Molecular modeling of human cytochrome P450 2W1 and its interactions with substrates.

The human cytochrome P450 2W1 (CYP2W1) was categorized into the so-called "orphan" CYPs because of its unknown enzymatic function. However, recent studies showed that the recombinant CYP2W1 exhibited broad catalytic activity towards several chemicals. Furthermore, this enzyme was selectively expressed in some forms of cancers, whereas a very low expression was found in human normal issues. These render CYP2W1 as a potential drug target for cancer therapy. At present, however, little information is available on the active site topology and the substrate binding modes of CYP2W1. In this study, the three-dimensional model of CYP2W1 was constructed using the homology modeling method. Two known substrates, benzphetamine and indole, were then docked into the active site, and refined by molecular dynamics simulations. The interaction energy between the substrates and the enzyme was calculated and analyzed by using the MM-GBSA method. The results indicated that the constructed CYP2W1 model can account for the regioselectivity of this enzyme towards the known substrates and van der Waals interactions were the driving force for the substrate binding. Several key residues were identified to be responsible for the binding of indole and benzphetamine with CYP2W1. These findings provide useful information for the detailed characterization of the biological roles of CYP2W1 and structure-based drug design of this enzyme.

Ubiquinone synthesis in mitochondrial and microsomal subcellular fractions of Pneumocystis spp.: differential sensitivities to atovaquone.

The lung pathogen Pneumocystis spp. is the causative agent of a type of pneumonia that can be fatal in people with defective immune systems, such as AIDS patients. Atovaquone, an analog of ubiquinone (coenzyme Q [CoQ]), inhibits mitochondrial electron transport and is effective in clearing mild to moderate cases of the infection. Purified rat-derived intact Pneumocystis carinii cells synthesize de novo four CoQ homologs, CoQ7, CoQ8, CoQ9, and CoQ10, as demonstrated by the incorporation of radiolabeled precursors of both the benzoquinone ring and the polyprenyl chain. A central step in CoQ biosynthesis is the condensation of p-hydroxybenzoic acid (PHBA) with a long-chain polyprenyl diphosphate molecule. In the present study, CoQ biosynthesis was evaluated by the incorporation of PHBA into completed CoQ molecules using P. carinii cell-free preparations. CoQ synthesis in whole-cell homogenates was not affected by the respiratory inhibitors antimycin A and dicyclohexylcarbodiimide but was diminished by atovaquone. Thus, atovaquone has inhibitory activity on both electron transport and CoQ synthesis in this pathogen. Furthermore, both the mitochondrial and microsomal fractions were shown to synthesize de novo all four P. carinii CoQ homologs. Interestingly, atovaquone inhibited microsomal CoQ synthesis, whereas it had no effect on mitochondrial CoQ synthesis. This is the first pathogenic eukaryotic microorganism in which biosynthesis of CoQ molecules from the initial PHBA:polyprenyl transferase reaction has been unambiguously shown to occur in two distinct compartments of the same cell.

Metabolism of N,N',N"-triethylenethiophosphoramide by CYP2B1 and CYP2B6 results in the inactivation of both isoforms by two distinct mechanisms.

The anticancer drug N,N,"N"-triethylenethiophosphoramide (tTEPA) inactivated CYP2B6 and CYP2B1 in the reconstituted system in a time-, concentration-, and NADPH-dependent manner indicative of mechanism-based inactivation. The KI value for the inactivation of CYP2B1 was 38 microM, the kinact was 0.3 min(-1), and the t1/2 value was 2.5 min. Spectral carbon monoxide (CO) binding and high-performance liquid chromatography heme studies of the tTEPA-inactivated CYP2B1 suggest that the loss in the enzymatic activity was primarily due to the binding of a reactive tTEPA intermediate to the 2B1 apoprotein. Inactivation by tTEPA in the presence of 7-ethoxycoumarin, an alternate substrate, reduced the rate of inactivation of CYP2B1. Incubations with tTEPA and NADPH resulted in greater than 90% loss in the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation and testosterone hydroxylation activity of CYP2B1. In contrast, benzphetamine metabolism was significantly less inhibited (47%). CYP2B6 was inactivated by tTEPA with a KI value of 50 microM, a k inact value of 0.1 min(-1), and a t1/2 value of 14 min. However, unlike CYP2B1, the tTEPA-inactivated human isoform showed losses in the cytochrome P450 (P450) CO spectrum, the pyridine hemochrome spectrum, and in the amount of native heme that were comparable with the loss in the 7-EFC and benzphetamine activity, suggesting that activity loss was brought about by a tTEPA-reactive intermediate damaging the CYP2B6 heme. CYP2B6 could only be protected from the tTEPA-dependent inactivation by the 2B6-specific substrate bupropion but not by other substrates of CYP2B such as benzphetamine, testosterone, or 7-ethoxycoumarin. The data indicate that tTEPA metabolism by these two 2B isoforms results in inactivation of the P450s by two distinct mechanisms.

Heme and apoprotein modification of cytochrome P450 2B4 during its oxidative inactivation in monooxygenase reconstituted system.

The mechanism of the cytochrome P450 2B4 modification by hydrogen peroxide (H2O2) formed as a result of partial coupling of NADPH-dependent monooxygenase reactions has been studied in the monooxygenase system reconstituted from the highly purified microsomal proteins: cytochrome P450 2B4 (P450) and NADPH-cytochrome P450 reductase in the presence of detergent Emulgen 913. It was found, that H2O2-mediated P450 self-inactivation during benzphetamine oxidation is accompanied by heme degradation and apoenzyme modification. The P450 heme modification involves the heme release from the enzyme under the action of H2O2 formed within P450s active center via the peroxycomplex decay. Additionally, the heme lost is destroyed by H2O2 localized outside of enzyme's active center. The modification of P450 apoenzyme includes protein aggregation that may be due to the change in the physico-chemical properties of the inactivated enzyme. The modified P450 changes the surface charge that is confirmed by the increasing retention time on the DEAE column. Oxidation of amino acid residues (at least cysteine) may lead to the alteration into the protein hydrophobicity. The appearance of the additional ionic and hydrophobic attractions may lead to the increase of the protein aggregation. Hydrogen peroxide can initiate formation of crosslinked P450 dimers, trimers, and even polymers, but the main role in this process plays nonspecific radical reactions. Evidence for the involvement of hydroxyl radical into the P450 crosslinking is carbonyl groups formation.

Cytochrome P450-dependent drug oxidation activities in liver microsomes of various animal species including rats, guinea pigs, dogs, monkeys, and humans.

Levels of cytochrome P450 (P450 or CYP) proteins immunoreactive to antibodies raised against human CYP1A2, 2A6, 2C9, 2E1, and 3A4, monkey CYP2B17, and rat CYP2D1 were determined in liver microsomes of rats, guinea pigs, dogs, monkeys, and humans. We also examined several drug oxidation activities catalyzed by liver microsomes of these animal species using eleven P450 substrates such as phenacetin, coumarin, pentoxyresorufin, phenytoin, S-mephenytoin, bufuralol, aniline, benzphetamine, ethylmorphine, erythromycin, and nifedipine; the activities were compared with the levels of individual P450 enzymes. Monkey liver P450 proteins were found to have relatively similar immunochemical properties by immunoblotting analysis to the human enzymes, which belong to the same P450 gene families. Mean catalytic activities (on basis of mg microsomal protein) of P450-dependent drug oxidations with eleven substrates were higher in liver microsomes of monkeys than of humans, except that humans showed much higher activities for aniline p-hydroxylation than those catalyzed by monkeys. However, when the catalytic activities of liver microsomes of monkeys and humans were compared on the basis of nmol of P450, both species gave relatively similar rates towards the oxidation of phenacetin, coumarin, pentoxyresorufin, phenytoin, mephenytoin, benzphetamine, ethylmorphine, erythromycin, and nifedipine, while the aniline p-hydroxylation was higher and bufuralol 1'-hydroxylation was lower in humans than monkeys. On the other hand, the immunochemical properties of P450 proteins and the activities of P450-dependent drug oxidation reactions in dogs, guinea pigs, and rats were somewhat different from those of monkeys and humans; the differences in these animal species varied with the P450 enzymes examined and the substrates used. The results presented in this study provide useful information towards species-related differences in susceptibilities of various animal species regarding actions and toxicities of drugs and xenobiotic chemicals.

The role of cytochrome P450 3A (CYP3A) isoform(s) in oxidative metabolism of testosterone and benzphetamine in human adult and fetal liver.

Testosterone metabolism was studied in human adult and fetal liver microsomes. In fetal livers 6 beta-hydroxylase (6 beta OH) activity (1-2% of adult activity) and 2 alpha-hydroxylase (2 alpha OH) activity (about 40% of adult activity) were present. Also some fetal livers produced two unknown metabolites. Androstenedione was formed in all fetal livers studied (10-20% of adult activity). Testosterone hydroxylations at 6 beta-, 2 beta-, 15 alpha- and 15 beta-positions were associated with CYP3A isoform(s) in adult liver, because they were strongly inhibited by midazolam, a known substrate for CYP3A4 and by anti-CYP3A4 antibody. Fetal liver activities were consistently inhibited less than the activities in adult livers. The formation of androstenedione was not affected by these inhibitors in fetal or adult liver microsomes. Benzphetamine N-demethylase activity in the fetal livers was about 40% of adult activity. Anti-CYP3A4 antibody had no effect on that activity in fetal or in adult liver microsomes, whereas a monoclonal antibody 1-68-11 (generated against rat CYP2C11) slightly inhibited benzphetamine N-demethylase activity in adult liver. This study indicates that human fetal and adult liver are dissimilar in their testosterone metabolism pattern. The formation of androstenedione from testosterone in fetal liver may have a physiological role. Testosterone hydroxylases are less inhibited by anti-CYP3A4 antibody, midazolam and progesterone in fetal than in adult liver.

Characterization of the cytochrome P-450 monooxygenase system in nonciliated bronchiolar epithelial (Clara) cells isolated from mouse lung.

The nonciliated bronchiolar epithelial (Clara) cell of the mouse is highly susceptible to toxicants that undergo metabolic activation, presumably because this cell type has high levels of cytochrome P-450 monooxygenases. As a first step in further defining the role of Clara cells in pulmonary xenobiotic activation and detoxication, we have isolated Clara cells (75 to 80% purity) and characterized them morphologically and biochemically. The identity of Clara cells, confirmed by transmission electron microscopy, was based on several features, including abundant agranular endoplasmic reticulum, large mitochondria, and dense secretory granules. Immunocytochemistry of isolated mouse cells showed that the majority were positive with antibodies against three major components of the pulmonary cytochrome P-450 monooxygenase system, cytochrome P-450 isozymes 2 (IIB), 5 (IVB), and NADPH cytochrome P-450 reductase, purified from rabbit lung. The isolated cells also showed a positive reaction with an antibody against the cytochrome P-450 isozyme that is active in the stereoselective metabolism of naphthalene, cytochrome P-450 mN (mN). Immunocytochemistry using the antibody against cytochrome P-450 isozyme 6 (IA1), purified from rabbit lung, showed no reaction in the isolated cells. The presence of intact cytochrome P-450 protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blots of homogenates of isolated cell preparations. The N-demethylation of benzphetamine and epoxidation of naphthalene occurred at easily measurable rates in incubations of isolated Clara cells. In contrast, diols, quinones, and monohydroxylated benzo(a)pyrene metabolites, analyzed by high performance liquid chromatography, were undetectable in extracts of Clara cells incubated with 3H-labeled substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic oxidation of the carcinogens 4-aminobiphenyl and 4,4'-methylene-bis(2-chloroaniline) by human hepatic microsomes and by purified rat hepatic cytochrome P-450 monooxygenases.

Metabolic N-oxidation and ring-oxidation of carcinogenic arylamines by hepatic cytochromes P-450 are generally regarded as critical activation and detoxification pathways, respectively. Two arylamines with known human exposure, 4-aminobiphenyl (ABP) and 4,4'-methylene-bis(2-chloroaniline) (MOCA), have been examined as substrates for 10 different purified rat hepatic cytochromes P-450 and for human liver microsomal preparations from 22 individuals. Metabolites were analyzed by high-performance liquid chromatography and flow scintillation techniques. As reported for certain other carcinogenic arylamines, the isosafrole-inducible isozyme, P-450ISF-G, had the highest catalytic activity for ABP N-oxidation (13.6 nmol/min/nmol P-450), but P-450BNF-B, P-450UT-A, P-450UT-F, and P-450PB-B also showed appreciable activity. Ring-oxidation of ABP occurred only to a minor extent. In contrast, N-oxidation of MOCA was preferentially catalyzed by the phenobarbital-inducible enzymes, P-450PB-B and P-450PB-D (9.0 and 6.6 nmol/min/nmol P-450, respectively). MOCA ring-oxidation and methylene carbon oxidation showed varied cytochrome P-450 selectivity and accounted for 14 to 79% of total oxidation products. There was a 44-fold variation in rates of ABP N-oxidation in the 22 human liver microsomal preparations, while rates of N-oxidation of MOCA varied only 8-fold. Ring/methylene carbon-oxidation of MOCA accounted for 6-19% of total oxidation products in the case of the human microsomal preparations, whereas ring-oxidation of ABP accounted for less than 7% of total oxidation. In addition, there was a strong correlation (R = 0.90) between rates of ABP N-oxidation and phenacetin O-deethylation, which is considered a human genetic polymorphism. Moreover, both the ABP N-oxidation and phenacetin O-deethylation activities of human liver microsomes showed a good correlation (R = 0.72) with the levels of cytochrome P-450 immunochemically related to rat P-450ISF-G. These data indicate that specific inducible and constitutive cytochromes P-450 are involved in the metabolic activation and detoxification of the carcinogens ABP and MOCA. Therefore, individual profiles of cytochromes P-450, affected by both environmental and genetic factors, may be significant determinants of individual susceptibility to arylamine carcinogenesis.

Cytochrome P-450-dependent covalent binding of carbon disulfide to rat liver microsomal protein in vitro and its prevention by reduced glutathione.

Carbon disulfide, a hepatotoxic solvent, is metabolized by liver microsomal enzymes to reactive sulfur atoms which get bound to the microsomal enzymes, causing inhibition of the enzyme system. These studies were carried out to examine whether glutathione can protect the liver enzymes from the sulfur binding and against carbon disulfide toxicity. When liver microsomes isolated from phenobarbital-pretreated rats were incubated with 35S-CS2, NADPH and glutathione, almost 60% decrease in sulfur binding to microsomal protein was observed under the experimental conditions. It was further observed that the addition of glutathione to microsomal incubations resulted in almost complete recovery of the activity of the enzyme system as measured by cytochrome P-450 concentration and benzphetamine metabolism. The data suggest that the presence of glutathione in sufficient amount in the liver of subject exposed to CS2 may significantly decrease the liver toxicity of this highly toxic compound.

Mechanistic biotransformations of some amphetamine drugs.

Amphetamine, fenfluramine and benzphetamine were the drugs investigated for the isolation of toxic metabolites using the biochemical mechanism of cytochrome P-450 monooxygenase mediated reaction. NH3 derived from amphetamine should be innocuous unless the in vivo ammonia detoxifying mechanism is overwhelmed thus culminating in ammonia intoxication in cerebral tissues with consequent concomitant convulsion. +CF3 electrophile derived from fenfluramine is potentially reactive with nucleophiles of proteins, carbohydrates, lipids, DNA and RNA. The derivation of .CF3 was discussed. Methylbenzylamine was derived from benzphetamine. This, in the nitrosating environment of the gastrointestinal tract, could yield the carcinogenic methylbenzylnitrosamine.

Efficacy of activated charcoal and other agents in the reduction of hepatotoxic effects of a single dose of aflatoxin B1 in chickens.

Liver injury caused by a toxic dose of aflatoxin B1 (AFB1) (6 mg/kg, p.o.) in experimental chickens was measured by observing changes in hepatic microsomal cytochrome P-450 and the activity of microsomal benzphetamine N-demethylase and serum glutamic oxaloacetic transaminase (SGOT). However, simultaneous administration of activated charcoal, reduced glutathione (GSH), cysteine, selenium, beta-carotene or fisetin with aflatoxin B1 considerably reduced the toxic injury to liver as measured by the above parameters.

Alterations of microsomal monooxygenase system and carcinogen metabolism by streptozotocin-induced diabetes in rats.

The effects of diabetes on the liver microsomal monooxygenase enzymes and carcinogen metabolism have been studied in rats. Treatment with streptozotocin causes a marked enhancement in microsomal N-nitrosodimethylamine (NDMA) demethylase activity. The enhancement is due mainly to the induction of a high affinity NDMA demethylase (Km, approximately 0.05 mM) which is accompanied by the induction of a protein species with mol. wt. of 50,000. The treatment also induces aniline hydroxylase whose activity is in parallel with NDMA demethylase. Streptozotocin-induced diabetes also increases the metabolism of N-nitrosomethylethylamine but not that of N-nitrosomethylaniline or N-nitrosomethylbenzylamine. On the other hand, diabetes decreases the metabolism of benzo[a]pyrene, benzphetamine, and ethylmorphine. The result suggest that diabetes causes an alteration of the composition of cytochrome P-450 isozymes; the forms efficient in metabolizing NDMA are increased while certain other forms of cytochrome P-450 are decreased.

Role of colonic cytochrome P-450 in large bowel carcinogenesis.

Rat colon mucosa microsomes contain a competent mixed function oxidase system that hydroxylates the N-methyl drugs benzphetamine and ethylmorphine, the O-alkyl drugs p-nitroanisole and p-nitrophenetole and the polycyclic carcinogen benzo[alpha]pyrene. The colon system's hydroxylation activities can be selectively induced by pretreatment with phenobarbital or beta-naphthoflavone and can be selectively inhibited by SKF-525A or 7,8-benzoflavone. The colon microsomal system has been solubilized with the non-ionic detergent Renex 690 and resolved by column chromatography into its components cytochrome P-450 and cytochrome P-450 reductase. Colon cytochrome P-450 and cytochrome P-450 reductase can be recombined to reconstitute hydroxylation activity. The colon system is also able to activate carcinogens to mutagenic metabolites as demonstrated in the Ames test system. In addition, the activity of the colon system is markedly increased by pretreatment with gastrointestinal hormones.

Studies on aflatoxin B1 toxicity in female rats pretreated with an oral contraceptive agent.

PIP: The effect of oral administration of the contraceptive agent Ovral-28 (100 mcg/day) on the hepatotoxic effects of aflatoxin B1 was studied in female rats. The animals received Ovral-28 for 16 days prior to the ip administration of a single dose of aflatoxin B1. Pretreatment with the contraceptive agent diminished the hepatic necrosis observed in animals receiving aflatoxin B1 only. Pretreatment also completely arrested the decrease in the level of hepatic reduced glutathione and partially reversed the decreased activity of the hepatic drug-metabolizing enzyme system induced by aflatoxin B1.^ieng