Genotoxic and mutagenic activation of aflatoxin B1 by constitutive forms of cytochrome P-450 in rat liver microsomes

Cytochrome P-450-mediated activation of aflatoxin B1 (AFB1) to genotoxic and mutagenic products which subsequently cause induction of an umu gene expression in Salmonella typhimurium TA1535/pSK1002 has been studied in a rat liver microsomal or reconstituted monooxygenase system. Liver microsomes from male Sprague-Dawley rats had a 1.5-fold higher activity to catalyze AFB1 than did those from female rats. In addition, the activation was not increased in liver microsomes from rats pretreated with phenobarbital, 3-methylcholanthrene, a polychlorinated biphenyl mixture, or dexamethasone, suggesting that the constitutive forms of cytochrome P-450 have important roles for the activation of AFB1 in rat liver microsomes. Using 15 forms of cytochrome P-450 purified from liver microsomes of untreated and phenobarbital- and 3-methylcholanthrene-treated rats, three isozymes from untreated male rats and one isozyme from untreated female rats were found to have high reactivities in metabolizing AFB1 to genotoxic products. Cytochrome P-450 forms isolated from inducer-treated rats were relatively less active. The close correlation between induction of umu gene expression and mutagenicity with Ames/S. typhimurium TA98 system by activated metabolites of AFB1 in the reconstituted monooxygenase system suggested that the constitutive forms of cytochrome P-450 had major roles for genotoxic and mutagenic activation of AFB1 in rat liver microsomes.     

In vivo and in vitro effects of beta-naphthoflavone on cytochrome P-450-dependent testosterone hydroxylase activities in liver microsomes

Hepatic microsomes from beta-naphthoflavone-treated adult male rats exhibit a 60-86% loss of testosterone 7 alpha-, 16 alpha-, and 6 beta-hydroxylase activities compared to those of untreated animals. Studies were conducted to determine if the loss of mono-oxygenase activities reflected the presence of beta-naphthoflavone in microsomes from treated animals or, rather, involved a change in the constitutive forms of cytochrome P-450 involved in testosterone hydroxylation. In vitro preincubation of microsomes from untreated animals with NADPH and beta-naphthoflavone resulted in a selective 30-40% loss of 16 alpha- and 6 beta-hydroxylase activities, results which suggest that a metabolite of beta-naphthoflavone may selectively inhibit testosterone hydroxylase activities. Hepatic microsomes from untreated sexually immature and adult rats and from beta-naphthoflavone-treated adult male rats were solubilized and each resolved into three or four cytochrome P-450 fractions by ion-exchange chromatography. The various cytochrome fractions were distinguished by their CO-difference spectra and reconstituted catalytic activity. Cytochrome P-450 fractions from beta-naphthoflavone-treated adult male rats exhibited only 10% of the 16 alpha- and 2 beta-hydroxylase activities of the analogous fractions from untreated adult male microsomes. The catalytic activity of the fractions from beta-naphthoflavone-treated adults was similar to that of cytochrome P-450 from sexually immature male rats. These data indicate that in vivo administration of beta-naphthoflavone results in a selective loss of androgen-dependent cytochrome P-450 testosterone hydroxylase activity.     

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

Relative participation of flavin-containing mono-oxygenase and cytochrome P-450 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. 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. Pretreatment of guinea-pigs with phenobarbital and 3-methylcholanthrene did not enhance the metabolism of methamphetamine. Pretreatment of rats with phenobarbital but not 3-methylcholanthrene increased slightly the N-demethylation of methamphetamine by liver microsomes. 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.     

Inhibition of hepatic mixed-function oxidase activity in vitro and in vivo by various thiono-sulfur-containing compounds.

Ten thiono-sulfur-containing compounds of varying structure were administered by intraperitoneal injection to untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated adult male rats. Six hr later, the concentration of hepatic cytochrome P-450 and the ability of the hepatic microsomes to metabolize benzphetamine were examined. In the untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated groups, two, four and four compounds, respectively, significantly decreased the concentration of cytochrome P-450 in the hepatic microsomes. A similar effect on benzphetamine metabolism was also seen. When examined 48 hr after the administration of the ten thiono-sulfurcontaining compounds, four, five and seven of the compounds decreased both the levels of hepatic cytochrome P-450 and the rate of benzphetamine metabolism in the untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated animals respectively. Eight of the thiono-sulfur-containing compounds were incubated in the presence of NADPH with hepatic microsomes isolated from untreated, phenobarbital-pretreated or 3-methylcholanthrene-pretreated animals. All of the compounds examined significantly decreased the concentration of cytochrome P-450 in the microsomes from each treatment group. Similar reductions in benzphetamine metabolism were also seen. When these same compounds were incubated with microsomes in the absence of NADPH, no significant reduction of cytochrome P-450 or benzphetamine metabolism was seen. When the oxygen analogs of six of the thiono-sulfur compounds were administered in vivo or incubated with hepatic microsomes either in the presence or absence of NADPH, no significant reduction of cytochrome P-450 or benzphetamine metabolism was seen.     

Loss of rat liver microsomal cytochrome P-450 during methimazole metabolism. Role of flavin-containing monooxygenase

The role of flavin-containing monooxygenase (FMO) in the decrease in cytochrome P-450 content during the microsomal metabolism of methimazole (N-methyl-2-mercaptoimidazole) was investigated by heat inactivation of FMO. Incubation of liver microsomes from untreated Fischer 344 rats with NADPH and methimazole resulted in a 25% loss of cytochrome P-450 detectable as its ferrous-carbon monoxide complex. The same extent of cytochrome P-450 loss was observed with 1 and 20 mM methimazole, suggesting saturation of the process. There was no significant loss of cytochrome P-450 when microsomal FMO was heat-inactivated prior to incubation with NADPH and methimazole. Heat pretreatment of the microsomes did not affect cytochrome P-450 concentrations and cytochrome P-420 was not observed. These results indicate that FMO-catalyzed metabolism of methimazole is necessary for the loss of cytochrome P-450 in microsomes from untreated rats. Sulfite and N-methylimidazole, the ultimate products of methimazole metabolism, did not cause a significant loss of cytochrome P-450. There was no loss of cytochrome P-450 when glutathione was included in the incubation with methimazole, suggesting that cytochrome P-450 loss was due to an interaction with oxygenated metabolites of methimazole formed by FMO. Losses of cytochrome P-450 were also observed after incubation of microsomes from phenobarbital- (31%) of beta-naphthoflavone-pretreated rats (44%) with NADPH and methimazole. In contrast to microsomes from untreated rats, heat inactivation of FMO did not prevent the loss of cytochrome P-450 in microsomes from the pretreated rats. These results indicate that both phenobarbital and beta-naphthoflavone induce isozymes of cytochrome P-450 capable of directly activating methimazole.     

Enhancement by cyanide of aniline p-hydroxylation activity in rat liver microsomes

Aniline p-hydroxylation activity of rat liver microsomes was found to be enhanced, rather than inhibited, at aniline concentrations higher than about 3 mM. The cyanide-induced enhancement increased as the oxygen tension was increased. The activation by cyanide was, however, significantly diminished with liver microsomes from phenobarbital- and 3-methylcholanthrene-pretreated rats. The enhancement was also decreased when liver microsomes were fortified with NADPH-cytochrome P-450 reductase. Aniline hydroxylation by reconstituted systems consisting of partially purified preparations of several species of cytochrome P-450 and the reductase was inhibited by cyanide, though the degree of inhibition was dependent on the species of cytochrome P-450 used for reconstitution. In several respects, the cyanide-induced enhancement of aniline hydroxylation is different from the enhancement caused by acetone and 2,2'-bipyridine, but is similar to the activation by ethylisocyanide.     

Peroxidative N-oxidation and N-dealkylation reactions of pargyline

The hydrogen peroxide-supported oxidation of pargyline in rat-liver microsomes was investigated and compared to that promoted by cytochrome P-450 in the presence of an NADPH-generating system. The metabolic conversions promoted by hydrogen peroxide and cytochrome P-450 comprised N-demethylation, N-depropargylation, N-debenzylation and N-oxidation. For the hydrogen peroxide-cytochrome P-450-promoted oxidation, cyanide, but not carbon monoxide, was an effective inhibitor of all the reactions. Similarly, 2,4-dichloro-6-phenyl phenoxyethylamine (DPEA) inhibited all reactions, particularly N-demethylation and N-oxidation more extensively than the NADPH-dependent microsomal oxidation. Using microsomes from rats pretreated with phenobarbital caused no increase in the metabolites above the levels seen with microsomes from untreated animals. Various other peroxidase systems which were investigated were essentially unable to promote oxidation of pargyline.     

The interaction between two forms of cytochrome P-450 during drug oxidation in the reconstituted system containing limited amount of NADPH-cytochrome P-450 reductase

The activities of drug oxidation in a reconstituted system which contains two forms of cytochrome P-450 and a limiting amount of NADPH-cytochrome P-450 reductase were determined. Cytochrome P-450 (termed MC P-4481 and MC P-4482) purified from liver microsomes of 3-methyl-cholanthrene-treated rats was active in both 2- and 4-hydroxylation of biphenyl but cytochrome P-450 (termed PB P-450) purified from liver microsomes of phenobarbital-treated rats was active in 4-hydroxylation of biphenyl only. PB P-450, MC P-4481 and MC P-4482 were most active toward benzphetamine N-demethylation, aniline hydroxylation and 7-ethoxycoumarin O-deethylation, respectively. PB P-450 inhibited the activity of biphenyl 2-hydroxylation supported by MC P-4481 or MC P-4482. On the contrary, no inhibition of PB P-450 supported benzphetamine N-demethylation was observed when MC P-4481 or MC P-4482 was added to the system containing PB P-450 and limited amount of the reductase. The apparent Km of PB P-450 for the reductase obtained from double reciprocal plot of the reductase concentration and the activity of biphenyl hydroxylase or benzphetamine N-demethylation was lower than that of MC P-4481 or MC P-4482. These and other results suggest that there is a certain hierarchy among the cytochrome P-450 species for receiving electrons from reductase.     

The effect of age on inducibility of various types of rat liver cytochrome P-450.

1. The content and specific activities of inducible cytochrome P-450 enzymes were determined in liver microsomes of rats of various ages after maximal induction with phenobarbital, isosafrole of 3-methylcholanthrene, and in untreated animals. 2. With age an increase in liver weight was observed both in untreated rats and in maximally induced ones; the microsomal protein content/g of liver decreased with age in untreated animals but not in induced ones. Total cytochrome P-450 content/mg microsomal protein remained unchanged with age in all experimental groups. 3. Immunologically detectable levels of cytochrome P4501A1/1A2 and 2B1/2B2 remain unchanged with age both in untreated animals and in maximally induced ones. 4. Several cytochrome P-450 activities showed an age-related decrease in untreated animals, but no change with age was observed in the activities of cytochrome P4501A1, 2A2 and 2B1/2B2 in rat liver microsomes. This indicates that ageing affects only the activity of some constitutive forms of cytochrome P-450 in male rats, but not the activity of inducible types of P-450. 5. Although previous results indicated decreased inducibility of the cytochrome P-450 mRNA levels with age, the present study clearly demonstrates that this is not reflected in decreased enzyme levels or activities after maximal induction. From this it is concluded that the decreased mRNA levels might rather be reflected in a decreased rate at which maximal induction can be achieved.     

Responses of cytochrome P-450 isozymes of rat lung to in vivo exposure to ozone

The effects of 2 weeks exposures of rats to 0.2 ppm O3 on pulmonary cytochrome P-450 isozymes were investigated. Two main types of cytochrome P-450 isozymes (cytochromes P-450FI and P-450FII) were separated from lung microsomes of control rats on an anion exchange cellulose column. Antibody raised against cytochrome P-450b, which is the main isozyme of liver obtained from phenobarbital-treated rats, cross-reacted with pulmonary cytochrome P-450FII, but not with cytochrome P-450FI. Ozone exposures caused increases in the content of both cytochromes P-450FI and P-450FII to the same extent two times greater than those of control rats without changes in immunological properties of these isozymes. No pulmonary cytochrome P-450 isozyme other than cytochromes P-450FI and P-450FII was observed in eluate through anion exchange column chromatography. These results indicate that increases in the content of pulmonary cytochrome P-450 of rats exposed to O3 can be ascribed to increase in constitutive types of the isozymes, but not to induction of other types of isozyme.     

Selective inactivation of four rat liver microsomal androstenedione hydroxylases by chloramphenicol analogs

The steroid androstenedione has been shown to be a valuable tool for the study of the selective inactivation of cytochrome P-450 isozymes in intact rat liver microsomes. The validity of this approach was investigated using microsomes, purified cytochrome P-450 isozymes, antibodies to particular cytochromes P-450, and the known mechanism-based inactivator chloramphenicol. Enzyme inactivation and antibody inhibition studies show that microsomes from both phenobarbital- and non-phenobarbital-treated rats are needed to accurately monitor the inactivation of the major phenobarbital-inducible cytochrome P-450 isozyme (PB-B) and of the major constitutive androstenedione 16 alpha-hydroxylase (UT-A). Similar experiments indicate that, although isozyme P-450g does catalyze the 6 beta-hydroxylation of androstenedione in a reconstituted system, this cytochrome appears to make only a minimal contribution to microsomal 6 beta-hydroxylase activity, which reflects instead the activity of pregnenolone-16 alpha-carbonitrile-induced isozymes. With these parameters investigated, initial enzyme inactivation studies showed that the antibiotic chloramphenicol caused different rates of NADPH-dependent enzyme inactivation among the four androstenedione hydroxylases monitored (16 beta greater than 6 beta greater than 16 alpha greater than 7 alpha). Based on these data, 12 chloramphenicol analogs were examined, and the results with these compounds show that their selectivity as cytochrome P-450 inactivators is a function of at least three structural features: 1) the number of halogen atoms, 2) the presence of a para-nitro group on the phenyl ring, and 3) substitutions on the ethyl side chain. For example, the compound N-(2-phenethyl)dichloroacetamide was shown to reversibly inhibit but not inactivate the cytochrome(s) P-450 responsible for androstenedione 6 beta-hydroxylase activity, whereas N-(2-p-nitrophenethyl) and N-(1,2-diphenethyl)dichloroacetamide rapidly inactivated the 6 beta-hydroxylase. The ability to monitor the activity of multiple isozymes with a single substrate should allow the development of a systematic approach to the design of selective inactivators of rat liver cytochromes P-450.     

Immunological and enzymatic comparison of hepatic cytochrome P-450 fractions from phenobarbital-, 3-methylcholanthrene-, β-naphtoflavone- and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats

A comparison of the cytochrome P-450 forms induced in rat liver microsomes by phenobarbital on the one hand, and 3-methylcholanthrene, β-naphtoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin on the other hand, was performed using specific antibodies: anti-P-450 B₂ PB IG (against the phenobarbital-induced cytochrome P-450) and anti-P-450 B₂ BNF IG (against the β-naphtoflavone-induced cytochrome P-450). On DEAE-cellulose chromatography, four cytochrome P-450 fractions were separated, called P-450 A (non-adsorbed), P-450 Ba, P-450 Bb and P-450 Bc, from control, phenobarbital-, 3-methylcholanthrene, /gb-naphtoflavone- and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats. Cytochrome P-450 A fractions appeared to be unmodified by the inducers, whereas the specifically induced cytochrome P-450 forms were always recovered in Bb fractions. The P-450 Bb fractions induced by 3-methylcholanthrene, β-naphtoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin exhibited common antigenic determinants, comparable catalytic activities (benzphetamine, N-demethylase, benzo[a]pyrene hydroxylase) and similar mol. wts. Moreover, the inhibition patterns by the two antibodies of benzphetamine N-demethylase and benzo[a]pyrene hydroxylase activities catalysed by 3-methylcholanthrene, β-naphtoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin microsomes or by the corresponding P-450 Bb fractions in a reconstituted system were quite identical. By these different criteria, β-naphtoflavone, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin seem to induce a common cytochrome P-450 species in rat liver.     

Selective inactivation of mouse liver cytochrome P-450IIIA by cannabidiol

Cannabidiol (CBD) inhibits hepatic drug metabolism in mice, particularly those activities known to be catalyzed by the cytochrome P-450IIIA (P-450IIIA) subfamily. CBD treatment (120 mg/kg) inhibited more than 75% of hepatic 6 beta-testosterone hydroxylase and erythromycin N-demethylase activities (functional markers of P-450IIIA) after 2 hr. An isozyme of the P-450IIIA subfamily (Mr 49,960) was purified to apparent homogeneity from hepatic microsomes of untreated mice and was found to catalyze testosterone hydroxylation at the 2 beta-, 6 beta-, and 15 beta-positions exclusively. Incubation of this isozyme with CBD in a reconstituted system resulted in a time- and concentration-dependent inactivation, with almost complete loss of P-450 chromophore and corresponding increase in P-420 content. NH2-terminal sequence analysis of the isozyme revealed an 86% similarity to the corresponding sequence of rat P-450IIIA2, a constitutive P-450 isozyme in the male rat liver. Pretreatment of mice with dexamethasone markedly (6-fold) increased the steroid-inducible P-450IIIA-dependent activities 6 beta-testosterone hydroxylation and erythromycin N-demethylation. CBD treatment of dexamethasone-pretreated animals failed to inhibit these activities, indicating that the steroid-inducible P-450IIIA was refractory to CBD-mediated inactivation. 3-Methylcholanthrene-inducible P-450IA and phenobarbital-inducible P-450IIB also appear to be refractory to CBD-mediated inactivation. On the other hand, erythromycin N-demethylase activity increased 4-fold after phenobarbital pretreatment and, as in untreated animals, was comparably inhibited by CBD, demonstrating its susceptibility to this drug. Thus, CBD appears to inactivate the P-450IIIA isozymes that are constitutively present in hepatic microsomes of untreated mice and/or inducible by phenobarbital pretreatment but not those that are steroid inducible.     

Rat liver cytochrome P-450 isozymes as catalysts of aldrin epoxidation in reconstituted monooxygenase systems and microsomes

To explore which rat liver cytochrome P-450 species are involved in aldrin epoxidation, we have studied the catalytic activities of a series of cytochrome P-450 isozymes purified from untreated and inducer-treated Sprague-Dawley rats. Of ten cytochrome P-450 forms analyzed, seven isozymes, listed in order of decreasing activity, catalyzed aldrin epoxidation: P-450UT-A, P-450PB-C, P-450UT-H, P-450PB-B, P-450PCN-E, P-450UT-F, and P-450PB-D. P-450UT-I, P-450BNF-B, and P-450ISF-G were not very active at all. A novel aldrin metabolite, endo-dieldrin, was formed by cytochrome P-450UT-F in a 6-fold excess over dieldrin, which is the exo-isomer. The activity of aldrin epoxidase furthermore was assayed in liver microsomes from Sprague-Dawley rats of diverse physiological status and after pretreatment with various inducers resulting in a peculiar pattern of cytochrome P-450 isozymes. Untreated animals, at an age of 3 weeks, showed similar enzyme activities in both genders. During maturation, the activity of males increased by 3-fold, while the activity in females did not significantly change during this period. Pretreatment with pregnenolone-16-alpha-carbonitrile or dexamethasone strongly increased the activity in females. Pretreatment with dexamethasone did not increase the activity of males. A 50% depression of epoxidase activity was noted for males pretreated with 5,6-benzoflavone. Phenobarbital pretreatment increased the activity of females by 12-fold and of males by 2-fold. Males responded to pretreatment with polychlorinated biphenyls in a strain dependent fashion: enzyme activity was increased 2-fold in Sprague-Dawley rats but was not altered in Wistar rats. "Theoretical" values of microsomal epoxidase activity were calculated for weanling and adult Sprague-Dawley rats from turnover numbers and published data on the relative abundance of aldrin epoxidizing P-450 isozymes (Waxmann et al., Biochemistry 24, 4409, 1985). These values agreed with the activities determined. A similar statement can be made for male rats of both strains pretreated with inducers, when the ratio of enzyme activity of pretreated to control animals was used as a basis of comparison. The activity ratio of females pretreated with pregnenolone-16-alpha-carbonitrile, dexamethasone and phenobarbital, however, was much higher than the ratio calculated. Our results reveal that aldrin epoxidation is a reaction indicative of male specific and of phenobarbital-inducible cytochrome P-450 isozymes in rat liver.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies on the metabolism of aminopyrine, antipyrine and theophylline using monoclonal antibodies to cytochrome P-450 isozymes purified from rat liver

We investigated the role played by monoclonal antibody defined classes of cytochrome P-450 in the metabolism of antipyrine, aminopyrine and theophylline. Two enzyme inhibitory monoclonal antibodies (MAb 1-7-1 and MAb 2-66-3) raised to two forms of cytochrome P-450 were used. Microsomes were prepared from the livers of untreated, 3-methylcholanthrene (MC)-treated, and phenobarbital (PB)-treated male Wistar rats. Addition of either monoclonal antibody to hepatic microsomes from untreated rats had a negligible effect on the metabolism of aminopyrine, antipyrine or theophylline. These results indicate that the constitutive enzymes responsible for metabolism of these three drugs differ from the MAb inhibitable enzymes responsible for transformation of these drugs in induced microsomes. In microsomes from MC- and PB-treated rats, however, the two MAbs differentially inhibited individual pathways. For example, at 20 mM aminopyrine, as much as 55% of 4-amino-antipyrine (4-AA) formation arose from the family of cytochrome P-450 isozymes that were not inhibited for 4-AA formation at 4 mM aminopyrine and 4-methylaminoantipyrine (4-MAA) formation at either concentration. Thus, the enzyme that functions at 20 mM aminopyrine in 4-MAA formation differs from that which functions at 4 mM aminopyrine in the formation of 4-AA or 4-MAA. Addition of MAbs to induced microsomes revealed at least four isozymes with overlapping specificities involved in antipyrine and theophylline metabolism. Each MAb-inhibitable pathway and the isozymes associated with it were classified into one of three epitope families: those pathways inhibited by both MAbs, those inhibited only by the MAb raised against PB-inducible P-450 isozymes, and those inhibited only by the MAb raised against 3-MC-inducible P-450 isozymes. A fourth group of pathways consisted of those unaffected by addition of either monoclonal antibody. Analysis of metabolism with these two MAbs suggests more extensive heterogeneity of the isozymes that biotransform these drugs than previously recognized.     

High magnitude hepatic cytochrome P-450 induction by an N-substituted imidazole antimycotic, clotrimazole

A 4-fold induction of hepatic microsomal cytochrome P-450 following 3 days of treatment of rats with clotrimazole (75 mg/kg), a potent monooxygenase inhibitor, greatly exceeded that evident from similar phenobarbital and dexamethasone treatment. The clotrimazole-induced microsomes exhibited a pattern of monooxygenase activities similar to that seen in microsomes from both phenobarbital- and dexamethasone-treated animals. Precautions were necessary to determine both monooxygenase activities and the full amount of cytochrome P-450 present in microsomes because of interference by residual clotrimazole in the microsomes.     

Tolbutamide hydroxylation by human, rabbit and rat liver microsomes and by purified forms of cytochrome P-450

Tolbutamide hydroxylation has been investigated in human, rabbit and rat liver microsomes and by six purified forms of hepatic rabbit cytochromes P-450. These studies were carried out to investigate whether an appropriate animal model could be developed for the human cytochrome(s) P-450 metabolizing tolbutamide. Selective induction was used in rats and rabbits to indicate the isozymes primarily responsible for tolbutamide hydroxylation in these species. Microsomal tolbutamide hydroxylase activity was significantly induced only by phenobarbital pretreatment in the rat which induces P-450 forms b (P-450IIB1) and/or e (P-450IIB2). Only pretreatment of rabbits with rifampicin, which induces cytochrome P-450 form 3c (P-450IIIA6), significantly increased the microsomal hydroxylation of tolbutamide. However, the increase in tolbutamide hydroxylase activity in rifampicin-induced microsomes (congruent to 50%) appears low compared to known levels of induction of P-450IIIA6 following rifampicin pretreatment (5-10-fold). These data suggest that P-450IIIA6 is at least partially involved in tolbutamide hydroxylation in rabbit liver but that other form(s) may be relatively more important. Reconstitution experiments with six purified forms of rabbit cytochrome P-450 indicated that the highest activity occurred with P-450IIIA6 (form 3c). As isozymes from different gene families or subfamilies appeared to metabolize tolbutamide in the three species studied, catalytic similarities between the P-450s with respect to inhibition was further investigated in microsomes using sulfaphenazole, alpha-naphthoflavone and mephenytoin. These studies showed that the catalytic characteristics in relation to inhibition differ markedly between species. Hence, it appears that the animal model approach is not likely to be successful in the identification and characterization of the cytochrome P-450 form(s) metabolizing tolbutamide in humans.     

Metabolism of lidocaine by purified rat liver microsomal cytochrome P-450 isozymes

The metabolism of lidocaine was studied using rat liver microsomes or a reconstituted lidocaine monooxygenase system with one of eight forms of cytochrome P-450 purified from liver microsomes from untreated- (P450 UT-2 and UT-5), phenobarbital- (P450 PB-1, PB-2, PB-4, and PB-5) or 3-methylcholanthrene- (P450 MC-1 and MC-5) treated rats. A reverse phase high-performance liquid chromatography system capable of simultaneously assaying four major lidocaine metabolites, namely, monoethylglycinexylidide (MEGX), 3-hydroxylidocaine (3-OH LID), methylhydroxylidocaine (Me-OH LID) and glycinexylidide (GX), was employed to determine the rate of formation of each metabolite. Untreated microsomes generated MEGX, Me-OH LID, and 3-OH LID, but the formation of GX was not detected. In male rat liver microsomes, MEGX was the major metabolite of lidocaine when a concentration of 1 mM was employed. The formation of MEGX and Me-OH LID was increased significantly (P less than 0.01) by microsomes from phenobarbital-treated rats, and the formation of 3-OH LID was increased with 3-methylcholanthrene. The study with the reconstituted system with purified cytochrome P-450 isozymes revealed that all eight forms of cytochrome P-450 used have an ability to N-deethylate lidocaine to form MEGX. Among these isozymes, cytochrome P450 PB-4 and P450 UT-2 showed a higher turnover number for the formation of MEGX. Me-OH LID was formed exclusively by P450 PB-5, and 3-OH LID exclusively by P450 MC-1. Selectivity of cytochrome P450 PB-5 for aromatic methyl hydroxylation of lidocaine was confirmed by an inhibition study; formation of Me-OH LID by microsomes of rats treated with phenobarbital was inhibited completely by antibody against P450 PB-5. It was concluded that different cytochrome P-450 isozymes metabolize lidocaine with a different rate and different position selectivities. Since a specific substrate of cytochrome P450 PB-5 (P-450e) is not known, lidocaine may be a useful substrate for the identification of P450 PB-5.     

The metabolism of 1-phenyl-2-(N-methyl-N-furfurylamino)propane (furfenorex) in the rat in vivo and in vitro

The metabolism of 1-phenyl-2-(N-methyl-N-furfurylamino)propane (furfenorex) was studied in the rat in vivo and in vitro. Nine metabolites with only traces of the unchanged drug were obtained from urine after oral administration of furfenorex to rats. The major metabolite was an acidic compound, isolated and identified as 1-phenyl-2-(N-methyl-N-gamma-valerolactonylamino)propane. Amphetamine, methamphetamine and their hydroxylated metabolites were excreted as minor metabolites. Metabolites excreted in two days after administration of the drug amounted to about 20% of dose. The acidic metabolite, a major metabolite in vivo, was not detected after incubation of furfenorex with rat-liver microsomes. The major metabolic routes of furfenorex in vitro were N-demethylation and N-defurfurylation which produced 1-phenyl-2-(N-furfurylamino)propane (furfurylamphetamine) and methamphetamine, respectively. The formation of furfurylamphetamine and methamphetamine were catalysed by rat-liver microsomes supplemented with NADPH and O2, and were inhibited by either SKF 525-A or CO. The formation of both metabolites were inhibited by 2-methyl-1,2-bis-(3-pyridyl)-1-propanone (metyrapone), but not by 7,8-benzoflavone. They were enhanced by pretreatment of rats with phenobarbitone, but not with 3-methylcholanthrene. These data suggested that N-demethylation and N-defurfurylation of furfenorex were mainly mediated by cytochrome P-450 but not cytochrome P-448.     

Clotrimazole induction of cytochrome P-450: dose-differentiated isozyme induction

Treatment of male rats for 3 days with the N-substituted imidazole, clotrimazole, produced up to a 4-fold induction of hepatic microsomal cytochrome P-450. The monooxygenase activities induced varied with the dose administered. At low doses (less than 25 mg/kg), p-nitroanisole demethylase and aniline hydroxylase activities were induced. Only at higher doses were other monooxygenase activities (erythromycin and ethylmorphine demethylases and cytochrome P-450 metabolic-intermediate complex formation from troleandomycin) induced. Microsomal UDP-glucuronosyltransferase activity toward morphine was induced at low doses in a manner similar to that of p-nitroanisole demethylase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of microsomes indicated that low doses of clotrimazole caused the intensification of a 48,000 molecular weight protein band, whereas at high doses, there was a marked intensification of an additional 50,500 molecular weight protein, the same molecular weight band as was intensified in phenobarbital- and dexamethasone-induced microsomes. The observations suggest a phenomenon of "dose-differentiated" isozyme induction for cytochrome P-450.