Hydroxyl Radical Scavenging Agents Research Articles

Radicals from “Good's” buffers

Oxidative deposition of iron in ferritin or the autoxidation of iron in the absence of protein produces radicals from Good's buffers. Radical species are formed from the piperazine ring-based buffers Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Epps 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, and Pipes 1,4-piperazinediethanesulfonic acid, but not from Mes (4-morpholineethanesulfonic acid) which contains a morpholine ring. The radicals all have half-lives around 10 min and display very similar electron paramagnetic resonance spectra consisting of at least 30 lines. The Hepes radical can be formed by the addition of potassium superoxide directly to the buffer and its production during iron(II) autoxidation is inhibited by superoxide dismutase (EC 1.15.1.1). Catalase (EC 1.11.1.6) accelerates the decay of the EPR spectrum. Thus the buffer radicals are secondary radical species produced from oxygen radicals formed during the iron catalyzed Haber-Weiss process. The deoxyribose/thiobarbituric acid assay for hydroxyl radical production shows that Hepes is an effective hydroxyl radical scavenging agent. The Hepes radical can also be formed electrolytically at a potential of +0.8 V (vs standard hydrogen electrode). Oxidation of Hepes at pH 10 during the autoxidation of iron(II) or by the addition of hydrogen peroxide produces a nitroxide radical. These results indicate that piperazine ring Good buffers should be avoided in studies of redox processes in biochemistry.

NAD(P)H-dependent oxidation of the hydroxyl radical scavenging agent 2-keto-4-thiomethylbutyric acid (KMBA) by microsomal fractions from the hepatopancreas of the red swamp crayfish, Procambarus clarkii

A secondary pathway of the microsomal mixed-function oxidase system in which the terminal oxidase is expressed as hydrogen peroxide (H 2O 2) production is well documented in mammalian systems. 1 H 2O 2 is a precursor of hydroxyl radical (·OH), a potent oxidant that has been implicated in numerous pathologies and in the metabolism of various xenobiotic substrates. 2 The ability of ·OH to oxidize 2-keto-4-thiomethylbutyric acid (KMBA) was used as a means to study the ·OH-generating oxidase pathway of P. clarkii hepatopancreas microsomes. Both a post-mitochondrial supernatant (S9) and microsomes catalyzed the oxidation of KMBA in a reaction that was dependent upon NAD(P)H, stimulated by added iron-EDTA and inhibited by superoxide dismutase (SOD), catalase and competing ·OH scavenging agents. The H 2O 2/·OH generating oxidase activity of P. clarkii microsomal fractions was mainly NADH-dependent, which is in contrast to the rat system which is mainly NADPH-dependent. This oxidase activity should be considered in evaluating xenobiotic metabolism by this aquatic invertebrate.

Differential effects of the cytochrome P-450/ reductase ratio on the oxidation of ethanol and the hydroxyl radical scavenging agent 2-keto-4-thiomethylbutyric acid (KMBA)

NADPH-cytochrome P-450 reductase catalyzes a low rate of oxidation of hydroxyl radical scavenging agents such as ethanol and 2-keto-4-thiomethylbutyric acid (KMBA), in a reaction markedly stimulated by the addition of ferric-EDTA. The effect of various ratios of cytochrome P-450 (pheno-barbital-inducible isozyme)/reductase on the oxidation of ethanol and KMBA was determined. There was essentially no increase in KMBA oxidation over the range of ratios from 0.5 to 5 as compared to the reductase-catalyzed rate. High ratios actually caused some decrease in KMBA oxidation, which was especially notable under conditions of increased rates of hydroxyl radical generation (presence of increasing amounts of ferric-EDTA). This decrease at high P-450/reductase ratios could reflect tight coupling of reductase to P-450-PB, therefore decreasing electron transfer from reductase to ferric-EDTA, or could involve non-specific scavenging of OH by P-450-PB. Indeed, native, but not boiled, P-450 inhibited KMBA oxidation by the xanthine oxidase system. By contrast, the oxidation of ethanol was stimulated at all concentrations of P-450-PB, and this increase was not sensitive to desferrioxamine. However, under conditions of high rates of OH production, the ethanol oxidation profile tended to resemble the KMBA oxidation profile with respect to the effect of P-450-PB, whereas the two profiles were different under conditions of low rates of OH production. These results suggest that P-450-PB does not catalyze the oxidation of OH scavengers or promote the production of OH, even at ratios of P-450/reductase approaching those found with intact microsomes and even in the presence of excess iron-EDTA, whereas ethanol is directly oxidized by P-450-PB, as are typical drug substrates. However, the P-450-PB-dependent oxidation of ethanol can be masked under conditions in which OH production is increased.

Redox modification of sodium-calcium exchange activity in cardiac sarcolemmal vesicles.

Na-Ca exchange activity in bovine cardiac sarcolemmal vesicles was stimulated up to 10-fold by preincubating the vesicles with 1 microM FeSO4 plus 1 mM dithiothreitol (DTT) in a NaCl medium. The increase in activity was not reversed upon removing the Fe and DTT. Stimulation of exchange activity under these conditions was completely blocked by 0.1 mM EDTA or o-phenanthroline; this suggests that the production of reduced oxygen species (H2O2, O2-.,.OH) during Fecatalyzed DTT oxidation might be involved in stimulating exchange activity. In agreement with this hypothesis, the increase in exchange activity in the presence of Fe-DTT was inhibited 80% by anaerobiosis and 60% by catalase. H2O2 (0.1 mM) potentiated the stimulation of Na-Ca exchange by Fe-DTT under both aerobic and anaerobic conditions; H2O2 also produced an increase in activity in the presence of either FeSO4 (1 microM) or DTT (1 mM), but it had no effect on activity by itself. Superoxide dismutase did not block the effects of Fe-DTT on exchange activity; however, the generation of O2-. by xanthine oxidase in the presence of an oxidizable substrate stimulated activity more than 2-fold. Hydroxyl radical scavenging agents (mannitol, sodium formate, sodium benzoate) did not attenuate the stimulation of activity observed with Fe-H2O2. Exchange activity was also stimulated by the simultaneous presence of glutathione (GSH; 1-2 mM) and glutathione disulfide (GSSG; 1-2 mM). Neither GSH nor GSSG was effective by itself and either 0.1 mM EDTA or o-phenanthroline blocked the effects on transport activity of the combination of GSH + GSSG. Treatment of the GSH and GSSG solutions with Chelex ion-exchange resin to remove contaminating transition metal ions reduced (by 40%) the degree of stimulation observed with GSH + GSSG. Full stimulating activity was restored to the Chelex-treated GSH and GSSG solutions by the addition of 1 microM Fe2+; Cu2+ was less effective than Fe2+ whereas Co2+ and Mn2+ were without effect. In the presence of 1 microM Fe2+, GSH alone produced a slight increase in transport activity, but this was markedly enhanced by the addition of Chelex-treated GSSG. The results indicate that stimulation of exchange activity requires the presence of both a reducing agent (DTT, GSH, O-.2, or Fe2+) and an oxidizing agent (H2O2, GSSG, and perhaps O2) and that the effects of these agents are mediated by metal ions (e.g. Fe2+).(ABSTRACT TRUNCATED AT 400 WORDS)

Evaluation of microsomal pathways of oxidation of alcohols and hydroxyl radical scavenging agents with carbon monoxide and cobalt protoporphyrin IX

Rat liver microsomes catalyze the oxidation of hydroxyl radical scavenging agents by an irondependent process, and can oxidize alcohols by pathways dependent on, as well as independent of, .OH. Experiments were carried out to evaluate which microsomal components participate in the production of OH, and in the two pathways of oxidation of alcohols. Cobalt protoporphyrin IX treatment of rats resulted in a decrease in microsomal oxidation of aminopyrine, .OH scavengers, and alcohols. However, this treatment not only lowered the content of cytochrome P-450, but also decreased the activity of NADPH-cytochrome P-450 reductase. Carbon monoxide, metyrapone and SKF-525A also inhibited the oxidation of aminopyrine but did not affect oxidation of OH scavengers. Desferrioxamine, a potent iron chelator, inhibited the oxidation of .OH scavengers but not aminopyrine. The oxidation of alcohols was partly sensitive to desferrioxamine and partly sensitive to carbon monoxide, thus showing similarities to the oxidation of .OH scavengers and drugs. These results suggest that the desferrioxamine-sensitive, .OH-dependent pathway of alcohol oxidation is mediated by the reductase, in analogy to results with .OH scavengers, whereas the desferrioxamine-resistant pathway of alcohol oxidation is mediated by cytochrome P-450, in analogy to results with aminopyrine. By the use of desferrioxamine or carbon monoxide, either of the two alcohol-oxidizing pathways can be inhibited independently of each other.

Ethanol oxidation by hydroxyl radicals: role of iron chelates, superoxide, and hydrogen peroxide.

Oxygen-derived free radicals such as the hydroxyl radical (.OH) have been shown to mediate the oxidation of ethanol by a variety of oxy radical-generating systems. Among these are microsomal electron transport systems (both intact and purified, reconstituted systems), the coupled oxidation of hypoxanthine or xanthine by xanthine oxidase, and the model iron-ascorbate system. The sequence of reactions leading to the oxy radical-dependent oxidation of ethanol as well as other hydroxyl radical-scavenging agents by these various systems is believed to proceed through the formation of a common intermediate, namely, hydrogen peroxide (H2O2), after dismutation of the superoxide anion radical (O2-.). The presence of iron, especially chelated iron, greatly enhances the production of .OH by serving as an oxidant for O2-. or a reductant for H2O2. Experiments were carried out to evaluate the role of iron, the chelating agent, O2-., and H2O2 in the oxidation of ethanol by a variety of in vitro systems (chemical, enzymatic, and intact membrane bound) that can produce oxy radicals via different mechanisms. The generation of .OH by all the systems studied was sensitive to catalase, which indicates that H2O2 is the precursor of .OH. Superoxide radical appears to be the reducing agent in the hypoxanthine-xanthine oxidase system, indicating an iron-catalyzed Haber-Weiss reaction. In the ascorbate, reductase, and microsomal systems, superoxide radical does not appear to be the reducing agent. However, superoxide radical probably is the precursor of H2O2. While iron plays an important role in the production of .OH by the various systems, the effect of iron depends on the nature of the iron chelate.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of iron chelates in hydroxyl radical production by rat liver microsomes, NADPH-cytochrome P-450 reductase and xanthine oxidase

Uninduced rat liver microsomes and NADPH-Cytochrome P-450 reductase, purified from phenobarbital-treated rats, catalyzed an NADPH-dependent oxidation of hydroxyl radical scavenging agents. This oxidation was not stimulated by the addition of ferric ammonium sulfate, ferric citrate, or ferric-adenine nucleotide (AMP, ADP, ATP) chelates. Striking stimulation was observed when ferric-EDTA or ferric-diethylenetriamine pentaacetic acid (DTPA) was added. The iron-EDTA and iron-DTPA chelates, but not unchelated iron, iron-citrate or iron-nucleotide chelates, stimulated the oxidation of NADPH by the reductase in the absence as well as in the presence of phenobarbital-inducible cytochrome P-450. Thus, the iron chelates which promoted NADPH oxidation by the reductase were the only chelates which stimulated oxidation of hydroxyl radical scavengers by reductase and microsomes. The oxidation of aminopyrine, a typical drug substrate, was slightly stimulated by the addition of iron-EDTA or iron-DTPA to the microsomes. Catalase inhibited potently the oxidation of scavengers under all conditions, suggesting that H 2O 2 was the precursor of the hydroxyl radical in these systems. Very high amounts of superoxide dismutase had little effect on the iron-EDTA-stimulated rate of scavenger oxidation, whereas the iron-DTPA-stimulated rate was inhibited by 30 or 50% in microsomes or reductase, respectively. This suggests that the iron-EDTA and iron-DTPA chelates can be reduced directly by the reductase to the ferrous chelates, which subsequently interact with H 2O 2 in a Fenton-type reaction. Results with the reductase and microsomal systems should be contrasted with results found when the oxidation of hypoxanthine by xanthine oxidase was utilized to catalyze the production of hydroxyl radicals. In the xanthine oxidase system, ferric-ATP and -DTPA stimulated oxidation of scavengers by six- to eightfold, while ferric-EDTA stimulated 25-fold. Ferric-desferrioxamine consistently was inhibitory. Superoxide dismutase produced 79 to 86% inhibition in the absence or presence of iron, indicating an iron-catalyzed Haber-Weiss-type of reaction was responsible for oxidation of scavengers by the xanthine oxidase system. These results indicate that the ability of iron to promote hydroxyl radical production and the role that superoxide plays as a reductant of iron depends on the nature of the system as well as the chelating agent employed.

The effect of EDTA and iron on the oxidation of hydroxyl radical scavenging agents and ethanol by rat liver microsomes

Rat liver microsomes catalyzed an NADPH-dependent oxidation of dimethylsulfoxide, 2-keto-4-thiomethylbutyrate and ethanol. The addition of EDTA and iron (ferric)-EDTA increased the oxidation of the hydroxyl radical scavenging agents and ethanol. Unchelated iron had no effect; therefore, appropriately chelated iron is required to stimulate microsomal production of hydroxyl radicals. Catalase strongly inhibited control rates as well as EDTA or iron-EDTA stimulated rates of hydroxyl radical production whereas superoxide dismutase had no effect. The rate of ethanol oxidation was ten- to twenty-fold greater than the rate of oxidation of hydroxyl radical scavengers in the absence of EDTA or iron-EDTA, suggesting little contribution by hydroxyl radicals in the pathway of ethanol oxidation. In the presence of EDTA or iron-EDTA, the rate of ethanol oxidation increased, and under these conditions, hydroxyl radicals appear to play a more significant role in contributing toward the overall oxidation of ethanol.

Increased microsomal oxidation of hydroxyl radical scavenging agents and ethanol after chronic consumption of ethanol

The oxidation of ethanol by rat liver microsomes is increased after chronic ethanol consumption. Previous experiments indicated that hydroxyl radicals play a role in the mechanism whereby microsomes oxidize ethanol. Experiments were therefore carried out to evaluate the role of these radicals in ethanol oxidation by microsomes from ethanol-fed rats, and to determine whether the increase in ethanol oxidation by these induced microsomes correlates with an increase in the generation of hydroxyl radicals. Rat liver microsomes from ethanol-fed rats catalyzed the oxidation of two typical hydroxyl radical scavenging agents, dimethylsulfoxide and 2-keto-4-thiomethylbutyric acid, at rates which were two- to threefold greater than rates found with control microsomes. This increased rate of oxidation of hydroxyl radical scavengers was similar to the increased rate of microsomal oxidation of ethanol. Azide, which inhibits contaminating catalase in microsomes, increased the oxidation of dimethyl sulfoxide and 2-keto-4-thiomethylbutyric acid by both microsomal preparations. This suggests that H 2O 2 may serve as the microsomal precursor of the hydroxyl radical. Cross competition for oxidation between ethanol and the hydroxyl radical scavenging agents was observed. Moreover, the oxidation of ethanol, dimethyl sulfoxide, or 2-keto-4-thiomethylbutyric acid was inhibited by other compounds which interact with hydroxyl radicals, e.g., benzoate, and the free-radical, spin-trapping agent, 5,5-dimethyl-1-pyrroline- N-oxide. These results suggest that the increase in the rate of ethanol oxidation found with microsomes from ethanol-fed rats may be due, at least in part, to an increase in the rate of production of hydroxyl radicals by these induced microsomes. Increased production of oxyradicals may possibly result in oxidative damage to the liver cell as a result of ethanol consumption.

NADPH-dependent production of oxy radicals by purified components of the rat liver mixed function oxidase system. I. Oxidation of hydroxyl radical scavenging agents.

Isolated microsomes catalyze an NADPH-dependent oxidation of typical hydroxyl radical scavenging agents. To determine which microsomal components participate in the oxidation of the scavengers, experiments were carried out with purified NADPH-cytochrome P-450 reductase and cytochrome P-450 isolated from phenobarbital-treated rats. The production of ethylene from 4-ketothiomethylbutyrate or of formaldehyde from either dimethyl sulfoxide or tertiary butyl alcohol was measured in the presence of NADPH plus reductase or NADPH plus reductase plus cytochrome P-450. The reductase-dependent system itself catalyzed the oxidation of the three scavengers. Addition of cytochrome P-450 had no effect on the rates of oxidation of the scavengers. Relative rates of oxidation of the scavengers reflected the amount of the reductase in the assay system. Varying the amount of cytochrome P-450 at a fixed concentration of the reductase did not result in rates different from that observed in the absence of cytochrome P-450. Dilauroyl phosphatidylcholine appeared to have a stimulatory role in these oxidations, whereas cytochrome P-450 reductase and NADPH were obligatory components. The reductase-dependent oxidation of the scavengers was inhibited by superoxide dismutase, catalase, and competing hydroxyl radical scavenging agents suggesting that superoxide, hydrogen peroxide, and an oxygen radical with the oxidizing power of the hydroxyl radical played a role in these oxidations. Further, these oxidations were inhibited by the iron-chelator desferrioxamine but stimulated by either EDTA or by iron. These results suggest that an iron-catalyzed Haber-Weiss reaction might be involved in the mechanism by which purified cytochrome P-450 reductase mediates the oxidation of typical hydroxyl radical scavengers. The results demonstrated that NADPH-cytochrome P-450 reductase may represent an important locus of oxygen activation leading to the production of a highly oxidizing species characteristic of the hydroxyl radical.

Pyrazole and 4-methylpyrazole inhibit oxidation of ethanol and dimethyl sulfoxide by hydroxyl radicals generated from ascorbate, xanthine oxidase, and rat liver microsomes

Pyrazole and 4-methylpyrazole, which are potent inhibitors of alcohol dehydrogenase, inhibited the oxidation of ethanol and of dimethyl sulfoxide by two model hydroxyl radical-generating systems. The systems used were the iron-catalyzed oxidation of ascorbic acid and the coupled oxidation of xanthine by xanthine oxidase. Pyrazole and 4-methylpyrazole were more effective inhibitors at lower substrate concentrations than at higher substrate concentrations; the oxidation of ethanol was inhibited to a greater extent than the oxidation of dimethyl sulfoxide. These results are consistent with competition between pyrazole or 4-methylpyrazole with the substrates for the generated hydroxyl radicals. Pyrazole and 4-methylpyrazole appear to be equally effective in reacting with hydroxyl radicals. An approximate rate constant of about 8 × 10 9 m −1 s −1 was calculated from the inhibition curves, indicating that pyrazole and 4-methylpyrazole are potent scavengers of the hydroxyl radical. Previous studies have implicated a role for hydroxyl radicals in the microsomal pathway of ethanol oxidation. In the presence of azide (to inhibit catalase), pyrazole and 4-methylpyrazole inhibited the NADPH-dependent microsomal oxidation of ethanol, as well as several other hydroxyl radical-scavenging agents. This inhibition by pyrazole and by 4-methylpyrazole may reflect a mechanism involving competition for hydroxyl radicals generated by the microsomes. However, the kinetics of inhibition by pyrazole were mixed, not competitive, and pyrazole and 4-methylpyrazole also inhibited aminopyrine demethylase activity. Pyrazole has been shown by others to interact with cytochrome P-450. It is suggested that pyrazole and 4-methylpyrazole affect microsomal oxidation of ethanol via effects on the mixed-function oxidase system and via competition for the generated hydroxyl radicals. In view of these results, low concentrations of pyrazole and 4-methylpyrazole should be used in studies on pathways of alcohol metabolism, and caution should be made in interpreting the actions of these compounds when used at high concentrations.

Oxidation of isopropanol by rat liver microsomes: Possible role of hydroxyl radicals

Isopropanol, a branched chain alcohol, served as a substrate for the microsomal alcohol oxidizing system. Isopropanol oxidation required NADPH; H 2O 2 or an H 2O 2-generating system did not effectively support isopropanol oxidation, indicating that isopropanol was not a substrate for the peroxidatic activity of catalase. The addition of azide by itself or H 2O 2 (in the presence of azide and NADPH) stimulated isopropanol oxidation, suggesting a pivotal, indirect role for H 2O 2 in the system. H 2O 2 may serve as a precursor of hydroxyl radicals. Accordingly, the oxidation of isopropanol was inhibited by hydroxyl radical scavenging agents, namely dimethylsulfoxide, mannitol, benzoate and 2-keto-4-thiomethylbutyric acid. Fe-EDTA, which is known to increase hydroxyl radical generation, stimulated the oxidation of isopropanol. The stimulation by Fe-EDTA was blocked by competing hydroxyl radical scavengers. Model hydroxyl radical generating systems such as the coupled oxidation of xanthine by xanthine oxidase, especially in the presence of Fe-EDTA, or the autoxidation of ascorbate in the presence of Fe-EDTA could also oxidize isopropanol. These results indicate that (a) rat liver microsomes are capable of oxidizing branched chain alcohols, and (b) hydroxyl radicals or a species with the oxidizing power of the hydroxyl radical, generated from microsomal electron transfer, may play a role in isopropanol oxidation.

Oxidative demethylation of t-butyl alcohol by rat liver microsomes

Tertiary butyl alcohol has often been used experimentally as a “non-metabolizable” alcohol. In this report, evidence is presented that t-butanol serves as a substrate for rat liver microsomes and that it is oxidatively demethylated to yield formaldehyde. The apparent K m for t-butanol is 30 mM while V max is about 5.5 nmol per min per mg microsomal protein. Formaldehyde production is stimulated by azide, which prevents destruction of H 2O 2 by catalase. Hydroxyl radical scavenging agents, such as benzoate, mannitol, and 2-keto-4-thiomethylbutyrate, suppress formaldehyde production. Therefore, the microsomal reaction pathway appears to involve the interaction of t-butanol with hydroxyl radicals generated from H 2O 2 by the microsomes. Formaldehyde is also produced when t-butanol is incubated with model hydroxyl radical-generating systems such as the iron-EDTA-stimulated oxidation of xanthine by xanthine oxidase or the iron-EDTA-catalyzed autoxidation of ascorbate. These results indicate that t-butanol cannot be used to distinguish metabolically-linked from non-metabolically-linked actions of ethanol.

Role of hydroxyl radicals in the iron-ethylenediaminetetraacetic acid mediated stimulation of microsomal oxidation of ethanol.

The microsomal oxidation of ethanol or 1-butanol was increased by ferrous ammonium sulfate-ethylenediaminetetraacetic acid (1:2) (Fe-EDTA) (3.4-50 microM). The increase was blocked by hydroxyl radical scavenging agents such as dimethyl sulfoxide or mannitol. The activities of aminopyrine demethylase or aniline hydroxylase were not affected by Fe-EDTA. The accumulation of H2O2 was decreased in the presence of Fe-EDTA, consistent with an increased utilization of H2O2. Other investigators have shown that Fe-EDTA increases the formation of hydroxyl radicals in systems where superoxide radicals are generated. The stimulation by Fe-EDTA appears to represent a pathway involving hydroxyl radicals rather than catalase because (1) stimulation occurred in the presence of azide, which inhibits catalase, (2) stimulation occurred in the presence of 1-butanol, which is not an effective substrate for catalase, and (3) stimulation was blocked by hydroxyl radical scavenging agents, which do not affect catalase-mediated oxidation of ethanol. A possible role for contaminating iron in the H2O or buffers could be ruled out since similar results were obtained with or without chelex-100 treatment of these solutions. The stimulatory effect by Fe-EDTA required microsomal electron transfer with NADPH, and H2O2 could not replace the NADPH-generating system. In the absence of microsomes or catalase, Fe-EDTA also stimulated the coupled oxidation of ethanol during the oxidation of xanthine by xanthine oxidase. These results suggest that during microsomal electrom transfer, conditions may be appropriate for a Fenton type or a modified Haber-Weiss type of reaction to occur, leading to the production of hydroxyl radicals.

Microsomal metabolism of hydroxyl radical scavenging agents: Relationship to the microsomal oxidation of alcohols

Previous studies provided indirect evidence that hydroxyl radicals are involved in the oxidation of primary aliphatic alcohols by rat liver microsomes. In the current study, three ·OH scavengers were used as chemical probes to evaluate ·OH production by microsomes. The scavengers and their products were 3-thiomethylpropanal (methional) and 2-keto-4-thiomethylbutyric acid, which yield ethylene gas, and dimethylsulfoxide, which yields methane gas. We observed that microsomes actively generate the appropriate hydrocarbon gas from each scavenger when electron transport is initiated with NADPH. Hydrocarbon gas production is augmented by 0.5 m m azide, an agent which inhibits catalase and, thereby, permits H 2O 2 to accumulate. However, no metabolism of scavengers occurs when H 2O 2 is added in the absence of microsomes. These results are consistent with a presumed role for H 2O 2 as a precursor of hydroxyl radicals. In addition, no metabolism of scavengers occurs when azide and H 2O 2 are added either to boiled microsomes or to intact microsomes in the absence of electron transport (NADPH-generating system omitted). Therefore, both H 2O 2 and simultaneous electron transport are required. Ethanol inhibits the metabolism of the scavengers. Similarly, the scavengers inhibit the oxidation of ethanol to acetaldehyde; inhibition in the presence of azide is competitive. These latter results indicate a competition between the scavengers and ethanol for metabolically generated ·OH in microsomes. The specificity of this interaction is evident from the observation that the scavengers do not affect the activities of microsomal aminopyrine demethylase or aniline hydroxylase. Two model ·OH-generating systems (Fenton's reagent and iron-EDTA-ascorbate) were also studied and they produced acetaldehyde from ethanol and hydrocarbon gases from the scavengers. These results, as a whole, tend to verify a role for ·OH in the microsomal oxidation of alcohols.

Production of hydroxyl radicals and their role in the oxidation of ethanol by a reconstituted microsomal system containing cytochrome P-450 purified from phenobarbital-treated rats

Ethanol oxidation by a reconstituted system composed of cytochrome P-450 purified from liver microsomes of phenobarbital-treated rats, NADPH-cytochrome c reductase, phospholipid and NADPH was inhibited by a series of hydroxyl radical scavenging agents. Inhibition was competitive with respect to ethanol and was specific in the sense that the metabolism of aminopyrine or benzphetamine by the reconstituted system was not affected by the scavengers. The generation of ethylene gas from 2-keto-4-thiomethylbutyric acid in an ethanol-sensitive manner provided chemical evidence consistent with the ability of the reconstituted system to generate hydroxyl radicals. These results suggest that the oxidation of ethanol by the reconstituted system reflects the interaction of ethanol with hydroxyl radicals generated during NADPH oxidation.

Chemical Evidence for Production of Hydroxyl Radicals During Microsomal Electron Transfer

Rat liver microsomes generate methane from dimethyl sulfoxide and ethylene from either methional or 2-keto-4-thiomethylbutyric acid during electron transfer initiated by reduced nicotinamide-adenine dinucleotide phosphate (NADPH). Hydrocarbon gas production is suppressed by hydroxyl radical scavenging agents. Azide, an inhibitor of catalase, augments the production of hydrocarbon gases. These observations constitute chemical evidence for the generation of hydroxyl radicals by microsomes.