Endogenous formation of protein adducts with carcinogenic aldehydes - Implications for oxidative stress

In the present study, we characterize the covalent modification of a protei n by crotonaldehyde, a representative carcinogenic aldehyde, and describe t he endogenous production of this aldehyde in vivo, The crotonaldehyde prefe rentially reacted with the lysine and histidine residues of bovine serum al bumin and generated a protein-linked carbonyl derivative. Upon incubation w ith the histidine and lysine derivatives, crotonaldehyde predominantly gene rated beta -substituted butanal adducts of histidine and lysine and N-epsil on-(2,5-dimethyl-3-formyl-3,4-dehydropiperidino)lysine (dimethyl-FDP-lysine ) as the putative carbonyl derivatives generated in the crotonaldehyde-modi fied protein. To verify the endogenous formation of crotonaldehyde in vivo, we raised the monoclonal antibody (mAb82D3) against the crotonaldehyde-mod ified protein and found that it cross-reacted with the protein-bound 2-alke nals, such as crotonaldehyde, 2-pentenal, and 2-hexenal. The anti-2-alkenal antibody recognized multiple crotonaldehyde-lysine adducts, including dime thyl-FDP-lysine and an unknown product, which showed the greatest immunorea ctivity with the antibody. On the basis of the chemical and spectroscopic e vidence, the major antigenic product was determined to be a novel Schiff ba se-derived crotonaldehyde-lysine adduct, N-epsilon- (5-ethyl-2-methylpyridi nium)lysine (EMP-lysine), It was found that the lysine residues that had di sappeared in the protein treated with crotonaldehyde were partially recover ed by EMP-lysine. The presence of immunoreactive materials with mAb82D3 in vivo was demonstrated in the kidney of rats exposed to the renal carcinogen , ferric nitrilotriacetate. In addition, the observations that the metal-ca talyzed oxidation of polyunsaturated fatty acids in the presence of protein s resulted in an increase in the antigenicity of the protein indicated that lipid peroxidation represents a potential pathway for the formation of cro tonalde-hyde/2-alkenals in vivo, These data suggest that the formation of c arcinogenic aldehydes during lipid peroxidation may be causally involved in the pathophysiological effects associated with oxidative stress.