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.

Less is more: a methodological assessment of extraction techniques for per- and polyfluoroalkyl substances (PFAS) analysis in mammalian tissues.

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants. Studying the bioaccumulation in mammalian tissues requires a considerable effort for the PFAS extraction from complex biological matrices. The aim of the current work was to select and optimize the most efficient among common extraction strategies for eleven perfluoroalkyl acids (PFAA). Primary extractions from wild boar tissues (liver, kidney, and lung) were performed with methanol at neutral, acidic, or alkaline conditions, or with methyl-tert-butyl ether (MTBE) after ion-pairing with tetrabutylammonium (TBA) ions. A second purification step was chosen after comparing different solid-phase extraction (SPE) cartridges (Oasis WAX, ENVI-Carb, HybridSPE Phospholipid) and various combinations thereof or dispersive SPE with C18 and ENVI-Carb material. The best extraction efficiencies of the liquid PFAA extraction from tissue homogenates were achieved with methanol alone (recoveries from liver 86.6-114.4%). Further purification of the methanolic extracts using dispersive SPE or Oasis WAX columns decreased recoveries of most PFAA, whereas using pairs of two SPE columns connected in series proved to be more efficient albeit laborious. Highest recoveries for ten out of eleven PFAA were achieved using ENVI-Carb columns (80.3-110.6%). In summary, the simplest extraction methods using methanol and ENVI-Carb columns were also the most efficient. The technique was validated and applied in a proof of principle analysis in human tissue samples.

Risks of misinterpretation of biomarker measurements in spot urine adjusted for creatinine - A problem especially for studies comparing plant based with omnivorous diets.

Biomarker measurements in spot urine are often adjusted for creatinine to control for dilution resulting from individual hydration. We here report on results of a study involving age- and sex-matched vegans and omnivores (n = 36 each). The daily urinary excretion of 2,3-dihydroxypropylmercapturic acid (DHPMA, a diet-independent endogenous C3-metabolite used as an example compound) was found not to be different in vegans and omnivores (median 433 µg/24 h each), however, creatinine-adjusted levels were 26% lower in omnivores (median 285 µg/g creatinine) than in vegans (median 383 µg/g creatinine, p = 0.003). This difference results from the higher urinary excretion of creatinine in the omnivores compared to vegans (median 1.51 vs. 1.21 g/24 h, p = 0.009). Linear regression showed - besides the fat-free mass - a significant impact of the factor diet (vegans vs. omnivores). This may be due to the consumption of meat and fish as exogenous sources of creatinine. A literature search revealed broad evidence for this interpretation, as creatinine is formed from creatine during heating of meat and fish. Accordingly, consumption leads to temporary increase of serum/plasma creatinine and urinary creatinine excretion, resulting in higher levels in omnivores compared to vegans/vegetarians. An adjustment of the urinary DHPMA concentrations using specific gravity revealed 13% lower values in omnivores (median 225 µg/L) than in vegans (median 260 µg/L, p = 0.07). Compared to creatinine-adjustment, adjustment for specific gravity introduces a smaller but still obvious difference between omnivores and vegans. Especially with respect to future studies comparing vegans, vegetarians and omnivores, researchers should be aware of the risks of severe misinterpretations if biomarker measurements in spot urine are adjusted for creatinine.

Stable isotope ratios of nitrogen and carbon as biomarkers of a vegan diet.

PURPOSE: Dietary biomarkers can potentially overcome the limitations of self-reported dietary data. While in ecology and archaeology, stable isotope ratios of carbon and nitrogen are widely used as biomarkers, this is not the case in nutrition research. Since the abundance of the 13C and the 15N isotope differ in food sources from plant and animal origin, stable isotope ratios of carbon and nitrogen (δ13C and δ15N) may differ in human biological material. Here, we investigated the stable isotope ratios of nitrogen and carbon in serum and urine from vegans and omnivores. METHOD: Measurement of δ15N and δ13C in serum and 24 h urine was performed by Elemental Analyzer-Isotope Ratio Mass Spectrometer in the cross-sectional study "Risks and Benefits of a Vegan Diet". The study included 36 vegans and 36 omnivores with a median age of 37.5 years (matched for age and sex), who adhered to their diet for at least 1 year. RESULTS: Both δ15N and δ13C were significantly lower in both the serum and 24 h urine of vegans compared to omnivores. δ15N either in serum or urine had 100% specificity and sensitivity to discriminate between vegans and omnivores. Specificity of δ13C was also > 90%, while sensitivity was 93% in serum and 77% in urine. CONCLUSION: δ15N both in serum and urine was able to accurately identify vegans and thus appears to be a promising marker for dietary habits.

3-MCPD as contaminant in processed foods: State of knowledge and remaining challenges.

3-Chloro-1,2-propanediol (3-MCPD) and its fatty acid esters (FE) are present as contaminants in different processed foods. Based on the available toxicological data the potential risk of 3-MCPD and its FE to human health was assessed by risk assessment authorities, including the European Food Safety Authority (EFSA). Considering the available data, EFSA concluded that 3-MCPD is a non-genotoxic compound exhibiting secondary carcinogenic effects in rodents. A tolerable daily intake of 2 µg/kg body weight and day was derived by EFSA for free and ester-bound 3-MCPD in 2018. However, there are still different pending issues that have remained unclear until now. Here, we summarize the current knowledge regarding 3-MCPD and its FE with a focus on pending issues regarding exposure assessment via biomarkers as well as the identification of (toxic) metabolites formed after exposure to FE of 3-MCPD and their modes of action.

Transdermal absorption of 13C4-perfluorooctanoic acid (13C4-PFOA) from a sunscreen in a male volunteer - What could be the contribution of cosmetics to the internal exposure of perfluoroalkyl substances (PFAS)?

Per- and polyfluoroalkyl substances (PFAS) are a complex group of man-made chemicals with high stability and mobility leading to ubiquitous environmental contamination and accumulation especially of some long-chain perfluoroalkyl acids (PFAA) in humans. While dietary intake is the main route of exposure, transdermal uptake from cosmetic products usually is considered negligible. However, PFAS are present in a part of these products, and recent epidemiological studies have provided evidence for relevant uptake via this route. The crucial question is whether PFAA in cosmetic products can cross the human skin barrier. A defined amount (110 µg) of 13C4-perfluorooctanoic acid (13C4-PFOA) was mixed into a sunscreen (30 g) which was applied on the whole skin of a volunteer. The plasma concentrations of 13C4-PFOA were determined in serial blood samples taken over 115 days using UHPLC-MS/MS and 13C2-PFOA as internal standard. After application, 13C4-PFOA plasma levels increased continuously, reaching levels of 3, 56 and 118 ng/L after 6 h, 3 days and 10 days, respectively. A maximum level of 132 ng/L was measured 22 days after application, representing 9.4 % of the PFOA level resulting from the volunteer's background exposure (1400 ng/L, equivalent to 1.4 ng/mL). In the following weeks, the levels slightly decreased with an estimated half-life of 1.8 years. The best estimate for the fraction absorbed may be 1.6 % of the dose, using a volume of distribution of 0.17 L/kg body weight. For PFOA mixed into a sunscreen, this experimental approach demonstrates a significant uptake of a PFAA via transdermal absorption in humans. In the past, some cosmetic products contained relevant PFAA levels as contaminants/impurities of PFAS added as active ingredients. Depending on these levels and the use (frequency, skin area involved), it is plausible that this route of exposure has contributed to the internal exposure to PFAA, as already suggested by epidemiological observations.

Comparison of Five Oxidative Stress Biomarkers in Vegans and Omnivores from Germany and Finland.

When the amount of reactive oxygen species produced by human metabolism cannot be balanced by antioxidants, this phenomenon is commonly referred to as oxidative stress. It is hypothesised that diets with high amounts of plant food products may have a beneficial impact on oxidative stress status. However, few studies have examined whether a vegan diet is associated with lower oxidative stress compared to an omnivorous diet. The present cross-sectional study aimed to compare the levels of five oxidative stress biomarkers in vegans and omnivores. Data of 36 vegans and 36 omnivores from Germany and of 21 vegans and 18 omnivores from Finland were analysed. HPLC coupled with mass spectrometry or fluorescence detection and ELISA methods were used to measure the oxidative stress biomarkers malondialdehyde (MDA), protein carbonyls and 3-nitrotyrosine in plasma and 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 8-iso-prostaglandin F2α (8-iso-PGF2α) in 24 h urine. Analyses of variance and covariance, considering potential confounders, were used. Vegans and omnivores showed no differences in MDA and protein carbonyl concentrations. In Finnish but not in German vegans, the concentrations of 3-nitrotyrosine were lower compared to those in omnivores (p = 0.047). In Germany, vegans showed lower excretion levels of 8-iso-PGF2α than omnivores (p = 0.002) and with a trend also of 8-OHdG (p = 0.05). The sensitivity analysis suggests lower 8-iso-PGF2α excretion levels in women compared to men, independently of the dietary group. The present study contributes to expanding our knowledge of the relationship between diet and oxidative stress and showed that 3-nitrotyrosine, 8-OHdG and 8-iso-PGF2α tended to be lower in vegans. Furthermore, studies are recommended to validate the present findings.

Simultaneous quantification of eight hemoglobin adducts of genotoxic substances by isotope-dilution UHPLC-MS/MS.

Various genotoxic carcinogens ubiquitously present in the human environment or respective reactive metabolites form adducts in DNA and proteins, which can be used as biomarkers of internal exposure. For example, the mass spectrometric determination of Val adducts at the N-termini of hemoglobin (Hb) peptide chains after cleavage by an Edman degradation has a long tradition in occupational medicine. We developed a novel isotope-dilution UHPLC-MS/MS method for the simultaneous quantification of Val adducts of eight genotoxic substances in Hb after cleavage with fluorescein-5-isothiocyanate (FIRE procedure™). The following adducts were included [sources in square brackets]: N-(2,3-dihydroxypropyl)-Val [glycidol], N-(2-carbamoylethyl)-Val [acrylamide], N-(2-carbamoyl-2-hydroxyethyl)-Val [glycidamide], N-((furan-2-yl)methyl)-Val [furfuryl alcohol], N-(trans-isoestragole-3'-yl)-Val [estragole/anethole], N-(3-ketopentyl)-Val [1-penten-3-one], N-(3-ketooctanyl)-Val [1-octene-3-one], and N-benzyl-Val [benzyl chloride], each of which was quantified with a specific isotope-labeled standard. The limits of quantification were between 0.014 and 3.6 pmol/g Hb (using 35 mg Hb per analysis); other validation parameters were satisfactory according to guidelines of the U.S. Food and Drug Administration. The quantification in erythrocyte samples of human adults (proof of principle) showed that the median levels of Hb adducts of acrylamide, glycidamide, and glycidol were found to be significantly lower in six non-smokers (25.9, 12.2, and 4.7 pmol/g Hb, respectively) compared to those of six smokers (69.0, 44.2, and 8.6 pmol/g Hb, respectively). In summary, the method surpasses former techniques of Hb adduct quantification due to its simplicity, sensitivity, and accuracy. It can be extended continuously with other Hb adducts and will be used in epidemiological studies on internal exposure to carcinogens.

Detection of a Hemoglobin Adduct of the Food Contaminant Furfuryl Alcohol in Humans: Levels of N-((Furan-2-yl)methyl)-valine in Two Epidemiological Studies.

SCOPE: Furfuryl alcohol is a heat-induced food contaminant, classified as possibly carcinogenic to humans. The proximal carcinogen 2-sulfoxymethylfuran leads to adduct formation in DNA and proteins (e.g., N-((furan-2-yl)methyl)-Val (FFA-Val) in hemoglobin). METHODS AND RESULTS: This study analyzed human erythrocyte samples from two studies for the presence of FFA-Val: the Risks and Benefits of a Vegan Diet study (RBVD; 72 adults) and the ENVIRonmental influence ON early AGEing birth cohort study (ENVIRONAGE; 100 mother-newborn pairs). In the RBVD study, FFA-Val levels are lower in vegans compared to omnivores (median 13.0 vs 15.8 pmol g-1 hemoglobin, p = 0.008), and lower in non-smokers compared to smokers (median 14.1 vs 17.0 pmol g-1 hemoglobin, p = 0.003). In the birth cohort, FFA-Val levels are distinctly higher in maternal compared to newborn samples (median 15.2 vs 2.2 pmol g-1 hemoglobin, p < 0.001). CONCLUSIONS: FFA-Val, hitherto detected only in blood samples of mice, is quantifiable in all human samples, indicating a general exposure to furfuryl alcohol. The low adduct levels in blood samples from newborn children suggested that the placenta is a barrier to furfuryl alcohol. Dietary habits and tobacco smoking are two main influencing factors on the formation of FFA-Val, which may be of use as a biomarker of exposure to furfuryl alcohol.

Alkenylbenzenes in Foods: Aspects Impeding the Evaluation of Adverse Health Effects.

Alkenylbenzenes are naturally occurring secondary plant metabolites, primarily present in different herbs and spices, such as basil or fennel seeds. Thus, alkenylbenzenes, such as safrole, methyleugenol, and estragole, can be found in different foods, whenever these herbs and spices (or extracts thereof) are used for food production. In particular, essential oils or other food products derived from the aforementioned herbs and spices, such as basil-containing pesto or plant food supplements, are often characterized by a high content of alkenylbenzenes. While safrole or methyleugenol are known to be genotoxic and carcinogenic, the toxicological relevance of other alkenylbenzenes (e.g., apiol) regarding human health remains widely unclear. In this review, we will briefly summarize and discuss the current knowledge and the uncertainties impeding a conclusive evaluation of adverse effects to human health possibly resulting from consumption of foods containing alkenylbenzenes, especially focusing on the genotoxic compounds, safrole, methyleugenol, and estragole.

Perfluorobutanoic acid (PFBA): No high-level accumulation in human lung and kidney tissue.

Perfluorobutanoic acid (PFBA) belongs to the complex group of synthetic perfluoroalkyl substances (PFAS) which have led to ubiquitous environmental contamination. While some of the long-chain compounds accumulate in the human body, the short-chain compound PFBA was found to have a relatively short half-life in blood of a few days, in agreement with relatively low PFBA serum/plasma levels of roughly 0.01 ng/ml in European studies. Surprisingly, very high median levels of PFBA of 807 and 263 ng/g tissue for human lung and kidney autopsy samples, respectively, were reported in a paper of Pérez et al. (2013). This would question the concept of PFAS blood analysis reflecting the body burden of these compounds. To verify the results of high PFBA tissue accumulation in humans, we have analyzed PFBA in a set of 7 lung and 9 kidney samples from tumor patients with a different method of quantification, using high-resolution mass spectrometry with the accurate mass as analytical parameter. The only human sample with a quantifiable amount of PFBA (peak area more than twice above the analytical background signals) contained approximately 0.17 ng/g lung tissue. In the light of our results and considering the analytical problems with the short-chain compound PFBA exhibiting only one mass fragmentation, it appears to be likely that PFBA is not accumulating on a high level in human lung and kidney tissue. In general, the analysis of short-chain PFAS in complex matrices like food or tissue is very challenging with respect to instrumental quantification and possible sample contamination.

Bioactivation of estragole and anethole leads to common adducts in DNA and hemoglobin.

Estragole and anethole are secondary metabolites occurring in a variety of commonly used herbs like fennel, basil, and anise. Estragole is genotoxic and carcinogenic in rodents, which depends on the formation of 1'-sulfoxyestragole after hydroxylation and subsequent sulfoconjugation catalyzed by CYP and SULT, respectively. It was hypothesized recently that anethole may be bioactivated via the same metabolic pathways. Incubating estragole with hepatic S9-fractions from rats and humans, specific adducts with hemoglobin (N-(isoestragole-3-yl)-valine, IES-Val) and DNA (isoestragole-2'-deoxyguanosine and isoestragole-2'-deoxyadenosine) were formed. An isotope-dilution technique was developed for the quantification of IES-Val after cleavage with fluorescein isothiocyanate (FITC) according to a modified Edman degradation. The same adducts, albeit at lower levels, were also detected in reactions with anethole, indicating the formation of 3'-hydroxyanethole and the reactive 3'-sulfoxyanethole. Finally, we conducted a pilot investigation in which IES-Val levels in human blood were determined during and after the consumption of an estragole- and anethole-rich fennel tea for four weeks. A significant increase of IES-Val levels was observed during the consumption phase and followed by a continuous decrease during the washout period. IES-Val may be used to monitor the internal exposure to the common reactive genotoxic metabolites of estragole and anethole, 1'-sulfoxyestragole and 3'-sulfoxyanethole, respectively.

Metabolites of 2- and 3-Monochloropropanediol (2- and 3-MCPD) in Humans: Urinary Excretion of 2-Chlorohydracrylic Acid and 3-Chlorolactic Acid after Controlled Exposure to a Single High Dose of Fatty Acid Esters of 2- and 3-MCPD.

SCOPE: Fatty acid esters of 2-monochloropropane-1,3-diol (2-MCPD) and 3-monochloropropane-1,2-diol (3-MCPD) are formed during the deodorization of vegetable oils. After lipase-catalyzed hydrolysis in the intestine, 2- and 3-MCPD are absorbed, but their ensuing human metabolism is unknown. METHODS AND RESULTS: The compounds 2-chlorohydracrylic acid (2-ClHA) and 3-chlorolactic acid (3-ClLA) resulting from oxidative metabolism of 2-MCPD and 3-MCPD, respectively, are identified and quantified in human urine samples. An exposure study with 12 adults is conducted to determine the urinary excretion of 2-ClHA and 3-ClLA. The participants eat 12 g of hazelnut oil containing 24.2 mg kg-1 2-MCPD and 54.5 mg kg-1 3-MCPD in the form of fatty acid esters. Average daily amounts of "background" excretion before the exposure are 69 nmol 2-ClHA and 3.0 nmol 3-ClLA. The additional mean excretion due to the uptake of the hazelnut oil amounts to 893 nmol 2-ClHA (34.0% of the 2-MCPD dose) and 16.4 nmol 3-ClLA (0.28% of the 3-MPCD dose). CONCLUSIONS: The products of oxidative metabolism of 2- and 3-MCPD, 2-ClHA, and 3-ClLA, are described for the first time in humans. Due to the lack of specificity, the metabolites may not be used as exposure biomarkers to low doses of bound 2- and 3-MCPD, respectively.

Urinary Excretion of 2/3-Monochloropropanediol (2/3-MCPD) and 2,3-Dihydroxypropylmercapturic Acid (DHPMA) after a Single High dose of Fatty Acid Esters of 2/3-MCPD and Glycidol: A Controlled Exposure Study in Humans.

SCOPE: 2- and 3-monochloropropanediol (2/3-MCPD) and glycidol are absorbed in the intestine after lipase-catalyzed hydrolysis of their fatty acid esters. METHODS AND RESULTS: In an exposure study with 12 non-smoking participants, the complete urinary excretion of the metabolite 2,3-dihydroxypropylmercapturic acid (DHPMA) and of 2/3-MCPD is measured on four consecutive days before and after consumption of 50 g glycidyl ester-rich palm fat or 12 g 2/3-MCPD ester-rich hazelnut oil. After controlled exposure, urinary excretion rates of 2/3-MCPD per hour strongly increase, followed by a decrease with average half-lives of 5.8 h (2-MCPD) and 3.6 h (3-MCPD). After consumption of hazelnut oil, mean excretion rates are 14.3% (2-MCPD) and 3.7% (3-MCPD) of the study doses. The latter rate is significantly higher (4.6%) after consumption of palm fat, indicating partial conversion (about 5%) of glycidol to 3-MCPD under the acidic conditions in the stomach. The average daily "background" exposure is estimated to be 0.12 and 0.32 µg per kg body weight (BW) for 2-MCPD and 3-MCPD, respectively. The relatively high and constant urinary excretion of DHPMA does not reflect the controlled exposure. CONCLUSION: Urinary excretion of 2- and 3-MCPD is suitable as biomarker for the external exposure to the respective fatty acid esters.

Levels of the hemoglobin adduct N-(2,3-Dihydroxypropyl)-valine in cord and maternal blood: Prenatal transfer of glycidol in the ENVIRONAGE birth cohort.

BACKGROUND: Glycidol, a probable human carcinogen, is a reactive chemical released in the gastrointestinal tract from glycidyl fatty acid esters, which are heat-induced dietary contaminants. OBJECTIVES: To investigate the prenatal transfer of glycidol, a specific hemoglobin adduct was measured as a biomarker for internal glycidol exposure in paired cord and maternal blood samples. METHODS: In 100 mother-newborn pairs from the Belgian ENVIRONAGE (ENVIRonmental influence ON AGEing in early life) birth cohort, we studied the correlation between levels of the glycidol-derived hemoglobin adduct N-(2,3-dihydroxypropyl)-valine (2,3-diHOPr-Val) in paired cord and maternal blood samples. The adduct levels were determined after cleavage with a modified Edman degradation by using ultra-high performance liquid chromatography-tandem mass spectrometry and an isotope-labeled reference standard. RESULTS: 2,3-DiHOPr-Val was detectable in all 100 maternal blood samples and in 96 cord blood samples (LOD =0.5 pmol 2,3-diHOPr-Val/g hemoglobin), with medians of 5.4 (range: 2.3-29.2) and 1.6 (range: LOD - 8.9) pmol/g hemoglobin), respectively. In blood samples of mothers who smoked during pregnancy and in the cord blood samples of their newborns (n = 6), the median 2,3-diHOPr-Val levels were 16.7 (range: 6.4-29.2) and 6.2 (range: LOD - 8.6) pmol/g hemoglobin, respectively. The median ratio of 2,3-diHOPr-Val levels of cord to maternal blood was 0.35 (range: 0.19-1.14) (n = 49). The Spearman correlation coefficient between 2,3-diHOPr-Val levels in cord and maternal blood samples was 0.63 (p < 0.001) among all mother-newborn pairs and 0.59 (p < 0.001) among mother-newborn pairs of non-smoking mothers. DISCUSSION: Maternal data confirm widespread exposure to glycidol, also in non-smokers. Neonatal levels indicate prenatal exposure to glycidol, due to an obviously relatively unhindered passive transfer through the placental barrier. Possible health effects of fetal (and postnatal) glycidol exposure in children may be addressed in epidemiological studies.

Detection of N-Acetyl-S-[3'-(4-methoxyphenyl)allyl]-l-Cys (AMPAC) in Human Urine Samples after Controlled Exposure to Fennel Tea: A New Metabolite of Estragole and trans-Anethole.

Fennel and other herbs contain the secondary plant metabolites estragole and trans-anethole, of which estragole is carcinogenic in rodents. It is metabolically activated by cytochrome P450-catalyzed conversion to 1'-hydroxyestragole and subsequent sulfo conjugation to the genotoxic 1'-sulfoxyestragole. The current study followed the hypothesis that the reactive sulfate ester may be detoxified by glutathione conjugation, leading to the urinary excretion of a resultant mercapturic acid. We identified the assumed downstream metabolite N-acetyl-S-[3'-(4-methoxyphenyl)allyl]-l-Cys (AMPAC) in human urine samples after consumption of fennel tea. An isotope-dilution technique for its quantification by ultraperformance liquid chromatography-tandem mass spectrometry and [13C3,15N]AMPAC in urine samples was developed. The method was applied to determine the AMPAC concentration in urine samples following uptake of 500 mL of fennel tea containing 2.2 mg of estragole by 12 healthy participants (six females and six males). Before drinking the tea, the urinary AMPAC concentration was below the limit of detection. In most of the participants, the highest amounts of urinary AMPAC were found in the first-hour urine after exposure. The excretion by first-order kinetics (range of t1/2 = 0.78-1.54 h; mean ± SD: 1.13 ± 0.21 h) led to a nearly complete clearance within 8 h in all participants. The total AMPAC excreted was in the range of 93-1076 ng, reflecting pronounced interindividual variations of enzymes taking part in estragole metabolism. Importantly, AMPAC was also formed in one volunteer following oral uptake of a single dose of isolated trans-anethole, albeit to a much smaller extent compared to estragole. AMPAC may be of future use as a human biomarker for the internal exposure to the carbocation formed from either 1'-sulfoxyestragole or 3'-sulfoxyisoestragole, the reactive sulfate ester metabolites of estragole and trans-anethole, respectively.

Mass Spectrometric DNA Adduct Quantification by Multiple Reaction Monitoring and Its Future Use for the Molecular Epidemiology of Cancer.

The formation of DNA adducts is considered essential for tumor initiation. Quantification of DNA adducts may be achieved by various techniques of which LC-MS/MS-based multiple reaction monitoring has become the most prominent in the past decade. Adducts of single nucleosides are analyzed following enzymatic break-down of a DNA sample following adduct enrichment usually by solid-phase extraction. LC-MS/MS quantification is carried out using stable isotope-labeled internal reference substances. An upcoming challenge is the use of DNA adducts as biomarkers either for internal exposure to electrophilic genotoxins or for the approximation of cancer risk. Here we review recent studies in which DNA adducts were quantified by LC-MS/MS in DNA samples from human matrices. We outline a possible way for future research to aim at the development of an 'adductome' approach for the characterization of DNA adduct spectra in human tissues. The DNA adduct spectrum reflects the inner exposure of an individual's tissue to electrophilic metabolites and, therefore, should replace the conventional and inaccurate external exposure in epidemiological studies in the future.

Hemoglobin adducts of furfuryl alcohol in genetically modified mouse models: Role of endogenous sulfotransferases 1a1 and 1d1 and transgenic human sulfotransferases 1A1/1A2.

Furfuryl alcohol (FFA) is a heat-induced food contaminant. Conversion by sulfotransferases (SULT) yields 2-sulfoxymethylfuran, which is prone to react with DNA and proteins. In order to monitor the internal FFA exposure we developed a technique for the mass spectrometric quantification of the adduct N-((furan-2-yl)methyl)-valine (FFA-Val) after cleavage from the N-termini of hemoglobin. In the current study the method was applied to investigate the influence of different SULT forms on the adduct formation in wild-type mice and three genetically modified mouse models treated with FFA. Two lines were devoid of endogenous Sult1a1 or Sult1d1, while another mouse line carried a transgene of human SULT1A1/1A2 in the Sult1a1/1d1 double knockout background. The Sult1d1 knockout did not influence adduct formation, whereas the lack of Sult1a1 reduced mean FFA-Val levels by 80% and 58% in male and female mice, respectively, in comparison to FFA-treated wild-type mice. The levels of FFA-Val in the humanized mice were elevated by factors of 2.7 (males) and 2.2 (females) as compared to the wild-type, indicating that SULT1A1/1A2 play a central role for FFA bioactivation also in humans. The excellent correlation between adduct levels in hepatic DNA and hemoglobin (r2 = 0.97) indicated that 2-sulfoxymethylfuran of hepatic origin is sufficiently stable to enter circulation and pass the cellular membrane of erythrocytes. This is a prerequisite for the application of FFA-Val as a biomarker of internal FFA exposure.

A hemoglobin adduct as a biomarker for the internal exposure to the rodent carcinogen furfuryl alcohol.

Furfuryl alcohol is a common food contaminant, which is formed by acid- and heat-catalyzed degradation of fructose and glucose. Its carcinogenic effect in rodents originates most likely from sulfotransferase (SULT)-catalyzed conversion into the mutagenic sulfate ester 2-sulfoxymethylfuran. In this study, a protein adduct biomarker was sought for the medium-term internal exposure to furfuryl alcohol. A UPLC-MS/MS screening showed that the adduct N-((furan-2-yl)methyl)-Val (FFA-Val) at the N-terminus of hemoglobin is a valid target analyte. The Val cleavage by fluorescein isothiocyanate-mediated Edman degradation yielded 3-fluorescein-1-(furan-2-ylmethyl)-5-(propan-2-yl)-2-thioxoimidazolidin-4-one (FFA-Val-FTH), which was characterized by 1H and 13C NMR spectroscopy. An isotope-dilution method for the quantification of FFA-Val-FTH by UPLC-MS/MS was developed. It was used to study the adduct formation in furfuryl alcohol-treated FVB/N mice and the influence of ethanol and the alcohol dehydrogenase (ADH) inhibitor 4-methylpyrazole on the adduct levels. The administration of 400 mg/kg body weight furfuryl alcohol alone led to 12.5 and 36.7 pmol FFA-Val/g Hb in blood samples of male and female animals, respectively. The co-administration of 1.6 g ethanol/kg body weight increased FFA-Val levels by 1.4-fold in males and by 1.5-fold in females. The co-administration of 100 mg 4-methylpyrazole/kg body weight had a similar effect on the adduct levels. A high correlation was observed between adduct levels in hemoglobin and in hepatic DNA samples determined in the same animal experiment. This indicated that FFA-Val is a valid biomarker for the internal exposure to 2-sulfoxymethylfuran, which may be suitable to monitor furfuryl alcohol exposure also in humans.