Evidence for complexation of P-450 IIC6 by an orphenadrine metabolite

Removal of the orphenadrine metabolite from its complex with rat liver P-450 IIB1 is associated with a discrepancy in the reactivation of IIB1 activity. Two possible explanations are that either (1) NADPH-P-450-reductase is inaccessible to the restored IIB1, or (2) complexation of other P-450s may occur. Exogenous P-450 reductase increased all pathways of steroid hydroxylation (1.9 to 3.6-fold) but did not enhance reactivation of IIB1-dependent steroid 16 beta-hydroxylation. Instead, P-450 IIC6-dependent progesterone 21-hydroxylase activity was increased after dissociation to 122% of control. IIC6 activity was also inhibited in vitro in microsomes from phenobarbital-induced rats (ki = 151 microM). Thus, orphenadrine appears to complex P-450 IIC6 as well as IIB1 in rat liver.     

Purification and characterization of the rat liver microsomal cytochrome P-450 involved in the 4-hydroxylation of debrisoquine, a prototype for genetic variation in oxidative drug metabolism

Genetic polymorphism in oxidative drug metabolism is perhaps best exemplified in the case of debrisoquine 4-hydroxylase activity, where the incidence of deficient metabolism ranges from 1% to 30% in various populations and this defect is also linked to an impaired ability to metabolize a number of other drugs effectively. Sprague-Dawley (SD) rats possess this activity, but females of the DA strain do not, although total cytochrome P-450 (P-450) levels are similar. We have purified, by using debrisoquine 4-hydroxylase activity as an assay, a minor P-450 to electrophoretic homogeneity from male SD rats and designate this as P-450UT-H. P-450UT-H differs from eight other purified rat liver P-450s as judged by peptide mapping and immunochemical analysis and thus appears to be isozymic with these other P-450s. P-450UT-H exhibited considerably more debrisoquine 4-hydroxylase activity than any of the other purified P-450s and, on a total P-450 basis, more than total microsomal P-450. Antibodies raised against P-450UT-H specifically recognized P-450UT-H and inhibited more than 90% of the debrisoquine hydroxylase activity present in SD rat liver microsomes. The level of P-450UT-H in SD rat liver microsomes accounted for less than 10% of the total P-450, as judged by immunochemical quantitation. These assays also indicated that the level of P-450UT-H in female DA rat liver microsomes is only about 5% of that in male or female SD rat liver microsomes, consonant with the view that deficiency of this form of P-450 is responsible for the defective debrisoquine 4-hydroxylase activity in the former animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunochemical similarity of P-450 HFLa, a form of cytochrome P-450 in human fetal livers, to a form of rat liver cytochrome P-450 inducible by macrolide antibiotics

A protein immunochemically related to P-450 HFLa, a form of cytochrome P-450 purified from human fetal livers, was detected in rat liver microsomes. The content of the immunoreactive protein in rat liver microsomes was increased by treatments with phenobarbital, pregnenolone 16 alpha-carbonitrile (PCN), erythromycin, erythromycin estolate, and oleandomycin but not with 3-methylcholanthrene, imidazole, ethanol, isosafrole, josamycin, midecamycin, or miocamycin. The activity of erythromycin N-demethylase correlated with the content of the immunoreactive protein in rat liver microsomes (r = 0.72). In addition, anti-P-450 HFLa IgG inhibited erythromycin N-demethylase in liver microsomes from erythromycin- or oleandomycin-pretreated rats. Furthermore, the content of the immunoreactive protein highly correlated with that of P-450 PB-1, which is distinct from Waxman's terminology, and is one of the forms of PCN-inducible cytochrome P-450s (r = 0.95). From these results and the results reported so far, it seems possible that P-450 HFLa is one of the forms of cytochrome P-450 inducible by glucocorticoids.     

Oxidation of uroporphyrinogen by methylcholanthrene-induced cytochrome P-450. Essential role of cytochrome P-450d.

We have previously shown that uroporphyrinogen is oxidized to uroporphyrin by microsomes (microsomal fractions) from 3-methylcholanthrene-pretreated chick embryo liver [Sinclair, Lambrecht & Sinclair (1987) Biochem. Biophys. Res. Commun. 146, 1324-1329]. We report here that a specific antibody to chick liver methylcholanthrene-induced cytochrome P-450 (P-450) inhibited both uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in chick-embryo liver microsomes. 3-Methylcholanthrene-pretreatment of rats and mice markedly increased uroporphyrinogen oxidation in hepatic microsomes as well as P-450-mediated ethoxyresorufin de-ethylation. In rodent microsomes, uroporphyrinogen oxidation required the addition of NADPH, whereas chick liver microsomes required both NADPH and 3,3',4,4'-tetrachlorobiphenyl. Treatment of rats with methylcholanthrene, hexachlorobenzene and o-aminoazotoluene increased uroporphyrinogen oxidation and P-450d, whereas phenobarbital did not increase either. The contribution of hepatic P-450c and P-450d to uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in methylcholanthrene-induced microsomes was assessed by using specific antibodies to P-450c and P-450d. Uroporphyrinogen oxidation by methylcholanthrene-induced rat liver microsomes was inhibited up to 75% by specific antibodies to P-450d, but not by specific antibodies to P-450c. In contrast, ethoxyresorufin de-ethylation was inhibited only 20% by anti-P450d but 70% by anti-P450c. Methylcholanthrene-induced kidney microsomes which contain P-450c but non P-450d did not oxidize uroporphyrinogen. These data indicate that hepatic P-450d catalyses uroporphyrinogen oxidation. We suggest that the P-450d-catalysed oxidation of uroporphyrinogen has a role in the uroporphyria caused by hexachlorobenzene and other compounds.     

Constitutive testosterone 6 beta-hydroxylase in rat liver

The cytochrome P-450 that was purified from hepatic microsomes of male rats treated with phenobarbital and designated P450 PB-1 (Funae and Imaoka (1985) Biochim. Biophys. Acta 842, 119-132) had high testosterone 6 beta-hydroxylation activity (turnover rate, 13.5 nmol of product/min/nmol of P-450) in a reconstituted system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5, and a 1:1 mixture of lecithin and phosphatidylserine in the presence of sodium cholate. In ordinary conditions in the reconstituted system with cytochrome P-450, reductase, and dilauroylphosphatidylcholine, P450 PB-1 had little 6 beta-hydroxylase activity. The catalytic activities toward testosterone of two major constitutive forms, P450 UT-2 and P450 UT-5, were not affected by cytochrome b5, phospholipid, or sodium cholate. P450 PB-1 in rat liver microsomes was assayed by immunoblotting with specific antibody to P450 PB-1. P450 PB-1 accounted for 24.4 +/- 5.6% (mean +/- SD) of the total spectrally-measured cytochrome P-450 in hepatic microsomes of untreated adult male rats, and was not found in untreated adult female rats. P450 PB-1 was induced twofold with phenobarbital in male rats. P450 PB-1 was purified from untreated male rats and identified as P450 PB-1 from phenobarbital-treated rats by its NH2-terminal sequence, peptide mapping, and immunochemistry. These results showed that P450 PB-1 is a constitutive male-specific form in rat liver. There was a good correlation (r = 0.925) between the P450 PB-1 level and testosterone 6 beta-hydroxylase activity in rat liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Incorporation of heme to microsomal cytochrome P-450 in the absence of protein biosynthesis

Determination of the heme and protein portions of phenobarbital (PB)-inducible and 3-methylcholanthrene inducible forms of cytochrome P-450, P-450(PB-1), and P-450(MC-1), in the liver microsomes of drug-treated animals indicated the presence of 20-30% of apo-cytochrome P-450 in both cases. Inhibition of protein synthesis by cycloheximide injection to the rats did not significantly inhibit the incorporation of delta-amino[14C]levulinic acid (ALA) into the heme of P-450(PB-1) or P-450(MC-1) in the liver, indicating that the heme incorporation into microsomal cytochrome P-450 is not tightly coupled with the synthesis of the apo-cytochrome. When heme-labeled cytosol prepared from [14C]ALA-injected rats was incubated with non-radioactive microsomes in vitro, a significant amount of labeled heme was incorporated into microsomal P-450(PB-1), whereas the incorporation into P-450(MC-1) was much less. The in vitro transfer of heme from cytosol to microsome-bound cytochrome P-450 was stimulated by the addition of an NADPH-generating system to the incubation mixtures, and inhibited when the microsomes were solubilized with sodium cholate and Emulgen-913. Although the in vitro incubation of heme-labeled microsomes with non-radioactive cytosol resulted in some release of labeled heme from the microsomes, no reversible transfer of heme between cytochrome P-450 molecules bound to separate microsomal vesicles was detected when heme-labeled microsomes were incubated with non-radioactive microsomes in the presence and absence of cytosol.     

Comparison of the formation of benzo[a]pyrene diolepoxide-DNA adducts in vitro by rat and human microsomes: evidence for the involvement of P-450IA1 and P-450IA2

The involvement of cytochrome P-450 isozymes in the activation of benzo[a]pyrene (BP) by human placental and liver microsomes was studied in vitro using monoclonal antibodies (Mab) toward the major 3-methylcholanthrene (MC)-inducible and phenobarbital-inductible rat liver P-450 isozymes (Mab 1-7-1 and Mab 2-66-3, respectively). Microsomes from human placenta and liver and rat liver were incubated with BP and DNA, and BP-diolepoxide-DNA (BPDE-DNA) adducts were measured by synchronous fluorescence spectrophotometry (SFS). The only BP metabolite giving the same fluorescence peak as chemically modified BPDE-DNA was BP-7,8-dihydrodiol. Five (smokers) out of 29 human placentas (smokers and nonsmokers), and five out of nine human livers were able to metabolically activate BP to BPDE-DNA adducts in this system. The Mab 1-7-1 totally inhibited the formation of BPDE-DNA adducts in placental microsomal incubations. Inhibition using rat or human liver microsomes was 50-60% and about 90%, respectively. The Mab 2-66-3 had no effect in any of the microsome types. Adduct formation was inhibited more strongly and at lower concentrations of Mab 1-7-1 compared with the inhibition of AHH activity. This study is a clear indication of the major role of P-450IA1 (P-450c) in human placenta and probably P-450IA2 (P-450d) in human liver in BP activation, while other isozymes also take part in the activation in rat liver. Furthermore, this clearly indicates that AHH activity and BP activation are not necessarily associated.     

Aflatoxin B1 metabolism by 3-methylcholanthrene-induced hamster hepatic cytochrome P-450s

We have studied the activation of aflatoxin B1 by hamster liver microsomes and purified hamster cytochrome P-450 isozymes using a umu mutagen test. The hamster liver microsomes or S-9 fractions were much more active than rat liver microsomes or S-9 fractions in the activation of umu gene expression by aflatoxin B1 metabolites. 3-Methyl-cholanthrene treatment increased aflatoxin B1 activation by hamster liver microsomes. Two major 3-methylcholanthrene-inducible cytochrome P-450 isozymes, P-450 MC1 (IIA) and P-450 MC4 (IA2), were purified from 3-methylcholanthrene-treated hamster liver microsomes, and the metabolism of aflatoxin B1 by these two cytochromes was studied. In the reconstituted enzyme system, both P-450 MC1 and P-450 MC4 were highly active in the activation of aflatoxin B1, and antibodies against these P-450s specifically inhibited these activities. Antibody against P-450 MC1 inhibited the activation of aflatoxin B1 by 20% in the presence of 3-methyl-cholanthrene-treated hamster liver microsomes. In contrast, antibody against P-450 MC4 stimulated the activity by 175%. These results indicated that hamster P-450 MC1 might convert aflatoxin B1 to more toxic metabolite(s), whereas P-450 MC4 might convert aflatoxin B1 to less toxic metabolite(s), than aflatoxin B1 in liver microsomes. The metabolite(s) produced by both hamster cytochrome P-450 MC1 and MC4 were genotoxic in the umu mutagen test.     

Expression and function of three cytochrome P-450 isozymes in rat extrahepatic tissues

Western blots using a polyclonal and a monoclonal antibody raised against rat liver cytochrome P-450b indicate tissue-specific expression of low levels of cytochrome P-450's b and e. P-450b and P-450e were expressed very selectively in, respectively, lung and adrenal microsomes of untreated rats but neither isozyme was detected in the corresponding kidney or small intestine microsomes. The regioselectivity of microsomal metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) as well as the sensitivity to inhibition by anti P-450b/e IgG established that low levels of "b-like" P-450's are functional in lung and adrenal microsomes from uninduced rats, but not in microsomes from the kidney or small intestine. Functional P-450c was also detected at low levels in liver, lung, kidney, and adrenals of untreated rats. Among the extrahepatic tissues examined, DMBA metabolism was the highest in rat adrenal microsomes. However, only 30% of this activity was due to P-450's b, e, or c. Phenobarbital (PB) treatment of rats increased microsomal DMBA metabolism in all extrahepatic tissues examined. The selectivity of this increase for 12-methyl hydroxylation of DMBA and the near complete inhibition by anti-P-450b/e are consistent with induction of P-450e even though P-450b was preferentially induced in each of the extrahepatic tissues examined. The levels of expression of P-450b were increased by PB in all sets of adrenal, lung, and intestinal microsomes and in three out of six sets of kidney microsomes. The levels of P-450e were also increased by PB in all sets of adrenal microsomes. Following PB treatment, P-450e became immunoquantifiable (greater than 2 pmol/mg protein) in three of six sets of lung and kidney microsomes but remained below detection in all sets of intestinal microsomes. Based on the activity of purified P-450e, undetectable levels (less than 1 pmol/mg protein) could account for increased DMBA metabolism in this tissue. The high constitutive level of P-450b in the lung (approximately 40 pmol/mg), was remarkably inactive in DMBA metabolism and was only slightly increased by PB treatment (50%). In contrast, PB treatment caused a 2.5- to 10-fold increase in 12-methyl hydroxylation of DMBA that was highly sensitive to anti-P-450b/e. A protein comigrating with P-450e was well above detection (6-7 pmol/mg) in two of six preparations of lung microsomes that showed highest induction of this activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of rat and human liver microsomal cytochrome P-450 forms involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug metabolism

The metabolism of the dihydropyridine calcium antagonist and vasodilator nifedipine has been reported to exhibit polymorphism among individual humans (Kleinbloesem, C. H., van Brummelen, P., Faber, H., Danhof, M., Vermeulen, N. P. E., and Breimer, D.D. (1984) Biochem. Pharmacol. 33, 3721-3724). Nifedipine oxidation has been shown to be catalyzed by cytochrome P-450 (P-450) enzymes. Reconstitution, immunoinhibition, and induction studies with rat liver indicated that the forms designated P-450UT-A and P-450PCN-E are the major contributors to microsomal nifedipine oxidation. The P-450 which oxidizes nifedipine (P-450NF) was purified to electrophoretic homogeneity from several human liver samples. Antibodies raised to P-450NF were highly specific as judged by immunoblotting analysis and inhibited greater than 90% of the nifedipine oxidase activity in human liver microsomes. A monoclonal antibody raised to the human P-450 preparation reacted with both human P-450NF and rat P-450PCN-E. Immunoblotting analysis of 39 human liver microsomal samples using anti-P-450NF antibodies revealed the same 52,000-dalton polypeptide, corresponding to P-450NF, with only one of the microsomal samples showing an additional immunoreactive protein. The level of nifedipine oxidase activity was highly correlated with the amount of P-450NF thus detected using either polyclonal (r = 0.78) or monoclonal (r = 0.65) antibodies, suggesting that the amount of the P-450NF polypeptide may be a major factor in influencing the level of catalytic activity in humans as well as rats. Cytochrome b5 enhanced the catalytic activity of reconstituted P-450NF, and anti-cytochrome b5 inhibited nifedipine oxidase activity in human liver microsomes. P-450NF also appears to be a major contributor to human liver microsomal aldrin epoxidation, d-benzphetamine N-demethylation, 17 beta-estradiol 2- and 4-hydroxylation, and testosterone 6 beta-hydroxylation, the major pathway for oxidation of this androgen in human liver microsomes.     

The characteristics of the microsomal hydroxylation of tolbutamide

The in vitro metabolism of tolbutamide to the hydroxymethyl derivative was studied using hepatic microsomal homogenates. The hydroxymethyl metabolite was quantitated by HPLC. The hepatic microsomal hydroxylase was completely inhibited by carbon monoxide and was NADPH dependent. Metyrapone, alpha-naphthoflavone, phenelzine, mercuric chloride, and nitrogen significantly inhibited the reaction indicating the involvement of the cytochrome P-450 monooxygenase. Species variation showed that the order of hepatic microsomal activity was rat greater than rabbit much greater than guinea pig much greater than mouse and hamster. The reaction increased with time up to 40 min and followed Michaelis-Menten kinetics in rat liver microsomes with apparent Km and Vmax values of 224.4 microM and 359.9 pmol.mg-1.min-1, respectively. The reaction was induced by phenobarbital but was depressed after pretreatment with 3-methylcholanthrene and isosafrole. However, expression of the hydroxylase activity per nanomoles of cytochrome P-450 showed that the activity was much higher in liver microsomes of isosafrole pretreated rats. These results indicate the involvement of different isozymes of cytochrome P-450 in the microsomal hydroxylation of tolbutamide.     

Differential expression and function of three closely related phenobarbital-inducible cytochrome P-450 isozymes in untreated rat liver

The levels of expression of cytochromes P-450b and P-450e (both inducible by phenobarbital (PB) and differing by only 14 of 491 amino acids) in liver microsomes from untreated male rats were separately quantitated by Western blotting with a polyclonal antibody raised against P-450b that is equally effective against P-450e (anti P-450b/e). A protein with mobility identical to P-450e was detected in all microsomal samples. Microsomes from uninduced livers of individual male rats from five different strains exhibited only minor interstrain and interindividual variability in the expression of P-450e (17 +/- 5 pmol P-450e/mg microsomal protein) with the exception of the Brown Norway strain (8.5 +/- 0.5 pmol P-450e/mg). Expression of P-450b varied widely from undetectable levels (less than 2 pmol/mg) in most Sprague-Dawley rats to about 50% of P-450e levels in Fischer and Brown Norway strains. Anti P-450b/e inhibited total metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) by uninduced microsomes, to an extent dependent on rat strain (15-30%), predominantly through inhibition of formation of 12-hydroxymethyl-7-methyl BA (12HOMMBA) (65-85%), the major metabolite of purified P-450e. A specific activity for P-450e-dependent DMBA metabolism was calculated from four sets of microsomes where the P-450b content was either undetectable or very low (0.7-1.0 nmol/nmol P-450e/min-1). Comparable calculated activities were, however, obtained from other untreated rat liver microsomes where P-450b levels were significant. Polymorphism in P-450b was detected but did not affect total P-450b expression or the sensitivity of DMBA metabolism to anti P-450b/e. A fourth band of greater mobility than P-450b (apparent Mr less than 50,000), was also recognized by anti P-450b/e. The intensity of this band did not vary among individual rats or among the different strains and therefore did not correlate with the sensitivity of microsomal DMBA metabolism to anti P-450b/e. A monoclonal antibody (MAb) against P-450b (2-66-3) recognized P-450's b, b2, and e on Western blots but did not react with this higher mobility band. MAb 2-66-3 and two other MAbs produced against P-450b inhibited 12-methylhydroxylation of DMBA by untreated rat liver microsomes to the same extent as anti P-450b/e. Following PB induction, P-450b was induced to about double the level of P-450e in most rat strains examined.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification of cholesterol 7 alpha-hydroxylase from human and rat liver and production of inhibiting polyclonal antibodies

Cholesterol 7 alpha-hydroxylase, the cytochrome P-450-dependent and rate-controlling enzyme of bile acid synthesis, was purified from rat and human liver microsomes. The purified fractions were assayed in a reconstituted system containing [4-14C]cholesterol, and cholesterol 7 alpha-hydroxylase activities in these fractions increased 500-600-fold relative to whole microsomes. Polyacrylamide gel electrophoresis of rat microsomes followed by immunoblotting with polyclonal rabbit antisera raised against purified cholesterol 7 alpha-hydroxylases revealed two peaks at molecular masses of 47,000 and 49,000 daltons for both rat and human fractions. Increasing amounts of rabbit anti-rat and anti-human antibodies progressively inhibited rat microsomal cholesterol 7 alpha-hydroxylase activity up to 80%. In contrast, monospecific antibodies raised against other purified cytochrome P-450 enzymes (P-450f, P-450g, and P-450j) did not inhibit rat or human cholesterol 7 alpha-hydroxylase activity. Immunoblots of rat microsomes with the rabbit anti-rat cholesterol 7 alpha-hydroxylase antibody demonstrated that the antibody reacted quantitatively with the rat microsomal enzyme. Microsomes from cholesterol-fed rats showed increased cholesterol 7 alpha-hydroxylase mass, whereas treatment with pravastatin, an inhibitor of hydroxy-methylglutaryl-coenzyme A reductase, reduced enzyme mass. Microsomes from starved rats contained slightly less cholesterol 7 alpha-hydroxylase protein than chow-fed control rats. These results indicate a similarity in molecular mass, structure, and antigenicity between rat and human cholesterol 7 alpha-hydroxylases; demonstrate the production of inhibiting anti-cholesterol 7 alpha-hydroxylase antibodies that can be used to measure the change in cholesterol 7 alpha-hydroxylase enzyme mass under various conditions; and emphasize the unique structure of cholesterol 7 alpha-hydroxylase with respect to other cytochrome P-450-dependent hydroxylases.     

Purification and characterization of cholesterol 7 alpha-hydroxylase from rat liver microsomes

Cholesterol 7 alpha-hydroxylase (cholesterol, NADPH: oxygen oxidoreductase, 7 alpha-hydroxylating, EC 1.14.13.17) was purified from liver microsomes of cholestryramine-fed male rats by using high-performance ion-exchange chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 52,000), and its dithionite-reduced CO complex exhibited an absorption maximum at 450 nm. The specific content of the enzyme was 9 nmol of cytochrome P-450/mg of protein. Upon reconstitution with NADPH-cytochrome P-450 reductase, the enzyme showed a high activity of cholesterol 7 alpha-hydroxylation with the turnover number of 50 min-1 at 37 degrees C. The reaction was inhibited neither by aminoglutethimide nor by metyrapone, but inhibited markedly by iodoacetamide and disulfiram. The reaction was also inhibited significantly by CO. The enzyme catalyzed hydroxylation of cholesterol with strict regio- and stereoselectivity and was inert toward other sterols which are intermediates in the conversion of cholesterol to bile acids, i.e. 7 alpha-hydroxy-4-cholesten-3-one (12 alpha-hydroxylation), 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol (25-hydroxylation), and taurodeoxycholate (7 alpha-hydroxylation). Unlike other cytochromes P-450 isolated from rat liver microsomes, the enzyme showed no activity toward testosterone and xenobiotics such as 7-ethoxycoumarin and benzo[a] pyrene. The NH2-terminal amino acid sequence of the enzyme was Met-Phe-Glu-Val(Ile)-Ser-Leu-, which was distinct from those of any other cytochromes P-450 of rat liver microsomes hitherto reported. These results indicate that the enzyme is a novel species of cytochrome P-450 so far not isolated from liver microsomes.     

Benzo[a]pyrene metabolism and induction of enzyme-altered foci in regenerating rat liver

The metabolism of benzo[a]pyrene (BP) in regenerating rat liver and the induction of enzyme-altered foci (EAF) in the liver of partially hepatectomized rats, treated with BP and promoted with 2-acetylaminofluorene (2-AAF)/CCl4 was investigated. The aim was to examine factors that might be of importance for the tumorigenicity of BP in the regenerating rat liver, such as cytochrome P-450 activity and glutathione levels. In regenerating rat liver, obtained 18 h after partial hepatectomy (PH), the amount of microsomal cytochrome P-450 was reduced by 20% whereas the level of glutathione was elevated by 15% and the cytosolic glutathione transferase activity towards chlorodinitrobenzene and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydro-BP (BPDE) was unaffected. Microsomes from these animals had a reduced capacity to activate (-)-trans-7,8-dihydroxy-7,8-dihydro-BP (BPD) to DNA-binding products but the pattern of BP metabolites was similar to that observed with control rat liver microsomes. Treatment of rats with 3-methylcholanthrene (MC, 50 mg/kg body wt.) increased cytochrome P-450 levels and glutathione transferase activity towards both substrates. Regenerating livers from these animals retained their cytochrome P-450 level and enzymatic activity towards BP and BPD. Regenerating rat liver microsomes from MC-treated animals were about 35 times more efficient in activating BPD than microsomes from uninduced, partially hepatectomized animals. Intraperitoneal administration of BP (50 mg/kg body wt.) 18 h after PH induced EAF in rats subsequently promoted with 2-AAF/CCl4. Pretreatment of rats with MC 66 h before PH and 84 h before BP administration, increased the number of EAF. In accordance with results by Tsuda et al. (Cancer Res., 40 (1980) 1157-1164), these studies demonstrate that BP is tumorigenic in regenerating rat liver, despite a reduced ability of the liver to activate this compound. Furthermore, MC, an inducer of certain cytochrome P-450 species ("aryl hydrocarbon hydroxylase"), potentiates the effect of BP.     

Cytochrome P-450 and oxygen toxicity. Oxygen-dependent induction of ethanol-inducible cytochrome P-450 (IIE1) in rat liver and lung

The ethanol-inducible form of cytochrome P-450 (P-450IIE1) has previously been shown to exhibit an unusually high rate of oxidase activity with the subsequent formation of reactive oxygen species, e.g., hydrogen peroxide, and to be the main contributor of microsomal oxidase activity in liver microsomes from acetone-treated rats [Ekstr?m & Ingelman-Sundberg (1989) Biochem. Pharmacol. (in press)]. The results here presented indicate that oxygen exposure of rats causes an about 4-fold induction of P-450IIE1 in rat liver and lung microsomes. The induction in liver was not accompanied by any measurable increase in the P-450IIE1 mRNA levels, but the enhanced amount of P-450IIE1 accounted for 60% of the net 50% increase in the level of hepatic P-450 as determined spectrophotometrically. The induction of P-450IIE1 was maximal after 60 h of O2 exposure, and concomitant increases in the rates of liver microsomal CCl4-dependent lipid peroxidation, O2 consumption, NADPH oxidation, O2- formation, H2O2 production, and NADPH-dependent microsomal lipid peroxidation were seen. Liver microsomes from oxygen-treated rats had very similar properties to those of microsomes isolated from acetone-treated rats with respect to the P-450IIE1 content and catalytic properties, but different from those of thyroxine-treated animals. Treatment of rats with the P-450IIE1 inducer acetone in combination with oxygen exposure caused a potentiation of the NADPH-dependent liver and lung microsomal lipid peroxidation and decreased the survival time of the rats. The results reached indicate a role for cytochrome P-450 and, in particular, for cytochrome P-450IIE1 in oxygen-mediated tissue toxicity.     

Theophylline metabolism by rat liver microsomal monooxygenases

Theophylline metabolism has been studied in a reconstituted monooxygenase system with purified forms of cytochrome P-450: P-450a, P-450b, P-450d and P-450k as well as in liver microsomes of control and 3-methylcholanthrene-induced rats. Cytochrome P-450 isoforms, P-450a and P-450b, had no effect on theophylline metabolism, whereas forms P-450d and P-450k induced the synthesis of 1.3-dimethyluric acid (1.3-DMA) at the rates of 900 and 330 pmol/min/nmol of protein, respectively. The catalytic activity of these isoforms was fully inhibited by homologous monospecific antibodies. P-450c catalyzed the formation of a nonidentified metabolite. In microsomes of control animals antibodies specifically directed to cytochrome P-450k suppressed the rate of 1.3-DMA synthesis by 73%, whereas antibodies specifically raised against P-450c+d--by 11%. In microsomes of methylcholanthrene-induced animals the rate of 1.3-DMA synthesis was increased two-fold. This activity was inhibited by 61% by antibodies to cytochrome P-450k and by 18% by anti-P-450c+d antibodies.     

Isolation and characterization of multiple forms of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced Wistar rats

Chromatography on 1.8-diaminooctyl-Sepharose and DEAE-Sephacel resulted in 4 fractions of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced Wistar rats. All the four fractions differed in terms of their absorption maxima in the CO-reduced state, Mr and catalytic activity. Only one cytochrome fraction (cytochrome P-450 C) possessed a high activity upon benz(a)pyrene hydroxylation. All cytochrome P-450 forms were characterized by a low rate of aminopyrine N-demethylation. Antibodies against cytochrome P-450 C (P-448) (anti-P-448) were raised. Cytochromes of fractions A, B1 and B2 in the Ouchterlony reaction of double immunodiffusion did not give precipitation bands with anti-P-448. Neither of the four cytochrome P-450 forms interacted with the antibodies raised against cytochrome P-450 isolated from liver microsomes of rats induced with phenobarbital. The procedure developed is applicable to the isolation of multiple forms of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced rats. Using rocket immunoelectrophoresis, cytochrome P-450 C possessing a high (as compared to benz(a)pyrene metabolism) activity (18 nmol/min/nmol cytochrome) and a high (60-70%) content in 3-methylcholanthrene-induced rat liver microsomes was shown to give a relatively high yield.     

Induction and regulation of cytochrome P450 K-5 (lauric acid hydroxylase) in rat renal microsomes by starvation

The effects of starvation on rat renal cytochrome P-450s were studied. The content of spectrally measured cytochrome P-450 in the renal microsomes of male rats increased 2-fold with 72 h starvation, but cytochrome b5 and NADPH-cytochrome P-450 reductase were not induced. 7-Ethoxycoumarin O-dealkylation and aniline hydroxylation activities of the renal microsomes of control male rats were very low but were induced 2.5-3-fold by 72 h starvation. Aminopyrine N-demethylation and lauric acid hydroxylation activities were induced 1.5-2-fold by 72 h starvation. The changes in catalytic activities suggested that the contents of individual cytochrome P-450s in the renal microsomes were altered by starvation. The contents of some cytochrome P-450s were measured by Western blotting. P450 DM (P450IIE1), a typical form of cytochrome P-450 induced by starvation in rat liver, was barely detected in rat kidney and was induced 2-fold by 72 h starvation. P450 K-5, a typical renal cytochrome P-450 and lauric acid hydroxylase, accounted for 81% of the spectrally measured cytochrome P-450 in the renal microsomes of control male rats and was induced 2-fold by 72 h starvation. P450 K-5 was not induced in rat kidney by treatment with chemicals such as acetone or clofibrate. The renal microsomes of male rats contained 6-times as much P450 K-5 as those of female rats. These results suggest that P450 K-5 is regulated by an endocrine factor.     

Interaction of 9-hydroxyellipticine with two forms of partially purified rabbit lung cytochrome P-450. Inhibition of lung microsomal benzo[a]pyrene hydroxylase.

9-Hydroxyellipticine (9-OHE), a potent inhibitor of rat liver monooxygenase activities, binds to the various forms of partially purified lung cytochromes P-450 from untreated and 3-methylcholanthrene (3-MC)-treated rabbits. The spectral data (lambda max: 428 nm (ox.), 447 nm (red.), Ks: 10 microM and 5 muM for cytochrome I and cytochrome II from 3-MC-treated rabbits respectively) resemble those obtained with cytochrome P-450 purified from liver of Aroclor 1254-pretreated rats (lambda max: 428 nm (ox.), 445 nm (red.), Ks: 8 microM). 9-OHE has been shown to inhibit the benzo[a]pyrene hydroxylase activity of rat and rabbit lung microsomes. The inhibitory effect was higher towards the 3-MC-induced lung microsomes than with the control microsomes. However, the lung microsomes, as well as the liver microsomes of rabbits were less sensitive to inhibition by 9-OHE than the corresponding microsomes from rats. These results suggest that rabbit and rat cytochromes P-450 have subtle structural differences.