Dinoflagellate genomes are large (up to 200 pg) and are encoded in histoneless chromosomes that are quasi-permanently condensed. This unique combination of chromosomal characteristics presents additional topological and cell cycle control problems for a eukaryotic cell, potentially exhibiting novel regulatory requirements of topoisomerase II. The heterotrophic dinoflagellate Crypthecodinium cohnii was used in this study. The topoisomerase II activities throughout its cell cycle were investigated by DNA flow cytometry following enzyme deactivation. Fluorescence microscopy was also used for studying the chromosome morphology of the treated cells. Two classes of topoisomerase II inhibitors were applied in our study, both of which caused G1 delay as well as G2/M arrest in the C. cohnii cell cycle. At high doses, the topoisomerase poisons amsacrine and ellipticine induced DNA fragmentation in C. cohnii cells. Topoisomerase II activities, as measured by the ability to decatenate kinetoplastid DNA (kDNA), are normally detected throughout the cell cycle in C. cohnii. Our results suggest that the requirement of type II topoisomerase activities during the G1 phase of the cell cycle may relate to the unwinding of quasi-permanently condensed chromosomes for the purpose of transcription. This was also the first time that topoisomerase II activity in dinoflagellate cells was detected.

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Most known algal toxins act on ion channels either directly or indirectly, resulting in a change in intracellular ion concentrations when administered to targeted cells. The present project developed the working conditions for the use of fluorescent dyes in monitoring changes in membrane potential, intracellular calcium, and intracellular sodium levels in mammalian cell lines. Using these conditions, we were able to demonstrate specific changes in fluorescent signals in response to several purified toxins. We were also able to generate algal extracts which, when administered to the developed fluorimetric assays, were able to elicit different pattern of changes in membrane potential, intracellular calcium, and intracellular sodium levels. The differential pattern of responses induced by the different algal toxins in the three fluorimetric assays serve as a proof of concept for the use of multiplex fluorimetric assays in the laboratory monitoring of algal toxins.