Urinary Mercapturic Acids Research Articles

Faster liquid chromatography-tandem mass spectrometry method for analysis of isomeric urinary mercapturic acid metabolites of crotonaldehyde, methacrolein, and methyl vinyl ketone

We report a significantly more rapid method for quantitation of the urinary mercapturic acids of the isomeric toxicants crotonaldehyde, methacrolein, and methyl vinyl ketone. The major innovation of this novel method is detection by liquid chromatography-negative atmospheric pressure chemical ionization-tandem mass spectrometry-selected reaction monitoring as opposed to detection by negative electrospray ionization in our previously reported method. The new method also uses an improved Raptor Biphenyl HPLC column. The total chromatographic analysis time was reduced to about 8 min compared to 35 min in our previously published method. Accuracy, precision, and ruggedness of the new method were established, and its suitability for the analysis of urine samples from 2500 cigarette smokers and non-smokers was confirmed. The improved method is practical for quantitation of these important toxicants in clinical studies requiring analysis of thousands of urine samples.

Kinetic Considerations in the Interpretation of Biomonitoring of 1,3-Butadiene Exposure by Determination of Urinary Mercapturic Acids.

1,3-Butadiene (BD) is classified as a human carcinogen, and occupational exposure should be minimized. This study examined the effectiveness of personal protective equipment (PPE) during the clean-up and repair of a storage tank containing sludge contaminated with BD. A total of 66 workers participated, providing repeat urine samples before and after the shift. Overall, 1286 samples were analyzed for 1,2-dihydroxy-4-(N-acetylcysteinyl)butane (DHBMA) and the isomers 2-hydroxy-1-(N-acetylcysteinyl)-3-butene and 1-hydroxy-2-(N-acetylcysteinyl)-3-butene (MHBMA). Both DHBMA and MHBMA are urinary metabolites of BD and serve as biomarkers for recent BD exposure. Established correlations between the urinary concentrations of these biomarkers and airborne BD levels allowed for exposure assessment. However, conclusions regarding the exceedances of occupational exposure limits can vary depending on whether DHBMA or MHBMA levels are considered. This study investigated this discrepancy by estimating the apparent urinary half-lives of DHBMA and MHBMA using sequential individual post- and pre-shift samples. The results indicated that the longer urinary half-life of MHBMA (19.7 ± 3.1 h) led to its accumulation during the work week, in contrast to DHBMA, which has a shorter half-life (10.3 ± 1.9 h) and showed limited accumulation. When the kinetic information was used to adjust for the MHBMA build-up over the week, the discrepancy with DHBMA resolved, confirming that exposure limit values were not exceeded and validating the effectiveness of the PPE used. In the context of biomonitoring, this study provides valuable insights into biomarker selection based on specific objectives. MHBMA is recommended for scenarios with uncertain exposure timing and activities, whereas DHBMA is the preferred biomarker for evaluating the effectiveness of protective measures in known exposure settings.

Internal exposure to heat-induced food contaminants in omnivores, vegans and strict raw food eaters: biomarkers of exposure to acrylamide (hemoglobin adducts, urinary mercapturic acids) and new insights on its endogenous formation

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.

Determinants of exposure to acrylamide in European children and adults based on urinary biomarkers: results from the “European Human Biomonitoring Initiative” HBM4EU participating studies

Little is known about exposure determinants of acrylamide (AA), a genotoxic food-processing contaminant, in Europe. We assessed determinants of AA exposure, measured by urinary mercapturic acids of AA (AAMA) and glycidamide (GAMA), its main metabolite, in 3157 children/adolescents and 1297 adults in the European Human Biomonitoring Initiative. Harmonized individual-level questionnaires data and quality assured measurements of AAMA and GAMA (urine collection: 2014–2021), the short-term validated biomarkers of AA exposure, were obtained from four studies (Italy, France, Germany, and Norway) in children/adolescents (age range: 3–18 years) and six studies (Portugal, Spain, France, Germany, Luxembourg, and Iceland) in adults (age range: 20–45 years). Multivariable-adjusted pooled quantile regressions were employed to assess median differences (β coefficients) with 95% confidence intervals (95% CI) in AAMA and GAMA (µg/g creatinine) in relation to exposure determinants. Southern European studies had higher AAMA than Northern studies. In children/adolescents, we observed significant lower AA associated with high socioeconomic status (AAMA:β = − 9.1 µg/g creatinine, 95% CI − 15.8, − 2.4; GAMA: β = − 3.4 µg/g creatinine, 95% CI − 4.7, − 2.2), living in rural areas (AAMA:β = − 4.7 µg/g creatinine, 95% CI − 8.6, − 0.8; GAMA:β = − 1.1 µg/g creatinine, 95% CI − 1.9, − 0.4) and increasing age (AAMA:β = − 1.9 µg/g creatinine, 95% CI − 2.4, − 1.4; GAMA:β = − 0.7 µg/g creatinine, 95% CI − 0.8, − 0.6). In adults, higher AAMA was also associated with high consumption of fried potatoes whereas lower AAMA was associated with higher body-mass-index. Based on this large-scale study, several potential determinants of AA exposure were identified in children/adolescents and adults in European countries.

Levels of Urinary Mercapturic Acids of Acrolein, Methacrolein, Crotonaldehyde, and Methyl Vinyl Ketone in Relationship to Chronic Obstructive Pulmonary Disease in Cigarette Smokers of the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS).

Cigarette smoking is an established cause of chronic obstructive pulmonary disease (COPD). Numerous studies implicate acrolein, which occurs in relatively high concentrations in cigarette smoke and reacts readily with proteins, as one causative factor for COPD in smokers. Far less is known about the possible roles in COPD of the related α,β-unsaturated carbonyl compounds of cigarette smoke crotonaldehyde, methacrolein, and methyl vinyl ketone. In the study reported here, we analyzed mercapturic acids of these α,β-unsaturated compounds in the urine of 413 confirmed cigarette smokers in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS)─202 with COPD and 211 without COPD. The mercapturic acids analyzed were 3-hydroxypropyl mercapturic acid (3-HPMA) from acrolein, 3-hydroxy-1-methylpropyl mercapturic acid (HMPMA-1) from crotonaldehyde, 3-hydroxy-2-methylpropyl mercapturic acid (HMPMA-2) from methacrolein, and 3-hydroxy-3-methylpropyl mercapturic acid (HMPMA-3) from methyl vinyl ketone. In models adjusting for age, sex, race, pack years of tobacco use, and BMI, all four mercapturic acids were increased in individuals with COPD but not significantly. Stratified by the GOLD status, there were increased levels of the metabolites associated with GOLD 3-4 compared to that with GOLD 0, with the methacrolein metabolite HMPMA-2 reaching statistical significance (adjusted odds ratio 1.23 [95% CI: 1.00-1.53]). These results highlight the possible role of methacrolein, which has previously received little attention in this regard, as a causative factor in COPD in cigarette smokers.

Global Profiling of Urinary Mercapturic Acids Using Integrated Library-Guided Analysis.

Urinary mercapturic acids (MAs) are often used as biomarkers for monitoring human exposures to occupational and environmental xenobiotics. In this study, we developed an integrated library-guided analysis workflow using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry. This method includes expanded assignment criteria and a curated library of 220 MAs and addresses the shortcomings of previous untargeted approaches. We employed this workflow to profile MAs in the urine of 70 participants─40 nonsmokers and 30 smokers. We found approximately 500 MA candidates in each urine sample, and 116 MAs from 63 precursors were putatively annotated. These include 25 previously unreported MAs derived mostly from alkenals and hydroxyalkenals. Levels of 68 MAs were comparable in nonsmokers and smokers, 2 MAs were higher in nonsmokers, and 46 MAs were elevated in smokers. These included MAs of polycyclic aromatic hydrocarbons and hydroxyalkenals and those derived from toxicants present in cigarette smoke (e.g., acrolein, 1,3-butadiene, isoprene, acrylamide, benzene, and toluene). Our workflow allowed profiling of known and unreported MAs from endogenous and environmental sources, and the levels of several MAs were increased in smokers. Our method can also be expanded and applied to other exposure-wide association studies.

Study of urinary mercapturic acids as biomarkers of human acrylonitrile exposure

Exposure to the vinyl monomer acrylonitrile (AN) is primarily occupational. AN is also found in cigarette smoke. AN can be detoxified to form N-acetyl-S-(2-cyanoethyl)-cysteine (CEMA) or activated to 2-cyanoethylene oxide (CEO) and detoxified to form N-acetyl-S-(1-cyano-2-hydroxyethyl)-cysteine (CHEMA) and N-acetyl-S-(2-hydroxyethyl)-cysteine (HEMA). These urinary mercapturic acids (MAs) are considered to be potential biomarkers of AN exposure. This study assessed personal AN exposure, urinary MAs (CEMA, CHEMA, and HEMA), and cotinine (a biomarker of cigarette smoke) in 80 AN-exposed and 23 non-exposed factory workers from urine samples provided before and after work shifts. Unambiguous linear correlations were observed between levels of urinary CEMA and CHEMA with personal AN exposures, indicating their potential as chemically-specific biomarkers for AN exposures. AN exposure was the dominant factor in MA formation for AN-exposed workers, whereas urinary cotinine used as a biomarker showed that cigarette smoke exposure was the primary factor for non-exposed workers. The CHEMA/CEMA and (CHEMA+HEMA)/CEMA ratios in this human study differ from those in similar studies of AN-treated rats and mice in literature, suggesting a possible dose- and species-dependent effect in AN metabolic activation and detoxification.

Comparison of urinary mercapturic acid excretions in users of various tobacco/nicotine products.

Urinary mercapturic acids (MAs) are detoxification products for electrophiles occurring in the human body. They are suitable biomarkers of exposure to directly acting electrophilic chemicals or to chemicals which generate the electrophile during its metabolism. We determined the urinary excretion of 19 MAs in habitual users of combustible cigarettes (CCs), electronic cigarettes (ECs), heated tobacco products (HTPs), oral tobacco (OT), and nicotine replacement therapy (NRT) products, and nonusers (NUs) of any tobacco/nicotine products. The 19 MAs are assumed to be physiologically formed primarily from 15 toxicants with three of them belonging to IARC Group 1 (human carcinogen), seven to Group 2A (probable human carcinogen), four to Group 2B (possible human carcinogen), and one to Group 3 (not classifiable as carcinogen). Smoking (CC) was found to be associated with significantly elevated exposure to ethylene oxide (or ethylene), 1,3-butadiene, benzene, dimethylformamide, acrolein, acrylamide, styrene, propylene oxide, acrylonitrile, crotonaldehyde, and isoprene compared with the other user groups and NU. Users of HTPs revealed slight elevation in the MAs related to acrolein, acrylamide, and crotonaldehyde compared with the other non-CC groups. Vaping (EC) was not found to be associated with any of the MAs studied. In conclusion, the determination of urinary MAs is a useful tool for assessing the exposure to toxicants (mainly potential carcinogens) in users of various tobacco/nicotine products. Our data also give cause to clarify the role of vaping (EC) in urinary excretion of DHPMA (precursor: glycidol).

Characterization of primary glutathione conjugates with acrylamide and glycidamide: Toxicokinetic studies in Sprague Dawley rats treated with acrylamide

Acrylamide (AA) is classified as a probable human carcinogen and is ubiquitous in foods processed at high temperatures. The carcinogenicity of AA has been attributed to its active metabolite, glycidamide (GA). Both AA and GA can spontaneously or enzymatically conjugate with glutathione (GSH) to form their corresponding GSH conjugates. Profiling AA-glutathione conjugate (AA-GSH) and GA-glutathione conjugates (2 isomers: GA2-GSH and GA3-GSH) in serum would better illustrate AA detoxification compared with urinary metabolite analysis. However, the lack of AA-, GA2, and GA3-GSH study remains a critical data gap. Our study aimed to investigate the toxicokinetics of AA-, GA2-and GA3-GSH in Sprague Dawley rats treated with 0.1 mg/kg, 1.0 mg/kg, or 5.0 mg/kg AA. Blood samples were collected for LC-MS/MS analysis of the GSH conjugate products. Within 24 h of treatment, we observed rapid formation, elimination, and linear kinetics of AA-, GA2-and GA3-GSH. The ∑GA-GSH AUC/AA-GSH AUC ratios were 0.14–0.29, similar to ∑GA/AA AUC in serum but different from ∑GA/AA-derived urinary mercapturic acids in rodents. Our analysis of AA- and GA-GSHs values represents direct detoxification of AA and GA in vivo. This study advances our understanding of sex and inter-species differences in AA detoxification and may refine the existing kinetic models for a more relevant risk extrapolation.

New data on the metabolism of chloromethylisothiazolinone and methylisothiazolinone in human volunteers after oral dosage: excretion kinetics of a urinary mercapturic acid metabolite (\u201cM-12\u201d)

Methylisothiazolinone (MI) as well as the mixture of chloromethylisothiazolinone/methylisothiazolinone [MCI/MI (3:1)] are biocides that are used in a variety of products of every-day life. Due to the skin sensitizing properties of these biocides, their use has come under scrutiny. We have previously examined the human metabolism of MI and MCI after oral dosage of isotope-labelled analogues in human volunteers and confirmed N-methylmalonamic acid to be a major, but presumably unspecific human urinary metabolite. In the present study, we have investigated the urinary kinetics of a mercapturic acid metabolite of MI and MCI using the same set of samples. Four human volunteers received 2 mg of isotopically labelled MI and MCI separately and at least 2 weeks apart. Consecutive urine samples were collected over 48 h and were examined for the content of the (labelled) 3-mercapturic acid conjugate of 3-thiomethyl-N-methyl-propionamide (“M-12”), a known metabolite in rats. On a molar basis, M-12 represented 7.1% (3.0–10.1%) of the dose excreted in urine after dosage of MI. Excretion of this mercapturate was fast with a mean half-life of 3.6 h. Surprisingly, for MCI the mercapturate M-12 represented only 0.13% of the dose excreted in urine. Thus, this biomarker is highly specific for exposures to MI and might be used to distinguish between different exposure patterns of these biocides [use of MI or MCI/MI (3:1)] in the general population.

Quantification of 3-Hydroxypropyl Mercapturic Acid in the Urine of Patients with Breast Cancer to Monitor Cyclophosphamide Toxicity

The alkylating agent cyclophosphamide is used in chemotherapy regimens for various type of cancer. However, cyclophosphamide may lead to toxic side effects on the bladder, namely hemorrhagic cystitis, which can cause hematuria, and, potentially, bladder cancer. These effects are caused by acrolein, a byproduct of cyclophosphamide metabolism. In this study, a method to quantify 3-hydroxypropyl mercapturic acid (3-HPMA) in urine was developed. 3-HPMA is a stable metabolite of acrolein that serves as biomarker of acrolein. Urine samples were collected 4 hours after cyclophosphamide administration and analyzed to determine the risk of hematuria. 3-HPMA was analyzed by reverse-phase LC-MS/MS using a triple quadrupole electrospray ionization mass spectrometer in the positive-ion mode. The mobile phase was a 90:10 (vol/vol) mixture of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Multiple reaction monitoring mode was used, with m/z 222.10 → 90.97 for 3-HPMA and 164.10 → 122.02 for the internal standard N-acetyl cysteine (NAC). Samples were prepared by acidification and dilution. The analytical method produced a linear response within the concentration range of 40-10,000 ng/mL. The method was validated in accordance with 2018 FDA guidelines and applied to quantify 3-HPMA in the urine of 40 patients with breast cancer. The measured concentrations ranged from 820.3 to 5596.1 ng/mg creatinine. Seven patients identified with hematuria had low 3-HPMA concentrations of 4445.824 ± 411.17 ng/mg creatinine, and 33 patients without hematuria had low 3-HPMA concentrations of 2419.4 ± 1171.8 ng/mg creatinine. The method was applicable for the quantification of 3-HPMA in human urine. Large variations in 3-HPMA concentrations were found in 40 patients with breast cancer treated with cyclophosphamide, with a significant difference (P < 0.05) observed between patients with hematuria and those without hematuria.

Urinary biomonitoring of subjects with different smoking habits. Part I: Profiling mercapturic acids

BackgroundWhile tobacco smoke contains thousands of chemicals, some of which are carcinogenic to humans, the content of electronic cigarette smoke is less known. This work aimed to assess and compare the exposure associated with different smoking habits by profiling urinary mercapturic acids as biomarkers of toxic compounds. MethodsIn this pilot study, sixty-seven healthy adults with different smoking habits were investigated: 38 non-smokers (NS), 7 electronic cigarette users (ECU), and 22 traditional tobacco smokers (TTS). Seventeen urinary mercapturic acids, metabolites of 1,3-butadiene (DHBMA, MHBMA), 4-chloronitrobenze (NANPC), acrolein (3-HPMA), acrylamide (AAMA, GAMA), acrylonitrile (CEMA), benzene (SPMA), crotonaldehyde (CMEMA, HMPMA), ethylating agents (EMA), methylating agents (MMA), ethylene oxide (HEMA), N,N-dimethylformamide (AMCC), propylene oxide (2-HPMA), styrene (PHEMA), and toluene (SBMA), were quantified, along with urinary nicotine and cotinine. ResultsMedian urinary cotinine was 0.4, 1530 and 1772 μg/L in NS, ECU and TTS, respectively. Most mercapturic acids were 2–165 fold-higher in TTS compared to NS, with CEMA, MHBMA, 3-HPMA and SPMA showing the most relevant increases. Furthermore, some mercapturic acids were higher in ECU than NS; CEMA and 3-HPMA, in particular, showed significant increases and were 1.8 and 4.9 fold-higher, respectively. ConclusionsThis study confirms that tobacco smoking is a major source of carcinogenic chemicals such as benzene and 1,3-butadiene; electronic cigarette use is a minor source, mostly associated with exposure to chemicals with less carcinogenic potential such as acrylonitrile and acrolein.

Urinary Mercapturic Acids to Assess Exposure to Benzene and Other Volatile Organic Compounds in Coke Oven Workers.

Coke production was classified as carcinogenic to humans by the International Agency for Research on Cancer. Besides polycyclic aromatic hydrocarbons, coke oven workers may be exposed to benzene and other volatile organic compounds (VOCs). The aim of this study was to assess the exposure to several VOCs in 49 coke oven workers and 49 individuals living in the same area by determining urinary mercapturic acids. Active tobacco smoking was an exclusion criterion for both groups. Mercapturic acids were investigated by a validated isotopic dilution LC-MS/MS method. Linear models were built to correct for different confounding variables. Urinary levels of N-acetyl-S-phenyl-L-cysteine (SPMA) (metabolite of benzene), N-acetyl-S-(2-hydroxy-1/2-phenylethyl)-L-cysteine (PHEMA) (metabolite of styrene), N-acetyl-S-(2-cyanoethyl)-L-cysteine (CEMA) (metabolite of acrylonitrile), N-acetyl-S-[1-(hydroxymethyl)-2-propen-1-yl)-L-cysteine and N-acetyl-S-(2-hydroxy-3-buten-1-yl)-L-cysteine (MHBMA) (metabolites of 1,3-butadiene) were 2–10 fold higher in workers than in controls (p < 0.05). For SPMA, in particular, median levels were 0.02 and 0.31 µg/g creatinine in workers and controls, respectively. Among workers, coke makers were more exposed to PHEMA and SPMA than foremen and engine operators. The comparison with biological limit values shows that the exposure of workers was within 20% of the limit values for all biomarkers, moreover three subjects exceeded the restrictive occupational limit value recently proposed by the European Chemicals Agency (ECHA) for SPMA.

Resolution and Quantitation of Mercapturic Acids Derived from Crotonaldehyde, Methacrolein, and Methyl Vinyl Ketone in the Urine of Smokers and Nonsmokers.

Using improved HPLC analysis conditions, we report the separation of three isomers of mercapturic acid conjugates previously assigned in the literature only to 3-hydroxy-1-methylpropylmercapturic acid (HMPMA-1), a human urinary metabolite of crotonaldehyde. The new conditions, employing a biphenyl column cooled to 5 °C and eluted with a gradient of formic acid, acetonitrile, and methanol, allow the analysis of human urinary mercapturic acids derived not only from crotonaldehyde but also from its isomers methacrolein (3-hydroxy-2-methylpropyl mercapturic acid, HMPMA-2) and methyl vinyl ketone (3-hydroxy-3-methylpropyl mercapturic acid, HMPMA-3). The mercapturic acids were detected and quantified by LC-ESI-MS/MS using the corresponding stable isotope labeled mercapturic acids as internal standards. The analysis was validated for accuracy and precision and applied to urine samples collected from cigarette smokers and nonsmokers. Smokers had significantly higher levels of all three mercapturic acids than did nonsmokers. The results demonstrated that HMPMA-3 from methyl vinyl ketone comprised the major portion of the peaks previously ascribed in multiple studies to HMPMA-1. HMPMA-1 had concentrations intermediate between those of HMPMA-2 and HMPMA-3 in both smokers and nonsmokers. This study reports the first quantitation of HMPMA-2 and HMPMA-3 in human urine. The observation of higher levels of HMPMA-3 than in the other two mercapturic acids suggests a previously unrecognized potential significance of methyl vinyl ketone as a toxicant in smokers and nonsmokers.

Non-targeted mercapturic acid screening in urine using LC-MS/MS with matrix effect compensation by postcolumn infusion of internal standard (PCI-IS).

While the targeted analysis of mercapturic acid (MA) metabolites in human urine is used to assess exposure to selected chemicals, this compound class has only rarely been addressed in non-target screening utilizing diagnostic neutral loss liquid chromatography tandem mass spectrometry (LC-MS/MS). Additionally, this type of analysis is severely affected bymatrix effects (MEs) causing poor comparability of samples and distortion of signal intensities. However, MEs have been neglected in urinary MA non-target screening so far. Therefore, we developed a non-target screening method relying on neutral loss scanning for MAs using post column infusion of an isotope-labelled standard. For signal correction, we synthesized a structural analogue to MAs, N-acetyl-S-methyl-homocysteine-D3, lacking the characteristic neutral loss of the MAs. For method development, 16 structurally different model MA compounds and 20 spiked urine samples were used. Twelve out of the 16 model compounds could be analysed by the developed method. We found severe matrix effects (largely signal suppression) for the spiked model compounds, with only 34% of all peaks' intensities changing by less than a factor of two. This could be compensated by the post column internal standard infusion with now 68% of all peaks' intensities changing by less than a factor of two. For three compounds, an over-compensation was observed resulting in an increase of signal of up to a factor of 16. In the 20 urine samples, altogether 558 native MAs (between 74 and 175 per sample) could be detected after ME compensation. These results indicate that a large number of so far uncharacterized MAs are present in urine, which yield a potential for biomarker discovery and pattern characterisation. Graphical Abstract.

Glutathione Conjugation and Protein Adduction Derived from Oxidative Debromination of Benzbromarone in Mice.

Benzbromarone (BBR), a uricosuric agent, has been known to induce hepatotoxicity, and its toxicity has a close relation to cytochrome P450-mediated metabolic activation. An oxidative debromination metabolite of BBR has been reported in microsomal incubations. The present study attempted to define the oxidative debromination pathway of BBR in vivo. One urinary mercapturic acid (M1) and one glutathione (GSH) conjugate (M2) derived from the oxidative debromination metabolite were detected in BBR-treated mice after solid phase extraction. M1 and M2 shared the same chromatographic behavior and mass spectral identities as those detected in N-acetylcysteine/GSH- and BBR-fortified microsomal incubations. The structure of M1 was characterized by chemical synthesis, along with mass spectrometry analysis. In addition, hepatic protein modification that occurs at cysteine residues (M'3) was observed in mice given BBR. The observed protein adduction reached its peak 4 hours after administration and occurred in a dose-dependent manner. A GSH conjugate derived from oxidative debromination of BBR was detected in livers of mice treated with BBR, and the formation of the GSH conjugate apparently took place earlier than the protein adduction. In summary, our in vivo work provided strong evidence for the proposed oxidative debromination pathway of BBR, which facilitates the understanding of the mechanisms of BBR-induced hepatotoxicity. SIGNIFICANCE STATEMENT: This study investigated the oxidative debromination pathway of benzbromarone (BBR) in vivo. One urinary mercapturic acid (M1) and one glutathione (GSH) conjugate (M2) derived from the oxidative debromination metabolite were detected in BBR-treated mice. M1 and M2 were also observed in microsomal incubations. The structure of M1 was characterized by chemical synthesis followed by mass spectrometry analyses. More importantly, protein adduction derived from oxidative debromination of BBR (M'3) was observed in mice given BBR, and occurred in dose- and time-dependent manners. The success in detection of GSH conjugate, urinary N-acetylcysteine conjugate, and hepatic protein adduction in mice given BBR provided solid evidence for in vivo oxidative debromination of BBR. The studies allowed a better understanding of the metabolic activation of BBR.

Dose-dependent detoxication of the airborne pollutant benzene in a randomized trial of broccoli sprout beverage in Qidong, China

Airborne pollutants have collectively been classified as a known human carcinogen and, more broadly, affect the health of hundreds of millions of people worldwide. Benzene is a frequent component of air pollution, and strategies to protect individuals against unavoidable exposure to this and other airborne carcinogens could improve the public's health. Earlier clinical trials in Qidong, China, demonstrated efficacy in enhancing the detoxication of benzene using a broccoli sprout beverage. A randomized, placebo-controlled, multidose trial of a broccoli sprout beverage was designed to determine the lowest effective concentration that enhances benzene detoxication adjudged by enhanced excretion of the urinary biomarker, S-phenylmercapturic acid (SPMA). Following informed consent, 170 subjects were randomly assigned in 5 blocks of 34 each to drink either a placebo beverage (n=55) or 1 of 3 graded concentrations of a broccoli sprout beverage [full (n=25), one-half (n=35), and one-fifth (n=55)] for 10 consecutive days. Concentrations of SPMA arising through induced benzene conjugation with glutathione were quantified by MS in sequential 12-h overnight urine collections during the intervention. MS was also used to quantify urinary sulforaphane metabolites in each dosing regimen that resulted in a median 24-h urinary output of 24.6, 10.3, and 4.3 µmol, respectively, confirming a dose-dependent de-escalation of the inducing principle within the beverage. A statistically significant increase in benzene mercapturic acids in urine was found for the high-dose group (+63.2%) during the 10-d period. The one-half dose (+11.3%) and one-fifth dose groups (-6.4%) were not significantly different from placebo controls. An intervention with a broccoli sprout beverage enhanced the detoxication of benzene, an important airborne pollutant, when dosed at a concentration evoking a urinary elimination of ∼25 µmol sulforaphane metabolites per day, and it portends a practical and frugal population-based strategy to attenuate associated long-term health risks of air pollution. This trial was registered at clinicaltrials.gov as NCT02656420.

An LC-MS/MS method to profile urinary mercapturic acids, metabolites of electrophilic intermediates of occupational and environmental toxicants

IntroductionMercapturic acids are urinary metabolites of occupational and environmental toxicants. The aim of this work was to develop and validate an analytical assay for the determination of several urinary mercapturic acids to be used as biomarkers of exposure. MethodAn isotope dilution tandem mass spectrometric method, coupled with reversed-phase liquid chromatography, was developed for the analysis of mercapturic acids derived from several compounds, including those of benzene, toluene, 1,3-butadiene, styrene, acrylonitrile, 4-chloronitrobenzene, acrylamide, acrolein, propylene oxide, N,N-dimethylformamide, crotonaldehyde, ethylene oxide, and methylating and ethylating agents. Samples were prepared by simple filtration after dilution. A validation was carried out, including the assessment of calibration curves, sensitivity, accuracy, precision, process efficiency, and stability, along with external verification. The assay was applied to the analysis of 14 end-of-shift urine samples from unexposed workers and gasoline station attendants. ResultsThe chromatographic run lasted 18 min. Limits of quantitation ranged from 0.01 to 3.2 μg/L; precision, expressed as relative standard deviation, ranged from 0.6 to 20.9%; and accuracy ranged from 93.4 to 114.9% of theoretical values. The use of deuterated internal standards was suitable for control of the matrix effect. The assay allowed the simultaneous quantitation of urinary mercapturic acids at different concentration ranges. The external verification exercise produced good results. The application of the assay to urine samples from workers revealed differences in mercapturic acid profiles in agreement with the expected patterns of exposure. ConclusionThis high-throughput method is valid and useful for the quantitation of urinary mercapturic acids, and is suitable for human biomonitoring of occupational and environmental exposure.

LC-MS Method Development for Simultaneous Determination of Trans-3′-hydroxycotinine and Three Mercapturic Acids in Urine

The negative impact of tobacco smoke on the human body is due to a wide range of harmful substances including volatile organic compounds (VOCs). Some VOCs of tobacco smoke metabolize in human organism into mercapturic acids (MAs). The determination of the amount of MAs in readily available biological fluids, for example in urine, allows to assess the level of exposure of these VOCs in a particular person. It is useful to assess the impact of individual VOCs on the body together with the assessment of the intake of nicotine. The intake of nicotine can be determined by the content of its metabolites in the urine, in particular by the content of trans-3′-hydroxycotinine (tH-Cot). A joint assessment of the concentrations of trans-3′-hydroxycotinine and MAs in urine allows obtaining selective information about effects of different VOCs and nicotine on the smoker’s body. We have developed a liquid chromatography–mass spectrometry (LC-MS) method for simultaneous quantifying of tH-Cot and three MAs: N-Acetyl-S-(3-hydroxypropyl)cysteine (HPMA), N-Acetyl-S-(3-hydroxypropyl-1-methyl)-L-cysteine (HMPMA), N-Acetyl-S-(2-cyanoethyl)-L-cysteine (CEMA). We used this method to quantify the levels of MAs and tH-Cot in the urine of a group of 15 smokers just before and 5 days after smoking cessation. For all studied compounds, we have found statistically significant changes in concentration on the fifth day of smoking cessation. The method developed can be used to jointly assess the levels of exposure to nicotine and VOCs in the study of various tobacco products.

Investigation of the presence in human urine of mercapturic acids derived from phenanthrene, a representative polycyclic aromatic hydrocarbon

Polycyclic aromatic hydrocarbons (PAH) are environmental carcinogens implicated as causes of cancer in certain industrial settings and in cigarette smokers. PAH require metabolic activation to exert their carcinogenic effects. One widely accepted pathway of metabolic activation proceeds through formation of “bay region” diol epoxides which are highly reactive with DNA and can cause mutations. Phenanthrene (Phe) is the simplest PAH with a bay region and an excellent model for the study of PAH metabolism. In previous studies in which [D10]Phe was administered to smokers, we observed higher levels of [D10]Phe-tetraols derived from [D10]Phe-diol epoxides in subjects who were null for the glutathione-S-transferase M1 (GSTM1) gene. We hypothesized that Phe-epoxides, the primary metabolites of Phe, were detoxified by glutathione conjugate formation, which would result ultimately in the excretion of the corresponding mercapturic acids in urine. We synthesized the four stereoisomeric mercapturic acids that would result from attack of glutathione on Phe-epoxides followed by normal processing of the conjugates. We also synthesized the corresponding dehydrated metabolites and sulfoxides. These 12 standards were used in liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry analysis of urine samples from smokers and creosote workers, the latter exposed to unusually high levels of PAH. Only the sulfoxide derivatives were consistently detected in the urine of creosote workers; none of the compounds was detected in the urine of smokers. These results demonstrate a new pathway of PAH-mercapturic acid formation, but do not provide an explanation for the role of GSTM1 null status on Phe-tetraol formation.