Excretion Of Mercapturic Acids Research Articles

Mercapturic acids, protein adducts, and DNA adducts as biomarkers of electrophilic chemicals.

The possibilities and limitations of using mercapturic acids and protein and DNA adducts for the assessment of internal and effective doses of electrophilic chemicals are reviewed. Electrophilic chemicals may be considered as potential mutagens and/or carcinogens. Mercapturic acids and protein and DNA adducts are considered as selective biomarkers because they reflect the chemical structure of the parent compounds or the reactive electrophilic metabolites formed during biotransformation. In general, mercapturic acids are used for the assessment of recent exposure, whereas protein and DNA adducts are used for the assessment of semichronic or chronic exposure. 2-Hydroxyethyl mercapturic acid has been shown to be the urinary excretion product of five different reactive electrophilic intermediates. Classification of these electrophiles according to their acid-base properties might provide a tool to predict their preference to conjugate with either glutathione and proteins or with DNA. Constant relationships appear to exist in the cases of 1,2-dibromoethane and ethylene oxide between urinary mercapturic acid excretion and DNA and protein adduct concentrations. This suggests that mercapturic acids in some cases may also play a role as a biomarker of effective dose. It is concluded that simultaneous determination of mercapturic acids, protein and DNA adducts, and other metabolites can greatly increase our knowledge of the specific roles these biomarkers play in internal and effective dose assessment. If the relationship between exposure and effect is known, similar to protein and DNA adducts, mercapturic acids might also be helpful in (individual) health risk assessment.

Potential tolerance against bromobenzene-induced acute hepatotoxicity due to prior subchronic exposure

Groups of adult male Sprague-Dawley rats were treated i.p. with 0, 0.3 and 0.5 mmole bromobenzene (BB)/kg per day in corn oil, 5 days a week for 4 weeks. Thereafter, one half of each of these groups was treated i.p. with a single acute toxic dose of 2.5 mmole BB/kg. Urines were then collected for 24 h and the animals were then sacrificed. The hepatotoxicity induced by an acute dose of BB was significantly reduced due to prior subchronic exposure to BB at 0.5 mmol/kg, but not so at 0.3 mmol/kg. These data indicate a potential tolerance against acute hepatotoxicity of BB due to prior subchronic exposure. A significant increase in the urinary excretion of thioethers or mercapturic acids of BB combined with a significant increase in the urinary level of p-bromocatechol due to prior subchronic treatment with 0.5 mmol BB/kg relative to those due to acute treatment alone was observed. Thus enhanced bromobenzene metabolism could partly explain such potential tolerance against its acute hepatotoxicity. Such protection may also be related to certain cellular events which might occur subsequent to metabolic activation of BB.

Environmental and biological monitoring of non-occupational exposure to 1,3-dichloropropene

Voluntary bystanders, simulating a situation of non-occupational exposure to Z- and E-1,3-dichloropropene (Z- and E-DCP), were exposed during field application of this nematocide in the Dutch flower-bulb culture. Environmental monitoring revealed that mean respiratory exposure concentrations of Z- and E-DCP varied from non-detectable levels to 1.12 mg/m3 8-h time-weighted average (TWA) for Z-DCP and to 0.91 mg/m3 8-h TWA for E-DCP. Biological monitoring was executed by determining urinary mercapturic acid metabolites of Z- and E-DCP according to a method recently validated in occupationally exposed applicators. A linear relationship between respiratory exposure to Z- and E-DCP and the urinary excretion of both mercapturic acids was observed in bystanders. Dermal uptake did not contribute significantly to the internal dose of Z- or E-DCP. The urinary mercapturic acid of Z-DCP was a more sensitive parameter for the detection of exposure than was respiratory air monitoring. In future studies it would be worthwhile to determine the extent of exposure of real bystanders to DCP on the basis of urinary mercapturic acid excretion.

Thioether excretion in urine of applicators exposed to 1,3-dichloropropene: a comparison with urinary mercapturic acid excretion.

The excretion of thioethers in urine of applicators occupationally exposed to the soil fumigant 1,3-dichloropropene (DCP) was determined by the thioether assay. The mercapturic acid metabolite of E-1,3-dichloropropene, N-acetyl-S-(E-3-chloropropenyl-2-)-L-cysteine (E-DCP-MA), was the reference compound in the thioether assay. The mean recovery of E-DCP-MA was 58.5% (coefficient of variation (CV) 9%, n = 4). In non-exposed men mean background of urinary thioethers was 6.05 mmol SH/mol creatinine (n = 56). In applicators exposed to soil fumigants containing DCP, urinary excretion of thioethers followed first order elimination kinetics. Urinary half lives of elimination of thioethers were 8.0 (SD 2.5) hours based on excretion rates and 9.5 (SD 3.1) hours based on creatinine excretion. The urinary half life of elimination of thioethers was almost twofold higher compared with half lives of elimination of the mercapturic acids of Z- and E-1,3-dichloropropene. The post- minus pre-shift thioether concentrations in urine and the cumulative urinary thioether excretions correlated well with exposure to DCP. In urine samples the mean thioether concentration was 1.38 higher than mean DCP mercapturic acid concentration. This suggests the presence of unidentified thioether metabolite(s) due to exposure to soil fumigants containing DCP. According to the present data, an eight hour time weighted average exposure to the Dutch occupational exposure limit of 5 mg/m(3) DCP results in a post- minus pre-shift thioether concentration of 9.6 mmol SH/mol creatinine (95% confidence interval (95%CI) 7.4-11.8 mmol SH/mon creatinine) and in a cumulative thioether excretion of 139 micromol SH (95% CI 120-157 micromol SH). It is concluded that the thioether assay can be used to assess comparatively high levels of exposure to DCP.

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

Mononuclear lymphocytes were isolated from the blood of 12 individuals, who had been exposed to the vapour of the soil fumigant 1,3-dichloropropene (DCP). Western blot experiments were performed on the crude lymphocyte homogenates, using a monoclonal antibody against human hepatic glutathione S-transferase (GST) isoenzyme mu, to determine the presence or absence of mu-class isoenzymes mu and/or psi. Nine of the individuals were found to be positive for mu and/or psi, the remaining three individuals being negative. In addition, all individuals showed a positive staining on immunoblot of a protein of somewhat lower molecular mass than the hepatic standard. This protein was bound by the S-hexylglutathione affinity column, and presumably constitutes a new mu-class isoenzyme, which is not subject to genetic polymorphism. Determination of the specific activities of individual human GST isoenzymes towards Z-(cis-) and E-(trans-)-DCP demonstrated that mu-class isoenzymes show a considerably higher specific activity with Z-DCP than alpha-class or pi-class isoenzymes. In addition, mu-class isoenzymes were found to be 2- to 3-fold more active with Z-DCP than with E-DCP. Their activity towards E-DCP was similar to the specific activity of alpha-class isoenzymes. Genetic polymorphism for mu-class isoenzymes could thus be a determinant in the extent of excretion of mercapturic acids from Z- and E-DCP. The urinary excretion of Z- and E-DCP mercapturic acids and the respiratory exposure to Z- and E-DCP were determined for nine and eight phenotyped individuals, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary excretion of mandelic, phenylglyoxylic, and specific mercapturic acids in rats exposed repeatedly by inhalation to various concentrations of styrene vapors

Adult male Sprague-Dawley rats were exposed by inhalation to various concentrations of styrene vapors (25, 50, 100, or 200 ppm) 6 h/day, 5 days/week, for 4 consecutive weeks. The concentrations were varied from day to day according to a random pattern allowing treated animals to be exposed five times to each concentration of styrene. Each day, the following urinary metabolites were analysed from samples collected during exposure (0-6 h) and after exposure (6-24 h): mandelic acid; phenylglyoxylic acid; and two mercapturic acids, N-acetyl-S-(1-phenyl-2-hydroxyethyl)-L-cysteine (M1) and N-acetyl-S-(2-phenyl-2-hydroxyethyl)-L-cysteine (M2). Various parameters of renal toxicity and hepatic microsomal and cytosolic enzyme activities were also measured. The results show that there is a very good relationship between the excretion of all four styrene metabolites and the degree of daily exposure to styrene over the entire period of urine collection, with correlation coefficients ranging from 0.82 to 0.98. The correlation was poor for mandelic acid during the 0-6 h period. There was no evidence that repeated exposure to styrene caused renal toxicity, nor induced hepatic microsomal enzyme activities; cytosolic glutathione S-transferase activity was increased moderately by 1.5 times. Thus, under conditions of exposure to styrene likely to be found in the workplace, all four metabolites measured were good indicators of styrene exposure throughout the length of the experiment. Since mercapturic acids result from the conjugation of styrene oxide with glutathione, the data suggest that measurement of these metabolites offers the possibility to monitor internal exposure to a toxic electrophilic compound more directly.

Liver toxicity due to 1,2-dichloropropane in the rat

The effect of 1,2-dichloropropane on rat liver was studied after short (5 days) and long term (4 weeks) i.p. administration. Animals were injected daily with 10-500 mg/kg body wt 1,2-dichloropropane and biochemical and histological changes of liver were investigated. Treatment was monitored by measuring urinary mercapturic acid excretion. A significant increase of mercapturate excretion was observed at all dose levels, with no further increase during the treatment; at lower doses a return to baseline values occurred within 48 h after the end of treatment. Mercapturate excretion at the end of weeks 2, 3 and 4 of treatment was significantly lower than that observed at the end of week 1. The liver reduced glutathione content was different after single or repeated injections. A dose-dependent decrease of liver reduced glutathione was observed after a single injection and a dose-dependent increase after 4 weeks. The liver biochemical pattern after 4 weeks of treatment (characterized by a decrease of cytochrome P-450 and by an increase of reduced glutathione and glutathione S-transferase activity) suggests a hyperplastic evolution of the liver cells, probably a repair mechanism induced by early depletion of reduced glutathione. Light microscopy confirms that the prevalent alterations are regenerative in type (atypical mitosis and hyperplastic nodules). Areas of focal necrosis are isolated, and trend to disappear after long term treatment.

Influence of a cysteine prodrug, L-2-oxothiazolidine-4-carboxylic acid, on the urinary elimination of mercapturic acids of ethylene oxide, dibromoethane, and acrylonitrile: a dose-effect study.

Metabolic disposition of ethylene oxide, dibromoethane, and acrylonitrile in rats after acute exposure was studied by examining the relationship between dose and urinary metabolites, and by establishing the influence of a glutathione precursor, L-2-oxothiazolidine-4-carboxylic acid (OTCA), on the above relationship. Respective urinary metabolites, hydroxyethylmercapturic acid, cyanoethylmercapturic acid, thiocyanate, and ethylene glycol, were quantified to estimate the extent to which each compound was metabolized. The animals were given either ethylene oxide (0.34, 0.68, or 1.36 mmol/kg), dibromoethane (0.2, 0.4, or 0.6 mmol/kg), or acrylonitrile (0.10, 0.38, or 0.76 mmol/kg). Urine samples were collected at 24 h. The metabolic biotransformation of all three chemicals to their respective mercapturic acids was strongly indicative of saturable metabolism. Administration of OCTA (4-5 mmol/kg) enhanced gluthathione availability and increased excretion of urinary mercapturic acids at the higher doses of the chemicals. This study indicates that OTCA increases the capacity for detoxification via the glutathione pathway thereby partially correcting the nonlinearity between the administered dose of ethylene oxide, dibromoethane, and acrylonitrile and the amount of certain urinary metabolites.

Mercapturic acid excretion by rats following inhalation exposure to 1,3-dichloropropene

Rats were exposed to 1,3-dichloropropene (DCP), a commonly used agricultural nematicide, by inhalation to assess the relationship between DCP concentration and the urinary excretion of the mercapturic acid of cis-DCP (3C-NAC). The nose-only exposure system that was used for simultaneously exposing up to four rodents is decribed. This apparatus provided for generation and monitoring of relative humidity and test vapor concentration. Animals were exposed for 1 hr to concentrations of up to 789 ppm DCP. Urine was collected for 24 hr after exposure. The quantity of 3C-NAC contained in the urine collections exhibited an exposure concentration-dependent increase from 0 to 284 ppm DCP. However, the amount of 3C-NAC was no greater for animals exposed to 398 or 789 ppm DCP than for animals exposed to 284 ppm DCP.

Mercapturic Acid Excretion by Rats following Inhalation Exposure to m1,3-Dichloropropene

Mercapturic Acid Excretion by Rats following Inhalation Exposure to m1,3-Dichloropropene

The contribution of bromobenzene to our current understanding of chemically-induced toxicities

The metabolism and toxicity of bromobenzene has been investigated for well over one hundred years. The urinary excretion of mercapturic acids was first reported in 1879, in animals treated with bromobenzene. Bromobenzene has since proven to be a valuable tool in efforts to unravel the complexities involved in chemical- induced toxicities. For example, the importance of metabolic activation via the cytochrome(s) P-450; the role of glutathione in the detoxification of reactive metabolites; and the toxicological significance of covalent binding, enzyme inactivation and lipid peroxidation have all been illustrated in studies with bromobenzene. Thus, many of the principles involved in chemical-induced toxicity have been exemplified in studies with bromobenzene. These studies have provided substantial insight into the role of chemically reactive metabolites in the genesis of xenobiotic-mediated cytotoxicity.

Urinary excretion of mercapturic acids and thiocyanate in rats exposed to acrylonitrile: influence of dose and route of administration

Adult male Sprague-Dawley rats were exposed acutely to acrylonitrile (ACN) according to 1 of 3 different routes of administration: inhalation (6 h), i.v., or i.p. Urinary metabolites measured 24 h after administration were 2-cyanoethylmercapturic acid (CMA), 2-hydroxyethylmercapturic acid (HMA) and thiocyanate (TCN). In all 3 series of experiments, the relationship between excretion of total urinary metabolites and the degree of exposure was reasonably linear. However, there was a marked influence of the route of administration on the pattern of metabolic excretion. For example, after i.p. and i.v. injection CMA was the most important metabolite while after inhalation it was TCN. Our results also indicate an important effect of the dose on the pattern of excretion for all types of administration.

Effect of a cysteine prodrug, L-2-oxothiazolidine-4-carboxylic acid, on the metabolism and toxicity of bromobenzene: an acute study.

The effect of a cysteine prodrug, L-2-oxothiazolidine-4-carboxylic acid (OTCA), on certain aspects of the metabolism and toxicity of bromobenzene administered acutely to mice was investigated by (i) characterizing the influence of OTCA on the metabolic profile of low and high bromobenzene dose at 0-6, 6-12, and 12-24 h, (ii) determining the effective doses range and administration time for OTCA, as well as the optimum period for urine sampling; and (iii) measuring the efficacy of OTCA for protection against bromobenzene induced toxicity. Coadministration of OTCA and bromobenzene enhanced the urinary excretion of mercapturic acid and phenolic metabolites, during 6-12 h, by approximately 152 and 193%, respectively. Maximum efficacy was observed when OTCA (16.0 mmol/kg) was administered concomitantly with bromobenzene (4.0 mmol/kg). Finally, OTCA administration was found to afford substantial protection against elevation of plasma transaminases used as indices of bromobenzene-induced hepatotoxicity. N-acetylcysteine, another cysteine prodrug, had essentially similar effects on the metabolism and toxicity of bromobenzene. Thus, administration of cysteine prodrugs enhances the urinary excretion of several metabolites of bromobenzene and affords protection against bromobenzene-induced hepatotoxicity.

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 degrees C, pH 7.4). 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Dose-dependent metabolism of trichloroethylene and its relevance to hepatotoxicity in rats

Fasted male Sprague-Dawley rats pretreated with phenobarbital received an intraperitoneal injection of 0.00, 0.25, 0.50, 0.75, 1.00, 1.50, or 2.00 ml/kg of trichloroethylene (TRI) in corn oil. The metabolic fate of TRI was monitored by measuring its major urinary metabolites 24 hr following the dosing. The hepatotoxic response of TRI was evaluated by determination of the serum transaminase activities (SGOT and SGPT) 24 hr after dosing. Treatment of rats with TRI up to 0.5 ml/kg did not affect the transaminase responses, but significant response was manifested at 0.75 ml/kg dose. Further continuous increases in such responses were induced on exposure to higher doses. Dose-dependent urinary excretions of trichloroethanol and trichloroacetic acid were observed and reached an apparent saturation at 1-1.5 ml/kg dose of TRI. The percentage of dose excreted as trichloroethanol was decreased from 16% at 0.25 ml or 2.8 mmole/kg to 8% at 2 ml or 22.3 mmole/kg dose. The percentage of trichloroacetic acid was decreased from 5 to 2% for the same dose interval. A maximum of only 29% depletion of hepatic glutathione was observed at 2 hr following 1 ml/kg dose of TRI. Similarly, the excretion of urinary mercapturic acid of TRI or thioether was insignificant at any dose level. These results suggest that the conjugation of hepatic glutathione with the electrophilic intermediate of TRI does not seem to be an important determinant for TRI hepatotoxicity nor the major detoxification pathway of its reactive intermediate. TRI produced also a dose-dependent decrease in hepatic microsomal monooxygenase activities as well as cytochrome P-450 content. All these results, when combined, show that there exists an apparent saturable metabolism of TRI involving its activation/deactivation pathways which correspond to an apparent threshold or minimal toxic dose, about 1 ml/kg or 11.15 mmole/kg of TRI for its hepatotoxicity.

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.

Investigations into the mechanism of coumarin-induced hepatotoxicity in the rat.

The administration of single doses of coumarin to the rat was found to produce hepatic centrilobular necrosis and also to depress a number of hepatic enzyme activities within 24 h. Coumarin-induced liver damage was diminished by pretreatment with cobaltous chloride but potentiated by the administration of diethyl maleate. Hepatic reduced non-protein sulphydryl levels were rapidly depleted following coumarin treatment whereas urinary mercapturic acid excretion was enhanced suggesting the formation of a coumarin metabolite or metabolites hitherto undetected in this species. In in vitro studies [3-14C]coumarin was converted by rat hepatic microsomes to reactive intermediates which became bound covalently to microsomal proteins. Additional studies established that the formation of reactive metabolites was a cytochrome P-450 dependent process and that macromolecular binding could be inhibited by sulphydryl compounds (including reduced glutathione) and hepatic cytosol fractions. These results demonstrate that coumarin-induced hepatotoxicity in the rat is likely to be mediated via one or more reactive metabolites generated by cytochrome P-450 dependent enzymes and that reduced glutathione and other thiol agents constitute a detoxification pathway.

Urinary mercapturic acid in chemical workers and in control subjects.

Mercapturic acid urinary excretion was followed in a control group and in chemical workers exposed to toxic and mutagenic compounds. Chemical workers had a significantly lower excretion of mercapturic acid when compared with controls. Smoking effects were not significant statistically. No correlation was found between urinary mutagenic activity and mercapturic acid urinary excretion.

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.

Isolation and identification of mercapturic acid metabolites of phenyl substituted acrylate esters from urine of female rats.

The urinary mercapturic acid excretion by female rats of methyl atropate (alpha-phenyl methyl acrylate) and methyl cinnamate (beta-phenyl methyl acrylate) has been studied. On the basis of the structures of these mercapturic acids the conclusion can be drawn that these compounds arise from a conjugation of glutathione with the acrylic esters in a Michael fashion. Previous administration of (tri-orthotolyl) phosphate (TOTP), a carboxy esterase inhibitor, enhances the capacity of the acrylate esters to alkylate glutathione in vivo. The amount increased from 1.5 to 22.8% of dose (1.0 mmol/kg) for methyl cinnamate and from 10.4 to 14.8% of dose (0.2 mmol/kg) for methyl atropate. Upon inhibition of the esterase activity the major actual mercapturic acid is a conjugate of the acrylate in which the ester function is retained. In the absence of an esterase inhibition the excreted mercapturic acid is a formal conjugate of the free acrylic acid (Fig. 1). No mercapturic acids could be detected which might arise from glutathione conjugation of a, beta-epoxyesters. Such epoxides are potential primary metabolites of unsaturated esters. They were not detected by in vitro experiments. Therefore, the intermediacy of glycidic esters in the biotransformation of these acrylic esters may be considered as highly unlikely.