Electron-transport cytochrome P-450 system is involved in the microsomal metabolism of the carcinogen chromate

The kinetics of chromate reduction by liver microsomes isolated from rats pretreated with phenobarbital or 3-methylcholanthrene with NADPH or NADH cofactor have been followed. Induction of cytochrome P-450 and NADPH-cytochrome P-450 reductase activity in microsomes by phenobarbital pretreatment caused a decrease in the apparent chromate-enzyme dissociation constant, Km, and an increase in the apparent second-order rate constant, kcat/Km, but did not affect the kcat of NADPH-mediated microsomal metabolism of chromate. Induction of cytochrome P-448 in microsomes by 3-methylcholanthrene pretreatment did not affect the kinetics of NADPH-mediated reduction of chromate by microsomes. The kinetics of NADH-mediated microsomal chromate reduction were unaffected by the drug treatments. The effects of specific enzyme inhibitors on the kinetics of microsomal chromate reduction have been determined. 2'-AMP and 3-pyridinealdehyde-NAD, inhibitors of NADPH-cytochrome P-450 reductase and NADH-cytochrome b5 reductase, inhibited the rate of microsomal reduction of chromate with NADPH and NADH. Metyrapone and carbon monoxide, specific inhibitors of cytochrome P-450, inhibited the rate of NADPH-mediated microsomal reduction of chromate, whereas high concentrations of dimethyl-sulfoxide (0.5 M) enhanced the rate. These results suggest that the electron-transport cytochrome P-450 system is involved in the reduction of chromate by microsomal systems. The NADPH and NADH cofactors supply reducing equivalents ultimately to cytochrome P-450 which functions as a reductase in chromate metabolism. The lower oxidation state(s) produced upon chromate reduction may represent the ultimate carcinogenic form(s) of chromium. These studies provide evidence for the role of cytochrome P-450 in the activation of inorganic carcinogens.     

Chromate metabolism in liver microsomes

The carcinogenicity and mutagenicity of various chromium compounds have been found to be markedly dependent on the oxidation state of the metal. The carcinogen chromate was reduced to chromium(III) by rat liver microsomes in vitro. Metabolism of chromate by microsomal enzymes occurred only in the presence of either NADPH or NADH as cofactor. The chromium(III) generated upon metabolism formed a complex with the NADP⁺ cofactor. Significant binding of chromium to DNA occurred only when chromate was incubated in the presence of microsomes and NADPH. Specific inhibitors of the mixed function oxidase enzymes, 2′-AMP, metyrapone, and carbon monoxide, inhibited the rate of reduction of chromate by microsomes and NADPH. The possible relationship of metabolism of chromate and its interaction with nucleic acids to its carcinogenicity and mutagenicity is discussed.     

Metabolism of nitroxide spin labels in subcellular fraction of rat liver. I. Reduction by microsomes

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of seven nitroxides in microsomes obtained from rat liver. The nitroxides were chosen to provide information on the effects of the type of charge, lipophilicity and the ring on which the nitroxide group is located. Important variables that were studied included adding NADH, adding NADPH, induction of enzymes by intake of phenobarbital and the effects of oxygen. Reduction to nonparamagnetic derivatives and oxidation back to paramagnetic derivatives were measured by electron-spin resonance spectroscopy. In general, the relative rates of reduction of nitroxides were similar to those observed with intact cells, but the effects of the various variables that were studied often differed from those observed in intact cells. The rates of reduction were very slow in the absence of added NADH or NADPH. The relative effect of these two nucleotides changed when animals were fed phenobarbital, and paralleled the levels of NADPH cytochrome c reductase, cytochrome P-450, cytochrome b5 and NADH cytochrome c reductase; results with purified NADPH-cytochrome c reductase were consistent with these results. In microsomes from uninduced animals the rate of reduction was about 10-fold higher in the absence of oxygen. The products of reduction of nitroxides by microsomes were the corresponding hydroxylamines. We conclude that there are significant NADH- and NADPH-dependent paths for reduction of nitroxides by hepatic microsomes, probably involving cytochrome c reductases and not directly involving cytochrome P-450. From this, and from parallel studies now in progress in our laboratory, it seems likely that metabolism by microsomes is an important site of reduction of nitroxides. However, mitochondrial metabolism seems to play an even more important role in intact cells.     

The interaction of chromium with nucleic acids

Native and denatured calf thymus DNA, and homopolyribonucleotides were compared with respect to chromium and protein binding after an in vitro incubation with rat liver microsomes, NADPH, and chromium(VI) or chromium(III). A significant amount of chromium bound to DNA when chromium(VI) was incubated with the native or the denatured form of DNA in the presence of microsomes and NADPH. For both native and denatured DNA the amount of protein bound to DNA increased with the amount of chromium bound to DNA. Denatured DNA had much higher amounts of chromium and protein bound than native DNA. There was no interaction between chromium(VI) and either form of DNA in the absence of the complete microsomal reducing system. The binding of chrornium(III) to native or denatured DNA was small and relatively unaffected by the presence of microsomes and NADPH. The binding of chromium and protein to polyriboadenylic acid (poly(A)), polyribocytidylic acid (poly(C), polyri-boguanylic acid (poly(G)) and polyribouridylic acid (poly(U)) was determined after incubation with chromium(VI) in the presence of microsomes and NADPH. The magnitude of chromium and protein binding to the ribo-polymers was found to be poly(G) ? poly(A) ? poly(C) ? poly(U). These results suggest that the metabolism of chromium(VI) is necessary in order for chromium to interact significantly with nucleic acids. The metabolically-produced chromium preferentially binds to the base guanine and results in DNA-protein cross-links. These findings are discussed with respect to the proposed scheme for the carcinogenicity of chromium(VI). Keywords: DNA-protein cross-links — Chromium-guanine interaction-Microsomal reduction of chromate     

Metabolism of nitroxide spin labels in subcellular fraction of rat liver: I. Reduction by microsomes

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of seven nitroxides in microsomes obtained from rat liver. The nitroxides were chosen to provide information on the effects of the type of charge, lipophilicity and the ring on which the nitroxide group is locted Important variables that were studied included adding NADH, adding, induction of enzymed by intake of phenobarbital and the effects of oxygen. Reduction of nonparamagnetic derivatives and oxidation to paramagnetic derivatives were measured by electron-spin resonance spectroscopy. In general, the relative rates of reduction of nitroxides were similar to those observed with intact cells, but the effects of the various variables that were studied often differed from those observed in intact cells. The rates of reduction were very slow in the absence of added NADh or NADPH. The relative effect of these two nucleotides changed when animals were fed phenobarbital and paralleled the levels of NADPH cytochrome c reductase, cytochrome P-450, cytochrome b₅ and NADH cytochrome c reductase; results with purified NADPH-cytochrome c reductase were consistent with these results. In microsomes from uninduced animals the rate of reduction was about 10-fold higher in the absence of oxygen. The products of reduction of nitroxides by microsomes were the corresponding hydroxylmines. We conclude that there are significant NADH- and NADPH-dependent paths for reduction of nitroxides by hepatic microsomes, probably involving cytochrome c reductases and not directly involving cytochrome P-450. From this, and from parallel studies now in progress in our laboratory, it seems likely that metabolism by microsomes is an important site of reduction of nitroxides. However, mitochondrial metabolism seems to play an even more important role in intact cells.     

Microsomal metabolism of carbon tetrachloride: participation of pyridine nucleotide synergism

The role of pyridine nucleotide synergism in CCl4 metabolism was evaluated for its potential contribution to enhanced lipid peroxidation. Male Sprague-Dawley rats receiving either no treatment (control) or treatment with phenobarbital (PB) were used to prepare hepatic microsomes. Metabolism was evaluated in the presence and absence of an NADPH generator system and in the presence and absence of NADH. The generator system produced a greater extent of metabolism for both control and PB microsomes. NADH-catalyzed CCl4 metabolism occurred to a similar extent in control and PB microsomes, amounting to 9-10% and 5-6% of the NADPH rate in control and PB microsomes, respectively. Synergism by NADH occurred at the lowest concentrations of NADPH, apparently decreasing the Km for NADPH and having little effect on the Vmax. Addition of NAD+ produced synergism, as did the addition of 5' AMP, an inhibitor of nucleotide pyrophosphatase. Thus, the synergistic increase in CCl4 metabolism produced by NADH may occur in part from an increased availability of NADPH, as a result of decreased degradation, rather than by electron donation from NADH.     

One-electron reduction of carcinogen chromate by microsomes, mitochondria, and Escherichia coli: identification of Cr(V) and .OH radical.

Earlier studies have shown that a long-lived Cr(V) species is produced during the reduction of chromate (Cr(VI] by microsomes/NADPH, mitochondria, and other cellular constituents and that this Cr(V) species plays a significant role in the mechanism of Cr(VI) toxicity. The present work indicates that this species is a Cr(V) complex involving the diol moieties of NADPH as the ligand. Additionally, ESR spin trapping investigations show that the hydroxyl (.OH) radical is also generated in the reduction process. Hydrogen peroxide (H2O2) enhances the .OH generation but suppresses the Cr(V)-NADPH complex formation. Catalase decreases the .OH radical generation and enhances the Cr(V)-NADPH formation. Measurements under anaerobic atmosphere show decreased .OH radical generation, indicating that during the cellular Cr(VI) reduction process molecular oxygen is reduced to H2O2, which reacts with the Cr(V)-NADPH complex to generate the .OH radical via a Fenton-like mechanism.     

Studies of the metabolism of carbon disulfide by rat liver microsomes

Carbon disulfide has been examined as a substrate for the hepatic mixed function oxidase enzyme system. These studies have revealed that carbonyl sulfide is formed when carbon disulfide is incubated with rat liver microsomes in the presence of NADPH. No carbonyl sulfide is formed in the absence of NADPH. The formation of carbonyl sulfide is inhibited when the reaction is carried out in an atmosphere containing carbon monoxide. The other product of the reaction leading to carbonyl sulfide formation appears to be a highly reactive form of sulfur which covalently binds to the microsomal membrane. A chemical mechanism for the mixed function oxidase catalyzed metabolism of carbon disulfide to carbonyl sulfide is proposed.     

Inhibition of hepatic aldehyde dehydrogenase by carbon tetrachloride: an in vitro study

In vitro inhibition of rat liver mitochondrial and microsomal aldehyde dehydrogenase (ALDH) under conditions of active CCl4 metabolism was investigated. Incubation of microsomes or mitochondria in the presence of NADPH alone caused significant, time-dependent inhibition of mitochondrial and microsomal ALDH. EDTA partially protected ALDH from inhibition. Incubation of microsomes or microsomes plus mitochondria in the presence of NADPH and CCl4 resulted in marked inhibition of microsomal and mitochondrial ALDH activity. The inhibition was both dose- and time-dependent and was relatively less in the presence of EDTA. It is proposed that the inhibition of membrane-bound ALDH may be one of the early events responsible for the genesis of CCl4-hepatotoxicity.     

Toxicological implications of the mixed-function oxidase catalyzed metabolism of carbon disulfide.

The results of these studies have indicated that the decrease in the activity of the hepatic mixed-function oxidase enzyme system and the concentration of cytochrome P-450 seen on incubation of carbon disulfide (CS2) with rat liver microsomes in the presence of NADPH is the result of the binding of the sulfur atom released in the mixed-function oxidase catalyzed metabolism of CS2 to carbonyl sulfide (COS). Moreover, it appears that COS is further metabolized by the mixed-function oxidase enzyme system to CO2 and that, analogous to the metabolism of CS2 to COS, the sulfur atom released in this reaction also binds to the microsomes and inhibits benzphetamine metabolism and decreases the concentration of cytochrome P-450 detectable as its carbon monoxide complex. The results of these studies also suggest that the decrease in the concentration of cytochrome P-450 and the liver damage seen on in vivo administration of CS2 to phenobarbital pretreated rats, is due to the mixed-function oxidase catalyzed release and binding of the sulfur atoms of CS2. The decrease in the concentration of cytochrome P-450 seen on incubation of CS2 with rat liver microsomes in the presence of NADPH does not appear to be the result of destruction of the heme group or its dissociation from the apoenzyme since the total amount of protoheme is unchanged in microsomes which have been incubated with CS2 and NADPH as compared to those not incubated with these compounds.     

New concepts of microsomal metabolism of the antibiotic adriamycin

Experimental and literary data are presented which are at variance with the known conception of adriamycin (AD) shunting the chain of microsomal electron transfer. AD, carminomycin, rubomycin, mitomycin C, and coenzyme Q9 are shown to interact with NADPH, in the absence of enzymes, with the nucleotide oxidation. A new scheme of AD metabolism is suggested, according to which AD in the hydroquinone form enters the chain of electron transfer in microsomes between NADPH and flavoprotein.     

Ethanol and drug metabolism in mouse liver microsomes subsequent to lipid peroxidation-induced destruction of cytochrome P-450

Preincubation of mouse liver microsomes with NADPH resulted in malondialdehyde formation, destruction of cytochrome P-450, and decreased rates of aniline hydroxylation and N-demethylation of aminopyrine and ethylmorphine. These phenomena were more pronounced in phosphate than in Tris buffer. No reduction in rates of NADPH-linked oxidation of ethanol or in the activities of NADPH oxidase and NADPH-cytochrome c reductase was observed. While addition of EDTA to preincubation mixtures prevented lipid peroxidation, loss of cytochrome P-450, and inactivation of the drug-metabolizing capacity of microsomes, it did not alter ethanol oxidation rates and the activities of NADPH oxidase and NADPH-cytochrome c reductase. These findings argue against the involvement of cytochrome P-450 in the microsomal ethanol-oxidizing system.     

Oxidation of ethanol by hepatic microsomes of acatalasemic mice

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

The effects of unsaturated fatty acids on hepatic microsomal drug metabolism and cytochrome P-450

1. The effects of unsaturated fatty acids on drug-metabolizing enzymes in vitro were measured by using rat and rabbit hepatic 9000g supernatant fractions. 2. Unsaturated fatty acids inhibited the hepatic microsomal metabolism of ;type I' drugs with inhibition increasing with unsaturation: arachidonic acid>linolenic acid>linoleic acid>oleic acid. Inhibition was independent of lipid peroxidation. Linoleic acid competitively inhibited the microsomal O-demethylation of p-nitroanisole and the N-demethylation of (+)-benzphetamine. 3. The hepatic microsomal metabolism of ;type II' substrates, aniline and (-)-amphetamine, was not affected by unsaturated fatty acids. 4. The rate of reduction of p-nitrobenzoic acid and Neoprontosil was accelerated by unsaturated fatty acids. 5. Linoleic acid up to 3.5mm did not decelerate the generation of NADPH by rat liver soluble fraction, nor the activity of NADPH-cytochrome c reductase of rat liver microsomes. Hepatic microsomal NADPH oxidase activity was slightly enhanced by added linoleic acid. 6. No measurable disappearance of exogenously added linoleic acid occurred when this fatty acid was incubated with rat liver microsomes and an NADPH source. 7. The unsaturated fatty acids used in this study produced type I spectra when added to rat liver microsomes, and affected several microsomal enzyme activities in a manner characteristic of type I ligands.     

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.     

Description of a 96-well plate assay to measure cytochrome P4503A inhibition in human liver microsomes using a selective fluorescent probe

The standard method to evaluate CYP3A inhibition is to study the conversion of the specific CYP3A probe testosterone to its 6 beta-hydroxy metabolite in human liver microsomes, in the absence and presence of potential inhibitors. Quantification of the 6 beta-hydroxy metabolite is achieved by HPLC resulting in a tedious and time-consuming assay. In order to increase the P450 inhibition throughput, efforts were made to find a CYP3A probe that would produce a fluorescent metabolite. This paper reports the discovery of DFB as a potential CYP3A fluorescent probe. DFB was significantly metabolized in human microsomes (approximately 1-2 nmol/(min. mg protein)) to give the fluorescent compound DFH. The involvement of CYP3A in the metabolism of DFB was determined using multiple approaches. First, incubations conducted with microsomes made from cell lines expressing single CYPs (Gentest Supersomes) indicated that CYP3A played a major role in the metabolism of DFB. Secondly, immunoinhibition studies conducted with CYP3A antibody resulted in >95% inhibition of DFB metabolism in HLM. Thirdly, inhibition studies with specific CYP1A1, 1A2, 2C8/9, 2C19, 2D6, and 2E1 chemical inhibitors did not suppress DFB activity in HLM. However, ketoconazole, miconazole, nicardipine, and nifedipine, all known CYP3A inhibitors, completely abolished the formation of DFH in HLM. The potency of several inhibitors determined using DFB and testosterone as CYP3A probes was consistent (R = 0.98). Finally, a good agreement was obtained for the formation of DFH and production of 6 beta-hydroxytestosterone when DFB and testosterone were incubated separately with various human liver microsome preparations (R = 0.94, N = 11). In order to use DFH as a fluorescent CYP3A marker in a 96-well plate format, it was important to remove the excess of NADPH at the end of the incubation because the fluorescence of NADPH interferes with DFH detection. This was achieved by adding oxidized glutathione and glutathione reductase to convert NADPH to NADP(+) which is not fluorescent. The liquid-handling steps were fully automated in a 96-well plate format and a template was designed to generate IC(50) curves and to address potential fluorescent interferences from the test compounds. The assay was found to be reproducible (intraday variability <10% and interday variability indicated less than a 2-fold variation in the IC(50) values) and is now routinely used in our laboratory to evaluate CYP3A inhibition of NCEs.     

Metabolism and mutagenic activation of benzo(a)pyrene by subcellular fractions from mussel (Mytilus galloprovincialis) digestive gland and sea bass (Discenthrarcus labrax) liver

1. The formation of B(a)P diols, phenols and bacterial mutagens by mussel subcellular fractions is dependent on NADPH whereas B(a)P quinones, the major metabolites, appear to be produced by radical reactions and chiefly in the absence of NADPH.2. B(a)P metabolism in sea bass liver fractions is totally dependent on NADPH and insensitive to radical scavengers excepted tocopherol inhibition of B(a)P mutagenesis.3. The 9–10 and 7–8 epoxides formed by sea bass microsomes have a high affinity for EH which readily metabolized all those epoxides to diols.4. Alpha-naphtoflavone inhibits sea bass B(a)P metabolism at high concentration (100 μM) whereas it increases it at low concentration (20 μM).     

The stimulation of microsomal azoreduction by flavins

The reduction of the azo dye, amaranth, by rat liver microsomes is inhibited about 90% by carbon monoxide, suggesting that the reaction largely depends on cytochrome P-450. Reducing equivalents for this reaction are supplied by NADPH. This reaction is stimulated by riboflavin, FMN and FAD, as well as by methylviologen. A large fraction of the stimulated reaction is not blocked by CO, indicating that there is a pathway of electron transfer which is dependent of cytochrome P-450. Rat liver microsomes can reduce FAD, with reducing equivalents supplied by NADPH. The FADH₂ thus produced is quickly oxidized by amaranth so that two FADH₂ are oxidized for every amaranth reduced. The same stoichiometry is observed with photochemically prepared FADH₂, formed in the absence of microsomes.     

Effects of pregnenolone-16 alpha-carbonitrile on drug metabolizing enzymes in hypophysectomized female rats

Treatment of intact and hypophysectomized female rats with pregnenolone-16 alpha-carbonitrile (PCN) resulted in a significant increase in hepatic aryl hydrocarbon hydroxylase (AHH) activity. However, the total cytochrome P-450 concentration, as measured by CO difference spectra, was increased to a greater extent in hypophysectomized rats than in intact rats. Total cytochrome P-450 was found to be 0.82 +/- 0.16 vs 2.43 +/- 0.31 nmoles/mg protein for control and PCN-treated hypophysectomized rats, respectively, and 0.68 +/- 0.23 vs 1.28 +/- 0.05 nmoles/mg protein for control and PCN-treated intact rats respectively. The concentration of metyrapone complex in microsomes from intact control and PCN-treated rats was found to be 0.4 +/- 0.11 vs 1.88 +/- 0.23 M respectively. Treatment of hypophysectomized rats with PCN resulted in an approximate 10-fold increase in the concentration of the metyrapone complex (0.42 +/- 0.15 M for control and 4.46 +/- 0.44 M for PCN-treated). Microsomal NADPH and NADPH cytochrome c reductase activities were also altered by PCN-treatment. Aminopyrine demethylase activity was stimulated approximately three-fold by PCN treatment in both intact and hypophysectomized rats. Benzphetamine demethylase activity was not significantly affected by PCN treatment. The results of these studies suggest that the absence of the pituitary gland can markedly influence PCN induction of cytochrome P-450 in the liver in female rats. PCN also differentially affects microsomal mixed-function oxidase activities associated with drug and xenobiotic metabolism.     

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.