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.     

Carbon radicals in the metabolism of alkyl hydrazines

The metabolism of phenelzine (2-phenylethylhydrazine) by rat liver microsomes yields phenylacetaldehyde, 2-phenylethanol, and ethylbenzene. A carbon radical is formed during the oxidative metabolism of phenelzine that reacts with the prosthetic heme of cytochrome P-450 and irreversibly inactivates the enzyme. The radical has been spin-trapped, isolated, and shown by mass spectrometry to be the 2-phenylethyl radical. The metal-free pophyrin derived from the prosthetic heme group has been isolated and identified as N-(2-phenylethyl)protoporphyrin IX. The metabolism of phenelzine, an alkyl hydrazine, thus yields a carbon radical that inactivates cytochrome P-450, is converted to a hydrocarbon by hydrogen atom abstraction, and reacts with spin traps or (presumably) alternative cellular targets.     

Microsomal metabolism of ciprofloxacin generates free radicals

Ciprofloxacin (CPFX) is a widely used fluoroquinolone antibiotic with a broad spectrum of activity. However, clinical experience has shown a possible incidence of undesirable adverse effects including gastrointestinal, skin, hepatic, and central nervous system (CNS) functions, and phototoxicity. Several examples in the literature data indicate that free radical formation might play a role in the mechanism of some of these adverse effects, including phototoxicity and cartilage defects. The purpose of this study is to investigate free radical formation during the metabolism of CPFX in hepatic microsomes using electron spin resonance (ESR) spectroscopy and spin trapping technique. We then investigate the effects of a cytochrome P450 inhibitor, SKF 525A, Trolox, and ZnCl2 on CPFX-induced free radical production. Our results show that CPFX induces free radical production in a dose- and time-dependent manner. The generation of 4-POBN/radical adduct is dependent on the presence of NADPH, CPFX, and active microsomes. Furthermore, free radical production is completely inhibited by SKF 525A, Trolox, or ZnCl2.     

Induction of free radicals in hepatocytes,mitochondria and microsomes of rats by ochratoxin A and its analogs

Oxidative damage may be one of the manifestations of cellular damage in the toxicity of ochratoxin A (OA). OA; its three natural analogs, OB, OC and Oα; and three synthetic analogs, the ethyl amide of OA (OE-OA), O-methylated OA (OM-OA), and the lactone-opened OA (OP-OA) were used to study free radical generation in hepatocytes, mitochondria and microsomes from rats. Electron paramagnetic resonance spectroscopy (EPR) using α-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (4-POBN) as a spin trapping agent showed an enhanced free radical generation due to the addition of NADPH to the microsomes. An EPR signal was not observed in the mitochondria and hepatocyte samples when they were treated with a variety of agents. Addition of OM-OA together with NADPH and Fe³⁺ to the microsomes resulted in a strong EPR signal compared with the other analogs, whereas the signal could be quenched by the addition of catalase. OM-OA does not have a dissociable phenolate group and does not chelate Fe³⁺. The spin adduct hyperfine splitting constants indicated the presence of α-hydroxyethyl radicals resulting from generated hydroxyl radicals, which were trapped by 4-POBN. The results also suggested that the production of hydroxyl radicals by OA does not require a dissociable phenolate group or the prior formation of an OA-Fe complex.     

DNA alterations induced by the carbon-centered radical derived from the oxidation of 2-phenylethylhydrazine

The possible significance of carbon-centered radicals in hydrazine-induced carcinogenesis is explored by studies of the interaction between the 2-phenylethyl radical and DNA. The radical is efficiently generated during oxidation of phenelzine (2-phenylethylhydrazine) promoted by oxyhemoglobin or ferricyanide, as demonstrated by spin-trapping experiments and analysis of the reaction products. In the ferricyanide promoted oxidation, ethylbenzene formation accounts for about 40% of the initial drug concentration, from 5 to 100 mM phenelzine. By contrast, product formation in the presence of oxyhemoglobin depends on the enzyme concentration due to the fact that the prosthetic heme is destroyed during catalytic turnover. Covalent binding of the 2-phenylethyl radical to oxyhemoglobin is demonstrated by experiments with 2-[3H]phenelzine, where tritium incorporation to the protein is inhibited by the spin-trap, alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone. The 2-phenylethyl radical is also able to alkylate DNA as suggested by electrophoretic studies with plasmid DNA, and proved by experiments with 2-[3H]-phenelzine. The carbon-centered radical has a preference for attacking guanine residues as demonstrated by the use of sequencing techniques with 32P-DNA probes. The results indicate that the 2-phenylethyl radical is an important product of phenelzine oxidation and that this species can directly damage protein and DNA.     

Free Radical Metabolism of Alcohols by Rat Liver Microsomes

By using e.s.r. spectroscopy coupled with the spin trapping technique we have detected the formation of free radical intermediates by rat liver microsomes incubated with either ethanol, 2-propanol or 2-butanol in the presence of a NADPH regenerating system and 4-pyridyl-l-oxide-t-butyl nitrone (4-POBN) as spin trap. The e.s.r. spectra have been identified as due to the hydroxyalkyl free radical adducts of 4-POBN.

The free radical formation depends upon the activity of the microsomal monoxygenase system and is blocked by omitting NADP⁺ from the incubation mixture, by anaerobic incubation or by enzyme denaturation. The involvement of hydroxyl radicals (OH) produced through a Fenton-type reaction from endogenously formed hydrogen peroxide is suggested by the opposite effects exerted on the e.s.r. signal intensity by azide and catalase. Consistently, iron chelation by desferrioxamine inhibits the free radical formation, while the supplementation of EDTA-iron increases it by several fold. Inhibitors of cytochrome P₄₅₀-dependent monoxygenase system reduce to various extents the production of free radical intermediates suggesting that reactive oxygen species might be formed at the active site of cytochrome P₄₅₀ where they react with alkyl alcohol molecules.

The data presented support the hypothesis that free radical species are generated during the microsomal metabolism of alcohols and suggest the possibility that ethanol-derived radicals might play a role in the pathogenesis of the liver lesions consequent upon alcoholic abuse.     

Spin trapping of free radical species produced during the microsomal metabolism of ethanol

Liver microsomes incubated with a NADPH regenerating system, ethanol and the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) produced an electron spin resonance (ESR) signal which has been assigned to the hydroxyethyl free radical adduct of 4-POBN by using 13C-labelled ethanol. The free radical formation was dependent upon the activity of the microsomal monoxygenase system and increased following chronic feeding of the rats with ethanol. The production of hydroxyethyl free radicals was stimulated by the addition of azide, while catalase and OH. scavengers decreased it. This suggested that hydroxyl radicals (OH.) produced in a Fenton-type reaction from endogenously formed hydrogen peroxide were involved in the free radical activation of ethanol. Consistently, the supplementation of iron, under various forms, also increased the intensity of the ESR signal which, on the contrary, was inhibited by the iron-chelating agent desferrioxamine. Microsomes washed with a solution containing desferrioxamine and incubated in a medium treated with Chelex X-100 in order to remove contaminating iron still produced hydroxyethyl radicals, although at a reduced rate. Under these conditions the free radical formation was apparently independent from the generation of OH. radicals, whereas addition of cytochrome P-450 inhibitors decreased the hydroxyethyl radical formation, suggesting that a cytochrome P-450-mediated process might also be involved in the activation of ethanol. Reduced glutathione (GSH) was found to effectively scavenge the hydroxyethyl radical, preventing its trapping by 4-POBN. The data presented suggest that ethanol-derived radicals could be generated during the microsomal metabolism of alcohol probably through two different pathways. The detection of ethanol free radicals might be relevant in understanding the pathogenesis of the liver lesions which are a consequence of alcohol abuse.     

A search for oxygen-centered free radicals in the lipoxygenase/linoleic acid system

Studies of the oxygenation of linoleic acid by soybean lipoxygenase utilizing electron spin resonance spectroscopy and oxygen uptake have been undertaken. The spin trap, alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (4-POBN) was included in the lipoxygenase system to capture short-lived free radicals. Correlation of radical adduct formation rates with oxygen uptake studies indicated that the major portion of radical adduct formation occurred when the system was nearly anaerobic. Incubations containing [17O]oxygen with nuclear spin of 5/2 did not have additional ESR lines as would be expected if an oxygen-centered 4-POBN-lipid peroxyl radical adduct were formed indicating that the trapped radical must be reassigned as a carbon-centered species. To establish the presence of [17O2]oxygen in our incubations, a portion of the gas from the lipoxygenase/linoleate experiments was used to prepare the 4-POBN-superoxide radical adduct utilizing a superoxide producing microsomal/paraquat/NADPH 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.     

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.     

Oxygen-based free radical generation by ferrous ions and deferoxamine

Deferoxamine accelerates the autooxidation of iron as measured by the rapid disappearance of Fe2+, the associated appearance of Fe3+, and the uptake of oxygen. Protons are released in the reaction. The formation of H2O2 was detected by the horseradish peroxidase-catalyzed oxidation of scopoletin, and the formation of hydroxyl radicals (OH.) was suggested by the formation of the OH. spin trap adduct (DMPO/OH). with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and the generation of the methyl radical adduct on the further addition of dimethyl sulfoxide. (DMPO/OH). adduct formation was inhibited by catalase but not by superoxide dismutase. The oxidant formed converted iodide to a trichloroacetic acid-precipitable form (iodination) and was bactericidal to logarithmic phase Escherichia coli. Both iodination and bactericidal activity was inhibited by catalase and by OH. scavengers, but not by superoxide dismutase. Iodination was optimal in 5 x 10(-4) M acetate buffer, pH 5.0, and when the Fe2+ and deferoxamine concentrations were equimolar at 10(-4) M. Fe2+ could not be replaced by Fe3+, Co2+, Zn2+, Ca2+, Mg2+, or Mn2+, or deferoxamine by EDTA, diethylenetriaminepentaacetic acid, or bathophenanthroline. These findings indicate that Fe2+ and deferoxamine can act as an oxygen radical generating system, which may contribute to its biological effects in vitro and in vivo.     

Kinetic studies on spin trapping of superoxide and hydroxyl radicals generated in NADPH-cytochrome P-450 reductase-paraquat systems. Effect of iron chelates

Electron spin resonance (ESR) studies on spin trapping of superoxide and hydroxyl radicals by 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) were performed in NADPH-cytochrome P-450 reductase-paraquat systems at pH 7.4. Spin adduct concentrations were determined by comparing ESR spectra of the adducts with the ESR spectrum of a stable radical solution. Kinetic analysis in the presence of 100 microM desferrioxamine B (deferoxamine) showed that: 1) the oxidation of 1 mol of NADPH produces 2 mol of superoxide ions, all of which can be trapped by DMPO when extrapolated to infinite concentration; 2) the rate constant for the reaction of superoxide with DMPO was 1.2 M-1 s-1; 3) the superoxide spin adduct of DMPO (DMPO-OOH) decays with a half-life of 66 s and the maximum level of DMPO-OOH formed can be calculated by a simple steady state equation; and 4) 2.8% or less of the DMPO-OOH decay occurs through a reaction producing hydroxyl radicals. In the presence of 100 microM EDTA, 5 microM Fe(III) ions nearly completely inhibited the formation of the hydroxyl radical adduct of DMPO (DMPO-OH) as well as the formation of DMPO-OOH and, when 100 microM hydrogen peroxide was present, produced DMPO-OH exclusively. Fe(III)-EDTA is reduced by superoxide and the competition of superoxide and hydrogen peroxide in the reaction with Fe(II)-EDTA seems to be reflected in the amounts of DMPO-OOH and DMPO-OH detected. These effects of EDTA can be explained from known kinetic data including a rate constant of 6 x 10(4) M-1 s-1 for reduction of DMPO-OOH by Fe(II)-EDTA. The effect of diethylenetriamine pentaacetic acid (DETAPAC) on the formation of DMPO-OOH and DMPO-OH was between deferoxamine and EDTA, and about the same as that of endogenous chelator (phosphate).     

Cytochrome P-450 inactivation by 3-alkylsydnones. Mechanistic implications of N-alkyl and N-alkenyl heme adduct formation

Incubation of 3-(2-phenylethyl)-4-methylsydnone (PMS) with liver microsomes from phenobarbital-pretreated rats or with reconstituted cytochrome P-450b results in loss of the enzyme chromophore. Chromophore loss is NADPH-dependent even though the sydnone decomposes by an oxygen- but not enzyme-dependent process to give pyruvic acid and, presumably, the (2-phenylethyl)diazonium cation. N-(2-Phenylethyl)protoporphyrin IX and N-(2-phenylethenyl)protoporphyrin IX have been isolated from the livers of rats treated with PMS. Both deuteriums are retained in the N-(2-phenylethyl) adduct derived from 3-(2-phenyl[1,1-2H]ethyl)-4-methylsydnone, but one deuterium is lost in the N-(2-phenylethenyl) adduct. The N-(2-phenylethyl) to N-(2-phenylethenyl) adduct ratio is increased by deuterium substitution. No spectroscopically detectable intermediates precede chromophore loss in incubations of reconstituted cytochrome P-450b with PMS. Electron paramagnetic resonance (EPR)-spin trapping studies show that carbon radicals are formed in incubations of the sydnones with liver microsomes but by a process that is independent of chromophore destruction. It is proposed that the 2-phenylethyl radical formed by electron transfer to the sydnone-derived (2-phenylethyl)diazonium cation adds to the prosthetic heme group to give the N-(2-phenylethyl) adduct. This alkylation reaction is similar to that observed with (2-phenylethyl)hydrazine. Autoxidation of the Fe-CH(CH2Ph)-N bridged species expected from insertion of 2-phenyldiazoethane into one of the heme Fe-N bonds is proposed to explain the unprecedented introduction of a double bond into the N-(2-phenylethenyl) adduct.     

Isolation and identification of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone radical adducts formed by the decomposition of the hydroperoxides of linoleic acid, linolenic acid, and arachidonic acid by soybean lipoxygenase.

alpha-(4-Pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) radical adducts, which are formed in the reactions of soybean lipoxygenase with linoleic acid, arachidonic acid, and linolenic acid, were isolated using HPLC-ESR spectroscopy. Both linoleic acid and arachidonic acid gave one radical adduct, whereas in the case of linolenic acid, two radical adducts were isolated. These radical adducts all showed virtually identical uv spectra with lambda max at 292 and 220 nm in hexane. The absence of absorbance with lambda max at 234 nm indicates that a conjugated diene structure is not contained in these radical adducts. The mass spectra of the radical adducts formed from linoleic and arachidonic acids were identical and contained a molecular ion of m/z 264, consistent with the trapping of the pentyl radical by 4-POBN. Indeed, authentic 4-POBN pentyl radical adduct obtained from the reaction between pentylhydrazine and 4-POBN gave the same mass spectrum as the product obtained from the reaction of linoleic acid and arachidonic acid with 4-POBN. The two 4-POBN radical adducts formed in the linolenic acid reaction were shown by mass spectrometry to be isomers of pentenyl radicals. The 4-POBN-pentyl radical adduct was also detected in the reaction mixture of 13-hydroperoxy-linoleic acid, soybean lipoxygenase, and 4-POBN, indicating that the pentyl radical and pentenyl radical are formed by the decomposition of the hydroperoxides.     

In vivo identification of aflatoxin-induced free radicals in rat bile

Aflatoxin B1 (AFB1) is a potent hepatocarcinogen. We have recently detected [via electron spin resonance (ESR) spectroscopy] free radicals in vivo in rat bile following AFB1 metabolism using the spin trapping [alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (4-POBN)] technique. The aim of the present study was to identify the trapped free radical intermediates from the in vivo hepatic metabolism of AFB1. Rats were treated simultaneously with AFB1 (3 mg/kg i.p.) and the spin trapping agent 4-POBN (1 g/kg i.p.), and bile was collected over a period of 1 h at 20 min intervals. On-line high performance liquid chromatography (HPLC) coupled to ESR was used to identify an arachidonic acid-derived radical adduct of 4-POBN in rat bile, and a methyl adduct of 4-POBN from the reaction of hydroxyl radicals with carbon-13-labeled dimethyl sulfoxide ((13)C-DMSO). The effect of metabolic inhibitors, such as desferoxamine mesylate (DFO), an iron chelator, 2-dimethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF) 525A, a cytochrome P-450 inhibitor, and gadolinium chloride (GdCl(3)), a Kupffer cell inactivator, on in vivo aflatoxin-induced free radical formation were also studied. It was found that there was a significant decrease in radical formation as a result of DFO, SKF525A and GdCl(3) inhibition. Trapped 4-POBN radical adducts were also detected in rat bile following the in vivo metabolism of aflatoxin-M1, one of the hydroxylated metabolites of AFB1.     

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.     

Oxidation of 7-dehydrocholesterol by a mouse liver microsomal system dependent on reduced pyridine nucleotides

Aerobic incubation of 7-dehydrocholesterol with mouse liver microsomes in the presence of a detergent, an iron salt, and NADH or NADPH resulted in the conversion of the sterol to more polar products. In the presence of Fe(3+) or low levels of Fe(2+) the reaction was dependent upon reduced pyridine nucleotide and a microsomal enzyme system. At high levels of Fe(2+) or in the presence of Fe(2+) or Fe(3+) and ascorbic acid, nonenzymatic oxidation of 7-dehydrocholesterol occurred in the absence of NADH or NADPH. Chromatograms of products resulting from the enzyme-dependent and enzyme-independent reactions were similar. The enzymatic reaction was inhibited by certain chelating agents, by antioxidants, and by menadione, phenazine methosulfate, and ferricyanide. Low concentrations of EDTA stimulated the reaction and high concentrations inhibited it. In the complete system sterol oxidation was correlated with the peroxidation of microsomal lipids, but peroxidation of microsomal lipids proceeded more rapidly when either the sterol, the detergent, or both were omitted. Ergosterol was resistant to oxidation under conditions that caused extensive loss of 7-dehydrocholesterol. Microsomes from tissues other than liver were relatively inactive.     

The inhibitory effects in vitro of phenothiazines and other drugs on lipid-peroxidation systems in rat liver microsomes, and their relationship to the liver necrosis produced by carbon tetrachloride

1. The effects of several phenothiazine derivatives on lipid-peroxidation systems in rat liver microsomes were studied and the results are considered in relation to the hepatotoxic action of carbon tetrachloride. 2. The lipid-peroxidation system coupled to NADPH(2) oxidation and stimulated by an ADP-Fe(2+) mixture is strongly inhibited in vitro by promethazine (50% inhibition at 29mum). Chlorpromazine and Stelazine also inhibit the peroxidation system but are less effective than promethazine. 3. The effects of promethazine on three other systems involving oxygen uptake (sulphite oxidation, orcinol oxidation and mitochondrial succinate oxidation) were also studied. Promethazine does not inhibit these systems to the same extent as it does the NADPH(2)-ADP-Fe(2+) lipid-peroxidation system. 4. Promethazine also produces an inhibition of the NADPH(2)-ADP-Fe(2+) system in liver microsomes after administration in vivo. It is concluded that the inhibition involves the interaction of the drug (or a metabolite of it) with the microsomal electron-transport chain. 5. Several other compounds known to protect the rat against liver necrosis after the administration of carbon tetrachloride were tested for inhibitory action on the NADPH(2)-ADP-Fe(2+) system. No clear correlation was observed between effectiveness in vivo as a protective agent and inhibitory effects on the NADPH(2)-ADP-Fe(2+) system in vitro. 6. Promethazine was found to inhibit the stimulation of lipid peroxidation produced in rat liver microsomes by low concentrations of carbon tetrachloride. This effect occurs at a concentration similar to that observed in vivo after administration of a normal clinical dose.     

Role of hydroxyl radical in superoxide induced microsomal lipid peroxidation: Protective effect of anion channel blocker

Treatment of bovine pulmonary artery smooth muscle microsomes with the superoxide radical generating system hypoxanthine plus xanthine oxidase stimulated iron release, hydroxyl radical production and lipid peroxidation. Pretreatment of the microsomes with deferoxamine or dime thy lthiourea markedly inhibited lipid peroxidation, and prevented hydroxyl radical production without appreciably altering iron release. The superoxide radical generating system did not alter the ambient superoxide dismutase activity. However,addition of exogenous superoxide dismutase prevented superoxide radical induced iron release,hydroxyl radical production and lipid peroxidation. Simultaneous treatment of the microsomes with deferoxamine, dimethylthiourea or superoxide dismutase prevented hydroxyl radical production and liqid peroxidation. While deferoxamine or dimethylthiourea did not appreciably alter iron release, superoxide dismutase prevented iron release. However, addition of deferoxamine, dimethylthiourea or superoxide dismutase even 2 min after treatment did not significantly inhibit lipid peroxidation, hydroxyl radical production and iron release. Pretreatment of microsomes with the anion channel blocker 4,4’- dithiocyano 2,′- disulphonic acid stilbine did not cause any discernible change in chemiluminiscence induced by the superoxide radical generating system but markedly inhibited lipid peroxidation without appreciably altering iron release and hydroxial radical production.     

Spin trapping of a free radical intermediate formed during microsomal metabolism of hydrazine

A radical formed during oxidative metabolism of hydrazine in rat liver microsomes was spin-trapped with α-phenyl-t-butylnitrone. The trapped species was identified as hydrazine radical by comparison of its ESR parameters and mass spectrum with those of the adduct formed during CuCl₂ catalyzed oxidation of hydrazine. The requirement for oxygen and NADPH in the microsomal oxidation and the occurrence of a typical binding spectrum by difference spectroscopy suggest the involvement of the participation of the cytochrome P-450 enzyme system in the formation of hydrazine radical which must be a precursor of diimide during microsomal oxidation of hydrazine.