Contamination with retinoic acid receptor agonists in two rivers in the Kinki region of Japan

This study was conducted to investigate the agonistic activity against human retinoic acid receptor (RAR) alpha in the Lake Biwa Yodo River and the Ina River in the Kinki region of Japan. To accomplish this, a yeast two-hybrid assay was used to elucidate the spatial and temporal variations and potential sources of RAR alpha agonist contamination in the river basins. RAR alpha agonistic activity was commonly detected in the surface water samples collected along two rivers at different periods, with maximum all-trans retinoic acid (atRA) equivalents of 47.6 ng-atRA/L and 23.5 ng-atRA/L being observed in Lake Biwa Yodo River and Ina River, respectively. The results indicated that RAR alpha agonists are always present and widespread in the rivers. Comparative investigation of RAR alpha and estrogen receptor alpha agonistic activities at 20 stations along each river revealed that the spatial variation pattern of RAR alpha agonist contamination was entirely different from that of the estrogenic compound contamination. This suggests that the effluent from municipal wastewater treatment plants, a primary source of estrogenic compounds, seemed not to be the cause of RAR alpha agonist contamination in the rivers. Fractionation using high performance liquid chromatography (HPLC) directed by the bioassay found two bioactive fractions from river water samples, suggesting the presence of at least two RAR alpha agonists in the rivers. Although a trial conducted to identify RAR alpha agonists in the major bioactive fraction was not completed as part of this study, comparison of retention times in HPLC analysis and quantification with liquid chromatography mass spectrometry analysis revealed that the major causative contaminants responsible for the RAR alpha agonistic activity were not RAs (natural BAR ligands) and 4-oxo-RAs, while 4-oxo-RA5 were identified as the major BAR agonists in sewage in Beijing, China. These findings suggest that there are unknown RAR alpha agonists with high activity in the rivers. (C) 2010 Elsevier Ltd. All rights reserved.