Effects of copper on a reconstructed freshwater rainbow trout gill epithelium: Paracellular and intracellular aspects

Abstract

The barrier properties and intracellular responses of a primary cultured trout gill epithelium (containing both mitochondria-rich and pavement cells) were examined over 24 h of copper (Cu) exposure (0, 200 and 1000 μg/L) in apical fresh water. Transepithelial resistance (TER) and mRNA abundance of tight junction proteins zonula occludens-1, occludin, cingulin, claudin-8d and -28b were examined as endpoints of barrier function and the paracellular pathway. Intracellular endpoints analyzed were Cu accumulation, Na⁺ content, carbonic anhydrase activity and mRNA abundance of carbonic anhydrase (ca-II) and Na⁺/K⁺ ATPase (nka α1a and nka α1b isoforms). After a brief initial drop in TER in the 1000 μg Cu/L treatment, Cu at both levels increased TER over the first 6 h of exposure but there were no differences among groups from 12 h onwards. After 24 h of Cu exposure, there were no differences in mRNA abundance of any of the tight junction proteins examined. Cu accumulation occurred at 1000 μg Cu/L (5.5-fold increase), but no depletion of Na⁺ content. Carbonic anhydrase activity decreased significantly (by 76%), however Cu exposure did not alter the transcript abundance of ca-II, nka α1a, or nka α1b. This study provides a first report of carbonic anhydrase sensitivity to Cu exposure in a cultured model gill epithelium. We conclude that Cu impacts the permeability of this model during the early stages of exposure and that the use of carbonic anhydrase inhibition as an endpoint of metal toxicity in this model preparation may be useful for future mechanistic investigations and environmental monitoring.

Keywords: Carbonic anhydrase; Na+/K+ ATPase; Tight junction proteins.