Covalent binding of 14C- and 35S-labeled thiocarbamides in rat hepatic microsomes.

The covalent binding of a series of 14C- or 35S-labeled benzimidazole-2-thione (MBI) derivatives to rat liver microsomal proteins was studied to determine the mechanisms of hepatic monooxygenase oxidation of model anti-hyperthyroid compounds. All thiocarbamides tested (including methimazole) produced an NADPH-dependent loss of cytochrome P450 (P450) chromophore which could be prevented by the addition of glutathione (GSH). The covalent binding of MBI to liver microsomal proteins from dexamethasone (DEX)-pretreated rats was enhanced 10-fold with NADPH, unaffected by P450 inactivation with 1-aminobenzotriazole (ABT) and attenuated by GSH addition. Heat treatment of microsomes to inactivate the flavin-containing monooxygenase (FMO) decreased the observed binding. Equivalent amounts of [35S]- and [14C]MBI were covalently bound to hepatic microsomal proteins, suggesting retention of both the carbon and sulfur portions of the molecule in the MBI/protein adduct. Thiophilic reagents effected release of covalently bound [14C]- and [35S]MBI in equal amounts suggesting the presence of disulfide bonds between an MBI-derived sulfenic acid and microsomal protein thiols. Coincubation with bovine serum albumin (BSA) resulted in NADPH-dependent binding of [14C]-MBI to BSA sulfhydryls which was blocked by prior treatment of BSA with iodoacetamide. 1-Methyl-benzimidazole-2-thione (MMBI) also covalently bound to microsomal proteins and BSA but at levels lower than with MBI. P450, however, appeared to be more important than FMO in the metabolism of MMBI based on the effects of microsome heat pretreatment or ABT addition. In addition, ca. 1.5-fold more 35S- than 14C-label became bound. The covalent binding of [35S]1,3-dimethyl-benzimidazole-2-thione (DMMBI) to microsomal proteins was ca. six times greater than that of [14C]DMMBI. ABT, catalase and superoxide dismutase had a minimal effect on [35S]DMMBI binding, while FMO inactivation decreased binding by ca. 30%. These findings suggest that both monooxygenases contribute significantly to the hepatic metabolism of thiocarbamides. However, FMO activates thiocarbamides primarily to sulfenic acids, whereas P450 appears to produce both sulfenic acid and other reactive sulfur-derived metabolites. Thiol groups of P450 and other proteins are the molecular targets for these reactive species formed during the hepatic metabolism of anti-hyperthyroid drugs.     

Tertiary Amines Related to Brompheniramine: Preferred Conformations for N-Oxygenation by the Hog Liver Flavin-Containing Monooxygenase

The metabolism of racemic, (D)- and (L)-brompheniramine, a widely used antihistamine, was studied with microsomes and with highly purified flavin-containing monooxygenase (FMO) from hog liver. In addition, a number of other similar tertiary amines were evaluated as substrates for FMO activity from hog liver and the kinetic constants obtained were compared with brompheniramine. Although some N-demethylation was observed, the major metabolite of brompheniramine and the other tertiary amines examined in hog liver microsomes was the metabolite containing an aliphatic nitrogen N-oxide. Brompheniramine was extensively N-oxygenated by the highly purified FMO from hog liver. N-Oxygenation of brompheniramine in both microsomes and with highly purified FMO from hog liver was enantioselective. The K _m for N-oxygenation of (D)-brompheniramine was markedly lower than the K _m for (L)-brompheniramine. (E)- and (Z)-zimeldine are less conformationally flexible model compounds of brompheniramine, and these compounds were also examined and were found to be stereoselectively N-oxygenated by the highly purified FMO from hog liver. The similarities and differences in K _m and V _max values were evaluated in terms of possible conformations of the substrates determined by SYBYL molecular mechanics calculations. Distance map data indicated that FMO preferentially accommodated selected conformations of tertiary amines. Thus, (D)-bromopheniramine and (Z)-zimeldine presumably have the aliphatic tertiary amine nitrogen atom and aromatic ring center at a defined distance and geometry and were more efficiently N-oxygenated than their respective isomers.     

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 microM), 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 Km and Vmax values, it would appear that the relative contributions of the two systems depend on the concentration of ABZ.     

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 [3-14C]coumarin to polar and covalently bound products by hepatic microsomes from the rat, Syrian hamster, gerbil and humans.

The metabolism of 0.19 and 2.0 mM-[3-14C]coumarin to polar products and covalently bound metabolites has been studied with hepatic microsomes from the rat, Syrian hamster, Mongolian gerbil and humans. [3-14C]Coumarin was metabolized by liver microsomes from all species to a number of polar products and to metabolite(s) that became covalently bound to microsomal proteins. The polar products included 3-, 5- and 7-hydroxycoumarins, o-hydroxyphenylacetaldehyde and o-hydroxyphenylacetic acid. Coumarin 7-hydroxylation was observed in all species except the rat. With 0.19 mM-[3-14C]coumarin, 7-hydroxycoumarin was the major metabolite in human liver microsomes, whereas in the other species with 0.19 mM substrate and in all species with 2.0 mM substrate o-hydroxyphenylacetaldehyde was the major metabolite. Of the three animal species studied the gerbil most resembled humans as this species also had a high coumarin 7-hydroxylase activity. The administration of Aroclor 1254 to the rat and Syrian hamster induced both microsomal cytochrome P-450 content and [3-14C]coumarin metabolism. With liver microsomes from all species a good correlation between rates of [3-14C]coumarin metabolism and covalent binding was observed at both substrate concentrations. However, in view of the known species difference between the rat and Syrian hamster in coumarin-induced hepatotoxicity, the present data are not consistent with microsomal coumarin metabolite covalent binding being an indicator of potential liver damage.     

Use of thiocarbamides as selective substrate probes for isoforms of flavin-containing monooxygenases.

The oxidation of thiourea, phenylthiourea, 1,3-diphenylthiourea, 1,3-bis-(3,4-dichlorophenyl)-2-thiourea and 1,1-dibenzyl-3-phenyl-2-thiourea was measured in reactions catalyzed by purified pig liver flavin-containing monooxygenase (FMO-1) and by microsomal fractions isolated from pig, guinea pig, chicken, rat and rabbit tissues. The reactions, followed by measuring substrate-dependent thiocholine oxidation [Guo and Ziegler, Anal Biochem 198: 143-148, 1991], were carried out in the presence of 2 mM 1-benzylimidazole to minimize potential interference from reactions other than those catalyzed by isoforms of the flavin-containing monooxygenase (FMO). While at saturating substrate concentrations the Vmax for purified FMO-1 catalyzed oxidation of all five thiocarbamides was essentially constant, velocities for the microsomal catalyzed reactions varied not only with tissue and species but also with the van der Waals' surface area of the thiocarbamide. Rat liver, rat kidney and rabbit liver microsomes failed to catalyze detectable oxidation of thiocarbamides larger than 1,3-diphenylthiourea and lung microsomes from a female rabbit only accepted substrates smaller than 1,3-diphenylthiourea. On the other hand, liver microsomes from chickens, pigs and guinea pigs catalyzed the oxidation of larger thiocarbamides, but the rates decreased with increasing substrate size and chicken liver microsomes showed no detectable activity with the largest thiocarbamide tested. To define more precisely the parameters affecting thiocarbamide substrate specificity of microsomal preparations, activities present in detergent extracts of guinea pig liver microsomes were separated into three distinct fractions. The substrate specificities of these partially purified fractions were different and consistent with the difference observed with microsomal catalyzed reactions. This strongly suggests that thiocarbamides that differ in size may be useful probes for measuring the number of activities of FMO isoforms in crude tissue preparations.     

Oxidation of ranitidine by isozymes of flavin-containing monooxygenase and cytochrome P450

Rat and human liver microsomes oxidized ranitidine to its N-oxide (66-76%) and S-oxide (13-18%) and desmethylranitidine (12-16%). N- and S-oxidations of ranitidine were inhibited by metimazole [flavin-containing monooxygenase (FMO) inhibitor] to 96-97% and 71-85%, respectively, and desmethylation of ranitidine was inhibited by SKF525A [cytochrome P450 (CYP) inhibitor] by 71-95%. Recombinant FMO isozymes like FMO1, FMO2, FMO3 and FMO5 produced 39, 79, 2180 and 4 ranitinine N-oxide and 45, 0, 580 and 280 ranitinine S-oxide pmol x min(-1) x nmol(-1) FMO, respectively. Desmethyranitinine was not produced by recombinant FMOs. Production of desmethylranitidine by rat and human liver microsomes was inhibited by tranylcypromine, a-naphthoflavon and quinidine, which are known to inhibit CYP2C19, 1A2 and 2D6, repectively. FMO3, the major form in adult liver, produced both ranitidine N- and S-oxides at a 4 to 1 ratio. FMO1, expressed primarily in human kidney, was 55- and 13-fold less efficient than the hepatic FMO3 in producing ranitidine N- and S-oxides, respectively. FMO2 and FMO5, although expressed slightly in human liver, kidney and lung, were not efficient producers of ranitidine N- and S-oxides. Thus, urinary contents of ranitidine N-oxide can be used as the in vivo probe to determine the hepatic FMO3 activity.     

Stereoselective N-oxygenation of zimeldine and homozimeldine by the flavin-containing monooxygenase

The metabolism of (Z)- and (E)-zimeldine and (Z)- and (E)-homozimeldine in hepatic rat and hog microsomes is described. The major metabolite observed in all cases examined was the tertiary amine N-oxide and it was formed at a rate 7-20 times that of norzimeldine or homonorzimeldine. N-Oxygenation requires NADPH and is stimulated by n-octylamine. Thiobenzamide and methimazole significantly inhibit N-oxide formation whereas heat pretreatment of microsomes completely abolishes N-oxide formation, strongly suggesting that zimeldine N-oxygenation if solely dependent on the flavin-containing monooxygenase. Hog liver microsomes N-oxygenate the Z-allylic and homoallylic tertiary amines in marked preference to the E-isomers, whereas rat liver microsomes N-oxygenate E-isomers to a greater extent than Z-isomers. Thus, opposite stereoselectivity for zimeldine N-oxygenation occurs in rat liver and hog liver microsomes.     

Effect of inducers of cytochrome P-450 on the metabolism of [3-14C]coumarin by rat hepatic microsomes

1. The metabolism of [3-14C]coumarin has been studied in rat hepatic microsomes and with two purified cytochrome P-450 isoenzymes. 2. [3-14C]Coumarin was converted by liver microsomes to several polar products including 3- and/or 5-hydroxycoumarin, omicron-hydroxyphenylacetic acid and a major unidentified novel coumarin metabolite. 3. [3-14C]Coumarin was also converted to reactive metabolite(s) as indicated by covalent binding to proteins, and by the depletion of reduced glutathione added to the microsomal incubations. 4. [3-14C]Coumarin metabolism to polar and covalently bound metabolites by rat liver microsomes was induced by pretreatment with phenobarbitone, 3-methylcholanthrene, beta-naphthoflavone, Aroclor 1254 and isosafrole; but not by dexamethasone or nafenopin. 5. The profile of [3-14C]coumarin metabolism to polar products was similar in control and pretreated liver microsomes and in incubations with purified cytochrome P450 IA1 and P450 IIB1 isoenzymes. 6. The results indicate that coumarin is a substrate for isoenzymes of the cytochrome P450 IA and P450 IIB subfamilies. The bioactivation of coumarin by rat hepatic microsomes is postulated to result in the formation of a coumarin 3,4-epoxide intermediate which may rearrange to 3-hydroxycoumarin, be further metabolized to a coumarin 3,4-dihydrodiol, or form a glutathione conjugate.     

Enzymic hydrolysis of nicotinate esters: comparison between plasma and liver catalysis.

1. The enzymic hydrolysis of a wide series of nicotinic acid esters was investigated using human and rat plasma, and purified hog liver carboxylesterase, and compared with previously published data from rat liver microsomes. Esterase activities were always found to obey Michaelis-Menten kinetics. 2. Rat liver microsomal and plasma enzyme velocities were six orders of magnitude smaller than those of purified hog liver carboxylesterase, and three orders smaller than human plasma activities, but the Km values were of the same magnitude. 3. The binding of nicotinate esters to human plasma esterases, and purified hog liver carboxylesterase, appears to depend mainly on hydrophobic and steric factors.     

Suppression of flavin-containing monooxygenase by overproduced nitric oxide in rat liver.

Effects of excessive nitric oxide (NO) produced in vivo by an i.p. injection of bacterial lipopolysaccharide (LPS) on hepatic microsomal drug oxidation catalyzed by flavin-containing monooxygenase (FMO) were determined. At 6 and 24 h after the LPS injection, liver microsomes were isolated and FMO activities were determined by using FMO substrates like thiobenzamide, trimethylamine, N,N-dimethylaniline, and imipramine. Liver microsomal FMO activities of LPS-treated rats were decreased significantly for all these substrates. Microsomal content of FMO1 (the major form in rat liver) in LPS-treated rats as determined by immunoblotting, was severely decreased as well. In support of this, hepatic content of FMO1 mRNA was decreased by 43.6 to 67.3%. However, the hepatic content of inducible NO synthase (iNOS) mRNA was increased by 2.6- to 5.4-fold and the plasma nitrite/nitrate concentration was increased by about 30-fold in the LPS-treated rats. When this overproduction of NO in the LPS-treated rats was inhibited in vivo by a single or repeat doses of either a general NOS inhibitor N(G)-nitro-L-arginine or a specific iNOS inhibitor aminoguanidine, the FMO1 mRNA levels were not severely depressed (70-85% of the control level). Attendant with the reduction of plasma nitrite/nitrate concentration by single and repeated doses of NOS inhibitors, activity and content of FMO1 in liver microsomes isolated from these NOS inhibitor cotreated rats were restored partially (in single-dose inhibitors) or completely (in repeat doses). In contrast to these NO-mediated in vivo suppressive effects on the mRNA and enzyme contents of FMO1 as well as the FMO activity, the NO generated in vitro from sodium nitroprusside did not inhibit the FMO activities present in microsomes of rat and rabbit liver as well as those present in rabbit kidney and lung. Combined, the excessive NO produced in vivo (caused by the LPS-dependent induction of iNOS) suppresses the FMO1 mRNA and enzyme contents as well as the FMO activities without any direct in vitro effect on the activities of premade FMO enzyme. These findings suggest that NO is an important mediator involved in the suppression of FMO1 activity in vivo. Thus, together with the previously reported suppression on the cytochrome P-450 activities, the overproduced NO in the liver caused by induction of iNOS under conditions of endotoxemia or sepsis suppresses FMO and appears to be responsible for the decreased drug oxidation function observed generally under conditions of systemic bacterial or viral infections.     

Facile N-oxygenation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine by the flavin-containing monooxygenase. A convenient synthesis of tritiated [methyl-3H]-4-phenyl-2,3-dihydropyridinium species

A rapid, efficient procedure useful for the radiosynthesis of [Me-3H]-MPDP+ ([methyl-3H]-4-phenyl-2,3-dihydropyridinium species) is described. Hog liver microsomes or the highly purified flavin-containing monooxygenase from hog liver quantitatively biotransforms [Me-3H]-MPTP to its corresponding radiolabeled N-oxide. For the small-scale synthesis required for radiolabeling procedures, this enzymatic process is superior to H2O2-mediated N-oxygenation of MPTP. In the presence of 0.5 mM NADPH, 4.5 mM n-octylamine, and 2 microCi [Me-3H]-MPTP, the only product detected in extracts from incubations performed with hog liver microsomes or purified hog liver flavin-containing monooxygenase is [Me-3H]-MPTP N-oxide. [Me-3H]-MPTP N-oxide is almost completely converted to [Me-3H]-MPDP+ by the action of trifluoroacetic anhydride. This procedure has the advantage of using a commercially available tritiated starting material, efficient transformations, and easily accomplished purification to afford a rapid synthesis of [Me-3H]-MPDP+.     

Activities of xenobiotic metabolizing enzymes in rat placenta and liver in vitro

In order to assess whether the placental metabolism of xenobiotic compounds should be taken into consideration for physiologically-based toxicokinetic (PBTK) modelling, the activities of seven phase I and phase II enzymes have been quantified in the 18-day placenta of untreated Wistar rats. To determine their relative contribution, these activities were compared to those of untreated adult male rat liver, using commonly accepted assays. The enzymes comprised cytochrome P450 (CYP), flavin-containing monooxygenase (FMO), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), esterase, UDP-glucuronosyltransferase (UGT), and glutathione S-transferase (GST). In contrast to liver, no activities were measurable for 7-ethylresorufin-O-dealkylase (CYP1A), 7-pentylresorufin-O-dealkylase (CYP2B), 7-benzylresorufin-O-dealkylase (CYP2B, 2C and 3 A), UGT1, UGT2 and GST in placenta, indicating that the placental activity of these enzymes was well below their hepatic activity. Low activities in placenta were determined for FMO (4%), and esterase (8%), whereas the activity of placental ADH and ALDH accounted for 35% and 40% of the hepatic activities, respectively. In support of the negligible placental CYP activity, testosterone and six model azole fungicides, which were readily metabolized by rat hepatic microsomes, failed to exhibit any metabolic turnover with rat placental microsomes. Hence, with the possible exception of ADH and ALDH, the activities of xenobiotic-metabolizing enzymes in rat placenta are too low to warrant consideration in PBTK modelling.     

Flavin-containing monooxygenase-dependent stereoselective S-oxygenation and cytotoxicity of cysteine S-conjugates and mercapturates.

The metabolism of cysteine S-conjugates of both cis- and trans-1,3-dichloropropene in the presence of rat kidney microsomes and purified flavin-containing monooxygenase from hog liver was investigated in vitro. Preliminary studies with isolated rat kidney cells demonstrated that cysteine S-conjugates were quite toxic to the cells in a process which was consistent with a role of the flavin-containing monooxygenase in the bioactivation of the nephrotoxins. Putative S-oxide metabolites of cysteine S-conjugates were chemically synthesized, and diastereomers were separated and identified by spectroscopic means. The metabolic products of cysteine S-conjugates were identified by comparing the chemical properties of the metabolites with authentic synthetic cysteine S-conjugate S-oxides. Surprisingly, S-conjugate S-oxygenase activity was not observed with rat kidney microsomes but was present when cysteine S-conjugates were incubated with the highly purified flavin-containing monooxygenase from hog liver. The kinetic parameters indicated that considerable S-oxygenase stereoselectivity and structural selectivity was observed: cis cysteine S-conjugates were preferred substrates and N-acetylation of cysteine S-conjugates decreased substrate activity. S-Oxygenation was considerably diastereoselective and diastereoselectivity was much greater for cysteine S-conjugates with higher Vmax values. Cysteine S-conjugate S-oxides were not indefinitely stable, and under certain conditions, the S-oxides underwent a [2,3]-sigmatropic rearrangement to acrolein. Formation of acrolein or other electrophilic products from S-(chloropropenyl)cysteine conjugate S-oxides may contribute to the renal effects observed for S-(chloropropenyl)cysteine conjugates.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo effect of ozone inhalation on xenobiotic metabolism of lung and liver of rats

Male Jcl:Wistar rats were exposed to 0.4 and 0.8 ppm O3 daily for 7 h for 14 d to examine the effect of O3 on xenobiotic metabolism of lung and liver microsomes. An exposure to 0.4 ppm O3 did not affect the microsomal xenobiotic metabolism of either lung or liver. On the other hand, 0.8 ppm O3 increased significantly the NADPH-cytochrome P-450 reductase activity and the cytochrome P-450 content of lung microsomes. The activities of lung benzo[a]pyrene hydroxylase and 7-ethoxycoumarin O-deethylase also increased significantly on d 7, and remained at a higher level by the d 14. These results show that exposures to 0.8 ppm O3 induce the xenobiotic metabolizing systems in the lung. In the liver, after the first day of exposure to 0.8 ppm O3, a significant reduction occurred in all components of the electron-transport systems examined as well as in the microsomal protein. A significant decrease was also observed in benzo[a]pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, and aniline hydroxylation. The decreased activities recovered in the following period of exposure. In contrast, the p-nitroanisole N-demethylase activity was not altered during the 14-d exposures. These results suggest that some isozymes of the hepatic cytochrome P-450 are sensitive to O3 inhalation but other(s) are resistant.     

Qualitative and quantitative differences in the induction and inhibition of hepatic benzo[a]pyrene metabolism in the rat and hamster

The present study compared the induction and inhibition of the metabolism of the prototype polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP), in rat and hamster liver microsomes. The production of total polar metabolites was quantitated by separating 3H-metabolites from [3H]-BaP using reverse-phase thin-layer chromatography. The rate of hepatic microsomal BaP metabolism was similar in the rat and hamster (0.81 vs 0.72 nmol/min/nmol cytochrome P-450 respectively). In the rat, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 5 micrograms/kg, i.p.) and 3-methylcholanthrene (3-MC; 50 mg/kg, i.p., X 3 days) pretreatments doubled the rate of BaP metabolism, whereas phenobarbital pretreatment (PB; 80 mg/kg, i.p., X 3 days) had no effect. In contrast, hamster hepatic microsomal BaP metabolism was elevated 2.3-fold by PB pretreatment, whereas TCDD and 3-MC pretreatments had no effect. Isosafrole pretreatment (ISO; 150 mg/kg, i.p., X 3 days) elevated the rate by almost 2-fold in each species. Another cytochrome P-448-mediated activity, 7-ethoxyresorufin O-deethylase (EROD), was induced by the same compounds that induced BaP metabolism in the rat. In hamster liver microsomes, in contrast to BaP metabolism, EROD was induced by TCDD and 3-MC but not PB or ISO pretreatments. The results suggest differences in the substrate specificity of the cytochromes P-448-450 induced by TCDD, 3-MC and PB in these species. This was supported by the different selectivity of the in vitro inhibitors, metyrapone and 7,8-benzoflavone, towards BaP metabolism and EROD in hepatic microsomes from TCDD- or PB-pretreated rats and hamsters. Reverse-phase HPLC analysis indicated that, while 3-hydroxy-BaP was the major metabolite formed by the untreated rat, untreated hamster liver microsomes formed predominantly BaP-4,5-diol. Microsomes from TCDD-treated rats generated elevated levels of all BaP-diols, diones and 3-hydroxy-BaP, with the major metabolites being BaP-9,10- and BaP-7,8-diols. In contrast, the metabolite profile from TCDD-pretreated hamsters was unchanged from the control. PB-treated hamster microsomes produced elevated levels of BaP-diones and 3-hydroxy-BaP. However, the major hepatic metabolite formed by PB-pretreated hamsters was BaP-4,5-diol, while BaP-9,10- and BaP-7,8-diols were not detected. The results of the study indicate differences in the induced cytochrome P-450s and the generation of toxic BaP metabolites in the liver of the rat and hamster.     

The effect of cimetidine on in vitro and in vivo microsomal drug metabolism in the rat

The effect of cimetidine on rat liver microsomal drug metabolism in vitro and in vivo was studied. Cimetidine inhibits aminopyrine N-demethylation and benzo[a]pyrene hydroxylation in a noncompetitive manner with inhibition constants between 1 and 10 mM. Benzo[a]pyrene hydroxylation in liver microsomes from 3-methylcholanthrene-pretreated rats is not appreciably inhibited by cimetidine indicating some specificity in terms of different cytochrome P-450 forms. Cimetidine gives rise to a type II spectral change with a spectral dissociation constant of about 0.1 mM. The prolonged administration of cimetidine does not result in the induction of hepatic drug metabolism. Pretreatment of rats with cimetidine prolongs aminopyrine half-life and hexobarbital sleeping time. These results demonstrate that cimetidine is an in vitro inhibitor of microsomal drug metabolism in the rat and this inhibition leads to pharmacokinetic drug-drug interactions in vivo.     

Hepatic microsomal metabolism of indole to indoxyl, a precursor of indoxyl sulfate.

The aim of our study was to determine which microsomal cytochrome P450 isozyme(s) were responsible for the microsomal oxidation of indole to indoxyl, an important intermediate in the information of the uremic toxin indoxyl sulfate. Indole was incubated together with an NADPH-generating system and rat liver microsomes. Formation of indigo, an auto-oxidation product of indoxyl, was used to determine the indole-3-hydroxylation activity. Apparent Km and Vmax values of 0.85 mM and 1152 pmol min(-1) mg(-1) were calculated for the formation of indoxyl from indole using rat liver microsomes. The effects of various potential inducers and inhibitors on the metabolism of indole to indoxyl by rat liver microsomes were studied to elucidate the enzymes responsible for metabolism. Studies with general and isozyme-specific P450 inhibitors demostrated that P450 enzymes and not FMO are responsible for the formation of indoxyl. In the induction studies, rate of indoxyl formation in the microsomes from untreated vs induced rats correlated nearly exactly with the CYP2E1 activity (4-nitrophenol 2-hydroxylation). These results suggests that CYP2E1 is the major isoform for the microsomal oxidation of indole to indoxyl.     

Inhibition of hepatic microsomal drug metabolism by the steroid hydroxylase inhibitor SU-10'603

SU-10'603 is a pyridine derivative that has been widely used as a steroid 17-hydroxylase inhibitor. Studies were done to compare the effects of SU-10'603 with those of the structurally related compound, metyrapone, on hepatic microsomal drug metabolism in vitro in rats and guinea pigs. In rat liver microsomes, SU-10'603 produced a concentration-dependent (0.01 to 1.0 mM) inhibition of ethylmorphine demethylation, aniline hydroxylation, and benzo[a]pyrene hydroxylation. A concentration of 0.1 to 0.2 mM decreased the metabolism of all three substrates by approximately 50%. SU-10'603 was a more potent inhibitor of ethylmorphine metabolism than metyrapone, and its relative potency was even greater with respect to aniline and benzo[a]pyrene metabolism. Similar results were obtained with guinea pig liver microsomes. SU-10'603 and metyrapone produced type II spectral changes in hepatic microsomes, but the apparent affinity of SU-10'603 for cytochrome(s) P-450 was greater than that of metyrapone. Both compounds inhibited the binding of type I substrates to microsomal cytochromes P-450; SU-10'603 was the more potent inhibitor. The results indicate that SU-10'603 is a potent inhibitor of hepatic microsomal monooxygenases whose mechanism of action is similar to that of metyrapone.     

Identification and characterization of hepatic carboxylesterases hydrolyzing hydrocortisone esters

The present study has provided evidence for the existence of three distinct carboxylesterases involved in the hydrolysis of steroid esters, where two enzymes are possibly responsible for the metabolism of hydrocortisone hemisuccinate (HCHS) at pH 5.5 and 8.0, and a third enzyme for the metabolism of hydrocortisone acetate (HCAC) at pH 8.0, in isolated rat liver microsomes. The activity of all three enzymes in rat liver was induced significantly by the administration of phenobarbital while no such function in enzyme activity was observed in animals receiving 3-methylcholanthrene or benzo[a] pyrene under similar experimental conditions. The increase in the activity of HCHS esterase I (HCHS-E1) active at pH 5.5, HCHS esterase II (HCHS-E2) active at pH 8.0, and HCAC esterase (HCAC-E) was approximately 7 to 8, 3- and 3-fold respectively. On the other hand, the degree of induction of nonspecific microsomal carboxylesterase acting on p-nitrophenylacetate (PNPA) was significantly less. The Km values for the hydrolysis of HCHS at pH 5.5 and 8.0 and HCAC by rat liver microsomes obtained from control rats were 2.45, 2.02 and 1.6 mM, respectively, and these Km values were not changed significantly in preparations obtained from rats treated with phenobarbital. The distinct in vitro responses displayed by hepatic microsomal steroid esterases to various inhibitors were able to distinguish three different enzymes which also differed from nonspecific carboxylesterases. The activity of HCAC-E was inhibited by NaAsO2 and AgNO3 while that of HCHS-E1 and HCHS-E2 remained unaffected. Selective inhibition of HCHS-E1 by NaF, HgCl2 and p-chloromercuribenzoate and that of HCHS-E2 by NiSO4 indicated the possible existence of different enzymes or isozymes of a carboxylesterase catalyzing HCHS hydrolysis. The effects elicited by the inhibitors on the activity of PNPA esterase were different from those observed with steroid esterases. Furthermore, the present study has also indicated species variations in the distribution of steroid esterases in the livers of rat, mouse, dog and cat.