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Gentronix Extends Toxicology Screening Services to Apoptosis

Published: Tuesday, August 13, 2013
Last Updated: Monday, August 12, 2013
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Company adds apoptosis testing to provide follow-up for micronucleus positive results.

Apoptosis, or programmed cell death, is used by the body to remove unwanted, damaged or senescent cells from tissues. Gentronix has announced the expansion of its service testing portfolio with the launch of Apoptosis Suite: a set of in vitro assays to detect apoptosis induction during micronucleus assessment.

New compounds being developed as pharmaceuticals, agrichemicals etc are subject to genotoxicity assessment in order to identify potential carcinogenicity.

However, positive genotoxicity results alone need not terminate the development of a compound. One reason for this is that some genotoxicity tests have recognized mechanisms for the generation of misleading, or ‘false positive’ results.

For example, the widely used micronucleus test can produce positive results as a consequence of ‘apoptosis’, which can be induced by both genotoxic and non-genotoxic mechanisms.

Thus when a compound produces a positive micronucleus test result, it is valuable to understand the context of the result in relation to cytotoxicity and apoptosis induction.

Activation of apoptotic pathways at similar doses to those inducing micronucleus formation can provide valuable weight of evidence in de-risking the positive result. This would then trigger the application of other genotoxicity test methods, such as the Ames test, GreenScreen HC or BlueScreen HC assays.

A positive Apoptosis suite result, combined with a negative Ames test or GreenScreen HC / BlueScreen HC result would suggest apoptosis may be the cause of a misleading positive result in the in vitro micronucleus test.

The Apoptosis Suite comprises of: an Annexin-V Affinity Assay, a Caspase 3/7 Assay and general cytotoxicity measures covering cellular proliferation and viability.

Phosphatidylserine (PS) is a phospholipid that is normally only present on the inner leaflet of cell membranes, but during apoptosis it is translocated to the extracellular membrane leaflet, marking the cell as a target for phagocytosis.

Fluorescently labelled recombinant Annexin-V preferentially binds to the translocated PS and is detected using flow cytometry. Caspases are proteases that play a key role in apoptosis in mammalian cells.

Adding a luminogenic Caspase 3/7 substrate results in cell lysis followed by caspase cleavage with an attendant ‘glow-type’ luminescence that is proportional to the amount of caspase activity present.

The combination of these assays, alongside general cellular toxicity measures, provides strong evidence for the presence or absence of an apoptotic mechanism.

Dr Steve Beasley, Commercial Director at Gentronix, commented: “Our Apoptosis Suite is a powerful tool to help determine the context of a positive genotoxicity result identified during early micronucleus test screening. This latest addition to our service offering highlights our commitment to expanding our range of assays and services in line with our customers’ needs. It enables toxicologists to better assess screening results and where necessary modify candidate compounds to eliminate genotoxicity, while still keeping the desired active properties.”

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