Metabolism-dependent covalent binding of (S)-[5-3H]nicotine to liver and lung microsomal macromolecules

Incubation of (S)-[5-3H]nicotine with rabbit liver microsomes in the presence of dioxygen and NADPH results in the formation of metabolites that bind covalently to microsomal macromolecules (250-550 pmol/mg of protein/hr). The partition ratio [(S)-nicotine metabolized/(S)-nicotine equivalents covalently bound] ranged between 250:1 and 500:1. The addition of SKF 525-A, cytochrome c, or n-octylamine inhibited both (S)-nicotine metabolism and covalent binding whereas phenobarbital pretreatment increased the rates of metabolism and covalent binding. Sodium cyanide, which forms stable adducts with the cytochrome P-450-generated iminium ion metabolites of (S)-nicotine and a variety of other tertiary amines, inhibited covalent binding but also decreased the rate of (S)-nicotine metabolism. The metabolism-dependent covalent binding of (S)-nicotine and its conversion to the delta 1',5'-iminium species were observed also in microsomal incubations prepared from rabbit lung and human liver tissues.     

Metabolism-dependent covalent binding of S(−)-3H-nicotine to lung microsomes in vitro

Incubation of S(?)-³H-nicotine with rabbit lung microsomes in the presence of dioxygen and NADPH results in the formation of metabolites that bind covalently to microsomal macromolecules. The addition of cytochrome P-450 monooxygenase inhibitors, α-methylbenzyl aminobenzotriazole and aroclor 1260, inhibited both (S)-nicotine metabolism and covalent binding. The relative rates of oxidation of nicotine Δ^1′,5′ iminium ion to cotinine indicates that lung 100,000×g supernatant catalyzed this oxidation approximately 18 times slower than that of liver system based on mg of protein, and increased covalent interactions. Since the activity of lung iminium oxidase appears much lower than the liver, it is tempting to speculate that localized concentrations of nicotine Δ^1′,5′ iminium ion in the lung will survive for a longer period of time. These results support that cytochrome P-450 catalyzed oxidation of nicotine leads to the formation of reactive and electrophilic intermediates capable of chemical interactions with biomacromolecules.     

Enantioselective S-oxygenation of 2-aryl-1,3-dithiolanes by rabbit lung enzyme preparations

Pulmonary microsomes, highly purified pulmonary flavin-containing monooxygenase, and highly purified pulmonary cytochrome P-450IIB-4 from pregnant female rabbits catalyze the NADPH-dependent S-oxygenation of a series of 2-aryl-1,3-dithiolanes. The S-oxide is the only detectable product formed during the short time period of the enzymatic reactions. Studies on the biochemical mechanism for S-oxygenation of 2-aryl-1,3-dithiolanes suggest that this reaction is catalyzed preferentially by the flavin-containing monooxygenase, although cytochromes P-450 also contribute to S-oxygenation. This conclusion is based on the effects of a cytochrome P-450 inhibitor, aminobenzotriazole, as well as on studies of the stereoselectivity of the reaction. Although both purified rabbit pulmonary cytochrome P-450IIB-4 and purified flavin-containing monooxygenase have identical diastereoselectivity, producing the (trans)-S-oxide, these monooxygenases possess opposite S-oxygenation enantioselectivity. Pulmonary cytochrome P-450IIB-4 S-oxygenates 2-aryl-1,3-dithiolanes almost exclusively at the pro-S-sulfur atom, whereas pulmonary flavin-containing monooxygenase S-oxygenates 2-aryl-1,3-dithiolanes exclusively at the pro-R-sulfur atom. 2-Aryl-1,3-dithiolane S-oxides are S-oxygenated a second time on the S'-sulfide sulfur atom but only by rabbit lung microsomes and pulmonary flavin-containing monooxygenase and not by cytochrome P-450IIB-4. That pulmonary flavin-containing monooxygenase only catalyzes formation of (trans)- and not (cis)-2-aryl-1,3-dithiolane S-oxide formation suggests that the active site of pulmonary flavin-containing monooxygenase exerts great steric limitations on 2-aryl-1,3-dithiolane S-oxygenation.     

The role of flavin-containing monooxygenase in the N-oxidation of the pyrrolizidine alkaloid senecionine

The pyrrolizidine alkaloid, senecionine, is N-oxidized by purified pig liver flavin-containing monooxygenase but not by purified rabbit lung flavin-containing monooxygenase. The activity of the pig liver enzyme toward senecionine was linear with time and amount of enzyme. The oxygenation was not due to some indirect mechanism, such as O2- release from the enzyme, as scavengers of activated oxygen had no effect on product formation. The Km of purified pig liver flavin-containing monooxygenase for senecionine was 0.3 mM. Although senecionine is a substrate for the pig liver enzyme, studies performed with rat liver microsomes suggest that, in this species, cytochromes P-450 catalyze the majority of senecionine-N-oxidation. These experiments included inhibition by chemical inhibitors of P-450, treatment of the microsomes with elevated temperatures, inhibition by anti-NADPH-cytochrome P-450 reductase antibody, the effect of dexamethasone on N-oxidation, and relative amounts of flavin-containing monooxygenase determined by immunoquantitation. These results demonstrate that flavin-containing monooxygenase can be involved in the detoxication of pyrrolizidine alkaloids via N-oxidation, but the relative contribution of flavin-containing monooxygenase and cytochromes P-450 may be species and tissue dependent.     

Enantioselective S-oxygenation of para-methoxyphenyl-1,3-dithiolane by various tissue preparations: effect of estradiol

Liver, kidney, and lung microsomes prepared from nonpretreated female Sprague-Dawley rats catalyze the NADPH- and oxygen-dependent S-oxygenation of para-methoxyphenyl-1,3-dithiolane. Studies on the biochemical mechanism of dithiolane S-oxygenation in liver, kidney, and lung microsomes suggest that this reaction is catalyzed in a diastereoselective and enantioselective fashion by the flavin-containing monooxygenase and, to a lesser extent, the cytochromes P-450. This conclusion is based on results examining the effects of selective cytochrome P-450 inhibitors and positive effectors, microsome heat-inactivation treatment, and alternate substrates for the flavin-containing monooxygenase. Liver and kidney microsomes prepared from ovarectomized female rats tended to have decreased S-oxygenase activity, compared with nonpretreated female rats, whereas ovarectomized rats pretreated with estradiol had markedly lower S-oxygenase activity. In contrast, lung microsomal S-oxygenase activity, which is low in pulmonary microsomes from nonpretreated female rats, increases 2-4-fold after ovariectomization and estradiol pretreatment. In female Sprague-Dawley rats, estradiol pretreatment is mainly responsible for the large decrease (or increase) in S-oxygenase activity observed in the tissues examined, although it is unlikely that estradiol alone controls flavin-containing monooxygenase S-oxygenase activity.     

Stereochemical studies on the cytochrome P-450 catalyzed oxidation of (S)-nicotine to the (S)-nicotine delta 1'(5')-iminium species

Mammals metabolize the tobacco alkaloid (S)-nicotine primarily to the lactam (S)-cotinine by a pathway involving an initial cytochrome P-450 catalyzed two-electron oxidation at the prochiral 5'-carbon atom. The stereochemical course of this oxidation was examined with human microsomal preparations and the E and Z diastereomers of (S)-nicotine-5'd1. The metabolically generated delta 1'(5')-iminium ion intermediate was trapped and analyzed as the corresponding diastereomeric 5'-cyano derivatives by a capillary column GC-EIMS selected ion monitoring assay. The results of these studies established that this biotransformation proceeds with the stereoselective abstraction of the 5'-pro-E proton, that is, the C-5' proton trans to the bulky pyridyl group. The observed stereoselectivity was independent of proton vs. deuteron abstraction. Additionally, the extent of (S)-cotinine formation was minor and did not influence the stereochemical composition of the metabolically derived alpha-cyano amines. Studies with male Dutch rabbit liver microsomal preparations gave similar results. These findings suggest that the structure of the complex formed between (S)-nicotine and the active site of cytochrome P-450 is highly ordered and dictates the stereochemical course of the reaction pathway.     

Stereoselective S-oxygenation of 2-aryl-1,3-dithiolanes by the flavin-containing and cytochrome P-450 monooxygenases

The reaction of NalO4, highly purified flavin-containing monooxygenase (EC 1.14.13.8), and microsomes from hog liver with 2-aryl-1,3-dithiolanes and 2-aryl-1,3-dithiolane S-oxides was investigated. The initial rates determined for the microsome- and purified flavin-containing monooxygenase-catalyzed rate of S-oxidation of para-substituted 2-aryl-1,3-dithiolanes were similar, demonstrating that S-oxidation of these substrates occurred with similar velocities at saturating concentrations of substrate and, at least for the first S-oxidation, the reaction was insensitive to the nature of the para-substituent. The diastereoselectivity of S-oxygenation of 2-aryl-1,3-dithiolanes was determined and, in general, a marked preference for addition of oxygen to the sulfide sulfur atom was observed to occur trans to the aryl groups. In all cases examined, enantioselective enzymatic S-oxidation was observed. For S-oxide formation in microsomes, the data provided evidence for a minor role of cytochrome P-450 in S-oxide formation, but the flavin-containing monooxygenase was mainly responsible for production of S-oxide. In contrast to previous reports, the enantioselectivity of S-oxidation catalyzed by highly purified cytochrome P-450IIB-1 and cytochrome P-450IIB-10 was not always opposite to that catalyzed by hog liver flavin-containing monooxygenase activity. 2-Aryl-1,3-dithiolane S-oxides were also oxidized a second time by NalO4, microsomes, or highly purified flavin-containing monooxygenase from hog liver but not cytochrome P-450IIB-1 or P-450IIB-10. The rate of the second oxidation was 10-15-fold slower than the corresponding first S-oxidation and S,S'-dioxide formation was markedly dependent on the electronic nature of the para-substituent (Hammett correlation rho value of -1.3 and -1.1 for microsomes and highly purified flavin-containing monooxygenase from hog liver, respectively). The large dependence of the rate of S,S'-dioxide formation on the nature of the para-substituent demonstrates that velocity values at saturating concentrations of S-oxide were not the same for all 2-aryl-1,3-dithiolane S-oxides and suggests that the chemical nature of the 2-aryl-1,3-dithiolane S-oxide contributes to the rate-determining step of this enzymatic reaction.     

Regio- and stereochemical studies on the alpha-carbon oxidation of (S)-nicotine by cytochrome P-450 model systems

Results from previous studies indicate that rabbit liver microsomal cytochrome P-450 catalyzes the C-5' two-electron oxidation of (S)-nicotine stereoselectivity with preferential loss of the pro-(E)-hydrogen atom trans to the pyridine ring. We now have examined the regio- and stereochemical features of the oxidation of (S)-nicotine by peroxides in the presence of various hemoproteins and by electrochemical and photochemical methods. None of these systems gave rise to the stereochemical outcomes observed with the cytochrome P-450 mediated reaction. The results of these studies are interpreted as additional evidence for the formation of a highly ordered complex between (S)-nicotine and cytochrome P-450 that directs the regio- and diasterioselective alpha-carbon oxidation of this substrate.     

Oxidation of nicotine and chelating agent by mercury(II)-compounds

The dehydrogenation of (S)-nicotine (1) with mercuric acetate in diluted acetic acid yields no cotinine (3), but reacts only to the iminium stage, resulting far predominant the 5'-iminium structure without affecting the chiral center at 2', therefore the reduction with borohydride nearly quantitatively gives rise to (S)-nicotine (1). For the preparation of cotinine (3) the best method proves the oxidation of (S)-1 with the equimolecular complex Hg(II)-EDTA in pure water. With preliminary alkalization of the preparation an oxidation also of the liberated EDTA to iminodiacetic acid (10) and oxalic acid (15) occurs. This side reaction increase with an excess of chelating agent, which makes the precipitation of mercury as measuring system for control of the dehydrogenation invalid. Surprising is the nearly complete failure of the dehydrogenation to the tertiary carbenium ion and the consecutive reaction of the secondary carbenium ion 5, which in equilibrium with its carbinolamine 5a is again dehydrogenated with Hg(II)-EDTA to the lactam 3 with retention of the configuration.     

S-oxygenation of 7 alpha-thiomethylspironolactone by the flavin-containing monooxygenase

Liver microsomes and highly purified flavin-containing monooxygenase from uninduced hogs catalyze the NADPH and oxygen-dependent S-oxygenation of 7 alpha-thiomethylspironolactone (7 alpha-TMSL), the major urinary metabolite of spironolactone, an effective antimineralocorticoid in humans. Studies on the biochemical mechanism of S-oxygenation of 7 alpha-TMSL suggests that this reaction is catalyzed exclusively by the flavin-containing monooxygenase and not by cytochrome P-450. This conclusion is based on the effects of selective cytochrome P-450 inhibitors as well as positive effectors and alternate substrates for the flavin-containing monooxygenase. The modest degree of stereoselective S-oxygenation of 7 alpha-TMSL may suggest steric inhibition of oxidation by the flavin-containing monooxygenase.     

N-alkylaminobenzotriazoles as isozyme-selective suicide inhibitors of rabbit pulmonary microsomal cytochrome P-450

To produce potent, isozyme-selective suicide inhibitors of cytochrome P-450 (P-450), a series of N-alkylated 1-aminobenzotriazole (ABT) derivatives was synthesized; these included the N-methyl, N-butyl (BuBT), N-benzyl (BBT), and N-alpha-methylbenzyl (alpha MB) analogues of ABT. The suicide inhibitors showing the greatest potency and isozyme selectivity were BBT and alpha MB, compounds which included molecular features for P-450 inactivation (the ABT moiety) and similarity to benzphetamine. ABT and its N-alkylated derivatives were tested as suicide inhibitors in rabbit lung microsomes, whose P-450 monooxygenase system has been well characterized in both untreated and beta-naphthoflavone- or 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated animals. ABT (10 mM) destroyed up to 99% of the total P-450 content of lung microsomes of untreated rabbits. At equimolar concentrations (10 microM), ABT was less effective than the N-alkylated compounds for the inhibition of P-450 isozyme 2-catalyzed benzphetamine N-demethylation (BND); in fact, BuBT, BBT, and alpha MB completely inhibited BND activity at this concentration and destroyed less than 40% of total pulmonary P-450. However, these compounds also inactivated 69-85% of isozyme 6-catalyzed 7-ethoxyresorufin O-deethylation. The most potent and isozyme-selective suicide inhibitor prepared was alpha MB: at 1 microM this compound inhibited approximately 80% of isozyme 2-catalyzed and 20% of isozyme 6-catalyzed monooxygenase activity but spared P-450 isozyme 5; at 2.5 microM it caused a near-complete loss (96 +/- 2%) of BND activity. The partition ratio of alpha MB, i.e., the molar ratio of inhibitor present to that of the P-450 destroyed, was 11 +/- 2, further demonstrating the potency of this compound. Experiments with BBT- and sodium phenobarbital-treated rats showed that the mechanism for suicidal inactivation of P-450 by this N-alkylated compound was by benzyne release, the same mechanism demonstrated earlier for the parent compound ABT.     

Enantioselective N-oxygenation of verapamil by the hepatic flavin-containing monooxygenase

The chemical and enzymatic N-oxygenation of verapamil was investigated. Verapamil N-oxide is readily synthesized by chemical means. It is not indefinitely stable, however, and undergoes Cope-type elimination to produce 3,4-dimethoxystyrene and a hydroxylamine. The major stable metabolite observed during the metabolism of verapamil with rat and hog liver microsomes and purified flavin-containing monooxygenase is 3,4-dimethoxystyrene. 3,4-Dimethoxystyrene is formed at a rate 4 times that of nor-verapamil. Studies suggest that N-oxygenation is catalyzed largely by the flavin-containing monooxygenase and N-demethylation is catalyzed by cytochrome P-450. This conclusion is based on the effects of cytochrome P-450 inhibitors and positive effectors for the flavin-containing monooxygenase as well as on studies with the purified enzyme. In the presence of rat and hog liver microsomes, significant stereoselectivity in N-oxygenation of verapamil is observed (S/R ratio of 3.1 and 4.1, respectively). With purified hog and rat hepatic flavin-containing monooxygenase, the stereoselectivity for verapamil N-oxygenation (S/R ratio of 10.1 and 6.6, respectively) suggests a role for this enzyme in the stereoselective first-pass metabolism of verapamil.     

Metabolism of diallyl disulfide by human liver microsomal cytochromes P-450 and flavin-containing monooxygenases.

The metabolism of diallyl disulfide (DADS), a garlic sulfur compound, was investigated in human liver microsomes. Diallyl thiosulfinate (allicin) was the only metabolite observed and its formation followed Michaelis-Menten kinetics with a Km = 0.61 +/- 0.2 mM and a Vmax = 18.5 +/- 4.2 nmol/min/mg protein, respectively (mean +/- S.E. M., n = 4). Both flavin-containing monooxygenase and the cytochrome P-450 monooxygenases (CYP) were involved in DADS oxidation, but the contribution of CYP was predominant. The cytochrome P-450 isoforms involved in this metabolism were investigated using selective chemical inhibitors, microsomes from cells expressing recombinant CYP isoenzymes, and studying the correlation of the rate of DADS oxidation with specific monooxygenase activities of human liver microsomes. Diethyldithiocarbamate and tranylcypromine inhibited allicin formation, whereas other specific inhibitors had low or no effect. Most of the different human microsomes from cells expressing CYP were able to catalyze this reaction, but CYP2E1 showed the highest affinity with a substantial activity. Furthermore, allicin formation by human liver microsomes was correlated with p-nitrophenol hydroxylase activity, a marker of CYP2E1, and tolbutamide hydroxylase activity, a marker of CYP2C9. Among these approaches only CYP2E1 was identified in each case, which suggested that DADS is preferentially metabolized to allicin by CYP2E1 in human liver. However the minor participation of other CYP forms and flavin-containing monooxygenases is likely.     

Stereoselectivity in the N'-oxidation of nicotine isomers by flavin-containing monooxygenase

N'-Oxidation of nicotine isomers by porcine liver flavin-containing monooxygenase shows a clear stereoselectivity in the formation of the diastereomeric N'-oxides. (S)-(-)-Nicotine exhibited no stereoselectivity in the formation of cis-1'R,2'S- and trans-1'S,2'S-products, whereas with (R)-(+)-nicotine, only the trans-1'R,2'R-N'-oxide was formed. The concentration of each isomer required for half maximal activity differs significantly, and access of (S)-(-)-nicotine to the active site appears to be more restricted than for (R)-(+)-nicotine as judged from the observed Km values (Km = 181 and 70 microM, respectively, for the (S)-(-)- and (R)-(+)-isomers). These results indicate that a region adjacent to the active site may sterically prohibit binding of (R)-(+)-nicotine when the N'-methyl and pyridyl groups are in a cis-orientation. N-Methylnicotinium ion (both R- and S-isomers) is not a substrate for either porcine flavin monooxygenase, guinea pig liver microsomes, or ram seminal vesicular microsomes.     

Rabbit lung flavin-containing monooxygenase. Purification, characterization, and induction during pregnancy

A flavin-containing monooxygenase has been purified to apparent homogeneity from lung microsomes of pregnant rabbits and characterized with respect to a number of physical and catalytic parameters. The apparent molecular weight, as determined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 59,000, and the lung microsomal flavoprotein was shown to contain 14 nmol of FAD/mg of protein. Addition of NADP+ to the oxidized flavoprotein produced a shift in the spectrum characteristic of the flavin-containing monooxygenase from porcine liver, and addition of small amounts of NADPH to the oxidized rabbit lung enzyme produced a stable spectral intermediate consistent with that of a 4a-peroxyflavin. Rabbit lung flavin-containing monooxygenase differed markedly from the porcine liver enzyme in exhibiting a broader pH optimum from 8.5-10.5, by not being inhibited by concentrations of sodium cholate as high as 1% and by withstanding, in the absence of NADPH, incubation at 45 degrees for at least 10 min with no significant loss of activity. Unlike the pig liver enzyme, purified rabbit lung enzyme was not activated by n-octylamine and, in fact, n-octylamine stimulated NADPH oxidation. A number of compounds known to be substrates of the pig liver enzyme, including benzphetamine, chlorpromazine, and imipramine, are not substrates for the rabbit lung enzyme, whereas prochlorperazine and trifluoperazine are excellent substrates. Antibodies to rabbit lung flavin-containing monooxygenase were raised in guinea pig and utilized for the immunoquantitation of this enzyme throughout gestation. The activity (as determined by N,N-dimethylaniline-N-oxidation) and amount of rabbit lung flavin-containing monooxygenase were maximally induced (5-fold) on the 28th day of gestation. Liver microsomes from rabbit did not contain any of the lung form of flavin-containing monooxygenase at any time during gestation, as evidenced by results from Western blotting. These results demonstrate that, at least in rabbit, flavin-containing monooxygenase can exist as more than a single form. The physiological significance of the induction of this enzyme during pregnancy is not known.     

Phencyclidine iminium ion. NADPH-dependent metabolism, covalent binding to macromolecules, and inactivation of cytochrome(s) P-450

The phencyclidine iminium ion (PCP-Im+), a potentially reactive 2,3,4,5-tetrahydropyridinium species, is formed by the cytochrome(s) P-450-catalyzed alpha-carbon oxidation of phencyclidine (PCP), a commonly abused psychotomimetic agent. Incubation of PCP-Im+ with liver microsomes obtained from phenobarbital-induced rabbits resulted in over 50% loss of microsomal N-demethylase activity and 30% reduction in cytochrome(s) P-450 content. These effects were concentration-dependent, irreversible, and exhibited pseudo-first order kinetics, characteristics of a mechanism-based enzyme inactivation process. Incubation of 3H-PCP-Im+ with liver microsomes resulted in covalent binding of radioactive material to macromolecules by a process that also was NADPH-dependent. PCP-Im+ was metabolized by liver microsomes in the presence of NADPH and this metabolism was inhibited by SKF 525A and carbon monoxide. HPLC analysis has led to the preliminary characterization of an oxidized metabolite of PCP-Im+ which also is formed from PCP. These results support the proposal that this tetrahydropyridinium metabolite of PCP is biotransformed in a cytochrome(s) P-450-catalyzed reaction to form reactive species capable of covalent interactions with biomacromolecules.     

Oxidation of N-methylthiobenzamide and N-methylthiobenzamide S-oxide by liver and lung microsomes

The in vitro oxidation of N-[14C]methylthiobenzamide (NMTB) and N-[14C]methylthiobenzamide S-oxide (NMTBSO) by rat lung and liver microsomes was studied. In the presence of an NADPH-generating system, NMTB was rapidly converted to NMTBSO and small amounts of N-methylbenzamide (NMBA) and covalently bound metabolites (CVB). Under similar conditions, NMTBSO was converted to NMBA and CVB. Studies with metabolic inhibitors indicate that both the S-oxidation of NMTB and its further conversion to NMBA and CVB, probably via enzymatic oxidation to the S,S-dioxide, are catalyzed by both cytochromes P-450 and the FAD-containing monooxygenase (MFMO). Based on differential effects of inhibitors with lung vs liver microsomes, it would appear that in lung microsomes the MFMO plays a significantly greater role than cytochrome P-450 in the oxidation of NMTB and NMTBSO, whereas in the liver the contribution of these two pathways is more nearly equal. 1-Methyl-1-phenyl-3-benzoylthiourea, which blocks the in vivo pneumotoxicity of both NMTB and NMTBSO, also inhibited their in vitro microsomal metabolism and CVB, suggesting that these oxidations are obligatory steps in the expression of toxicity by these compounds.     

Selective mechanism-based inactivation of the major phenobarbital-inducible P-450 cytochrome from rabbit liver by phencyclidine and its oxidation product, the iminium compound

Phencyclidine, 1-(1-phenylcyclohexyl)piperidine, was found in this study to be a mechanism-based inactivating agent for cytochrome P-450 form 2, the major phenobarbital-inducible cytochrome of rabbit liver microsomes. This process is highly selective for P-450 form 2, both in microsomes and in the reconstituted enzyme system, in that forms 3a, 3b, 4, and 6 are not affected. However, phencyclidine iminium ion, an oxidative metabolite, inactivates both P-450 form 2 and form 3b in a metabolism-dependent manner. Both phencyclidine and its iminium ion give biphasic kinetics of inactivation with similar rate constants, which supports the hypothesis that the iminium ion is an intermediate in the inactivation of P-450 form 2 by the parent compound. The absorption of the oxidized cytochrome and of the ferrous-carbonyl complex in the visible spectrum are both decreased upon inactivation by phencyclidine, indicating modification of the heme moiety. Several modified hemes produced by the action of phencyclidine were isolated by HPLC.     

6-methylhydroxylation of the anti-cancer agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) by flavin-containing monooxygenase 3.

The involvement of flavin-containing monooxygenase (FMO) in the 6-methylhydroxylation of the experimental anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) was investigated by use of human liver microsomes and microsomes containing cDNA-expressed FMOs. The involvement of FMO in the formation of 6-methyl hydroxylate of DMXAA, 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA) in human liver microsomes was indicated by the fact that this biotransformation was sensitive to heat treatment, increased at pH 8.3, and inhibited by methimazole. Only FMO3 formed 6-OH-MXAA at a similar rate to that in cDNA-expressed cytochromes P-450 (CYP)1A2. The results of this study indicate that human FMO3 has the capacity to form 6-OH-MXAA, but plays a lesser important role for this reaction than CYP1A2 that has been demonstrated to catalyse 6-OH-MXAA formation.     

Immunochemical analysis of a cytochrome P-450IA1 homologue in human lung microsomes

The monoclonal antibody MAb 1-7-1, which specifically binds to cytochromes P-450IA1 and P-450IA2 in 3-methylcholanthrene-induced rat liver microsomes, was used to identify a cytochrome P-450IA1 homologue in human lung microsomes. Although MAb 1-7-1 had similar affinity constants for human and rat microsomes, the amount bound to human lung microsomes was severalfold lower than that bound to microsomes from untreated rat or rabbit lung and much lower than the amount bound to 3-methylcholanthrene-induced rat lung or liver microsomes. The amount bound to untreated baboon lung microsomes was similar to that bound to human lung microsomes. Three cytochrome P-450IA1-catalyzed activities, 7-ethoxyresorufin O-deethylase, 7-ethoxycoumarin, O-deethylase, and aryl hydrocarbon hydroxylase, were measurable in human lung microsomes, but the cytochrome P-450IA2-dependent activity acetanilide 4-hydroxylase was not. MAb 1-7-1 inhibited, and its binding correlated strongly with, 7-ethoxyresorufin O-deethylase activity (r = 0.92, p less than 0.01) in human lung microsomes. 7-Ethoxyresorufin O-deethylase activities in human lung were similar to those measured in untreated baboon lung but considerably lower than those present in untreated rabbit lung, untreated or 3-methylcholanthrene-induced rat lung and liver, or human liver. We conclude that MAb 1-7-1 recognizes a cytochrome P-450IA1 homologue in human lung and that no cytochrome P-450IA2 homologue is detected. Cytochrome P-450IA1 is expressed in human lung at relatively low levels, similar to those observed in untreated primate (baboon) lung. The majority of the 19 human lung samples examined do not exhibit a permanent polycyclic aromatic hydrocarbon-induced state with respect to this isozyme.