Steroid Hydroxylation Research Articles

Importance of Amino Acid Residue 474 for Substrate Specificity of Canine and Human Cytochrome P450 3A Enzymes

Canine cytochromes P450 3A12 and 3A26 are identical in sequence at 481 of 503 amino-acid positions but exhibit different substrate specificities. A recent study utilizing chimeric enzymes and site-directed mutagenesis identified three residues (187, 368, and 369) that contribute to differences in steroid hydroxylation and also indicated the presence of additional determinants of specificity among the 44 carboxyl terminal residues. Therefore, three 3A26 multiple mutants (I187T-S368P-V369I-S467P, I187T-S368P-V369I-S474P, and I187T-S368P-V369I-R476K-I477L-T479A-R480Q) were constructed. Insertion of 3A12 residue Pro-474 into 3A26 I187T-S368P-V369I resulted in metabolite profiles with testosterone, androstenedione, and progesterone very similar to 3A12. Substitution of Pro-474 with Ser in P450 3A12 or human 3A4 significantly increased 2β-hydroxylase activity with all three steroids. Residue 474 was also found to be an important contributor to diazepam metabolism by the canine and human enzymes. The results provide further evidence for the role of steric constraints exerted by the enzyme in P450 3A-mediated oxidations.

Identification of the rat adrenal zona fasciculata/reticularis specific protein, inner zone antigen (IZAg), as the putative membrane progesterone receptor

Using immunological methods, a protein specific to the inner zones of the rat adrenal cortex, and called inner zone antigen (IZAg), was previously shown to have two interrelated forms of 26 kDa (IZAg1) and 55-60 kDa (IZAg2), and to have an action on steroid hydroxylation. After two-dimensional gel electrophoresis, and immunoaffinity column purification, N-terminal amino-acid analysis showed that the first 12 amino acids were identical to those of a recently described putative membrane located progesterone receptor (PPMR). RT-PCR was then used to generate the cDNA of this protein, using RNA extracted from rat adrenals. A glutathione S-transferase (GST)-fusion construct was expressed in Escherichia coli, and shown to generate an immunoreactive product of molecular mass consistent with its identification as IZAg1. More detailed examination of the distribution of this protein, not only in the zona fasciculata/reticularis of the adrenal cortex, but also in the Leydig cell, kidney and liver, suggest it may have a role in steroid hormone synthesis and/or metabolism.

Metabolic activation of mitomycin C by NADPH-ferredoxin reductase in vitro

Mammalian NADPH-ferredoxin reductase (EC 1.18.1.2) functions in the mitochondrial electron transport chain for cytochrome P-450-dependent steroid hydroxylation. Significant homology of three-dimensional structure exists in the surroundings of FAD between NADPH-ferredoxin reductase and NADH-cytochrome b 5 reductase. The latter is involved in the bioreduction of mitomycin C (MC), a prototype antitumor agent. In this study, we assessed the capacity of NADPH-ferredoxin reductase to activate MC. Mitomycin C increased the NADPH oxidase activity of NADPH-ferredoxin reductase. In the absence of ferredoxin, the K m value of NADPH-ferredoxin reductase for MC was 73.5 ± 2.3 μM. While in the presence of 500 nM ferredoxin, a Lineweaver-Burk plot exhibited a biphasic curve. NADPH-ferredoxin reductase-mediated reduction of MC resulted in the formation of an alkylated complex of 4-(p-nitrobenzyl) pyridine and an increase in plasmide DNA single-strand breaks under hypoxic conditions. With the addition of 500 nM ferredoxin, the amount of the alkylated complex of 4-(p-nitrobenzyl) pyridine and the plasmide DNA single-strand breaks increased by 40% and 37%, respectively. However, neither alkylated complex of 4-(p-nitrobenzyl) pyridine nor DNA strand breaks was observed in the presence of SOD and catalase under aerobic conditions. These findings demonstrate that NADPH-ferredoxin reductase is capable of catalyzing the bioactivation of mitomycin C under hypoxic conditions in vitro.

Adrenodoxin Reductase-Adrenodoxin Complex Structure Suggests Electron Transfer Path in Steroid Biosynthesis

The steroid hydroxylating system of adrenal cortex mitochondria consists of the membrane-attached NADPH-dependent adrenodoxin reductase (AR), the soluble one-electron transport protein adrenodoxin (Adx), and a membrane-integrated cytochrome P450 of the CYP11 family. In the 2.3-A resolution crystal structure of the Adx.AR complex, 580 A(2) of partly polar surface are buried. Main interaction sites are centered around Asp(79), Asp(76), Asp(72), and Asp(39) of Adx and around Arg(211), Arg(240), Arg(244), and Lys(27) of AR, respectively. In particular, the region around Asp(39) defines a new protein interaction site for Adx, similar to those found in plant and bacterial ferredoxins. Additional contacts involve the electron transfer region between the redox centers of AR and Adx and C-terminal residues of Adx. The Adx residues Asp(113) to Arg(115) adopt 3(10)-helical conformation and engage in loose intermolecular contacts within a deep cleft of AR. Complex formation is accompanied by a slight domain rearrangement in AR. The [2Fe-2S] cluster of Adx and the isoalloxazine rings of FAD of AR are 10 A apart suggesting a possible electron transfer route between these redox centers. The AR.Adx complex represents the first structure of a biologically relevant complex between a ferredoxin and its reductase.

Molecular cloning of NADPH-cytochrome P450 oxidoreductase from silkworm eggs. Its involvement in 20-hydroxyecdysone biosynthesis during embryonic development.

Using RT-PCR, a cDNA fragment of NADPH-cytochrome P450 oxidoreductase from silkworm, Bombyx mori, was cloned from three-day-old nondiapause eggs. RACE was used to isolate the ends of the DNA. The full-length cDNA obtained was composed of 3471 bp with an open reading frame encoding a protein of 687 amino-acid residues with a relative molecular mass of 77 700. The protein, fused with glutathione S-transferase, was expressed in Escherichia coli and purified to homogeneity. The fused protein not only had NADPH-dependent cytochrome c-reducing activity, but also acted as an electron carrier from NADPH to bovine adrenal 21-hydroxylase P450 in the steroid hydroxylation reaction, confirming that the protein is the silkworm NADPH-cytochrome P450 oxidoreductase. Ecdysone 20-hydroxylase activity in the nondiapause egg microsomes increased until the fourth day after oviposition, and then decreased, little being detected on the ninth day. An antibody raised against the P450 reductase inhibited the ecdysone hydroxylation. Immunoblot analyses of the microsomes indicated that the P450 reductase protein appeared distinctly in the three-day-old nondiapause eggs and, in contrast to the developmental pattern of ecdysone hydroxylase activity, continued to increase as the embryos developed. These results suggest that ecdysone hydroxylation in the early stage of embryogenesis is dependent on the presence of both P450 reductase and ecdysone 20-hydroxylase P450, but its gradual reduction in the later stage may be due to the decrease in the level of ecdysone 20-hydroxylase P450.

Induction of hepatic cytochrome p450 activities by toxaphene in rat and japanese quail

Several organochlorine compounds can induce hepatic cytochrome P450 activities. We exposed female Japanese quail and female rats to a single oral dose of technical toxaphene ranging from 0.012 to 40 mg/kg body weight to investigate a possible dose-response relationship for P450 activity induction. The hepatic microsomal alkoxyresorufin dealkylation and steroid hydrox-ylation activities (testosterone, 17β-estradiol) were determined. Only in the highest dose groups (40 mg/kg) of rats and Japanese quail were some P450 activities induced. Pentoxyresorufin-O-dealkylation, formation of 15β-hydroxytestosterone, and formation of 2-hydroxyestradiol were induced significantly (p ≤ 0.05) in rat hepatic microsomes with a factor of 3.9, 3.1, and 2.4, respectively. In Japanese quail hepatic microsomes, the formation rates of 6β-, 15α-, 16β-hydroxytestosterone and androstenedione were induced significantly (p ≤ 0.05) with a factor of 2.4, 6.5, 3.3, and 1.6, respectively. It is concluded that exposure to toxaphene can lead to specific P450 activity induction in rats and Japanese quail but only at doses near reported LD50 values. Because reported toxaphene levels in biota are lower compared with liver residue levels that we have measured in our study, we conclude that it is unlikely that P450 activity induction occurs in wildlife higher in the food chain due to environmental toxaphene exposure.

Allelic variants of human cytochrome P450 1A1 (CYP1A1): effect of T461N and I462V substitutions on steroid hydroxylase specificity.

Steroid hydroxylation specificities were determined for the wild-type and the two allelic variants of the polymorphic human cytochrome P450 1A1 (CYP1A1) that were associated with amino acid exchanges near the active site of the enzyme. All three variants were expressed in insect cells using recombinant baculoviruses. Each variant protein was spectrally and enzymatically active, as judged by the ability of the prepared microsomes to catalyse O-dealkylation of ethoxyresorufin and pentoxyresorufin in cumene hydroperoxide-mediated reactions. With progesterone and testosterone as substrate, all variants of CYP1A1 exhibited high, but different steroid hydroxylation activities (8-40 pmol hydroxysteroid/min/pmol CYP1A1, i.e. approximately 800-4000 pmol/min/mg microsomal protein). All three variants exclusively catalysed 6beta-hydroxylation of both steroids. In addition, towards progesterone as substrate, all variants also catalysed 16alpha-hydroxylations with approximately half of the rate of 6beta-hydroxylation activity. With progesterone as substrate for hydroxylation in 6beta position, CYP1A1 T461N had the lowest catalytic efficiency (Vmax/Km) followed by the CYP1A1 I462V variant and the wild-type enzyme. For 16alpha-hydroxylation of progesterone, the catalytic efficiencies of the three variants are not statistically significantly different. With testosterone as substrate the CYP1A1 1462V variant catalysed 6beta-hydroxylation with an efficiency considered not significantly different compared to the wild-type, although both the apparent Km and Vmax were significantly decreased. In contrast, the CYP1A1 T461N variant exhibited significantly decreased catalytic efficiencies compared to both the 1462V variant and the wild-type enzyme. These results indicate that all three naturally occurring allelic variants of human CYP1A1 hydroxylate steroid hormones with varying efficiencies in a stereo- and regioselective manner, whereby the CYP1A1 T461N variant exhibited the lowest catalytic efficiency.

Purification of cytochrome P450 from filamentous fungus Rhyzopus nigricans

It has been reported in our previous studies that steroid hydroxylation system of Rhizopus nigricans involves cytochrome P450 and NADPH-cytochrome P450 reductase in the electron transport system. Both enzymes are membrane bound and are located in the microsomal preparations of progesterone induced fungal mycelia. In order to identify and characterize the cytochrome P450 component of fungal monoxygenase system, microsomal proteins from induced mycelia were subjected to HIGH Q anion exchange and MONO P (FPLC) anion exchange chromatography. Four fractions containing cytochrome P450 have been resolved on MONO P column. They exhibit CO difference spectra and type II difference spectra with ketoconazole.

Characterization of plasma membrane fraction from filamentous fungus Rhizopus nigricans

In the filamentous fungus Rhizopus nigricans a steroid hydroxylating multienzyme system is inducible by progesterone and by several other steroids. The biological signal carried by progesterone might be mediated by receptors, located either in the plasma membrane or inside the cell. To elucidate the first possibility, plasma membrane fraction was examined for the presence of progesterone receptors. The isolation of plasma membrane from fungal homogenate containing different other membranes is difficult because of the rigid cell wall. Three different membrane fractions were prepared by differential centrifugation of the fungal homogenate and characterized by plasma membrane and mitochondrial membrane marker enzymes, H+-ATPase and mit-ATPase, respectively. The same fractions were examined for the presence of specific progesterone-binding molecules. Two of these fractions comprising the highest level of plasma membrane enzyme activity contained also the highest level of specific progesterone-binding compounds: 27,6 fmol/mg protein and 18,8 fmol/mg protein. The correlation between plasma membrane marker enzyme activity and the amount of progesterone-binding proteins in plasma membrane fraction of Rhizopus nigricans might indicate the involvement of these molecules in the induction process.

Glucocorticoid-dependent maintenance of CYP2C11-dependent oxidation in male rat liver in vivo.

1. Hormonal factors participate in the regulation of xenobiotic metabolising enzymes in liver. Hepatic xenobiotic oxidation capacity is decreased in adrenalectomised rats, which directly implicates adrenal hormones in the control of cytochrome P450 (CYP) expression. In addition, recent studies in cultured hepatocytes have demonstrated that low concentrations of glucocorticoid upregulate the male-specific CYP2C11, which is a major enzyme that catalyses xenobiotic and steroid hydroxylations in rat liver. The present study evaluated whether glucocorticoid or mineralocorticoid may be the adrenal factor that contributes to the in vivo expression of CYP2C11 in liver. 2. Adrenalectomy of male rats selectively decreased CYP2C11-dependent 2alpha-/16alpha-hydroxylation of testosterone and other steroid substrates to 60-70% of control, whereas activities mediated by other constitutive CYPs were unaffected. The decrease in CYP2C11 activity was due to impaired protein expression in liver after adrenalectomy. Administration of dexamethasone (DEX; 0.2 mg/kg i.p. daily for 6 days) restored CYP2C11 activity and protein, whereas the mineralocorticoid deoxycorticosterone (DOC) and adrenocorticotropic hormone (ACTH) were ineffective. 3. These findings establish that glucocorticoids have a partial role in the maintenance of CYP2C11 expression and associated microsomal oxidation in liver and provide a physiological correlate for similar observations made in vitro in hepatocyte culture.

The Importance of SRS-1 Residues in Catalytic Specificity of Human Cytochrome P450 3A4

The structural basis for the regioselective hydroxylation of Δ-4-3-ketosteroids by human CYP3A4 was investigated. Prior studies had suggested that the chemical reactivity of the allylic 6β-position might have a greater influence than steric constraints by the enzyme. Six highly conserved CYP3A residues from substrate recognition site 1 were examined by site-directed mutagenesis. F102A and A117L showed no spectrally detectable P450. V101G and T103A exhibited a wild-type progesterone metabolite profile. Of five mutants at residue N104, only N104D yielded holoenzyme and exhibited the same steroid metabolite profile as wild-type. Of four mutants at position S119 (A, L, T, V), the three hydrophobic ones produced 2β-OH rather than 6β-OH progesterone or testosterone as the major metabolite. Kinetic analysis showed S50 values similar to wild-type for S119A (progesterone) and S119V (testosterone), whereas the Vmax values for 2β-hydroxysteroid formation were increased in both cases. All four mutants exhibited an altered product profile for 7-hexoxycoumarin side-chain hydroxylation, whereas the stimulation of steroid hydroxylation by α-naphthoflavone was similar to the wild-type. The results indicate that the highly conserved residue S119 is a key determinant of CYP3A4 specificity and reveal an important role of the active site topology in steroid 6β-hydroxylation.

2-Methylisoborneol disposition in three strains of catfish: absence of biotransformation.

2-Methylisoborneol (MIB) and structurally related terpenoid compounds are responsible for millions of dollars of lost revenue to catfish farmers. In an attempt to determine enzymatic pathways of biotransformation and elimination of MIB, the in vitro metabolism of MIB was examined in the Ulvade strain of channel catfish (Ictalurus punctatus). Although cytochrome P450 (CYP) activities were observed and correlated with expression of specific isoforms (i.e. steroid hydroxylation and CYP3A expression), no metabolites of MIB were observed. To determine whether extrahepatic biotransformation may be occurring the in vivo metabolism and disposition of 14C-MIB was examined in Uvalde, USDA-103 channel catfish, and a channel catfish X blue catfish (Ictalurus furcatus) hybrid species. Confirming in vitro hepatic studies, no metabolites were observed in plasma from animals treated with an intra-arterial dose of 14C-MIB. 14C-MIB elimination was predicted using a two compartment model in each strain of fish. There was no significant difference in terminal half-lives between strains but possible differences in total body clearance and apparent volumes of distribution which may be related to higher lipid content in the hybrids. Results of these studies indicate biotransformation has no involvement in MIB elimination and that other physiological processes may play a more significant role in MIB disposition within Ictalurid fish species.

INDUCTION OF HEPATIC CYTOCHROME P450 ACTIVITIES BY TOXAPHENE IN RAT AND JAPANESE QUAIL

Several organochlorine compounds can induce hepatic cytochrome P450 activities. We exposed female Japanese quail and female rats to a single oral dose of technical toxaphene ranging from 0.012 to 40 mg/kg body weight to investigate a possible dose-response relationship for P450 activity induction. The hepatic microsomal alkoxyresorufin dealkylation and steroid hydrox-ylation activities (testosterone, 17β-estradiol) were determined. Only in the highest dose groups (40 mg/kg) of rats and Japanese quail were some P450 activities induced. Pentoxyresorufin-O-dealkylation, formation of 15β-hydroxytestosterone, and formation of 2-hydroxyestradiol were induced significantly (p ≤ 0.05) in rat hepatic microsomes with a factor of 3.9, 3.1, and 2.4, respectively. In Japanese quail hepatic microsomes, the formation rates of 6β-, 15α-, 16β-hydroxytestosterone and androstenedione were induced significantly (p ≤ 0.05) with a factor of 2.4, 6.5, 3.3, and 1.6, respectively. It is concluded that exposure to toxaphene can lead to specific P450 activity induction in rats and Japanese quail but only at doses near reported LD50 values. Because reported toxaphene levels in biota are lower compared with liver residue levels that we have measured in our study, we conclude that it is unlikely that P450 activity induction occurs in wildlife higher in the food chain due to environmental toxaphene exposure.

Purification of cytochrome P450 from filamentous fungus Rhyzopus nigricans.

It has been reported in our previous studies that steroid hydroxylation system of Rhizopus nigricans involves cytochrome P450 and NADPH-cytochrome P450 reductase in the electron transport system. Both enzymes are membrane bound and are located in the microsomal preparations of progesterone induced fungal mycelia. In order to identify and characterize the cytochrome P450 component of fungal monoxygenase system, microsomal proteins from induced mycelia were subjected to HIGH Q anion exchange and MONO P (FPLC) anion exchange chromatography. Four fractions containing cytochrome P450 have been resolved on MONO P column. They exhibit CO difference spectra and type II difference spectra with ketoconazole.

Characterization of plasma membrane fraction from filamentous fungus Rhizopus nigricans.

In the filamentous fungus Rhizopus nigricans a steroid hydroxylating multienzyme system is inducible by progesterone and by several other steroids. The biological signal carried by progesterone might be mediated by receptors, located either in the plasma membrane or inside the cell. To elucidate the first possibility, plasma membrane fraction was examined for the presence of progesterone receptors. The isolation of plasma membrane from fungal homogenate containing different other membranes is difficult because of the rigid cell wall. Three different membrane fractions were prepared by differential centrifugation of the fungal homogenate and characterized by plasma membrane and mitochondrial membrane marker enzymes, H+-ATPase and mit-ATPase, respectively. The same fractions were examined for the presence of specific progesterone-binding molecules. Two of these fractions comprising the highest level of plasma membrane enzyme activity contained also the highest level of specific progesterone-binding compounds: 27,6 fmol/mg protein and 18,8 fmol/mg protein. The correlation between plasma membrane marker enzyme activity and the amount of progesterone-binding proteins in plasma membrane fraction of Rhizopus nigricans might indicate the involvement of these molecules in the induction process.

G-Protein coupled progesterone receptors in the plasma membrane of fungus Rhizopus nigricans.

We have demonstrated simultaneous existence of progesterone receptors and GTPase activity in the membranes prepared from the filamentous fungus Rhizopus nigricans. The results obtained with pertussis toxin treated fungal mycelium suggest that these receptors do not couple to Gi-Go-proteins and play a role in the induction of steroid hydroxylating enzyme system by steroid substrates in the fungus.

Lack of mechanism-based inactivation of rat hepatic microsomal cytochromes P450 by doxorubicin

Administration of the antineoplastic doxorubicin to rodents causes depression of hepatic cytochrome P450 (CYP) dependent biotransformation, an effect that has been partially attributed to the ability of doxorubicin to stimulate microsomal lipid peroxidation. Since doxorubicin can be bioactivated by the CYP/NADPH-CYP reductase system to products that bind covalently to microsomal protein, we hypothesized that doxorubicin functions as a mechanism-based inactivator of hepatic microsomal CYPs and (or) NADPH-CYP reductase under conditions in which doxorubicin-stimulated NADPH-dependent lipid peroxidation is minimized. In vitro studies were conducted with hepatic microsomes isolated from untreated and phenobarbital-treated male rats. Unlike the positive control carbon tetrachloride, doxorubicin (10 microM) did not stimulate NADPH-dependent lipid peroxidation in microsomal incubations containing EDTA (1.5 mM). Doxorubicin did not cause NADPH-dependent loss of microsomal CYP, heme, or steroid hydroxylation activities selective for CYP2A, CYP2B, CYP2C11, and CYP3A. The positive control 1-aminobenzotriazole caused marked NADPH-dependent decreases in all of these parameters. Neither doxorubicin nor 1-aminobenzotriazole caused NADPH-dependent loss of NADPH-CYP reductase activity, and neither compound altered the immunoreactive protein levels of CYP2B, CYP2C11, CYP3A, and NADPH-CYP reductase. These results indicate that a pharmacologically relevant concentration of doxorubicin does not cause direct mechanism-based inactivation of hepatic microsomal CYPs or NADPH-CYP reductase, suggesting that the ability of doxorubicin to depress hepatic CYP-mediated biotransformation in vivo is due to lipid peroxidation mediated heme destruction, altered heme metabolism, and (or) decreased expression of selected CYP enzymes.

Biotransformation of steroids by the fission yeast Schizosaccharomyces pombe.

The fungal biotransformation of steroids is of applied interest due to the economic importance of such stereo- and regiospecific reactions and also in the context of ergosterol pathway engineering to produce vitamin D and steroidal products. In Schizosaccharomyces pombe no steroid hydroxylation as is found in filamentous fungi was observed, but a cytosolic NAD(H)/NADP(H)-dependent hydroxysteroid dehydrogenase activity was identified. Progesterone was reduced at the delta 4 double bond (in vivo only) as well as at the C-3 and C-20 keto groups. Testosterone and 4-androstene-3,17-dione were interconverted and 5 alpha-pregnane-3,20-dione and 5 beta-pregnane-3,20-dione were reduced to 3-hydroxy products. The reactions were sometimes reversible and showed regio- and stereo specificity. In S. pombe more than one steroid dehydrogenase homologue is likely to occur, as has been observed in Saccharomyces cerevisiae. Our findings indicate that genes encoding soluble proteins should be examined as candidates for actual steroid dehydrogenase activity.

Use of chimeric enzymes and site-directed mutagenesis for identification of three key residues responsible for differences in steroid hydroxylation between canine cytochromes P-450 3A12 and 3A26.

Canine cytochromes P-450 3A12 and 3A26 differ by 22 out of 503 amino acid residues. Chimeric constructs and site-directed mutants were used to identify the residues responsible for the much higher rates of steroid hydroxylation by 3A12. Six initial 3A12/3A26 hybrids were generated using convenient restriction sites, and site-directed mutagenesis was used to restore full 3A12 activity to two of the hybrids. One pair of 3A12/3A26 chimeras indicated that the first four residue differences between 3A12 and 3A26 were at least partially responsible for the differences in progesterone hydroxylation. Conversion in one of the hybrids of the Ile-187 residue found in 3A26 to the Thr in 3A12 conferred 3A12 levels of progesterone 6beta-hydroxylase activity. Analysis of another chimera identified key residues within an internal PstI fragment (codons 331-459) containing six amino acid residue differences. Subsequent site-directed mutagenesis of 3A26 residues Ser-368 and Val-369 to Pro and Ile, respectively, restored the rate of formation of 6beta-hydroxyprogesterone by the hybrid to that of 3A12. The simultaneous conversion of 3A26 residues 187, 368, and 369 to those of 3A12 conferred greater than a third of the progesterone 6beta-hydroxylase activity and all of the testosterone and androstenedione 6beta-hydroxylase activity of 3A12. Addition of the carboxyl terminal 44 3A12 residues to the 3A26 triple mutant doubled progesterone 6beta-hydroxylase activity. This is the first study to use catalytically distinct cytochromes P-450 3A from the same species in the elucidation of structure-function relationships.

Lack of mechanism-based inactivation of rat hepatic microsomal cytochromes P450 by doxorubicin

Administration of the antineoplastic doxorubicin to rodents causes depression of hepatic cytochrome P450 (CYP) dependent biotransformation, an effect that has been partially attributed to the ability of doxorubicin to stimulate microsomal lipid peroxidation. Since doxorubicin can be bioactivated by the CYP/NADPH-CYP reductase system to products that bind covalently to microsomal protein, we hypothesized that doxorubicin functions as a mechanism-based inactivator of hepatic microsomal CYPs and (or) NADPH-CYP reductase under conditions in which doxorubicin-stimulated NADPH-dependent lipid peroxidation is minimized. In vitro studies were conducted with hepatic microsomes isolated from untreated and phenobarbital-treated male rats. Unlike the positive control carbon tetrachloride, doxorubicin (10 microM) did not stimulate NADPH-dependent lipid peroxidation in microsomal incubations containing EDTA (1.5 mM). Doxorubicin did not cause NADPH-dependent loss of microsomal CYP, heme, or steroid hydroxylation activities selective for CYP2A, CYP2B, CYP2C11, and CYP3A. The positive control 1-aminobenzotriazole caused marked NADPH-dependent decreases in all of these parameters. Neither doxorubicin nor 1-aminobenzotriazole caused NADPH-dependent loss of NADPH-CYP reductase activity, and neither compound altered the immunoreactive protein levels of CYP2B, CYP2C11, CYP3A, and NADPH-CYP reductase. These results indicate that a pharmacologically relevant concentration of doxorubicin does not cause direct mechanism-based inactivation of hepatic microsomal CYPs or NADPH-CYP reductase, suggesting that the ability of doxorubicin to depress hepatic CYP-mediated biotransformation in vivo is due to lipid peroxidation mediated heme destruction, altered heme metabolism, and (or) decreased expression of selected CYP enzymes.