Metabolism of benzene in rat hepatocytes. Influence of inducers on phenol glucuronidation

Alterations of benzene metabolism in liver markedly influence benzene toxicity at extrahepatic target tissues. Therefore, generation of 11 phase I and II metabolites of benzene (including phenol, hydroquinone, catechol, benzene-1,2-dihydrodiol, their sulfates and glucuronides, and phenylglutathione) was compared in hepatocytes from 3-methylcholanthrene (MC)- or phenobarbital-treated rats and from untreated controls. At 0.1 mM benzene, total metabolism appeared to be unchanged by treatment with inducers. Phenylsulfate (35%), phenylglucuronide (15%), and phenylglutathione (12%) represented the major metabolites in hepatocytes from untreated controls. With hepatocytes from MC-treated rats, a pronounced shift from phenylsulfate to phenylglucuronide (increase to 34%) was observed, while the formation of unconjugated phenol, hydroquinone, and catechol was decreased (from 16 to 10%). A similar shift from sulfation to glucuronidation was seen in similar studies with phenol. Lineweaver-Burk analysis of microsomal phenol UDP-glucuronosyltransferase activity suggested that MC-treatment induced a high affinity isozyme (KM = 0.14 mM), in addition to the low affinity isozyme (KM = 3.1 mM) present in liver microsomes from untreated and phenobarbital-treated rats. It is concluded that induction by MC of a high affinity hepatic phenol UDP-glucuronosyltransferase effectively shifts benzene metabolism toward formation of less toxic metabolites. This shift may reduce toxic risks at extrahepatic target tissues.     

Characterization of the benzene monooxygenase system in rabbit bone marrow

The microsomal fraction of bone marrow contains cytochrome P-450 (39 +/- 11 pmoles/mg microsomal protein) and monooxygenase activity could be demonstrated by the O-dealkylation of 7-ethoxycoumarin (114 +/- 65 pmoles/(min X mg microsomal protein] and the hydroxylation of benzene to phenol (51 +/- 8.6 pmol/45 min X mg microsomal protein). This monooxygenase system differs from that in liver in various aspects. The conversion of benzene to phenol calculated as molecular activity was about 4 times higher than in liver and no induction by phenobarbital could be observed. Aroclor 1254 induced the cytochrome P-450 content about twofold but lowered the O-dealkylation activity of 7-ethoxycoumarin in contrast to liver. Pretreatment with benzene did not change the O-dealkylation in bone marrow, but had a stimulating effect on benzene monooxygenation and covalent binding of 14C-benzene metabolites. From these results we conclude that the bone marrow monooxygenase system develops its own pattern of cytochrome P-450 isoenzymes. Especially after chronic exposure to benzene this system can convert this chemical to phenol and secondary metabolites. The similar behaviour of phenol formation and covalent binding strengthens the hypothesis of a common pathway for metabolism and toxicity but the active intermediate still remains unknown.     

Cytochrome P450 forms in fish: Catalytic,immunological and sequence similarities

1. Hepatocytes isolated from the adult male NMRI mouse or Wistar rat were incubated for 1?h with 0·5 mM ¹⁴C-benzene, the supernatant was separated from the cells, and analysed for benzene metabolites. Separately, formation of sulphate conjugates during benzene metabolism was studied in hepatocytes in the presence of ³⁵S-sulphate. In addition sulphate conjugation of the benzene metabolites hydroquinone and 1,2,4-trihydroxybenzene was investigated in mouse liver cytosol supplemented with 3′-phosphoadenosine-5′-phospho-³⁵S-sulphate.

2. Two novel metabolites, not detectable in rat hepatocyte incubations, were found in mouse hepatocytes, and were identified as 1,2,4-trihydroxybenzene sulphate and hydroquinone sulphate. Formation of the ³⁵S-labelled conjugates could be demonstrated in incubations of mouse liver cytosol with hydroquinone or 1,2,4-trihydroxybenzene supplemented with 3′-phosphoadenosine-5′-phospho-³⁵S-sulphate, and in mouse hepato-cytes incubated with benzene and ³⁵S-sulphate.

3. In comparison with hepatocytes from the Wistar rat, hepatocytes from the NMRI mouse were almost three times more effective in metabolizing benzene. The higher formation of hydroquinone, and the formation of trihydroxybenzene sulphate and hydroquinone sulphate, mainly contributed to the higher rate of benzene metabolism.

4. In conclusion, qualitative and quantitative differences in benzene metabolism may contribute to the higher susceptibility of mouse towards the myelotoxic and leucaemogenic action of benzene.     

Hematotoxicity and concentration-dependent conjugation of phenol in mice following inhalation exposure to benzene

Benzene is metabolized to one or more hematotoxic species. Saturation of benzene metabolism could limit the production of toxic species. Saturation of phase II enzymes involved in the conjugation of the phenolic metabolites of benzene also could affect the hematotoxicity of benzene. To investigate the latter possibility, we exposed male Swiss mice, via the inhalation route, to various concentrations of benzene for 6 h per day for 5 days. Following termination of the final exposure the mice were killed and the levels of phenylsulfate and phenylglucuronide in the blood determined. Spleen weights were recorded and the number of white blood cells counted. At low benzene exposure concentrations phenylsulfate is the major conjugated form of phenol in the blood. At high exposure concentrations, phenylglucuronide is the predominant species. The reductions in spleen weight and white blood cell numbers correlated with the concentration of phenylsulfate in the blood, but are most probably not causally related.     

Age-related changes in disposition and metabolism of benzene in male C57BL/6N mice.

Benzene disposition and metabolism were examined as a function of age in male C57BL/6N mice aged 3 and 18 months. Mice received a single oral dose of either 10 or 200 mg/kg 14C-benzene (approximately 25 microCi/kg). Excretion of 14C-derived benzene radioactivity (RA) was monitored in urine, feces, and as exhaled 14CO2 from 0 to 72 hr, and as exhaled unmetabolized benzene from 0 to 6 hr. At 10 mg/kg 14C-benzene, urinary elimination was the major route of excretion in both 3- and 18-month mice. Urinary excretion of 14C-derived benzene RA was significantly decreased in 18- vs. 3-month mice at 4, 6, 24, and 48 hr, while fecal excretion was significantly increased at 72 hr. Elimination of 14C-benzene as 14CO2 and unmetabolized 14C-benzene was also increased in 18- vs. 3-month mice at this dose. Hydroquinone glucuronide (HQG), phenylsulfate (PS), and muconic acid (MUC) were the major urinary metabolites at 10 mg/kg 14C-benzene in both 3- and 18-month mice, representing approximately 40, 28, and 15% of an administered dose of 14C-benzene. Smaller amounts of phenyl glucuronide (4.0%), pre-phenyl mercapturic acid (1.2%), and catechol glucuronide (0.5%) were also detected. No significant differences were found with age in the percentage of an administered dose of benzene excreted as the various metabolites at 10 mg/kg. At 200 mg/kg 14C-benzene, the total percentage of 14C-derived benzene RA eliminated in urine within 72 hr was not significantly different with age, but elimination at early time points (4, 6, and 8 hr) was significantly decreased in 18- vs. 3-month mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

An interaction of benzene metabolites reproduces the myelotoxicity observed with benzene exposure

Benzene-induced myelotoxicity can be reproduced by the coadministration of two principal metabolites, phenol and hydroquinone. Coadministration of phenol (75 mg/kg) and hydroquinone (25-75 mg/kg) twice daily to B6C3F1 mice for 12 days resulted in a significant loss in bone marrow cellularity in a manner exhibiting a dose-response. One explanation for this potentiation is that phenol stimulates the peroxidase-dependent metabolism of hydroquinone. Addition of phenol to incubations containing horseradish peroxidase, H2O2, and hydroquinone resulted in a stimulation of both hydroquinone removal and benzoquinone formation. Stimulation occurred with phenol as low as 100 microM and with very low concentrations of horseradish peroxidase. When boiled rat liver protein was added to identical incubations containing [14C]hydroquinone, the level of radioactivity recovered as protein bound increased by 37% when phenol was added. Similar results were observed when [14C]hydroquinone was incubated in the presence of activated human leukocytes. Hydroquinone binding was increased by approximately 70% in the presence of phenol. Phenol-induced stimulation of hydroquinone metabolism and benzoquinone formation represents a likely explanation for the bone marrow suppression associated with benzene toxicity.     

Species comparison of hepatic and pulmonary metabolism of benzene

Benzene is an occupational hazard and environmental toxicant found in cigarette smoke, gasoline, and the chemical industry. The major health concern associated with benzene exposure is leukemia. Studies using microsomal preparations from human, mouse, rabbit, and rat to determine species differences in the metabolism of benzene to phenol, hydroquinone and catechol, indicate that the rat is most similar, both quantitatively and qualitatively, to the human in pulmonary microsomal metabolism of benzene. With hepatic microsomes, rat is most similar to human in metabolite formation at the two lower concentrations examined (24 and 200 microM), while at the two higher concentrations (700 and 1000 microM) mouse is most similar in phenol formation. In all species, the enzyme system responsible for benzene metabolism approached saturation in hepatic microsomes but not in pulmonary microsomes. In pulmonary microsomes from mouse, rat, and human, phenol appeared to competitively inhibit benzene metabolism resulting in a greater proportion of phenol being converted to hydroquinone when the benzene concentration increased. The opposite effect was seen in hepatic microsomes. These findings support the hypothesis that the lung plays an important role in benzene metabolism, and therefore, toxicity.     

Stereo- and regioselectivity account for the diversity of dehydroepiandrosterone (DHEA) metabolites produced by liver microsomal cytochromes P450.

The purpose of this study was to quantify the oxidative metabolism of dehydroepiandrosterone (3beta-hydroxy-androst-5-ene-17-one; DHEA) by liver microsomal fractions from various species and identify the cytochrome P450 (P450) enzymes responsible for production of individual hydroxylated DHEA metabolites. A gas chromatography-mass spectrometry method was developed for identification and quantification of DHEA metabolites. 7alpha-Hydroxy-DHEA was the major oxidative metabolite formed by rat (4.6 nmol/min/mg), hamster (7.4 nmol/min/mg), and pig (0.70 nmol/min/mg) liver microsomal fractions. 16alpha-Hydroxy-DHEA was the next most prevalent metabolite formed by rat (2.6 nmol/min/mg), hamster (0.26 nmol/min/mg), and pig (0.16 nmol/min/mg). Several unidentified metabolites were formed by hamster liver microsomes, and androstenedione was produced only by pig microsomes. Liver microsomal fractions from one human demonstrated that DHEA was oxidatively metabolized at a total rate of 7.8 nmol/min/mg, forming 7alpha-hydroxy-DHEA, 16alpha-hydroxy-DHEA, and a previously unidentified hydroxylated metabolite, 7beta-hydroxy-DHEA. Other human microsomal fractions exhibited much lower rates of metabolism, but with similar metabolite profiles. Recombinant P450s were used to identify the cytochrome P450s responsible for DHEA metabolism in the rat and human. CYP3A4 and CYP3A5 were the cytochromes P450 responsible for production of 7alpha-hydroxy-DHEA, 7beta-hydroxy-DHEA, and 16alpha-hydroxy-DHEA in adult liver microsomes, whereas the fetal/neonatal form CYP3A7 produced 16alpha-hydroxy and 7beta-hydroxy-DHEA. CYP3A23 uniquely formed 7alpha-hydroxy-DHEA, whereas other P450s, CYP2B1, CYP2C11, and CYP2D1, were responsible for 16alpha-hydroxy-DHEA metabolite production in rat liver microsomal fractions. These results indicate that the stereo- and regioselectivity of hydroxylation by different P450s account for the diverse DHEA metabolites formed among various species.     

Identification of N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine as a urinary metabolite of benzene, phenol, and hydroquinone

The metabolite 2-(S-glutathionyl)hydroquinone is formed when a microsomal incubation mixture containing either benzene or phenol is supplemented with glutathione. This metabolite is derived from the conjugation of benzoquinone, an oxidation product of hydroquinone. However, neither the glutathione conjugate or its mercapturate, N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine, have been identified as metabolites resulting from in vivo metabolism of benzene, phenol, or hydroquinone. To determine if a hydroxylated mercapturate is produced in vivo, we treated male Sprague-Dawley rats with either benzene (600 mg/kg), phenol (75 mg/kg), or hydroquinone (75 mg/kg) and collected the urine for 24 hr. HPLC coupled with electrochemical detection confirmed the presence of a metabolite that was chromatographically and electrochemically identical to N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine. The metabolite was isolated from the urine samples and treated with diazomethane to form the N-acetyl-S-(2,5-dimethoxyphenyl)-L-cysteine methyl ester derivative. The mass spectra obtained from these samples were identical to that of an authentic sample of the derivative. The results of these experiments indicate that benzene, phenol, and hydroquinone are metabolized in vivo to benzoquinone and excreted as the mercapturate, N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine.     

Metabolism of 3-methylindole in human tissues.

Bioactivation of 3-methylindole (3MI), a highly selective pneumotoxin in goats, was investigated in human lung and liver tissues in order to provide information about the susceptibility of humans to 3MI toxicity. Human lung microsomes were prepared from eight organ transplantation donors and liver microsomes from one of the donors were utilized. The 3MI turnover rate with human lung microsomes was 0.23 +/- 0.06 nmol/mg/min, which was lower than the rate with the human liver microsomes (7.40 nmol/mg/min). The activities were NADPH dependent and inhibited by 1-aminobenzotriazole, a potent cytochrome P-450 suicide substrate inhibitor. Covalent binding of 3MI reactive intermediates to human tissues was determined by incubation of 14C-3MI and NADPH with human lung and liver microsomal proteins. Although human lung microsomes displayed measurable covalent binding activity (2.74 +/- 2.57 pmol/mg/min), the magnitude of this reaction was only 4% as large as that seen with human liver microsomes (62.02 pmol/mg/min). However, the covalent binding was protein dependent and also was inhibited by 1-aminobenzotriazole. Therefore, the bioactivation of 3MI to covalently binding intermediates is catalyzed by cytochrome P-450 in human pulmonary tissues. These activities were compared to those activities measured with tissues from goats. Proteins from goat and human pulmonary and hepatic microsomal incubations were incubated with radioactive 3MI, and radioactive proteins were analyzed by SDS-PAGE and HPLC and visualized by autoradiography and radiochromatography, respectively. The results showed that a 57-kDa protein was clearly the most prominently alkylated target associated with 3MI reactive intermediates.(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)     

Metabolism of [14C]phenol by sheep, pig and rat.

1. Following an oral dose of [14C]phenol (12.5 or 25 mg/kg) to sheep, pig and rat, urinary elimination of radioactivity was rapid, 80-90% dose being excreted in the first 8 h. 2. In anaesthetized, ureter-cannulated rats, 70-80% of an intraduodenal dose was eliminated in 2 h; 2% dose was excreted as phenol conjugates in the urine within 10 min. 3. The major urinary metabolites from phenol (25 mg/kg) were phenylglucuronide and phenylsulphate. In the sheep, pig and rat, the glucuronide accounted for 49%, 83% and 42% respectively, of the total urinary metabolites and sulphate accounted for 32%, 1% and 55%. Conjugates of quinol were minor urinary metabolites (less than 7%) in all three species. 4. In sheep some 12% of the urinary metabolites was conjugated with phosphate; this metabolite was not found in rat or pig.     

Influence of strain differences in mice on the metabolism and toxicity of benzene

Chronic benzene exposure results in a progressive depression of bone marrow function and is thought to be caused by a metabolite of benzene (Snyder and Kocsis, 1975; Goldstein and Laskin, 1977). Several reports concerning differences in xenobiotic metabolism and toxicity among inbred strains of mice prompted us to study benzene metabolism and toxicity in C57BL/6 and DBA/2 mice. DBA/2 mice were more susceptible to benzene than C57BL/6 mice. No differences in the total amount of urinary benzene metabolites produced were found between the strains; however, differences in the relative amounts of specific metabolites were noted. DBA/2 mice produced more phenylglucuronide but less ethereal sulfate conjugates than C57BL/6 mice. Hydrolysis of the urinary conjugates revealed that DBA/2 mice excreted more phenol, but less hydroquinone than C57BL/6 mice. Multiple dose studies revealed that the more resistant C57BL/6 mice contained less water soluble benzene metabolites in bone marrow, liver, kidney, blood, spleen, and lung than DBA/2 mice. C57BL/6 mice also contained less covalently bound metabolites in bone marrow, blood, spleen, and muscle than DBA/2 mice following multiple doses of benzene. V_max values for UDPGA utilization in C57BL/6 mice were almost six times the V_max values observed for DBA/2 mice. Furthermore, V_max values for phenylsulfate formation in C57BL/6 mice were three times the V_max values for DBA/2 mice. It was concluded that the difference in susceptibility to benzene between C57BL/6 and DBA/2 mice was not the result of a single factor, buth rather, the sum total of a number of metabolic events.     

Glutathione adduct formation with microsomally activated metabolites of the pulmonary alkylating and cytotoxic agent, 3-methylindole

Incubations with goat lung and liver microsomes were conducted to trap with exogenous glutathione (GSH) the electrophilic intermediate produced via cytochrome P-450-dependent metabolic activation of 3-methylindole (3MI). Microsomal incubation mixtures with [14C]3MI, a NADPH-generating system, and [3H]GSH produced a dual-labeled adduct which was isolated by reverse-phase high-performance liquid chromatography. Reactive 3MI intermediates were also trapped with cysteine. Adduct formation increased in proportion to the concentration of either thiol. Covalent binding of activated 3MI metabolites to microsomal protein was inversely related to adduct production. There were both qualitative and quantitative differences in the formation of GSH adducts by lung and liver microsomes. In the presence of 2 mM GSH, the adduct was produced at a rate of 1.8 nmol/mg protein/min by lung microsomes but only at 0.1 nmol/mg protein/min by hepatic microsomes. The addition of cytosolic fractions containing glutathione S-transferase activity increased GSH adduct formation by approximately 30%. These results support the view that electrophilic 3MI intermediates are trapped by conjugation with GSH, and that organ-selective toxicity is primarily due to much faster rates of cytochrome P-450 oxidation of 3MI in the lung than in the liver.     

Phenol-induced stimulation of hydroquinone bioactivation in mouse bone marrow in vivo: possible implications in benzene myelotoxicity

The coadministration of phenol and hydroquinone has been shown to produce myelotoxicity in mice similar to that observed following benzene exposure. One explanation of this phenomenon may be that phenol enhances the peroxidase-dependent metabolic activation of hydroquinone in the mouse bone marrow. Here we report that radiolabeled [14C]hydroquinone and [14C]phenol bind covalently to tissue macromolecules of blood, bone marrow, liver and kidney, when administered intraperitoneally to the mouse in vivo. Substantially more radiolabeled hydroquinone was covalently bound 18 h after administration as compared with that bound after 4 h. Phenol, when administered together with [14C]hydroquinone, significantly stimulated the covalent binding of [14C]hydroquinone oxidation products to blood (P less than 0.001) and bone marrow (P less than 0.05) macromolecules, but had no significant effect on covalent binding of [14C]hydroquinone oxidation products to liver and kidney macromolecules (P greater than 0.05). Catechol, on the other hand, had no effect on the binding of [14C]hydroquinone oxidation products in either bone marrow, kidney or liver (P greater than 0.05). When hydroquinone was administered together with [14C]phenol, a stimulation of the covalent binding of phenol oxidation products to bone marrow macromolecules also occurred (P less than 0.05). In addition, hydroquinone co-administration increased the covalent binding of [14C]phenol oxidation products in kidney and blood (P less than 0.05), but significantly decreased the covalent binding in liver (P less than 0.05). These results suggest that altered pharmacokinetics play a major role in the hydroquinone-dependent stimulation of covalent binding of [14C]phenol oxidation products to extrahepatic tissue macromolecules in vivo. The mechanism underlying the phenol-induced stimulation of binding of [14C]hydroquinone by phenol in blood and bone marrow remains unclear, but stimulation of peroxidase-mediated hydroquinone metabolism may be responsible. The latter may therefore play an important role in benzene-induced myelotoxicity.     

Effect of exposure concentration, exposure rate, and route of administration on metabolism of benzene by F344 rats and B6C3F1 mice

To determine the effect of exposure concentration and the route of administration on benzene metabolism, male F344/N rats and B6C3F1 mice were orally exposed to 1, 10, and 200 mg benzene/kg, and by inhalation for 6 hr to 5, 50, and 600 ppm benzene vapor. The effect of different exposure rates on the metabolism of benzene was determined by exposing rodents over different time intervals to the same total amount of benzene [constant concentration X time factor (C X T) = 300 ppm.hr]. Water-soluble metabolites constituted greater than 90% of the metabolite dose to the tissues and were used as a measure of the metabolism of benzene via different pathways. Water-soluble metabolites were measured in the blood, urine, liver, lung, and bone marrow from animals killed following oral exposures and during and following inhalation exposures. The total "dose" to the tissue of individual metabolites was determined by the area under the curve (AUC). The results indicated a shift in metabolism from putative toxification pathways to detoxification pathways as the exposure concentration or oral dose increased. In mice, hydroquinone glucuronide and muconic acid (markers of toxification metabolic pathways) represented a greater percentage of the administered dose at low doses than at high doses. At high doses, phenylglucuronide and prephenylmercapturic acid (detoxification products) increased as a percentage of the administered dose. This same metabolic shift was observed in rats, except that hydroquinone glucuronide was a minor metabolite of benzene at all concentrations. The AUC of phenylsulfate (detoxification pathway) was proportional to the exposure concentration in both species. Within the range of C X T factors studied, the rate of the inhalation exposure to benzene did not affect the AUC of metabolites in tissues of rats; however, a high dose rate (600 ppm 0.5 hr) in mice caused a shift in metabolism to phenyl conjugates. The comparison of oral and 6-hr inhalation exposures indicated that, in terms of metabolite dose to tissues, there is no simple relationship between these two routes of administration. An oral dose and an inhalation exposure concentration which produce an equal dose of one metabolite produce very different doses of another metabolite. These studies demonstrated a species difference in benzene metabolism, as well as a metabolic shift in benzene metabolic pathways as the exposure concentration was increased.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metabolism of the beta-carbolines, harmine and harmol, by liver microsomes from phenobarbitone- or 3-methylcholanthrene-treated mice. Identification and quantitation of two novel harmine metabolites

This study extends an investigation of the metabolism of the beta-carbolines, harmine and harmol, by untreated, phenobarbitone-induced, or 3-methylcholanthrene (MC)-induced mouse liver microsomes to identify two MC-inducible metabolites of harmine and to quantitate their rates of formation using 3H-labeled substrate. An HPLC system was devised to separate harmine and its metabolites. The major metabolite with MC-induced microsomes was identified by mass spectroscopy and by NMR to be 6-hydroxy-7-methoxyharman and was produced at an initial reaction rate of 11 nmol/min/mg of microsomal protein (27-fold induction). The other novel metabolite, 3- or 4-hydroxy-7-methoxyharman (the position of the hydroxyl group could not be definitively assigned by NMR) was produced at an initial reaction rate of 3.8 nmol/min/mg of microsomal protein (32-fold induction) which was similar to the rate of formation of the other metabolite, harmol, determined previously. All three metabolites were further metabolized to unidentified metabolites. Protein binding of [3H]harmine and [3H]harmol was measured and shown to be metabolism dependent. It was also noted that the alkali conditions used for optimal extraction stimulated the protein binding.     

Metabolism of the Flame Retardant Plasticizer Tris(2-chloroethyl)phosphate by HUman and Rat Liver Preparations

Metabolism of the Flame Retardant Plasticizer Tris(2-chloroethyl)phosphateby Human and Rat Liver Preparations. CHAPMAN, D. E., MICHENER,S. R., AND POWIS, G. (1991) Fundam. Appl Toxrcol. 17, 2 15–224.Previous studies indicate that tris(2-chloroethyl)phosphate(TCP) preferentially produces hippocampal brain lesions in femaleversus male rats, and the expression of these lesions is inverselyrelated to the in vivo rate of TCP metabolism. In the presentstudies. TCP (0.17 mM in all incubations) was metabolized invivo by liver slices and microsomes from human and Fischer 344Nrat Liver to bis(2-chloroethy) hydrogen phosphate (BCP), 2-chloroethanol(CE), and three unidentified metabolites. The rate of TCP metabolismby male rat liver microsomes and liver slices was 0.049 nmol/min/mgprotein and 2.53 nmol/min/g liver, respectively. TCP metabolismby male rat liver microsomes was inhibited by 10 µM diisopropylfluorophosphate, 10 µM paraoxon and carbon monoxide. TCPdid not appear to be metabolized by female rat liver microsomes,but female rat liver slices metablized TCP at a rate of 1.51 nmol/mln/g liver. TCP was metabolized by male and female ratplasma at a rate of 0.156 and 0.169 nmol/ml plasma respectively.TCP was metabolized by male and female human liver microsomesat a rate of 0.027 and 0.031 nmol/mln/mg protein, respectively.TCP was metabolized by male and female human liver slices ata rate of 1.37 and 1.82 nmol/min/g liver, respectively. BCPand CE were the major metabolites formed in all studies, exceptfor liver slices and microsomes from two human male subjectsin which an unidentified metabolite constituted 29 to 38% ofthe total TCP metabolism. TCP was not metabolized by plasmaor whole blood from male or female human subjects. These resultssupport the previously reported sex-specific difference in TCPmetabolism by male and female Fischer 344N rats. However, nosex-specific difference In rates of TCP metabolism by male andfemale human liver microsomes or slices was observed.     

Hepatotoxicity of ketoconazole in Sprague-Dawley rats: glutathione depletion,flavin-containing monooxygenases-mediated bioactivation and hepatic covalent binding

1. This study has examined ketoconazole (KT)-induced hepatotoxicity in vivo and in vitro, using male Sprague-Dawley rats with [3 H]KT (1.5 µCi?mg ? 1) at 40 and 90?mg KT kg ? 1 doses. Blood and liver samples were collected from 0 to 24?h for alanine aminotransaminase (ALT), glutathione (GSH) and covalent binding analyses. 2. Covalent binding occurred as early as 0.5?h, peaked at 2?h (0.026 ± 0.01 nmol KT?mg ? 1 protein) and 8?h (0.088 ± 0.04 nmol KT?mg ? 1 protein) for 40 and 90?mg KT kg ? 1 doses, respectively. ALT levels increased at 0.5?h for the 40 and 90?mg KT kg ? 1 doses (44.3 and 56.4 U ml ? 1, respectively) relative to control, 22.7 U ml ? 1. At 24?h, the 90?mg KT kg ? 1 dose reduced hepatic GSH levels from 9.92 ± 1.1 to 4.76 ± 0.3 nmol GSH?mg ? 1 protein. 3. The role of the flavin-containing monooxygenases (FMO) utilized Sprague-Dawley microsomes with 1, 10 and 100 µM [3 H]KT. Maximum covalent binding occurring at 100 µM KT. Heat inactivation of microsomal FMO significantly decreased covalent binding by 75%, whereas 1 mM GSH significantly reduced covalent binding by 65%. 4. Thus, KT-induced hepatotoxicity is dose- and time-dependent and appears to be FMO mediated, in part, to metabolites that may react with protein and, possibly, GSH.     

Hepatotoxicity of ketoconazole in Sprague-Dawley rats: glutathione depletion,flavin-containing monooxygenases-mediated bioactivation and hepatic covalent binding

1. This study has examined ketoconazole (KT)-induced hepatotoxicity in vivo and in vitro, using male Sprague-Dawley rats with [(3)H]KT (1.5 micro Ci mg(-1)) at 40 and 90 mg KT kg(-1) doses. Blood and liver samples were collected from 0 to 24 h for alanine aminotransaminase (ALT), glutathione (GSH) and covalent binding analyses. 2. Covalent binding occurred as early as 0.5 h, peaked at 2 h (0.026 +/- 0.01 nmol KT mg(-1) protein) and 8 h (0.088 +/- 0.04 nmol KT mg(-1) protein) for 40 and 90 mg KT kg(-1) doses, respectively. ALT levels increased at 0.5 h for the 40 and 90 mg KT kg(-1) doses (44.3 and 56.4 U ml(-1), respectively) relative to control, 22.7 U ml(-1). At 24 h, the 90 mg KT kg(-1) dose reduced hepatic GSH levels from 9.92 +/- 1.1 to 4.76 +/- 0.3 nmol GSH mg(-1) protein. 3. The role of the flavin-containing monooxygenases (FMO) utilized Sprague-Dawley microsomes with 1, 10 and 100 micro M [(3)H]KT. Maximum covalent binding occurring at 100 micro M KT. Heat inactivation of microsomal FMO significantly decreased covalent binding by 75%, whereas 1 mM GSH significantly reduced covalent binding by 65%. 4. Thus, KT-induced hepatotoxicity is dose- and time-dependent and appears to be FMO mediated, in part, to metabolites that may react with protein and, possibly, GSH.