An eco-friendly solvent-free reaction based on peptide probes: design an extraction-free method for analysis of acrylamide under microliter volume.

Acrylamide is a group 2A carcinogen and potential endocrine disruptor that can enter the ecosystem by various routes and has recently become a dangerous pollutant. This widely used chemical can enter the human body via air inhalation, food or water consumption, or skin contact. In this study, we developed a peptide probe for the detection of acrylamide by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) after its micro-tagging with a peptide. Direct detection of acrylamide by MALDI-TOF MS is not feasible due to its poor ionization in the MALDI interface, which hinders its analysis by the technique. After microwave irradiation for 2 min, the formed acrylamide-peptide derivative was detected easily by MALDI-TOF MS without the need for extraction procedures. The procedure does not involve organic solvents and a water-soluble peptide that allows detection of acrylamide in small sample volumes with a limit of detection (LOD) of 0.05 ng/µL. The relative standard deviation (RSD) and relative error (RE) of the measurements were < 6.7% for intra- and inter-day assays. Gel-washing solutions from a polyacrylamide gel experiment were used as a model to study the efficiency of the developed method. Finally, we used the proposed method for the detection of free acrylamide in small volumes of lung epithelial cells (a model to test the air inhalation of acrylamide under a tiny volume of sample) and human urine. The developed method will enable rapid acrylamide detection in environmental and biological samples via a green approach based on microwave-assisted derivatization in water alongside the use of a less toxic derivatization reagent, reusable target plate, and miniaturization protocols.

Are AAMA and GAMA Levels in Urine after Childbirth a Suitable Marker to Assess Exposure to Acrylamide from Passive Smoking during Pregnancy?-A Pilot Study.

INTRODUCTION: Acrylamide (AA) is a "probably carcinogenic to humans" monomer that can form in heated starchy food and in tobacco smoke. N-Acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA) and N-Acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine (GAMA), acrylamide metabolites in urine, are recognized as good markers of exposure to acrylamide. AIM: The aim of the study is a preliminary assessment whether the levels of AAMA and GAMA in urine after childbirth are good markers of acrylamide exposure due to passive smoking during pregnancy. MATERIAL AND METHOD: The study group consisted 67 non-smokers and 10 passive-smoker women during pregnancy. AAMA and GAMA levels in urine samples were determined using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). RESULTS: The median AAMA levels in urine of non-smoking and passively smoking women were 30.7 µg/g creatinine and 25.2 µg/g creatinine, respectively. Much lower values were determined for GAMA: 11.4 µg/g creatinine and 10.3 µg/g creatinine, respectively. There is no significant difference between AAMA and GAMA content in urine samples between both groups of women as well as in the anthropometric parameters of newborns between those two groups of mothers. CONCLUSION: Our pilot study did not confirm that postpartum AAMA and GAMA concentrations in urine are good markers of exposure to acrylamide from passive smoking during pregnancy. It is probably due to the different ways of acrylamide absorption from tobacco smoke by active and passive smokers. Exposure of pregnant women to acrylamide from passive smoking requires further research.

Association between Acrylamide Metabolites and Cardiovascular Risk in Children With Early Stages of Chronic Kidney Disease.

Cardiovascular disease (CVD) begins early in children with chronic kidney disease (CKD). Reduced nitric oxide (NO) bioavailability has been associated with increased CVD in CKD patients. Children tend to have more exposure to acrylamide, one of the most common toxins in food. We aimed to determine whether urinary levels of acrylamide metabolites N-acetyl-S-(2-carbamoylethyl)-cysteine (AAMA) and N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-cysteine (GAMA) are associated with CV risk markers in children with CKD. Data on 112 children and adolescents ages three to 18 years old with CKD stage G1-G4 are reported. We observed that 24 h ambulatory blood pressure monitoring (ABPM) abnormalities were greater, and left ventricular (LV) mass and ambulatory arterial stiffness index (AASI) were higher in children with CKD stage G2-G4 versus G1. Patients with CKD stage G2-G4 had a lower urinary acrylamide level, but a higher AAMA-to-GAMA ratio than those with CKD stage G1. Urinary acrylamide level was negatively associated with high systolic blood pressure (SBP) and diastolic BP (DBP) load on 24 h ABPM. Lower urinary levels of acrylamide, AAMA, and GAMA were correlated with LV mass. Additionally, GAMA are superior to AAMA related to NO-related parameters, namely citrulline and symmetric dimethylarginine (SDMA). This study suggests that determinations of urinary acrylamide level and its metabolites in the early stages of pediatric CKD may identify patients at risk of CVD. Further studies should clarify mechanisms underlying acrylamide exposure to define the treatment for protection against CVD.

Rapid Simultaneous Determination of Cascade Metabolites of Acrylamide in Urine for Toxicokinetics Profiles and Short-Term Dietary Internal Exposure.

The current study developed an ultrahigh-performance liquid chromatography tandem mass spectrometry method to simultaneously analyze cascade metabolites of acrylamide in urine of rats and humans, including acrylamide, glycidamide, N-acetyl-S-(2-carbamoylethyl)-l-cysteine (AAMA), N-acetyl-S-(2-carbamoylethyl)-l-cysteine-sulfoxide, N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-l-cysteine, and N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)-l-cysteine. A tandem solid-phase extraction procedure was novelly used to purify all metabolites at once from human urine. The rapid analysis showed high sensitivity with LOD and LOQ ranges of 0.1-0.8 and 0.4-5.8 ng/mL, respectively, and achieved acceptable within-laboratory reproducibility (RSD < 12.0%) and spiking recovery (92.2%-117.3%) within 8 min per sample. Approximately 70.7 and 63.0% of ingested acrylamide were recovered during the toxicokinetics analysis from urine of male and female rats, respectively. For nonsmoking participants, the urinary levels of acrylamide and glycidamide were higher in men than women, whereas the urinary concentration of AAMA showed the opposite behavior. The current analysis provides methodological support of cascade metabolites of acrylamide for the dietary short-term internal exposure assessment of acrylamide.

Study Design, Rationale and Procedures for Human Biomonitoring of Hazardous Chemicals from Foods and Cooking in Korea.

Objectives: A nationwide biomonitoring program identified the long-term trends of environmental exposures to hazardous chemicals in the general population and found geographical locations where body burdens of an exposed group significantly differed from those of the general population. The purpose of this study is to analyze the hazardous compounds associated with foods and cooking in the nationwide general population for evaluation of the environmental exposures and health risk factors and for the establishment of the reference levels at the national level. Methods: During 2009-2010, the National Institute of Food and Drug Safety Evaluation (NIFDS) conducted a nationwide human biomonitoring study, including a questionnaire survey and environmental exposure assessments for specific hazardous compounds from foods and cooking among the general population in South Korea. Results: A total of 2139 individuals voluntarily participated in 98 survey units in South Korea, including 889 (41.6%) men and 1250 women (58.4%). Bio-specimens (serum and urine) and questionnaires were collected from the study population. Acrylamides, heterocyclic amines (HCAs), phenols, and phthalates were analyzed from urine, and perfluorinated compounds (PFCs) and organic chloride pesticides (OCPs) were analyzed from serum samples. The information on exposure pathway and geographical locations for all participants was collected by questionnaire interviews, which included demographic characteristics, socioeconomic status, history of family diseases, conditions of the indoor and outdoor environment, lifestyles, occupational history, and food and dietary information. Conclusion: We describe the design of the study and sampling of human biospecimen procedures including bio-sample repository systems. The resources produced from this nationwide human biomonitoring study and survey will be valuable for use in future biomarkers studies and for the assessment of exposure to hazardous compounds associated with foods and cooking.

Association of urinary acrylamide concentration with lifestyle and demographic factors in a population of South Korean children and adolescents.

Acrylamide (AA) has been identified as probably carcinogenic to humans and thus represents a potential public health threat. This study aimed to determine the urinary concentrations of AA and N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA) in a nationally representative sample (n = 1025) of children and adolescents (age range 3-18 years) in South Korea. The AA and AAMA detection rates and geometric mean concentrations were 97%, 19.1 ng/mL, and 98.7%, 26.4 ng/mL, respectively. Although urinary AA levels did not vary widely by age (17.2 ng/mL at 3-6 years, 19.9 ng/mL at 7-18 years), the urinary concentration of AAMA increased with age (18.3 ng/mL at 3-6 years, 30.4 ng/mL at 7-18 years). A multiple linear regression analysis revealed that the urinary levels of AA and AAMA varied significantly by sex, with the adjusted proportional changes indicating rates of 1.47- to 1.48-fold higher at 3-6 years and 1.36- to 1.68-fold higher at 7-18 years among males relative to females. Furthermore, the urinary levels of AA and AAMA correlated with the consumption of certain foods (doughnuts, hotdogs, popcorn, and nachos) among male subjects aged 7-18 years. The urinary concentrations of AA and AAMA increased significantly with the smoking status and passive smoking exposure, with adjusted proportional changes of 1.51 to 1.71-fold higher among smokers relative to non-smokers in the age range of 7-18 years. Exposure to smoking for > 30 min led to adjusted proportional increases in AA and AAMA of 1.51 and 1.77 times in the non-smoking group aged 3-6 years and a 1.52-fold increase in AAMA in the non-smoking group aged 7-18 years. In conclusion, the urinary levels of AA and AAMA were found to associate with age, sex, smoking, and food consumption in a population of Korean children and adolescents.

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.

Feasibility of using urinary N7-(2-carbamoyl-2-hydroxyethyl) Guanine as a biomarker for acrylamide exposed workers.

Acrylamide (AA), a probable human carcinogen, is a widely-used industrial chemical but is also present in tobacco smoke and carbohydrate-rich foods processed at high temperatures. AA is metabolized to glycidamide (GA) to cause the formation of DNA adducts. N7-(2-carbamoyl-2-hydroxyethyl) guanine (N7-GAG), the most abundant DNA adduct induced by GA, was recently detected in urine of smokers and non-smokers. In this study, we assessed the variability of AA exposure and biomarkers of AA exposure in urine samples repeatedly collected from AA-exposed workers and explored the half-life of N7-GAG. A total of 8 AA-exposed workers and 36 non-exposed workers were recruited. Pre-shift and post-shift urine samples were collected from the exposed group in parallel with personal sampling for eight consecutive days and from the control group on day 1 of the study. Urinary N7-GAG and the mercapturic acids of AA and GA, namely N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA) and N-(R,S)-acetyl-S-(1-carbamoyl-2-hydroxyethyl)-L-cysteine (GAMA) were analyzed using on-line solid phase extraction-liquid chromatography-electrospray ionization/tandem mass spectrometry methods. We found that N7-GAG levels in urine were significantly higher in exposed workers than in controls and that N7-GAG level correlated positively with AAMA and GAMA levels. Results from this study showed that AAMA and GAMA possibly remain the more preferred biomarkers of AA exposure and that N7-GAG levels could be elevated by occupational exposures to AA and serve as a biomarker of AA-induced genotoxicity for epidemiological studies.

Exposure assessment of process-related contaminants in food by biomarker monitoring.

Exposure assessment is a fundamental part of the risk assessment paradigm, but can often present a number of challenges and uncertainties. This is especially the case for process contaminants formed during the processing, e.g. heating of food, since they are in part highly reactive and/or volatile, thus making exposure assessment by analysing contents in food unreliable. New approaches are therefore required to accurately assess consumer exposure and thus better inform the risk assessment. Such novel approaches may include the use of biomarkers, physiologically based kinetic (PBK) modelling-facilitated reverse dosimetry, and/or duplicate diet studies. This review focuses on the state of the art with respect to the use of biomarkers of exposure for the process contaminants acrylamide, 3-MCPD esters, glycidyl esters, furan and acrolein. From the overview presented, it becomes clear that the field of assessing human exposure to process-related contaminants in food by biomarker monitoring is promising and strongly developing. The current state of the art as well as the existing data gaps and challenges for the future were defined. They include (1) using PBK modelling and duplicate diet studies to establish, preferably in humans, correlations between external exposure and biomarkers; (2) elucidation of the possible endogenous formation of the process-related contaminants and the resulting biomarker levels; (3) the influence of inter-individual variations and how to include that in the biomarker-based exposure predictions; (4) the correction for confounding factors; (5) the value of the different biomarkers in relation to exposure scenario's and risk assessment, and (6) the possibilities of novel methodologies. In spite of these challenges it can be concluded that biomarker-based exposure assessment provides a unique opportunity to more accurately assess consumer exposure to process-related contaminants in food and thus to better inform risk assessment.

Metabonomic analysis of quercetin against the toxicity of acrylamide in rat urine.

This research aims to determine whether quercetin has protective effects against the toxicity of acrylamide (AA) using metabonomic technology. Randomly, the rats were assigned into a control group, AA treatment group, quercetin treatment group and quercetin plus AA treatment group. Quercetin and AA were administered to rats daily via gavage and drinking water for 16 weeks, respectively. To detect the metabonomic profiles of urine, ultra-performance liquid chromatography/mass spectrometry was used. A total of 15 metabolites, including biomarkers of AA exposure (GAMA, AAMA, and iso-AAMA) and quercetin exposure (quercetin and isorhamnetin), were identified. In comparison with the control group, the intensities of GAMA, AAMA, iso-AAMA, 1-salicylate glucuronide, vinylacetylglycine, PE(20:1(11Z)/14:0), 7-ketodeoxycholic acid, cysteic acid, p-cresol sulfate, and l-cysteine in the AA-treated group were statistically significantly increased (p < 0.01), and the intensities of 2-indolecarboxylic acid, 3-acetamidobutanal, and kynurenic acid in the AA-treated group were statistically significantly decreased (p < 0.01). The above-mentioned metabolites were significantly ameliorated in the quercetin (50 mg per kg bw) plus AA-treated group compared with the AA-treated group (p < 0.01 or p < 0.05). However, the intensities of these metabolites in the quercetin (50 mg per kg bw) plus AA-treated groups were still significantly different from those of the control group (p < 0.01 or p < 0.05). The above results suggest that quercetin has a partial protective effect on AA-induced toxicity. The protective effects include regulation of fatty acid metabolism and amino acid metabolism and enhancing the antioxidant defense system.

Metabolomics analysis of urine from rats administered with long-term, low-dose acrylamide by ultra-performance liquid chromatography-mass spectrometry.

1. To study the toxic effect of chronic exposure to acrylamide (AA) at low-dose levels, we applied metabolomics approach based on ultra-performance liquid chromatography/mass spectrometry (UPLC-MS). A total of 40 male Wistar rats were randomly assigned to different groups: control, low-dose AA (0.2 mg/kg.bw), middle-dose AA (1 mg/kg.bw) and high-dose AA (5 mg/kg.bw). The rats continuously received AA via drinking water for 16 weeks. Rat urine samples were collected at different time points for measurement of metabolomic profiles. 2. Thirteen metabolites, including the biomarkers of AA exposure (AAMA, GAMA and iso-GAMA), were identified from the metabolomic profiles of rat urine. Compared with the control group, the treated groups showed significantly increased intensities of GAMA, AAMA, iso-GAMA, vinylacetylglycine, 1-salicylate glucuronide, PE (20:1(11Z)/14:0), cysteic acid, L-cysteine, p-cresol sulfate and 7-ketodeoxycholic acid, as well as decreased intensities of 3-acetamidobutanal, 2-indolecarboxylic acid and kynurenic acid in rat urine. Notably, three new candidate biomarkers (p-cresol sulfate, 7-ketodeoxycholic acid and 1-salicylate glucuronide) in rat urine exposed to AA have been found in this study. 3. The results indicate exposure to AA disrupts the metabolism of lipids and amino acids, induces oxidative stress.

Toxicokinetics and internal exposure of acrylamide: new insight into comprehensively profiling mercapturic acid metabolites as short-term biomarkers in rats and Chinese adolescents.

Acrylamide is classified as a probable carcinogen to humans and generated from Maillard reaction. Currently, the short-term exposure to acrylamide was evaluated via external diet sources in vitro or two main mercapturic acid metabolites: N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA) and N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine (GAMA) in vivo. In the present work, we comprehensively profiled four mercapturic acid metabolites and evaluated their internal exposure in rats and Chinese adolescents. The cumulative excretion of mercapturic acid metabolites contributes 38.4-73.0 and 43.8-63.6 % of total in vivo metabolites of acrylamide in male and female rats, respectively, when 1, 10, and 50 mg/kg bw of acrylamide were orally administered. Toxicokinetic study revealed that the conversion of acrylamide into glycidamide and glutathione coupling process is highly related to the gender and oral gavage dose via evaluating kinetic parameters, accumulative excretion percentages, and molar ratios of oxidative to reductive metabolism. In human study, a total of 101 Chinese adolescents (41 men and 60 women) were enrolled and served with a meal of potato chips, corresponding to a single-dose (12.6 µg/kg bw) exposure to acrylamide. Toxicokinetic work showed that AAMA is an early and predominant metabolite appearing as a biomarker in urine. N-acetyl-S-(2-carbamoylethyl)-L-cysteine-sulfoxide (AAMA-sul), an oxidative product from AAMA, exhibits a higher peak concentration than GAMA and N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)-L-cysteine (iso-GAMA) during the whole 48-h toxicokinetic period. The internal exposure via four mercapturic acid metabolites is associated with the gender and body mass index characteristics. Thus, current study aims at mercapturic acid metabolites as urinary biomarkers and provides comprehensive insights into the short-term internal exposure to acrylamide.

Towards a biological monitoring guidance value for acrylamide.

Acrylamide is classified as a potential human carcinogen and neurotoxicant. Biological monitoring is a useful tool for monitoring worker exposure. However, other sources of exposure to acrylamide (including cigarette smoke and diet) also need to be considered. This study has performed repeat measurements of the urinary mercapturic acids of acrylamide (AAMA) and its metabolite glycidamide (GAMA) and determined globin adducts in 20 production-plant workers at a UK acrylamide production facility. The relationship between biomarker levels and environmental monitoring data (air levels and hand washes) was investigated. Good correlations were found between all of the biomarkers (r(2)=0.86-0.91) and moderate correlations were found between the biomarkers and air levels (r(2) = 0.56-0.65). Our data show that urinary AAMA is a reliable biomarker of acrylamide exposure. Occupational hygiene data showed that acrylamide exposure at the company was well within the current UK Workplace Exposure Limit. The 90th percentile of urinary AAMA in non-smoking production-plant workers (537 µmol/mol creatinine (n = 59 samples)) is proposed as a possible biological monitoring guidance value. This 90th percentile increased to 798 µmol/mol if smokers were included (n = 72 samples). These values would be expected following an airborne exposure of less than 0.07 mg/m(3), well below the current UK workplace exposure limit of 0.3mg/m(3). Comparison of biomarker levels in non-occupationally exposed individuals suggests regional variations (between UK and Germany), possibly due to differences in diet.

Urinary concentrations of acrylamide (AA) and N-acetyl-S-(2-carbamoylethyl)-cysteine (AAMA) and associations with demographic factors in the South Korean population.

Acrylamide (AA) and N-acetyl-S-(2-carbamoylethyl)-cysteine (AAMA) are important urinary biomarkers of acrylamide exposure in human biomonitoring, because AA is classified as a probable carcinogen in humans. In this study, urinary AA and AAMA were assessed in the South Korean adult population aged 18-69, based on the Korean National Human Biomonitoring Survey conducted in 2009. Urinary metabolites in samples were analyzed with LC-MS/MS system. Relying on data from 1873 representative South Korean adults, the population-weighted geometric means of urinary AA and AAMA concentrations were 6.8 ng/ml (95% CI: 6.4-7.3), and 30.0 ng/ml (95% confidence interval (CI): 28.2-31.8), respectively. The creatinine-adjusted geometric means of AA and AAMA were 6.2 µg/g creatinine (95% CI: 5.8-6.7) and 26.4µg/g creatinine (95% CI: 24.9-28.0), respectively. When covariates for predictors of urinary metabolites were adjusted simultaneously in a log-linear multiple regressions, the strongest predictors of urinary AA were education (OR=1.08-1.28; 95% CI: 1.11-1.48; p=0.0024) and age (OR=0.66-0.84; 95% CI: 0.54-0.97; p=0.0003), and those of urinary AAMA were smoking status (OR=1.16-2.63; 95% CI: 0.98-3.08; p=0.001) and education (OR=1.12-1.19; 95% CI: 1.02-1.38; p=0.0425). The ratio of current/never smokers for urinary AA was 1.3, whereas the same ratio for urinary AAMA was 3.0. These findings suggested that most South Koreans had detectable levels of AA and AAMA (98.7% and 99.4%, respectively) in their urine and that the body burden of AA and AAMA varied according to demographic, geographic, and lifestyle (smoking) factors.

Profiling of mercapturic acids of acrolein and acrylamide in human urine after consumption of potato crisps.

SCOPE: Acrolein (AC) and acrylamide (AA) are food contaminants generated by heat treatment. We studied human exposure after consumption of potato crisps by monitoring excretion of mercapturic acids (MAs) in urine. METHODS AND RESULTS: MA excretion was monitored in human urine collected up to 72 h after ingestion of a test meal of experimental (study 1: 1 mg AA/150 g) or commercially available (study 2: 44 µg AA plus 4.6 µg AC/175 g) potato crisps. MA contents were analysed after purification via SPE using HPLC-ESI-MS/MS. On the basis of the area under the curve values of MAs excreted in urine, the total excretion of AC-related MAs exceeded that of AA-related MAs up to 12 times in study 1 and up to four times in study 2. Remarkably, AC content of potato crisps of study 2 was found to be only about 1/10 the AA content, as determined by isotope dilution headspace GC/MS. CONCLUSION: Our results indicate substantially higher exposure to AC from potato crisps than to AA. Total AC in such foods may encompass bioavailable AC forms not detected by headspace GC/MS. Both findings may also apply to other heat processed foods.

N7-glycidamide-guanine DNA adduct formation by orally ingested acrylamide in rats: a dose-response study encompassing human diet-related exposure levels.

Acrylamide (AA) is formed during the heating of food and is classified as a genotoxic carcinogen. The margin of exposure (MOE), representing the distance between the bench mark dose associated with 10% tumor incidence in rats and the estimated average human exposure, is considered to be of concern. After ingestion, AA is converted by P450 into the genotoxic epoxide glycidamide (GA). GA forms DNA adducts, primarily at N7 of guanine (N7-GA-Gua). We performed a dose-response study with AA in female Sprague-Dawley (SD) rats. AA was given orally in a single dosage of 0.1-10 000 µg/kg bw. The formation of urinary mercapturic acids and of N7-GA-Gua DNA adducts in liver, kidney, and lung was measured 16 h after application. A mean of 37.0 ± 11.5% of a given AA dose was found as mercapturic acids (MAs) in urine. MA excretion in urine of untreated controls indicated some background exposure from endogenous AA. N7-GA-Gua adduct formation was not detectable in any organ tested at 0.1 µg AA/kg bw. At a dose of 1 µg/kg bw, adducts were found in kidney (around 1 adduct/10(8) nucleotides) and lung (below 1 adduct/10(8) nucleotides) but not in liver. At 10, respectively, 100 µg/kg bw, adducts were found in all three organs, at levels close to those found at 1 µg AA/kg, covering a range of about 1-2 adducts/10(8) nucleotides. As compared to DNA adduct levels from electrophilic genotoxic agents of various origin found in human tissues, N7-GA-Gua adduct levels within the dose range of 0.1-100 µg AA/kg bw were at the low end of this human background. We propose to take the background level of DNA lesions in humans more into consideration when doing risk assessment of food-borne genotoxic carcinogens.

Occupational exposure to acrylamide in closed system production plants: air levels and biomonitoring.

The aim of this study was to evaluate biomarkers of acrylamide exposure, including hemoglobin adducts and urinary metabolites in acrylamide production workers. Biomarkers are integrated measures of the internal dose, and it is total acrylamide dose from all routes and sources that may present health risks. Workers from three companies were studied. Workers potentially exposed to acrylamide monomer wore personal breathing-zone air samplers. Air samples and surface-wipe samples were collected and analyzed for acrylamide. General-area air samples were collected in chemical processing units and control rooms. Hemoglobin adducts were isolated from ethylenediamine teraacetic acid (EDTA)-whole blood, and adducts of acrylamide and glycidamide, at the N-terminal valines of hemoglobin, were cleaved from the protein chain by use of a modified Edman reaction. Full work-shift, personal breathing zone, and general-area air samples were collected and analyzed for particulate and acrylamide monomer vapor. The highest general-area concentration of acrylamide vapor was 350 µg/cm(3) in monomer production. Personal breathing zone and general-area concentrations of acrylamide vapor were found to be highest in monomer production operations, and lower levels were in the polymer production operations. Adduct levels varied widely among workers, with the highest in workers in the monomer and polymer production areas. The acrylamide adduct range was 15-1884 pmol/g; glycidamide adducts ranged from 17.8 to 1376 p/mol/g. The highest acrylamide and glycidamide adduct levels were found among monomer production process operators. The primary urinary metabolite N-acetyl-S-(2-carbamoylethyl) cysteine (NACEC) ranged from the limit of detection to 15.4 µg/ml. Correlation of workplace exposure and sentinel health effects is needed to determine and control safe levels of exposure for regulatory standards.

Association of CYP2E1, GST and mEH genetic polymorphisms with urinary acrylamide metabolites in workers exposed to acrylamide.

This study elucidates the association of acrylamide metabolites, N-acetyl-S-(2-carbamoylethyl)-cysteine (AAMA), N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)-cysteine (GAMA2), and N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-cysteine (GAMA3) in urine with genetic polymorphisms of the metabolic enzymes cytochrome P450 2E1 (CYP2E1), microsomal epoxide hydrolase (mEH) in exon 3 and exon 4, glutathione transferase theta (GSTT1) and mu (GSTM1), involved in the activation and detoxification of acrylamide (AA) in humans. Eighty-five workers were recruited, including 51 AA-exposed workers and 34 administrative staffs serve as controls. Personal air sampling was performed for the exposed workers. Each subject provided pre- and post-shift urine samples and blood samples. Urinary AAMA, GAMA2 and GAMA3 levels were simultaneously quantified using liquid chromatography-electronspray ionization/tandem mass spectrometry (LC-ESI-MS/MS). CYP2E1, mEH (in exon 3 and exon 4), GSTT1, and GSTM1 were analyzed using polymerase chain reaction (PCR). Our results reveal that AA personal exposures ranged from 4.37 × 10⁻³ to 113.61 µg/m³ with a mean at 15.36 µg/m³. The AAMA, GAMA2, and GAMA3 levels in the exposed group significantly exceeded those in controls. The GAMAs (the sum of GAMA2 and GAMA3)/AAMA ratios, potentially reflecting the proportion of AA metabolized to glycidamide (GA), varied from 0.003 to 0.456, and indicate high inter-individual variability in the metabolism of AA to GA in this study population. Multivariate regression analysis demonstrates that GSTM1 genotypes significantly modify the excretion of urinary AAMA and the GAMAs/AAMA ratio, exon 4 of mEH was significantly associated with the urinary GAMAs levels after adjustment for AA exposures. These results suggest that mEH and/or GSTM1 may be associated with the formation of urinary AAMA and GAMAs. Further study may be needed to shed light on the role of both enzymes in AA metabolism.

Biological effects of acrylamide after daily ingestion of various foods in comparison to water: a study in rats.

SCOPE: Acrylamide (AA), classified as a genotoxic carcinogen, is generated by heating foods. We studied whether the food matrix modulates bioavailability and/or biotransformation and investigated kinetics and biological effectiveness of AA in rats. METHODS AND RESULTS: AA was given to the animals at a daily intake level of AA containing foods for up to 9 days, resulting in an exposure of 50 or 100 µg AA/kg body weight (b.w.)/day. Positive controls received the same dosages of AA in water, negative controls just water. As biomarkers urinary mercapturic acids, hemoglobin adducts, plasma levels of AA and glycidamide (GA) and DNA integrity in white blood cells and hepatocytes were measured. Altogether, no significant differences in bioavailability of AA from water and the different food matrices were observed. Only with bread crust, biomarkers indicated a slightly reduced bioavailability. Monitoring glycidamide valine adduct adducts did not provide evidence for treatment-related significantly enhanced GA-haemoglobin adduct formation in blood although glycidamide mercapturic acid excretion in urine indicated significant GA formation. CONCLUSIONS: The results suggest AA at dietary intake levels, exceeding estimated human mean intake by a factor of at least 100 to become detoxified in Sprague-Dawley rats to a major extent through glutathione coupling.

Development of a physiologically-based toxicokinetic model of acrylamide and glycidamide in rats and humans.

Physiologically-based toxicokinetic ("pharmacokinetic") (PBPK or PBTK) modeling can be used as a tool to compare internal doses of acrylamide (AA) and its metabolite glycidamide (GA) in humans and rats. An earlier PBTK model for AA and GA in rats was refined and extended to humans based on new data. With adjustments to the previous parameters, excellent fits to a majority of the data for male Fisher 344 rats were obtained. Kinetic parameters for the human model were estimated based on fit to available human data for urinary metabolites of AA, and levels of hemoglobin adducts of AA and GA measured in studies in which human volunteers ingested known doses of AA. The simulations conducted with the rat and human models predicted that rats and humans ingesting comparable levels of AA (in mg/kg day) would have similar levels of GA in blood and tissues. This finding stands in contrast to the default approach that assumes a 3.2-fold increase in human risk due to pharmacokinetic differences between rats and humans. This model was used in a companion paper to estimate safe levels of ingested AA.