The involvement of cytochrome P-450 in hepatic microsomal steroid hydroxylation reactions supported by sodium periodate, sodium chlorite, and organic hydroperoxides.

The mechanism of steroid hydroxylation in rat liver microsomes has been investigated by employing NaIO4, NaClO2, and various organic hydroperoxides as hydroxylating agents and comparing the reaction rates and steroid products formed with those of the NADPH-dependent reaction. Androstenedione, testosterone, progesterone, and 17beta-estradiol were found to act as good substrates. NaIO4 was by far the most effective hydroxylating agent followed by cumene hydroperoxide, NADPH, NaClO2, pregnenolone 17alpha-hydroperoxide, tert-butyl hydroperoxide, and linoleic acid hydroperoxide. Androstenedione was chosen as the model substrate for inducer and inhibitor studies. The steroid was converted to its respective 6beta-, 7alpha, 15-, and 16alpha-hydroxy derivatives when incubated with microsomal fractions fortified with hydroxylating agent. Evidence for cytochrome P-450 involvement in androstenedione hydroxylation included a marked inhibition by substrates and modifiers of cytochrome P-450 and by reagents which convert cytochrome P-450 to cytochrome P-420. The ratios of the steroid products varied according to the type of hydroxylating agent used and were also modified by in vivo phenobarbital pretreatment. It was suggested that multiple forms of cytochrome P-450 exhibiting different affinities for hydroxylating agent are responsible for these different ratios. Horse-radish peroxidase, catalase, and metmyoglobin could not catalyze androstenedione hydroxylation. Addition of NaIO4, NaClO2, cumene hydroperoxide and other organic hydroperoxides to microsomal suspensions resulted in the appearance of a transient spectral change in the difference spectrum characterized by a peak at about 440 nm and a trough at 420 nm. The efficiency of these oxidizing agents in promoting steroid hydroxylation in microsomes appeared to be related to their effectiveness in eliciting the spectral complex. Electron donors, substrates, and modifiers of cytochrome P-450 greatly diminished the magnitude of the spectral change. It is proposed that NaIO4, NaClO2, and organic hydroperoxides promote steroid hydroxylation by forming a transient ferryl ion (compound I) of cytochrome P-450 which may be the common intermediate hydroxylating species involved in hydroxylations catalyzed by cytochrome P-450.     

Rat hepatic cytochrome P-450 isoenzyme 2c. Identification as a male-specific, developmentally induced steroid 16 alpha-hydroxylase and comparison to a female-specific cytochrome P-450 isoenzyme

Rat hepatic cytochrome P-450 isoenzyme 2c, purified to homogeneity from uninduced, adult rat liver (Waxman, D.J., Ko, A., and Walsh, C. (1983) J. Biol. Chem. 258, 11937-11947), was shown to exhibit a unique NH2-terminal amino acid sequence as well as distinctive peptide maps and immunochemical properties when compared to seven other purified rat liver P-450 isoenzymes. P-450 2c was an efficient monooxygenase catalyst with several xenobiotic substrates; P-450 2c also catalyzed 16 alpha- and 2 alpha-hydroxylations of testosterone, androst-4-ene-3,17-dione and progesterone (total turnover = 7-9 min-1 P-450(-1) at 25 microM steroid substrate) with the ratio of 2 alpha to 16 alpha hydroxylation varying from less than or equal to 0.02 to 1.6 depending on the steroid's C-17 substituent. Six different microsomal steroid hydroxylase activities characteristic of purified P-450 2c and sensitive to specific inhibition by anti-P-450 2c antibody were induced at puberty in male but not female rat liver. Microsomal steroid hydroxylations catalyzed by other P-450 isoenzymes exhibited age and sex dependencies distinct from those of the P-450 2c-mediated activities. Immunochemical analyses confirmed that this sex dependence and developmental induction reflected alterations in P-450 2c polypeptide levels. Attempts to chromatographically detect P-450 2c in either immature male or adult female microsomes were unsuccessful and led to purification of P-450 2d (female), a catalytically distinct and female-specific form. Peptide mapping and immunochemical analyses suggested significant structural homologies between the two sex-specific isoenzymes, P-450 2c and P-450 2d (female). A significant suppression of P-450 2c levels (up to 70-80%) was observed upon administration of several classical P-450 inducers. These studies establish that P-450 2c corresponds to the male-specific and developmentally-induced steroid 16 alpha-hydroxylase of rat liver and suggest that the expression of P-450 2c versus P-450 2d (female) may provide a biochemical basis for the sex differences characteristic of rat liver xenobiotic metabolism.     

Regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by cytochrome P-450 isozymes purified from phenobarbital-induced rat liver

The regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by five isozymes of cytochrome P-450 purified from phenobarbital-induced rat liver were studied in a reconstituted monooxygenase system using testosterone (T) and androst-4-ene-3,17-dione (delta 4-A) as substrates. P-450 PB-3, an isozyme exhibiting low catalytic activity with many xenobiotic substrates, catalyzed efficient (turnover = 15.7 to 18.5 min-1 P-450-1 at 25 microM substrate) and highly stereoselective B-ring hydroxylations of both steroid substrates, with the corresponding 7 alpha- and 6 alpha-hydroxy alcohols formed in ratios of approximately 20 to 30:1, respectively. P-450 PB-2c metabolized testosterone to a mixture of 16 alpha OH-T, 2 alpha OH-T, and delta 4-A (product ratio = 1.0/0.78/0.33; turnover = 10.2 min-1 P-450-1). PB-2c is present in significantly larger amounts in mature male rats as compared to immature males, and probably catalyzes the male-specific testosterone 16 alpha-hydroxylase activity known to be induced at puberty and subject to endocrine control. P-450 PB-4, the major phenobarbital-induced isozyme in rat liver, catalyzed efficient D-ring hydroxylations, yielding 16 beta OH- delta 4-A as the predominant product with delta 4-A as substrate (turnover = 12.0 min-1 P-450-1) and a mixture of 16 beta OH-T, 16 alpha OH-T, and delta 4-A (the latter compound presumably formed via 17 alpha hydroxylation) with testosterone as substrate (turnover = 5.2 min-1 P-450-1). P-450 isozymes PB-1 and PB-5 hydroxylated both steroids with essentially the same regioselectivity as PB-4 but at only 5 to 10% the catalytic rate. Cytochrome b5 stimulated most of these steroid hydroxylations up to 2-fold with no change in regio- or stereoselectivity. The identification of specific steroid metabolites as diagnostic of particular P-450 isozymes should be useful for the assessment of isozymic contributions to microsomal activities and, in addition, facilitate comparisons of P-450 isozymes isolated in different laboratories.     

Human liver microsomal steroid metabolism: identification of the major microsomal steroid hormone 6 beta-hydroxylase cytochrome P-450 enzyme

Cytochrome P-450-dependent steroid hormone metabolism was studied in isolated human liver microsomal fractions. 6 beta hydroxylation was shown to be the major route of NADPH-dependent oxidative metabolism (greater than or equal to 75% of total hydroxylated metabolites) with each of three steroid substrates, testosterone, androstenedione, and progesterone. With testosterone, 2 beta and 15 beta hydroxylation also occurred, proceeding at approximately 10% and 3-4% the rate of microsomal 6 beta hydroxylation, respectively, in each of the liver samples examined. Rates for the three steroid 6 beta-hydroxylase activities were highly correlated with each other (r = 0.95-0.97 for 25 individual microsomal preparations), suggesting that a single human liver P-450 enzyme is the principal microsomal 6 beta-hydroxylase catalyst with all three steroid substrates. Steroid 6 beta-hydroxylase rates correlated well with the specific content of human P-450NF (r = 0.69-0.83) and with its associated nifedipine oxidase activity (r = 0.80), but not with the rates for debrisoquine 4-hydroxylase, phenacetin O-deethylase, or S-mephenytoin 4-hydroxylase activities or the specific contents of their respective associated P-450 forms in these same liver microsomes (r less than 0.2). These correlative observations were supported by the selective inhibition of human liver microsomal 6 beta hydroxylation by antibody raised to either human P-450NF or a rat homolog, P-450 PB-2a. Anti-P-450NF also inhibited human microsomal testosterone 2 beta and 15 beta hydroxylation in parallel to the 6 beta-hydroxylation reaction. This antibody also inhibited rat P-450 2a-dependent steroid hormone 6 beta hydroxylation in uninduced adult male rat liver microsomes but not the steroid 2 alpha, 16 alpha, or 7 alpha hydroxylation reactions catalyzed by other rat P-450 forms. Finally, steroid 6 beta hydroxylation catalyzed by either human or rat liver microsomes was selectively inhibited by NADPH-dependent complexation of the macrolide antibiotic triacetyloleandomycin, a reaction that is characteristic of members of the P-450NF gene subfamily (P-450 IIIA subfamily). These observations establish that P-450NF or a closely related enzyme is the major catalyst of steroid hormone 6 beta hydroxylation in human liver microsomes, and furthermore suggest that steroid 6 beta hydroxylation may provide a useful, noninvasive monitor for the monooxygenase activity of this hepatic P-450 form.     

25-hydroxylation of C27-steroids and vitamin D3 by a constitutive cytochrome P-450 from rat liver microsomes

A constitutive cytochrome P-450 catalyzing 25-hydroxylation of C27-steroids and vitamin D3 was purified from rat liver microsomes. The enzyme fraction contained 16 nmol of cytochrome P-450/mg of protein and showed only one protein band with a minimum molecular weight of 51,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified cytochrome P-450 catalyzed 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, and 1 alpha-hydroxyvitamin D3 up to 50 times more efficiently, and 25-hydroxylation of vitamin D3 about 150 times more efficiently than the microsomes. The cytochrome P-450 showed no detectable 25-hydroxylase activity towards vitamin D2 and was inactive in cholesterol 7 alpha-hydroxylation as well as in 12 alpha- and 26-hydroxylations of C27-steroids. It catalyzed hydroxylations of testosterone and demethylation of ethylmorphine at the same rates as, or lower rates than, microsomes. The 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and vitamin D3 with the purified cytochrome P-450 was not stimulated by addition of phospholipid or cytochrome b5 to the reconstituted system. Emulgen inhibited 25-hydroxylase activity towards both substrates. The possibility that 25-hydroxylation of C27-steroids and vitamin D3 is catalyzed by the same species of cytochrome P-450 is discussed.     

Iodosylbenzene derivatives as oxygen donors in cytochrome P-450 catalyzed steroid hydroxylations.

The mechanism of cytochrome P-450 catalyzed steroid hydroxylations in rat liver microsomes has been investigated by employing derivatives of iodosylbenzene as oxygen donors. The model steroid substrate androstenedione which was hydroxylated in positions 7 alpha, 6 beta, and 16 alpha was used in reactions supported by NADPH, iodosylbenzene, and iodosylbenzene derivatives. Evidence for cytochrome P-450 involvement in iodosylbenzene-sustained androstenedione hydroxylation included inhibition by substrates and modifiers of cytochrome P-450. The most efficient oxygen donors were (diacetoxyiodo)-2-nitrobenzene greater than (diacetoxyiodo)-2-chlorobenzene greater than 2-nitroiodosylbenzene greater than (dinitratoiodo)-2-nitrobenzene greater than (diacetoxyiodo)benzene greater than (diacetoxyiodo)-2-methoxybenzene greater than 4-(diacetoxyiodo)toluene greater than iodosylbenzene. The capacity of the oxidation agents to serve as oxygen donors in cytochrome P-450 dependent steroid hydroxylation is probably dependent upon several factors such as the tendency of iodosyl compounds to associate, which decreases coordination with the heme iron, the presence of bulky substituents in the 2 position (decreases association), and the presence of electron-withdrawing substituents (tends to decrease coordination with the heme iron). The rates of 7 alpha, 6 beta, and i6 alpha hydroxylation of androstenedione catalyzed by (diacetoxyiodo)-2-nitrobenzene were 108-, 130-, and 167-fold higher, respectively, than the rates of the NADPH-supported reactions. These results strongly suggest that the rate-limiting step in NADPH-sustained cytochrome P-450 catalyzed reactions is the rate of reduction of cytochrome P-450.     

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.     

Heterogeneity of hepatic mixed function oxidases

The effects of NADH and increasing concentrations of potassium phosphate buffer, potassium chloride and potassium thiocyanate on several hydroxylations catalyzed by rat liver microsomes were studied. All the hydroxylations were stimulated by NADH in the presence of suboptimal concentrations of NADPH. The 7α-hydroxylation of cholesterol, the 12α-hydroxylation of 7α-hydroxy-4-cholesten-3-one and the 16-hydroxylation of palmitic acid were inhibited by increasing concentrations of potassium phosphate buffer, potassium chloride and potassium thiocyanate to a greater extent than any of the other hydroxylations studied. This finding and the previous finding that these three hydroxylations are not stimulated by phenobarbital treatment suggest differences between these hydroxylations and most other microsomal hydroxylations in liver. The possibility is discussed that different types of cytochrome P-450 may be involved.     

Sodium periodate, sodium chlorite, and organic hydroperoxides as hydroxylating agents in steroid hydroxylation reactions catalyzed by adrenocortical microsomal and mitochondrial cytochrome P450.

This study has investigated the mechanism of steroid hydroxylation in bovine adrenocortical microsomes and mitochondria by employing NaIO₄, NaClO₂, and various organic hydroperoxides as hydroxylating agents and comparing the reaction rates and steroid products formed with those of the NADPH-dependent reaction. In the microsomal hydroxylating system, progesterone, 17α-hydroxyprogesterone, and androstenedione were found to act as substrates. Progesterone was chosen as the model substrate and was converted mainly to the 21-hydroxylated derivative in the presence of microsomal fractions fortified with hydroxylating agent. Using saturating levels of hydroxylating agent, NaIO₄ was found to be the most effective in promoting progesterone hydroxylation followed by cumene hydroperoxide, t-butyl hydroperoxide, NADPH, NaClO₂, and pregnenolone 17α-hydroperoxide. Evidence for cytochrome P₄₅₀ involvement included a marked inhibition of the activity by substrates and modifiers of cytochrome P₄₅₀ and by reagents that convert cytochrome P₄₅₀ to cytochrome P₄₂₀. Steroid hydroxylation was studied in adrenocortical mitochondria that had been previously depleted of endogenous pyridine nucleotides by aging for 1 h at 30 dgC in a phosphate-supplemented medium. Androstenedione was converted to its respective 6β-, 11β-, 16β-, and 19-hydroxylated derivatives when incubated with aged mitochondrial fractions fortified with hydroxylating agent whereas progesterone was hydroxylated in the 1β-, 6β-, and 15β- positions. These hydroxylations were completely abolished by preheating the mitochondria for 5 min at 95 dgC prior to assay, indicating the enzymic nature of the reactions. Deoxycorticosterone and deoxycortisol were effective substrates for NADPH-dependent enzymic 11β-hydroxylation but were extensively degraded nonenzymically to unidentified products in the presence of NaIO₄ and hydroxylating agents other than NADPH and consequently could not be utilized as substrates in these reactions. Using androstenedione as substrate, NaIO₄ was the most effective hydroxylating agent, followed by cumene hydroperoxide, NaClO₂, t-butyl hydroperoxide, and NADPH. These hydroxylations were inhibited by substrates and modifiers of cytochrome P₄₅₀ and by reagents that convert cytochrome P₄₅₀ to cytochrome P₄₂₀. A mechanism for steroid hydroxylation in adrenocortical microsomes and mitochondria is proposed in which the ferryl ion (compound I) of cytochrome P₄₅₀ functions as the common “activated oxygen” species.     

Cytochrome P-450-linked monooxygenase systems of bovine brain mitochondria and microsomes

1. Cytochrome P-450-linked monooxygenase of mitochondria and microsomes of the cerebrum, cerebellum and pituitary gland of bovine brain were investigated biochemically and immunochemically. 2. The cytochromes P-450scc and P-450(11) beta, NADPH-ferredoxin reductase and adreno-ferredoxin were detected and their enzymatic activities of steroid hydroxylations were found in the bovine brain.     

Inhibition of steroid 5 alpha-reductase and its effects on testosterone hydroxylation by rat liver microsomal cytochrome P-450

It has been shown previously that liver microsomal steroid 5 alpha-reductase activity increases with age in female but not male rats, which coincides with a female-specific, age-dependent decline in the cytochrome P-450-dependent oxidation of testosterone to 1 beta-, 2 alpha-, 2 beta-, 6 alpha-, 6 beta-, 7 alpha-, 15 beta-, 16 alpha-, 16 beta-, and 18-hydroxytestosterone and androstenedione. To determine whether the increase in steroid 5 alpha-reductase activity is responsible for the decrease in testosterone oxidation, we have examined the effects of the steroid 5 alpha-reductase inhibitor, 4-MA (17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one), on the pathways of testosterone oxidation catalyzed by rat liver microsomes. We have also determined which hydroxytestosterone metabolites are substrates for steroid 5 alpha-reductase. At concentrations of 0.1 to 10 microM, 4-MA completely inhibited steroid 5 alpha-reductase activity without inhibiting the pathways of testosterone oxidation catalyzed by liver microsomes from rats of different age and sex, and from rats induced with phenobarbital or pregnenolone-16 alpha-carbonitrile. 4-MA (10 microM) had little or no effect on the oxidation of testosterone catalyzed by liver microsomes from mature male rats (which have low steroid 5 alpha-reductase activity). In contrast, the hydroxylated testosterone metabolites formed by liver microsomes from mature female rats (which have high steroid 5 alpha-reductase activity) accumulated to a much greater extent in the presence of 4-MA. Evidence is presented that 4-MA increases the accumulation of hydroxytestosterones by two mechanisms. First, 4-MA inhibited the 5 alpha-reduction of those metabolites (such as 6 beta-hydroxytestosterone) that were found to be excellent substrates for steroid 5 alpha-reductase. In the absence of 4-MA, these metabolites eventually disappeared from incubations containing liver microsomes from mature female rats. Second, 4-MA inhibited the formation of 5 alpha-dihydrotestosterone, which otherwise competed with testosterone for oxidation by cytochrome P-450. This second mechanism explains why 4-MA increased the accumulation of metabolites (such as 7 alpha-hydroxytestosterone) that were found to be poor substrates for steroid 5 alpha-reductase. Despite its marked effect on the accumulation of hydroxylated testosterone metabolites, 4-MA had no effect on their initial rate of formation by liver microsomes from either male or female rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Purification and characterization of cytochromes P-450 from liver microsomes of 3-methylcholanthrene-treated crab-eating monkeys

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.     

Leukotriene B4 omega-hydroxylase in rat liver microsomes: identification as a cytochrome P-450 that catalyzes prostaglandin A1 omega-hydroxylation, and participation of cytochrome b5

The omega-hydroxylation of leukotriene B4 (LTB4) by rat liver microsomes requires NADPH and molecular oxygen, suggesting that the hydroxylation is catalyzed by a cytochrome P-450 (P-450)-linked monooxygenase system. The reaction is inhibited by CO, and the inhibition is reversed by irradiation of light at 450 nm in a light-intensity-dependent manner. The extent of the reversal is strongly dependent on the wavelength of the light used, the 450-nm light is most efficient. The finding provides direct evidence for the identification of the LTB4 omega-hydroxylase as a P-450. The P-450 seems to be also responsible for prostaglandin A1 (PGA1) omega-hydroxylation, but not for lauric acid omega-hydroxylation. The LTB4 omega-hydroxylation is competitively inhibited by PGA1, but not affected by lauric acid. The Ki value for PGA1 of 38 microM agrees with the Km value for PGA1 omega-hydroxylation of 40 microM. LTB4 inhibits the PGA1 omega-hydroxylation by rat liver microsomes in a competitive manner with the Ki of 43 microM, which is consistent with the Km for the LTB4 omega-hydroxylation of 42 microM. An antiserum raised against rabbit pulmonary PG omega-hydroxylase (P-450p-2) inhibits slightly the omega-hydroxylations of LTB4 and PGA1, while it has stronger inhibitory effect on lauric acid omega-hydroxylation. In addition to NADPH-cytochrome P-450 reductase, cytochrome b5 appears to participate in the LTB4 omega-hydroxylating system, since the reaction is inhibited by an antibody raised against the cytochrome b5 as well as one raised against the reductase.     

Hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by rat prostate microsomes: potent inhibition by imidazole-type antimycotic drugs and lack of inhibition by steroid 5 alpha-reductase inhibitors.

5 alpha-Dihydrotestosterone, the principal androgen mediating prostate growth and function in the rat, is formed from testosterone by steroid 5 alpha-reductase. The inactivation of 5 alpha-dihydrotestosterone involves reversible reduction to 5 alpha-androstane-3 beta,17 beta-diol by 3 beta-hydroxysteroid oxidoreductase followed by 6 alpha-, 7 alpha-, or 7 beta-hydroxylation. 5 alpha-Androstane-3 beta,17 beta-diol hydroxylation represents the ultimate inactivation step of dihydrotestosterone in rat prostate and is apparently catalyzed by a single, high-affinity (Km approximately 0.5 microM) microsomal cytochrome P450 enzyme. The present studies were designed to determine if 5 alpha-androstane-3 beta,17 beta-diol hydroxylation by rat prostate microsomes is inhibited by agents that are known inhibitors of androgen-metabolizing enzymes. Inhibitors of steroid 5 alpha-reductase (4-azasteroid analogs; 10 microM) or inhibitors of 3 beta-hydroxysteroid oxidoreductase (trilostane, azastene, and cyanoketone; 10 microM) had no appreciable effect on the 6 alpha-, 7 alpha-, or 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol (10 microM) by rat prostate microsomes. Imidazole-type antimycotic drugs (ketoconazole, clotrimazole, and miconazole; 0.1-10 microM) all markedly inhibited 5 alpha-androstane-3 beta,17 beta-diol hydroxylation in a concentration-dependent manner, whereas triazole-type antimycotic drugs (fluconazole and itraconazole; 0.1-10 microM) had no inhibitory effect. The rank order of inhibitory potency of the imidazole-type antimycotic drugs was miconazole greater than clotrimazole greater than ketoconazole. In the case of clotrimazole, the inhibition was shown to be competitive in nature, with a Ki of 0.03 microM. The imidazole-type antimycotic drugs inhibited all three pathways of 5 alpha-androstane-3 beta,17 beta-diol hydroxylation to the same extent, which provides further evidence that, in rat prostate microsomes, a single cytochrome P450 enzyme catalyzes the 6 alpha-, 7 alpha-, and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol. These studies demonstrate that certain imidazole-type compounds are potent, competitive inhibitors of 5 alpha-androstane-3 beta,17 beta-diol hydroxylation by rat prostate microsomes, which is consistent with the effect of these antimycotic drugs on cytochrome P450 enzymes involved in the metabolism of other androgens and steroids.     

The effect of pyridine and pyridine-N-oxide on the monooxygenase system of rat liver microsomes]

Effects of pyridine and pyridine-N-oxide on the monooxygenase system of rat liver microsomes have been studied. Pyridine (200 mg/kg) increased total cytochrome P-450 content and activated metabolism of some specific substrates 24 hours after injection. There was an increase in the degree of p-nitrophenol and chlorzoxazone hydroxylation due to increasing ethanol-induced cytochrome P-450IIE1 content. Pyridine was also able to induce cytochrome P-450IIB1 in rat microsomes; this reaction was accompanied by acceleration of 7-pentoxyresorufin 0-dealkylation. Cytochrome P-450IA1 appearance in liver microsomes was associated with increasing content of cytochrome P-450IA2. Dealkylation rates for specific substrates (7-ethoxyresorufin and 7-methoxyresorufin) were also increased. Similar to pyridine, pyridine-7-oxide induced cytochromes P-450IIE1, P-450IIB1/B2, and P-450IA1/A2, resulting in activation of specific substrate metabolism. Hence, pyridine and its derivative pyridine-N-oxide can be regarded as effective inducers of cytochrome P-450.     

Evaluation of alpha-cyano ethers as fluorescent substrates for assay of cytochrome P450 enzyme activity

We have previously reported the synthesis of four alpha-cyano-containing ethers based on 2-naphthaldehyde (2-NA) as cytochrome P450 (P450) fluorescent substrates. Activity detection was based on the formation of fluorescent 2-NA following substrate hydrolysis. A major limitation of these substrates was the need to remove NADPH, a required cofactor for P450 oxidation, before measuring 2-NA fluorescence. In this article, we report the synthesis of a new series of novel P450 substrates using 6-dimethylamino-2-naphthaldehyde (6-DMANA), which has a green fluorescent emission that is well separated from the NADPH spectrum. A major advantage of the 6-DMANA substrates is that NADPH removal is not required before fluorescence detection. We used eight alpha-cyano ether-based substrates to determine the O-dealkylation activity of human, mouse, and rat liver microsomes. In addition, substrate activities were compared with the commercial substrate 7-ethoxyresorufin (7-ER). The catalytic turnover rates of both the 6-DMANA- and 2-NA-based substrates were in some cases threefold faster than the catalytic turnover rate of 7-ER. The 2-NA-based substrates had greater turnover than did the 6-DMANA-based substrates. Murine and rat liver microsomes prepared from animals that had been treated with various P450 inducers were used to examine for isozyme-selective turnover of the substrates. The vastly improved optical properties and synthetic flexibility of the alpha-cyano ether compounds suggest that they are possibly good general P450 substrates.     

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. I. Properties of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) monomers.

Oligomers and monomers of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) isolated from the liver microsomes of phenobarbital-treated rabbits were examined for physicochemical properties and catalytic activities. As measured using laser correlation spectroscopy the particle sizes of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers were 14.8 +/- 1.7 and 19.2 +/- 1.4 nm, respectively. Twenty-four-hour incubation with Emulgen 913 at 4 degrees C at a molar ratio of 1:100 led to the monomerization of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers, the particle sizes diminishing to 6.1 +/- 1.3 and 5.2 +/- 0.4 nm, respectively. The thermal stability of NADPH-cytochrome P450 reductase monomers was the same as that of oligomers, whereas cytochrome P450 LM2 monomers were less thermostable than oligomers and cytochrome P450 in microsomes. Similar to cytochrome P450 LM2 oligomers and the microsomal hemoprotein, cytochrome P450 LM2 monomers formed complexes with type I and II substrates, but with Kd values higher than those of microsomes and cytochrome P450 LM2 oligomers. Kinetic parameters (Vmax and Km) of H2O2- and cumene hydroperoxide-dependent oxidation of benzphetamine and aniline in the presence of cytochrome P450 LM2 oligomers, monomers, and microsomes were determined. Peroxidase activities of the oligomers and monomers were the same, but were lower than those of microsomes. Thus the substitution of protein-protein interactions in cytochrome P450 LM2 oligomers with protein-detergent interactions in the monomers did not influence the catalytic properties of the hemoprotein.     

Comparative kinetic studies of the dealkylation reaction and steroid hydroxylations in various oxygen and carbon monoxide:oxygen atmospheres

The time-course kinetics of the cytochrome P-450-catalyzed dealkylations of the exogenous compounds benzphetamine, ethylmorphine, codeine, and 7-ethoxycoumarin were compared to the hydroxylation of the endogenous compound testosterone. Using liver microsomes from phenobarbital-induced rats, the time course of the demethylations of ethylmorphine, codeine, and especially benzphetamine was characterized by a fast initial phase of enzymatic activity and then a steady decline in the rate throughout the remainder of the reaction. In contrast, under the same experimental conditions, both the dealkylation of 7-ethoxycoumarin and the hydroxylation of testosterone showed no initial fast phase of activity and a constant rate of product formation for most of the remainder of the time course. The difference also held for the carbon monoxide inhibition studies in which the degree of inhibition of the demethylation reactions by a variety of CO:O₂ mixtures was time dependent, in contrast to the constant, time-independent degree of CO inhibition of the other two reactions. The kinetics of the demethylation reactions could not be explained by enzyme destruction, back reaction, or product adduct formation and were further confirmed by measurements of the rate of O₂ utilization and NADPH oxidation. The complexity of the demethylation reaction should be taken into consideration in any detailed studies of the monooxygenation reaction system.     

A comparison of aroclor 1254-induced and uninduced rat liver microsomes to human liver microsomes in phenytoin O-deethylation, coumarin 7-hydroxylation, tolbutamide 4-hydroxylation, S-mephenytoin 4'-hydroxylation, chloroxazone 6-hydroxylation and testosterone 6beta-hydroxylation

Aroclor 1254-induced rat liver homogenate supernatant (liver S-9) is routinely used as an exogenous metabolic activation system for the evaluation of mutagenicity of xenobiotics. The purpose of this study is to evaluate whether results obtained with Aroclor 1254-induced liver microsomes would be relevant to human. Aroclor 1254-induced and uninduced rat liver microsomes were compared to human liver microsomes in the metabolism of substrates which are known to be selectively metabolized by the major human cytochrome P450 (CYP) isoforms. The activities studied and the major CYP isoforms involved were as follows: phenacetin O-deethylation (CYP1A2); coumarin 7-hydroxylation, (CYP2A6); tolbutamide 4-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19); dextromethorphan O-demethylation (CYP2D6); chloroxazone 6-hydroxylation (CYP2E1); and testosterone 6beta-hydroxylation (CYP3A4). We found that both induced and uninduced rat liver microsomes were active in all the pathways studied with the exception of coumarin 7-hydroxylation. Coumarin 7-hydroxylation was observed with human liver microsomes but not the rat liver microsomes. Aroclor-1254 was found to induce all activities measured, with the exception of coumarin 7-hydroxylation. Dextromethorphan O-deethylation activity was higher in the rat liver microsomes than the human liver microsomes. Testosterone 6beta-hydroxylation activity was found to be similar between the human liver microsomes and the induced rat liver microsomes. Our results suggest that experimental data obtained with Aroclor 1254-induced rat liver microsomes may not always be relevant to human.