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 may be the result of bioactivation. Oxidation by P450s yields the electrophile TCE oxide. We previously analyzed N(6)-acyllysine adducts formed from the reaction of TCE oxide with proteins [Cai, H., and Guengerich, F. P. (2000) Chem. Res. Toxicol. 13, 327-335]; however, we had been unable to measure ester adducts under the prolonged conditions of proteolysis and derivatization. Protein amino acid adducts were directly observed by mass spectrometry during the reaction of TCE oxide with the model polypeptides insulin and adrenocorticotropic hormone (ACTH, residues 1-24). The majority (80%) of the protein adducts were unstable under physiological conditions and had a collective t(1/2) of approximately 1 h, suggesting that they are ester type adducts formed from reactions of Cys, Ser, Tyr, or Thr residues with intermediates formed 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 stabilities 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 two model enzymes for which the literature suggests roles of a Lys, rabbit muscle aldolase and glucose-6-phosphate dehydrogenase. However, in the cases of papain (essential Cys residue in the active site), alpha-chymotrypsin (critical Ser residue), and D-amino acid oxidase (essential Cys and Tyr residues), time-dependent recoveries of enzyme activity were observed following reaction with TCE oxide or either of two model nucleophiles (dichloroacetyl chloride and acetic formic anhydride), paralleling the kinetics of removal of adducts from insulin and ACTH. Formation of adducts ( approximately 2%) was 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 formed from the reaction of TCE oxide with macromolecules and their biological effects.
Cai
H
H
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Guengerich
F P
FP
D000324:Adrenocorticotropic Hormone / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002273:Carcinogens / Q000378:metabolism / Q000633:toxicity*; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018736:DNA Adducts / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000633:toxicity; D004852:Epoxy Compounds / Q000378:metabolism / Q000633:toxicity*; D005634:Fructose-Bisphosphate Aldolase / Q000037:antagonists & inhibitors / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism; D007328:Insulin / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D013058:Mass Spectrometry; D009132:Muscles / Q000201:enzymology; D009705:Nucleosides / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits
D002273:Carcinogens; D018736:DNA Adducts; D004791:Enzyme Inhibitors; D004852:Epoxy Compounds; D007328:Insulin; D009705:Nucleosides; D009841:Oligonucleotides; C036489:trichloroepoxyethane; D000324:Adrenocorticotropic Hormone; D004247:DNA; D005954:Glucosephosphate Dehydrogenase; D005634:Fructose-Bisphosphate Aldolase; D008239:Lysine