Metabolic Activation of 2-Methylfuran to Acetylacrolein and Its Reactivity toward Cellular Proteins.

2-Methylfuran (2-MF) is a process-related contaminant found primarily in heat-treated foods, such as coffee or canned food. The oxidative metabolic activation of 2-MF is supposed to follow the pathway established for furan, which is known to generate the highly reactive metabolite butenedial (BDA). In the case of 2-MF, generation of the BDA homologue 3-acetylacrolein (AcA) is to be expected. 2-MF metabolism to AcA was investigated in two model systems: commercial microsomal preparations and primary rat hepatocytes (pRH). To scavenge the generated 2-MF, two model nucleophils, N-acetyl-l-cysteine (AcCys) and N-α-acetyl-l-lysine (AcLys), were used, and the formation of the corresponding adducts was measured in the supernatants. The metabolic activation of 2-MF to AcA was studied using human liver microsomes as well as rat liver microsomes. Incubation of 2-MF in Supersomes allowed to identify the cytochrome P450 isoenzyme primarily responsible for 2-MF. In addition, primary rat hepatocytes were incubated with 2-MF or AcA and AcLys adduct of AcA (N-α-acetyl-l-lysine-acetylacrolein, AcLys-AcA) determined in the cell supernatants by UHPLC-MS/MS. In model experiments, AcA formed adducts with AcCys and AcLys. The structures of both adducts were characterized. For incubations in biological activating systems, CYP 2E1 was found to be a key enzyme for the conversion of 2-MF to AcA in Supersomes. When pRH were incubated with 2-MF and AcA, AcLys-AcA was detected in the cell supernatants in a time- and dose-dependent manner. The results showed that AcA was indeed formed at the cellular level. In contrast to the AcLys-AcA adduct, no N-acetyl-l-cysteine-acetylacrolein (AcCys-AcA) adduct could be detected in pRH. AcA was determined as a reactive metabolite of 2-MF in vitro, and its adduct formation with nucleophilic cellular components was evaluated. The metabolites were characterized, and AcLys-AcA was identified as potential biomarker.

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.

Fractionation of Extracts from Black Chokeberry, Cranberry, and Pomegranate to Identify Compounds That Influence Lipid Metabolism.

Polyphenols show a spectrum of bioactive effects, including an influence on lipid metabolism. In this study, we performed activity-guided fractionations of black chokeberry (aronia), cranberry, and pomegranate extracts to identify the biologically active compounds. The extracts were prepared from fruit juice concentrates with the adsorbent resin Amberlite XAD-7 and were separated into a copigment and an anthocyanin fraction, followed by fractionation into a polymer and monomeric fraction by means of hexane precipitation. For further fractionation of the cranberry and pomegranate copigment fractions, high-performance countercurrent chromatography (HPCCC) was used. The compounds in each fraction were identified by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS), and the quantification was performed by ultra high-performance liquid chromatography-diode array detector (UHPLC-DAD) analyses. Each of the (sub-)fractions was tested in three in vitro assays: phosphodiesterase 3B (PDE) activity, lipid accumulation, and lipolysis in 3T3-L1 cells. The results showed that various fractions and subfractions can inhibit lipid accumulation and PDE activity as well as increase lipolysis, particularly copigments. Overall, our results indicate an influence of polyphenol-rich (sub-)fractions on the lipid metabolism.