title: Adduction of the chloroform metabolite phosgene to lysine residues of human histone H2B.
abstract: Human exposure to trihalomethanes such as chloroform has been associated with both cancer and reproductive toxicity. While there is little evidence for chloroform mutagenicity or DNA adduct formation, in vivo studies in rats have demonstrated adduction to histones and other nuclear proteins. Histones play a key role in controlling DNA expression particularly through the acetylation of lysine residues in their N-termini. Therefore, we studied the reaction of phosgene, the major active metabolite of chloroform, with the N-terminus of human histone H2B (Hpep, Pro-Glu-Pro-Ala-Lys-Ser-Ala-Pro-Ala-Pro-Lys-Lys-Gly-Ser-Lys-Lys-Ala-Val-Thr-Lys-Ala-Gln-Lys) in a model chemical system. The aim of this study was to assess whether phosgene is able to form irreversible adducts with this peptide and to investigate which residues are most susceptible. Hpep was reacted with a range of phosgene concentrations (0.03-36 mM) at 37 degrees C, pH 7.4. The products of these reactions, analyzed by matrix-assisted laser desorption ionization MS, showed that up to three CO moieties could be adducted to the peptide. The singly and doubly adducted peptides were purified by HPLC and then hydrolyzed with trypsin to produce a series of fragments that were analyzed by HPLC-MS. The tryptic products showed that adduction occurred principally at lysine residues, and that all seven lysine residues of the peptide were subject to adduction. Collision-induced dissociation analysis using ion trap MS-MS of the tryptic fragment [Pro-Glu-Pro-Ala-Lys-Ser-Ala-Pro-Ala-Pro-Lys + CO] and of the full-length singly adducted peptide supported the role of lysine residues in adduction; the data also indicated that the N-terminal proline and the serine residues are susceptible. Addition of glutathione to the reaction mixture only partially attenuated adduct formation and allowed production of another adducted species, i.e., Hpep-CO-glutathione. The occurrence of such reactions to the N-termini of histones, if confirmed by in vivo studies, could help to explain the mechanism of chloroform carcinogenicity.
authors.affiliation: Section on Clinical Pharmacology, Division of Medicine, Faculty of Medicine, Imperial College, Hammersmith Campus, Du Cane Road, London W12 0NN, UK. laura.fabrizi@iss.it
authors.forename: Laura
authors.initials: L
authors.lastname: Fabrizi
authors.forename: Graham W
authors.initials: GW
authors.lastname: Taylor
authors.forename: Benito
authors.initials: B
authors.lastname: Cañas
authors.forename: Alan R
authors.initials: AR
authors.lastname: Boobis
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Edwards
mesh_terms: D000595:Amino Acid Sequence; D002725:Chloroform / Q000378:metabolism*; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D010705:Phosgene / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014357:Trypsin / Q000378:metabolism
chemical_list: D006657:Histones; D009842:Oligopeptides; D010446:Peptide Fragments; D010705:Phosgene; D002725:Chloroform; D014357:Trypsin; D008239:Lysine