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

Abstract

While pharmaceuticals are now routinely detected in aquatic environments, we know little of the biological activity their presence might provoke. It is estimated that nearly 40% of all marketed pharmaceuticals are G protein-coupled receptors (GPCRs) acting pharmaceuticals. Here, we applied an in-vitro assay, called the TGFα shedding assay, to measure the biological activities of GPCRs-acting pharmaceuticals present in effluents from municipal wastewater treatment plants in the United Kingdom (UK) and Japan from 2014 to 2016. The results indicated that compounds were present in the wastewater with antagonistic activities against angiotensin (AT1), dopamine (D2), adrenergic (β1), acetylcholine (M1), and histamine (H1) receptors in both countries. The most consistent and powerful antagonistic activity was against the H1, D2, and AT1 receptors at up to microgram-antagonist-equivalent quantity/L. Chemical analysis of the same UK samples was also conducted in parallel. Comparing the results of the bioassay with the chemical analysis indicated (1) the existence of other D2 or M1 receptor antagonists besides sulpiride (D2 antagonist) or pirenzepine (M1 antagonist) in wastewater and (2) that there might be a mixture effect between agonist and antagonistic activities against β1 receptor. GPCR-acting pharmaceuticals should be paid more attention in the environmental monitoring and toxicity testing in future studies.

Wastewater‐derived antagonistic activities of G protein‐coupled receptor‐acting pharmaceuticals in river water

“…We selected AT1, D2, β1, M1 and H1 receptors (Table ), because strong antagonistic activities against these receptors were detected in effluent from WWTPs in Japan in our previous study (Ihara et al, ; Zhang et al, ). For each GPCR, known corresponding antagonists were used as positive controls in the bioassays and as reference compounds for quantification of activity (Table ).…”

“…For each GPCR, known corresponding antagonists were used as positive controls in the bioassays and as reference compounds for quantification of activity (Table ). The activity of all tested antagonists for each receptor had already been quantified by the TGFα shedding assay in our previous study (Zhang et al, ).…”

“…Concentration that gave a 20% reduction of agonist‐induced alkaline phosphatase‐tagged transforming growth factor α release in the corresponding concentration‐dependent inhibition curve. Data cited from our previous study (Zhang et al, ).…”

“…The principle of the TGFα shedding assay of antagonistic activity is agonist‐induced accumulation of alkaline phosphatase‐tagged TGFα (AP‐TGFα), a reporter enzyme, in the medium collected from cultured cells (i.e., conditioned medium). The TGFα shedding assay was conducted as previously described (Ihara et al, , Zhang et al, ). In brief, a GPCR‐expressing plasmid is transiently transfected into a cultured cell line (HEK 293 cells).…”

“…In 2012, the in vitro transforming growth factor‐α (TGFα) shedding assay, which is a high‐throughput and sensitive assay designed to detect both agonism and antagonism of GPCRs, was developed (Inoue et al, ). We have demonstrated that the TGFα shedding assay is useful for detecting biological activity of GPCR‐acting pharmaceuticals in wastewater: assays of effluents of municipal WWTPs in Japan and the UK extracted by solid‐phase extraction (SPE) detected antagonistic activities of several classes of GPCR‐acting pharmaceuticals (Ihara et al, ; Zhang et al, ). In particular, activities against receptors of angiotensin (AT1), dopamine (D2), adrenergic family members (β1), muscarinic acetylcholine (M1) and histamine (H1) were detected at up to several μg/L of antagonist equivalent quantity (EQ).…”