New rapid cell-based assays for beta blocker and nonsteroidal anti-inflammatory drug determination in wastewater effluents
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1 New rapid cell-based assays for beta blocker and nonsteroidal anti-inflammatory drug determination in wastewater effluents
2 Biosensor cell lines analyte fluorescent protein Sensor Reporter signal Time range: seconds
3 Pharmaceuticals commonly found in the environment Beta blocker ß-adrenergic receptor NSAIDs Cyclooxygenase Antihypertensives Metoprolol Propranolol Bisoprolol Atenolol Sotalol Enalapril Losartan Furosemide Analgesics Diclofenac Ibuprofen Naproxen Indometacin Ketoprofen Identification of the resulting biological effect of unknown mixtures
![2) Subject: [Danio rerio] 597 aa (AAK33030.](/docs-images/85/91530199/images/4-2.jpg "1) Diclofenac Target: Cyclooxigenase (Danio rerio) Effects caused")
4 Target protein similarity Diclofenac LOEC µg/l prostaglandin G/H synthase 1 isoform 1 Query: [Homo sapiens] 599 aa (NP_ ) Subject: [Danio rerio] 597 aa (AAK ) Diclofenac Target: Cyclooxigenase (Danio rerio) Effects caused by compounds with same MOA
5 MOA (mode-of-action) based cellular assays Reporter cell lines (reporter gene induction) Response: hours Biosensor cell lines (immediate fluorescence signal) Response: seconds
6 Beta blocker biosensor cell line Sensor Reporter camp ratio (470/535) Measurement: Em470/Em535 Excitation: 420 nm
7 Beta blocker in vitro assay Response Concentration-response curve Metoprolol IC 50 : 32 nm Metoprolol GC-MS/MS Metoprolol equivalent quantity (MetEQ) in vitro Assay 1.2 µg/l 4.2 µg/l
8 Beta blocker in vitro assay Beta blocker IC 50 [nm] Factor MetEQ Metoprolol Propranolol Sotalol Bisoprolol 8.8+2, Atenolol Metoprololsäure 426x
9 Wastewater treatment plant effluents Beta blocker in vitro Assay WWTP effluent (SPE purified) Metoprolol LC-MS/MS MetoEQ In vitro Assay February March WWTP effluent Beta blocker (µg/l) LC-MS/MS MetoEQ total MetoEQ February 2016 Bisoprolol (0.28) Metoprolol (1.2) 1.2 Propranolol (<BG) 0 Atenolol (<BG) 0 March 2016 Bisoprolol (0.4) Metoprolol (1.2) 1.2 Propranolol (0.05) 0.23 Atenolol (0.07) 0.04
10 Wastewater treatment plant effluents Beta blocker in vitro Assay WWTP effluent Metoprolol LC-MS/MS total MetoEQ LC-MS/MS MetEQ in vitro Assay February March MOA in vitro assay measures 1.6 fold beta blocker activity compared to the chemical analysis of beta blockers Additional beta blocker compounds not analysed by chemical analysis? Synergistic effects?
11 NSAID biosensor cell line Sensor Reporter Cox-1 ratio (395/485) Measurement: Em528 (Excitation 395nm) Em528 (Excitation 485nm)
12 NSAID in vitro assay Response Concentration-response curve Diclofenac IC 50 : 6.3 nm Diclofenac GC-MS/MS Diclofenac Äquivalent in vitro Assay 2.2 µg/l 3.5 µg/l
13 Wastewater treatment plant effluents NSAID in vitro Assay WWTP effluent Diclofenac LC-MS/MS DicloEQ in vitro Assay March MOA in vitro assay measures 1.6 fold NSAID activity compared to the chemical analysis of diclofenac Additional NSAID compounds not analysed by chemical analysis? WWTP effluent NSAID LC-MS/MS March 2016 Diclofenac 2.2 Ibuprofen 1.1 Naproxen 1.4
14 Wastewater treatment plant effluents NSAID in vitro Assay NSAID IC 50 [nm] Factor DicloEQ Diclofenac Naproxen Ibuprofen WWTP effluent NSAID LC-MS/MS DicloEQ LC-MS/MS DicloEQ in vitro Assay March 2016 Diclofenac Naproxen n.a. Ibuprofen n.a. total NSAID MOA in vitro assay measures 1.4 NSAID activity compared to the chemical analysis of NSAIDs
15 MOA based in vitro assays using biosensor cell lines Appropriate throughput capacity to allow for routine application Specificity Sensitivity Robustness Reliability Matrix effects / proven suitability for environmental samples Application: Aim: Monitoring of WWTP effluent, surface waters Assessment of sewage treatment technologies Development of MOA based biosensor cell lines for the monitoring of additional pharmaceuticals
16 Kevin Bernhard Cordula Stahl Regina Martens SIZ Zellkulturtechnik, Mannheim Marco Scheurer DVGW Water Technology Center, Karlsruhe Rita Triebkorn Heinz Köhler University of Tübingen, Animal Physiological Ecology Ina Ebert Jean Bachmann German Environment Agency (UBA) - Dessau-Roßlau Work was funded by UBA (FKZ , , )
17 Many thanks for your interest and attention.
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