Recycling and antioxidant activity of tocopherol homologs of differing hydrocarbon chain lengths in liver microsomes

Tocopherols (vitamin E) function as inhibitors of lipid peroxidation in biomembranes by donating a hydrogen atom to the chain propagating lipid radicals, thus giving rise to chromanoxyl radicals of the antioxidant. We have shown that alpha-tocopherol homologs differing in the lengths of their hydrocarbon side chains (alpha-Cn) manifest strikingly different antioxidant potencies in membranes. The antioxidant activity of tocopherol homologs during (Fe2+ + ascorbate)- or (Fe2+ + NADPH)-induced lipid peroxidation in rat liver microsomes increased in the order alpha-tocopherol (alpha-C16) less than alpha-C11 less than alpha-C6 less than alpha-C1. Chromanoxyl radicals generated from alpha-tocopherol and its more polar homologs by an enzymatic oxidation system (lipoxygenase + linolenic acid) can be recycled in rat liver microsomes by NAD-PH-dependent electron transport or by ascorbate. The efficiency of recycling increased in the same order: alpha-tocopherol (alpha-C16) less than alpha-C11 less than alpha-C6 less than alpha-C1. Thus the high efficiency of regeneration of short-chain homologs of vitamin E may account for their high antioxidant potency.     

NADPH-dependent inhibition of lipid peroxidation in rat liver microsomes.

Microsomal NADPH-driven electron transport is known to initiate lipid peroxidation by activating oxygen in the presence of iron. This pro-oxidant effect can mask an antioxidant function of NADPH-driven electron transport in microsomes via vitamin E recycling from its phenoxyl radicals formed in the course of peroxidation. To test this hypothesis we studied the effects of NADPH on the endogenous vitamin E content and lipid peroxidation induced in liver microsomes by an oxidation system independent of iron: an azo-initiator of peroxyl radicals, 2,2'-azobis (2,4-dimethylvaleronitrile), (AMVN), in the presence of an iron chelator deferoxamine. We found that under conditions NADPH: (i) inhibited lipid peroxidation; (ii) this inhibitory effect was less pronounced in microsomes from vitamin E-deficient rats than in microsomes from normal rats; (iii) protected vitamin E from oxidative destruction; (iv) reduced chromanoxyl radicals of vitamin E homologue with a 6-carbon side-chain, chromanol-alpha-C-6. Thus NADPH-driven electron transport may function both to initiate and/or inhibit lipid peroxidation in microsomes depending on the availability of transition metal catalysts.     

Production of superoxide radical in reductive metabolism of a synthetic food-coloring agent, indigocarmine, and related compounds

Indigocarmine, which is widely used as a synthetic colouring agent for foods and cosmetics in many countries, was reduced to its leuco form and decolorized by rat liver microsomes with NADPH under anaerobic conditions. The reductase activity was enhanced in liver microsomes of phenobarbital-treated rats, and inhibited by diphenyliodonium chloride, a NADPH-cytochrome P450 reductase (P450 reductase) inhibitor, but was not inhibited by SKF 525-A or carbon monoxide. Indigocarmine reductase activity was exhibited by purified rat P450 reductase. In contrast, when indigocarmine was incubated with rat liver microsomes and NADPH under aerobic conditions, superoxide radical was produced and its production was inhibited by superoxide dismutase and diphenyliodonium chloride. When indigocarmine was incubated with purified rat P450 reductase in the presence of NADPH, superoxide radical production was enhanced 17.7-fold (similar to the enhancement of indigocarmine-reducing ability) as compared with that of rat liver microsomes. A decrease of one molecule of NADPH was accompanied with formation of about two molecules of superoxide radical. P450 reductase exhibited little reductase activity towards indigo and tetrabromoindigo, which also afforded little superoxide radical under aerobic conditions. These results indicate that indigocarmine is reduced by P450 reductase to its leuco form, and superoxide radical is produced by autoxidation of the leuco form, through a mechanism known as futile redox cycling.     

Generation and recycling of radicals from phenolic antioxidants

Hindered phenols are widely used food preservatives. Their pharmacological properties are usually attributed to high antioxidant activity due to efficient scavenging of free radicals. Butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) also cause tissue damage. Their toxic effects could be due to the production of phenoxyl radicals. If phenoxyl radicals can be recycled by reductants or electron transport, their potentially harmful side reactions would be minimized. A simple and convenient method to follow phenoxyl radical reactions in liposomes and rat liver microsomes based on an enzymatic (lipoxygenase + linolenic acid) oxidation system was used to generate phenoxyl radicals from BHT and its homologues with substitutents in m- and p-positions. Different BHT-homologues display characteristic ESR signals of their radical species. In a few instances the absence of phenoxyl radical ESR signals was found to be due to inhibition of lipoxygenase by BHT-homologues. In liposome or microsome suspensions addition of ascorbyl palmitate resulted in disappearance of the ESR signal of phenoxyl radicals with concomittant appearance of the ascorbyl radical signal. After exhaustion of ascorbate, the phenoxyl radical signal reappears. Comparison of the rates of ascorbyl radical decay in the presence or absence of BHT-homologues showed that temporary elimination of the phenoxyl radical ESR signal was due to their reduction by ascorbate. Similarly, NADPH or NADH caused temporary elimination of ESR signals as a result of reduction of phenoxyl radicals in microsomes. Since ascorbate and NADPH might generate superoxide in the incubation system used, SOD was tested. SOD shortened the period, during which the phenoxyl radicals ESR signal could not be observed. Both ascorbyl palmitate and NADPH exerted sparing effects on the loss of BHT-homologues during oxidation. These effects were partly diminished by SOD. These data indicate that reduction of phenoxyl radicals was partly superoxide-dependent. It is concluded that redox recycling of phenoxyl radicals can occur by intracellular reductants like ascorbate and microsomal electron transport.     

Antioxidant effects of ubiquinones in microsomes and mitochondria are mediated by tocopherol recycling

Ubiquinones and tocopherols (vitamin E) are intrinsic lipid components which have a stabilizing function in many membranes attributed to their antioxidant activity. The antioxidant effects of tocopherols are due to direct radical scavenging. Although ubiquinones also exert antioxidant properties the specific molecular mechanisms of their antioxidant activity may be due to: (i) direct reaction with lipid radicals or (ii) interaction with chromanoxyl radicals resulting in regeneration of vitamin E. Lipid peroxidation results have now shown that tocopherols are much stronger membrane antioxidants than naturally occurring ubiquinols (ubiquinones). Thus direct radical scavenging effects of ubiquinols (ubiquinones) might be negligible in the presence of comparable or higher concentrations of tocopherols. In support of this our ESR findings show that ubiquinones synergistically enhance enzymic NADH- and NADPH-dependent recycling of tocopherols by electron transport in mitochondria and microsomes. If ubiquinols were direct radical scavengers their consumption would be expected. Further proving our conclusion HPLC measurements demonstrated that ubiquinone-dependent sparing of tocopherols was not accompanied by ubiquinone consumption.     

Free radical recycling and intramembrane mobility in the antioxidant properties of alpha-tocopherol and alpha-tocotrienol.

d-Alpha-tocopherol (2R,4'R,8'R-Alpha-tocopherol) and d-alpha-tocotrienol are two vitamin E constituents having the same aromatic chromanol "head" but differing in their hydrocarbon "tail": tocopherol with a saturated and toctrienol with an unsaturated isoprenoid chain. d-Alpha-tocopherol has the highest vitamin E activity, while d-alpha-tocotrienol manifests only about 30% of this activity. Since vitamin E is considered to be physiologically the most important lipid-soluble chain-breaking antioxidant of membranes, we studied alpha-tocotrienol as compared to alpha-tocopherol under conditions which are important for their antioxidant function. d-Alpha-tocotrienol possesses 40-60 times higher antioxidant activity against (Fe2+ + ascorbate)- and (Fe2+ + NADPH)-induced lipid peroxidation in rat liver microsomal membranes and 6.5 times better protection of cytochrome P-450 against oxidative damage than d-alpha-tocopherol. To clarify the mechanisms responsible for the much higher antioxidant potency of d-alpha-tocotrienol compared to d-alpha-tocopherol, ESR studies were performed of recycling efficiency of the chromanols from their chromanoxyl radicals. 1H-NMR measurements of lipid molecular mobility in liposomes containing chromanols, and fluorescence measurements which reveal the uniformity of distribution (clusterizations) of chromanols in the lipid bilayer. From the results, we concluded that this higher antioxidant potency of d-alpha-tocotrienol is due to the combined effects of three properties exhibited by d-alpha-tocotrienol as compared to d-alpha-tocopherol: (i) its higher recycling efficiency from chromanoxyl radicals, (ii) its more uniform distribution in membrane bilayer, and (iii) its stronger disordering of membrane lipids which makes interaction of chromanols with lipid radicals more efficient. The data presented show that there is a considerable discrepancy between the relative in vitro antioxidant activity of d-alpha-tocopherol and d-alpha-tocotrienol with the conventional bioassays of their vitamin activity.     

Identification of radicals formed in the reaction mixtures of rat liver microsomes with ADP, Fe3+ and NADPH using HPLC EPR and HPLC EPR MS

The reaction of rat liver microsomes with Fe(3+), ADP and NADPH was examined using EPR, HPLC-EPR and HPLC-EPR-MS combined use of spin trapping technique. A prominent EPR spectrum (alpha(N) = 1.58 mT and alpha(H)beta = 0.26 mT) was observed in the complete reaction mixture. The EPR spectrum was hardly observed for the complete reaction mixture without rat liver microsomes. The radicals appear to be derived from microsomal components. The EPR spectrum was also hardly observed in the absence of Fe(3+). Addition of some iron chelators such as EDTA, citrate and ADP resulted in the dramatic change in the EPR intensity. Iron ions seem to be essential for this reaction. For the complete reaction mixture with boiled microsomes, a weak EPR spectrum was observed, suggesting that enzymes participate in the reaction. Five peaks were separated on the HPLC-EPR elution profile of the complete reaction mixture of rat liver microsomes with ADP, Fe(3+) and NADPH. The retention times of the peaks 1 to 5 were 19.4, 22.5, 27.3, 29.8 and 31.4 min, respectively. To identify the radical adducts, HPLC-EPR-MS analyses were performed for the three prominent peaks. The HPLC-EPR-MS analyses showed that a new radical adduct, 4-POBN/1-hydroxypentyl radical, in addition to 4-POBN/ethyl radical adducts, forms in a reaction mixture of rat liver microsomes with ADP, Fe(3+) and NADPH.     

Genetic polymorphism of the CYP1A1, CYP2E1, GSTM1 and GSTT1 genes and lung cancer susceptibility in a north Indian population

The present study investigates in a experimental system in vitro the relationship between the non-enzymatic (ascorbate-Fe2+) and enzymatic (NADPH) lipid peroxidation in rat liver microsomes and nuclei. Chemiluminescence was measured as cpm/mg protein during 180 min at 37 degrees C. Approximately 50-55% of the fatty acids located in rat liver microsomes and nuclei are polyunsaturated with a prevalence of C18:2 n6 and C20:4 n6. The values of total light emission during the non-enzymatic and enzymatic lipid peroxidation were highest in microsomes than in nuclei. A significant decrease of C20:4 n6 and C22:6 n3 in rat liver microsomes and nuclei was observed during the lipid ascorbate-Fe2+-dependent peroxidation, whereas a significant decrease of C20:4 n6 in rat liver microsomes was observed during enzymatic lipid peroxidation. Over the time course studies, analysis of chemiluminescence in microsomes and nuclei demonstrated that the lipid peroxidation in the presence of ascorbate-Fe2+ reach a maximum at 50 and 30 min, respectively, whereas in the presence of NADPH it reachs a maximum at 20 min in both organelles. In liver microsomes and nuclei the peroxidizability index (pi) which indicates the degree of vulnerability to degradation of a selected membrane showed statistically significant differences between control versus ascorbate-Fe2+ when microsomes or nuclei were compared. Our results indicate that non-enzymatic (ascorbate-Fe2+) and enzymatic (NADPH) lipid peroxidation are operative in rat liver microsomes and nuclei but the sensitivities of both organelles to lipid peroxidation evidenced by chemiluminescence was greater in the presence of ascorbate-Fe2+ when compared with NADPH.     

The mechanism of microsomal and mitochondrial nitroreductase. Electron spin resonance evidence for nitroaromatic free radical intermediates.

Electron spin resonance spectra are observed during the enzymatic reduction of many nitrophenyl derivatives by rat hepatic microsomes or mitochondria. The spectra indicate that nitroaromatic anion radicals are present and are freely rotating in aqueous solution at a steady-state concentration of 0.1-6 muM. The rate of formation of p-nitrobenzoate (NBZO) dianion radical in microsomal incubates is consistent with the radical being an obligate intermediate in the reduction of NBZO to p-aminobenzoic acid. A model system consisting of NBZO, NADPH, and FMN, but no heme-containing compounds, also reduced NBZO to the NBZO dianion free radical. The steady-state concentration of the anion radicals in microsomal systems is not altered by CO. This observation, together with the results from the model system, suggests that the formation of nitroaromatic anion radicals is mediated through a flavine and not cytochrome P-450. The oxidation of the anion radical intermediate by O2 to the parent nitro compound is proposed to account for the well-known O2 inhibition of microsomal nitroreductase.     

Direct detection of radical generation in rat liver nuclei on treatment with tumour-promoting hydroperoxides and related compounds

EPR spin trapping has been employed to directly detect radical production in isolated rat nuclei on exposure to a variety of hydroperoxides and related compounds which are known, or suspect, tumour promoters. The hydroperoxides, in the absence of reducing equivalents, undergo oxidative cleavage, generating peroxyl radicals. In the presence of NADPH (and to a lesser extent NADH) reductive cleavage of the OO bond generates alkoxyl radicals. These radicals undergo subsequent rearrangements and reactions (dependent on the structure of the alkoxyl radical), generating carbon-centred radicals. Acyl peroxides and peracids appear to undergo only reductive cleavage of the OO bond. With peracids this cleavage can generate aryl carboxyl (RCO₂·) or hydroxyl radicals (HO·); with acyl peroxides, aryl carboxyl radicals are formed and, in the case of t-butyl peroxybenzoate, alkoxyl radicals (RO·). The radicals detected with each peroxide are similar in type to those detected in the rat liver microsomal fraction, although the extent of radical production is lower. The subsequent reactions of the initially generated radicals are similar to those determined in homogenous chemical systems, suggesting that they are in free solution. Experiments with NADPH/NADH, heat denaturation of the nuclei and various inhibitors suggest that radical generation is an enzymatic process catalysed by haemproteins, in particular cytochrome P-450, and that NADPH/cytochrome P-450 reductase is involved in the reductive cleavage of the OO bond. The generation of these radicals by the rat liver nuclear fraction is potentially highly damaging for the cell due to the proximity of the generating source to DNA. Several previous studies have shown that some of the radicals detected in this study, such as aryl carboxyl and aryl radicals, can damage DNA, via various reactions which results in the generation of strand breaks and adducts to DNA bases: these processes are suggested to play an important role in the tumour promoting activity of these hydroperoxides and related compounds.     

Formation of a tyrosyl radical intermediate in Proteus mirabilis catalase by directed mutagenesis and consequences for nucleotide reactivity.

Proteus mirabilis catalase (PMC) belongs to the family of NADPH binding catalases. The function of NADPH in these enzymes is still a matter of debate. This study presents the effects of two independent phenylalanine mutations (F194 and F215), located between NADPH and heme in the PMC structure. The phenylalanines were replaced with tyrosines which we predicted could carry radicals in a NADPH-heme electron transfer. The X-ray crystal structures of the two mutants indicated that neither the binding site of NADPH nor the immediate environment of the residues was affected by the mutations. Measurements using H2O2 as a substrate confirmed that the variants were as active as the native enzyme. With equivalent amounts of peroxoacetic acid, wild-type PMC, F215Y PMC, and beef liver catalase (BLC) formed a stable compound I, while the F194Y PMC variant produced a compound I which was rapidly transformed into compound II and a tyrosyl radical. EPR studies showed that this radical, generated by the oxidation of Y194, was not related to the previously observed radical in BLC, located on Y369. In the presence of excess NADPH, compound I was reduced to a resting enzyme (k(obs) = 1.7 min(-1)) in a two-electron process. This was independent of the enzyme's origin and did not require any thus far identified tyrosyl radicals. Conversely, the presence of a tyrosyl radical in F194Y PMC greatly enhanced the oxidation of reduced beta-nicotinamide mononucleotide under a steady-state H2O2 flow with observable compound II. This process could involve a one-electron reduction of compound I via Y194.     

Tocopheroxyl radical persistence and tocopherol consumption in liposomes and in vitamin E-enriched rat liver mitochondria and microsomes

Substantial loading of rat liver mitochondrial and microsomal membranes with D-alpha-tocopherol was achieved by dietary supplementation with no adverse effects of this loading being apparent, e.g. on treadmill exercise endurance. The tocopheroxyl radical was readily detected by ESR in the enriched microsomes and mitochondria. Continuous enzymatic oxidation with horseradish peroxidase and a hydrophilic phenol, to favor selective oxidation of tocopherol without the involvement of lipid peroxidation, allowed the tocopheroxyl radical to be observed for up to 1 h in liposomes of dioleoylphosphatidylcholine and for about 15 min in the subcellular membranes. Total alpha-tocopherol decreased throughout this period, but a significant residual fraction remained after all the ESR signal of tocopheroxyl had disappeared. Decay kinetics of the tocopheroxyl radical ESR signal produced by a burst of intense UV irradiation consisted of a rapid initial phase and a slower exponential decay. A more narrow and more persistent ESR signal, not yet chemically identified, was observed after the tocopheroxyl radical had disappeared under prolonged oxidation. Ascorbic acid prevented formation of the tocopheroxyl radical until the ascorbyl radical ESR signal had decayed, whereas uric acid, up to saturating concentration in phosphate buffer, had no effect.     

Nitrobenzyl radical metabolites from microsomal reduction of nitrobenzyl chlorides

The o-, m-, and p-nitrobenzyl chlorides are reduced aerobically and anaerobically by NADPH and rat hepatic microsomes. Under aerobic conditions, these nitro anion radicals reduce oxygen to superoxide as demonstrated by oxygen consumption and spin trapping of superoxide with 5,5-dimethyl-1-pyrroline N-oxide. At low oxygen concentration, the p- and o-nitro anion radicals undergo intramolecular electron transfer and decompose to carbon-centered nitrobenzyl radicals, which can be spin-trapped with t-nitrosobutane. The p-nitrobenzyl (o-nitrobenzyl) radical adduct was characterized by a nitrogen hyperfine splitting of 16.5 G (17.1 G) and two equivalent beta-hydrogen hyperfine splittings of 10.6 G (14.4 G). The spin trap 5,5-dimethyl-1-pyrroline N-oxide also yields adducts characteristic of carbon-centered free radicals. This unimolecular decomposition is much faster than the disproportionation decay, which is characteristic of most nitro anion radicals, and the primary o- and p-nitrobenzyl chloride anion radicals never achieve detectable concentrations. The nitrobenzyl radical trapping is not inhibited by metyrapone or CO. In contrast, the m-nitrobenzyl anion radical does achieve a detectable steady-state concentration, which is increased 20% by either metyrapone or a CO atmosphere.     

Spin-trapping of the trichloromethyl radical produced during enzymic NADPH oxidation in the presence of carbon tetrachloride or bromotrichloromethane.

Utilizing the spin-trapping agent phenyl-t-butyl nitrone, a free radical has been detected which is produced from carbon tetrachloride or bromotrichloromethane during the enzymic oxidation of NADPH by rat liver microsomes. The presence of NADPH is obligatory for generation of the radical. The formation of the trichloromethyl radical-phenyl-t-butyl nitrone adduct is an enzymic process, as evidenced by the inhibition of its formation in systems containing heated microsomes and in systems containing p-hydroxymercuribenzoate. A computer-simulated ESR spectrum for the trichloromethyl adduct of phenyl-t-butyl nitrone can reproduce the essential features of the spectrum of the spin-trapped radical produced enzymically from CCl4. A mechanism is proposed for the formation of the trichloromethyl radical from CCl4 or BrCCl3.     

Effect of the microsomal system on interconversions between hydroquinone, benzoquinone, oxygen activation, and lipid peroxidation

Our previous results indicated that cytochrome P450 destruction by benzene metabolites was caused mainly by benzoquinone (Soucek et al., Biochem. Pharmacol. 47 (1994) 2233-2242). The aim of this study was to investigate the interconversions between hydroquinone, semiquinone, and benzoquinone with regard to both spontaneous and enzymatic processes in order to test the above hypothesis. We have also studied the participation of hydroquinone and benzoquinone in OH radicals formation and lipid peroxidation as well as the role of ascorbate and transition metals. In buffered aqueous solution, hydroquinone was slowly oxidized to benzoquinone via a semiquinone radical. This conversion was slowed down by the addition of NADPH and completely stopped by microsomes in the presence of NADPH. Benzoquinone was reduced to semiquinone radical at a significantly higher rate and this conversion was stimulated by NADPH and more effectively by microsomes plus NADPH while semiquinone radical was quenched there. In microsomes with NADPH. both hydroquinone and benzoquinone stimulated the formation of OH radicals but inhibited peroxidation of lipids. Ascorbate at 0.5-5 mM concentration also produced significant generation of OH radicals in microsomes. Neither hydroquinone nor benzoquinone did change this ascorbate effect. On the contrary, 0.1-1.0 mM ascorbate stimulated peroxidation of lipids in microsomes whereas presence of hydroquinone or benzoquinone completely inhibited this deleterious effect of ascorbate. Iron-Fe2+ apparently played an important role in lipid peroxidation as shown by EDTA inhibition, but it did not influence OH radical production. In contrast, Fe3+ did not influence lipid peroxidation, but stimulated OH radical production. Thus, our results indicate that iron influenced the above processes depending on its oxidation state, but it did not influence hydroquinone/benzoquinone redox processes including the formation of semiquinone. It can be concluded that interconversions between hydroquinone and benzoquinone are influenced by NADPH and more effectively by the complete microsomal system. Ascorbate, well-known antioxidant produces OH radicals and peroxidation of lipids. On the other hand, both hydroquinone and benzoquinone appear to be very efficient inhibitors of lipid peroxidation.     

Oxygen- and carbon-centered free radical formation during carbon tetrachloride metabolism. Observation of lipid radicals in vivo and in vitro

Free radical reactions involved in the metabolism of carbon tetrachloride by rat liver have been considered to be a cause of at least part of the injury resulting from exposure to this halocarbon. In an earlier study employing electron spin resonance and spin-trapping techniques, we demonstrated that trichloromethyl (13.CCl3) radicals are readily observed in rat liver microsomes metabolizing 13CCl4, and that the same radical could be shown to form in vivo in the liver of intact rats given a single dose of 13CCl4. This report describes the production of lipid dienyl (L.) and oxygen-centered lipid radicals (LO. or LOO., or both) in in vitro systems metabolizing 13CCl4, and also the formation of lipid dienyl radicals (L.) in liver of intact animals exposed to CCl4. The radicals appear to be produced in a sequence of reactions governed among other things by the oxygen tension in the system. The lipid radicals (L.) which form in intact liver of CCl4-treated rats are apparently the result of an attack on lipids of the endoplasmic reticulum by 13.CCl3 radicals formed by reductive cleavage to CCl4 and are the initial intermediates in the process of lipid peroxidation. These investigations demonstrate that while the events occurring in liver microsomes in vitro appear to parallel those which take place in intact liver in vivo, the conditions in vivo make the spin-trapping studies of radicals in intact animals much more selective than it is in vitro for a given spin trap, and requires the use of more than one type of spin-trapping agent to detect different radical species in vivo.     

Inhibition of lipid peroxidation by heme-nonapeptide derived from cytochrome c

Heme-nonapeptide, derived from cytochrome c, inhibited both the NADPH- and NADH-dependent lipid peroxidation of brain microsomes but, in the case of liver microsomes, this inhibitory effect manifested itself in the presence of SKF-525A (a specific blocker of cytochrome P-450) only. Heme-nonapeptide prevented the transient accumulation of lipid peroxides in microsomes during lipid peroxidation. The oxygen consumption of microsomes in the presence of NADPH or NADH was stimulated by heme-nonapeptide. From these results we concluded that, in vitro, there are two independent mechanisms of lipid peroxidation in liver microsomes. It is suggested that, in vivo, the heme-peptide-sensitive mechanism, observed in brain microsomes, is more important.     

Increased microsomal interaction with iron and oxygen radical generation after chronic acetone treatment

In vivo administration of acetone influences a variety of reactions catalyzed by rat liver microsomes. The effect of chronic treatment with acetone (1% acetone in the water for 10-12 days) on interaction with iron and subsequent oxygen radical generation by liver microsomes was evaluated. Microsomes from the acetone-treated rats displayed elevated rates of H2O2 generation, an increase in iron-dependent lipid peroxidation, and enhanced chemiluminescence upon the addition of t-butylhydroperoxide. The ferric EDTA-catalyzed production of formaldehyde from DMSO or of ethylene from 2-keto-4-thiomethylbutyrate was increased 2-fold after acetone treatment. This increase in hydroxyl radical generation was accompanied by a corresponding increase in NADPH utilization and was sensitive to inhibition by catalase and a competitive scavenger, ethanol, but not to superoxide dismutase. In vitro addition of acetone to microsomes had no effect on oxygen radical generation. Associated with the chronic acetone treatment was a 2-fold increase in the microsomal content of cytochrome P-450 and in the activity of NADPH-cytochrome-P-450 reductase. It appears that increased oxygen radical generation by microsomes after chronic acetone treatment reflects the increase in the major enzyme components which comprise the mixed-function oxidase system.     

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.     

Ascorbyl free radical and dehydroascorbate formation in rat liver endoplasmic reticulum

The mechanism of ascorbate oxidation was studied in rat liver microsomes. A continuous consumption of the added ascorbate was observed, which was accompanied with a prompt appearance of ascorbyl free radical and dehydroascorbate. Microsomes sustained steady-state level of ascorbyl free radical and dehydroascorbate till ascorbate was present in the medium. Ascorbyl free radical formation was diminished when microsomes had been pretreated with heat or trypsine. It was also decreased by addition of quercetin, econazole or metal chelators, including the copper specific neocuproine. Enzymatic (superoxide dismutase, catalase) and nonenzymatic (dimethyl sulfoxide, mannitol) antioxidants did not modify the microsomal production of ascorbyl free radical. Investigation of the subcellular distribution of ascorbate oxidation showed that the microsomal fraction of liver had the highest activity. The decrease of ascorbate oxidation after protease treatment and the negligible increase upon permeabilization of microsomal vesicles showed that a membrane protein is responsible for the activity, which is exposed to the outer surface of the endoplasmic reticulum. The results indicate the presence of a primary enzymatic ascorbate oxidation in rat liver endoplasmic reticulum which is able to generate dehydroascorbate, an important source of the oxidizing environment in the endoplasmic reticulum.