Metabolism Of Benzphetamine Research Articles

Interactions between CYP2E1 and CYP2B4: Effects on Affinity for NADPH-Cytochrome P450 Reductase and Substrate Metabolism

Studies in microsomal and reconstituted systems have shown that the presence of one cytochrome P450 isoform can significantly influence the catalytic activity of another isoform. In this study, we assessed whether CYP2E1 could influence the catalytic activity of CYP2B4 under steady-state turnover conditions. The results show that CYP2E1 inhibits CYP2B4-mediated metabolism of benzphetamine (BNZ) with a K(i) of 0.04 µM. However, CYP2B4 is not an inhibitor of CYP2E1-mediated p-nitrophenol hydroxylation. When these inhibition studies were performed with the artificial oxidant tert-butyl hydroperoxide, CYP2E1 did not significantly inhibit CYP2B4 activity. Determinations of the apparent K(M) and k(cat) of CYP2B4 for CPR in the presence of increasing concentrations of CYP2E1 revealed a mixed inhibition of CYP2B4 by CYP2E1. At low concentrations of CYP2E1, the apparent K(M) of CYP2B4 for CPR increased up to 23-fold with virtually no change in the k(cat) for the reaction, however, at higher concentrations of CYP2E1, the apparent K(M) of CYP2B4 for CPR decreased to levels similar to those observed in the absence of CYP2E1 and the k(cat) also decreased by 11-fold. Additionally, CYP2E1 increased the apparent K(M) of CYP2B4 for BNZ by 8-fold and the apparent K(M) did not decrease to its original value when saturating concentrations of CPR were used. While the individual apparent K(M) values of CYP2B4 and CYP2E1 for CPR are similar, the apparent K(M) of CYP2E1 for CPR in the presence of CYP2B4 decreased significantly, thus suggesting that CYP2B4 enhances the affinity of CYP2E1 for CPR and this may allow CYP2E1 to out-compete CYP2B4 for CPR.

Mechanistic Analysis of the Inactivation of Cytochrome P450 2B6 by Phencyclidine: Effects on Substrate Binding, Electron Transfer, and Uncoupling

Phencyclidine (PCP) is a mechanism-based inactivator of cytochrome P450 (P450) 2B6. We have analyzed several steps in the P450 catalytic cycle to determine the mechanism of inactivation of P450 2B6 by PCP. Spectral binding studies show that binding of benzphetamine, a type I ligand, to P450 2B6 was significantly affected as a result of the inactivation, whereas binding of the inhibitor n-octylamine, a type II ligand, was not compromised. Binding of these ligands to P450 2B6 occurs in two phases. Stopped-flow spectral analysis of the binding kinetics of benzphetamine to PCP-inactivated 2B6 revealed a 15-fold decrease in the rate of binding during the second phase of the kinetics (k(1) = 5.0 s(-1), A(1) = 30%; k(2) = 0.02 s(-1), A(2) = 70%, where A(2) indicates the fractional magnitude of the second phase) compared with the native enzyme (k(1) = 8.0 s(-1), A(1) = 58%; k(2) = 0.3 s(-1), A(2) = 42%). Analysis of benzphetamine metabolism by the inactivated protein using liquid chromatography/electrospray ionization/mass spectrometry showed that the rates of formation of nor-benzphetamine and hydroxylated nor-benzphetamine were decreased by 75 and 69%, respectively, whereas the rates of formation for amphetamine, hydroxybenzphetamine, and methamphetamine showed slight but statistically insignificant decreases after the inactivation. The rate of reduction of P450 2B6 by NADPH and reductase was decreased by 6-fold as a result of the modification by PCP. In addition, the extent of uncoupling of NADPH oxidation from product formation, a process leading to futile production of H(2)O(2), increased significantly during the metabolism of ethylbenzene as a result of the inactivation.

Cytochrome b5 Inhibits Electron Transfer from NADPH-Cytochrome P450 Reductase to Ferric Cytochrome P450 2B4

Experiments demonstrating that cytochrome (cyt) b5 inhibits the activity of cytochrome P450 2B4 (cyt P450 2B4) at higher concentrations suggested that cyt b5 was occupying the cyt P450 reductase-binding site on cyt P450 2B4 and preventing the reduction of ferric cyt P450 (Zhang, H., Im, S.-C., and Waskell, L. (2007) J. Biol. Chem. 282, 29766-29776). In this work experiments were undertaken with manganese-containing cyt b5 (Mn-cyt b5) to test this hypothesis. Because Mn-cyt b5 does not undergo oxidation state changes under our experimental conditions, interpretation of the experimental results was unambiguous. The rate of electron transfer from cyt P450 reductase to ferric cyt P450 2B4 was decreased by Mn-cyt b5 in a concentration-dependent manner. Moreover, reduction of cyt P450 2B4 by cyt P450 reductase was incomplete in the presence of Mn-cyt b5. At a Mn-cyt b(5):cyt P450 2B4:cyt P450 reductase molar ratio of 5:1:1, the rate of reduction of ferric cyt P450 was decreased by 10-fold, and only 30% of the cyt P450 was reduced, whereas 70% remained oxidized. It could be demonstrated that Mn-cyt b5 had its effect by acting on cyt P450, not the reductase, because the reduction of cyt c by cyt P450 reductase in the presence of Mn-cyt b5 was not effected. Furthermore, under steady-state conditions in the cyt P450 reconstituted system, Mn-cyt b5, which lacks the ability to reduce oxyferrous cyt P450 2B4, was unable to stimulate the activity of cyt P450. Mn-cyt b5 only inhibited the cyt P450 2B4 activity. In conjunction with site-directed mutagenesis studies and experiments that strongly suggested that cyt b5 competed with cyt P450 reductase for binding to cyt P450, the current investigation demonstrates unequivocally that cyt b5 inhibits the activity of cyt P450 2B4 by preventing cyt P450 reductase from binding to cyt P450, a prerequisite for electron transfer from cyt P450 reductase to cyt P450 and catalysis.

Cytochrome b5 Increases the Rate of Product Formation by Cytochrome P450 2B4 and Competes with Cytochrome P450 Reductase for a Binding Site on Cytochrome P450 2B4

The kinetics of product formation by cytochrome P450 2B4 were compared in the presence of cytochrome b(5) (cyt b(5)) and NADPH-cyt P450 reductase (CPR) under conditions in which cytochrome P450 (cyt P450) underwent a single catalytic cycle with two substrates, benzphetamine and cyclohexane. At a cyt P450:cyt b(5) molar ratio of 1:1 under single turnover conditions, cyt P450 2B4 catalyzes the oxidation of the substrates, benzphetamine and cyclohexane, with rate constants of 18 +/- 2 and 29 +/- 4.5 s(-1), respectively. Approximately 500 pmol of norbenzphetamine and 58 pmol of cyclohexanol were formed per nmol of cyt P450. In marked contrast, at a cyt P450:CPR molar ratio of 1:1, cyt P450 2B4 catalyzes the oxidation of benzphetamine congruent with100-fold (k = 0.15 +/- 0.05 s(-1)) and cyclohexane congruent with10-fold (k = 2.5 +/- 0.35 s(-1)) more slowly. Four hundred picomoles of norbenzphetamine and 21 pmol of cyclohexanol were formed per nmol of cyt P450. In the presence of equimolar concentrations of cyt P450, cyt b(5), and CPR, product formation is biphasic and occurs with fast and slow rate constants characteristic of catalysis by cyt b(5) and CPR. Increasing the concentration of cyt b(5) enhanced the amount of product formed by cyt b(5) while decreasing the amount of product generated by CPR. Under steady-state conditions at all cyt b(5):cyt P450 molar ratios examined, cyt b(5) inhibits the rate of NADPH consumption. Nevertheless, at low cyt b(5):cyt P450 molar ratios <or=1:1, the rate of metabolism of cyclohexane and benzphetamine is enhanced, whereas at higher cyt b(5):cyt P450 molar ratios, cyt b(5) progressively inhibits both NADPH consumption and the rate of metabolism. It is proposed that the ability of cyt b(5) to enhance substrate metabolism by cyt P450 is related to its ability to increase the rate of catalysis and that the inhibitory properties of cyt b(5) are because of its ability to occupy the reductase-binding site on cyt P450 2B4, thereby preventing reduction of ferric cyt P450 and initiation of the catalytic cycle. It is proposed that cyt b(5) and CPR compete for a binding site on cyt P450 2B4.

Roles of the threonine 407, aspartic acid 417, and threonine 419 residues in P450 2B1 in metabolism

We have previously observed that the quadruple (S407T-N417D-A419T-K473M) and triple (S407T-N17D-A419T) mutants of the chimeric construct of P450 2B1/2B2 do not undergo mechanism-based inactivation by 17α-ethynylestradiol (17EE) and tert-butyl 1-methyl-2-propynyl ether ( tBMP). The ability of these mutants to metabolize 17EE, benzphetamine, and testosterone has been investigated. The profile for 17EE metabolism by both mutants was characteristic of both wild-types. The two mutants metabolized testosterone to form androstenedione with no formation of the hydroxy products as was seen with both the wild-types. Benzphetamine metabolism by the mutants showed that both mutants exhibited an increased tendency to catalyze demethylation rather than debenzylation. In the presence of the alternate oxidants cumene hydroperoxide and tert-butyl hydroperoxide, the wild-type 2B1 was not inactivated by 17EE. Metabolism of 17EE by 2B1 supported by these alternate oxidants revealed differences in the metabolites that may be related to the inability of 2B1 to be inactivated under these conditions.

THE NATURALLY OCCURRING CYTOCHROME P450 (P450) 2B6 K262R MUTANT OF P450 2B6 EXHIBITS ALTERATIONS IN SUBSTRATE METABOLISM AND INACTIVATION

The polymorphic human cytochrome P450 (P450) 2B6 is primarily responsible for the metabolism of several clinically relevant drugs including bupropion, cyclophosphamide, propofol, and efavirenz. Although a number of single nucleotide polymorphisms have been found in the P450 2B6 gene, the influence of these variants on the metabolism of substrates and on the response to known inactivators of P450 2B6 has not been examined. We have compared the metabolism of different substrates of P450 2B6 (P450 Delta2B6) and the effects of mechanism-based inactivators with that observed with the polymorphic P450 Delta2B6 K262R in a reconstituted monooxygenase system (reconstituted system). Metabolism of bupropion by P450 Delta2B6 K262R resulted in increased production of hydroxybupropion compared with P450 Delta2B6. However, production of formaldehyde from the metabolism of benzphetamine by the P450 Delta2B6 K262R mutant was significantly less than that of the wild-type isozyme. P450 Delta2B6 K262R formed fewer benzphetamine metabolites compared with the wild type. N,N',N''-Triethylenethiophosphoramide (tTEPA) and bergamottin decreased the ability of both enzymes to hydroxylate bupropion and to O-deethylate 7-hydroxy-4-(trifluoromethyl)coumarin (7-EFC). Incubation with 17-alpha-ethynylestradiol decreased bupropion hydroxylation and 7-EFC O-deethylation with the wild-type enzyme but had no effect on the mutant. The kinetics for inactivation of the variant by tTEPA and bergamottin were determined using 7-EFC. The KI values for inactivation of the variant were significantly greater than those determined for the wild-type enzyme. These data demonstrate a functional difference between P450 Delta2B6 and the allelic variant P450 Delta2B6 K262R.

The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.

Cytochrome b(5) (cyt b(5)) is a 15-kDa amphipathic protein with a cytosolic amino-terminal catalytic heme domain, which is anchored to the microsomal membrane by a hydrophobic transmembrane alpha-helix at its carboxyl terminus. These two domains are connected by an approximately 15-amino acid linker domain, Ser(90)-Asp(104), which has been modified by site-directed mutagenesis to investigate whether the length or sequence of the linker influences the ability of cyt b(5) to bind ferric cytochrome P450 2B4 and donate an electron to oxyferrous (cyt P450 2B4), thereby stimulating catalysis. Because shortening the linker by 8 or more amino acids markedly inhibited the ability of cyt b(5) to bind cyt P450 2B4 and stimulate catalysis by this isozyme, it is postulated 7 amino acids are sufficient to allow a productive interaction. All mutant cyts b(5) except the protein lacking the entire 15-amino acid linker inserted normally into the microsomal membrane. Alternatively, lengthening the linker by 16 amino acids, reversing the sequence of the amino acids in the linker, and mutating conserved linker residues did not significantly alter the ability of cyt b(5) to interact with cyt P450 2B4. A model for the membrane-bound cyt b(5)-cyt P450 complex is presented.

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.

Theoretical study of the ligand-CYP2B4 complexes: effect of structure on binding free energies and heme spin state.

The molecular origins of temperature-dependent ligand-binding affinities and ligand-induced heme spin state conversion have been investigated using free energy analysis and DFT calculations for substrates and inhibitors of cytochrome P450 2B4 (CYP2B4), employing models of CYP2B4 based on CYP2C5(3LVdH)/CYP2C9 crystal structures, and the results compared with experiment. DFT calculations indicate that large heme-ligand interactions (ca. -15 kcal/mol) are required for inducing a high to low spin heme transition, which is correlated with large molecular electrostatic potentials (approximately -45 kcal/mol) at the ligand heteroatom. While type II ligands often contain oxygen and nitrogen heteroatoms that ligate heme iron, DFT results indicate that BP and MF heme complexes, with weak substrate-heme interactions (ca. -2 kcal/mol), and modest MEPS minima (>-35 kcal/mol) are high spin. In contrast, heme complexes of the CYP2B4 inhibitor, 4PI, the product of benzphetamine metabolism, DMBP, and water are low spin, have substantial heme-ligand interaction energies (<-15 kcal/mol) and deep MEPS minima (<-45 kcal/mol) near their heteroatoms. MMPBSA analysis of MD trajectories were made to estimate binding free energies of these ligands at the heme binding site of CYP2B4. In order to initially assess the realism of this approach, the binding free energy of 4PI inhibitor was computed and found to be a reasonable agreement with experiment: -7.7 kcal/mol [-7.2 kcal/mol (experiment)]. BP was determined to be a good substrate [-6.3 kcal/mol (with heme-ligand water), -7.3 kcal/mol (without ligand water)/-5.8 kcal/mol (experiment)], whereas the binding of MF was negligible, with only marginal binding binding free energy of -1.7 kcal/mol with 2-MF bound [-3.8 kcal/mol (experiment)], both with and without retained heme-ligand water. Analysis of the free energy components reveal that hydrophobic/nonpolar contributions account for approximately 90% of the total binding free energy of these substrates and are the source of their differential and temperature-dependent CYP2B4 binding. The results indicate the underlying origins of the experimentally observed differential binding affinities of BP and MF, and indicate the plausibility of the use of models derived from moderate sequence identity templates in conjunction with approximate free energy methods in the estimation of ligand-P450 binding affinities.

Molecular cloning and characterization of mouse CYP2J6, an unstable cytochrome P450 isoform

A cDNA encoding a new cytochrome P450 was cloned from a mouse liver library. Sequence analysis revealed that this 2046-bp cDNA encodes a 501-amino acid polypeptide that is 72–94% identical to other CYP2J subfamily P450s and is designated CYP2J6. Northern analysis demonstrated that CYP2J6 transcripts are abundant in the small intestine and present at lower levels in other mouse tissues. In situ hybridization revealed that CYP2J6 mRNAs are present in luminal epithelial cells of the gastrointestinal mucosa. The CYP2J6 cDNA was expressed in Sf9 cells using baculovirus. The heterologously expressed CYP2J6 protein displayed a typical P450 CO-difference spectrum; however, the protein was unstable as evidenced by the loss of the Soret maxima at 450 nm and the appearance of a 420 nm peak when CYP2J6-expressing cells were disrupted by mechanical homogenization, sonication, or freeze–thaw. Immunoblotting of mouse microsomes with the anti-human CYP2J2 IgG, which cross-reacts with rodent CYP2Js, demonstrated the presence of multiple distinct murine CYP2J immunoreactive proteins in various tissues. Immunoblotting with an antibody to a CYP2J6-specific peptide detected a prominent 55–57 kDa protein in Sf9 cell extracts expressing recombinant CYP2J6 but did not detect a protein of similar molecular mass in mouse small intestinal microsomes. Mixing experiments demonstrated that recombinant CYP2J6 is degraded rapidly in the presence of small intestinal microsomes consistent with proteolysis at highly sensitive sites. Sf9 cells, which express both CYP2J6 and NADPH-P450 oxidoreductase, metabolized benzphetamine but not arachidonic acid. We conclude that CYP2J6 is an unstable P450 that is active in the metabolism of benzphetamine, but not arachidonic acid.

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The capacity of four native P-450 enzymes to interact with cytochrome b5 was compared and discussed in terms of spin shift and metabolism. Two-dimensional electrophoresis was employed as a tool to aid in characterization of the different enzymes isolated from liver microsomes of the untreated rat. RLM5 had a pl of 7.4 and RLM3 had a pl of 7.1. Two new forms isolated by this laboratory, RLM2 and RLM5a, differed from other forms of cytochrome P-450 characterized to date. The pl values of these forms were 7.35 and 7.6, respectively. The interaction of all four enzymes with cytochrome b5 differed. Cytochrome b5 caused a major low to high spin transition when added to RLM5. The latter hemoprotein was 28% high spin at 25 degrees C and was shifted to 55% high spin by cytochrome b5. RLM5a shifted from 4% high spin to 15% high spin under comparable conditions. In contrast, RLM2 and RLM3 were both minimally influenced by cytochrome b5, reaching only 8% high spin. Cytochrome b5 did not appreciably influence the rates of metabolism of aminopyrine, benzphetamine, testosterone, or p-nitroanisole with RLM2 or RLM3. However, with RLM5 and RLM5a, rates of aminopyrine and benzphetamine demethylation and testosterone hydroxylation were increased to about 130% with RLM5 and up to 200% with RLM5a. The demethylation of p-nitroanisole was stimulated by cytochrome b5, 3.5-fold with RLM5 and 14-fold with RLM5a. In no case was the ratio of monohydroxy metabolites of testosterone altered by the addition of cytochrome b5, indicating an effect on Vmax rather than Km.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of amphetamine and methamphetamine following administration of benzphetamine.

Interpretation of urine drug-testing results is a challenging endeavor for several reasons. Effects of pH, dilution, legitimate and illicit sources of the drugs, and, perhaps the most challenging, the possibility of the methamphetamine and/or amphetamine being the result of the use of some other drug. Although it is known that 14 different compounds are metabolized to methamphetamine or amphetamine or both, there is little information on the metabolic profile of many of these compounds, making interpretation of results difficult. Benzphetamine, administered as a single Didrex tablet, was given to 10 subjects (7 male and 3 female) and urine samples collected for the next 7 days. Gas chromatography-mass spectrometry results showed 3 of the 10 subjects did not have a single urine sample that exceeded a 500-ng/mL cutoff for amphetamine or methamphetamine. The other subjects had between one and six samples that tested positive at or above that level. Two subjects excreted more methamphetamine than amphetamine, whereas the other eight excreted greater amounts of amphetamine than methamphetamine. The observed ratio between amphetamine and methamphetamine was significantly different than what would be expected from the use of methamphetamine. Results of this study indicate the metabolism of benzphetamine to desmethylbenzphetamine is a major pathway in the metabolism of the drug. Enantiomer analysis of the methamphetamine and amphetamine revealed only the d-enantiomer. Results of this study add significant information useful to interpret the possibility of benzphetamine as the origin of methamphetamine and amphetamine in urine samples.

Reconstitution Premixes for Assays Using Purified Recombinant Human Cytochrome P450, NADPH-Cytochrome P450 Reductase, and Cytochromeb5

The development of enzyme and buffer premixes forin vitrobiotransformation assays is described. The protein premixes contain a mixture of three recombinant human proteins, cytochrome P450 (P450) 3A4, NADPH-P450 reductase, cytochromeb5, and liposomes. The buffer premix contains reagents which, when diluted, provide for optimal metabolic activity with selected P450 3A4 substrates. P450 3A4 premixes were competent in the oxidation of known substrates including testosterone, midazolam, nifedipine, erythromycin, benzphetamine, and amitriptyline. Premixes stored at −80°C for 2 months and those that underwent an additional five freeze/thaw cycles were able to hydroxylate testosterone at turnover rates similar to freshly prepared reconstitution mixes. In addition, premixes stored unfrozen at 4°C for 2 weeks showed no significant loss in the rate of testosterone 6β-hydroxylation by P450 3A4. Premixes prepared with and without reduced glutathione, a component which had previously been found to be important for P450 3A4 reactions, were equally efficient at carrying out testosterone hydroxylation under these conditions. Kinetic parameters determined for the metabolism of testosterone, amitriptyline, nifedipine, and benzphetamine using P450 3A4 premixes were compared with human pooled microsomes and insect microsomes prepared from cells infected with a baculovirus containing two cDNA inserts coding for P450 3A4 and NADPH-P450 reductase. Each format gave differentVmaxandKmvalues indicating different catalytic efficiencies. Analysis of P450 1A2 premixes which contained different lipid concentrations indicated thatVmaxandKmcould be altered. The availability of human P450 recombinant enzymes and the development of the P450 premixes that remain active after being stored frozen should allow for rapid identification of novel P450 substrates and inhibitors and the development of large-scale screening assays.

Regulation ofCYP3A9Gene Expression by Estrogen and Catalytic Studies Using Cytochrome P450 3A9 Expressed inEscherichia coli

Sexual dimorphism in the expression ofCYP3A9,a novel form ofCYP3Afrom rat brain, is shown for the first time in rat brain as well as in rat liver.CYP3A9expression is female specific in rat liver as judged by its 10-fold higher expression in females than in males.CYP3A9gene expression was inducible by estrogen treatment both in male and in female rats. Ovariectomy of adult female rats elicited a drastic reduction on the mRNA level ofCYP3A9which could be fully restored by estrogen replacement. These results suggest that estrogen may play an important role in the female-specific expression of theCYP3A9gene. P450 3A9 recombinant protein was expressed inEscherichia coliby means of the pCWOri+ expression vector and the MALLLAVF amino terminal sequence modification. This construct gave a high level of expression (130 nmol P450 3A9/liter culture) and the recombinant protein of the modified P450 3A9 was purified to electrophoretic homogeneity with a specific content of 10.1 nmol P450/mg protein from solubilized fractions through two chromatographic steps. The purified P450 3A9 protein was active in the metabolism of imipramine, erythromycin, benzphetamine, and ethylmorphine as well as 17β-estradiol in a reconstituted system containing lipid and rat NADPH-P450 reductase. Of special interest is the finding that P450 3A9 can catalyze the formation of desipramine with a turnover number of 4.9 nmol/min/nmol P450, suggesting the possible involvement of this isoform in the metabolism of imipramine in brain. Optimal reconstitution conditions for P450 3A9 activities required a lipid mixture (1:1:1 mixture ofl-α-dilauroyl phosphatidylcholine,l-α-dioleoyl phosphatidylcholine, and phosphatidylserine) and GSH.

Mechanistic Studies of 9-Ethynylphenanthrene-Inactivated Cytochrome P450 2B1

The mechanism of inactivation of the major phenobarbital-inducible cytochrome P450 of rat liver, P450 2B1, by 9-ethynylphenanthrene (9EPh) has been investigated. Matrix-assisted laser desorption ionization–mass spectrometry analysis of the cyanogen bromide-generated peptides from 9EPh-inactivated P450 2B1 confirmed the addition of a phenanthrylacetyl group to the peptide corresponding to residues 290 to 314. When this peptide was further digested with pepsin, the site of attachment could be assigned to one of the amino acids in the peptide Phe297 to Leu307 [Roberts, E. S., Hopkins, N. E., Zaluzec, E. J., Gage, D. A., Alworth, W. L., and Hollenberg, P. F. (1995)Arch. Biochem. Biophys.323, 000–000]. The inactivation by 9EPh resulted in a 90–95% loss in the NADPH-supported deethylation of 7-ethoxy-4-trifluoromethylcoumarin (EFC), but had no effect on the iodosobenzene- or cumene hydroperoxide-supported metabolism of EFC. The loss of NADPH-supported activity was not affected by the addition of cytochrome b5or the presence of excess levels of reductase. The magnitude of the Type 1 spectral change upon the addition of benzphetamine was decreased with the 9EPh-modified protein. There was no decrease in the ability of modified 2B1 to form the steady-state level of the CO-reduced complex either enzymatically with NADPH and reductase or chemically with sodium dithionite, but the rate of reduction by reductase under anaerobic conditions was 57% that of native protein in the absence of substrate and 35% that of native protein in the presence of substrate. The 9EPh-modified 2B1 had an overall slower rate of NADPH oxidation, H2O2formation, and formaldehyde formation during metabolism of benzphetamine compared to native 2B1. The ratio of H2O2to HCHO was 1.0:1.0 for the native and 1.6:1.0 for the modified protein. The ability of the modified protein to form the steady-state level of the oxygen–iron complex in the presence of cyclohexane was decreased. These results are consistent with the idea that the covalent modification of one of the residues in the peptide Phe297 to Leu307 by the phenanthrylacetyl group impairs the reduction of P450 2B1 by reductase and also causes the uncoupling of NADPH utilization and oxygen consumption from product formation.

Oxygen radical formation during cytochrome P450-catalyzed cyclosporine metabolism in rat and human liver microsomes at varying hydrogen ion concentrations.

The role of pH in uncoupling the electron-flux between oxidoreductase and cytochrome P450 (P450) or P450 and cyclosporine (CyA) and resulting in the generation of oxygen radicals was investigated in vitro in rat and human liver microsomal preparations. Since the electron-flux from NADPH to cytochrome c via oxidoreductase showed a fairly constant reduction activity from pH 7.0-9.5, the generation of oxygen radicals at the level of P450-Cyclosporine (instead of oxidoreductase-P450) was investigated. The effects of increasing pH on oxygen radical formation was measured by the thiobarbituric acid assay (TBA) and the adrenochrome reaction. The trends in oxygen radical production were correlated with benzphetamine metabolism (production of formaldehyde) and CyA metabolism (analyzed by high performance liquid chromatography). The TBA assay showed increased MDA-detected lipid peroxidation (unrelated to autooxidation) at pH < 8.0 and pH > 8.0 (rat and human, respectively) while the adrenochrome reaction showed decreased oxygen radical production. When these results were compared to benzphetamine (a substrate of P450 2B and 3A) metabolism and CyA (a substrate of P450 3A) metabolism, increased metabolism followed the pH-dependent trend of MDA-detected lipid peroxidation. Benzphetamine metabolism with formaldehyde production and depletion of parent compound during CyA metabolism were increased at pH < 8.0 in the rat samples and at pH > 8.0 in the human samples. This parallel relation suggests that the increased metabolism of CyA at lower pH in rats and higher pH in humans may be the result of favorable interactions of P450 with Cyclosporine that also result in increased oxygen radical-related lipid peroxidation.

The Stoichiometry of the Cytochrome P-450-catalyzed Metabolism of Methoxyflurane and Benzphetamine in the Presence and Absence of Cytochrome b5

The complete stoichiometry of the metabolism of the cytochrome b5 (cyt b5)-requiring substrate, methoxyflurane, by purified cytochrome P-450 2B4 was compared to that of another substrate, benzphetamine, which does not require cyt b5 for its metabolism. Cyt b5 invariably improved the efficiency of product formation. That is, in the presence of cyt b5 a greater percentage of the reducing equivalents from NADPH were utilized to generate substrate metabolites, primarily at the expense of the side product, superoxide. With methoxyflurane, cyt b5 addition always resulted in an increased rate of product formation, while with benzphetamine the rate of product formation remained unchanged, increased or decreased. The apparently contradictory observations of increased reaction efficiency but decrease in total product formation for benzphetamine can be explained by a second effect of cyt b5. Under some experimental conditions cyt b5 inhibits total NADPH consumption. Whether stimulation, inhibition, or no change in product formation is observed in the presence of cyt b5 depends on the net effect of the stimulatory and inhibitory effects of cyt b5. When total NADPH consumption is inhibited by cyt b5, the rapidly metabolized, highly coupled (approximately equal to 50%) substrate, benzphetamine, undergoes a net decrease in metabolism not counterbalanced by the increase in the efficiency (2-20%) of the reaction. In contrast, in the presence of the slowly metabolized, poorly coupled (approximately equal to 0.5-3%) substrate, methoxyflurane, inhibition of total NADPH consumption by cyt b5 was never sufficient to overcome the stimulation of product formation due to an increase in efficiency of the reaction.

Difference between in vivo and in vitro effects of propofol on defluorination and metabolic activities of hamster hepatic cytochrome P450-dependent mono-oxygenases

We have compared the in vivo and in vitro effects of propofol on cytochrome P450-dependent monooxygenase activities in hamster liver microsomes. Propofol (Diprivan) 10 mg/100 g body weight was injected i.p. twice a day for 2 weeks to induce cytochrome P450 enzymes. Liver microsomes were prepared by differential centrifugation. Metabolism of the cytochrome P450-dependent mono-oxygenase system was evaluated by measuring aniline hydroxylation, benzphetamine demethylation and benzo(a)pyrene hydroxylation. Defluorination of enflurane was assayed by detecting free fluoride metabolites. At similar concentrations as in the in vivo group, propofol in vitro exhibited concentration-dependent inhibition of metabolism of benzphetamine and benzo(a)pyrene. Aniline hydroxylation and defluorination of enflurane were inhibited to 78% of control with propofol 0.25 mmol litre-1. In propofol-treated hamsters, there was only minimal inhibitory or inductive effects on either mono-oxygenase activities or capacity for defluorination. This difference between the in vitro and in vivo effects of propofol on cytochrome P450 mono-oxygenase activities emphasizes the need for care when comparing in vitro and clinical data.

Isoflurane and cytochrome b5 stimulation of 2-chloro-1,1-difluoroethene metabolism by reconstituted rat CYP2B1 and CYP2C6

Isoflurane stimulates the metabolism of 2-chloro-1,1-difluoroethene (CDE) in liver microsomes from phenobarbital-treated rats or rabbits. The P450 isozymes involved and the mechanism by which such stimulation occurs have not been clarified. The present study examined the effects of isoflurane ami cytochrome b 5 on CDE metabolism in reconstituted systems containing purified rat CYP2B1 orCYP2C6. Under similar incubation conditions, CYP2B1 defluorinated CDE at approximately five times the rate of CYP2C6. Isoflurane was a potent stimulator of CDE metabolism, increasing it nearly 5-fold when catalyzed by CYP2B1, but only 2-fold when catalyzed by CYP2C6. Isoflurane had no stimulatory effect on benzphetamine metabolism by CYP2B1 or CYP2C6. Cytochrome b 5 was not required for isoflurane-facilitated CDE metabolism; however, the addition of cytochrome b 5 to CYP2B1 increased CDE metabolism 71 and 44%, in the absence and presence of isoflurane, respectively. In reconstituted CYP2B1, isoflurane generated a type I difference spectrum of approximately twice the magnitude of CDE and stimulated NADPH consumption more so than CDE. The same quantity of NADPH was consumed when CDE was present with isoflurane as compared with isoflurane alone. These data support the hypothesis that isoflurane stimulates CDE metabolism by a mechanism involving increased P450 reduction via direct isoflurane interaction with P450.

Influence of Ionic Strength on the P450 Monooxygenase Reaction and Role of Cytochrome b 5 in the Process

A stimulatory effect of increased salt content on the metabolism of benzphetamine, 7-ethoxycoumarin, and coumarin by rabbit liver microsomes, CYP2B4 and rabbit CYP1A2, was seen, indicating that the effect was not specific for either substrate or form of cytochrome P450. The stimulation was not due to an action on the cytochrome P450 itself as increased salt concentration minimally affected the substrate turnover when cumene hydroperoxide was used as the source of active oxygen. The elevation of ionic strength increased the coupling efficiency of the monooxygenase reaction with benzphetamine as substrate. Cytochrome b 5 also can increase the monooxygenase coupling efficiency. At low ionic strength cytochrome b 5 did not much influence the reduction of P450, but the rate constant of the cytochrome b 5 reduction was increased about 15-fold by its binding to cytochrome P450. A stimulatory effect of cytochrome b 5 on benzphetamine oxidation was seen at low ionic strength, but it was lost at elevated ionic strength as the binding of cytochrome b 5 to cytochrome P450 was weakened. At the higher ionic strength cytochrome b 5 competes with cytochrome P450 for the reductase, an action that slows cytochrome P450 reduction. Based upon these observations, plus those in the literature, a scheme is suggested that proposes the stimulatory effect of cytochrome b 5 on the cytochrome P450-mediated monooxygenation reaction is due to an increase in the efficiency of the electron transfer reaction: With cytochrome b 5 bound to cytochrome P450, two electrons can be provided from the reductase to the P450-b 5 complex in a single interaction, obviating the need for a second interaction with the reductase.