In 2008 Gentronix launches a new S9 protocol to extend the application of the GreenScreen HC assay to include the detection of genotoxic metabolites.
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:
The first successful demonstration of the use of exogenous metabolic activation in the GreenScreen HC assay was reported in the Hastwell validation paper. Gentronix has since 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 (MS in preparation). 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:
We have carried out an initial evaluation of the S9 method using a group of 50 compounds including non-genotoxins, genotoxins and pro-genotoxins. 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.
Further validation and protocol information is available under the cover of a confidentiality agreement and will be published in due course. Please contact Gentronix for more details.