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 H₂O₂ 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 H₂O₂ 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.     

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 mm azide, an agent which inhibits catalase and, thereby, permits H₂O₂ to accumulate. However, no metabolism of scavengers occurs when H₂O₂ is added in the absence of microsomes. These results are consistent with a presumed role for H₂O₂ as a precursor of hydroxyl radicals. In addition, no metabolism of scavengers occurs when azide and H₂O₂ are added either to boiled microsomes or to intact microsomes in the absence of electron transport (NADPH-generating system omitted). Therefore, both H₂O₂ 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.     

Microbial oxidation of methanol: Properties of crystallized alcohol oxidase from a yeast, Pichia sp

Alcohol oxidase (alcohol:oxygen oxidoreductase) was crystallized from a methanolgrown yeast, Pichia sp. The crystalline enzyme is homogenous as judged from polyacrylamide gel electrophoresis. Alcohol oxidase catalyzed the oxidation of short-chain primary alcohols (C₁ to C₆), substituted primary alcohols (2-chloroethanol, 3-chloro-1-propanol, 4-chlorobutanol, isobutanol), and formaldehyde. The general reaction with an oxidizable substrate is as follows: Primary alcohol + O₂ → aldehyde + H₂O₂ Formaldehyde + O₂ → formate + H₂O₂. Secondary alcohols, tertiary alcohols, cyclic alcohols, aromatic alcohols, and aldehydes (except formaldehyde) were not oxidized. The K_m values for methanol and formaldehyde are 0.5 and 3.5 mm, respectively. The stoichiometry of substrate oxidized (alcohol or formaldehyde), oxygen consumed, and product formed (aldehyde or formate) is 1:1:1. The purified enzyme has a molecular weight of 300,000 as determined by gel filtration and a subunit size of 76,000 as determined by sodium dodecyl sulfate-gel electrophoresis, indicating that alcohol oxidase consists of four identical subunits. The purified alcohol oxidase has absorption maxima at 460 and 380 nm which were bleached by the addition of methanol. The prosthetic group of the enzyme was identified as a flavin adenine dinucleotide. Alcohol oxidase activity was inhibited by sulfhydryl reagents (p-chloromercuribenzoate, mercuric chloride, 5,5′-dithiobis-2-nitrobenzoate, iodoacetate) indicating the involvement of sulfhydryl groups(s) in the oxidation of alcohols by alcohol oxidase. Hydrogen peroxide (product of the reaction), 2-aminoethanol (substrate analogue), and cupric sulfate also inhibited alcohol oxidase activity.     

Lactoperoxidase-catalyzed lipid peroxidation of microsomal and artificial membranes

Lactoperoxidase, in the presence of H₂O₂, I^?, and rat liver microsomes, will peroxidize membrane lipids, as evidence by malondialdehyde formation. Fe³⁺ assists in the formation of malondialdehyde. Fe³⁺ can be added at the end of the reaction period as well as at the beginning with equal effectiveness, suggesting that it only acts to assist in the conversion of lipid peroxides, previously formed by lactoperoxidase, to malondialdehyde. The addition of EDTA to the microsomal reaction mixture results in a 40% decrease in malondialdehyde formation. The antioxidant butylated hydroxytoluene will completely block the formation of malondialdehyde. Malondialdehyde formation is not dependent upon the production of superoxide, singlet oxygen, or hydroxyl radicals. Peroxidation of membrane lipids by this system is equally effective in both intact microsomes and in liposomes, indicating that iodination of microsomal protein is not required for lipid peroxidation to occur.     

Oxygen free radical scavenger properties of dehydroepiandrosterone

The microsomes from dehydroepiandrosterone (DHEA)-supplemented animals are good hydroxyl radical scavengers, as demonstrated through electron spin resonance and deoxyribose degradation. The ability of DHEA-supplemented microsomes to react with superoxide radical was also demonstrated through the inhibition of nitro-blue tetrazolium reduction determined by superoxide radicals produced in a hypoxanthine–xanthine oxidase system. DHEA-enriched microsomes, obtained from acutely DHEA-treated rats, become resistant to iron-dependent lipid peroxidation triggered by H₂O₂/FeSO₄ and ascorbate/FeSO₄. The direct addition of DHEA to microsomes from untreated rats failed to prevent iron-dependent lipid peroxidation, even if the microsomes were preincubated with DHEA for up to 15 min, indicating that in vivo transformation is required before antioxidant action can be exerted. © 1998 John Wiley & Sons, Ltd.     

Electron acceptor function of O2 in radical N-demethylation reactions catalyzed by hemeproteins

In aerobic solutions, O₂ consumption correlated well with N-demethylation of N,N-dimethyl-$\text{p}$-toluidine catalyzed by horseradish peroxidase, in the presence or absence of H₂O₂. In the absence of added H₂O₂, superoxide dismutase stimulated, and catalase inhibited, both reactions; in the presence of H₂O₂, argon inhibition of formaldehyde production increased with increasing concentration of horseradish peroxidase. These results provide evidence for competing reactions of the enzymatically-generated substrate radical: oxidation by O₂ increases formaldehyde production, while radical dimerization decreases the yield of this product. Implications of these findings for similar reactions catalyzed by microsomal cytochrome P-450 are suggested.     

Experimental approaches to free radicals and ageing

Abstract

Probucol, a clinically used cholesterol lowering and antioxidant drug, was investigated for possible protection against lipid peroxidation and DNA damage induced by iron nitrilotriacetate (Fe-NTA) plus hydrogen peroxide (H₂O₂). Fe-NTA is a potent nephrotoxic agent and induces acute and subacute renal proximal tubular necrosis by catalyzing the decomposition of H₂O₂-derived production of hydroxyl radicals, which are known to cause lipid peroxidation and DNA damage. Fe-NTA is associated with a high incidence of renal adenocarcinoma in rodents. Lipid peroxidation and DNA damage are the principal manifestation of Fe-NTA induced toxicity, which could be mitigated by probucol. Incubation of renal microsomal membrane and/or calf thymus DNA with H₂O₂ (40 mM) in the presence of Fe-NTA (0.1 mM) induces renal microsomal lipid peroxidation and DNA damage to about 2.4-fold and 5.9-fold, respectively, as compared to control (P < 0.05). Induction of renal microsomal lipid peroxidation and DNA damage was inhibited by probucol in a concentration-dependent manner. In lipid peroxidation protection studies, probucol treatment showed a concentration-dependent inhibition (10–34% inhibition; P <0.05) of Fe-NTA plus H₂O₂-induced lipid peroxidation as measured by thiobarbituric acid reacting species' (TBARS) formation in renal microsomes. Similarly, in DNA damage protection studies, probucol treatment also showed a concentration-dependent strong inhibition (36–71% inhibition; P < 0.05) of DNA damage. From these studies, it was concluded that probucol inhibits peroxidation of microsomal membrane lipids and DNA damage induced by Fe-NTA plus H₂O₂. However, because the lipid peroxidation and DNA damage studied here are regarded as early markers of carcinogenesis, we suggest that probucol may be developed as a cancer chemopreventive agent against renal carcinogenesis and other adverse effects of Fe-NTA exposure in experimental animals, in addition to being a cholesterol-lowering drug, useful for the control of hypercholestrolemia.     

Hydroxyl radical production from hydrogen peroxide and enzymatically generated paraquat radicals: Catalytic requirements and oxygen dependence

The ability of paraquat radicals (PQ^+.) generated by xanthine oxidase and glutathione reductase to give H₂O₂-dependent hydroxyl radical production was investigated. Under anaerobic conditions, paraquat radicals from each source caused chain oxidation of formate to CO₂, and oxidation of deoxyribose to thiobarbituric acid-reactive products that was inhibited by hydroxyl radical scavengers. This is in accordance with the following mechanism derived for radicals generated by γ-irradiation [H. C. Sutton and C. C. Winterbourn (1984) Arch. Biochem. Biophys.235, 106–115] PQ^+. + Fe³⁺ (chelate) → Fe²⁺ (chelate) + PQ⁺⁺ H₂O₂ + Fe²⁺ (chelate) → Fe³⁺ (chelate) + OH^? + OH.. Iron-(EDTA) and iron-(diethylenetriaminepentaacetic acid) (DTPA) were good catalysts of the reaction; iron complexed with desferrioxamine or transferrin was not. Extremely low concentrations of iron (0.03 μm) gave near-maximum yields of hydroxyl radicals. In the absence of added chelator, no formate oxidation occurred. Paraquat radicals generated from xanthine oxidase (but not by the other methods) caused H₂O₂-dependent deoxyribose oxidation. However, inhibition by scavengers was much less than expected for a reaction of hydroxyl radicals, and this deoxyribose oxidation with xanthine oxidase does not appear to be mediated by free hydroxyl radicals. With O₂ present, no hydroxyl radical production from H₂O₂ and paraquat radicals generated by radiation was detected. However, with paraquat radicals continuously generated by either enzyme, oxidation of both formate and deoxyribose was measured. Product yields decreased with increasing O₂ concentration and increased with increasing iron(DTPA). These results imply a major difference in reactivity between free and enzymatically generated paraquat radicals, and suggest that the latter could react as an enzyme-paraquat radical complex, for which the relative rate of reaction with Fe³⁺ (chelate) compared with O₂ is greater than is the case with free paraquat radicals.     

Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent formation and breakdown of hydrogen peroxide during mixed function oxidation reactions in liver microsomes.

Conditions for the recovery of H₂O₂ from microsomes and for determination of the rate and extent of H₂O₂ formation during oxidation of NADPH by liver microsomes have been investigated. H₂O₂ was determined by two methods that are applicable to conditions existing during microsomal mixed function oxidation reactions, provided that contaminating catalase activity is inhibited by azide and that interference by other mixed function oxidation reactions can be excluded. To estimate the formation of H₂O₂ in absence of azide, H₂O₂ was determined indirectly by the production of HCHO during oxidation of cold and ¹⁴C-labeled methanol and an excess of exogenous catalase. As additional catalase-independent decomposition of H₂O₂ also occurs during oxidation of NADPH, the kinetics of H₂O₂ formation in microsomes is influenced by two independent processes. H₂O₂ will be produced under optimal conditions i.e., at V when O₂ and NADPH are in excess. Addition or formation of increasing amounts of H₂O₂ raises the substrate (H₂O₂) concentration and will enhance the rate of breakdown of H₂O₂.     

Inactivation of the human CuZn superoxide dismutase during exposure to O-2 and H2O2

Human copper-zinc superoxide dismutase undergoes inactivation when exposed to O₂^? and H₂O₂ generated during the oxidation of acetaldehyde by xanthine oxidase at pH 7.4 and 37° C. In contrast, human manganese superoxide dismutase is not inactivated under the same conditions. Catalase and Mn-superoxide dismutase protect CuZn superoxide dismutase from inactivation. Similar protection is observed with hydroxyl radical (OH.) scavengers, such as formate and mannitol. In contrast, other OH. scavengers such as ethanol and tert-butyl alcohol, have no protective action. The latter results indicate that “free OH.” is not responsible for the inactivation. Furthermore, H₂O₂ generated during the oxidation of glucose by glucose oxidase, i.e., without production of O₂^?, does not induce CuZn superoxide dismutase inactivation. A mechanism accounting for this $\text{O}{}_2{}^{\mathrm{?}}\text{H}{}_2\text{O}{}_2$-dependent inactivation of CuZn superoxide dismutase is proposed.     

Oxidation of Tris to One-Carbon Compounds in a Radical-Producing Model System,in Microsomes,in Hepatocytes and in Rats

The buffer substance tris(hydroxymethyl)aminomethane (Tris) is converted to formaldehyde in an hydroxyl radical producing model system and in rat liver microsomes, and to CO₂ in rat hepatocytes and in the intact rat. In microsomes, formaldehyde formation from Tris is inhibited by catalase, by the antioxidant propylgallate and by the iron chelator deferoxamine, formaldehyde formation is stimulated by the addition of Fe (II) EDTA. In hepatocytes, the formation of [¹⁴C] CO₂ from [¹⁴C] Tris is inhibited by propylgallate and by the iron chelator o-phenanthroline and is stimulated by the presence of a xanthine oxidase system plus Fe (II) EDTA in the medium. In the intact rat, the administration of [¹⁴C] Tris results in the exhalation of [¹⁴C] CO². The results indicate that an oxidant formed via a Fenton-type reaction, possibly the hydroxyl radical, may be involved in the formation of one-carbon compounds from Tris.     

Superoxide and hydrogen peroxide production and NADPH oxidation stimulated by nitrofurantoin in lung microsomes: possible implications for toxicity.

The addition of nitrofurantoin to aerobic incubation mixtures containing rat lung microsomes strongly enhanced the generation of adrenochrome from epinephrine. Adrenochrome formation in this system was blocked by superoxide dismutase, but not by catalase. Hydrogen peroxide production was also strongly enhanced by nitrofurantoin in these preparations; superoxide dismutase did not significantly alter the amount of H₂O₂ measured, but no H₂O₂ was detected in incubation mixtures in the presence of catalase. Nitrofurantoin enhanced the oxidation of NADPH in lung microsomal suspensions under aerobic conditions; the enhancement was unaffected by catalase but was partially prevented by superoxide dismutase. Neither adrenochrome formation nor H₂O₂ production were enhanced by nitrofurantoin under anaerobic (N₂) conditions, but NADPH oxidation in the presence of nitrofurantoin was greater under anaerobic conditions than under aerobic conditions. These results are consistent with the view that the redox cycling of nitrofurantoin in lung microsomes in the presence of oxygen results in the consumption of NADPH and the production of activated oxygen species, emphasizing some $\text{in vitro}$ metabolic similarities with the lung-toxic herbicide, paraquat.     

Cytochrome P450 oxidase activity and its role in NADPH dependent lipid peroxidation

A comparison is made between microsomal NADPH-dependent H₂O₂ production and malondialdehyde (MDA) formation in rat liver microsomes, obtained from phenobarbital pretreated rats. An increase in H₂O₂ formation was observed during NADPH-dependent disposition (10 min) of 100 μM diazepam (33%) and 2 mM hexobarbital (69%). In contrast orphenadrine (100 μM) and its mono-N-demethylated metabolite tofenacine (100 μM) decreased the H₂O₂ formation (35% and 55%, respectively). However, all these substrates were found to inhibit NADPH-dependent lipid peroxidation (60 min), estimated by measuring MDA formation, to various extents. These data strongly suggest that the oxidase activity of cytochrome P450 (H₂O₂ production) is not involved in a rate-limiting step in NADPH-dependent lipid peroxidation.     

Ethylene from 2-keto-4-thiomethyl butyric acid: the Haber-Weiss reaction

2-Keto-4-thiomethyl butyric acid is cooxidized, with production of ethylene, by the xanthine oxidase reaction. Ethylene production was inhibited by either superoxide dismutase or catalase indicating an essential role for both O₂^? and H₂O₂. Ethylene production was dependent upon iron complexes, while a variety of other metals were found ineffective. At pH 7.8 ethylenediaminetetraacetate-iron was much more effective in facilitating ethylene production, in the xanthine oxidase system, than was diethylenetriaminepentaacetate-iron; yet these two chelates were equally effective in ethylene production dependent upon the Fenton reaction. pH was an important variable in determining the activity of iron chelates in facilitating ethylene production in the xanthine oxidase system. The effectiveness of hydroxyl radical scavengers, in preventing ethylene production, was directly related to their abilities to scavenge OH · and this was the case in the presence of any of the several chelating agents tested. The proximal oxidant, responsible for ethylene production, thus appears to be OH ·, rather than some metal-oxy complex.     

Oxidation of ethanol by hepatic microsomes of acatalasemic mice

Hepatic microsomes of acatalasemic Cs^b mice subjected to heat inactivation displayed decreased catalatic activity but NADPH dependent microsomal ethanol oxidation (MEOS) remained active and unaffected. Even without heat inactivation, in the Cs^b strain, the NADPH dependent metabolism of ethanol was much more active than the H₂O₂ mediated one whereas microsomes of Cs^a control mice displayed equal rates of H₂O₂ and NADPH dependent ethanol oxidation. Addition of catalase to liver microsomes in vitro abolished this difference whereas the catalase inhibitor azide established in the Cs^a mice a pattern similar to that of the Cs^b, namely a much more active NADPH dependent than a H₂O₂ mediated ethanol oxidation. The selective persistence in the Cs^b mice of NADPH dependent ethanol oxidation contrasting with the reduction in the H₂O₂ mediated metabolism of ethanol supports the existence of a microsomal ethanol oxidizing system independent of catalase.     

Free Radical Scavenging and Antioxidative Properties of ‘Silibin’ Complexes on Microsomal Lipid Peroxidation

The antioxidant properties of silibin complexes, the water-soluble form silibin dihemisuccinate (SDH), and the lipid-soluble form, silibin phosphatidylcholine complex known as IdB 1016, were evaluated by studying their abilities to react with the superoxide radical anion (O₂^.−), and the hydroxyl radical (OH^.). In addition, their effect on pulmonary and hepatic microsomal lipid peroxidation had been investigated. Superoxide radicals were generated by the PMS-NADH system and measured by their ability to reduce NBT. IC₅₀ concentrations for the inhibition of the NBT reduction by SDH and IdB 1016 were found to be 25 μM and 316 μM respectively. Both silibin complexes had an inhibitory effect on xanthine oxidase activity. SDH reacted rapidly with OH^. radicals at approximately diffusion controlled rate and the rate constant was found to be (K=8·2×10⁹ M ⁻¹ s⁻¹); it appeared to chelate Fe²⁺ in solution. In hepatic microsomes, when lipid peroxidation was induced by Fe²⁺, SDH inhibited by 39·5 per cent and IdB 1016 by 19·5 per cent, whereas when lipid peroxidation was induced by CuOOH, IdB 1016 exerted a better protective effect than SDH (29·4 per cent and 19·4 per cent inhibition, respectively). In both microsomal systems lipid peroxidation proceeded through a thiol depletion mechanism which could be restored in the presence of silibin complexes. Low levels of lipid peroxidation in pulmonary microsomes point out the differences between in-vitro lipid peroxidation occurring in microsomes of different tissues. The results support the free radical scavenger and antioxidative properties of silibin when it is complexed with a suitable molecule to increase its bioavailabilty. © 1997 John Wiley & Sons, Ltd.     

Structural determinants for alcohol substrates to be oxidized to formaldehyde by rat liver microsomes.

Glycerol can be oxidized to formaldehyde by rat liver microsomes and by cytochrome P450. The ability of other alcohols to be oxidized to formaldehyde was determined to evaluate the structural determinants of the alcohol which eventually lead to this production of formaldehyde. Monohydroxylated alcohols such as 1- or 2-propanol did not produce formaldehyde when incubated with NADPH and microsomes. Geminal diols such as 1,3-propanediol, 1,3-butanediol, or 1,4-butanediol also did not yield formaldehyde. However, vicinal diols such as 1,2-propanediol or 1,2-butanediol produced formaldehyde. With 1,2-propanediol, the residual two-carbon fragment was found to be acetaldehyde, while with 1,2-butanediol, the residual three-carbon fragment was propionaldehyde. Oxidation of 1,2-propanediol to formaldehyde plus acetaldehyde involved interaction with an oxidant derived from H2O2 plus nonheme iron, since production of the two aldehydic products was completely prevented by catalase or glutathione plus glutathione peroxidase and by chelators such as desferrioxamine or EDTA. The oxidant was not superoxide or hydroxyl radical. Product formation was fivefold lower when NADH replaced NADPH, and was inhibited by substrates, ligands, and inhibitors of cytochrome P450. A charged glycol such as alpha-glycerophosphate (but not the geminal beta-glycerophosphate) was readily oxidized to formaldehyde, suggesting that interaction of the glycol with the oxidant was occurring in solution and not in a hydrophobic environment. These results indicate that the carbon-carbon bond between 1,2-glycols can be cleaved by an oxidant derived from microsomal generated H2O2 and reduction of non-heme iron, with the subsequent production of formaldehyde plus an aldehyde with one less carbon than the initial glycol substrate.     

Spin-trapping studies of hydroxyl radical production involved in lipid peroxidation.

Evidence presented in this report suggests that the hydroxyl radical (OH.), which is generated from liver microsomes is an initiator of NADPH-dependent lipid peroxidation. The conclusions are based on the following observations: 1) hydroxyl radical production in liver microsomes as measured by esr spin-trapping correlates with the extent of NADPH induced microsomal lipid peroxidation as measured by malondialdehyde formation; 2) peroxidative degradation of arachidonic acid in a model OH · generating system, namely, the Fenton reaction takes place readily and is inhibited by thiourea, a potent OH · scavenger, indicating that the hydroxyl radical is capable of initiating lipid peroxidation; 3) trapping of the hydroxyl radical by the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide prevents lipid peroxidation in liver microsomes during NADPH oxidation, and in the model system in the presence of linolenic acid. The possibility that cytochrome P-450 reductase is involved in NADPH-dependent lipid peroxidation is discussed. The optimal pH for the production of the hydroxyl radical in liver microsomes is 7.2. The generation of the hydroxyl radical is correlated with the amount of microsomal protein, possibly NADPH cytochrome P-450 reductase. A critical concentration of EDTA (5 × 10^?5m) is required for maximal production of the hydroxyl radical in microsomal lipid peroxidation during NADPH oxidation. High concentrations of Fe²⁺-EDTA complex equimolar in iron and chelator do not inhibit the production of the hydroxyl radical. The production of the hydroxyl radical in liver microsomes is also promoted by high salt concentrations. Evidence is also presented that OH radical production in microsomes during induced lipid peroxidation occurs primarily via the classic Fenton reaction.     

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 × 10⁹m^?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.     

Superoxide-dependent formation of hydroxyl radicals: Detection of hydroxyl radicals by the hydroxylation of aromatic compounds

A mixture of xanthine or hypoxanthine and xanthine oxidase generates the superoxide radical, O₂^$\text{?}$, and H₂O₂. In the presence of iron salts, O₂^$\text{?}$ and H₂O₂ can interact to produce the hydroxyl radical, OH·. Superoxide-dependent formation of OH· can be measured by its ability to hydroxylate salicylate as followed by an improved colorimetric assay described in this paper. A more accurate analysis of OH· can be obtained using its ability to hydroxylate phenol, the hydroxylated products being separated and measured after derivatization using gas-liquid chromatography and electron-capture detection. The derivatization and separation techniques are described.