Urinary Mercapturic Acids Research Articles

Influence of Caffeine on Allyl Alcohol-Induced Hepatotoxicity in Rats* I. In Vivo Study

Cotreatment of rats with a low hepatotoxic dose (30.7 mg/kg, i.p.) of allyl alcohol (AA) and a higher, but nontoxic, dose (150 mg/kg, oral) of caffeine (CF) potentiated the hepatotoxicity of AA. This was verified by significantly higher levels of plasma alanine aminotransferase (ALT) activity and histopathologically greater severity of lesions in the periportal hepatocytes than those due to AA alone. Treatment of rats with 4-methylpyrazole (4-MP) (0.5 mmol/kg, i.p.) (an inhibitor liver alcohol dehydrogenase) for 30 minutes, followed by similar cotreatment with AA and CF, completely prevented the elevation of plasma levels of ALT and histological damage induced by cotreatment with CF and AA 24 hours following their administration. Severe liver damage induced by cotreatment with CF and AA was further, markedly enhanced by phenobarbital pretreatment (80 mg/kg, i.p., 3 days). Thus, extensive necrosis of periportal hepatocytes was noted, as well as edema and accumulation of inflammatory cells in the necrotic foci caused by such pretreatment. The depression of hepatic nonprotein sulfhydryls resulting from CF plus AA was much more severe than that caused by AA or CF alone and appeared as early as 30 minutes after administration. However, much less marked depletion of protein thiols was observed following similar treatments. Significant increase in lipid peroxidation (as measured by melondialdehyde [MDA] formation) was also observed in rat liver but only 24 hours after administration. The production ofMDA in the rat liver was significantly higher after administration of AA plus CF than after administration of AA alone. Pretreatment of rats with phenobarbital further significantly enhanced the formation of 2,4-dinitrophenylhydrazine (DNP)-reactive metabolite(s) (measured as DNP-acrolein adduct equivalents) in rat liver induced by AA (30.7 mg/kg) plus CF (150 mg/kg) within 1 hour following such treatment. Cotreatment with AA and a higher dose of CF resulted in significantly higher excretion of urinary thioethers or mercapturic acids than in rats treated with AA alone. Thus, these data suggest that an increased bioactivation pathway of acrolein involving a P450 mixed-function oxidase system caused by CF may be involved in such potentiating effects of CF on AA-induced hepatotoxicity in rats.

Importance of genetic polymorphisms of drug-metabolizing enzymes for the interpretation of biomarkers of exposure to styrene

The objective of this study was to test the infiuence of genetic polymorphisms for metabolic enzymes (CYP2E1, mEH, GSTM1 and GSTT1) implicated in the biotransformation of styrene in humans on the interpretation of urinary biomarkers of exposure. Thirty workers from a fibreglass-reinforced plastics factory took part in the study. Ambient styrene concentration was determined during the whole workshift by passive sampling. Urine was collected at the end of the shift for the determination of mandelic acid (MA) and phenylglyoxylic acid (PGA) (major biotransformation pathway), N-acetyl-S-(1-phenyl-2-hydroxy)ethyl-L-cysteine (M1) and N-acetyl-S-(2-phenyl-2-hydroxy)ethyl-L-cysteine (M2) (minor metabolic pathway) and creatinine. The average airborne styrene concentration of 18.2 ppm (range: 0.9-68.9 ppm) was very close to the current threshold limit value (TLV-TWA) recently adjusted by ACGIH from 50 to 20 ppm. There was a better correlation between external and internal exposure as estimated by urinary MA + PGA (r=0.92; p<0.0001) compared with urinary M1 + M2 (r=0.74; p<0.0001). To investigate to what extent genetic polymorphisms in metabolic enzymes could explain interindividual variations observed in the concentration of urinary biomarkers related to a given external exposure, two 'metabolic indexes' (derived from the ratio between the sum of urinary metabolites for a specific pathway and ambient styrene concentration) were calculated for each worker and compared for different allelic combinations. Monovariate analyses showed that GSTM1 polymorphism was clearly the most significant parameter infiuencing urinary concentrations of mercapturic acids. Based on GSTM1 allelic status, two different biological exposure indexes (BEIs) for M1 + M2 in post-shift urinary samples corresponding to a 20 ppm styrene concentration are proposed (GSTM1null: 1330 µg g-1 creatinine, GSTM1+: 2878 µg g-1 creatinine). Multivariate regression analyses were also performed and revealed that the presence of the rare CYP2E1*1B allele linked to TaqI polymorphism (A1/A2) was associated with increased urinary concentrations of metabolites from both pathways. Two previously described polymorphisms for the EPHX gene were also tested but seemed not really relevant for interpretation of biomarkers. In conclusion, while CYP2E1 genotyping, particularly assessment of the CYP2E1*1B allelic status, is useful for a more accurate interpretation of the concentration of urinary biomarkers, GSTM1 genotyping is absolutely necessary when considering a biological monitoring programme based on determination of urinary mercapturic acids.

Identification of the dimethylbenzyl mercapturic acid in urine of rats administered with 1,2,4-trimethylbenzene

A study was undertaken of the mercapturic acid metabolism of 1,2,4-trimethylbenzene in the rat. Of three regioisomeric dimethylbenzyl mercapturic acids, i.e. 2,4-, 2,5- and 3,4-dimethylbenzyl isomers, the third isomer was not found in the urinary mercapturic acid isolated by preparative HPLC, from the comparison of NMR spectrum of the isolate with those of authentic specimens. The urinary mercapturate was then assigned to 2,4- and/or 2,5-dimethylbenzyl isomers. The excretion rate of the mercapturic acid was 14–20% of dose as 2,4-dimethylbenzyl isomer.

Determination of mercapturic acids in urine by solid-phase extraction followed by liquid chromatography-electrospray ionization mass spectrometry.

A novel method for the determination of five kinds of mercapturic acids, found in urine as metabolites of alkylbenzenes, based on liquid chromatography-electrospray ionization mass spectrometry is described. A solid-phase extraction procedure was used for the extraction of the mercapturic acids from urine and the separation was performed on a reversed-phase C30 column. The detection limits were in the range 2.4-3.2 ng ml-1.

A new method for the analysis of styrene mercapturic acids by liquid chromatography/electrospray tandem mass spectrometry.

A new method based on liquid chromatography/tandem mass spectrometry has been developed for the direct determination of specific urinary mercapturic acids arising from the conjugation of (R)-and (S)-enantiomers of styrene 7,8-oxide with glutathione (GSH), i.e. (R,R)- and (S,R)-N-acetyl-S-(1-phenyl-2-hydroxyethyl)cysteine (R,R-M1 and S,R-M1) and (R,R)- and (S,R)-N-acetyl-S-(2-phenyl-2-hydroxyethyl)-cysteine (R,R-M2 and S,R-M2). The four diastereoisomers were separated on a C18-DB (7.5 cm, 3 microm) column using variable proportions of 20 mM aqueous ammonium formate buffer and methanol at a flow-rate of 0.5 mL/min. The analytes were ionized by electrospray, in negative-ion mode. Operating in selected-reaction monitoring mode, linearity of the MS response versus analyte concentration was established over 4 orders of magnitude, the detection limits being 0.7-1.0 microg/L for all the mercapturates. Precision of the method determined at 50 microg/L (n = 12), expressed as relative standard deviation, was respectively 3.1, 4.8 and 6.9% within the run, intra-day and inter-day. The corresponding figures at 1.0 mg/L (n = 12) were respectively 2.0, 3.6 and 5.5%. The method was applied to the quantitative analysis of conjugated metabolites in urine samples from workers occupationally exposed to styrene. The diastereoisomers R,R-M1 and S,R-M2 accounted respectively for 50 and 40% of total mercapturates, whereas the proportion of R,R-M2 was 7% and only minor amounts of S,R-M1 were detectable. Styrene mercapturates represented a minor fraction of total styrene metabolites, less than 1% on average. The ratio mercapturates/main metabolites (mandelic + phenylglyoxylic acid) showed a bimodal distribution, the medians of the two subgroups being 0.2 and 1%, respectively. Such subgroups are probably characterized by the genetic polymorphisms of the drug-metabolizing enzymes to be identified.

Genotoxic markers among butadiene polymer workers in China

While 1,3-butadiene is carcinogenic in rodents, cancer causation in humans is less certain. We examined a spectrum of genotoxic outcomes in 41 butadiene polymer production workers and 38 non-exposed controls, in China, to explore the role of butadiene in human carcinogenesis. Because in vitro studies suggest that genetic polymorphisms in glutathione S-transferase enzymes influence genotoxic effects of butadiene, we also related genotoxicity to genetic polymorphisms in GSTT1 and GSTM1. Among butadiene-exposed workers, median air exposure was 2 p.p.m. (6 h time-weighted average), due largely to intermittent high level exposures. Compared with unexposed subjects, butadiene-exposed workers had greater levels of hemoglobin N-(2,3,4-trihydroxybutyl)valine (THBVal) adducts (P < 0.0001) and adduct levels tended to correlate, among butadiene-exposed workers, with air measures (P = 0.03). Butadiene-exposed workers did not differ, however, from unexposed workers with respect to frequency of uninduced or diepoxybutane-induced sister chromatid exchanges, aneuploidy as measured by fluorescence in situ hybridization of chromosomes 1, 7, 8 and 12, glycophorin A variants or lymphocyte hprt somatic mutation. Also among the exposed, greater THBVal levels were not associated with increases in uninduced sister chromatid exchanges, aneuploidy, glycophorin A or hprt mutations. Butadiene-exposed workers had greater lymphocyte (P = 0.002) and platelet counts (P = 0.07) and lymphocytes as a percentage of white blood cells were moderately correlated with greater THBVal levels (Spearman's phi = 0.32, P = 0.07). Among butadiene-exposed workers, neither GSTM1 nor GSTT1 genotype status predicted urinary mercapturic acid butanediol formation, THBVal adducts, uninduced sister chromatid exchanges, aneuploidy or mutations in the glycophorin A or hprt genes. Overall, the study demonstrated exposure to butadiene in these workers, by a variety of short-term and long-term measures, but did not show specific genotoxic effects, at the chromosomal or gene levels, related to that exposure.

Identification of the dimethylbenzyl mercapturic acid in urine of rats treated with 1,2,3-trimethylbenzene

The structure was investigated of the mercapturic acid excreted in urine of rats after the ip administration of 1,2,3-trimethylbenzene. Of the two regioisomeric mercapturic acids, ie N-acetyl-S-(2,3-dimethylbenzyl)-L-cysteine and N-acetyl-S-(2,6-dimethyl-benzyl)-L-cysteine, only the former was isolated by preparative HPLC and identified, by comparison with an authentic specimen. The excretion rate of the mercapturate was estimated to be ∼ 5% of dose, not a substantial metabolic route.

Quantification in Patient Urine Samples of Felbamate and Three Metabolites: Acid Carbamate and Two Mercapturic Acids

Previously we proposed and provided evidence for the metabolic pathway of felbamate (FBM), which leads to the reactive metabolite, 3-carbamoyl-2-phenylpropion-aldehyde. This aldehyde carbamate was suggested to be the reactive intermediate in the oxidation of 2-phenyl-1,3-propanediol monocarbamate to the major human metabolite 3-carbamoyl-2-phenylpropionic acid. In addition, the aldehyde carbamate was found to undergo spontaneous elimination to 2-phenylpropenal, commonly known as atropaldehyde. Moreover, atropaldehyde was proposed to play a role in the development of toxicity during FBM therapy. Evidence for atropaldehyde formation in vivo was reported with the identification of modified N-acetyl-cysteine conjugates of atropaldehyde in both human and rat urine after FBM administration. Identification of the atropaldehyde-derived mercapturic acids in urine after FBM administration is consistent with the hypothesis that atropaldehyde is formed in vivo and that it reacts with thiol nucleophiles. Based on the hypothesis that the potential for toxicity will correlate to the amount of atropaldehyde formed, we sought to develop an analytic method that would quantify the amount of relevant metabolites excreted in patient urine. We summarize the results of an LC/MS method used to quantify FBM, 3-carbamoyl-2-phenylpropionic acid and two atropaldehyde-derived mercapturic acids in the patient population. Analysis was performed on 31 patients undergoing FBM therapy. The absolute quantities of FBM and three metabolites were measured. This method demonstrated sufficient precision for the identification of patients exhibiting "abnormal" levels of atropaldehyde conjugates and may hold potential for patient monitoring.

High performance liquid chromatographic determination of mercapturic acids in urine of rats administered with m− or p-xylene

The determination of urinary mercapturic acid, N-acetyl-S-( m-xylyl)-L-cysteine or N-acetyl-S-( p-xylyl)-L-cysteine, was undertaken by HPLC after i.p. administration of m − or p-xylene to rats in order to confirm the structure of thioether which was known to be excreted in small amounts related with the metabolism of m − or p-xylene. The excretion rates of these mercapturic acids were much lower than those previously estimated from thioether determinations. The present study clearly demonstrates the mercapturic acid pathway operates only to limited degree as a metabolic route of m − or p-xylene.

Allylmercapturic acid as urinary biomarker of human exposure to allyl chloride.

OBJECTIVE: To evaluate the use of urinary mercapturic acids as a biomarker of human exposure to allyl chloride (3-chloropropene) (AC). During three regular shut down periods in a production factory...

Synthesis and characterization of m- and p-methylbenzyl-mercapturic acids derived from m- and p-xylenes

Chemical synthesis and physical properties of two mercapturic acids suggested as urinary metabolites of m- and p-xylenes ace described. These compounds may be used for the identification and quantivative determination by high-performance liquid chromatography of the corresponding mercapturic acids in urine.

Urinary Mercapturic Acids as Markers for the Determination of Isothiocyanate Release from Glucosinolates in Rats Fed a Cauliflower Diet

Glucosinolates, found in plants of the genus Brassica, release physiologically active metabolites, such as isothiocyanates and nitriles, in the digestive tract under the action of plant or bacterial myrosinase. Isothiocyanates are excreted in the urine as their mercapturic acid derivatives. To study the influence of diet on isothiocyanate release, two groups of eight rats were offered either a cauliflower-based diet or a standard laboratory rat diet. After 10 days' adaptation to the diets release of allyl isothiocyanate (AITC) from a single oral dose of sinigrin, a glucosinolate commonly occurring in brassicas, was quantified. Each rat was dosed with 50 μmol of either sinigrin or AITC by stomach tube and urine was collected at 0, 2, 6, 24 and 48 h after administration. Three days later, rats which had previously received sinigrin were administered with AITC and vice versa. The concentrations of urinary mercapturic acids of isothiocyanates were determined by HPLC. Recoveries of the mercapturic acid derivative of AITC in each animal were used to estimate AITC production in sinigrin-dosed rats. Estimates of AITC release from sinigrin showed that a greater proportion of administered sinigrin was hydrolysed to AITC in cauliflower-fed rats than in rats fed a control diet (0·412 vs 0·134; P〈0·001). The approach should allow more rigorous investigation of the influence of dietary factors on glucosinolate hydrolysis in vivo. © 1997 SCI.

Urinary Mercapturic Acids as Markers for the Determination of Isothiocyanate Release from Glucosinolates in Rats Fed a Cauliflower Diet

Urinary Mercapturic Acids as Markers for the Determination of Isothiocyanate Release from Glucosinolates in Rats Fed a Cauliflower Diet

Urinary Mercapturic Acids as Markers for the Determination of Isothiocyanate Release from Glucosinolates in Rats Fed a Cauliflower Diet

Glucosinolates, found in plants of the genus Brassica, release physiologically active metabolites, such as isothiocyanates and nitriles, in the digestive tract under the action of plant or bacterial myrosinase. Isothiocyanates are excreted in the urine as their mercapturic acid derivatives. To study the influence of diet on isothiocyanate release, two groups of eight rats were offered either a cauliflower-based diet or a standard laboratory rat diet. After 10 days' adaptation to the diets release of allyl isothiocyanate (AITC) from a single oral dose of sinigrin, a glucosinolate commonly occurring in brassicas, was quantified. Each rat was dosed with 50 μmol of either sinigrin or AITC by stomach tube and urine was collected at 0, 2, 6, 24 and 48 h after administration. Three days later, rats which had previously received sinigrin were administered with AITC and vice versa. The concentrations of urinary mercapturic acids of isothiocyanates were determined by HPLC. Recoveries of the mercapturic acid derivative of AITC in each animal were used to estimate AITC production in sinigrin-dosed rats. Estimates of AITC release from sinigrin showed that a greater proportion of administered sinigrin was hydrolysed to AITC in cauliflower-fed rats than in rats fed a control diet (0.412 vs 0.134; P<0.001). The approach should allow more rigorous investigation of the influence of dietary factors on glucosinolate hydrolysis in vivo.

Determination of urinary mercapturic acids of styrene in man by high-performance liquid chromatography with fluorescence detection.

A method for the determination of urinary N-acetyl-S-(1-phenyl-2-hydroxyethyl)-L-cysteine (M1) and N-acetyl-S-(2-phenyl-2-hydroxyethyl)-L-cysteine (M2) in man was developed. Clean-up of urine samples was obtained by a chromatographic technique, using a short reversed-phase precolumn; purified samples were then deacetylated with porcine acylase I for 16 h at 37 degrees C and deproteinized by centrifugal ultrafiltration. Derivatization was performed with o-phthaldialdehyde and 2-mercaptoethanol and the fluorescent derivatives were separated on a reversed-phase analytical column with a gradient mobile phase consisting of 50 mM acetate buffer (pH 6.5) and methanol. The retention times of the diastereoisomers of M1 (M1-"S" and M1-"R") were 52.8 and 73.7 min, respectively: M2 diastereoisomers eluted as a single peak at 70.5 min. The fluorescence detector was set at 330 nm (excitation) and 440 nm (emission). The detection limit (at a signal-to-noise ratio of three) was about 7 micrograms/1. The method was applied to 25 urine samples from workers exposed to styrene. A relationship was found between urinary mandelic and phenylglyoxylic acids and mercapturic acids specific for styrene. Urine samples from ten non-exposed subjects showed no detectable amounts of analytes.

Urinary mercapturic acid diastereoisomers in rats subchronically exposed to styrene and ethanol.

Styrene is stereoselectively oxidized by cytochrome P450 to its reactive metabolite, styrene oxide. The (R)- and (S)-enantiomers of styrene oxide can be conjugated with glutathione (GSH) to both (R)- and (S)-diastereoisomers of the specific mercapturic acids, N-acetyl-S-(1-phenyl-2-hydroxyethyl)-L-cysteine (M1) and N-acetyl-S-(2-phenyl-2-hydroxyethyl)-L-cysteine (M2). Several investigations have indicated different toxic potential of the (R)- and (S)-configurations of styrene oxide and its GSH- and N-acetyl-conjugates. In this study the mercapturic acid diastereoisomers were measured in the urine of rats exposed to styrene in combination with ethanol, a good inducer of styrene metabolism. Male Sprague-Dawley rats were given an isocaloric liquid diet containing ethanol (5% w/v) for 3 weeks. Starting from the 2nd week, the animals were also exposed to styrene vapours (300 ppm, 6 h/day, 5 days/week) in a dynamic exposure chamber. Both the (R)- and (S)-diastereoisomers of the M1 and M2 as well as the conventional biomarkers, mandelic acid (MA) and phenylglyoxylic acid (PGA) were measured in urinary samples. Approximately 30 and 25% reduction of the levels of brain non-protein sulfhydryls (NPS) was observed in the animals given styrene and ethanol, respectively, while the combined ethanol and styrene treatment resulted in a 60% decrease. Ethanol consumption also resulted in higher urinary levels of the M1-R, M1-S and M2 metabolites associated with increased M1-R/S ratio and higher urinary MA excretion compared to animals treated with styrene. These results suggest that the urinary mercapturic acid diastereoisomers may be used as a noninvasive tool to examine stereoselective patterns of styrene metabolism in vivo, as well as their alterations caused by ethanol. These compound-specific mercapturic acids may also be valuable indicators of styrene-induced disorders of GSH homeostasis in nonaccessible organs.

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 beta -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 mumol trichloroacetic acid + trichloroethanol and 0.45 mumol 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.

Dose-dependent kinetics and metabolism of 1,2-dichlorobenzene in rat: effect of pretreatment with phenobarbital.

1. Toxicity of halobenzenes has been ascribed mainly to their epoxides, but recent studies with bromobenzene have shown that secondary quinone metabolites are also involved in the alkylation of hepatic proteins. However, the relative contribution of the quinones and the epoxides to the toxicity of halobenzenes is still unclear. In order to investigate the relation between metabolism and toxicity of 1,2-dichlorobenzene (1,2-DCB), the biotransformation, tissue distribution, blood kinetics, and excretion at three different oral dose levels (5, 50 and 250 mg/kg) of the radiolabelled compound were investigated in the male Wistar rat. A toxic dose level (250 mg/kg, as demonstrated by Allis et al. 1992) was included. 2. The major route of elimination (75-85%) was renal excretion. Excretion via the faeces ranged from 19% for the low dose to 7% for the high-dose level. Excretion was nearly complete within 24 h for the low and mid-dose level, and within 48 h for the high-dose level. Pretreatment with phenobarbital resulted in a more rapid excretion for the high-dose level and an overall higher urinary excretion. Biliary excretion was 50-60%, indicating a considerable enterohepatic circulation. 3. Highest concentrations of radioactivity after a low dose were found in fat, liver and kidney at 6 h after administration, and then declined rapidly. 4. The maximum concentration of radioactivity in blood was reached at 6-8 h for the low and mid-dose level, and at 24 h for the high-dose level. The concentration of parent chemical was essentially constant during 3 and 6 h for the mid- and high-dose level respectively, and then declined. 1,2-DCB could only be detected in blood in the first 2 h after administration of the 5-mg/kg dose. 5. The major route of biotransformation was via the glutathione pathway and 60% of the urinary metabolites were mercapturic acids. In addition, the major metabolites in bile were conjugates of glutathione. Other major metabolites in urine were the sulphate conjugates of 2,3- and 3,4-dichlorophenol (DCP). No significant differences in metabolic profiles were observed between the different doses. Induction with phenobarbital resulted in the increased excretion of sulphate conjugates (30% in the induced rat, 20% in the control rat), mainly the conjugate of 3,4-DCP. 6. The mercapturic acids in urine and glutathione conjugates in bile were epoxide-derived, whereas no quinone- or hydroquinone-derived metabolites were observed. Therefore the hepatotoxicity of 1,2-dichlorobenzene is assumed to be related, at least partly, to the presence of the intermediate arene oxide. A high dose of 1,2-DCB will result in depletion of GSH, followed by oxidative stress and possible binding to macromolecules.

Urinary mercapturic acids as markers for the estimation of isothiocyanate release in the digestive tract of rats following oral adminstration of glucosinolates

Glucosinolates are a group of plant thioglucosides, found in a range of plant genera, notably theBrassicae. Glucosinolates assume agricultural importance because of their presence in rapeseed meal and forage brassica crops whose nutritional value they may limit. Glucosinolates are hydrolysed under the action of plant or microbial myrosinase to release a range of toxic metabolites (see Duncan, 1991). For example, sinigrin, a commonly found glucosinolate, breaks down to allyl isothiocyanate (AITC) and allyl cyanide as well as a number of lesser components. The course of hydrolysis is influenced by characteristics of the hydrolysis environment such as pH and the presence of metallic ions and enzymic co-factors. Because of the varied nature of the effects of different glucosinolate metabolites, the course of hydrolysis is a key influence on the ultimate toxicity of glucosinolates. Little is known, however, of the identity of metabolites arising in the digestive tract of livestock under different dietary circumstances. This is due, in part, to the technical difficulties associated with measuring concentrations of the compounds in the digestive tract. An alternative approach to the study of glucosinolate hydrolysisin vivois to measure concentrations of ultimate excretory products and relate this to events earlier in the catabolic pathway. For example, isothiocyanates are excreted as their mercapturic acid derivatives in rats and thus provide potentially useful markers for estimating rates of release of isothiocyanates from sinigrinin vivo(Mennickeet al. 1983). In the experiment reported here, this approach was validated in rats and used to measure rates of release of AITC from sinigrin in animals consuming diets with or without added myrosinase.

Urinary mercapturic acids as markers for the estimation of isothiocyanate release in the digestive tract of rats following oral adminstration of glucosinolates

Glucosinolates are a group of plant thioglucosides, found in a range of plant genera, notably the Brassicae. Glucosinolates assume agricultural importance because of their presence in rapeseed meal and forage brassica crops whose nutritional value they may limit. Glucosinolates are hydrolysed under the action of plant or microbial myrosinase to release a range of toxic metabolites (see Duncan, 1991). For example, sinigrin, a commonly found glucosinolate, breaks down to allyl isothiocyanate (AITC) and allyl cyanide as well as a number of lesser components. The course of hydrolysis is influenced by characteristics of the hydrolysis environment such as pH and the presence of metallic ions and enzymic co-factors. Because of the varied nature of the effects of different glucosinolate metabolites, the course of hydrolysis is a key influence on the ultimate toxicity of glucosinolates. Little is known, however, of the identity of metabolites arising in the digestive tract of livestock under different dietary circumstances. This is due, in part, to the technical difficulties associated with measuring concentrations of the compounds in the digestive tract. An alternative approach to the study of glucosinolate hydrolysis in vivo is to measure concentrations of ultimate excretory products and relate this to events earlier in the catabolic pathway. For example, isothiocyanates are excreted as their mercapturic acid derivatives in rats and thus provide potentially useful markers for estimating rates of release of isothiocyanates from sinigrin in vivo (Mennicke et al. 1983). In the experiment reported here, this approach was validated in rats and used to measure rates of release of AITC from sinigrin in animals consuming diets with or without added myrosinase.