Echinoderm coelomocytes as a cellular model in toxicity testing and biomonitoring

Abstract

In the studies described in this manuscript, a new cellular model system has been developed and two fluorometric assays have been assessed for their applicability in biomonitoring and in vitro toxicity testing. Echinoderm coelomocytes were chosen as the cellular system because of their ease of sampling, and because their immunofunction makes effects on these cells likely to cause adverse effects on the host organism. Primary cell cultures of coelomocytes were established in the 96-well microtiter plate format by removal of coelomic fluid, dilution to suitable cell density in culture medium phosphate buffered saline and application in wells without further processing. The 96-well format is suitable for high sample number and small sample size, thus allowing the high throughput screening that is desirable in biomonitoring and toxicity testing. Two fluorometric assays, the alamar BlueTM and CFDA-AM cytotoxicity assay and the multixenobiotic resistance (MXR) accumulation assay, were optimised and applied on the cultured coelomocytes. MXR is believed to serve as a cellular first line of defence against numerous substances, and is therefore expected to be highly relevant for cell viability and function. The biomonitoring study was conducted in Kaštela Bay, Croatia, on coelomocytes from the sea cucumber Holothuria tubulosa. Cells taken from individuals collected at a heavily polluted site was compared to cells taken from individuals at a relatively pristine site. In the toxicity testing study, Asterias rubens coelomocytes in culture were exposed to different toxicants for 96 hours, before the assays were run. Both assays provided significant results in biomonitoring and toxicity testing. Used in combination, coelomocyte primary culture and the two fluorometric assays may constitute a rapid, cost-effective, easily performed procedure for biomonitoring and toxicity testing.