International Research Contract Report: Evaluation of a Radio-Sensitive DNA Bioassay.
Final rept. 1 Sep 98-31 Aug 99
COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANIZATION MELBOURNE (AUSTRALIA)
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The project combined a range of complex elements that all needed to succeed to produce an optimal result, i.e., a sensitive radiation bioassay. The specialized cell line provided a reliable and consistent biological test target model. The cell synchrony experiments provided reliable data and demonstrated the ability to precisely separate G1 cells from asynchronous populations of L5178Y SS cells. Data from cell flow cytometry CFC confirmed the successful elutriation and yield of narrow distributions of DNA content in G1 cells. Results also demonstrated the ability of the centrifugal elutriation technique to separate out populations of cells in later stages of the cell cycle. This offers a valuable tool for the design of enhanced sensitivity test systems that may need to examine the effects of stressors on specific susceptible stages in the cell cycle. Careful and rigorous analysis of the application of the comet assay for DNA single strand breaks found the results to be inconsistent when using the established routine laboratory protocol. Inconsistencies in the data for DNA content acquired by the comet assay and the effect on calculation of tail moment parameter reduced our level of confidence in this part of the assay. Our study identified a major problem that could not be resolved within the time and resource constraints of the current project. These findings give cause for some concern regarding claims for sensitivity of the comet assay technique and the status of publications in this area of DNA damage. There may be good reason to doubt the reliability of data given in some publications on comet assays that do not disclose information on DNA content or valid zero-dose values. It is apparent that most researchers are either unaware of the limitation or choose to ignore it.