Insights into Lipidomic Perturbations in Zebrafish Tissues upon

Article pubs.acs.org/est

Insights into Lipidomic Perturbations in Zebrafish Tissues upon Exposure to Microcystin-LR and Microcystin-RR ̂

̂

Shruti Pavagadhi,†,‡ Siria Natera,§,∥ Ute Roessner,§,∥ and Rajasekhar Balasubramanian†,‡,* †

Singapore-Delft Water Alliance and ‡Department of Civil and Environmental Engineering National University of Singapore, Block E1A, #07-03 No.1 Engineering Drive 2, Singapore 117576 § Metabolomics, Australia, School of Botany, The University of Melbourne, 30 Flemington Road, Victoria 3010, Australia ∥̂ Australian Centre for Plant Functional Genomics, School of Botany, The University of Melbourne, 3010 Victoria, Australia S Supporting Information *

ABSTRACT: This work represents the first study of its kind that was conducted to evaluate changes in lipid metabolic networks following a balneation exposure of adult zebrafish to MCLR (microcystin-leucinearginine) and MCRR (microcystin-arginine-arginine) at a sublethal dose (10 μg L−1) for a period of 30 days. Following the exposure to MCLR and MCRR, gills, liver, intestine, and brain tissues were harvested for metabolite extraction. Extracted metabolites were detected using qTOF-LC-MS (timeof-flight-liquid chromatography−mass spectrometry). Metabolites were identified using Kegg pathways. The identified metabolites are shown on lipid biochemical maps to demonstrate major perturbations in the metabolic machinery. Results showed that most of the metabolic pathways under the lipid class were affected in different tissues of zebrafish following the exposure to MCLR and MCRR (10 μg L−1 for 30 days). The kind and flux of metabolic perturbations varied among different tissues of the organs after the exposure to MCLR and MCRR with the tissues of gills being the most affected. Among the various lipid pathways, cholesterol synthesis was affected significantly as observed from the highest number of perturbed metabolites in that pathway. Cholesterol is responsible for synthesis of steroid hormones and bile acids, which have been recognized as endocrine signaling molecules. Disruption in the synthesis of these compounds following MCLR/MCRR exposure suggests that MCs are capable of causing endocrine disruption among aquatic organisms even under sublethal conditions. Apart from cholesterol synthesis, various other metabolic pathways belonging to the class of essential fatty acids and lipid oxidation were also observed to be perturbed following a balneation exposure of zebrafish to MCLR/MCRR.

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INTRODUCTION Harmful algal blooms (HABs) have emerged as a worldwide concern due to their increased frequency of occurrence, severity, and their toxicity to all living forms.1 HABs release toxic secondary metabolites upon lysis, known as cyanotoxins, into aquatic systems.2 Among cyanotoxins, microcystins (MCs) are particularly prevalent in eutrophic waters following bloom lysis. MCs have beenstudied extensively by the research community globally.3−5 MCs have been reported in various water bodies with a wide range of concentrations (19−141 μg l−1) throughout the world.6−8 MCs in the water phase (extracellular MCs) can break down slowly at high temperature (40 °C) at either very low (9) pH.9 The typical half-life of MCs in natural waters is 10 weeks. As a consequence, MCs tend to persist in the aquatic environment for a long period of time, and can get into aquatic organisms, leading to their possible accumulation in the aquatic food web. Certain groups of organisms such as fish species (such as Phytoplanktivorous (Hypophthalalmichthys molitrix; silver carp), herbivorous (Parabramis pekinensis; white amur bream), omnivorous (Carassius auratus; gold fish); carnivorous (Chanodichthys erythropterus; predatory carp) were found to © 2013 American Chemical Society

accumulate MCs (specifically MCLR (microcystin-leucinearginine) and MCRR (microcystin-arginine-arginine), resulting in disturbances at the ecological levels affecting the entire food chain.10−12 The MCs that are widely studied are potent acute liver toxins with LD50 in mouse in the range of 50−500 μg kg−1 (intraperitoneal injection), which can act as tumor promoters.2 The molecular basis of the tumor promotion is the inhibition of protein phosphatases 1 and 2A (PP-1 and PP-2A),2,13,14 two of the important enzymes from protein phosphatases (PPP) family responsible for control of many diverse cellular processes.15 PPPs are a group of enzymes that are found ubiquitously, and are responsible for the dephosphorylation of various proteins and enzymes in a cell. Protein phosphorylation and dephosphorylation are required for the regulation of a large number of cellular and metabolic activities. Hence, these enzymes are essential for many biochemical pathways. These biochemical pathways consist mainly of lipid, protein and sugar Received: Revised: Accepted: Published: 14376

January 27, 2013 October 18, 2013 October 23, 2013 October 23, 2013 dx.doi.org/10.1021/es4004125 | Environ. Sci. Technol. 2013, 47, 14376−14384

Environmental Science & Technology

Article

The fish were acclimated to the laboratory conditions for 10 days prior to the experiments. They were maintained in 20 L tanks containing aquarium water (distilled water containing 1000 mg/L sea salt, 200 mg/L CaCl2) with acclimation being performed in a temperature controlled room at 25 ± 1 °C, with a 12-h light: 12 h dark cycle. Fish were fed once a day with commercial fish food. Experimental Design. Zebrafish were exposed to MCLR and MCRR dissolved in aquarium water at a concentration of 10 μg l−1 for a period of 30 days. The concentration of MCLR and MCRR for the exposure was decided based on the previous experiments conducted by our research group, namely, the biochemical changes upon a balneation exposure (dose− response study)20 and the analytical measurements done for detection of MCLR in real water samples.26 Upon exposure of zebrafish to MCLR and MCRR at 10 μg l−1, it was found that the biochemical enzyme activities (glutathione-S-transferase (GST), glutathione peroxidise (GPx), gluthatione reductase (GR), and superoxide dismutase (SOD)) were altered significantly in the zebrafish organs. Briefly, stock solutions of MCLR and MCRR were prepared (10 mg l−1) and were diluted to obtain the required concentrations. Water in the fish tanks was replaced with fresh water spiked with the toxins on a daily basis. Dosing concentrations for MCLR and MCRR (10 μg l−1) were confirmed by measurements using liquid chromatograph, composed of an HP100 liquid chromatograph (Agilent Technologies, U.S.) interfaced with a triple quadrupole MS/ MS (Applied Biosystems, U.S.). Analytical separation was achieved on a Zorbax Extend-C18 5 μm, 2.1 × 150 mm (Agilent technologies, Germany). The injection volume was 10 μL. The mobile phase consisted of 0.1% formic acid, HCHO (solvent A) and methanol, CH3OH (solvent B). A gradient elution was used, starting with water: methanol at 90:10 from 0 to 6 min, and switching to 5:95 up to 10 min before returning to the original conditions to re-equilibrate the system. The capillary voltage was set at 89 V and the cone voltage at 4 V. The desolvation gas (nitrogen) temperature and the gas flow-rate were set at 350 °C and 615 l/h, respectively. The ion source temperature was set at 120 °C. LC-MS-MS was operated in the positive ion mode. MCLR and MCRR were monitored by using the MS instrument in the SRM (Single reaction monitoring) mode: m/z 995.6; fragment ion at 135.1 and m/z 520; fragment ion 135.1, respectively. At the end of the exposure period, zebrafish (n = 5 per group) were collected and the organs (gills, liver, intestine, and brain) were harvested, snap frozen and then lyophilized. Nonpolar Metabolite Extraction. Following lyophilization, tissue samples were sequentially extracted using a series of solvents in order to separate the polar and non polar tissue components to reduce matrix interferences during analyses. Briefly, methanol (HPLC grade) was added to the lyophilized samples and then homogenized on ice. Five μL of the internal standard mix was added to the samples at this stage. Homogenized tissue samples were then centrifuged at 22 000g for 10 min at 4 °C. This process was repeated again with residual pellets obtained from the first centrifugation step. Supernatants from both the extraction steps were pooled together. To the pooled supernatant, 500 μL of milli-Q water and 400 μL of chloroform were added which resulted in a biphasic mixture which was then vortexed briefly and centrifuged (22 000g; 10 min at room temperature). After centrifugation, the reaction mixture was divided into two clear

metabolic networks, which are likely to be disrupted upon inhibition of protein phosphatases by MCs. Perturbation in the metabolic and biochemical networks, as induced by MCs, can lead to increased production of ROS (reactive oxygen species) and hence oxidative stress as has been reported by many researchers based on in vivo toxicity measurements using different fish species.16−19 A recent study conducted in our laboratory using zebrafish also revealed that extracellular MCs can cause oxidative stress, even at chronic, sublethal doses.20 However, to-date, no systematic study has been conducted on the perturbation of lipidomic networks in zebrafish tissues upon exposure to MCLR and MCRR, to the best of our knowledge. Studying the metabolic pathways would not only provide a better understanding of toxicity mechanisms involved with MCs, but also lead to the identification of potential biomarkers that can be used for risk management when HABs occur in natural waters. With this goal in mind, we initiated a systematic study to evaluate the adverse effects of extracellular MCs on lipid metabolic networks in zebrafish tissues under balneation conditions (i.e., fish bathed in water containing MCs). MCLR and MCRR were selected as model MCs for the present set of experiments based on our previous study.20 MCLR is considered to be the most commonly occurring and lethal toxin.21 A provisional safety guideline of 1.0 μg l−1 MCLR in drinking water was recommended by WHO.22 Toxicity of MCLR based on intraperitoneal LD50 in laboratory mouse or rat injections was found to be 50 μg kg−1. Another common variant known is MCRR which has a LD50 of 300 μg kg−1.2 It can be seen that MCRR is less toxic than MCLR. However, both are released by the same strains of cyanobacteria,23 and often occur together in a bloom episode.24 Zebrafish were exposed to MCLR and MCRR at 10 μg l−1 for a period of 30 days. Following the exposure, the fish tissues were harvested for metabolite extraction. Lipid metabolic networks were mapped to study and identify the perturbations that occurred in the zebrafish tissues following an exposure to MCLR and MCRR separately.

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EXPERIMENTAL SECTION Reagents. MCLR and MCRR (≥95% HPLC grade) were obtained from Alexis Biochemicals (Switzerland). Methanol and formic acid were obtained from Merck (U.S.). Methanol (HPLC grade), 100% ethanol (molecular grade), chloroform (HPLC grade), tetrahydrofuran (HPLC grade), butanol (HPLC grade) and ammonium formate were obtained from Merck (Germany). Milli-Q water (18 M′Ω) used in all experiments was obtained through a Milli-Q (Millipore, Bedford, MA) water purification system. Internal lipid standard mixture (9 deuterated lipid standards (16:0 D31−18:1 PE; 16:0 D31 Ceramide; Cholesterol (d7); 16:0 D31−18:1 PI; 16:0 D31−18:1 PG; 16:0 D31−18:1 PS; 16:0 D31−18:1 PC; 16:0 D31 SM; Sphingosine (d7)) for non polar metabolite analyses was provided by Bio21 Institute, University of Melbourne, Australia (Node, Metabolomics Australia). Fish. We have selected zebrafish (adult) that are commonly found in a tropical freshwater for this work because of two main reasons. First, zebrafish represent an important vertebrate model organism that is widely used in scientific research.25 Second, these fish can easily be maintained in the laboratory conditions since they are small, and can be maintained at low husbandry cost in large numbers. We procured adult specimen from the mainland tropical fish farm, Singapore. They were transported to our laboratory within water tanks. 14377

dx.doi.org/10.1021/es4004125 | Environ. Sci. Technol. 2013, 47, 14376−14384


Article

with the Mass Hunter Qualitative software database search algorithm (Agilent, U.S.).

layers with an interphase composed of proteins and other debris. Upper polar phase and interphase consisting of methanol and water was carefully removed. The residual interphase consisting of proteins and amino acids was discarded. The chloroform phase was thus retained and transferred to a fresh tube. To recover nonpolar metabolites completely, another 300 μL of chloroform was added to the polar phase supernatant, vortexed briefly and centrifuged (22 000g ; 10 min at room temperature). After centrifugation, the clean non polar chloroform phase was pooled with the previous chloroform phase. The extracts were dried down subsequently using speedvac (Thermo scientific, U.S.). Metabolite Analyses. The dried extracts were reconstituted using 100 μL of 50:50 methanol/butanol containing 10 mM ammonium formate and were briefly vortexed. The samples were then incubated in a shaking heater block for 25 min at 30 °C before being centrifuged at 19 500g for 3 min at ambient temperature. The resulting supernatant was transferred to glass vial inserts in amber LC-MS vials for instrumental analysis. For the LC-MS analyses, a HPLC (Agilent 1200 series; Binary pump with thermostatted autosampler) system was coupled to a Q-TOF Mass Spectrometer (Agilent 6520 Q-TOF LC/MS) operating in ESI+ and ESI− ion modes. Separation of metabolites was achieved on a Ascentis Express RP Amide column, 2.1 × 50 mm, 2.7 μm (Supelco, Sigma Aldrich, U.S.). The injection volume was 5 μL. The mobile phase consisted of water/methanol/tetrahydrofuran 50:20:30 in 10 mM ammonium formate (Solvent A) and water/methanol/tetrahydrofuran 5:20:75 in 10 mM ammonium formate (Solvent B). A gradient elution was used, starting with A/B at 100:0 at 0 min and switching to 0:100 at 8 min and holding for 2.2 min before returning to the original conditions to re-equilibrate the system. The capillary voltage was set at 4000 V. The drying gas (nitrogen) temperature and flow-rate were set at 325 °C and 10 l min−1, respectively. The nebulizer pressure was set at 45 psi. LC-MS Data Pretreatment. The acquired mass spectra were calibrated and converted into mzXML (Extensible Markup Language) file format to carry out further LC-MS data analyses. MZmine 2.2 27 was used for preprocessing of LCMS spectra obtained from tissue extracts. Normalization and other preprocessing steps were done using a centroid peak detector algorithm and the RANSAC aligner. Statistical Analysis. Statistical analyses were performed using the open source program R.28 For lipid metabolites data, differences in the intensities of metabolite ions between control fish, MCLR-exposed fish and MCRR-exposed fish were tested by one-way ANOVA with multiple comparisons. In order to correct for multiple comparisons, false discovery rate (FDR) q values were calculated,29 with a significant threshold set at q< 0.25.30 Metabolite Identification. Accurate masses were used to search within the databases for metabolite identification. metDAT, a workflow-based free online pipeline for mass spectrometry data processing, analysis, and interpretation, 31 was used for pathway analysis. The following databases were searched through metDAT/KEGG (http://www.genome.jp/ kegg/pathway.html), PubChem compound database (http:// pubchem.ncbi.nlm.nih.gov), and METLIN database (http:// metlin.scrips.edu) for pathway analysis. The mass features identified, as being significantly altered, were also searched using a combination of KEGG and Metabolomics Australia inhouse reference (authentic standards) compound library along

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RESULTS AND DISCUSSION HPLC-ESI-qTOF-MS was applied to obtain metabolic patterns from non polar extracts of zebrafish tissues (a representative base peak chromatogram obtained from the instrument is provided in the Supporting Information, SI, refer to Figure S1). Zebrafish were exposed to extracellular MCLR and MCRR at 10 μg l−1 for a period of 30 days, following which gill, liver, intestine, and brain tissues (n = 5) were harvested and extracted for metabolite analyses. Tissues from control fish, MCLRexposed fish and MCRR-exposed fish were harvested and extracted for nonpolar metabolites. The present study aimed at detecting and identifying only lipid class metabolites in the extracts from exposed zebrafish tissues. Following preprocessing and statistical treatment of the analytical data obtained, metabolites were identified based on fragmentation patterns and accurate mass using different databases as listed earlier. The metabolites that satisfied the criteria of significance, i.e., p < 0.05 (after correction) and fold change >2 were selected for database search from different experimental groups. On the basis of these criteria, the metabolites that satisfied the conditions were shown on different lipid pathway maps to understand the possible implications of the balneation exposure at biochemical levels. Fourteen biochemical pathways appear to have been affected significantly in lipid metabolism among different organs (Table 1). Out of these pathways, five pathways belonged to a class of essential fatty acids and related compounds (linoleic acid metabolism, α-linoleic acid metabolism, arachidonic acid metabolism, eiconosoid metabolism, and prostaglandins). Three pathways belonged to fatty acid metabolism (fatty acid Table 1. Specific Pathways Affected in Lipid Metabolism in Various Zebrafish Organs Following a Balneation Exposure to MCLR and MCRR lipid metabolism fatty acid oxidation glycerophospholipid metabolism fatty acid elongation linoleic acid metabolism

bile acid synthesis biosynthesis of unsaturated fatty acid arachidonic acid metabolism

α-linoleic acid metabolism secondary bile synthesis prostaglandins eiconosoid metabolism steroid biosynthesis

steroid hormone biosynthesis

cholesterol synthesis 14378

organs

experimental group

gills gills gills gills intestine brain intestine liver intestine gills brain intestine liver liver intestine intestine gills brain liver gills intestine brain gills

LR LR LR LR LR LR RR RR LR, RR LR RR, LR LR LR RR LR LR,RR LR

LR, RR

LR, RR



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Figure 1. Lipid maps-Interlinkage between different lipid biochemical pathways.

Table 2. Significant metabolites (p < 0.05, after correction; fold change >2) as detected and identified from gill tissues of zebrafish neutral mass

common name

ESI+ ion mode 384.262 ethynodiol diacetate 286.210 androstenedione 399.367 L-palmitoylcarnitine 386.569 cholesterol 458.459 5,10-methylene-THF 853.664 3-hydroxybutyryl-CoA 861.688 hexanoyl-CoA ESI− ion mode 352.223 prostaglandin H2; 11α-epidioxy15-hydroxyprosta-5,2.13 13-dienoate; PGH2 334.214 Prostaglandin A2; PGA2; Medullin 320.235 (15S)-15-hydroxy-5,8,11-cis-13trans- eicosatetraenoate; 15(S)-HETE 312.231 9(S)-HPODE 382.119 farnesyl diphosphate 320.234 5,6-EET a

retention time

fold change

9.07

2.19

C27H44O

4.77 5.63 1.41 2.34 9.25 9.98

2.02 2.53 2.20 2.13 2.12 2.45

C19H26O2 C23H45NO4 C27H46O1 C20H23N7O6 C25H42N7O18P3S1 C27H46N7O17P3S

sterprogesterone receptor agonistoid biosynthesis/steroid biosynthesis steroid biosynthesis fatty acid metabolism steroid biosynthesis glycerophospholipid metabolism fatty acid oxidation fatty acid oxidation; fatty acid elongation

1.11

5.46

C20H32O5

arachidonic acid metabolism

72

1.24 2.66

2.56 4.56

C20H30O4 C20H32O3

arachidonic acid metabolism arachidonic acid metabolism

55

3.19 1.66 3.56

5.67 5.34 6.57

C18H32O4 C15H28O7P2 C20H32O3

linoleic acid metabolism steroid biosynthesis/cholesterol synthesis arachidonic acid metabolism

68

chemical formula

pathway

score (out of 100) 12−41 38 25 a a a a

a

a

Identity uncertain; possibility of more than one compound being found at the same mass.

belongs to a special class of lipid molecules called phospholipids (glycerophospholipid metabolism) was also observed to be affected following the MCLR and MCRR exposure. All these metabolic pathways do not function in isolation, but are interconnected and interrelated to each other. Perturbation in

oxidation, fatty acid elongation, and biosynthesis of unsaturated fatty acids). Five pathways belonged to steroid and bile synthesis (primary bile acid synthesis, secondary bile acid synthesis, cholesterol synthesis, steroid biosynthesis, and steroid hormone synthesis). In addition, one pathway that 14379



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Table 3. Significant Metabolites (p < 0.05, after Correction; Fold Change >2) as Detected and Identified from Intestine Tissues of Zebrafish neutral mass

common name

retention time

fold change

chemical formula

9.09 1.98 2.60

5.67 4.56 3.56

C20H40O2 C26H43NO4 C22H32O2

0.24 5.75

3.42 5.46

C21H28O5 C20H32O5

ESI+ ion mode 312.260 eicosanoic acid; arachidic acid 433.304 glycolithocholate 328.236 stanozolol 360.227 352.226 a

cortisone (5Z)-(15S)-11α-hydroxy-9,15dioxoprostanoate

score (out of 100)

pathway linoleic acid metabolism; fatty acid biosynthesis bile acid synthesis progesterone, androgen and estrogen receptor agonists/ steroid biosynthesis prostaglandins, glucocorticoid synthesis eiconosoid pathway; prostaglandin synthesis

a

30 22−74 a

31


Table 4. (a) Significant Metabolites (p < 0.05, after Correction; Fold Change >2) as Detected and Identified from Liver Tissues of Zebrafish; (b) Significant Metabolites (p < 0.05, after Correction; Fold Change >2) as Detected and Identified from Brain Tissues of Zebrafish (a) neutral mass ESI+ ion mode 432.362 ESI− ion mode 382.272 276.209 (b) neutral mass

common name calcitetrol 7 dehydrocholesterol 6,9,12,15-octadecatetraenoic acid common name

ESI+ ion mode 455.221 20-hydroxyleukotriene E4 350.232 3α,20α,21-Trihydroxy-5β-pregnane11-one 386.327 5β-cholestan-3-one 386.401 lathosterol a

retention time

fold change

chemical formula

pathway

3.45

7.54

C27H44O4

bile acid synthesis

2.74 4.86 retention time

4.56 3.44 fold change

C27H42O C18H28O2 chemical formula

1.82 5.69

5.46 2.12

C23H37NO6S C20H30O5

arachidonic acid metabolism steroid hormone synthesis

49 26

1.41 1.39

3.45 4.56

C27H46O C27H46O

steroid hormone synthesis steroid hormone synthesis; bile acid synthesis

77 77

steroid biosynthesis α-linolenic acid metabolism pathway

score (out of 100) 71 a

82 score (out of 100)


route of exposure would mainly lead to the toxin uptake by gills (inhalation) and to some extent (or equally) through dermal surface.34 This route of exposure implies very active absorption of MCLR and MCRR through the gills and distribution in the body bypassing the first-pass metabolism. Normally, the firstpass effect (also known as first-pass metabolism or presystemic metabolism) is a phenomenon whereby the concentration of a chemical species following its entry into fish is greatly reduced before it reaches the systemic circulation.35,36 The first pass usually takes places when the exposure route is oral. In the present study, fish exposure to toxins is mainly through dermal (via skin) and inhalation (via gills), in which case liver is bypassed. The MCs are therefore taken up by the gills and systemic circulation and then distributed to the different organ systems including liver. Liver is a main detoxifying organ for MCs since liver contains glutathione (GSH), which is known to form a conjugate with MCs (GST enzyme transfers the GSH moiety) and produce soluble complexes which can then be eliminated via excretory routes.37 Kondo et al. (1996) demonstrated that MCs are transformed in the liver cytosol (animal studies) to a more polar metabolite with a dose- and exposure time-dependent depletion in the glutathione pool.38 A recent study from our research group20 also reported increase in GST (glutathione-S-transferase) activities upon continued balneation exposure for more than 4 days (MCLR/MCRR) in liver tissues of zebrafish. GST conjugates with MCs resulting in a more polar metabolite which is then eliminated from the body of an organism.

one biochemical pathway would lead to concomitant changes in other pathways thus affecting the overall metabolism of an organism. These changes at biochemical levels would affect general physiological processes in an organism, resulting in changes at higher levels of the population pyramid.32 Inhibition of PP 1 and PP −2A, following the exposure of zebrafish to MCs, results in cascading events of metabolic disruption starting from glycogen and glucose-related pathways and consequently affecting the lipid networks. Figure 1 illustrates the linkages between different lipid class pathways that were identified to be perturbed in zebrafish tissues following the exposure to MCLR and MCRR. It was also observed that MCLR exposure resulted in increased perturbations of metabolites and hence disrupted more lipid biochemical pathways as compared to MCRR exposure at the same dose (Table 1). Since it is known that MCLR is more toxic than MCRR, variations thus observed in the metabolic perturbations could be attributed to the differences in the toxicity potencies of the two MCs. However, both of them are synthesized and released by the same cyanobacteria, i.e., Microcystis aeroginosa and often occur together in a bloom episode.26,33 Apart from the metabolic variations as observed after exposure of zebrafish tissues to MCLR and MCRR, it was also noted that tissues of gills had the highest number of perturbed metabolites as compared to those in other tissues (Tables 2−4). This observation could be explained based on the route of exposure involved in the present study. Balneation 14380



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Since liver is the major detoxification organ for MCs,38 any exposure route that by-passes liver for metabolism would thus increase the bioavailability of MCs to other organs. Increased bioavailability and residence time would in turn accelerate the rate of metabolic perturbations thus resulting in disturbance in the entire biochemical cycle in the exposed fish. Tables 2−4 provide different pathways that belong to the class of lipids which were affected in various zebrafish tissues following the balneation exposure to MCLR and MCRR. As shown in Table 1, a total of 14 pathways linked to lipid metabolism were affected as a consequence of metabolite perturbations in zebrafish tissues. Five of these identified pathways belong to the class of essential fatty acids and related compounds (linoleic acid metabolism, α-linoleic acid metabolism, arachidonic acid metabolism, eiconosoid metabolism, and prostaglandins). Linoleic acid, α-linoleic acid and arachidonic acid are essential fatty acids which are required for the functioning of important biological processes in fish apart from acting as fuel.39 It is well established that fish require these long-chain highly unsaturated fatty acids (HUFA) for normal growth and development, including reproduction. 40 As mentioned, these fatty acids play important physiological roles in fish as components of membrane phospholipids and as precursors of biologically active eicosanoids.41,42 Prostaglandins, the subclass of eicosanoids, are members of lipid compounds which have been shown to play essential roles in the development and reproductive behavior of fish.43,44 In addition, eicosanoids in general might also influence the immune system of fish.45 In particular, prostaglandins have been reported to affect the immunosuppressive behavior.45 Arachidonic acid is the major eicosanoid precursor in fish cells,46 which is also present in the phospholipids that (especially, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositides) are integral component of all membranes in the body’s cells. Arachidonic acid is particularly abundant in the brain, muscles, and liver.47 In addition to being involved in cellular signaling as a lipid secondary messenger (involved in the regulation of signaling enzymes, such as PLC-γ, PLC-δ, and PKC-α, -β, and -γ isoforms), arachidonic acid is a key inflammatory intermediate.46 Owing to the important physiological roles of these essential fatty acids, i.e., arachidonic acid, linoleic acid, and α-linoleic acid, they are known to play a key role in maintaining the overall health of the fish.48,49 Thus, any disturbance in the metabolism of these essential and important fatty acids could affect a vast number of cellular and biological processes. Prostaglandin synthesis and eicosanoid metabolism were also affected upon exposure to MCLR and MCRR in the present study (Tables 1−4) which could be due to the disruption of essential fatty acid synthesis as described above; these fatty acids are precursor molecules for prostaglandin synthesis. Apart from affecting essential fatty acids and their related compounds, zebrafish exposure to MCLR and MCRR also resulted in perturbation of cholesterol biosynthesis (Table 1). The perturbation in cholesterol synthesis further affected bile acid synthesis and steroid hormones synthesis downstream. Figure 2 presents the conserved cholesterol biosynthesis pathway as described for zebrafish50 with the list of perturbed metabolites as observed in the present study. As mentioned earlier, molecular basis of MCs toxicity is inhibition of PP-1 and PP-2A. These phosphatases are responsible for normal functioning of glycolytic and glucose-related pathways. End product of glucose breakdown (through pyruvate dehydrogenase complex), i.e., acetyl-CoA,

Figure 2. Cholesterol biosynthesis pathway in zebrafish (adapted from Thorpe et al., 2004). Solid blocks in the schematic represent the perturbed metabolites as found in the present study.

serves to maintain a balance between glucose and fat metabolism. Apart from maintaining the balance, acetyl CoA also serves as a precursor molecule for cholesterol synthesis which is required for production of bile and steroid hormones. 50 Both steroids hormones and bile acids are endocrine signaling molecules, which have very important roles to play in maintaining important physiological functions in fish. 51As can be seen from the figure, perturbations in cholesterol synthesis lead to adverse effects for synthesis of other important biochemical compounds as well. Steroid hormones such as progesterone, testosterone, and estradiol are derived from cholesterol, as mentioned previously. Steroid hormones can be classified into five groups by the receptors to which they bind: glucocorticoids, mineralocorticoids, androgens, estrogens, and progestogens.52 Steroid hormones help control metabolism, inflammation, immune functions, salt and water balance, development of sexual characteristics, and the ability to withstand illness and injury.53 Most of the male and female reproductive hormones belong to the class of steroid hormones.52 Perturbation of the metabolic pathways involved in their synthesis could have serious ecological consequences, for instance, change in sexual characteristics and behavior. A number of chemical compounds, known for their endocrine disrupting action (endocrine disrupters), can affect the action of these steroid hormones.54 Endocrine disrupters usually act as steroid agonists and bind to the receptor proteins. It has been reported that MCLR tends to mimic an endocrine disruptor in human breast carcinoma cells and zebrafish larva.55 Weak estrogenic effects on larval zebrafish have been observed, as demonstrated by up-regulation of genes produced in the liver in response to Microcystis aeroginosa.56 Endocrine disruption caused by MCs could thus have adverse impacts on aquatic ecosystems as a whole, and also affect the terrestrial environment, including birds and mammals. Bile acids function as physiological detergents that facilitate the absorption and digestion of lipid-soluble nutrients.57 Any impairment in bile acid synthesis could cause disturbance in the 14381



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entire lipid metabolism as a whole.58 In the present set of experiments, lipid oxidation, fatty acid elongation, and synthesis of unsaturated fatty acids were found to be perturbed in different organs, as shown in Tables 2−4. Apart from regular lipids, special lipids (phospholipids) were also observed to be affected by the balneation exposure. Glycerophospholipid, which is a member of phospholipids family, was affected in the case of fish exposed to MCRR. These lipids are components of biological membranes, and are essential for the integrity of the cell membranes. If the pathways concerning these phospholipids are altered, then they could in turn result in altered and impaired cell functions. Bile acids were initially considered to play a simple dietary role after meal, but recent evidence suggest that these acids are endocrine signaling molecules that activate multiple nuclear and membrane receptor-mediated signaling pathways to regulate integrative metabolism and energy balance.59,60 Hence, apart from disrupting the entire lipid metabolism, irregularities in the synthesis of bile acids can also impair other signaling pathways as well that could lead to alterations in the entire metabolome. From the results obtained in the present study, it is evident that both MCLR and MCRR have a potential to disrupt major lipid metabolic pathways in zebrafish tissues (representative pathway maps as obtained from KEGG are provided in the SI, refer to Figures S2−S4). Some of the affected lipid metabolites such as steroid hormones could have major ecotoxicological implications as described previously. MCs are known to inhibit phosphatases which are involved in the activation and deactivation of enzymes involved in major biochemical pathways. However, till-date research has not been carried out to explore the nature and extent of metabolic implications that could arise as a result of this enzyme inhibition. This is the first report that presents preliminary insights into the interlinked metabolic disruptions that resulted from the balneation exposure of MCLR and MCRR to zebrafish. However, more in-depth and detailed investigations are required to confirm and validate these findings to provide further insights into metabolic changes taking place in fish after exposure to MCs. MCs have been known as tumor promoters under acute conditions which often lead to fatal consequences. Results obtained from the present study reveal the possible effects of MCLR and MCRR under chronic conditions where no apparent lethality is observed. More investigations are warranted in this research area to understand the effects of such an exposure on other classes of metabolites such as polar metabolites such as carbohydrates and amino acids. Data obtained from these experiments could help the scientific community in gaining a better understanding of the integrative metabolic changes resulting from MCs exposure. Research is underway in our laboratory to further enhance the current state of knowledge in the area of metabolomics in order to identify potential biomarkers following the chronic and subchronic exposure of zebrafish to other MCs such as MCYR (MCTyrosine-Arginine), MCLA (MC-Leucine-Alanine) and MCLF (MC-Leucine-Phenyl alanine). In addition, we are exploring the use of high-throughput omic platforms such as transcriptomics and proteomics to gain more insights into toxicity mechanisms of MCs under chronic conditions.

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ASSOCIATED CONTENT

S Supporting Information *

Representative pathway maps (as obtained from KEGG). This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +65-6516-5135; fax: +65-6774-4272; e-mail: ceerbala@ nus.edu.sg. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Singapore-Delft Water Alliance (SDWA) for the financial support extended to this project and Professor Gong Zhiyuan of the Department of Biological Sciences for sharing his laboratory facilities in support of the fish exposure studies.

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REFERENCES

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