Hypo activity of cytochrome P-450 after triacetyloleandomycin administration

In a preceding communication we reported that triacetyloleandomycin (TAO) induces its own transformation into a metabolite forming a stable complex with the iron (II) of reduced cytochrome P-450; the complex is unstable in the ferric state and disrupted by potassium ferricyanide. In this communication, we report the effects of TAO administration on various monooxygenase activities. Repeated administration of TAO, 1 mmole/kg i.p. daily for 4 days, markedly prolonged the hexobarbital sleeping time, decreased the in vivo disappearance rate of hexobarbital from the liver, and decreased the in vitro activity of hexobarbital hydroxylase, benzo(a)pyrene hydroxylase, ethylmorphine N-demethylase and the in vitro conversion of 17β-estradiol into water-soluble metabolites. Hypoactivity of musomal enzymes was not detected 1 hr after a single dose and was moderate 24 hr after a single dose of TAO, 1 mmole/kg i.p. In vitro, addition of 0.3 mM TAO to the incubation mixture decreased hexobarbital hydroxylase activity by only 6% in control musomes or musomes from rats killed 1 hr after a single dose of TAO, but decreased it by 30% in musomes from rats killed 24 hr after a single dose of TAO and by 60% in musomes from rats killed after repeated doses of TAO. Addition of 50 μM potassium ferricyanide to the musomes did not modify monooxygenase activities in control musomes but increased them in musomes from rats treated with repeated doses of TAO. It is concluded that the administration of TAO decreases several monooxygenase activities and that hypoactivity is at least partly related to the in vivo formation, in TAO-induced rats, of an inactive cytochrome P-450-TAO metabolite complex.     

Alcohol-inducible cytochrome P-450 (P-450ALC)

Of the family of P-450 cytochromes occurring in rabbit liver microsomes, only isozyme 3 a (P-450_ALC) is induced by alcohol administration and is effective in catalyzing the reaction: ethanol+0₂+NADPH+H⁺ acetaldehyde +2H₂O+NADP⁺. As judged by immuno-chemical quantitation, P-450_ALC is also induced in the animals by other diverse agents, including imidazole, trichlorethylene, acetone, pyrazole, and isoniazid. Evidence has been obtained for the occurrence of a protein immuno-chemically related to P-450_ALC in human liver microsomes and of a similar alcohol-inducible protein in the rat and in the normal and alcohol dehydrogenase-deficient deer-mouse. P-450_ALC catalyzes the activation of foreign compounds such as acetaminophen, various nitrosamines, and carbon tetrachloride and is therefore believed to play an important role in the enhanced toxicity of these substances accompanying alcohol administrationDedicated to Professor Dr. Herbert Remmer on the occasion of his 65th birthday     

Induction of cytochrome P-450 isozymes upon repeated administration of nifedipine

In experiments on male Wistar rats it has been found that nifedipine administration at a dose of 10 mg/kg body weight i.p. daily for 20 days did not significantly increase the total amount of cytochrome P-450 but markedly increased the 7 alpha-, 16 beta- and 6 beta-hydroxylation of androstenedione in liver microsomes, suggesting the induction of cytochromes P-450IIA1, P-450IIB1, and P-450IIIA1, respectively. The induction of cytochrome P-450IIIB1 was also confirmed immunochemically with polyclonal antibodies against cytochrome P-450IIB1/B2.     

2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated depression of rat testicular heme synthesis and microsomal cytochrome P-450

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces hirsutism, alopecia, and chlorance, symptoms that suggest a possible alteration of endocrine function. Therefore, the effects of TCDD on rat testicular cytochrome P-450 content were investigated. Forty-eight hours after a single, oral dose of TCDD (25 μg/kg) testicular microsomal cytochrome P-450 levels were depressed by approximately 24%. Microsomal cytochrome P-450 continued to decrease to 62% of control levels at 4 days and remained at approximately the same levels 7 days following treatment. Testicular microsomal heme content exhibited a similar pattern after administration of TCDD. No alterations in testicular δ-aminolevulinic acid (ALA) synthase were detected. The incorporation of [¹⁴C]ALA into microsomal heme was decreased to approximately 36% of control values at 24 hr after TCDD administration. Testicular weights were not altered during the 7-day experimental period. These data suggest that TCDD depresses cytochrome P-450 levels in the rat testis through an inhibition of the synthesis of testicular heme.     

Imprinting of hepatic microsomal cytochrome P-450 enzyme activities and cytochrome P-450IIC11 by peripubertal administration of testosterone in female rats.

The influence of peripubertal exposure to physiological doses of testosterone on the adult androgen responsiveness of hepatic microsomal cytochrome P-450 was investigated. Male and female Sprague-Dawley rats were sham-operated or gonadectomized before puberty, at 25 days of age. They were injected subcutaneously with testosterone enanthate (5 mumol/kg/day) during the pubertal time period, on days 35-49. Responsiveness to this same dose of testosterone was tested by administering the compound during adulthood, on days 81-89. The females provided a model that had not been exposed to neonatal androgen imprinting, in contrast to the males. Testosterone 2 alpha-hydroxylase activity and cytochrome P-450IIC11, which are normally expressed only in adult males, were expressed in the gonadectomized females administered testosterone during puberty with no further exposure to the hormone for the next 40 days. The levels found were similar to those in the gonadectomized male group. When the combined pubertal and adult testosterone regimen was used, a synergistic effect was produced; the 2 alpha-hydroxylase activity reached control male levels in both gonadectomized and sham-operated females and, in addition, cytochrome P-450IIC11 attained control male levels in the gonadectomized females. Testosterone 6 beta-hydroxylase and erythromycin N-demethylase activities were used as indicators of the cytochrome P-450IIIA subfamily. These activities were significantly increased only in the females treated with testosterone during both the pubertal and adult periods, reaching control male levels of 6 beta-hydroxylation. A similar effect, but in the opposite direction, was found with testosterone 7 alpha-hydroxylase, an enzyme activity indicative of cytochrome P-450IIA1. A decrease in this enzyme was produced in the females administered testosterone during both time periods, resulting in levels equivalent to those found in control males. In general, a highly significant interaction was found between the pubertal and adult treatment periods for the females, indicating a chronic effect of the pubertal exposure. The experiments with castrated males did not result in synergistic interactions, although there was some evidence of an additive effect. The results of this study support the hypothesis that the peripubertal period is a time during which testosterone imprinting of both increased basal levels and adult androgen responsiveness of some hepatic cytochrome P-450 enzymes can occur in the female rat.     

Polymorphism of human cytochrome P-450

The cytochrome P-450 forms involved in debrisoquine 4-hydroxylation (P-450DB), phenacetin O-deethylation (P-450PA), S-mephenytoin 4-hydroxylation (P-450MP), and nifedipine 1,4-oxidation (P-450NF) have been purified to electrophoretic homogeneity from human liver microsomes. All of these reactions show in vivo polymorphism in humans. Evidence for the roles of the purified proteins in these processes comes from in vitro reconstitution and immunoinhibition studies. The rat orthologs of these enzymes are as follows--P-450DB: P-450UT-H; P-450PA: P-450ISF-G; P-450MP: P-450UT-I; P-450NF: P-450PCN-E. Only in the case of P-450UT-H is the primary rat ortholog the same cytochrome P-450 which catalyses the catalytic reaction under consideration. Reconstitution and immunochemical studies establish that the following reactions are catalysed by the individual P-450s--P-450DB: debrisoquine 4-hydroxylation, sparteine delta 5-oxidation, bufuralol 1'-hydroxylation, encainide O-demethylation, and propanolol 4-hydroxylation; P-450PA: phenacetin O-deethylation; P-450MP: S-mephenytoin 4-hydroxylation and tolbutamide methyl hydroxylation; P-450NF: oxidation of nifedipine and 16 other substituted dihydropyridines, estradiol 2- and 4-hydroxylation, aldrin epoxidation, benzphetamine N-demethylation and 6 beta-hydroxylation of testosterone, androstenedione and cortisol. A cDNA clone has been isolated that corresponds to rat P-450UT-H, as shown by a number of criteria. Studies with this probe establish that the sex and strain variation in debrisoquine 4-hydroxylase and related activities is related to differences in the levels of a 2.0 kb length mRNA present.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of cytidine nucleotides and level of cytochrome P-450 in rat liver after administration of carbon tetrachloride

Within 2 h after the administration of CCl₄ (2 ml/kg of body weight) to rats, the utilization of labeled orotic acid for the synthesis of cytidine nucleotides in the acid-soluble pool of the liver increased. The specific activity of the uracil moiety of this pool remained unchanged, whereas the specific activity of liver RNA uridine nucleotides slightly increased within 8 h after the administration of CCl₄. The specific activity of liver RNA cytosine is similar to that of cytidine nucleotides of the acid-soluble pool. The specific activity of DNA thymine was decreased during this interval and the specific activity of DNA cytosine reflected the specific activities of the cytidine components of acid-soluble pool. The enhancement of utilization of labeled orotic acid for the synthesis of cytidine nucleotides in the acid-soluble pool and in RNA was followed by a depression of the level of microsomal cytochrome P-450 in the liver. Liver microsomal cytochrome b₅ was depressed at later time intervals after the administration of CCl₄. The increased utilization of labeled orotic acid for the synthesis of microsomal cytochrome P-450 was observed in the liver also after the administration of CS₂ or CoCl₂.     

Interspecies homology of liver microsomal cytochrome P-450. A form of dog cytochrome P-450 (P-450-D1) crossreactive with antibodies to rat P-450-male

P-450-male is a male specific form of cytochrome P-450 in rat liver microsomes. Cytochrome P-450 crossreactive with anti-P-450-male antibodies was purified to an electrophoretical homogeneity from liver microsomes of male beagle dogs. The specific content of the purified cytochrome P-450 (P-450-D1) was 16.9 nmol/mg protein. The apparent monomeric molecular weight of P-450-D1 was 48,000, which was smaller than P-450-male (51,000). P-450-D1 showed similarities in spectral properties, N-terminal amino acid sequence, and catalytic activities with some limited exceptions: P-450-D1 did not catalyze 2 alpha-hydroxylation of testosterone and progesterone and catalyzed 21-hydroxylation of progesterone. Based on these results, we propose that P-450-D1 is a form of cytochrome P-450 in the same gene subfamily as P-450-male.     

Polymorphism of cytochrome P-450 in humans

A number of different cytochrome P-450 proteins are found in human liver and other tissues. These enzymes oxidize drugs and carcinogens as well as endogenous chemicals such as steroids and eicosanoids. As Peter Guengerich explains, activities of individual cytochrome P-450 enzymes vary among individuals as a result of both genetic and environmental influences; in some cases, the mechanisms are known. Such variation can have major influences in determining drug toxicity, inborn errors of steroid metabolism, and possibly cancer risk.     

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.     

Inhibition of rat testicular heme synthesis and depression of microsomal cytochrome P-450 by estradiol benzoate

Twenty-four hours after a single dose (50 μg, s.c.) of estradiol benzoate (EB), rat testicular microsomal heme and cytochrome P-450 were decreased to 72 and 76% of control levels respectively. Treatment of rats with human chorionic gonadotropin (hCG) resulted in elevated levels of microsomal heme and cytochrome P-450 and increased activity of δ-aminolevulinic acid (ALA) synthase (EC 2.3.1.37). However, the hCG-mediated elevations of testicular microsomal heme and cytochrome P-450 content failed to occur in animals treated with EB. To investigate the possibility that the observed effect of EB was mediated through the pituitary, studies were conducted with hypophysectomized animals. The increased microsomal heme and cytochrome P-450 content mediated by hCG in hypophysectomized animals was again prevented by administration of EB. The elevated activity of testicular mitochondrial ALA synthase produced by hCG in both intact and hypophysectomized animals was not affected by EB. Incorporation of [¹³C]ALA into microsomal heme was depressed 60% 12 hr following a single dose of EB (50 μg, s.c.). These data suggest that EB depresses testicular microsomal heme and cytochrome P-450 content by inhibiting the synthesis of heme at an enzymatic reaction other than ALA synthase.     

Effect of DI-n-pentyl phthalate treatment on testicular steroidogenic enzymes and cytochrome P-450 in the rat

Treatment of young male rats with dipentyl phthalate (DPP) produced significant decreases in testicular cytochrome P-450, cytochrome P-450 dependent microsomal steroidogenic enzymes (17 alpha-hydroxylase, 17-20 lyase) and in the maximal binding of a natural substrate (progesterone) to testis microsomes. No effect was demonstrated by this compound on hepatic cytochrome P-450 content. Treatment of animals with a phthalate ester not causing testicular atrophy (diethyl phthalate; DEP) produced no significant changes in any of the parameters measured. This effect on the enzymes responsible for androgen production may be important as a mechanism of action involved in the development of phthalate-induced testicular damage.     

Formation of an inactive cytochrome P-450 Fe(II)-metabolite complex after administration of troleandomycin in humans

In rats, it has been shown that troleandomycin induces its own transformation into a metabolite forming an inactive complex with reduced cytochrome P-450. To determine whether similar effects occur in humans, we studied hepatic microsomes from 6 untreated patients and 6 patients treated with troleandomycin, 2 g per os daily for 7 days. In the treated patients, NADPH-cytochrome c reductase activity was increased by 48%; total cytochrome P-450 concentration was also increased, but 33% of total cytochrome P-450 was complexed by a troleandomycin metabolite. The cytochrome P-450 Fe(II)-metabolite complex exhibited properties identical to those of the inactive complex formed in rats: it exhibited a Soret peak at 456 nm, was unable to bind CO, and was destroyed by addition of 50 μM potassium ferrciyanide.We also measured the clearance of antipyrine in 6 other subjects. This clearance was decreased by 45% when measured again on the seventh day of the troleandomycin treatment. We conclude that repeated administration of troleandomycin induces microsomal enzymes, produces an inactive cytochrome P-450 Fe(II)-metabolite complex, and decreases the clearance of antipyrine in humans.