Tetsuya Kamataki

Sex difference of cytochrome P-450 in the rat: Purification, characterization, and quantitation of constitutive forms of cytochrome P-450 from liver microsomes of male and female rats☆

One of each constitutive form of cytochrome P-450 from liver microsomes of adult male and female rats was purified essentially following the same method to an apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights estimated by the electrophoresis were 52,000 and 50,000 for forms of cytochrome P-450, P-450-male, and P-450-female, purified from male and female rats, respectively. In addition, the purified preparations of P-450-male and P-450-female showed properties different from each other with respect to spectral characteristics and catalytic activities. In Ouchterlony double diffusion plates, partially purified rabbit immunoglobulin G (IgG) raised against P-450-male and P-450-female showed very weak or no cross-reactivity with P-450-female and P-450-male, respectively. From these results, P-450-male was confirmed to be a form distinct from P-450-female. The anti-P-450-male and anti-P-450-female antibodies, which had been further purified by immunoadsorption, did not form any apparent precipitation bands with liver microsomes from untreated female and male rats, respectively. Supporting this, radial immunodiffusion analysis for P-450-male and P-450-female with an agarose gel impregnated with the rabbit antibodies showed that P-450-male and P-450-female appear in liver microsomes rather specifically depending on the sex hormones. Based on these results, sex differences in drug metabolism in the rat were confirmed as explicable, at least in part, by the presence of distinct forms of cytochrome P-450 in microsomes of male and female rats.

Purification and properties of cytochrome P-450 from homogenates of human fetal livers

A form of cytochrome P-450, namely P-450HFLa of human fetal livers, was purified to a specific content of 12.6 nmol/mg protein. The cytochrome P-450 preparation was electrophoretically homogeneous and had an apparent monomeric molecular weight of 51,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cytochrome showed catalytic activities as oxidations of N-methylaniline, ethylmorphine, N,N-dimethylaniline, N,N-dimethylnitrosamine, benzphetamine, aminopyrine, aniline, p-nitroanisole, and 7-ethoxycoumarin to various extents. In fetal liver homogenate, the amount of cytochrome P-450 that reacted with the antiserum to P-450HFLa accounted for more than 36% of the total cytochrome P-450 in three different fetal livers. On the other hand, the amount of P-450HFLa was less than 5% of the total cytochrome P-450 in adult liver microsomes.

Cytochrome P-450 as a determinant of sex difference of drug metabolism in the rat

1. Recent advances in the studies of sex-related differences of drug metabolism in the rat are described. Experiments with novel substrates have provided new insights into the sex differences of drug metabolism. 2. One of each form of cytochrome P-450, P-450-male and P-450-female, from liver microsomes of male and female rats, respectively, were purified to electrophoretic homogeneity. 3. These forms of cytochrome P-450 are distinguishable from each other in their catalytic, physical and immunochemical properties. 4. The use of antibodies to the two forms of cytochrome P-450 shows that P-450-male and P-450-female, present in rat-liver microsomes, are dependent on sex hormones. 5. In addition, evidence for the involvement of multiple forms of cytochrome P-450 in the occurrence of the sex difference has been presented.

Isolation and characterization of active metabolites of tryptophan-pyrolysate mutagen, TRP-P-2, formed by rat liver microsomes.

The mutagenic compound derived from the pyrolysis of tryptophan, 3-amino-1-methyl-5H-pyrido-[4,3b]indole (Trp-P-2) was metabolized by rat liver microsomes to more than four metabolites, separable by high performance liquid chromatography. Among these metabolites, two metabolites, M-3 and M-4 were directly active in increasing the frequency of mutation in Salmonella typhimurium TA98. Treatments of rats with polychlorinated biphenyl (PCB) or 3-methylcholanthrene dramatically induced the activity of liver microsomes to form these active metabolites, while treatment with phenobarbital was without effect. A major active metabolite (M-3) formed the pentacyano-ammine ferroate, which is known to be formed by reaction of sodium pentacyano-ammine ferroate with some hydroxylamines. Further this metabolite was oxidized to the minor active metabolite (M-4) with potassium ferricyanide or gamma-manganese dioxide, and was reduced back to Trp-P-2 with titanium trichloride. These results indicated that the major active metabolite of Trp-P-2, which is formed by cytochrome P-450, is the 3-hydroxyamino derivative.

Sex difference in the O-dealkylation activity of 7-hydroxycoumarin O-alkyl derivatives in liver microsomes of rats

Abstract Sex differences in the O-dealkylation activities of O-alkyl derivatives of 7-hydroxycoumarin were compared using liver microsomes from male and female rats. The sex difference (male>female) in the O-depropylation activity was found to be greater than the sex differences in O-demethylation and O-deethylation activities. The magnitude of sex difference seen in the O-depropylation activity was diminished after pretreatment of rats with spironolactone, phenobarbital, isosafrole or 3-methylcholanthrene. The latter two inducers were much more effective than the former in enhancing the activity. The sex differences in the O-dealkylation activities were also seen when the activities were measured in the presence of cumene hydroperoxide instead of NADPH. The sex difference of the O-depropylation activity remained essentially unchanged by the fortification of male and female microsomes with purified NADPH-cytochrome c (P-450) reductase. The difference was also seen when reconstituted with cyto- chrome P-450 partially purified from both male and female rats by means of ω-amino-n-octyl Sepharose 4B and hydroxylapatite columns. The addition of 7,8-benzoflavone to the incubation mixture resulted in the increased magnitude of sex difference in the O-depropylation activity. From these results, we confirm that one or more cytochrome P-450 species other than a cytochrome P-450 species sensitive to 7,8-benzoflavone are present in male microsomes in higher amounts than in female microsomes.

Pituitary regulation of sex-specific forms of cytochrome P-450 in liver microsomes of rats.

Effects of hypophysectomy and treatment with testosterone or estradiol on the sex-specific forms of cytochrome P-450, P-450-male and P-450-female, were examined. The amounts of P-450-male as well as drug oxidation activities were decreased by hypophysectomy of male rats. In female rats, drug oxidation activities were increased by hypophysectomy, which was associated with the disappearance of P-450-female and the appearance of P-450-male. Treatment of hypophysectomized female rats with testosterone or estrodiol effected minor changes in the amounts of P-450-male.

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

Abstract 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.

Mutagenic activation of aflatoxin B1 by several forms of purified cytochrome P-450

Metabolic activation by several forms of purified cytochrome P-450 of aflatoxin B1 to a product(s) mutagenic to Salmonella typhimurium TA100 was examined. Of the 5 forms of cytochrome P-450 purified from liver microsomes of untreated and PCB-treated male rats, a constitutive form purified from untreated male rats, P-450-male, and a high-spin form of cytochrome P-450, P-448-H, from PCB-treated rats were highly active.

Evidence for the involvement of multiple forms of cytochrome P-450 in the occurrence of sex-related differences of drug metabolism in the rat

Multiple forms of cytochrome P-450 in liver microsomes of untreated male and female rats could be divided into several fractions by the use of omega-amino-n-octyl Seph. 4B and DE-52 columns. Male cytochrome P-450 fractions (I-b - I-e) differed from female fractions (I-b - I-e) with respect to absorption peaks in carbon monoxide difference spectra and 7-propoxycoumarin O-depropylation activities. Although male and female I-a fractions showed quite similar protein bands on SDS-polyacrylamide gel electrophoresis, some protein bands in other male fractions (I-b - I-e) were absent in corresponding female fractions. Immunochemical examinations using immunoglobulin G raised to cytochrome P-450 purified from untreated male rats also showed that liver microsomes from male and female rats contain different forms of cytochrome P-450. Based on these results, we propose that sex-related differences of drug metabolizing activities in liver microsomes are caused by multiple forms of cytochrome P-450.

Partial purification and characterization of cytochrome P-450 responsible for the occurrence of sex difference in drug metabolism in the rat

Abstract Cytochrome P-450 in cholate-solubilized microsomes from untreated male and female rats was divided into two fractions (I and II) by ω-amino- n -octyl Sepharose 4B columns. A marked sex difference in the O-depropylation of 7-propoxycoumarin was seen between the I fractions from male and female rats in which male rat fraction I exhibited higher activity than that of female rats. Addition of cytochrome b5 resulted in about 2-fold increase in the O-depropylation activities of the I fractions from male and female rats but not in those of the II fractions. Based on these and other results, we propose that at least one of multiple forms of cytochrome P-450 responsible for the occurrence of the sex difference in drug metabolism requires cytochrome b5 for maximal activity.