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Article
Xenobiotics Produce Distinct Metabolomic Responses in Zebrafish Larvae (Danio rerio) Susie Shih Yin Huang, Jonathan P. Benskin, Bharat Chandramouli, Heather Butler, Caren C. Helbing, and John R. Cosgrove Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01128 • Publication Date (Web): 27 May 2016 Downloaded from http://pubs.acs.org on June 7, 2016
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Environmental Science & Technology
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Xenobiotics Produce Distinct Metabolomic Responses in Zebrafish Larvae (Danio rerio)
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Susie S.Y. Huang1*, Jonathan P. Benskin1,2, Bharat Chandramouli1, Heather Butler1, Caren C. Helbing3,
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John R. Cosgrove1
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Stockholm, Sweden
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AXYS Analytical Services Ltd., Sidney, BC, Canada
Department of Environmental Science and Analytical Chemistry (ACES), Stockholm University,
Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada
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* Corresponding author
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2045 Mills Road West
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Sidney, BC, Canada V8L 5X2
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Tel: +1-250-655-5800
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Fax: +1-250-5811
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E-mail:
[email protected] 1 ACS Paragon Plus Environment
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Abstract
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Sensitive and quantitative protocols for characterizing low-dose effects are needed to meet the
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demands of 21st century chemical hazard assessment. To test the hypothesis that xenobiotic exposure at
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environmentally relevant concentrations produces specific biochemical fingerprints in organisms,
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metabolomic perturbations in zebrafish (Danio rerio) embryo/larvae were measured following 24-hour
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exposures to 13 individual chemicals covering a wide range of contaminant classes. Measured
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metabolites (208 in total) included amino acids, biogenic amines, fatty acids, bile acids, sugars, and
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lipids. The 96 to 120 hours post-fertilization developmental stage was the most appropriate model for
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detecting xenobiotic-induced metabolomic perturbations. Metabolomic fingerprints were largely
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chemical- and dose-specific and were reproducible in multiple exposures over a 16 month period.
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Furthermore, chemical-specific responses were detected in the presence of an effluent matrix;
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importantly, in the absence of morphological response. In addition to improving sensitivity for detecting
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biological responses to low-level xenobiotic exposures, these data can aid the classification of novel
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contaminants based on the similarity of metabolomic responses to well-characterized ‘model’
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compounds. This approach is clearly of use for rapid, sensitive, and specific analyses of chemical effect
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on organisms, and can supplement existing methods, such as the Zebrafish Embryo Toxicity assay (OECD
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TG236), with molecular-level information.
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Environmental Science & Technology
Introduction
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The introduction of the European Union’s Regulation on Registration, Evaluation, Authorization
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and Restriction of Chemicals (REACH) places an increased demand on the use of animals for toxicity
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testing. The Zebrafish Embryo Toxicity assay (ZFET; Organization of Economic Cooperation and
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Development (OECD) assay #TG236), which involves acute toxicity testing in zebrafish (Danio rerio; ZF)
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embryos, was recently introduced as an alternative to adult fish toxicity testing.1 ZFET vastly reduces the
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number of animals used for testing since teleost up to 120 hours post-fertilization (hpf) do not fall into
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regulatory frameworks dealing with animal experimentation, such as EU Directive 2010/63/EU and the
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Canadian Council of Animal Care. The limitation of ZFET is that it measures morphological endpoints
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which are often indistinguishable among substances with different modes-of-action (MOAs) and
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importantly, may not be observable in low-dose exposures. New approaches which supplement existing
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toxicological assays, such as ZFET, with highly specific information on a chemical’s MOA, would add
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tremendous value to predictive toxicity testing.
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Changes in RNA, proteins, and/or metabolite levels in animals exposed to a toxicant can provide
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information on the molecular initiating events and the affected biological pathways that lead to adverse
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outcomes.2 These molecular biomarkers may provide a useful means of carrying out effects-directed
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analysis in environmental samples. Recently, toxicogenomic profiles of 11 toxicants were characterized
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in a ZF model at dose concentrations that did not induce observable changes in morphology.3 This
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molecular “effect-based” profiling approach can be a rapid and cost effective alternative to conventional
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methods of identifying active substances associated with adverse effects.
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Metabolomics is the highly multiplexed analysis of endogenous small molecules (metabolites)
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within a cell, tissue or biofluid. As endogenous metabolite concentrations are determined by the
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organism's biochemical processes, metabolomics is regarded as a comprehensive evaluation of
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biological response.4 The technique is gaining ground in studies on organism-environment interactions
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since, in principle, metabolite measurements represent the functional metabolic phenotype of an
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exposed organism. Furthermore, metabolomics is considered to be more statistically powerful in
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detecting robust effects compared with other “omics” technologies.5 Metabolomics is becoming a
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popular tool for characterising biological responses to environmental stressors in a variety of aquatic
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species. In teleost, metabolomics has been used to study the effects of temperature stress,6 anoxia,7 in-
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stream migration and spawning,8 environmental contaminants,9–12 as well as characterization of early
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developmental stages.13
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Gene expression barcoding of xenobiotic responses in ZF has been attempted previously3 but to
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our knowledge metabolomics has not been similarly applied. In the work presented here, we
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hypothesized that sub-lethal exposure of ZF to individual chemicals could be fingerprinted using
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metabolomics. Among the existing classes of contaminants, we tested this hypothesis using a suite of 13
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diverse xenobiotics, including endocrine disrupting compounds (EDCs), petroleum-derived compounds,
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performance chemicals, pharmaceuticals and personal care products (PPCPs), and heavy metals.
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Metabolomic analysis was carried out using a targeted approach for 208 primary metabolites. As the
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first toxicometabolomic fingerprinting study of its kind, we characterized fundamental parameters of
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the assay, including the effect of dose, developmental stage, co-exposure to multiple substances, and
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the presence of an environmental matrix (wastewater treatment plant effluent; WWTP). This approach
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can provide a powerful means of classifying novel contaminants based on the similarity of their
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metabolomic response to well-characterized substances, while supplementing functional data
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generated by OECD TG236 with information on molecular phenotype.
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Experimental
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Chemicals
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Chemicals were selected for their environmental relevance, toxicity, and to encompass a diverse
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range of known MOAs (Table S1). Background information on individual chemicals and the justification
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for their inclusion can be found in the SI. Nominal concentrations were selected to induce minimal
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phenotypic effects in zebrafish and other teleost species based on available half maximal effective
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concentrations (EC50), lowest observable effect levels (LOEL), and no observable effect levels (NOEL) in
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the available literature (Table S1). The dosing concentrations used were adjusted from those reported in
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the literature to account for differences in developmental stages of the exposed animals, duration of the
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exposure, and experimental conditions. For PPCPs and heavy metals, the lowest dose selected is
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comparable to effluent levels14 or consumer safety guidelines.15
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Ethanol was used as the carrier (0.05-0.1%) for all chemicals except for heavy metals and
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effluent groups where no carrier was used. All chemicals and reagents were obtained from Sigma-
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Aldrich (USA) unless stated otherwise (SI). Chemical concentrations are reported as measured values in
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all results shown.
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Exposures
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Five biological replicates comprised a treatment group. Each replicate consisted of 80 zebrafish
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(ZF) at either the embryonic (24-48 hpf) or larval (96-120 hpf) stage. All exposures were carried out for
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24 h. No apparent toxic effects were observed during and after the exposure period in either the control
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(embryo media [HE3] and carrier control [CC]) or treatment groups at either life stage. Water hardness
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for the embryo media used in the heavy metal exposures was calculated to be 4.14 mg/L as CaCO3. In
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general, the measured concentrations were in the range of their respective nominal values as reported
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in Figure 2. The non-detects are reported as less than their limit of detection (LOD). Since these were
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post-exposed exposure media, any decreases observed were likely due to animal uptake and losses to
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evaporation or container adsorption. A detailed description of the fish husbandry, embryo collection,
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exposure procedures, and chemical analysis of post-exposure media is provided in the Supporting
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Information.
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Metabolite Analysis
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A suite of 208 metabolites was measured from whole body ZF embryo/larvae, including 21
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amino acids (AA), 21 biogenic amines (BAs), 4 bile acids, ∑hexose, 17 faTy acids (FAs), 40 acylcarniUnes
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(ACs), 89 phosphatidylcholine (PCs), and 15 sphingomyelins (SMs). A full list of the analytes, internal
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standards, and abbreviations is provided in Table S3. Instrumental analysis was performed on an Agilent
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1100 high performance liquid chromatography (HPLC) system coupled to an API4000 triple quadruple
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mass spectrometer (Applied Biosystems/Sciex, Concord, ON, Canada). A detailed description of the
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metabolite extraction, instrumental analysis, quality control, and statistical analyses can be found in the
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Supporting Information.
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Results and Discussion
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Morphological effects
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There were no significant differences in hatchability, phenotype, and mortality in zebrafish (ZF)
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embryos exposed at 24-48 hpf. However, in the larvae, the highest concentration of CuSO4 (7.1 μM) and
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CdCl2 (5 μM) induced lateral recumbence in approximately 10% of the exposed animals. Cu and Cd have
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previously been reported to induce locomotor deficit in ZF larvae. For example, Johnson et al.16 reported
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that a 72 h exposure to 3.8 μM Cu reduced neuromast abundance in 120 hpf ZF, an effect associated
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with a reduced ability to orientate in a current. The dose used is comparable to the 3.1 μM Cu dose in
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the present study, when differences in water hardness and exposure period are accounted for. Exposure
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to Cd has also been shown to affect muscle development and axon growth in ZF embryos resulting in
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reduced motility.17 No significant mortality or other observable phenotypic changes (>5%) were noted in
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the remaining ZF larvae exposures; thus, the chemical concentration ranges used in the current study
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are sublethal.
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Stage-specific metabolomic response
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For triiodothyronine (T3), perfluorooctanesulfonic acid (PFOS), bisphenol A (BPA), refined
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merichem (RM), and triclosan (TCS), the metabolomic responses were compared at two different
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developmental stages (embryonic and larval). Since the embryonic stage coincides with the onset of
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organogenesis,18 we expected this developmental stage to be more sensitive to chemical exposure.
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Surprisingly, this was not the case. The number of affected metabolites in the 24-48 hpf exposure
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experiments ranged from one in PFOS and RM exposed embryos to 15 in BPA-treated embryos (mostly
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acylcarnitines) (Table 1; top). TCS did not induce any significant metabolite changes in the embryos.
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Amino acids, bile acids, SMs, and hexose were not affected by any chemical groups at this life stage. In
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contrast, the larvae displayed a number of significantly changed metabolites (Padj
