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Validation of metabolomics for toxic mechanism of action screening with the earthworm Lumbricus rubellus

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Abstract

One of the promises of environmental metabolomics, together with other ecotoxicogenomic approaches, is that it can give information on toxic compound mechanism of action (MOA), by providing a specific response profile or fingerprint. This could then be used either for screening in the context of chemical risk assessment, or potentially in contaminated site assessment for determining what compound classes were causing a toxic effect. However for either of these two ends to be achievable, it is first necessary to know if different compounds do indeed elicit specific and distinct metabolic profile responses. Such a comparative study has not yet been carried out for the earthworm Lumbricus rubellus. We exposed L. rubellus to sub-lethal concentrations of three very different toxicants (CdCl2, atrazine, and fluoranthene, representing three compound classes with different expected MOA), by semi-chronic exposures in a laboratory test, and used NMR spectroscopy to obtain metabolic profiles. We were able to use simple multivariate pattern-recognition analyses to distinguish different compounds to some degree. In addition, following the ranking of individual spectral bins according to their mutual information with compound concentrations, it was possible to identify both general and specific metabolite responses to different toxic compounds, and to relate these to concentration levels causing reproductive effects in the worms.

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Acknowledgements

We thank Slawomir Zukowski for valuable assistance in developing the code for analysis of mutual information. The Natural Environment Research Council is acknowledged for funding.

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Authors and Affiliations

  1. Department of Biomolecular Medicine, Division of Surgery, Oncology, Reproductive Biology, and Anaesthetics, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, London, SW7 2AZ, UK

    Qi Guo, Jasmin K. Sidhu, Timothy M. D. Ebbels, Faisal Rana & Jacob G. Bundy

  2. Centre for Ecology and Hydrology, Maclean Building, Benson Lane, Crowmarsh Gifford, Wallingford, OX10 8BB, UK

    David J. Spurgeon & Claus Svendsen

  3. Pharmaceutical Science Division, King’s College London, School of Biomedical & Health Sciences, Franklin Wilkins Building, Stamford Street, London, SE1 9NH, UK

    Stephen R. Stürzenbaum

  4. University of Cardiff, School of Biosciences, Main Building, Park Place, Cardiff, CF10 3TL, UK

    Peter Kille & A. John Morgan

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  1. Qi Guo
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  2. Jasmin K. Sidhu
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  3. Timothy M. D. Ebbels
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  4. Faisal Rana
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  6. Claus Svendsen
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  10. Jacob G. Bundy
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Correspondence to Jacob G. Bundy.

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Qi Guo and Jasmin K. Sidhu contributed equally to this paper.

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Fig. S1

600 MHz 1H NMR spectrum of typical Lumbricus rubellus tissue extract. A: entire spectrum. B–E: expansions of peak-containing regions. Asterisks indicate resonances from unassigned compound mentioned in text (PDF 876 kb)

Fig. S2

Validation plots for PLS models shown in Fig. 1A–C, based on 20 permutations of the sample order. Green symbols represent R2 and blue symbols Q2. Abscissa represents correlation of the permuted data order with the original order. A: AZ. B: Cd. C: FA (PDF 663 kb)

Fig. S3

PLS models (predicted vs. observed for training sets) and validation plots for 3 compounds against concentrations measured in tissues. A: AZ. B: Cd. C: FA (PDF 1321 kb)

Fig. S4

Principal components analysis of NMR spectral data for three compound exposures (replicated doses only). Eight axes were fitted. The top and bottom vertices of the diamonds represent the 95% confidence limits for the group means; all data points are also shown on the chart. Controls: grey. Cd: blue. FA: red. AZ: black (PDF 706 kb)

Fig. S5

Plots of individual spectral variable relationships against AZ, Cd, or FA (for both soil and tissue concentrations) for the variables identified in Fig. 4 in the manuscript (PDF 2822 kb)

Fig. S6

Metabolites related to cocoon production rate: validation plot for PLS regression model shown in Fig. 6 (PDF 105 kb)

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Guo, Q., Sidhu, J.K., Ebbels, T.M.D. et al. Validation of metabolomics for toxic mechanism of action screening with the earthworm Lumbricus rubellus . Metabolomics 5, 72–83 (2009). https://doi.org/10.1007/s11306-008-0153-z

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