Carbonic anhydrase activity in the digestive gland of Mytilus galloprovincialis: the story of a novel biomarker?

Aim: Carbonic anhydrase is a zinc metalloenzyme catalysing the reversible hydration of CO2. Its activity is virtually ubiquitous in nature and is involved in a wide variety of physiological processes. The aim of the present work was to study the in vitro and in vivo sensitivity of CA to chemical pollutants in Mytilus galloprovincialis, widely used as sentinel organism in marine pollution biomonitoring, with the aim to develop a new biomarker of exposure/effect to chemical contamination. Methods: CA activity was measured applying a simple and low cost electrometric method. Briefly, enzymatic activity units were calculated from the rate of H+ production in the reaction mixture (where CO2 as substrate was present) against a blank containing the specific CA inhibitor acetazolamide. Results: As first step, we demonstrated the presence of a significant CA activity in M. galloprovincialis tissues and its sensitivity to in vitro exposure to several heavy metals. As second step, we demonstrated a significant tissue-specific alteration of CA activity following in vivo Cadmium exposure, with a significant increase in the digestive gland. As third step, immunoprecipitation and western blot analysis on mussel digestive gland revealed a protein that immunoreacts with the antibody at a molecular mass of approximate 42 kDa (the sequencing analysis is in progress). Interestingly the 42 KDa band, ascribed to CA, appeared strongly increased in the digestive gland of cadmium exposed mussel (14 day exposure), thus leading to an induction of CA expression in accordance with the observed enzymatic activity increase. As final step, the laboratory resulted was validated with field experiments. Conclusion: Obtained results represent a good starting point for future potential biomarker application of the CA activity in the sentinel organism M. galloprovincialis