Further development of the beta-lactamase MutaGen assay and evaluation by comparison with Ames fluctuation tests and the umu test.
A rapid, high-throughput bacterial mutagenicity test system has been developed (MutaGen test) that detects reversions of inactivating base-pair substitutions and frameshifts in a TEM-1 class A beta-lactamase (ampicillinase) gene. To quickly and sensitively detect mutagens, the system utilises a series of plasmids that contain the mutated ampicillinase gene and the mucAB operon. Inactivating mutations in the ampicillinase gene include frameshifts integrated into repetitive GC-sequences and G-runs known to be mutagenic hot-spots, and base-pair substitutions inserted in or around the beta-lactamase active site. Frameshift mutations completely inactivated the enzyme only when located downstream of the active-site serine (Ser68). Previous (reporter gene based) assays with this system have detected reversion to ampicillin resistance by luminescence driven by induction of the tet-promotor controlled lacZ gene. In the present study, we describe the construction and evaluation of 19 additional potential tester strains. We also developed conditions for detecting reversions by pH shift using bromocresol purple and by directly detecting the enzymatic activity of beta-lactamase using nitrocefin. A 384-well format version of the pH shift MutaGen test was used to assay more than 20 chemicals. The responses in the assay were compared with responses for the same chemicals in the umu test and Ames fluctuation assays. The results indicate that the MutaGen test has high specificity for detecting specific mutations and, in some instances, better sensitivity than the other tests. Since the test is easy to conduct, sterile working conditions are not necessary, and the mutagenicity results are available either within one working day or overnight, the assay shows promise for the rapid screening of potentially genotoxic substances.
Reifferscheid
Georg
G
Department of Environmental and Molecular Genotoxicity (AMMUG), University of Mainz, Germany. reifferscheid@bafg.de
Arndt
Christian
C
Schmid
Claudia
C
D001419:Bacteria / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D001681:Biological Assay; D001962:Bromcresol Purple / Q000633:toxicity; D002511:Cephalosporins / Q000633:toxicity; D004784:Environmental Monitoring / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D009152:Mutagenicity Tests / Q000379:methods; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics*
D002511:Cephalosporins; D001962:Bromcresol Purple; D001618:beta-Lactamases; C021720:nitrocefin