Bacterial genotoxicity – SOS/umu

Aim: Detection of bacterial genotoxicity in a 96-well screening system.

Background information: Test items showing genotoxic activity are considered to have the potential to be human carcinogens and/or mutagens, this being an important liability for drug development.

In the SOS/umu assay, Salmonella typhimurium bacterial strain TA1535/pSK1002 carries plasmid pSK1002 which bears umuD and umuC genes (involved in the SOS pathway of DNA repair) fused with lacZ, the structural gene for b-galactosidase. Exposure to DNA-damaging agents induces the transcription of the umuDC operon fused gene producing a hybrid protein with ß-galactosidase activity. The b-galactosidase metabolization of the colorless substrate ortho-Nitrophenyl-β-galactoside (ONPG) into galactose and the yellowish coloured ortho-nitrophenol, allows the colorimetric quantitative assessment of the enzymatic activity, that correlates with DNA damage.

This miniaturized bacterial genotoxicity screening system shows a high correlation with the Ames test and allows the early screening of a high number of test items, in a reduced time and with a limited amount of compound (Oda, 1985; Reifferscheid, 1996; Walmsley, 2005).

Experimental procedure

  • Top concentration: 1000 µg/mL, unless limited by solubility
  • 4 h exposure period; 37ºC
  • Positive control: 2-aminofluorene (2-AF), genotoxin requiring metabolic activation

Delivered results

  • Bacterial cytotoxicity (Absorbance at 600 nm)
  • Absolute genotoxic activity (Absorbance at 420 nm)
  • Relative genotoxic activity: absolute genotoxic activity corrected by cytotoxicity
  • Positive when “Relative genotoxic activity” > 2-fold the vehicle control value

Concentrations indicated in the x-axes correspond to test item. For the positive control (2-AF), tested concentrations are (from left to right):  0.039, 0.078, 0.156, 0.313, 0.625, 1.25, 2.50, 5, 10, 20 and 40 µg/mL.