The VITOTOX test, an SOS bioluminescence Salmonella typhimurium test to measure genotoxicity kinetics.
A new test to detect genotoxicity, that we refer to as the VITOTOX test, was developed. Four gene fusions that are based on the Escherichia coli recN promoter were constructed and evaluated for their SOS response-dependent induction. The wild-type recN promoter, a derivative mutated in the second LexA binding site, a derivative with a mutated -35 region, and a derivative from which both the second LexA binding site and the -35 region were mutated, were cloned upstream of the promoterless Vibrio fischeri luxCDABE operon of pMOL877, in such a way that lux became under transcriptional control of the recN promoter derivatives. The inducibility by the SOS response of the promoter constructs was tested in both E. coli and in the Ames test Salmonella typhimurium strains TA98, TA100 and TA104. In all strains, the highest sensitivity and induction was observed with the plasmids pMOL1067 and pMOL1068, that contain the lux operon under control of the recN promoter mutated in the second LexA binding site, or a recN promoter with a mutated -35 region, respectively. Therefore, strains containing pMOL1067 or pMOL1068 were further used for genotoxicity testing. With the VITOTOX test, genotoxicity was detected within 1-4 h. The VITOTOX test is very sensitive: for most products tested, the minimal detectable concentration (MDC) values were considerably lower (5 to > 100 times) than those described for the Ames test and the SOS chromotest. A good correlation was observed with the results from the Ames tests, but certain PAHs that are not mutagenic in the Ames test were genotoxic in the VITOTOX test. With the VITOTOX strains, the kinetics of SOS induction can be determined. This feature made it possible to distinguish between compounds in mixtures of genotoxic products so long as they had different induction kinetics.
van der Lelie
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Environment Division, Flemish Institute for Technological Research (VITO), Mol, Belgium. vdlelied@vito.be
Regniers
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Borremans
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Provoost
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Verschaeve
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D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D004262:DNA Restriction Enzymes; D003851:Deoxyribonucleases / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D008163:Luminescent Measurements; D016297:Mutagenesis, Site-Directed; D009152:Mutagenicity Tests; D009153:Mutagens / Q000494:pharmacology*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000378:metabolism; D015183:Restriction Mapping; D013014:SOS Response, Genetics / Q000187:drug effects; D012486:Salmonella typhimurium / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000378:metabolism; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D017931:DNA Primers; D004262:DNA Restriction Enzymes; D003851:Deoxyribonucleases; D009153:Mutagens; D011994:Recombinant Proteins; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein; C047879:RecN protein, Bacteria