Detection of Physiological Activities of G Protein-Coupled Receptor-Acting Pharmaceuticals in Wastewater

Abstract

Although pharmaceuticals are generally found at very low levels in aquatic environments, concern about their potential risks to humans and aquatic species has been raised because they are designed to be biologically active. To resolve this concern, we must know whether the biological activity of pharmaceuticals can be detected in waters. Nearly half of all marketed pharmaceuticals act by binding to the G protein-coupled receptors (GPCRs). In this study, we measured the physiological activity of pharmaceuticals in wastewater. We applied the in vitro transforming growth factor-α (TGFα) shedding assay, which accurately and sensitively detect GPCR activation, to investigate the agonistic/antagonistic activities of wastewater extracts against receptors for angiotensin (AT1), dopamine (D2, D4), adrenergic family members (α1B, α2A, β1, β3), acetylcholine (M1, M3), cannabinoid (CB1), vasopressin (V1A, V2), histamine (H1, H2, H3), 5-hydroxytryptamine (5-HT1A, 5-HT2C), prostanoid (EP3), and leukotriene (BLT1). As a result, antagonistic activity against AT1, D2, α1B, β1, M1, M3, H1, and V2 receptors was detected at up to several μg/L for the first time. Agonistic activity against α2A receptor was also detected. The TGFα shedding assay is useful for measuring the physiological activity of GPCR-acting pharmaceuticals in the aquatic environment.

Quantification of Pharmaceutical Related Biological Activity in Effluents from Wastewater Treatment Plants in UK and Japan

“…We selected AT1, D2, β1, M1, and H1 receptors (Table 1), because strong antagonistic activities against these receptors were detected in effluent from WWTPs in Japan in our previous study. 20 We also selected a number of receptors in the same classes as these (D4, β3, M3, and H2), in order to compare the receptor specificity of the biological activity of the effluent extracts.…”

“…receptor in effluent extracts. 20 However, in this study, valsartan was used as a standard instead of OM. Because OM is a pro-drug for olmesartan, its active form, OM is not appropriate as a standard.…”

“…EC , IC 20 , agonist EQ, and antagonist EQ of wastewater extracts were calculated using the linear concentration-effect curves approach as previously described 14,[32][33][34] with slight modification (SI Methods S5 and Figure S2). Briefly, the linear part of the concentrationeffect curves was used to determine the EC 20…”

“…These results indicate that activities were attributable to highly selective GPCR-acting pharmaceuticals, but not to nonreceptor-mediated pathway, such as adsorption of the agonist by large organic molecules, as previously described. 20…”

“…d: Inhibition of AP-TGFα release was not observed at the test concentration.In vitro TGFα shedding assayThe principle of the TGFα shedding assay for agonistic activity is agonist-induced accumulation of alkaline phosphatase-tagged TGFα (AP-TGFα), a reporter enzyme, in the media harvested from cultured cells (i.e., conditioned medium (CM)). The TGFα shedding assay was conducted as previously described19,20 with slight modifications (SI Methods S3).Briefly, GPCR-expressing plasmid was transiently transfected into a cultured cell line (HEK 293 cells). By selecting the GPCR expression plasmid in cells, we can measure agonistic and antagonistic activities against each GPCR.…”