Ethylene oxide, a widely-used chemical intermediate and fumigant, is carcinogenic to animals and is """"""""probably carcinogenic to humans,"""""""" according to the IARC. The initiating event is presumed to be DNA damage, and repair is probably a major cellular defense. Thus, ethylene oxide is an excellent model for the study of environmental exposure. In this project, we will: 1) Determine the nature of the DNA modifications which are caused by ethylene oxide; 2) Determine what bacterial and mammalian enzymes repair these lesions; and 3) Develop an assay for DNA damage which could be used to monitor the risk of ethylene oxide exposure. The chemical nature of the nucleoside modifications produced by ethlylene oxide will be determined by incubating deoxyribonucleosides with ethylene oxide and separating the reaction mixtures by HPLC. Structures of the modified deoxynucleosides will be determined by comparison with known standards already available in this laboratory, and by ultraviolet, NMR, and mass spectrometry. DNA will be reacted with ethylene oxide in buffered aqueous solution at 37 degree C and digested enzymatically. Digests will be separated by HPLC, and modified peaks will be identified in comparison with known markers. To investigate the repair of ethylene oxide-induced DNA lesions, nick-translated DNA's will be prepared from 14C-deoxynucleoside triphosphates, and modified with ethylene oxide. Removal or repair of these modified bases will be studied with specific, bacterial repair enzymes and calf thymus extracts. A sensitive method will be developed for determining the extent of modification in lymphocyte DNA. In collaboration with other laboratories, monoclonal antibodies will be developed to modified nucleosides; at the same time, we will develop a GC/MS assay as an alternate analytical method. Ultimately, it will be possible to standardize these methods to monitor human exposure because the nitrosoureas currently used in cancer chemotherapy produce many of the same DNA modifications that ethylene oxide presumably causes in exposed personnel.
