Quantification of midkine gene expression in Patella caerulea (Mollusca, Gastropoda) exposed to cadmium

The release of cadmium into many coastal areas represents a threat to ecosystems and human health; cadmium is carcinogenic in mammals and in both marine invertebrates and vertebrates. The use of molluscs to assess the ecologic risk associated with contaminants is strongly recommended on account of their ecological role and on their highly conserved control and regulatory pathways that are often homologous to vertebrate systems. We previously identified a midkine family protein in the limpet Patella caerulea; the midkine is a recently discovered cytokines family with unequivocal informative value on repairing injury and neoplastic processes in mammals. Here we report on midkine (mdk) and atubulin (a-tub) gene expression patterns in P. caerulea exposed to cadmium. Limpets, collected on two occasions from a breakwater at a marina (Tyrrhenian Sea) were exposed to sublethal cadmium concentrations (0.5 and 1 mg l 1 Cd) over a 10-day exposure period. RNA was extracted from the viscera of unexposed and exposed specimens. Real time TaqMan RT-PCR was performed to measure the relative mdk and a-tub gene expression levels. A remarkable mdk over-expression was observed in all exposed animals with respect to unexposed ones; mdk over-expression was significantly higher in both treatments when compared with un-treatment (mean expression levels: 23- and 38-fold, for 0.5 and 1 mg l 1 Cd treatment, respectively; ANOVA, for both P < 0.01). The study also indicates that the mdk up-regulation was significantly Cd-concentration dependent (P < 0.05). A significant up-regulation of the constitutive a-tub gene was also observed in 1 mg l 1 Cd-treated animals (mean expression level: 4-fold; ANOVA, P < 0.05). In conclusion, these data provide the first evidence paving the way for the use of the midkine as a promising new biomarker of effect in the environment risk assessment policy.