Introduction:
Many pharmaceuticals only present a genotoxicity hazard after metabolism and such chemicals are called pro-mutagens or pro-genotoxins. Generally, although not exclusively, the liver is considered responsible for the “metabolic activation” (MA) of promutagens. However, none of the in vitro genotoxicity test systems currently available has the inherent metabolic activity of the liver. In order to gain an early insight into MA effects prior to animal studies, in vitro tests are carried out in the presence of commercially prepared metabolic activation systems, such as the post-mitochondrial supernatant S9 (usually of rodent origin). The spectrum of potential metabolic conversions varies between S9 source species, and is greatly influenced by chemical pre-treatment regimes for enzyme induction. Metabolic activation in vitro also varies according to the concentration of S9 used, incubation time with the test chemical and the presence of other enzyme co-factors in the S9 mix.
The enzymatic activity in S9 preparations is heat-labile and the preparation itself can be toxic to cultured mammalian cells. These properties constrain protocol development in all S9 MA assays. In addition S9 extracts are red, fluorescent, and contain particulate matter, properties which have to be accommodated in assays that rely upon optically measured endpoints
Using S9 extracts in the GreenScreen HC assay:
Gentronix has developed a flow cytometry method for the measurement of the GFP signal as an alternative to bulk cell spectrophotometry in a microplate reader. This technique successfully reproduces the results from compounds tested without MA in the original validation study. Flow cytometry allows users to exclude interference from particulate matter and to dramatically reduce interference due to colour and fluorescence. This has allowed the successful development and evaluation of the GreenScreen HC S9 assay.
Cells are incubated in 96 well microplates for 3 hours in the presence of the test compound and 1% S9 mix. After plate washing to remove S9, the cells are resuspended in fresh recovery medium for 45 hours. Following data collection in the flow cytometer, a data processing template generates positive / negative genotoxicity decisions alongside a clear graphical output.
Validation of the S9 protocol:
An evaluation of the S9 method using a group of 50 compounds including non-genotoxins, genotoxins and pro-genotoxins has recently been published. Using a single protocol and treatment regime, the assay has detected the genotoxicity of metabolites from a high proportion of the pro-genotoxins tested, including common S9 control compounds from other assays such as benzo[a]pyrene and cyclophosphamide.
Click here to download a flyer on this assay....
Cross Platform Validation: A version for use with microplate readers.
Understanding that not everyone has ready access to, or working knowledge of, a flow cytometer, Gentronix have also launched a version of the GreenScreen HC S9 assay which can be used entirely with microplate readers. The assay uses a fluoresent cell stain to assess cell density rather than optical absorbance. Data analysis is straightforward and in practical terms this adaptation requires no more time than the flow cytotmetric protocol. Results for standard chemicals are comparable with those from the flow cytometer.
Click here to download an application note on GreenScreen HC S9 Cross-platform Protocols....
The GreenScreen HC S9 assay is now available either as reagent kits or as part of the Gentronix contract research service offering. Please contact Gentronix for more details.