Stable Isotope Dilution Analysis (SIDA) to Determine Metabolites of Furan and 2-Methylfuran in Human Urine Samples: A Pilot Study.

Furan and 2-methylfuran (2-MF) are food contaminants that are classified as potentially carcinogenic to humans. The main source of exposure for adults via food is coffee consumption. Furan and 2-MF are volatile, which complicates exposure assessment because their content measured in food prior to consumption does not afford a reliable dosimetry. Therefore, other ways of exposure assessment need to be developed, preferably by monitoring exposure biomarkers, e.g., selected metabolites excreted in urine. In this study, cis-2-buten-1,4-dial (BDA)-derived urinary furan metabolites Lys-BDA (l-2-amino-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)hexanoic acid), AcLys-BDA (l-2-(acetylamino)-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)hexanoic acid) and GSH-BDA (N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-l-cysteinyl-glycine cyclic sulfide), as well as acetyl acrolein (AcA, 2-oxo-pent-2-enal)-derived metabolites Lys-AcA (l-2-(acetylamino)-6-(2,5-dihydro-5-methyl-2-oxo-1H-pyrrol-1-yl)-hexanoic acid) and AcLys-AcA (l-2-amino-6-(2,5-dihydro-5-methyl-2-oxo-1H-pyrrol-1-yl)-hexanoic acid) and their stable isotopically labeled analogs, were synthesized and characterized through NMR and MS, and a stable isotope dilution analysis (SIDA) with UPLC-ESI-MS/MS was established. As a proof of concept, urinary samples of a four-day human intervention study were used. In the frame of this study, ten subjects ingested 500 mL of coffee containing 0.648 µmol furan and 1.059 µmol 2-MF. Among the furan metabolites, AcLys-BDA was the most abundant, followed by Lys-BDA and GSH-BDA. Exposure to 2-MF via the coffee brew led to the formation of Lys-AcA and AcLys-AcA. Within 24 h, 89.1% of the ingested amount of furan and 15.4% of the ingested amount of 2-MF were detected in the urine in the form of the investigated metabolites. Therefore, GSH-BDA, Lys-BDA, AcLys-BDA, Lys-AcA and AcLys-AcA may be suitable as short-term-exposure biomarkers of furan and 2-MF exposure.

Characterization of brewer's spent grain extracts by tandem mass spectrometry and HPLC-DAD: Ferulic acid dehydrodimers, phenolamides, and oxylipins.

Brewer's spent grain (BSG) is a major by-product of the brewing industry which is generated in high amounts. In recent years, sustainable food production has become more and more important. BSG mainly used as cattle feed has gained high interest due to not only its valuable ingredients such as fiber and proteins but also secondary metabolites remaining in BSG after the brewing process and known for many biological effects. In the present study, various methods were applied, such as acetone extraction (A), alkaline hydrolysis followed by ethyl acetate extraction (HE), and acetone extraction of alkaline hydrolysis residue (HA). Compounds present in the respective bioactive extracts were characterized by mass spectrometry to identify the active compounds. Various hydroxycinnamic acid derivatives as well as oxylipins and some dicarboxylic acids, such as azelaic acid, were present in HE and HA extracts. In contrast, some catechins and phenolamides, such as numerous hordatines, as well as oxylipins and phospholipids were detected in A extracts. Quantification using HPLC-DAD revealed hordatine contents up to 172.2 ± 2.1 µg p-coumaric acid equivalents/mg extract. Hydroxycinnamic acid derivatives content accounted for up to 48% of the total extract (HE extracts) but only around 3% of the total HA extracts. In summary, all extracts contained secondary plant metabolites belonging to different classes, ranging from hydroxycinnamic acids to phenolamides, such as not only hordatines but also oxylipins, which were identified for the first time in BSG.

Polyphenol-Rich Beverage Consumption Affecting Parameters of the Lipid Metabolism in Healthy Subjects.

Polyphenols are a diverse and widely distributed class of secondary metabolites, which possess numerous beneficial properties including a modulation of glucose and lipid metabolism. This placebo-controlled human intervention study was performed to explore effects of polyphenol-rich beverage (PRB) uptake on lipid metabolism, as well as DNA integrity. In this case, 36 healthy men were randomly divided to consume either 750 mL of a PRB (containing 51% chokeberry, cranberry, and pomegranate) or a placebo drink daily for eight weeks. Only PRB consumption was found to decrease fat and protein intakes significantly compared to the preceding one-week washout period. During the intervention with PRB an increased fat-free mass was shown after four weeks, whereas a significant elevation in body weight and leptin was observed in placebo group. Blood lipids were not significantly altered after PRB consumption, while triglyceride levels increased after placebo drink intake. In platelets, a significant inhibition of phosphodiesterase (PDE) activity was observed, more pronounced in test group. Consuming the PRB decreased total DNA strand breaks in whole blood as well as H2O2-induced breaks in isolated lymphocytes. Overall, our study suggested beneficial effects on lipid metabolism by reduced energy intake, modulation of biomarkers such as PDE activity and improved DNA integrity associated with PRB consumption.

p53 triggers mitochondrial apoptosis following DNA damage-dependent replication stress by the hepatotoxin methyleugenol.

Liver cancer is one of the most frequent tumor entities worldwide, which is causally linked to viral infection, fatty liver disease, life-style factors and food-borne carcinogens, particularly aflatoxins. Moreover, genotoxic plant toxins including phenylpropenes are suspected human liver carcinogens. The phenylpropene methyleugenol (ME) is a constituent of essential oils in many plants and occurs in herbal medicines, food, and cosmetics. Following its uptake, ME undergoes Cytochrome P450 (CYP) and sulfotransferase 1A1 (SULT1A1)-dependent metabolic activation, giving rise to DNA damage. However, little is known about the cellular response to the induced DNA adducts. Here, we made use of different SULT1A1-proficient cell models including primary hepatocytes that were treated with 1'-hydroxymethyleugenol (OH-ME) as main phase I metabolite. Firstly, mass spectrometry showed a concentration-dependent formation of N2-MIE-dG as major DNA adduct, strongly correlating with SULT1A1 expression as attested in cells with and without human SULT1A1. ME-derived DNA damage activated mainly the ATR-mediated DNA damage response as shown by phosphorylation of CHK1 and histone 2AX, followed by p53 accumulation and CHK2 phosphorylation. Consistent with these findings, the DNA adducts decreased replication speed and caused replication fork stalling. OH-ME treatment reduced viability particularly in cell lines with wild-type p53 and triggered apoptotic cell death, which was rescued by pan-caspase-inhibition. Further experiments demonstrated mitochondrial apoptosis as major cell death pathway. ME-derived DNA damage caused upregulation of the p53-responsive genes NOXA and PUMA, Bax activation, and cytochrome c release followed by caspase-9 and caspase-3 cleavage. We finally demonstrated the crucial role of p53 for OH-ME triggered cell death as evidenced by reduced pro-apoptotic gene expression, strongly attenuated Bax activation and cell death inhibition upon genetic knockdown or pharmacological inhibition of p53. Taken together, our study demonstrates for the first time that ME-derived DNA damage causes replication stress and triggers mitochondrial apoptosis via the p53-Bax pathway.

A Benchmark analysis of acrylamide-derived DNA adducts in rat hepatocytes in culture measured by a new, highly sensitive method.

Acrylamide (AA) is a carcinogen formed during thermal food processing and can cause tumors in rodents while its carcinogenic potency in humans is unclear. Metabolism of AA, preferentially in the liver, leads to glycidamide (GA) forming N7-GA-guanine (N7-GA-Gua) as the major AA-derived DNA adduct in rodents. Here, a novel method allowing high sensitivity by avoidance of major matrix effects was applied to analyze N7-GA-Gua levels in nuclear DNA from rat hepatocytes in primary culture. We could thus for the first time detect a background level of 5-10 adducts/108 nucleosides in untreated hepatocytes. Incubation with AA did not result in a statistically significant increase in adduct levels over background up to a substrate concentration of 500 µM although a trend to slightly higher adduct levels was observed at and above 200 µM AA. At concentrations > 500 µM significant increases in N7-GA-Gua levels were found. When Benchmark concentration (BMC) modeling was applied to the data, non-linear concentration-response curves were obtained suggesting that AA started to cause measurable increases over background of N7-GA-Gua levels above certain concentrations only. Calculation of the composite BMCL10 (Lower Bound of a 95 % confidence interval) of a BMC leading to a 10 % increase of N7-GA-Gua levels over background resulted in a value of 6.35 µM AA after 24 h. A concentration below this value cannot be expected to lead to an increase in N7-GA-Gua of more than 10 % over the background seen in untreated hepatocytes.

Acrylamide-derived DNA adducts in human peripheral blood mononuclear cell DNA: Correlation with body mass.

Acrylamide (AA) is a carcinogen formed during thermal food processing and can cause tumors in rodents while its carcinogenic potency in humans is unclear. Metabolic conversion of AA leads to glycidamide (GA) forming N7-GA-guanine (N7-GA-Gua) as the major DNA adduct in rodents while no such adducts were found in human tissues so far. In a cohort of 56 healthy volunteers adduct levels were determined in peripheral blood mononuclear cell (PBMC) DNA and anthropometric, dietary, and biochemical parameters were measured or inquired using a questionnaire. In the majority of PBMC DNA samples the levels found were above one adduct/108 nucleosides not being correlated to dietary habits including coffee consumption, or to blood glucose levels or hemoglobin HbA1c. However, adduct levels were significantly correlated with the body mass index (BMI) and showed a continuous increase over three BMI classes. Our findings indicate a background of AA-derived DNA adducts present in humans in PBMC related to body mass rather than to certain dietary or lifestyle factors.

A New UPLC-qTOF Approach for Elucidating Furan and 2-Methylfuran Metabolites in Human Urine Samples after Coffee Consumption.

We have investigated urine samples after coffee consumption using targeted and untargeted approaches to identify furan and 2-methylfuran metabolites in urine samples by UPLC-qToF. The aim was to establish a fast, robust, and time-saving method involving ultra-performance liquid chromatography-quantitative time-of-flight tandem mass spectrometry (UPLC-qToF-MS/MS). The developed method detected previously reported metabolites, such as Lys-BDA, and others that had not been previously identified, or only detected in animal or in vitro studies. The developed UPLC-qToF method detected previously reported metabolites, such as lysine-cis-2-butene-1,4-dial (Lys-BDA) adducts, and others that had not been previously identified, or only detected in animal and in vitro studies. In sum, the UPLC-qToF approach provides additional information that may be valuable in future human or animal intervention studies.

Assessment and characterization of DNA adducts produced by alkenylbenzenes in fetal turkey and chicken livers.

Formation of DNA adducts by five alkenylbenzenes, safrole, methyl eugenol, eugenol, and asarone with either α- or ß-conformation, was analyzed in fetal avian livers in two in ovo models. DNA reactivity of the carcinogens safrole and methyl eugenol was previously demonstrated in the turkey egg model, whereas non-genotoxic eugenol was negative. In the current study, alkenylbenzenes were also tested in the chicken egg model. Injections with alkenylbenzenes were administered to fertilized turkey or chicken eggs for three consecutive days. Three hours after the last injection, liver samples were evaluated for DNA adduct formation using the 32P-nucleotide postlabeling assay. DNA samples from turkey livers were also analyzed for adducts using mass spectrometry. In both species, genotoxic alkenylbenzenes safrole, methyl eugenol, α- and ß-asarone produced DNA adducts, the presence and nature of which, with exception of safrole, were confirmed by mass spectrometry, validating the sensitivity of the 32P-postlabeling assay. Overall, the results of testing were congruent between fetal turkey and chicken livers, confirming that these organisms can be used interchangeably. Moreover, data obtained in both models is comparable to genotoxicity findings in other species, supporting the usefulness of avian models for the assessment of genotoxicity as a potential alternative to animal models.

Formation and fate of DNA adducts of alpha- and beta-asarone in rat hepatocytes.

While alpha-asarone (aA) and beta-asarone (bA) are genotoxic and were shown to be carcinogenic the mechanisms underlying these effects are not understood. Major metabolites of both compounds are epoxides which are mutagenic in the Ames test. We investigated their reactivity towards nucleosides and identified epoxide-derived DNA adducts with 2'-deoxyadenosine (dA) and 2'-deoxyguanosine (dG) using UPLC-UV/VIS, LC-MS/MS and NMR spectroscopy. The adducts were characterized as N6-1'-hydroxy-dihydro-asarone-dA and N2-1'-hydroxy-dihydro-asarone-dG. Chemical synthesis of these adducts, isotope labeled standards and development of a sensitive and specific isotope dilution mass spectrometric method allowed the quantification of DNA adducts formed in primary rat hepatocytes incubated with aA or bA over up to 48 h. We observed a concentration-dependent, nearly linear formation of DNA adducts, which was higher for bA than for aA. In time course experiments, the amount of DNA adducts reached a maximum within the first 6 h. Over the next 42 h, the amount of DNA adducts decreased, however DNA adducts were still detectable even at the lowest substrate concentration of 10 µM. These results clearly show that aA and bA are able to form epoxide-derived DNA adducts in mammalian cells which may be responsible for their genotoxic, mutagenic and carcinogenic mode of action.

A neutral halogen bonding macrocyclic anion receptor based on a pseudocyclopeptide with three 5-iodo-1,2,3-triazole subunits.

The converging arrangement of iodine atoms along its confined cavity causes a cyclic pseudopeptide with three 5-iodo-1,2,3-triazole subunits to interact with halides, in particular with chloride, in 2.5 vol% water/DMSO.

Comparative investigation of the mutagenicity of propenylic and allylic asarone isomers in the Ames fluctuation assay.

α-, ß- and γ-asarone are naturally occurring phenylpropenes that occur in different plant families, mainly in Aristolochiaceae, Acoraceae and Lauraceae. Plants containing asarones are used as flavouring ingredients in alcoholic beverages (bitters), traditional phytomedicines and the rhizome of e.g. Acorus calamus is used to prepare tea. Although α- and ß-asarone show a potential in the treatment of several diseases, previous studies have shown carcinogenicity in rodents (duodenum, liver). However, the mechanism of action remained unclear. Studies on the mutagenicity of propenylic α- and ß-asarone are inconsistent and data on carcinogenicity and genotoxicity of allylic γ-asarone are lacking completely. Thus, the present study determined the mutagenicity of the three asarone isomers using the Ames fluctuation assay with and without exogenous metabolic activation (S9 mix) in the standard Salmonella typhimurium strains TA98 and TA100. A concentration dependent increase in mutagenicity could be verified for α- and ß-asarone in strain TA100 in the presence of rat liver homogenate. The side-chain epoxides of α- and ß-asarone, major metabolites formed in liver microsomes, caused mutations in TA100, supporting the hypothesis that epoxidation of the side chain plays a key role in mutagenicity of the propenylic alkenylbenzenes. The allylic γ-asarone, not undergoing detectable side-chain epoxidation in liver microsomes, was supposed to be activated via side-chain hydroxylation and further sulphonation, a typical pathway for other allylic alkenylbenzenes like estragole or methyleugenol. However, neither y-asarone nor 1'-OH-γ-asarone showed any mutagenic effect even in the human SULT-expressing Salmonella strains (TA100-hSULT1A1 and TA100-hSULT1C2), while 1'-OH-methyleugenol used as a positive control was mutagenic under these conditions. These results indicate that the propenylic asarones are genotoxic via metabolic formation of side-chain epoxides while the side-chain hydroxylation/sulphonation pathway is either not operative or does not lead to mutagenicity with the allylic γ-asarone.

Hepatic metabolism of carcinogenic ß-asarone.

ß-Asarone (1) belongs to the group of naturally occurring phenylpropenes like eugenol or anethole. Compound 1 is found in several plants, e.g., Acorus calamus or Asarum europaeum. Compound 1-containing plant materials and essential oils thereof are used to flavor foods and alcoholic beverages and as ingredients of many drugs in traditional phytomedicines. Although 1 has been claimed to have several positive pharmacological effects, it was found to be genotoxic and carcinogenic in rodents (liver and small intestine). The mechanism of action of carcinogenic allylic phenylpropenes consists of the metabolic activation via cytochrome P450 enzymes and sulfotransferases. In vivo experiments suggested that this pathway does not play a major role in the carcinogenicity of the propenylic compound 1 as is the case for other propenylic compounds, e.g., anethole. Since the metabolic pathways of 1 have not been investigated and its carcinogenic mode of action is unknown, we investigated the metabolism of 1 in liver microsomes of rats, bovines, porcines, and humans using (1)H NMR, HPLC-DAD, and LC-ESI-MS/MS techniques. We synthesized the majority of identified metabolites which were used as reference compounds for the quantification and final verification of metabolites. Microsomal epoxidation of the side chain of 1 presumably yielded (Z)-asarone-1',2'-epoxide (8a) which instantly was hydrolyzed to the corresponding erythro- and threo-configurated diols (9b, 9a) and the ketone 2,4,5-trimethoxyphenylacetone (13). This was the main metabolic pathway in the metabolism of 1 in all investigated liver microsomes. Hydroxylation of the side chain of 1 led to the formation of three alcohols at total yields of less than 30%: 1'-hydroxyasarone (2), (E)- and (Z)-3'-hydroxyasarone (4 and 6), with 6 being the mainly formed alcohol and 2 being detectable only in liver microsomes of Aroclor 1254-pretreated rats. Small amounts of 4 and 6 were further oxidized to the corresponding carbonyl compounds (E)- and (Z)-3'-oxoasarone (5, 7). 1'-Oxoasarone (3) was probably also formed in incubations with 1 but was not detectable, possibly due to its rapid reaction with nucleophiles. Eventually, three mono-O-demethylated metabolites of 1 were detected in minor concentrations. The time course of metabolite formation and determined kinetic parameters show little species-specific differences in the microsomal metabolism of 1. Furthermore, the kinetic parameters imply a very low dependence of the pattern of metabolite formation from substrate concentration. In human liver microsomes, 71-75% of 1 will be metabolized via epoxidation, 21-15% via hydroxylation (and further oxidation), and 8-10% via demethylation at lower as well as higher concentrations of 1, respectively (relative values). On the basis of our results, we hypothesize that the genotoxic epoxides of 1 are the ultimate carcinogens formed from 1.