A new short-term toxicity assay using Aspergillus awamori with recombinant aequorin gene

BACKGROUND: Most currently available short-term toxicity assays are based on bacterial cells. Therefore there is a need for novel eukaryotic microbial bioassays that will be relevant to higher eukaryotes such as animals and plants. Ca(2+ )is a universal intracellular signalling molecule found in all organisms from prokaryotes to highly specialized animal cells. In fungi calcium has been demonstrated to be involved in control of many important processes. The recombinant aequorin gene from the jellyfish Aequorea victoria responsible for the expression of the Ca(2+)-sensitive aequorin photoprotein has been cloned in the filamentous fungus Aspergillus awamori. This has allowed real life monitoring of [Ca(2+)](c )changes in living fungal cells. When subjected to different physico-chemical stimuli fungal cells respond by transiently changing the concentration of free Ca(2+ )in the cytosol ([Ca(2+)](c)) and the pattern of these changes (Ca(2+ )signature) is specific to each particular stimulus. Therefore it was interesting to investigate whether different environmental toxicants would be able to affect the pattern of [Ca(2+)](c )changes in a reproducible and dose dependant manner. RESULTS: Toxicity bioassay has been developed to monitor changes [Ca(2+)](c )of the recombinant fungus in the presence of toxicants representing heavy metals – Cr(6+ )and Zn(2+ )and a phenolic polar narcotic -3,5-DCP. The fungus responds to toxicants by a decrease in the amplitude of [Ca(2+)](c )response to 5 mM external CaCl(2 )and an increase in Ca(2+ )final resting levels and recovery time. CONCLUSION: A novel toxicity bioassay utilizing eukaryotic cells has been developed based on filamentous fungi transformed with the recombinant aequorin gene. A range of parameters characterising changes in [Ca(2+)](c )has been identified, e.g. Amplitude, Length of Transient, Final Resting Level and Recovery Time. These parameters can be used to determine the toxicity of a range of chemicals to eukaryotic cells in a 96-well microtitre plate method.