Dihydroxylated mercapturic acid metabolites of bromobenzene.

Bromobenzene is metabolized to electrophilic epoxides and quinones which covalently bind to protein sulfur nucleophiles, yet no quinone-derived mercapturic acid metabolites of bromobenzene have been reported. To search for them, phenobarbital-induced Sprague-Dawley rats were dosed (0.5-1.5 mmol/kg, ip) with either bromobenzene, 2-, 3-, or 4-bromophenol (BP), 3- or 4-bromocatechol (BC), or 2-bromohydroquinone (BHQ). Urine (0-24 h) was alkaline permethylated (2 N NaOH/CH3I/80 degrees C), and the resulting thioanisole derivatives were analyzed by GC/MS. Three dimethoxythioanisoles and eight bromodimethoxythioanisoles were formed by alkaline permethylation of urine of rats treated with bromobenzene or 3-BP; alkaline permethylation of urine from rats in other treatment groups afforded characteristic subsets of these derivatives. The major thioanisole from all groups except 3-BC or 4-BC was 2,5-dimethoxythioanisole, which arises from (2,5-dihydroxyphenyl)mercapturic acid. The latter was isolated from rat urine and is the first debrominated metabolite of bromobenzene reported to date. Its formation from both 4-bromophenol and BHQ requires two parallel mechanisms for bromine loss: (1) nucleophilic addition to 1,4-benzoquinone formed by hydroxylative debromination of 4-bromophenol and (2) nucleophilic displacement of bromine from 2-bromo-1,4-benzoquinone by sulfur. The yields of quinone-derived mercapturic acids formed from bromobenzene are quite low (less than 1% of dose) compared to the high yields of epoxide-derived mercapturic acids (40% of dose). Potential reasons for this are discussed.     

Premercapturic acid metabolites of bromobenzene derived via its 2,3- and 3,4-oxide metabolites

1. A new premercapturic acid metabolite of bromobenzene was isolated from the urine of beta-naphthoflavone-induced rats; using 1H-n.m.r., FAB mass spectrometry and chemical degradation it was identified as S-(2-hydroxy-3-bromocyclohexa-3,5-dienyl)-N-acetylcysteine. 2. Two regioisomeric premercapturic acids apparently derived from bromobenzene-3,4-oxide were isolated as an inseparable 1:1 mixture from the urine of phenobarbital-induced rats and characterized by similar means. 3. Acid dehydration of bromobenzene 3,4- and 4,3-premercapturic acids (mixture) afforded only p-bromophenylmercapturic acid, whereas acid dehydration of 3,2-premercapturic acid gave both o- and m-bromophenylmercapturic acids. This implies a shift of sulphur in acid dehydration of the 3,4- and 3,2- but not the 4,3-premercapturic acids. 4. Base dehydration of the 3,4- and 4,3-premercapturic acid mixture gave a mixture of p- and m-bromophenylmercapturic acids, whereas base dehydration of the 3,2-premercapturic acid gave only m-bromophenylmercapturic acid. This indicates these premercapturic acids dehydrate by direct elimination without rearrangment. 5. The 3,2-premercapturic acid was detected only in the urine of BNF-induced animals, whereas the 3,4- and 4,3-premercapturic acids were detected in the urines of untreated as well as PB- and BNF-induced animals. 6. Together with earlier reports of the isolation of the 2,3-dihydrodiol, the isolation of the 3,2-premercapturic acid as a urinary metabolite of bromobenzene implies that bromobenzene-2,3-oxide is a discrete metabolite of bromobenzene and not merely a hypothetical intermediate.     

Premercapturic acid metabolites of bromobenzene derived via its 2,3- and 3,4-oxide metabolites

1. A new premercapturic acid metabolite of bromobenzene was isolated from the urine of β-naphthoflavone-induced rats; using ¹H-n.m.r., FAB mass spectrometry and chemical degradation it was identified as S-(2-hydroxy-3-bromocyclohexa-3,5-dienyl)-N-acetylcysteine.

2. Two regioisomeric premercapturic acids apparently derived from bromobenzene-3,4-oxide were isolated as an inseparable 1 :1 mixture from the urine of phenobarbital-induced rats and characterized by similar means.

3. Acid dehydration of bromobenzene 3,4- and 4,3-premercapturic acids (mixture) afforded only p-bromophenylmercapturic acid, whereas acid dehydration of 3,2-premer-capturic acid gave both o- and m-bromophenylmercapturic acids. This implies a shift of sulphur in acid dehydration of the 3,4- and 3,2- but not the 4,3-premercapturic acids.

4. Base dehydration of the 3,4- and 4,3-premercapturic acid mixture gave a mixture of p- and m-bromophenylmercapturic acids, whereas base dehydration of the 3,2-premercapturic acid gave only m-bromophenylmercapturic acid. This indicates these premercapturic acids dehydrate by direct elimination without rearrangement.

5. The 3,2-premercapturic acid was detected only in the urine of BNF-induced animals, whereas the 3,4- and 4,3-premercapturic acids were detected in the urines of untreated as well as PB- and BNF-induced animals.

6. Together with earlier reports of the isolation of the 2,3-dihydrodiol, the isolation of the 3,2-premercapturic acid as a urinary metabolite of bromobenzene implies that bromobenzene-2,3-oxide is a discrete metabolite of bromobenzene and not merely a hypothetical intermediate.     

Biotransformation of 1,2-dibromopropane in rats into four mercapturic acid derivatives

1,2-Dibromopropane was administered orally in doses of 50-350 mg/kg to male Wistar rats. Four mercapturic acids were identified in urine by GC/MS, viz. N-acetyl-S-(2-oxopropyl)-L-cysteine (I), N-acetyl-S-(2-hydroxypropyl)-L-cysteine (II), N-acetyl-S-(1-carboxyethyl)-L-cysteine (III), and N-acetyl-S-(2-bromo-2-propenyl)-L-cysteine (IV). Mercapturic acid IV was a minor metabolite which could only be measured at doses of 200 mg/kg or higher. In 24 hr, urinary excretion of mercapturic acids amounted to about 36% of the dose (11% I, 21% II, 4% III, 0.2% IV). No dose dependency was found up to the highest dose. A unified scheme is proposed for the metabolism of 1,2-dibromopropane in the rat, which accounts for the identified mercapturic acids. The role of direct glutathione conjugation in the route leading to the major metabolite II, presumably involving thiiranium ion formation, is discussed. This route probably is biologically not very important because of the absence of detectable activity of 1,2-dibromopropane toward glutathione S-transferases in vitro, the very low mutagenicity of 1,2-dibromopropane, and the high mutagenic activity of N-acetyl-S-(2-bromopropyl)-L-cysteine methyl ester which was studied as a model compound for direct conjugation.     

Mechanism of formation of mercapturic acids from aromatic aldehydes in vivo

Male adult Wistar rats dosed i.p. with o-substituted benzaldehydes (o-F, o-Cl, and o-Br = V, VI, and VII) excreted mercapturic acids in urine. These acids were identified as N-acetyl-S-(ortho-substituted benzyl)cysteines (I, II, III). The total mercapturic acid excretion as % dose (2.0 mmol/kg, n = 4) was 1.2 +/- 0.4, 6.8 +/- 0.9, and 10.4 +/- 2.0 for V, VI, and VII. p-Cl-benzaldehyde administered in the same dose showed a non-significant urinary thioether excretion. The aim of the investigation was to prove in vivo a postulated metabolic pathway of substituted benzaldehydes via sulphate esters to mercapturic acids. After a single administration of the sodium salts of o- and p-Cl-benzylsulfuric acid a significant increase in mercapturic acid excretion of 21.2 +/- 1.8% and 14.5 +/- 1.2% of dose (2.0 mmol/kg, n = 4) was found. By pretreatment with pyrazole the mercapturic acid excretion increased after administration of o-Cl-benzyl alcohol (IX) whereas a significant decrease was found after administration of o-Cl-benzaldehyde (VI). After simultaneous administration of ethanol with IX and VI an increase in mercapturic acid excretion was observed. After previous administration of pentachlorophenol a significant decrease in urinary mercapturic acid excretion for IX and VI was found. These findings are in accordance with a metabolic pathway of substituted benzaldehydes via benzyl alcohols, subsequently sulphate esters to the corresponding benzylmercapturic acids.     

Excretion of mercapturic acids in human urine after occupational exposure to 2-chloroprene

A pilot study was conducted for human biomonitoring of the suspected carcinogen 2-chloroprene. For this purpose, urine samples of 14 individuals occupationally exposed to 2-chloroprene (exposed group) and of 30 individuals without occupational exposure to alkylating substances (control group) were analysed for six potential mercapturic acids of 2-chloroprene: 4-chloro-3-oxobutyl mercapturic acid (Cl-MA-I), 4-chloro-3-hydroxybutyl mercapturic acid (Cl-MA-II), 3-chloro-2-hydroxy-3-butenyl mercapturic acid (Cl-MA-III), 4-hydroxy-3-oxobutyl mercapturic acid (HOBMA), 3,4-dihydroxybutyl mercapturic acid (DHBMA) and 2-hydroxy-3-butenyl mercapturic acid (MHBMA). In direct comparison with the control group, elevated levels of the mercapturic acids Cl-MA-III, MHBMA, HOBMA and DHBMA were found in the urine samples of the exposed group. Cl-MA-I and Cl-MA-II were not detected in any of the samples, whereas HOBMA and DHBMA were found in all analysed urine samples. Thus, for the first time, it was possible to detect HOBMA and Cl-MA-III in human urine. The mercapturic acid Cl-MA-III could be confirmed as a specific metabolite of 2-chloroprene in humans providing evidence for the intermediate formation of a reactive epoxide during biotransformation. The main metabolite, however, was found to be DHBMA showing a distinct and significant correlation with the urinary Cl-MA-III levels in the exposed group. The obtained results give new scientific insight into the course of biotransformation of 2-chloroprene in humans.     

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     

Directing role of organic anion transporters in the excretion of mercapturic acids of alkylated polycyclic aromatic hydrocarbons.

Excretion of mercapturic acids of a xenobiotic is a good indicator for the formation of electrophilic intermediates. However, the route of excretion, urine or feces, is important for usage of a given mercapturic acid as a biomarker in humans. In the present study we investigated the excretion routes of 1-methylpyrenyl mercapturic acid (MPMA) and 1,8-dimethylpyrenyl mercapturic acid (DMPMA) formed from the corresponding benzylic alcohols in rats. Whereas MPMA was primarily excreted in urine (72% of the total urinary and fecal level), DMPMA clearly preferred the fecal route (88%). We then examined interactions of these mercapturic acids with renal basolateral organic anion transporters (OATs) using HEK293 cells stably expressing human OAT1 and OAT3. The uptake rates of MPMA by OAT1- and OAT3-expressing cells were 2.8- and 1.7-fold, respectively, higher than that by control cells. MPMA was a competitive inhibitor of p-aminohippurate uptake by OAT1 and estrone sulfate uptake by OAT3 with K(i) values of 14.5 microM and 1.5 microM, respectively. In contrast, DMPMA was not transported by OAT1 and only modestly transported by OAT3 (1.25-fold over control). Thus, we suspect that the substrate specificities, alone or together with other factors, played a directing role in the excretion of MPMA and DMPMA. Although the mechanistic link requires verification, our results clearly show that a minute structural difference (the presence or absence of an additional methyl group in an alkylated four-ring polycyclic hydrocarbon) can strongly affect the interaction with transporter proteins and direct the excretion route of mercapturic acids.     

Mechanism of formation of mercapturic acids from (1-bromoethyl)benzene and (2-bromoethyl)benzene in the rat.

1. Three hypotheses have been proposed for the mechanism of metabolism of alkylhalides to hydroxy-alkylmercapturic acids, two of which involve the intermediate step of dehydrohalogenation and formation of an epoxide. 2. After injection of (1-bromoethyl)benzene in rat, the only mercapturic acid appearing in the urine was N-acetyl-S-1-phenylethylcysteine. After injecting (2-bromoethyl)benzene in the rat only N-acetyl-S-2-phenylethylcysteine and N-acetyl-S-(2-phenyl-2-hydroxyethyl)cysteine were found in the urine. 3. Since the principal mercapturic acid formed from both styrene and styrene oxide could not be detected in the urine of rats receiving either 1- or 2-bromoethyl benzene, the intermediate formation of styrene or styrene oxide from the arylalkylhalides does not occur.     

Dose-dependent metabolism of fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (compound A), an anesthetic degradation product, to mercapturic acids and 3,3,3-trifluoro-2-(fluoromethoxy)propanoic acid in rats.

The volatile anesthetic sevoflurane is degraded in anesthesia machines to fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (FDVE), to which humans are exposed. FDVE is metabolized in rats and humans to two alkane and two alkene glutathione S-conjugates that are hydrolyzed to the corresponding cysteine S-conjugates. The latter are N-acetylated to mercapturic acids, or bioactivated by renal cysteine conjugate beta-lyase to metabolites which may react with cellular macromolecules or hydrolyze to 3,3,3-trifluoro-2-(fluoromethoxy)propanoic acid. FDVE causes nephrotoxicity in rats, which evidence suggests is mediated by renal uptake of FDVE S-conjugates and metabolism by beta-lyase. Although pathways of FDVE metabolism have been described qualitatively, the purpose of this investigation was to quantify FDVE metabolism via mercapturic acid and beta-lyase pathways. Fischer 344 rats underwent 3-h nose-only exposure to FDVE (0 +/- 0, 46 +/- 19, 98 +/- 7, 150 +/- 29, and 220 +/- 40 ppm), and urine was collected for 24 h. Urine concentrations of the mercapturates, N-acetyl-S-(1,1,3,3, 3-pentafluoro-2-fluoromethoxypropyl)-L-cysteine and N-acetyl-S-(1-fluoro-2-fluoromethoxy-2-(trifluoromethyl)vinyl)-L- cysteine, the beta-lyase-dependent metabolite 3,3, 3-trifluoro-2-(fluoromethoxy)propanoic acid, and its degradation product trifluorolactic acid, were determined by GC/MS. There was dose-dependent urinary excretion of the alkane mercapturate N-acetyl-S-(1,1,3,3,3-pentafluoro-2-fluoromethoxypropyl)-L- cysteine and 3,3,3-trifluoro-2-(fluoromethoxy)propanoic acid, while excretion of the alkene mercapturate N-acetyl-S-(1-fluoro-2-fluoromethoxy-2-(trifluoromethyl)vinyl)-L- cysteine plateaued at higher FDVE exposures. The alkane:alkene mercapturic acid excretion ratio was between 2:1 and 4:1. Trifluorolactic acid was only rarely observed. Urine excretion of the beta-lyase-dependent metabolite 3,3, 3-trifluoro-2-(fluoromethoxy)propanoic acid was 10-fold greater than that of the combined mercapturates. Results show that FDVE cysteine S-conjugates undergo facile metabolism via renal beta-lyase, particularly in comparison with detoxication by mercapturic acid formation. The quantitative assay developed herein may provide a biomarker for FDVE exposure and relative metabolism via toxification and detoxifying pathways, applicable to animal and human investigations.     

Formation of mercapturic acids from acrylonitrile, crotononitrile, and cinnamonitrile by direct conjugation and via an intermediate oxidation process

After administration of acrylonitrile, crotononitrile and cinnamonitrile to rats, two types of mercapturic acids were isolated from urine and identified by mass and NMR spectroscopy as N-acetyl-S-(2-cyanoethyl)-L-cysteine (I) and N-acetyl-S-(2-hydroxyethyl)-L-cysteine (II) (methyl-substituted in the case of crotonitrile and phenyl-substituted in the case of cinnamonitrile). After pretreatment of rats with the cytochrome P-450 inhibitor 1-phenylimidazole, no trace of mercapturic acid II was found, whereas a higher amount of mercapturic acid I was excreted. It is suggested that the first type of products result from direct addition of glutathione, whereas the second group of metabolites (II), in which the cyano group has been replaced by a hydroxyl group, are formed via an intermediate epoxide. Substituents on the double bond had a considerable influence on the ratio of the two mercapturic acids formed, and thus presumably on the amount metabolized via an oxidative process: the ratio of the cyano (I) to hydroxy (II) mercapturic acid was 72:28 for AN; introduction of a methyl or a phenyl group resulted in ratios of 91:9 and 98:2, respectively.     

Nephrotoxicity of mercapturic acids of three structurally related 2,2-difluoroethylenes in the rat. Indications for different bioactivation mechanisms

The biotransformation and the hepato- and nephrotoxicity of the mercapturic acids (N-acetyl-1-cysteine S-conjugates) of three structurally related 2,2-difluoroethylenes were investigated in vivo in the rat. All mercapturic acids appeared to cause nephrotoxicity, without any measureable effect on the liver. The mercapturic acid of tetrafluoroethylene (TFE-NAC) appeared to be the most potent nephrotoxin, causing toxicity upon an i.p. dose of 50 mumol/kg. The mercapturic acids of 1,1-dichloro-2,2-difluoroethylene (DCDFE-NAC) and 1,1-dibromo-2,2-difluoroethylene (DBDFE-NAC) were nephrotoxic at slightly higher doses, i.e. at 75 and 100 mumol/kg, respectively. In the urine of TFE-NAC-treated rats significant amounts of difluoroacetic acid (DFAA) could be detected. With increasing doses, the relative amount of DFAA in urine increased progressively (5-18% of dose). In urine of rats treated with DCDFE-NAC and DBDFE-NAC, however, the corresponding dihaloacetic acids, dichloroacetic acid and dibromoacetic acid, could not be detected. Formation of DFAA and pyruvate could also be observed during in vitro metabolism of the cysteine conjugate of tetrafluoroethylene (TFE-CYS) by rat renal cytosol. Inhibition by aminooxyacetic acid (AOA) pointed to a beta-lyase dependency for the DFAA-formation. Next to DFAA and pyruvate, also formation of hydrogen sulfide and thiosulfate could be detected. These results suggest that TFE-CYS is bioactivated to a significant extent to difluorothionacyl fluoride, which most likely is subsequently hydrolysed to difluorothio(no)acetic acid and difluoroacetic acid. According to formation of pyruvate, the cysteine conjugates derived from DCDFE-NAC and DBDFE-NAC also were efficiently metabolized by rat renal beta-lyase. However, the formation of corresponding dihaloacetic acids, dichloroacetic acid and dibromoacetic acid, could not be detected in vitro at all. Only very small amounts of hydrogen sulfide and thiosulfate were detected. These results suggest that bioactivation of the latter two conjugates to a dichloro- or dibromothionoacyl fluoride represents only a minor route. Because of better leaving group abilities of chloride and bromide compared to fluoride, rearrangement of the initially formed ethanethiol to a thiirane might be favoured. Based on the present in vivo and in vitro data, it is concluded that the nephrotoxicity of the structurally related mercapturic acids of 2,2-difluoroethylenes is dependent on halogen substitution and presumably the result of at least two different mechanisms of bioactivation.     

Mercapturic acid urinary metabolites of 3-butene-1,2-diol as in vivo evidence for the formation of hydroxymethylvinyl ketone in mice and rats

3-Butene-1,2-diol (BDD), a major metabolite of 1,3-butadiene (BD), can readily be oxidized to hydroxymethylvinyl ketone (HMVK), a Michael acceptor. In previous studies, 4-(N-acetyl-l-cystein-S-yl)-1,2-dihydroxybutane (DHB), a urinary metabolite of BD that was used to assess human BD exposure, was suggested to be a metabolite of HMVK, but DHB formation from BDD and the formation of the DHB precursor 4-(N-acetyl-l-cystein-S-yl)-1-hydroxy-2-butanone (HB) have not been previously investigated. In the current study, four HMVK-derived mercapturic acids [DHB, HB, 3-(N-acetyl-l-cystein-S-yl)propan-1-ol (POH), and 3-(N-acetyl-l-cystein-S-yl)propanoic acid (PA)] were identified in the urine of mice and rats given BDD (284-2272 micromol/kg, i.p.) based on GC/MS analyses and comparisons with synthetic standards after esterification and silylation of the carboxyl and hydroxyl groups, respectively. The combined amounts of the mercapturic acids excreted after BDD exposure were dose-dependent and were mostly similar between mice and rats given equivalent doses of BDD. The mercapturic acids accounted for a greater fraction of the administered BDD dose as the dose was lowered, suggesting that HMVK formation represents a prominent route for BDD metabolism in both mice and rats. The major mercapturic acid excreted by mice was DHB, whereas rats excreted equivalent amounts of DHB and HB. The levels of POH or PA were significantly lower in both species relative to DHB or HB. The observed species differences in the excretion of DHB and HB were thought to be due to differences in the capacity of mice and rats to reduce HB to DHB.     

Mechanism of formation of mercapturic acids from (1-bromoethyl)benzene and (2-bromoethyl)benzene in the rat

1. Three hypotheses have been proposed for the mechanism of metabolism of alkylhalides to hydroxy-alkylmercapturic acids, two of which involve the intermediate step of dehydrohalogenation and formation of an epoxide.

2. After injection of (1-bromoethyl)benzene in rat, the only mercapturic acid appearing in the urine was N-acetyl-S-1 -phenylethylcysteine. After injecting (2-bromoethyl)benzene in the rat only N-acetyl-S-2-phenylethylcysteine and N-acetyl-S-(2-phenyl-2-hydroxyethyl)cysteine were found in the urine.

3. Since the principal mercapturic acid formed from both styrene and styrene oxide could not be detected in the urine of rats receiving either 1- or 2-bromoethyl benzene, the intermediate formation of styrene or styrene oxide from the arylalkylhalides does not occur.     

Identification and quantitative determination of four different mercapturic acids formed from 1,3-dibromopropane and its 1,1,3,3-tetradeutero analogue by the rat

1,3-Dibromopropane (1,3-DBP) was administered i.p. in doses ranging from 5.6 to 54 mg to male Wistar rats. Four different mercapturic acids, viz. N-acetyl-S-3-bromopropyl-(MA I), N-acetyl-S-3-chloropropyl-(MA II), N-acetyl-S-2-carboxyethyl-(MA III) and N-acetyl-S-3-hydroxypropyl(-1-)cysteine (MA IV) were synthesized and identified as metabolites in urine by g.l.c.-mass spectrometry. 1,1,3,3-Tetradeutero-1,3-dibromopropane was used to study the mechanism of formation of the mercapturic acids in more detail. It was found that in the formation of MA IV a reactive episulphonium ion could be involved. Gas chromatographic quantification of the mercapturic acids (mercapturic acid assay) was correlated with a spectrophotometric thioether determination of the metabolites (thioether test). At doses up to 30 mg of 1,3-DBP, excretion of mercapturic acids was virtually complete in 24 h urine and amounted to about 19% of the dose (11.3% MA I, 4.9% MA II, 2.6% MA III and 0.2% MA IV). From excretion rate curves a half-time t1/2 was calculated as being about 4.5 h. A plateau in the dose-excretion curve was observed at 1,3-DBP doses higher than 40 mg, probably caused by glutathione depletion.     

Bioactivation of diclofenac via benzoquinone imine intermediates-identification of urinary mercapturic acid derivatives in rats and humans.

The metabolism of diclofenac has been reported to produce reactive benzoquinone imine intermediates. We describe the identification of mercapturic acid derivatives of diclofenac in rats and humans. Three male Sprague-Dawley rats were administered diclofenac in aqueous solution (pH 7) at 50 mg/kg by intraperitoneal injection, and urine was collected for 24 h. Human urine specimens were obtained, and samples were pooled from 50 individuals. Urine samples were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). Two metabolites with MH(+) ions at m/z 473 were detected in rat urine and identified tentatively as N-acetylcysteine conjugates of monohydroxydiclofenac. Based upon collision-induced fragmentation of the MH(+) ions, accurate mass measurements of product ions, and comparison of LC/MS/MS properties of the metabolites with those of synthetic reference compounds, one metabolite was assigned as 5-hydroxy-4-(N-acetylcystein-S-yl)diclofenac and the other as 4'-hydroxy-3'-(N-acetylcystein-S-yl)diclofenac. The former conjugate also was detected in the pooled human urine sample by multiple reaction-monitoring LC/MS/MS analysis. It is likely that these mercapturic acid derivatives represent degradation products of the corresponding glutathione adducts derived from diclofenac-2,5-quinone imine and 1',4'-quinone imine, respectively. Our data are consistent with previous findings, which suggest that oxidative bioactivation of diclofenac in humans proceeds via benzoquinone imine intermediates.     

Glutathione conjugation of chlorobenzylidene malononitriles in vitro and the biotransformation to mercapturic acids in rats

The glutathione conjugation of 2-chloro-, 3-chloro-, 4-chloro- and 2,6-dichlorobenzylidene malononitrile (chloroBMNs) was investigated in vitro. In incubation mixtures containing rat liver cytosol (9000 g), the decrease in the initial amount of glutathione due to the various chloroBMNs ranged from 40 to 60% and occurred both enzymatically and spontaneously at physiological conditions (37°C, pH7.4). 2,6-DichloroBMN, however, depleted glutathione largely spontaneously (38±3%). The steric hindrance of the two chlorosubstituents probably plays an important role during the glutathione-S-transferase catalyzed reaction.The hydrolysis of the chloroBMNs to the corresponding chlorobenzaldehydes and malononitrile was studied in a mixture of buffer pH 7.4 and ethanol. The rate of hydrolysis of 2,6-dichloroBMN was slower than those of the related chloroBMNs. This means that 2,6-dichloroBMN will be the most stable compound in the presence of water.Only IP administration of 2-chloroBMN (CS) to adult male Wistar rats gave enhancement of urinary thioether excretion. A thioether could be isolated and was identified as the N-acetyl-S-[2-chlorobenzyl]-L-cysteine. The quantity of this benzylmercapturic acid in the urine of rats amounted to 4.4% dose (0.07 mmol/kg, n=12).After IP administration of 2-chloro- and 3-chlorobenzaldehyde to rats benzylmercapturic acid excretion in the urine was found to be 7.6 and 1.1% of the dose, respectively. Administration of the related 4-chloro- and 2,6-dichlorobenzaldehyde, however, resulted in _no urinary mercapturic acid excretion.It is very likely that in rats the initial biotransformation of chloroBMNs is mainly hydrolysis to corresponding chlorobenzaldehydes, leading in the case of 3-chloro-, 4-chloro- and 2,6-dichloroBMN to no mercapturic acid excretion in the urine.Nevertheless, 2,6-dichloroBMN will be the most reactive compound with proteins and therefore the best haptene in comparison with the related chloroBMNs.This work was financially supported by a grant from the Dutch Foundation for Medical Research FUNGO, grant no. 13-28-57     

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.     

Specificity of aminoacylase III-mediated deacetylation of mercapturic acids.

Trichloroethylene (TCE) and other halogenated alkenes are known environmental contaminants with cytotoxic and nephrotoxic effects, and are potential carcinogens. Their metabolism via the mercapturate metabolic pathway was shown to lead to their detoxification. The final products of this pathway, mercapturic acids or N-acetyl-l-cysteine S-conjugates, are secreted into the lumen in the renal proximal tubule. The proximal tubule may also deacetylate mercapturic acids, and the resulting cysteine S-conjugates are transformed by cysteine S-conjugate beta-lyases to nephrotoxic reactive thiols. The specificity and rate of mercapturic acid deacetylation may determine the toxicity of certain mercapturic acids; however, the exact enzymologic processes involved are not known in detail. In the present study we characterized the kinetics of the recently cloned mouse aminoacylase III (AAIII) toward a wide spectrum of halogenated mercapturic acids and N-acetylated amino acids. In general, the V(max) value of AAIII was significantly larger with chlorinated and brominated mercapturic acids, whereas fluorination significantly decreased it. The enzyme deacetylated mercapturic acids derived from the TCE metabolism including N-acetyl-S-(1,2-dichlorovinyl)-l-cysteine (NA-1,2-DCVC) and N-acetyl-S-(2,2-dichlorovinyl)-l-cysteine (NA-2,2-DCVC). Both mercapturic acids induced cytotoxicity in mouse proximal tubule mPCT cells expressing AAIII, which was decreased by an inhibitor of beta-lyase, aminooxyacetate. The toxic effect of NA-2,2-DCVC was smaller than that of NA-1,2-DCVC, indicating that factors other than the intracellular activity of AAIII mediate the cytotoxicity of these mercapturic acids. Our results indicate that in proximal tubule cells, AAIII plays an important role in deacetylating several halogenated mercapturic acids, and this process may be involved in their cyto- and nephrotoxicity.     

Isolation and identification of mercapturic acids of cinnamic aldehyde and cinnamyl alcohol from urine of female rats

Rats dosed with cinnamic aldehyde (I) excreted two mercapturic acids in the urine. The major one was identified as N-acetyl-S-(1-phenyl-3-hydroxypropyl)cysteine (V). The minor one was identified as N-acetyl-S-(1-phenyl-2-carboxy ethyl)cysteine (VI). The ratio appeared to be VVI=41. The hydroxy mercapturic acid (V) was also isolated from urine of rats dosed with cinnamyl alcohol (II). The total mercapturic acid excretion as percentage of the dose was 14.8±1.9% for cinnamic aldehyde (250 mg/kg) (n = 4) and 8.8±1.7% for cinnamyl alcohol (n = 4) (125 mg/kg). Inhibition of the alcohol dehydrogenase by pyrazole (206 mg/kg) diminished the thioether excretion of cinnamyl alcohol to 3.3±1.4% of the dose (n = 8). Cinnamic aldehyde has been proposed to be an intermediate in the mercapturic acid formation of cinnamyl alcohol.