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Gustavsson, Linda, 2006. Evaluation of cytotoxicity assays and a high-througput variant of the alkaline Comet assay in mouse lymphoma L5178Y cells. SLU, Dept. of Biomedical Sciences and Veterinary Public Health, Uppsala. Uppsala: SLU, Dept. of Biomedical Sciences and Veterinary Public Health

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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.

Main title:Evaluation of cytotoxicity assays and a high-througput variant of the alkaline Comet assay in mouse lymphoma L5178Y cells
Authors:Gustavsson, Linda
Supervisor:Czene, Stefan
Examiner:UNSPECIFIED
Series:UNSPECIFIED
Volume/Sequential designation:UNSPECIFIED
Year of Publication:2006
Level and depth descriptor:Other
Student's programme affiliation:BTEMP Biotechnology Programme 240 HEC
Department:(VH) > Dept. of Biomedical Sciences and Veterinary Public Health
Keywords:Comet assay, ML cells, cytotoxicity
URN:NBN:urn:nbn:se:slu:epsilon-s-7339
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-s-7339
Subjects:SLU > (VH) > Dept. of Biomedical Sciences and Veterinary Public Health
Language:English
Deposited On:02 Oct 2017 09:30
Metadata Last Modified:02 Oct 2017 09:30

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