Purification of cytochrome P-450 from liver microsomes of phenobarbital-treated guinea pigs

Cytochrome P-450 was purified from phenobarbital-treated guinea pigs to a specific content of 19.8 nmoles per mg of protein, and was free of cytochrome b₅ and NADPH-cytochrome $\text{c}$ reductase. The purified cytochrome P-450 gave a single protein band on sodium dodecylsulfate-polyacrylamide gel electrophoresis, and an apparent molecular weight of about 49,000 was estimated. Benzphetamine N-demethylation activity could be reconstituted by mixing the purified cytochrome, NADPH-cytochrome $\text{c}$ reductase and phosphatidylcholine.     

N-aralkylated derivatives of 1-aminobenzotriazole as isozyme-selective, mechanism-based inhibitors of guinea pig hepatic cytochrome P-450 dependent monooxygenase activity

The mechanism-based inactivation of hepatic cytochrome P-450 by the suicide inhibitor 1-aminobenzotriazole and two of its derivatives, N-benzyl-1-aminobenzotriazole and N-alpha-methylbenzyl-1-aminobenzotriazole, was investigated in microsomes from untreated, phenobarbital-induced, and beta-naphthoflavone-induced guinea pigs. Microsomal 7-ethoxyresorufin O-deethylase, 7-pentoxyresorufin O-dealkylase, and benzphetamine N-demethylase activities, and cytochrome P-450 content were determined following incubation with 1-aminobenzotriazole and its analogues. The loss of hepatic cytochrome P-450 content and monooxygenase activity was dependent on inhibitor concentration and required NADPH. N-Benzyl-1-aminobenzotriazole and N-alpha-methylbenzyl-1-aminobenzotriazole were more potent inhibitors of monooxygenase activity than the parent compound in microsomes from untreated and phenobarbital-induced guinea pigs. In microsomes from phenobarbital-induced guinea pigs, N-alpha-methylbenzyl-1-aminobenzotriazole (10 microM) was highly selective for the inactivation of the major cytochrome P-450 isozyme catalyzing 7-pentoxyresorufin O-dealkylation (the guinea pig ortholog of P-450IIB1) compared with those isozymes catalyzing 7-ethoxyresorufin O-deethylation or benzphetamine N-demethylation (88 +/- 3% loss of activity vs. 35 +/- 11 and 13 +/- 7%, respectively). N-Benzyl-1-aminobenzotriazole was also selective for the inactivation of 7-pentoxyresorufin O-dealkylase activity, but to a lesser degree (56 +/- 6 vs. 31 +/- 8 and 21 +/- 8%, respectively). In hepatic microsomes from untreated guinea pigs, the two N-substituted analogues were selective for the inhibition of 7-pentoxyresorufin O-dealkylation compared with benzphetamine N-demethylation, but not 7-ethoxyresorufin O-deethylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lack of mechanism-based inactivation of rat hepatic microsomal cytochromes P450 by doxorubicin.

Administration of the antineoplastic doxorubicin to rodents causes depression of hepatic cytochrome P450 (CYP) dependent biotransformation, an effect that has been partially attributed to the ability of doxorubicin to stimulate microsomal lipid peroxidation. Since doxorubicin can be bioactivated by the CYP/NADPH-CYP reductase system to products that bind covalently to microsomal protein, we hypothesized that doxorubicin functions as a mechanism-based inactivator of hepatic microsomal CYPs and (or) NADPH-CYP reductase under conditions in which doxorubicin-stimulated NADPH-dependent lipid peroxidation is minimized. In vitro studies were conducted with hepatic microsomes isolated from untreated and phenobarbital-treated male rats. Unlike the positive control carbon tetrachloride, doxorubicin (10 microM) did not stimulate NADPH-dependent lipid peroxidation in microsomal incubations containing EDTA (1.5 mM). Doxorubicin did not cause NADPH-dependent loss of microsomal CYP, heme, or steroid hydroxylation activities selective for CYP2A, CYP2B, CYP2C11, and CYP3A. The positive control 1-aminobenzotriazole caused marked NADPH-dependent decreases in all of these parameters. Neither doxorubicin nor 1-aminobenzotriazole caused NADPH-dependent loss of NADPH-CYP reductase activity, and neither compound altered the immunoreactive protein levels of CYP2B, CYP2C11, CYP3A, and NADPH-CYP reductase. These results indicate that a pharmacologically relevant concentration of doxorubicin does not cause direct mechanism-based inactivation of hepatic microsomal CYPs or NADPH-CYP reductase, suggesting that the ability of doxorubicin to depress hepatic CYP-mediated biotransformation in vivo is due to lipid peroxidation mediated heme destruction, altered heme metabolism, and (or) decreased expression of selected CYP enzymes.     

Mechanistic studies of cytochrome P450 2B1 inactivation by xanthates

Xanthates have previously been shown to inactivate the phenobarbital-inducible rat cytochrome P450 2B1 as well as its human homologue P450 2B6. The inactivation was mechanism-based and the loss in enzymatic activity was due to covalent binding of a reactive xanthate intermediate to the P450 2B1 apoprotein. In this report, we investigated various mechanistic events to elucidate the individual step(s) in the P450 catalytic cycle that are compromised due to the inactivation by xanthates. Different xanthates displayed typical type I binding spectra and the spectral binding constants were in the low-millimolar range. A dramatic loss in 7-ethoxy-4-(trifluoromethyl)coumarin activity was observed when P450 2B1 was incubated with five different xanthates in the presence of NADPH. With the exception of the C14 xanthate, virtually no loss of absorbance at 418 or 450 nm in the reduced-CO complex was observed. Long-chain xanthates were able to affect the rate of the first electron transfer in the P450 catalytic cycle by stabilizing the heme in its low-spin state. n-Octyl xanthate (C8) metabolism led to very little observable oxy-ferro intermediate complex formation. The alternate oxidant tert-butyl hydroperoxide was able to support the inactivation reaction of C8 in the absence of reductase or NADPH. The rates of reduction of native, C8-exposed, and C8-inactivated P450 2B1 were measured. The C8-inactivated P450 had a 62% lower rate of reduction in the absence or presence of benzphetamine compared to the native enzyme. Product formation of the three enzyme preparations was quantified with benzphetamine as the substrate. The C8-inactivated P450 2B1 exhibited a much lower rate of NADPH consumption and formation of formaldehyde. However, the ratio of H2O2 to formaldehyde production increased from 1:1 for the native enzyme to 2.8:1 for the inactivated P450. Together these observations indicate that the covalent modification of P450 2B1 by a reactive intermediate of xanthates reduces the rate of the first electron transfer by the reductase and also leads to uncoupling of electron transfer from product formation by diverting a greater proportion of the electrons to H2O2 formation.     

Induction and suppression of cytochrome P450 isoenzymes and generation of oxygen radicals by procymidone in liver,kidney and lung of CD1 mice

Although chronic administration of procymidone (a widely used dicarboximide fungicide) leads to an increased incidence of liver tumors in mice, short-term genotoxicity studies proved negative. As cytochrome P450 (CYP) induction has been linked to non-genotoxic carcinogenesis, we investigated whether procymidone administration causes induction of CYP-dependent monooxygenases in liver, kidney and lung microsomes of male Swiss Albino CD1 mice after single or repeated (daily for three consecutive days) i.p. treatment with either 400 or 800 (1/10 or 1/20 of the DL(50)) mgkg(-1) b.w. procymidone. CYP content and CYP3A1/2, 1A1, 1A2, 2B1/2, 2E1, 2A, 2D9 and 2C11 supported oxidations were studied using either the regio- and stereo-selective hydroxylation of testosterone as multibiomarker or highly specific substrates as probes of various CYPs. While a single dose was uneffective, multiple procymidone administration lead to marked inductions of various monooxygenases: CYP3A1/2 in liver and lung (as measured by N-demethylation of aminopyrine and testosterone 6 beta-hydroxylase); CYP2E1 in liver (p-nitrophenol hydroxylation); CYP1A1 in liver and kidney (deethylation of ethoxyresorufin). Several hydroxylations were induced in the liver, including the CYP2A-linked 7 alpha (14-fold) as well as 6 alpha (22-fold), 6 beta, 16 beta and 2 beta hydroxylases. The pattern of inductions/suppressions recorded in the three different tissues suggests that procymidone exerts complex effects on the CYP profile. Tissue-specific trends included a large number of inductions in the liver and suppressions in the lung. The main inductions were corroborated by immunoblotting analyses and Northern blotting showed that inductions of CYP3A1/2, CYP2E1 and CYP1A1/2 were paralleled by increased mRNA levels. It was also found that CYP over-expression generates large amounts of reactive oxygen species (ROS), especially in liver. These data may explain why in vitro short-term genotoxicity studies on procymidone were negative, whereas in vivo long-term carcinogenesis studies turned out positive: long-term CYP induction (e.g. oxygen centered free radicals over-production) can have a co-carcinogenic and/or promoting potential.     

Occurrence and biological function of cytochrome P450 monooxygenases in the actinomycetes

Many species within the order Actinomycetales contain one or more soluble cytochrome P450 monooxygenases, often substrate-inducible and responsible for a variety of xenobiotic transformations. The individual cytochromes exhibit a relatively broad substrate specificity, and some strains have the capacity to synthesize large amounts of the protein(s) to compensate for low catalytic turnover with some substrates. All three of the Streptomyces cytochromes sequenced to date are exclusive members of one P450 family, CYP105. In several instances, monooxygenase activity arises from induction of a P450 and associated ferredoxin, or of a P450 only, suggesting that some essential electron donor proteins (reductase and ferredoxin) are not co-ordinately regulated with the cytochrome. The overall properties of these systems suggest an adaptive strategy whose twofold purpose is to maintain a competitive advantage via the production of secondary metabolites, and, whenever possible, to utilize unusual growth substrates by introducing metabolites from these reactions into the more substrate-specific primary metabolic pathways.     

Selective, competitive and mechanism-based inhibitors of human cytochrome P450 2J2

Twenty five derivatives of the drugs terfenadine and ebastine have been designed, synthesized and evaluated as inhibitors of recombinant human CYP2J2. Compound 14, which has an imidazole substituent, is a good non-competitive inhibitor of CYP2J2 (IC(50)=400nM). It is not selective towards CYP2J2 as it also efficiently inhibits the other main vascular CYPs, such as CYP2B6, 2C8, 2C9 and 3A4; however, it could be an interesting tool to inhibit all these vascular CYPs. Compounds 4, 5 and 13, which have a propyl, allyl and benzo-1,3-dioxole terminal group, respectively, are selective CYP2J2 inhibitors. Compound 4 is a high-affinity, competitive inhibitor and alternative substrate of CYP2J2 (K(i)=160+/-50nM). Compounds 5 and 13 are efficient mechanism-based inhibitors of CYP2J2 (k(inact)/K(i) values approximately 3000Lmol(-1)s(-1)). Inactivation of CYP2J2 by 13 is due to the formation of a stable iron-carbene bond which occurs upon CYP2J2-catalyzed oxidation of 13 with a partition ratio of 18+/-3. These new selective inhibitors should be interesting tools to study the biological roles of CYP2J2.     

Prediction and analysis of the modular structure of cytochrome P450 monooxygenases

Background  

Cytochrome P450 monooxygenases (CYPs) form a vast and diverse family of highly variable sequences. They catalyze a wide variety of oxidative reactions and are therefore of great relevance in drug development and biotechnological applications. Despite their differences in sequence and substrate specificity, the structures of CYPs are highly similar. Although being in research focus for years, factors mediating selectivity and activity remain vague.     

Immunochemical detection of cytochrome P450C-M/F and NADPH-cytochrome P450 reductase in rat liver and kidney

The localization and distribution of NADPH-cytochrome P450 reductase and cytochrome P450C-M/F were investigated immunohistochemically in the liver and the kidney of untreated rats employing both an unlabelled antibody peroxidase-antiperoxidase method and a peroxidase labelled primary antibody technique. In both immunohistochemical procedures, the reductase and P450C-M/F were detected in hepatocytes throughout the liver. In contrast, the reductase and P450C-M/F in the kidney were only detectable in the proximal tubule cells.     

Differential regulation of hepatic cytochrome P450 monooxygenases in streptozotocin-induced diabetic rats

The present investigation was carried out to study the expression of major cytochrome P450 (CYP) isozymes in streptozotocin-induced diabetes with concomitant insulin therapy. Male Sprague-Dawley rats were randomly assigned to untreated control, streptozotocin-induced diabetic, insulin-treated groups and monitored for 4 weeks. Uncontrolled hyperglycemia in the early phase of diabetes resulted in differential regulation of cytochrome P450 isozymes. CYP1B1, CYP1A2, heme oxygenase (HO)-2 proteins and CYP1A2-dependent 7-ethoxyresorufin O-deethylase (EROD) activity were upregulated in the hepatic microsomes of diabetic rats. Insulin therapy ameliorated EROD activity and the expression of CYP1A2, CYP1B1 and HO-2 proteins. In addition, CYP2B1 and 2E1 proteins were markedly induced in the diabetic group. Insulin therapy resulted in complete amelioration of CYP2E1 whereas CYP2B1 protein was partially ameliorated. By contrast, CYP2C11 protein was decreased over 99% in the diabetic group and was partially ameliorated by insulin therapy. These results demonstrate widespread alterations in the expression of CYP isozymes in diabetic rats that are ameliorated by insulin therapy.     

Expression of four phenobarbital-inducible cytochrome P-450s in liver, kidney, and lung of rats

Specific antibodies were prepared against cytochromes P450 PB-1, PB-2, PB-4, and PB-5 purified from hepatic microsomes of male rats treated with phenobarbital. With these antibodies, the levels of these four cytochrome P450s in hepatic, renal, and pulmonary microsomes of male rats that were untreated, treated with phenobarbital, or treated with 3-methylcholanthrene were examined. P450 PB-1 and PB-2 were present in moderate amounts in hepatic microsomes of untreated male rats and were induced 2- to 3-fold with phenobarbital. Also, the expression of these forms was suppressed by 3-methylcholanthrene. These forms were not detected in the renal or pulmonary microsomes of untreated rats or rats treated with phenobarbital or 3-methylcholanthrene. P450 PB-4 and PB-5 were found in the hepatic microsomes of untreated male rats at a low level but were induced with phenobarbital more than 50-fold. P450 PB-4 and PB-5 were not detected in renal microsomes; only P450 PB-4 or a closely related form was present in the pulmonary microsomes of untreated male rats, and its level was not changed by phenobarbital treatment. The constitutive presence of P450 PB-4 in pulmonary microsomes was confirmed by the investigation of testosterone metabolism. Purified P450 PB-4 had high testosterone 16 alpha- and 16 beta-hydroxylation activity in a reconstituted system. The testosterone 16 beta-hydroxylation activity of hepatic microsomes was induced with phenobarbital, and more than 90% of the testosterone 16 beta-hydroxylation activity of hepatic microsomes from rats treated with phenobarbital was inhibited by anti-P450 PB-4 antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conversion and synthesis of chemicals catalyzed by fungal cytochrome P450 monooxygenases: A review

Cytochrome P450s (also called CYPs or P450s) are a superfamily of heme-containing monooxygenases. They are distributed in all biological kingdoms. Most fungi have at least two P450-encoding genes, CYP51 and CYP61, which are housekeeping genes that play important roles in the synthesis of sterols. However, the kingdom fungi is an interesting source of numerous P450s. Here, we review reports on fungal P450s and their applications in the bioconversion and biosynthesis of chemicals. We highlight their history, availability, and versatility. We describe their involvement in hydroxylation, dealkylation, oxygenation, C═C epoxidation, C–C cleavage, C–C ring formation and expansion, C–C ring contraction, and uncommon reactions in bioconversion and/or biosynthesis pathways. The ability of P450s to catalyze these reactions makes them promising enzymes for many applications. Thus, we also discuss future prospects in this field. We hope that this review will stimulate further study and exploitation of fungal P450s for specific reactions and applications.     

Modification of cytochrome P450 1A2 enzymes by the mechanism-based inactivator 2-ethynylnaphthalene and the photoaffinity label 4-azidobiphenyl.

2-Ethynylnaphthalene (2EN) had previously been demonstrated to be a mechanism-based inactivator of rat cytochrome P450 (P450) 1A2 [Hammons, G.J., Alworth, W.L., Hopkins, N.E., Guengerich, F. P., & Kadlubar, F. F. (1989) Chem. Res. Toxicol. 2, 367-374]. In this work 2EN was also demonstrated to be a useful inactivator of rabbit P450 1A2 (k(inactivation) 0.094 min-1, K(i) 11 microM) but it did not inactivate human P450 1A2, although the sequences of the three proteins are approximately 80% identical. Rat and rabbit P450 1A2 were modified by incubation with NADPH-P450 reductase, NADPH, and [3H]2EN to levels of 0.35 and 0.47 nmol of adduct (nmol of P450)-1, respectively. In each case only a single tryptic peptide was labeled; recovery of labeled peptides was low under the acidic HPLC conditions. The rabbit P450 1A2 peptide FQELMAAVGR (positions 175-184) and the rat P450 1A2 peptide L(S)QQYGDVLQIR (positions 67-78) were identified. 4-Azidobiphenyl (4-N3BP) was developed as a photoaffinity label for P-450 1A2 proteins because of its similarity to 4-aminobiphenyl, a known substrate for the enzymes. 4-N3BP was shown to be photolyzed with 350-nm light and radioactive label could be incorporated into rat P450 1A2. Labeling of the protein was found to be saturable with increasing concentrations of 4-N3BP and up to 0.59 nmol of label could be incorporated (nmol P450 1A2)-1. The substrate 4-aminobiphenyl and the competitive inhibitor 7,8-benzoflavone blocked photolabeling of P450 1A2 with 4-N3BP, and 4-N3BP inhibited N-hydroxylation of 4-aminobiphenyl by P450 1A2 in the usual enzyme assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

The involvement of cytochrome P-450 in the NADH-dependent O-demethylation of p-nitroanisole in phenobarbital-treated rabbit liver microsomes.

Addition of $\text{p}$-nitroanisole to a reaction mixture containing phenobarbital-pretreated rabbit liver microsomes brings about an increase the reoxidation rate of NADH-reduced cytochrome b₅. Addition of partially purified cytochrome b₅ to a solution containing microsomes results in a marked increase in both NADH- and NADPH-dependent O-demethylation of $\text{p}$-nitroanisole. $\text{p}$-Nitroanisole also increases the rate of NADH mediated cytochrome P-450 reduction. From these and other results described in the Discussion section, we confirm that electrons required for NADH-dependent O-demethylation of $\text{p}$-nitroanisole is transfered from NADH to cytochrome P-450 via cytochrome b₅ and that cytochrome P-450 is the enzyme which catalyzes $\text{p}$-nitroanisole O-demethylation.     

The role of cytochrome P450 lysine residues in the interaction between cytochrome P450IA1 and NADPH-cytochrome P450 reductase.

Cytochrome P450IA1 (purified from hepatic microsomes of beta-naphthoflavone-treated rats) has been covalently modified with the lysine-modifying reagent acetic anhydride. Different levels of lysine residue modification in cytochrome P450IA1 can be achieved by varying the concentration of acetic anhydride. Modification of lysine residues in P450IA1 greatly inhibits the interaction of P450IA1 with NADPH-cytochrome P450 reductase. Modification of 1.0 and 3.3 mol lysine residues per mole P450IA1 resulted in 30 and 95% decreases, respectively, in 7-ethoxycoumarin hydroxylation by a reconstituted P450IA1/reductase complex. However, modification of 3.3 mol lysine residues per mole P450IA1 decreased only cumene hydroperoxide-supported P450-dependent 7-ethoxycoumarin hydroxylation by 30%. Spectral and fluorescence studies showed no indication of global conformational change of P450IA1 even with up to 8.8 mol lysine residues modified per mole P450IA1. These data suggest that at least three lysine residues in P450IA1 may be involved in the interaction with reductase. Identification of lysine residues in P450IA1 possibly involved in this interaction was carried out by [14C]acetic anhydride modification, trypsin digestion, HPLC separation, and amino acid sequencing. The lysine residue candidates identified in this manner were K97, K271, K279, and K407.     

Prostaglandin involvement in lung C-fiber activation by substance P in guinea pigs.

Airway hyperresponsiveness is a cardinal feature of asthma. Lung C-fiber activation induces central and local defense reflexes that may contribute to airway hyperresponsiveness. Initial studies show that substance P (SP) activates C fibers even though it is produced and released by these same C fibers. SP may induce release of other endogenous mediators. Bradykinin (BK) is an endogenous mediator that activates C fibers. The hypothesis was tested that SP activates C fibers via BK release. Guinea pigs were anesthetized, and C-fiber activity (FA), pulmonary insufflation pressure (PIP), heart rate, and arterial blood pressure were monitored before and after intravenous injection of capsaicin (Cap), SP, and BK. Identical agonist challenges were repeated after infusion of an antagonist cocktail of des-Arg9-[Leu8]-BK (10(-3) M, B1 antagonist), and HOE-140 (10(-4) M, B2 antagonist). After antagonist administration, BK increased neither PIP nor FA. Increases in neither PIP nor FA were attenuated after Cap or SP challenge. In a second series of experiments, Cap and SP were injected before and after infusion of indomethacin (1 mg/kg iv) to determine whether either agent activates C fibers through release of arachidonic acid metabolites. Indomethacin administration decreased the effect of SP challenge on FA but not PIP. The effect of Cap on FA or PIP was not altered by indomethacin. In subsequent experiments, C fibers were activated by prostaglandin E2 and F2alpha. Therefore, exogenously applied SP stimulates an indomethacin-sensitive pathway leading to C-fiber activation.     

Mechanism-based inactivation of cytochrome P450 3A4 by L-754,394

Mechanism-based inactivation of human liver P450 3A4 by L-754,394, a Merck compound synthesized as a potential HIV protease inhibitor, was investigated using recombinant P450 3A4. Enzyme inactivation was characterized by a small partition ratio (3.4 or 4.3 +/- 0.4), i.e., the total number of metabolic events undergone by the inhibitor divided by the number of enzyme inactivating events, lack of reversibility upon extensive dialysis, no decrease in the characteristic 450-nm species relative to control, and covalent modification of the apoprotein. The major and minor products formed during the inactivation of P450 3A4 were the monohydroxylated and the dihydrodiol metabolites of L-754,394, respectively. L-754,394 that had been adducted to P450 3A4 was hydrolyzed under the conditions used for SDS-PAGE, Ni(2+) affinity chromatography, and proteolytic digestion. In addition, the modification was not stable to the acidic conditions of HPLC separation and CNBr digestion. The labile nature of the peptide adduct and the nonstoichiometric binding of the inactivating species to P450 3A4 precluded the direct identification of a covalently modified amino acid residue or the peptide to which it was attached. However, Tricine SDS-PAGE in combination with MALDI-TOF-MS and homology modeling, allowed I257-M317 to be tentatively identified as an active site peptide, while prior knowledge of the stability of N-, O-, and S-linked conjugates of activated furans implicates Glu307 as the active site amino acid that is labeled by L-754, 394.     

The involvement of cytochrome P450 monooxygenases in methanol elimination in Drosophila melanogaster larvae

Methanol is one of the most common short-chain alcohols in fermenting fruits, the natural food of the fruit fly, Drosophila melanogaster. The larvae cope continuously with methanol at various concentrations in order to survive and develop. In the present article, we found toxicities of dietary methanol and formaldehyde were enhanced by piperonyl butoxide, but not by 3-amino-1, 2, 4-triazole, 4-methylpyrazole, diethylmeleate, and triphenyl phosphate, when assessing by the combination index method. These results reveal that cytochrome P450 monooxygenases (CYPs), rather than catalases, alcohol dehydrogenases, glutathione S-transferases, and esterases, participate in methanol metabolism. Moreover, methanol exposure dramatically increased CYP activity. The ratios of the CYP activities in treated larvae to those in control reached, respectively, up to 3.0-, 3.9-, and 2.7-fold, at methanol concentrations of 22.6, 27.9, and 34.5 mg/g diet. In addition, methanol exposure greatly up-regulated the mRNA expression level of five Cyp genes, which were Cyp304a1, Cyp9f2, Cyp28a5, Cyp4d2, and Cyp4e2. Their resulting proteins were suggested as the candidate enzymes for methanol metabolism in D. melanogaster larvae.