of Work:
The aim of this project is to investigate the mechanism(s) of tetranitromethane (TNM) pulmonary toxicity and carcinogenicity. Of particular interest is the investigation of the molecular reaction sites of TNM with nucleic acids and/or nuclear and membrane proteins. In addition, the potential reactions of TNM with DNA and with membrane tyrosine residues will be evaluated. The biochemical reactions of TNM with nucleic acids and proteins were investigated utilizing an acellular in vitro system with purified substrates (DNA, nucleic acids, peptides, amino acids) and measuring reaction products by HPLC, capillary electrophoresis and PAGE. DNA and nucleobase standards were reacted in vitro with TNM at 37 degrees C for 24 hours. DNA was hydrolyzed to normal and adducted bases which were separated and analyzed by capillary electrophoresis/mass spectrometry. DNA adducts increased for each base in the order of guanine > adenine > cytosine > thymidine. Adducts identified included guanine + C(NO2), guanine + NO2, and guanine + NO. Studies are in progress to investigate the formation of DNA adducts in cultured lung cells treated with TNM. In vitro studies conducted to date indicate that TNM does not cause DNA strand breaks or DNA-protein adducts in lung cells treated in vitro. Because TNM specifically nitrates tyrosine residues on proteins, the effects of TNM on phosphorylation and dephosphorylation of membrane tyrosine residues, and subsequent effects on cell proliferation are being investigated. Preliminary in vitro studies indicate that phosphorylation of nitro-tyrosine is a very slow reaction.
