The Influence of Lead on Different Proteins in Gill Cells From the Freshwater Bivalve, Corbicula fluminea, From Defense to Repair Biomarkers

Abstract

The objective of this study was to evaluate the influence of lead (Pb) on regulatory proteins linked to mechanisms of animal adaptation to polluted environments (using in vivo and in vitro tests) and to validate the in vitro assay as a tool for environmental assessment. Specimens of the bivalve Corbicula fluminea were exposed to nominal concentrations of Pb 5 mg l(-1) for 96 h. Isolated gill cells were exposed to three concentrations (1, 10, and 100 μM) for 5 h. Metal toxicity was evaluated by cell viability (trypan blue exclusion). We also analyzed Na+/K+ adenosine triphosphatase (ATPase) and carbonic anhydrase activity. Additionally, the multixenobiotic-resistance (MXR) phenotype was evaluated by the accumulation of rhodamine B (RB). Immunolabeling was used to quantify the expression of P-glycoproteins (C219) and proteins involved in ion transport, water movement, and cellular repair using antibodies against Na+/K+ ATPase, aquaporin 1, and heat-shock protein 70 (Hsp70). Pb was shown to be toxic in both in vivo and in vitro tests, in which cellular viability significantly decreased by approximately 25%. Cellular viability in the in vivo assays was determined by gill cell isolation after the entire animal was exposed to Pb. We observed that Na+/K+ ATPase activity was inhibited by 70%. Also, the expression of the MXR phenotype significantly increased in our in vivo tests. A statistically significant difference was observed in the expression of all proteins in the in vitro assays, whereas only Hsp70 increased in vivo. Employing these analyses, we could validate the sensitivity of the in vitro tests and can propose our in vitro model as a possible tool for environmental assessment.