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The urinary mercapturic acids N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA) and N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine (GAMA) are short-term biomarkers of exposure from acrylamide and its metabolite glycidamide, respectively. The medium-term exposure to acrylamide and glycidamide is monitored by the adducts N-(2-carbamoylethyl)-Val (AA-Val) and N-(2-carbamoyl-2-hydroxyethyl)-Val (GA-Val) in hemoglobin (Hb), respectively. Three questions were addressed by application of these biomarkers in two diet studies including 36 omnivores, 36 vegans and 16 strict raw food eaters (abstaining from any warmed or heated food for at least four months): first, what is the internal acrylamide exposure following a vegan or a raw food diet in comparison to that in omnivores? Second, did the exposure change between 2017 and 2021? And third, what is the stability over time of AAMA/GAMA excretion compared to that of AA-Val/GA-Val levels in Hb between both time points? Median urinary AAMA excretion per day in non-smoking omnivores, vegans and raw food eaters were 62.4, 85.4 and 15.4 µg/day, respectively; the corresponding median AA-Val levels were 27.7, 39.7 and 13.3 pmol/g Hb, respectively. Median levels in strict raw food eaters were about 25% (AAMA excretion) and 48% (AA-Val) of those in omnivores. In comparison to 2017, AAMA and GAMA excretion levels were hardly altered in 2021, however, levels of AA-Val and GA-Val in 2021 slightly increased. There was a weak correlation between AAMA excretion levels determined four years apart (rS = 0.30), and a moderate correlation between levels of AA-Val (rS = 0.55) in this timeframe. Our data in strict raw food eaters confirm a significant endogenous formation to acrylamide in a size range, which is—based on the levels of AA-Val—distinctly higher than reported previously based on levels of urinary AAMA excretion. The relatively lower AAMA excretion in raw food eaters likely represents a lower extent of glutathione conjugation due to missing hepatic first-pass metabolism in case of endogenous formation of acrylamide, which leads to a higher systemic exposure. [ABSTRACT FROM AUTHOR]

The formation of carbonyls and epoxides in e-cigarette (EC) aerosol is possible due to heating of the liquid constituents. However, high background levels of these compounds have inhibited a clear assessment of exposure during use of ECs. An EC containing an e-liquid replaced with 10% of 13C-labeled propylene glycol and glycerol was used in a controlled use clinical study with 20 EC users. In addition, five smokers smoked cigarettes spiked with the described e-liquid. Seven carbonyls (formaldehyde, acetaldehyde, acrolein, acetone, crotonaldehyde, methacrolein, propionaldehyde) were measured in the aerosol and the mainstream smoke. Corresponding biomarkers of exposure were determined in the user's urine samples. 13C-labeled formaldehyde, acetaldehyde and acrolein were found in EC aerosol, while all seven labeled carbonyls were detected in smoke. The labeled biomarkers of exposure to formaldehyde (13C-thiazolidine carboxylic acid and 13C-N-(1,3-thiazolidine-4-carbonyl)glycine), acrolein (13C3-3-hydroxypropylmercapturic acid) and glycidol (13C3-dihydroxypropylmercapturic acid) were present in the urine of vapers indicating an EC use-specific exposure to these toxicants. However, other sources than vaping contribute to a much higher extent by several orders of magnitude to the overall exposure of these toxicants. Comparing data for the native (unlabeled) and the labeled (exposure-specific) biomarkers revealed vaping as a minor source of user's exposure to these toxicants while other carbonyls and epoxides were not detectable in the EC aerosol. [ABSTRACT FROM AUTHOR]

Acrylamide (AA) is a heat-induced food contaminant considered as genotoxic carcinogen. The present study investigated the influence of nutritional and lifestyle preferences on human AA exposure. A 10-day human study was performed with ten volunteers without nutritional preferences (omnivores) and ten vegans. Volunteers self-reported their daily routine and dietary habits. Overall mean AA intake, calculated from contents of diet duplicates, was 0.32 ± 0.19 µg/kg body weight (bw)/day with marked inter-day and inter-volunteer variabilities. Vegans ingested more AA (0.38 ± 0.23 µg/kg bw/day) than omnivore volunteers without dietary restrictions (0.26 ± 0.10 µg/kg bw/day). Excretion kinetics of urinary AA-related mercapturic acids N-acetyl-S-(2-carbamoylethyl)-l-cysteine and N-acetyl-S-(2-hydroxy-2-carbamoylethyl)-l-cysteine were essentially concordant with the respective dietary AA intake. Disproportionately enhanced AA-related biomarker excretion could be traced back to reportedly inadvertent, passive exposure to tobacco and/or fire smoke, as evidenced by the respective urinary exposure biomarkers, cotinine and N-acetyl-S-(2-cyanoethyl)-l-cysteine. Although the study is based on the comparison of small volunteer groups, the results confirm the association of AA exposure biomarkers with documented dietary preferences and lifestyle factors. Some additional contribution of endogenous background AA exposure was demonstrated individually. Disproportionately enhanced AA exposure is suggested to result from passive exposure to tobacco and/or barbecue smoke. [ABSTRACT FROM AUTHOR]

The aim of the present study was to explore the relation of controlled dietary acrylamide (AA) intake with the excretion of AA-related urinary mercapturic acids (MA), N-acetyl- S-(carbamoylethyl)- l-cysteine (AAMA) and N-acetyl- S-(1-carbamoyl-2-hydroxyethyl)- l-cysteine (GAMA). Excretion kinetics of these short-term exposure biomarkers were monitored under strictly controlled conditions within a duplicate diet human intervention study. One study arm (group A, n = 6) ingested AA via coffee (0.15-0.17 µg/kg bw) on day 6 and in a meal containing an upper exposure level of AA (14.1-15.9 μg/kg bw) on day 10. The other arm (group B) was on AA minimized diet (washout, 0.05-0.06 µg/kg bw) throughout the whole 13-day study period. On day 6, these volunteers ingested CD-AA (1 μg/kg bw). In both arms, urinary MA excretion was continuously monitored and blood samples were taken to determine hemoglobin adducts. Ingestion of four cups of coffee resulted in a slightly enhanced short-term biomarker response within the background range of group B. At the end of the 13-day washout period, group B excreted an AAMA baseline level of 0.14 ± 0.10 µmol/d although AA intake was only about 0.06 µmol/d. This sustained over-proportional AAMA background suggested an endogenous AA baseline exposure level of 0.3-0.4 µg/kg bw/d. The excretion of CD-AA was practically complete within 72-96 h which rules out delayed release of AA (or any other MA precursor) from deep body compartments. The results provide compelling support for the hypothesis of a sustained endogenous AA formation in the human body. [ABSTRACT FROM AUTHOR]

Acrylamide (AA), classified as class 2A carcinogen (probably carcinogenic to humans) by the International Agency for Research on Cancer (IARC), is formed during heating of food from reducing carbohydrates and asparagine by Maillard reaction chemistry. After dietary uptake, AA is in part metabolically converted into the proximate genotoxic phase I metabolite glycidamide (GA). GA reacts with nucleophilic base positions in DNA, primarily forming N7-(2-carbamoyl-2-hydroxyethyl)guanine ( N7-GA-Gua) adducts. In a competing phase II biotransformation pathway AA, as well as its phase I metabolite GA, is coupled to glutathione (GSH). The GSH coupling products are further biotransformed and excreted via urine as mercapturic acids (MA), N-acetyl- S-(2-carbamoylethyl)cysteine (AAMA), and N-acetyl- S-(2-hydroxy-2-carbamoylethyl)cysteine (GAMA). In the present study, hepatic biotransformation pathways and DNA adduct formation were studied in primary rat hepatocytes, incubated with AA (0.2-2,000 μM) for up to 24 h. Contents of AA-GSH, GA, AAMA, and GAMA were measured in the cell culture medium after solid phase extraction (SPE). N7-GA-Gua adducts in DNA of hepatocytes were determined by HPLC-ESI-MS/MS after lysis of the cells and neutral thermal hydrolysis. Formation of AA-GSH was linear with AA concentration and incubation time and became detectable already at 0.2 μM (4 h). In contrast to AA, GA was not detected before 16 h incubation at 10-fold higher AA concentration (2 μM). In summary, the rate of AA-GSH formation was found to be about 1.5-3 times higher than that of GA formation. N7-GA-Gua adducts were found only at the highest AA concentration tested (2,000 μM). [ABSTRACT FROM AUTHOR]

We investigated the human metabolism of AA to the mercapturic acids N-acetyl-S-(2-carbamoylethyl)- l-cysteine (AAMA) and N-(R/S)-acetyl-S-(2-carbamoyl-2-hydroxyethyl)- l-cysteine (GAMA) which are derived from AA itself and from its oxidative genotoxic metabolite glycidamide (GA), respectively. A healthy male volunteer received a single dose of about 1 mg deuterium-labelled acrylamide (d3-AA), representing 13 μg/kg body weight, in drinking water. Urine samples before dosing and within 46 h after the dose were analysed for d3-AAMA and d3-GAMA by LC-ESI-MS/MS. A first phase of increase in urinary concentration was found to last 18 h with a broad plateau between 8 and 18 h for AAMA, and 22 h for GAMA. Elimination half-lives of both AAMA and GAMA were estimated to be approximately 3.5 h for the first phase and more than 10 h up to few days for the second phase. Total recovery in urine after 24 h was about 51% as the sum of AAMA and GAMA and hereby well in accordance with former studies in rats. After 2 days AAMA, accounting for altogether 52% of the total AA dose, was the major metabolite of AA in humans. GAMA, accounting for 5%, appeared as a minor metabolite of AA. In humans we found a urinary ratio of 0.1 for GAMA/AAMA compared to previously reported values of 0.2 for rats and 0.5 for mice. Therefore, the metabolic fate of AA in humans was more similar to that in rats than in mice as already demonstrated in terms of the haemoglobin adducts. Consequently a genotoxic potency of AA mediated by GA could be supposed to be comparable in rats and humans. [ABSTRACT FROM AUTHOR]

It was the aim of this study to ascertain whether xylenes form phenylmercapturic acids via aromatic epoxides in the human metabolism. Aromatic epoxides are suspected to exert mutagenic properties. Therefore we developed an LC/MS/MS procedure for the determination of these mercapturic acids. Using this method we were able to detect dimethylphenylmercapturic acid (DPMA) in urine samples of persons occupationally exposed to xylenes. The unequivocal LC/MS/MS detection of phenylmercapturic acid metabolites of xylene thanks to authentic standards was verified with an independent GC/MS method. Xylene concentrations in the air of the workplaces ranged between 0.7 and 58.1 ppm (median 12.6 ppm). The excretion of methylhippuric acid in the urine samples of the workers (n=27) ranges from 19.8 to 2332.5 mg/l (median 450.9 mg/l). DPMA was detected in only 9 samples of 27 exposed persons. According to a rough calculation DPMA is only formed in a ratio of 0.0003% respective to the xylene main metabolite MHA. That means that even under occupationally relevant xylene exposure potential mutagenic potencies should be negligible. DPMA in urine is not sensitive enough for general biomonitoring purposes due to the low ratio of excretion. MHA should therefore be used for biomonitoring from now on. Furthermore, our results show that irrespective of the structure of xylene isomers there is no preferences in metabolism. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 V∶VI=4∶1. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A mercapturic acid attached to the aromatic ring of toluene was for the first time detected in human urine as a metabolite of toluene. Since the metabolism of toluene is usually considered to take place at the side-chain, this gives, besides the biosynthesis of cresols, a further hint of a metabolic conversion of the aromatic system. We examined a group of 33 workers occupationally exposed to toluene, determining the concentrations of toluene in ambient air and in whole blood, o-cresol and hippuric acid in urine and p-toluylmercapturic acid (p-TMA) in urine. All blood and urine samples were collected post-shift. The renal excretion of S- p-toluylmercapturic acid showed highly significant correlations with established parameters of a biological monitoring of toluene. The median ambient air concentration was 63 ppm, ranging from 13 to 151 ppm, the median concentration of toluene in whole blood was 804 μg/l, corresponding to median urinary concentrations for o-cresol of 2.3 mg/l, hippuric acid of 2.3 g/l and p-TMA of 20.4 μg/l. p-TMA was not detectable in urine samples of a control group of 10 non-exposed persons. Both the German Biological Tolerance Values (BAT-values) for toluene in blood (1000 μg/l) and o-cresol in urine (3 mg/l) correspond to a mean p-TMA elimination of ∼50 g/l, and thus are in agreement with each other. According to our results p-TMA reflects internal toluene exposure diagnostically sensitive and specifical. With the developed analytical procedure we determined a median benzylmercapturic acid (BMA) concentration of 190 μg/l in the urine samples of the toluene exposed persons. We also determined a median BMA concentration of 30 μg/l in the control samples of non-exposed persons. However, these results are preliminary and require further confirmation as the reliability of the method was determined only for p-TMA. [ABSTRACT FROM AUTHOR]

The formation of cysteine S-conjugates is thought to play an important role in the nephrotoxicity of haloalkenes such as trichloroethene, tetrachloroethene and hexachlorobutadiene. Glutathione S-conjugates formed from these haloalkenes in the liver are processed to the corresponding cysteine S-conjugates, which may be N-acetylated to mercapturic acids and may be accumulated in the kidney. Haloalkene-derived cysteine S-conjugates are also substrates for cysteine conjugate β-lyases and reactive intermediates are formed in this reaction. The equilibrium between cysteine S-conjugate and mercapturic acid thus influences the extent of β-lyase dependent bioactivation and subsequently the nephrotoxicity of S-conjugates. In this study,␣we compared the rates of N-acetylation in vitro and the biotransformation, excretion and nephro toxicity of S-(1,2-dichlorovinyl)- l-cysteine (1,2-DCVC), S-(2,2-dichlorovinyl)- l-cysteine (2,2-DCVC), S-(1,2,2-trichlorovinyl)- l-cysteine (TCVC) and S-(1,2,3,4,4-pentachlorobutadienyl)- l-cysteine (PCBC) in rats after i.v. injection (40 μmoles/kg). Marked differences in the extent of enzymatic N-acetylation were observed; N-acetylation was most efficient with 2,2-DCVC and least efficient with 1,2-DCVC. In urine, within 48 h, most of the given 2,2-DCVC (77% of the recovered dose) and 1,2-DCVC (92%) were recovered as the corresponding mercapturic acids. In contrast, a higher percentage of cysteine S-conjugate and less of the mercapturic acid were recovered in urine after administration of PCBC and TCVC (50 and 23% of dose as mercapturic acid), respectively. Histopathological examination of the kidneys and urine clinical chemistry showed marked differences in the extent of renal damage. Necroses of the proximal tubules were found after TCVC, PCBC and 1,2-DCVC administration in male, but not in female rats. These differences in nephrotoxicity do not correlate with the balance of acetylation/deacetylation. The higher toxicity observed in male rats may indicate the involvement of other parameters such as uptake mechanisms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 μ, to determine the presence or absence of mu-class isoenzymes μ, and/or ψ. Nine of the individuals were found to be positive for μ and/or ψ, 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. Urinary excretion levels (corrected for the time weighted average 8-h exposure), the urinary ratio and the elimination half-lives of the mercapturic acids of Z- and E-DCP were compared with the data on the mu-phenotype. No statistically significant differences were observed between mu-class positive and mu-class negative individuals. [ABSTRACT FROM AUTHOR]

Z- and E-1,3-dichloropropene, mutagenic geometric isomers and major constituents of commercial soil fumigants, were found to be metabolized to mercapturic acid derivatives by the rat. Extremely small quantities of mixtures of the parent compounds were administered intraperitoneally to the rat and the isomeric urinary mercapturic acids were quantified in three ways. Gas chromatographic procedures with nitrogen selective, sulphur selective and mass spectrometric detection, using negative chemical ionization with single ion detection, were evaluated with respect to selectivity and sensitivity. Applying the former two techniques, urinary mercapturic acids could still be quantified following 5 μg doses of each of the dichloropropene isomers. With gas chromatography-negative chemical ionization mass spectrometry, only mercapturic acid metabolites arising from 25 μg doses and higher could be quantified because of interference from endogenous compounds. These results suggest that all three analytical methods can be used to determine exposure of men to soil fumigants containing low levels of 1,3-dichloropropene. [ABSTRACT FROM AUTHOR]

Acrylamide (AA) is carcinogenic in animals and classified by the International Agency for Research on Cancer as probably carcinogenic in humans. Regarding the AA contents of food the diet significantly contributes to the overall AA burden of the general population. However, it is unclear to which degree the diet, apart from smoking, contributes to the internal AA exposure. Therefore the influence of an AA-free diet on the excretion of urinary mercapturic acid metabolites derived from AA in three healthy volunteers fasting for 48 h was examined. Urinary AA mercapturic acid metabolites were considerably reduced after 48 h of fasting. The levels were even well below the median level in non-smokers. This confirms that the diet is the main source of environmental AA exposure in humans, apart from smoking. Other possible AA sources could be of minor quantitative importance only. [ABSTRACT FROM AUTHOR]