The sensitivity of carbonic anhydrase to metal exposure in the model organisms Mytilus galloprovincialis: in vitro, in vivo and in field approach

Carbonic anhydrase (CA) is a ubiquitous metalloenzyme involved in a number of physiological processes. Its sensitivity to chemical pollutants has been recently recognized. The work was aimed to study the CA sensitivity to metal exposure in the digestive gland of the model organism Mytilus galloprovincialis under in vitro, in vivo and in field conditions, with reference to the functional involvement of CA in the lysosomal response to pollutant exposure. The study was carried out by immunofluorescence confocal microscopy, electrometric assay of CA activity, spectrofluorimetric and confocal analysis of the lysosomal system. Under in vitro exposure to CdCl2 or CuCl2, CA activity was dose-dependently inhibited with an IC50 of 8.7 10-5M for copper and 1.1 10-3M for cadmium. On the other hand, under in vivo chronic exposure to CuCl2 (0.3 10-6 M) or CdCl2 (0.54 10-6 M) for 14 days, CA showed a significant upregulation, paralleled by the increased fluorescence of LysoSensor green charged cells, indicative of lysosome proliferation/increase in size. The metal induced lysosomal activation was prevented by the in vivo exposure to the specific CA inhibitor acetazolamide, demonstrating a key role of CA in the pollutant induced lysosomal activation. The response of CA upregulation paralleled by lysosomal activation was validated in the field by an active biomonitoring approach in coastal marine sites interested by metal contamination. In conclusion, data showed the complexity and multi-aspect nature of the CA sensitivity to metals, which can be CA inhibitors at higher concentrations and modulator of CA expression at lower concentrations typical of chronic exposure. In this condition CA upregulation can be functional to the prolonged increased requirement of H+ under lysosomal activation.