Reduced nicotinamide adenine dinucleotide-dependent reduction of tertiary amine N-oxide by liver microsomal cytochrome P-450.

NADH-supported reduction of tertiary amine N-oxides in rat liver microsomes was investigated with imipramine N-oxide, tiaramide N-oxide and N, N-dimethylaniline N-oxide as substrate in the presence of NADH. The reductase activity is sensitive to carbon monoxide. NADH-cytochrome b₅ reductase or cytochrome b₅, solubilized by trypsin or subtilisin, showed no N-oxide reductase activity. The NADH-dependent reduction of tertiary amine N-oxides was markedly inhibited by antibody to NADH-eytochrome b₅ reductase and antibody to cytochrome b₅. These results confirmed that NADH-dependent N-oxide reduction was catalyzed by cytochrome P-450 and the reducing equivalent for the N-oxide reduction was transferred from NADH to cytochrome P-450 mainly via NADH-cyto-chrome b₅ reductase and cytochrome b₅ in the microsomal membranes. NADH-dependent N-oxide reductase is also sensitive to oxygen and 4 μM oxygen gave 50 per cent inhibition with imipramine N-oxide. Kinetic study shows that K_m values for the reduction of imipramine N-oxide, tiaramide N-oxide and N, N-dimethylaniline N-oxide were 0.05 mM, 0.14 mM and 0.16 mM, respectively. NADH-dependent N-oxides reductase activity is affected by azo and nitroso compounds and hydrazide, although the degree of inhibition was rather weak compared with those of NADPH-dependent activity. Furthermore, NADH-dependent reduction of tertiary amine N-oxide was only slightly affected by n-octylamine, 2,4-diehloro-6-phenylphenoxyethylamine and aniline. NADH-dependent N-oxide reductase activity in liver microsomes was less sensitive to phenobarbital or 3-methyleholanthrene pretreatment than NADPH-dependent activity. Some characteristics of NADH-dependent N-oxide reductase activity were discussed and compared with those of NADPH-dependent activity.     

Characteristics of the microsomal N-hydroxylation of benzamidine to benzamidoxime

1. A simple and fast h.p.l.c. analysis of benzamidoxime formed by microsomal N-hydroxylation of benzamidine is presented which is well suited for the determination of the N-oxygenation activity of microsomal enzymes.

2. Optimal reaction conditions were determined. The apparent K_m and V_max values were, respectively, 1–61 mM and 0.38nmol benzamidoxime/min per mg protein.

3. The effects of the inducers phenobarbital, 3-methylcholanthrene and benzamidine itself on hepatic benzamidine metabolizing activity in rabbits were determined.

4. Neither superoxide anion nor hydrogen peroxide is directly involved in the N-hydroxylation reaction.

5. The direct involvement of cytochrome P-450 in the N-hydroxylation of benzamidine is supported by the observation that inhibitors of cytochrome P-450, in particular carbon monoxide, markedly decreased the rate of N-oxygenation.     

Non-enzymatic reduction of aliphatic tertiary amine N-oxides mediated by the haem moiety of cytochrome P450

1. The mechanism of reduction of aliphatic tertiary amine N-oxides to tertiary amines in liver microsomes was examined and a novel type of reduction by cytochrome P450 was found. 2. Rat liver microsomes exhibited a significant N-oxide reductase activity toward brucine N-oxide and imipramine N-oxide in the presence of both NAD(P)H and FAD under anaerobic conditions. These N-oxide reductase activities were inhibited by carbon monoxide or air. However, the activities were not abolished by boiling the microsomes; indeed, in the case of brucine N-oxide, the activity was enhanced. 3. The activity toward brucine N-oxide was also observed after the conversion of cytochrome P450 to cytochrome P420. Cytochrome P4502B1 alone exhibited the reductase activity in the presence of both NAD(P)H and FAD. After the removal of haem from cytochrome P4502B1, the activity was observed in the haem moiety, but not in the cytochrome P450 apoprotein. 4. Photochemically reduced FAD was effective in the reduction in place of NAD(P)H and FAD. 5. The N-oxide reduction appears to proceed non-enzymatically, catalysed by the haem group of cytochrome P450 in the presence of a reduced flavin.     

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.     

Microsomal N-Oxygenation of Adenine to Adenine 1-N-Oxide

During investigations on the N-oxygenation of adenine ( 1 ) the enzymatic formation of adenine 1-N-oxide 3 was demonstrated for the first time. The identity of this metabolite was confirmed by its chromatographic behaviour and UV-spectrum recorded after HPLC separation. Adenine 1-N-oxide ( 3 ) and similar oxygenated derivatives of adenine were synthesized as reference substances. The enzymatic formation of 3 exhibits the typical characteristics of a reaction catalysed by microsomal mono-oxygenases. In induction experiments, an increase in the rate of formation of 3 after pretreatment with phenobarbital was observed. A participation of those isoenzymes of the cytochrome P-450 enzyme system which can be induced by phenobarbital is assumed.     

Microsomal N-Hydroxylation of Dibenzylamine

1. The properties of the rabbit liver microsomal enzyme system(s) catalysing the formation of N,N-dibenzylhydroxylamine as the major metabolite of dibenzylamine have been investigated.

2. The system consists of NADPH- and NADH-dependent components which are differentiated by their different pH optima and sensitivity towards cyanide.

3. The effect of various metabolic inhibitors on the N-oxidation process in vitro are investigated.

4. The N-oxidation of the parent amine was inhibited by CO, SKF 525-A, and inhibitors known to interact with microsomal cytochrome P-450. Phenobarbitone pre-treatment stimulates further metabolism of the hydroxylamine.     

On the sulphoxidation of cimetidine and etintidine by rat and human liver microsomes

1. Sulphoxidation of cimetidine and etintidine was investigated by in vitro assays with liver microsomes from untreated 5,6-benzoflavone- and phenobarbital-pretreated rats as well as with human liver microsomes. The formation rate of cimetidine sulphoxide and etintidine sulphoxide with liver microsomes of normal or pretreated rats reached to 1.1 and 0.9 nmol/min mg microsomal protein, respectively.

2. Inhibition experiments with carbon monoxide and n-octylamine indicated that this sulphoxidation is catalyzed by cytochrome(s) P-450, whereas flavin-containing monooxygenase and/or non-enzymatic reactions (via peroxides) seems not to be involved: no inhibition was observed by methimazole, N,N-dimethylaniline, preheating or glutathione and EDTA.

3. With human liver microsomes the cytochrome P-450-dependent sulphoxidation accounted for no more than 40% of the total oxidation.     

Studies on the metabolism of dimethylnitrosamine in vitro by rat-liver preparations.: II. Inhibition by substrates and inhibitors of monoamine oxidase

1. The metabolism of dimethylnitrosamine (DMN) to formaldehyde by rat-hepatic postmitochondrial supernatant fractions has been compared with the activities of several cytochrome P-450-dependent mixed-function oxidase enzymes and the Ziegler mixedfunction amine oxidase enzyme (EC 1.14.13.8).

2. A variety of monoamine oxidase (MAO, EC 1.4.3.4) inhibitors of diverse chemical structure inhibited the metabolism of DMN. In parallel studies a number of MAO substrates, but not their deaminated products, also inhibited DMN metabolism, whereas substrates of diamine oxidase were ineffective.

3. At concentrations which inhibited DMN metabolism several MAO substrates and inhibitors did not inhibit the N-oxidation of N, N-dimethylaniline and an inhibitor and an activator of the Ziegler enzyme had no corresponding effect on DMN metabolism.

4. The metabolism of DMN and a number of MAO enzyme activities were stable to storage under conditions where mixed-function oxidase enzymes were not.

5. These results are consistent with the suggestion that DMN may, at least in part, be metabolized by hepatic enzyme(s) not dependent on cytochrome P-450 and that a microsomal amine oxidase enzyme, unrelated to the Ziegler enzyme, may be involved in the hepatic degradation of this nitrosamine. The present data does, however, suggest a role for microsomal NADPH-cytochrome c reductase in hepatic DMN metabolism.     

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 ρ-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.     

Relationship between lipid composition and drug metabolizing capacity of human liver

Summary The relationship between hepatic glycerolipids and microsomal drug-metabolizing enzymes was studied in liver biopsies from 41 subjects. The series included obese, diabetic, epileptic and chronic alcoholic patients, all of whom were hospitalized for suspected hepatic ailments (fatty liver, hepatitis or cirrhosis). Therapy with enzyme-inducing anticonvulsants was associated with high phospholipid and cytochrome P-450 and low triacylglycerol concentration in the liver. In patients with fatty liver or cirrhosis low phospholipid and cytochrome P-450 and high triacylglycerol concentrations were observed. There was a significant correlation (r (Pearsons's product moment correlation coefficient) =0.91) between the hepatic phospholipid and cytochrome P-450 concentration. The cytochrome P-450 concentration was inversely related (r=–0.74) to the triacylglycerol concentration. The positive correlation between hepatic phospholipids and drug-metabolizing enzymes could be interpreted as indicating that in human liver phospholipid and cytochrome P-450 synthesis share common regulators, or that phospholipids are necessary for the maximum rate of cytochrome P-450 synthesis.     

“Detoxication,a most Stimulating Concept of Toxicology”

1. Large, independent variations occur among New Zealand White rabbits in the 21-and 6β-hydroxylation of progesterone as catalysed by liver microsomes.

2. These reactions are catalysed respectively by two electrophoretically distinct types of rabbit-liver microsomal cytochrome P-450, 1 and 3b, as judged by their catalytic efficiency and the capacity of specific monoclonal antibodies to extensively inhibit the respective microsomal hydroxylases.

3. The relatively large variations in progesterone 6β-hydroxylase activity do not appear to be associated with differences in microsomal content of cytochrome P-450 3b, whereas differences in the microsomal concentration of cytochrome P-450 1 may underlie variations in 21-hydroxylase activity.

4. Preparations of cytochrome P-450 3b contain at least two catalytically distinct subforms, one of which catalyses both 6β- and 16α-hydroxylation of progesterone with a low K_m while the other subform catalyses predominantly 16α-hydroxylation with a significantly greater K_m.

5. The two catalytic subforms of cytochrome P-450 3b can be independently modulated in vitro by positive and negative effectors that can arise in vivo from the metabolism of progesterone.

6. The 6β-hydroxylase subform of cytochrome P-450 3b is not expressed in a genetically defined strain of rabbits, IIIVO/J, indicating a heritable basis for the differential expression of the two subforms of cytochrome P-450 3b.

7. These results indicate that the extent of cytochrome P-450 multiplicity may be greater than is evident from the isolation of electrophoretically distinct forms of cytochrome P-450, and that small differences in structure may underlie large differences in catalytic properties.

8. It is not known whether the differences among outbred New Zealand White rabbits in the expression of either cytochrome P-450 1 or the subforms of cytochrome P-450 3b reflect regulatory phenomena or genetic polymorphism.     

The in vitro N-oxygenation of N-tert.alkyl-substituted benzamidine

Studies of the N-Oxygenation of N-(tert.Alkyl)benzamidines in vitro N-tert.Butyl- and N-tert.octylbenzamidine ( 1a and b ) and their potential N-oxygenated metabolites, the amidoximes 2a and b , have been synthesized and characterized. N-tert.Butylbenzamidine 1a is N-oxygenated to the amidoxime 2a by aerobic incubation with non-induced microsomal fractions of rabbit liver homogenates and NADPH. This transformation supports the hypothesis that benzamidines undergo N-oxygenation by the cytochrome P-450 enzyme system when N-dealkylation is not possible (absence of hydrogen atoms in α-position to the amidine nitrogen atoms).     

Inhibition of mono-oxygenase activities by 1,1,1-trichloropropene 2,3-oxide, an inhibitor of epoxide hydrase, in rat liver microsomes.

Addition of 1,1,1-trichloropropene 2,3-oxide (TCPO), an inhibitor of microsomal epoxide hydrase, to rat liver microsomes caused a type I spectral change, and its magnitude was increased by pretreatment of animals with phenobarbital (PB) but not with 3-methylcholanthrene and polychlorinated biphenyls. TCPO inhibited aminopyrine N-demethylation competitively and prevented covalent binding of 2,4,2',4'-tetrachlorobiphenyl to macromolecules catalyzed by liver microsomes, although it stimulated benzolalpyrene hydroxylation significantly. It is suggested that TCPO interacts with cytochrome P-450, especially a species of the cytochrome which is inducible by PB administration, and thus inhibits mono-oxygenase activities of liver microsomes.     

Cytochrome P-450 and oxidative metabolism in molluscs

1. Cytochrome P-450 and the associated components and oxidative activities of a mixed-function oxidase (MFO) system are localized primarily in the microsomes of the digestive gland of molluscs.

2. Cytochrome P-450 and putative cytochrome P-450-catalysed oxidative activities, measured in vitro and/or in vivo, have variously been detected in 23 species of mollusc.

3. Cytochrome P-450 and other MFO components and activities may be increased by exposure to xenobiotics, but the results are variable and no correlation is obvious between changes in cytochrome P-450 content and measured MFO activities (benzo[a]pyrene hydroxylase (BPH) and 7-ethoxycoumarin O-deethylase (ECOD)).

4. Type II binding compounds (clotrimazole, miconazole, ketoconazole, metyrapone and pyridine) give type II difference spectra with mussel digestive gland microsomal P-450, whereas type I binding compounds (testosterone, 7-ethoxycoumarin, α-naphthoflavone, SKF525-A) give apparent reverse type I difference spectra.

5. The existence of multiple or particular forms (P450 IVA or LAw) of cytochrome P-450 is indicated from enzyme kinetics and inhibition studies, seasonality, purification studies and cDNA probes.

6. Microsomal MFO activities are observed even in the absence of added or generated NADPH, and the NADPH-independent BPH, ECOD and N,N-dimethylaniline N-demethylase activities are inhibited by reducing agents, including NADPH.

7. The major metabolites of microsomal benzo[a]pyrene metabolism are quinones.

8. One-electron oxidation is considered to be one possible mechanism of molluscan cytochrome P-450 catalytic action.     

The interactions of hydrazine derivatives with rat-hepatic cytochrome P-450

1. The ability of different classes of hydrazine derivatives to modify cytochrome P-450 function during turnover as judged by loss of absorbance at 416 nm, loss of CO-reactive cytochrome P-450, or destruction of haem has been studied.

2. Addition of monosubstituted hydrazines to rat-liver microsomes caused considerable loss of CO-reactive cytochrome P-450 and haem destruction; monosubstituted hydrazides caused mainly loss of CO-reactive cytochrome P-450, most likely due to abortive complex formation. Metabolism of 1,1-disubstituted hydrazines by microsomal cytochrome P-450 resulted in loss of CO-reactive cytochrome P-450 only, with no haem destruction. The 1,2-disubstituted hydrazines and hydrazides, procarbazine and iproniazid, acted similarly to the monosubstituted hydrazines, while 1,2-dimethyl-hydrazine elicited no response, cither in observable spectral changes or loss of CO-reactive cytochrome P-450.

3. Synthetic diazene intermediates of phenylhydrazine and N-aminopiperidine reacted rapidly with microsomal cytochrome P-450 to form a spectral intermediate resembling the putative iron porphyrin-diazenyl complex. The decomposition of certain iron porphyrin-diazenyl derivatives apparently leads to destruction of the porphyrin prosthetic group, most likely due to haem alkylation.     

Induction of cytochrome P-450 isozymes by chromanamine derivatives in rat liver

1. Pretreatment of rats with 6-(3-picolyl)amino-2,2,5,8-tetramethylchromane (PATC) for 7 days resulted in a significant increase in the activities of benzphetamine N-demethylase, p-nitroanisole O-demethylase and aniline hydroxylase in liver microsomes prepared 24?h after the last treatment.

2. Analysis by Western blot showed that PATC induces cytochrome P-450 b, P-450 c and P-450 d, which are the major forms of cytochrome P-450 in liver microsomes of rats when pretreated with phenobarbital and 3-methylcholanthrene.

3. Exposure of liver sections to the antibodies to cytochrome P-450 b and P-450 c resulted in intense immunostaining within the centrilobular regions, but produced staining of considerably weaker intensity in the perilobular region. Semiquantitative immunochemical analysis, by image analyser, of cytochrome P-450 b and P-450 c showed that centrilobular hepatocytes were stained more intensively than perilobular hepatocytes.

4. These results indicate that PATC induces cytochromes P-450 b and P-450 c, in the centrilobular hepatocytes to a greater degree than those in the perilobular hepatocytes.

5. Co-administration of PATC with pentobarbital caused a significant increase in pentobarbital sleeping time. Furthermore, PATC was found to cause a decrease in the activity of benzphetamine N-demethylase in liver microsomes prepared 30?min after treatment with the drug.     

Formation of two major nicotine metabolites in livers of guinea pigs

Using antibody against NADPH-cytochrome P-450 reductase and several effectors of cytochrome P-450 and FAD-containing monooxygenase, we investigated nicotine metabolites formed by these two enzymes. When [³H]nicotine was metabolized by the combination of liver microsomes of guinea pigs and partially purified aldehyde oxidase, three distinct spots corresponding to nicotine, cotinine and nicotine-1'-oxide were observed on fluorograms of thin-layer chromatography. Antibody against NADPH-cytochrome P-450 reductase inhibited the formation of cotinine but not nicotine-1'-oxide. Metyrapone and n-octylamine inhibited the cotinine formation, while methimazole prevented the formation of nicotine-1'-oxide. These results show that microsomal electron transport systems participate in the formation of nicotine-1'-oxide and strongly suggest the involvement of FAD-containing monooxygenase in the formation of nicotine-1'-oxide.     

Meeting of Drug Metabolism Group Extrahepatic Metabolism

1. Four non-acidic primary metabolites of N-(5-pyrrolidinopent-3-ynyl)succinimide (BL 14) were identified and quantified using g.l.c. and mass spectrometry. The metabolites are α-hydroxy-N-(5-pyrrolidinopent-3-ynyl)succinimide (A), N-(5-(2-oxopyrrolidino)-pent-3-ynyl)succinimide (B), N-(2-hydroxy-5-pyrrolidinopent-3-ynyl)succinimide (C) and N-(5-pyrrolidinopent-3-ynyl)succinimide N'-oxide (E), the latter analysed after reduction to the parent amine.

2. In rat liver preparations, all metabolites are formed by microsomal, NADPH-dependent enzyme systems, but with different characteristics. The response to inhibitors such as CO and SKF 525A indicates participation of cytochrome P-450 enzymes in the formation of all metabolites. Phenobarbital pretreatment markedly enhances propynylic hydroxylation (C) but has little or no effect on the other metabolic pathways. Succinimide hydroxylation (A) exhibits a pH optimum at 7±0, while the formation of metabolites B and C increases at pH values between 6±4 and 7±7.

3. Kinetic studies on the formation of metabolites A-C revealed differences in the Michaelis constant, while the V_max values were similar. Succinimide hydroxylation (A) is most efficient with a K_m of 3±7 × 10^?5 M, compared with a K_m of 1±7 × 10^?3 M for propynylic hydroxylation (C).

4. The formation of metabolites B and E conforms to the corresponding mechanisms for lactam and N-oxide formation for other xenobiotics. The formation of metabolites A and C represents two extremities, reflected in their different responses to phenobarbital pretreatment, pH changes and in their different K_m values. Although little can be discerned about the mechanisms from the literature, the enzymes cataly sing both reactions appear to be cytochromes.     

Induction of drug metabolism. VI. Effects of phenobarbital and 3-methylcholanthrene administration on N-demethylating enzyme systems of rough and smooth hepatic microsomes

The induction of the microsomal hemoproteins, cytochromes P-450 and Pi-450, and of N-demethylase activities in hepatic microsomal subfractions from rats were studied at various times after administration of phenobarbital or 3-methylcholanthrene. After a single dose of phenobarbital, N-demethylase activity and cytochrome P-450 levels increased initially only in rough microsomes (RER) whereas a single dose of 3-methylcholanthrene caused almost simultaneous increases of the two enzymes in both RER and smooth microsomes (SER). The increases in N-demethylase activities during this early period of induction by 3-methylcholanthrene were paralleled by a change in P-450 hemoprotein from cytochrome P-450 to cytochrome P₁-450 in both microsomal subfractions, but the total amount of P-450 hemoprotein remained essentially unchanged. These results add to existing evidence that phenobarbital and 3-methylcholanthrene produce their inductive effects by different mechanisms and raise the possibility that cytochrome P₁-450 may be synthesized in both RER and SER.     

Sulphoxidation of albendazole by the FAD-containing and cytochrome P-450 dependent mono-oxygenases from pig liver microsomes

1. Two distinct microsomal pathways involved in the metabolism of albendazole (ABZ) to albendazole-sulphoxide (SO. ABZ) by pig liver microsomes have been identified and quantified.

2. The binding of ABZ to microsomal cytochrome P-450 (Type I spectrum. Ks = 25.5/μM), the decrease of the rate of sulphoxidation by antibody against NADPH cytochrome c reductase, and the use of purified cytochrome P-450 A demonstrated the contribution of a cytochrome P-450-dependent mono-oxygenase to the metabolism of ABZ.

3. The involvement of FAD-containing mono-oxygenase (FMO) was shown by thermal pretreatment of microsomes, n-octylamine activation of the reaction, and by using purified pig liver FMO.

4. From K_m and V_max values, it would appear that the relative contributions of the two systems depend on the concentration of ABZ.