Membrane destabilisation of haemocytes measured by uptake of fluorescent probes

Lysosomal stability measured as neutral red retention (NRR) in haemocytes of mussels has been used to measure impact of environmental pollution. In this study we have tested whether membrane destabilisation also may be measured in haemocytes by a fluorescent plate reader after addition of the fluorescent probes BODIPY FL verapamil (BFLV) and ethidium homodimer-1 (EthD-1). Blue mussels (Mytilus edulis) have been exposed to pyrene, Cu and Zn, and the ratio of fluorescence calculated after different time intervals. Healthy granulated haemocytes efficiently take up BFLV to their lysosomes, while uptake is decreased in haemocytes of exposed mussels. EthD-1 is taken up generally with time and the fluorescence will increase upon binding to nucleic acids; however, damaged cells take up EthD-1 to a greater extent. Thereby, the ratio of the fluorescent probes enhances the signal. The effect was verified by fluorescence microscope studies. The results with pyrene demonstrated significant reduction in ratio of fluorescence after treatment with 0.4, 1, and 2.5 mg/l for 1 week, as did 2 mg/l Cu and Zn. The results have been compared with the NRR assay, demonstrating similar sensitivity. A comparison of the methods has also been performed with mussels collected from a metal-polluted field site. The measured fluorescence ratio may be an alternative to the NRR assay, providing automated reading.