| Summary: | 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.
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