Detection of Physiological Activities of G Protein-Coupled Receptor

Jan 4, 2015 - Although pharmaceuticals are generally found at very low levels in aquatic environments, concern about their potential risks to humans a...
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Detection of Physiological Activities of G Protein-Coupled ReceptorActing Pharmaceuticals in Wastewater Masaru Ihara,*,† Asuka Inoue,‡,§ Seiya Hanamoto,† Han Zhang,† Junken Aoki,‡,∥ and Hiroaki Tanaka† †

Research Center for Environmental Quality Management, Graduate School of Engineering, Kyoto University, Otsu, Shiga 520-0811, Japan ‡ Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan § PRESTO, Japan Science and Technology Agency (JST), Tokyo 102-0076, Japan ∥ CREST, Japan Science and Technology Agency (JST), Tokyo 102-0076, Japan S Supporting Information *

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 proteincoupled 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 (5HT1A, 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.

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INTRODUCTION Over recent years, growing numbers of human pharmaceuticals have been detected in effluents from wastewater treatment plants (WWTPs) and river water.1−6 Although these are generally found at very low levels (e.g., ng/L to μg/L) in these waters, concerns about their potential risks to humans and aquatic species have been raised because they are designed to be biologically active.7−11 To determine whether pharmaceuticals in aquatic environments pose risks to aquatic organisms, we must know the extent to which such organisms may be exposed to those pharmaceuticals, and whether such exposure is likely to result in physiological responses, as determined by the pharmaceuticals’ respective modes of action (MoAs). It is possible to measure the concentrations of selected pharmaceuticals by chemical analysis, but such concentrations do not indicate the physiological activity of the pharmaceuticals in waters. For example, even if the concentration of each substance is low, through additivity and/or synergistic compounds with similar MoAs might produce a strong enough physiological activity to harm aquatic organisms. The physiological activity of © XXXX American Chemical Society

pharmaceuticals which act in environmental waters on nuclear steroid hormone receptors such as 17α-ethinylestradiol, has been able to be measured by in vitro detection methods, based on their MoA (i.e., interaction with the hormone receptors).8 However, until now, to the best of our knowledge, the physiological activity, in such environments, of other classes of pharmaceuticals, with different MoAs, has hitherto not been measured; and one of the reasons is the lack of detection methods applicable to environmental waters. One potential means to identify MoA-specific biological responses initiated by pharmaceuticals in the environment would be to look at cell membrane receptors. Membrane receptors act like an inbox for messages in the form of peptides, lipids, and proteins. G protein-coupled receptors (GPCRs) are the largest group of these cell surface receptors in eukaryotes, and participate in various physiological and pathophysiological Received: November 2, 2014 Revised: December 28, 2014 Accepted: January 4, 2015

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DOI: 10.1021/es505349s Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology Table 1. GPCRs Used in This Study, And Their Agonists and Antagonistsa receptor class

name

agonist used [abbr.]

antagonist used [abbr.]

Angiotensin II

AT1

Angiotensin II [ANG II]

Olmesartan Medoxomil [OM]

Dopamine

D2 D4

Dopamine [DA]

Sulpiridea [SUL] Olanzapineb [OLA]

Adrenoceptors

α1B α2A β1 β3

Norepinephrine [NE]

Carvedilol [CAR] Mirtazapine [MIR] Metoprolol [MET] METc

Acetylcholine

M1 M3

Acetylcholine [ACh]

Pirenzepine [PIR] PIRc

Cannabinoid

CB1

CP-55940 [CP] (research agent)

AM251 (research agent)

Histamine

H1 H2 H3

Histamine [HIS]

Diphenhydramine [DIP] Famotidene [FAM] DIPc

Isoproterenol [ISO]

Vasopressin

V1A V2

Vasopressin [VAS]

Tolvaptane [TOL]c TOL

5-Hydroxytryptamine

5-HT1A 5-HT2C

Serotonin [5-HT]

NAD 299 (research agent) OLAb

Prostanoid

EP3

Prostaglandin E2 [PGE2]

―d

Leukotriene

BLT1

Leukotriene B4 [LTB4]

―d

a

Unless otherwise noted, all information about the antagonistic pharmaceuticals is drawn from Katzung et al. (2012).13 Agonists used in this study were endogenous unless otherwise noted. Antagonists were marketed pharmaceuticals unless otherwise noted. Receptors that are main targets for pharmaceuticals are bolded. a: Gingrich and Caron (1993).17 b: Bymaster et al. (1996).18 c: For these receptors, we utilized antagonists for other GPCRs in the same receptor class. d: For these GPCRs, antagonists were not commercially available.

adrenergic family members (α1B, α2A, β1, and β3), acetylcholine (M1 and M3), cannabinoid (CB1), vasopressin (V1A and V2), histamine (H1, H2, and H3), 5-hydroxytryptamine (5HT1A and 5-HT2C), prostanoid (EP3), and leukotriene (BLT1). In addition, we measured the activity of a known agonist and corresponding antagonist for each GPCR, and quantified the activity detected in the wastewater extracts as agonist or antagonist equivalent quantities (EQs). We simultaneously analyzed pirenzepine (an antagonist for M1 receptor) by chemical analysis. We compared the EQ for pirenzepine, as measured by the TGFα shedding assay, with its concentration, and investigated whether the measured antagonistic activity in the wastewater could be explained by the presence of this known pharmaceutical.

processes. It is estimated that nearly half of all marketed pharmaceuticals act by binding to GPCRs,12 for example, antihypertensives, antipsychotics, antidepressants, antiallergenics, and antiasthmatics.12,13 Once agonists bind to them, GPCRs change their conformation and transduce intracellular signaling, primarily through activation of the Gα subunit of heterotrimeric G proteins, which are classified into four subfamilies (Gs, Gi, Gq, and G12/13).14 Recently, Dr. Inoue and colleagues developed an in vitro method, a transforming growth factor-α (TGFα) shedding assay which accurately and sensitively detects activation of most GPCRs that couple with any of the four G proteins.15 In the assay, GPCR activation is measured as ectodomain shedding of a membrane-bound proform of alkaline phosphatase-tagged TGFα (AP-TGFα) mediated by a TNFα-converting enzyme (TACE), and its release into a conditioned medium. The TGFα shedding assay can very simply and rapidly detect the activation and inhibition of GPCRs that couple with any of the four Gα-induced signals in the same format, and could therefore allow us to detect the physiological activity of GPCR-acting pharmaceuticals in environmental waters. In this study, we applied the TGFα shedding assay to measure the physiological activity of pharmaceuticals in wastewaters. We investigated the extent to which GPCRs were activated or inhibited by wastewater extracts. To do this, we selected receptors associated with common pharmaceutical MoAs; for angiotensin II (AT1), dopamine (D2 and D4),

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MATERIALS AND METHODS

Sampling and Sample Treatment for the TGFα Shedding Assay. Water samples were collected from two wastewater treatment plants (WWTPs), located in different parts of Japan, both using the conventional activated sludge treatment process. Secondary treated effluent was collected at WWTP A in 2013, and final effluent after chlorination was collected at WWTP B in 2014. A total 9 L of each sample was collected in amber glass bottles, to which 1 g/L ascorbic acid was added as a preservative. After collection, the samples were transported to our laboratory, where they were filtered and B

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Figure 1. Principle of the TGFα shedding assay and data processing. (a) Schematic of agonistic activity assay: Upon stimulation by an agonist, an activated GPCR induces TACE-dependent ectodomain shedding and releases membrane-bound pro-AP-TGFα. (b) Schematic of antagonistic activity assay. (c) Protocol of the agonist (upper panels) and antagonist (bottom panels) activity tests. After transfection with plasmids, transfected cells were reseeded in a 96-well plate. (d) Representative data for agonist and antagonistic activity using AT1 (top and bottom panels, respectively). Relative percentage AP activity in conditioned medium in the vehicle-treated condition (leftmost bar in each bar graph) was set as the baseline (dashed lines), and the percentage of AP activity in conditioned medium was defined as percentage AP-TGFα release (y axis in the dose−response curve). ANG II, angiotensin II; OM, olmesartan medoxomil. Values represent the mean ± SEM (n = 6−9).

extracted within 6 h. The samples were stored at 4 °C before filtration. Samples for the TGFα shedding assay were extracted by solid-phase extraction (SPE) as described.16 In brief, 1 L of sample was passed through a glass fiber filter (pore size 1 μm; GF/B, Whatman, Maidstone, Kent, UK), and the filtrate was passed through an SPE cartridge (Oasis HLB, 200 mg/6 cc, 30 μm particle size, Waters Corp., Milford, MAA), with a total nine cartridges being used for each 9 L sample. The cartridge was dried for 2 h under gentle air pressure. Then 10 mL of methanol was used to elute the compounds trapped on the cartridge. The eluates from the nine cartridges were combined together, and evaporated to dryness under a gentle nitrogen stream at 37 °C. The residue was immediately dissolved in 3.6 and 4.5 mL of Milli-Q water (Millipore Corp., Billerica, MA) containing 1% dimethyl sulfoxide, for the secondary effluent from WWTP A (2500 times concentrated) and the final effluent from WWTP B (2000 times concentrated), respectively. These wastewater extracts were serially diluted, from 10 to 10 000 times dilution, with Hank’s Balanced Salt Solution containing 5 mM HEPES (pH 7.4), and then applied to the TGFα shedding assay. The concentrations of wastewater extracts during cell exposure were defined in terms of the relative enrichment factor (REF: the ratio of the enrichment factor (from the SPE step) to the dilution factor of the wastewater extracts in the TGFα shedding assay). For example, in the case of the secondary effluent from WWTP A, the REF

ranged from 0.25 to 250 times concentration. All samples were stored at −30 °C until the TGFα shedding assay. The Milli-Q water was also treated in parallel as a blank control, which was confirmed, by TGFα shedding assay, to have no agonistic or antagonistic activity. Chemical Analysis of Pirenzepine. The final effluent from WWTP B was collected for chemical analysis in parallel with the samples for the TGFα shedding assay, and extracted by the SPE process as described above. The concentration of pirenzepine was measured using ultraperformance liquid chromatography coupled with tandem mass spectrometry (UPLC/MS/MS) (Supporting Information (SI) Methods S1). Selection of GPCRs. We selected 19 GPCRs: AT1, D2, D4, α1B, α2A, β1, β3, M1, M3, CB1, H1, H2, H3, V1A, V2, 5HT1A, 5-HT2C, EP3, and BLT1 (Table 1). Notably, AT1, D2, α1B, α2A, β1, M1, H1, H2, V2, 5-HT2C, and EP3 are the main targets of marketed pharmaceuticals.13 We also selected a number of receptors in the same classes as these (D4, β3, M3, H3, V1A, and 5-HT1A), in order to compare the receptor specificity of the physiological activity of the wastewater extracts. For each GPCR, known agonists and corresponding antagonists were used as positive controls for the activity tests, and as reference compounds for activity quantification (Table 1 and SI Methods S2). Details of the selection of GPCRs, agonists, and antagonists used in this study are described in SI Methods S3. C

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Environmental Science & Technology Plasmids. All the GPCRs and Gα proteins (chimeric Gα proteins or Gα16) used in this study were of human origin, and were constructed as previously described.15 Gα proteins were used to enhance the AP-TGFα release response in some GPCRs. By additional expression of the chimeric Gα proteins or the promiscuous Gα16 protein, GPCRs that were otherwise insufficient to induce the AP-TGFα shedding responses became detectable.15 Details of the plasmids and Gα proteins are described in SI Methods S4. The combinations of GPCRs and Gα proteins, and the Gα signaling pathways which could be activated or inhibited by wastewater extracts, are shown in SI Table S1. The AP-TGFα expression vector19 was kindly provided by Dr. Higashiyama of Ehime University. TGFα Shedding Assay. The chemicals used in the TGFα shedding assay are described in Table 1 and SI Methods S2. The principle of the TGFα shedding assay for agonistic activity is agonist-induced ectodomain shedding of membrane-bound pro-AP-TGFα, a reporter enzyme (Figure 1a). The released AP-TGFα can be quantified by measuring alkaline phosphatase (AP) activity in a conditioned medium (CM) from the optical density of yellow-colored para-nitrophenyl (p-NP). The principle of the TGFα shedding assay for antagonistic activity is the inhibition of agonist-induced AP-TGFα release by antagonists (Figure 1b). If AP-TGFα is not released into the CM, but remains on the cell surface, the cell surface instead changes to yellow. The procedure of the TGFα shedding assay for agonistic activity is described in detail in SI Methods S5. The overview of the procedure is shown in Figure 1c (upper). In brief, after transfection with plasmids, transfected cells were reseeded in a 96-well plate, and then exposed to a test agonist or wastewater extract. The CM was separated, and then para-nitrophenylphosphate (p-NPP, from Sigma-Aldrich Co., St. Louis, MO), a substrate for alkaline phosphatase, was added to both the CM and the remaining cells. Before (background) and after incubation, the intensity at 405 nm (OD405) was measured. The CM becomes yellow (p-NP) by the activity of the released AP-TGFα. Representative data for color change of CM and remaining cells by stimulation with an agonist are shown in Figure 1d (upper). In the TGFα shedding assay for the AT1 receptor, the CM color change increases with the concentration of angiotensin II (ANG II), while the yellow of the cells decreases in inverse proportion. If agonistic pharmaceuticals are present in the wastewater extract, the released AP-TGFα accumulates in the CM, which becomes yellow. AP-TGFα release was calculated (Figure 1d; SI Methods S6), and dose− response data were analyzed using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA). All assays were performed in triplicate for all GPCRs. When the AP-TGFα release from a given wastewater extract reached >25% of the maximum AP-TGFα release induced by the corresponding agonist (e.g., ANG II for AT1 receptor), it was defined as “detected”. Only when the AP-TGFα release reached >50% was the agonistic activity of the wastewater extract quantified as the agonist equivalent quantity (AEQ) value (see below). The procedure of the TGFα shedding assay for antagonistic activity was similar to that for agonistic activity, with slight modifications (Figure 1c, lower). Here, cells were pretreated with the test antagonist or wastewater extract 5 min before stimulation with a known agonist corresponding to the tested GPCR. If the agonist-induced AP-TGFα release is inhibited, AP-TGFα remains on the cell surface, and the cells become yellow. Representative data for color change of CM and cells

are shown in Figure 1d (lower). In the TGFα shedding assay for the AT1 receptor, the inhibition of the CM color change induced by ANG II depends on the concentration of olmesartan medoxomil, while the yellow of the cells increases in inverse proportion. If antagonistic pharmaceuticals are present in the wastewater extracts, agonist-induced ectodomain shedding should be inhibited. As a result, AP-TGFα remains on the cell surface, and the cells become yellow. The detailed procedure is described in SI Methods S5. AP-TGFα release was calculated (Figure 1d; SI Methods S6). From the reduction in agonist-induced AP-TGFα release, the antagonistic effects of the wastewater extracts were determined as an antagonist equivalent quantity (AntEQ). All assays were performed in triplicate for all GPCRs. When agonist-induced AP-TGFα release was inhibited by a given wastewater extract by >25%, it was defined as “detected”. Only when it was inhibited by >50% was AntEQ calculated (see below). For each assay, the activity of the known agonist corresponding to the GPCR (Table 1) and 12-O-tetradecanoylphorbol-13-acetate (TPA), a well-known inducer of TGFα shedding, was analyzed in parallel as a positive control. TPA activates protein kinase-C (PKC), which is involved in the Gαq signaling pathway.20 To detect the AP-TGFα release induced solely by the specific interaction between an agonist in wastewater extract and a GPCR, we investigated the dilution range in which AP-TGFα release was not observed without a GPCR (mock transfection conditions). In the same way, to detect a decrease in AP-TGFα release solely owing to the specific interaction between an antagonist in wastewater extract and a GPCR, we investigated the dilution range in which TPA-induced AP-TGFα release was not inhibited, under mock transfection conditions (SI Methods S7). The cytotoxicity of each extract was analyzed under the same conditions as for agonistic/antagonistic activity measurement (SI Methods S8), and we confirmed that the number of living cells was not statistically different (P < 0.05) from the vehicle exposure control in all dilutions of extracts. Thus, by selecting the GPCR expression plasmid in cells, we can measure agonistic and antagonistic activity against each GPCR. The TGFα shedding assay was performed with both secondary effluent from WWTP A and final effluent from WWTP B. Calculation of EC25, EC50, IC25, IC50, Agonist and Antagonist Equivalent Quantity, And Limit of Detection. AEQ and AntEQ were calculated based on the EC50 and IC50 values, respectively. In some receptors, antagonistic activities were clear, but did not reach 50% inhibiton (see Results and Discussions). To distingush this clear but slightly weak inhibition from no response, we arbitrarily set 25% inhibition as the cutoff for detection of antagonistic activity in the environment. In the same way, we set 25% activation as an arbitrary cutoff for detection of agonistic activity. For each agonist used, the half-maximum effective concentration (EC50(agonist)) was calculated from the sigmoid dose− response curves. For each wastewater extract, the REF that gave 50% of the maximum AP-TGFα release induced by the corresponding agonist (EC50(extract)) was calculated. The AEQ (ng-agonist/L) was determined as EC50(agonist)/EC50(extract). In the case of GPCRs for which agonistic activity was detected in the wastewater extracts, assays consisting of three technical replicates were performed at least twice, and a total of 6−9 data sets were obtained. EC50(extract)s were determined from each D

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Environmental Science & Technology Table 2. EC50 of Agonists and IC50 of Antagonists GPCR AT1 D2 D4 α1B α2A β1 β3 M1 M3 CB1 H1 H2 H3 V1A V2 5-HT1A 5-HT2C EP3 BLT1

agonist Angiotensin II Dopamine Dopamine Norepinephrine Norepinephrine Isoproterenol Isoproterenol Acetylcholine Acetylcholine CP-55940 Histamine Histamine Histamine Vasopressin Vasopressin Serotonin Serotonin Prostaglandin E2 Leukotriene B4

EC50(agonist) (M)a 2.7 7.7 1.6 4.1 7.8 2.8 2.9 4.2 5.4 7.7 6.2 8.1 9.6 1.8 7.9 1.5 5.1 3.2 1.3

× × × × × × × × × × × × × × × × × × ×

−10

10 10−9 10−8 10−8 10−9 10−8 10−6 10−8 10−9 10−9 10−9 10−8 10−8 10−8 10−10 10−7 10−10 10−10 10−9

antagonist Olmesartan Medoxomil Sulpiride Olanzapine Carvedilol Mirtazapine Metoprolol Metoprolol Pirenzepine Pirenzepine AM251 Diphenhydramine Famotidine Diphenhydramine Tolvaptan Tolvaptan NAD299 Olanzapine ―c ―c

IC50(antagonist) (M)a 1.7 1.5 5.4 7.2 1.1 1.2 6.9 1.8 2.5 1.5 3.2 2.5 >1.0 6.1 8.4 4.5 2.2

× × × × × × × × × × × × × × × × ×

10−8 10−7 10−8 10−9 10−6 10−7 10−5 10−8 10−6 10−8 10−7 10−7 10−4b 10−8 10−9 10−9 10−7

a

Values represent the mean (n = 6−9). Variations in the mean are shown in SI Table S2. bInhibition of AP-TGFα release was detected (25% inhibition), but did not reach the quantification limit (50% inhibition) at the test concentration. cFor these GPCRs, antagonists were not commercially available.

variations in the IC50(antagonist)s shown in SI Table S2) and used to calculate the AntEQs of the wastewater extracts (Table 3, and SI Table S3). Some antagonists were applied to multiple receptors belonging to the same class. For example, metoprolol was applied to β1 and β3 receptors. Its IC50(antagonist) against the β3 receptor (6.9 × 10−5 M) was much higher than that against the β1 receptor (1.2 × 10−7 M) (Table 2). These results agree well with the fact that metoprolol is a selective antagonist against the β1 receptor. The IC50(antagonist) values also show that pirenzepine, diphenhydramine, and tolvaptan are highly selective for the M1, H1, and V2 receptors, respectively. These results agree well with known antagonist−receptor binding specificities,13 and show that the TGFα shedding assay could detect the specificity of receptor−antagonist binding affinities. Differences in the IC50(antagonist) values led to differences in the LODs of antagonistic activity of the wastewater extracts, among GPCRs of the same class (Table 3). The LOD of antagonistic activity against the β3 receptor, based on the metoprolol (MET)-equivalent quantity (7.3 × 104 ng-MET/L in the secondary effluent from WWTP A), was much higher than that against the β1 receptor (1.1 × 102 ngMET/L), which means that if a selective antagonist such as metoprolol is causative, antagonistic activity could be detected with β1 but not with β3. Thus, we could ascertain whether antagonistic activity in wastewater extracts is receptor specific by comparing the results for GPCRs of the same class. Agonistic and Antagonistic Activity in the Wastewater Extracts. First, we confirmed the dilution range of the wastewater extracts in which neither AP-TGFα release nor inhibition of TPA-induced AP-TGFα release was observed under mock transfection conditions (SI Figure S2). The secondary effluent from WWTP A showed no activity under these conditions at all dilutions, similarly to the Milli-Q water extract (top and middle panels), and cytotoxicity was not observed. On the basis of these results, we conducted the TGFα shedding assay on the secondary effluent from WWTP A

data set, and AEQs were calculated based on the respective EC50(extract)s. AEQs were presented as the mean ± SEM (n = 6− 9). For each GPCR, the limit of detection (LOD) for the agonistic activity test was determined as the EC25(agonist) divided by the highest sample extract REF (250 for secondary effluent from WWTP A, 63.2 for final effluent from WWTP B) tested in the TGFα shedding assay. The IC50 value of each wastewater extract (IC50(extract): the REF that gave a 50% reduction of agonist-induced AP-TGFα release) was determined from the concentration-dependent inhibition curve of each extract. The IC50 value of the corresponding antagonist (IC50(antagonist)) was determined similarly. The AntEQ (ng-antagonist/L) for each GPCR was determined as IC50(antagonist)/IC50(extract). In the case of GPCRs for which antagonistic activity was detected in wastewater extracts, assays were performed at least twice, and total 6−9 data sets were obtained. IC50(extract)s were determined for each data set, and AntEQs were calculated based on each IC50(extract)s. AntEQs were presented as the mean ± SEM (n = 6−9). For each GPCR, the LOD for the antagonistic activity assay was determined as IC25(antagonist) divided by the highest sample extract REF tested in the TGFα shedding assay.

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RESULTS AND DISCUSSION Activity of Known Agonists and Antagonists. The activity of all the tested agonists was detected using the TGFα shedding assay. The concentration−response curves are shown in SI Figure S1 (Agonist). From these curves, the EC50(agonist) values were calculated (Table 2, with variations in the EC50(agonist)s shown in SI Table S2) and used to calculate the AEQs of the wastewater extracts (Table 3, and SI Table S3). The test antagonists decreased the agonist-induced AP-TGFα release for all the GPCRs (SI Figure S1, Antagonist). From the concentration−inhibition curves, the IC50(antagonist) values for the respective antagonists were calculated (Table 2, with E

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Figure 2. Concentration−response curves and concentration−inhibition curves of the secondary effluent from WWTP A. AP-TGFα release responses of each GPCR in agonistic activity test (blue) and antagonistic activity test (red) of secondary effluent of WWTP A. The amount of agonist used in the antagonist test is shown for each GPCR. Bold dashed lines, either 50% of the maximum response (E) or 50% reduction of the agonist-induced AP-TGFα release (A, B, D, F, H, I, K, and O); thin dashed lines, either 25% of the maximum response (G and R) or 25% reduction of AP-TGFα release (I and O). Values represent the mean ± SEM (n = 3−9). For most data points, error bars are smaller than the symbols. Arrows with numbers in the graphs indicate the average EC50(extract) or IC50(extract) (n = 6−9). ANG II: angiotensin II; DA: dopamine; NE: norepinephrine; ISO: isoproterenol; ACh: acetylcholine; HIS: histamine; VAS; vasopressin; 5-HT: serotonin; PGE2: prostaglandin E2; LTB4: leukotriene B4.

specific. Neither cytotoxicity nor nonreceptor-mediated pathways were the reason for the decrease in endogenous agonistinduced AP-TGFα release, and therefore selective GPCR-acting pharmaceuticals were causative (see below). Antagonistic activity against the AT1, D2, M1, and H1 receptors was also detected in the final effluent from WWTP B (SI Figure S4: A, B, H, and K, respectively; red plots and fitted lines). The results for the final effluent from WWTP B also support receptor specificity in the antagonistic activity of the wastewater. In contrast to the results for agonistic activity, antagonistic activity was detected against several GPCRs. These results indicate that pharmaceuticals with antagonistic activity could pose a greater threat to aquatic organisms than those with agonistic activity; and based on the information on the DrugBank online database (SI Table S4),21,22 most of the currently marketed GPCR-acting pharmaceuticals are antagonists. Agonist and Antagonist Equivalent Quantities for the Wastewater Extracts. From the concentration−inhibition curves of antagonistic activity of the wastewater extracts, we calculated the AntEQ values for the secondary effluent from WWTP A (Table 3, AntEQ): >103 ng/L for the AT1, D2, β1, and H1 receptors, >102 ng/L for the M1 receptor, and >10 ng/ L for the α1B receptor. The AntEQ values for the final effluent from WWTP B were also calculated (SI Table S3). For example, PIR-EQ for M1 receptor for the final effluent from WWTP B was roughly determined as follows: 1.8 × 10−8 M (Table 2, IC50(antagonist) for M1 receptor and pirenzepine) divided by 44 (SI Figure S4, the average IC50(extract) for M1), and then multiplied by the molecular weight of PIR (446.85 g/ mol). The precise calculation steps are explained in SI Table S3.

at all dilutions (the maximum REF; 250). On the other hand, the final effluent from WWTP B showed a weak AP-TGFα release at the highest REF under mock transfection condition (bottom panel, arrow). Thus, we conducted the TGFα shedding assay on the final effluent from WWTP B below a REF value of 63.3. The concentration−response curves of agonistic activity, and the concentration−inhibition curves of antagonistic activity, for the secondary effluent from WWTP A were obtained from the results of the TGFα shedding assay (Figure 2). The Milli-Q water extract gave no response with all the tested GPCRs (SI Figure S3), which showed that all the agonistic and antagonistic activity was wastewater-specific. Agonistic activity was observed only with the α2A receptor (Figure 2, E, blue plots and fitted line). Because AP-TGFα release was not observed with the α1B receptor, which belongs to the same receptor class and shares the same endogenous agonist (norepinephrine), it seems that an α2A receptorspecific agonist was present in the secondary effluent from WWTP A. Agonistic activity was not observed in the final effluent from WWTP B (SI Figure S4). In the antagonistic activity test, >50% inhibition of endogenous agonist-induced AP-TGFα release was observed with AT1, D2, α1B, β1, M1, and H1 receptors (Figure 2, red plots and fitted lines). Interestingly, no antagonistic activity was observed against receptors in the same class, which shared the same endogenous agonists (D4, α2A, β3, H2, and H3), or inhibition was weak (M3 receptor) (C, E, G, L, M, and I, respectively). Inhibition of the V2 receptor was also detected, but inhibition of the V1A receptor was not (O and N, respectively). These results show that antagonistic activity against AT1, D2, α1B, β1, M1, H1, and V2 was receptor F

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Environmental Science & Technology

These results clearly show that the antagonistic activity of the wastewater extracts against these receptors was receptor specific, and that promiscuous inhibition of the Gαq signaling pathway was not a factor. Similarly, the antagonistic activity against the AT1 and M1 receptors was not an outcome of promiscuous inhibition of the Gα12/13 signaling pathway, because the CB1, V1A, 5-HT2C, and EP3 receptors were not inhibited. Taken together, the study’s results show that the antagonistic activity of the wastewater extracts is attributable to highly selective GPCR-acting pharmaceuticals. Comparison of the TGFα Shedding Assay EQ and Chemical Analysis Concentration. The pirenzepine EQ for the final effluent from WWTP B was 2.1 × 102 ng-PIR/L (described above, SI Table S3), 10 times the concentration by chemical analysis (21 ng/L). This result indicates that pharmaceuticals with similar MoAs in wastewater might have additive and/or synergistic effects, and emphasizes the necessity for an in vitro assay, such as the TGFα shedding assay, to detect the gross physiological activity of pharmaceuticals present in waters. Prioritization of Pharmaceuticals in Research Involving Environmental Monitoring and Toxicity Testing. In this study, antagonistic activity against the AT1, D2, α1B, β1, M1, M3, H1, and V2 receptors was detected for the first time, at levels up to several μg/L in the secondary effluent from WWTP A (Table 3). Antagonistic activity against the AT1, D2, β1, and M1 receptors was also detected in another secondary effluent sample from WWTP A, taken on a different day (data not shown). Agonistic activity against the α2A receptor was also detected. β-receptor antagonists have been ubiquitously detected in wastewaters and surface waters.5,23 Recently, chemical analyses of wastewaters or surface waters have detected pharmaceuticals antagonistic to histamine receptors,2,24−26 dopamine receptors,6,27−29 muscarinic acetylcholine receptor,6,27 and angiotensin II receptor.30−32 More attention should be paid to these classes of pharmaceuticals in future environmental monitoring and toxicity testing (SI Discussion S1). In addition, to investigate the adverse effects of pharmaceuticals on aquatic organisms, investigation of the physiological activity of pharmaceuticals in receiving waters, using the TGFα shedding assay, is needed in future studies (SI Discussion S2). This study has demonstrated the usefulness of the TGFα shedding assay for measuring the physiological activity of GPCR-acting pharmaceuticals in the aquatic environment. However, the TGFα shedding assay does not detect the physiological activity of commonly used pharmaceuticals which affect GPCRs indirectly (e.g., acetylcholinesterase inhibitors, nonsteroidal anti-inflammatory drugs, selective serotonin reuptake inhibitors, and serotonin and norepinephrine reuptake inhibitors). To assess the physiological activity of these pharmaceuticals in waters, other in vitro assays, capable of detecting this activity, must be employed in testing environmental waters.

Table 3. Summary of Agonistic and Antagonistic Activity of the Secondary Effluent from WWTP A

Values represent the mean ± SEM (n = 6−9). Receptors that are main targets for pharmaceuticals are bolded. Red arrows: antagonistic activity; blue arrow: agonistic activity. ―: Unless otherwise noted, below the limit of detection. aOver the limit of detection, but below the limit of quantification. bFor these GPCRs, antagonists were not commercially available, and therefore LODs could not be calculated. NA: Not analyzed. For these GPCRs, agonistic activity was not analyzed because of insufficient sample volume.

Receptor Specificity of the Agonistic Activity and Antagonistic Activity Detected in This Study. We excluded the possibility that the decrease in the agonistinduced AP-TGFα release was due to cytotoxicity or nonreceptor-mediated pathways. If cytotoxicity were the cause, the same absolute reduction should have been observed in all the tested GPCRs, but it was not. In fact, cytotoxicity was not observed (SI Figure S2). We also confirmed that the antagonistic activity against the AT1 receptor was mitigated by higher doses of ANG II (10 nM) (data not shown), which further indicates that the decrease in AP-TGFα release was not due to cytotoxicity. In addition, if a nonreceptor-mediated pathway such as adsorption of the agonist by large dissolved organic matter in the wastewater extracts was responsible, the same absolute reduction should have been observed in GPCRs of the same class, as these receptors share the same agonist (e.g., dopamine for D2 and D4, isoproterenol for β1 and β3), but it was not. Nor was promiscuous inhibition of the Gα signaling pathway responsible for the decrease. With the exception of the H2 and EP3 receptors, all GPCRs used in this study interact with Gαq signaling pathways (SI Table S1). Only the AT1, D2, α1B, β1, M1, M3, H1, and V2 receptors were inhibited by the wastewater extracts (Figure 2, and Table 3).

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ASSOCIATED CONTENT

S Supporting Information *

Gα signaling pathways which could be activated or inhibited by wastewater extracts, variations in EC50 and IC50, summary of the agonistic and antagonistic activity of the final effluent from WWTP B, types of GPCR-acting pharmaceuticals, the results of mock transfection conditions experiments, dose−response curves of known agonists antagonistic pharmaceuticals, dose− G

DOI: 10.1021/es505349s Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology response curves of the Milli-Q water and the final effluent of WWTP B, methods for other experiments, and, discussion of potential adverse effects of pharmaceuticals, and of the environmental relevance of the study’s findings. This material is available free of charge at http://pubs.acs.org/. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-77-527-6220; fax: +81-77-527-9869; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Iguchi for his useful comments on this paper. We thank Ms. K. Nakano and Ms M.O. Ihara for technical assistance. This work was supported by grants from the Lake Biwa−Yodo River Water Quality Preservation Organization; the Ministry of Education, Culture, Sport, Science and Technology of Japan; and UK−Japan cooperative project of the Ministry of the Environment, Japan; PRESTO (to I.A.) and CREST (to J.A.), Japan Technology and Science Society, Japan.

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DOI: 10.1021/es505349s Environ. Sci. Technol. XXXX, XXX, XXX−XXX