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.

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.

Association of genetic polymorphisms with mercapturic acids in the urine of young healthy subjects before and after exposure to outdoor air pollution.

We aimed to identify the relationship between variations in metabolic genes and human urinary changes in mercapturic acids (MAs), including CEMA, HMPMA, SPMA, HPMA and HEMA, before and after air pollution exposure. Genotype detection for 47 relevant single nucleotide polymorphisms (SNPs) collected by literature research was performed. Five MAs expression levels in the urinary samples of 50 young healthy individuals with short-term exposure to clean, polluted and purified air at five time points were detected by targeted online solid-phase extraction liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS), followed with associations of SNPs with MAs changes. Difference in MAs between polluted and clean/purified air was significantly associated with 21 SNPs mapped into 9 genes. Five SNPs in GSTP1 showed the most prominent association with the changes in SPMA expression, indicating that those SNPs in GSTP1 and SPMA might serve as biomarkers for susceptibility and the prognosis of lung cancer.

Assessment of the potential vaping-related exposure to carbonyls and epoxides using stable isotope-labeled precursors in the e-liquid.

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.

Biomonitoring of nutritional acrylamide intake by consumers without dietary preferences as compared to vegans.

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.

Sensorineural hearing loss and volatile organic compound metabolites in urine.

PURPOSE: Oxidative stress in the auditory system contributes to acquired sensorineural hearing loss. Systemic oxidative stress, which may predict auditory oxidative stress, can be assessed by measuring volatile organic compound metabolite concentrations in urine. The purpose of this retrospective study was to determine if hearing decreased in those with higher concentrations of urinary volatile organic compound metabolites. MATERIALS AND METHODS: Audiometric, demographic, and metabolite concentration data were downloaded from the 2011-2012 cycle of the U.S. National Health and Nutritional Examination Survey. Participants were first grouped by reported noise exposure. For each metabolite, an analysis of covariance was used to look for differences in age-adjusted hearing loss among urinary volatile organic compound metabolite concentration groups. Participants were grouped into quartiles based on concentration for each metabolite separately because many individuals were at the lower limit of concentration detection for several metabolites, leading to a non-normal distribution. RESULTS: Age-adjusted high-frequency pure-tone thresholds were significantly (FDR < 0.05) increased by about 3 to 4 dB in high concentration quartile groups for five metabolites. All five metabolites were glutathione-dependent mercapturic acids. The parent compounds of these metabolites included acrylonitrile, 1,3 butadiene, styrene, acrylamide, and N,N-dimethylformamide. Significant associations were only found in those with no reported noise exposure. CONCLUSIONS: Urinary metabolites may help to explain susceptibility to oxidative stress-induced hearing loss.

Biomarker monitoring of controlled dietary acrylamide exposure indicates consistent human endogenous background.

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 13C3D3-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 13C3D3-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.

[Biomarkers of internal exposure to toxicologically relevant contaminants in food]. / Biomarker der internen Exposition gegenüber toxikologisch relevanten Kontaminanten in Lebensmitteln.

The assessment of health risks resulting from the intake of genotoxic carcinogens in food depends essentially on a valid exposure assessment. The reliability of the external exposure estimation is restricted by various factors, e. g. inaccurate data from dietary protocols and variations of food contaminant contents. As an alternative, the individual internal exposure to genotoxic substances may be described by specific biomarkers in different matrices. For example, mercapturic acids formed after glutathione conjugation of electrophilic metabolites can be detected in the urine. This typically reflects the exposure to the parent compound over a period of one to two days. The determination of adducts in the blood proteins serum albumin (SA) and hemoglobin (Hb) allows for conclusions to be drawn about the external exposure within the last three weeks (SA) or within the last four months (Hb). Protein adducts are used routinely in occupational medicine as biomarkers of internal exposure to substances in the ambient air of the workplace. The availability of increasingly sensitive analytical techniques also makes it possible to detect numerous adducts in proteins from human blood samples that are formed after the continuous intake of very small doses of toxic substances from foods. Here, we present the current state of science exemplified by protein adducts of the food contaminants acrylamide, aflatoxin B1 and glycidol. The biomarker can be used in the future to investigate previously unknown relationships between internal exposure and disease incidences.

Urinary Metabolome Analysis Reveals Potential Microbiota Alteration and Electrophilic Burden Induced by High Red Meat Diet: Results from the French NutriNet-Santé Cohort and an In Vivo Intervention Study in Rats.

SCOPE: High red and processed meat consumption is associated with several adverse outcomes such as colorectal cancer and overall global mortality. However, the underlying mechanisms remain debated and need to be elucidated. METHODS AND RESULTS: Urinary untargeted Liquid Chromatography-Mass Spectrometry (LC-MS) metabolomics data from 240 subjects from the French cohort NutriNet-Santé are analyzed. Individuals are matched and divided into three groups according to their consumption of red and processed meat: high red and processed meat consumers, non-red and processed meat consumers, and at random group. Results are supported by a preclinical experiment where rats are fed either a high red meat or a control diet. Microbiota derived metabolites, in particular indoxyl sulfate and cinnamoylglycine, are found impacted by the high red meat diet in both studies, suggesting a modification of microbiota by the high red/processed meat diet. Rat microbiota sequencing analysis strengthens this observation. Although not evidenced in the human study, rat mercapturic acid profile concomitantly reveals an increased lipid peroxidation induced by high red meat diet. CONCLUSION: Novel microbiota metabolites are identified as red meat consumption potential biomarkers, suggesting a deleterious effect, which could partly explain the adverse effects associated with high red and processed meat consumption.

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).

Harmonization of acronyms for volatile organic compound metabolites using a standardized naming system.

Increased interest in volatile organic compound (VOC) exposure has led to an increased need for consistent, systematic, and informative naming of VOC metabolites. As analytical methods have expanded to include many metabolites in a single assay, the number of acronyms in use for a single metabolite has expanded in an unplanned and inconsistent manner due to a lack of guidance or group consensus. Even though the measurement of VOC metabolites is a well-established means to investigate exposure to VOCs, a formal attempt to harmonize acronyms amongst investigators has not been published. The aim of this work is to establish a system of acronym naming that provides consistency in current acronym usage and a foundation for creating acronyms for future VOC metabolites.

Quantitative NanoLC/NSI+-HRMS Method for 1,3-Butadiene Induced bis-N7-guanine DNA-DNA Cross-Links in Urine.

1,3-Butadiene (BD) is a common environmental and industrial chemical widely used in plastic and rubber manufacturing and also present in cigarette smoke and automobile exhaust. BD is classified as a known human carcinogen based on evidence of carcinogenicity in laboratory animals treated with BD by inhalation and epidemiological studies revealing an increased risk of leukemia and lymphohematopoietic cancers in workers occupationally exposed to BD. Upon exposure via inhalation, BD is bioactivated to several toxic epoxides including 3,4-epoxy-1-butene (EB), 3,4-epoxy-1,2-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB); these are conjugated with glutathione and excreted as 2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene/1-(N-acetyl-L-cystein-S-yl)-2-hydroxybut-3-ene (MHBMA), 4-(N-acetyl-L-cystein-S-yl)-1,2-dihydroxybutane (DHBMA), and 1,4-bis-(N-acetyl-L-cystein-S-yl)butane-2,3-diol (bis-BDMA). Exposure to DEB generates monoalkylated DNA adducts, DNA-DNA crosslinks, and DNA-protein crosslinks, which can cause base substitutions, genomic rearrangements, and large genomic deletions. In this study, we developed a quantitative nanoLC/NSI+-HRMS methodology for 1,4-bis-(gua-7-yl)-2,3-butanediol (bis-N7G-BD) adducts in urine (LOD: 0.1 fmol/mL urine, LOQ: 1.0 fmol/mL urine). This novel method was used to quantify bis-N7G-BD in urine of mice treated with 590 ± 150 ppm BD for 2 weeks (6 h/day, 5 days/week). Bis-N7G-BD was detected in urine of male and female BD-exposed mice (574.6 ± 206.0 and 571.1 ± 163.4 pg/mg of creatinine, respectively). In addition, major urinary metabolites of BD, bis-BDMA, MHBMA and DHBMA, were measured in the same samples. Urinary bis-N7G-BD adduct levels correlated with DEB-derived metabolite bis-BDMA (r = 0.80, Pearson correlation), but not with the EB-derived DNA adducts (EB-GII) or EB-derived metabolites MHBMA and DHBMA (r = 0.24, r = 0.14, r = 0.18, respectively, Pearson correlations). Urinary bis-N7G-BD could be employed as a novel non-invasive biomarker of exposure to BD and bioactivation to its most mutagenic metabolite, DEB. This method will be useful for future studies of 1,3-butadiene exposure and metabolism.

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

BACKGROUND: While 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. METHODS: In 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. RESULTS: Median 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. CONCLUSIONS: This 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.

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

BACKGROUND: 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. OBJECTIVES: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

New diaryl ω-(isothiocyanato)alkylphosphonates and their mercapturic acids as potential antibacterial agents.

Thirty-four novel, diaryl ω-(isothiocyanato)alkylphosphonates with chlorine atom and methoxy, dimethoxy, methylsulfanyl, or methoxycarbonyl groups at ortho, meta, or para positions of the phenyl ring, and with an unbranched alkyl chain (n = 2-6) were designed and synthesized in a one-pot reaction in 11-76% yields. All isothiocyanates thus generated were evaluated for the first time for antibacterial activity on Pseudomonas aeruginosa and Staphylococcus aureus bacterial strains, and had satisfactory antibacterial activity in most cases. The highest activity, similar to that of reference gentamicin activity against S. aureus, was seen in compounds 9 and 13 (1.5 ±â€¯0.1 and 2.5 ±â€¯0.2 µM, respectively), whereas for P. aeruginosa more than half of tested compounds proved to be more effective than gentamicin. Additionally, selected isothiocyanates (9, 13, 18, and 23) were transformed in 52-73% yields into mercapturic acids 42-45, which also exhibited satisfactory antibacterial effect against S. aureus strain.

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

INTRODUCTION: Mercapturic 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. METHOD: An 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. RESULTS: The 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. CONCLUSION: This 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.

Mercapturate Pathway in the Tubulocentric Perspective of Diabetic Kidney Disease.

BACKGROUND: The recent growing evidence that the proximal tubule underlies the early pathogenesis of diabetic kidney disease (DKD) is unveiling novel and promising perspectives. This pathophysiological concept links tubulointerstitial oxidative stress, inflammation, hypoxia, and fibrosis with the progression of DKD. In this new angle for DKD, the prevailing molecular mechanisms on proximal tubular cells emerge as an innovative opportunity for prevention and management of DKD as well as to improve diabetic dysmetabolism. SUMMARY: The mercapturate pathway (MAP) is a classical metabolic detoxification route for xenobiotics that is emerging as an integrative circuitry detrimental to resolve tubular inflammation caused by endogenous electrophilic species. Herein we review why and how it might underlie DKD. Key Messages: MAP is a hallmark of proximal tubular cell function, and cysteine-S-conjugates might represent targets for early intervention in DKD. Moreover, the biomonitoring of urinary mercapturates from metabolic inflammation products might be relevant for the implementation of preventive/management strategies in DKD.

Sustained Human Background Exposure to Acrolein Evidenced by Monitoring Urinary Exposure Biomarkers.

SCOPE: This study investigates a potential correlation between the intake of heat-processed food and the excretion of the acrolein (AC) biomarkers N-acetyl-S-(3-hydroxypropyl)-l-cysteine (HPMA) and N-acetyl-S-(carboxyethyl)-l-cysteine (CEMA) based on two human studies. METHODS AND RESULTS: Human exposure to AC is monitored using the AC-related mercapturic acids HPMA and CEMA in the urine of a) non-smoking volunteers under defined living conditions and b) of non-smoking volunteers on unrestricted or vegan diet under free living conditions. Free living volunteers in part show markedly enhanced urinary excretions of HPMA and CEMA. The intake of heat-processed food does not influence AC-related biomarker excretion. Incidentally enhanced urinary exposure biomarker levels appear to suggest AC exposure possibly from open fire, barbecuing, or tobacco smoke. However, kinetics of urinary biomarkers related to tobacco and other potential smoke exposure, do not correlate with those observed for HPMA and CEMA. CONCLUSION: This study is the first to convincingly show a sustained and substantial background exposure to AC in non-smoking humans, clearly independent from uptake of heat-processed foods. The data strongly point to endogenous AC generation by pathways of mammalian and/or microbial metabolism as yet not taken into consideration.

LC-MS/MS procedure for the simultaneous determination of N-acetyl-S-(1-naphthyl)cysteine and N-acetyl-S-(2-napthyl)cysteine in human urine.

A two-dimensional liquid chromatographic electrospray-ionization tandem mass spectrometric (LC/LC-ESI-MS/MS) procedure for the simultaneous determination of the expected mercapturic acids of naphthalene (1- and 2-naphthylmercapturic acids; 1- and 2-NpMA) and of the well-established parameter for benzene biomonitoring (S-phenylmercapturic acid; PhMA) in human urine was developed, validated and applied to human urine samples. Apart from sample acidification, the enrichment of analytes and sample clean-up as well as the separation of all analytes were completely automated using both a restricted access material column (RAM C18) and a core-shell biphenyl material. Sensitive, specific and reliable detection of all target substances, with limits of detection ranging from 0.03 to 0.04 µg/L, was achieved using structurally well matching isotope-labelled internal standard substances for each analyte. Intraday and interday precision were determined, ranging from 2.2 to 4.3%, and mean accuracy from 98.4 to 100.8%. Due to the low limits of detection, the good precision and accuracy of the developed procedure, it is well suited for application in biomonitoring studies to evaluate the validity of mercapturic acids as biomarkers after naphthalene exposure.