Lysosomal reaction to xenobiotics in mussel hemocytes using BODIPY-FL-verapamil

Many cellular and sub-cellular biomarkers associated with mussel (Mytilus edulis) digestive gland and kidney have been characterised. The lysosomal compartment of these tissues have been recognised as being particularly sensitive, exhibiting pollutant induced responses which could be potentially used as a ‘biomarker’. However, relatively few studies have investigated the lysosomal response within molluscan hemocytes. This study was conducted to test whether lysosomal reactions, in live hemocytes isolated from mussels, can be used as a biomarker of pollutant exposure and deleterious effect. Lysosomal responses to a number of hydrocarbons, including anthracene and phenanthrene, and to the amphiphilic heterocylic chemical, chlorpromazine, were examined. The supravital dye neutral red (NR) was used to examine lysosomal membrane fragility, following xenobiotic exposure. NR was also used to verify the lysosomal compartment as the reported accumulation site of a new molecular probe, BODIPY-FL-verapamil (BFLV). The use of BFLV, with confocal laser microscopy and image analysis enabled visualisation and quantification of lysosomal distribution and perturbation. BFLV showed that exposure of molluscan hemocytes to xenobiotics (20 ppb–10 ppm) induced the formation of pathologically enlarged lysosomes. The internal trafficking of lysosomes was shown to be severely compromised after exposure to chlorpromazine. Exposed molluscan hemocytes exhibited significantly reduced lysosomal retention times, for neutral red. Preliminary data is presented demonstrating the opportunity for these non-destructive biomarker techniques to detect pollution gradients in situ.