VITOTOX assay

The VITOTOX test is a bacterial genotoxicity test. The test is based on bacteria that contain the lux operon of Vibrio fischeri under transcriptional control of the mutated recN promoter, which is controlled by the bacterial SOS-system (TA 104-recN2-4 strain or Genox strain). After incubation of the cells with a genotoxic product, the recN promoter will become derepressed, resulting in expression of the lux operon. This expression results in light production in function of the genotoxicity. Some products act directly on the light production (e.a aldehydes) or enhance the metabolism of the bacteria, creating false-positive results. Therefore a bacterial strain with a constitutively expressed lux operon (pr1-lux fusion = continuous light production) is also used as an internal control (pr1 or Cytox strain). If the light production increases in this Cytox strain, the test compound affects the lux gene in a different way than by damaging the DNA. Furthermore, a decrease in light production would indicate a cytotoxic response. The bacteria in both strains are treated with the test substance in the presence and absence of a metabolizing induced rat liver S9 fraction (for detection of metabolites). Light measurements are performed in a luminometer at constant temperature every 5 minutes in each well of a black 96-well plate during a 4-hour period after addition of the test samples to the bacteria. The signal to noise ratio (S/N) or, specifically, the light production of exposed bacteria divided by the light production of non-exposed bacteria is calculated for each measurement. S/N is calculated for both strains separately and the ratio between the maximum S/N values of the genox over the cytox strain. A substance is considered genotoxic when: - max S/N (genox)/max S/N (cytox) >1.5 - max S/N in genox shows a good dose–effect relationship - max S/N (genox)/Max S/N (cytox) shows a good dose–effect relationship. A substance is considered cytotoxic when S/N in the cytox strain decreases and becomes considerably lower than 0.8. If both strains are (strongly) induced there is no evidence for genotoxicity.