Novel Markers for Toxic Stress Risk Assessment

1. Novel effect markers for risk assessment of toxic stressMechthild Schmitt-Jansen1, Fred Sans Piché1, Karen Hanisch1, Christina Klünder1, Ulrike Gündel1, Rolf Altenburger1, Helmut Segner21Helmholtz Centre for Environmental Research - UFZ, Leipzig, Germany2University of Bern, Switzerland

2. Introduction ‘Omics-techniques Current status: examples Future challenges outline

3. chemicals in the environment ex postex ante e.g. ~ 70000 REACH compounds e.g. ~ 20000 megasites in Europe Site specific assessment Prospective assessment

4. Chemicals with unknown mode-of-action- New groups of substances, e.g. pharmaceuticals, nanoparticles- Multiple modes-of-action Predicting long-term effects from acute effect data Mixtures in the environment Cross species extrapolations chemicals in the environmentchallenges

5. unknown effects “… there are known unknowns; … but there are also unknown unknowns – the ones we don’t know we don’t know”. e.g. Effect-Directed Analysis We just find toxicants reflected by a bioanalytical tool

6. Ethinylestradiol  feminization of fish (Desbrow et al. 1998; Larsson et al. 1999) Diclofenac decline in vulture populations in India (Oaks et al. , Nature, 2004) examples

7. complexity in effectsneed of new advanced tools for effect assessment of chemicals in the environment reductionistic  holistic

8. toxicogenomics (‘omics – techniques) definition Global detection and analysis of gene expression, proteins or metabolites of an organism http://www.biology.ucsc.edu/mcd/research/index.html www.palmgren.dk.SPPSnews/0408/PlaCe3.jpg open / unbiased approach looking at several hundred / thousand of signals in parallel aiming to capture the complete picture of an organism

9. Goodacre, Journal of Experimental Botany, 2005

10. some data E.coli > 440 metabolites, > 4400 genes S. cerevisiae > 700 metabolites, > 6200 genes eukaryotes > 200,000 proteins (according to Roessner, 2007) • All connected in pathways • Potential to identify multiple markers for toxicity

11. http://www.biology.ucsc.edu/mcd/research/index.html novel workflow required Experimental design Measurement 2-DE gel chromatogram Gene array Data processing Multi-variate statistics data evaluation Biological interpretation Pathway-analysis

12. challenge implement ‘omics – techniques in ecotoxicology Publications (web of science)

13. link ‘omics to classical parameters Metabolome Proteome High sensitivity of metabolic markers growth Kluender, C., Sans-Piché, F., Riedl, J., Altenburger, R., Härtig, C., Laue, G., Schmitt-Jansen, M., (2009): A metabolomics approach to assessing phytotoxic effects on the green alga Scenedesmus vacuolatus, Metabolomics, 5 (1), 59-71. Sans-Piché F., Kluender C., Altenburger R. & Schmitt-Jansen M. (2010) Anchoring metabolic changes to phenotypic effects in the chlorophyte Scenedesmus vacuolatus under chemical exposure. Marine Environmental Research. (in press)

14. ‘omics to identify candidate biomarkers controls ethanol 740 Spots Gündel U. et al. Submitted: Characterisation of cathepsins as candidate biomarkers for toxic exposure and abnormal development in zebrafish (Danio rerio) embryos

15. comparison of cathepsins with classical OECD-observation parameters for the fish embryo test

16. priorisation of measures ‘omics for assessing the health status of local fish populations at all sites immune-related pathways affected Site 1 Site 2 0 25 32 6 11 13 49 Site 3 H. Segner

17. conclusions • several ‘omics techniques established • proof-of-principle studies • perspective for assessment of the health status of organisms  priorization of further activities  development of new designed tests  high sensitivity (NOMetabolimicsEC << NOEC growth)

18. methodological / ecotoxicological - model organisms of ecotoxicology; environmental species, BQE - relate alterations in pathways to modes-of-action - discriminate multiple stressors - implementing ‘omics results in current ecotoxicological approaches, e.g. EDA or concepts of mixture toxicity future challenges

19. regulatory- standardization of methods and quality assurance- relate ‘omics outcomes to biological/ecological results, e.g. status of BQEs- development of predictive tools- diagnosis of environmental samples at sites of moderate ecological status in investigative monitoring - implementing ‘omics tools in weight-of-evidence approaches future challenges

20. „the successful incorporation of toxicogenomics into regulatory frameworks may someday be regarded as the most important intellectual and practical contribution from this generation of ecotoxicologists“ (Ankley et al., 2006, ES&T)

21. Thank you for your attention!

22. Methodological-`’omics goes systems biology´ (pathways)-cross species extrapolations (sequencing of model organisms of ecotoxicology; environmental species)-costs and work load (development of new analytical and bioinformatic tools, e.g. 454-sequencer)-development of high-throughput methods Future challenges

23. Ecotoxicological-relate alterations in pathways to modes-of-action-Differentiate pharmaceutical (compensatory) from adverse effects-concentration-time-response relationships-development of predictive tools-implementing ‘omics results in current ecotoxicological approaches, e.g. EDA or concepts of mixture toxicity Future challenges

24. Regulatory-standardization of methods-quality assurance-relate ‘omics outcomes to biological /ecological results-development of predictive tools-diagnosis of environmental samples (sediments, organisms)-implementing ‘omics tools in weight-of-evidence approaches Future challenges

25. Transcriptomics for assessing the health status of local fish populations 1) 2) Site 1 Site 2 0 25 32 6 11 13 49 Site 3 • Congruence of GO categories among the three site comparisons: • 6 pathways in common • all 6 pathways immune-related

26. 12 12 10 8 8 10 4 4 6 6 14 8 14 6 10 14 12 4 0 0 time-concentration-response relationships in the chloropyhte Scenedesmus vacuolatus Variation in time Prometryn (0.1 µmol L-1) Variation in concentration Prometryn (0.002 - 0.262 µmol L-1) Prin2 (16.7%) Prin1 (53.4%)

27. Example: pyrene effects on earth worms Jones et al., 2008