Subcutaneous NaAs 3+ exposure increases metallothionein mRNA and protein expression in juvenile winter flounder

Abstract

We investigated the effect of NaAs 3+ exposure on liver metallothionein (MT) mRNA and protein expression in juvenile winter flounder ( Pleuronectes americanus). Sexually immature flounder (age 18 months) were subcutaneously implanted with silastic tubing containing 0 (vector control), 1.5, 3.0, 6.0 and 16.0 μmol g −1 fish wt. NaAs 3+. Liver MT mRNA and protein were significantly elevated within 24 h of exposure at the 1.5 μmol g −1 dose before there was significant liver As accumulation or mortality. All four NaAs 3+ treatments caused a significant rise in MT mRNA and protein and the mRNA demonstrated the clearest dose response and greatest fold change (19 fold at 16.0 μmol g −1 dose). MT protein reached its maximum (1.8 times the control) at the 1.5 μmol g −1 dose. This maximum was maintained across all four treatments and decreased slightly at the 16.0 μmol g −1 dose where there was 40% mortality. The level of liver As content among fish with maximal MT expression was similar to that of fish which recently succumbed to NaAs 3+ toxicity. This links saturation of the MT response with mortality and provides a framework for assessing the significance of MT measurements in feral flounder. The experiment was repeated (minus the 16.0 μmol g −1 dose) in a smaller (and younger) group of fish (12 months old) which expressed a two-fold lower liver Zn and MT protein content. This relatively low level of pre-existing MT theoretically widened the MT response window such that the MT maximum was not attained until the 6.0 μmol g −1 dose. These fish also did not show a significant rise in either MT mRNA or protein at the sublethal dose (1.5 μmol g -1). This lack of change appeared to reflect the higher level of baseline liver As which also did not increase at the 1.5 μmol g −1 dose. Reevaluation of the earlier experiment revealed a 1.5 fold increase in liver As content at the 1.5 μmol g −1 dose that was not significant due to high variance. This suggests that MT is a better biomarker of NaAs 3+ exposure because there is less variance associated with either MT mRNA or protein measurements. These results lend merit to MTOs usefulness as a biomarker of metal exposure and provides the foundation for linking NaAs 3+ exposure with MT expression and possibly metal-induced carcinogenesis in fish.