Metabolism by rat liver cytosol of illudin S, a toxic substance of Lampteromyces japonicus. II. Characterization of illudin S-metabolizing enzyme.

1. Enzyme systems responsible for formation of cyclopropane ring-cleavage metabolites (M1 and M2) of illudin S in rat liver were characterized. 2. The enzymes were localized in the cytosol fraction and utilized NADPH alone as electron donor; they were not affected by oxygen and had low pH optima. 3. Formation of metabolites M1 and M2 was inhibited completely by dicumarol (10(-4) M), an inhibitor of DT-diaphorase. 4. Menadione (10(-4) M) and quercetin (10(-4) M) both inhibited formation of M1 and M2 by 35% and 15%, respectively, but quinacrine, barbital, pyrazole and p-chloromercuribenzoic acid had no significant effect. 5. Results show that the enzyme systems may differ from DT-diaphorase, aldehyde oxidase, xanthine oxidase, ketone reductase, aldose reductase, aldehyde reductase and alcohol dehydrogenase, known cytosolic enzymes responsible for xenobiotic metabolism.     

Metabolism of illudin S, a toxic principle of Lampteromyces japonicus, by rat liver. I. Isolation and identification of cyclopropane ring-cleavage metabolites

1. Illudin S, a toxic principle of the basidiomycete Lampteromyces japonicus, was incubated with rat liver 9000 g supernatant and its metabolites studied. 2. Two metabolites, M1 and M2, were isolated and identified as cyclopropane ring-cleavage compounds by n.m.r., i.r. and mass spectral analyses. Moreover, M2 contained a chlorine atom. 3. On the basis of detailed analyses of the 2D n.m.r. spectra and differential nuclear Overhauser effect experiments, the previous assignments of the cyclopropane carbons of illudin S were revised.     

Effect of toluene and xylenes on liver glutathione and their urinary excretion as mercapturic acids in the rat

Administration of toluene and xylenes to rats caused a decrease in liver glutathione concentration. The effect was most pronounced after the administration of o-xylene. 26% of the initial glutathione level was found three hours after treatment with o-xylene (4.0 mmoles/kg).No in vitro conjugation of o-xylene with glutathione was observed, neither spontaneously nor in the presence of 105,000 g supernatant from rat liver homogenate, containing glutathione S-transferases. Thus, a metabolite of o-xylene, which is not formed during incubation with 105,000 g supernatant, reacts with glutathione.A thioether was isolated from urine of rats given o-xylene; the compound was identified as o-methylbenzyl mercapturic acid by GC-MS and NMR. Chromatographic evidence was found for the presence of benzyl mercapturic acid in the urine of toluene-treated rats. The amounts of mercapturic acids excreted in the urine after administration of toluene, p-xylene, m-xylene, and o-xylene were 0.4–0.7, 0.6, 1.3, and 10–21% of the dose, respectively.These results demonstrate the involvement of a thusfar unknown pathway in the biotransformation of toluene and xylenes.     

Identification of urinary mercapturic acids formed from acrylate,methacrylate and crotonate in the rat

1. After administration to rats of methyl acrylate (I), methyl methacrylate (II) and methyl crotonate (III), urinary mercapturic acids were isolated and identified as the dicarboxylic acids N-acetyl-S-(2-carboxyethyl)cysteine (IV, R = H), N-acetyl-S-(2-carboxypropyl)cysteine (V, R = H) and N-acetyl-S-(1-methyl-2-carboxyethyl)cysteine (VI, R = H) and for a minor part as their monomethyl esters IV (R = CH₃), V (R = CH₃) and VI (R = CH₃).

2. After a single dose of the acrylates (I), (II) and (III) (0·14mmol/kg), the excretion of the thioethers amounted to 6·6 ± 0·6, 0·0, and 2·0 ± 0·6% dose respectively.

3. After 18?h previous administration of the carboxylesterase inhibitor tri-o-tolyl phosphate (0·34mmol/kg) the excretion of the thioethers amounted to 40·6 ± 2·1, 11·0 ± 3·3, and 16·0 ± 2·0% dose.

4. For methyl acrylate (I) the ratio of the excreted dicarboxylic acid and monomethyl ester was 20:1. After previous administration of tri-o-tolyl phosphate this ratio was 1 : 2.     

Urinary excretion of mercapturic acids in chimpanzees and rats

The urinary excretion of mercapturic acids has been considered as an indicator for human exposure to environmental chemicals. To evaluate this concept, the excretion of urinary mercapturic acids was determined in chimpanzees and rats after the oral administration of single doses of naphthalene and diethylmaleate. The excretion rate of endogenous thioethers in the urine of untreated chimpanzees and rats was 18.0 ± 1.1 and 94.4 ± 2.8 μmol/kg/24 hr respectively. The value for man was nearly the same as found in chimpanzees. After the administration of naphthalene (30, 75, and 200 mg/kg) a dose-dependent increase of the excretion rate of urinary mercapturic acids up to 408 μmol/kg/24 hr was observed in the rat. In the chimpanzees the same treatment failed to increase the urinary thioether concentrations. The administration of diethylmaleate (30, 75, and 200 mg/kg) led to a dose-dependent increase in the excretion of urinary mercapturic acids in both species. In rats this increase was about twice that of chimpanzees. The results suggest that the chimpanzee is a relevant model for man to study the urinary excretion of mercapturic acids deriving from electrophilic compounds, whereas the rat is not. Experiments with [¹⁴C]-naphthalene indicate that the species differences observed are due to differences in the glutathione conjugation.     

Metabolism and urinary excretion of esmolol in humans

The urinary excretion patterns of esmolol, a short-acting beta blocker, and its major metabolite were investigated in eight healthy men after intravenous infusion of 50, 100, 200, and 300 micrograms/kg/min of esmolol for six hours and 150 micrograms/kg/min for 24 hours. Esmolol and the metabolite concentrations in urine were determined by high-performance liquid chromatography. The mean urinary recoveries of the unchanged drug were 0.64%, 0.67%, 0.69%, 0.77%, and 0.98% after the 50, 100, 150, 200, and 300 micrograms/kg/min dose, respectively. Recovery of the metabolite was independent of dose, and the overall mean recovery accounted for 73% of administered dose. The results of this study indicate that esmolol is extensively metabolized, and the extent of the metabolism is not dose related in the dosage range used. The renal route plays a very minor role in the elimination of the drug but is important for the elimination of the metabolite.     

Metabolism and urinary excretion of benzhexol in humans

1. Benzhexol and three of its metabolites excreted in urine in man have been investigated by g.l.c.--mass spectrometry. 2. Three isomeric hydroxylated metabolites were identified as the 1-(hydroxycyclohexyl)-1-phenyl-3-piperidinopropan-1-ols. 3. The amounts of benzhexol and its identified metabolites have been semiquantitatively determined after a single oral dose in two healthy adults. Approx. 56% of the dose was excreted as the hydroxylated metabolites. The levels of benzhexol excreted were too low to be measured by the techniques used.     

A note on individual differences in the urinary excretion of optical enantiomers of styrene metabolities and of styrene-derived mercapturic acids in humans

 Urine samples from 20 male workers in the polyester industry exposed by inhalation to styrene concentrations ranging from 29 to 41 ppm were investigated. Excretion products of styrene metabolism, mandelic acid and mercapturic acids, were purified from the urine over an extraction column packed with Porapak Q, with subsequent ether elution. The optical enantiomers R- and S-mandelic acid were then determined by thin layer chromatography (TLC) using chiral plate material and selective staining with vanadium pentoxide. Quantitative analysis of these compounds was performed using commercial reference substances. Styrene-specific mercapturic acids were analyzed by a modified TLC method, using synthesized reference substances. The concentration of racemic mandelic acid in the individual urine samples ranged from 80 to 1610 mg/l, and the ratio of the R- and S- enantiomers ranged from 0.7 to 2.2. These individual variations are not explained by differences in individual styrene exposure levels, or by differences in the concentration of the urine samples (in relation to creatinine excretion). Styrene-specific mercapturic acids were detected in the urine of only 1 of the 20 workers, at a concentration much lower than expected from previous investigations by others in humans and laboratory animals, in which less specific analytical methods had been used. The results point to marked interindividual differences in metabolism of styrene, probably related to enzyme polymorphisms. Received: 15 June 1994/Accepted: 3 November 1994     

Acrylamide exposure via the diet: influence of fasting on urinary mercapturic acid metabolite excretion in humans

Acrylamide (AA) is carcinogenic in animals and classified by the International Agency for Research on Cancer as probably carcinogenic in humans. Regarding the AA contents of food the diet significantly contributes to the overall AA burden of the general population. However, it is unclear to which degree the diet, apart from smoking, contributes to the internal AA exposure. Therefore the influence of an AA-free diet on the excretion of urinary mercapturic acid metabolites derived from AA in three healthy volunteers fasting for 48 h was examined. Urinary AA mercapturic acid metabolites were considerably reduced after 48 h of fasting. The levels were even well below the median level in non-smokers. This confirms that the diet is the main source of environmental AA exposure in humans, apart from smoking. Other possible AA sources could be of minor quantitative importance only.     

Metabolism and urinary excretion of a new quinoline anticancer drug, TAS-103, in humans

1. TAS-103, a novel condensed quinoline derivative, has been developed as an anticancer drug targeting topoisomerases I and II. 2. The purpose of the present study was to characterize the metabolism and urinary excretion of TAS-103 after the intravenous infusion of a single dose to patients in Phase I clinical trials. 3. Five metabolites were detected using high-performance liquid chromatography (HPLC) photodiode array and a precursor scan by liquid chromatography mass spectrometry mass spectrometry (LC/MS/MS). 4. Structures of the five metabolites were determined using the results of enzymatic hydrolysis and the analysis of production mass spectra obtained by LC/MS/MS, and by comparing HPLC retention times and UV, mass and production mass spectra of authentic standards. 5. The metabolites were identified as demethyl-TAS-103 glucuronide (DM-TAS-103-G), TAS-103 glucuronide (TAS-103-G), TAS-103 glucuronide N-oxide (NO-TAS-103-G), demethyl-TAS-103 (DM-TAS-103) and TAS-103 N-oxide (NO-TAS-103). 6. The mean total amount of TAS-103 and TAS-103-G in urine was only 6.03% of the dose, suggesting that urine is not the main elimination route. TAS-103 was extensively metabolized, and a small percentage of the parent drug (0.41%) was found in urine.     

Metabolism and urinary excretion of a new quinoline anticancer drug,TAS-103, in humans

1. TAS-103, a novel condensed quinoline derivative, has been developed as an anticancer drug targeting topoisomerases I and II. 2. The purpose of the present study was to characterize the metabolism and urinary excretion of TAS-103 after the intravenous infusion of a single dose to patients in Phase I clinical trials. 3. Five metabolites were detected using high-performance liquid chromatography (HPLC) photodiode array and a precursor scan by liquid chromatography mass spectrometry mass spectrometry (LC/MS/MS). 4. Structures of the five metabolites were determined using the results of enzymatic hydrolysis and the analysis of production mass spectra obtained by LC/MS/MS, and by comparing HPLC retention times and UV, mass and production mass spectra of authentic standards. 5. The metabolites were identified as demethyl-TAS-103 glucuronide (DM-TAS103-G), TAS-103 glucuronide (TAS-103-G), TAS-103 glucuronide N -oxide (NO-TAS103-G), demethyl-TAS-103 (DM-TAS-103) and TAS-103 N -oxide (NO-TAS-103). 6. The mean total amount of TAS-103 and TAS-103-G in urine was only 6.03% of the dose, suggesting that urine is not the main elimination route. TAS-103 was extensively metabolized, and a small percentage of the parent drug (0.41%) was found in urine.     

Metabolism and urinary excretion of chlormethiazole in humans.

1. Chlormethiazole and five of its metabolites excreted in urine in man have been investigated by g.l.c.-mass spectrometry. 2. Four metabolites have been identified by comparison with authentic compounds as 5-acetyl-4-methylthiazole, 5-(1-hydroxyethyl)-4-methylthiazole, 5-(2-hydroxyethyl)-4-methylthiazole and 4-methyl-5-thiazoleacetic acid; 4-methyl-5-thiazoleacetaldehyde is proposed for the other metabolite. 3. The amounts of chlormethiazole and its identified metabolites excreted in urine have been quantitatively determined after a single oral dose in three healthy adults. Approximately 16% of the dose was excreted as chlormethiazole, 5-acetyl-4-methylthiazole, 5-(1-hydroxyethyx)-4-methylthiazole and 4-methyl-5-thiazoleacetic acid.     

Metabolism and excretion of imrecoxib in rat

The metabolism and excretion of imrecoxib, a novel and moderately selective cyclooxygenase-II inhibitor, were investigated in rat. The structures of metabolites were identified by mass spectrometry (MSn) and nuclear magnetic resonance. Metabolic profiles of imrecoxib in urine, bile and faeces were obtained by HPLC and LC/MSn, and cumulative excretion was determined by LC/MSn. Imrecoxib was extensively metabolized in rat after intravenous administration, with less than 2% of the dose excreted as parent drug in either urine or faeces. The major metabolic pathway was that the 4'-methyl group of imrecoxib was first oxidized to the 4'-hydroxymethyl metabolite (M4), followed by additional oxidation to 4'-carboxylic acid metabolite (M2). The dihydroxylated metabolite, 4'-hydroxymethyl-5-hydroxyl imrecoxib (M3), was further oxidized to 4'-hydroxymethyl-5-carbonyl metabolite (M5), and glucuronide conjugates of M2-4 were formed. After intravenous (5 mg kg-1) administration, the majority of the dose was recovered in the faeces. The dose was primarily excreted as the carboxylic acid metabolite in addition to the 4'-hydroxymethyl metabolite. The carboxylic acid metabolite was mainly excreted in faeces, while the 4'-hydroxymethyl metabolite was mainly excreted in urine.     

Simultaneous determination of urinary mandelic, phenylglyoxylic, and mercapturic acids of styrene by high-performance liquid chromatography

We have developed a relatively simple and reproducible HPLC procedure for the determination of urinary metabolites of styrene. Urine samples (pH 2) are extracted with ethyl acetate, the organic layer is evaporated to dryness, and the residues are dissolved in water-methanol (1:1). Samples are analyzed by HPLC with a C18 reversed-phase column and a water (pH 6, 5mM tetrabutylammonium dihydrogen phosphate)-acetonitrile gradient and ultraviolet detection at 225 nm. Using this method, it is possible to determine simultaneously mandelic and phenylglyoxylic acids, N-acetyl-S-(1-phenyl-2-hydroxyethyl)-L-cysteine (M1), and N-acetyl-S-(2-phenyl-2-hydroxyethyl)-L-cysteine (M2). The internal standard is p-hydroxybenzoic acid. Additional validation data are obtained by analysis of urine samples obtained from rats treated with a wide range of styrene doses.     

Biotransformation of trichloroethene: dose-dependent excretion of 2,2,2-trichloro-metabolites and mercapturic acids in rats and humans after inhalation

 Chronic bioassays with trichloroethene (TRI) demonstrated carcinogenicity in mice (hepatocellular carcinomas) and rats (renal tubular cell adenomas and carcinomas). The chronic toxicity and carcinogenicity is due to bioactivation reactions. TRI is metabolized by cytochrome P450 and by conjugation with glutathione. Glutathione conjugation results in S-(dichlorovinyl) glutathione (DCVG) and is presumed to be the initial biotransformation step resulting in the formation of nephrotoxic metabolites. Enzymes of the mercapturic acid pathway cleave DCVG to the corresponding cysteine S-conjugate, which is, after translocation to the kidney, cleaved by renal cysteine S-conjugate β-lyase to the electrophile chlorothioketene. After N-acetylation, cysteine S-conjugates are also excreted as mercapturic acids in urine. The object of this study was the dose-dependent quantification of the two isomers of N-acetyl-S-(dichlorovinyl)-L-cysteine, trichloroethanol and trichloroacetic acid, as markers for the glutathione- and cytochrome P450-mediated metabolism, respectively, in the urine of humans and rats after exposure to TRI. Three male volunteers and four rats were exposed to 40, 80 and 160 ppm TRI for 6 h. A dose-dependent increase in the excretion of trichloroacetic acid, trichloroethanol and N-acetyl-S-(dichlorovinyl)-L-cysteine after exposure to TRI was found both in humans and rats. Amounts of 3100 μmol trichloroacetic acid+trichloroethanol and 0.45 μmol mercapturic acids were excreted in urine of humans over 48 h after exposure to 160 ppm TRI. The ratio of trichloroacetic acid+trichloroethanol/mercapturic acid excretion was comparable in rats and humans. A slow rate of elimination with urine of N-acetyl-S-(dichlorovinyl)-L-cysteine was observed both in humans and in rats. However, the ratio of the two isomers of N-acetyl-S-(dichlorovinyl)-L-cysteine was different in man and rat. The results confirm the finding of the urinary excretion of mercapturic acids in humans after TRI exposure and suggest the formation of reactive intermediates in the metabolism of TRI after bioactivation by glutathione also in humans. Received: 22 June 1995 / Accepted: 5 October 1995     

Isosorbide, isomannide and isoidide dinitrate: urinary excretion in the rat

In rats given the dinitrates of isosorbide, isomannide and isoidide orally, urinary excretion products were studied by gas chromatography and thin layer chromatography. The unchanged dinitrates are excreted in small amounts; the different mononitrates and denitrated alcohols are also present; the mononitrates are conjugated to a large extent. After administration of isosorbide dinitrate, isoidide mononitrate and isoidide are excreted in the urine, and after isomannide dinitrate administration, 5-isosorbide mononitrate and isosorbide can be found. This inversion of configuration from the endo- to the exo-position could be due to the formation of a carbonyl intermediate or to a backside displacement mechanism.     

Genetic deficiency of human class mu glutathione S-transferase isoenzymes in relation to the urinary excretion of the mercapturic acids of Z- and E-1,3-dichloropropene

Mononuclear lymphocytes were isolated from the blood of 12 individuals, who had been exposed to the vapour of the soil fumigant 1,3-dichloropropene (DCP). Western blot experiments were performed on the crude lymphocyte homogenates, using a monoclonal antibody against human hepatic glutathione S-transferase (GST) isoenzyme , to determine the presence or absence of mu-class isoenzymes , and/or . Nine of the individuals were found to be positive for and/or , the remaining three individuals being negative. In addition, all individuals showed a positive staining on immunoblot of a protein of somewhat lower molecular mass than the hepatic standard. This protein was bound by the S-hexylglutathione affinity column, and presumably constitutes a new mu-class isoenzyme, which is not subject to genetic polymorphism. Determination of the specific activities of individual human GST isoenzymes towards Z-(cis-) and E-(trans-)-DCP demonstrated that mu-class isoenzymes show a considerably higher specific activity with Z-DCP than alpha-class or pi-class isoenzymes. In addition, mu-class isoenzymes were found to be 2- to 3-fold more active with Z-DCP than with E-DCP. Their activity towards E-DCP was similar to the specific activity of alpha-class isoenzymes. Genetic polymorphism for mu-class isoenzymes could thus be a determinant in the extent of excretion of mercapturic acids from Z- and E-DCP. The urinary excretion of Z- and E-DCP mercapturic acids and the respiratory exposure to Z- and E-DCP were determined for nine and eight phenotyped individuals, respectively. Urinary excretion levels (corrected for the time weighted average 8-h exposure), the urinary ratio and the elimination half-lives of the mercapturic acids of Z- and E-DCP were compared with the data on the mu-phenotype. No statistically significant differences were observed between mu-class positive and mu-class negative individuals.