ZeGlobalTox: An Innovative Approach to Address Organ Drug Toxicity Using Zebrafish

Toxicity is one of the major attrition causes during the drug development process. In that line, cardio-, neuro-, and hepatotoxicities are among the main reasons behind the retirement of drugs in clinical phases and post market withdrawal. Zebrafish exploitation in high-throughput drug screening is becoming an important tool to assess the toxicity and efficacy of novel drugs. This animal model has, from early developmental stages, fully functional organs from a physiological point of view. Thus, drug-induced organ-toxicity can be detected in larval stages, allowing a high predictive power on possible human drug-induced liabilities. Hence, zebrafish can bridge the gap between preclinical in vitro safety assays and rodent models in a fast and cost-effective manner. ZeGlobalTox is an innovative assay that sequentially integrates in vivo cardio-, neuro-, and hepatotoxicity assessment in the same animal, thus impacting strongly in the 3Rs principles. It Reduces, by up to a third, the number of animals required to assess toxicity in those organs. It Refines the drug toxicity evaluation through novel physiological parameters. Finally, it might allow the Replacement of classical species, such as rodents and larger mammals, thanks to its high predictivity (Specificity: 89%, Sensitivity: 68% and Accuracy: 78%).


Introduction
The direct costs of bringing a new drug to the market are continuously increasing. Nowadays, the estimated costs are higher than US $1 billion per drug, and most of that is spent in the clinical phases [1]. On the other hand, the pharmaceutical industry has multiplied its investments in R&D. However, this increase does not correlate well with an increased success rate in marketing new drugs. This is partly due to the high rate of compound failure during clinical trials, where only around 10% of the molecules entering phase 1 clinical trials are ultimately approved by the United States Food and Drug Administration (FDA) [2,3]. Lack of efficacy or drug safety (toxicity) are the major factors in drug attrition, with the lack of efficacy being the leading cause of drug attrition during clinical trials and unanticipated toxicity being the most common cause of post market withdrawal [3][4][5][6]. To reduce the (teratogenicity) could mask possible organ-toxicities appearing later in development. To counteract this prospect, we included a preliminary Acute Toxicity assay performed with five logarithmic concentrations which follows the Organisation for Economic Cooperation and Development (OECD) guidelines (OECD 236) ( Figure 1A). This preliminary assay allowed for the identification of non-mortal/non-teratogenic concentrations-no observed effect concentration (NOEC)-to use in the following assays. The hypothesis is that NOEC could affect organ physiology (organ toxicity), while uncoupled from putative developmental toxicity side-effects affecting organ development or function. Another consideration was to start drug incubations at 96 hours post fertilization (hpf), when the analysed organs are close to or already developed ( Figure 1B). The overall aim is to understand the drug impact on organ physiology and function rather than early embryogenesis. The third consideration was to organize the sequential organ evaluation during the experimental and drug incubation time.
Among the three organs, the heart is developed earlier. In addition, cardiotoxic effects are observable shortly after compound incubation. Thus, cardiotoxicity evaluation was chosen first. Neurotoxicity was analysed second through the drug impact on motor behaviour (locomotion), which is a fundamental readout of CNS function. Although both autonomous swimming and liver development are completed by 5 dpf, phenotypes promoted by hepatotoxic effects require a longer drug exposure. In addition, part of the hepatotoxicity evaluation required fixed larvae. Then, hepatotoxicity was the final parameter evaluated. The integrated experimental pipeline is displayed in Figure 1. preliminary Acute Toxicity assay performed with five logarithmic concentrations which follows the Organisation for Economic Cooperation and Development (OECD) guidelines (OECD 236) ( Figure  1A). This preliminary assay allowed for the identification of non-mortal/non-teratogenic concentrations-no observed effect concentration (NOEC)-to use in the following assays. The hypothesis is that NOEC could affect organ physiology (organ toxicity), while uncoupled from putative developmental toxicity side-effects affecting organ development or function. Another consideration was to start drug incubations at 96 hours post fertilization (hpf), when the analysed organs are close to or already developed ( Figure 1B). The overall aim is to understand the drug impact on organ physiology and function rather than early embryogenesis. The third consideration was to organize the sequential organ evaluation during the experimental and drug incubation time.
Among the three organs, the heart is developed earlier. In addition, cardiotoxic effects are observable shortly after compound incubation. Thus, cardiotoxicity evaluation was chosen first. Neurotoxicity was analysed second through the drug impact on motor behaviour (locomotion), which is a fundamental readout of CNS function. Although both autonomous swimming and liver development are completed by 5 dpf, phenotypes promoted by hepatotoxic effects require a longer drug exposure. In addition, part of the hepatotoxicity evaluation required fixed larvae. Then, hepatotoxicity was the final parameter evaluated. The integrated experimental pipeline is displayed in Figure 1.

Test Compounds
In order to validate our ZeGlobalTox platform, 24 compounds were evaluated; including four drugs used as positive toxic controls. To calculate the ZeGlobalTox predictive potential, compounds were chosen according to their known toxicity in humans, as displayed in known molecule toxicity

Test Compounds
In order to validate our ZeGlobalTox platform, 24 compounds were evaluated; including four drugs used as positive toxic controls. To calculate the ZeGlobalTox predictive potential, compounds were chosen according to their known toxicity in humans, as displayed in known molecule toxicity databases such as TOXNET (Hazardous Substances Data Bank, HSDB), Side effects (EMBL), drugs.com, and ema.europa. The four selected control drugs have been selected due to their reported toxicities both in humans and zebrafish. Haloperidol, a known anti-dopaminergic antipsychotic drug, has been used as our positive cardiotoxic drug because it has been described to produce hERG blockade, QT interval prolongation, and arrhythmias both in humans and zebrafish [19][20][21]. MPTP, a prodrug to 1-methyl-4-phenylpyridinium (MPP + ) first synthesized as an analgesic, has been shown to cause permanent Parkinson's symptoms by destroying dopaminergic neurons in the substantia nigra [22]. Indeed, it has been used to model Parkinson's disease in various animal models including zebrafish [23]. Hence, we used MPTP as the neurotoxic positive control drug. Finally, ethanol and acetaminophen (APAP, paracetamol) were used as our positive hepatotoxicity drugs. Both are well known molecules producing liver injury (extensively reviewed in [24,25]), with steatosis as a major side-effect for ethanol and liver malfunction and necrosis of liver tissue as the main toxic effect from paracetamol. Both hepatotoxic effects have also been reported in zebrafish larvae [26][27][28][29] (Table 1).

Acute Tox Analysis
As explained above, the Acute Tox test was performed to determine the maximum drug concentration in which no mortality or gross teratogenic effects were observed (Non Observed Effect Concentration; NOEC). As a positive toxic drug we chose Diethylaminobenzaldehyde (DEAB), a Retinoid Acid inhibitor that promoted mortality and teratogenicity in a reproducible concentration dependent curve. 1% DMSO, which is the constant solvent concentration in all conditions, was used as the negative control. Mortality curves for all compounds at 96 hpf (blue line; Figure 2A-T), compared with DEAB curves (red line; Figure 2A-T), are shown in Figure 2. Additionally, the results from this analysis are displayed in Table 2.

Cardiotoxicity Analysis
Four cardiac parameters were evaluated to assess whether a compound was cardiotoxic: heart rate (beats per minute, BPM), QTc prolongation, ejection fraction (EJF), and cardiac arrest. Haloperidol

Cardiotoxicity Analysis
Four cardiac parameters were evaluated to assess whether a compound was cardiotoxic: heart rate (beats per minute, BPM), QTc prolongation, ejection fraction (EJF), and cardiac arrest. Haloperidol has been described as cardiotoxic in humans and zebrafish [19] and was used as our cardiotoxic control. 1% DMSO was used as the negative control.
Cardiotoxicity evaluation was performed from 96 hpf, when the zebrafish heartbeat is already stabilised [36,37], so the analysis might not be altered by unstable beating. Zebrafish hearts were video-recorded at 4 h after drug incubation (100 hpf) and analysed using the ZeCardio ® β software ( Figure 3A). has been described as cardiotoxic in humans and zebrafish [19] and was used as our cardiotoxic control. 1% DMSO was used as the negative control. Cardiotoxicity evaluation was performed from 96 hpf, when the zebrafish heartbeat is already stabilised [36,37], so the analysis might not be altered by unstable beating. Zebrafish hearts were video-recorded at 4 h after drug incubation (100 hpf) and analysed using the ZeCardio ® β software (Figures 3A).  Eight compounds-haloperidol, cisapride, docetaxel, dofetilide, pindolol, riluzole, trifluoperazine HCL, and vincristine-decreased the heart rate when compared to DMSO-only ( Figure 3B). Longer cardiac arrest was promoted by the same compounds as well ( Figure 3E). Inversely, zebrafish larvae treated with ciprofloxacin and D-(+)-glucose showed increased heart rates but no differences were observed in the duration of the cardiac arrest, when compared to the DMSO-only treated larvae ( Figure 3B,E).
QTc interval prolongation was detected in larvae treated with haloperidol and pindolol, while ciprofloxacin and D-(+)-glucose showed a shorter QTc interval than the DMSO treated group ( Figure 3C). Finally, no differences in ejection fraction were detected in any of the 24 compounds tested ( Figure 3D).

Locomotor Activity Analysis
By 5 dpf, zebrafish larvae perform spontaneous swimming and their visual system is fully developed [31,38,39]. Therefore, behavioural experiments were performed from this time point ( Figure 4A). Deviations in total distance moved, in response to photo-visual stimulation, were analysed as a direct measurement of neurotoxicity. Thus, drugs increasing or decreasing total distance moved when compared to the DMSO-only group were considered neurotoxic. As the positive neurotoxic control we used MPTP, which has been identified as a neurotoxic drug in humans and zebrafish [40].
Decreased motility was detected in MPTP, paracetamol, and trifluoperazine-HCL treated larvae, while (±)-epinephrine HCL, docetaxel, pindolol, and vincristine groups showed increased motility when compared to the DMSO ( Figure 4B). Eight compounds-haloperidol, cisapride, docetaxel, dofetilide, pindolol, riluzole, trifluoperazine HCL, and vincristine-decreased the heart rate when compared to DMSO-only ( Figure 3B). Longer cardiac arrest was promoted by the same compounds as well ( Figure 3E). Inversely, zebrafish larvae treated with ciprofloxacin and D-(+)-glucose showed increased heart rates but no differences were observed in the duration of the cardiac arrest, when compared to the DMSO-only treated larvae ( Figure 3B,E).
QTc interval prolongation was detected in larvae treated with haloperidol and pindolol, while ciprofloxacin and D-(+)-glucose showed a shorter QTc interval than the DMSO treated group ( Figure 3C). Finally, no differences in ejection fraction were detected in any of the 24 compounds tested ( Figure 3D).

Locomotor Activity Analysis
By 5 dpf, zebrafish larvae perform spontaneous swimming and their visual system is fully developed [31,38,39]. Therefore, behavioural experiments were performed from this time point ( Figure 4A). Deviations in total distance moved, in response to photo-visual stimulation, were analysed as a direct measurement of neurotoxicity. Thus, drugs increasing or decreasing total distance moved when compared to the DMSO-only group were considered neurotoxic. As the positive neurotoxic control we used MPTP, which has been identified as a neurotoxic drug in humans and zebrafish [40].

Hepatotoxicity Analysis
Zebrafish liver development is fast and can be divided in three main stages: specification, differentiation, and hepatic outgrowth (reviewed in [41]). By 5 dpf, the liver is fully functional and consists of two lobes, with an overall oblong shape [42]. Since hepatotoxic effects are mainly due to metabolic processes, which require a certain time to be executed, experiments were performed at 132 hpf. As the positive control we used paracetamol and ethanol, which have been shown to produce hepatotoxicity in humans and zebrafish [43].

Hepatotoxicity Analysis
Zebrafish liver development is fast and can be divided in three main stages: specification, differentiation, and hepatic outgrowth (reviewed in [41]). By 5 dpf, the liver is fully functional and consists of two lobes, with an overall oblong shape [42]. Since hepatotoxic effects are mainly due to metabolic processes, which require a certain time to be executed, experiments were performed at 132 hpf. As the positive control we used paracetamol and ethanol, which have been shown to produce hepatotoxicity in humans and zebrafish [43].

Hepatomegaly and Liver Necrosis Evaluation
Zebrafish larvae were fixated and photographed after 36 h of drug incubation (96-132 hpf; Figure 5A). The transgenic zebrafish line Tg(cmlc2:GFP; fabp10:RFP; ela31:EGFP) expressed RFP protein in all liver cells. The analysis of fluorescence intensity allowed for the detection of drugs affecting liver size or the number of hepatocytes [44]. Thus, drugs reducing the number of hepatocytes (necrosis) translated into reduced RFP area, whereas drugs increasing liver size (hepatomegalia) corresponded with increased RFP area. In that regard, liver areas of the 24 compounds were analysed and compared with those obtained using the DMSO-only group. Three drugs including paracetamol, flupirtine, and methyldopa showed decreased RFP area signal, whereas finasteride and fusidic acid treatments increased the area of the RFP signal ( Figure 5B).  Figure 5A). The transgenic zebrafish line Tg(cmlc2:GFP; fabp10:RFP; ela31:EGFP) expressed RFP protein in all liver cells. The analysis of fluorescence intensity allowed for the detection of drugs affecting liver size or the number of hepatocytes [44]. Thus, drugs reducing the number of hepatocytes (necrosis) translated into reduced RFP area, whereas drugs increasing liver size (hepatomegalia) corresponded with increased RFP area. In that regard, liver areas of the 24 compounds were analysed and compared with those obtained using the DMSO-only group. Three drugs including paracetamol, flupirtine, and methyldopa showed decreased RFP area signal, whereas finasteride and fusidic acid treatments increased the area of the RFP signal ( Figure 5B).

Steatosis and Yolk Lipid Accumulation Evaluation
During the first week of development, the unique source of energy for the zebrafish embryo and larva is the yolk sac. Zebrafish yolk consists of 70% neutral lipid, which is metabolized mainly in

Steatosis and Yolk Lipid Accumulation Evaluation
During the first week of development, the unique source of energy for the zebrafish embryo and larva is the yolk sac. Zebrafish yolk consists of 70% neutral lipid, which is metabolized mainly in the liver [45]. Thus, yolk lipid accumulation can be used as an endpoint for liver function since, if impaired, the yolk metabolism and absorption is delayed, which results in higher lipid retention [46]. On the other hand, drug-induced steatosis (hepatocyte lipid accumulation) is an off-target liver effect which can be used to prioritize compounds for development [47,48]. Hence, drugs affecting lipid metabolism in human hepatocytes might be identified by using zebrafish livers [26,49].
In order to assess drugs producing steatosis and yolk lipid accumulation, and subsequent to the RFP filtered images being acquired, zebrafish larvae were stained with Oil Red O. Larvae and were sorted into steatosis positive or negative, yolk lipid accumulation, or both (see materials and methods). Percentages were calculated for each drug and compared with those obtained in the DMSO-only group ( Figure 5D).

Discussion
Cardio-, neuro-, and hepatotoxicity are the most relevant organ-toxicities promoting drug attrition during preclinical, clinical, and post market stages [10]. Previous studies have shown the relevance of using zebrafish for predicting the possible impact of drugs in those three organs individually [21,29,[50][51][52][53][54][55][56]. However, no previous studies have integrated the analysis of these three organ-toxicities in the same animal; a procedure that reduces animal usage, experimental time and costs, and the quantity of the tested compound.
Results obtained through the ZeGlobalTox assay show high sensitivity, specificity, and accuracy values when we compare the zebrafish experimental data with known human toxicity outputs (Table 3). This is indeed a promising conclusion, given the need for predictive and cost-effective procedures required to narrow down the number of compounds reaching expensive and time-consuming mammalian and clinical studies. Altogether, we propose ZeGlobalTox could be used to reduce the time and costs of drugs for being approved, together with improving 3Rs policies during the whole drug discovery process. Nonetheless, we will discuss below a number of aspects to be considered in order to improve this approach.  FP  TN  TN  Digoxigenin  FN  TN  FP  Docetaxel  TP  TP  FN  Dofetilide  TP  TN  TN  Finasteride  TN  TN  FP  Flupirtine  TN  TN  TP  Fusidic Acid  TN  TN  TP  Isoniazid  TN  FN  TP  Table 3. Cont. TN  TN  TN   L-Glutamine   TN  TN  TN  Methyldopa  TN  FN  TP  NaCl  TN  TN  TN  Pindolol  TP  TP  TN  Riluzole  TP  TN  FN  Suramin  TN  FN  TN  Trifluoperazine  HCL  TP  TP  Four endpoints were analysed for cardiotoxicity evaluation-BPM, QTc, EJF, and cardiac arrest. A drug was considered cardiotoxic when one of these parameters was found to be statistically different when compared to the DMSO-only group. Our analysis has detected cardiotoxic end-phenotypes in 9 out of 12 human cardiotoxic compounds present in the study. However, from 6 drugs reported to produce QTc prolongation in humans-haloperidol, (±)-epinephrine HCL, ciprofloxacin, cisapride, dofetilide, and trifluoperazine HCL-only haloperidol treated larvae displayed that phenotype. Furthermore, pindolol, not reported to produce QTc prolongation in humans, showed increased QTc in zebrafish larvae. This latter phenotype might be explained by pindolol non-selective blockage of heart ß-receptors. Interestingly, bradycardia was detected in 4 out of 6 drugs producing QTc prolongation in humans. This is consistent with results presented by Wen et al. [57], which showed a correlation between drugs producing QTc prolongation (in dogs) and bradycardia in zebrafish. On the other hand, tachycardia was observed in D-(+)-glucose treated zebrafish larvae. Although glucose is generally innocuous for humans, cardiotoxicity has been reported in hyperglycemic patients and patients suffering from diabetes [58][59][60]. A correlation between high blood glucose levels and poorer outcomes after cardiac arrest has also been described [61]. Therefore, high doses of glucose might also be related to increased cardiotoxicity risk in humans. We hypothesize that tachycardia detected in zebrafish might be due to the need for eliminating/compensating high glucose concentrations as fast as possible. Cardiotoxic false negatives (FN) such as paracetamol, ethanol, and (±)-epinephrine HCL could be explained by differences among human and zebrafish physiology or by the ZeGlobalTox procedure, where cardiotoxic effects are analysed only 4 h after drug incubation. Most cardiotoxic effects can be detected shortly after compound incubation; however, these three compounds might require a longer exposure to reproduce their known cardiotoxic effects. This seems certain for ethanol and paracetamol, since their human cardiotoxic impact is observed as a late effect after drug poisoning. In fact, paracetamol has been reported to have no impact on the heart rate in zebrafish larvae [50,57], to the point that Wen et al. [54] included this drug as a negative cardiotoxic drug [57]. In summary, we support zebrafish as a powerful tool for predicting drug-induced cardiotoxic liabilities in humans, including typical repolarization and depolarization end-phenotypes such as QTc or EJC. However, our experimental methodology-drug exposure timing, chosen drug concentration, image acquisition, and image analysis-might require some further improvement to facilitate a more accurate detection of some of the analysed parameters.

L-Cysteine
Regarding neurotoxicity assessment, motor behaviour might be affected by neurotoxic, but also by non-neurotoxic compounds that affect the function of the nervous system, such as hypnotic or neuroactive drugs [52,54,60]. This ambivalence could have promoted the identification of a larger percentage of false positives (FP). However, we observed high specificity, since no false positives have been detected. On the other hand, better sensitivity is indeed required because five compounds known to produce some kind of neurotoxicity in humans did not alter larvae locomotion significantly. Thus, we suggest locomotion results should be interpreted cautiously. Indeed, we propose that drugs altering zebrafish locomotion should be tagged with a red-flag, since they could signal a possible Central Side Effect impact. However, drugs not influencing larvae locomotion cannot be tagged safe for neurotoxicity, since they might be neurotoxic without affecting locomotor neural pathways. In that regard, future ZeGlobalTox experimental versions might include a more comprehensive assessment of neural tissue after drug incubation-neuronal mortality, axonal growth defects, etc.
Previous studies have shown the robustness of zebrafish for hepatotoxicity prediction [43,44]. This robustness is supported by a high degree of genetic conservation for the enzymes and pathways required in drug metabolism, such as ARH receptors, CYP enzymes, or Adh isoinzymes, which are present, and functional, from early developmental stages, including our experimental window [62][63][64]. Three phenotypic endpoints were analysed for hepatotoxicity evaluation: liver area, steatosis, and yolk lipid retention. A drug was considered hepatotoxic when at least one of these parameters was statistically different when compared to the DMSO-only group. Consistent with previous studies, we show that paracetamol reduces liver size and increases yolk lipid accumulation. Thus, by reducing the hepatocyte number and/or viability, paracetamol was reducing zebrafish liver size and impairing its function, which led to a decreased lipid metabolism and therefore, its accumulation in the yolk. On the other hand, larvae treated with 2% ethanol showed steatosis but no impact on the liver size or yolk lipid accumulation. Steatosis promoted by 2% ethanol has been extensively reported [26,49,65]. However, there are controversial results regarding the ethanol impact on liver size. Gong et al. [44] identified a reduction in liver size, but Sadler et al. showed hepatomegaly [26,27,49]. In our hands, 2% ethanol did not significantly affect the liver area. However, we detected more rounded livers (shape differences). This phenotype agreed with [26] and might be indicative of an inflammatory process, which later leads to hepatomegaly. Larvae treated with (±)-epinephrine HCL, digoxigenin, and finasteride were found to produce significant higher percentages of hepatic steatosis when compared to the DMSO-only treated group. (±)-epinephrine is not reported to be hepatotoxic in humans. However, it is known that high levels of epinephrine stimulate lipolysis in adipose tissue liberating free fatty acids to the blood, which are then absorbed by the liver that converts them to triglycerides [64,66]. Furthermore, (±)-epinephrine stimulates the breakdown of glycogen in the liver releasing glucose [67]. Glucose can also be converted to fatty acids and finally into triglycerides [68]. Thus, high (±)-epinephrine concentrations might lead to an excessive accumulation of triglycerides in hepatocytes producing, as a side effect, hepatic steatosis in zebrafish larvae. Finasteride and digoxigenin are both extensively metabolized in the liver. Finasteride is a 5-alpha reductase inhibitor that is metabolized via the cytochrome P450 system (CYP 3A4). No severe hepatotoxicity or clinical liver injury has been reported. However, some publications report mild transient serum aminotransferase elevations occurring during finasteride therapy [69]. Digoxigenin is a steroid that when attached to sugars forms glycosides. Digoxigenin is metabolized in the liver via the human liver alcohol dehydrogenase [70]. Thereby, hepatic steatosis, observed after digoxigenin treatment might be originated by a similar mechanism to that seen after ethanol 2% treatment. Consistent with that, in our approach, both treatments cause steatosis in the same percentage ( Figure 5C). Finally, regarding MPTP, its hepatotoxicity in humans is not known, however it has been reported to be hepatotoxic in rat livers or isolated hepatocytes [71,72]. Consistent with these studies, steatosis was observed in MPTP treated larvae. All in all, the predictive power of zebrafish hepatotoxicity assessment, may be greater than most in silico or in vitro approaches that are traditionally used [73].
Overall, our results show ZeGlobalTox to be a reliable method to red flag a toxic compound according to its putative general organ liability. On its current methodological version-preliminary AcuteTox, drug concentration, drug exposure timing, and typology of end-phenotypes-it yields an overall high sensitivity, specificity, and accuracy at identifying specific organ toxicities. However, we acknowledge some adjustments need to be implemented to more accurately segment the general organ-toxicities into specific end-phenotypes (i.e., General cardiotoxicity vs. specific QTc prolongation). Moreover, the exposure to NOEC might yield some false negatives. In that sense, testing more than one concentration might provide a better understanding of a possible drug-induced organ liability, if that phenotype requires a higher than NOEC concentration to be triggered. In spite of those possible drawbacks, we expect our results will further support the use of zebrafish as an appropriate model to be exploited in the early phases of drug discovery/development. In that regard, zebrafish could become the chosen model to bridge the gap between low predictive but high throughput in vitro studies and high predictive but expensive and time-consuming in vivo mammalian studies.

Zebrafish Maintenance
Zebrafish embryos were obtained by mating adult fish through standard methods. All experiments were performed on zebrafish larvae from 4 dpf until 5.5 dpf, with the exception of the Acute Toxicity test (see "zebrafish exposure conditions" below). Transgenic zebrafish (Danio rerio) Tg(cmlc2:GFP; fabp10:RFP;ela31:EGFP) was obtained by crossing individual transgenic lines and were kept according to established standard procedures. Tg(cmlc:GFP) [50] expresses Green Fluorescent Protein (GFP) in cardiomyocytes, Tg(fabp10:RFP) [74] expresses Red Fluorescent Protein (RFP) in hepatocytes, and Tg(ela31:EGFP) [75] expresses enhanced Green Fluorescent Protein (EGFP) in pancreatic cells. In the present study, pancreatic toxicity was not analysed, but since it was not affecting the current image analysis, and it might become useful in future experiments, the pancreatic reporter line was kept inside the complete transgene line.

Drug Exposure Conditions
Mortality and Developmental toxicity were assessed through an Acute Toxicity test, adapted from specific OECD guidelines (FET: Fish Embryo Toxicity; OECD 236). Thus, 20 wild type (wt) zebrafish embryos per condition were incubated with tested compounds from 3 to 96 hpf. The test was performed in 5 logarithmic concentrations per drug (from 0.1 µM to 1 mM). Each larva was analysed for mortality, body deformity, edema, tail detachment, pigmentation, heart activity, heart edema, and motor activity. For every compound, a no observed effect concentration (NOEC) was identified to use in the following experiments. The concentrations for drugs used as organo-toxic positive controls were obtained from previous publications and in-house validation: paracetamol (2600 µM; [46]), EtOH (2%; [26]), MPTP (100 µM; [30]), and haloperidol (10 µM; [21]). DMSO 1% was used as the negative control in all experiments.

Cardiotoxicity Evaluation in Zebrafish Larvae
After 4 h of drug incubation (100 hpf), zebrafish larvae were anesthetized by immersion in 0.7 µM tricaine methanesulfonate (A4050, Sigma-Aldrich, Saint Louis, MO, USA)/E3 solution. 10 µM haloperidol treated embryos were used as positive cardiotoxic controls. The 1% DMSO treated embryos were used as negative cardiotoxic controls. Embryos were positioned in an agarose based mold to allow their appropriate orientation under the fluorescence stereo microscope (Olympus MVX10). Individual fluorescent hearts were recorded during 60 s each ( Figure 6A). Videos were acquired with a high-speed recording camera (Hamamatsu C11440 ORCA-flash 2.8) and analysed with the ZeCardio ® β software to extract different cardiac parameters-heart rate, cardiac arrest, QTc prolongation, and Ejection Fraction (EJF) ( Figure 6B).  Figure 6A). Videos were acquired with a high-speed recording camera (Hamamatsu C11440 ORCA-flash 2.8) and analysed with the ZeCardio ® β software to extract different cardiac parameters-heart rate, cardiac arrest, QTc prolongation, and Ejection Fraction (EJF) ( Figure 6B). ZeCardio ® β software, developed by ZeClinics and currently in β status, provides a graphical user interface (GUI) that facilitates the semi-automatic analysis of living heart videos. Interactive analysis of the different parameters functions as follows: The user draws a line along the heart axis, from ventricle to atrium, to initiate the calculation. At the ventricle and atrium, an additional line perpendicular to the heart axis (first line) is automatically displayed ( Figure 6C). All lines can be subjected to modification of their angles and lengths. From the line selections, two outputs are ZeCardio ® β software, developed by ZeClinics and currently in β status, provides a graphical user interface (GUI) that facilitates the semi-automatic analysis of living heart videos. Interactive analysis of the different parameters functions as follows: The user draws a line along the heart axis, from ventricle to atrium, to initiate the calculation. At the ventricle and atrium, an additional line perpendicular to the heart axis (first line) is automatically displayed ( Figure 6C). All lines can be subjected to modification of their angles and lengths. From the line selections, two outputs are generated: (i) A kymograph for each of the lines that allows, on one hand, the visual inspection and easy identification/validation of phenotypes ( Figure 6D) and, on the other hand, it is used for individual beat detection ( Figure 6E); (ii) A numerical output that is displayed in the ZeCardio ® GUI.
Heart beat frequency for each chamber is detected and frequencies are presented in the GUI as a mean. A plot distribution is used for assessing beat stability over time ( Figure 6F). In the same fashion, chamber specific cardiac arrest is measured as the longest beating pause ( Figure 6H). No beating chambers and/or incorrect bpm can be manually flagged when detected.

Neurotoxicity Evaluation in Zebrafish Larvae
Immediately after heart video acquisition, larvae are washed with E3 medium to remove tricaine methanesulfonate from the solution. Fresh drug solution is added and the larvae are transferred individually in a volume of 150 µL to 96 well plates.
Neurotoxicity is analysed at 120 hpf by locomotion assessment using the EthoVision XT 11.5 software and the DanioVision device from Noldus Information Technologies, Wageningen, The Netherlands. This closed system consists of a camera placed above a chamber with circulating water and a temperature sensor set at 28 • C. The 96-well plates are placed in the chamber, which can then be illuminated with white light using the software. Larvae are then left for 20 min under these conditions and with the lights on to help their acclimation. Finally, larvae locomotion is measured during 50 under the following light/dark conditions: 10 darkness-10 Light-10 darkness-10 Light-10 darkness. Total distance moved (in mm) is acquired under this light/dark trial. Due to circadian rhythms, all locomotion assays were performed from 13:00 pm onwards to ensure steady activity of the zebrafish [76].
Neurotoxicity is assessed by comparing locomotion differences among the tested compounds (solved in DMSO) and the negative control group (DMSO 1%-only).

Liver Area Analysis
Fixed larvae are observed under an Olympus MVX10 fluorescent stereo microscope and photographed with a digital camera (Olympus DP71) and the cell'D software. RFP filtered images of the liver were taken and their areas were analysed using the FIJI software for hepatomegaly and necrosis detection.

Oil Red O Staining
Oil Red O is a lysochrome dye used for the staining of neutral triglycerides and lipids. In order to detect the presence of steatosis and yolk lipid retention, zebrafish larvae were stained with Oil Red O (O0625-25G, Sigma-Aldrich, Saint Louis, MO, USA) as described in [77]. Briefly, the skin pigment from fixed larvae is removed by incubating with bleaching solution (for 10 mL: 6 mL H 2 O, 0.25 mL 20× SSC, 0.5 mL formamide, 3.3 mL H 2 O 2 ) during 20 min at RT. Then, the larvae are 5× washed with PBS. Bleached embryos are first submerged in 85% Propylene glycol (PG) (134368-1L, Sigma-Aldrich, Saint Louis, MO, USA) for 10 min and then in 100% PG for another 10 min before staining them with Oil Red O 0.5% in 100% PG (overnight, at RT and with gentle rocking). Oil Red O stained embryos are washed in 100% PG for 30 min, 50 min in 85% PG, and 40 min in 85% PG with an equal volume of PBS. Finally, embryos are washed 1× with PBS before adding 80% glycerol (G7757-500ML, Sigma-Aldrich, Saint Louis, MO, USA). Bright field images are taken to detect both steatosis and yolk lipid accumulation. For steatosis, larvae are considered positive when 3 or more round lipid droplets are visible within the hepatic parenchyma ( Figures 5E and 7; [77]). Yolk lipid retention is considered positive when a red strong signal is observed in the yolk area ( Figure 5F).
Embryos were incubated with ethanol 2% as the positive control for steatosis [26,49] and with APAP 2600 µM as the positive control for necrosis and yolk lipid accumulation [46]. DMSO 1% treated larvae were used as the negative control group.  Figures 5E and 7; [77]). Yolk lipid retention is considered positive when a red strong signal is observed in the yolk area ( Figure 5F). Embryos were incubated with ethanol 2% as the positive control for steatosis [26,49] and with APAP 2600 µM as the positive control for necrosis and yolk lipid accumulation [46]. DMSO 1% treated larvae were used as the negative control group.

Statistical Analysis
Data were analysed using the IBM SPSS Statistics version 20.0 software (Armonk, NY, USA). Data are presented as mean ± standard error (SE). Prior to the analyses, the Shapiro-Wilk test was used to assess the normality of the distribution of the dependent variables. Not normally distributed variables were transformed using Templeton's two-step method for transforming continuous variables to normal variables [78]. Statistical analysis of the data for the cardiotoxic and neurotoxic parameters as well as for liver size measurements were performed using One-way ANOVA followed by the Dunnett test. Fisher's exact test was used for data analysis of the steatosis and yolk lipid retention. Results were statistically compared between drug-treated groups and untreated (DMSO) group. Differences were considered statistically significant when p < 0.05. Author Contributions: Carles Cornet designed the project, performed the experiments, analyzed the data and contributed to the manuscript preparation. Simone Calzolari conceived and designed the project, performed the experiments, and contributed to the manuscript preparation. Rafael Miñana-Prieto performed the experiments. Sylvia Dyballa designed the ZeCardio software, contributed to its development, and contributed to writing the manuscript. Els van Doornmalen contributed to the experimental design and analysis of the results. Helma Rutjes provided reagents, and contributed to the project design and data analysis. Thierry Savy developed the ZeCardio software. Davide D'Amico and Javier Terriente conceived of the project, coordinated the work, and contributed to the manuscript preparation.

Conflicts of Interest:
The authors declare the following conflict of interest: All authors, except Els van

Statistical Analysis
Data were analysed using the IBM SPSS Statistics version 20.0 software (Armonk, NY, USA). Data are presented as mean ± standard error (SE). Prior to the analyses, the Shapiro-Wilk test was used to assess the normality of the distribution of the dependent variables. Not normally distributed variables were transformed using Templeton's two-step method for transforming continuous variables to normal variables [78]. Statistical analysis of the data for the cardiotoxic and neurotoxic parameters as well as for liver size measurements were performed using One-way ANOVA followed by the Dunnett test. Fisher's exact test was used for data analysis of the steatosis and yolk lipid retention. Results were statistically compared between drug-treated groups and untreated (DMSO) group. Differences were considered statistically significant when p < 0.05. experiments, and contributed to the manuscript preparation. Rafael Miñana-Prieto performed the experiments. Sylvia Dyballa designed the ZeCardio software, contributed to its development, and contributed to writing the manuscript. Els van Doornmalen contributed to the experimental design and analysis of the results. Helma Rutjes provided reagents, and contributed to the project design and data analysis. Thierry Savy developed the ZeCardio software. Davide D'Amico and Javier Terriente conceived of the project, coordinated the work, and contributed to the manuscript preparation.

Conflicts of Interest:
The authors declare the following conflict of interest: All authors, except Els van Doornmalen, Helma Rutjes, and Thierry Savy, are currently employed by Zeclinics.