New insight into the molecular mechanism of protein cross-linking induced by cis-2-butene-1,4-dial, the metabolite of furan: Formation of 2-substituted pyrrole cross-links involving the cysteine and lysine residues.

Furan is an environmental pollutant also present in heat-treated food. The compound is toxic and carcinogenic to rats, and classified as a possible human carcinogen. Mechanisms laying behind the furan carcinogenicity remain unclear, however scientific data indicate the involvement of bioactivation catalysed by cytochrome P450 2E1. The resulted initial metabolite of furan, cis-2-butene-1,4-dial (BDA) is an extremely reactive conjugated dialdehyde able to damage important cellular components such as glutathione, proteins and nucleic acids. Earlier works showed that BDA induces protein cross-linking leading to the formation of modifications containing substituted pyrrole ring, and that the cysteine and lysine residues are prone to undergo such processes. The resulted cross-linked protein adducts are important for research aimed at exploring biomarkers of furan exposure. Due to furan's high volatility, biomarker-based methods appear to be a reliable approach to measure a level of exposure to this chemical. To date, numerous urinary and hepatocyte metabolites were identified. However, all the metabolites contain 3-substituted pyrrole ring resulted from the 1,4-addition of the cysteine thiol function to BDA followed by the condensation with the lysine free amino group. In this work we provide evidence that also 2-substituted pyrrole cross-links are formed at the physiological pH, when BDA is subjected to the reaction with N-acetylcysteine and N-acetyllysine, N-acetyllysine or lysine, respectively. The 2-substituted cross-links arise from the initial 1,2-addition of N-acetylcysteine to BDA accomplished by the condensation with lysine or its N-acetylated derivatives. Formation of the 2-substituted pyrroles sheds new light on the reactivity of cis-2-butene-1,4-dial towards amino acids, and contributes to complete characterisation of the compound chemistry. Our studies show that the 2- and 3-substituted cross-links are indistinguishable based on their MS and MS/MS spectra, and that for reliable qualitative and quantitative measurements the use of isotope-substituted synthetic internal standards is necessary. In the light of these facts, our results are important for research into furan exposure biomarkers.