Oxidative activation of the thiophene ring by hepatic enzymes. Hydroxylation and formation of electrophilic metabolites during metabolism of tienilic acid and its isomer by rat liver microsomes

Tienilic acid (TA) is metabolized by liver microsomes from phenobarbital-treated rats in the presence of NADPH with the major formation of 5-hydroxytienilic acid (5-OHTA) which is derived from the regioselective hydroxylation of the thiophene ring of TA. During this in vitro metabolism of TA, reactive electrophilic intermediates which bind irreversibly to microsomal proteins are formed. 5-Hydroxylation of TA and activation of TA to reactive metabolites which covalently bind to proteins both required intact microsomes, NADPH and O2 and are inhibited by metyrapone and SKF 525A, indicating that they are dependent on monooxygenases using cytochromes P-450. Microsomal oxidation of an isomer of tienilic acid (TAI) bearing the aroyl substituent on position 3 (instead of 2) of the thiophene ring also leads to reactive intermediates able to bind covalently to microsomal proteins. Covalent binding of TAI, as that of TA, depends on cytochrome P-450-dependent monooxygenases and is almost completely inhibited in the presence of sulfur containing nucleophiles such as glutathione, cysteine or cyteamine. These results show that 5-OHTA, which has been reported as the major metabolite of TA in vivo in humans, is formed by liver microsomes by a cytochrome P-450-dependent reaction. They also show that two thiophene derivatives, TA and TAI, bind to microsomal proteins after in vitro metabolic activation, TAI giving a much higher level of covalent binding than TA (about 5-fold higher) and a much higher covalent binding: stable metabolites ratio (4 instead of 0.5).     

Cytochrome P-450-dependent formation of alkylating metabolites of the 2-chloroethylnitrosoureas MeCCNU and CCNU

Rat liver microsomes catalyzed the biotransformation of the clinically important nitrosourea anticancer agents 1-(2-chloroethyl)-3-(trans-4-methyl-cyclohexyl)-1-nitrosourea (MeCCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) to alkylating metabolites that bound covalently to microsomal protein and to DNA. The enzyme-mediated microsomal alkylation required NADPH and oxygen and was inhibited by carbon monoxide, indicating the participation of a cytochrome P-450-dependent monooxygenase. Additional studies with inhibitors such as piperonyl butoxide and with the inducers 3-methylcholanthrene and phenobarbital were consistent with this view. In contrast to these observations on the formation of alkylating metabolites, carbamylation reactions were not affected significantly by microsomal metabolism. Reduced glutathione, cysteine or N-acetylcysteine decreased the microsomal alkylation by MeCCNU and produced a corresponding increase in the formation of polar metabolites that was resolved by HPLC as three distinct N-acetylcysteine-MeCCNU adducts. The addition of semicarbazide to the reaction decreased microsomal alkylation by 30%, indicating that the formation of the alkylating species may proceed via an aldehyde intermediate. Renal microsomes were not found to catalyze the alkylation reaction. Moreover, MeCCNU inhibited the renal slice accumulation of p-aminohippuric acid only in the presence of liver microsomes and NADPH, suggesting that a liver metabolite may be responsible for the renal toxicity of the parent nitrosourea.     

Comparison of the arachidonic acid and NADPH-dependent microsomal metabolism of naphthalene and 2-methylnaphthalene and the effect of indomethacin on the bronchiolar necrosis

Naphthalene and 2-methylnaphthalene cause a highly organo- and species-selective lesion of the pulmonary bronchiolar epithelium in mice. Naphthalene- but not 2-methylnaphthalene-induced pulmonary bronchiolar injury is blocked by prior administration of the cytochrome P-450 monooxygenase inhibitor piperonyl butoxide, thus suggesting that metabolism by enzymes other than the P-450 monooxygenase inhibitor piperonyl butoxide, thus suggesting that metabolism by enzymes other than the P-450 monooxygenases may be important in 2-methylnaphthalene-induced lung injury. Since many of the polycyclic aromatic hydrocarbons are metabolized by the prostaglandin endoperoxide synthetase system and because detectable xenobiotic metabolizing activity has been associated with the prostaglandin synthetases in the Clara cell, the studies reported here were done to compare NADPH-versus arachidonate-dependent metabolism of naphthalene and 2-methylnaphthalene in vitro and to determine whether indomethacin, a potent inhibitor of prostaglandin biosynthesis, was capable of blocking the in vivo toxicity of these two aromatic hydrocarbons. The NADPH-dependent metabolism of naphthalene and 2-methylnaphthalene to covalently bound metabolites in lung or liver microsomal incubations occurred at easily measurable rates. Renal microsomal NADPH-dependent metabolism of either substrate was not detected. The formation of covalently bound naphthalene or 2-methylnaphthalene metabolites was dependent upon NADPH and was inhibited by the addition of reduced glutathione, piperonyl butoxide, and SKF 525A. Covalent binding of radioactivity from [14C]2-methylnaphthalene also was strongly inhibited by incubation in a nitrogen atmosphere or at 2 degree. The arachidonic acid-dependent formation of reactive metabolites from naphthalene or 2-methylnaphthalene was undetectable in microsomal incubations from lung, liver or kidney. Indomethacin, 1 hr before and 6 hr after the administration of 300 mg/kg naphthalene or 2-methylnaphthalene, failed to block the pulmonary bronchiolar injury induced by these aromatic hydrocarbons. These studies suggest that the major enzymes involved in the metabolic activation of naphthalene or 2-methylnaphthalene in vitro are the cytochrome P-450 monooxygenases and that cooxidative metabolism by the prostaglandin synthetases appears to play little role in the formation of reactive metabolites in vitro.     

Reductive metabolism and activation of benznidazole

Benznidazole (Bz) (N-benzyl-2-nitro-1-imidazole-acetamide) is a drug used against Chagas' disease. Rat liver microsomal and cytosolic fractions, but not mitochondria, exhibited Bz nitroreductase activity under anaerobic conditions in the presence of NADPH. Microsomal nitroreductase activity was enhanced by FAD and was inhibited totally by oxygen and partially by carbon monoxide. Liver cystosol fraction was able to reduce Bz nitrogroups in the presence of either N-methylnicotinamide or hypoxanthine as substrates. These enzyme activities were inhibited by menadione or allopurinol respectively. Under every experimental condition leading to enzymatic reduction of Bz nitrogroups and its inhibition or enhancement, reactive metabolites that bind covalently to proteins were also produced. This covalent binding was effectively prevented by reduced glutathione. Results suggest the participation of cytochrome P-450 and cytochrome c reductase in liver microsomal processes and of xanthine oxidase and aldehyde oxidase in liver cytosolic processes of Bz nitroreduction and activation to reactive metabolites that bind covalently to proteins. Possible pharmacological and toxicological implications of the described observations were discussed.     

Effect of isoniazid administration on selected rat and mouse hepatic microsomal mixed- function oxidases and in vitro [14C]acetylhydrazine-derived covalent binding

The effect of isoniazid on selected microsomal mixed-function oxidase and activities and on the microsomal metabolism of its own metabolite, acetylhydrazine, to a highly reactive compound which covalently binds to intracellular macromolecules was characterized in male C57BL6 mice and male Sprague-Dawley rats. In comparison with controls, isoniazid pretreatment of rats significantly increased the sp. act. of acetanilide 4-hydroxylase and the in vitro [¹⁴C]acetylhydrazine-derived covalent binding to hepatic microsomes but significantly decreased the sp. act. benzo[a]pyrene hydroxylase and testosterone 16α-hydroxylase. Isoniazid treatment of mice had no effect on any of these parameters except for a significant reduction is sp. act. of testosterone 7α-hydroxylase. Thus the pathway of isoniazid metabolism leading to the formation of reactive metabolites of acetylhydrazine is enhanced by isoniazid pretreatment in rats but not in mice. The presence of similar routes of isoniazid metabolism in man may account for the 8.7–24% incidence of subclinical hepatocellular damage observed in patients receiving isoniazid alone in the chemoprophlaxis of tuberculosis.     

Bioactivation of tetrachloroethylene. Role of glutathione S-transferase-catalyzed conjugation versus cytochrome P-450-dependent phospholipid alkylation

The metabolism of [14C]tetrachloroethylene (Tetra) and its metabolite S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC) was investigated with in vitro systems to substantiate metabolic pathways of Tetra deduced from in vivo experiments. In the presence of NADPH, rat hepatic microsomal fractions metabolized Tetra to soluble metabolites, which were identified as trichloroacetic acid and oxalic acid by gas chromatography/mass spectroscopy and a metabolite largely bound to microsomal macromolecules. The majority of the alkylated macromolecules were identified as N-trichloroacetylated phospholipids by high performance liquid chromatography and GC/MS. When Tetra was incubated with hepatic microsomes and cytosol in the presence of 10 mM glutathione, but in the absence of NADPH, the formation of a polar metabolite other than trichloroacetic acid and oxalic acid was observed. This metabolite was identified, after hydrolysis to the corresponding cysteine conjugate, as S-(1,2,2-trichlorovinyl)-glutathione (TCVG). Microsomal GSH S-transferases catalyzed TCVG formation more efficiently than cytosolic GSH S-transferases; the competitive substrate 1-chloro-2,4-dinitrobenzene inhibited TCVG formation. In the presence of both NADPH and GSH, TCVG formation in microsomes was decreased, indicating that oxidative metabolism and GSH conjugation of Tetra are competitive reactions. The Tetra metabolite TCVC was cleaved by bacterial cysteine conjugate b-lyase to dichloroacetic acid and pyruvate. The obtained results substantiate the postulated pathways of Tetra biotransformation and demonstrate that both oxidative and conjugative reactions occur in hepatic Tetra metabolism. Phospholipid alkylation, which occurs during oxidative metabolism, may be a deactivation reaction, whereas TCVG formation, renal metabolism to TCVC, and cleavage of TCVC by b-lyase under formation of mutagenic intermediates may contribute to the nephrocarcinogenic effect of Tetra.     

Metabolic activation of hydralazine by rat liver microsomes

There is evidence to suggest that the oxidative metabolism of hydralazine (HP), an antihypertensive drug, may represent a toxic pathway which could account for some of the adverse effects of the drug. Experiments were done to determine whether the hepatic oxidative metabolism of HP is associated with the formation of reactive metabolites. In the presence of NADPH, HP was metabolized by rat liver microsomes to three major oxidation products, phthalazine, phthalazinone (PZ), and a dimer compound. Under similar incubation conditions, radioactivity derived from [14C]HP was covalently bound to microsomal protein. Metabolite formation and covalent binding increased following pretreatment of rats with phenobarbital. In contrast, pretreatment with 3-methylcholanthrene or with the monooxygenase inhibitor, piperonyl butoxide, slightly decreased both metabolite formation and covalent binding. Electron spin resonance (ESR) analyses indicated that nitrogen-centered radicals were formed when rat liver microsomes were incubated with HP under conditions similar to those required for covalent binding and for the production of the oxidative metabolites. In addition, reduced glutathione (GSH) caused concentration-dependent decreases in the production of phthalazine, PZ, and the dimer, in the covalent binding of HP to microsomal protein, and in the formation of nitrogen-centered radicals. The results of these investigations indicate that the oxidative metabolism of HP by rat liver microsomes is highly correlated with the formation of nitrogen-centered radicals and the production of metabolites that become covalently bound to microsomal protein. These observations support the hypothesis that the oxidation of HP generates reactive metabolites which may contribute to the toxicity of the drug.     

Activation of 14C-toluene to covalently binding metabolites by rat liver microsomes

14C-Toluene was incubated with rat liver microsomes in the presence of an NADPH-generating system and metabolites were concentrated on cyclohexyl cartridges. The metabolites were separated by reverse phase HPLC and identified by comparing the retention time to standards. 14C-Toluene was converted to 14C-benzylalcohol, 14C-cresols, and an unidentified 14C-metabolite. Some of the radioactivity was found to bind covalently to microsomal macromolecules, preferentially to proteins. The binding was proportional to incubation time and microsomal protein concentration and required NADPH and molecular oxygen. The binding was greatly diminished when microsomes were heat denatured. The binding process was partially inhibited by carbon monoxide and SKF 525-A. When microsomes from phenobarbital- and 3-methylcholanthrene-treated rats were employed, binding was enhanced by 8- and 4-fold, respectively. The binding process was effectively diminished by the presence of reduced glutathione or cysteine in the incubation mixture and was not affected by lysine. Styrene oxide greatly enhanced binding. UDP-glucuronic acid, superoxide dismutase, and ascorbic acid also diminished the binding to some degree. It was concluded that toluene undergoes a hepatic microsomal monooxygenase-mediated activation, and the resultant reactive metabolites binds covalently to microsomal proteins.     

2-Acetamido-p-benzoquinone: a reactive arylating metabolite of 3'-hydroxyacetanilide

The covalent binding to protein of 3'-hydroxyacetanilide (3HAA), its primary metabolite 2',5'-dihydroxyacetanilide (2,5DHAA), and a putative secondary metabolite thereof, 2-acetamido-p-benzoquinone (APBQ), was studied in hepatic microsomal preparations from phenobarbital-pretreated mice. All compounds were found to bind irreversibly to microsomal protein, APBQ being by far the most effective member of the group. In the case of 3HAA, binding was dependent upon the presence in incubation media of the co-factor NADPH, indicating that metabolism of 3HAA was necessary for the generation of a reactive intermediate. In contrast, NADPH decreased by more than 2-fold the binding of both 2,5DHAA and APBQ. The free radical spin-trapping agent alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) did not reduce the binding of 3HAA to protein. These results support the contention that metabolic activation of 3HAA is a two-step process which involves initial aromatic hydroxylation to give the substituted hydroquinone, 2,5DHAA, followed by a second oxidation reaction (which may not be enzyme-mediated) to produce the benzoquinone derivative, APBQ. This quinone is a reactive, electrophilic intermediate which may either undergo reduction back to 2,5DHAA or bind covalently to cellular macromolecules.     

Hydroxylation of 4,4'-methylenebis(2-chloroaniline) by canine, guinea pig, and rat liver microsomes

An in vitro microsomal mixed function oxidase enzyme system was used to study the phase I metabolism of 4,4'-methylenebis(2-chloroaniline) (MBOCA) by dog, guinea pig, and rat liver. TLC with color development and autoradiography, and HPLC with detection by UV absorbance and radioactivity flow monitoring were utilized to isolate metabolites. Reference standards of the N-oxidized metabolites were prepared by oxidation of MBOCA with 3-chloroperoxybenzoic acid and structures confirmed by mass spectrometry and proton NMR. These were utilized to identify the N-hydroxy and nitroso metabolites of MBOCA isolated from the microsomal incubations by comparison of their HPLC retention times and mass spectra. The structure of the o-hydroxy metabolite (ring, ortho to the amine) isolated from the microsomal incubations was elucidated by mass spectrometry and proton NMR. N- and o-hydroxylations of MBOCA were shown to increase with incubation time, microsomal protein, substrate, and NADPH concentration, and were inhibited by 2,3-dichloro-6-phenylphenoxyethylamine, an inhibitor of the microsomal mixed function oxidase enzyme system. Guinea pig liver microsomes oxidized MBOCA to the N-hydroxy metabolite predominantly, whereas the dog liver formed predominantly the o-hydroxylated metabolite, with significant amounts of the hydroxylamine as well. The rat liver formed lesser amounts of the N- and o-hydroxylated metabolites, but larger numbers of other polar compounds.     

Covalent binding of procainamide in vitro and in vivo to hepatic protein in mice

Procainamide (PA) formed reactive metabolites capable of covalently binding to protein both in vivo and in vitro. The in vitro covalent binding of PA to washed hepatic microsomal protein prepared from control male mice was dependent upon mixed-function oxidase activity. The binding was proportional with time and protein concentration. Glutathione and SKF 525-A inhibited the in vitro covalent binding by 88 and 51%, respectively. The addition of NaF to the incubation medium produced a concentration-dependent decrease in covalent binding. Covalent binding of N-acetylprocainamide in vitro was 90% less than that of procainamide and was not increased by NaF. The in vivo covalent binding of PA to hepatic protein in male mice was increased with phenobarbital and 3-methylcholanthrene pretreatment, resulting in increase in binding of 29 and 56%, respectively, compared to control mice. Pretreatment of mice with SKF 525-A inhibited binding by 39%. Depletion of hepatic glutathione with diethyl maleate pretreatment increased the amount of covalent binding in vivo. Bioactivation of PA by hepatic microsomal enzymes in the mouse produces a metabolite capable of covalent interactions with cellular macromolecules.     

Metabolic activation of 1-naphthol by rat liver microsomes to 1,4-naphthoquinone and covalent binding species

1-Naphthol was metabolized by rat liver microsomes, in the presence of an NADPH-generating system, both to methanol-soluble metabolites including 1,4-naphthoquinone and an uncharacterized product(s) (X) and also to covalently bound products. NADH was much less effective as an electron donor than NADPH. Metyrapone, SKF 525-A and carbon monoxide all inhibited the metabolism of 1-naphthol to 1,4-naphthoquinone and to covalently bound products suggesting the involvement of cytochrome P-450 in at least one step in the metabolic activation of 1-naphthol to reactive products. Ethylene diamine, which reacts selectively with 1,2-naphthoquinone but not 1,4-naphthoquinone, did not affect the covalent binding whereas glutathione, which reacts with both naphthoquinones, caused an almost total inhibition of covalent binding. These and other results suggested that 1,4-naphthoquinone, or a metabolite derived from it, was responsible for most of the covalent binding observed and that little if any of the binding was due to 1,2-naphthoquinone.     

Lack of hepatic microsomal metabolism of deoxynivalenol and its metabolite, DOM-1

Rat hepatic microsomal preparations were used to study the metabolism of deoxynivalenol (DON) and its metabolite 3 alpha,7 alpha,15-trihydroxytrichothec-9,12-dien-8-one (DOM-1). The N-demethylation of ethylmorphine was monitored to assess the viability of the mixed-function oxidase. DON was incubated with microsomes and an NADPH-generating system. Samples were removed from the incubation system and analysed for DON using an HPLC equipped with a UV detector. After incubation for 30 min, there was no evidence of disappearance of DON or of the presence of new metabolites; neither was microsomal NADPH oxidation altered by the addition of DON. Rat and pig hepatic microsomal preparations were used to assess DON glucuronidation, using p-nitrophenol disappearance to check the viability of the microsomal glucuronidating system. When DON was incubated with microsomes and 14C-labelled uridine 5'-diphosphoglucuronic acid, no radioactivity was detected in the TLC zone where the glucuronide was expected. Three rats and one pig were dosed orally with 2 mg DON/kg and samples of their urine and faeces were extracted and incubated with beta-glucuronidase or with buffer only. No differences in DON or DOM-1 concentrations were detected between samples incubated with or without beta-glucuronidase. These results suggest that DON was neither bioactivated to a more toxic product nor oxidized to a less toxic compound by the rat hepatic mixed-function oxidase system. Likewise, DOM-1 was not reactivated or metabolized by this system. Neither DON nor DOM-1 glucuronides were formed either in in vitro liver systems or in vivo.     

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.     

The in vitro hepatic microsomal metabolism of N-benzyladamantanamine in rats

The metabolism of N-benzyladamantanamine (NBAD) was studied in vitro using rat hepatic microsomal preparations. The substrate and proposed metabolites were synthesized and characterized using spectroscopic techniques and separated using a reverse phase HPLC system. NBAD was incubated with rat microsomal preparations, extracted into DCM in the presence of NaCl and evaporated under a stream of nitrogen. The results from HPLC studies showed that NBAD produced the corresponding nitrone and hydroxylamine. This experiment also revealed that dealkylation occurred. No metabolites were observed which corresponded to authentic amide or oxaziridine. The reactions required a microsomal enzyme source and NADPH as a cofactor. The results indicate that the nitrone observed as a metabolite of NBAD is not an intermediate leading to the formation of an oxaziridine and hence an amide, under careful experimental conditions excluding light.     

In vitro hepatic microsomal metabolism of N-benzyl-N-methylaniline.

In the present study, the in vitro microsomal metabolism of a tertiary aniline, N-benzyl-N-methylaniline (NBNMA) was studied to determine whether this compound produces an amide derivative (benzoyl) together with N-dealkylation and C- and N-oxidation products as metabolites. The preparations of the corresponding potential metabolites were undertaken and were separated using TLC and HPLC. Incubations were performed using rat microsomal preparations fortified with NADPH. The substrate and its potential metabolites were extracted into dichloromethane in the presence of NaCl and examined by TLC and HPLC-UV. The results indicated that NBNMA did not produce the corresponding amide (benzoyl derivative) or N-oxide metabolite but was dealkylated to the corresponding secondary amine. Two p-hydroxylated phenolic metabolites were also observed. These findings support the concept that nitrones are essential intermediate metabolites for the formation of amides from secondary aromatic amines (chemical rearrangement to amide via an oxaziridine intermediate). The carbinolamine produced from NBNMA does not seem stable enough to allow further oxidation to the amide and therefore this intermediate is broken down to the dealkylation products. N-Dealkylations and p-hydroxylations are major metabolic reactions following in vitro hepatic microsomal metabolism of the benzylic tertiary aniline, NBNMA.     

Metabolic activation of the tricyclic antidepressant amineptine--I. Cytochrome P-450-mediated in vitro covalent binding

Incubation of [14C]amineptine (1 mM) with hamster liver microsomes resulted in the irreversible binding of an amineptine metabolite to microsomal proteins. Covalent binding measured in the presence of various concentrations of amineptine (0.0625-1 mM) followed Michaelis-Menten kinetics. Pretreatment with phenobarbital increased not only the Vmax, but also the Km, for this binding. Covalent binding required NADPH and molecular oxygen and was decreased when the incubation was made in the presence of inhibitors of cytochrome P-450 such as piperonyl butoxide (4 mM), SKF 525-A (4 mM) or carbon monoxide (80:20 CO-O2 atmosphere). In contrast, binding was increased when microsomes from untreated hamsters were incubated in the presence of 0.5 mM 1,1,1-trichloropropene 2,3-oxide, an inhibitor of epoxide hydrolase. Metabolic activation also occurred in kidney microsomes. In vitro covalent binding to kidney microsomal proteins required NADPH and was decreased by piperonyl butoxide (4 mM) but was not increased by pretreatment with phenobarbital. We conclude that amineptine is activated by hamster liver and kidney microsomes into a chemically reactive metabolite that covalently binds to microsomal proteins.     

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 in vitro hepatic microsomal metabolism of methyl 2-(2(3H)-benzoxazolone-3-yl)acetate in rats

The in vitro hepatic microsomal metabolism of methyl 2-(2(3H)-benzoxazolone-3-yl)acetate (I) was studied using hepatic washed rat microsomal preparations fortified with NADPH. The substrate (I) and its potential hydrolytic metabolite 2-(2(3H)-benzoxazolone-3-yl)acetic acid (II) and 2(3H)-benzoxazolone (III), a potential dealkylation metabolite, were separated using a reverse phase HPLC system which consisted of a C18 column and a mobile phase of acetonitrile: 0.02 M phosphate buffer (30:70, final pH 7) at a flow rate of 1 ml/min with UV detection at 254 nm. The substrate (I) was incubated with rat microsomal preparations, extracted into DCM, and finally evaporated under nitrogen. The results from HPLC studies showed that (I) was metabolised to (II) and (III) by rat microsomes in the presence of NADPH.     

In vivo and in vitro effects of helenalin on mouse hepatic microsomal cytochrome P450

Helenalin, a natural plant product with significant antitumor activities, decreased male BDF1 mouse hepatic microsomal cytochrome P450 contents in vivo and in vitro. A single i.p. dose of 25 mg helenalin/kg body weight significantly (P less than 0.05) decreased microsomal cytochrome P450 contents and inhibited cytochrome P450-dependent mixed-function oxidase activities within 1-2 hr post-exposure. Helenalin (1.0 mM) decreased microsomal cytochrome P450 contents in vitro by 11% in the absence of NADPH and by 32% in the presence of NADPH. These in vitro and in vivo decreases in cytochrome P450 were accompanied by comparable decreases in total microsomal heme contents. Helenalin (1.0 mM) increased mouse hepatic microsomal oxygen consumption and NADPH utilization by 3.2 and 5.4 nmol/min/mg protein respectively. Helenalin (1.0 mM) significantly (P less than 0.05) increased microsomal lipid peroxidation in vitro, and this helenalin-induced increase in lipid peroxidation was inhibited completely by the addition of 0.05 mM EDTA. However, microsomal cytochrome P450 contents were equally affected by helenalin in the presence or absence of EDTA, suggesting that lipid peroxidation did not contribute to the helenalin-induced decrease in cytochrome P450. The addition of 0.05 mM hemin to microsomes treated in vitro with 1.0 mM helenalin resulted in a 58% recovery of cytochrome P450 contents. This ability of hemin to reconstitute cytochrome P450 in helenalin-treated microsomes suggests that helenalin produced a selective loss of heme from the cytochrome P450 holoprotein, and that the resulting cytochrome P450 apoprotein remained intact after helenalin treatment. The increased loss of microsomal cytochrome P450 produced by helenalin in the presence of NADPH suggests that a helenalin metabolite may be responsible for heme loss and the in vitro destruction of cytochrome P450.