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UWS Academic Portal Physiological and behavioral effects of exposure to environmentally relevant concentrations of prednisolone during zebrafish (Danio rerio) embryogenesis McNeil, Paul L.; Nebot, Carolina; Sloman, Katherine Published in: Environmental Science and Technology DOI: 10.1021/acs.est.6b00276 E-pub ahead of print: 28/04/2016 Document Version Peer reviewed version Link to publication on the UWS Academic Portal Citation for published version (APA): McNeil, P. L., Nebot, C., & Sloman, K. (2016). Physiological and behavioral effects of exposure to environmentally relevant concentrations of prednisolone during zebrafish (Danio rerio) embryogenesis. Environmental Science and Technology, 50(10), 5294-5304. https://doi.org/10.1021/acs.est.6b00276 General rights Copyright and moral rights for the publications made accessible in the UWS Academic Portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Take down policy If you believe that this document breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 05 Feb 2020

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Page 1: UWS Academic Portal Physiological and behavioral …...4 95 physiological processes such as circadian rhythmicity16, metabolism17,18 and growth19,20 96 during early life stages. 97

UWS Academic Portal

Physiological and behavioral effects of exposure to environmentally relevantconcentrations of prednisolone during zebrafish (Danio rerio) embryogenesisMcNeil, Paul L.; Nebot, Carolina; Sloman, Katherine

Published in:Environmental Science and Technology

DOI:10.1021/acs.est.6b00276

E-pub ahead of print: 28/04/2016

Document VersionPeer reviewed version

Link to publication on the UWS Academic Portal

Citation for published version (APA):McNeil, P. L., Nebot, C., & Sloman, K. (2016). Physiological and behavioral effects of exposure toenvironmentally relevant concentrations of prednisolone during zebrafish (Danio rerio) embryogenesis.Environmental Science and Technology, 50(10), 5294-5304. https://doi.org/10.1021/acs.est.6b00276

General rightsCopyright and moral rights for the publications made accessible in the UWS Academic Portal are retained by the authors and/or othercopyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated withthese rights.

Take down policyIf you believe that this document breaches copyright please contact [email protected] providing details, and we will remove access to thework immediately and investigate your claim.

Download date: 05 Feb 2020

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Physiological and behavioural effects of exposure to environmentally relevant 1

concentrations of prednisolone during zebrafish (Danio rerio) embryogenesis 2

3

Paul L. McNeila*

, Carolina Nebotb and Katherine A. Sloman

a. 4

5

a Institute of Biomedical and Environmental Health Research, School of Science and Sport, 6

University of the West of Scotland, Paisley, UK 7

bDepartment of Analytical Chemistry, Nutrition and Bromatology, Faculty of Veterinary 8

Medicine, University of Santiago de Compostela, Lugo, Spain 9

10

*Corresponding author: 11

School of Science and Sport 12

University of the West of Scotland 13

Paisley, 14

Scotland, PA1 2BE 15

Email: [email protected] 16

Tel +44 (0)141 848 3252 17

Fax +44 (0)141 848 3289 18

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Abstract 31

32

The presence of synthetic glucocorticoids within the aquatic environment has been 33

highlighted as a potential environmental concern as they may mimic the role of endogenous 34

glucocorticoids during vertebrate ontogeny. Prednisolone is a commonly prescribed synthetic 35

glucocorticoid which has been repeatedly detected in the environment. This study 36

investigated the impact of environmentally relevant concentrations of prednisolone (0.1, 1 & 37

10 µg/l) during zebrafish embryogenesis using physiological and behavioural endpoints 38

which are known to be mediated by endogenous glucocorticoids. The frequency of 39

spontaneous muscle contractions (24 hpf) was significantly reduced by prednisolone and 0.1 40

µg/l increased the distance embryos swam in response to a mechanosensory stimulus (48 41

hpf). The percentage of embryos hatched significantly increased following prednisolone 42

treatment (1 & 10 µg/l), while growth and mortality were unaffected. The onset of heart 43

contraction was differentially affected by prednisolone while heart rate and oxygen 44

consumption both increased significantly throughout embryogenesis. No substantial effect on 45

the axial musculature was observed. Morphological changes to the lower jaw were detected at 46

96 hpf in response to 1 µg/l of prednisolone. Several parameters of swim behaviour were also 47

significantly affected. Environmentally relevant concentrations of prednisolone therefore 48

alter early zebrafish ontogeny and significantly affect embryo behaviour. 49

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Introduction 63

64

The presence of pharmaceuticals within the aquatic environment has emerged as a significant 65

contributing factor to the degradation of aquatic ecosystems.1,2

However, the majority of 66

toxicological studies which have attempted to assess the environmental impact of 67

pharmaceuticals have largely overlooked synthetic glucocorticoids (SGs).2,3

Synthetic 68

glucocorticoids are used in treating a variety of autoimmune and inflammatory diseases4 and 69

are often excreted without being transformed into inactive metabolites3. The addition of 70

chlorine and fluorine molecules is a common feature of SGs which increases their stability 71

and so may also increase their longevity within the environment.4 Breakdown of SGs can also 72

yield secondary compounds which are in fact more toxic than parent molecules.5 Considering 73

the large quantities of these compounds which are prescribed annually6, and the relatively 74

low removal rates of specific SGs from sewage management sites7, chronic exposure to SGs 75

may potentially produce significant toxic effects during early development. Therefore, SGs 76

have been highlighted as a priority for future toxicological testing.8 77

78

Prednisolone is the second most commonly prescribed SG in the UK8 and has been detected 79

within the aquatic environment at relatively low concentrations9. Schriks et al.

6 found 80

concentrations of prednisolone within industrial and hospital waste water ranged between 81

approximately 0.25 and 2 µg/l. Prednisolone has also been reported to have a relatively low 82

removal rate from sewage treatment plants compared to other commonly occurring SGs; 83

around 20% less.7 Prednisolone was first prioritised for toxicological analysis by Besse and 84

Garric10

based upon the potential environmental risk and yet few studies have since attempted 85

to assess its toxicity. Concentrations of prednisolone as low as 1 µg/l have been shown to 86

significantly decrease the number of leukocytes within adult fathead minnows (Pimephales 87

promelas) and significantly increase plasma glucose levels.11

These changes are analogous to 88

those effects associated with cortisol, the principal glucocorticoid in fish.12

Synthetic 89

glucocorticoids therefore appear to mimic the role of endogenous hormones. Synthetic 90

glucocorticoids are potent glucocorticoid receptor (GR) agonists which allows them to 91

directly affect the transcription and expression of specific GR mediated genes.13-15

The 92

effects of SGs may be particularly pronounced during early ontogeny which is dependent 93

upon endogenous glucocorticoid signalling; cortisol is known to regulate several key 94

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4

physiological processes such as circadian rhythmicity16

, metabolism17,18

and growth19,20

95

during early life stages. 96

97

Endogenous glucocorticoids play a particularly prominent role during musculoskeletal 98

development in vertebrates and are responsible for regulating muscle mass21,22

and bone 99

density23

. Sustained exposure to elevated glucocorticoid levels is associated with skeletal 100

muscle atrophy22

, an increase in the expression of myostatin, a negative regulator of muscle 101

mass21

and increased bone resorption and decreased osteoblast production, leading to 102

osteoporosis24

. Knockout of GR in zebrafish (Danio rerio) causes a delay in the formation of 103

somites which display a misshapen phenotype and is associated with a reduction in the 104

expression of several bone morphogenetic proteins.25

The SG dexamethasone significantly 105

alters craniofacial development during embryogenesis in zebrafish due to altered matrix 106

metalloproteinase (MMP) expression.13,14

107

108

Non-functional zebrafish GR morphants are also associated with a reduced swim activity 109

phenotype.26

Glucocorticoid mediated alterations to the development of the musculoskeletal 110

system may therefore have a direct impact upon swimming behaviour. In addition, indirect 111

effects may also occur. Paralysis, associated with neuromuscular disorders and muscular 112

myopathies is connected with a reduction in the mineralisation of bone.27,28

Indeed, 113

contraction of the muscle fibres is not only required for subsequent development of the 114

muscle tissue29

but is also necessary during ossification30

. Studies have demonstrated that 115

swim training may accelerate the rate of ossification during development in fish.31,32

116

Therefore, the musculoskeletal system may not only be susceptible to SGs directly through 117

developmental changes but also indirectly caused by knock on effects via alterations to 118

individual components of the musculoskeletal system and behaviour. Consequently, 119

analysing changes to the ontogeny of swim behaviour may offer an insightful means of 120

assessing the impact of SGs on the musculoskeletal system. 121

122

The zebrafish embryological model offers several advantages to investigate the effects of 123

prednisolone during early development. The onset of endogenous glucocorticoid production 124

i.e. cortisol, has already been characterised33,34

, the expression of those hormones responsible 125

for regulating cortisol production has also been studied35

and GRs identified36

. Pikulkaew et 126

al.37

found that the maternally inherited gr transcript is the most abundant transcript inherited 127

which encodes for nuclear and membrane steroid receptors. Glucocorticoid signalling is 128

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therefore vital during early zebrafish development and is essential for several specific 129

developmental processes including neural, vascular and visceral organ development.37

The 130

development of the musculoskeletal system of zebrafish has also been extensively 131

characterised38-41

and has been utilised as a model system to investigate muscular dystrophies 132

in other vertebrates42,43

. In addition, the rapid development of the axial musculature and 133

associated skeleton facilitates an extensive behavioural repertoire during embryogenesis in 134

zebrafish. 135

136

Understanding the physiological effects of prednisolone exposure during early ontogeny in 137

zebrafish may simultaneously reveal the potential environmental impact of SG 138

pharmaceuticals within the aquatic environment, as well as contributing to our understanding 139

of the function of endogenous glucocorticoids during vertebrate development. Therefore, the 140

aim of this study was to explore the potential physiological and behavioural impact of 141

exposure to environmentally relevant concentrations of prednisolone during zebrafish 142

embryogenesis. We use Balon's44

classification of early life stages of fishes, in which the 143

transition from embryo to larva is defined by the onset of exogenous feeding. All 144

experimentation was conducted using embryos. 145

146

147

Methods 148

149

Embryo collection 150

Adult zebrafish from an existing stock at the University of the West of Scotland (UWS) were 151

maintained in 25 l glass aquaria (28±1°C; 12:12 light-dark) in a 400 l re-circulatory system 152

which used charcoal filtered tap water. Fish were fed twice daily with flake (AQUARIAN) 153

and once daily with Artemia sp. nauplii ad libitum. Males and females were separated prior to 154

spawning when they were mixed within a breeding net allowing collection of embryos. 155

156

157

Exposure protocol 158

Embryos were exposed to 0.1, 1 and 10 µg/l of prednisolone (Sigma, CAS 50-24-8) which 159

represents the upper limits of environmentally relevant concentrations of SGs6. Ethanol was 160

used as a carrier solvent and stock solutions were made in 100% ethanol (Analytical grade, 161

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Sigma, CAS 64-17-5) immediately prior to use and were kept in the dark (4°C). Water and 162

ethanol controls (0.01% ethanol) were also tested. All embryos were continually exposed 163

following fertilisation (from the 2-cell stage) in groups of 50 embryos using static exposures 164

with partial renewal (50%) every 24 h. The 50% of solution which was removed daily was 165

frozen (-20°C) for chemical analysis. A 100 ml volume of system water (water from adult 166

fish systems) was used as an exposure medium for the majority of testing, however, for the 167

purposes of improving survivability into later stages of development, groups of embryos 168

(including controls) which were used to investigate musculoskeletal and behavioural 169

parameters were exposed to prednisolone from the 2-cell stage in 50 ml of embryo media (5 170

mM NaCl, 0.17 mM KCl, 0.33 mM MgSO4.7H20, 0.33 mM CaCl2.2H2O and 10-5

% of 171

methylene blue in distilled water)45

.All embryos were maintained at 28±1°C using water 172

baths and any embryos which failed to develop normally were removed daily and mortality 173

was recorded. 174

175

In the event that dechorination was required to measure specific endpoints before hatching 176

had occurred, only dechorinated embryos were used. Similarly, in the event that freely 177

hatched embryos were required, only freely hatched embryos were used. In both cases, this 178

ensured comparisons between treatments were made on individuals which were at the same 179

stage of development. Replication varied between the individual parameters measured and is 180

therefore stated in the relevant section of the methods, however, a minimum of three beaker 181

replicates of each experimental treatment were tested for all parameters. 182

183

184

Chemical analysis 185

Water samples were analysed at the University of Santiago de Compostela (USC), Spain via 186

liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a previously 187

developed method for analysing pharmaceuticals including corticosteroids46

. Water samples 188

(25 ml) were filtered under vacuum using a 0.47-µm glass microfiber filter, from Filter-Lab 189

(La Rioja, ES). Filtered samples were acidified with 0.1 N-HCl solution to a pH of 3 and 10 190

µl of the IS solution (1 µg/ml) were added. The samples were shaken and transferred into 191

Strata®-X SPE cartridges that were conditioned with 4 ml of methanol (CAS 67-56-1) and 4 192

ml of Milli-Q water. The samples were loaded into the cartridges at a flow rate of 1 ml/min. 193

Cartridges were dried under vacuum for 30 min. The sample vessels were rinsed with 4 ml of 194

methanol which was transferred to the SPE cartridges and was left to soak for 5 min; the 195

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eluent was collected in a 10 ml conical graduated glass Pyrex® tube (16.5×110 mm) with 4 196

ml additional methanol. The final extract was evaporated under nitrogen at 45°C. The dried 197

extracts were reconstituted with 200 µl of 0.1% formic acid (CAS 64-18-1) in methanol, 198

vortexed and transferred into a 300 µl glass insert and fused into an amber 2 ml screw top 199

standard vial (12×32 mm). The extracts were stored at -20°C until further analysis by LC-200

MS/MS. 201

202

Samples were analysed on a LC-MS/MS system consisting of an HPLC 1100 separation 203

module from Agilent Technologies (Waldbronn, Germany), a Qtrap 2000™ mass 204

spectrometer from Applied Biosystems/MDS Sciex (Toronto,Canada) equipped with a 205

TurboIonSpray® source, and the Software Analyst 1.4.1 from Applied Biosystems. The 206

chromatographic analyses were performed by injecting 10 µl of extract into a Synergi 2.5 µm 207

Polar-RP 100A column (50×2.0 mm) connected to a Polar-RP security-guard cartridge 208

(4.0×2.0 mm), both obtained from Phenomenex (Macclesfield, UK). Separation of analytes 209

was achieved using a gradient mixture of two components, 0.1% formic acid in acetonitrile 210

(CAS 75-05-8) and 0.1% formic acid in water. The gradient programme is reported in 211

Iglesias et al.46

212

213

Mass-spectrometry (MS) measurements were performed using positive electrospray, and drug 214

identification was performed using two multiple reaction monitoring transitions and their 215

retention times. The following MS parameters were utilised for prednisolone measurements: 216

declustering potential, 21; entrance potential, 6; collision cell entrance potential, 14; collision 217

energy, 29 and cell exit potential, 4. A source temperature of 400°C, a vacuum gauge of 218

22×104 Pa, an ion spray voltage of 5.5 V and a curtain gas pressure of 17×10

4 Pa were 219

maintained throughout. The first ion source was set at 38×104

Pa, and the second ion source 220

at 34×104 Pa. For all monitored transitions, the dwell time was 10 ms. 221

222

The limits of detection and quantification were 12.5 and 25 ng/l. Preliminary testing found 223

that samples collected at 0 h were 0.1±0.05, 1.1±0.63 and 8.2±0.45 µg/l. No prednisolone 224

was detected within either control group. Unfortunately, the majority of samples collected 225

throughout the experiment defrosted in transit between UWS and USC which caused 226

degradation of the samples. Results are therefore discussed in terms of nominal 227

concentrations of prednisolone at the start of exposure. 228

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229

230

Pre-hatch activity 231

The onset of pre-hatch activity was monitored at 15, 16 and 17 hours post fertilisation (hpf). 232

Embryos were visualised in the chorion within the beakers in which they were maintained 233

under a dissection microscope. Care was taken not to cause any sudden vibrations along the 234

bench which could cause a startle response during observation. Embryos were observed for 1 235

minute after an initial 1 minute acclimation period. The percentage of embryos which 236

displayed stereotypic body contractions at each time point was recorded. Four beaker 237

replicates of each experimental treatment were visualised (n = 4). The frequency of 238

spontaneous contractions at 24 hpf was also recorded using four embryos from each of the 239

four treatment replicates (n = 16). Embryos were observed for 3 minutes after an initial 1 240

minute acclimation period and the mean number of contractions was calculated per minute 241

(contractions per minute, cpm). 242

243

244

Touch evoked escape response 245

Methods were adapted from Smith et al.47

. Individual freely-hatched embryos (48 hpf) were 246

placed into a 6 cm diameter petri dish filled with clean embryo media at 28°C and were 247

viewed under a JVC TH-C1480B colour camera connected to a desktop PC. Embryos were 248

centred under the field of view and were filmed from prior to the onset of the 249

mechanosensory stimulus. The mechanosensory stimulus was applied to the tail of the 250

embryo using a blunt needle tip. Imaging ceased once the embryo had ceased swimming. Still 251

images were taken throughout the touch response (every 25 frames), including the initial and 252

final positions of the embryo. Images were then analysed using ImageJ48

to calculate distance 253

travelled between consecutive images. Total distance travelled (mm) was then calculated. 254

Four embryos from each of three treatment replicates were tested (n = 12). 255

256

257

Hatching & growth 258

Four replicates of each experimental treatment were screened at 24 h intervals to record the 259

number of embryos hatched over the first 96 h of development (n = 4). Length and yolk-sac 260

area was analysed as an indicator of growth at 24, 48 and 72 hpf. Four embryos from each of 261

four treatment replicates were terminally anaesthetised using buffered MS-222 (0.08%). 262

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Images of the embryos were taken using a Leica MZFIII photo-microscope and Leica Image 263

Manager 50 and viewed using ImageJ48

. The length of embryos, the distance from the most 264

anterior part of the head to the tip of the tail following the path of the developing spinal 265

cord49

, and the area of the yolk-sac (including the yolk-extension) were measured (n = 16) at 266

each time point. Embryos were dechorionated at 24 hpf to facilitate photography and 267

morphometric analysis while freely-hatched embryos were used at 48 and 72 hpf. 268

269

270

Cardiac development 271

Single embryos (20 hpf) were placed into individual wells of a 96 well plate filled with 360 272

µl of the appropriate concentration of prednisolone or control media. The entire plate was 273

covered to prevent evaporation while it was maintained in an incubator at 25°C. Embryos 274

were viewed at 1 h intervals over the course of 4 h and the percentage of embryos which 275

displayed a visible heart beat was recorded. Maintaining embryos at 25°C reduced the rate of 276

development and enabled any effect of prednisolone on the onset of heart beat to be 277

highlighted. Four embryos from each of the six treatment replicates were used to calculate the 278

percentage of embryos with a visible heart beat (n = 6). Heart rate was recorded at 24, 48 and 279

72 hpf at 28°C. Individual embryos were visualised using a digital EUROMEX 6.0 M Pixel 280

camera attached to a NOVEX Holland B-series microscope connected to a desktop PC. 281

Embryos at 24 hpf were visualised within the chorion. At 48 and 72 hpf, freely-hatched 282

embryos were mounted in a 6% methylcellulose solution on a microscope slide to maintain 283

their position during recording. A drop of water was added on top of the methylcellulose to 284

prevent desiccation during observation. Embryos were left to acclimate within the 285

methylcellulose for 15 minutes prior to visualisation on top of a microscope slide heater to 286

provide a constant temperature. An additional 1 minute acclimation period was provided 287

while positioned on the microscope stage. Heart rate in beats per minute (bpm) was 288

calculated as the mean number of beats during three 20 s periods, multiplied by three. Heart 289

rate was measured using four embryos from three replicates of each treatment (n = 12). 290

291

292

Metabolism 293

Oxygen consumption was used as an indicator of metabolic rate and was measured using 294

methods modified from Sloman18

at 24, 48 and 72 hpf. Oxygen consumption was measured 295

using sealed respiratory chambers consisting of 20 ml blackened glass vials containing a 296

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magnetic stirrer separated from the main body of the respirometer by a horizontal mesh 297

partition. Magnetic fleas prevented an oxygen gradient forming within the chamber. Each 298

respiratory chamber contained three embryos from the same replicate, and two respiratory 299

chambers were tested for each of four treatment replicates (n = 8). Chambers were filled with 300

system water and were maintained at 28±1°C in water baths. Water samples (0.1 ml) were 301

taken from each chamber after a 4 h period and were immediately injected into a 302

thermostatted cell (Strathkelvin Instruments, Glasgow, UK), containing an oxygen electrode 303

connected to an oxygen meter. Embryos were then removed from the chambers and weighed 304

on a balance. Calibration of the oxygen electrode used air-saturated water from the water 305

baths as 100% saturation and a 2% solution of sodium sulphite (in 0.01 M sodium borate) as 306

a 0% standard. The oxygen saturation values were then converted into µmol of oxygen which 307

were adjusted for temperature and air-pressure and used to calculate mass specific oxygen 308

consumption. Blank chambers served as controls to account for bacterial oxygen 309

consumption. 310

311

312

Skeletal muscle development 313

Morphometric measurements were taken at 72 hpf. Anaesthetised embryos (0.04% MS-222) 314

were visualised laterally under a dissection microscope attached to a EUROMEX 6.0 M Pixel 315

camera connected to a desktop PC. The area of the anal (AN), pre (PA) and post-anal (PSA) 316

somites (Figure 1A) was calculated in addition to the angle connecting these segments. Six 317

embryos from three treatment replicates were photographed (n = 18). Muscle integrity was 318

visualised via birefringence using methods adapted from Smith et al.47

. Polarised lens filters 319

were positioned between the embryo and the microscope lens. The upper filter was then 320

rotated to align both filters at 90° to eliminate any light which was not perpendicular to the 321

muscle fibres (Figure 1B). The mean intensity of the skeletal muscle was divided by the area 322

of muscle, and then normalized to control values. Four embryos were photographed from 323

each of four treatment replicates (n = 16). All images were processed using Image J. 324

325

326

Craniofacial morphogenesis 327

Developmental changes to the anterior skeleton of embryos were investigated at 96 hpf. 328

Embryos were euthanatized using an overdose of MS-222 (0.08%), fixed overnight at 4°C in 329

4% phosphate-buffered formaldehyde, washed in PBS with 0.1% Tween-20 (PBT) and then 330

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bleached in 3% hydrogen peroxide/ 1% potassium hydroxide. Embryos were then rinsed with 331

PBT and transferred into 0.1% alcian blue solution (1% concentrated hydrochloric acid, 70% 332

ethanol) and stained overnight. After rinsing several times with acidic ethanol (5% 333

concentrated hydrochloric acid, 70% ethanol) embryos were rehydrated through an HCl-334

EtOH series (75%, 50%, 25% and 0%) and stored in glycerol. Embryos were then 335

photographed ventrally to capture the anterior portion of the developing skeleton using a 336

EUROMEX 6.0 M Pixel camera (Figure 1C). Criteria for assessing skeletal development 337

were adopted from Hillegass et al.14

. The intercranial distance (ICD), lower jaw length (LJL), 338

and ceratohyal cartilage length (CCL) were measured using ImageJ. Four embryos were 339

photographed from each of four treatment replicates (n = 16). 340

341

342

Swimming behaviour 343

Swimming behaviour of individual embryos was assessed at 120 hpf in groups of five 344

embryos which were placed into a 6 cm diameter petri dish marked with a grid floor (4.5 345

mm2; approx. one body length) filled with clean embryo media (28°C). After an initial 2 346

minute acclimation period within the dish, activity was recorded for 10 minutes. Behaviour 347

was recorded using a JVC TH-C1480B colour camera positioned above the test chamber. The 348

percentage of time spent swimming, distance swum (no. of body lengths swum; bls) and the 349

average swim speed (body lengths per second; blsps) was calculated. Five embryos were 350

filmed from each of four treatment replicates (n = 20). 351

352

353

Statistical analysis 354

All statistical analysis was conducted using SPSS v.18. Data were checked for normality and 355

homogeneity of variances using Kolmogorov-Smirnov and Levene’s tests, respectively. 356

Where data were not normally distributed, or in the case of percentage data, transformations 357

were conducted to allow parametric testing. No significant effect of replicate was found and 358

so data were combined within treatments. Two-way ANOVAs were used to determine the 359

effects of treatment and time on all measurements. Where significant interactions occurred 360

within the ANOVA model, time and treatment effects were further analysed using one-way 361

ANOVAs with Fisher’s LSD test post hoc. Non-parametric equivalents were used where 362

necessary (Scheirer-Ray-Hare (SRH), Kruskal- Wallis test (KW) and pairwise Mann-363

Whitney U). 364

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365

366

Results 367

368

Pre-hatch activity 369

There was no significant difference in the onset of spontaneous contractions in response to 370

prednisolone in pre-hatched embryos, however, all concentrations tested significantly 371

reduced the frequency of spontaneous contractions at 24 hpf (Table 1, Figure 2A). 372

373

Touch evoked escape response 374

The touch-evoked escape response of freely-hatched embryos at 48 hpf increased following 375

treatment with 0.1 µg/l of prednisolone (Table 1, Figure 2B). 376

377

Hatching & growth 378

Exposure to 1 and 10 µg/l prednisolone induced precocious hatching at 48 and 72 hpf (Table 379

1, Figure 3), although prednisolone did not significantly alter length or yolk-sac area during 380

embryogenesis. Mortality did not vary during the first 24 hours of development between 381

treatments and no mortality was observed after this period. 382

383

Cardiac development 384

A significantly higher proportion of embryos exhibited a visible heart beat following 0.1 µg/l 385

of prednisolone at 23 hpf, while 10 µg/l of prednisolone significantly reduced the onset of 386

heart beat at 23 and 24 hpf (Table 1, Figure 4A). In contrast, heart rate was significantly 387

increased by all concentrations of prednisolone between 24 and 72 hpf (Table 1, Figure 4B). 388

389

Metabolism 390

The rate of oxygen consumption between 24 and 72 hpf was significantly increased by all 391

concentrations of prednisolone but at specific time points (Table 1, Figure 5). 392

393

Skeletal muscle development 394

The area of pre-anal somites were reduced in response to 0.1 µg/l of prednisolone, without 395

affecting the angle of attachment between somites or the integrity of skeletal muscle (Table 396

1). 397

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398

Craniofacial morphogenesis 399

There was also an overall enlargement of the cranial skeleton following 1.0 µg/l of 400

prednisolone at 96 hpf, as all indices of craniofacial morphogenesis were significantly 401

increased (Table 1, Figure 6). 402

403

Swimming behaviour 404

Finally, prednisolone significantly reduced the percentage of time embryos spent swimming 405

(10 µg/l), the distance swum (0.1, 1.0 and 10 µg/l) and the average swim speed (1.0 and 10.0 406

µg/l) at 120 hpf (Table 1, Figure 7). 407

408

409

Discussion 410

411

Exposure to environmental concentrations of prednisolone during embryogenesis resulted in 412

significant physiological and behavioural effects throughout ontogeny. While the onset of 413

spontaneous muscular contractions did not change in response to prednisolone treatment, the 414

frequency of contractions were significantly reduced (Figure 2A). Exposure to 0.1 µg/l of 415

prednisolone also increased the touch evoked escape response to a mechanosensory stimuli at 416

48 hpf (Figure 2B). No effect of the higher concentrations of prednisolone on the escape 417

response was observed and it may be prudent to interpret the fact that an effect was only seen 418

at the lowest dose with caution. Regardless, zebrafish embryos which exhibit defects in 419

skeletal muscle development are typically associated with a diminished escape response47

420

which suggests that the developing musculature has not been negatively affected by 421

prednisolone at this stage. Spontaneous contractions, as well as the startle response, are 422

regulated by the neural circuitry within the spinal cord50-53

and the Mauthner neurons in 423

particular are associated with regulating the escape response following mechanosensory 424

stimulus54

. Whether the neural circuitry has been altered in response to prednisolone however 425

requires specific investigation. 426

427

The increase in the percentage of embryos hatched following prednisolone treatment (Figure 428

3) agrees with Wilson et al.55

who found that exposure to dexamethasone also resulted in 429

precocious hatching in zebrafish embryos. Hatching is facilitated by a combination of 430

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enzymatic digestion of the chorion and mechanical damage due to embryonic activity.56

431

Since the frequency of pre-hatch activity significantly decreased, the increased hatching is 432

more likely to be related to changes in the release of proteolytic enzymes. Cloud57

also 433

hypothesised that exposure to exogenous steroids led to changes in the release of hatching 434

enzymes. No statistically significant change in either length or yolk-sac area was found which 435

suggests that growth was not detrimentally affected by prednisolone. This is in contrast to 436

previous studies have shown diminished growth rates and reduced yolk-sac volumes in 437

response to elevated glucocorticoid exposure.19,20

438

439

The onset of heart contraction was significantly altered by prednisolone (Figure 4A). While 440

the heart is the first organ to fully develop during vertebrate embryogenesis58

, embryonic and 441

early larval stages do not require a functional cardiovascular system for oxygen delivery59

. 442

The “prosynchronotropy” hypothesis proposes that the early onset of cardiovascular 443

contraction is primarily related to cardiac morphogenesis60

, which has been demonstrated 444

experimentally by Hove et al.61

. An earlier onset of heart contraction in response to 0.1 µg/l 445

of prednisolone could therefore suggest an overall acceleration of cardiac development. 446

However, exposure to 10 µg/l prednisolone also produced a delay in the onset of heart 447

contraction. Biphasic physiological responses to xenobiotics are not uncommon although the 448

precise underlying mechanisms are not clear, especially since both concentrations increased 449

heart rate (Figure 4B). This could suggest that the developmental processes which regulate 450

cardiogenesis and the physiological mechanisms which control heart rate are differentially 451

regulated by glucocorticoids. Glucocorticoid signalling during early zebrafish ontogeny has 452

been recently shown to significantly affect the structure and function of the heart in 453

embryos.62

In addition, exposure to dexamethasone (100 µM) increases the size of embryonic 454

hearts and enhances cardiac performance. Interestingly, injection of cortisol into zebrafish 455

embryos causes heart deformities and suppression of essential cardiac genes.63

Wilson et al.62

456

also found that zebrafish GR knockouts displayed diminished heart maturation. The intensity 457

of glucocorticoid signalling therefore appears to differentially alter heart development and 458

further study is necessary to elucidate the precise mechanisms of glucocorticoid mediated 459

effects on cardiac development. 460

461

Embryos exposed to prednisolone consumed more oxygen throughout the first 72 hours of 462

development (Figure 5). This is in agreement with findings reported by Giesing et al.17

and 463

Sloman18

and who found that exposure to endogenous glucocorticoids increased oxygen 464

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15

consumption during embryonic development in stickleback (Gasterosteus aculeatus) and 465

brown trout (Salmo trutta) respectively. The increase in oxygen consumption may be related 466

to an increase in metabolic rate as glucocorticoids are essential regulators of metabolism in 467

teleosts.64

Metabolic enzymes are sensitive to SG exposure during early development in 468

zebrafish, e.g. phosphoenolpyruvate carboxykinase, which controls the rate limiting step in 469

gluconeogenesis, increases in response to dexamethasone.65

Similarly, prednisolone (1 µg/l) 470

is known to increase plasma glucose concentrations in adult fathead minnow.11

Increased 471

metabolic rate could also accelerate organogenesis66

which is prioritised over growth during 472

ontogeny67

and would facilitate an increase in hatching and heart maturation. 473

474

Although there was an almost 10% reduction in birefringence in response to 10 µg/l of 475

prednisolone, in a manner consistent with a myopathic phenotype47

, no statistically 476

significant difference between treatments was found. Nor were they any significant changes 477

in the angle of attachment between adjacent somites. However, exposure to 0.1 µg/l of 478

prednisolone resulted in significantly smaller pre-anal somites at 72 hpf. Given the relatively 479

small change in the area of somites between treatments (approx. 5%), and no observable 480

change in muscle integrity, the significance of these changes are likely to be minor since 481

changes were localised to individual somites at specific concentrations of prednisolone. 482

Swimbladder inflation was also unaffected by prednisolone treatment (data not shown) which 483

suggests that this small change in somite size did not affect swim-up behaviour and is 484

therefore likely to be physiologically insignificant. 485

486

Craniofacial morphogenesis was significantly altered in response to 1 µg/l of prednisolone at 487

96 hpf (Figure 6). Intercranial distance, LJL and CCL all increased, which suggests an overall 488

enlargement of the cranial skeleton. Prednisolone has previously been shown to induce bone 489

loss in zebrafish embryos but at far greater concentrations than those utilised in this study.68

490

Dexamethasone also significantly alters craniofacial development in zebrafish embryos by 491

altering the expression of MMPs which culminates in significantly longer heads.13,14

Whether 492

prednisolone may also affect axial skeletal development requires additional investigation, 493

however, it is unlikely that the increase in skull size led to the observed changes in swim 494

behaviour, since 0.1 µg/l and 10 µg/l of prednisolone also caused behavioural effects, despite 495

failing to alter skeletal development. 496

497

498

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16

Embryos at 120 hpf exhibited significantly reduced swim activity in response to prednisolone 499

treatment (Figure 7). This coincides with behavioural changes in response to dexamethasone 500

exposure69

and from studies which have prevented normal glucocorticoid signalling during 501

zebrafish development26,55

. Reductions in spontaneous activity during pre-hatch and free-502

swimming stages could suggest common physiological mechanisms may be susceptible to 503

prednisolone throughout development. Alternatively, changes during early ontogeny may 504

persist into free swimming stages. It may also be possible that higher concentrations of 505

prednisolone may affect additional physiological systems which regulate behaviour; 506

increasing concentrations of prednisolone produced differences in the swim profile of free 507

swimming embryos. 508

509

Given the parity between these findings and those from previous studies which have 510

investigated glucocorticoid signalling during zebrafish ontogeny, we tentatively hypothesis 511

that prednisolone toxicity may be primarily mediated through binding to the glucocorticoid 512

receptor and subsequent modulation of glucocorticoid regulated pathways. Glucocorticoid 513

signalling is responsible for regulating hundreds of genes; knockout of the maternally derived 514

gr mRNA results in several developmental abnormalities within neural, vascular and visceral 515

tissues.37

Exposure to SGs during early development may similarly interfere with multiple 516

processes and would therefore suggest multiple molecular pathways may be responsible for 517

the observed developmental changes. However, commonality with such studies may point to 518

disruption to the hypothalamic-pituitary-interrenal (HPI) axis, which controls the release of 519

cortisol and thereby regulates homeostasis12

, as a logical starting point for future 520

investigation. Cortisol is known to mediate several essential physiological processes such as 521

metabolism and growth during early life stages. In addition, the persistent behavioural 522

changes throughout development observed in this study also bear semblance to how changes 523

in cortisol levels affect the neurological programming of individuals and subsequent 524

behavioural phenotypes.70,71

Glucocorticoids are believed to serve an active role during 525

neurogenesis70,71

and could have subsequent effects on brain monoamines72

which are 526

important neural regulators of behavioural phenotypes in zebrafish73

. 527

528

529

The results from this study (summarised in Table 2) contribute to the growing body of 530

evidence that SGs can alter several physiological processes during early development in fish, 531

in a manner consistent with the effects of endogenous glucocorticoid signalling. Such 532

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17

changes may have significant consequences upon the future development, performance and 533

survival of zebrafish. Further investigations are therefore required to identify the underlying 534

mechanisms for these changes during development and the potential long term implications 535

also need to be clarified. 536

537

538

539

540

541

542

543

544

545

546

547

548

549

550

551

552

553

554

555

556

557

558

559

560

561

562

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References 563

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Table legends 763

764

Table 1: Summary of statistical analysis for all parameters measured. P values in bold 765

indicate significant effects. 766

767

Table 2: Summary of significant developmental effects following prednisolone treatment. ↓ 768

indicates statistically significant decrease compared to controls; ↑, significant increase 769

compared to controls; -, no significant change. ICD, intercranial distance; LJL, lower jaw 770

length; CCL, ceratohyal length; hpf, hours post fertilisation. 771

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Figure legends 793

794

Figure 1: (A) Control embryo (72 hpf) showing somites analysed for morphometric analysis, 795

pre-anal (PA), anal (AN) and post-anal (PSA). (B) Control embryo (72 hpf) under polarized 796

light. Scale bars = 0.5 mm. (C) Control embryo at 96 hpf stained with 0.1% alcian blue. 797

Numbers correspond with defined parameters; 1, lower jaw length (LJL); 2, intercranial 798

distance (ICD); 3, ceratohyal cartilage length (mm). Parameters 1 and 3 begin from the 799

dashed line. Scale bar = 250 µm. 800

801

Figure 2: (A) Mean number of spontaneous contractions per minute at 24 hpf (n = 16). 802

Letters denote significant differences between treatments at specific time points (P < 0.05). 803

Where bars share letters there is no significant difference. Means ± SEM. (B) Distance 804

travelled during touch invoked escape response at 48 hpf (n = 12). Letters denote significant 805

differences between treatments (P< 0.05). Means ± SEM. 806

807

Figure 3: The percentage of hatched embryos over the first 96 h of development (n = 4). 808

Letters denote significant differences between treatments at specific time points (P < 0.05). 809

Where bars share letters there is no significant difference. Means ± SEM. 810

811

Figure 4: (A) The percentage of embryos which displayed a visible heart beat during the first 812

25 h of development in response to prednisolone (n = 6). (B) Heart rate (beats per minute, 813

bpm) of embryos at 24, 48 and 72 hpf in response to prednisolone (n = 16). Letters denote 814

significant differences between treatments at specific time points (P < 0.05). Where bars 815

share letters there is no significant difference. Means ± SEM. 816

817

Figure 5: The oxygen consumption (µmol.g-1

h-1

) of embryos at 24, 48 and 72 hpf in response 818

to prednisolone (n = 8). Letters denote significant differences between treatments at specific 819

time points (P < 0.05). Where bars share letters there is no significant difference. Means ± 820

SEM. 821

822

Figure 6: Craniofacial measurements (mm) at 96 hpf (n = 16). Letters denote significant 823

differences between treatments at specific time points (P < 0.05). Where bars share letters 824

there is no significant difference. Means ± SEM. 825

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826

Figure 7: (A) Percentage of time swimming at 120 hpf (n = 20). (B) Activity (number of body 827

lengths swum) at 120 hpf (n = 20). (C) Average swim speed at 120 hpf (n = 20). Letters 828

denote significant differences between treatments (P< 0.05). Means ± SEM. 829

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Table 1 856

857

858

859

860

861

862

863

Developmental

endpoints Test Test statistic P-value

Pre-hatch activity Onset ANOVA F4, 19 = 0.424 P = 0.789

Frequency ANOVA F4,79= 7.268 P < 0.001

Touch evoked escape

response ANOVA F4,56= 3.437 P = 0.016

Hatching & growth Hatching 2-Way ANOVA

Time, F1, 39 = 280.154

Treatment, F4, 119 = 13.206

Time*Treatment, F4, 39= 4.279

P < 0.001

P < 0.001

P = 0.007

Length 2-Way ANOVA

Treatment, F4, 230 = 1.865

Time, F2, 230 = 174.667

Treatment*Time, F8, 230 = 0.822

P = 0.118

P < 0.001

P = 0.584

Yolk-sac area Scheirer-Ray-

Hare test

Treatment, F4, 231, = 8.574

Time, F2, 231 = 89.84

Treatment*Time, F8, 231 = 4.577

P = 0.373

P < 0.001

P = 0.805

Cardiac development Onset of heart

beat 2-Way ANOVA

Time, F1, 60 = 82.147

Treatment, F 4, 60 = 11.532

Time*Treatment, F 4, 60 = 4.557

P < 0.001

P < 0.001

P = 0.003

Heart rate 2-Way ANOVA

Time, F1, 119 = 527.366

Treatment, F4, 119 = 26.055

Time*Treatment, F4, 119= 2.571

P < 0.001

P < 0.001

P = 0.042

Metabolism Oxygen

consumption 2-Way ANOVA

Time, F2, 119 = 30.586

Treatment, F4, 119 = 8.667

Time*Treatment, F8, 119 = 8.667

P < 0.001

P < 0.001

P < 0.001

Skeletal muscle

development Area of somites ANOVA

Pre-anal, F4, 89 = 2.547

Anal, F4, 89 = 1.148

Post-anal, F4, 89 = 2.954

P = 0.045

P = 0.340

P = 0.024

Angle of

attachment ANOVA

Pre-anal, F4, 89 = 1.047

Anal, F4, 89 = 1.284

Post-anal, F4, 89 = 1.747

P = 0.388

P = 0.283

P = 0.147

Muscle integrity ANOVA F4, 74 = 0.363 P = 0.834

Craniofacial

morphogenesis 2-Way ANOVA

Area, F2, 206 = 817.249

Treatment, F4, 206 =10.098

Area*Treatment, F8, 206 = 0.606

P< 0.001

P< 0.001

P = 0.772

Swimming behaviour Time swimming Kruskal-Wallis χ² (4) = 13.344 P = 0.01

Distance swum ANOVA F4, 97 = 16.258 P < 0.001

Swim speed ANOVA F4, 97 = 2.682 P = 0.036

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Table 2 864

865

866

867

868

869

870

871

Concentration of

prednisolone (µg/l)

Developmental

endpoints

Stage of

development

(hpf)

0.1 1.0 10.0

Pre-hatch activity Onset 15, 16, 17 -,-,- -,-,- -,-,-

Frequency 24 ↓ ↓ ↓

Touch evoked escape

response

48 ↑ - -

Hatching & growth Hatching 48, 72, 96 -,-,- -,↑,- -,↑,-

Length 24, 48, 72 -,-,- -,-,-, -,-,-,

Yolk-sac area 24, 48, 72 -,-,- -,-,-, -,-,-,

Cardiac

development Onset of heart beat

23, 24, 25 ↑,-,- -,-,- ↓,↓,-

Heart rate 24, 48, 72 ↑,↑,- ↑,↑,↑ ↑,↑,↑

Metabolism Oxygen

consumption

24, 48, 72 -,↑,- ↑,↑,- ↑,↑,↑

Skeletal muscle

development Area of somites Pre-anal 72 ↓ - -

Anal 72 - - -

Post-anal 72 - - -

Angle of

attachment

72 - - -

Muscle integrity 72 - - -

Craniofacial

morphogenesis ICD

96 - ↑ -

LJL 96 - ↑ -

CCL 96 - ↑ -

Swimming behaviour Time swimming 120 - - ↓

Distance swum 120 ↓ ↓ ↓

Swim speed 120 - ↓ ↓

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1

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PSA

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Figure 2A 898

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Figure 3 932

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Figure 4A 954

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Figure 5 977

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Figure 6 991

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Figure 7A 1014

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Figure 7B 1021

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