Much of the work in toxicology has focused on delineating the effects of a single chemical entity often at high doses and over short time periods. However, humans are more often exposed to multiple chemicals over long time periods and at lower doses than generally used experimentally. Thus, there is a need to understand potential interactions of exposure to multiple chemical entities at both the cellular and whole organism level. The current request proposes to take advantage of recent developments in analysis of gene expression with high density microarrays to explore the use of this technology to identify alterations associated with exposure to multiple chemicals. This work will build on recent findings showing that the cytotoxicity of the metabolically activated, systemic pulmonary injurant, 1- nitronaphthalene, is considerably enhanced by preexposure to ozone. Two approaches will be utilized. Arrays of genes coding for both Phase I and Phase II metabolizing enzymes, enzymes involved in the synthesis and degradation of glutathione, several heat shock proteins and housekeeping genes will be prepared. mRNA isolated from control and treated (nitronaphthalene, ozone and nitronaphthalene plus ozone) rat lung will be used as a template for synthesis of cDNA labeled with fluorescent tags (CY-3 (control) and CY-5 (treated)) and these will be hybridized to the arrayed targets to determine whether treatments cause up or down regulation of genes likely to control the metabolic activation or detoxication of nitronaphthalene. Parallel quantitative histopathology studies will be done to confirm the severity of the pulmonary lesion in all treatment groups. In the second approach, clones from a control rat lung library will be arrayed on glass slides and screened against labeled mRNA from control (CY-3) and treated (CY-5) animals. Clones showing up or down regulation will be sequenced for identification. These studies will test the validity of using DNA arrays to rapidly screen changes in gene expression in response to mixtures of lung toxicants. The combination of dose and time course response studies which include detailed examination of tissues by histopathology will define cellular/molecular events that occur in response to chemical exposure and are expected to explore the validity of using DNA arrays to screen potential chemical interactions. By examining library clones, these studies may identify new genes whose regulation is altered by chemical exposure.
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