Nmol Of P-450 Research Articles

Role of cytochrome P-450 in the stimulation of microsomal production of reactive oxygen species by ferritin

Microsomes can remove iron from ferritin by a superoxide-dependent reaction. The released iron can then catalyze formation of a variety of reactive oxygen species. Experiments were carried out to evaluate the role of cytochrome P-450 in the release of iron from ferritin, and whether induction of certain P-450 isoforms alters ferritin-dependent reactive oxygen radical production. Rats were treated with phenobarbital, 3-methylcholanthrene, 4-methylpyrazole, or saline to produce microsomes with varying P-450 content and composition. Oxidations of 2.7′-dichlorofluorescein diacetate to a fluorescent product and chemiluminescence were used as indices of production of reactive oxygen species. The extreme sensitivity of these reactions to trolox, a potent chain-breaking oxidant, indicates the involvement of lipid peroxidation products in these reactions. In the absence of ferritin, formation of reactive oxygen species was higher in microsomes from the treated rats compared to saline controls when results were expressed on a per mg protein basis but not per nmol P-450, suggesting that the increased content of total P-450 (2-fold increases) rather than the population of isoforms was responsible for ·h, increase. Superoxide dismutase had no effect on the non-ferritin catalyzed reactions. Ferritin increased production of reactive oxygen species with all the microsomal preparations; the increase by ferritin was completely prevented by superoxide dismutase. The net increase by ferritin was higher in microsomes from the treated rats compared to saline controls, but this, again, largely reflected the increased content, rather than the type of isoforms of P-450 present. Similar results were obtained with either NADPH or NADH as microsomal reductants, although NADPH was much more effective in supporting ferritin-dependent reactive oxygen formation. In microsomes from phenobarbital-treated rats, anti-CYP2B1/B2 IgG completely prevented the NADPH- and NADH-dependent increases in reactive oxygen formation produced by ferritin. Anti-cytochrome b 5 IgG produced partial inhibition of the ferritin-stimulation. These results indicate that P-450, and to a lesser extent, cytochrome b 5, play a role in the ferritin-dependent increase in formaion of reactive oxygen species with either NADPH or NADH, most likely reflecting the requirement of these enzymes for microsomal production of superoxide anion.

Metabolic activation of the potent carcinogen dibenzo[a,l]pyrene by human recombinant cytochromes P450, lung and liver microsomes.

The metabolic activation of dibenzo[a,l]pyrene (DB[a,l]P), recently considered the most potent carcinogen among all polycyclic aromatic hydrocarbons, to the 11,12-dihydrodiol, a precursor of the ultimate carcinogens, the 11,12-diol-13,14-epoxides, was investigated using eleven human recombinant cytochrome P450s, as well as human lung and liver microsomes. Of all human P450s, 1A1 was the most active in the metabolism of DB[a,l]P (310 pmol/min, nmol P450) and had 5-23-fold higher catalytic activity than other P450s examined. The order of activity in the formation of the 11,12-dihydrodiol was as follows: 1A1 (116 pmol/min, nmol P450) > 2C9 (29) > 1A2 (22) > 2B6 (18) > 3A4 (16) > others (< or = 5). The Km of 1A1 for DB[a,l]P and Vmax for the formation of 11,12-dihydrodiol were 3.9 microM and 0.13/min, respectively. Liver microsomes from 14 individuals were shown to metabolize DB[a,l]P and the rates for production of 11,12-dihydrodiol ranged from 4 to 71 pmol/min, nmol P450. Lung microsomes from six organ donors formed the 11,12-dihydrodiol at a rate from 0.1 to 1.3 pmol/min, mg of microsomal protein. These findings describe the potential of individual P450s present in liver and lung to contribute to the metabolic activation and the carcinogenicity of DB[a,l]P.

Microsomal oxidation of N,N-diethylformamide and its effect on P450-dependent monooxygenases in rat liver.

N,N-Diethylformamide (DEF) is a hepatotoxic polar solvent in which metabolism has not been investigated. In this study we examined the following: (a) the oxidative metabolism of DEF using both liver microsomes from rats pretreated with selected P450 inducers and purified P450 enzyme (2B1, 2E1, 2C11); and (b) the effect of administration of DEF and its metabolite, the monoethylformamide (MEF), on induction and/or inhibition of the P450 isoforms in rats. DEF was deethylated by microsomal P450-dependent oxidation forming acetaldehyde and MEF according to Michaelis-Menten kinetic parameters. Microsomes from rats pretreated with acetone and pyrazole (selective P4502E1 inducers) or rats pretreated with dexamethasone and 200 mg/kg DEF were able to deethylate DEF in a biphasic manner, showing a low Km component with a Vmax of about 0.2 nmol/(min.mg of protein) and a Km between 70 microM and 250 microM. The low Km component was not present in control microsomes or in microsomes from rats treated with phenobarbital, beta-naphthoflavone, or clofibrate, where linear Kinetics were observed. The use of purified P4502E1 and 2C11 in a reconstituted system showed that 2E1, which oxidized DEF with a Vmax of 4.5 nmol/(min.nmol of P450) and a Km of 0.7 mM, can partially account for the low Km DEF deethylase, whereas 2C11, which oxidized DEF with a Vmax of 4.8 nmol/(min.nmol of P450) and a Km of 17 mM, might be the high Km deethylase. The purified 2B1 was barely able to deethylate DEF. A confirmation of the role of 2E1 in DEF metabolism was obtained by using various selective inhibitors of P450 isoforms and immunoprecipitation experiments with anti P4502E1 IgG. The low Km component of DEF deethylation in acetoneor pyrazole-induced microsomes was strongly inhibited (approximately 90%) by diethyldithiocarbamate, 4-methylpyrazole, and anti-2E1 IgG, but in 200 mg/kg DEF-induced microsomes the inhibition was partial, suggesting that other P450(s) may be involved. Administration of DEF 200 mg/kg ip for 4 days induced hepatic microsomal P4502E1-dependent aniline hydroxylase, P4502B1/2-linked pentoxyresorufin O-depentylase, 16 beta-testosterone hydroxylase P4503A1/2-associated erythromycin N-demethylase, and 6 beta-testosterone hydroxylase. Alternatively, the same dose regimen of MEF induced only the aniline hydroxylase and depressed the 3A1/2-linked activities. Immunoblot experiments verified these data. These findings indicate that DEF, at low concentrations, is predominantly oxidized by P4502E1 and that this enzyme may be induced in rodents by repeated MEF or DEF treatment, thereby increasing their own metabolism and potentially their cytotoxicity through the formation of ethyl isocyanate.

The Relation between Two Molecular Species of P-450 in Adult Testis and 17α-Hydroxylase and 17,20-Lyase Activities

Two P-450s from adult pig testis were purified to specific contents of 11.2 and 12.0 nmol P-450/mg protein and shown to have minimum molecular weights of 45,000 and 46,000, respectively. The absorption spectra were typical of P-450s. The P-450s were separated from the two fractions by CM-C50 Sephadex column chromatography. One P-450 (Mr = 45000) exhibited 17,20-lyase activity of 6.78 nmol of androstenedione/min/nmol P-450, on incubation with 17α-hydroxyprogesterone as a substrate. The other P-450 (Mr = 46,000) exhibited no 17,20-lyase activity. Both P-450s exhibited 17α-hydroxylase activity that amounted to 10 nmol of steroid products. Accordingly, the two molecular species of P-450 are thus markedly different in 17,20-lyase activity toward 17α-hydroxyprogesterone.

Activation of alkylhydrazines to free radical intermediates by ethanol-inducible cytochrome P-4502E1 (CYP2E1)

Electron spin resonance (EPR) spectroscopy analysis using the spin trapping agent 4-pyridyl-l-oxide-t-butyl nitrone (4-POBN) was used to measure the formation of free radical intermediates during NADPH-dependent oxidation of 1-methyl-, 1-ethyl-, and 1-isopropylhydrazine in rat liver microsomes and in reconstituted enzyme systems. The experiments in microsomes revealed that the specific activation of the hydrazines, as measured by the EPR signal intensities, was about two-fold higher, when expressed per nmol of P-450, in microsomes from rats treated with ethanol (EtOH) as compared to membranes isolated from either phenobarbital (PB)-, β-naphthoflavone (β-NF)-treated or control rats. Furthermore, kinetic experiments revealed that EtOH-microsomes had an apparent affinity for 1-ethylhydrazine about one order of magnitude higher than PB-microsomes. In reconstituted vesicular systems composed of phospholipids, NADPH cytochrome P-450 reductase and P-450, the intensities of EPR signals produced by the formation of the methyl-, ethyl- and isopropyl-free radicals, were 3- to 5-fold more intense in membrane vesicles containing ethanol-inducible CYP2E1 than phenobarbital-inducible CYP2B1. By contrast, CYP1A2, CYP2B4 and CYP2C4 were inefficient catalysts of radical formation. Desferrioxamine, catalase and superoxide dismutase did not influence the extent of ethyl radicals formed in EtOH-microsomes, indicating that hydroxyl radicals are not involved in the CYP2E1-dependent activation of 1-ethylhydrazine. Addition of cytochrome b 5, an efficient donor of the second electron to P-450 and hence an inhibitor of the formation of the oxy-cytochrome P-450 complex, increased to be consistent with the results, did not influence the amount of ethyl radicals trapped. In liver microsomes from untreated rats selective substrates of CYP2E1, such as diethyl-dithiocarbamate and p-nitrophenol, as well as anti-CYP2E1-IgG, inhibited the free radical formation from 1-ethylhydrazine by about 60%. The anti-CYP2E1 IgG used significantly inhibited ethyl radical production also in human liver microsomes incubated with 1-ethylhydrazine and 4-POBN. Taken together, these results indicate that CYP2E1, as compared to other rat liver cytochromes P-450, is an efficient catalyst of transformation of alkylhydrazines to free radical intermediates, a finding that might be of importance in the development of the toxicity of these compounds.

Hepatotoxicity and P-4502E1-dependent metabolic oxidation of N,N-dimethylformamide in rats and mice

A comparative biochemical and histological study on the hepatotoxicity of a single dose of N,N-dimethylformamide (DMF) and N-methylformamide (NMF) in control and acetone-treated SD male rats and CD-1 male mice was performed. In control and acetone-pretreated rats, neither DMF nor NMF caused hepatic damage or elevation of plasma transaminases. In contrast, in acetonized but not in control mice, DMF administration yielded some evidence of liver necrosis and elevation of ALAT (alanine-amino transferase) activity. After a DMF dose of 1000 mg/kg, ALAT activity was found 1215 +/- 832 mU/ml and 47 +/- 18 mU/ml in acetonized and control mice, respectively. NMF treatment was hepatotoxic in control mice and lethal in acetonized mice. In control mice, an NMF dose of 600 mg/kg increased ALAT activity from a basal value of 35 +/- 5 to 2210 +/- 1898 mU/ml. When the oxidative metabolism of DMF was investigated, microsomes from both rats and mice preinduced by acetone increased the demethylation rate of DMF 7 to 10-fold compared to that (about 0.25 nmol/min per mg protein) of the corresponding control microsomes. The enzymatic affinities for DMF oxidation, however, were different: in mice the Km (0.05 mM) was one order of magnitude lower than that (0.56 mM) found in rats. The experiments performed with purified rat and mouse P-450 2E1 in a reconstituted system confirmed that the P-450 2E1 isoforms are very active catalysts towards DMF oxidation (the turnover was about 10 nmol/min per nmol P-450 for both enzymes) but with a strikingly different affinity. Whereas the Km for mouse P-450 2E1 was 0.08 +/- 0.03 mM, that for rat P-450 2E1 was 1.1 +/- 0.2 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of aflatoxin B 1 by mouse CYP2A enzymes and cytotoxicity in recombinant yeast cells

The ability of three highly homologous mouse liver CYP2A enzymes to activate aflatoxin B 1 was studied by expressing them in recombinant AH22 Saccharomyces cerevisiae yeast cells. The reconstituted monooxygenase complex with CYP2A5 purified from yeast cell microsomes produced epoxide at a rate of 17.2 nmol/min per nmol P450 in the presence of 50 μM aflatoxin B 1 while CYP2A4 had about 10% and P4507 α only 1.5% of this activity. However, K m values were 530 and 10 μM and V max values 12.5 and 14.3 nmol/min per nmol P450 for CYP2A4 and CYP2A5, respectively. When recombinant yeast cells were exposed to aflatoxin B 1 LC 50 concentrations were 7.5 ± 5.5 μM for CYP2A4, 0.45 ± 0.10 μM for CYP2A5 and > 320 μM for P4507 α expressing yeast cells. Aflatoxin B 1-DNA adduct levels in the same yeast cells were 50, 890 pmol/mg DNA and below detection limit when 3.0 μM aflatoxin B 1 was used in the incubation mixture. Coumarin an inhibitor for CYP2A4 and a substrate for CYP2A5 diminished the toxicity of aflatoxin B 1 in a dose-dependent manner for these recombinant yeast cells. These data demonstrate that (1) highly homologous mouse CYP2A enzymes activate aflatoxin B 1 in a different manner and (2) that recombinant yeast cells expressing mammalian CYP enzymes are a useful and inexpensive system to test the role of different enzymes in aflatoxin B 1 toxicity. The data also indicate that mouse CYP2A5 and its counterpart in other species could have a significant role in aflatoxin B 1 toxicity in organs where it is expressed at high levels.

Expression of Modified Human Cytochrome P450 1A1 in Escherichia coli: Effects of 5′ Substitution, Stabilization, Purification, Spectral Characterization, and Catalytic Properties

Human cytochrome P450 (P450) 1A1 is primarily an extrahepatic enzyme and is important because of its roles in the activation of polycyclic hydrocarbons and other xenobiotic chemicals. Purification of active enzyme from human tissues has not been successful. We report the expression and purification of the recombinant enzyme from Escherichia coli. A full-length cDNA of human cytochrome P450 1A1 and several modified constructs were engineered into a pCW vector and used to transform E. coli cells. Little expression was observed with the native sequence and several modified constructs, but successful expression (20-25 nmol membrane-bound P450 1A1 per liter of culture) was achieved with a construct in which the Ala codon GCT was placed in the second position and the 5′-terminal codons were maximized for AT content and minimized for the potential of secondary structure formation of the mRNA transcript. α-Naphthoflavone was found to protect against denaturation by detergents during solubilization and was added to buffers used for purification. The recombinant P450 1A1 was purified to electrophoretic homogeneity after two ion-exchange chromatography steps in ∼50% yield. N-Terminal amino acid sequence analysis verified the expected first 21 residues, with the exception of the terminal Met. The isolated human ferric P450 1A1 was predominantly in the high spin state, in contrast to the orthologous rat and rabbit enzymes. Recombinant P450 1A1 catalyzed 7-ethoxyresorufin O-deethylation and benzo[ a]pyrene 3-hydroxylation with K m values of 0.58 and 15 μM and V max values of 8.3 and 2.5 nmol min −1 (nmol P450 1A1) −1, respectively. The successful expression and purification of human P450 1A1 should increase the availability of this enzyme and the generation of antibodies for further biochemical and other biological studies.

Atypical metabolism of deprenyl and its enantiomer, (S)-(+)-N,alpha-dimethyl-N-propynylphenethylamine, by cytochrome P450 2D6.

Debrisoquine 4-hydroxylase is a unique cytochrome P450 that effects oxidation of protonated substrates at sites distal from the basic nitrogen. A basic tenet of the several models that have been proposed for the active site of P450 2D6 is that oxidation occurs at distances of approximately 5 or approximately 7 A from the protonated site. In this study, the metabolism of both stereoisomers of deprenyl, a therapeutically valuable monoamine oxidase B inhibitor, was shown to produce N-demethylation and N-depropargylation of the sole basic nitrogen in the molecule by recombinant cytochrome P450 2D6. N-Demethylation of L-(-)-deprenyl leading to nordeprenyl was favored by approximately 13:1 over N-depropargylation which produced methamphetamine. The Km and kcat values for formation of methamphetamine, the minor metabolite, were 56 +/- 5 microM and 0.63 +/- 0.063 nmol of methamphetamine min-1 (nmol of P450)-1, respectively; the kcat for nordeprenyl formation was approximately 8.2 nmol of nordeprenyl min-1 (nmol of P450)-1. Although these pathways would be the anticipated processes for monoamine oxidases and most cytochrome P450s, this mode of biotransformation is not predicted by current active site models and represents a novel pathway for P450 2D6. Statistical analysis indicates that the therapeutically important L-(-)-isomer was preferentially metabolized [kcat/Km (-)/(+) ratio = 2.66]. Competitive inhibition of deprenyl metabolism by both quinidine and quinine with an approximate 10(3) differential confirms that this metabolic pathway is P450 2D6 mediated.(ABSTRACT TRUNCATED AT 250 WORDS)

The demethylenation of methylenedioxymethamphetamine (“ecstasy”) by debrisoquine hydroxylase (CYP2D6)

The metabolism of methylenedioxymethamphetamine (MDMA, “ecstasy”) was examined in a microsomal preparation of the yeast Saccharomyces cerevisiae expressing human debrisoquine hydroxylase, CYP2D6. Only one product, dihydroxymethylamphetamine (DHMA), was detected in the incubation mixture, and this product accounted for all of the substrate consumption at low concentration (10μM). Mean ± SD values of apparent K m ( μM) and V max (nmol/min per nmol P450) for the demethylenation of (+) and (−)-MDMA at low concentrations (1–1000 μM) were 1.72, 0.12 and 6.45, 0.10 and 2.90, 0.10 and 7.61, 0.06, respectively. At high concentrations (> 1000 μM) substrate inhibition was noted, with K i values of 14.2 and 28.2 mM, respectively, for the (+) and (−) enantiomers. Incubation of MDMA isomers with human liver microsomes indicated that their demethylenation is deficient in the poor metabolizer phenotype. Thus, MDMA is converted to the catecholamine DHMA by CYP2D6, and this may give rise to genetically-determined differences in toxicity.

Bisdihydrodiols, rather than dihydrodiol oxides, are the principal microsomal metabolites of tumorigenic trans-3,4-dihydroxy-3,4-dihydrodibenz[a,h]anthracene.

Several studies on metabolism and biological activity of tumorigenic dibenz[a,h]anthracene (DBA) and its derivatives have led to the conclusion that the M-region dihydrodiol, trans-3,4-dihydroxy-3,4-dihydro-DBA (DBA-3,4-dihydrodiol), is the precursor of the ultimate mutagenic and tumorigenic metabolite of DBA with the presumed structure of a bay-region dihydrodiol oxide. Incubations of DBA-3,4-dihydrodiol (50 microM) with the microsomal hepatic fraction of Sprague-Dawley rats pretreated with Aroclor 1254 yielded more than 13 metabolites upon separation by HPLC. anti-3,4-Dihydroxy-1,2-epoxy-1,2,3,4-tetrahydro-DBA [0.27 nmol/(nmol of P450.15 min)] could be identified for the first time by UV spectroscopy, by cochromatography with the synthetic reference compound, and by its nonenzymatic hydrolysis to r-1,t-2,t-3,c-4-tetrahydroxy-1,2,3,4-tetrahydro-DBA, while firm evidence for the presence of the diastereomeric syn-dihydrodiol oxide was not obtained. Major microsomal metabolites of the M-region dihydrodiol were however three bisdihydrodiols: trans,trans-3,4:8,9-tetrahydroxy-3,4,8,9-tetrahydro-DBA [0.32 nmol/(nmol of P450.15 min)], trans,trans-3,4:10,11-tetrahydroxy-3,4,-10,11-tetrahydro-DBA [DBA-3,4:10,11-bisdihydrodiol; 1.44 nmol/(nmol of P450.15 min)], and trans,trans-3,4:12,13-tetrahydroxy-3,4,12,13-tetrahydro-DBA [0.70 nmol/(nmol of P450.15 min)], whose structures were verified by UV and mass spectrometry as well as cochromatography with synthetic reference compounds and by the observation that they were not formed when epoxide hydrolase was inhibited (1,1,1-trichloro-2-propene oxide, 1 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

A highly sensitive tool for the assay of cytochrome P450 enzyme activity in rat, dog and man: Direct fluorescence monitoring of the deethylation of 7-ethoxy-4-trifluoromethylcoumarin

The O-deethylation of 7-ethoxy-4-trinuoromethylcoumarin (EFC) by liver microsomes has been assessed as a method for monitoring the activity of cytochrome P450. The principle advantage of this substrate is the formation of a fluorescent product 7-hydroxy-4-trifluoromethylcoumarin (HFC) which can be assayed directly in the reaction medium. For rat microsomes the deethylated product was confirmed as the main metabolite, the reaction rate was linear with respect to both time and microsomal protein concentration and was independent of small changes in the added co-factors. A linear formation rate for the deethylated metabolite was also confirmed with dog and human microsomes. The intraassay precision for rat, dog and human microsomes was 3, 5 and 4%, respectively. Hanes transformations of the dog and human data showed two phases, in contrast to a linear decline seen for the rat. Hybrid parameters for V max and K m , calculated from the apparently linear portions of these curves, gave interday SD for the V max of rat, dog and man of 2. 14 and 4%, respectively, and approximately 15% for the K m, in all species. The V max, in rat, dog and human microsomes was 1.4 ± 0.2, 4.3 ± 1.5 and 0.9 ± 0.5 nmol HFC min nmol P450, respectively. The K m , was 11.0 ± 3.1, 67 ± 19 and 6.8 ± 2.5 μM, respectively. Direct evidence that at least two isoenzymes (cytochrome P450 1A2 and 2E1) metabolize EFC was obtained by experiments with competitive, suicide and immuno-inhihitors. Compared with ethoxycoumarin, the involvement of P450 2E1 in 0-deethylation seemed similar in the rat. In conclusion, EFC provides a straightforward and reproducible assay for microsomal enzyme activity, requiring at most 25 pmol mL of cytochrome P450.

Purification from Adult Pig Testicular P-450 and 17α-Hydroxylase Activity of P-450 Containing Liposomal Membranes

A cytochrome P-450 from adult pig testicular microsomes was purified to a specific content of 12 nmol P-450/mg protein. P-450 has a minimum molecular weight of 46000 ± 1000, as judged on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Adult testicular P-450 is prepared in the low-spin form with an absorbance maximum at 417 nm. The substrate-induced difference spectrum with progesterone is a typical I difference spectrum. P-450 was incorporated into liposomal membranes composed of phosphatidylcholine, and 17 α-hydroxylase activity was shown to amount to 15.5 nmol product/min/nmol of P-450. Furthermore, testicular cytochrome b 5 did not increase the 17 α-hydroxy1ase activity, and the activity was largely inhibited by the addition of sodium cholate, Emulgen 913 and testicular lipid.

Characterization of cytochromes P450 in liver and kidney of rats treated with di-(2-ethylhexyl)phthalate.

A polyclonal antibody was made to a liver cytochrome P450 purified from di-(2-ethyl-hexyl)phthalate (DEHP)-treated Sprague-Dawley rats and was used to identify the CYP4A forms in liver and kidney cortex microsomes of control rats and rats treated with this peroxisome proliferator. Three clearly separated major protein bands were recognized on western blots in liver microsomes of control male rats or male rats treated with a single dose of DEHP, which, based on the description of relative mobility, tissue specificity, and sex dependent expression of CYP4A forms (Sundseth and Waxman (1992). J. Biol. Chem., 267, 801-810), correspond to the migration pattern of forms 4A1, 4A2, and 4A3 in clofibrate-treated rats. The administration of DEHP for 2 or 3 days caused a loss of resolution of two of the protein bands. The protein band corresponding to 4A2 was absent in liver or kidney cortex microsomes of DEHP-treated or control female rats and was not always visible in the livers of control male rats. The purified P450DEHP supported the hydroxylation of arachidonic acid at both the 19- and 20-carbon atoms with turnover rates of 1.4 +/- 0.2 and 22.7 +/- 2.5 nmoles per minute per nmol P450, respectively. No measurable amounts of hydroxylated products were obtained when prostaglandin E1, leukotriene B4, or testosterone were used as substrates. Another member of the CYP4 family, 4B1 from rabbit lung microsomes, was also recognized by this antibody on western blot analysis; however, rabbit lung form 4A4 showed only minimal cross-reactivity with this antibody.

Expression of Modified Human Cytochrome P450 3A4 in Escherichia coli and Purification and Reconstitution of the Enzyme

A full-length human cytochrome P450 (P450) 3A4 cDNA clone and four derivatives in which the N-terminus was modified were inserted into a pCW vector and used to transform Escherichia coli DH5α cells. Little expression was seen with the native sequence; the highest level of expression (range of 40-110 membrane-bound nmol P450 liter −1) was achieved with a construct (NF14) in which residues 3-12 were deleted. In all of the constructs P450 was found primarily in the membranes. The modified P450 3A4 (construct NF14) showed typical P450 hemoprotein spectra. The protein was purified to electrophoretic homogeneity in a five-step procedure [nominally 23 nmol P450 (mg protein) −1]. For most purposes it was found to be more practical to purify the modified P450 3A4 to ∼70% homogeneity [nominally 15 nmol P450 (mg protein) −1] in a simple two-step process. The modified P450 3A4 (NF14) or P450 3A4 purified from human liver could be mixed with rabbit liver NADPH-P450 reductase to achieve catalytic activities nearly as high as those found in human liver microsomes (on a nmol P450 basis), but the optimal reconstitution conditions included not only a mixture of phosphatidylserine, L-α-dilauroyl- and L-α-dioleoyl- sn-glycero-3-phosphocholines, cholate, and cytochrome b 5 suggested by others but also glutathione during the preincubation. Several other thiols were found not to substitute in this role. Good catalytic activity was seen for nifedipine oxidation, testosterone 6β-hydroxylation, and the 8,9-epoxidation and 3α-hydroxylation of aflatoxin B 1, reactions previously ascribed to the enzyme. These procedures provide a relatively convenient and reliable means of producing, purifying, and reconstituting a catalytically active and useful derivative of P450 3A4, a human P450 enzyme that has many roles in the oxidation of drugs and other xenobiotic chemicals.

Characterization of a phenobarbital-inducible cytochrome P-450, NADPH-cytochrome P-450 reductase and reconstituted cytochrome P-450 mono-oxygenase system from rat brain. Evidence for constitutive presence in rat and human brain

Cytochrome P-450 was purified to apparent homogeneity from the brain microsomes of phenobarbital-treated rats. The specific content of the purified P-450 was 12.7 nmol/mg of protein. NADPH-cytochrome P-450 reductase (reductase) was also purified to apparent homogeneity from brain microsomes. The specific content was 34.7 mumol of cytochrome c reduced/min per mg of protein. The reduced carbon monoxide spectrum of purified P-450 exhibited a peak at 450 nm. Both the P-450 and the reductase moved as single bands on SDS/PAGE. The molecular masses of the purified P-450 and the reductase were determined to be 53.3 and 72.0 kDa respectively. The purified brain P-450 cross-reacted with antibodies to rat liver P-450IIB1/IIB2 when examined by Western immunoblotting, but no immunological similarity was observed with rat liver P-450IA1/IA2 or P-450IIE1. Purified rat brain reductase cross-reacted with antibodies to rat liver reductase. Further, immunoblot experiments with untreated rat and human brain microsomes using antisera to the purified rat brain P-450 and reductase indicated that these forms of P-450 and NADPH-cytochrome P-450 reductase exist constitutively in rat and human brain. Purified rat brain P-450 was reconstituted with purified NADPH-cytochrome P-450 reductase, deoxycholate and dilauroyl glyceryl 3-phosphocholine. NADPH-dependent N-demethylation of aminopyrine and morphine was observed in the reconstituted system. The catalytic-centre activities were 80.25 and 38.2 nmol of formaldehyde formed/min per nmol of P-450 respectively. The reconstituted system had a comparatively lower catalytic-centre activity for 7-ethoxycoumarin O-de-ethylase (10.5 nmol of product formed/min per nmol of P-450).

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 effects of age on the pharmacokinetics and biotransformation of theophylline in vivo and in vitro in the Mongolian gerbil ( Meriones unguiculatus)

The effect of post maturational aging on the in vivo disposition of theophylline was examined in the Mongolian gerbils ( Meriones unguiculatus) aged 30–39 (old), 12–18 (middle-aged) and 3 (young) months following a 20 mg/kg i.p. dose. Biotransformation of theophylline was also examined in liver microsomes from non-induced and 3-methylcholanthrene induced gerbils. Analysis of theophylline plasma kinetics showed decreased clearance, increased half-life and increased volume of distribution in old vs. young animals. Clearance to the 1,3-dimethyluric acid metabolite was similar for all age groups, while clearance to the 1-methyluric acid metabolite was significantly lower in the middle-aged group compared to that of young and old gerbils. Urinary recovery of 1-methylurate was increased in old vs. young and middle-aged animals while recovery of theyphylline was decreased. 3-Methylcholanthrene inductions resulted in decreased recovery of theophylline and increased recovery of 1,3-dimethylurate and 1-methylurate in young and middle-aged gerbils compared to non-induced controls. Decreased microsomal protein content was observed in old vs. young and middle-aged gerbils and an age-related decrease in cytochrome P-450 content (nmol P-450/ g liver) was also observed. The rate of dimethylurate formation was decreased 37% in microsomes fromold vs. young and middle-aged gerbils. increase in the rate of 1,3-dimethylurate formation in young and middle-aged gerbils, respectively. The results of these experiments indicate that the Mongolian gerbil may be useful for the study of the biochemical mechanisms underlying age-related changes in the biotransformation and kinetics of theophylline.

Benzo[ e]pyrene elicits changes in the biochemical activities and chromatographic behavior of murine hepatic cytochromes P-450 that are distinct from those induced by 2,3,7,8-tetrachlorodibenzo- p-dioxin

The objective of this study was to examine the potential for a specific ligand of carcinogen binding protein (CBP) to induce changes in the overall character of hepatic microsomal cytochromes P-450 (P450) and to compare potential changes with those induced by an Ah receptor ligand. Benzo[e]pyrene (BeP) was previously shown to bind CBP with high affinity and Ah receptor with low affinity. In contrast, 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) binds Ah receptor avidly and CBP weakly. Hepatic microsomes were prepared from C57BL/6J (B6) and DBA/2J (D2) mice treated with corn oil, BeP or TCDD. Relative to corn oil controls, pretreatment of B6 mice with BeP or TCDD increased the nmol P450/mg microsomal protein content 26 and 28%, respectively. In D2 mice, nmol P450/mg microsomal protein was increased 23% in the BeP pretreatment, while TCDD pretreatment had no effect relative to the corn oil controls. For the O-alkyl ethers of resorufin, rates of metabolism (per nmol P450) were affected differently in B6 and D2 by BeP pretreatment. Pentoxyresorufin O-dealkylase activity was reduced to 44% of control activity in B6 mice and increased 39% relative to controls in D2 mice. BeP pretreatment had no effect on ethoxyresorufin O-dealkylase activity in B6 mice, while this activity was decreased to 58% of controls in D2 mice. Additionally, benzyloxyresorufin O-dealkylase activity was reduced to 65% of control levels in B6 mice and not affected in D2 mice. Methoxyresorufin O-dealkylase activity was reduced in both strains to an average of 55% of control values. As expected, TCDD pretreatment resulted in increases of all O-dealkylations measured in both strains of mouse. For both inbred strains of mouse, anion exchange chromatography revealed a P450 peak associated with BeP pretreatment that was not present in chromatograms generated with corn oil or TCDD pretreatments. Results of enzyme linked immunosorbant assays also indicated that the pattern of P450 isoenzyme expression associated with BeP pretreatment was distinct from that associated with TCDD pretreatment. Overall, these data show that treatment with a specific ligand of CBP induces changes the biochemical activities and chromatographic behavior of P450 isozymes in murine hepatic microsomes. Moreover, they indicate that changes in P450 occurring after treatment with a CBP ligand are distinct from those changes that are associated with treatment with an Ah receptor ligand (TCDD). Differences between B6 and D2 strains suggest that the hepatic P450 changes occurring in response to pretreatment with a CBP ligand may be influenced by the presence of Ah receptor.

Modulation of aflatoxin B 1 biotransformation in rabbit pulmonary and hepatic microsomes

Aflatoxin B 1 (AFB 1) is a carcinogenic mycotoxin that requires activation to the corresponding 8,9-epoxide for activity. In addition to being present in foodstuffs, AFB 1 can contaminate respirable grain dusts and thus the respiratory system is a potential target for carcinogenesis. In the present study, we have investigated the role of polycyclic aromatic hydrocarbon-inducible forms of cytochrome P-450 in the pulmonary and hepatic microsomal activation ([ 3H]AFB 1-DNA binding) and detoxification ([ 3H]AFM 1 and [ 3H]AFQ 1 formation) of [ 3H]AFB 1. In rabbit lung microsomes, the apparent V max for [ 3H]AFM 1 formation was increased significantly when values were expressed per mg microsomal protein or per nmol P-450 present. In liver microsomes, the apparent V max for DNA binding and [ 3H]AFM 1 formation were increased by β-naphthoflavone (BNF) treatment (to 2.3 and 3.3 times control, respectively) when expressed per mg protein, but when expressed per nmol P-450, only AFM 1 formation was significantly increased. The apparent K m values for both these reactions were unaffected. The apparent V max for [ 3H]AFQ 1 formation was not affected by BNF treatment, but the apparent K m was increased to 4.5 times control. Boiling of microsomes or omitting the NADPH-generating system decreased DNA binding, AFM 1 formation and AFQ 1 formation by 89–97%, while addition of 1.0 mM SKF-525A inhibited these reactions by 46–57%. Addition of 1.0 mM α-naphthoflavone (ANF) had no effect on the biotransformation of [ 3H]AFB 1 in lung microsomes of control rabbits, but significantly decreased AFM 1 formation (by 31%) in lung microsomes from BNF-treated animals (other reactions were unaffected). In liver microsomes from BNF treated rabbits, 1.0 mM ANF inhibited DNA binding of [ 3H]AFB 1 by 68%, while there was no effect in control microsomes. ANF significantly inhibited AFM 1 formation in liver microsomes from both control and BNF-treated animals (by 87–97% and 67–78% at 1.0 mM and 2.0 μM, respectively), but had no effect on AFQ 1 formation in liver microsomes from animals in either treatment group. These results indicate an important role for the 1A subclass of P-450 isozymes in the biotransformation of AFB 1 to AFM 1 in rabbit lung and liver, and a minor role in AFB 1 activation in liver.