Reaction of trichloroethylene oxide with proteins and DNA: Instability of adducts and modulation of functions

Trichloroethylene (TCE) shows several types of toxicities, some of which ma y be the result ofbioacbivation. Oxidation by P450s yields the electrophile TCE oxide. We previously analyzed N-6-acyllysine adducts formed from the r eaction of TCE oxide with proteins [Cai, H., and Guengerich, F. P, (2000) C hem. Res. Toxicol. 13, 327-335]; however, we had been unable to measure est er adducts under the prolonged conditions of proteolysis and derivatization . Protein amino acid adducts were directly observed by mass spectrometry du ring the reaction of TCE oxide with the model polypeptides insulin and adre nocorticotropic hormone (ACTH, residues 1-24). The majority (80%) of the pr otein adducts were unstable under physiological conditions and had a collec tive t(1/2) of similar to1 h, suggesting that they are ester type adducts f ormed from reactions of Cys, Ser, Tyr, or Thr residues with intermediates f ormed in TCE oxide hydrolysis. Synthetic O-acetyl-L-Ser and O-acetyl-L-Tyr had half-lives of 1 h and 10 min at pH 8.0, respectively, similar to the st abilities of the protein adducts. The effects of TCE oxide adduct formation on catalytic activities were examined with five model enzymes. No recovery of catalytic activity was observed during the reaction of TCE oxide with t wo model enzymes for which; the literature suggests roles of a Lys, rabbit muscle aldolase and glucose-6-phosphate dehydrogenase. However, in the case s of papain (essential Cys residue in the active site), alpha -chymotrypsin (critical Ser residue), and D-amino acid oxidase (essential Cys and Tyr re sidues), time-dependent recoveries of enzyme activity were observed followi ng reaction with TCE oxide or either of two model nucleophiles (dichloroace tyl chloride and acetic formic anhydride), paralleling the kinetics of remo val of adducts from insulin and ACTH. Formation of adducts (similar to2%) w as detected in the direct reaction of TCE oxide with 2'-deoxyguanosine, but not with the other three nucleosides found in DNA. During the reaction of TCE oxide with a synthetic 8-mer oligonucleotide, formation of adducts was observed by mass spectrometry. However, the adducts had a t(1/2) of 30 min at pH 8.5. These results indicate the transient nature of the adducts forme d from the reaction of TCE oxide with macromolecules and their biological e ffects.