Genotoxicity Testing at Gentronix
Gentronix specialises in genotoxicity testing for a range of industries, at both a screening and regulatory level. In addition to classical genotoxicity assays such as the Ames and Micronucleus test, Gentronix has developed BlueScreen™ HC and GreenScreen® HC which are novel, patented, systems that, unlike earlier assays, detect all known classes of genotoxin.
Gentronix’s research efforts focus on the creation, development and validation of novel technologies and methodologies, primarily for application in in vitro genotoxicity testing.
To date our innovations have focussed on delivering fast, accurate in vitro genotoxicity assessment, most recently in human-derived cells. At the heart of both the GreenScreen HC and BlueScreen HC in vitro genotoxicity assays is a reporter for the expression of the human GADD45a gene. Key to these assays is the central role of GADD45a in the cell’s genotoxic stress response and the faithful regulation of this gene in p53-competent host cells. It is these core elements that underpin the accuracy of these assays for the prediction of in vivo genotoxicity and genotoxic carcinogenicity.
The use of GFP (GreenScreen HC) and luciferase (BlueScreen HC) light-emitting reporters in these assays allows them to be miniaturised to microplate format and hence increases test chemical throughput, whilst minimising the amount of test chemical required for testing.
Developing a product or service with a novel technology requires data to demonstrate reliability and reproducibility, as well as evidence to support the prediction model and applicability of the assay. We pride ourselves in performing and publishing evaluation studies of substantial sets of test chemicals, as well as organising international ring trials to assess assay transferability and reproducibility.
GADD45a: “at the apex of the DNA damage response”
Why did Gentronix choose to base its in vitro screening genotoxicity tests around a gene called GADD45a?
- GADD45a was first described by Al Fornace. It was the first gene to be identified as a target of p53 (the ‘guardian of the genome’). ‘GADD’ stands for ‘Growth Arrest and DNA Damage’ and it is the only GADD gene up-regulated in response to ionising radiation as well as other genotoxic stress stimuli.
- It has been shown to have key roles in cell cycle regulation, DNA repair and apoptosis .
- Genetically modified mice without the gene survive; however, they are sensitive to induced DNA damage.
- Fibroblasts show centrosome amplification and multiple spindle poles, leading to chromosome mis-segregation and aneuploidy.
The GADD45a protein is a small globular protein. It modifies DNA accessibility in damaged chromatin and associates with nuclear factors involved in the mitotic checkpoint.
GADD45a is one of the most robustly induced mRNAs by genotoxins.
Gentronix developed GFP and luciferase reporter assays for GADD45a by replacing DNA in the native human GADD45a gene between ATG ‘Start’ and Exon 3. Both upstream and downstream regulatory regions and response elements (e.g. the intron 3 p53 response element) were retained for faithful regulation of GADD45a expression.