Developing an off-on fluorescence sensor based on red copper nanoclusters wrapped by sulfhydryl and polymer double ligands for sensitive detection of N-acetyl-L-cysteine.

N-acetyl-L-cysteine (NAC) as a class of thiols is commonly used in the treatment of lung diseases, detoxification and prevention of liver damage. In this paper, 4-mercaptobenzoic acid (4-MBA) coated and polyvinylpyrrolidone (PVP) attached copper nanoclusters (4-MBA@PVP-CuNCs) were successfully synthesized using a simple one-pot method with an absolute quantum yield of 10.98 %, and its synthetic conditions (like effects of single/double ligands and temperature) were studied intensively. Then Hg2+ could quench the fluorescence of the 4-MBA@PVP-CuNCs and its fluorescence was restored with the addition of NAC. Based on the above principles, an off-on switching system was established to detect NAC. That is, the 4-MBA@PVP-CuNCs-Hg probe was prepared by adding Hg2+ to switch off the fluorescence of the CuNCs by static quenching, and then NAC was added to switch on the fluorescence of the probe based on the chelation of NAC and Hg2+. Moreover, the effects of metal ion types and mercury ion doses for the probe construction were also further discussed. The method showed excellent linearity in the range of 0.05-1.25 µM and low detection limit of 16 nM. Meanwhile, good recoveries in real urine, tablets and pellets were observed, which proved the reliability of the method and provided a convenient, fast and sensitive method for NAC detection.

Synthesis and characterization of the trans- and cis-isomers of N-acetyl-S-(4-hydroxy-2-buten-1-yl)-L-cysteine and their attempted detection in human urine.

1,3-Butadiene (BD) is a carcinogenic air pollutant. N-acetyl-S-(4-hydroxy-2-buten-1-yl)-L-cysteine (MHBMA3 or 4HBeMA), an urinary BD metabolite with unspecified configuration, is considered the most sensitive BD biomarker and has been used in routine biomonitoring since 2012. However, two issues remain unaddressed: why its concentrations are unusually high relative to other urinary BD biomarkers and why some authors reported no detection of the biomarker whereas other authors readily quantitated it. To address the issues, we synthesized and structurally characterized the authentic trans- and cis-isomers of MHBMA3 (designated NE and NZ, respectively), developed an isotope-dilution LC-MS/MS method for their quantification, and examined 67 urine samples from barbecue restaurant personnel (n = 47) and hotel administrative staff (n = 20). The restaurant personnel were exposed to barbecue fumes, which contain relatively high concentrations of BD. The results showed that NE and NZ had highly similar NMR spectra, and were difficult to be well separated chromatographically. The NMR data showed that the MHBMA3 isomer investigated in most previous studies was NE. We did not detect NE and NZ in any samples; however, an interfering peak with varying heights was observed in most samples. Notably, under the chromatographic conditions used in the literature, the peak exhibited indistinguishable retention time from that of NE. Thus, it is highly likely that the interfering peak has been mis-identified as NE in previous studies, providing a reasonable explanation for the high MHBMA3 concentration in urine. The contradiction in the presence of MHBMA3 in urine was also caused by the mis-identification, because the researchers who reported the absence of MHBMA3 were actually detecting NZ. Thus, we clarified the confusion on MHBMA3 in previous studies through correctly identifying the two MHBMA3 isomers. The presence of NE and NZ in human urine warrants further investigations.

Benzene Exposure in Workers From a Waste Oil Regeneration Plant During Ordinary Activities by Air and Biological Monitoring.

BACKGROUND: In the regeneration of waste oil, a strategical technological process for the European Union circular economy action plan, exhausted oils are regenerated to produce high performing oil bases. Aim of this work was to assess the exposure to benzene in plant workers during ordinary activities. METHODS: 59 workers, potentially exposed to benzene, and 9 administrative workers from an Italian plant were monitored for the whole work shift with personal air samplers; urinary benzene (BEN-U) and S-phenyl mercapturic acid (SPMA) were measured by mass spectrometry methods in end-shift urine samples. Different job tasks were identified among workers. RESULTS: Median (minimum-maximum) airborne exposures to benzene were <0.9 (<0.9-6.3) and <0.9 (<0.9-0.9) µg/m3, BEN-U and SPMA levels were 0.094 (<0.015-3.095) µg/L and 0.15 (<0.10-9.67) µg/g crt and 0.086 (0.034-0.712) µg/L and <0.10 (<0.10-3.19) µg/g creatinine in workers and administrative workers, respectively. No differences were found among job tasks and between workers and administrative workers, while higher levels were found in smokers than in non-smokers. For all job tasks, the exposure to benzene was always below occupational limit values. CONCLUSIONS: This study has investigated for the first time the exposure to benzene of workers employed in the re-refining of exhaust oil. The results showed that normal production activities in regenerating used oils do not pose a risk of exposure to benzene in workers.

Leukemia risk assessment of exposure to low-levels of benzene based on the linearized multistage model.

Objectives: To assess leukemia risk in occupational populations exposed to low levels of benzene. Methods: Leukemia incidence data from the Chinese Benzene Cohort Study were fitted using the Linearized multistage (LMS) model. Individual benzene exposure levels, urinary S-phenylmercapturic acid (S-PMA) and trans, trans-muconic acid (t, t-MA) were measured among 98 benzene-exposed workers from factories in China. Subjects were categorized into four groups by rounding the quartiles of cumulative benzene concentrations (< 3, 3-5, 5-12, ≥12 mg/m3·year, respectively). The risk of benzene-induced leukemia was assessed using the LMS model, and the results were validated using the EPA model and the Singapore semi-quantitative risk assessment model. Results: The leukemia risks showed a positive correlation with increasing cumulative concentration in the four exposure groups (excess leukemia risks were 4.34, 4.37, 4.44 and 5.52 × 10-4, respectively; Ptrend < 0.0001) indicated by the LMS model. We also found that the estimated leukemia risk using urinary t, t-MA in the LMS model was more similar to those estimated by airborne benzene compared to S-PMA. The leukemia risk estimated by the LMS model was consistent with both the Singapore semi-quantitative risk assessment model at all concentrations and the EPA model at high concentrations (5-12, ≥12 mg/m3·year), while exceeding the EPA model at low concentrations (< 3 and 3-5 mg/m3·year). However, in all four benzene-exposed groups, the leukemia risks estimated by these three models exceeded the lowest acceptable limit for carcinogenic risk set by the EPA at 1 × 10-6. Conclusion: This study demonstrates the utility of the LMS model derived from the Chinese benzene cohort in assessing leukemia risk associated with low-level benzene exposure, and suggests that leukemia risk may occur at cumulative concentrations below 3 mg/m3·year.

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.

Validation of putative biomarkers of furan exposure through quantitative analysis of furan metabolites in urine of F344 rats exposed to stable isotope labeled furan.

Humans are chronically exposed to furan, a potent liver toxicant and carcinogen that occurs in a variety of heat-processed foods. Assessment of human exposure based on the furan content in foods is, however, subject to some uncertainty due to the high volatility of furan. Biomarker monitoring is thus considered an alternative or complementary approach to furan exposure assessment. Previous work suggested that urinary furan metabolites derived from the reaction of cis-2-butene-1,4-dial (BDA), the reactive intermediate of furan, with glutathione (GSH) or amino acids may serve as potential biomarkers of furan exposure. However, some metabolites were also reported to occur in urine of untreated animals, indicating either background contamination via animal feed or endogenous sources, which may limit their suitability as biomarkers of exposure. The overall aim of the present study was to accurately establish the correlation between external dose and concentration of furan metabolites in urine over time and to discriminate against endogenous formation and furan intake via feed. To this end, the furan metabolites GSH-BDA (N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-L-cysteinylglycine), NAcLys-BDA (R-2-(acetylamino)-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid), NAcCys-BDA-NAcLys (N-acetyl-S-[1-[5-(acetylamino)-5-carboxypentyl]-1H-pyrrol-3-yl]-L-cysteine) and NAcCys-BDA-NAcLys sulfoxide (N-acetyl-S-[1-[5-(acetylamino)-5-carboxypentyl]-1H-pyrrol-3-yl]-L-cysteine sulfoxide) were simultaneously analyzed by stable isotope dilution ESI-LC-MS/MS as unlabeled and [13C4]-furan dependent metabolites following oral administration of a single oral dose of isotopically labelled [13C4]-furan (0.1, 1, 10, 100 and 1000 µg/kg bw) to male and female F344/DuCrl rats. Although a linear correlation between urinary excretion of [13C4]-furan-dependent metabolites was observed, analysis of unlabeled NAcLys-BDA, NAcCys-BDA-NAcLys and NAcCys-BDA-NAcLys sulfoxide revealed substantial, fairly constant urinary background levels throughout the course of the study. Analysis of furan in animal feed excluded feed as a source for these background levels. GSH-BDA was identified as the only furan metabolite without background occurrence, suggesting that it may present a specific biomarker to monitor external furan exposure. Studies in humans are now needed to establish if analysis of urinary GSH-BDA may provide reliable exposure estimates.

Specific CpG sites methylation is associated with hematotoxicity in low-dose benzene-exposed workers.

Benzene is a broadly used industrial chemicals which causes various hematologic abnormalities in human. Altered DNA methylation has been proposed as epigenetic biomarkers in health risk evaluation of benzene exposure, yet the role of methylation at specific CpG sites in predicting hematological effects remains unclear. In this study, we recruited 120 low-level benzene-exposed and 101 control male workers from a petrochemical factory in Maoming City, Guangdong Province, China. Urinary S-phenylmercapturic acid (SPMA) in benzene-exposed workers was 3.40-fold higher than that in control workers (P < 0.001). Benzene-induced hematotoxicity was characterized by reduced white blood cells counts and nuclear division index (NDI), along with an increased DNA damage and urinary 8-hydroxy-2'-deoxyguanosine (all P < 0.05). Methylation levels of TRIM36, MGMT and RASSF1a genes in peripheral blood lymphocytes (PBLCs) were quantified by pyrosequencing. CpG site 6 of TRIM36, CpG site 2, 4, 6 of RASSF1a and CpG site 1, 3 of MGMT methylation were recognized as hot CpG sites due to a strong correlation with both internal exposure and hematological effects. Notably, integrating hot CpG sites methylation of multiple genes reveal a higher efficiency in prediction of integrative damage compared to individual genes at hot CpG sites. The negative dose-response relationship between the combined methylation of hot CpG sites in three genes and integrative damage enabled the classification of benzene-exposed individuals into high-risk or low-risk groups using the median cut-off value of the integrative index. Subsequently, a prediction model for integrative damage in benzene-exposed populations was built based on the methylation status of the identified hot CpG sites in the three genes. Taken together, these findings provide a novel insight into application prospect of specific CpG site methylation as epi-biomarkers for health risk assessment of environmental pollutants.

Urinary Excretion of Mercapturic Acids of the Rodent Carcinogen Methyleugenol after a Single Meal of Basil Pesto: A Controlled Exposure Study in Humans.

Methyleugenol (ME), found in numerous plants and spices, is a rodent carcinogen and is classified as "possibly carcinogenic to humans". The hypothesis of a carcinogenic risk for humans is supported by the observation of ME-derived DNA adducts in almost all human liver and lung samples examined. Therefore, a risk assessment of ME is needed. Unfortunately, biomarkers of exposure for epidemiological studies are not yet available. We hereby present the first detection of N-acetyl-l-cysteine conjugates (mercapturic acids) of ME in human urine samples after consumption of a popular ME-containing meal, pasta with basil pesto. We synthesized mercapturic acid conjugates of ME, identified the major product as N-acetyl-S-[3'-(3,4-dimethoxyphenyl)allyl]-l-cysteine (E-3'-MEMA), and developed methods for its extraction and LC-MS/MS quantification in human urine. For conducting an exposure study in humans, a basil cultivar with a suitable ME content was grown for the preparation of basil pesto. A defined meal containing 100 g of basil pesto, corresponding to 1.7 mg ME, was served to 12 participants, who collected the complete urine at defined time intervals for 48 h. Using d6-E-3'-MEMA as an internal standard for LC-MS/MS quantification, we were able to detect E-3'-MEMA in urine samples of all participants collected after the ME-containing meal. Excretion was maximal between 2 and 6 h after the meal and was completed within about 12 h (concentrations below the limit of detection). Excreted amounts were only between 1 and 85 ppm of the ME intake, indicating that the ultimate genotoxicant, 1'-sulfooxy-ME, is formed to a subordinate extent or is not efficiently detoxified by glutathione conjugation and subsequent conversion to mercapturic acids. Both explanations may apply cumulatively, with the ubiquitous detection of ME DNA adducts in human lung and liver specimens arguing against an extremely low formation of 1'-sulfooxy-ME. Taken together, we hereby present the first noninvasive human biomarker reflecting an internal exposure toward reactive ME species.

Biomarkers of exposure in urine of active smokers, non-smokers, and vapers.

The exposure to smoking related products has been evaluated through urine illness risk marker determination through the analysis of urine samples of smokers and vapers. Biomarkers and their metabolites such as N-acetyl-S-(2-cyanoethyl)-L-cysteine (CEMA), N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine (DHBMA), N-acetyl-S-[1-(hydroxymethyl)-2-propen-1-yl)-L-cysteine (MHBMA), N-acetyl-S-(3-hydroxypropyl)-L-cysteine (3HPMA), 2R-N-acetyl-S-(4-hydroxybutan-2-yl)-L-cysteine (HMPMA), and N-acetyl-S-(3-carboxy-2-propyl)-L-cysteine (CMEMA) together with nicotine and cotinine were identified and quantified by LC-HRMS and LC-MS/MS, and data found normalized to the creatinine level. One hundred two urine samples were collected from smokers, non-smokers, and vapers, spanning an age range from 16 to 79 years. Results obtained showed that CEMA was only detected in urine samples from smokers and MHBMA was in the same order of magnitude in all the urine samples analyzed. HMPMA was found in the urine of vapers at the same order of concentration as in non-smokers. 3HPMA in vapers was lower than in the urine of smokers, presenting an intermediate situation between smokers and non-smokers. On the other hand, DHBMA in vapers can reach similar values to those found for smokers, while CMEMA shows concentrations in the urine of vapers higher than in the case of non-smokers and traditional smokers, requiring new research to link this metabolite to the use of electronic cigarettes and possible alternative metabolomic routes. In general, this study seems to verify that traditional smoking practice constitutes a major source of carcinogenic chemicals compared with substitutive practices, although those practices are not free of potential harm.

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.

Isotope Labeling Mass Spectrometry to Quantify Endogenous and Exogenous DNA Adducts and Metabolites of 1,3-Butadiene In Vivo.

Human exposure to known carcinogen 1,3-butadiene (BD) is common due to its high concentrations in automobile exhaust, cigarette smoke, and forest fires, as well as its widespread use in the polymer industry. The adverse health effects of BD are mediated by epoxide metabolites such as 3,4-epoxy-1-butene (EB), which reacts with DNA to form 1-hydroxyl-3-buten-1-yl adducts on DNA nucleobases. EB-derived mercapturic acids (1- and 2-(N-acetyl-l-cysteine-S-yl)-1-hydroxybut-3-ene (MHBMA) and N-acetyl-S-(3,4-dihydroxybutyl)-l-cysteine (DHBMA)) and urinary N7-(1-hydroxyl-3-buten-1-yl) guanine DNA adducts (EB-GII) have been used as biomarkers of BD exposure and cancer risk in smokers and occupationally exposed workers. However, low but significant levels of MHBMA, DHBMA, and EB-GII have been reported in unexposed cultured cells, animals, and humans, suggesting that these metabolites and adducts may form endogenously and complicate risk assessment of butadiene exposure. In the present work, stable isotope labeling in combination with high-resolution mass spectrometry was employed to accurately quantify endogenous and exogenous butadiene metabolites and DNA adducts in vivo. Laboratory rats were exposed to 0.3, 0.5, or 3 ppm of BD-d6 by inhalation, and the amounts of endogenous (d0) and exogenous (d6) DNA adducts and metabolites were quantified in tissues and urine by isotope dilution capillary liquid chromatography/high resolution electrospray ionization tandem mass spectrometry (capLC-ESI-HRMS/MS). Our results reveal that EB-GII adducts and MHBMA originate exclusively from exogenous exposure to BD, while substantial amounts of DHBMA are formed endogenously. Urinary EB-GII concentrations were associated with genomic EB-GII levels in tissues of the same animals. Our findings confirm that EB-GII and MHBMA are specific biomarkers of exposure to BD, while endogenous DHBMA predominates at sub-ppm exposures to BD.

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.

Machine learning prediction of exposure to acrylamide based on modelling of association between dietary exposure and internal biomarkers.

The ubiquitous occurrence of acrylamide in various thermal processing food products poses a potential health risk for the public. An accurate exposure assessment is crucial to the risk evaluation of acrylamide. Machine learning emerging as a powerful computational tool for prediction was employed to establish the association between internal exposure and dietary exposure to acrylamide among a Chinese cohort of middle-aged and elderly population (n = 1,272). Five machine learning regression models were constructed and compared to predict the daily dietary acrylamide exposure based on urinary biomarkers including N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA), N-acetyl-S-(2-carbamoylethyl)-L-cysteine-sulfoxide (AAMA-sul), N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine (GAMA), and N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)-L-cysteine (iso-GAMA). Other important covariates such as age, gender, physical activities, and total energy intake were also considered as predictors in the models. Average dietary intake of acrylamide among Chinese elderly participants was 8.9 µg/day, while average urinary contents of AAMA, AAMA-sul, GAMA, and iso-GAMA were 52.2, 19.1, 4.4, and 1.7 nmol/g Ucr (urine creatinine), respectively. Support vector regression (SVR) model showed the best prediction performance with a R of 0.415, followed by light gradient boosting machine (LightGBM) model (R = 0.396), adjusted multiple linear regression (MLR) model (R = 0.378), neural networks (NN) model (R = 0.365), MLR model (R = 0.363), and extreme gradient boosting (XGBoost) model (R = 0.337). The present study firstly correlated dietary exposure with internal exposure to acrylamide among Chinese elderly population, providing an innovative perspective for the exposure assessment of acrylamide.

Characterization of the association between cigarette smoking intensity and urinary concentrations of 2-hydroxyethyl mercapturic acid among exclusive cigarette smokers in the National Health and Nutrition Examination Survey (NHANES) 2011-2016.

BACKGROUND: 2-Hydroxyethyl mercapturic acid (2HEMA, N-acetyl-S-(2-hydroxyethyl)-L-cysteine) is a urinary metabolite of several volatile organic compounds including acrylonitrile and ethylene oxide, which are found in cigarette smoke. METHODS: We measured 2HEMA concentrations in urine specimens collected during the National Health and Nutrition Examination Survey (2011-2016) from eligible participants aged >12 years (N = 7,416). We developed two multiple linear regression models to characterize the association between cigarette smoking and 2HEMA concentrations wherein the dependent variable was 2HEMA concentrations among participants who exclusively smoked cigarettes at the time of specimen collection and the independent variables included sex, age, race/ethnicity, creatinine, diet, and either cigarettes smoked per day (CPD) or serum cotinine. RESULTS: We detected 2HEMA in 85% of samples tested among exclusive cigarette smokers, and only 40% of specimens from non-smokers. When compared to exclusive cigarette smokers who smoked 1-9 CPD, smoking 10-19 CPD was associated with 36% higher 2HEMA (p < 0.0001) and smoking >19 CPD was associated with 61% higher 2HEMA (p < 0.0001). Additionally, 2HEMA was positively associated with serum cotinine. CONCLUSIONS: This study demonstrates that cigarette smoking intensity is associated with higher urinary 2HEMA concentrations and is likely a major source of acrylonitrile and/or ethylene oxide exposure.

In Vitro and In Vivo Analysis of Ochratoxin A-Derived Glucuronides and Mercapturic Acids as Biomarkers of Exposure.

Ochratoxin A (OTA) is a widespread food contaminant, with exposure estimated to range from 0.64 to 17.79 ng/kg body weight (bw) for average consumers and from 2.40 to 51.69 ng/kg bw per day for high consumers. Current exposure estimates are, however, associated with considerable uncertainty. While biomarker-based approaches may contribute to improved exposure assessment, there is yet insufficient data on urinary metabolites of OTA and their relation to external dose to allow reliable estimates of daily intake. This study was designed to assess potential species differences in phase II biotransformation in vitro and to establish a correlation between urinary OTA-derived glucuronides and mercapturic acids and external exposure in rats in vivo. In vitro analyses of OTA metabolism using the liver S9 of rats, humans, rabbits and minipigs confirmed formation of an OTA glucuronide but provided no evidence for the formation of OTA-derived mercapturic acids to support their use as biomarkers. Similarly, OTA-derived mercapturic acids were not detected in urine of rats repeatedly dosed with OTA, while indirect analysis using enzymatic hydrolysis of the urine samples prior to LC-MS/MS established a linear relationship between urinary glucuronide excretion and OTA exposure. These results support OTA-derived glucuronides but not mercapturic acids as metabolites suitable for biomonitoring.

Magnetic molecularly imprinted polymers for extraction of S-phenylmercapturic acid from urine samples followed by high-performance liquid chromatography.

In this study, magnetic molecularly imprinted polymers (MMIPs) were prepared and used as sorbents for extraction of S-phenylmercapturic acid (S-PMA) from urine samples, followed by high-performance liquid chromatography ultraviolet-visible (HPLC-UV/Vis) analysis. The MMIPs were synthesized by the copolymerization reaction of (phenylthio) acetic acid (template molecule), methacrylic acid (functional monomers) and ethylene glycol dimethacrylate (cross-linkers). The morphology, structure property and surface groups of the prepared MMIPs were characterized by scan electron microscopy, transmission electron microscopy, infrared spectroscopy, X-ray diffraction pattern, thermogravimetric analyses, Brunauer-Emmett-Teller and vibrating sample magnetometer. The selectivity of the MMIPs was investigated in the presence of interferents. Various parameters affecting the S-PMA extraction efficiency were investigated, including MMIPs amount, pH, sample volume, desorption solvent, as well as extraction and desorption time. The obtained optimal parameters were as follows: MMIPs amount (20 mg), pH (3.0), sample volume (5 mL), desorption solvent (methanol/acetic acid [9/1, v/v]), extraction time (30 minutes) and desorption time (2 minutes). The method was validated according to the Food and Drug Administration Guidance for Industry on Bioanalytical Method Validation. The calibration curve for the analyte was linear in the concentration range of 0.030-1.0 mg/L (r = 0.9995). The LOD and LOQ of the method were 0.0080 and 0.0267 mg/L, respectively. The enrichment factor of the MMIPs was 5. The relative standard deviations of intra- and inter-day tests were in the range of 3.8-5.1% and 3.9-6.3%, respectively. The recoveries at three different concentrations of 0.10, 0.50 and 0.80 mg/L ranged between 95.2% and 98.6%. In addition, the MMIPs could be reused for at least eight times. The proposed method was successfully applied to the determination of S-PMA in urine samples. In addition, this developed method could be used as a tool in the early screening and clinical diagnosis of benzene intoxication.

Psychosocial impairment following mild blast-induced traumatic brain injury in rats.

Traumatic brain injury (TBI) is associated with increased risk for mental health disorders, impacting post-injury quality of life and societal reintegration. TBI is also associated with deficits in psychosocial processing, defined as the cognitive integration of social and emotional behaviors, however little is known about how these deficits manifest and their contributions to post-TBI mental health. In this pre-clinical investigation using rats, a single mild blast TBI (mbTBI) induced impairment of psychosocial processing in the absence of confounding physical polytrauma, post-injury motor deficits, affective abnormalities, or deficits in non-social behavior. Impairment severity correlated with acute upregulations of a known oxidative stress metabolite, 3-hydroxypropylmercapturic acid (3-HPMA), in urine. Resting state fMRI alterations in the acute post-injury period implicated key brain regions known to regulate psychosocial behavior, including orbitofrontal cortex (OFC), which is congruent with our previous report of elevated acrolein, a marker of neurotrauma and 3-HPMA precursor, in this region following mbTBI. OFC of mbTBI-exposed rats demonstrated elevated mRNA expression of metabotropic glutamate receptors 1 and 5 (mGluR1/5) and injection of mGluR1/5-selective agonist in OFC of uninjured rats approximated mbTBI-induced psychosocial processing impairment, demonstrating a novel role for OFC in this psychosocial behavior. Furthermore, OFC may serve as a hotspot for TBI-induced disruption of psychosocial processing and subsequent mental health disorders.

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

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.

Ethnic differences in excretion of butadiene-DNA adducts by current smokers.

1,3-Butadiene (BD) is a known human carcinogen used in the synthetic polymer industry and also found in cigarette smoke, automobile exhaust and wood burning smoke. BD is metabolically activated by cytochrome P450 monooxygenases (CYP) 2E1 and 2A6 to 3,4-epoxy-1-butene (EB), which can be detoxified by GST-catalyzed glutathione conjugation or hydrolysis. We have previously observed ethnic differences in urinary levels of EB-mercapturic acids in white, Japanese American and Native Hawaiian smokers. In the present study, similar analyses were extended to urinary BD-DNA adducts. BD-induced N7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) adducts were quantified in urine samples obtained from smokers and non-smokers belonging to three racial/ethnic groups: white, Japanese American and Native Hawaiian. After adjusting for sex, age, nicotine equivalents, body mass index and batch, we found that Japanese American smokers excreted significantly higher amounts of urinary EB-GII than whites [1.45 (95% confidence interval: 1.12-1.87) versus 0.68 (95% confidence interval: 0.52-0.85) fmol/ml urine, P = 4 × 10-5]. Levels of urinary EB-GII in Native Hawaiian smokers were not different from those in whites [0.67 (95% confidence interval: 0.51-0.84) fmol/ml urine, P = 0.938]. There were no racial/ethnic differences in urinary EB-GII adduct levels in non-smokers. Racial/ethnic differences in urinary EB-GII adduct levels in smokers could not be explained by GSTT1 gene deletion or CYP2A6 enzymatic activity. Urinary EB-GII adduct levels in smokers were significantly associated with concentrations of BD metabolite dihyroxybutyl mercapturic acid. Overall, our results reveal that urinary EB-GII adducts in smokers differ across racial/ethnic groups. Future studies are required to understand genetic and epigenetic factors that may be responsible for these differences.

Household tobacco smoke exposure and acrylonitrile metabolite levels in a US pediatric sample.

This study seeks to determine if members of a pediatric (<18 years) sample who have high levels of the urinary volatile organic compound (VOC) metabolite of acrylonitrile, N-Acetyl-S-(2-cyanoethyl)-l-cysteine (CYMA), are significantly more likely to be living in a household with indoor tobacco smoke exposure. A weighted logistic regression was used to compare pediatric participants in the highest quartile of CYMA levels (≥ 4.56 ng/mL) with those whose CYMA levels were not in the highest quartile. 411 pediatric participants were identified in the NHANES data for analysis. Those in the highest quartile of recorded CYMA values were more likely to be living with active indoor smokers (69.35 %) than those who were not in the highest quartile (32.72 %). Having one indoor smoker (adjusted-OR: 2.53, 95 % CI: 1.01-6.34) or 2+ indoor smokers (adjusted-OR: 4.25, 95 % CI: 1.84-9.81) were both associated with a pediatric participant having a CYMA value in the highest quartile.