Mercury- and copper-induced lysosomal membrane destabilisation depends on [Ca2+]i dependent phospholipase A2...

Heavy metals are environmental pollutants able to produce different cellular effects, such as an alteration of Ca2+ homeostasis and lysosomal membrane destabilisation. The latter is one of the most used stress indices in biomonitoring programs. Recently, it has been demonstrated that cytosolic calcium increase can modulate lysosomal membrane destabilisation via activation of Ca(2+)-dependent phospholipase A2 (cPLA2). The aim of this work was to investigate the possible involvement of Ca(2+)-activated PLA2 in lysosomal membrane destabilisation induced by heavy metals in mussel haemolymph cells. We have studied the effects of Hg2+ and Cu2+ on free cytosolic calcium using Fura2/AM-loaded cells and lysosomal membrane destabilisation using neutral red (NR) staining. Hg2+ induced a [Ca2+]i rise from 100 to 780 nM in 30 min, and a lysosome destaining of 70% after 60 min that indicates destabilisation of lysosomal membranes. Both effects were reduced in a Ca(2+)-free medium, suggesting a cause-effect relationship. Exposure to Cu2+ produced the same effects, but with an intensity of about 50% respect to Hg2+. Metal-induced lysosomal destabilisation was also reduced in cells pre-exposed to a specific Ca(2+)-dependent cPLA2 inhibitor (AACOCF3). Conversely, haemocyte pretreatment with a Ca(2+)-independent PLA2 inhibitor (bromoenol-lactone (BEL)) did not prevent the destabilizing effect of heavy metals on lysosomes. Exposure to heavy metals also produced an increase in lysosomal volume of 1.8-2-folds, that was prevented by pre-incubation with AACOCF3 but not with BEL. These data indicate an involvement of cPLA2 in lysosomal membrane destabilisation induced by heavy metals.