Bioluminescent Salmonella reverse mutation assay: a screen for detecting mutagenicity with high throughput attributes

Abstract

Here, we describe the development and evaluation of a novel bioluminescent high-throughput Salmonella reverse mutation assay applicable to the screening of large numbers of small molecules. The bioluminescent Salmonella assay utilizes genetically engineered standard Salmonella tester strains TA98 and TA100 expressing the lux(CDABE) operon from Xenorhabdus luminescence. In principle, the assay employs bioluminescence as a sensor of changes in bacterial metabolism associated with starvation or energy depletion effectively identifying colonies of histidine-independent revertant cells in a high-throughput fashion. The assay provides highly concordant data with the outcome in the standard Salmonella plate incorporation reverse mutation assay. Since the results of the standard Salmonella plate assay are required by various regulatory agencies for approval of new drugs, the bioluminescent Salmonella assay can be effectively used for prioritization of compounds in pharmaceutical drug discovery as well as the evaluation of environmental and industrial chemicals. Because of its high throughput attributes, the assay permits effective, fast and economical screening of a large series of structural analogs enabling the investigation of structure-activity relationships.

Genetic Toxicity:In VitroApproaches for Medicinal Chemists

“…While practical for candidate selection, this is still not suitable for profiling assessments. A more recent development is the Pfizer "BioLum" test [1]. In this assay, bacterial mutation assays are incorporated into the Salmonella tester strains of a gene encoding a light-emitting protein under the control of the constitutively expressed kanamycin resistance gene.…”

“…Cells that can form even small colonies (for the Ames test, revertant mutant colonies selected for histidine prototrophy) will emit light and are readily counted using electronic imaging systems. This allows higher cell densities to be plated in microplate format with the concomitant reduction in use of plastic ware and test article [1]. Initial validation work was performed in 24-well microplates with a throughput of around 20 compounds per week in the authors' laboratories.…”

Genetic Toxicity: In Vitro Approaches for Hit and Lead Profiling

“…The resolution of these devices means that very much smaller colonies can be counted. This in turn allows for higher cell densities to be plated, in microplate format -with the concomitant reduction in use of plastic ware and test article [1]. Initial validation work was performed in 24-well microplates with a throughput of around 20 compounds/week, though the authors felt that the simple protocol and amenability to automation should mean that significantly higher throughputs are possible.…”

High-throughput approaches for genotoxicity testing in drug development: recent advances

“…The modified Ames test has been validated with 105 compounds that have previously been evaluated in the standard Ames test. 13 The simplicity and the minimal time requirement serve as major advantages for the high-throughput forms of the Ames test. However, on the other hand, a major drawback lies in the inability to predict mutagenicity/genotoxicity in vivo completely.…”