Exposure to certain classes of industrial toxicants has been shown to result in oxidative damage. The herbicide paraquat has been shown to exhibit its lung toxicity through well studied and characterized oxidative mechanisms. Metals are also capable of causing oxidative injury, and the role of oxidative damage in metal-induced fibrosis and carcinogenesis is currently an active area of research. Oxidative damage is usually examined by studying lipid peroxidation or DNA damage; however, proteins are also potential targets but have not been as well studied because techniques of comparable sensitivity are lacking. We propose to develop polyclonal antibody techniques for detection of oxidatively damaged proteins in the following manner. The formation of carbonyl moieties has been demonstrated to result from oxidative damage to proteins, such as occurs from exposure to oxidants in industrial environments. The techniques to be developed involve reaction of the protein carbonyl moieties derived from oxidized proteins with 2,4-dinitrophenylhydrazine and subsequent detection with an antibody. Proteins., both purified and from fractions of mouse tissue, will be oxidized in vitro using several sources of oxidants, including metal-ions of occupational importance. Detection of the carbonyl groups will be compared from results obtained with the polyclonal antibody technique to results from established spectrophotometric techniques. We also propose to assess the utility of the immunoassay to detect oxidized proteins generated in vivo. For these studies, mice will be treated with paraquat, a model lung toxicant which produces oxidative damage, and we will develop methods to detect the carbonyl groups formed using our polyclonal antibody technique in samples from tissue homogenates or subcellular fractions and determine the protein fractions most susceptible to oxidative damage. It is hoped that the preliminary data obtained in this study will lead to the development of a simple, inexpensive immunoassay which can easily assess an important toxicological endpoint for occupational oxidant exposure.
