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.

Internal exposure to heat-induced food contaminants in omnivores, vegans and strict raw food eaters: biomarkers of exposure to 2- and 3-monochloropropanediol (urinary excretion) and glycidol (hemoglobin adduct N-2,3-dihydroxypropyl-Val).

Fatty acid esters of 2/3-monochloropropanediol (2/3-MCPD) and glycidol are formed mainly during heat processing (deodorization) of vegetable oils, and are hydrolyzed by lipases in the gastrointestinal tract leading to the absorption of 2/3-MCPD and glycidol. The International Agency for Research on Cancer (IARC) has classified 3-MCPD as possibly and glycidol as probably carcinogenic to humans. The aims of the current work were to clarify the exposure to 2/3-MCPD and glycidol associated with different dietary habits (omnivore, vegan, raw-food eating), and the exposure development between 2017 and 2021 in German study participants. The questions were addressed using the daily urinary excretion of 2/3-MCPD and the hemoglobin adduct N-(2,3-dihydroxypropyl)-Val (DHP-Val) formed from glycidol as biomarkers of exposure, which were determined in two dietary studies including 36 omnivores, 36 vegans and 16 strict raw food eaters (abstaining from any heated food for at least four months). The median urinary excretion of 2- and 3-MCPD in non-smoking omnivores and vegans was 0.87 and 1.35 µg/day (2-MCPD), respectively, and 0.79 and 1.03 µg/day (3-MCPD), respectively. The 2/3-MCPD concentrations in urine samples of raw food eaters were usually below the limit of detection. The median DHP-Val levels in non-smoking vegans and omnivores were 3.9 pmol/g Hb each, and 1.9 pmol/g Hb in raw food eaters. Between 2017 and 2021, the exposure to 3-MCPD and glycidol did not change, however, the median 2-MCPD excretion decreased (p = 0.02, omnivores and vegans combined). The correlation between daily excretions of 2/3-MCPD determined 4 years apart was weak, whereas a moderate correlation was observed for DHP-Val (rS = 0.66) in this timeframe. In conclusion, the exposure to glycidol in omnivores and vegans was alike, whereas the 2/3-MCPD exposure was somewhat (albeit not significantly) higher in vegans. While 2/3-MCPD were hardly detectable in urine samples of raw food eaters, the median DHP-Val level (about 50% of those in omnivores) indicates a glycidol source independent of the dietary exposure.

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.

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.

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.

Correction to: Mode of action-based risk assessment of genotoxic carcinogens.

The author would like to thank N. Bakhiya, S. Hessel-Pras, B. Sachse, and B. Dusemund for their support in the chapter about pyrrolizidine alkaloids.

Mode of action-based risk assessment of genotoxic carcinogens.

The risk assessment of chemical carcinogens is one major task in toxicology. Even though exposure has been mitigated effectively during the last decades, low levels of carcinogenic substances in food and at the workplace are still present and often not completely avoidable. The distinction between genotoxic and non-genotoxic carcinogens has traditionally been regarded as particularly relevant for risk assessment, with the assumption of the existence of no-effect concentrations (threshold levels) in case of the latter group. In contrast, genotoxic carcinogens, their metabolic precursors and DNA reactive metabolites are considered to represent risk factors at all concentrations since even one or a few DNA lesions may in principle result in mutations and, thus, increase tumour risk. Within the current document, an updated risk evaluation for genotoxic carcinogens is proposed, based on mechanistic knowledge regarding the substance (group) under investigation, and taking into account recent improvements in analytical techniques used to quantify DNA lesions and mutations as well as "omics" approaches. Furthermore, wherever possible and appropriate, special attention is given to the integration of background levels of the same or comparable DNA lesions. Within part A, fundamental considerations highlight the terms hazard and risk with respect to DNA reactivity of genotoxic agents, as compared to non-genotoxic agents. Also, current methodologies used in genetic toxicology as well as in dosimetry of exposure are described. Special focus is given on the elucidation of modes of action (MOA) and on the relation between DNA damage and cancer risk. Part B addresses specific examples of genotoxic carcinogens, including those humans are exposed to exogenously and endogenously, such as formaldehyde, acetaldehyde and the corresponding alcohols as well as some alkylating agents, ethylene oxide, and acrylamide, but also examples resulting from exogenous sources like aflatoxin B1, allylalkoxybenzenes, 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx), benzo[a]pyrene and pyrrolizidine alkaloids. Additionally, special attention is given to some carcinogenic metal compounds, which are considered indirect genotoxins, by accelerating mutagenicity via interactions with the cellular response to DNA damage even at low exposure conditions. Part C finally encompasses conclusions and perspectives, suggesting a refined strategy for the assessment of the carcinogenic risk associated with an exposure to genotoxic compounds and addressing research needs.

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.

The hemoglobin adduct N-(2,3-dihydroxypropyl)-valine as biomarker of dietary exposure to glycidyl esters: a controlled exposure study in humans.

Fatty acid esters of glycidol (glycidyl esters) are heat-induced food contaminants predominantly formed during industrial deodorization of vegetable oils and fats. After consumption, the esters are digested in the gastrointestinal tract, leading to a systemic exposure to the reactive epoxide glycidol. The compound is carcinogenic, genotoxic and teratogenic in rodents, and rated as probably carcinogenic to humans (IARC group 2A). Assessment of exposure from occurrence and consumption data is difficult, as lots of different foods containing refined oils and fats may contribute to human exposure. Therefore, assessment of the internal exposure using the hemoglobin adduct of glycidol, N-(2,3-dihydroxypropyl)-valine (2,3-diHOPr-Val), may be promising, but a proof-of-principle study is needed to interpret adduct levels with respect to the underlying external exposure. A controlled exposure study was conducted with 11 healthy participants consuming a daily portion of about 36 g commercially available palm fat with a relatively high content of ester-bound glycidol (8.7 mg glycidol/kg) over 4 weeks (total amount 1 kg fat, individual doses between 2.7 and 5.2 µg/kg body weight per day). Frequent blood sampling was performed to monitor the 2,3-diHOPr-Val adduct levels during formation and the following removal over 15 weeks, using a modified Edman degradation and ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Results demonstrated for the first time that the relatively high exposure during the intervention period was reflected in corresponding distinct increases of 2,3-diHOPr-Val levels in all participants, following the expected slope for hemoglobin adduct formation and removal over time. The mean adduct level increased from 4.0 to 12.2 pmol 2,3-diHOPr-Val/g hemoglobin. By using a nonlinear mixed model, values for the adduct level/dose ratio (k, mean 0.082 pmol 2,3-diHOPr-Val/g hemoglobin per µg glycidol/kg body weight) and the adduct lifetime (τ, mean 104 days, likely the lifetime of the erythrocytes) were determined. Interindividual variability was generally low. 2,3-DiHOPr-Val was therefore proven to be a biomarker of the external dietary exposure to fatty acid esters of glycidol. From the background adduct levels observed in our study, a mean external glycidol exposure of 0.94 µg/kg body weight was estimated. This value is considerably higher than current estimates for adults using occurrence and consumption data of food. Possible reasons for this discrepancy are discussed (other oral or inhalational glycidol sources, endogenous formation, exposure to other chemicals also forming the adduct 2,3-diHOPr-Val). Further research is necessary to clarify the issue.