HIGHLY SENSITIVE UMU TEST SYSTEM FOR THE DETECTION OF MUTAGENIC NITROARENES IN SALMONELLA-TYPHIMURIUM NM3009 HAVING HIGH O-ACETYLTRANSFERASE AND NITROREDUCTASE ACTIVITIES

A highly sensitive umu test system for the detection of genotoxic activities of a variety of mutagenic nitroarenes has been developed using a new tester strain, Salmonella typhimurium NM3009 having high 0-acetyltransferase (O-AT) and nitroreductase (NR) activities. The NM3009 was constructed by subcloning both the O-AT and NR genes into plasmid vector pACYCl 84, and the resulting plasmid was introduced into the parent tester strain S. typhimurium TA1535/pSK1 002 harboring an umuC'-'/ocZ fusion gene. The induction of umuC gene expression could be monitored by measuring the cellular beta-galactosidase activity produced by fusion gene. The purpose of the study was to evaluate whether the newly developed strain NM3009 is highly sensitive toward nitroarene compounds. The sensitivity of the strain NM3009 was compared with those of the parent TA1535/pSK1 002 strain, the NR-overexpressing strain NM1011, the NR-deficient strain NM1000, the O-AT-overexpressing strain NM2009, and the O-AT-defective strain NM2000. The newly developed NM3009 strain had about 13-fold and 3-fold higher activities for N-AT and NR, respectively, than the original S. typhimurium TA1535/pSK1002 strain. Among six strains tested, NM3009 showed the highest sensitivity toward such chemicals as 1 -nitronaphthalene, 2-nitrofluorene, 3,7-dinitrofluoranthene, 3-nitrofluoranthene, 5-nitroacenaphthene, 2-nitronaphthalene, 1-nitropyrene, 1,6-dinitropyrene, 3,9-dinitrofluoranthene, 4,4-dinitrobiphenyl, 1,8-dinitropyrene, m-dinitrobenzene, 2,4-dinitrotoluene, and 1,3-dinitropyrene. We have also found that the order of sensitivities to induce umuC gene expression toward a variety of nitroarenes was NM3009 > NM2009 > NM1011 > TA1535/pSK1002 > NM2000 > NM1000. These results suggest that the newly developed tester strain NM3009 is of great use for the detection of genotoxic activities of numerous carcinogenic and mutagenic chemicals including nitroarenes, which require NR and/or O-AT for the activation.