The time and material consumption associated with the industry standard Ames mutagenicity assay prohibits its use as a high throughput screening method for industrial, human health, and environmental applications. HESS, Inc. has initiated the development of a marketable rapid, high-throughput genotoxicity assay that will apply the principle of mutagen-induced recovery of growth capacity (reverse mutation) employed in the Ames assay. However, a mutant beta-galactosidase gene will be utilized for the dual purpose of selective growth in lactose media and sensitive detection of mutagenesis with fluorescent substrates. Preliminary experiments funded by HESS, Inc. have demonstrated the proposed assay's potential for detecting mutagens more rapidly and more sensitively than previously possible with the Ames assay. Sensitive detection of mutagenically induced revertants allows for the miniaturization and incorporation of the assay into a 96- or 384-well format for rapid simultaneous genotoxicity screening of multiple test materials. Further optimization and development of this assay is expected to match the reliability of the Ames assay in positively identifying mutagens and reduce the time needed to determine mutagenic potential. Phase I funding is being sought to pursue the following specific aims: (1) production and forward assessment of mutant forms of beta-galactosidase alpha-peptide against mutagens of known mechanisms, and (2) optimization of fluorescent beta-galactosidase detection to improve sensitivity and accelerate screening of mutagens. Demonstration of feasibility during phase I experimentation will establish a path for expanding the utility of the proposed assay in subsequent development by producing an integrated and comprehensive method of simultaneously detecting diverse mechanisms of mutagenesis in a rapid highly efficient manner. Phase II efforts will include the following activities: (1) expansion of the methodology to employ the assay in environmental, pharmaceutical and industrial applications, (2) development of computer software to integrate and analyze the assay data, and (3) design and manufacture of a marketable test kit.
