The mechanism of NADPH-dependent lipid peroxidation. The propagation of lipid peroxidation.

NADPH-dependent lipid peroxidation occurs in two distinct sequential radical steps. The first step, initiation, is the ADP-perferryl ion-catalyzed formation of low levels of lipid hydroperoxides. The second step, propagation, is the iron-catalyzed breakdown of lipid hydroperoxides formed during initiation generating reactive intermediates and products characteristic of lipid peroxidation. Propagation results in the rapid formation of thiobarbituric acid-reactive material and lipid hydroperoxides. Propagation can be catalyzed by ethylenediamine tetraacetate-chelated ferrous ion, diethylenetriamine pentaacetic acid-chelated ferrous ion, or by ferric cytochrome P-450. However, cytochrome P-450 is destroyed during propagation.     

Microsomal lipid peroxidation: the role of NADPH--cytochrome P450 reductase and cytochrome P450

The role of NADPH--cytochrome P450 reductase and cytochrome P450 in NADPH- and ADP--Fe3(+)-dependent lipid peroxidation was investigated by using the purified enzymes and liposomes prepared from either total rat-liver phospholipids or a mixture of bovine phosphatidyl choline and phosphatidyl ethanolamine (PC/PE liposomes). The results suggest that NADPH- and ADP--Fe3(+)-dependent lipid peroxidation involves both NADPH--cytochrome P450 reductase and cytochrome P450. Just as in the case of cytochrome P450-linked monooxygenations, the role of these enzymes in lipid peroxidation may be to provide two electrons for O2 reduction. The first electron is used for reduction of ADP--Fe3+ and subsequent addition of O2 to the perferryl radical (ADP--Fe3(+)-O2-), which then extracts an H atom from a polyunsaturated lipid (LH) giving rise to a free radical (LH.) that reacts with O2 yielding a peroxide free radical (LOO.). The second electron is then used to reduce LOO. to the lipid hydroperoxide (LOOH). In the latter capacity, reduced cytochrome P450 can be replaced by EDTA--Fe2+ or by the superoxide radical as generated through redox cycling of a quinone such as menadione.     

NADPH- and iron-dependent lipid peroxidation inhibit aromatase activity in human placental microsomes

During pregnancy placenta is the most significant source of lipid hydroperoxides and other reactive oxygen species (ROS). The increased production of lipid peroxides and other ROS is often linked to pre-eclampsia. It is already proved that placental endoplasmic reticulum may be an important place of lipid peroxides and superoxide radical production. In the present study we revealed that NADPH- and iron-dependent lipid peroxidation in human placental microsomes (HPM) inhibit placental aromatase--a key enzyme of estrogen biosynthesis in human placenta. We showed that significant inhibition of this enzyme is caused by small lipid peroxidation (TBARS (thiobarbituric acid-reactive substances)<4nmol/mg microsomal protein (m.p.)). More intensive lipid peroxidation (TBARS>9nmol/mg microsomal protein) diminishes aromatase activity to value being less than 5% of initial value. NADPH- and iron-dependent lipid peroxidation also causes disappearance of cytochrome P450 parallel to observed aromatase activity inhibition. EDTA, alpha-tocopherol, MgCl(2) and superoxide dismutase (SOD) prevent aromatase activity inhibition and cytochrome P450(AROM) degradation. Mannitol and catalase have not effect on TBARS synthesis, aromatase activity and cytochrome P450 degradation. In view of the above we postulate that the inhibition of aromatase activity observed is mainly a consequence of cytochrome P450(AROM) degradation induced by lipid radicals. The role of hydroxyl radical in cytochrome P450 degradation is negligible in our experimental conditions. The results presented here also suggest that the inhibition of aromatase activity can also take place in placenta at in vivo conditions.     

A possible role of cAMP dependent phosphorylation of hepatic microsomal cytochrome P450: a mechanism to increase lipid peroxidation in response to hormone

Enzymatic lipid peroxidation in hepatocytes is believed to involve cytochrome P450. cAMP dependent phosphorylation of cytochrome P450 was found to increase the NADPH dependent production of malondialdehyde (lipid peroxidation) by about 30%. The cytochrome P450 inhibitor cyanide abolished this activity. The presence of spermine decreased the cytochrome P450 dependent lipid peroxidation in non-phosphorylated microsomes, phosphorylation partially reversed this effect. Thus, phosphorylation of cytochrome P450 and the associated increased lipid peroxidation may be a hormone dependent response to pathological conditions e.g. stress Phosphorylation was observed to subtly alter other properties of cytochrome P450. The rate of 7-ethoxycoumarin deethylase activity was reduced and the microwave power required to saturate the EPR spectrum of the low spin cytochrome P450 was decreased. It is hypothesized that phosphorylation of cytochrome P450 alters the interaction between the components of the cytochrome P450 system, which may enhance production of free radical species, initiating lipid peroxidation.     

Protective effect of N-acetylcysteine in isoniazid induced hepatic injury in growing rats

Status of oxidative/antioxidative profile was the mechanistic approach to inumerate the nature of protection by N-acetylcysteine (NAC) in isoniazid (INH) exposed experimental animals. Analysis of lipid peroxidation, thiol levels, cytochrome P450, superoxide dismutase (SOD), catalase, glutathione peroxidase, reductase and transferase were estimated in liver along with the body and liver weight of animals and histological observations. Isoniazid exposure to animals resulted in no change in body and liver weights. Thiols, lipid peroxidation, catalase, SOD glutathione peroxidase, reductase, transferase and cytochrome P450 levels were altered with INH exposure. Supplementation of NAC with INH protected the animals against hepatotoxic reactions by minimizing the free radical induced tissue injury and overall maintenance of the endogenous scavengers of free radicals.     

Oxygen radical formation during cytochrome P450-catalyzed cyclosporine metabolism in rat and human liver microsomes at varying hydrogen ion concentrations

The role of pH in uncoupling the electron-flux between oxidoreductase and cytochrome P450 (P450) or P450 and cyclosporine (CyA) and resulting in the generation of oxygen radicals was investigatedin vitro in rat and human liver microsomal preparations. Since the electron-flux from NADPH to cytochrome c via oxidoreductase showed a fairly constant reduction activity from pH 7.0–9.5, the generation of oxygen radicals at the level of P450-Cyclosporine (instead of oxidoreductase-P450) was investigated. The effects of increasing pH on oxygen radical formation was measured by the thiobarbituric acid assay (TBA) and the adrenochrome reaction. The trends in oxygen radical production were correlated with benzphetamine metabolism (production of formaldehyde) and CyA metabolism (analyzed by high performance liquid chromatography). The TBA assay showed increased MDA-detected lipid peroxidation (unrelated to autooxidation) at pH<8.0 and pH>8.0 (rat and human, respectively) while the adrenochrome reaction showed decreased oxygen radical production. When these results were compared to benzphetamine (a substrate of P450 2B and 3A) metabolism and CyA (a substrate of P450 3A) metabolism, increased metabolism followed the pH-dependent trend of MDA-detected lipid peroxidation. Benzphetamine metabolism with formaldehyde production and depletion of parent compound during CyA metabolism were increased at pH<8.0 in the rat samples and at pH>8.0 in the human samples. This parallel relation suggests that the increased metabolism of CyA at lower pH in rats and higher pH in humans may be the result of favorable interactions of P450 with Cyclosporine that also result in increased oxygen radical-related lipid peroxidation.Abbreviations CCl₄ carbon tetrachloride - CyA cyclosporin A - EDTA ethylenediaminetetraacetic acid - HPLC high performance liquid chromatography - MDA malondialdehyde - MFO mixed function oxidase - MICROS microsomes - NADPH nicotinamide adenine dinucleotide phosphate - TBA thiobarbituric acid This work was supported by Grant No. CA-53191 from the National Cancer Institute DHHW     

An aqueous extract of Rubus chingii fruits protects primary rat hepatocytes against tert-butyl hydroperoxide induced oxidative stress

Different in vitro free radical generating systems were used to assess the antioxidative activity of aqueous extracts of the five herbal components of Wu-zi-yan-zong-wan, a traditional Chinese medicinal formula with a long history of use for tonic effects. Fructus Rubi [Rubus chingii (Rosaceae) fruits] was found to be the most potent. It was further investigated using the primary rat hepatocyte system. tert-Butyl hydroperoxide (t-BHP) was used to induce oxidative stress. Being a short chain analog of lipid hydroperoxide, t-BHP is metabolized into free radical intermediates by the cytochrome P450 system in hepatocytes, which in turn, initiate lipid peroxidation, glutathione depletion and cell damage. Pre-treatment of hepatocytes with Fructus Rubi extract (50 microg/ml to 200 microg/ml) for 24 h significantly reversed t-BHP-induced cell viability loss, lactate dehydrogenase leakage and the associated glutathione depletion and lipid peroxidation. The amount of reactive oxygen species formed was also decreased as visualized by the fluorescence probe 2',7'-dichlorofluorescin diacetate. These results suggested that Fructus Rubi was useful in protecting against t-BHP-induced oxidative damage and may also be capable of attenuating cytotoxicity of other oxidants.     

Nadph-initiated cytochrome P450-dependent free iron-independent microsomal lipid peroxidation: Specific prevention by ascorbic acid

In this paper we demonstrate that ascorbic acid specifically prevents NADPH-initiated cytochrome P450 (P450)-mediated microsomal lipid peroxidation in the absence of free iron. Lipid peroxidation has been evidenced by the formations of conjugated dienes, lipid hydroperoxide and malondialdehyde. Other scavengers of reactive oxygen species including superoxide dismutase, catalase, glutathione, -tocopherol, uric acid, thiourea, mannitol, histidine, -carotene and probucol are ineffective to prevent the NADPH-initiated P450-mediated free iron-independent microsomal lipid peroxidation. Using a reconstituted system comprised of purified NADPH-P450 reductase, P450 and isolated microsomal lipid or pure L--phosphatidylcholine diarachidoyl, a mechanism has been proposed for the iron-independent microsomal lipid peroxidation and its prevention by ascorbic acid. It is proposed that the perferryl moiety P450 Fe³⁺. O₂ initiates lipid peroxidation by abstracting methylene hydrogen from polyunsaturated lipid to form lipid radical, which then combines with oxygen to produce the chain propagating peroxyl radical for subsequent formation of lipid peroxides. Apparently, ascorbic acid prevents initiation of lipid peroxidation by interacting with P450 Fe³⁺. O₂. (Mol Cell Biochem 166: 35-44, 1997)     

Involvement of inducible nitric oxide synthase in hydroxyl radical-mediated lipid peroxidation in streptozotocin-induced diabetes

Free radical production is implicated in the pathogenesis of diabetes mellitus, where several pathways and different mechanisms were suggested in the pathophysiology of the complications. In this study, we used electron paramagnetic resonance (EPR) spectroscopy combined with in vivo spin-trapping techniques to investigate the sources and mechanisms of free radical formation in streptozotocin-induced diabetic rats. Free radical production was directly detected in the diabetic bile, which correlated with lipid peroxidation in the liver and kidney. EPR spectra showed the trapping of a lipid-derived radical. Such radicals were demonstrated to be induced by hydroxyl radical through isotope-labeling experiments. Multiple enzymes and metabolic pathways were examined as the potential source of the hydroxyl radicals using specific inhibitors. No xanthine oxidase, cytochrome P450s, the Fenton reaction, or macrophage activation were required for the production of radical adducts. Interestingly, inducible nitric oxide synthase (iNOS) (apparently uncoupled) was identified as the major source of radical generation. The specific iNOS inhibitor 1400W as well as L-arginine pretreatment reduced the EPR signals to baseline levels, implicating peroxynitrite as the source of hydroxyl radical production. Applying immunological techniques, we localized iNOS overexpression in the liver and kidney of diabetic animals, which was closely correlated with the lipid radical generation and 4-hydroxynonenal-adducted protein formation, indicating lipid peroxidation. In addition, protein tyrosine nitration occurred in the diabetic target organs. Taken together, our studies support inducible nitric oxide synthase as a significant source of EPR-detectable reactive intermediates, which leads to lipid peroxidation and may contribute to disease progression as well.     

Cholate solubilization of liver microsomal membrane components which promote NADPH-supported lipid peroxidation.

NADPH-supported lipid peroxidation monitored by malondialdehyde (MDA) production in the presence of ferric pyrophosphate in liver microsomes was inactivated by heat treatment or by trypsin and the activity was not restored by the addition of purified NADPH-cytochrome P450 reductase (FPT). The activity was differentially solubilized by sodium cholate from microsomes, and the fraction solubilized between 0.4 and 1.2% sodium cholate was applied to a Sephadex G-150 column and subfractionated into three pools, A, B, and C. MDA production was reconstituted by the addition of microsomal lipids and FPT to specific fractions from the column, in the presence of ferric pyrophosphate and NADPH. Pool B, after removal of endogenous FPT, was highly active in catalyzing MDA production and the disappearance of arachidonate and docosahexaenoate, and this activity was abolished by heat treatment and trypsin digestion, but not by carbon monoxide. The rate of NADPH-supported lipid peroxidation in the reconstituted system containing fractions pooled from Sephadex G-150 columns was not related to the content of cytochrome P450. p-Bromophenylacylbromide, a phospholipase A2 inhibitor, inhibited NADPH-supported lipid peroxidation in both liver microsomes and the reconstituted system, but did not block the peroxidation of microsomal lipid promoted by iron-ascorbate or ABAP systems. Another phospholipase A2 inhibitor, mepacrine, poorly inhibited both microsomal and pool-B'-promoted lipid peroxidation, but did block both iron-ascorbate-driven and ABAP-promoted lipid peroxidation. The phospholipase A2 inhibitor chlorpromazine, which can serve as a free radical quencher, blocked lipid peroxidation in all systems. The data presented are consistent with the existence of a heat-labile protein-containing factor in liver microsomes which promotes lipid peroxidation and is not FPT, cytochrome P450, or phospholipase A2.     

Ethanol-Induced Oxygen Radical Formation and Lipid Peroxidation in Rat Brain: Effect of Chronic Alcohol Consumption

Abstract: The effect of chronic and in vitro ethanol exposure on brain oxygen radical formation and lipid peroxidation was analyzed. Ethanol induces a dose-dependent increase in lipid peroxidation in brain homogenates. The peroxidative effects of alcohol seem to be related to both cytochrome P450 and the ethanol-inducible form of cytochrome P450 (CYP2E1), because preincubation with metyrapone (an inhibitor of cytochrome P450) or with an antibody against CYP2E1 abolished the ethanol-increased lipid peroxidation. Using the formation of dichlorofluorescein, we also demonstrated that both in vitro and chronic alcohol exposure significantly enhanced the formation of oxygen radical species in synaptosomes. Chronic alcohol treatment also leads to an induction of cytochrome P450 (230%), NADPH cytochrome c reductase (180%), NADPH oxidation (184%), and CYP2E1 in brain microsomes. In addition, this treatment produced a decrease in the GSH/GSSG ratio in brain and significantly enhanced the levels of superoxide dismutase and catalase activities. This mechanism could be involved in the toxic effects of ethanol on brain and membrane alterations occurring after chronic ethanol intake.     

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.     

Role of cytochrome P450 in phospholipase A2- and arachidonic acid-mediated cytotoxicity

Phospholipases A2 (PLA2) comprise a set of extracellular and intracellular enzymes that catalyze the hydrolysis of the sn-2 fatty acyl bond of phospholipids to yield fatty acids and lysophospholipids. The PLA2 reaction is the primary pathway through which arachidonic acid (AA) is released from phospholipids. PLA2s have an important role in cellular death that occurs via necrosis or apoptosis. Several reports support the hypothesis that unesterified arachidonic acid in cells is a signal for the induction of apoptosis. However, most of the biological effects of arachidonic acid are attributable to its metabolism by mainly three different groups of enzymes: cytochromes P450, cyclooxygenases, and lipoxygenases. In this review we will focus on the role of cytochrome P450 in AA metabolism and toxicity. The major pathways of arachidonic acid metabolism catalyzed by cytochrome P450 generate metabolites that are subdivided into two groups: the epoxyeicosatrienoic acids, formed by CYP epoxygenases, and the arachidonic acid derivatives that are hydroxylated at or near the omega-terminus by CYP omega-oxidases. In addition, autoxidation of AA by cytochrome P450-derived reactive oxygen species produces lipid hydroperoxides as primary oxidation products. In some cellular models of toxicity, cytochrome P450 activity exacerbates PLA2- and AA-dependent injury, mainly through the production of oxygen radicals that promote lipid peroxidation or production of metabolites that alter Ca2+ homeostasis. In contrast, in other situations, cytochrome P450 metabolism of AA is protective, mainly by lowering levels of unesterified AA and by production of metabolites that activate antiapoptotic pathways. Several lines of evidence point to the combined action of phospholipase A2 and cytochrome P450 as central in the mechanism of cellular injury in several human diseases, such as alcoholic liver disease and myocardial reperfusion injury. Inhibition of specific PLA2 and cytochrome P450 isoforms may represent novel therapeutic strategies against these diseases.     

Reconstituted microsomal lipid peroxidation: ADP-Fe3+-dependent peroxidation of phospholipid vesicles containing NADPH-cytochrome P450 reductase and cytochrome P450

A reconstituted lipid peroxidation system consisting of rat liver microsomal NADPH-cytochrome P450 reductase and cytochrome P450 incorporated into phospholipid vesicles was developed and characterized. Peroxidation of the vesicles required NADPH and ADP-Fe3+, just as in the NADPH-dependent peroxidation of microsomes. The peroxidation of the vesicles was inhibited 30-50% by superoxide dismutase, depending upon their cytochrome P450 content: those with higher cytochrome P450 contents exhibited greater rates of malondialdehyde formation which were less sensitive to inhibition by superoxide dismutase. When cytochrome P450 was incorporated into vesicles, EDTA-Fe3+ was not required for lipid peroxidation, distinguishing this system from the one previously described by Pederson and Aust [Biochem. Biophys. Res. Comm. 48, 789; 1972]. Since at least 50% of the malondialdehyde formation in the vesicular system was not inhibited by superoxide dismutase, alternative means of iron reduction (O2-.-independent) were examined. It was found that rat liver microsomes or a reconstituted mixed function oxidase system consisting of NADPH-cytochrome P450 reductase and cytochrome P450 in dilauroylphosphatidylcholine micelles reduced ADP-Fe3+ under anaerobic conditions.     

Hydroxyl radical production in a purified NADPH--cytochrome c (P-450) reductase system.

Using the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) we have demonstrated that hydroxyl radicals are generated indirectly from purified preparations of rat liver microsomal NADPH-cytochrome c (P-450) reductase during NADPH oxidation. Hydroxyl radical formation is completely inhibited by p-chloromercuribenzoate, but not by metyrapone. In addition, hydroxyl radical DMPO adduct formation is blocked by added linolenic acid which, in turn, is peroxidatively degraded into malondialdehyde, suggesting that hydroxyl radicals formed from purified NADPH-cytochrome c (P-450) reductase are capable of initiating lipid peroxidation. A mechanism for the indirect production of hydroxyl radicals from NADPH-cytochrome P-450 reductase is discussed.     

Zinc suppression of free radicals induced in cultures of rat hepatocytes by iron, t-butyl hydroperoxide, and 3-methylindole

The effect of zinc on lipid peroxidation initiated by either ferric-nitrilotriacetate, t-butyl hydroperoxide, or 3-methylindole was studied using primary monolayer cultures of rat liver parenchymal cells. The malondialdehyde content of the cells and culture medium was used to estimate the extent of lipid peroxidation. As the zinc concentration of the culture medium was increased from 1 to 48 microM, peroxidation was diminished. Cellular zinc and metallothionein levels were proportionally increased by supplemental zinc. Zinc supplementation of the medium inhibited NADPH-cytochrome c reductase activity and stimulated glutathione peroxidase activity. The uptake of iron into the hepatocytes was significantly reduced as the level of zinc was raised, suggesting that zinc antagonizes uptake of chelated iron into isolated hepatocytes and in this way blocks iron-induced peroxidation. Furthermore, induction of metallothionein synthesis by zinc may contribute to the reduction in free radicals. Spectra from electron spin resonance studies, using phenylbutylnitrone as a spin-trapping reagent, demonstrated that free radical production was inversely related to the zinc concentration of the culture medium. Spin trap data suggest that metallothionein added to lysed cells in vitro decreases free radical production. Studies using the spin trap, 3,3,5,5-tetramethylpyrroline-N-oxide indicated that cumulatively the predominant radical present in the cultures was a phenyl radical with hydroperoxide or methylindole. Collectively, our data demonstrate that zinc inhibits free radical production and lipid peroxidation in cultured hepatocytes. The mode of action of zinc could occur via free radical scavenging by zinc-induced metallothionein and/or by processes related to cytochrome P-450 and glutathione peroxidase, since these were also found to be sensitive to zinc supplementation levels of the culture medium.     

The NADPH- and iron-dependent lipid peroxidation in human placental microsomes

In pregnant females, placenta is the most important source of lipid hydroperoxides and other reactive oxygen species (ROS). The increased production of lipid peroxides is often linked to preeclampsia. In our study, we revealed that NADPH- and iron-dependent lipid peroxidation in human placental microsomes (HPM) occurred. In the presence of Fe²⁺ ion, HPM produced small amounts of thiobarbituric acid-reactive substances (TBARS) – a final product of lipid peroxidation. NADPH caused a strong increase of iron stimulated TBARS formation. TBARS formation was inhibited by superoxide dismutase, butylated hydroxytoluene and α-tocopherol but not by mannitol or catalase. TBARS and superoxide radical production was inhibited in similar manner by cytochrome P450 inhibitors. The results obtained led us to the following conclusions: (1) microsomal lipid peroxidation next to mitochondrial lipid peroxidation may by an important source of lipid hydroperoxides in blood during pregnancy and (2) superoxide radical released by microsomal cytochrome P450 is an important factor in NADPH- and iron-dependent lipid peroxidation in HPM.     

Spectroscopic characterization of the iron-oxo intermediate in cytochrome P450

From analogy to chloroperoxidase from Caldariomyces fumago, it is believed that the electronic structure of the intermediate iron-oxo species in the catalytic cycle of cytochrome P450 corresponds to an iron(IV) porphyrin-pi-cation radical (compound I). However, our recent studies on P450cam revealed that after 8 ms a tyrosine radical and iron(IV) were formed in the reaction of ferric P450 with external oxidants in the shunt pathway. The present study on the heme domain of P450BM3 (P450BMP) shows a similar result. In addition to a tyrosine radical, a contribution from a tryptophan radical was found in the electron paramagnetic resonance (EPR) spectra of P450BMP. Here we present comparative multi-frequency EPR (9.6, 94 and 285 GHz) and M?ssbauer spectroscopic studies on freeze-quenched intermediates produced using peroxy acetic acid as oxidant for both P450 cytochromes. After 8 ms in both systems, amino acid radicals occurred instead of the proposed iron(IV) porphyrin-pi-cation radical, which may be transiently formed on a much faster time scale. These findings are discussed with respect to other heme thiolate proteins. Our studies demonstrate that intramolecular electron transfer from aromatic amino acids is a common feature in these enzymes. The electron transfer quenches the presumably transiently formed porphyrin-pi-cation radical, which makes it extremely difficult to trap compound I.     

Hydrogen peroxide-mediated inactivation of microsomal cytochrome P450 during monooxygenase reactions

Cytochrome P450 can undergo inactivation following monooxygenase reactions in liver microsomes of untreated, phenobarbital and 3-methylcholanthrene-treated rats and rabbits. The acceleration of cytochrome P450 loss in the presence of catalase inhibitors (sodium azide, hydroxylamine) indicates that hydrogen peroxide is involved in hemoprotein degradation. It was revealed that cytochrome P450 is inactivated mainly by H₂O₂ formed through peroxy complex breakdown, whereas H₂O₂ formed via the dismutation of superoxide anions produces a slight inactivating effect. The hydrogen peroxide added outside or formed by a glucose-glucose oxidase system has less of an inactivating effect than H₂O₂ produced within the cytochrome P450 active center. Self-inactivation of cytochrome P450 during oxygenase reactions is highly specific. Other components of the monooxygenase system, such as cytochrome b₅, NADH- and NADPH-specific flavorproteins, undergo no inactivation. The alterations in phospholipid content and in the rate of lipid peroxidation were not observed as well. The inactivation of cytochrome P450 by H₂O₂ is the result of heme loss or destruction without cytochrome P420 formation. Such. a mechanism operates with different substrates and cytochrome P450 species catalyzing the partially coupled monooxygenase reactions.     

Spin trapping and its application in the study of lipid peroxidation and free radical production with liver microsomes.

Lipid peroxidation in microsomes was studied using a spin-trapping technique. Free radical adducts of phenyltertiarybutylnitrone (PBN) were produced as detected by electron spin resonance during induced lipid peroxidation of microsomes with a system consisting of NADPH, Fe²⁺, and pyrophosphate. The adducts were identified as intermediates of the substrates added to the microsomal system and not OH · or HO₂ radicals. The production of the adduct parallels the NADPH-dependent formation of malondialdehyde (MDA). Analyses of the electron spin resonance hyperfine splitting constants allowed in some instances identification of the adducts. Purified preparations of cytochrome P-450 mimic the results of the microsomes. The carcinogens dimethyl and diethylnitrosoamine were metabolized in this system yelding reactive free radicals and free NO, suggesting an alternate mechanism for the activity of these compounds as ultimate carcinogens.