DEVELOPMENT OF RISK ASSESSMENT METHODS ON HAZARD IDENTIFICATION USING CATALASE MUTANT GENE AS A PREDICTIVE INDEX

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This research was aimed at developing a risk assessment method for potential hazards by using catalase mutant strains. At first, we successfully constructed catalase mutant E coli strains by transforming plasmids encoding the catalase cDNA from catalase mutant mice (Cs^b, Cs^c) and its corresponding wild type (Cs^a) into catalase-deficient Escherichia coli UM255. There was a significant difference in catalase activities among each catalase-deficient E. coli mutant. The level of catalase in Csa appeared higher than that in the others, the catalase level in Cs^c was 65.1% of Cs^a, Cs^b was 61.7%, and UM 255 was 8.8% of Cs^a. Susceptibility to H_2O_2 showed a clear dose dependency with negative correlation to catalase activity levels. The susceptibility order was UM255>Cs^b>Cs^c>Cs^a. To assess the cytotoxicity of the chemicals, we employed colony-forming efficiency test and zones of inhibition test, the results demonstrated that survival of catalase-deficient E. coli mutants was signific … More antly reduced by treatment of hydroquinone, pyrogallol and L-Dopa in a dose-dependent manner and the cell viabilities underexposure to these chemicals were also different significantly among each catalase mutant strain, showing the following orders : Cs^a>Cs^c>Cs^b>UM255. The cytotoxicities induced by hydroquinone, pyrogallol and L-Dopa were also confirmed by zone of inhibition test, in which UM255 was the most susceptible, showing the largest zone of growth inhibition surrounding the paper disc, followed by Cs^b, Cs^c and Cs^a. The toxicities of these chemicals were completely blocked by catalase and ascorbic acid in a concentration-dependent relation. Treatment by some heavy metals (Hg, Cr, Cd, As, Ni, Fe, Cu) also significantly reduced the survival of these strains in a dose-dependent manner, suggesting H_2O_2 was increasingly produced in the process of hydroquinone cytotoxicity and catalase played a protective role in inhibiting the cell damage. The results support the usefulness of these newly established strains for hazard identification of oxidative chemicals in a risk assessment process. Less