A total of 16 water samples from four rivers in Japan were examined for their agonistic activities against five human nuclear receptors (estrogen receptor [ER] alpha, thyroid hormone receptor alpha, retinoic acid receptor [RAR] alpha, retinoid X receptor alpha, and vitamin D receptor) by using a yeast two-hybrid assay. The results suggest that the river environment is contaminated with endocrine disrupting chemicals (EDCs) that can interact with a variety of nuclear receptors and that contamination with those that have RAR agonistic activity may be more serious than contamination with well-known EDCs that act as ER agonists.

RARa agonistic activityRiver Surface water Yeast two-hybrid assay a b s t r a c t This study was conducted to investigate the agonistic activity against human retinoic acid receptor (RAR) a 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 RARa agonist contamination in the river basins. RARa 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 RARa agonists are always present and widespread in the rivers. Comparative investigation of RARa and estrogen receptor a agonistic activities at 20 stations along each river revealed that the spatial variation pattern of RARa 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 RARa 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 RARa agonists in the rivers. Although a trial conducted to identify RARa 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 RARa agonistic activity were not RAs (natural RAR ligands) and 4-oxoRAs, while 4-oxo-RAs were identified as the major RAR agonists in sewage in Beijing, China.These findings suggest that there are unknown RARa agonists with high activity in the rivers. A v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / w a t r e s w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 2 4 0 9 -2 4 1 80043-1354/$ -see front matter ª

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