Scholarly article on topic 'Large-Scale Assessment of the Zebrafish Embryo as a Possible Predictive Model in Toxicity Testing'

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Academic research paper on topic "Large-Scale Assessment of the Zebrafish Embryo as a Possible Predictive Model in Toxicity Testing"

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Large-Scale Assessment of the Zebrafish Embryo as a Possible Predictive Model in Toxicity Testing

Shaukat Ali1, Harald G. J. van Mil1,2, Michael K. Richardson1*

1 institute of Biology, Sylvius Laboratory, Leiden University, Leiden, The Netherlands, 2 Mathematical Institute, Leiden University, Leiden, The Netherlands

Abstract

Background: In the drug discovery pipeline, safety pharmacology is a major issue. The zebrafish has been proposed as a model that can bridge the gap in this field between cell assays (which are cost-effective, but low in data content) and rodent assays (which are high in data content, but less cost-efficient). However, zebrafish assays are only likely to be useful if they can be shown to have high predictive power. We examined this issue by assaying 60 water-soluble compounds representing a range of chemical classes and toxicological mechanisms.

Methodology/PrincipalFindings: Over 20,000 wild-type zebrafish embryos (including controls) were cultured individually in defined buffer in 96-well plates. Embryos were exposed for a 96 hour period starting at 24 hours post fertilization. A logarithmic concentration series was used for range-finding, followed by a narrower geometric series for LC50 determination. Zebrafish embryo LC50 (log mmol/L), and published data on rodent LD50 (log mmol/kg), were found to be strongly correlated (using Kendall's rank correlation tau and Pearson's product-moment correlation). The slope of the regression line for the full set of compounds was 0.73403. However, we found that the slope was strongly influenced by compound class. Thus, while most compounds had a similar toxicity level in both species, some compounds were markedly more toxic in zebrafish than in rodents, or vice versa.

Conclusions: For the substances examined here, in aggregate, the zebrafish embryo model has good predictivity for toxicity in rodents. However, the correlation between zebrafish and rodent toxicity varies considerably between individual compounds and compound class. We discuss the strengths and limitations of the zebrafish model in light of these findings.

Citation: Ali S, van Mil HGJ, Richardson MK (2011) Large-Scale Assessment of the Zebrafish Embryo as a Possible Predictive Model in Toxicity Testing. PLoS ONE 6(6): e21076. doi:10.1371/journal.pone.0021076

Editor: Ferenc Mueller, University of Birmingham, United Kingdom

Received March 22, 2011; Accepted May 18, 2011; Published June 28, 2011

Copyright: © 2011 Ali et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the Smart Mix Programme of the Netherlands Ministry of Economic Affairs and the Netherlands Ministry of Education, Culture and Science (M.K.R.) under grant number SSM06010, the University of Azad Jammu and Kashmir, Pakistan (S.A.) under project No. F-3/PD/Main & Mirpur/ 369/2007. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: m.k.richardson@biology.leidenuniv.nl

Introduction

There is an unmet need for low-cost, high-throughput animal models in some fields of biomedical research such as drug screening and toxicity assessment [1,2]. The zebrafish embryo is emerging as one such model [1]. It has been proposed as a bridge between simple assays based on cell culture, and biological validation in whole animals such as rodents [1]. The zebrafish cannot replace rodent models but is complementary to them, being particularly useful for rapid, high-throughput, low-cost assays, as for example in the early (pre-regulatory) stages of the drug development pipeline [3].

Among the attractive features of the zebrafish embryo model are its small size, small volume of compound consumed and rapid development. The organogenesis of major organs is completed at 5 days post fertilization (dpf) [4]. Also, many fundamental cellular and molecular pathways involved in the response to chemicals or stress are conserved between the zebrafish and mammals [5]. Genomic sequencing has shown extensive homology between zebrafish and other vertebrate species (including humans), and some aspects of brain patterning, structure and function are also conserved [6-9]. We have shown for example that the glucocor-

ticoid receptor of the zebrafish is functionally closer to that of the human than is its mouse cognate [10]. The availability of genomic tools in the zebrafish provides an advantage over other teleosts such as the fathead minnow (Pimephales promelas) used, for example, in environmental toxicity assessment in the United States [11]. Indeed, zebrafish embryos may be a suitable replacement for some of these adult fish toxicity tests [12].

The zebrafish is increasing being used in toxicological studies [reviewed by: 13,14]. Example include the use of adult zebrafish for the testing of lead and uranium [15], malathion [16], colchicine [17], anilines [18], and metronidazole [19]; and the use of juveniles for testing agricultural biocides [20]. Zebrafish embryos are also being used in toxicity studies [reviewed by: 21]. Examples include the use of zebrafish embryos for testing nanoparticles [22,23].

Although the body plans of zebrafish are in many aspects similar to those of mammals, there are important differences. The fish is ectothermic, and lacks cardiac septa, synovial joints, cancellous bone, limbs, lungs and other structures [24-26]. Therefore, some toxic effects seen in humans are difficult to model in the zebrafish. Furthermore, the zebrafish embryo remains inside the chorion at least up to 48 hpf [27]. In pre-hatching embryos, therefore, the

Table 1. Concentration-dependent mortality at 5 dpf after 96 h exposure.

Cumulative % mortality after 96 h exposure

Compounds logarithmic series (mg/L){ geometric series* ± SEM

0 1 10 100 1000 C0 C1 C2 C3 C4 C5

1 Aconitine 0 0 0 63 100¥ 060 6762 9861 10060 10060 10060

2 Atropine 0 0 0 0 100 060 060 060 1760 7361 10060

3 Berberine chloride 0 0 0 25 100 060 060 1762 5464 10060 10060

4 Colchicine 0 0 0 100 100 060 060 461 4262 9861 10060

5 Coniine 0 0 0 100 100 060 060 060 261 10060 10060

6 a-Lobeline hydrochloride 0 0 0 100 100 060 060 662 83 62 10060 10060

7 Morphine hydrochloride 0 0 0 0 0 060 060 060 060 2560 9460

8 Nicotine 0 0 0 100 100 060 461 861 5464 10060 10060

9 Quinine sulfate 0 0 0 88 94 060 060 060 060 060 4261

10 (—)-Scopolamine hydrobromide trihydrate 0 0 0 6 6 060 060 261 461 1961 7762

11 Strychnine hydrochloride 0 0 0 100 100 060 2962 4061 6761 10060 10060

12 Theobromine 0 0 0 50 100¥ 060 1362 1562 3860 5864 10060

13 (+)-Tubocurarine chloride hydrate 0 0 0 0 100 060 060 660 3561 10060 10060

14 Yohimbine hydrochloride 0 0 0 75 100 060 1362 1362 2361 2961 81 60

15 Amygdalin 0 0 25 94 100 060 060 261 861 1763 40 62

16 Arbutin 0 0 0 100 100 060 060 961 48 6 7 5066 6765

17 Convallatoxin 0 0 0 78 100¥ 060 6965 786 5 9661 10060 10060

18 Coumarin 0 0 0 0 100 060 1762 2363 4061 9861 10060

19 Digitoxin 0 25 100 100¥ 100¥ 060 2761 9461 10060 10060 10060

20 Gentamycin sulfate 0 0 0 6 100 060 2961 3461 6761 92 60 92 60

21 Glycyrrhizin 0 0 6 100 100 060 060 1261 3562 6966 9461

22 Hesperidin 0 0 0 69 100¥ 060 060 862 1063 6361 81 62

23 Kanamycin monosulfate 0 6 13 38 38 060 261 261 1561 4661 79 65

24 Naringin 0 0 0 63 94 060 060 261 662 1062 7766

25 Neohesperidin 0 0 0 100 100¥ 060 060 060 060 060 3461

26 Ouabain octahydrate 0 0 0 19 100 060 261 661 65 63 9661 9661

27 Phloridzin dihydrate 0 0 0 0 100 060 060 261 661 1261 65 63

28 Rutin hydrate 0 0 0 0 0 060 060 862 862 1061 73 66

29 Streptomycin sulfate 0 0 0 6 31 060 060 060 060 1360 7361

30 Cadmium(II) chloride 0 38 38 100 100 060 1960 2561 6063 8461 10060

31 Copper(II) nitrate trihydrate 0 0 13 100 100 060 060 261 1360 3860 10060

32 Lead acetate trihydrate 0 0 0 94 100 060 2566 33 6 8 3568 9461 9461

33 Lithium chloride 0 0 0 0 0 060 060 1561 6065 10060 10060

34 Chloramphenicol 0 0 0 0 94 060 060 060 1261 9461 10060

35 Ethanol 0 0 0 0 0 060 060 060 060 060 21 61

36 Glycerol 0 0 0 0 0 060 060 060 060 060 9861

37 Tween 80 0 0 0 0 100 060 060 261 71 64 10060 10060

38 Acetic acid 0 0 0 38 100 060 060 461 6761 10060 10060

39 Salicylic acid 0 0 6 100 100 060 060 060 1062 10060 10060

40 Sodium oxalate 0 0 0 0 94 060 060 33 6 2 5261 7763 9861

41 Trichloroacetic acid 0 0 6 56 100 060 060 3368 60620 10060 10060

42 Ampicillin sodium 0 0 0 0 38 060 060 060 1960 1960 3561

43 Cyclophosphamide monohydrate 0 0 0 0 0 060 060 73 6 2 9661 10060 10060

44 Paracetamol 0 0 0 0 100 060 060 060 861 90610 10060

45 Phenacetin 0 0 0 0 94 060 060 060 261 83 62 10060

46 Benserazide hydrochloride 0 0 0 0 6 060 060 261 862 31 62 8663

Table 1. Cont.

Cumulative % mortality after 96 h exposure Compounds logarithmic series (mg/L){ geometric series* ± SEM

0 1 10 100 1000 C0 C1 C2 C3 C4 C5

47 Chlorpromazine hydrochloride 0 0 94 100 100 060 060 060 461 6761 10060

48 Isoniazid 0 0 0 6 38 0 6 0 0 6 0 2 61 26 1 67 6 4 94 62

49 Phenelzine sulfate 0 0 19 100 100 060 461 2361 10060 10060 10060

50 Ethambutol dihydrochloride 0 0 0 0 0 0 6 0 0 6 0 0 60 17 6 3 73 64 100 60

51 Verapamil hydrochloride 0 0 0 100 100 060 060 261 1062 4267 10060

52 Phenol 0 0 0 100 100 060 060 060 060 3862 10060

53 Sodium azide 0 100 100 100 100 0 6 0 0 60 10 6 2 90 63 100 60 100 60

54 Dimethyl sulfoxide 0 0 0 0 0 060 060 060 060 461 10060

55 Formaldehyde 0 0 50 100 100 060 060 060 1561 7161 10060

56 Phenformin hydrochloride 0 0 0 13 100 0 6 0 2 61 6 62 17 6 2 92 62 100 60

57 Ropinirole hydrochloride 0 0 0 0 100 060 861 861 2161 9661 10060

58 Amitriptyline hydrochloride 0 0 63 100 100 0 6 0 4 61 6 6 2 40 62 100 60 100 60

59 Sodium dodecyl sulfate 0 0 94 100 100 0 6 0 8 6 6 2 33 6 8 58 6 5 9261 100 60

60 Barbital sodium 0 0 0 0 6 060 060 060 1363 5060 9063

({)This was a one-time range-finding experiment and so there is no SEM;

(*)a different geometric scale was used for different compounds because of the variations in toxicity found with the logarithmic range-finding. The values given are the mean percentage mortality from three replicates; the geometric series concentrations C0, C1, etc. are given for each compound in Table S2. For each concentration for each compound, N = 48 (3 replications x16) embryos; ^percentage mortality was found but at these high concentrations, compounds were precipitated out of solution. doi:10.1371/journal.pone.0021076.t001

chorion (a membrane perforated by channels of 0.5-0.7 mm in diameter), may provide a barrier to diffusion of compounds [28-31].

The evolutionary divergence of zebrafish and mammals is around 445 million years ago [32] and so it is by no means certain that we will necessarily share the same sensitivity to toxic substances. Therefore, there is a need for validation of the model using compounds that have a known effect in other species [33]. One study has reported, using 18 toxic compounds, that toxicity in zebrafish was well-correlated with values reported from rodent studies [34]. The zebrafish embryo system has also been compared, as a toxicology screen, with the aquatic crustacean Daphnia magna [35]. Such studies are an important step towards the kind of comparative toxicity database represented by the well-known 'Registry of Cytotoxicity' which examines the predictive power of cell assays [36].

Our aim here is to determine the toxicity of 60 compounds from diverse pharmacological and chemical classes, and examine the strength of correlation between zebrafish embryo LC50 and data from the literature on rodent LD50. Compounds are added to the water in which the embryos develop, and so we focus here on water soluble compounds to avoid any confounding effects of carrier solvents.

Materials and Methods

Ethics statement

All animal experimental procedures were conducted in accordance with local and international regulations. The local regulation is the Wet op de dierproeven (Article 9) of Dutch Law (National) and the same law administered by the Bureau of Animal Experiment Licensing, Leiden University (Local). This local regulation serves as the implementation of Guidelines on the

protection of experimental animals by the Council of Europe, Directive 86/609/EEC, which allows zebrafish embryos to be used up to the moment of free-living (approximately 5-7 days after fertilisation). Because embryos used here were no more than 5 days old, no licence is required by Council of Europe (1986), Directive 86/609/EEC or the Leiden University ethics committee.

Animals

Male and female adult zebrafish (Danio rerio) of AB wild type were purchased from Selecta Aquarium Speciaalzaak (Leiden, the Netherlands) who obtain stock from Europet Bernina International BV (Gemert-Bakel, the Netherlands). Fish were kept at a maximum density of 100 individuals in glass recirculation aquaria (L 80 cm; H 50 cm, W 46 cm) on a 14 h light: 10 h dark cycle (lights on at 08.00). Water and air were temperature controlled (25±0.5°C and 23°C, respectively). The fish were fed twice daily with 'Sprirulina' brand flake food (O.S.L. Marine Lab., Inc., Burlingame, USA) and twice a week with frozen food (Dutch Select Food, Aquadistri BV, the Netherlands).

Defined embryo buffer

To produce a defined and standardized vehicle for these experiments, we used 10% Hank's balanced salt solution (made from cell-culture tested, powdered Hank's salts, without sodium bicarbonate, Cat. No H6136-10X1L, Sigma-Aldrich, St Louis, MO) at a concentration 0.98 g/L in Milli-Q water (resistivity = 18.2 MV?cm), with the addition of sodium bicarbonate at 0.035 g/L (Cell culture tested, Sigma Cat S5761), and adjusted to pH 7.46. A similar medium has been used previously [3739].

Table 2. Zebrafish embryo LC50 values found in this study, and the corresponding rodent LD50 values based on the literature.

Compounds Zebrafish Embryo LC50 (mg/L ±SEM) Zebrafish Embryo LC50 (mmol/L ±SEM) Rodent LD50 (mg/kg) Rodent LD50 (mmol/kg)

1 Aconitine 34.361.5 0.05 6 0.00 1« 0.002

2 Atropine 607.86 7.7 2.1060.03 500n 1.73

3 Berberine chloride 129.263.6 0.3560.01 60(*) 0.16

4 Colchicine 41.560.7 0.1060.00 5.9(*) 0.02

5 Coniine 55.1 60.2 0.4360.00 80(*) 0.63

6 a-Lobeline hydrochloride 30.960.9 0.0860.00 39.9(*) 0.11

7 Morphine hydrochloride 9915.1 60.8 23.3960.00 745(*) 1.76

8 Nicotine 35.1 60.5 0.2260.00 50(#) 0.31

9 Quinine sulfate 562.46 9.5 1.4460.02 800(*) 2.04

10 (—)-Scopolamine hydrobromide trihydrate 11465.16166.1 26.1660.38 1413(*) 3.22

11 Strychnine hydrochloride 20.860.58 0.0660.00 2.73(*) 0.01

12 Theobromine 150.461.83 0.83 6 0.01 530(*) 2.94

13 (+)-Tubocurarine chloride hydrate 414.263.6 0.6160.01 33(*) 0.05

14 Yohimbine hydrochloride 93.061.5 0.2460.00 55(*) 0.14

15 Amygdalin 268.5619.6 0.5960.04 250(*) 0.55

16 Arbutin 120.9613.0 0.4460.05 500(*) 1.84

17 Convallatoxin 36.663.5 0.0760.01 15.2(*) 0.03

18 Coumarin 241.268.9 1.65 6 0.06 293(*) 2.01

19 Digitoxin 0.560.06 0.001 60.00 4.1(*) 0.01

20 Gentamycin sulfate 253.366.5 0.4460.01 384(*) 0.67

21 Glycyrrhizin 55.863.0 0.0760.00 589(*) 0.70

22 Hesperidin 77.663.2 0.1360.01 1000(*) 1.64

23 Kanamycin monosulfate 1787.5616.8 3.0760.03 1 700(*) 2.92

24 Naringin 850.16 78.5 1.4660.14 2000(*) 3.45

25 Neohesperidin 199.561.2 0.3360.00 1000(*) 1.64

26 Ouabain octahydrate 184.164.8 0.2560.01 3.75(*) 0.01

27 Phloridzin dihydrate 793.265.1 1.6860.01 500(*) 1.06

28 Rutin hydrate 8722.96164.24 14.29 60.27 2000(*) 3.28

29 Streptomycin sulfate 3164.0 6 35.4 2.1760.02 600(*) 0.41

30 Cadmium(II) chloride 27.960.1 0.066.00 88(#) 0.18

31 Copper(II) nitrate trihydrate 58.761.2 0.2460.00 940(*) 3.89

32 Lead acetate trihydrate 62.461.1 0.1660.00 174(*) 0.46

33 Lithium chloride 3324.26143.6 78.4263.39 1165n 27.48

34 Chloramphenicol 525.06 7.4 1.62 6 0.02 400n 1.24

35 Ethanol 36212.06501.8 786.02610.89 14008.3(#) 304.07

36 Glycerol 23357.46282.1 253.5863.06 12619(#) 137.00

37 Tween 80 323.4610.1 0.2560.01 25021(#) 19.10

38 Acetic acid 186.361.0 3.1060.02 3309.3<#) 55.11

39 Salicylic acid 46.761.2 0.3460.01 184(*) 1.33

40 Sodium oxalate 372.262.9 2.7860.02 155.4(#) 1.16

41 Trichloroacetic acid 66.464.7 0.4160.03 270(*) 1.65

42 Ampicillin sodium 6068.56114.9 16.34 60.31 5314n 14.31

43 Cyclophosphamide monohydrate 1777.4626.1 6.3760.09 1930.9(#) 6.92

44 Paracetamol 535.8617.1 3.5460.11 367« 2.43

45 Phenacetin 309.968.4 1.7360.05 634(*) 3.54

46 Benserazide hydrochloride 4747.9628.7 16.1760.10 5000(*) 17.02

47 Chlorpromazine hydrochloride 7.060.04 0.02 6 0.00 20(,) 0.06

48 Isoniazid 1297.5638.0 9.4660.28 1250(*) 9.12

tt PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e21076

Table 2. Cont.

Compounds Zebrafish Embryo LC50 (mg/L ±SEM) Zebrafish Embryo LC50 (mmol/L ±SEM) Rodent LD50 (mg/kg) Rodent LD50 (mmol/kg)

49 Phenelzine sulfate 11.56Q.13 Q.Q56Q.QQ 125« Q.53

5Q Ethambutol dihydrochloride 6325.96197.2 22.82 6Q.71 68QQ(*) 24.53

51 Verapamil hydrochloride 81.1 64.8 Q.176Q.Q1 1 Q8(#) Q.22

52 Phenol 86.46Q.8 Q.926Q.Q1 112« 1.19

53 Sodium azide 1.46Q.Q4 Q.Q26Q.QQ 19« Q.29

54 Dimethyl sulfoxide 2Q964.66158.1 268.33 62.Q2 19691.3(#) 252.Q3

55 Formaldehyde 12.76Q.1 Q.426Q.QQ 42« 1.4Q

56 Phenformin hydrochloride 5Q8.3617.6 2.1Q6Q.Q7 4Q7n 1.69

57 Ropinirole hydrochloride 437.361Q.2 1.476Q.Q3 396n 1.33

58 Amitriptyline hydrochloride 8.Q6Q.1 Q.Q36Q.QQ 21« Q.Q7

59 Sodium dodecyl sulfate 3.66Q.3 Q.Q16Q.QQ 118« Q.41

6Q Barbital sodium 39Q2.563Q.5 18.93 6Q.15 31Q1(#) 15.Q4

(i)from Chemical Identification/Dictionary database at http://toxnet.nlm.nih.gov/cgibin/sis/search/; (#)from [36].

doi:1Q.1371/journal.pone.QQ21Q76.t0Q2

Egg water

Egg water was made from 0.21 g 'Instant Ocean®' salt in 1 L of Milli-Q water with resistivity of 18.2 Mfl-cm.

Embryo care

Eggs were obtained by random pairwise mating of zebrafish. Three adult males and four females were placed together in small breeding tanks (Ehret GmbH, Emmendingen, Germany) the evening before eggs were required. The breeding tanks (L 26 cm; H 12.5 cm, W 20 cm) had mesh egg traps to prevent the eggs from being eaten. The eggs were harvested the following morning and transferred into 92 mm plastic Petri dishes (50 eggs per dish) containing 40 ml fresh embryo buffer. Eggs were washed four times to remove debris. Further, unfertilized, unhealthy and dead embryos were identified under a dissecting microscope and removed by selective aspiration with a pipette. At 3.5 hpf, embryos were again screened and any further dead and unhealthy embryos were removed. Throughout all procedures, the embryos and the solutions were kept at 28±0.5°C, either in the incubator or a climatised room. All incubations of embryos were carried out under a light cycle of 14 h light: 10 h dark (lights on at 08.00). All pipetting was done manually, with an 8-channel pipetter.

Viability of early embryos

There are reports of an early ''mortality wave'' in zebrafish embryos cultured under certain conditions [for examples, see: 40,41]. In order to assess this mortality wave in our facilities, and to avoid taking embryos during such a die-off, we raised cleaned embryos in 92 mm Petri dish (60 eggs per dish) containing 40 ml Hank's buffer alone, or egg water alone. We scored the fertilisation rate and mortality of embryos at 4, 8, and 24 hpf (see below) in these two media.

Evaporation of buffer from 96-well plate

Evaporation rate of buffer from the 96-well plates (Costar 3599, Corning Inc., NY) was determined as follows. In each well of the plate, 250 mL of freshly prepared buffer was dispensed at 0 h. As

for all 96-well plate experiments reported in this study, the lids were in place but were not sealed with a sealing mat or film (because preliminary studies indicated that all embryos die within sealed plates). The plates were kept at 28±0.5°C without refreshing the buffer (static non-replacement regime) and weighed at daily intervals on a digital balance. Results were calculated as mean from four different plates. Buffer volume from some individual wells in different regions of the plate were also weighed at 4 days to determine the impact of well location on the evaporation rate.

Test compounds

We used water-soluble compounds representing a range of different chemical classes and biochemical activities (Table S1). The required dilution was always freshly prepared in buffer just prior to assay on zebrafish embryos.

Mortality scoring

Mortality rate (Table 1) was recorded at 48, 72, 96 and 120 hpf in both logarithmic series and geometric series using a dissecting stereomicroscope. Embryos were scored as dead if they were no longer moving, the heart was not beating and the tissues had changed from a transparent to an opaque appearance.

Range-finding

To determine a suitable range of concentrations for testing, we performed range-finding using a logarithmic series (0.0, 1.0, 10.0, 100.0 and 1000 mg/L) as recommended in standard protocols [11]. Zebrafish embryos of 24 hpf from Petri dish were gently transferred using a sterile plastic pipette into 96-well microtitre plates. A single embryo was plated per well, so that dead embryos would not affect others, and also to allow individual embryos to be tracked for the whole duration of the experiment. A static non-replacement regime was used. Thus there was no replacement or refreshment of buffer after the addition of compound. Each well contained 250 mL of either freshly prepared test compound; or vehicle (buffer) only as controls. We used 16 embryos for each concentration and 16 embryos as controls for each drug.

Figure 1. Cumulative mortality and infertility of zebrafish in buffer or egg water. Embryos were kept in 92 mm Petri dishes with 40 ml of either buffer or egg water, 60 eggs per dish. Each error bar represents ±SEM of N = 420 embryos each for buffer and egg water. A, cumulative infertility and early mortality in buffer. B, the same, in egg water. There is no significant difference between the two media in terms of survival and fertilization percentage. doi:10.1371/journal.pone.0021076.g001

Geometric series and LC50 determination

After the range finding experiments, a series of concentrations lying in the range between 0% and 100% mortality were selected. The actual concentrations used are shown in Table S2. The concentrations were in a geometric series in which each was 50% greater than the next lowest value [11]. Each geometric series of

concentrations for each compound was repeated three times (in total 48 embryos per concentration and 48 embryos for vehicle for each drug). LC50 (expressed in mg/L of buffer) was determined based on cumulative mortality obtained from three independent experiments at 120 hpf using Regression Probit analysis with SPSS Statistics for windows version 17.0 (SPSS Inc., Chicago, USA). Thus the embryos

10. 8. 6. 42- ^

oJ--------------

24 h 48 h 72 h 96 h

Timepoints

Figure 2. Rate of evaporation from 96-well plates at 28.0°C. Buffer was dispensed in four different 96-well plates. A, cumulative average percentage buffer loss per plate. All wells were initially filled with 250 mL buffer. B, percentage buffer loss after 96 h, per well, as a function of well position. The letters A-H and the numbers 1-12 correspond to the standard coordinates embossed into 96-well plates. All wells were initially filled with 250 mL buffer. Only the wells with grey columns were measured. doi:10.1371/journal.pone.0021076.g002

are exposed to the drug for 96 h. The LC50 in mg/L was converted into LC50 mmol/L to make relative toxicity easier to examine.

Rodent data

The sources of LD50 data from rodents (rats and mice) are shown in Table 2.

Statistical analysis

Statistical analyses were performed using GraphPad Prism for Windows (version 5.03) or R (v. 2.12). One way ANOVA and

Newman-Keuls Multiple Comparison test was employed for survival rate. Correlation and ANCOVA models were used to investigate the relationship between LC50 in zebrafish embryos and published LD50 values in rodents.

Results and Discussion

We have examined the toxicity, in zebrafish embryos, of a 96 h exposure (during the period 24 hpf to 5 dpf) to 60 compounds of differing biochemical classes. Our logarithmic and geometric

4-2 - I 0 1 2 Rodent Log LD50 (mmol/kg)

Figure 3. Correlation between zebrafish embryo Log LC50 and rodent Log LD50 for the 60 compounds tested in this study. Zebrafish embryo LC50 was determined based on cumulative mortality after 96 h exposure of compounds from three independent experiments and rodent LD50 was taken from the literature. Key: blue, regression line; solid black lines, 0.25 and 0.75 quartiles; dashed line, perfect correlation line. The slope of the regression line (blue) is 0.73403. doi:10.1371/journal.pone.0021076.g003

concentration series both showed concentration-dependent mortality. LC50 values were determined, and compared with rodent LD50 values from the literature.

Infertility and spontaneous early mortality of eggs/ embryos

We found that, in controls (buffer only), 5% of eggs were unfertilised, and a further 9% represented embryos that died spontaneously in the first 24 hpf. This is similar to the spontaneous mortality of 5-25% reported elsewhere for early zebrafish development [40]. We find no significant difference between these values when Hank's buffer was used as the medium, and when egg water was used (Figure 1A, B). In order to avoid this natural early mortality we began our assays at 24 hpf. This also makes our study consistent with a previous one, in which the zebrafish was exposed to different compounds at 24 hpf to find the correlation between zebrafish and rodent toxicities [34].

It could be argued that, by beginning exposure at 24 h, we are missing out on early developmental toxicity effects, such as the action of compounds on gastrula stages. However, this is likely to be a trade-off because other compounds mainly cause embryo death at these early stages. For example, a recent study [42] showed that exposure of zebrafish embryos at early stages (dome to 26-somite) to ethanol resulted in high mortality, while exposure at later stages (prim-6 and prim-16) led to a high incidence of abnormal embryos. Other examples of compounds which are more toxic to larval stages than to embryonic and adult stages of freshwater fish species are copper and cadmium [43-45]. Finally,

it is known that presence of chorion at early stages acts as a possible barrier to diffusion of compounds [29,30,42].

Rate of evaporation from 96-well plates at 28.0°C

In our study, we did not replace the buffer. Therefore, we decided to check how much water would be lost during this period by evaporation from the 96-well plate (with its lid in place). We found that, by 96 h of incubation at 28.0°C, 9.46% of the buffer had evaporated (Figure 2A). Further investigation showed that the rate of evaporation was higher in the external rows and columns, and highest of all in the four corner wells (Figure 2B). In view of this evaporation pattern, we filled all the 96-wells with buffer, but did not plate embryos into wells A1-H1 and A12-H12. A way of mitigating the effects of this rate of evaporation would be to use dynamic replacement of buffer, as in a microfluidic chip [39], or static replacement (e.g. daily refreshing). Nonetheless, static non-replacement, as used here, is a popular technique for zebrafish embryo culture, and was used in a recent toxicity study [46].

Concentration response and LC50 of compounds

For all compounds, mortality at 5 dpf was concentration-dependent (Table 1). This was true for both logarithmic and geometric series. By contrast, controls (vehicle only) showed 0% mortality. The LC50 values are shown in Table 2.

Correlation between zebrafish embryo log LC50 and rodent log LD50

To examine the ability of zebrafish assays to predict toxicity in rodents, we analysed a correlation between our zebrafish embryo

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Others

Rodent Log LD5o{mmol/kg)

Figure 4. Linear regression model: rodent log LD50 and zebrafish embryo log LC50. The effect of the different compounds on the slope and intercept of the ANCOVA model. Although we must consider the effect of the unknown error in the rodent LD50 values, the different compound classes seem to cluster in different regions in the graph. doi:10.1371/journal.pone.0021076.g004

log LC50 values, and rodent log LD50 from the literature. The comparison is shown graphically in Figure 3. A correlation test produced Spearman's rank correlation of 0.7688 (p<0.001) and a Pearson's product-moment correlation 0.7832 (df= 178, p<0.001) between zebrafish embryo LD50 and rodent log LD50 for the whole set of compounds. These values of correlation indicate that zebrafish LC50 and rodent LD50 values co-vary. This is consistent with a previous report [34] that the toxicity of 18 compounds in

zebrafish embryos was well-correlated with values reported from rodent studies. It is also in line with another study [46] suggesting that zebrafish embryos could be used as a predictive model for the developmental toxicity of compounds.

Toxicity by compound class

We next developed a statistical model that examines the similarity between zebrafish and rodent toxicity values when the

Table 3. Statistical analysis of regression per group of compound using the ANCOVA model described in the text.

Coefficients Estimate Std. error t-value p-value Significance level

Intercept: Others -0.43 0.08704 -4,930 1.96E-06 #

Intercept: Alcohols -0.64 0.35187 -1,813 0.071546

Intercept: Alkaloids 0.03 0.11947 0.24 0.810735

Intercept: Amides -0.08 0.49548 -0.168 0.866425

Intercept: Carboxylic acids -0.17 0.23275 -0.751 0.45351

Intercept: Glycosides -0.2 0.10027 -1,970 0.050426

Slope: Others 1.27 0.08803 14,456 2.00E-16 *

Slope: Alcohols 1.24 0.21852 -0.139 0.889249 *

Slope: Alkaloids 0.56 0.13171 -5,427 1.97E-07 *

Slope: Amides 1.06 0.63408 -0.326 0.744924

Slope: Carboxylic acids 0.36 0.27869 -3,279 0.001265 *

Slope: Glycosides 0.77 0.13576 -3,684 0.000309 *

doi:10.1371/journal.pone.0021076.t003

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compounds are clustered into chemically similar groups. To do this, we mapped zebrafish values to rodent values, taking account of specific variances in intercept and slope, due to those groupings. The groupings were alcohols, alkaloids, amides, carboxylic acids, glycosides and the remaining compounds (others). We designed an ANCOVA with the values [zebrafish embryo log LC50] as dependent variables, and [rodent log LD50] and [compound type] as independent variables. Table 3 shows the statistics of our ANCOVA model, while the dataset is displayed graphically in Figure 4. As can be seen, there is a significant effect of compound type on intercept and slope.

The slope for amides (Table 3) does not differ significantly from

I.0, indicating a very similar toxicity in zebrafish and rodents. By contrast, 'others' and alcohols have a slope significantly greater than 1.0, indicating that they are generally less toxic in zebrafish than in rodents. The groups carboxylic acids, glycosides and alkaloids have a slope significantly less than 1.0 indicating that they are more toxic in zebrafish than in rodents (Table 3).

If we look at the relative toxicity ([zebrafish LC50 mmol/L] 4 [rodent LD50 mmol/kg]) of individual compounds we see the following examples of compounds that have a similar toxicity in the two sepcies: coumarin (0.95), benserazide hydrochloride (1.06), phenformin hydrochloride (1.11) and theobromine (1.11). Examples of compounds less toxic in zebrafish than in rodents are aconitine (0.01), ouabain octohydrate (0.02), tubocurarine hydro-chloride (0.07), morphine hydrochloride (0.08) and colchicine (0.13). At the other extreme are compounds more toxic in zebrafish than in rodents including: Tween80 (103.01), sodium dodecyl sulfate (98.33), lead acetate trihydrate (29.49) and copper (II) nitrate trihydrate (19.40).

Among the alcohols, the general trend is a lower toxicity in zebrafish than in rodents. Tween 80 is an exception to this trend because it is much more toxic to zebrafish. This could be because of its surfactant properties, a suggestion supported by the comparably high relative toxicity to zebrafish (98.33) that we find for another surfactant tested, sodium dodecyl sulphate (SDS). Our LC50 for SDS in 96 h exposure to zebrafish embryos was 3.6 mg/ L. This is similar to the dose of SDS that causes pathological changes in the gills of the teleost Thalassoma pavo [47]. Copper also

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Conclusions

Our findings show that the zebrafish embryo is a tool that offers potential in the evaluation of drug safety. However, we show that the predictivity varies between the class of compound studied. More work is required to examine how the covariance of zebrafish and rodent toxicity is influenced by such factors as compound type, absorption, metabolism and mechanism of toxicity.

Supporting Information

Table S1 Summary of compounds used in this study for toxicity evaluation in zebrafish embryo.

Table S2 Concentrations used in geometric series.

Acknowledgments

We thank Peter J. Steenbergen for expert technical assistance and zebrafish breeding.

Author Contributions

Conceived and designed the experiments: SA MKR HGJvM. Performed the experiments: SA. Analyzed the data: SA MKR HGJvM. Contributed reagents/materials/analysis tools: MKR. Wrote the paper: SA MKR.

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