Covalent modification of cytochrome c by reactive metabolites of furan.

Metabolism of the hepatotoxicant furan leads to protein adduct formation in the target organ. The initial bioactivation step involves cytochrome P450-catalyzed oxidation of furan, generating cis-2-butene-1,4-dial (BDA). BDA reacts with lysine to form pyrrolin-2-one adducts. Metabolic studies indicate that BDA also reacts with glutathione (GSH) to generate 2-(S-glutathionyl)butanedial (GSH-BDA), which then reacts with lysine to form GSH-BDA-lysine cross-links. To explore the relative reactivity of these two reactive intermediates, cytochrome c was reacted with BDA in the presence and absence of GSH. As judged by MALDI-TOF mass spectrometry, BDA reacts extensively with cytochrome c to form adducts that add 66 Da to the protein, consistent with the formation of pyrrolinone adducts. Addition of GSH to the reaction mixture reduced the overall extent of adduct formation. The mass of the adducted protein was shifted by 355 Da as expected for GSH-BDA-protein cross-link formation. LC-MS/MS analysis of the tryptic digests of the alkylated protein indicated that the majority of adducts occurred on lysine residues, with BDA reacting less selectively than GSH-BDA. Both types of adducts may contribute to the toxic effects of furan.