Comparison of monooxygenase activities and cytochrome P-450 isozyme concentrations in human liver microsomes

Concentrations of three human liver microsomal cytochrome P-450 isozymes and 20 different monooxygenase activities were determined in human liver microsomal preparations. The results of correlation analysis suggest that: there are important variations in the amounts of the three cytochrome P-450 isozymes measured, particularly P-450(8) and P-450(9); aldrin epoxidase, d-benzphetamine N-demethylase, and S-warfarin 4-hydroxylase activities are linked to cytochrome P-450(5); aryl hydrocarbon (benzo(a)pyrene) hydroxylase and 4-nitroanisole-O-demethylase activities are linked to P-450(8); hydroxylations at the 4'-, 6-, 7-, and 8-positions of R-warfarin are closely linked to each other but are not correlated with other measured monooxygenase activities or P-450 isozyme levels; and P-450(9) is not related to any of the catalytic activities tested. Thus, certain monooxygenase activities can be attributed to specific cytochrome P-450 isozymes. This approach should be useful in suggesting the roles of different cytochromes P-450 in drug metabolism in man which can be further examined using in vitro and in vivo methods.     

Involvement of the cytochrome P-450IID subfamily in minaprine 4-hydroxylation by human hepatic microsomes.

4-Hydroxylation of minaprine was measured on microsomal fractions prepared from 25 different human liver samples. In vitro formation of 4-hydroxyminaprine exhibited a large interindividual variability. Indeed, minaprine 4-hydroxylase activity ranged between 0.033 and 0.421 nmol/min/mg microsomal protein. Two samples presented a particularly low enzyme activity. Minaprine 4-hydroxylation followed Michaelis-Menten kinetics with KM and Vmax values of 5.26 microM and 0.478 nmol/min/mg microsomal protein, respectively, for one particular representative sample. The effects of various compounds (substrates or inhibitors of cytochrome P-450 isoforms) on 4-hydroxyminaprine formation were investigated. Selective substrates for P-450IA [benzo(a)pyrene, theophylline, and phenacetin], IIC (hexobarbital), IIE (aniline), and IIIA (erythromycin, nifedipine, and troleandomycin) cytochrome subfamilies did not inhibit 4-hydroxyminaprine formation. The nonspecific cytochrome P-450 inhibitor, cimetidine, slightly inhibited minaprine 4-hydroxylation. The classical substrates of the P-450IID cytochrome subfamily (debrisoquine, propranolol, and sparteine) inhibited minaprine 4-hydroxylation, as did the known P-450IID specific inhibitor, quinidine. These compounds inhibited minaprine 4-hydroxylase with Ki values of 16.5 (debrisoquine), 14.4 (propranolol), 61.9 (sparteine), and 0.146 microM (quinidine). 4-Hydroxyminaprine formation rate was shown not to be correlated with the activity of both erythromycin N-demethylase (r = 0.29, non-significant) and aniline hydroxylase (r = -0.15, NS). In contrast, minaprine 4-hydroxylase was well correlated with both debrisoquine 4-hydroxylase activity (r = 0.501, p less than 0.05) and immunoquantified cytochrome P-450IID6 (r = 0.579, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of cytochrome P-450 and NADPH-cytochrome c (P-450) reductase from human liver microsomes.

Cytochrome P-450 and NADPH-cytochrome c (P-450) reductase were purified to 10.6 nmoles per mg of protein and 19.9 units per mg of protein, respectively, from human liver microsomes. The purified cytochrome was assumed to be in a low spin state as judged by the absolute spectrum. n-Octylamine and aniline produced type II difference spectra and SKF 525-A and benzphetamine type I spectra when bound to the purified cytochrome P-450. The purified human cytochrome P-450 catalyzed laurate oxidation as determined by NADPH oxidation but not aniline hydroxylation, benzphetamine N-demethylation and 7-ethoxycoumarin O-deethylation when reconstituted with the reductases purified from human and rat liver microsomes. The human cytochrome P-450, however, catalyzed drug oxidations when cumene hydroperoxide was used as the oxygen source. The purified human NADPH-cytochrome c (P-450) reductase contained FAD and FMN at a ratio of 1:0.76. The reductase was capable of supporting 7-ethoxycoumarin O-deethylation activity of cytochrome P-448 purified from 3-methylcholanthrene-treated rat liver microsomes.     

Postnatal development of constitutive forms of cytochrome P-450 in liver microsomes of male and female rats

In a previous paper (1), we reported on the purification of constitutive forms of cytochrome P-450, namely P-450-male and P-450-female, from liver microsomes of male and female rats, respectively. Immunochemical examinations of these hemoproteins showed that P-450-male and P-450-female were detectable specifically in respective liver microsomes of adult male and female rats. The synthesis of P-450-male was apparently dependent on testosterone, and that of P-450-female was dependent on estradiol.Thus, in this study we examined the postnatal development of P-450-male and P-450-female. We show herein that P-450-female is primarily synthesized before the occurrence of P-450-male in male rats. This and other results support the view that the synthesis of unknown pre-existing forms of cytochrome P-450 is depressed in association with the appearance of P-450-male and P-450-female during postnatal periods before sexual maturation.     

Purification and characterization of a mouse liver cytochrome P-450 induced by cannabidiol

A cytochrome P-450 isozyme (Mr = 51,600) was purified to apparent homogeneity from hepatic microsomes of mice pretreated with cannabidiol (CBD), a major constituent of marijuana. The isozyme exhibited high pentoxyresorufin O-dealkylase, hexobarbital hydroxylase, and 16 alpha- and 16 beta-testosterone hydroxylase activities and formed a Fe+2-metyrapone complex, properties characteristic of the major hepatic cytochrome P-450s previously purified from phenobarbital (PB)-pretreated animals. In addition, the CBD-induced cytochrome P-450 was immunoreactive with an antibody raised against the major rat hepatic PB-inducible cytochrome P-450 and exhibited an NH2-terminal amino acid sequence greater than 90% homologous with that of the PB-inducible rat liver isozyme. Because of the many similarities between the CBD-induced isozyme and certain other isozymes previously purified from PB-pretreated animals, a cytochrome P-450 isozyme was purified from PB-pretreated mice by a chromatographic procedure similar to that employed for purification of the CBD-induced isozyme. The PB-inducible isozyme was indistinguishable from the CBD-inducible cytochrome P-450 on the bases of apparent molecular weight, absorption spectra, NH2-terminal amino acid sequence, peptide mapping, immunoreactivity, and catalytic activity. Although the CBD- and PB-inducible P-450 isozymes appear to be qualitatively very similar, PB appears to be a quantitatively better inducer of the isozyme. Thus, CBD exposure results in the induction of an isozyme that is refractory to CBD-mediated inactivation, thereby apparently altering the cytochrome P-450 isozymal composition of mouse hepatic microsomes.     

Purification and characterization of microsomal cytochrome P-450s

1. Eight different forms of cytochrome P-450 have been purified to electrophoretic homogeneity. Electrophoretic, spectral and catalytic properties of these cytochrome P-450s are presented and comparison is made with preparations presented elsewhere in the literature.

2. The levels of these forms of cytochrome P-450 present in liver microsomes of rats treated with various compounds have now been quantified. Several forms of cytochrome P-450 are induced, in a more or less coordinate manner, while levels of other cytochrome P-450s are lowered, during administration of commonly used inducing agents.

3. The role of cytochrome P-450 purification and characterization studies in the understanding of the total field is discussed, along with directions in which future research is needed.     

Purification and characterization of microsomal cytochrome P-450s

1. Eight different forms of cytochrome P-450 have been purified to electrophoretic homogeneity. Electrophoretic, spectral and catalytic properties of these cytochrome P-450s are presented and comparison is made with preparations presented elsewhere in the literature. 2. The levels of these forms of cytochrome P-450 present in liver microsomes of rats treated with various compounds have now been quantified. Several forms of cytochrome P-450 are induced, in a more or less coordinate manner, while levels of other cytochrome P-450s are lowered, during administration of commonly used inducing agents. 3. The role of cytochrome P-450 purification and characterization studies in the understanding of the total field is discussed, along with directions in which future research is needed.     

Purification and characterization of human liver microsomal cytochrome P-450 2A6

Cytochrome P-450 (P-450) 2A6 was purified by chromatography of human liver microsomes. The final preparation was electrophoretically homogeneous and contained 16 nmol of P-450/mg of protein. The amino-terminal amino acid sequence of the protein (first 13 residues) matched that of the reported cDNA exactly. The UV-visible spectrum indicated that the isolated hemoprotein was in the low-spin form. The protein was recognized by rabbit antibodies raised against rat P-450 2A1, and a rabbit antiserum against the P-450 2A6 preparation was also prepared. With these antibodies, it was estimated that P-450 2A6 accounted for a maximum of 1% of the total P-450 present in the human liver microsomes; the level varied greater than 100-fold among the 20 samples examined. Purified P-450 2A6 catalyzed coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation at rates similar to those measured in the human liver sample used to prepare P-450 2A6, and these two microsomal activities were strongly inhibited by the antibodies. The purified P-450 2A6 enzyme also catalyzed low levels of 4,4'-methylene-bis(2-chloroaniline) (MOCA) N-oxidation and activation of aflatoxin B1, 6-aminochrysene, 2-amino-3-methylimidazo[4,5-f]quinoline, and 2-amino-3,5-dimethylimidazo [4,5-f]quinoline to genotoxic products; the antibody inhibited the activity of purified P-450 2A6 towards aflatoxin B1 and 6-aminochrysene but did not inhibit these reactions in human liver microsomes (MOCA N-oxidation was inhibited approximately 20%). Human P-450 2A6 did not catalyze testosterone 7 alpha-hydroxylation, a characteristic activity of the related rat P-450 2A1 protein. These results emphasize the need to characterize individual P-450 enzymes in order to understand their functions in the context of more complex systems.     

Nilutamide inhibits mephenytoin 4-hydroxylation in untreated male rats and in human liver microsomes

1. The effects of nilutamide (an anti-androgen with a hydantoin moiety) on the 4-hydroxylation of mephenytoin were studied in rat liver microsomes. Nilutamide, at a concentration expected in human liver (100uM) during prolonged administration of nilutamide, inhibited by 40% mephenytoin (0-3 mM) 4-hydroxylase activity in liver microsomes from untreated male rats, but not in microsomes from untreated female rats, or in microsomes from dexamethasone-treated male or female rats.

2. Administration to male rats of nilutamide, in doses (20mg/kg i.p. twice daily) known to reproduce plasma concentrations observed in human therapeutics, decreased by 60% the 24?h urinary excretion of 4-hydroxymephenytoin after administration of mephenytoin (15mg/kg oral).

3. Nilutamide (100uM) markedly inhibited mephenytoin 4-hydroxylase activity in human liver microsomes. Inhibition kinetics were consistent with mixed inhibition. It is concluded that nilutamide inhibits mephenytoin 4-hydroxylase activity in untreated male rats and in human liver microsomes. It is suggested that inhibition is likely to occur in vivo in humans receiving therapeutic doses of nilutamide.     

Purification of cytochrome P-450 and NADPH cytochrome p-450 reductase from human liver

Two methods for the purification of cytochromes-P450 from microsomes of human liver are described. Method A: Cyt-P450 were solubilized from microsomes using a non ionic detergent, the Lubrol. The Cyt-P450 were purified by affinity, hydrophobicity followed by ion-exchange chromatography on DEAE-5PW column (HPLC) with an overall yield of 18% and a specific activity of 10 nmole/mg of protein. The recovery of NADPH Cyt-P450 reductase by method A (affinity) is about 60% with a specific activity of 16.2 U.I./mg of protein. Method B: Cyt-P450 were solubilized from microsomes using a zwitterionic detergent, the CHAPS. Cyt-P450 were filtered and separated by chromatofocusing on Mono-P column (HPLC). By this method it was possible to increase strongly the specific activity keeping a yield of 50% of Cyt-P450. Also it was possible to apply this method to small samples of human liver like biopsies (0.5 to 2.5 g).     

A high-spin form of cytochrome P-450 highly purified from polychlorinated biphenyl-treated rats. Catalytic characterization and immunochemical quantitation in liver microsomes

A high-spin form of cytochrome P-450 (termed PCB P-448-H) was purified from liver microsomes of polychlorinated biphenyl (PCB)-treated rats to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This high-spin form of cytochrome P-450 was distinguishable from a low-spin form of cytochrome P-450 (PCB P-448-L) purified from microsomes of PCB-treated rats by the criteria of molecular weights, peptide mapping, and immunochemical properties. In addition, PCB P-448-H catalyzed the hydroxylation of acetanilide (position 4) and biphenyl (positions 2 and 4), and N-hydroxylation of the promutagens 3-amino-1-methyl-5H-pyrido(4,3-b)indole (Trp-P-2), 2-amino-6-methyl-dipyrido(1,2-alpha:3',2'-d)imidazole (Glu-P-1), 2-aminofluorene, and 4-aminobiphenyl at much faster rates than did PCB P-448-L. These promutagens and aflatoxin B1 were efficiently metabolized to mutagens by this high-spin form of hemoprotein. Rabbit immunoglobulin G (IgG) raised against PCB P-448-H inhibited the microsomal O-depropylation activity of p-propoxyaniline. Radial immunodiffusion assay with the IgG showed that PCB P-448-H was one of the major forms of cytochrome P-450 in liver microsomes of PCB-treated rats. On the basis of these results, we propose that this high-spin form of cytochrome P-450 is a key enzyme activating a variety of environmental promutagens in the 9000 x g supernatant fraction of PCB-treated rats, which has been widely used as an activation system in routine mutation tests.     

Evaluation of the involvement of a male specific cytochrome P-450 isozyme in senescence-associated decline of hepatic drug metabolism in male rats

The major "male specific" species of cytochrome P-450 (P-450ml) was purified and an antibody against it used to evaluate the involvement of this isozyme in alterations of drug metabolism in senescence. P-450ml exhibited strikingly high imipramine (IM) N-demethylase activity while it showed no IM 2-hydroxylation, which is an alternate pathway of IM metabolism in rat liver microsomes. The antibody to P-450ml inhibited 80% of imipramine N-demethylation in young male rats. In old male rats, which have been shown to have lower IM N-demethylase activity, a 60% inhibition was observed. The inhibitable portion of this activity in old male rats is about one third of that in young rats, but the remaining portion not inhibited by this antibody is almost identical in young and old rats. IM 2-hydroxylation on the other hand was not inhibited by this antibody at all. It also inhibited about 30% of diazepam(DZ) N-demethylation in young rats but showed no inhibition in old rats, resulting in the loss of the age difference in the remaining portion. DZ 3-hydroxylation was not inhibited by this antibody, in spite of the fact that it showed a markedly higher activity in young male than in young female rats with a subsequent reduction in old age in male rats. This study provides the first direct evidence that differences in the amount of the major male specific P-450 isozyme (P-450ml) are responsible for the age- and sex-associated differences in some of the drug metabolizing activities. It also became apparent that P-450ml may not be the only isozyme responsible for these differences.     

Metabolic activation of environmental carcinogens and mutagens by human liver microsomes. Role of cytochrome P-450 homologous to a 3-methylcholanthrene-inducible isozyme in rat liver

The metabolic activation of procarcinogens and promutagens by human liver microsomal cytochrome P-450 has been investigated by means of a newly developed method measuring the induction of umu gene in Salmonella typhimurium TA1535/pSK1002 [T. Shimada and S. Nakamura, Biochem. Pharmac. 36, 1979 (1987)]. The chemicals examined were aflatoxin B1 (AFB1), eight carcinogenic heterocyclic aromatic amines isolated from protein and amino acid pyrolysates, and 2-aminoanthracene. Liver microsomes from six patients catalyzed the metabolic activation of these chemicals; 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ) and AFB1 were most actively bioactivated, followed by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-aminoanthracene (2-AA) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline. At least two forms of human cytochrome P-450 may be involved in the activation of these procarcinogens. This suggestion was supported by the following lines of evidence: (a) addition of non-ionic detergent Emulgen 913 to the incubation mixture caused a more profound inhibition of microsome-catalyzed activation of AFB1 than of MeIQ, IQ and 2AA, (b) 7,8-benzoflavone stimulated the activation of AFB1 by about 2.5-fold, whereas it inhibited significantly the reactions with MeIQ, IQ and 2AA, and (c) polyclonal antibodies against a 3-methylcholanthrene-inducible form of rat cytochrome P-450 (P-450d) caused a marked inhibition of the metabolic activation of MeIQ, IQ and 2-AA by human liver microsomes though they did not show any effects on the microsomal activation of AFB1. Data are also presented showing that none of the reactions catalyzed by human liver microsomes were inhibited by antibodies to a phenobarbital-inducible form of rat cytochrome P-450 (P-450b). These results suggest that the human cytochrome P-450 isozyme that is immunochemically similar and, thus, homologous to rat P-450d plays a major role in the metabolic activation of several procarcinogens examined, and that the activation of AFB1 is catalyzed by another and, possibly, not phenobarbital-inducible form(s) of human cytochrome P-450.     

Oxidation of troglitazone to a quinone-type metabolite catalyzed by cytochrome P-450 2C8 and P-450 3A4 in human liver microsomes.

Troglitazone, a new oral antidiabetic drug, is reported to be mostly metabolized to its conjugates and not to be oxidized by cytochrome P-450 (P-450) enzymes. Of fourteen cDNA-expressed human P-450 enzymes examined, CYP1A1, CYP2C8, CYP2C19, and CYP3A4 were active in catalyzing formation of a quinone-type metabolite at a concentration of 10 microM troglitazone, whereas CYP3A4 had the highest catalytic activity at 100 microM substrate. In human liver microsomes, rates of the quinone-type metabolite formation (at 100 microM) were correlated well with rates of testosterone 6beta-hydroxylation (r = 0.98), but those at 10 microM troglitazone were not correlated with any of several marker activities of P-450 enzymes. Quercetin efficiently inhibited quinone-type metabolite formation (at 10 microM troglitazone) in human samples that contained relatively high levels of CYP2C, whereas ketoconazole affected these activities in liver microsomes in which CYP3A4 levels were relatively high. Anti-CYP2C antibodies strongly inhibited quinone-type metabolite formation (at 10 microM troglitazone) in CYP2C-rich human liver microsomes (by approximately 85%); the intensity of this effect depended on the human samples and their P-450 status. The results suggest that in human liver both CYP2C8 and CYP3A4 have major roles in quinone-type metabolite formation and that the hepatic contents of these two P-450 forms determine which P-450 enzymes play major roles in individual humans. CYP3A4 may be expected to play a role in formation of quinone-type metabolite from troglitazone even at a low concentration in humans.     

Purification and aminopyrine monooxygenase activity of liver microsomal cytochrome P-450 from alloxan-induced diabetic rats.

We purified two diabetes-inducible and insulin-sensitive forms of cytochrome P-450, named P-450AL-1 and AL-2, from the liver microsomes of alloxan-diabetic male rats, using sodium cholate solubilization, octylamino-Sepharose 4B chromatography, and HPLC with diethylaminoethyl-5PW and hydroxyapatite columns. The purified forms gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 50,000 for P-450AL-1 or 48,500 for P-450AL-2. The CO-reduced spectral maximum of these forms was at 452 nm for P-450AL-1 and 451 nm for P-450AL-2. The two purified forms had the low-spin state of heme in the oxidized form. Both P-450AL-1 and AL-2 were active in the metabolism of aniline, benzphetamine, and 7-ethoxycoumarin. However, the catalytic activity of P-450AL-2 for these substrates was obviously higher than that of AL-1. The NH2-terminal sequences of P-450AL-1 and AL-2 differed from each other, and did not agree with those of the other P-450 forms purified from diabetic rats previously. Furthermore, we examined the metabolism of aminopyrine in a reconstituted system with the purified cytochromes P-450. The diabetes-inducible forms of P-450 had high aminopyrine 3-hydroxylation and low N-demethylation activities. These findings provide clear evidence supporting our previous results, which have shown an increase in 3-hydroxymethyl-2-methyl-4-dimethylamino-1-phenyl-3-pyrazolin-5-on e and a decrease in 4-monomethylaminoantipyrine in intact diabetic rats.     

Comparative study of cytochrome P-450 in liver microsomes. A form of monkey cytochrome P-450, P-450-MK1, immunochemically cross-reactive with antibodies to rat P-450-male

Cytochrome P-450, designated as P-450-MK1, which is cross-reactive with antibodies to rat P-450-male, was purified to an electrophoretical homogeneity from liver microsomes of the untreated male crab-eating monkey. The molecular weight of P-450-MK1 was estimated to be 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The oxidized form of P-450-MK1 showed a peak at 418 nm, indicating that this cytochrome is in a low spin state. The carbon monoxide-bound reduced form showed a peak at 451 nm. The first 22 amino acid residues of the NH2-terminal sequence of P-450-MK1 was fairly homologous to those of P-450-male (75% identity, not including unidentified amino acid residues). Unlike the P-450-male, P-450-MK1 did not exhibit catalytic activities for testosterone 2 alpha- and 16 alpha-hydroxylations and catalyzed testosterone 6 beta-hydroxylation. It is, therefore, suggested that although the spectral and immunochemical properties and the N-terminal amino acid sequence of P-450-MK1 were similar to those of P-450-male, the physiological functions of P-450-MK1 may be somewhat different from those of P-450-male. Comparison of the physico-chemical properties of P-450-MK1 with those of P-450-D1 and P-450-HM2, which are cross-reactive with anti-P-450-male antibodies, purified from liver microsomes of dogs and humans, respectively, are also discussed.