L13 Effect-directed Analysis.pdf

8-6-2021 HUMAN AND ENVIRONMENTAL TOXICOLOGY L13‐EFFECT DIRECTED ANALYSIS MARJA LAMOREE 1 Learning goals In this lecture you will learn about Effect-Directed Analysis (EDA). Afterwards you are able t...

8-6-2021 HUMAN AND ENVIRONMENTAL TOXICOLOGY L13‐EFFECT DIRECTED ANALYSIS MARJA LAMOREE 1 Learning goals In this lecture you will learn about Effect-Directed Analysis (EDA). Afterwards you are able to: Describe what EDA is and draw a schematic representation of the concept Indicate the conditions that would merit to carry out an EDA study Explain what happens during fractionation of an extract Explain the role of both high resolution mass spectrometry and toxicity assay in the identification and identity confirmation steps of EDA Describe how the effects of specific active chemicals can be masked and how to circumvent this Explain the importance of chemical, toxicological databases and mass spectral databases for the identification of compounds; you should be able to mention a few ‹#› Het begint met een idee Department Environment & Health 1  8-6-2021 Effect-Directed Analysis (EDA)  A combination of bioassay and fractionation methods and chemical analysis  A bioassay is directing the identification process Cause-effect relationships – relationship between bioassay response and toxicant  Analytical chemistry is used: To reduce the complexity of the sample To identify the responsible toxicants Concept of EDA List of toxicants List of suspects Extract Confirmation Biological analysis Fractionation Identification Chemical analysis Department Environment & Health 2  8-6-2021 Toxicity characterisation and EDA Toxicity characterisation Chemical Toxicity profiling identification In vivo screening In vitro screening -Priority chemicals -Bacteria Sample -Basin specific chemicals -Algae -Invertebrates Extraction -Fish No Clean -up Bioassays Yes Crude fractionation Bioassays Only hot fractions Fine fractionation Bioassays Effect-directed analysis Only hot fractions Identification Confirmation Identification key toxicant(s) Identification of active compounds: Effect-Directed Analysis (EDA) concept Bioassay Bioassay confirmation Bioassay Bioassay Y xY x x x Y Y Y x x x complex mixture of compounds 1st 2nd Fractionation Fractionation Chemical identification 3  8-6-2021 Effect directed analysis (EDA) bioassay extraction clean up test fractionation EXTRACT CLEANED TOXIC SAMPLE EXTRACT EXTRACT polar bioassay bioassay fractionation tests testen functional apolar groups TOXIC TOXIC FRACTION FRACTION FRACTIONS FRACTIONS 100 90 80 chemical Respons (% van maximum) 70 bioassay 60 analysis 50 test 40 30 20 IDENTIFICATION AND 10 0 CONFORMATION QUANTIFICATION 1.E-15 1.E-14 1.E-13 1.E-12 1.E-11 1.E-10 1.E-09 Concentratie (M) Case study 1: Toxicity profiling of Dutch sediments 4  8-6-2021 Toxicity profiling using a battery of in vitro bioassays l l l ti e le le l l ta ta le e ta ta ta bl ab ab ab to to to ca to to sta st st st Lo x S9 S9 X X to U U R ox X X u+ u- ro AL TT AL U U ot um ic um AL AL ic r -C -C M -C -C ER DR M ER DR Oosterschelde 1 1 1 1 1 1 1 1 1 1 Oosterschelde 2 16 3 7 1 1 1 1 1 1 Zierikzee buiten 65 20 2 1 4 13 1100 2 1 Zierikzee binnen 1178 99 25 1 1 13 1 1 3 EDA Veerse Meer 88 12 36 1 2 204 2 6 5 Haringvliet 953 68 68 1 5 38 1 1 1 Bruinisse 69 72 25 1 3 9 1 1 5 Dintel Sluizen 485 58 18 1 4 21 1 1 1 Moerdijk 1144 84 13 1 2 61 1 1 3 Nieuwe Maas 118 11 3 1 1 2 1 1 1 Nieuwe Waterweg 92 2 2 1 1 2 1 3 1 Rotterdam IJssel Haven 381 102 15 1 2 24 1 1 1 Rotterdam 2e Petroleumhaven 919 70 26 2 3 12 14 1 1 Biesbosch 1 393 63 9 1 1 16 1 2 1 Biesbosch 2 422 46 8 1 1 12 1 4 1 Compound identification? Houtman et al. (2004) ET&C 23:32-40 Source identification??? EDA approach 5  8-6-2021 Fractionation Zierikzee inner harbour sediment extract Fractionation with RP-HPLC Estrogenic activity: ER-CALUX Dioxin-like activity: DR-CALUX Identification of chemicals in “hot” fractions Estrogenic activity Chemical target analysis (GC-MS) of natural hormones α-estradiol β-estradiol Estrone Dioxin-like toxicity  GC-MS screening Identification of compounds by mass spectrum  Many PAHs 12 6  8-6-2021 EDA in sediment from Zierikzee inner harbour E f f e c t g e s t u u r d e a n a l y s e : S t o f - e n b r o n i d e n t i f i c a t i e b ta le t b to to t t ie ta ta to to b ta to s s ta X X a s 9 t ox 9 c X X s U U S t ox -S o U U AL AL TR + ro L L L u u ro ic A A m -C -C m T ic -C -C M u u R R M R R D E E D O o s t e r s c h e l d e 1 1 1 1 1 1 1 1 1 1 O o s t e r s c h e l d e 2 1 6 3 7 1 1 1 1 1 1 Z i e r i k z e e b u i t e n 6 5 2 0 2 1 4 1 3 1 1 0 0 2 1 Z i e r i k z e e b i n n e n 1 1 7 8 9 9 2 5 1 1 1 3 1 1 3 V e e r s e M e e r 8 8 1 2 3 6 1 2 2 0 4 2 6 5 H a r i n g v l i e t 9 5 3 6 8 6 8 1 5 3 8 1 1 1 B r u i n i s s e 6 9 7 2 2 5 1 3 9 1 1 5 D i n t e l S l u i z e n 4 8 5 5 8 1 8 1 4 2 1 1 1 1 M o e r d i j k 1 1 4 4 8 4 1 3 1 2 6 1 1 1 3 N i e u w e M a a s 1 1 8 1 1 3 1 1 2 1 1 1 N i e u w e W a t e r w e g 9 2 2 2 1 1 2 1 3 1 R o t t e r d a m I J s s e l H a v e n 3 8 1 1 0 2 1 5 1 2 2 4 1 1 1 R o t t e r d a m 2 e P e t r o l e u m h a v e n 9 1 9 7 0 2 6 2 3 1 2 1 4 1 1 B i e s b o s c h 1 3 9 3 6 3 9 1 1 1 6 1 2 1 B i e s b o s c h 2 4 2 2 4 6 8 1 1 1 2 1 4 1 76% of estrogenic response by natural complex extraction hormones mixture bioassay analysis 38% of dioxin-like fractionation bioassay response by toxicant polycyclic aromatic hydrocarbons (PAHs) Houtman et al. (2006), Chemosphere 65:2244-2252 Case study 2 Toxicity profiling of European sediments 7  8-6-2021 European rivers  Elbe (Germany)  In vivo assays  Scheldt (Belgium) Snail  Llobregat (Spain) Daphnia Microtox  Water  Sediment  In vitro assays Dioxin-like toxicity Endocrine assays Genotoxicity Snail bioassay Eenhoorn 8  8-6-2021 Toxicity profiling of European sediments lu s in in s in AS ta plu o o xm m m R C S xp ag ag an uC t iY AB YE TT 98 98 Mi Mi AR DR AR An TA Um TA TA TA Par 100x DL Ll4 SRV SE Eenhoorn SHW STR Anti Hormonal Genotoxicity Ah- Thy- biot Disruption based roid Sediment ~40 gram Sieved (125 μm) and freeze dried Sediment EDA ASE GPC 0-16.5 min 16.5-24 min 24-29 min 29-36 min RP-LC Non- 1. 2. 3. 4. 5. polar NP-LC NP-LC NP-LC Bioassay: (anti-)androgenicity 1. 2. 3. 4. 5. 6. 7. 8. 1. 2. 3. 4. 5. 6. 7. 8. 1. 2. 3. 4. 5. 6. 7. 8. 18 9  8-6-2021 EDA scheme Sediment EDA, (anti)androgenicity Weiss et al. (2009) Anal Bioanal Chem 394:1385-1397 Bioassay results Active fractions: 3 distinct groups of compounds with increasing polarity Response in subfraction higher than in the original indicates masking effect Weiss et al. 2009, Anal Bioanal Chem 394:1385–1397 20 10  8-6-2021 LTQ-Orbitrap identification strategy Elemental 1. SIEVE composition Xcalibur LTQ- 2. Orbitrap 3. Excel 4. Chemical NIST formula 7. 5. 6. AR-Calux 8. Purchase tentatively Confirmation of EPI correct log Kow identified compounds suite 9. List of Confirmed compounds Weiss et al. 2011, Anal Bioanal Chem 400:3141–3149 LTQ-Orbitrap data evaluation 1/3 of tested compounds confirmed 14807 12849 Yes! 10000 1539 419 259 1000 74 21 100 10 8 10 1 SIEVEd Ratio (active/non-active) Peak Tentatively Tested Analytical Bioassay peaks 100 check identified Confirmed confirmed 11  8-6-2021 LTQ-Orbitrap results  Androgenic Tris(2-chloroisopropyl) O 7H-Benz[de]anthracen-7-one phosphate O (82-05-3) PAH found in urban airborne Anabolic steroid (13674-84-5) Flame ClP O O Cl particulate matter. retardant. O Androstenone (18339-16-7)  Anti-androgenic Tonalide (1506-02-1) Cl Traseolide (68140-48-7) Galoxolide (1222-05-5 ) A steroid found in both male and female sweat and urine. PAHs Tetralin derivate musk. Indane derivate musk. Isochroman derivate musk. O Pheromone male O pigs Fragrances (polycylic musks) O O Phosphorous flame retardants Nandrolone (434-22-0) Nonyl phenols An anabolic steroid, present naturally in the human body. Phthalates O O O P O Phosphoric acid, tris(2- O ethylhexyl) ester (78-42-2) Industrial high production volume chemical. lubricant O additives etc. Weiss et al. 2011, Analytical Bioanalytical Chemistry Collaborators on this EDA Jana Weiss1, Eszter Simons1, Timo Hamers1, Marja Lamoree1, Bert van Hattum1, Pim Leonards1, Gerard Stroomberg2, Ronald de Boer2, Joan Staeb2, Jos Hermens2, Sander van Vliet2, Joop Bakker2, Jan Balaam3, Sara Pacitto3, Paul Roberts3, Andy Smith3 and Angela Ward3, Sander van der Linden4, Eric de Deckere5, Chris van Liefferinge5, Vicky Leloup5 , Martine van den Heuvel- Greve6, Miren Lopez de Alda7, Rikke Brix7, Peter Korytar8, Christiaan Kwadijk8, Isabel Muñoz9, Anton Kocan10, Pavel Jurajda11, Zdenek Adamek11, Mirek Machala12, Georg Streck13 1VU University Amsterdam; 2Waterdienst, 3CEFAS, 4BDS, 5University Antwerpen, 6Deltares, 7CSIC, 8Imares, 9UdB, 10SZU, 11IVB, 12VRI, 13 UFZ 24 12  8-6-2021 Polar bear plasma EDA  1998 and 2008  n=31 Jenny Bytingsvik Photo: J. Bytingsvik Eszter Simon Target chemical analysis with GC- Bioscreening with T4*-TTR binding assay MS Known thyroid hormone disruptors Target analyzed chemicals could not fully explain the measured effects EDA to identify active compounds Sample selection ΣT4-Equivalents = [a]xREPa+[b]xREPb …+ [z]xREPz Hydroxylated penta-,hexa- and heptaCBs PFASs 40-50 % unexplained activity 13  8-6-2021 Identification – Library based LC-MS data Compiled SAMPLE mass lists  Occurrence (blood, LC-MS environment) data BLANKS  TTR-binding compounds  Persistent compounds  High production volume chemicals Chemical formula List of suspects & Compound name Isotope Cluster Analysis Data Analysis 4.0 Edit Chromatogram Traces Isotope Cluster Analysis plot Manual Integration Smart Formula Manually List of suspects 14  8-6-2021 Identification of thyroid hormone disrupters UNKNOWN Photo: J. Bytingsvik COMPOUNDS OH-PCBs Nonylphenol Mass library Isotope Cluster screening Analysis    Occurrence TH disruptors Persistent +  High production volume Simon et al., ES&T 47 (2013) 8902 High Throughput EDA Extract Spotter technology for fractionation (384-1536 well Fractionation plates, array slides) Bioassay Mass spec Identification Miniaturization and application of bioassays to expand the HT-EDA platform Fast (automated) workflows for identification of Chemicals of Emerging Concern 15  8-6-2021 FractioMate™ Collaboration of Jeroen Kool, Willem Jonker, Rob ten Broek and Electronic and Mechanical Instrumentation Engineering group Fractionation strategies 10% LC LC column column 90% ToF-MS FractioMate Replicates Dilutions Multiple endpoints 26 s fractions 1 64 fractions 1x ER 2 3x AR 3 10x anti-AR Higher fraction resolution: 9 s fractions helps to correlate bio-activity to 192 fractions chromatographic peak shape risk of sensitivity reduction Zwart et al., EST 2018 16  8-6-2021 Sample origin Samples showed agonistic and/or antagonistic activity on Androgen receptor Estrogen receptor Arylhydrocarbon receptor Waste water treatment plant Amersfoort Eijsden River Meuse Miniaturized cell based assays AR-EcoScreen H4IIE DR-Luc 150 96-well format 150 96-well format 384-well format 384-well format Response (%) Response (%) 100 100 50 50 0 0 -13 -12 -11 -10 -9 -8 -10 -9 -8 -7 -6 DHT log(M) TCDD log(M) VM7Luc4E2 150 96-well format 384-well format 96‐well format 384‐well format F‐test Response (%) 100 EC50 Slope EC50 Slope P‐value AR‐EcoScreen 150 pM -45.3 170 pM -46.18 0.7717 H4IIE DR‐Luc 12.3 nM -17.59 18.2 nM -18.24 0.3807 50 VM7Luc4E2 4.7 pM -27.51 3.5 pM -34.43 0.0746 0 -14 -13 -12 -11 -10 -9 Miniaturization obtained by simply E2 log(M) downscaling the assay volumes proportionally and selecting a well plate type for optimal detector signal Zwart et al., EST 2018 17  8-6-2021 Identification pipeline Exact mass Chemspider or MS data Structures ± 0.002 mDa PubChem database Isotopic distribution Molecular formula Retention time LogD Fragmentation pattern Compound spectrum Candidate structures PubChem BioAssays database Chemical confirmation Confirmed Potentially bioactive Biological confirmation hits candidates Zwart et al., ES&T 2018 MS and activity – river Meuse at Eijsden MS base peak chromatogram Class Speedisk, 64 fractions Type Drugs * Amitriptyline ß-estradiol Antidepressant Estrogen Carbamazepine Antidepressant Celecoxib NSAID Clopidogrel Antiplatelet drug Miconazole Anti-fungal drug Personal care products Silicone rubber, 64 fractions DEET Mosquito repellent DHHB UV filter * Galaxolide Fragrant Oxybenzone UV filter Pesticides Diazinon Insecticide Propiconazole Fungicide Fenpropidin Fungicide Silicone rubber, 192 fractions Additives Tributyl phosphate Plasticizer * Tris(2-butoxyethyl)phosphate Flame retardant Other 4-dimethylaminobenzophenone Intermediate Piperine Alkaloid Zwart et al., EST 2018 18  8-6-2021 LC×LC in EDA with an AChE assay Link of toxicity to chemical identity loop 1 waste 2DLC 1D interface 20% pump 1 auto‐ ESI‐TOF‐ column (HPLC) sampler MS 2D 80% column UV fraction collector pump 2 detector (UPLC) loop 2 evaporation AChE assay plate reader CentriVap active 4×96 well fractions plates Application to WWTP effluent sample 4x96 well plates LCxLC-ESI (+)-ToF MS of WWTP effluent extract AChE inhibition (%, n=3) of the 384 fractions of the WWTP effluent extract 2D retention time alignment for confirmation of compound identity Ouyang et al., Analytical Chemistry 67 (2016) 179-191 19  8-6-2021 Database for suspect screening Database source Description n° compounds CPCat Chemical and product 43,599 KEMI Swedish Chemical Agency 14,510 BUMA Building materials 457 Norman Emerging substances 966 Impurities/ by-products in 320 commercial chemicals P&B compounds “Howard&Muir papers” Pharmaceuticals in the 274 environment in house database Plastic additives, flame 110 retardants Indoor dust Compounds present in 433 indoor dust (literature) ECHA Compounds available from 18,370 ECHA website Total raw database 79,039 Total database after removal of duplicates and salts 34,395 Database for suspect screening Database source Description n° compounds CPCat Chemical and product 43,599 KEMI Swedish Chemical Agency 14,510 BUMA Building materials 457 Norman Emerging substances 966 Impurities/ by-products in 320 commercial chemicals P&B compounds “Howard&Muir papers” Pharmaceuticals in the 274 environment in house database Plastic additives, flame 110 retardants Internship Non-Target Compounds Screening present inat Aqualab Zuid Indoor dust indoor dust (literature) 433 7-10 months, starting in July, partly as a holiday job ECHA Compounds available from 18,370 ECHA website Total raw database 79,039 Total database after removal of duplicates and salts 34,395 20  8-6-2021 Outlook – results from projects RoutinEDA and HBM4EU Metabolic activation Expanding the scope: - more bioassays/endpoints - human samples Further miniaturization QTOF-MS for identification Summary of topics Concept of EDA Examples: sediment in NL, Europe, polar bear plasma, surface water and sewage treatment plant effluent Miniaturization of bioassays High throughput fractionation into multiwell plates High resolution mass spectrometry for identification [email protected] ‹#› Het begint met een idee Department Environment & Health 21