| Popis: |
The comet assay is widely used nowadays as its main applications include genotoxicity testing, human biomonitoring, DNA repair studies, environmental biomonitoring and clinical studies. It appeals to researchers because of its simplicity, sensitivity, and versatility. Both the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) and the European Food Safety Authority (EFSA) have included the in vivo comet assay in their step-wise approach for genotoxicity testing. Furthermore, last year the Organisation for Economic Co-operation and Development (OECD) finally adopted the guideline to carry out the in vivo mammalian alkaline comet assay in fresh tissue. Although the OECD Guideline takes into account the possibility of freezing tissues or cell nuclei for later analysis, the laboratory should demonstrate competency in freezing methodologies and confirm low ranges of % tail DNA in target tissues of vehicle treated animals, and that positive responses can still be detected. Freezing procedures for tissues have been described in the literature using different methods, but there is currently no agreement on how to best freeze and thaw tissues. The possibility of freezing the samples may be very helpful when handling too many samples, to integrate the comet assay with other toxicological studies, or to combine its endpoint with other genotoxicity endpoints. Therefore our aim was to test different methods for freezing liver samples, in order to be able to store the samples for later alkaline comet assay analysis. Liver pieces were obtained directly at necropsy and 4 different procedures were followed to freeze them: 1.- flash freezing in liquid nitrogen and storage at -80 °C; 2.- flash frozing in isopentane cooled in liquid nitrogen and storage at -80 °C; 3.- immersion in RNA later® solution at 4 °C overnight and storage at -80 °C ; 4.- immersion in Merchant buffer at 4 °C overnight and storage at -80 °C. Besides, we also tried cutting the tissue and homogenizing it using a metal sieve, and storing the resulting cell suspension at -80 °C both with and without using Mr. Frosty™, before and after filtering through a 100 μM nylon filter and centrifuging it. To process the frozen samples, they were taken out of the freezer and immediately put on ice; the tissue was then washed in a beaker with ice-cold Merchant buffer, transferred to another beaker with ice-cold Merchant buffer, cut into pieces with scissors and filtered through a metal sieve. The cell suspension was then filtered through a 100 μM nylon filter and centrifuged. Besides, we also tested the thawing protocol described by Jackson et al. (2013), with the samples which had been flash frozen by inmmersion in liquid nitrogen. None of the different methods used was capable of giving good results, except immersing the liver samples in liquid nitrogen, followed by Jackson’s et al. (2013) thawing protocol, suggesting that the thawing process may be as critical as the freezing process. To sum up, these results highlight the importance of deepening the possibility to perform the comet assay with frozen tissue. |
