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 of benzimidazoline-2-thiones by rat hepatic microsomes and hog liver flavin-containing monooxygenase.

The metabolism of benzimidazoline-2-thione (I) and the 1-methyl (II) and 1,3-dimethyl (III) derivatives was studied to elucidate the mechanisms of hepatic oxidation for this class of thionosulfur-containing xenobiotics. NADPH-dependent metabolism of I, II, and III to the corresponding benzimidazoles Ia, IIa, and IIIa, respectively, was observed in dexamethasone-pretreated rat hepatic microsomes. III was the only thiocarbamide converted to an amide metabolite (IIIb). The effects of heat and 1-aminobenzotriazole pretreatment suggested that rat hepatic microsomal metabolism of I was catalyzed by the flavin-containing monoxygenase (FMO) only and that of II and III by both FMO and cytochrome P450 isozymes (P450). Addition of 5.0 mM glutathione (GSH) blocked formation of all metabolites from I, II, and III. Highly purified hog liver FMO catalyzed formation of all metabolites observed in rat hepatic microsomal systems. Incubation of III with either rat liver microsomes or with highly purified hog liver FMO in the presence of [18O]water led to ca. 50% incorporation of [18O] into IIIb. When [18O] molecular oxygen was used, ca. 8% incorporation of [18O] into IIIb was observed. Highly purified hog liver FMO also converted I-III to chemically reactive species that covalently bound to protein thiols. In the presence of hog liver FMO, the covalent binding pattern of radiolabeled I-III to bovine serum albumin was essentially identical to that observed for rat hepatic microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microsomal metabolism and covalent binding of [3H/14C]-bromobenzene. Evidence for quinones as reactive metabolites

1. The metabolism and covalent binding of [3H/14C]bromobenzene has been investigated using liver microsomes from untreated and phenobarbital (PB)-pretreated rats. A model has been developed to relate the observed 3H/14C ratios in the covalently bound residues to the type of metabolite (epoxide versus quinone) responsible for their formation. 2. With control microsomes metabolism was linear for 60 minutes, but with PB microsomes the time course showed a short-lived burst of rapid metabolism followed by a long phase with an overall rate comparable to control. With both types of microsomes covalent binding was synchronous with metabolism. 3. The normalized 3H/14C ratios of recovered substrate and water-soluble metabolites was 1.0, whereas that of the covalently bound material was only 0.5. Such extensive loss of tritium implies that a considerable portion of the covalent binding arises from bromobenzene metabolites more highly oxidized than an epoxide (e.g. quinones). 4. The normalized 3H/14C ratios for bromobenzene metabolites covalently bound to liver proteins in vivo (total and microsomal) was the same as with microsomes in vitro (0.5). However, for the lung and kidney the 3H/14C ratios were considerably higher (0.71 and 0.62), indicating that differences between tissues in vivo may be greater than between liver microsomes in vitro and in vivo.     

Metabolism of prazepam by rat liver microsomes: stereoselective formation and N-dealkylation of 3-hydroxyprazepam

The metabolism of prazepam (7-chloro-1-(cyclopropylmethyl)-1,3-dihydro-2H-1, 4-benzodiazepin-2-one) (PZ) was studied using liver microsomes prepared from untreated, phenobarbital (PB)-treated, and 3-methylcholanthrene (3MC)-treated male Sprague-Dawley rats. Relative rates of PZ metabolism by liver microsomes prepared from rats were PB-treated greater than untreated greater than 3MC-treated. Metabolites of PZ were separated by normal phase high performance liquid chromatography and the relative amounts of major metabolites were found to be N-desalkylprazepam (also known as N-desmethyldiazepam and nordiazepam) greater than 3-hydroxy-PZ (3-OH-PZ) greater than oxazepam. Enantiomers of 3-OH-PZ were resolved by high performance liquid chromatography on an analytical column packed with Pirkle's chiral stationary phase, (R)-N-(3,5-dinitrobenzoyl)phenylglycine covalently bonded to spherical particles of gamma-aminopropylsilanized silica. 3-OH-PZ formed in the metabolism of PZ by liver microsomes prepared from rats was found to have 3R/3S enantiomer ratios of 84:16 (untreated), 85:15 (PB-treated), and 84:16 (3MC-treated), respectively. Relative rates of N-dealkylation of PZ by three rat liver microsomal preparations were PB-treated greater than untreated greater than 3MC-treated. N-Dealkylation of 3-OH-PZ by rat liver microsomes was substrate enantioselective; the 3S-enantiomer was N-dealkylated faster than the 3R-enantiomer. The results indicated that both C3-hydroxylation of PZ and N-dealkylation of 3-OH-PZ catalyzed by rat liver microsomes were stereoselective, resulting in the formation of a 3-OH-PZ highly enriched in the 3R-enantiomer.     

Studies on the in vivo metabolism of hydralazine in the rat

A single dose of [1-14C]hydralazine is extensively metabolized in the rat as no unchanged drug is excreted in the urine, the major route of elimination of the drug. However, only a small proportion of the dose could be accounted for as known metabolites. The lack of expired 14CO2 suggests that the phthalazine ring is metabolically stable. The metabolites were qualitatively but not quantitatively similar to those excreted by human subjects. The three major urinary metabolites were found to be 3-methyl-s-triazolo[3,4a] phthalazine and acid-labile conjugates of hydralazine and 1-hydrazinophthalazin-4-one. There were also small amounts of s-triazolo[3,4-a]-phthalazine, 3-hydroxymethyl-s-triazolo[3,4-a]-phthalazine, hydrazine, and phthalazin-1-one. Induction of the microsomal enzymes by pretreatment with 3-methylcholanthrene reduced the excretion of the acetylated metabolite 3-methyl-s-triazolo[3,4-a]phthalazine, of conjugates of hydralazine and 1-hydrazinophthalazin-4-one. Pretreatment with phenobarbital reduced excretion of 3-methyl-s-triazolo[3,4-a]phthalazine. Conversely, inhibition of the microsomal enzymes by pretreatment with piperonyl butoxide increased the excretion of 3-methyl-s-triazolo[3,4-a]phthalazine and decreased the excretion of 1-hydrazinophthalazin-4-one conjugates. [14C]Hydralazine or a metabolite was covalently bound to tissue protein, particularly in the aorta, lungs, and spleen. Induction of the microsomal enzymes with 3-methylcholanthrene reduced and inhibition of the microsomal enzymes increased the binding to the aorta. In conclusion, the covalent binding of [14C]hydralazine or a metabolite to protein does not appear to be mediated by the microsomal enzymes in the rat and the metabolism of hydralazine in the rat shows considerable quantitative differences from that in man.     

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.     

In vitro covalent binding of new brain tracer, para-125I-amphetamine, to rat liver and lung microsomes

p-125I-amphetamine (I-Amp) is retained significantly in liver and lung during brain tomoscintigraphy. To attempt to explain this clinical observation, we have investigated the interaction of I-Amp with rat liver and lung microsomal proteins. Studies using spectral shift technique indicate that low concentration of I-Amp gives a type I complex and high concentration appears very stable type II complex with cytochrome P-450 Fe III. In the presence of NADPH, I-Amp gives rise to a 455 nm absorbing complex with similar properties to the Fe-RNO complexes. This complex formation was greatly enhanced with phenobarbital treated liver microsomes. The in vitro binding study shows that I-Amp and/or its metabolites was covalently bound to macromolecules in the presence of the molecular oxygen and NADPH-generating system. Incubation in the presence of glutathione, cystein and radical scavengers decreases binding. Mixed function oxydase (MFO) inhibitors diminish the amount of covalent binding and alter the extent of metabolite formation. The total covalent binding level increased with liver microsomes from PB pretreated rats as it was observed with the 455nm complex formation. The radioactivity distribution on microsomal proteins was examinated with SDS polyacrylamide gel electrophoresis and autoradiography. This experiment proves that the radiolabelled compounds are bound on the cytochrome P-450. The radioactivity bound increased when the PB induced rat liver microsomes were used. All these results indicate that I-Amp was activated by an oxydative process dependent on the MFO system which suggests a N-oxydation of I-Amp and the formation of reactive entities which covalently bind to proteins.     

Adducts of 3-methylindole and glutathione: species differences in organ-selective bioactivation

Lung and liver microsomes of several species were evaluated for potential to form activated metabolites of 3-methylindole (3MI). Microsomes were incubated with [14C]3MI and glutathione (GSH). Electrophilic 3MI metabolites were trapped and quantitated as GSH adducts by HPLC, and by determining the amounts of activated intermediates which became covalently bound to microsomal protein. The highest rates of 3MI-GSH adduct formation by the lung were detected in microsomes of the goat, followed in decreasing order by pulmonary microsomes from the horse, monkey, mouse, and rat, respectively. In contrast, hepatic 3MI-GSH adduct production was highest in microsomes from the rat, followed by mouse, monkey, goat, and horse microsomes, respectively. These results suggest that the species and organ-selective toxicity of 3MI are primarily caused by differences in rates of oxidative metabolism of 3MI to an electrophilic intermediate.     

Activation of acetaminophen oxidation in rat liver microsomes by caffeine

Addition of caffeine in vitro stimulated the oxidative metabolism of acetaminophen by rat liver microsomes, resulting in increased formation of acetaminophen-glutathione (GSH) conjugates and increased covalent binding of acetaminophen to microsomal protein. This metabolic enhancement by caffeine was most prominent using liver microsomes from phenobarbital (PB)-treated rats. Liver microsomes obtained from rats treated with ethanol-oxidized acetaminophen at much faster rates than microsomes from control, PB-treated or 3-methylcholanthrene (3-MC)-treated animals. The stimulatory effect of caffeine was, however, minimal in liver microsomes obtained from ethanol-treated rats.     

Covalent binding of [14C]methoxychlor metabolite(s) to rat liver microsomal components

[14C]Methoxychlor was incubated with NADPH-fortified liver microsomes from male rats, and covalent binding to microsomal components was determined. The binding process was markedly enhanced when microsomes from phenobarbital-treated rats were employed. However, when microsomes from methylcholanthrene-treated rats were used the level of binding was not significantly affected. Incubation in the presence of glutathione, cysteine, or ascorbate markedly diminished binding. Metyrapone and SKF 525-A, inhibitors of hepatic cytochrome P-450-linked monooxygenase activity, inhibited the binding. Also, ethylmorphine and hexobarbital, alternate substrates of the monooxygenase system, inhibited binding. There was no binding to microsomal components in the absence of NADPH or oxygen. TCPO (1,1,1-trichloropropane-2,3-oxide), an inhibitor of epoxide hydrase activity, failed to enhance the binding process. However, N,N'-diphenyl-p-phenylenediamine (NDP) and n-propyl gallate (PG), both free radical scavengers, decreased binding at micromolar concentrations without altering the extent of formation of polar [14C]methoxychlor metabolites. It was concluded that methoxychlor undergoes a hepatic microsomal monooxygenase(s)-mediated activation and that the resultant reactive metabolites (possibly free radicals) bind covalently to microsomal components. By contrast, the binding resulting from the incubation of an impure mixture of polar [14C]methoxychlor metabolites with liver microsomes did not require NADPH and O2 and was not affected by NDP, Pg, ascorbate, or heat-treatment of microsomes. This finding suggested that the binding subsequent to the initial metabolic activation of methoxychlor does not require further enzymatic transformation. However, whether the binding with metabolites represents the same chemical species as the binding with [14C]methoxychlor remains to be established.     

The role of CYP2E1 and 2B1 in metabolic activation of benzene derivatives

CYP2B1 and 2E1 oxidized toluene, aniline and monochlorobenzene (MCB) to water-soluble metabolites and to products covalently binding to microsomal proteins from male Wistar rats at high efficiency. Oxidation of benzene to covalently binding metabolites was catalysed by CYP2B1 and 2E1 more effectively than the formation of water-soluble metabolites, especially at low benzene levels. Thus, the formation of covalently binding products was inversely related but formation of soluble metabolites was proportional to benzene concentration. 1,4-Benzoquinone was responsible for the majority of covalent binding to microsomal proteins, being suppressed by ascorbate; 1,4-semiquinone was not important, since α-tocopherol did not inhibit the covalent binding and ESR showed its rapid decay, if NADPH was available. Specific antibodies and inhibitors confirmed the role of CYP2B1 and 2E1 induction. Covalent binding of benzene to DNA was largely due to benzene oxide; ∼50% was due to N-7 guanine adduct. CYP2E1 oxidizing benzene via phenol to 1,4-hydroquinone appeared to mediate its further oxidation to 1,4-benzoquinone, which also occurred spontaneously, but was reversed in a reducing environment of microsomes with NADPH. Production of OH radicals in microsomes with NADPH was greatly stimulated by HQ and less by BQ, especially in CYP2E1 induced microsomes, although the quinones themselves failed to produce OH radicals. The quinones could act by stimulation of the CYP futile cycle. Therefore, CYP2B1 and 2E1 in rats appeared essential for metabolic activation of benzene derivatives to potentially genotoxic products; BQ dominated the covalent binding of benzene to proteins, whereas DNA adducts were largely due to benzene oxide. Received: 12 March 1996/Accepted: 5 July 1996     

Rat liver microsomal lipid peroxidation produced during the oxidative metabolism of ethacrynic acid

Thiobarbituric acid reactive substances (TBARS) were produced in rat liver microsomal suspension incubated with ethacrynic acid (loop diuretic drug) and NADPH. Two oxidative metabolites of ethacrynic acid with dicarboxylic acid and hydroxylated ethyl group, respectively, were formed in the reaction mixture. The oxidative metabolism of ethacrynic acid was inhibited by cytochrome P450 inhibitors. The formation of TBARS was remarkably depressed by inhibitors like diethyldithiocarbamate and disulfiram. These results indicate that lipid peroxidation occurred in rat liver microsomes through the oxidative metabolism of ethacrynic acid.     

Metabolism of benzo[a]pyrene by guinea pig adrenal and hepatic microsomes

Studies were carried out to compare the metabolism of benzo[a]pyrene (BP) by adrenal and hepatic microsomes obtained from adult male guinea pigs. Adrenal microsomes produced fluorescent metabolites (primarily phenols) approximately three to four times more rapidly than hepatic microsomes, but the differences in the rates were considerably smaller when total BP metabolism was assessed using an isotopic assay. The apparent discrepancy between the two assays is attributable to differences in the profiles of BP metabolites produced by adrenal and liver. Separation of metabolites by high pressure liquid chromatography revealed that adrenal microsomes converted BP to primarily a phenolic metabolite with a retention time identical to that of 3-hydroxy-BP. Liver microsomes, by contrast, produced approximately equal amounts of compounds co-chromatographing with 3-hydroxy-BP and BP-4,5-dihydrodiol. Small amounts of other metabolites were also produced by adrenal and hepatic microsomes. Liver microsomes catalyzed the conversion of BP to metabolites that became covalently bound to exogenous DNA. The amount of binding was dependent upon the duration of incubation and concentration of microsomal protein. Adrenal microsomes, by contrast, did not promote BP binding to DNA. Inhibition of microsomal epoxide hydratase activity with trichloropropene oxide (TCPO) blocked the formation of dihydrodiol metabolites of BP by adrenal and liver microsomes. In the presence of TCPO, liver microsomes produced large amounts of a BP metabolite co-chromatographing with BP-4,5-oxide. TCPO also increased the rate of production of DNA-binding roetabolites by liver microsomes but had no effect on the formation of DNA-binding metabolites by adrenal microsomes. The results demonstrate major differences in the pathways of BP metabolism by guinea pig adrenal and hepatic microsomes. Although adrenal microsomes metabolize BP more rapidly than hepatic microsomes, far greater amounts of reactive metabolites are produced by the liver. Thus, adrenal metabolism of BP may be of little toxicological significance.     

Metabolic activation of nitrofurantoin--possible implications for carcinogenesis.

Although past investigations have indicated that nitrofurantoin is noncarcinogenic, the present studies demonstrate several features of the metabolism of the drug which are similar to those of other nitrofurans that are known carcinogens. Microsomal and soluble fractions from both rat liver and lung mediated the covalent binding of [¹⁴C]nitrofurantoin to tissue macromolecules in vitro. Oxygen strongly inhibited the binding in both the microsomal and soluble fractions, and carbon monoxide failed to inhibit binding in microsomal preparations, indicating nitrofurantoin was activated in both systems by nitroreduction and not by oxidation of the furan ring. An antibody against NADPH-cytochrome c reductase inhibited the microsomal nitroreduction and covalent binding of nitrofurantoin, while the addition of a flavin (FAD) markedly enhanced the covalent binding. Maximal rates of covalent binding were obtained in soluble fractions in the presence of NADH or hypoxanthine; covalent binding was inhibited in these fractions by allopurinol. an inhibitor of xanthine oxidase. Nitroreduction of nitrofurantoin was enhanced, but covalent binding was decreased, in liver microsomes from phenobarbital-pretreated rats. Phenobarbital did not alter nitroreduction or covalent binding of nitrofurantoin in lung microsomes or in soluble fractions from lung or liver. Reduced glutathione markedly decreased covalent binding of nitrofurantoin. in both the microsomal and the soluble fractions from liver and lung. but did not alter the rate of nitroreduction in any of the fractions. Radioactivity was covalently bound in several organs of rats given [¹⁴C]nitrofurantoin in vivo.     

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.     

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.     

Human and rat liver UDP-glucuronosyltransferases are targets of ketoprofen acylglucuronide.

Acylglucuronides formed from carboxylic acids by UDP-glucuronosyltransferases (UGTs) are electrophilic metabolites able to covalently bind proteins. In this study, we demonstrate the reactivity of the acylglucuronide from the nonsteroidal anti-inflammatory drug, ketoprofen, toward human and rat liver UGTs. Ketoprofen acylglucuronide irreversibly inhibited the glucuronidation of 1-naphthol and 2-naphthol catalyzed by human liver microsomes or by the recombinant rat liver isoform, UGT2B1, which is the main isoform involved in the glucuronidation of the drug. A decrease of about 35% in the glucuronidation of 2-naphthol was observed when ketoprofen acylglucuronide was produced in situ in cultured V79 cells expressing UGT2B1. Inhibition was always associated with the formation of microsomal protein-ketoprofen adducts. The presence of these covalent adducts within the endoplasmic reticulum of cells expressing UGT2B1 was demonstrated following addition of ketoprofen to culture medium by immunofluorescence microscopy with antiketoprofen antibodies. Immunoblots of liver microsomes incubated with ketoprofen acylglucuronide and probed with antiketoprofen antibodies revealed the presence of several protein adducts; among those was a major immunoreactive protein at 56 kDa, in the range of the apparent molecular mass of UGTs. The adduct formation partially prevented the photoincorporation of the UDP-glucuronic acid (UDP-GlcUA) analog, [beta-32P]5N3UDP-GlcUA, on the UGTs, suggesting that ketoprofen glucuronide covalently reacted with the UDP-GlcUA binding domain. Finally, UGT purification from rat liver microsomes incubated with ketoprofen glucuronide led to the isolation of UGT adducts recognized by both anti-UGT and antiketoprofen antibodies, providing strong evidence that UGTs are targets of this metabolite.     

The in vitro metabolism of hydralazine

Hydralazine was metabolized in vitro to phthalazine and phthalazine-1-one by microsomal enzymes requiring NADPH. Hydrazine was not detected as a metabolite under these in vitro conditions. Addition of 1-hydrazinophthalazin-4-one, a possible intermediate metabolite, decreased covalent binding to protein but also decreased metabolism. Phthalazine and phthalazin-1-one also decreased covalent binding to protein to a lesser extent. N-Acetylcysteine significantly decreased the level of phthalazine and phthalazin-1-one detected in microsomal incubations. The results are consistent with a reactive intermediate, but not 1-hydrazinophthalazin-4-one, being responsible for both covalent binding and production of phthalazine.     

Positional metabolism of benzo(a)pyrene in rat placenta and maternal liver. Comparison of induction effects

The biotransformation of [14C]benzo(a)pyrene (BP) was studied in vitro in the presence of microsomes prepared from isolated labyrinth and basal zone tissues of the rat placenta, as well as from maternal liver. Pregnant rats, day 14 of gestation, received beta-naphthoflavone (beta NF; 15 mg/kg, ip) or 3-methyl-cholanthrene (3MC; 30 mg/kg, ip). On day 15, placentae were dissected and microsomes were incubated with 17 microM [14C]BP and 2 mM NADPH. Metabolites formed in the incubation flasks were extracted and separated by HPLC utilizing a reverse phase column. Only trace BP metabolism occurred in basal zone microsomes from control, beta NF-, or 3MC-pretreated animals, as well as in labyrinth microsomes from control animals. In contrast, the preadministration of beta NF and 3MC increased labyrinth microsomal BP metabolism by 10- to 15-fold. Labyrinth and maternal liver microsomes from beta NF- and 3MC-treated animals actively converted BP to eight separate metabolites which co-chromatographed primarily with quinones and phenols. The overall formation of BP diol and phenolic metabolites by labyrinth microsomes was appreciably less than was observed for liver preparations. The very low activity of BP-4,5-oxide hydrolase in labyrinth microsomes compared to liver may in part explain the low level of formation of BP diols in placental microsomes. Labyrinth microsomes catalyzed the covalent binding of [3H]BP to calf thymus DNA, and this activity increased 5-fold following beta NF pretreatment. A comparison of induced tissues indicates that the amount of DNA binding in labyrinth microsomes is more extensive than would be expected by the level of total BP metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism and covalent binding of [14C]toluene by human and rat liver microsomal fractions and liver slices

The in vitro metabolism of [14C]toluene by liver microsomes and liver slices from male Fischer F344 rats and human subjects has been compared. Rat liver microsomes produced only benzyl alcohol from toluene. Liver microsomes from human subjects metabolized toluene to benzyl alcohol, benzaldehyde, and benzoic acid. Liver microsomes from one human donor also produced p-cresol and o-cresol. The overall rate of toluene metabolism by human liver microsomes was 9-fold greater than by rat liver microsomes. Human liver microsomal metabolism of benzyl alcohol to benzaldehyde required NADPH and was inhibited by carbon monoxide and high pH (pH 10). but was not inhibited by ADP-ribose or sodium azide. These results suggest that cytochrome P-450, rather than alcohol dehydrogenase, was responsible for the metabolism of benzyl alcohol to benzaldehyde. Human and rat liver slices metabolized toluene to hippuric acid and benzoic acid. The overall rate of toluene metabolism by human liver slices was 1.3-fold greater than by rat liver slices. Cresols and cresol conjugates were not detected in human or rat liver slice incubations. Covalent binding of [14C]toluene to human liver microsomes and slices was 21-fold and 4-fold greater than to the comparable rat liver preparations. Covalent binding did not occur in the absence of NADPH, was significantly decreased by coincubation with cysteine, glutathione, or superoxide dismutase, and was unaffected by coincubation with lysine. Protease and ribonuclease digestion decreased the amount of toluene covalently bound to human liver microsomes by 78% and 27% respectively. Acid washing of human liver microsomes had no effect on covalent binding.(ABSTRACT TRUNCATED AT 250 WORDS)