Major Form Of Cytochrome P-450 Research Articles

Cytochrome P450 expression system for high-throughput real-time detection of genotoxicity: Application to the study of human CYP1A2 variants

Individual variations in cytochrome P450-mediated metabolism are believed to contribute to individual susceptibility to chemical carcinogenesis. CYP1A2 is one of the major forms of cytochrome P450 involved in drug metabolism and bioactivation of carcinogens. We have applied a recently developed high-throughput Salmonella typhimurium TA1535 system for detection of DNA damaging agents to the study of CYP1A2 polymorphisms. Non-synonymous variants T83M [CYP1A2*9], S212C [CYP1A2*12], S298R [part of CYP1A2*21], G299S [CYP1A2*13], I314V [no allele designation], I386F [CYP1A2*4], C406Y [CYP1A2*5] and R456H [CYP1A2*8] were examined. The cDNAs for each of these variants and the wild-type were co-expressed with human NADPH cytochrome P450 oxidoreductase in the TA1535-based system. The bioactivation capacity of these CYP1A2 variants was investigated using three CYP1A2-dependent pro-mutagens, 1-aminopyrene (1AP), 2-aminoanthracene (2AA), and 2-amino-3-methylimidazo(4,5-f)quinoline (IQ). All CYP1A2 variants except R456H, T83M, and I386F gave positive responses with all three compounds. Variant R456H generated no detectable holoenzyme and no detectable response for any of the compounds; I386F did not bioactivate IQ; T83M did not bioactivate 1AP. Multivariate analysis indicated variant T83M to be substantially altered in catalytic properties when compared with wild-type CYP1A2; variants G299S and I386F are slightly but significantly different. These results corroborate our previous studies, indicating the effectiveness of this new high-throughput system, not only for examining the effect of CYP1A2 polymorphisms on pro-mutagen bioactivation, but also for obtaining insights on CYP1A2 function at the mechanistic level.

Expression of human cytochrome p450 3A4 gene in Schizosaccharomyces pombe

Heterologous expression systems can be utilized to great advantage in the study of cytochrome P450 enzymes. P450 3A4 is one of the major forms of cytochrome P450 found in liver. It is also involved in the metabolism of numerous widely used drugs and xenobiotics. In the present study human liver cytochrome P450 3A4 gene was transferred into the fission yeast Schizosaccharomyces pombe via two different S. pombe expression vectors carrying thiamine repressible promoter — nmt1 (pREP42) and constitutive promoter — adh1 (pART1). Heterologously expressed cytochrome P450 3A4 was detected in the cells grown in minimal (EMM) or rich medium (YEL) containing 0.5% (w/v) glucose. A typical cytochrome P450 peak for 3A4 was observed at 448 nm in microsomal fraction. The presence of heterologous expression of 3A4 form was also determined by SDS-PAGE and it molecular mass was identified as 52 kDa. The enzyme activity was confirmed by HPLC analysis, using testosterone as substrate.

Modulation of cytochrome P450 by inflammation in astrocytes

Activation of systemic host defense mechanisms results in the down-regulation of cytochrome P450 enzymes in the liver. This occurs for various induced and constitutive isoforms of cytochrome P450 in response to cytokines such as IFNs, IL-1, IL-6, and TNF-α, which are produced during infection. Although the levels of cytochrome P450 in brain regions are low, the enzymes are regionally distributed and may play a critical role in the activation or degradation of drugs and chemicals in localized areas. If activation of the immune response in the CNS by LPS modulates the activity of cytochrome P450 forms in the brain, this may alter normal metabolic pathways or contribute to drug or chemical toxicity. This hypothesis was addressed by examining the effect of LPS on a major cytochrome P450 form in isolated astrocytes obtained from newborn rats. These cells were shown to express CYP1A1/2 when induced by dibenz[ a, h]anthracene (DBA) as determined by enzyme activity, immunohistochemistry, and Western blotting. The treatment of these cells with LPS significantly attenuated the activity of these enzymes but had no effect on CYP1A1/2 protein levels as determined by Western blotting. The lack of effect by detoxified LPS indicated the requirement of the lipid A region on LPS to stimulate this response. Pentoxifylline (PNTX) prevented the LPS evoked decrease in CYP1A1/2 activity suggesting that cytokine release was a required component of this effect in astrocytes. These results indicate that stimulation of the immune response by LPS in isolated astrocytes decreases CYP1A1/2 activity. The release of cytokines is implicated in this effect and thought to participate in the functional inhibition of the enzyme as no effect on CYP1A1/2 protein levels was observed.

Regulation of aldosterone biosynthesis: the end of the road?

1. Because aldosterone is the minority hormone of the adrenal cortex, its proper function depends on protective physiological mechanisms. These include a particular site of aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex as well as a complex multifactorial control system, which adapts aldosterone production to acute and chronic changes in body sodium and potassium content, irrespective of pituitary adrenocorticotropic hormone (ACTH) secretion. 2. Over the past decade, an important element of these mechanisms has been identified in the form of the enzyme involved in the final steps of aldosterone biosynthesis. In species such as the human, rat and mouse, the conversion of deoxycorticosterone to aldosterone is catalysed by an isozyme (CYP11B2) of cytochrome P450(11) beta (CYP11B1). The gene encoding this enzyme is expressed only in the zona glomerulosa. Its transcription is enhanced by sodium deficiency and potassium intake, but is suppressed by long-term administration of high doses of ACTH. 3. In contrast, the gene encoding CYP11B1 (i.e. the major (non-aldosterone-producing) type of the enzyme) is expressed mainly in the zona fasciculata and its expression depends on physiological concentrations of ACTH. 4. In other animal species (cattle, pig, sheep), the major forms of cytochrome P450(11) beta have an inherent aldosterone-synthesizing activity, which is, however, selectively suppressed in mitochondria of zona fasciculata cells. 5. The elucidation of the mechanisms involved in this suppression or of those mediating alterations in CYP11B2 expression in response to physiological stimuli may be important areas of future research on the regulation of aldosterone biosynthesis.

Interlaboratory comparison of the assessment of P450 activities in human hepatic microsomal samples

1. Although the importance of in vitro technology in supporting drug development is widely accepted, there is no real consensus about which approaches should be taken, which substrates should be used, or on the reliability and application of in vitro data. Consequently, as part of a collaborative project to characterize human liver with respect to the major forms of cytochrome P450, an interlaboratory comparison of the analysis of samples for form-specific activities was undertaken. 2. Microsomal fractions were isolated from five different human liver samples taken from the liver bank maintained at the Royal Postgraduate Medical School (RPMS). Aliquots from the five samples were sent to the 11 collaborating laboratories for characterization using their in-house, form-specific assays for cytochrome P450 activities. Although each laboratory assayed protein concentration, total cytochrome P450 content and enzyme activities were calculated using the protein estimation generated by RPMS to eliminate this possible source of variability. 3. With the exception of one laboratory, all estimates of protein concentration were similar (coefficient of variation, CoV, 9-13%) and the rank-order of the five samples was consistent across the laboratories. There was greater variability in the estimates of total cytochrome P450 content (CoV 28-43%), although again rank order of the samples across laboratories was fairly consistent. 4. The various laboratories used a number of different probe substrates, together with a range of conditions (substrate concentration, time of incubation, amount of protein), to assay for activity of CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4. However, apart from the occasional outlier, the five samples were ranked for activity of all these forms of cytochrome P450 with a high degree of consistency by the various laboratories and the choice of substrate had no appreciable effect on the ranking of the samples. 5. While this interlaboratory comparison has shown that greater consistency in the approach to in vivo determinationof drug-metabolizing activity is desirable,therewas little indication that any particular approach or substrate was superior to the others.

Expression of CYP3A7, a human fetus-specific cytochrome P450, in cultured cells and in the hepatocytes of p53-knockout mice

CYP3A7 is a major form of cytochrome P450 in human fetal livers. To elucidate toxicological significance of CYP3A7 in fetal livers, CYP3A 7 cDNA was introduced into Chinese hamster lung (CHL) cells. Transformants carrying the CYP3A 7 gene were more sensitive to mycotoxins than parental CHL cells. In additional studies, we established a hepatocyte cell line from CYP3A7-transgenic/p53-knockout mice. In hepatocyte cells from CYP3A7-transgenic/p53-knockout mice, CYP3A7 mRNA was expressed and the catalytic activity of CYP3A7 protein was detected. The cells are expected to show cytotoxicity to mycotoxins and teratogens. These cell lines provide a valuable panel for studying the fetal toxicities of chemicals in humans.

Identification and characterization of lysosomal enzymes involved in the proteolysis of phenobarbital-inducible cytochrome P450.

We have studied lysosomal proteases capable of degrading a major form of cytochrome P450 (CYP2B1) which was purified from the liver microsomes of phenobarbital-treated rats. After incubation of CYP2B1 with extracts of triton-filled lysosomes (tritosomes), its proteolysis was measured by a quantitative immunoblot procedure. A CYP2B1 protein band with an apparent molecular mass of 53 kilodaltons (kDa) was degraded to 42-, 38-, and 29-kDa polypeptides after a short period of incubation. These proteolytic fragments disappeared on prolonged incubation. One milligram of tritosomal protein contained enough activity to hydrolyze approximately 67.3 micrograms CYP2B1 protein/min at pH 4.5. Approximately 85% of the hydrolyzing activity was localized in a soluble fraction of the tritosomes. The degradation of CYP2B1 was effectively inhibited by pepstatin A, an aspartic protease inhibitor, but not by phenylmethanesulfonyl fluoride (PMSF), o-phenanthroline and leupeptin. CYP2B1-hydrolyzing activity was coeluted with cathepsin D when the soluble fraction was chromatographed by means of Ultrogel Ac44 gel filtration. Cathepsin D, purified from rat livers, was able to degrade CYP2B1 at pH 4.5 which corresponds to the intralysosomal pH. These results indicate that cathepsin D is responsible for the major CYP2B1-hydrolyzing enzyme activity in lysosomes.

ヒト胎児肝チトクロームP-450.P-450HFLaの性質と特徴

One of the major forms of cytochrome P-450, named P-450 HFLa, of human fetal livers was purified and characterized. The cytochrome P-450 preparation had an apparent molecular weight of 51,500 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-Terminal amino acid sequence of P-450 HFLa was similar but not identical to that of P-450NF involved in nifedipine oxidation in adult human livers. P-450 HFLa catalyzed 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate (DHEA-sulfate) in a reconstituted system. The concentration of P-450 HFLa in liver homogenates from human fetuses highly correlated with the activity of DHEA-sulfate 16 alpha-hydroxylase. Furthermore, anti-P-450 HFLa antibodies inhibited the 16 alpha-hydroxylation. P-450 HFLa was also found to catalyze the mutagenic activation of aflatoxin B1 (AFB1) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). The antibodies to P-450 HFLa inhibited efficiently the mutagen-producing activities from AFB1 and IQ in human fetal livers. The nucleotide and the deduced amino acid sequences of lambda HFL33 containing the entire coding region for a form of cytochrome P-450 related to P-450 HFLa, were highly similar to but clearly distinct from those of NF25 and HLp complementary deoxyribonucleic acids. The oligonucleotide probes specific to the coding and 3'-noncoding region of lambda HFL 33 (oli-HFL and oli-HFL3', respectively) gave hybridizable bands with ribonucleic acid (RNA) from fetal but not adult livers. In contrast, an oligonucleotide probe specific to the coding region of NF 25 and HLp (oli-NF) gave hybridizable bands with RNA from only adult but not fetal livers.

5-2) MOLECULAR TOXICOLOGY ON CYTOCHROME P-450 IN HUMAN FETAL LIVERS

Among mammalian species, humans and monkeys are sole species which possess cytochrome P-450 in the liver in fetal life. Cytochrome P-450 catalyzes oxidations of a wide variety of compounds including steroids, drugs and toxicants. Thus, the purpose of this study was to examine the toxicological significance of human fetal cytochrome P-450. Firstly, we found that human fetal livers were capable of activating numbers of carcinogenic compounds. Secondly, the capacity of human fetal cytochrome P-450 to activate mutagens and carcinogens was confirmed using purified forms of cytochrome P-450. For this, we purified four forms of cytochrome P-450 from human fetal livers. Lastly, we could isolate a cDNA clone coding for P-450HFLa which is one of the major forms of cytochrome P-450 in human fetal livers. Utilizing synthesized cD-NAs as probes, we found that this form of cytochrome was expressed specifically in fetuses.

Immunohistochemical detection of cytochrome P450 isoenzymes in cultured human epidermal cells.

We used specific monoclonal antibodies (MAb) to human cytochrome P450 isoenzymes to determine the presence of these proteins in human epidermal cells. Two MAb (P450-5 and P450-8) recognize major forms of hepatic cytochrome P450 involved in biotransformation of xenobiotics. A third MAb, to cytochrome P450-9, is not fully characterized. The proteins were determined by the indirect immunoperoxidase technique after fixation with methanol and acetone. Biopsy materials for cultured keratinocytes, i.e., foreskin and hair follicles, contained the two major forms of cytochrome P450. In cultured keratinocytes derived from hair follicles the proteins were undetectable, whereas the keratinocytes derived from foreskin continued to express the two major forms of hepatic cytochrome P450. Cultured human fibroblasts and a human keratinocyte cell line (SVK14) showed staining similar to that of the foreskin keratinocytes. Cytochrome P450-9 was detectable only in human hepatocytes. The results indicate that, under the culture conditions applied, cultured human foreskin cells and the cell line SVK14 continue to express specific cytochrome P450 isoenzymes in culture, in contrast to hair follicle keratinocytes.

Isolation of cytochrome P450 from hepatopancreas microsomes of the spiny lobster, Panulirus argus, and determination of catalytic activity with NADPH cytochrome P450 reductase from vertebrate liver

One major form of cytochrome P450 has been isolated from the hepatopancreas of untreated spiny lobsters, Panulirus argus. This form, termed here D1, was purified to a specific content of 12.1 ± 1.8 nmol/mg protein. Two minor forms, termed D2 and D3 were partially purified to 4.6 ± 1.6 and 2.3 ± 0.2 nmol P450/mg protein, respectively. No NADPH-cytochrome P450 reductase activity was detected in spiny lobster hepatopancreas microsomes and no purification of spiny lobster reductase was achieved in this study. Very low NADPH-cytochrome c reductase activity was found in hepatopancreas microsomes and also in cytosol. Indirect evidence suggested that proteolysis of spiny lobster P450 reductase during the preparation of hepatopancreas microsomes may in part account for the lack of detectable monooxygenase activity in hepatopancreas microsomes. The catalytic activities of the D1 or D2 forms of spiny lobster P450 were measured by mixing D1 or D2 with NADPH-cytochrome P450 reductase isolated from pig or rat liver microsomes. D2 was very efficient in demethylating benzphetamine, with a turnover number of 122 per minute, and D1 was an efficient catalyst of progesterone 16 α-hydroxylation, with a turnover number of 43 per minute. Other good substrates for D1 and D2 forms were aminopyrine, testosterone, benzo(a)pyrene, and 7-ethoxycoumarin. Little activity was found with methyl-, ethyl-, pentyl-, or benzyl-phenoxazone ethers as substrates. The profile of metabolites formed by D1 or D2 with benzo(a)pyrene as substrate were more similar to those formed with uninduced rat liver microsomes than to those formed by liver microsomes from uninduced flatfish species.

Purification and Characterization of Cytochromes P-450 from Liver Microsomes of 3-Methylcholanthrene-Treated Crab-Eating Monkeys1

Two forms of cytochrome P-450 (P-450MC1 and P-450MC2) were purified from liver microsomes of crab-eating monkeys (Macaca irus) treated with 3-methylcholanthrene (MC). Monkey P-450MC1 preparation had a specific content of 14.0 nmol/mg protein and showed a main protein band with a minimum molecular weight of 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monkey P-450MC2 preparation had a specific content of 12.1 nmol/mg protein and a minimum molecular weight of 54,000. The carbon monoxide-reduced difference spectral peaks of monkey P-450MC1 and P-450MC2 were at 448 and 447 nm, respectively. In the reconstituted system, monkey P-450MC2 had high activities for benzo[a]pyrene 3-hydroxylation and 7-ethoxycoumarin O-deethylation. Monkey P-450MC1 had low activities toward these two substrates and a high activity for benzphetamine N-demethylation. Monkey P-450MC1 and P-450MC2 were detected by immunoblotting using an antibody prepared against rat cytochrome P-450c, which is a major form of cytochrome P-450 in liver microsomes of MC-treated rats. These results suggested that the molecular properties of cytochrome P-450 in liver microsomes of crab-eating monkeys treated with MC are similar to those in rats.

Induction of cytochrome P-450 isozymes by chromanamine derivatives in rat liver.

1. Pretreatment of rats with 6-(3-picolyl)amino-2,2,5,8-tetramethylchromane (PATC) for 7 days resulted in a significant increase in the activities of benzphetamine N-demethylase, p-nitroanisole O-demethylase and aniline hydroxylase in liver microsomes prepared 24 h after the last treatment. 2. Analysis by Western blot showed that PATC induces cytochrome P-450 b, P-450 c and P-450 d, which are the major forms of cytochrome P-450 in liver microsomes of rats when pretreated with phenobarbital and 3-methylcholanthrene. 3. Exposure of liver sections to the antibodies to cytochrome P-450 b and P-450 c resulted in intense immunostaining within the centrilobular regions, but produced staining of considerably weaker intensity in the perilobular region. Semiquantitative immunochemical analysis, by image analyser, of cytochrome P-450 b and P-450 c showed that centrilobular hepatocytes were stained more intensively than perilobular hepatocytes. 4. These results indicate that PATC induces cytochromes P-450 b and P-450 c, in the centrilobular hepatocytes to a greater degree than those in the perilobular hepatocytes. 5. Co-administration of PATC with pentobarbital caused a significant increase in pentobarbital sleeping time. Furthermore, PATC was found to cause a decrease in the activity of benzphetamine N-demethylase in liver microsomes prepared 30 min after treatment with the drug.

Effect of nutritional imbalances on cytochrome P-450 isozymes in rat liver

Male Sprague-Dawley rats were fed for six weeks either a control diet containing 22% casein (C) and 5% fat (F) or a low-protein diet (6% C, 5% F) or high-lipid diet (30% C, 30% F). A group of rats received a control diet containing 50 ppm of Phenoclor DP6. Three major forms of cytochrome P-450, UT 50, BP 3a and MC 2 were purified from livers of DP6-fed rats and only two forms, UT 50 and PB 3a, were purified from control and dietary groups. The amino acid composition and the catalytic activities towards all substrates tested were only significantly modified in the purified UT 50 P-450 isozyme from rats fed the low-protein diet. The N-terminal sequence analysis shows that cytochrome P-450 UT 50 (from control group) and UT 501 (from low-protein group) are two distinct proteins.

Multiplicity of steroid-inducible cytochromes P-450 in rat liver microsomes

Highly purified preparations of steroid-inducible cytochromes P-450 have been isolated from liver microsomes of adult male Sprague-Dawley rats treated with pregnenolone-16α-carbonitrile (PCN), phenobarbital (PB), or triacetyloleandomycin (TAO). The interrelationships among these preparations as well as their relationship to the major forms of cytochrome P-450 of this gene family identified in other laboratories have been evaluated by amino-terminal sequence analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, chromatography on DEAE-Sephacel, and Ouchterlony double-immunodiffusion. The results indicate that a cytochrome P-450 previously isolated in this laboratory and referred to as PCNa ( P. E. Graves, L. S. Kaminsky, and J. Halpert (1987), Biochemistry, 26, 3887–3894) is the major cytochrome P-450 induced in rats by TAO, and corresponds to P-450p ( K. A. Hostetler, S. A. Wrighton, P. Kremers, and P. S. Guzelian (1987), Biochem. J., 245, 27–33) and to P450PCN1 ( F. J. Gonzalez, B. J. Song, and J. P. Hardwick (1986), Mol. Cell. Biol., 6, 2969–2976). A second previously isolated cytochrome P-450 termed PCNb in this laboratory appears to be identical to PB PCN-E ( F. P. Guengerich, G. A. Dannan, S. T. Wright, M. V. Martin, and L. S. Kaminsky (1982) Biochemistry, 21, 6019–6030). PCNb as well as a third cytochrome P-450 termed PCNc isolated from PB-treated rats both correspond in amino-terminal sequence to the putative protein product of the pP450 PCN2 cDNA clone of Gonzalez et al. These results document at the protein level the multiplicity of steroid-inducible rat liver cytochromes P-450.

Induction of hepatic drug-metabolizing enzymes in rats treated with 1-nitropyrene

The inducing effects of 1-nitropyrene (1-NP) on the microsomal cytochrome P-450 system were studied in rats. Intraperitoneal administration of 1-NP led to increases in cytochrome P-450 content and aryl hydrocarbon hydroxylase, ethoxycoumarin, and ethoxyresorufin-O-deethylase activities. These increases were dose dependent. Cytochrome b5 content and aminopyrine and p-nitroanisole demethylase activities were not affected by treatment of rats with 1-NP. Substrate specificity, sensitivity to mixed-function oxidase inhibitors, and electrophoretic pattern of 1-NP-induced cytochrome(s) P-450 were compared to the major forms of cytochrome P-450 induced by phenobarbital and methylcholanthrene. Furthermore microsomes from 1-NP-induced rats showed greater ability to metabolize the chemical as compared with those from control animals; this result indicates that 1-NP induces a form(s) of cytochrome P-450 especially effective in the metabolism of the substance itself.

Purification and NH 2-terminal sequence of cytochrome P-450 from kidney microsomes of untreated male rats

The major form of cytochrome P-450, P-450K-5, was purified from kidney microsomes of untreated male rats with high-performance liquid chromatography with anion-exchange and hydroxylapatite columns. The monomeric molecular weight of P-450K-5 was 52000 on SDS-polyacrylamide gel electrophoresis and the CO-reduced absorption maximum was at 452 nm. P-450K-5 catalyzed the omega- and (omega-1)-hydroxylation of lauric acid, but was inefficient in the N-demethylation of benzphetamine and the O-dealkylation of 7-ethoxycoumarine. The NH2-terminal sequence of P-450K-5 was quite different from cytochrome P-450s purified from rat hepatic microsomes.

Purification and characterization of a form of cytochrome P-450 with high specificity for aflatoxin B 1 from 3-methylcholanthrene treated hamster liver

A form of cytochrome P-450 highly active in inducing mutagenicity of aflatoxin B1 was purified to a specific content of 15.1 nmol/mg of protein from 3-methylcholanthrene-treated hamster liver. This species of cytochrome P-450, having its absorption maximum at 448.5 nm in carbon monoxide-complex of reduced form and low spin ferric ion, is of molecular weight of 56,000 and distinctly different in physicochemical and catalytic properties from major forms of cytochrome P-450 purified from phenobarbital- or 3-methylcholanthrene-treated rat liver. In the induction of aflatoxin B1 mutagenicity, this hamster cytochrome P-450 is 50 times more potent than those from rat liver.

Drug interactions with macrolide antibiotics: specificity of pseudo-suicide inhibition and induction of cytochrome P-450.

Macrolide antibiotics like Erythromycin and Tri-acetyl oleandomycin (TAO) are metabolized to nitrosoderivatives which cause inactivation of Cytochrome P-450 by forming stable complex with the Iron of the hemoporphyrin. Several derivatives of erythromycin having lost their cladinose moiety are stronger inducer of liver cytochrome P-450 itself. The major form of cytochrome P-450 induced by all these macrolides in rat liver electrophoretically and immunologically indistinguishable from the major form induced by pregnenolone 16 alpha carbonitrile (PCN). This form is particularly able to metabolize macrolide and to lead to the corresponding 456 nm absorbing cytochrome P-450 complexes in vivo and in vitro.

Induction and catalytic activities of cytochromes P-450 b/ e and P-450 c in the liver microsomes of neonatal rats

1. 1. The activities of cytochrome P-450-dependent monooxygenases has been investigated in the liver microsomes of newborn rats (3–16 days after birth) induced with PB or 3-MC. 2. 2. It has been shown that the induction by PB and 3-MC results in the increase of both the total amount of cytochrome P-450 as determined by the CO-reduced spectrum and the amount of induced forms P-450 b/ e † † In designations of the major forms of cytochrome P-450 induced with PB and 3-MC we have used those of Thomas et al. (1981). Our cytochrome P-450 b/ e possessed the following properties: λ max of the CO-reduced spectrum is 450 nm, M r is 52,000, BPh- N-demethylation rate in the reconstituted system is 70 nmol formaldehyde min −1 nmol P-450 b/ e −1, its contents in PB-microsomes is 40–50% of the general one measured by the CO-reduced state. It is evident that anti- P-450 b used by us reacts with both P-450 b and P-450 e . P-450 c has the following characteristics: λ max of the CO-reduced spectrum is 447.5 nm, M r is 56,000, the rates of BP-hydroxylation and 7-ER-deethylation in reconstituted system are 17 and 7 nmol of product min −1 nmol P-450 c −1, respectively, the contents in 3-MC-microsomes is 60–70%. and P-450 c † respectively. 3. 3. In the course of induction of the specific forms of cytochrome P-450 BP-hydroxylase and 7-ER- O-deethylase activities increased at 3-MC-induction, while BPh- N-demethylase and BP-hydroxylase increased at PB-induction. 4. 4. Analysis of inhibition of monooxygenase reactions with antibodies has showed that only P-450 c was involved in metabolism of BP and 7-ER. Participation of P-450 b/ e in BPh N-demethylation was notably lower in the neonates in comparison to the adult rats. 5. 5. In the one-week-old rats induced with 3-MC a considerable rate of BP hydroxylation and 7-ER- O-deethylation (2–4.5 nmol of product min −1 mg −1) has been observed despite a small amount of P-450 (0.02–0.1 nmol/mg of protein). This fact shows the higher catalytic activity of this cytochrome P-450 in the neonates compared to similar characteristics of P-450 c in the 3-MC-induced microsomes. 6. 6. Metabolism of BP in the PB-microsomes of the neonatal rats was inhibited neither by anti- P-450 b/ e nor anti- P-450 c in contrast to the adults, where this reaction was inhibited by antibodies against P-450 b/ e .