Much effort has been expended in developing assays that enhance the likelihood of detecting environmental and occupational toxicants while minimizing the use of human and animal resources. Our laboratory has produced a very sensitive in vitro assay for genotoxicity that is based on the altered expression of the p53 and p53-regulated proteins which accompanies DNA damage. With this assay alterations in protein levels can be detected at doses of ionizing radiation that are 10-100 times lower than those required for the induction of micronuclei or reduction in colony-forming efficiency. The purpose of this research initiative is to develop a rapid and sensitive in vitro assay to assess genotoxicity from environmental and occupational exposures. The overall goal of the proposed research is to monitor changes in protein expression that occur in response to DNA damage from a wide assortment of environmental and occupational chemicals and to determine whether the changes predict genotoxic potential. Cell lines derived from rodent (FRLE) and human (A549) lung epithelial cells will be exposed to known genotoxicants and the specific protein expression quantified by multiparameter flow cytometry to detect fluorescent-immunolabeled proteins. This approach is quicker, it uses fewer cells, and it is 10-100 times more sensitive than a conventional Western blot analysis for quantifying proteins. The ability of the assay to detect DNA damage in cells exposed to low doses of other known genotoxicants will be determined. Initially, selected compounds classified by the International Agency for Research on Cancer as class 1, 2A, 2B, or 3 carcinogens and those compounds that have been tested in long-term animal bioassays will be selected for analysis. The flow cytometric protein assays will be correlated with results from the cytochalasin B block micronucleus assay and the colony-forming efficiency assay to determine relative sensitivity in terms of temporal and dose- response relationships. The different kinetic and temporal patterns of gene expression will also be correlated with the putative mechanism of action of the genotoxicants. The results obtained with the known genotoxicants will be related to results with X rays as a means of comparing genotoxicities between chemicals and between investigations. Ultimately, the assay will be adopted for assessing genotoxic risk for unknown mixtures and constituents of aerosols and will provide evidence of synergism or superadditivity among mixtures. The assay should be applicable to assessing new drugs and chemicals developed by the pharmaceutical and chemical industries.
| Johnson, N F; Jaramillo, R J (1997) p53, Cip1, and Gadd153 expression following treatment of A549 cells with natural and man-made vitreous fibers. Environ Health Perspect 105 Suppl 5:1143-5 |
| Johnson, N F; Carpenter, T R; Jaramillo, R J et al. (1997) DNA damage-inducible genes as biomarkers for exposures to environmental agents. Environ Health Perspect 105 Suppl 4:913-8 |
