Challenges in Toxicity Profiling

Transcription

1 Challenges in Toxicity Profiling Timo Hamers Profiling individual compounds E.g. suspect in crime case Caucasian male years old About 1.90 meter tall Robust physique Short, black hair Decent appearance Blue/grey blocked shirt with short sleeves Khaki-colored trousers Brown belt with chrome buckle Grey leather sandals Dark rectangular sunglasses Description Combination of characteristics Identification of the bad guys 2 1

2 Toxicity profiling: multiple characteristics of single compounds Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7 Compound 8 Compound 9 Compound 10 Compound 11 Compound 12 Biochemical/ physiological effects Toxic effects (individual) Effects on population Effects on community/ ecosystem 3 Biomonitoring using specific bioassays MODE OF ACTION Compound 1 Compound Compound 3 5 Compound 4 Compound 5 Compound 6 Compound 7 Compound 8 Compound Compound 10 2 Compound 11 1 Compound

3 Toxicity profiling of brominated flame retardants (BFRs) 5 5 Twelve different endocrine disrupting mechanisms of action Dioxinlike Anti-dioxinlike Androgenic Anti-androgenic Progestagenic Anti-progestagenic Estrogenic Anti-estrogenic Estrogenic Thyroidlike Anti-thyroid like Anti-thyroid like through AhR activation through AhR inactivation through AR activation through AR inactivation through PR activation through PR inactivation through ER activation through ER inactivation through inhibited sulfonation of E2 through mimicking T3 activity through inhibiting T3 activity through competition with T4 for binding protein TTR 6 3

4 7 Toxicity profiling of brominated flame retardants (BFRs) Compound ERago ERanta TTR DRago DRanta ARanta PRanta E2SULT TSago TSanta ARago PRago BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE BDE TBBPA TBP OH-BDE HBCD TM HBCD a HBCD b HBCD g TBBPA-DBPE ass Criterium % < effect < 3 1.0µM < EC50 < 10µM 4 0.1µM < EC50 < 1.0µM µM < EC50 < 0.1µM 7 Toxicity profiling of brominated flame retardants (BFRs) Hierarchical clustering Hamers et al. (2006) Tox. Sci. 92:

5 Toxicity profiling of metabolites Phenobarbital CYP2B induced liver homogenate Test compound min incubation extraction NADPH toxicity profiling 9 Metabolite profiling Compound PARENTS ERago ERanta TTR DRago Dranta ARanta PRanta E2SULT Tscreenago Tscreenanta ARago PRago Metabol METABOLITES ERago ERanta TTR DRago Dranta ARanta PRanta E2SULT Tscreenago Tscreenanta ARago PRago BDE CT? BDE CT? 3? 4 CT CT CT? BDE CT CT CT CT 1 1 BDE CT? BDE CT? BDE CT? 1 4 CT CT CT CT 1 1 BDE CT CT 1 1 BDE BDE BDE BDE BDE BDE TBBPA TBP ? OH-BDE CT HBCD TM HBCD alpha HBCD beta HBCD gamma TBBPA-DBPE Hamers et al. (2008) Mol. Nutr. Food Res. 52:

6 Toxicity profiling of non-dioxinlike PCBs PCB-19 PCB-74 PCB-118 PCB-138 PCB-28 PCB-80 PCB-122 PCB-153 PCB-47 PCB-95 PCB-125 PCB-168 PCB-51 PCB-100 PCB-126 PCB-170 PCB-52 PCB-101 PCB-128 PCB-180 PCB-53 PCB-104 PCB-136 PCB Ten different mechanisms of action Androgenic Anti-androgenic Estrogenic Anti-estrogenic Estrogenic Anti-thyroid Anti-thyroid Anti-thyroid Anti-thyroid Tumor promoting through AR activation through AR inactivation through ER activation through ER inactivation through inhibited sulfonation of E2 through competition with T4 for binding protein TTR through UGT1A1 induction through UGT1A6 induction through DI-1 induction through GJIC inhibition 12 6

7 Toxicity profiling of PCBs Hierarchical clustering Hamers et al. (2011) Tox. Sci. 121: Contribution of PCB congeners to overall toxic potency in human blood samples Hamers et al. (2011) Tox. Sci. 121:

8 Contribution of PCB congeners to overall toxic potency in human blood samples Hamers et al. (2011) Tox. Sci. 121: Toxicity profiling: a screening to get GRIP on compounds Group compounds with similar toxicity profiles Rank compounds based on Number of red flags Mode of action Identify Most important modes of action Representative compounds for each class Prioritize (asses of) Compounds for further research (asses of) Compounds for quality standard setting 16 8

9 Profiling complex mixtures E.g. Chemistry & Biology Department at IVM High Quality Contaminant Analysis Toxicity profiling Development of biomarkers and bioassays Effect Directed Analysis (EDA) Toxicogenomics Teaching Academic environment Description Combination of characteristics Hard to see individuals contribution Total assessment of the mixture Useful for quality assessment 17 Toxicity profiling: multiple characteristics of a complex mixture Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7 Compound 8 Compound 9 Compound 10 Compound 11 Compound 12 Biochemical/ physiological effects Toxic effects (individual) Effects on population Effects on community/ ecosystem 18 9

10 Biomonitoring using specific bioassays MODE OF ACTION Mixture 1 Mixture 2 Mixture 3 Mixture 4 Mixture 5 Mixture 6 Mixture 7 Mixture 8 Mixture 9 Mixture 10 Mixture 11 Mixture Chemical analysis vs Toxicity test of a mixture + - Chemical analysis Identity is known Concentration is known Not all compounds are analyzed Compounds <DL are not recognized Activity of the mixture is unknown Growing list of compounds Toxicity test Activity of the mixture is known All compounds contribute Composition of the mixture is unknown Responsible compounds are unknown 20 10

11 Biomonitoring using specific in vitro bioassays Hazard profile Ecological risk Molecular Cellular Organ Individual Population Community MODE OF ACTION LEVEL A B C D E F G H I J K Specific Toxicity in vitro Profile bioassays EDA 21 From Toxicity Profile to Hazard Profile Hazard profile MODE OF ACTION LEVEL A B C D E F G H I J K Molecular Cellular Toxicity Profile 22 11

12 Toxicity profiles of harbor sediments Location DR-CALUX total (pg TEQ/g dw) MODE OF ACTION DR-CALUX stable (pg TEQ/g dw) ER-CALUX total (pg EEQ/g dw) ER-CALUX stable (pg EEQ/g dw) TTR stable (pmol T4-eq/g dw) Microtox total (1/EC20) (1/mg dw) Oosterschelde Oosterschelde Zierikzee buiten Zierikzee binnen Veerse Meer Haringvliet Bruinisse Dintel Sluizen Moerdijk Nieuwe Maas Nieuwe Waterweg Rotterdam IJssel Haven Rotterdam 2e Petroleumhaven Biesbosch Biesbosch Microtox stable (1/EC20) (1/mg dw) umu-s9 total (ng 4NQOEQ/g dw) umu+s9 total (ng 2AAEQ/g dw) Houtman et al. (2004) ET&C 23:32-40 dioxin-like endocrine disrupting decreased respiration genotoxic 23 Response ratios location:watersystem-specific reference MODE OF ACTION 24 12

13 Hierarchical clustering: reference based hazard profiles MODE OF ACTION Hamers et al. (2010) IEAM 6: From toxicity to hazard Comparison to a reference toxicity profile Detection limit Reference profile Appointed on beforehand Based on lowest toxicity profile Based on good ecological quality Ecologically relevant effect levels 26 13

14 From Toxicity Profile to Ecological Risk: The famous so-what? question Ecological risk LEVEL Molecular Cellular Organ Individual Population Community 27 Bioassay response threshold MODE OF ACTION LEVEL Molecular Cellular Organ Individual Population Community Ecological relevance Ecological relevance?? 28 14

15 Example: threshold for estrogenic potency in sediments Fish are sensitive species Most studies with ethynylestradiol (EE2) Three key studies identified Multiple generation study (zebrafish) Lifecycle study (medaka) Field study (fathead minnow) 29 Effect EE2 on fathead minnow population EE2 5 ng/l, during summer season Kidd et al. (2007) PNAS 104:

16 Example: threshold for estrogenic potency in sediments No effect concentration in water (0.35 ng EE2/l) Freundlich isotherms No effect concentration in sediment (665 pg EE2/g dw) Relative potency EE2:E2 No effect concentration in bioassay (733 pg E2/g dw) EE2 is worst case reference compound Persistent in the environment Hardly metabolized by fish Steep Freundlich isotherm 31 Example: threshold for estrogenic potency in sediments Oosterschelde 1 Oosterschelde 2 Zierikzee buiten Zierikzee binnen Veerse Meer Haringvliet Bruinisse Dintel Sluizen Moerdijk Nieuwe Maas Nieuwe Waterweg Rotterdam IJssel Haven Rotterdam 2e Petroleumhaven Biesbosch 1 Biesbosch pg EE2EQ/g dw FATHEAD MINNOW 32 16

17 Similarly: threshold for dioxin-like potency in sediments 33 From Toxicity profiles to hazard and risk profiles Hazard-profile Reference location Toxicity profile Equivalent concentrations Risk-profile Threshold value DR-CALUX stable Reference ER-CALUX total Reference >1000 Location DR-CALUX stable (pg TEQ/g dw) ER-CALUX total (pg EEQ/g dw) Oosterschelde Oosterschelde Zierikzee buiten Zierikzee binnen Veerse Meer Haringvliet Bruinisse Dintel Sluizen Moerdijk Nieuwe Maas Nieuwe Waterweg 3 10 Rotterdam IJssel Haven Rotterdam 2e Petroleumhaven Biesbosch Biesbosch DR-CALUX stable Common tern ER-CALUX total fathead minnow >

18 From Toxicity Profile to Compound Identification MODE OF ACTION LEVEL A B C D E F G H I J K Molecular Cellular EDA Toxicity Profile 35 Who is the bad guy? 36 18

19 EDA: Splitting the bad guys from the good guys 37 Effect directed analysis (EDA) with in vitro bioassays Locatie DR-CALUX total DR-CALUX stable ER-CALUX total ER-CALUX stable TTR stable Microtox total Microtox stable umu-s9 total umu+s9 total Oosterschelde Oosterschelde Zierikzee buiten Zierikzee binnen Veerse Meer Haringvliet Bruinisse Dintel Sluizen Moerdijk Nieuwe Maas Nieuwe Waterweg Rotterdam IJssel Haven Rotterdam 2e Petroleumhaven Biesbosch Biesbosch EDA Compound identification? Source identification??? 38 19

20 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 O o s t e r s c h e l d e O o s t e r s c h e l d e Z i e r i k z e e b u i t e n Z i e r i k z e e b i n n e n V e e r s e M e e r H a r i n g v l i e t B r u i n i s s e D i n t e l S l u i z e n M o e r d i j k N i e u w e M a a s N i e u w e W a t e r w e g R o t t e r d a m I J s s e l H a v e n 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 B i e s b o s c h B i e s b o s c h /25/2012 Identification of active compounds: Effect-Directed Analysis complex mixture extraction bioassay analysis fractionation bioassay toxicant Brack, 2003 Anal Bioanal Chem 377: EDA in sediment from Zierikzee inner harbour L o ca t ie D R - C A L U X tot D R -C A L U X st a b E R - C A L U X t o t E R - C A L U X st a b T T R s ta ble M ic ro to x t o ta M icrotox stab u m u -S 9 to ta u m u + S 9 to t a EDA 76% of estrogenic response by natural hormones complex mixture extraction bioassay analysis fractionation bioassay toxicant 38% of dioxin-like response by polycyclic aromatic hydrocarbons (PAHs) Houtman et al. (2006) Chemosphere 65:

21 Biomonitoring using specific in vitro bioassays Hazard profile Ecological risk Molecular Cellular Organ Individual Population Community MODE OF ACTION LEVEL A B C D E F G H I J K EDA Toxicity Profile 41 Toxicity profiling: a safety net to get GRIP on mixtures Group locations with similar hazard profiles Rank locations based on distance to reference profile Assigned Good Ecological quality Risk threshold Identify Important modes of action Responsible compounds (EDA) Prioritize Hot Spots Compounds of interest 42 21

22 Current challenges in Toxicity Profiling Improve the translation of toxicity profiles into risk profiles? Include in vivo bioassays in the test battery? Include biomarker measurements in the test battery? 43 Sample Test matrix Test system Information Toxicity index Abiotic sample Whole sample representing in situ situation Worst case total extract In vivo bioassay Survival Growth Reproduction Biomarkers Toxicity profiles In vitro bioassay General responses Specific responses Biota Organism Direct examination Health index Biomarkers Health status 22

23 Current challenges in Toxicity Profiling Improve the translation of toxicity profiles into risk profiles Include in vivo bioassays in the test battery? Include biomarker measurements in the test battery? Take bioavailability better into account Biota samples Passive sampling 45 Sample Test matrix Test system Information Toxicity index Abiotic sample Whole sample representing in situ situation Worst case total extract In vivo bioassay Survival Growth Reproduction Biomarkers Toxicity profiles Passive sample Bioavailable extract In vitro bioassay General responses Specific responses Biota Bioavailable + Biotransformed extract Organism Direct examination Health index Biomarkers Health status 23

24 Current challenges in Toxicity Profiling Improve the translation of toxicity profiles into risk profiles Include in vivo bioassays in the test battery? Include biomarker measurements in the test battery? Take bioavailability better into account Biota samples Passive sampling What is reference situation? Based on detection limit, reference location, relevant effect levels Improve EDA efficiency Identification strategies 47 EDA strategy for data evaluation Selected masses~ 2% 1/3 of tested compounds confirmed SIEVEd Ratio (active/non-active) peaks < >100 Peak check Tentatively identified Tested Analytical Confirmed Bioassay confirmed Weiss et al Anal Bioanal Chem 400:

25 Future challenges in Toxicity Profiling Safety net that covers all toxicity syndromes Include new assays for neurotoxicity, immunotoxicity Include multiple endpoint techniques as omics Account for toxicokinetics Include a biotransformation model (e.g. rat liver homogenate) Include an uptake model (e.g. Caco-2 cells) 49 Caco2: transwell model for intestinal absorption Epithilium colon adenocarcinoma cell line Differentiate into enterocyte-like cells (small intestine) Grow as a polarized epithelial enterocyte monolayer Provide a physical and biochemical barrier to the passage of ions and small molecules 50 25

26 Future challenges in Toxicity Profiling Safety net that covers all toxicity syndromes Include new assays for neurotoxicity, immunotoxicity Include multiple endpoint techniques as omics Account for toxicokinetics Include a biotransformation model (e.g. rat liver homogenate) Include an uptake model (e.g. Caco-2 cells) Introduce high-throughput screening techniques TOXCAST like battery Omics Aggregate toxicity profiles to a transparent single-indicator value Dow Jones index for toxicity 51 Conclusions Toxicity profiles can serve as safety net Get GRIP on individual compounds and complex mixtures Applicability for quality assessment Compare to normal situation (what is normal?) Compare to threshold level for risk Identify responsible compounds Current challenges include Improvement of the steps above Taking bioavailability into account Future challenges include New endpoints Taking toxicokinetics into account High throughput techniques 52 26

27 Acknowledgements Institute for Environmental Studies (IVM) Pim Leonards Bert van Hattum Juliette Legler Marja Lamoree Jana Weiss Jorke Kamstra Peter Cenijn Deltares Dick Vethaak Cor Schipper 53 27