Studies on N-demethylation of methamphetamine by liver microsomes of guinea-pigs and rats: The role of flavin-containing mono-oxygenase and cytochrome P-450 systems

1. Relative participation of flavin-containing mono-oxygenase and cytochrome P-4S0 systems in N-hydroxylation of and formaldehyde release from methamphetamine were studied in vitro using liver microsomes of guinea-pigs and rats. In guinea pigs, only methimazole, an inhibitor of flavin-containing mono-oxygenase, significantly suppressed the above reactions.

2. Formaldehyde release from methamphetamine was significantly inhibited not only by methimazole but also by inhibitors of the cytochrome P-450 system in liver microsomes from rats, but not guinea-pigs.

3. Pretreatment of guinea-pigs with phenobarbital and 3-methylcholanthrene did not enhance the metabolism of methamphetamine.

4. Pretreatment of rats with phenobarbital but not 3-methylcholanthrene increased slightly the N-demethylation of methamphetamine by liver microsomes.

5. The results indicate that a marked species difference exists in the enzymes concerned with N-demethylation of methamphetamine. N-Oxidation predominates in guinea-pigs, whereas in rats, N-oxidation and C-oxidation of the methyl group participate equally as the initial reaction of the N-demethylation pathway.     

The measurement of FAD-containing mono-oxygenase activity in microsomes containing cytochrome P-450

1. Antibodies to NADPH-cytochrome P-450 reductase have been used to essentially abolish the contribution of cytochrome P-450 to xenobiotic metabolism by mammalian microsomes. This permits the determination of the activity of the FAD-containing monooxygenase and the stoichiometry between substrate, O₂ and NADPH, in the microsomal membrane, and in the absence of cytochrome P-450-dependent activity.

2. FAD-containing mono-oxygenase oxidation rates were determined for sulphur- and nitrogen-containing substrates, including: thiols; sulphides; thioamides; primary, secondary and tertiary amines; hydrazines.

3. Although the enzyme in mouse, rabbit, rat and pig microsomes displays similar substrate specificity, some catalytic characteristics are different between species and tissues.     

The role of cytochrome P-450 in the dual pathways of N-demethylation of N,N-dimethylaniline by hepatic microsomes

The role of cytochrome P-450 in the demethylation of N,N'-dimethylaniline (DMA) has been investigated by studying carbon monoxide (CO) inhibition and its reversal by light. Two distinct pathways of N-demethylation are present in hepatic microsomes from phenobarbital-(PB) and 3-methylcholanthrene-(3-MC) induced rats. Pathway A, the demethylation of DMA, is catalysed by a microsomal cytochrome P-450 system similar to the one we have reported for the dealkylation of benzphetamine and 3-ethylmorphine, in that there is a maximal, although weak, light reversal of CO inhibition by wavelengths of light around 445 nm. The second pathway consists of two enzyme steps (pathways B + C). Pathway B is the step in which the N-oxide of DMA, N,N'-dimethylaniline N-oxide (DMAO) is formed and this step is catalysed by the flavin amine mixed-function oxidase described by Ziegler and his co-workers. Pathway C, the demethylation of DMAO, requires a cytochrome P-450 with rather unusual properties. This reaction is inhibited by SKF 525A and CO, but is not significantly affected by the removal of oxygen even though CO strongly inhibits in this anaerobic atmosphere. Higher CO/O2 ratios (2:1 to 4:1) are required for inhibition equal to that of pathway A and light reversal of this inhibition is almost complete and maximally produced by wavelengths of light around 455 nm.     

The role of cytochrome P-450 in the dual pathways of N-demethylation of N,N'-dimethylaniline by hepatic microsomes

1. The role of cytochrome P-450 in the demethylation of N, N'-dimethyl aniline (DMA) has been investigated by studying carbon monoxide (CO) inhibition and its reversal by light.

2. Two distinct pathways of N-demethylation are present in hepatic microsomes from phenobarbital-(PB) and 3-methyIcholanthrene-(3-MC) induced rats.

3. Pathway A, the demethylation of DMA, is catalysed by a microsomal cytochrome P-450 system similar to the one we have reported for the dealkylation of benzphetamine and 3-ethylmorphine, in that there is a maximal, although weak, light reversal of CO inhibition by wavelengths of light around 445 nm.

4. The second pathway consists of two enzyme steps (pathways B + C).

5. Pathway B is the step in which the N-oxide of DMA, N, N'-dimethylaniline N-oxide (DMAO) is formed and this step is catalysed by the flavin amine mixed-function oxidase described by Ziegler and his co-workers.

6. Pathway C, the demethylation of DMAO, requires a cytochrome P-450 with rather unusual properties.

7. This reaction is inhibited by SKF 525A and CO, but is not significantly affected by the removal of oxygen even though CO strongly inhibits in this anaerobic atmosphere.

8. Higher CO/O₂ ratios (2 :1 to 4:1) are required for inhibition equal to that of pathway A and light reversal of this inhibition is almost complete and maximally produced by wavelengths of light around 455 nm.     

Enantioselective S-oxygenation by flavin-containing and cytochrome P-450 monooxygenases

The reaction of the modified Sharpless reagent, as well as microsomes and highly purified flavin-containing monooxygenase from hog liver, and cytochrome P-450IIB-1 from rat liver efficiently S-oxygenates 2-aryl-1,3-oxathiolanes with significant diastereoselectivity and enantioselectivity. The absolute configuration of the synthetic S-oxides and the enzyme-derived S-oxides was correlated by NMR analysis and by the sign of the Cotton effects obtained from circular dichroism studies. of the sulfides studied, the trans-S-oxide was the major diastereomer produced from monooxygenase-catalyzed biotransformations. In all cases examined, enantioselective S-oxygenation was observed although enantiomeric excess varied from 7 to 100%. In contrast to previous reports, the enantioselectivity of S-oxygenation catalyzed by cytochrome P-450IIB-1 was not always opposite to that of hog liver flavin-containing monooxygenase activity. The presence of the minor S-oxide diastereomers in each case was due to incomplete chiral processing by each monooxygenase and not to a competing achiral nonenzymatic process. The results suggest that the active site of hog liver flavin-containing monooxygenase places greater constraints than that of cytochrome P-450IIB-1 on substrate orientation, but in both cases trans-S-oxide formation is strongly preferred possibly due to steric interactions of the substrate and the active site.     

Participation of cytochrome P-450 isozymes in N-demethylation, N-hydroxylation and aromatic hydroxylation of methamphetamine

1. Five isozymes of cytochrome P-450 were purified from liver microsomes of phenobarbital-pretreated (P-450-SD-I and -II), 3-methylcholanthrene-pretreated (P-450-SD-III) and untreated rats (P-450-SD-IV and -V) to determine their catalytic activities in metabolic reactions of methamphetamine. 2. All the isozymes except P-450-SD-III showed considerably high N-hydroxylating activity of methamphetamine. The cytochromes P-450 initiate N-demethylation of this drug by two metabolic pathways, C-hydroxylation and N-hydroxylation. 3. Both N-demethylation and N-hydroxylation of methamphetamine were efficiently catalysed by the phenobarbital-inducible forms P-450-SD-I and -II and constitutive forms P-450-SD-IV and -V. 4. The constitutive forms P-450-SD-IV and -V revealed high catalytic activities of p-hydroxylation of methamphetamine, but phenobarbital- and 3-methylcholanthrene-inducible isozymes showed only low activities. 5. The present results indicate that the different extents of the metabolic intermediate complex formation with cytochrome P-450 (455 nm complex) in the microsomes from phenobarbital-, 3-methylcholanthrene-pretreated, and untreated rats is not attributable to the activities of the respective isozymes of cytochrome P-450 to form the precursor of the complex, N-hydroxymethamphetamine.     

Comparative studies of N-hydroxylation and N-demethylation by microsomal cytochrome P-450

The N-hydroxylation of representative aromatic amines by rabbit liver microsomes was mediated by cytochrome P-450 as demonstrated by the sensitivity to carbon monoxide and other cytochrome P-450 inhibitors. The rate of N-hydroxylation was increased by induction with phenobarbital. Involvement of isozyme LM2 (P-50IIB1) was demonstrated in reconstituted systems. Aromatic N-hydroxylation was substantially faster and more efficient than aliphatic N-hydroxylation, while N-demethylation of aromatic and aliphatic dimethylamines was comparable in rate and efficiency. Aliphatic N-hydroxylation showed no rate increase with increasing pH despite the predicted increase in the concentration of the neutral substrate. The relative rates of N-hydroxylation and N-demethylation were compared for a series of para-substituted aromatic amines. The rate of demethylation of para-substituted N,N-dimethylanilines, as measured both by product formation and by NADPH consumption, correlated with the electronic parameter sigma and with the Hansch lipophilicity parameter pi. N-Hydroxylation of a similar series of anilines did not show a dependence on the electronic parameter but was dependent on the lipophilicity parameter. The differing dependence on the electronic parameter suggests that there are different rate-determining processes of N-oxidation for these two reactions.     

Relative contribution of cytochromes P-450 and flavin-containing monoxygenases to the metabolism of albendazole by human liver microsomes

AIMS: Albendazole (ABZ; methyl 5-propylthio-1H-benzimidazol-2-yl carbamate) is a broad spectrum anthelmintic whose activity resides both in the parent compound and its sulphoxide metabolite (ABS). There are numerous reports of ABZ metabolism in animals but relatively few in humans. We have investigated the sulphoxidation of ABZ in human liver microsomes and recombinant systems. METHODS: The specific enzymes involved in the sulphoxidation of ABZ were determined by a combination of approaches; inhibition with an antiserum directed against cytochrome P450 reductase, the effect of selective chemical inhibitors on ABZ sulphoxidation in human liver microsomes, the capability of expressed CYP and FMO to mediate the formation of ABS, regression analysis of the rate of metabolism of ABZ to ABS in human liver microsomes against selective P450 substrates and regression analysis of the rate of ABS sulphoxidation against CYP expression measured by Western blotting. RESULTS: Comparison of Vmax values obtained following heat inactivation (3min at 45 degrees C) of flavin monoxygenases (FMO), chemical inhibition of FMO with methimazole and addition of an antiserum directed against cytochrome P450 reductase indicate that FMO and CYP contribute approximately 30% and 70%, respectively, to ABS production in vitro. Comparison of CLint values suggests CYP is a major contributor in vivo. A significant reduction in ABZ sulphoxidation (n = 3) was seen with ketoconazole (CYP3 A4; 32-37%), ritonavir (CYP3 A4: 34-42%), methimazole (FMO: 28-49%) and thioacetamide (FMO; 32-35%). Additive inhibition with ketoconazole and methimazole was 69 +/- 8% (n = 3). ABS production in heat - treated microsomes (3 min at 45 degrees C) correlated significantly with testosterone 6beta-hydroxylation (CYP3A4; P < 0.05) and band intensities on Western blots probed with an antibody selective for 3A4 (P < 0.05). Recombinant human CYP3 A4, CYP1A2 and FMO3 produced ABS in greater quantities than control microsomes, with those expressing CYP3A4 producing threefold more ABS than those expressing CYP1A2. Kinetic studies showed the Km values obtained with both CYP3A4 and FMO3 were similar. CONCLUSIONS: We conclude that the production of ABS in human liver is mediated via both FMO and CYP, principally CYP3A4, with the CYP component being the major contributor.     

Enrichment of cytochrome P-450 in human liver microsomes by the action of proteases

Human liver microsomes have been partially enriched in cytochrome P450 using a simple and rapid method. Microsomes were digested with protease XXVII (40 micrograms/nmole cyt. P450), then incubated with CHAPS, a zwitterionic detergent (25 mg/nmole cyt. P450) in combination with 0.07% protamine sulfate and the solubilized cyt. P450 was separated by ultracentrifugation. By this method, about 35% of total microsomal protein was solubilized with more than 80% of cyt. P450. This technique increases the specific activity of cyt. P450 by three fold.     

Involvement of cytochrome P-450c in alpha-naphthoflavone metabolism by rat liver microsomes

Metabolism of alpha-naphthoflavone (ANF) is increased markedly in rat liver microsomes by 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), two inducers of cytochromes P-450c and P-450d (P-450c and P-450d). Although several indirect lines of evidence in the literature suggest that ANF is metabolized by P-450c, Vyas et al. [J. Biol. Chem. 258:5649-5659 (1983)] reported that ANF metabolism by 3-MC-induced rat liver microsomes was only partially inhibited by antibodies against P-450c. Our laboratory has previously reported clastogenic effects of metabolites of ANF, and in the present study we reexamined the role of P-450c in ANF metabolism by both uninduced and TCDD-induced rat liver microsomes, using monospecific polyclonal antibodies to P-450c and P-450d. ANF metabolism was inhibited to different extents in TCDD-induced microsomes by different preparations of anti-P-450c. One lot of anti-P-450c produced only 50% inhibition of ANF metabolism in TCDD-induced microsomes, whereas another lot of anti-P-450c inhibited ANF metabolism by 80%. Anti-P-450d had no effect on ANF metabolism. Neither anti-P-450c nor anti-P-450d inhibited ANF metabolism in uninduced rat liver microsomes. In a reconstituted enzyme system, purified P-450c metabolized ANF 47 and 510 times more rapidly than P-450d and P-450b, respectively. Metabolites resulting from oxidation at 7,8- or 5,6-positions (7,8-dihydro-7,8-dihydroxy-ANF, 5,6-dihydro-5,6-dihydroxy-ANF, 5,6-oxide-ANF, and 6-hydroxy-ANF) were formed by all preparations of microsomes. An unknown toxic ANF metabolite was formed only with a reconstituted P-450c system and with 3-MC- or TCDD-induced microsomes. Our results indicate that P-450c is responsible for the majority of the metabolism of ANF in TCDD-induced microsomes, whereas other constitutive isozymes are responsible for the metabolism seen in uninduced liver microsomes. The variable inhibition of ANF metabolism with different lots of anti-P-450c probably reflects the differences in the proportion of antibodies to different epitopes important in the binding or metabolism of this substrate.     

One-electron reduction of mitomycin c by rat liver: role of cytochrome P-450 and NADPH-cytochrome P-450 reductase

1. The role of cytochrome P-450 in the one-electron reduction of mitomycin c was studied in rat hepatic microsomal systems and in reconstituted systems of purified cytochrome P-450. Formation of H2O2 from redox cycling of the reduced mitomycin c in the presence of O2 and the alkylation of p-nitrobenzylpyridine (NBP) in the absence of O2 were taken as parameters. 2. With liver microsomes from both 3-methylcholanthrene (MC)- and phenobarbital (PB)-pretreated rats, reverse type I difference spectra were observed, indicative of a weak interaction between mitomycin c and the substrate binding site of cytochrome P-450. Mitomycin c inhibited the oxidative dealkylation of aminopyrine and ethoxyresorufin in both microsomal systems. 3. Under aerobic conditions the H2O2 production in the microsomal systems was dependent on NADPH, O2 and mitomycin c, and was inhibited by the cytochrome P-450 inhibitors, metyrapone and SKF-525A. 4. Although purified NADPH-cytochrome P-450 reductase was also effective in reduction of mitomycin c and the concomitant reduction of O2, complete microsomal systems and fully reconstituted systems of cytochrome P-450b or P-450c and the reductase were much more efficient. 5. Under anaerobic conditions in the microsomal systems both reduction of mitomycin c (measured as the rate of substrate disappearance) and the reductive alkylation of NBP were dependent on cytochrome P-450. 6. The relative rate of reduction of mitomycin c by purified NADPH-cytochrome P-450 reductase was lower than that by a complete microsomal system containing both cytochrome P-450 and a similar amount of NADPH-cytochrome P-450 reductase. 7. It is concluded that although NADPH-cytochrome P-450 reductase is active in the one-electron reduction of mitomycin c, the actual metabolic locus for the reduction of this compound in liver microsomes under a relatively low O2 tension is more likely the haem site of cytochrome P-450.     

Inhibitory effects of silibinin on cytochrome P-450 enzymes in human liver microsomes

Silibinin, the main constituent of silymarin, a flavonoid drug from silybum marianum used in liver disease, was tested for inhibition of human cytochrome P-450 enzymes. Metabolic activities were determined in liver microsomes from two donors using selective substrates. With each substrate, incubations were carried out with and without silibinin (concentrations 3.7-300 microM) at 37 degrees in 0.1 M KH2PO4 buffer containing up to 3% DMSO. Metabolite concentrations were determined by HPLC or direct spectroscopy. First, silibinin IC50 values were determined for each substrate at respective K(M) concentrations. Silibinin had little effect (IC50>200 microM) on the metabolism of erythromycin (CYP3A4), chlorzoxazone (CYP2E1), S(+)-mephenytoin (CYP2C19), caffeine (CYP1A2) or coumarin (CYP2A6). A moderate effect was observed for high affinity dextromethorphan metabolism (CYP2D6) in one of the microsomes samples tested only (IC50=173 microM). Clear inhibition was found for denitronifedipine oxidation (CYP3A4; IC50=29 microM and 46 microM) and S(-)-warfarin 7-hydroxylation (CYP2C9; IC50=43 microM and 45 microM). When additional substrate concentrations were tested to assess enzyme kinetics, silibinin was a potent competitive inhibitor of dextromethorphan metabolism at the low affinity site, which is not CYP2D6 (Ki.c=2.3 microM and 2.4 microM). Inhibition was competitive for S(-)-warfarin 7-hydroxylation (Ki,c=18 microM and 19 microM) and mainly non-competitive for denitronifedipine oxidation (Ki,n=9 microM and 12 microM). With therapeutic silibinin peak plasma concentrations of 0.6 microM and biliary concentrations up to 200 microM, metabolic interactions with xenobiotics metabolised by CYP3A4 or CYP2C9 cannot be excluded.     

Metabolism of the "mixed" cytochrome P-450 inducer hexachlorobenzene by rat liver microsomes

Hexachlorobenzene (HCB) was metabolised by phenobarbital-induced liver microsomes from male rats to pentachlorobenzene, pentachlorophenol, tetrachloro-1,2-benzenediol and tetrachloro-1,4-benzenediol (1:88:2:9). Metabolites were identified and quantified by electron capture g.l.c. Structures were confirmed by selective ion monitoring g.l.c.-m.s. The formation of pentachlorophenol was dependent on the presence of NADPH and O2 and inhibited by CO, SKF 525A and metyrapone. Conversion of HCB to pentachlorophenol was stimulated by pretreatment of rats with phenobarbital (PB) but not by 3-methylcholanthrene (3-MC), or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, the conversion of pentachlorophenol to tetrachloro-1,4-benzenediol was markedly induced by 3-MC but poorly by PB. HCB, Aroclor 1254 and isosafrole stimulated both hydroxylations. The cytochrome P-450c inhibitor 9-hydroxyellipticine inhibited conversion of pentachlorophenol to tetrachlorobenzenediols by HCB and beta-naphthoflavone induced micromes. In addition to hydroxylation reactions, evidence was obtained for the conjugation of HCB with glutathione catalysed by a microsomal glutathione transferase. Radioactivity from [14C]HCB was bound to microsomal protein during aerobic incubations. Binding was inhibited by GSH and N-acetyl-cysteine. Preliminary studies suggested that the reactive species was derived from tetrachloro-1,4-benzoquinone. No correlation was found between levels of metabolites or covalent binding produced by the two sexes and the marked sex dependent hepatic porphyrogenic and carcinogenic effects of HCB.