Analysis of residual amino acid--DNA crosslinks induced in intact cells by nickel and chromium compounds.
Chinese hamster ovary cells were incubated with radioactive amino acids, the DNA was isolated by standard proteinase K/phenol/chloroform extraction and residual amino acids complexed to the DNA were examined as an index of metal induced DNA-protein crosslinks. Using this method, both chromate and nickel caused residual histidine and cysteine to be complexed with the DNA isolated from metal-treated cells. In the case of chromate, a number of amino acids were studied and Tyr, Thr and Cys were found to be complexed to DNA at a level (above the untreated control) that was statistically significant. Stability studies indicated that some of the chromate-induced DNA-protein complexes were mediated by direct participation of chromium(III), whereas others that were resistant to dissociation by EDTA and mercaptoethanol did not seem to involve direct chromium(III) participation. A significant portion of the cysteine complexed to DNA by chromate was believed to involve glutathione since treatment of cells with cycloheximide did not decrease chromate-induced cysteine-DNA crosslinks. In the case of nickel, most of the stable DNA-protein crosslinks did not involve direct metal participation and were probably oxidatively mediated by Ni(II)/Ni(III) redox cycling. These findings present new methodology for analysis of DNA-protein crosslinks by examination of residual amino acids associated with the DNA. This method should be highly sensitive and will yield important information about the mechanism of metal-induced DNA-protein crosslinks.
Lin
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X
New York University Medical Center, Nelson Institute of Environmental Medicine, NY 10016.
Zhuang
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Z
Costa
M
M
D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D002623:Chemistry Techniques, Analytical; D002840:Chromates / Q000378:metabolism / Q000494:pharmacology; D002857:Chromium / Q000494:pharmacology*; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D004249:DNA Damage; D004355:Drug Stability; D019286:Endopeptidase K; D007553:Isotope Labeling; D007700:Kinetics; D009532:Nickel / Q000494:pharmacology*; D019800:Phenol; D010636:Phenols; D012697:Serine Endopeptidases / Q000378:metabolism
D000596:Amino Acids; D002840:Chromates; D003432:Cross-Linking Reagents; D010636:Phenols; D002857:Chromium; D019800:Phenol; D009532:Nickel; D004247:DNA; D012697:Serine Endopeptidases; D019286:Endopeptidase K