Zebrafish Models for Human Acute Organophosphorus Poisoning

Terrorist use of organophosphorus-based nerve agents and toxic industrial chemicals against civilian populations constitutes a real threat, as demonstrated by the terrorist attacks in Japan in the 1990 s or, even more recently, in the Syrian civil war. Thus, development of more effective countermeasures against acute organophosphorus poisoning is urgently needed. Here, we have generated and validated zebrafish models for mild, moderate and severe acute organophosphorus poisoning by exposing zebrafish larvae to different concentrations of the prototypic organophosphorus compound chlorpyrifos-oxon. Our results show that zebrafish models mimic most of the pathophysiological mechanisms behind this toxidrome in humans, including acetylcholinesterase inhibition, N-methyl-D-aspartate receptor activation, and calcium dysregulation as well as inflammatory and immune responses. The suitability of the zebrafish larvae to in vivo high-throughput screenings of small molecule libraries makes these models a valuable tool for identifying new drugs for multifunctional drug therapy against acute organophosphorus poisoning.

Absorbance was read at 586 nm, and MDA content in each sample was extrapolated from the standard curve of 1,1,3,3-tetramethoxypropane (TMP) treated under similar conditions as samples. The final results were normalized by mg of total lipid contents for each sample and expressed as nmol mg protein -1 .

Pharmacological treatments
The following drugs, which were obtained from Sigma-Aldrich, were used in the

Behaviour analysis
Basal locomotor activity and VMR of 8 dpf zebrafish larvae were analysed and an EthoVision XT 9 video tracking system (Noldus Information Technology, Leesburg, VA). A dynamic subtraction method was applied, using a sampling rate of 60 images/s, dark contrast 20-250, current frame weight 1, subject-size 2-125000, and no subject contour dilatation. A minimum distance input with a filter of 10% of the total larva body, equivalent to 0.4 mm, was used to remove background noise. Larvae with any gross morphology defect or dead larvae were not included in this analysis. All measurements occurred in the afternoon between 1:00 and 6:00 pm, the optimal time interval for the stability of the basal activity.
Tracks were analysed for velocity (mm s-1), total distance moved (m) and mobility time (s) calculated for each dark or light period. Mobility time refers to the period where 20 to 60% of the total body of the larva is modified even if the central point does not change. All microplates were analysed at 20 ± 0.5°C with same detection and acquisition settings.
For TMR, startle responses were evoked in 8 dpf larvae by a light-touch stimulus applied to the rostral head skin using a glass capillary injection needle. Video recordings were initiated 2 min after moving the larva to the testing arena to allow sufficient time for locomotor activity to stabilize. All video recordings were made with a high-speed Photron Fastcam SA3 camera (Photron USA Inc., San Diego, CA, USA) at 512 × 512 pixel resolution using a Sigma 105 mm F2.8 EX DG lens at 1000 10/43 frames per second. Individual larvae were tested in 6 cm Petri dishes. The plate was illuminated from below by a LLUB White LED Backlight 50×50 (PHLOX, Aixen-Provence, France) adjusted to 300 W/cm 2 using a Gardasoft RT 220-20 LED Lighting Controller (Gardasoft, Cambridge, UK). The light intensity in the testing platform was measured using an ILT1400 radiometer (International Light Technologies Inc., Peabody, MA, USA). All behavioural measurements were made using the Flote software package. Briefly, this software performs tracking of the filmed larva and then performs an automated analysis of its body curvature to extract kinematic details of swimming movements. The curvature of the body was calculated for each frame and plotted over time using Microsoft Excel (Microsoft Corporation, Redmon, WA, USA).

Whole-mount immunofluorescence
Larvae were fixed in 4% PFA in PBS overnight at 4°C, dehydrated through a methanol gradient and stored in absolute methanol at -20°C. After rehydration, the larvae were depigmented (3% H2O2 and 1% KOH in water, 35 min) and permeabilized by immersion in -20°C acetone (12 min) and treatment with 0.1% collagenase (Sigma, C-9891) in PBS (pH 7.4) at room temperature for 20 min.
Then, the larvae were pre-incubated in blocking buffer (4% goat serum, 1% BSA, 1% DMSO, 0.8% Triton X-100, and 0.1% Tween-20 in PBS, pH 7.4) for 2 h and incubated overnight at 4°C in a mixture of mouse monoclonal anti-acetylated tubulin antibody (IgG2b; Sigma) at 1:1000 and mouse monoclonal F59 antibody (IgG1; DSHB, University of Iowa, Iowa) at 1:50. These antibodies were used to detect spinal axonal tracts and slow muscle fibres, respectively. Then, the larvae were washed for 3 h and incubated for 2 h in a mixture of secondary antibodies 11/43 (Alexa 488-conjugated goat anti-mouse IgG2b and Alexa 594-conjugated goat antimouse IgG1), at 1:300. After the larvae were washed for 3 h, they were imaged on a Nikon Eclipse 90i microscope fitted with a Nikon Intensilight C-HGFI unit.

Measurement of oxygen consumption in zebrafish larvae homogenates
Respiration of zebrafish homogenates (400-500 µg protein) was measured at 28°C Peak identities were confirmed by comparison with the retention times of standard adenine nucleotides. Extracts from larvae exposed to 5 M oligomycin plus 40 mM 2-DOG were used to validate the protocol (see Supplementary Figure   S10). 14/43

Supplementary Discussion
One of the problems to overcome when animals models are developed to increase our current understanding of the pathophysiological mechanisms involved in OPP is that when animals are exposed to high concentrations of OPs, death occurs very quickly, preventing the expression of any specific phenotype. Most of these early deaths in acute OPP result from acute respiratory failure 2 . Thus, to prevent death, animals are usually pre-treated with a combination of antidotes (atropine and oximes), thus introducing an important confounding factor in the interpretation of the results when the models are used for toxicodynamic studies. Respiratory gas exchange in adult fish occurs by diffusion over the gills 3 , and exposure to OPs has been reported to severely decrease oxygen uptake by the gills 4 . The higher resistance of our OPP models, which were developed in larvae instead of adult zebrafish, is probably related to the fact that at 7 dpf, the skin still appears capable of satisfying the oxygen requirements of the larvae 5 . The high resistance of zebrafish larvae to death by CPO allowed the expression of phenotypes with different grades of severity without the need to protect the larvae with antidotes.
In the present manuscript, zebrafish OPP severity was graded as mild (grade 1), moderate (grade 2) and severe (grade 3) according to morphological and behavioural criteria. Further analyses at molecular, subcellular, cellular and tissue levels confirmed a progressive increase in the OPP severity from grade 1 to grade 3. Different scoring systems have been proposed for predicting outcome in human OPP, including the Namba scale of poisoning, poisoning severity score, Glasgow coma scale, acute physiology and chronic health evaluation II and predicted mortality rate 6,7 . These systems are primarily based on the evaluation of clinical effects and mortality. Although some clinical signs and symptoms used in the 15/43 human scoring system cannot be analysed in zebrafish, most of the criteria used in the present study for grading severity are also used in humans. Thus, similar to the Namba scale 6 used in human OPP, a progressive increase in the severity of the motor problems and mortality from mild to severe poisoning was observed and correlated with a decrease in the cholinesterase activity. Our data show that the grading system used in the present manuscript is highly effective for predicting outcome (mortality and severity of the pathological effects at different levels of organization) in zebrafish OPP, strongly suggesting that even considering the differences in some analysed endpoints, this grading system is convenient for developing zebrafish models of human OPP with varying degrees of severity.
One of the most consistent effects we have found among the different grades of OPP is on the visual system. The cholinergic system plays a pivotal role in the physiology and development of a normal mammalian retina, where AChE is expressed very early 8 . When eyes from human volunteers were exposed to the AChE inhibitors sarin or physostigmine, different parameters of the visual function were altered, including sensitivity of the retina to light stimulus 9, 10 , and retinal detachment has been noted as a complication in the treatment of glaucoma with some AChE inhibitors used in human pharmacology 11 . Although the potential retinotoxic effect of OP compounds on humans is still under discussion, chlorpyrifos has been reported to be highly toxic for the retina in different mammalian models 12,13 . Consistently, grade 1 larvae exhibited impaired visual function, whereas retinotoxicity was found in grade 2 and 3 larvae.
The molecular initiating event (MIE) in the adverse outcome pathways resulting in OPP is AChE inhibition 14 . We have demonstrated that AChE inhibition is also the 16/43 MIE in our zebrafish models for OPP. Thus, a significant correlation was found between AChE inhibition and VMR in grade 1 larvae, and a full rescue of the phenotype was obtained when larvae were co-exposed with CPO and 2-PAM in GSH is not only one of the most important antioxidants but also involved in other essential biological processes including xenobiotic detoxification. In fact, GSH seems to play an essential role in CPO metabolism, and different GSH conjugates, such as GSCPO, have been identified in the metabolism of this compound 23,24 . The fact that the GSH precursor N-acetycysteine, but not any other antioxidant tested, decreased the severity of OPP in zebrafish suggests that this action might be mediated by an increase in the metabolism (GSH-conjugation) of CPO and not by an antioxidant action. Further studies, including additional OPs with different metabolic pathways, will be required to test this hypothesis.

Supplementary Figures
Supplementary Figure S1: Concentration-response analysis of AChE activity in zebrafish larvae exposed for 1 h to chlorpyrifos-oxon. Effect of chlorpyrifosoxon (CPO) on AChE activity in 7 days post-fertilization larvae was extremely severe as early as 1 h exposure. Thus, at that time, an inhibition of AChE activity higher than 99% was found in larvae treated with 0.5 M CPO. Regression