Generation of free radicals from organic hydroperoxide tumor promoters in isolated mouse keratinocytes. Formation of alkyl and alkoxyl radicals from tert-butyl hydroperoxide and cumene hydroperoxide

The organic hydroperoxides tert-butyl hydroperoxide and cumene hydroperoxide are tumor promoters in the skin of SENCAR mice, and this activity is presumed to be mediated through the activation of the hydroperoxides to free radical species. In this study we have assessed the generation of free radicals from organic hydroperoxides in the target cell (the murine basal keratinocyte) using electron spin resonance. Incubation of primary isolates of keratinocytes from SENCAR mice in the presence of spin traps (5,5-dimethyl-1-pyrroline N-oxide or 2-methyl-2-nitrosopropane) and either tert-butyl hydroperoxide or cumene hydroperoxide resulted in the generation and detection of radical adducts of these spin traps. tert-Butyl alkoxyl and alkyl radical adducts of 5,5-dimethyl-1-pyrroline N-oxide were detected shortly after addition of tert-butyl hydroperoxide, whereas only alkyl radical adducts were observed with cumene hydroperoxide. Spin trapping of the alkyl radicals with 2-methyl-2-nitrosopropane led to the identification of methyl and ethyl radical adducts following both tert-butyl hydroperoxide and cumene hydroperoxide exposures. Prior heating of the cells to 100 degrees C for 30 min prevented radical formation. The radical generating capacity of subcellular fractions of these epidermal cells was examined using 5,5-dimethyl-1-pyrroline N-oxide and cumene hydroperoxide, and this activity was confined to the 105,000 X g supernatant fraction.     

Hydroperoxide-metabolizing systems in rat liver.

1. Metabolism of added hydroperoxides was studied in hemoglobin-free perfused rat liver and in isolated rat hepatocytes as well as microsomal and mitochondrial fractions. 2. Perfused liver is capable of removing organic hydroperoxides [cumene and tert-butyl hydroperoxide] at rates up to 3--4 mumol X min-1 X gram liver-1. Concomitantly, there is a release of glutathione disulfide (GSSG) into the extracellular space in a relationship approx. linear with hydroperoxide infusion rates. About 30 nmol GSSG are released per mumol hydroperoxide added per min per gram liver. GSSG release is interpreted to indicate GSH peroxidase activity. 3. GSSG release is observed also with added H2O2. At rates of H2O2 infusion of about 1.5 mumol X min-1 X gram liver-1 a maximum of GSSG release is attained which, however, can be increased by inhibition of catalase with 3-amino-1,2,4-aminotriazole. 4. A contribution of the endoplasmic reticulum in addition to glutathione peroxidase in organic hydroperoxide removal is demonstrated (a) by comparison of perfused livers from untreated and phenobarbital-pretreated rats and (b) in isolated microsomal fractions, and a possible involvement of reactive iron species (e.g. cytochrome P-450-linked peroxidase activity) is discussed. 5. Hydroperoxide addition to microsomes leads to rapid and substantial lipid peroxidation as evidenced by formation of thiobarbituric-acid-reactive material (presumably malondialdehyde) and by O2 uptake. Like in other types of induction of lipid peroxidation, malondialdehyde/O2 ratios of 1/20 are observed. Cumene hydroperoxide (0.6 mM) gives rise to 4-fold higher rates of malondialdehyde formation than tert-butyl hydroperoxide (1 mM). Ethylenediamine tetraacetate does not inhibit this type of lipid peroxidation. 6. Lipid peroxidation in isolated hepatocytes upon hydroperoxide addition is much lower than in isolated microsomes or mitochondria, consistent with the presence of effective hydroperoxide-reducing systems. However, when NADPH is oxidized to the maximal extent as evidenced by dual-wavelength spectrophotometry, lipid peroxidation occurs at large amounts. 7. A dependence of hydroperoxide removal rates upon flux through the pentose phosphate pathway is suggested by a stimulatory effect of glucose in hepatocytes from fasted rats and by an increased rate of 14CO2 release from [1-14C]glucose during hydroperoxide metabolism in perfused liver.     

Butylated hydroxytoluene prevents cumene hydroperoxide-induced Ca2+ release from liver mitochondria by inhibiting pyridine nucleotide hydrolysis.

The mechanism by which the free radical scavenger butylated hydroxytoluene (BHT) prevents cumene hydroperoxide-induced Ca2+ release from rat liver mitochondria was studied. In Ca(2+)-loaded mitochondria cumene hydroperoxide induced a rapid oxidation and subsequent hydrolysis of the pyridine nucleotides. In the presence of BHT, pyridine nucleotide oxidation by cumene hydroperoxide occurred but was reversible as hydrolysis was prevented by BHT. However, the addition of BHT directly to rat liver submitochondrial particles did not inhibit NAD+ hydrolysis or the formation of ADP-ribose from NAD+. Thus, whilst BHT prevented NAD+ hydrolysis in isolated mitochondria, this appeared not to be due to a direct effect of BHT on the NADase. It is concluded that the mechanism of action of BHT on cumene hydroperoxide-induced Ca2+ release from mitochondria involves the inhibition of pyridine nucleotide hydrolysis by an indirect mechanism rather than the radical scavenging properties of BHT.     

The yeast glutaredoxins are active as glutathione peroxidases

The yeast Saccharomyces cerevisiae contains two glutaredoxins, encoded by GRX1 and GRX2, which are active as glutathione-dependent oxidoreductases. Our studies show that changes in the levels of glutaredoxins affect the resistance of yeast cells to oxidative stress induced by hydroperoxides. Elevating the gene dosage of GRX1 or GRX2 increases resistance to hydroperoxides including hydrogen peroxide, tert-butyl hydroperoxide and cumene hydroperoxide. The glutaredoxin-mediated resistance to hydroperoxides is dependent on the presence of an intact glutathione system, but does not require the activity of phospholipid hydroperoxide glutathione peroxidases (GPX1-3). Rather, the mechanism appears to be mediated via glutathione conjugation and removal from the cell because it is absent in strains lacking glutathione-S-transferases (GTT1, GTT2) or the GS-X pump (YCF1). We show that the yeast glutaredoxins can directly reduce hydroperoxides in a catalytic manner, using reducing power provided by NADPH, GSH, and glutathione reductase. With cumene hydroperoxide, high pressure liquid chromatography analysis confirmed the formation of the corresponding cumyl alcohol. We propose a model in which the glutathione peroxidase activity of glutaredoxins converts hydroperoxides to their corresponding alcohols; these can then be conjugated to GSH by glutathione-S-transferases and transported into the vacuole by Ycf1.     

Relaxation of supercoiled plasmid DNA by oxidative stresses in Escherichia coli.

The relaxation of plasmid DNA was observed after the visible light irradiation of Escherichia coli AB1157 harboring plasmid pBR322 or some other plasmids in the presence of a photosensitizing dye, such as toluidine blue or acridine orange, and molecular oxygen. Treatment of the cells with hydroperoxides, such as tert-butyl hydroperoxide, cumene hydroperoxide, and hydrogen peroxide, also caused the plasmid DNA relaxation in vivo. Relaxation was not observed in these treatments of purified pBR322 DNA in vitro. Plasmid DNA relaxation was also detected after near-UV irradiation. Far-UV irradiation did not induce such relaxation.     

Role of THR501 residue in substrate binding and catalytic activity of cytochrome P4501A1

A putative binding region for cumene hydroperoxide in the active site of cytochrome P4501A1 was identified using photoaffinity labeling. Thr501 was determined as the most likely site of modification by azidocumene used as the photoaffinity label (T. Cvrk and H. W. Strobel, (1998) Arch. Biochem. Biophys. 349, 95-104). To evaluate further the role of this amino acid residue a site-directed mutagenesis approach was employed. P4501A1 wild type and two mutants, P4501A1Glu501 and P4501A1Phe501, were expressed in and purified from Escherichia coli and used for kinetic analysis to confirm the role of Thr501 residue in cumene hydroperoxide binding. The mutation resulted in a two- to fourfold decrease in the rate of heme degradation in the presence of 0.5 mM cumene hydroperoxide. The mutations do not prevent or significantly alter binding of the tested substrates; however, binding of 2-phenyl-2-propanol (product generated from cumene hydroperoxide) to P4501A1Glu501 and P4501A1Phe501 exhibited four- and eightfold decreases, respectively, suggesting that the mutations strongly affected the affinity of cumene hydroperoxide for the P4501A1 active site. The kinetic analysis of cumene hydroperoxide-supported reactions showed that both mutants exhibit increased Km and decreased VMax values for all tested substrates. Furthermore, the mutations affected product distribution in testosterone hydroxylation. On the basis of P4501A1Glu501 and P4501A1Phe501 characterization, it can be concluded that Thr501 plays an important role in cumene hydroperoxide/P4501A1 interaction.     

Cumene hydroperoxide changes the type of conductivity of the mitochondrial membrane for K+

The release of K+ from mitochondria under hypotonic conditions is a non-electrogenic process with an activity maximum at alkaline values of pH. Under these conditions an addition of cumene hydroperoxide causes the appearance of a second K+ release maximum at acidic pH values. The stimulation of K+ release by cumene hydroperoxide is accompanied by a decrease of the transmembrane potential and the accumulation of lipid peroxidation products. The effects of cumene hydroperoxide are prevented by the free radical scavenger, ionol. The acceleration of K+ release is not due to the activation of the non-electrogenic mechanism. It is concluded that the cumene hydroperoxide-induced release of K+ is caused by an increase in the nonspecific ionic permeability of the inner mitochondrial membrane and is controlled by free radical reactions. The contribution of K+ permeability of both types to the hormone-induced changes in the ionic composition of the mitochondrial matrix is discussed.     

Mitochondrial damage as a mechanism of cell injury in the killing of cultured hepatocytes by tert-butyl hydroperoxide

The killing of cultured hepatocytes by tert-butyl hydroperoxide (TBHP) occurs by different mechanisms depending on the presence or absence of the antioxidant N,N'-diphenylphenylenediamine (DPPD). In either situation there is evidence of mitochondrial damage. The mitochondrial inner membrane potential is lost, a result determined by the release from the cells of the lipophilic cation [3H]triphenylmethylphosphonium (TPMP+). Deenergization of the mitochondria is accompanied by a loss of ATP. Oligomycin reduced ATP stores without release of TPMP+ or without effect on the viability of the hepatocytes over the same time course that TBHP killed the majority of the cells. Monensin, a H+/Na+ ionophore, potentiated the toxicity of tert-butyl hydroperoxide in the presence or absence of DPPD. By contrast, extracellular acidosis reduced the toxicity of tert-butyl hydroperoxide in the presence or absence of DPPD. Neither monensin nor extracellular acidosis affected the metabolism of tert-butyl hydroperoxide, the release of TPMP+, or the extent of the peroxidation of cellular lipids. These data document the presence of mitochondrial damage in hepatocytes intoxicated with TBHP in both the presence and absence of DPPD. Furthermore, the potentiation by monensin is readily explained by the proposal that mitochondrial deenergization is accompanied by an intracellular acidosis. Such acidosis tends to delay the development of lethal cell injury. The protective effect of extracellular acidosis supports this interpretation.     

The role of lipid peroxidation products in cumene hydroperoxide-induced Ca2+ efflux from mitochondria

Cumene hydroperoxide-induced calcium release from mitochondria has been studied. Activation of lipid peroxidation by increasing concentrations of cumene hydroperoxide does not enhance calcium efflux induced by low (up to 50 microM) concentration of cumene hydroperoxide. It is concluded that cumene hydroperoxide-induced calcium release depends mainly on processes coupled to hydroperoxide reduction by an endogenous enzyme system.     

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.     

Effects of cumene hydroperoxide on cellular cation composition in frog kidney proximal tubular cells

Effects of cumene hydroperoxide were studied on the peritubular membrane potential and cellular cation composition in frog kidney proximal tubular cells. After perfusion of isolated frog kidneys for 30 min with 1.3x10(-4) mol l(-1) cumene hydroperoxide Ringer solution, the peritubular membrane potential gradually declined. The ouabain-like effects were demonstrated on cell Na and K activities after 1 h of perfusion with cumene hydroperoxide. The peritubular apparent transference number for potassium was decreased. Intracellular pH was not altered in the presence of cumene hydroperoxide. Intracellular free Ca(2+) concentration increased slowly and moderately. The concentration of the malondialdehyde in the kidney homogenates, measured as an index of lipid peroxidation, was increased. A previously observable effect of cumene hydroperoxide on the peritubular membrane potential was prevented by oxygen radical scavengers.     

Decreased lipid peroxidation in the rat kidney during gestation

Renal malonaldehyde content and lipid peroxidation, induced by ascorbate, NADPH and cumene hydroperoxide, are significantly decreased during gestation in rats. Lipid peroxidation tends to reach normal levels in the kidney post partum. In the renal mitochondria lipid peroxidation without co-factors and that induced by cumene hydroperoxide, ascorbate and NADPH is decreased during pregnancy. However, in the microsomes, only lipid peroxidation induced by NADPH and cumene hydroperoxide is affected. The observed decrease in lipid peroxidation during gestation is reflected by low levels of total lipid and phospholipid. Endogenous inhibitors of lipid peroxidation also increase during pregnancy.     

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.     

Peroxidatic oxidation of benzo[a]pyrene and prostaglandin biosynthesis.

The arachidonic acid dependent oxidation of benzo[a]pyrene to a mixture of 3,6-, 1,6-, and 6,12-quinones has been studied by using enzyme preparations from sheep seminal vesicles. Maximal oxidation is observed at 100 microM benzo[a]pyrene and 150 microM arachidonic acid. The arachidonic acid dependent oxidation is peroxidatic and utilizes prostaglandin G2 (PGG2), generated in situ from arachidonate, as the hydroperoxide substrate. 15-Hydroperoxy-5,8,11,13-eicosatetraenoic acid is equivalent to PGG2 as a hydroperoxide substrate, but hydrogen peroxide, cumene hydroperoxide, and tert-butyl hydroperoxide are much poorer substrates. Arachidonic acid dependent benzo[a]pyrene oxidation by microsomal and solubilized enzyme preparations is markedly.     

Role of lipoxygenase in the O2-dependent activation of soluble guanylate cyclase from rat lung.

Guanylate cyclase activity in rat lung supernatant fractions is stimulated 3-4 fold by aerobic incubation at 30 degrees C for approx. 30 min ('O2-dependent activation'). This stimulation was blocked by 20 microM-eicosa-5,8,11,14-tetraynoic acid (ETYA), an inhibitor of lipoxygenase and cyclo-oxygenase, but not by aspirin or indomethacin, which are cyclo-oxygenase inhibitors. The enzyme activator(s) is presumed to be the fatty acid hydroperoxide(s) formed by lipoxygenase. Removal of lipoxygenase from the supernatant fraction by chromatography on Amberlite XAD-4 also prevented activation, which was restored by the addition of soya-bean lipoxygenase. Bovine serum albumin prevented O2-dependent activation or activation by soya-bean lipoxygenase, through its ability to bind the unsaturated fatty acid substrate of lipoxygenase. The lipoxygenase in the supernatant fraction is inhibited by endogenous glutathione peroxidase plus reduced glutathione (GSH); removal of GSH de-inhibits lipoxygenase and activates guanylate cyclase. This was effected by autoxidation, by cumene hydroperoxide (with GSH peroxidase) and by titration with N-ethylmaleimide (NEM). Activation by NEM was inhibited by serum albumin or ETYA, as was activation by low concentrations (less than 50 microM) of cumene hydroperoxide. Activation by higher concentrations was not so inhibited; therefore, cumene hydroperoxide can also activate by a direct effect on guanylate cyclase. A hypothesis for physiological activation is proposed.     

Oxidative stress during selenium deficiency in seedlings ofTrigonella foenum-graecum and mitigation by mimosine part II. glutathione metabolism

Adaptive alterations in glutathione (GSH) metabolism were studied during oxidative stress induced by selenium (Se) deficiency in germinating seedlings of Trigonella foenum-graecum grown for 72 h and the response to supplementation individually of Se or mimosine was explored. Growth enhancement with improved mitochondrial efficiency was elicited by supplementation of Se at 0.5-0.75 ppm or mimosine at 0.1-0.2 mM. Total thiol and protein levels of mitochondrial and soluble fractions, in general, did not vary significantly with supplementation of either Se or mimosine except that the mitochondrial protein levels in mimosine groups (0.1-0.2 mM) decreased by 20-30%. Mitochondrial glutathione peroxidase (GSH-Px) increased by twofold in activity toward H2O2, cumene hydroperoxide (CHP), and t-butyl hydroperoxide (tBHP) in Se groups, and by 50-60% increase toward H2O2 and CHP but by a twofold enhancement in enzyme activity with tBHP in mimosine groups. Soluble GSH-Px activity increased by 30-40% only in mimosine groups and remained unaltered in Se groups. Glutathione S-transferase activity (GST) in the soluble fraction of both Se and mimosine groups increased dramatically by fivefold to sixfold. Distinct differences were noted in the response of the stressed seedlings toward exposure to Se or mimosine and included a decline in glutathione reductase (GR) activity by 50-60% in both mitochondria and soluble fractions of Se groups and an increase in GR activity of the mitochondria by twofold and of the soluble enzyme activity by 30% in the mimosine groups. Mimosine exposure resulted in a dose-dependent decrease in the gamma-glutamyl transpeptidase levels, but, in contrast, a significant enhancement by 50% was noted in the Se group at 0.75 ppm. The results including the differential response of GR activity to Se or mimosine supplementation are reflective of an effective reductive environment in Se groups and increased turnover of GSH in the presence of mimosine.     

Ferrous ion-mediated cytochrome P-450 degradation and lipid peroxidation in adrenal cortex mitochondria.

The relationship between the degradation reaction of cytochrome P-450 and lipid peroxidation was studied utilizing bovine adrenal cortex mitochondria. The two reactions were found to be closely correlated in terms of their response to storage of the mitochondrial preparation, stimulation by Fe2+, inhibition by EDTA and their initiation by cumene hydroperoxide. Both reactions were also found not to be inhibited by catalase, superoxide dismutase, 1,4-diazabicyclo-(2,2,2)-octane and alcohols, indicating that H2O2, superoxide, singlet oxygen and hydroxyl radicals do not participate in these reactions. Yet, diphenylamine proved to be a powerful inhibitor for both reactions, suggesting the involvement of a radical species. Cumene hydroperoxide could induce these two reactions at below 0.1 mM concentrations in the presence of molecular oxygen. The chemiluminescence observed during the Fe2+-mediated lipid peroxidation reaction which was not inhibited by either superoxide dismutase or 1,4-diazabicyclo-(2,2,2)-octane, was biphasic: one was a rapid burst; and the other was a slowly increasing emission. The latter portion of the emission of light coincided with the formation of malondialdehyde. These results indicate that in adrenal cortex mitochondria the degradation of cytochrome P-450 is closely related to lipid peroxidation.     

Asymmetric catalysis with self-organized chiral lanthanum complexes: practical and highly enantioselective epoxidation of alpha,beta-unsaturated ketones

A highly efficient and practical method for obtaining alpha,beta-epoxy ketones with high optical purities was developed. The chiral lanthanum complex self-organized in situ from lanthanum triisopropoxide, (R)-BINOL, triarylphosphine oxide, and alkyl hydroperoxide (1:1:1:1) was found to catalyze the epoxidation of alpha,beta-unsaturated ketones with tert-butyl hydroperoxide or cumene hydroperoxide at room temperature to give the corresponding epoxy ketones in high enantioselectivities (up to >99% enantiomeric excess (ee)). A remarkably high asymmetric amplification, a positive nonlinear effect, was observed in the epoxidation of chalcone, which strongly suggests the formation of a dinuclear peroxide-involved mu-complex as the active catalyst.     

Inhibition of arachidonic acid incorporation into erythrocyte phospholipids by peracetic acid and other peroxides. Role of arachidonoyl-CoA: 1-palmitoyl-sn-glycero-3-phosphocholine acyl transferase

To explore possible mechanisms of the arachidonic acid deficiency of the red blood cell membrane in alcoholics, we compared the effect of ethanol and its oxidized products, acetaldehyde and peracetic acid, with other peroxides on the accumulation of [14C]arachidonate into RBC membrane lipids in vitro. Incubation of erythrocytes with 50 mM ethanol or 3 mM acetaldehyde had no effect on arachidonate incorporation. Pretreatment of erythrocytes with 10 mM hydrogen peroxide, 0.1 mM cumene hydroperoxide or 0.1 mM t-butyl hydroperoxide had little effect on [14C]arachidonate incorporation in the absence of azide. However, pretreatment of cells with N-ethylmaleimide, 0.1 mM peracetic acid or performic acid, with or without azide, inhibited arachidonate incorporation into phospholipids but not neutral lipids. In chase experiments, peracetate also inhibited transfer of arachidonate from neutral lipids to phospholipids. To investigate a possible site of this inhibition of arachidonate transfer into phospholipids by percarboxylic acids, we assayed a repair enzyme, arachidonoyl CoA: 1-palmitoyl-sn-glycero-3-phosphocholine acyl transferase (EC 2.3.1.23). As in intact cells, phospholipid biosynthesis was inhibited more by N-ethylmalemide and peracetic acid than by hydrogen peroxide, cumene hydroperoxide, and t-butyl hydroperoxide. Peracetic acid was the only active inhibitor among ethanol and its oxidized products studied and may deserve further examination in ethanol toxicity.     

Hydroperoxides selectively inhibit human erythrocyte membrane enzymes

Treatment of washed erythrocytes with tert-butyl hydroperoxide (0.5 mM, 10 min) inhibited basal Ca2+ + Mg2+-ATPase activity by 40% and calmodulin-stimulated activity by 54%. The inhibition was accompanied by the formation of methemoglobin and the aggregation of some membrane proteins into a high-molecular-weight polymer. Membranes, isolated from washed erythrocytes, showed a similar pattern of inhibition. Basal Ca2+ + Mg2+-ATPase activity was inhibited 50% at 10 min and 70% at 30 min while calmodulin-stimulated activity was inhibited 70% at 10 min and 84% at 30 min. Thiobarbituric acid-reactive products formed slowly during the first 10 min and then increased sharply between 10 and 30 min. The polymerization of membrane proteins was also observed during the tert-butyl hydroperoxide exposure. Inhibition of erythrocyte membrane enzymes was selective. The Na+ + K+-stimulated Mg2+ ATPase, like the Ca2+ + Mg2+-ATPase, was sensitive to membrane oxidation but the activities of Mg2+-ATPase and acetylcholinesterase were less inhibited by tert-butyl hydroperoxide. Acetylcholinterase was found to be very resistant to hydroperoxide treatment with less than 10% loss of activity. The effects of two other hyproperoxides on enzyme inhibition were studied also. Cumene hydroperoxide (0.5 mM) was found to be as potent as tert-butyl hydroperoxide but hydrogen peroxide at 10 mM did not produce thiobarbituric acid-reactive products or inhibit Ca2+ + Mg2+-ATPase activity until after 20 min. The selective effects of peroxides on these enzyme activities are discussed.