Urinary metabolite profile of phenyl and o-cresyl glycidyl ether in rats: identification of a novel pathway leading to N-acetylserine O-conjugates.

The urinary excretion of metabolites of phenyl glycidyl ether (PGE) and o-cresyl glycidyl ether (o-CGE) was investigated in rats. Urine was collected, in fractions, from rats intraperitoneally administered PGE or o-CGE in doses ranging from 0.033 to 1.0 mmol/kg. The metabolites were extracted from acidified urine with ethyl acetate or diethyl ether, and their identity was elucidated by GC/MS analysis. The epoxide of PGE can be inactivated by glutathione (GSH) conjugation or epoxide hydrolysis. After further metabolism, these routes lead to the urinary excretion of phenyl glycidyl ether mercapturic acid (PGEMA) and 3-(phenyloxy)lactic acid (POLA). The excretion of PGEMA and POLA was described before and is confirmed in this study. Additionally, a new metabolite was identified as N-acetyl-O-phenylserine (NAPS), which is proposed to be formed from POLA by subsequent oxidation, transamination, and N-acetylation. For PGEMA a linear dose-excretion relationship was found (r2 = 0.988), and the percentage of the dose excreted declined from 27% to 10% with increasing PGE dose. For NAPS also a linear dose-excretion relationship was found (r2 = 0.985), and NAPS accounted for 27% of the PGE dose. The excretion of PGEMA and NAPS was rather fast: 93% and 75%, respectively, of the respective total cumulative amounts excreted was already collected within 6 h after administration. The urinary metabolite profile of o-CGE was not investigated in rats before. Three urinary metabolites of o-CGE were identified, namely, 3-(o-cresyloxy)lactic acid (COLA), o-cresyl glycidyl ether mercapturic acid (o-CGEMA), and N-acetyl-O-(o-cresyl)serine (NACS), showing that the metabolite profiles of PGE and o-CGE are comparable. Up to a o-CGE dose of 0.333 mmol/kg, the excretion of o-CGEMA was linear (r2 = 0.997), while above this dose the excretion did not increase anymore. The percentage of the o-CGE dose excreted as o-CGEMA declined from 31% to 11% with increasing dose. Again 93% of the total cumulative amount of o-CGEMA excreted was collected within 6 h after administration of o-CGE. Analytical methods were developed for the quantitative determination of mercapturic acid metabolites of PGE and o-CGE. These methods were sufficiently sensitive for their determination in urine of rats administered PGE or o-CGE in the dose range applied. It is anticipated that the analytical methods developed are also sufficiently sensitive to investigate excretion of the mercapturic acid metabolites in humans occupationally exposed to low air concentrations (<6 mg/m3 of air, 8h-TWA) of PGE or o-CGE.

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 for AC, both types of variables were measured in 136 workers involved in maintenance operations. METHODS: Potential airborne exposure to AC was measured by personal air monitoring in the breathing zone. In total 205 workshifts were evaluated. During 99 workshifts no respiratory protection equipment was used. Mercapturic acid metabolites were measured in urinary extracts by gas chromatography-mass spectrometry (GC-MS). RESULTS: During 86 work shifts when no respiratory protection was used the air concentrations of AC were below the Dutch eight hour time weighted average (8 h-TWA) occupational exposure limit (OEL) of AC (3 mg/m3), whereas in 13 workshifts the potential exposure, as measured by personal air monitoring, exceeded the OEL (3.3 to 17 mg/m3). With the aid of GC-MS, 3-hydroxypropylmercapturic acid (HPMA) was identified as a minor and allylmercapturic acid (ALMA) as a major metabolite of AC in urine samples from the maintenance workers exposed to AC. The concentrations of ALMA excreted were in a range from < 25 micrograms/l (detection limit) to 3550 micrograms/l. The increases in urinary ALMA concentrations during the workshifts correlated well with the 8h-TWA air concentrations of AC (r = 0.816, P = 0.0001, n = 39). Based on this correlation, for AC a biological exposure index (BEI) of 352 micrograms ALMA/g creatinine during an eight hour workshift is proposed. In some urine samples unexpectedly high concentrations of ALMA were found. Some of these could definitely be attributed to dermal exposure to AC. In other cases garlic consumption was identified as a confounding factor. CONCLUSION: The mercapturic acid ALMA was identified in urine of workers occupationally exposed to airborne AC and the increase in ALMA concentrations in urine during a workshift correlated well with the 8 h-TWA exposure to AC. Garlic consumption, but not smoking, is a potential confounding factor for this biomarker of human exposure to AC.

3-Chloro-2-hydroxypropylmercapturic acid and α-chlorohydrin as biomarkers of occupational exposure to epichlorohydrin.

Until now no urinary biomarker of exposure was available to assess human exposure to epichlorohydrin (ECH). For this purpose the urinary excretion of mercapturic acids and α-chlorohydrin (α-CH), which are potential metabolites of ECH in humans was investigated. This study was undertaken in a chemical plant in which ECH is used in the production of glycidyl ethers. Urine samples were collected from 19 persons at the beginning and at the end of work-shifts and at the morning after the last work-shift. Respiratory air concentrations of ECH were determined by personal air monitoring (PAM) and were found to range from<0.03 to 1.1 mg/m(3) (8 h-TWA, median 0.09, n=23). The determined respiratory exposure to ECH was in all cases below the current occupational exposure limit of 4 mg/m(3) for ECH (8 h-TWA-OEL). In one additional case a dermal exposure to an unknown amount of technical ECH was noted. Urinary metabolites were isolated by ethyl acetate extraction or by lyophilization and determined by GC-MS. In ethyl acetate extracts of acidified urine samples of workers with potential occupational exposure to ECH, 3-chloro-2-hydroxypropylmercapturic acid (CHPMA) was identified with GC-MS and the concentrations measured ranged from<0.05 (detection limit) to 5.35 mmol/mol creatinine. The increase of the CHPMA excretion during the work-shifts, corrected for creatinine excretion, correlated well with the 8 h-TWA respiratory air concentrations of ECH (r(2)=0.94, n=7). For 8 individuals it was possible to assess an urinary half-life for the excretion of CHPMA (2.54±0.94 h). By extrapolating the relation between the ambient air concentrations of ECH and the urinary CHPMA excretions, an excretion of 6.2 mmol CHPMA/mol creatinine (tolerance levels of 95% C.I.: 5.1-7.3) is predicted if ECH exposure is at the level of the current OEL. The urinary excretion of two other known metabolites of ECH in rats, namely α-CH and 2,3-dihydroxypropylmercapturic acid (DHPMA) was also investigated. α-CH was identified in urine of workers exposed to low air concentrations of ECH but DHPMA could only be identified after the dermal exposure to technical ECH. In these latter samples CHPMA and α-CH were determined up to 167 and 6.3 mmol/mol creatinine, respectively. From this investigation it is concluded that urinary excretion of the mercapturic acid CHPMA is an appropriate biomarker of human exposure to ECH. A tentative biological exposure index (BEI) of 6 mmol CHPMA/mol creatinine for ECH during an 8 h work-shift is proposed.

Identification and quantitative determination of 3-chloro-2-hydroxypropylmercapturic acid and alpha-chlorohydrin in urine of rats treated with epichlorohydrin.

Epichlorohydrin (ECH) is used in many industrial processes. Different toxic effects of ECH were found in rodents. The metabolism of ECH was investigated before in rats using [14C]ECH. The aim of this investigation was the development of non-radioactive quantitative analytical methods for measuring two urinary metabolites of ECH, namely 3-chloro-2-hydroxypropylmercapturic acid (CHPMA) and alpha-chlorohydrin (alpha-CH). The identity of CHPMA and alpha-CH excreted in urine of rats treated with 5 to 35 mg/kg ECH was confirmed by GC-MS. The quantitative analysis of CHPMA, involving ethyl acetate extraction from acidified urine and subsequent methylation and analysis by gas chromatography-flame photometric detection (GC-FPD), showed a method limit of detection of 2 micrograms/ml. The analysis of alpha-CH based on ethyl acetate extraction and subsequent analysis by GC-ECD, showed a method limit of detection of 2 micrograms/ml. CHPMA and alpha-CH derivatives could be determined quantitatively down to concentrations of 0.5 and 0.4 micrograms/ml urine, respectively, by selected-ion monitoring GC-MS under EI conditions. Cumulative urinary excretion of CHPMA and alpha-CH by rats treated with ECH were found to be 31 +/- 10 and 1.4 +/- 0.6% (n = 13) of the ECH dose, respectively. For CHPMA, the dose-excretion relationship suggested partially saturated ECH metabolism. For alpha-CH, the doe-excretion relationship was linear. With fractionated urine collection it was found that approximately 74 and 84% of the total cumulative excretion of CHPMA and alpha-CH, respectively, took place within the first 6 h after administration of ECH. From these investigations it is concluded that the GC-FPD and GC-ECD based methods developed are sufficiently sensitive to measure urinary excretion of CHPMA and alpha-CH in urine from rats administered 5 to 35 mg/kg ECH. It is anticipated that the analysis of CHPMA and alpha-CH based on GC-MS may be sufficiently sensitive to investigate urinary excretion from humans occupationally exposed to ECH.

Biotransformation of allyl chloride in the rat. Influence of inducers on the urinary metabolic profile.

Allyl chloride (AC) is used as intermediate in the synthesis of epichlorohydrin (ECH). We investigated the biotransformation of AC in rats to select potential urinary biomarkers of exposure. For this purpose, we developed analytical methods to measure different selected urinary metabolites of AC. The earlier described urinary metabolites of AC [allyl mercapturic acid (ALMA) and 3-hydroxypropyl mercapturic acid (HPMA)], as well as two urinary metabolites of ECH [alpha-chlorohydrin (alpha-CH) and 3-chloro-2-hydroxypropyl mercapturic acid (CHPMA)], were determined in this study. After intraperitoneal administration of AC, in doses ranging from 66 to 590 mumol/kg, control rats excreted 30 +/- 6.5% of the AC dose as ALMA. HPMA was a minor urinary metabolite of AC (< 3% of the AC dose), and, for this metabolite, no clear dose-excretion relationship was found. Two other minor urinary metabolites were also found as well, namely CHPMA and alpha-CH, suggesting the formation of ECH. CHPMA excretion was linear from 66 to 330 mumol/kg AC and amounted to 0.21 +/- 0.08% of the AC dose. alpha-CH excretion was linear in the dose range used and was excreted for 0.13 +/- 0.02% of the AC dose. In addition, we investigated the influence of three different enzyme inducers on the urinary metabolite profile of AC, namely pyrazole, beta-naphthoflavone, and phenobarbital. Pyrazole only increased the urinary excretion of alpha-CH. beta-Naphthoflavone induction only enhanced the ALMA excretion significantly. Phenobarbital inducted both the excretion of CHPMA and alpha-CH. From these studies, we conclude that urinary excretion of ALMA, CHPMA, and alpha-CH can be used as biomarkers in humans potentially exposed to AC. However, ALMA seems to be the more appropriate biomarker, because enzyme induction may play a confounding role if CHPMA or alpha-CH is used.

Urinary excretion of N-acetyl-S-allyl-L-cysteine upon garlic consumption by human volunteers.

N-Acetyl-S-allyl-L-cysteine (allylmercapturic acid, ALMA) was previously detected in urine from humans consuming garlic. Exposure of rats to allyl halides is also known to lead to excretion of ALMA in urine. ALMA is a potential biomarker for exposure assessment of workers exposed to allyl halides. It is not known whether garlic consumption can lead to urinary concentrations of ALMA which may interfere with biological monitoring of exposure to allyl halides by determination of urinary ALMA. Therefore, this study was undertaken to determine the cumulative excretion and the excretion kinetics of ALMA in urine of humans consuming garlic. Six human volunteers were given orally two garlic tablets, each containing 100 mg garlic extract (each representing 300 mg fresh garlic). Three of the volunteers consumed additional garlic after the garlic tablet intake. Urine samples were collected up to 24 h after the intake of the garlic tablets. ALMA was identified in the urine using gas chromatography-mass spectrometry (GC-MS) and determined quantitatively with a limit of detection of 0.10 microgram/ml with gas chromatography with sulphur selective detection. The total amount of ALMA found in urine of volunteers who consumed two garlic tablets was 0.43 +/- 0.14 mg (n = 3). In the urine of the three volunteers who consumed not only two garlic tablets but also additional fresh garlic, a significantly higher amount of ALMA was excreted in the urine, 1.4 +/- 0.2 mg (n = 3). The elimination half-life of ALMA, estimated from urinary excretion rate versus time curves, was 6.0 +/- 1.3 h (n = 5). One volunteer, who ate additional garlic, showed an irregular elimination profile and was excluded from this estimation. The highest urinary concentration of ALMA found in this study was 2.2 micrograms/ml. In a preliminary biological monitoring study of exposure in workers with potential exposure to allyl chloride (AC) up to the occupational exposure limit of 1 ppm (8-h TWA), we recently found urinary ALMA concentrations up to 4 micrograms/ml. Based on the results presented here, we conclude that garlic consumption is a potential confounder when monitoring human exposure to allylhalides and other chemicals leading to ALMA excretion when ALMA is used as a biomarker of exposure.

Mechanism of the reaction of ebselen with endogenous thiols: dihydrolipoate is a better cofactor than glutathione in the peroxidase activity of ebselen.

The therapeutic effect of ebselen has been linked to its peroxidase activity. In the present study, the peroxidase activity of ebselen toward H2O2 with the endogenous thiols GSH and dihydrolipoate [L(SH)2] as cofactors was determined. When GSH was used, peroxide removal was described by a ter uni ping pong mechanism with Dalziel coefficients for GSH and H2O2 of 0.165 +/- 0.011 and 0.081 +/- 0.005 mM min, respectively. When L(SH)2 was used, peroxidase activity was independent of the concentration of L(SH)2 in the concentration range studied (5 microM to 2 mM) and peroxide removal was only dependent on the concentration of H2O2 and ebselen, with the second-order rate constant being 12.3 +/- 0.8 mM-1 min-1. To elucidate the difference between GSH and L(SH)2, the molecular mechanism of the peroxidase activity of ebselen was investigated, using UV spectrophotometry, high pressure liquid chromatography, 77Se NMR, and mass spectrometry. GSH was found to react quickly with ebselen to give a selenenyl sulfide, an adduct of GSH to ebselen. Subsequently, the GSH-selenenyl sulfide is converted into the diselenide of ebselen. Finally the diselenide reacts with a peroxide and ebselen is regenerated. The formation by GSH of the diselenide from the GSH-selenenyl sulfide of ebselen is slow and linearly dependent on the concentration of free thiol; however, no net consumption of GSH was observed. Furthermore, it is likely that a selenol is an intermediate in diselenide formation. After reaction between ebselen and L(SH)2 the diselenide of ebselen was immediately detected. The fast formation of the diselenide with L(SH)2 versus the slow formation of the diselenide with GSH accounts for our observation that L(SH)2 is a better cofactor than GSH in the peroxidase activity of ebselen. Our results suggest that the interaction between ebselen and L(SH)2 might be of major importance in the mechanism by which ebselen exerts its therapeutic effect.

Beta-adrenoceptor agonists do not reduce hydrogen peroxide production from superoxide radicals.

Recently, it has been reported that beta-adrenoceptor agonists containing a catechol moiety, reduce hydrogen peroxide production by alveolar macrophages, an effect suggested to be due to scavenging by the catecholamines of superoxide, the precursor of hydrogen peroxide. However, catecholamines interfere with the method used, in that study, to determine hydrogen peroxide formation. We re-examined the obtained results using two independent methods for measuring hydrogen peroxide that are not affected by catecholamines. We found that catecholamines, in a concentration up to 10(-5) M, had no effect on hydrogen peroxide formation out of superoxide. It was, furthermore, established that reduction of hydrogen peroxide formation by scavenging of superoxide by catecholamines is not very likely.