Reduction of 7-alkoxyresorufins by NADPH-cytochrome P450 reductase and its differential effects on their O-dealkylation by rat liver microsomal cytochrome P450

Antibody-inhibition experiments established that the induction of cytochrome P450c is largely responsible for the marked increase in liver microsomal 7-ethoxyresorufin O-dealkylation in rats treated with 3-methylcholanthrene, whereas the induction of cytochrome P450b and/or P450e is largely responsible for the marked increase in 7-pentoxy- and 7-benzyloxyresorufin O-dealkylation in rats treated with phenobarbital. When reconstituted with NADPH-cytochrome P450 reductase and lipid, purified cytochrome P450c catalyzed the O-dealkylation of 7-ethoxyresorufin at a rate of approximately 30 nmol/nmol P450/min, which far exceeded the rate catalyzed by either purified cytochromes P450b and P450e or microsomal cytochrome P450c. In contrast, purified cytochrome P450b and P450e were poor catalysts of the O-dealkylation of 7-pentoxy- and 7-benzyloxyresorufin. However, purified cytochrome P450b is an excellent catalyst of several other reactions, such as the N-demethylation of benzphetamine, the hydroxylation of testosterone, and the O-dealkylation of 7-ethoxycoumarin. The low rate of 7-pentoxyresorufin O-dealkylation catalyzed by purified cytochrome P450b did not reflect a requirement for cytochrome b5, and could not be ascribed to an artifact of the method used to measure the formation of resourufin. The catalytic activity of purified cytochrome P450b toward 7-pentoxyresorufin was consistently low over a range of substrate and lipid concentrations, and was not stimulated by sodium deoxycholate (which stimulates the N-demethylation of benzphatamine by purified cytochrome P450b). Evidence is presented which indicates that cytochrome P450c catalyzes the O-dealkylation of both the oxidized and reduced forms of 7-ethoxyresorufin, with perhaps a slight preference for the reduced form. In contrast, cytochrome P450b preferentially catalyzes the O-dealkylation of the oxidized form of 7-pentoxyresorufin. Conditions that favored formation of the reduced form of 7-ethoxyresorufin tended to stimulate its O-dealkylation by purified cytochrome P450c, whereas conditions that favored formation of the reduced form of 7-pentoxyresorufin decreased its rate of O-dealkylation by purified cytochrome P450b. Such conditions included a molar excess of NADPH-cytochrome P450 reductase over cytochrome P450, the presence of superoxide dismutase, and the presence of DT-diaphorase (liver cytosol).(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification of rat liver microsomal cytochrome P-450b without the use of nonionic detergent

Sodium cholate, Emulgen 911, and (3-[(-cholamidopropyl)-dimethyl- ammonio]-1-propanesulfonate) (CHAPS) were selected to examine the effects of ionic, nonionic, and zwitterionic detergents on testosterone hydroxylation catalyzed by four purified isozymes of rat liver microsomal cytochrome P-450, namely P-450a, P-450b, P-450c, and P-450h, in reconstituted systems containing optimal amounts of dilauroylphosphatidylcholine and saturating amounts of NADPH- cytochrome P-450 reductase (reductase). The major phenobarbital-inducible form of rat liver microsomal cytochrome P-450, designated P-450b, was extremely sensitive to the inhibitory effects of Emulgen 911, which is used in several procedures to purify this and other forms of cytochrome P-450. In contrast, sodium cholate and CHAPS had little effect on the catalytic activity of cytochrome P-450b, even at ten times the concentration of Emulgen 911 effecting 50% inhibition (IC-50). By substituting the zwitterionic detergent CHAPS for Emulgen 911, we purified cytochrome P-450b without the use of nonionic detergent. The protein is designated cytochrome P-450b^* to distinguish it from cytochrome P-450b purified with the use of Emulgen 911. NADPH-cytochrome P-450 reductase was also purified both with and without the use of nonionic detergent. The absolute spectra of cytochrome P-450b and P-450b^* were indistinguishable, as were the carbon monoxide (CO)- and metyrapone-difference spectra of the dithionite-reduced hemoproteins. When reconstituted with NADPH-cytochrome P-450 reductase and dilauroylphosphatidylcholine, cytochromes P-450b and P-450b^* catalyzed the N-demethylation of benzphetamine and aminopyrine, the 4-hydroxylation of aniline, the O-dealkylation of 7-ethoxycoumarin, the 3-hydroxylation of hexobarbital, and the 6-hydroxylation of zoxazolamine. Both hemo-proteins catalyzed the 16α- and 16β-hydroxylation of testosterone, as well as the 17-oxidation of testosterone to androstenedione. Both hemoproteins were poor catalysts of erythromycin demethylation and benzo[a]pyrene 3-/9-hydroxylation. The rate of biotransformation catalyzed by cytochrome P-450b^* was up to 50% greater than the rate catalyzed by cytochrome P-450b when reconstituted with either reductase or reductase^*. The activity of cytochrome P-450b and P-450b^* increased up to 50% when reconstituted with reductase^* instead of reductase. In addition to establishing the feasibility of purifying an isozyme of rat liver microsomal cytochrome P-450 without the use of nonionic detergent, these results indicate that the catalytic activity of cytochrome P-450 is not unduly compromised by residual contamination with the nonionic detergent Emulgen 911.     

Purification of a cytochrome P-450 from pig kidney microsomes catalysing the 25-hydroxylation of vitamin D3.

Cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 was purified from pig kidney microsomes. The enzyme fraction contained 7 nmol of cytochrome P-450/mg of protein and showed only one protein band with an apparent Mr of 50,500 upon SDS/polyacrylamide-gel electrophoresis. The purified cytochrome P-450 catalysed 25-hydroxylation of vitamin D3 up to 1,000 times more efficiently, and 25-hydroxylation of 1 alpha-hydroxyvitamin D3 up to 4000 times more efficiently, than the microsomes. The cytochrome P-450 required microsomal NADPH-cytochrome P-450 reductase for catalytic activity. Mitochondrial ferredoxin and ferredoxin reductase could not replace microsomal NADPH-cytochrome P-450 reductase. The enzyme preparation showed no detectable 25-hydroxylase activity towards vitamin D2 or 1 alpha-hydroxylase activity towards 25-hydroxyvitamin D3. CO inhibited the 25-hydroxylation by more than 85%. Mannitol, hydroquinone, catalase and superoxide dismutase did not affect the 25-hydroxylation. The possible role of the kidney microsomal cytochrome P-450 in the metabolism of vitamin D3 is discussed.     

Hydroxylations in biosynthesis of bile acids. Isolation of a cytochrome P-450 from rabbit liver mitochondria catalyzing 26-hydroxylation of C27-steroids

A cytochrome P-450 catalyzing 26-hydroxylation of C27-steroids was purified from liver mitochondria of untreated rabbits. The enzyme fraction contained 10 nmol of cytochrome P-450/mg of protein and showed only one protein band with a minimum Mr = 53,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified mitochondrial cytochrome P-450 showed apparent molecular weight similar to microsomal cytochromes P-450LM4 but differed in spectral and catalytic properties from these microsomal isozymes. The purified cytochrome P-450 catalyzed 26-hydroxylation of cholesterol, 5-cholestene-3 beta,7 alpha-diol, 7 alpha-hydroxy-4-cholesten-3-one, 5 beta-cholestane-3 alpha,7 alpha-diol, and 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol up to 1000 times more efficiently than the mitochondria. The cytochrome P-450 required both ferredoxin and ferredoxin reductase for catalytic activity. Microsomal NADPH-cytochrome P-450 reductase could not replace ferredoxin and ferredoxin reductase. The cytochrome P-450 was inactive in 7 alpha-, 12 alpha- and 25-hydroxylations of C27-steroids. The results suggest that mitochondrial 26-hydroxylation of various C27-steroids is catalyzed by the same species of cytochrome P-450.     

A conspicuous down-regulating effect of morphine on essential steroid hydroxylation reactions and certain drug N-demethylations.

This study was conducted to explore the potency of morphine to induce reductions of specific cytochrome P450 isoenzyme functions. Male Sprague-Dawley rats were treated with escalating doses (20-125 mg/kg per day) of morphine for 2 weeks in order to study the effects on the following cytochrome P450 catalyzed reactions: 16 alpha-hydroxylation of dehydroepienderosterone (DHA) and progesterone; 17 alpha- and 21-hydroxylation of progesterone; N-demethylation of ethymorphine, codeine and morphine as well as O-dealkylation of ethylmorphine and codeine. 16 alpha-Hydroxylation of DHA and progesterone and 17 alpha-hydroxylation of progesterone decreased to 18, 12 and 10% of control activities, respectively. The N-demethylation of ethylmorphine and codeine decreased to 34 and 43% of control activities, respectively. Morphine treatment had no effect on the 21-hydroxylation reactions or the O-dealkylation of ethylmorphine or codeine. A monoclonal antibody (Mab) against rat liver cytochrome P450 2 c/RLM 5 exerted a 66-73% inhibition of the N-demethylation of ethylmorphine and codeine, respectively, whereas the O-dealkylation reactions were not affected. This Mab inhibited the 16 alpha- and 17 alpha-hydroxylation of DHA and progesterone, whereas the 21-hydroxylation reactions were unaffected. The steroid hydroxylation reactions in rat adrenals were not altered upon morphine treatment. Our data suggest that a major part of the 16 alpha- and 17 alpha-steroid hydroxylations are catalyzed by the same (or closely related) cytochrome(s) P450 as the opioid N-demethylation reactions.     

A direct, highly sensitive fluorometric assay for a microsomal cytochrome P450-mediated O-demethylation using a novel coumarin analog as substrate

A highly sensitive fluorometric assay for the determination of monooxygenase activity in liver microsomes is described. The assay is based on the use of 3-chloro-7-methoxy-4-methylcoumarin which is demethylated to 3-chloro-7-hydroxy-4-methylcoumarin. The rate of formation of 3-chloro-7-hydroxy-4-methylcoumarin was recorded as an increase of fluorescence (lambdaA = 380 nm, lambdaF = 480 nm) with time. When 3-chloro-7-methoxy-4-methylcoumarin was incubated in the presence of MgCl2 and NADPH with rat liver microsomes, a continuous increase of the fluorescence could be measured. The reaction proceeded linearly for about 10 min and at least up to a concentration of 0.1 mg/ml of microsomal protein. Besides 3-chloro-7-hydroxy-4-methylcoumarin a hydroxylated derivative of the substrate was formed as a second metabolite during the incubation. Using an excitation wavelength of 380 nm and a fluorescence/emission wavelength of 480 nm, the fluorescence of this substance (lambdaA = 338 nm, lambdaF = 422 nm) amounted only to about 1% of the fluorescence of the main product. The use of 3-chloro-7-methoxy-4-methylcoumarin as substrate enables the fluorometric determination of the O-dealkylation activity of a cytochrome P450-dependent monooxygenase system in rat liver which is inducible by phenobarbital but not by 3-methylcholanthrene.     

Purification and NH2-terminal sequence of cytochrome P-450 from kidney microsomes of untreated male rats

The major form of cytochrome P-450, P-450K-5, was purified from kidney microsomes of untreated male rats with high-performance liquid chromatography with anion-exchange and hydroxylapatite columns. The monomeric molecular weight of P-450K-5 was 52000 on SDS-polyacrylamide gel electrophoresis and the CO-reduced absorption maximum was at 452 nm. P-450K-5 catalyzed the omega- and (omega-1)-hydroxylation of lauric acid, but was inefficient in the N-demethylation of benzphetamine and the O-dealkylation of 7-ethoxycoumarine. The NH2-terminal sequence of P-450K-5 was quite different from cytochrome P-450s purified from rat hepatic microsomes.     

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.     

Mouse liver phenobarbital-inducible P450 system: purification, characterization, and differential inducibility of four cytochrome P450 isozymes from D2 mouse

Three novel cytochrome P450 isozymes were purified from phenobarbital (PB)-treated D2 mouse liver microsomes and compared to the previously characterized coumarin 7-hydroxylase, P450Coh. The molecular masses were 56.5, 55, 51, and 49.5 kDa, and the peaks of the reduced CO complexes were at 450, 447.5, 451.5, and 449 nm for P450PBI, P450PBII, P450PBIII, and P450Coh, respectively. The NH2-terminal sequences suggest that these isozymes belong to the P450 gene subfamilies 2B, 1A, 2C, and 2A, respectively. On the basis of reconstituted activities and microsomal immunoinhibition studies, P450Coh was the sole catalyst of coumarin 7-hydroxylation. P450PBI was the major isozyme catalyzing the high Km 7-pentoxyresorufin O-dealkylation. This reaction was also mediated at a slower rate by the low Km isozyme, P450PBII. P450PBIII contributed significantly to the microsomal O-deethylation of 7-ethoxyresorufin and N-demethylation of benzphetamine. Western blotting and dot immunobinding analyse of microsomes showed that the induction patterns of the isozymes were different. PB and TCPO-BOP induced all isozymes variably: P450PBI (19- and 31-fold), P450PBII (2- and 3-fold), P450PBIII (9- and 4-fold), and P450Coh (about 2-fold). Pyrazole induced only P450Coh, while all other isozymes were decreased by 30 to 60%. The changes in the microsomal amounts of these isozymes correlated generally well with the variation in the immunoinhibitable enzyme activities. On the basis of the structural and catalytic properties, immunochemical characteristics, and induction profiles, all three isozymes were different from each other and from the previously characterized P450Coh. This mouse PB-inducible P450 model may be valuable in further studies on the induction mechanisms of PB and TCPOBOP.     

Characterization of three cytochrome P450s purified from renal microsomes of untreated male rats and comparison with human renal cytochrome P450

Three renal cytochrome P450s (P450 K-2, K-4, and K-5) were purified from renal microsomes of untreated male rats. Also, the human renal cytochrome P450 (P450 HK) was partially purified from renal microsomes and its properties were compared with those of the rat renal cytochrome P450s. The molecular weight of P450 K-2, K-4, and K-5 was 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The absolute spectrum of the oxidized forms indicated that they had the low-spin state of heme, and the CO-reduced spectral maxima of P450 K-2, K-4, and K-5 were at 449, 451, and 452 nm, respectively. NH2-terminal sequence analysis of P450 K-2, K-4, and K-5 showed that these forms were different from hepatic cytochrome P450s purified previously. P450 K-2, K-4, and K-5 catalyzed the O-dealkylation of 7-ethoxycoumarin but were not efficient in the hydroxylation of testosterone. Aminopyrine was metabolized by P450 K-2 and K-4 but not by P450 K-5. Lauric acid was metabolized efficiently by all of these forms in the presence of cytochrome b5. The regiospecificity of these forms toward lauric acid was different. P450 K-2 hydroxylated lauric acid only at the (omega-1)-position, not at the omega-position. P450 K-4 and K-5 hydroxylated lauric acid at both the omega- and (omega-1)-positions. The ratios of omega/(omega-1)-hydroxylation activity of P450 K-4 and K-5 were 2.5 and 7.8, respectively. Human P450 HK was purified 220-fold and its specific content was 2.0 nmol/mg of protein. The Soret maxima of P450 HK were at 418 nm for the oxidized form, 416 nm for the reduced form, and 450 nm for the CO-reduced form. P450 HK catalyzed the O-dealkylation of 7-ethoxycoumarin but was not efficient in aminopyrine N-demethylation or testosterone hydroxylation. P450 HK had high lauric acid omega- and (omega-1)-hydroxylation activities in the presence of cytochrome b5, especially omega-hydroxylation. These properties resembled those of P450 K-5 most closely. Anti-P450 K-5 antibody cross-reacted with P450 HK as well as P450 K-5 and only one band was stained on immunostained Western blotting for partially purified P450 HK. The molecular weight of P450 HK was 52,000 on Western blotting.     

Coumarin substrates for cytochrome P450 2D6 fluorescence assays

A set of nine 4-aminomethyl-7-alkoxycoumarin derivatives was synthesized and characterized as substrates for O-dealkylation by recombinant cytochrome P450 2D6, a major human enzyme involved in drug metabolism. Enzymatic O-dealkylation yields 7-hydroxycoumarins, which have useful fluorescence properties. The substrates, which differed in substitution at the amino and 7-hydroxy positions, varied in terms of catalytic efficiency of O-dealkylation and in their selectivity as substrates for cytochrome P450 2D6 in human liver microsomes. Several of the compounds are useful as cytochrome P450 2D6 substrates in single-phase, rapid-throughput assays.     

Preferential induction of the rat hepatic P450 I proteins by the food carcinogen 2-amino-3-methyl-imidazo[4,5-f]quinoline

1. Administration of the food carcinogen, 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) to rats gave rise to significant dose-dependent increases in the microsomal O-deethylations of ethoxycoumarin and ethoxyresorufin but had no effect on the O-dealkylation of pentoxyresorufin and the NADPH-dependent reduction of cytochrome c, and decreased the N-demethylation of dimethylnitrosamine. Microsomal cytochrome b5 and total cytochrome P-450 levels decreased following the administration of the carcinogen. 2. Hepatic microsomal preparations from IQ-treated animals were much more efficient than control in activating the premutagen 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole to mutagenic intermediates in the Ames test. 3. Immunoquantification of two of the major families of cytochrome P-450, namely P450 I and P450 II B, using ELISA techniques showed that treatment with IQ induced the apoprotein levels of the P450 I family but not of P450 II B. 4. Immunoblot analysis employing polyclonal antibodies against P450 I revealed that IQ induced both isoenzymes of this family, namely P450 I A1 and A2. 5. It is concluded that IQ is an inducer of the rat hepatic monooxygenases, selectively inducing the P450 I family as predicted by a computer-graphic analysis of its dimensions which showed that it is a large, essentially planar, molecule.     

A convenient assay for mephenytoin 4-hydroxylase activity of human liver microsomal cytochrome P-450

A simple and rapid method for the determination of (S)-mephenytoin 4-hydroxylase activity by human liver microsomal cytochrome P-450 has been developed. [Methyl-14C] mephenytoin was synthesized by alkylation of S-nirvanol with 14CH3I and used as a substrate. After incubation of [methyl-14C]mephenytoin with human liver microsomes or a reconstituted monooxygenase system containing partially purified human liver cytochrome P-450, the 4-hydroxylated metabolite of mephenytoin was separated by thin-layer chromatography and quantified. The formation of the metabolite depended on the incubation time, substrate concentration, and cytochrome P-450 concentration and was found to be optimal at pH 7.4. The Km and Vmax rates obtained with a human liver microsomal preparation were 0.1 mM and 0.23 nmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450, respectively. The hydroxylation activity showed absolute requirements for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH in a reconstituted monooxygenase system. Activities varied from 5.6 to 156 pmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450 in 11 human liver microsomal preparations. The basic system utilized for the analysis of mephenytoin 4-hydroxylation can also be applied to the estimation of other enzyme activities in which phenol formation occurs.     

Characterization of a major form of rat hepatic microsomal cytochrome P-450 induced by isoniazid

Cytochrome P-450j has been purified to electrophoretic homogeneity from isoniazid-treated adult male rats; and this enzyme appears to be a major protein induced in hepatic microsomes after administration of isoniazid, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hemoprotein has a minimum molecular weight of approximately 51,500, and the ferrous-carbonyl complex of cytochrome P-450j has a Soret maximum at 451-452 nm. The oxidized heme iron appears to be predominately in the high spin state as deduced from the Soret maximum at 395 nm. Ethylisocyanide binds to ferrous cytochrome P-450j to yield spectral maxima at approximately 458 and 430 nm with a resultant 458/430 ratio of 0.7 at pH 7.4. Cytochrome P-450j has no measurable catalytic activity for the metabolism of benzo[a]pyrene (3- and 9-hydroxylation), hexobarbital, testosterone, and 5 alpha-androstane-3 alpha,17 beta-diol-3,17-disulfate. Low, but detectable, catalytic activity is obtained for the metabolism of 7-ethoxycoumarin, benzphetamine, p-nitroanisole, zoxazolamine, and 2-hydroxylation of 17 beta-estradiol. In contrast, cytochrome P-450j effectively catalyzes p-hydroxylation of aniline with a turnover of 12.7 nmol/min/nmol cytochrome P-450j. Hydroxyl radical scavengers, Fe-EDTA, superoxide dismutase, and catalase have no effect on aniline p-hydroxylation catalyzed by cytochrome P-450j. Cytochrome P-450j is distinct from nine other rat hepatic microsomal cytochromes P-450 (P-450a-P-450i) previously purified in this laboratory, as well as different isozymes described by other investigators, based on several parameters including minimum molecular weight, spectral properties, and catalytic activity. In Ouchterlony double diffusion plates, antibodies against cytochromes P-450a-P-450f show no cross-reaction with cytochrome P-450j. Structural differences among cytochromes P-450a-P-450j are apparent from the NH2-terminal sequence of cytochrome P-450j, as well as the electrophoretic profiles of proteolytic digests of the hemoproteins.     

Reversible and time-dependent inhibition of the hepatic cytochrome P450 steroidal hydroxylases by the proestrogenic pesticide methoxychlor in rat and human

Methoxychlor, a currently used pesticide, is demethylated and hydroxylated by several hepatic microsomal cytochrome P450 enzymes. Also, methoxychlor undergoes metabolic activation, yielding a reactive intermediate (M*) that binds irreversibly and apparently covalently to microsomal proteins. The study investigated whether methoxychlor could inhibit or inactivate certain liver microsomal P450 enzymes. The regioselective and stereoselective hydrox-ylation of testosterone and the 2-hydroxylation of estradiol (E₂) were utilized as markers of the P450 enzymes inhibited by methoxychlor. Both reversible and time-dependent inhibition were examined. Coincubation of methoxychlor and testosterone with liver microsomes from phenobarbital treated (PB-microsomes) male rats, yielded marked diminution of 2α- and 16α-testosterone hydroxylation, indicating strong inhibition of P4502C11 (P450h). Methoxychlor moderately inhibited 2β-, 7α-, 15α-, 15β-, and 16β-hydroxylation and androstenedi-one formation. There was only a weak inhibition of 6β-ydroxylation of testosterone. The methox-ychlor-mediated inhibition of 6β-hydroxylation was competitive. By contrast, when methoxychlor was permitted to be metabolized by PB-microsomes or by liver microsomes from pregnenolone-16α-car-bonitrile treated rats (PCN-microsomes) prior to addition of testosterone, a pronounced time-dependent inhibition of 6β-hydroxylation was observed, suggesting that methoxychlor inactivates the P450 3A isozyme(s). The di-demethylated methoxychlor (bis-OH-M) and the tris-hydroxy (ca-techol) methoxychlor metabolite (tris-OH-M) inhibited 6β-hydroxylation in PB-microsomes competitively and noncompetitively, respectively; however, these methoxychlor metabolites did not exhibit a time-dependent inhibition. Methoxychlor inhibited competitively the formation of 7α-hydroxytestosterone (7α-OH-T) and 16α-hydroxy-testosterone (16α-OH-T) but exhibited little or no time-dependent inhibition of generation of these metabolites, indicating that P450s 2A1, 2B1/B2, and 2C11 were inhibited but not inactivated. Methoxychlor inhibited in a time-dependent fashion the 2-hydroxylation of E2 in PB-microsomes. However, bis-OH-M exhibited solely reversible inhibition of the 2-hydroxylation, supporting our conclusion that the inactivation of P450s does not involve participation of the demethylated metabolites. Both competitive inhibition and time-dependent inactivation of human liver P450 3A (6β-hydroxylase) by methoxychlor, was observed. As with rat liver microsomes, the human 6β-hydroxylase was inhibited by bis-OH-M and tris-OH-M competitively and noncompetitively, respectively. Testosterone and estradiol strongly inhibited the irreversible binding of methoxychlor to microsomal proteins. This might explain the “clean” competitive inhibition by methoxychlor of the 6β-OH-T formation when the compounds were coin-cubated. Glutathione (GSH) has been shown to interfere with the irreversible binding of methoxychlor to PB-microsomal proteins. The finding that the coincubation of GSH with methoxychlor partially diminishes the time-dependent inhibition of 6β-hydroxylation provides supportive evidence that the inactivation of P450 3A isozymes by methoxychlor is related to the formation of M*.     

Identification of human liver cytochromes P450 by using MALDI-TOF mass spectrometry

Proteomic approaches have been used for detection and identification of cytochromes P450 forms from highly purified membrane preparations of human liver. These included the protein separation by 2D-and/or 1D-electrophoresis and molecular scanning of a SDS-PAGE gel fragment in a range 45–66 kDa (this area corresponds molecular weights of cytochromes P450). The analysis of protein content was statistically evaluated by means of an original 1D-ZOOMER software package which allowed to carry out the processing of mass spectra mixture instead of individual mass spectra used by standard techniques. In the range 45–66 kDa we identified 13 microsomal membrane proteins including such cytochrome P450 forms as CYPs 1A2, 1B1, 2A6, 2E1, 2C8, 2C9, 2C10, 2D6, 3A4, 4A11, 4F2. Study of enzymatic activities of human liver microsomal cytochrome P450 isoforms CYP 1A, 2B, 3A, and 2E revealed the decrease in the rates of O-dealkylation and N-demethylation catalyzed by CYP 450 1A1/1A2 and 3A4 under pathological conditions, whereas 7-benzyloxyresorufin-O-debenzylase activity (which characterizes the total activity of CYP 2B and CYP 2C), the activities of CYP 2E1 (methanol oxidation), 7-pentoxyresorufin-O-dealkylation (CYP 2B), 7-ethoxy-and 7-methoxycoumarin-O-dealkylases (CYP 2B1) remained basically unchanged.     

Purification and characterization of two forms of 2,3,4,7,8-pentachlorodibenzofuran-inducible cytochrome P-450 in hamster liver

Two forms of cytochrome P-450 (P-450) from liver microsomes of hamsters treated with 2,3,4,7,8-pentachlorodibenzofuran (PenCDF), which possesses the potent acute toxicity and 3-methylcholanthrene (MC)-type inducing ability of liver microsomal monooxygenases in animals, were purified and characterized. These P-450 forms, designated as hamster P-450H and hamster P-450L, had the molecular masses of 52 and 50 kDa, respectively, and showed the absorption maximum of CO-reduced difference spectra at 446 nm. The absolute spectra of their oxidized forms indicated that hamster P-450H was in high-spin state and hamster P-450L was in low-spin state. A part of PenCDF injected into hamster was tightly bound to purified hamster P-450H at a ratio of 0.107 nmol PenCDF/nmol P-450. In a reconstituted system, both hamster P-450H and hamster P-450L showed relatively low catalytic activities for 3-hydroxylation of benzo[a]pyrene and O-deethylations of both 7-ethoxyresorufin and 7-ethoxycoumarin, while they both catalyzed 7 alpha- and 2 alpha-hydroxylations of testosterone effectively to a similar extent. Addition of cytochrome b5-to a reconstituted system accelerated the formation of 7 alpha-hydroxytestosterone 5.3-fold with hamster P-450L and 2.2-fold with hamster P-450H. In addition, hamster P-450H catalyzed estradiol 2-hydroxylation at a high rate but hamster P-450L did not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Highly sensitive high-performance liquid chromatographic assay for coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation by human liver cytochrome P450 enzymes

A highly sensitive method for the determination of coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation by human cytochrome P450 (P450 or CYP) enzymes was developed using high-performance liquid chromatography (HPLC). The newly developed HPLC method was found to be about 100-fold more sensitive than the previous spectrofluorimetric method in detecting the metabolite 7-hydroxycoumarin (umbelliferone). With this high sensitivity, the kinetics of coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation catalyzed by human liver microsomal and recombinant P450 enzymes were determined more precisely. With 36 different substrate concentrations in these two reactions, coumarin 7-hydroxylation was found to be catalyzed mainly by a single enzyme CYP2A6 and 7-ethoxycoumarin was oxidized by at least two enzymes CYP2E1 and CYP1A2 in human liver microsomes.     

Cytochrome P450 and steroid 21-hydroxylation in microsomes from beef adrenal cortex

Cytochrome P450 in beef adrenal cortex microsomal preparations reacted with progesterone and with 17-hydroxyprogesterone at pH 7.4 to produce Type I spectral changes. The magnitude of the spectral shift produced by addition of progesterone or 17-hydroxyprogesterone was related to the concentration of cytochrome P450 (over P450 concentration range of 0.1 to 0.3 μM). Prior saturation of cytochrome P450 with 17-hydroxyprogesterone prevented further spectral shift with the addition of progesterone. On the other hand, saturation of cytochrome P450 with progesterone decreases the expected shift with 17-hydroxyprogesterone by more than 50% but did not prevent the shift. The difference spectra were diminished by more than 50% at pH 9.0.The addition of NADPH resulted in loss of the spectral shifts and production of 21-hydroxylated products, predominantly DOC and 11-deoxycortisol. These reactions were not inhibited by their specific products. The rate of 21-hydroxylation was linearly related to microsomal protein (and microsomal P450) concentration. The 21-hydroxylation of progesterone was competitively inhibited by 17-hydroxyprogesterone; inhibition of the 21-hydroxylation of 17-hydroxyprogesterone by progesterone was not demonstrated.     

Spectral evidence for a liver mitochondrial cytochrome P450 involved in bile acid metabolism.

Optical difference spectroscopy of liver mitochondria has revealed the presence of a cytochrome P450 species by its ligand reactions with carbon monoxide, metyrapone and diethylphenylphosphine. Its concentration of 0.15 nmol/mg mitochondrial protein is high enough to be detectable by ESR also. A microsomal contamination of the mitochondria could be excluded. Mitochondrial cytochrome P450 forms an enzyme-substrate complex with 5beta-cholestane-3alpha, 7alpha, 12alpha-triol with Ks value very similar to the Km value of the 26-hydroxylation of this substrate. This supports the existence in liver mitochondria of a cytochrome P450-dependent 26-monooxygenase for bile acid precursors, as previously postulated by us on the basis of a photochemical action spectrum.