Differential role of CYP2E1 binders and isoniazid on CYP2E1 protein modification in NADPH-dependent microsomal oxidative reactions: free radical scavenging ability of isoniazid

We evaluated the effect of "weak" CYP2E1 binders (ethanol, acetone and glycerol) "tight" CYP2E1 binders (4-methylpyrazole, imidazole, isoniazid and pyridine) and CCl4 (suicide substrate of CYP2E1) on the NADPH-dependent production of microsomal reactive oxygen species (ROS), lipid peroxidation (LPO), and subsequent modification of microsomal and CYP2E1 proteins. The oxidation of 2',7'-dichlorofluorescin diacetate (DCFHDA) was used as an index of formation of microsomal ROS and LPO-derived reactive species. Microsomal LPO was determined by malondialdehyde (MDA) HPLC measurement. Addition of NADPH to rat liver microsomes initiated DCFHDA oxidation and MDA formation, leading to further selective modification of microsomal proteins and proteases-independent degradation of CYP2E1 protein. Iron chelators prevented these processes whereas hydroxyl radical scavengers showed weak effects, suggesting an important role of LPO. Among the tested CYP2E1 binders, only isoniazid strongly inhibited NADPH-dependent DCFHDA oxidation, LPO and modification of microsomal proteins. Other CYP2E1 binders showed weak inhibitory effects of these processes. Concerning NADPH-dependent modification of CYP2E1 protein, all of the tested CYP2E1 binders, except glycerol, prevented this process with a different potency (isoniazid > 4-methylpyrazole = imidazole = pyridine 3 > acetone > ethanol). "Tight" binders were more effective than "weak" binders. The CCl4 stimulated the DCFHDA oxidation, LPO and CYP2E1 protein modification. Among the tested CYP2E1 binders, only isoniazid effectively scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals. In microsomes isolated from CYP2E1 transfected HepG2 cells, isoniazid inhibited the CYP2E1-dependent DCFHDA oxidation whereas other CYP2E1 binders did not inhibit this reaction although these compounds strongly inhibited CYP2E1 activity. The present study demonstrates that CYP2E1 binders and isoniazid differentially inhibit LPO-catalyzed oxidative modification of CYP2E1 protein in NADPH-dependent microsomal reactions. It seems that CYP2E1 binders protect CYP2E1 from the oxidative modification mainly by binding to the active site of the enzyme, rather than by blocking the reactive species production. The strong protective effect of isoniazid can be attributed to its ability to scavenge free radicals. These effects of CYP2E1 binders are considered to contribute to the regulation of hepatic CYP2E1 protein levels via stabilization of the protein.     

Differential Role of CYP2E1 Binders and Isoniazid on CYP2E1 Protein Modification in NADPH-dependent Microsomal Oxidative Reactions: Free Radical Scavenging Ability of Isoniazid

We evaluated the effect of "weak" CYP2E1 binders (ethanol, acetone and glycerol) "tight" CYP2E1 binders (4-methylpyrazole, imidazole, isoniazid and pyridine) and CCl 4 (suicide substrate of CYP2E1) on the NADPH-dependent production of microsomal reactive oxygen species (ROS), lipid peroxidation (LPO), and subsequent modification of microsomal and CYP2E1 proteins. The oxidation of 2',7'-dichlorofluorescin diacetate (DCFHDA) was used as an index of formation of microsomal ROS and LPO-derived reactive species. Microsomal LPO was determined by malondialdehyde (MDA) HPLC measurement. Addition of NADPH to rat liver microsomes initiated DCFHDA oxidation and MDA formation, leading to further selective modification of microsomal proteins and proteases-independent degradation of CYP2E1 protein. Iron chelators prevented these processes whereas hydroxyl radical scavengers showed weak effects, suggesting an important role of LPO. Among the tested CYP2E1 binders, only isoniazid strongly inhibited NADPH-dependent DCFHDA oxidation, LPO and modification of microsomal proteins. Other CYP2E1 binders showed weak inhibitory effects of these processes. Concerning NADPH-dependent modification of CYP2E1 protein, all of the tested CYP2E1 binders, except glycerol, prevented this process with a different potency (isoniazid > 4-methylpyrazole=imidazole=pyridine &#100 acetone > ethanol). "Tight" binders were more effective than "weak" binders. The CCl 4 stimulated the DCFHDA oxidation, LPO and CYP2E1 protein modification. Among the tested CYP2E1 binders, only isoniazid effectively scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals. In microsomes isolated from CYP2E1 transfected HepG2 cells, isoniazid inhibited the CYP2E1-dependent DCFHDA oxidation whereas other CYP2E1 binders did not inhibit this reaction although these compounds strongly inhibited CYP2E1 activity. The present study demonstrates that CYP2E1 binders and isoniazid differentially inhibit LPO-catalyzed oxidative modification of CYP2E1 protein in NADPH-dependent microsomal reactions. It seems that CYP2E1 binders protect CYP2E1 from the oxidative modification mainly by binding to the active site of the enzyme, rather than by blocking the reactive species production. The strong protective effect of isoniazid can be attributed to its ability to scavenge free radicals. These effects of CYP2E1 binders are considered to contribute to the regulation of hepatic CYP2E1 protein levels via stabilization of the protein.     

The role of covalent binding and lipid peroxidation in liver damage by carbon tetrachloride]

4-[N-sodium-N-(5-ethyl-1,3,4-thiadiazol-2-yl)]- sulphanylamido-5-methoxy-1,2-benzoquinone selectively inhibiting lipid peroxidation (LPO) was used to study the hepatotoxic effect of carbon tetrachloride in vivo. It was found that inactivation of the liver microsomal oxidation system during the first few hours after CCl4 injection is due to covalent binding rather than LPO.     

The activity of mammalian peroxidases (lactoperoxidase and myeloperoxidase) and their compounds III toward 2-t-butyl-4-methoxyphenol (butylated hydroxyanisole) and its dimer (2,2'-dihydroxy-3,3'-di-t-butyl-5,5'-dimethoxydiphenyl).

Spectral evidence is presented which shows that butylated hydroxyanisole (BHA) and its dimer act as electron donors for lactoperoxidase (LPO) and myeloperoxidase (MPO) by two different pathways: peroxidative and oxidative. LPO compound II and MPO compound II are converted to native enzymes in their reactions with BHA without detectable intermediates. This confirms a normal peroxidatic oxidation of this commonly used antioxidant. We also report spectral data indicating the reductions of peroxidase compound III to the native state in reactions with BHA (LPO, MPO) or with di-BHA (LPO). This oxidative reaction has significant physiological relevance, ensuring return of peroxidases to the native state for re-entry into the normal peroxidatic cycle or into halogenating reactions.     

Effects of MPTP, MPP+, and paraquat on NADPH-dependent lipid peroxidation in mouse brain and lung microsomes

Both MPTP and MPP+ inhibited the NADPH-dependent microsomal LPO in mouse brain and lung. On the other hand, PQ significantly stimulated the LPO in brain microsomes in a dose-dependent manner. The herbicide, however, stimulated lung microsomal LPO only in a narrow concentration range, despite much higher P450 reductase activity in lung microsomes than that in brain microsomes. These findings suggest that the effect of PQ on microsomal LPO is different from those of the analogous neurotoxins, MPTP and MPP+, and is not uniform in brain and lung.     

Antioxidative activity of flavonoids in various systems of lipid peroxidation

The antioxidant action of flavonols in different systems of lipid peroxidation (LPO) was studied. Quercetin and rutin were found to inhibit NADPH and CCl4-dependent LPO in rat liver microsomes, however, in the case of CCl4-dependent LPO, rutin had a very poor antioxidant effect. Study of flavonols oxidation by products of the cytochrome c catalyzed destruction of linoleic acid hydroperoxide demonstrated that the differences in the antioxidant offects of quercetin and rutin can be due to their different capability to terminate free radical chain reactions. The antioxidant effect of rutin was shown to be largely due to the chelating properties of this compound.     

Cytochrome P-450-mediated oxidation of substrates by electron-transfer; role of oxygen radicals and of 1- and 2-electron oxidation of paracetamol

The mechanism by which the hepatic cytochrome P-450 (Cyt. P-450) containing mixed-function oxidase system oxidizes the analgesic drug paracetamol (PAR) to a hepatotoxic metabolite was studied. Since previous studies excluded the possibility of oxygenation of PAR, three other mechanisms, namely direct 1-electron oxidation by a Cyt. P-450-ferrous-dioxygen complex under concomitant formation of H2O2 to N-acetyl-p-semiquinone imine (NAPSQI), direct 2-electron oxidation by a Cyt. P-450-ferric-oxene complex to N-acetyl-p-benzoquinone imine (NAPQI) and indirect oxidation by active oxygen species released from Cyt. P-450, were considered. Indirect oxidation by active oxygen species was not involved, as active oxygen scavengers such as superoxide dismutase, catalase and DMSO did not affect the oxidation of PAR in hepatic microsomes. No reaction products characteristic for a direct 1-electron oxidation of PAR by Cyt. P-450 were observed: neither NAPSQI radical formation was detectable by ESR, nor PAR-dimer formation, nor stimulation of the microsomal H2O2 production was found to occur. In fact, PAR inhibited the spontaneous microsomal H2O2 formation. Studies on the reactions of NAPSQI with glutathione (GSH) revealed that NAPSQI hardly conjugated with GSH to a 3-glutathionyl-paracetamol conjugate (PAR-GSH) conjugate. The reactions of the elusive reactive metabolite formed during microsomal oxidation of PAR in the presence of GSH closely resembled those of synthetic NAPQI: both PAR-GSH and oxidized glutathione (GSSG) formation occurred. Furthermore, in agreement with a 2-electron oxidation hypothesis, iodosobenzene-dependent oxidation of PAR by cyt. P-450 in the presence of GSH resulted in the formation of the PAR-GSH conjugate. It is concluded that bioactivation of PAR by the Cyt. P-450 containing mixed-function oxidase system consists of a direct 2-electron oxidation to NAPQI.     

Temperature dependence of the microsomal oxidation of ethanol by cytochrome P450 and hydroxyl radical-dependent reactions

The temperature dependence and activation energies for the oxidation of ethanol by microsomes from controls and from rats treated with pyrazole was evaluated to determine whether the overall mechanism for ethanol oxidation by microsomes was altered by the pyrazole treatment. Arrhenius plots of the temperature dependence of ethanol oxidation by pyrazole microsomes were linear and exhibited no transition breaks, whereas a slight break was observed at about 20 +/- 2.5 degrees C with control microsomes. Energies of activation (about 15-17 kcal/mol) were identical for the two microsomal preparations. Although transition breaks were noted for the oxidation of substrates such as dimethylnitrosamine and benzphetamine, activation energies for these two substrates were similar for control microsomes and microsomes from the pyrazole-treated rats. The addition of ferric-EDTA to the microsomes increased the rate of ethanol oxidation by a hydroxyl radical (.OH)-dependent pathway. Arrhenius plots of the .OH-dependent oxidation of ethanol by both microsomal preparations were linear with energies of activation (about 7 kcal/mol) that were considerably lower than values found for the P450-dependent pathway. These results suggest that, at least in terms of activation energy, the increase in microsomal ethanol oxidation by pyrazole treatment is not associated with any apparent change in the overall mechanism or rate-limiting step for ethanol oxidation but likely reflects induction of a P450 isozyme with increased activity toward ethanol. The lower activation energy for the .OH-dependent oxidation of ethanol suggests that different steps are rate limiting for oxidation of ethanol by .OH and by P450, which may reflect the different enzyme components of the microsomal electron transfer system involved in these reactions.     

Oxidative and free-radical processes in the mechanism of toxic effect of anti-diabetic drugs dicarboxylic acids derivatives

Dynamics of changes in the processes of the lipids free-radical peroxide oxidation (LPO), state of the monooxygenase system (MOS) and system of anti-oxidant protection (SAOP) in the liver and blood of rats under multiple peroral and inhalatory expositions of anti-diabetic drugs--dicarboxylic acids derivatives: phensuccinalum (PhS) and diacamph (Dc) in toxic and sub-toxic doses is studied. It has been found that one of the key biochemical mechanisms which determines the early signs of the toxic damage in the cells and organism under the effect of certain doses/concentrations of PhS (500 and 100 mg/kg, 20.2 mg/m3) and Dc (1000 mg/kg) consists in intensification of the free-radical processes, including, in particular, LPO combined with activation of MOGS and inhibition of SAOP in microsomal fraction of the liver. The high significance of changes of researched parameters for an assessment or forecasting of toxicity of dicarboxylic acids derivates is shown. Unlike the liver, the PhS and Dc impact on the blood caused changes of compensatory-adaptive nature in the LPO and SAOP systems.     

The antioxidative activity of drugs in the liver microsomal system of rats]

The antioxidative properties of drugs--diethylcarbamazine citrate--DECC, dipyridamole-DP, levamisole and labinzarit--have been investigated in various microsomal lipid peroxidation (LPO) models: NADPH-, ascorbate- and CCl4-dependent. The most strong antioxidant of direct action turned out to be DP, DECC exhibited the antioxidative properties as a result of metabolic activity in monooxygenases system of rat liver microsomes. Levamisole and labinzarit turned out to be weak antioxidants. The control of microsomal membrane stability against Fe(2+)-ADP, NADPH-induced LPO, after being isolated from rat liver after the action of CCl4 without and with DECC, showed that DECC protected microsomal membranes from CCl4 in vivo and they remained stable against LPO in vitro.     

Participation of oxygen activated species in enzymatic lipid peroxidation in biomembranes]

The participation of oxygen activated species in the induction of lipid peroxidation (LPO) in the membrane systems containing cytochrome P-450 (liver microsomes) and in the membrane fragments devoid of this hemoprotein (brain and skeletal muscle microsomes) was studied. It was shown that the rate of NADH-dependent LPO does not depend on the presence of hemoproteins and the activity of NADH-specific flavoprotein in the membranes. On the other hand, the microsomal membranes of the liver with high specific contents of b5 and P-450 cytochromes and NADPH-specific flavoprotein, had the highest rates of NADPH-dependent LPO. It was found that the most effective inhibitors of free oxygen activated species in the case of NADPH- and NADH-dependent LPO in the microsomal fractions of liver, brain and skeletal muscles are the superoxide (O ./2) anion radical inhibitors. The singlet oxygen (1O2) quenchers inhibit only NADPH-dependent LPO in the liver, however, in a far lesser degree. The hydroxyl radical (OH) scavengers had no effect on enzymatic LPO in all systems studied.     

Lactoperoxidase-catalyzed oxidation of thiocyanate by hydrogen peroxide: 15N nuclear magnetic resonance and optical spectral studies

To establish the agent(s) responsible for the activity of the lactoperoxidase (LPO)/SCN-/H2O2 system, the oxidation of thiocyanate with hydrogen peroxide, catalyzed by lactoperoxidase, has been studied by 15N NMR and optical spectroscopy at different concentrations of thiocyanate and hydrogen peroxide and at different pHs. The formation of hypothiocyanite ion (OSCN-) as one of the oxidation products correlated well with the activity of the LPO/SCN-/H2O2 system and was maximum when the concentrations of the H2O2 and SCN- were nearly the same and the pH was less than 6.0. At [H2O2]/[SCN-] = 1, OSCN- decomposed very slowly back to thiocyanate. When the ratio [H2O2]/[SCN-] was above 2, formation of CN- was observed, which was confirmed by 15N NMR and also by changes in the optical spectrum of LPO. The oxidation of thiocyanate by H2O2 in the presence of LPO does not take place at pH greater than 8.0. Since thiocyanate does not bind to LPO above this pH, the binding of thiocyanate to LPO is considered to be prerequisite for the oxidation of thiocyanate. Maximum inhibition of oxygen uptake by Streptococcus cremoris 972 bacteria was observed when hydrogen peroxide and thiocyanate were present in equimolar amounts and the pH was below 6.0.     

Peroxide modification of membranes and isomorphic composition of cytochrome P-450 of rat liver microsomes during antioxidant deficiency]

Lipid peroxidation (LPO), physico-chemical properties of the membranes and isoformic composition of microsomal cytochrome P-450 from the rat liver were studied under conditions of antioxidant insufficiency (AOI) which was modelled by exclusion of alpha-tocopherol from the animals' ration. An insignificant accumulation of microsomal diene conjugates and schiff bases against a sharp increase of the ability to the prooxidant stimulated LPO in vitro took place. A significant decrease of membrane lipid microviscosity and a change in surface properties of microsomal membranes of rats with AOI was determined. Absence of alpha-tocopherol in the ration was accompanied by a significant change in the content of separate isoforms of cytochrome P-450 exhibited in growth of a polypeptide with m. w. 54 kDa and the lowering of proteins with m. w. 48 and 50 kDa. Less intensive quenching of tryptophan fluorescence by acrylamide was also revealed, which testified to a lower accessibility of the quencher to membrane proteins or their fluorophore sites. Modification of lipid composition and of physicochemical properties of the rat liver membrane microsomes which was observed at AOI was significantly correlated by pretreatment with the antioxidant 4-methyl-2,6-ditretbutylphenol (ionol).     

Aldehydic lipid peroxidation products derived from linoleic acid

Lipid peroxidation (LPO) processes observed in diseases connected with inflammation involve mainly linoleic acid. Its primary LPO products, 9-hydroperoxy-10,12-octadecadienoic acid (9-HPODE) and 13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE), decompose in multistep degradation reactions. These reactions were investigated in model studies: decomposition of either 9-HPODE or 13-HPODE by Fe(2+) catalyzed air oxidation generates (with the exception of corresponding hydroxy and oxo derivatives) identical products in often nearly equal amounts, pointing to a common intermediate. Pairs of carbonyl compounds were recognized by reacting the oxidation mixtures with pentafluorobenzylhydroxylamine. Even if a pure lipid hydroperoxide is subjected to decomposition a great variety of products is generated, since primary products suffer further transformations. Therefore pure primarily decomposition products of HPODEs were exposed to stirring in air with or without addition of iron ions. Thus we observed that primary products containing the structural element R-CH=CH-CH=CH-CH=O add water and then they are cleaved by retroaldol reactions. 2,4-Decadienal is degraded in the absence of iron ions to 2-butenal, hexanal and 5-oxodecanal. Small amounts of buten-1,4-dial were also detected. Addition of m-chloroperbenzoic acid transforms 2,4-decadienal to 4-hydroxy-2-nonenal. 4,5-Epoxy-2-decenal, synthetically available by treatment of 2,4-decadienal with dimethyldioxirane, is hydrolyzed to 4,5-dihydroxy-2-decenal.     

Iron (II) Ions Induced Oxidation of Ascorbic Acid and Glucose

Lipid peroxidation (LPO) of polyunsaturated fatty acids (PUFAs) is suspected to be involved in the generation of chronic diseases. A model reaction for LPO is the air oxidation of PUFAs initiated by Fe²⁺ and ascorbic acid. In the course of such model reactions glycolaldehyde (GLA) was detected as main aldehydic product. Since it is difficult to explain the generation of GLA by oxidation of PUFAs, it was suspected that GLA might be derived by oxidation of ascorbic acid. This assumption was verified by treatment of ascorbic acid with Fe²⁺.

Produced aldehydic compounds were trapped by addition of pentafluorobenzylhydroxylamine hydrochloride (PFBHA-HCl), trimethylsilylated and finally identified by gas chromatography/mass spectrometry (GC/MS). Oxidation of ascorbic acid with O₂ in presence of iron ions produced not only glycolaldehyde (GLA), but also glyceraldehyde (GA), dihydroxyacetone (DA) and formaldehyde. Glyoxal (GO) and malondialdehyde (MDA) were detected as trace compounds.

The yield of the aldehydic compounds was increased by addition of lipid hydroperoxides (LOOH) or H₂O₂. The buffer influenced the reaction considerably: Iron ions react with Tris buffer by producing dihydroxyace-tone (DA). Since ascorbic acid is present in biological systems and Fe²⁺ ions are obviously generated by cell damaging processes, the production of GLA and other aldehydic components might add to the damaging effects of LPO.

Glucose suffers also oxidation to short-chain aldehydic compounds in aqueous solution, but this reaction requires addition of equimolar amounts of Fe²⁺ together with equimolar amounts of H₂O₂ or 13-hydroperoxy-9-cis-11-trans-octadecadienoic acid (13-HPODE). Therefore this reaction, also influenced by the buffer system, seems to be not of biological relevance.     

Inhibition of cytochrome P-450-linked monooxygenase systems by naphthoquinones

Several naphthoquinones, except 2-hydroxy-1,4-naphthoquinone, were found to inhibit microsomal cytochrome P-450-linked monooxygenase activities in rabbit liver and human placenta. In particular, 5-hydroxy-1,4-naphthoquinone inhibited placental estrogen biosynthesis more effectively than it did hepatic drug oxidation reactions. There was little contribution by superoxide radicals to these enzyme inhibitions by naphthoquinones. Spectrophotometric studies revealed that naphthoquinones bind to the cytochrome P-450 component of the monooxygenase complex in both microsomal systems, suggesting that the inhibition is caused by direct interaction of these compounds with the heme.     

The role of an endogenous nonheme iron in microsomal redox reactions.

Microsomal membranes contain a nonheme iron which serves in vitro for the peroxidation of unsaturated lipids or the oxidation of several other chemicals. These redox reactions are reviewed in light of a recent identification of two or more iron-binding proteins in the microsomal milieu. Indirect evidence that the microsomal iron might serve in vivo for the synthesis of heme iron is also presented and discussed. Consistent with this, the newly identified iron proteins not only participate in redox reactions but also release their bound iron upon incubation with certain intermediates of heme synthesis.     

Metabolism of carbon tetrachloride to electrophilic chlorine by liver microsomes: exclusion of cytochrome P-450 catalyzed chloroperoxidase reaction

In this report, we have examined the origin of the electrophilic chlorine formed during the microsomal metabolism of carbon tetrachloride and the possibility that liver microsomal proteins catalyze chloroperoxidase or myeloperoxidase halogenation reactions. Studies with stable isotopes of chlorine show that at least 99% of the trapped chlorine originated from carbon tetrachloride. When hydrogen peroxide or cumene hydroperoxide was added to liver microsomes in the presence of chloride ion, no trapped chlorine was observed. Thus, cytochrome P-450 does not catalyze chloroperoxidase type chloride ion oxidation but instead catalyzes a reaction leading to cleavage of a carbon-chlorine bond with concomitant chlorine atom oxidation.     

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.     

Role of bilirubin as a natural antioxidant in regulating lipid peroxidation intensity in acute viral hepatitis

Elevation of the content of lipid peroxidation (LPO) products in blood serum of patients with acute virus hepatitis (VH) is caused by an increase in the patients' blood serum lipids rather than by the intensity of peroxide reactions in lipids. There is a reverse correlation between the content of LPO products and bilirubin level and a direct correlation between lipid antioxidant activity (AOA) and bilirubin level. Marked antioxidant action of bilirubin that compares very favourably with the action of ionol (4-methyl-2,6-ditretbutylphenol) was demonstrated in the model of oxidation of methyl oleate. It was shown that the rise of lipid AOA during VH might be completely attributed to the antioxidant properties of bilirubin. It is suggested that elevation of bilirubin level and associated increase of lipid AOA during VH can be viewed as a reaction aimed at a decrease of the level of toxic products of LPO and intensification of reparative processes in the liver.