages and the protective effect of protein-bound polysaccharide administered intraperitoneally and orally.

Our previous studies have shown that a protein-bound polysaccharide, polysaccharide krestin (PSK), can protect macrophages from lipoperoxidative injury induced by tert-butyl hydroperoxide (tbOOH) and oxidatively modified low density lipoprotein (O-LDL). PSK was administered intraperitoneally to mice, and the peritoneal macrophages harvested were used as an experimental model. PSK does not reveal any protective effect on macrophages injured by tbOOH when incubated in vitro. In order to elucidate its mode of action, in the present study, the protective effects of PSK were further investigated using morphological changes and viability as indices. It was shown that, compared with the non-PSK group, the viability of macrophages was much higher in the PSK group, and the morphological changes in the PSK group were much less than those in the non-PSK group. The protective effect of PSK administered intraperitoneally and orally on macrophages injured by tbOOH was comparable. Furthermore, the serum of the PSK treated mice had the same protective effect.

The mitochondrial permeability transition pore is modulated by oxidative agents through both pyridine nucleotides and glutathione at two separate sites.

We studied the modulation of the permeability transition pore (MTP), a cyclosporin-A-sensitive channel, in deenergized mitochondria. Rat liver mitochondria were incubated in a potassium gluconate medium and treated with uncoupler, valinomycin, oligomycin and A23187. Under these conditions the membrane and Donnan potentials are collapsed, and no ion gradients are maintained, allowing the study of the dependence of MTP opening on the Ca2+ concentration under a variety of oxidative conditions without the complexities arising from changes of the membrane potential and matrix pH, and from secondary-ion redistribution. Cross-linking of mitochondrial dithiols with arsenite or phenylarsine oxide, or treatment with tert-butylhydroperoxide leading to complete oxidation of glutathione, increased the sensitivity of MTP opening to Ca2+. This effect could be fully prevented by prior treatment of mitochondria with monobromobimane and restored by reduction with dithiothreitol. The effect of tert-butylhydroperoxide was not additive with that of AsO, and it was completely blocked by modification of reduced glutathione with 1-chloro 2,4-dinitrobenzene through glutathione-S-transferase, indicating that oxidized glutathione affects the pore through the AsO-reactive and PhAsO-reactive dithiol. Oxidation of mitochondrial pyridine nucleotides by a variety of treatments also increased the sensitivity of MTP opening to Ca2+ under conditions where glutathione was maintained in the reduced state. This effect could be fully prevented when reduced pyridine nucleotides levels were reestablished with 2-hydroxybutyrate but not by treatment with monobromobimane or dithiothreitol. The effects of dithiol cross-linking or oxidation, and of pyridine nucleotide oxidation on the MTP were additive. The contribution of each of these two oxidative events, when they were induced at the same time, could still be selectively blocked by monobromobimane and dithiothreitol. The effects of dithiol cross-linking or oxidation, and of pyridine nucleotide oxidation on the MTP were additive. The contribution of each of these two oxidative events,when they were induced at the same time, could still be selectively blocked by monobromobimane and dithiothreitol, or by 2-hydroxybutyrate, respectively. Complete oxidation of pyridine nucleotides did not affect the reactivity of the dithiol towards monobromobimane, indicating that it remained in the reduced state. After transient opening of the MTP, sensitivity to pyridine nucleotide oxidation was lost while sensitivity to dithiol cross-linking was retained. These data indicate that the dithiol belongs to an MTP regulatory component which is larger than the MTP cutoff of about 1500 Da, or is non-diffusible. Taken together, these findings indicate that the MTP is influenced by oxidation-reduction events at two separate sites that can be distinguished experimentally, and that these sites are not connected by common oxidation-reduction intermediates other than glutathione.

The effects of tert-butyl hydroperoxide on human erythrocyte membrane ion transport and the protective actions of antioxidants

The oxidising actions of tert-butyl hydroperoxide (tBH) (0–3 mmol/l) on human erythrocyte membrane ion transport have been studied using measurements of 86Rb + influx. Ouabain and bumetanide were used to distinguish Rb + flux via the sodium pump (Na,K-ATPase), Na,K,2Cl cotransporter and through residual membrane permeability. The protective actions of antioxidants and related molecules (vitamin E, vitamin E acetate, Trolox, butylated hydroxytoluene (BHT) and dithioerythritol (DTE)) were studied. The effects of tBH were concentration dependent and the mean residual (ouabain and bumetanide insensitive) Rb + permeability was increased by a factor of 8.5 (S.E.M. 2.2, n = 15) by a 5-min exposure to 2 mmol/l tBH. This action was almost completely prevented by co-incubation with Trolox or BHT, and partially prevented by the presence of vitamin E or DTE. Incubation with 2 mmol/l tBH for 5 min increased intracellular Na + by a factor of 1.8 (S.E.M. 0.1, n = 8) and reduced intracellular K + by a factor of 0.93 (S.E.M. 0.03, n = 8). These effects were prevented by Trolox and partially prevented by vitamin E, whereas DTE and vitamin E acetate were ineffective. Incubation with 2 mmol/l tBH for 5 min reduced the mean apparent sodium pump V max by 43% (S.E.M. 4, n = 8). This effect was completely prevented by Trolox and partially prevented by vitamin E. Vitamin E acetate had no effect. The mean bumetanide-sensitive Rb + influx via the Na,K,2CI cotransporter was reduced by 30% (S.E.M. 8.7, n = 25) by a 5-min exposure to 2 mmoll tBH. This action was variable and no significant actions of the antioxidants studied could be demonstrated. This study suggests that tBH-mediated oxidative damage occurs from a hydrophilic site and involves increased non-selective membrane cation permeability and inhibition of specific transport systems.

Hydroperoxide-induced nucleotide degradation and adenosine formation in isolated rat cardiomyocytes

The effect of tert-butyl hydroperoxide (tBH) on the nucleotide level in isolated cardiomyocytes from adult rats was examined. Incubation of myocytes with the hydroperoxide resulted in loss of both ATP and NAD from mitochondria and cytosol. In contrast, only the cytosolic level of NADP was increased. The effect on myocyte content of ADP and AMP was less pronounced. A significant increase in formation and release of adenosine also occurred in tBH-exposed cells. Treatment of cardiomyocytes with tBH resulted in a progressive decline of rod-shaped and viable cells, which was accompanied by an increase in numbers of hypercontracted and round myocytes in the incubation mixture. Electron microscopic examination showed an intense contraction of the cells. In summary our results demonstrate that plasma membrane damage and hypercontraction are early manifestations of tBH-induced injury to isolated cardiomyocytes. The depletion of ATP and formation of adenosine also suggest that degradation of adenine nucleotides may contribute to the cytotoxicity caused by tBH. Possible mechanisms involved in the ATP depletion are discussed.

Effects of membrane lipid peroxidation by tert butyl hydroperoxide on the sodium current in isolated feline ventricular myocytes.

Membrane lipid peroxidation is known to play a pivotal role in the genesis of coronary reperfusion arrhythmias in both experimental and clinical settings. To elucidate the electrophysiological mechanisms underlying these arrhythmias, the effects of tert butyl hydroperoxide (TBH) on the Na+ current (INa) in isolated feline ventricular myocytes were studied using whole-cell patch clamp techniques under 100% O2 bubbling. This agent at 20 mM inhibited INa from 2.2 +/- 1.3 to 1.7 +/- 1.0 nA (P < 0.01, n = 7) without changing time courses of INa inactivation. Twenty millimoles TBH shifted the steady-state inactivation curve for INa from -77.4 +/- 1.7 to -81.3 +/- 1.8 mV when measured at INa half inhibition voltage (P < 0.01, n = 7), but did not affect the slope factor. The kinetics of INa recovery from inactivation remained unchanged. These findings suggest that lipid peroxidation in the membrane by TBH reduces INa conductance and voltage-dependent INa availability, most likely as a result of structural damage to the Na+ channels.

Tert-Butyl Hydroperoxide-Induced [Ca2+]i in Thymus and Thymoma Cells

The effect of tert-butyl hydroperoxide (t-BOOH) on Ca2+ homeostasis in tumor thymocytes in comparison to normal thymus cells has been studied. t-BOOH induces an early Ca2+ increase which shows different kinetic and extent in normal and tumor thymocytes. However, evidence is reported that in both types of cells it is just an epiphenomenon not directly related to late injury event. The different effects of various compounds (EGTA, dithiothreitol, deferoxamine and glucose) on the early Ca2+ increase and the late LDH release induced by t-BOOH suggest the occurrence, only in tumor cells, of some undefined deleterious process related to Ca2+ movements.

Effect of tert-butyl hydroperoxide on cyclooxygenase and lipoxygenase metabolism of arachidonic acid in rabbit platelets

The effect of tert-butyl hydroperoxide (t-BOOH) on the formation of thromboxane (TX) B 2, 12-hydroxy-5, 8, 10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from exogenous arachidonic acid (AA) in washed rabbit platelets was examined. t-BOOH enhanced TXB 2 and HHT formation at concentrations of 8 μM and below, and at 50 μM it inhibited the formation, suggesting that platelet cyclooxygenase activity can be enhanced or inhibited by t-BOOH depending on the concentration. t-BOOH inhibited 12-HETE production in a dose-dependent manner. When the platelets were incubated with 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) instead of AA, t-BOOH failed to inhibit the conversion of 12-HPETE to 12-HETE, indicating that the inhibition of 12-HETE formation by t-BOOH occurs at the lipoxygenase step. Studies utilizing indomethacin (a selective cyclooxygenase inhibitor) and desferrioxamine (an iron-chelating agent) revealed that the inhibitory effect of t-BOOH on the lipoxygenase is not mediated through the activation of the cyclooxygenase and that this effect of t-BOOH is due to the hydroperoxy moiety. These results suggest that hydroperoxides play an important role in the control of platelet cyclooxygenase and lipoxygenase activities.