Changes in oxygen consumption induced by t-butyl hydroperoxide in perfused rat liver. Effect of free-radical scavengers.

The addition of t-butyl hydroperoxide to perfused rat liver elicited a biphasic effect on hepatic respiration. A rapid fall in liver oxygen consumption was initially observed, followed by a recovery phase leading to respiratory rates higher than the initial steady-state values of oxygen uptake. This overshoot in hepatic oxygen uptake was abolished by free-radical scavengers such as (+)-cyanidanol-3 or butylated hydroxyanisole at concentrations that did not alter mitochondrial respiration. (+)-Cyanidanol-3 was also able to facilitate the recovery of respiration, the diminution in the calculated rate of hydroperoxide utilization and the decrease in liver GSH content produced by two consecutive pulses of t-butyl hydroperoxide. It is suggested that the t-butyl hydroperoxide-induced overshoot in liver respiration is related to increased utilization of oxygen for lipid peroxidation as a consequence of free radicals produced in the scission of the hydroperoxide by cellular haemoproteins.     

Chlorophyll destruction in the presence of bisulfite and linoleic acid hydroperoxide

Chlorophyll was rapidly destroyed in the presence of bisulfite and linoleic acid hydroperoxide. Both bisulfite and linoleic acid hydroperoxide were required for chlorophyll destruction and both were consumed in the reaction; however, there was no oxygen requirement. Chlorophyll destruction occurred most readily in the slightly acidic pH region with little destruction occurring above pH 8. The free radical scavengers, hydroquinone and α-tocopherol, were very effective at inhibiting chlorophyll destruction, but the singlet oxygen quenchers, β-carotene, 2,5-dimethylfuran and 1,3-diphenylisobenzofuran, were only slightly effective. The addition of metal chelators indicated that metals were not participating in the reaction. The evidence indicates that chlorophyll was destroyed by a free radical mechanism. Based on the present results and that of others, it is suggested that chlorophyll was destroyed via oxidation by the alkoxy radical which was produced during the decomposition of linoleic acid hydroperoxide by bisulfite.     

Generation of free radicals from lipid hydroperoxides by Ni2+ in the presence of oligopeptides.

The generation of free radicals from lipid hydroperoxides by Ni2+ in the presence of several oligopeptides was investigated by electron spin resonance (ESR) utilizing 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap. Incubation of Ni2+ with cumene hydroperoxide or t-butyl hydroperoxide did not generate any detectable free radical. In the presence of glycylglycylhistidine (GlyGlyHis), however, Ni2+ generated cumene peroxyl (ROO.) radical from cumene hydroperoxide, with the free radical generation reaching its saturation level within about 3 min. The reaction was first order with respect to both cumene hydroperoxide and Ni2+. Similar results were obtained using t-butyl hydroperoxide, but the yield of t-butyl peroxyl radical generation was about 7-fold lower. Other histidine-containing oligopeptides such as beta-alanyl-L-histidine (carnosine), gamma-aminobutyryl-L-histidine (homocarnosine), and beta-alanyl-3-methyl-L-histidine (anserine) caused the generation of both cumene alkyl (R.) and cumene alkoxyl (RO.) radicals in the reaction of Ni2+ with cumene hydroperoxide. Similar results were obtained using t-butyl hydroperoxide. Glutathione also caused generation of R. and RO. radicals in the reaction of Ni2+ with cumene hydroperoxide but the yield was approximately 25-fold greater than that produced by the histidine-containing peptides, except GlyGlyHis. The ratio of DMPO/R. and DMPO/RO. produced with glutathione and cumene hydroperoxide was approximately 3:1. Essentially the same results were obtained using t-butyl hydroperoxide except that the ratio of DMPO/R. to DMPO/RO. was approximately 1:1. The free radical generation from cumene hydroperoxide reached its saturation level almost instantaneously while in the case of t-butyl hydroperoxide, the saturation level was reached in about 3 min. In the presence of oxidized glutathione, the Ni2+/cumene hydroperoxide system caused DMPO/.OH generation from DMPO without forming free hydroxyl radical. Since glutathione, carnosine, homocarnosine, and anserine are considered to be cellular antioxidants, the present work suggests that instead of protecting against oxidative damage, these oligopeptides may facilitate the Ni(2+)-mediated free radical generation and thus may participate in the mechanism(s) of Ni2+ toxicity and carcinogenicity.     

Organic hydroperoxide-dependent oxidation of ethanol by microsomes: lack of a role for free hydroxyl radicals

Organic hydroperoxides can replace NADPH in supporting the oxidation of ethanol by liver microsomes. Experiments were carried out to evaluate the role of hydroxyl radicals in the organic hydroperoxide-catalyzed reaction. Maximum rates of ethanol oxidation occurred in the presence of either 0.5 mM cumene hydroperoxide or 2.5 mM t-butyl hydroperoxide and were linear for 2 to 4 min. The Km for ethanol was about 12 mM and Vmax was about 8 nmol ethanol oxidized/min/mg microsomal protein. Besides ethanol, the organic hydroperoxides supported the oxidation of longer-chain alcohols (1-butanol), and secondary alcohols (isopropanol). The organic hydroperoxide-supported oxidation of alcohols was not affected by several hydroxyl-radical scavengers such as dimethylsulfoxide, mannitol, or 2-keto-4-thiomethylbutyrate which blocked NADPH-dependent oxidation of alcohols by 50% or more. Iron-EDTA, which increases the production of hydroxyl radicals, increased the NADPH-dependent oxidation of ethanol, whereas desferrioxamine, which blocks the production of hydroxyl radicals, inhibited the NADPH-dependent oxidation of ethanol. Neither iron-EDTA nor desferrioxamine had any effect on the organic hydroperoxide-supported oxidation of ethanol. Cumene-and t-butyl hydroperoxide did not support microsomal oxidation of hydroxyl-radical scavengers. These results suggest that, in contrast to the NADPH-dependent oxidation of ethanol, free-hydroxyl radicals do not play a role in the organic hydroperoxide-dependent oxidation of ethanol by microsomes. Ethanol appears to be oxidized by two pathways in microsomes, one which is dependent on hydroxyl radicals, and the other which appears to be independent of these oxygen radicals.     

t-Butyl hydroperoxide alters fatty acid incorporation into erythrocyte membrane phospholipid

Because the ability of cells to replace oxidized fatty acids in membrane phospholipids via deacylation and reacylation in situ may be an important determinant of the ability of cells to tolerate oxidative stress, incorporation of exogenous fatty acid into phospholipid by human erythrocytes has been examined following exposure of the cells to t-butyl hydroperoxide. Exposure of human erythrocytes to t-butyl hydroperoxide (0.5-1.0 mM) results in oxidation of glutathione, formation of malonyldialdehyde, and oxidation of hemoglobin to methemoglobin. Under these conditions, incorporation of exogenous [9,10-3H]oleic acid into phosphatidylethanolamine is enhanced while incorporation of [9,10-3H]oleic acid into phosphatidylcholine is decreased. These effects of t-butyl hydroperoxide on [9,10-3H]oleic acid incorporation are not affected by dissipating transmembrane gradients for calcium and potassium. When malonyldialdehyde production is inhibited by addition of ascorbic acid, t-butyl hydroperoxide still decreases [9,10-3H]oleic acid incorporation into phosphatidylcholine but no stimulation of [9,10-3H]oleic acid incorporation into phosphatidylethanolamine occurs. In cells pre-treated with NaNO2 to convert hemoglobin to methemoglobin, t-butyl hydroperoxide reduces [9,10-3H]oleic acid incorporation into phosphatidylcholine by erythrocytes but does not stimulate [9,10-3H]oleic acid incorporation into phosphatidylethanolamine. Under these conditions oxidation of erythrocyte glutathione and formation of malonyldialdehyde still occur. These results indicate that membrane phospholipid fatty acid turnover is altered under conditions where peroxidation of membrane phospholipid fatty acids occurs and suggest that the oxidation state of hemoglobin influences this response.     

Effects of Oxygen Radical Scavengers on the Inactivation of SS ΦX174 DNA by the Semi-Quinone Free Radical of the Antitumor Agent Etoposide

We have studied the effects of oxygen radical scavengers on the inactivation of ss ΦX174 DNA by the semi-quinone free radical of the antitumor agent etoposide (VP 16-213), which was generated from the ortho-quinone of etoposide at pH ≥ 7.4. A semi-quinone free radical of etoposide is thought to play a role in the inactivation of ss ΦDX174 DNA by its precursors 3',4'-ortho-quinone and 3',4'-ortho-dihydroxy-derivative. The possible role of oxygen radicals formed secondary to semi-quinone formation in the inactivation of DNA by the semi-quinone free radical was investigated using the hydroxyl radical scavengers t-butanol and DMSO. the spin trap DMPO, the enzymes catalase and superoxide dismutase, the iron chelator EDTA and potassium superoxide. Hydroxyl radicals seem not important in the process of inactivation of DNA by the semi-quinone free radical, since t-butanol, DMSO, catalase and EDTA had no inhibitory effect on DNA inactivation. The spin trapping agent DMPO strongly inhibited DNA inactivation and semi-quinone formation from the ortho-quinone of etoposide at pH ≥ 7.4 with the concomitant formation of a DMPO-OH adduct. This adduct probably did not arise from OH· trapping but from trapping of O₂^-. DMSO increased both the semi-quinone formation from and the DNA inactivation by the ortho-quinone of etoposide at pH ≥ 7.4. Potassium superoxide also stimulated ΦDX174 DNA inactivation by the ortho-quinone at pH ≤ 7. From the present study, it is also concluded that superoxide anion radicals probably play an important role in the formation of the semi-quinone free radical from the orthoquinone of etoposide, thus indirectly influencing DNA inactivation.     

Low-level chemiluminescence from Drosophila melanogaster fed with chemical mutagens polycyclic aromatic hydrocarbon quinones and a carcinogenic bracken fern

The spontaneous photon emission (chemiluminescence) from Drosophila melanogaster fed chemical mutagens, polycyclic aromatic hydrocarbon quinones, and a carcinogenic bracken fern was studied. The fly chemiluminescence was evidently enhanced by mutagen or carcinogen administration and was increased proportionally to the administered amount of tested compound. Strong chemiluminescence was observed especially at the larval stage. Living larvae emitted stronger chemiluminescence than their homogenate. The chemiluminescence from Drosophila melanogaster fed polycyclic aromatic hydrocarbon quinones showed a linear relation with the mutation frequency in the Drosophila wing spot test. The chemiluminescence from flies fed a bracken fern decreased by the addition of free radical scavengers and active oxygen quenchers. The phosphatidylcholine hydroperoxide concentration in the flies was increased proportionally with the chemiluminescence intensity. It seems that the free radical formation is stimulated as shown by the enhanced chemiluminescence in mutagen- or carcinogen-dosed flies, and as a result, lipid peroxide accumulation accompanies mutation in Drosophila melanogaster.     

The generation of hydroxyl and alkoxyl radicals from the interaction of ferrous bipyridyl with peroxides.

Reaction conditions by which the iron-chelate ferrous bipyridyl can be used as a Fenton reagent to generate specifically alkoxyl radical (.OR) from its corresponding alkyl hydroperoxide (ROOH) without producing hydroxyl radical (.OH) as a result of autoxidation are described. In this manner, the relative ability of common .OH-scavenging agents to react with .OH and various .OR species could be assessed. When .OH was generated from H2O2, 4-methylmercapto-2-oxobutyrate, ethanol and benzoate all were oxidized. When .OR (cumoxyl radical, t-butoxyl radical or ethoxyl radical) was generated specifically, each was found to oxidize 4-methylmercapto-2-oxobutyrate and ethanol. In contrast with .OH, however, none of the .OR radicals mediated the decarboxylation of benzoate. Cross-competition studies with the scavengers showed that, in contrast with the .OH-dependent reaction, the .OR-dependent oxidation of 4-methylmercapto-2-oxobutyrate and ethanol was not inhibited by benzoate. Rate constants for ferrous bipyridyl oxidation by ROOH and by H2O2 were found to be essentially the same, and therefore the differential oxidation of the various scavengers was not a reflection of iron-peroxide interaction, but rather an interaction between generated oxy radicals and the scavengers. In contrast with the H2O2 system, catalase did not inhibit the oxidation of 4-methylmercapto-2-oxobutyrate or ethanol by either the cumene hydroperoxide or the t-butyl hydroperoxide system, suggesting that the oxidizing species was not derived from H2O2. These results suggest that benzoate decarboxylation might serve as a more specific probe to detect the presence of .OH than either 4-methylmercapto-2-oxobutyrate or ethanol, which react readily with .OR.     

Chemiluminescence studies on the generation of oxygen radicals from the interaction of NADPH-cytochrome P-450 reductase with iron

The ability of NADPH-cytochrome P-450 reductase to interact with iron and generate oxygen radicals was evaluated by assaying for low level chemiluminescence. The basic reaction system which contained the reductase, an NADPH-generating system, ferric-EDTA as an electron acceptor, and t-butyl hydroperoxide as the oxidant acceptor, resulted in the production of chemiluminescence. Omission of any of these components resulted in a complete loss of chemiluminescence. The light emission was completely sensitive to inhibition by glutathione and butylated hydroxytoluene, partially sensitive (about 60% decrease) to catalase and hydroxyl radical scavengers, and relatively insensitive (about 20% decrease) to superoxide dismutase. The ability of other ferric chelates to replace ferric-EDTA in catalyzing the reductase-dependent chemiluminescence was evaluated. Ferric-citrate, -ADP, -ATP, and ferric-ammonium sulfate were ineffective in promoting chemiluminescence, whereas ferric-diethylenetriaminepentaacetic acid was even more effective than ferric-EDTA. Thus, the ferric chelates, which catalyze reductase-dependent chemiluminescence, are those which are efficient electron acceptors from the reductase and were previously shown to be those capable of catalyzing hydroxyl radical production by microsomes and the reductase. It is suggested that chemiluminescence results from (a) the direct interaction of the reduced iron chelate with the hydroperoxide (Fenton-type of reaction) to generate alkoxyl and peroxyl radicals, and (b) the generation of hydroxyl radicals, which subsequently react with the hydroperoxide to generate secondary radicals. The latter, but not the former, would be sensitive to inhibition by catalase and competitive hydroxyl radical scavengers, whereas both would be sensitive to antioxidants such as butylated hydroxytoluene. Chemiluminescence appears to be a versatile tool for studying the reductase-dependent generation of oxygen radicals and for the interaction of reductase with iron.     

The participation of hydroperoxides and oxygen radicals in the control of prostaglandin synthesis

Our results demonstrate that the organic hydroperoxide t-butyl-hydroperoxide (TBHP) influences the synthesis of prostaglandins in the human embryo lung fibroblast. TBHP inhibits or stimulates prostaglandin synthesis as a function of its concentration. Regardless of the concentration employed in these experiments however, TBHP stimulated the release of arachidonate from lipid stores. When the arachidonate release step in prostaglandin (PG) synthesis was bypassed by the addition of free arachidonate to the cell cultures, t-butyl hydroperoxide further stimulated PG synthesis, indicating that the hydroperoxide activates arachidonate conversion to prostaglandins. 4-Hydroxy-2,2,6,6-tetramethylpiperidinoxy radical, a scavenger of oxygen radicals, when added to cell cultures alone had no measurable effect on either arachidonate release or prostaglandin synthesis. When 4-hydroxy-2,2,6,6-tetramethylpiperidinoxy radical was administered to cell cultures in combination with t-butyl hydroperoxide, it increased prostaglandin synthesis while inhibiting arachidonate release by the hydroperoxide. The participation of hydroperoxides and trappers of oxygen radicals in the regulation of PG synthesis is not unique to lung fibroblasts. Endothelial cells from the vasculature as well as fibroblasts from the cornea also appear to be affected by these compounds with respect to prostaglandin synthesis.     

Protein kinase C involvement in lipid peroxidation and cell membrane damage induced by oxygen-based radicals in hepatocytes

We investigated the effects of oxygen-based radicals induced by t-butyl hydroperoxide or H2O2/Cu2+ on cultured hepatocytes. Radical exposure caused membrane lesions (blebs), lactate dehydrogenase release and lipid peroxidation (i.e. formation of malondialdehyde) in cells. As expected, radical scavengers (catalase, alpha-tocopherol) strongly inhibited these phenomena. A similar or even superior inhibitory effect was achieved by the protein kinase C (PKC) inhibitors H-7 and phloretin. These agents did not reveal notable radical scavenging properties as assessed by their ability to break down H2O2. The PKC stimulators 4 beta-phorbol-12-myristate-13 and 1-olyeoyl-2-acetyl-sn-glycerol intensified the detrimental actions of the radical-inducing agents. [3H]Phorbol-12,13-dibutyrate-binding studies showed that membrane association of PKC is markedly increased in hepatocytes after exposure to H2O2/Cu2+ or t-butyl hydroperoxide. These results suggest that PKC membrane translocation and activation may be important for mediating membrane damage and lipid peroxidation after cells are exposed to oxygen-based radicals.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. The relative roles of haem- and glutathione-dependent decomposition of t-butyl hydroperoxide and membrane lipid hydroperoxides in lipid peroxidation and haemolysis

Red cells exposed to t-butyl hydroperoxide undergo lipid peroxidation, haemoglobin degradation and hexose monophosphate-shunt stimulation. By using the lipid-soluble antioxidant 2,6-di-t-butyl-p-cresol, the relative contributions of t-butyl hydroperoxide and membrane lipid hydroperoxides to oxidative haemoglobin changes and hexose monophosphate-shunt stimulation were determined. About 90% of the haemoglobin changes and all of the hexose monophosphate-shunt stimulation were caused by t-butyl hydroperoxide. The remainder of the haemoglobin changes appeared to be due to reactions between haemoglobin and lipid hydroperoxides generated during membrane peroxidation. After exposure of red cells to t-butyl hydroperoxide, no lipid hydroperoxides were detected iodimetrically, whether or not glucose was present in the incubation. Concentrations of 2,6-di-t-butyl-p-cresol, which almost totally suppressed lipid peroxidation, significantly inhibited haemoglobin binding to the membrane but had no significant effect on hexose monophosphate shunt stimulation, suggesting that lipid hydroperoxides had been decomposed by a reaction with haem or haem-protein and not enzymically via glutathione peroxidase. The mechanisms of lipid peroxidation and haemoglobin oxidation and the protective role of glucose were also investigated. In time-course studies of red cells containing oxyhaemoglobin, methaemoglobin or carbonmono-oxyhaemoglobin incubated without glucose and exposed to t-butyl hydroperoxide, haemoglobin oxidation paralleled both lipid peroxidation and t-butyl hydroperoxide consumption. Lipid peroxidation ceased when all t-butyl hydroperoxide was consumed, indicating that it was not autocatalytic and was driven by initiation events followed by rapid propagation and termination of chain reactions and rapid non-enzymic decomposition of lipid hydroperoxides. Carbonmono-oxyhaemoglobin and oxyhaemoglobin were good promoters of peroxidation, whereas methaemoglobin relatively spared the membrane from peroxidation. The protective influence of glucose metabolism on the time course of t-butyl hydroperoxide-induced changes was greatest in carbonmono-oxyhaemoglobin-containing red cells followed in order by oxyhaemoglobin- and methaemoglobin-containing red cells. This is the reverse order of the reactivity of the hydroperoxide with haemoglobin, which is greatest with methaemoglobin. In studies exposing red cells to a wide range of t-butyl hydroperoxide concentrations, haemoglobin oxidation and lipid peroxidation did not occur until the cellular glutathione had been oxidized. The amount of lipid peroxidation per increment in added t-butyl hydroperoxide was greatest in red cells containing carbonmono-oxyhaemoglobin, followed in order by oxyhaemoglobin and methaemoglobin. Red cells containing oxyhaemoglobin and carbonmono-oxyhaemoglobin and exposed to increasing concentrations of t-butyl hydroperoxide became increasingly resistant to lipid peroxidation as methaemoglobin accumulated, supporting a relatively protective role for methaemoglobin. In the presence of glucose, higher levels of t-butyl hydroperoxide were required to induce lipid peroxidation and haemoglobin oxidation compared with incubations without glucose. Carbonmono-oxyhaemoglobin-containing red cells exposed to the highest levels of t-butyl hydroperoxide underwent haemolysis after a critical level of lipid peroxidation was reached. Inhibition of lipid peroxidation by 2,6-di-t-butyl-p-cresol below this critical level prevented haemolysis. Oxidative membrane damage appeared to be a more important determinant of haemolysis in vitro than haemoglobin degradation. The effects of various antioxidants and free-radical scavengers on lipid peroxidation in red cells or in ghosts plus methaemoglobin exposed to t-butyl hydroperoxide suggested that red-cell haemoglobin decomposed the hydroperoxide by a homolytic scission mechanism to t-butoxyl radicals.     

t-butyl hydroperoxide-induced perturbations of human erythrocytes as a model for oxidant stress

Erythrocytes were incubated with t-butyl hydroperoxide in the presence and absence of hemoglobin as a model system for oxidative stress and the alterations in the structure and integrity of the membranes were investigated. The results showed that in the presence of hemoglobin a significant modification in the membrane surface charge was induced but no such alteration was observed in peroxidized hemoglobin-free membranes. As increased hemoglobin oxidation occurred in the erythrocytes, membrane lipid peroxidation diminished, suggesting a protective role for methemoglobin in t-butyl hydroperoxide-induced lipid peroxidation. Electrophoresis on polyacrylamide gels showed modification of the cytoplasmic protein region but no high molecular weight aggregates formed at the concentrations of the hydroperoxide used in this work. The results suggest that the t-butyl hydroperoxide/normal erythrocyte system seems to be an instructive model for membrane perturbations characteristic of oxidative disorders.     

Sulfite-induced lipid peroxidation in chloroplasts as determined by ethane production

Ethane formation, as a measure of lipid peroxidation, was studied in spinach (Spinacia oleracea L.) chloroplasts exposed to sulfite. Ethane formation required sulfite and light, and occurred with concomitant oxidation of sulfite to sulfate. In the dark, both ethane formation and sulfite oxidation were inhibited. Ethane formation was stimulated by ferric or ferrous ions and inhibited by ethylenediamine tetraacetate. The photosynthetic electron transport modulators, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and phenazine methosulfate, inhibited both sulfite oxidation and ethane formation. Methyl viologen greatly stimulated ethane formation, but had little effect on sulfite oxidation. Methyl viologen, in the absence of sulfite, caused only a small amount of ethane formation in comparison to that produced with sulfite alone. Sulfite oxidation and ethane formation were effectively inhibited by the radical scavengers, 1,2-dihydroxybenzene-3,5-disulfonic acid and ascorbate. Ethanol, a hydroxyl radical scavenger, inhibited ethane formation only to a small degree; formate, which converts hydroxyl radical to superoxide radical, caused a small stimulation in both sulfite oxidation and ethane formation. Superoxide dismutase inhibited ethane formation by 50% when added at a concentration equivalent to that of the endogenous activity. Singlet oxygen did not appear to play a role in ethane formation, inasmuch as the singlet oxygen scavengers, sodium azide and 1,4-diazobicyclo-[2,2,2]-octane, were not inhibitory. These data are consistent with the view that O₂ is reduced by the photosynthetic electron transport system to superoxide anion, which in turn initiates the free radical oxidation of sulfite, and the free radicals produced during sulfite oxidation were responsible for the peroxidation of membrane lipids, resulting in the formation of ethane.     

Low density lipoprotein cytotoxicity induced by free radical peroxidation of lipid

Low density lipoprotein (LDL) has been reported to be injurious or toxic to cells in vitro. This injurious effect is, in some instances, due to oxidation of the lipid moiety of the lipoprotein. The objectives of this study were to determine if the oxidation rendering the lipoprotein toxic to human skin fibroblasts occurred by free radical mechanisms, and if so, which of the common free radical oxygen species were involved. The selective free radical blockers or scavengers employed included superoxide dismutase for superoxide, catalase for hydrogen peroxide, dimethylfuran for singlet molecular oxygen, and mannitol for hydroxyl radical. The presence during lipoprotein preparation of general free radical scavengers (vitamin E, butylated hydroxytoluene) or the divalent cation chelator ethylenediamine tetraacetic acid prevented the formation of cytotoxic low density lipoprotein, while the simultaneous presence of superoxide dismutase and catalase partially inhibited its formation. The results indicate that superoxide and/or hydrogen peroxide are involved in the formation of the toxic LDL lipid. The toxic action of oxidized LDL could not be prevented by inclusion of antioxidants in the culture medium, indicating that an oxidized lipid was responsible for cell injury rather than free radicals generated in culture by the action of oxidized LDL. Three separate assays for cell injury (enumeration of attached cells, cell loss of lactate dehydrogenase into the culture medium, and trypan blue uptake) indicated a sequence of events in which the fibroblasts are injured, die, and then detach.     

Free radical-mediated platelet activation by hemoglobin released from red blood cells.

It is known that the rate of thrombus formation depends on interaction between platelets and erythrocytes, but the mechanism of this process has remained obscure. We here show that nanomolar levels of hemoglobin released from damaged red blood cells can induce platelet aggregation. The molecular mechanism is not receptor-based, but involves oxidation of oxyhemoglobin by platelet-derived hydrogen peroxide, with subsequent generation of a small unknown free radical species, detected by ESR spectroscopy. Methemoglobin and carbon monoxide-treated hemoglobin are unable to cause platelet activation or radical formation. The aggregation of platelets induced by hemoglobin is completely blocked by catalase or radical scavengers. These findings indicate a role for a novel extracellular free radical second messenger in the activation of platelets.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. Effects of the hexose monophosphate shunt as mediated by glutathione and ascorbate.

Lipid peroxidation and haemoglobin degradation were the two extremes of a spectrum of oxidative damage in red cells exposed to t-butyl hydroperoxide. The exact position in this spectrum depended on the availability of glucose and the ligand state of haemoglobin. In red cells containing oxy- or carbonmono-oxy-haemoglobin, hexose monophosphate-shunt activity was mainly responsible for metabolism of t-butyl hydroperoxide; haem groups were the main scavengers in red cells containing methaemoglobin. Glutathione, via glutathione peroxidase, accounted for nearly all of the hydroperoxide metabolizing activity of the hexose monophosphate shunt. Glucose protection against lipid peroxidation was almost entirely mediated by glutathione, whereas glucose protection of haemoglobin was only partly mediated by glutathione. Physiological concentrations of intracellular or extracellular ascorbate had no effect on consumption of t-butyl hydroperoxide or oxidation of haemoglobin. Ascorbate was mainly involved in scavenging chain-propagating species involved in lipid peroxidation. The protective effect of intracellular ascorbate against lipid peroxidation was about 100% glucose-dependent and about 50% glutathione-dependent. Extracellular ascorbate functioned largely without a requirement for glucose metabolism, although some synergistic effects between extracellular ascorbate and glutathione were observed. Lipid peroxidation was not dependent on the rate or completion of t-butyl hydroperoxide consumption but rather on the route of consumption. Lipid peroxidation appears to depend on the balance between the presence of initiators of lipid peroxidation (oxyhaemoglobin and low concentrations of methaemoglobin) and terminators of lipid peroxidation (glutathione, ascorbate, high concentrations of methaemoglobin).     

In vivo thiyl free radical formation from hemoglobin following administration of hydroperoxides

Although free radical formation due to the reaction between red blood cells and organic hydroperoxides in vitro has been well documented, the analogous in vivo ESR spectroscopic evidence for free radical formation has yet to be reported. We successfully employed ESR to detect the formation of the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)/hemoglobin thiyl free radical adduct in the blood of rats dosed with DMPO and tert-butyl hydroperoxide, cumene hydroperoxide, ethyl hydrogen peroxide, 2-butanone hydroperoxide, 15(S)-hydroperoxy-5,8,11,13-eicosatetraenoic acid, or hydrogen peroxide. We found that pretreating the rats with either buthionine sulfoximine or diethylmaleate prior to dosing with tert-butyl hydroperoxide decreased the concentration of nonprotein thiols within the red blood cells and significantly enhanced the DMPO/hemoglobin thiyl radical adduct concentration. Finally, we found that pretreating rats with the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea prior to dosing with tert-butyl hydroperoxide enhanced the DMPO/hemoglobin thiyl radical adduct concentration and induced the greatest decrease in nonprotein thiol concentration within the red blood cells.     

Inhibition of protein hydroperoxide formation by protein thiols

Studies on plasma and cells exposed to hydroxyl and peroxyl radicals have indicated that there are few inhibitors of protein hydroperoxide formation. We have, however, observed a small variable lag period during bovine serum albumin (BSA) oxidation by 2-2' azo-bis-(2-methyl-propionamidine) HCl (AAPH) generated peroxyl radicals, where no protein hydroperoxide was formed. The addition of free cysteine to BSA during AAPH oxidation also produced a lag phase suggesting protein thiols could inhibit protein hydroperoxide formation. The selective reduction of thiols on BSA by beta-mercaptoethanol treatment caused the appearance of a lag period where no protein hydroperoxide was formed during the AAPH mediated oxidation. Increasing free thiol concentration on the BSA increased the lag period. Protein hydroperoxide formation began when the protein thiol concentration dropped below one thiol per BSA molecule. It is unlikely that the lag period is due to gross structural alteration of the reduced protein since blocking the free thiols with N-ethyl maleimide eliminated the lag in protein hydroperoxide formation. Protein thiols were found to be ineffective in inhibiting hydroxyl radical-mediated protein hydroperoxide formation during X-ray radiolysis. Evidence is given for protein thiol oxidation occurring via a free radical mediated chain reaction with both free cysteine and protein bound thiol. The data suggest that reduced protein thiol groups can inhibit protein hydroperoxide formation by scavenging peroxyl radicals.     

Inhibition of protein hydroperoxide formation by protein thiols

Abstract

Studies on plasma and cells exposed to hydroxyl and peroxyl radicals have indicated that there are few inhibitors of protein hydroperoxide formation. We have, however, observed a small variable lag period during bovine serum albumin (BSA) oxidation by 2-2′ azo-bis-(2-methyl-propionamidine) HCl (AAPH) generated peroxyl radicals, where no protein hydroperoxide was formed. The addition of free cysteine to BSA during AAPH oxidation also produced a lag phase suggesting protein thiols could inhibit protein hydroperoxide formation. The selective reduction of thiols on BSA by β-mercaptoethanol treatment caused the appearance of a lag period where no protein hydroperoxide was formed during the AAPH mediated oxidation. Increasing free thiol concentration on the BSA increased the lag period. Protein hydroperoxide formation began when the protein thiol concentration dropped below one thiol per BSA molecule. It is unlikely that the lag period is due to gross structural alteration of the reduced protein since blocking the free thiols with N-ethyl maleimide eliminated the lag in protein hydroperoxide formation. Protein thiols were found to be ineffective in inhibiting hydroxyl radical-mediated protein hydroperoxide formation during X-ray radiolysis. Evidence is given for protein thiol oxidation occurring via a free radical mediated chain reaction with both free cysteine and protein bound thiol. The data suggest that reduced protein thiol groups can inhibit protein hydroperoxide formation by scavenging peroxyl radicals.