Application of the single-cell gel electrophoresis (’COMET’) assay to lymphocytes exposed In Vitro to captan, a fungicide

• Main Author: Stuart, Gregory Roy
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/3618

This thesis describes the assembly and validation of a relatively developed assay of genotoxicity, the single-cell gel electrophoresis ('comet') assay, and its application to the detection of D N A damage associated with the exposure to captan, a fungicide, in human cells. The comet assay system was assembled using commercially available laboratory and image processing equipment. To facilitate the analysis of digital images from the comet assay, software was developed that was based on the public-domain program N IH Image. The comet assay system was validated in vitro using human peripheral blood lymphocytes and a known genotoxic agent, hydrogen peroxide, previously shown to give a positive response in the comet assay. The results from these experiments confirmed that this assay is capable of providing sensitive quantitative measures of the extent of induced D N A damage. Investigations of the amount of damage present in untreated lymphocyte D N A from different donors were also conducted. Once the comet assay system was validated it was applied to a study of the genotoxic effects of an agricultural fungicide, captan, which is widely used in British Columbia and may pose a risk to human health through occupational exposures. Both technical and analytical grades of captan were examined in vitro in the comet assay, using human lymphocytes. It was demonstrated that the comet assay was capable of detecting D N A strand breaks caused by captan at 0.1 micromolar concentrations. This result represents an increase in sensitivity of greater than an order of magnitude for detecting D N A strand breaks caused by captan, when compared to techniques previously employed, such as alkaline sucrose sedimentation.