Full length article New insights into the molecular mechanisms of chemical carcinogenesis: In vivo adduction of histone H2B by a reactive metabolite of the chemical carcinogen furan João Nunes a , Inês L. Martins a , Catarina Charneira a , Igor P. Pogribny b , Aline de Conti b , Frederick A. Beland b , M. Matilde Marques a , Cristina C. Jacob a , Alexandra M.M. Antunes a, * a Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal b Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA H I G H L I G H T S G R A P H I C A L A B S T R A C T  A furan-derived adduct was identi- fied in liver histone 2B of furan- treated rats.  This adduct was detected prior to the identification of epigenetic changes.  This adduct represents an early compound-specific biomarker of ex- posure.  The first evidence for in vivo occur- rence of carcinogen-modified histo- nes is provided.  New insights into the mechanisms of chemical carcinogenesis are provid- ed. A R T I C L E I N F O Article history: Received 17 September 2016 Received in revised form 27 October 2016 Accepted 31 October 2016 Available online 5 November 2016 Keywords: Histone adducts Chemically induced cancers Furan Biomarkers A B S T R A C T Furan is a rodent hepatocarcinogen ubiquitously found in the environment and heat-processed foods. Furan undergoes cytochrome P450 2E1-catalyzed bioactivation to cis-2-butene-1,4-dial (BDA), which has been shown to form an electrophilic conjugate (GSH-BDA) with glutathione. Both BDA and GSH-BDA yield covalent adducts with lysine residues in proteins. Dose- and time-dependent epigenetic histone alterations have been observed in furan-treated rats. While the covalent modification of histones by chemical carcinogens has long been proposed, histone-carcinogen adducts have eluded detection in vivo. In this study, we investigated if the covalent modification of histones by furan may occur in vivo prior to epigenetic histone alterations. Using a “bottom-up” methodology, involving the analysis of tryptic peptides by liquid chromatography – high resolution mass spectrometry, we obtained evidence for a cross-link between GSH-BDA and lysine 107 of histone H2B isolated from the livers of male F344 rats treated with tumorigenic doses of furan. This Abbreviations: BDA, cis-2-butene-1,4-dial; GSH, glutathione; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; LC–HRMS, liquid chromatography–high resolution mass spectrometry; NP-40, nonyl phenoxypolyethoxylethanol; PMSF, phenylmethylsulfonyl fluoride. * Corresponding author at: Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, 1049001 Lisboa, Portugal. E-mail address: alexandra.antunes@tecnico.ulisboa.pt (A.M.M. Antunes). http://dx.doi.org/10.1016/j.toxlet.2016.10.018 0378-4274/ã 2016 Elsevier Ireland Ltd. All rights reserved. Toxicology Letters 264 (2016) 106–113 Contents lists available at ScienceDirect Toxicology Letters journal homepage: www.elsev ier.com/locate /toxlet