Genotoxicity testing is commonly carried out in preclinical trials in mice, but the new methodology being developed by Litron should could bring this forward to the high-throughput screening stage and reduce the number of animals that are exposed to experimental drugs.
The company already has a micronucleus MicroFlow kit available for in vivo genotoxicity testing and is in the final development stages of the animal-free test with pharmaceutical companies and contract research organisations (CROs) currently beta-testing the product.
The micronucleus (MN) assay is being increasingly used as the 'gold standard' for detecting a chemical's genotoxic potential as an alternative to the more costly and time-consuming chromosome aberration assay.
A micronucleus is small nucleus that forms during the process of cell division. Normally genetic material will be divided equally between the two daughter cells.
However if a chromosome has been damaged and broken - for example after exposure to a genotoxic compound - the genetic fragment may fail to be included in either of the nuclei and will be left behind to form a micronucleus.
According to Steven Bryce, research and product development manager at Litron, these tests have traditionally been carried out by microscopic inspection. This is a very labour intensive way to determine the number of micronuclei in a sample, as a huge number of cells need to be counted.
While a microscopist may be able to study 4,000 cells in an hour, Litron's patented in vivo flow cytometry micronucleus test can count up to 20,000 cells in a minute.
Litron currently offers the in vivo test as both a service, with customers shipping the company blood samples, and a microflow kit so that pharmaceutical companies and contract research organisations (CROs) can run the tests themselves on their own flow cytometers.
The test uses different dyes to stain different cells and allow them to be differentiated as they pass through a flow cytometer.
"One of the dyes stains apoptotic cells that have historically caused problems in MN assay because they have fragmented nuclei like a micronucleated cell but this occurs by a different pathway that is not caused by genetic damage," said Bryce.
"Another dye, SYTOX green, stains the nuclei of all the cells and then the cells are lysed so they can be studied without the cell membrane."
The combination of dyes allows the proportion of apoptotic cells to be determined by their colour, with the number of miconuclei being differentiated from by the magnitude of their fluorescence response.
However, these in vivo tests still need the drugs to be tested on animals, so the company has been developing a micronucleus test that uses cultured cell lines to provide results more quickly.
The company initially developed the in vitro system using mouse L5178Y cells and published results in the journal Environmental and Molecular Mutagenesis .
Litron has published initial results of the animal-free testing method in the journal Mutation Research using a human TK6 cell culture, originally derived from the spleen of a five year-old male with hereditary spherocytosis.
The results showed that the method was directly transferable to human TK6 cells and the company is in the process of miniaturising the test for release in a standard 96 well plate for high-throughput applications.
