Epsilon Archive for Student Projects

Abstract

In the pharmaceutical industry, there is an increasing demand for rapid screening assays with a good predictivity for the regulatory tests. The Comet assay, or Single Cell Gel Electrophoresis Assay, is a fast, easy and relatively inexpensive method to visualize and measure DNA strand breaks in individual cells. In the early drug development, when the amount of the test substance is limited, the Comet assay must fulfil several criteria.

Since DNA damage is associated with cell death, cytotoxicity measurements should be evaluated concurrently with each Comet experiment. The purpose of the present study was to find a suitable cytotoxicity test in mouse lymphoma L5178Y (ML) cells and evaluate a high-throughput variant of the alkaline Comet assay (HT-ACA) in the same cell line. A modified protocol for the Comet assay, with inclusion of commercially available multi-well object slides was tested. The HT-ACA experiments were performed with four alkylating agents and the obtained results were then compared to Comet assay data previously obtained by the standard HT-ACA protocol of Comet Assay, currently in use at Safety Assessment, AstraZeneca.

Based on the results from experiments with different cytotoxicity tests, measurement of ATP levels was chosen as a suitable test in ML cells. The observed LOELs obtained from the modified HT-ACA were in good accordance with the standard HT-ACA protocol, albeit some differences were revealed. For instance, the modified version detected positive genotoxicity in concentrations two to five times lower than earlier had been found with the standard HT-ACA protocol. Even though the Comet assay has the potential to become a powerful complement to other genotoxicity tests, there are some bottlenecks in its performance, which need to be addressed.