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.     

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 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.     

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 of 2,6-dichlorobenzamide in rats and mice

1. Oral doses of 2,6-dichlorobenzamide (DCB) were excreted by rats as DCB, two monohydroxy-DCBs, 2-chloro-5-hydroxy-6-(methylthio)benzamide and 2-chloro-5-hydroxy-6-[S-(N-acetyl)cysteinyl]benzamide (mercapturic acid). 2. Biliary excretion (33% of the dose), enterohepatic circulation and intestinal micro-floral metabolism were involved in formation of 2-chloro-5-hydroxy-6-(methylthio)benzamide, and the mercapturic acid served as a precursor. 3. Whole body autoradiography and microautoradiography showed the accumulation of non-extractable residues from DCB in the nasal mucosa and contents of the large intestines of rats and mice dosed with 14C-labelled DCB.     

Metabolism of 2,6-dichlorobenzamide in rats and mice

1. Oral doses of 2,6-dichlorobenzamide (DCB) were excreted by rats as DCB, two monohydroxy-DCBs, 2-chloro-5-hydroxy-6-(methylthio)benzamide and 2-chloro-5-hydroxy-6-[S-(N-acetyl)cysteinyl]benzamide (mercapturic acid).

2. Biliary excretion (33% of the dose), enterohepatic circulation and intestinal microfloral metabolism were involved in formation of 2-chloro-5-hydroxy-6-(methylthio)benzamide, and the mercapturic acid served as a precursor.

3. Whole body autoradiography and microautoradiography showed the accumulation of non-extractable. residues from DCB in the nasal mucosa and contents of the large intestines of rats and mice dosed with ¹⁴C-labelled DCB.     

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     

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.     

Glutathione conjugation and bacterial mutagenicity of racemic and enantiomerically pure cis-and trans-methyl epoxycinnamates

This paper describes the ability of racemic, and enantiomerically pure cis-and trans-methyl epoxycinnamates (methyl 3-phenyl-2,3-epoxy-propanoates) to undergo glutathione conjugation and subsequent excretion as mercapturic acid and on the mutagenicities of these epoxy esters in the Ames assay. In incubation mixtures containing rat liver cytosol (9,000 g), the decrease of glutathione due to the epoxy esters occurred enzymatically. The highest glutathione depletion was found for the cis-epoxy cinnamic esters. Adult male rats administered a single i.p. dose of racemic trans- and cis-epoxy cinnamates (0.7 mmol/kg, n = 4) excreted thioethers in urine. Higher urinary thioether excretion was found after the cis-epoxy ester dosing. The structures of the thioether metabolites isolated from the urinary extracts were identified by TLC and confirmed by synthesis and mass spectrometry (FAB⁺). The thioethers appeared to be hydroxy mercapturic acids. The N-alkylating potential of the racemic epoxy esters was determined using 4-(p-nitrobenzyl)pyridine (=NBP). The trans-epoxy ester appeared to react much better with NBP than the cis-compound. Mutagenic effects of racemic trans-epoxy cinnamate as well as the enantiomerically pure trans-epoxy cinnamates were observed in the Ames test with S.typhimurium strains TA1535, TA1537, TA1538 and TA100 without metabolic activation. No mutagenic responses were detected using any of the epoxy cinnamates with S9 activation. By comparing the mutagenicity and the enzymatically catalyzed glutathione conjugation it follows that the activity of the respective enantiomeric methyl cinnamates goes in the opposite order. Glutathione conjugation plays a protective role in the detoxication in living organism of the potentially toxic methyl epoxy cinnamates.     

Glutathione conjugation of 1,2-dibromo-1-phenylethane in rats in vivo

The metabolism of 1,2-dibromo-1-phenylethane (DBPE) was studied in rats. Administration of DBPE orally, in doses of 0.25-1.25 mmol/kg (66-330 mg/kg), to male Wistar rats resulted in the excretion of a single mercapturic acid in urine. The methyl esters of three potential mercapturic acid metabolites were synthesized: N-acetyl-S-(2-oxo-2-phenylethyl)-L-cysteine methyl ester (O),N-acetyl-S-(2-hydroxy-1-phenylethyl)-L-cysteine methyl ester (I), and N-acetyl-S-(2-hydroxy-2-phenylethyl)-L-cysteine methyl ester (II). GC/MS analysis showed that the methyl ester of the excreted mercapturic acid was identical with II. Quantitative measurement of II in urine by GLC showed that, after 24 hr, excretion of the mercapturic acid was almost complete and amounted to 41% of the administered dose. At doses higher than 1.00 mmol/kg, the excretion no longer increased. Inhibition of the oxidative pathways by ip injection of 1-phenylimidazole resulted in an excretion decrease of about 40%. (Pre)treatment with diethyl maleate lowered the excretion of mercapturic acid by 30-60%. Glutathione conjugates synthesized from DBPE and styrene oxide were separated by HPLC. Both compounds can produce the same two pairs of diastereomers, viz. (R)- and (S)-(2-hydroxy-1-phenyl-ethyl)glutathione ((R)-1 and (S)-1), and (R)- and (S)-(2-hydroxy-2-phenylethyl)glutathione ((R)-2 and (S)-2). These could be separated in the order (R)-2, (R)-1, (S)-1, and (S)-2 within 20 min. This method was also applied to examine glutathione conjugates excreted in bile after DBPE administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the fate of the glutathione and cysteine conjugates of acetaminophen in mice

Studies were conducted in mice to examine the origin and fate of the amino acid-containing conjugates of acetaminophen (APAP). Collection of bile containing [14C]APAP metabolites (mainly the glutathione conjugate) in common duct-cannulated mice given a 250 mg/kg oral dose of the drug reduced by greater than 70% the urinary excretion of the cysteine and mercapturic acid conjugates of APAP. This confirmed previous reports which indicated that these urinary metabolites originated from the glutathione conjugate excreted in bile. The urinary excretion of cysteine and mercapturic acid conjugates was not altered, however, by ligation of the common bile duct in mice given APAP. Thus, biliary excretion of the glutathione conjugate is not obligatory for the appearance of cysteine and mercapturic acid conjugates in urine. Intravenous administration of purified glutathione conjugate to mice having a bile-duct cannula indicated that this conjugate did not appear in bile but appeared in urine primarily in the form of the cysteine conjugate. An identical pattern of excretion was observed after an iv dose of the purified cysteine conjugate of APAP to bile duct-cannulated mice. These results indicated that, if the glutathione conjugate leaves the liver via the blood, it is rapidly converted to the cysteine conjugate which is eliminated in urine. This conversion takes place at multiple sites in the body and evidence is presented to implicate both intestine and kidney in the process. The appearance of a small amount of glutathione conjugate in urine (16%) after an iv dose of the cysteine conjugate indicates that formation of the glutathione of APAP can occur by a route that does not involve direct conjugation of reactive metabolites of the drug with glutathione.     

Thioethers as urinary metabolites of thiophene and monobromothiophenes

1. Thiophene and its two monobromo derivatives were administered to rats and the amounts of thioether excreted in urine were measured by an assay based on Ellman's reagent. This assay, which involves extraction and hydrolysis, was validated by determining extraction and hydrolysis efficiencies for several authentic thioethers including N-acetyl-S-(2-thienyl)-L-cysteine, a previously reported metabolite of thiophene and 2-bromothiophene.

2. The thioethers present in urine of animals dosed with thiophenes have been examined chromatographically. Contrary to previous reports, the present work indicates that S-substituted, N-acetyl-L-cysteines (mercapturic acids) are not important thioether metabolites of thiophene in rats, and the small quantity of such compounds formed does not include either of the two simple S-thienyl derivatives.

3. The two monobromo thiophenes form higher proportions of thioethers than does thiophene, and one of these thioethers, arising from 3-bromothiophene, was identified, chromatographically, as N-acetyl-S-(3-thienyl)-L-cysteine.     

Mercapturic acid formation in the marmoset (Callithrix jacchus)

1. Benzylmercapturic acid is a major metabolite of [methylene-¹⁴C]benzyl chloride in the marmoset, as in the rat. The excretion of the minor metabolites benzylmercapturic acid sulphoxide and benzylcysteine accounted for a greater proportion of the dose than in the rat. Excretion of hippuric acid as a metabolite of benzyl chloride was variable in the marmoset.

2. Acetylation of S-benzyl- and S-pentyl-l-Cysteine to the corresponding mercapturic acids was extensive in the marmoset. Trace amounts of the sulphoxides of these acids were also excreted.     

Metabolism of illudin S,a toxic substance of Lampteromyces japonicus: urinary excretion of mercapturic acids in rat

1. The urinary excretion of the mercapturic acids of illudin S after oral administration to rat has been studied.

2. From Ic-ms/ms analysis of methanolic extracts of lyophilized rat urine, stereoisomeric mercapturic acids were detected.

3. The mercapturic acids excreted 3 days following administration amounted to approximately 0.39–0.73% of the administered dose.

4. In vitro glutathione conjugation of illudin S by subcellular fractions was also examined.

5. No significant increases in the formation of glutathione adducts were observed in any subcellular fractions examined.     

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.     

Urinary excretion of acrylonitrile and its metabolites in rats

Quantitative analysis of the dose-dependent urinary excretion of acrylonitrile and its metabolites was carried out in male Wistar rats following inhalation exposure of the animals to 1, 5, 10, 50 and 100 ppm acrylonitrile for 8 h. Quantitative analysis of acrylonitrile in urine was performed by gas chromatography. The urinary metabolites cyanoethyl mercapturic acid, S-carboxymethyl cysteine and hydroxyethyl mercapturic acid were measured by a modified amino acid analysis, and thiodiglycolic acid by GC-MS. The excretion pattern of the compound and its metabolites was dependent on the exposure level; it is concluded that urinary determination of the unmetabolized acrylonitrile and two of its metabolites, cyanoethyl mercapturic acid and thioglycolic acid, may be useful for biological monitoring of industrial exposure.     

Mercapturic acid formation in the marmoset (Callithrix jacchus)

Benzylmercapturic acid is a major metabolite of [methylene-14C]benzyl chloride in the marmoset, as in the rat. The excretion of the minor metabolites benzylmercapturic acid sulphoxide and benzylcysteine accounted for a greater proportion of the dose than in the rat. Excretion of hippuric acid as a metabolite of benzyl chloride was variable in the marmoset. Acetylation of S-benzyl- and S-pentyl-L-cysteine to the corresponding mercapturic acids was extensive in the marmoset. Trace amounts of the sulphoxides of these acids were also excreted.     

Metabolism of some methyl 2-cyano-3-phenyl-acrylates (methyl alpha-cyanocinnamates) in rats

1. The metabolism of 2-cyano-3-phenylacrylic acid (CPA) and some methyl 2-cyano-3-phenylacrylates (methyl alpha-cyanocinnamates) after i.p. administration to rats, was investigated. 2. The conjugation of CPA and methyl alpha-cyanocinnamates with L-cysteine, N-acetylcysteine and glutathione (GSH) in vitro was studied and the rate of hydrolysis of the double bond of the methyl alpha-cyanocinnamates determined. 3. CPA and the methyl alpha-cyanocinnamates caused a significant increase in urinary (24 h) thioether excretion, with no increase in SCN- excretion. 4. No thioethers, such as possible addition products of N-acetylcysteine with CPA or methyl alpha-cyanocinnamates, were isolated; such thioether compounds appeared to be very unstable. 5. Administration of the carboxylesterase inhibitor, tri-ortho tolyl phosphate, to rats resulted in increased urinary excretion of SCN- but no significant excretion of thioethers, after a single i.p. injection of methyl alpha-cyanocinnamates. 6. Following incubation of methyl alpha-cyanocinnamates with GSH under non-enzymic and enzymic conditions, the GSH was 100% recovered. However, incubation of CPA depleted GSH and N-acetylcysteine (non-enzymic). 7. A metabolic pathway for the metabolism of methyl alpha-cyanocinnamates to thioether adducts is proposed, which proceeds via the corresponding CPA. Ester hydrolysis and the carbon-carbon double bond hydrolysis are probably in vivo reactions of some importance.     

2-Chlorobenzylmercapturic acid,a metabolite of the riot control agent 2-chlorobenzylidene malononitrile (CS) in the rat

Adult male Wistar rats administered i.p. with 2-chlorobenzylidene malononitrile (CS) excreted one mercapturic acid in urine. The amount of mercapturic acid determined gaschromatographically was about 4% of the dose (0.07 mmol/kg, n = 12). The structure of the mercapturic acid methylester was identified by t.l.c. and confirmed by synthesis and mass-spectrography. The acid appeared to be 2-chlorobenzylmercapturic acid [N-acetyl-S-(2-chlorobenzyl)-L-cysteine]. CS and some of its metabolites were also tested in the Ames Salmonella/microsome assay. Both mutagenic and toxic effects were measured with strain TA 100 as the indicator organism. No mutagenic effects were found with any of the tested substances. At dosages of CS, higher than 1,000 micrograms/plate a bacteriotoxicity was revealed.     

Biotransformation of diethenylbenzenes. III: Identification of metabolites of 1,3-diethenylbenzene in rat.

1. Biotransformation of 1,3-diethenylbenzene (1) in rat gave four major metabolites, namely, 3-ethenylphenylglyoxylic acid (2), 3-ethenylmandelic acid (3), N-acetyl-S-[2-(3-ethenylphenyl)-2-hydroxyethyl]-L-cysteine (4) and N-acetyl-S-[1-(3-ethenylphenyl)-2-hydroxyethyl]-L-cysteine (5) were isolated from urine and identified by n.m.r. and mass spectrometry. 2. Four minor metabolites, 3-ethenylbenzoic acid (6), 3-ethenylphenylacetic acid (7), 3-ethenylbenzoylglycine (8) and 2-(3-ethenylphenyl)ethanol (9) were identified by g.l.c.-mass spectrometric analysis of urine extract derivatized in two different ways. 3. All identified metabolites are derived from 3-ethenylphenyloxirane (10), a reactive metabolic intermediate. No product of any metabolic transformation of second ethenyl group has been identified. However, several minor unidentified metabolites were detected by g.l.c.-mass spectrometry. 4. Total thioether excretion in 24 h urine after a single i.p. dose of 1 amounted to 28.3 +/- 3.5 dose (mean +/- SD). No significant differences in the thioether fraction were observed in the dose range 100-300 mg/kg. 5. Thioether metabolites consisted mainly of mercapturic acids 4 and 5. The ratio of metabolites 5 to 4 was 62:38. Each mercapturic acid consisted of two diastereomers. Their ratio, as determined by quantitative 13C-n.m.r. measurement was 95:5 and 79:21 for mercapturic acids 4 and 5, respectively.