Development of biomarkers of stress related to endocrine disruption in gastropods: Alkali-labile phosphates, protein-bound lipids and vitellogenin-like proteins

The occurrence in aquatic environments of compounds capable of acting as endocrine disruptors (ED) is of concern for wildlife. Elevated levels of the egg-yolk precursor protein vitellogenin (Vg) are widely accepted as a biomarker for xenoestrogen exposure in aquatic organisms. The purpose of this study was to develop a method for tracking changes in levels of Vg-like proteins in gastropods. Vg-like and egg-yolk proteins were analyzed in three freshwater gastropods having different modes of reproduction: Potamopyrgus antipodarum (asexual reproduction), Valvata piscinalis (hermaphroditism) and Lithoglyphus naticoides (sexual reproduction). Vitellogenin-like protein levels were examined by the alkali-labile phosphate (ALP) technique, a novel protein-bound lipid (PBL) assay, and by gradient gel electrophoresis with silver staining. The first phase of the study was dedicated to the development and optimization of an analytical method for detecting Vg-like proteins in these three gastropod species. In the second phase, the snails P. antipodarum and V. piscinalis were exposed to bisphenol A (BPA), octylphenol (OP) and tributyltin (TBT) for 14 and 28 days. Vg-like proteins were resolved in one major band at 250300 kDa for L. naticoides and V. piscinalis and in two bands at 100 and 30 kDa for P. antipodarum. After 14 days of exposure, all techniques showed an increase in Vg-like protein levels at 100 μg/L BPA and at 1 μg/L OP in P. antipodarum. A decrease in these proteins was observed with high concentrations of OP (100 μg/L) and TBT (≥5 ng/L). In V. piscinalis, a decrease in Vg-like proteins was shown after 14 days of exposure for OP ≥10 μg/L and TBT ≥5 ng/L; however, at 28 days, gel electrophoresis revealed an increase in these proteins. Histological observations showed significant necrosis in ovotestes of V. piscinalis with the three endocrine-disrupting compounds, while tissue modifications were not detected for P. antipodarum. A method for measuring Vg-like and egg-yolk proteins is proposed to track changes in both estrogenic and androgenic endocrine disruptors in gastropods. The potential use of these species as bioindicators of endocrine disruption in freshwater environments in terms of their reproduction mode is discussed.