| 摘要 |
Methods and kits for measuring mutant hypersensitivity assay using high-throughput screening methodology to evaluate the mechanisms of toxicity of chemicals. The assay is performed in multi-well plates, such as those having 96 wells, making the process conducive to testing many compounds in a short period of time. The assay is versatile in that it can test compounds for ability to cause, for example, DNA damage, ability to mutate genetic material (mutagenicity), the ability to cause cancer (carcenogenicity), cause protein or membrane damage, energy depletion, mitochondrial damage, as well as the more general genotoxicity. Thus, the term toxicity, as used in this disclosure, is intended to encompass all of these types of effects. Furthermore, the assay can detect oxidative stress, protein damage, cell cycle disruption, energy charge and depletion, microtubule disruption or onset of metabolic competency through overexpression of human gene inserts encoding metabolism genes or incorporation of S9 fraction. In a preferred embodiment of the present invention, wildtype (wt) yeast and respective mutants are dosed with the desired chemical and yeast growth is determined using turbidimetry. Dose response curves are generated and mutant sensitivity to the compound relative to its parent (relative sensitivity) calculated. Relative sensitivities which are statistically significant indicate a hypersensitivity of the mutant to the test compound. The assay therefore provides an inexpensive, reliable, short term toxicity test which is an excellent alternative to animal testing and which provides valuable information about the mechanism of action of a compound. The present invention has applications to the pharmaceutical industry, environmental testing and clinical studies. |
