OPEN ACCESS DOCUMENT

Information of the Journal in which the present paper is published:

  • ELSEVIER Journal Science of The Total Environment 2016, 545, 127-136
  • DOI: 10.1016/j.scitotenv.2015.12.097

TITLE PAGE:

Compounds alteringfat storage in Daphnia magna

Rita Jordão1,2,3, Elba Garreta 1, Bruno Campos1, Marco F. L. Lemos 2,3, Amadeu M.V.M. Soares2, Romà Tauler1, and Carlos Barata1*

1Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDAEA), Spanish Research Council (IDAEA, CSIC), Barcelona, Spain

2Centre for Environmental and Marine studies (CESAM), Department of Biology, University of Aveiro, Aveiro, Portugal

3Marine and Environmental Sciences Centre (MARE), ESTM,, Polytechnic Institute of Leiria, Peniche, Portugal

*Address correspondence to Carlos Barata, Institute of Environmental Assessment

and Water Research (IDAEA-CSIC), Jordi Girona 18, 08034 Barcelona, Spain.

Telephone: ± 34-93-4006100. Fax: ± 34-93-2045904.

E-mail:

Keywords: obesogen, lipid disruptor, nuclear receptor, arthropod, reproduction, juvenile receptor
Abstract

The analysis of lipid disruptive effects in invertebrates is limited by our poor knowledge of the lipid metabolic pathways. A recent study showed that tributyltin activated the ecdysteroid, juvenile hormone and retinoic X receptor signaling pathways, and disrupted the dynamics of neutral lipids in the crustacean Daphnia magna impairing the transfer of triacylglycerols to eggs and hence promoting their accumulation in post-spawning females. Tributyltin disruptive effects correlated with lower fitness for offspring and adults. The present study aims to addresses effects of existing compounds on storage lipids in post-spawning females and their health effects.D. magnaindividualswere exposed 12 chemicals that included vertebrate obesogens (tributyltin, triphenyltin, bisphenol A,nonylphenol, di-2-ethylhexyl phthalate), other contaminants known to affect arthropods (pyriproxyfen, fenarimol, methoprene, emamectin benzoate and fluoxetine), as well as the natural hormones methyl farnesoateand 20-hydroxyecdysone. Reproductive effects were also assessed. Quantitative changes in storage lipids accumulated in lipid droplets were studied using Nile red staining, which showed a close relationship with whole organism levels of triacylglycerols. Ten compounds alteredstorage lipids in a concentration related manner enhancing (tributyltin, bisphenol A, methyl farnesoate,pyriproxyfenand20-hydroxyecdysone) or decreasing (nonylphenol, fenarimol, emamectin benzoate, methoprene and fluoxetine) their levels in post-spawning females.Eight compounds that altered lipid levelsalso had detrimental effects on growth and/ or reproduction.

1.Introduction

The maintenance of energy reserves is vital, so animals stored energy as fat to survive food shortages. When food is not limiting disruption of lipid storage equilibrium may ultimately lead to obesity. Recent studies have suggested the involvement of endocrine disrupting chemicals in mammal obesity epidemic (Grün and Blumberg, 2006). Obesity increases the risk of coronary artery diseases, diabetes and related health detrimental effects, such as hypertension and lipidemia (Grün and Blumberg, 2006; Sharma and Staels, 2007). There are widely use chemicals that at low doses can promote weight gain. The list is extensive and includes chemicals such as phthalates, bisphenol A (BPA), alkylphenols and organotin pesticides (Grün and Blumberg, 2009).

Obesogenic effects have often been associated to the disruption of peroxisome proliferator-activated receptor (PPARγ) signaling pathway, that together with its heterodimeric partner retinoid X receptor (RXR), are master regulators of adipocyte differentiation and lipid metabolism in vertebrates (Grün and Blumberg, 2009). Although PPAR has not been described outside deuterostomes, a recent study showed that the crustacean Daphnia magna responds to the obesogen tributyltin, through the RXR signaling cascade, thus, increasing the scope of the search for obesogenic effects (Jordão et al., 2015). These authors showed that tributyltin altered the dynamics of neutral lipids impairing the transfer of triacylglycerols to eggs and hence promoting their accumulation inside of lipid droplets in post-spawning adult females. Lipid droplets were visualized using Nile red (Jordão et al., 2015). Tributyltin disruptive effects on lipids correlatedwith the lower fitness ofoffspring and adults. Gene transcripts indicated that tributyltin increases mRNA levels of the retinoic X receptor gene (RXR), and enhanced those of key genes belonging to the signaling pathways of the ecdysteroid (EcR) and methyl farnesoate hormone (MfR) receptors (Jordão et al., 2015).

However, there are other mechanisms of endocrine disruption of lipid regulation besides those based on PPAR and RXR signaling pathways (Grün and Blumberg, 2009). Obesogens may also disrupt neuroendocrine signaling pathways that control appetite (i.e. neuropeptide Y that stimulates appetite) and energy homeostasis among other processes. Psychiatric drugs such as serotonin reuptake inhibitors, bisphenol A, nonylphenol and some phthalates also affect the expression of neuropeptide Y an hence change feeding behaviour in rodents (Grün and Blumberg, 2009).

D. magna is a long established model species in regulatory toxicology with an important role in determining chemical safety criteria around the world, and is the most commonly used system for ecotoxicological testing worldwide. Daphnia shares the most number of genes with human than any other sequenced invertebrate (Colbourne et al., 2011), which also includes several molecular mechanisms that regulate fat storage. Daphnia has a functional RXR receptor that is activated with the model obesogen tributyltin, the cellular fuel gauge mechanisms (TOR), protein kinase complex regulating fatty acid catabolism and glycolysis (AMP-activated kinase), and neuroendocrine regulators such as insulin, serotonin, dopamine and neuropeptides that regulate food-related behavior and energy metabolism (Boucher et al., 2010; Campos et al., 2013; Campos et al., 2012a; Dircksen et al., 2011; McCoole et al., 2012; Sheng et al., 2012; Wang and LeBlanc, 2009; Wang et al., 2007). D. magna also shares additional potential mechanisms of fat regulationwith insects. In D. magna like in other crustacean and arthropods, storage lipid dynamics varied along the moult and reproduction cycle (Tessier and Goulden, 1982) that are regulated by the ecdysteroid and juvenile hormone receptor signaling pathways (hereafter referred as methyl farnesoate). D. magna has a functional HR96 receptor, which in Drosophila has a key role in dietary fat utilization and metabolism (Karimullina et al., 2012). Ecdysone, exert its effects through the interaction with EcR, known to heterodimerize with RXR and to bind to the promoters of ecdysone-regulated genes (LeBlanc 2007; Wang and LeBlanc 2009). Recent findings indicate that theMfR in Daphnia is a complex of two nuclear proteins of the bHLH-PAS family of transcription factors: the methoprene-tolerant receptorand steroid receptor activator protein (LeBlanc et al., 2013; Miyakawa et al., 2013). Tributyltin, which is an agonist of RXR together with methyl farnesoate and other juvenoids, enhanced the ecdysteroid-dependent activation of the EcR: RXR heterodimer (Wang and LeBlanc 2009). Transactivation assays also showed that the nuclear HR96 receptor was activated by juvenoids and it was repressed by psychiatric drugs (e.g. fluoxetine) that targeted neuroendocrine systems (Karimullina et al., 2012). Thus, vertebrate and non -vertebrate endocrine disrupting chemicals may alter lipid homeostasis in the crustacean D. magna by interacting with RXR, EcR, MfR receptors and/or neuroendocrine signaling pathways.

The quantity and quality of ingested food together with body size are key determinats of fat storage in Daphnia(Goulden and Place, 1993; Guisande and Gliwicz, 1992; Martin-Creuzburg and von Elert, 2009; Tessier and Goulden, 1982; Wacker and Martin-Creuzburg, 2007). Food intake also regulates the amount of resources invested to growth and reproduction and is the undelayed mechanisms behind many reported toxic effects on reproduction in Daphnia(Barata and Baird, 2000; Barata et al., 2004). This means that endocrine disruption of fat storage in D. magna and its life-history consequences have to be assessed at lower concentrations than those impairing growth and food intake.

The aim of this study is to assess chemical alterationof storage lipids (i.e. triacylglycerols) accumulated in lipid droplets in D. magna and its effects in life-history responses. Thedeparture hypothesis t is that many compounds may behave like tributyltin(Jordão et al., 2015), alteringthe transfer of storage lipids to eggs, promoting their accumulation inside lipid droplets in post-spawning females, and hence affecting growth and reproduction. It washypothesized that vertebrate and non -vertebrate chemicals that are known to interact with RXR, EcR, MfR receptors and/or neuroendocrine signaling pathways may alter lipid homeostasis in the crustacean D. magna. Nile red fluorescence assay was used to quantify storage lipids inside lipid droplets since this measure is closely related to accumulated triacylglycerols in lipid droplets (Jordão et al., 2015).

2. Material and Methods

2.1.Studied compounds

Studied compounds included two natural hormones (LeBlanc, 2007): the juvenile hormone methyl farnesoate (CAS 10485-70-8) and the moulting hormone 20-hydroxyecdysone (CAS 5289-74-7); pesticides that act as juvenoids like pyriproxyfen (CAS 95737-68-1), methoprene (CAS 40596-69-8); agonists of RXR like tributyltin (CAS 1461-22-9) and triphenyltin (CAS 639-58-7)(LeBlanc, 2007); the ecdysone agonist emamectin benzoate (CAS 155569-91-8) (Rodríguez et al., 2007); the ecdysone antagonist fenarimol (CAS 60168-88-9) (Mu and LeBlanc, 2004b); contaminants with reported obesogenic effects: nonylphenol (CAS 84852-15-3), bisphenol A (CAS 80-05-7),di-2-ethylhexyl phthalate(CAS 117-81-7) (Grün and Blumberg, 2006; Hao et al., 2013), and the fat regulator fluoxetine (CAS 56296-78-7) (Lemieux et al., 2011). All the compounds were obtained from Sigma Aldrich (U.S.A/Netherlands) except methyl farnesoate, which was supplied by Echelon Bioscience, Utah, U.S.A.

2.2. Experimental animals

All experiments were performed using the well-characterized single clone F of D. magna maintained indefinitely as pure parthenogenetic cultures(Barata and Baird, 1998).Photoperiod was set to 14 h light: 10 h dark cycle and temperature at 20 ±1 °C.Individual cultures were maintained in 100 ml of ASTM hard synthetic water at high food ration levels (5x105 cells/ml of Chlorella vulgaris, respectively), as described in Barata and Baird(1998).

2.3. Experimental design

Experiments were initiated with newborn neonates <4-8 h old obtained from synchronized females cultured individually at high food ration levels. Groups of five neonates were reared in 100 ml of ASTM hard water under high food ration conditions until the end of the third juvenile instar (about 4-8 h before moult for the third time). At this point, juveniles were exposed individually in 100 mL test medium to selected chemicals and used in lipid droplet (Nile red) and life-history experiments (figure S1). Exposures were conducted duringthe adolescent instar (i.e. 3 days), which is the instar where the first brood of eggs isformed in the ovaries. Females used for lipid droplet analyses were sampled just after their fourth moult and having released their first clutch of eggs into the brood pouch. For the life history trait analyses, exposed females were cultured for an additional instar period - till the next one in control conditions, without pollutants, to allow eggs to develop and be released as neonates in the first clutch after the fifth moult (the test procedure is depicted in figure S1). Test media were renewed every other day. The studied chemicals were assayed at concentrations not impairing survival, moulting or food acquisition. Therefore, for each compound toxicity tests were performed to establish the tested concentration rank.Three sets of experiments were performed:

2.3.1Toxicity tests

Toxicity tests were conducted using the experimental design as described above, which means that the tests were initiated with adolescent instar and ended when females just moult and deposited their first brood into their brood pouch. Measured responses included survival, delay on moulting and feeding rates. Feeding experimentswere limited to 24 h period between the third and fourth moult.. Under our culture conditions females usually enter the adolescent instar in their third moult. Feeding assays followed previous procedures (Barata et al., 2006). Briefly, groups of five adolescent instar females were transferred into test vessels filled with 100 mL of the appropriate treatment solution plus food. Concentration solutions of each compound are depicted in figure 1. Three replicate test vessels filled with the same culture medium but with no animals were used as blank replicates to measure that algal densities did not increase during feeding assays. The mean initial algal cell concentration of the experimental vessels were measured at the start and end of tests. Cell density was estimated from absorbance measurements at = 650 nm in a dual-beam spectrophotometer (Uvikon 941) using standard calibration curves based on at least 20 data points, with an r2> 0.98.

Figure 1. Feeding inhibition responses of the studied compounds. Each symbol means a single observation. Fitted regression curves are also depicted.Axis x is in log scale.

Individual feeding rates (cells x animal-1 x h-1) were determined as the change in cell density during 24 h according to the method given by (Allen et al., 1995) and converted to feeding inhibition relative to control treatments (%).Median concentration effect estimateswere obtained by fitting percentage values to eq. 1 and setting the Max value to 100.

2.3.2. Storage lipid assays

Effects of single exposures on the accumulation of storage lipids into lipid droplets were performed, using from 4 to 9 concentrations of the 12 studied compounds (x 10 replicates per treatment) to fully define concentration-response curves, which were dosed using acetone as a carrier (0.1 mL/L). A solvent control containing the same amount of acetone was then included for comparison purposes. Non- solvent controls were not used since previous studies showed that the tested acetone concentrations did not affect lipid droplets neither life-history responses (Jordão et al., 2015). At the end of exposures animals were sampled, their body length measured and then processed for lipid droplet quantification using Nile red fluorescence according to previous procedures (Jordão et al., 2015).

2.3.3 Life-history assays

The third experiment studied effects of exposures of the tested chemicals on the body length of first reproducing females and on the number and size of offspring of the first clutch. Following exposures, females were cultured individually in 100 mL of ASTM hard water at high food conditions without contaminants and the number and size of the offspring released in the first clutch wasassessed. Two to three exposure concentrations (shown in Table 3), were selected for each tested chemical that included measured low and high effects on storage lipid accumulation into lipid droplets. Treatments were replicated ten times. Body length measurements were performed from the head to the base of the spine using a Nikon stereoscope microscope (SMZ 150, Nikon, Barcelona, Spain) and the ImageJ software (

2.4 Nile red assay to quantify storage lipids into lipid droplets

Quantification of storage lipids into lipid droplets follow previous methods developed for D. magna(Jordão et al., 2015). Briefly,Nile red stock solutions were prepared in acetone and store protected from light. Prior to use, the working solution was obtained by dilutingthe stock solution to 1.5 µM in ASTM. Live individuals were then exposed to Nile red working solution in the dark for 1 h at 20 ºC. After incubation, animals were washed in ASTM and further processed to visualized or/and quantify fluorescence. The same animals were also used for visualization of lipid droplets. Images were takenin the surrounding areaof the midgut. Observations and fluorescence images were obtained using a Nikon SMZ1500 (Nikon, Japan) microscope and Nikon Intensilight C-HGFI with a GFP filter (EX 472/30, EM 520/35). The images were captured with a Nikon digital Sight DS-Ril camera and NIS Elements AR software (version 3.0). Quantification of Nile red fluorescence was conducted as follow: organisms wereplaced individually in 1.5 ml centrifuge tubes, the remaining water removed, and were then sonicated in 300 µl of isopropanol. The homogenized extract was then centrifuged at 10000 g. Two hundred µl of supernatant was used to measure Nile red fluorescence using an excitation/emission wavelength 530/590 nm and a microplate fluorescence reader (Synergy 2, BioTek, USA). For each quantification and treatment 10 blanks (animals not exposed to Nile red) were used to account for background levels of fluorescence, which were negligible.

2.5. Determination of triacylglycerols

Triacylglycerol levels in post-spawning females from controls and those exposed to low and high concentrations oftributyltin,bisphenol A, methyl farnesoate, 20-hydroxyecdysone , pyriproxyfen and fenarimolwere also measured and related to measured levels of Nile red (figure 4). We restrict analyses to the six chemicals that showed greatest variation in fluorescence. Concentrations of triacylglycerols were determined using a commercial Kit (Spinreact S.A., Sant Esteve De Bas, Spain) based on a peroxidase coupled method for the colorimetric detection of triacylglycerols followed hydrolysis, phosphorylation of glycerol and oxidation to produce hydrogen peroxide (McGowan et al., 1983). Briefly pools of five de-brooded adolescent D. magna individuals that just released their first clutch of eggs into the brood pouch were homogenized in 250 µl of 0.1 M phosphate buffer (pH 7.2) with 2, 6-di-tert-butyl-4-methylphenol (BHT) 0.01 %, as an antioxidant. A volume of the supernatant (20 µl) was then mixed with the assay reagents, incubated in the dark for 10 min and measured spectrophotometrically at 505 nm according to the manufacturer’s protocol. Quantification of triacylglycerol levels was performed from a triacylglycerol calibration standard curve of 8 points (r2>0.99) also following the manufacturer’s protocol.

2.6. Chemical analysis

Dissolved oxygen concentration (DO) was measured using an oxygen electrode model 1302 (Strathkelvin Instruments, Glasgow) and pH was measured using an epoxy-body combination electrode, coupled to a micro pH 2001 meter(Crison, Spain) and calibrated with standard pH buffer solutions (Sigma, Madrid, Spain). Mean oxygen levels were 96 ± 2.4 % of saturation and pH values 7.2 ± 0.2 for all experiments.

The water residue analyses of most tested compounds in freshly prepared and old (24 h) test solutions were restricted to two exposure levels that included low and high concentrations (Table S2). Actual triphenyltin and tributyltin concentrations in test solutions were measured as total Sn by means of Perkin Elmer model Elan 6000 inductively coupled plasma mass spectrometer (ICP-MS) (Barata et al., 2005). Stability of the rest compounds during the test was confirmed using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Fluoxetine residues were analysed following previous procedures(Campos et al., 2012a) using a Waters 2690 HPLC separations module (Mildford, MA, USA) equipped with a Purospher Star RP-18 endcapped column (125mm×2.0 mm, particle size 5 µm, Merck, Darmstadt, Germany) connected to a Micromass Quattro triple quadrupole mass spectrometer equipped with a Z-spray electrospray interface (Manchester, UK). The rest of chemical residues were measured using an Acquity Ultra Performance LC system (Waters, Mildford, MA, USA) connected to a Triple Quadruple Detector Acquity. The analysis was performed on an Acquity UPLC BEH C18 (1.7 µm, 2.1 x 100 mm) supplied by Waters (Waters, Mildford, MA, USA). The C18 column was equilibrated at 40°C. Chromatographic separation was carried out with a mobile phase consisting of methanol with 0.1% formic acid (eluent A) and MilliQ water with 0.1% formic acid (eluent B) at a flow rate of 0.3 mL/min. The elution started with 20% of eluent A, followed by a 4-min gradient to 75% of eluent A and a 2-min gradient to 100% of eluent A, held for 1 min, and then back to the initial conditions within 6 min. An injection volume of 50 µL was used.