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Risk assessment: comet assay and Pig-a mutation assay used together

The in vivo Pig-a Gene Mutation Assay detects mutations at the Pig-a gene that cause failure of “GPI anchors”. Without these anchors, specific markers on the exterior of cells will not be present. This assay measures the frequency of cells without these surface markers, also known as “Pig-a mutant cells”.  Litron Laboratories produce MutaFlow kits which use flow cytometry to measure the frequency of Pig-a mutant red blood cells.  For more information about the Pig-a mutation test, please visit www.litronlabs.com

The OECD has recently released a test guideline for the in vivo comet assay and, prior to this release, researchers were interested in the performance of a combined comet assay/Pig-a gene mutation study.  The authors were evaluating the potential of the combined comet assay /Pig-a gene mutation study and it is possible that this could be a standard combination of regulatory tests in the future.

The study was conducted with genotoxicant benzo[a]pyrene (BaP) at an exposure level known to induce germ cell mutation.

During this investigation, the scientists aimed to better understand:

(i) the strengths and weaknesses of the two methods applied in blood and their potential to predict germ cell mutagenicity, and

(ii) the involvement of reactive oxygen species (ROS) following in vivo BaP-exposure.

To explore the involvement of ROS on BaP genotoxicity, the investiagtors utilised a model deficient in a DNA glycosylas which was treated for three consecutive days with 50 mg BaP/kg/day. DNA damage in nucleated blood cells was measured four hours after the last treatment with the comet assay, with and without formamidopyrimidine DNA glycosylase (Fpg).  A Comet Assay IV, from Perceptive Instruments, system was used to analyse the results. Pig-a mutant phenotype blood erythrocytes were analysed two and four weeks after treatment.

It was reported that BaP-induced DNA lesions were not significantly increased in either version of the comet assay. The phenotypic mutation frequencies for immature and mature erythrocytes were significantly increased after two weeks. The scientist concluded that these effects were not affected by model genotype, suggesting oxidative damage may have a minor role in BaP genotoxicity, at least in the acute exposure situation studied here.

Following the investigation, the researchers concluded that “While both assays are promising tools for risk assessment, these results highlight the necessity of understanding the limitations regarding each assay's ability to detect chemicals’ genotoxic potential.”

If you would like more information on the Pig-a mutation assay, please contact Litron Laboratories.

If you would like more information on Comet Assay IV, please contact Perceptive Instruments. 

And, please read the full publication here:

Single cell gel electrophoresis (SCGE) and Pig-a mutation assay in vivo-tools for genotoxicity testing from a regulatory perspective: A study of benzo[a]pyrene in Ogg1−/− mice.Anne Graupner, Christine Instanes,   Stephen D. Dertinger,  Jill Mari Andersen, Birgitte Lindeman, Tonje Danielsen Rongved,Gunnar Brunborg,    Ann-Karin Olsen, Mutation Research/Genetic Toxicology and Environmental Mutagenesis. Volume 772, 15 September 2014, Pages 34–41.