Targeting the Redox Balance Pathway Using Ascorbic Acid in sdhb Zebrafish Mutant Larvae

Simple Summary Thus far, no curative therapies are available for malignant SDHB-associated phaeochromocytomas and paragangliomas (PPGLs). Therapy development is severely hampered by the limited availability of suitable animal models. In this study, we investigated the potential of the sdhbrmc200 zebrafish model to study SDHB-associated PPGLs using a drug screening approach. One of the key features of cancer initiation and progression is redox imbalance. First, we identified increased reactive oxygen species levels in homozygous sdhbrmc200 larvae at baseline. Next, we tested the effect of anti- and pro-oxidant ascorbic acid (Vitamin C) on these larvae. We validated the sdhbrmc200 zebrafish model as a powerful drug screening tool to provide valuable insights into pathomechanisms, which may lead to novel therapeutic targets and therapy development in the future. Abstract Patients with mutations in the β-subunit of the succinate dehydrogenase (SDHB) have the highest risk to develop incurable malignant phaeochromocytomas and paragangliomas (PPGLs). Therapy development is hindered by limited possibilities to test new therapeutic strategies in vivo. One possible molecular mechanism of SDHB-associated tumorigenesis originates in an overproduction of reactive oxygen species (ROS) due to mitochondrial dysfunction. Ascorbic acid (Vitamin C) has already been shown to act as anti-cancer agent in several clinical trials for various types of cancer. In this study, the potential of the sdhbrmc200 zebrafish model to study SDHB-associated PPGLs using a drug screening approach was investigated. First, we identified increased basal ROS levels in homozygous sdhb larvae compared to heterozygous and wild-type siblings. Using a semi high-throughput drug screening, the effectiveness of different dosages of anti- and pro-oxidant Vitamin C were assessed to evaluate differences in survival, ROS levels, and locomotor activity. Low-dosage levels of Vitamin C induced a decrease of ROS levels but no significant effects on lifespan. In contrast, high-dosage levels of Vitamin C shortened the lifespan of the homozygous sdhbrmc200 larvae while not affecting the lifespan of heterozygous and wild-type siblings. These results validated the sdhbrmc200 zebrafish model as a powerful drug screening tool that may be used to identify novel therapeutic targets for SDHB-associated PPGLs.


Introduction
The mitochondrial enzymatic succinate dehydrogenase (SDH) complex, also called mitochondrial complex II, has an essential role in ATP production. The dysfunction of the SDH complex is linked to several diseases, varying from severe neuromuscular disorders [1] to different types of cancer including phaeochromocytomas and paragangliomas (PPGLs), gastrointestinal stromal tumour, renal cell carcinoma (RCC), pituitary adenoma, and pancreatic neuroendocrine tumours [2,3]. To investigate whether sdhb rmc200 larval zebrafish mutants possess an unbalanced cellular redox state, whole-mount ROS-detection was used to determine ROS levels at baseline. At day 6 post fertilization (dpf), increased levels of ROS were observed in homozygous sdhb compared to their heterozygous sdhb and wild-type siblings (Figure 1). baseline. At day 6 post fertilization (dpf), increased levels of ROS were observed in homozygous sdhb compared to their heterozygous sdhb and wild-type siblings ( Figure 1).

Successful Design of Drug Screening Protocol
To test the effect of Vitamin C on zebrafish larvae, an optimal drug screening protocol was established ( Figure 2). First, the offspring of the incross of heterozygous adult sdhb mutants were collected. At day 2 post fertilization (dpf), hatched larvae were assembled in 48-well plates with either an E3 control egg medium or E3 medium supplemented with Vitamin C (20, 500, or 1000 mg⋅L −1 ). Different read-outs to assess the effect of the vitamin were developed. Lethality scores were performed to assess the effect of Vitamin C on overall survival. In addition, at day 6, ROS levels were measured to check the effect of supplementation of the anti-and pro-oxidant Vitamin C. As quick read-out for general health and toxicity, locomotor activity was evaluated using DanioVision at day 6.

High-Dosage Levels of Vitamin C Decreases Lifespan of Homozygous sdhb rmc200 Larvae
Heterozygous adult sdhb mutants were used to generate a mixed offspring following Mendelian inheritance. The larvae were checked at least twice a day to determine lethality. At baseline, homozygous sdhb larvae showed an enhanced mortality compared to Reactive oxygen species (ROS) measurements showed a significant increase in homozygous sdhb larvae (n = 17) compared to their heterozygous (n = 22) and wild-type siblings (n = 12) at 6 dpf. One-way ANOVA with Tukey's post hoc test, *** p < 0.001.

Successful Design of Drug Screening Protocol
To test the effect of Vitamin C on zebrafish larvae, an optimal drug screening protocol was established ( Figure 2). First, the offspring of the incross of heterozygous adult sdhb mutants were collected. At day 2 post fertilization (dpf), hatched larvae were assembled in 48-well plates with either an E3 control egg medium or E3 medium supplemented with Vitamin C (20, 500, or 1000 mg·L −1 ). Different read-outs to assess the effect of the vitamin were developed. Lethality scores were performed to assess the effect of Vitamin C on overall survival. In addition, at day 6, ROS levels were measured to check the effect of supplementation of the anti-and pro-oxidant Vitamin C. As quick read-out for general health and toxicity, locomotor activity was evaluated using DanioVision at day 6. baseline. At day 6 post fertilization (dpf), increased levels of ROS were observed in homozygous sdhb compared to their heterozygous sdhb and wild-type siblings ( Figure 1).

Successful Design of Drug Screening Protocol
To test the effect of Vitamin C on zebrafish larvae, an optimal drug screening protocol was established ( Figure 2). First, the offspring of the incross of heterozygous adult sdhb mutants were collected. At day 2 post fertilization (dpf), hatched larvae were assembled in 48-well plates with either an E3 control egg medium or E3 medium supplemented with Vitamin C (20, 500, or 1000 mg⋅L −1 ). Different read-outs to assess the effect of the vitamin were developed. Lethality scores were performed to assess the effect of Vitamin C on overall survival. In addition, at day 6, ROS levels were measured to check the effect of supplementation of the anti-and pro-oxidant Vitamin C. As quick read-out for general health and toxicity, locomotor activity was evaluated using DanioVision at day 6.

High-Dosage Levels of Vitamin C Decreases Lifespan of Homozygous sdhb rmc200 Larvae
Heterozygous adult sdhb mutants were used to generate a mixed offspring following Mendelian inheritance. The larvae were checked at least twice a day to determine lethality. At baseline, homozygous sdhb larvae showed an enhanced mortality compared to  Heterozygous adult sdhb mutants were used to generate a mixed offspring following Mendelian inheritance. The larvae were checked at least twice a day to determine lethality. At baseline, homozygous sdhb larvae showed an enhanced mortality compared to heterozygous and wild-type siblings ( Figure 3A,C,E). Low dosages of Vitamin C (20 mg·L −1 ) did not have an significant effect on the survival of homozygous sdhb, heterozygous sdhb, or wild-type larvae measured until two weeks of age. High dosage levels of Vitamin C (500 and 1000 mg·L −1 ) significantly decreased the survival rate of the homozygous sdhb mutants ( Figure 3F; p < 0.01) while not having a significant effect on heterozygous and wild-type siblings ( Figure 3B-D).

Effects of Vitamin C Treatment on ROS Levels
The effect of low-dosage and high-dosage levels of Vitamin C was measured compared to an untreated control group. In Figure 4, the ROS levels after the supplementation of either the E3 medium control medium (0 mg·L −1 ) or low-dosage levels of Vitamin C (20 mg·L −1 ) and high-dosage Vitamin C levels of Vitamin C (500 and 1000 mg·L −1 ) are shown. Low-dosage Vitamin C levels significantly decreased ROS levels in the heterozygous and homozygous sdhb mutant group compared to the untreated control group, while no significant differences were identified in the wild-type group without low-dosage levels of Vitamin C. High-dosage levels of Vitamin C possessed a more heterozygous effect in heterozygous and homozygous sdhb larvae. No significant differences were identified high-dosage levels of Vitamin C supplementation.

Effects of Vitamin C Treatment on ROS Levels
The effect of low-dosage and high-dosage levels of Vitamin C was measured compared to an untreated control group. In Figure 4, the ROS levels after the supplementation of either the E3 medium control medium (0 mg⋅L −1 ) or low-dosage levels of Vitamin C (20 mg⋅L −1 ) and high-dosage Vitamin C levels of Vitamin C (500 and 1000 mg⋅L −1 ) are shown. Low-dosage Vitamin C levels significantly decreased ROS levels in the heterozygous and homozygous sdhb mutant group compared to the untreated control group, while no significant differences were identified in the wild-type group without low-dosage levels of Vitamin C. High-dosage levels of Vitamin C possessed a more heterozygous effect in heterozygous and homozygous sdhb larvae. No significant differences were identified highdosage levels of Vitamin C supplementation. Reactive oxygen species (ROS) measurements after a low-and high-dosage Vitamin C treatment. Relative average fluorescence levels are shown normalised to control levels without Vitamin C supplementation. (A) Larvae were supplemented with 20 mg⋅L −1 Vitamin C homozygous sdhb (n = 22), heterozygous sibling (n = 33), and wild-type sibling (n = 21) compared to the control group (indicated with 0 mg⋅L −1 ) consisting of homozygous sdhb larvae (n = 24), heterozygous sibling (n = 38), and wild-type sibling (n = 18) from two different replicates measured at 6 dpf. Low-dosage levels of Vitamin C significantly decreased ROS levels in heterozygous and homozygous sdhb larvae compared to the untreated control group. (B) Larvae were supplemented with 500 mg⋅L −1 Vitamin C homozygous sdhb (n = 12), heterozygous sibling (n = 24), and wild-type sibling (n = 18); 1000 mg⋅L −1 Vitamin C homozygous sdhb (n = 8), heterozygous sibling (n = 22), and wildtype sibling (n = 22) compared to the control group (indicated with 0 mg⋅L −1 ) consisting of homozygous sdhb larvae (n = 10), heterozygous sibling (n = 14), and wild-type sibling (n = 16) from three different replicates measured at 6 dpf. Highdosage levels of Vitamin C did not alter ROS levels in all three genotypes compared to the untreated control group. Twotailed unpaired Student's t-test, *p < 0.05 and *** p < 0.001.

Behavioural Assessment as Quick Read-Out of Toxicity of the Larvae
We previously identified that homozygous sdhb mutant larvae possess a lower basal activity and reduced endurance compared to heterozygous and wild-type siblings [22]. We designed a short protocol (<5 min) with randomized tapping stimuli to induce a robust startle response. In Figure 5A, the maximum distance moved is plotted against time in seconds, all peaks reflect a startle response induced by the tapping stimulus. At baseline, homozygous sdhb mutant larvae possessed a decreased startle response compared to heterozygous and wild-type siblings. Low-dosage levels of Vitamin C induced a significantly decreased startle response in heterozygous sdhb and wild-type larvae but did not alter the startle response of homozygous sdhb larvae ( Figure 5B). Furthermore, both high-dosage levels of Vitamin C decreased the startle response of heterozygous sdhb larvae, but only 1000 mg⋅L −1 had a significant decreased in startle response in homozygous sdhb larvae and none of the high-dosage levels significantly affected the startle response of the wild-type larvae ( Figure 5C). , and wild-type sibling (n = 21) compared to the control group (indicated with 0 mg·L −1 ) consisting of homozygous sdhb larvae (n = 24), heterozygous sibling (n = 38), and wild-type sibling (n = 18) from two different replicates measured at 6 dpf. Low-dosage levels of Vitamin C significantly decreased ROS levels in heterozygous and homozygous sdhb larvae compared to the untreated control group. (B) Larvae were supplemented with 500 mg·L −1 Vitamin C homozygous sdhb (n = 12), heterozygous sibling (n = 24), and wild-type sibling (n = 18); 1000 mg·L −1 Vitamin C homozygous sdhb (n = 8), heterozygous sibling (n = 22), and wild-type sibling (n = 22) compared to the control group (indicated with 0 mg·L −1 ) consisting of homozygous sdhb larvae (n = 10), heterozygous sibling (n = 14), and wild-type sibling (n = 16) from three different replicates measured at 6 dpf. High-dosage levels of Vitamin C did not alter ROS levels in all three genotypes compared to the untreated control group. Two-tailed unpaired Student's t-test, * p < 0.05 and *** p < 0.001.

Behavioural Assessment as Quick Read-Out of Toxicity of the Larvae
We previously identified that homozygous sdhb mutant larvae possess a lower basal activity and reduced endurance compared to heterozygous and wild-type siblings [22]. We designed a short protocol (<5 min) with randomized tapping stimuli to induce a robust startle response. In Figure 5A, the maximum distance moved is plotted against time in seconds, all peaks reflect a startle response induced by the tapping stimulus. At baseline, homozygous sdhb mutant larvae possessed a decreased startle response compared to heterozygous and wild-type siblings. Low-dosage levels of Vitamin C induced a significantly decreased startle response in heterozygous sdhb and wild-type larvae but did not alter the startle response of homozygous sdhb larvae ( Figure 5B). Furthermore, both high-dosage levels of Vitamin C decreased the startle response of heterozygous sdhb larvae, but only 1000 mg·L −1 had a significant decreased in startle response in homozygous sdhb larvae and none of the high-dosage levels significantly affected the startle response of the wild-type larvae ( Figure 5C). . Both 500 and 1000 mg⋅L −1 concentrations of Vitamin C induced a decreased startle response in heterozygous sdhb larvae, while only the 1000 mg⋅L −1 concentration of Vitamin C induced a decreased startle response in homozygous sdhb larvae and none of the high-dosage levels of Vitamin C significantly altered the startle response of the wild-type larvae. Larvae were supplemented with 500 mg⋅L −1 Vitamin C homozygous sdhb (n = 73), heterozygous sibling (n = 139), and wild-type sibling (n = 55), 1000 mg⋅L −1 Vitamin C homozygous sdhb (n = 20), heterozygous sibling (n = 37), and wild-type sibling (n = 19) compared to the control group (indicated with 0 mg⋅L −1 ) consisting of homozygous sdhb larvae (n = 32), heterozygous sibling (n = 48), and wild-type sibling (n = 40) from at least two different replicates measured at 6 dpf. One-way ANOVA with Tukey's post hoc test, * p < 0.05 and *** p < 0.001.

Discussion
The development of novel therapeutic targets for metastatic SDHB-associated PPGLs is hampered by the limited availability of suitable in vivo models. In this study, we investigated the effects of Vitamin C in the sdhb rmc200 zebrafish model and thereby tested the suitability for drug screening experiments in this validated PPGL cancer model [22]. First, we revealed an imbalance in cellular redox homeostasis in homozygous sdhb zebrafish larvae by increased levels of ROS at baseline. Next, we successfully tested the effect of different dosages of Vitamin C treatment: aqueous solutions of Vitamin C were added to the swim water of the larvae, and we showed that although low-dosage levels (20 mg⋅L −1 ) of Vitamin C did not increase the lifespan of homozygous sdhb larvae, ROS was decreased. . Low-dosage levels of Vitamin C decreased the startle response of wild-type and heterozygous sdhb larvae while not affecting homozygous sdhb larvae. Larvae were supplemented with 20 mg·L −1 Vitamin C homozygous sdhb (n = 106), heterozygous sibling (n = 244), and wild-type sibling (n = 137) compared to the control group (indicated with 0 mg·L −1 ) consisting of homozygous sdhb larvae (n = 91), heterozygous sibling (n = 227), and wild-type sibling (n = 100) from five different replicates measured at 6 dpf. (C) Quantification of the average of the maximum velocity of three startle responses with or without supplementation of high-dosage levels of Vitamin C (500 and 1000 mg·L −1 ). Both 500 and 1000 mg·L −1 concentrations of Vitamin C induced a decreased startle response in heterozygous sdhb larvae, while only the 1000 mg·L −1 concentration of Vitamin C induced a decreased startle response in homozygous sdhb larvae and none of the high-dosage levels of Vitamin C significantly altered the startle response of the wild-type larvae. Larvae were supplemented with 500 mg·L −1 Vitamin C homozygous sdhb (n = 73), heterozygous sibling (n = 139), and wild-type sibling (n = 55), 1000 mg·L −1 Vitamin C homozygous sdhb (n = 20), heterozygous sibling (n = 37), and wild-type sibling (n = 19) compared to the control group (indicated with 0 mg·L −1 ) consisting of homozygous sdhb larvae (n = 32), heterozygous sibling (n = 48), and wild-type sibling (n = 40) from at least two different replicates measured at 6 dpf. One-way ANOVA with Tukey's post hoc test, * p < 0.05 and *** p < 0.001.

Discussion
The development of novel therapeutic targets for metastatic SDHB-associated PPGLs is hampered by the limited availability of suitable in vivo models. In this study, we investigated the effects of Vitamin C in the sdhb rmc200 zebrafish model and thereby tested the suitability for drug screening experiments in this validated PPGL cancer model [22]. First, we revealed an imbalance in cellular redox homeostasis in homozygous sdhb zebrafish larvae by increased levels of ROS at baseline. Next, we successfully tested the effect of Cancers 2021, 13, 5124 7 of 12 different dosages of Vitamin C treatment: aqueous solutions of Vitamin C were added to the swim water of the larvae, and we showed that although low-dosage levels (20 mg·L −1 ) of Vitamin C did not increase the lifespan of homozygous sdhb larvae, ROS was decreased. High-dosage levels of Vitamin C (500 and 1000 mg·L −1 ) significantly shortened the lifespan of homozygous sdhb larvae while not altering the lifespan of their heterozygous sdhb and wild-type siblings regardless of unchanged levels of ROS.
As previously reported, homozygous sdhb larvae have a shortened lifespan (maximal two weeks of age) compared to heterozygous sdhb and wild-type siblings [22]. Additionally, these homozygous sdhb larvae display key metabolic characteristics of SDHB-associated PPGLs such as impaired mitochondrial complex II function and vastly increased succinate levels [22]. The heterozygous sdhb larvae revealed no differences in mitochondrial function and metabolite levels compared to wild-types siblings.
Here, we identified increased ROS levels in homozygous sdhb larvae compared to heterozygous and wild-type siblings. Redox imbalance by increased levels of ROS is known to play a critical role in carcinogenesis [23][24][25], as has also been suggested for PPGLs [14,26,27]. Although no alternative relevant systemic Sdhb knockout animal model is available, different cell lines and graft models have been created. Our findings are in line with increased ROS levels in the mitochondria of SDHB-deficient mouse phaeochromocytoma cells [19], confirmed by two SDHB-silenced cell lines and one SDHC-mutated transgenic mouse cell line [17,28,29]. On the other hand, two other studies reported no increased ROS levels in cell lines silenced for SDHB [30,31], despite hypoxia-inducible factor (HIF) stabilisation. The usage of different cell lines and the variations of different assays for measuring ROS could be reasons for this discrepancy.
Zebrafish models possesses unique advantages for investigating the effect of drugs to unravel pathomechanisms and test the therapeutic efficacy of re-purposing drugs from related types of cancer such as neuroblastoma and RCC [32]. Zebrafish can produce a large number of offspring, rapidly develop, and still have a high grade of similarity with humans; approximately 70% of human genes have at least one obvious zebrafish orthologue [33]. The use of larval zebrafish as a model organism in semi high-throughput drug screens is rapidly expanding [34][35][36]. This drug screen approach enables one to test a high number of potential targets, evaluate toxicity, and evaluate compound efficiency to select the most promising drugs to be validated in pre-clinical tumour models. The read-outs we optimized for our drug screen are lethality measurements, which are the most important and direct values used to check effects on lifespan, a protocol to assess locomotion activity as read-out for toxicity and possible other negative side-effects, and ROS levels.
Vitamin C is a natural compound with a high safety profile that was previously positively tested in pre-clinical studies for non-PPGL types of cancer [37]. The efficiency of Vitamin C has also been assessed in clinical trials, such as renal cell carcinoma in a phase-II clinical trial [21]. Often, Vitamin C is used supplementary to other types of treatment such as chemotherapy and radiation therapy. The exact mechanism of its action remains unclear since multiple critical pathways are targeted including redox imbalance, epigenetic reprogramming, and oxygen-sensing regulation, thereby preventing ROS-mediated toxicity [21]. Pharmacological levels of Vitamin C aggravated the oxidative burden of SDHB-deficient PPGLs, leading to genetic instability and apoptotic cell death [19]. Furthermore, in a preclinical animal model with PPGL allografts, high-dosage levels of Vitamin C suppressed metastatic lesions and prolonged overall subject survival [19].
We investigated the effects of low-and high-dosage levels of Vitamin C as pro-and antioxidants in the sdhb zebrafish larvae. Low-dosage levels of Vitamin C induced a decrease of ROS levels in homozygous mutants but no significant effects on lifespan. In contrast, high-dosage levels of Vitamin C further shortened the lifespan of the homozygous sdhb larvae while not affecting heterozygous and wild-type siblings. This is in line with previous findings obtained in the allografted mice model treated, with high-dosage levels of Vitamin C inducing ROS-medicated toxicity in tumour cells [19]. We detected no increase in basal ROS levels in the homozygous sdhb larvae supplemented with high-Cancers 2021, 13, 5124 8 of 12 dosage levels of Vitamin C at 6 dpf. This discrepancy could be explained by the timing of the ROS measurement. We performed ROS measurements by using the fluorescent dye CM-H2DCFDA analysed by microscopy at 6 dpf. Fluorescence-activated cell sorting (FACs) analysis could optimise the quantification of the total ROS value in an entire larvae, and the measurements also could be performed at later time points to identify possible differences. Since in all other studies, HIF stabilisation was detected regardless of whether increased or normal ROS levels were detected, in follow up studies, other ROS indicators and HIF stabilisation could be measured as well.
Despite the fact that homozygous sdhb zebrafish larvae mimic the metabolic human tumour environment, the major limitation of using this zebrafish model is the absence of tumours at the age of 14 days. The translational value to predict the effect in human PPGLs therefore remains challenging and requires further investigation. For this, the allograft mice model and rat xenograft model are currently the best used alternatives [19,38]. Currently, the homozygous sdhb zebrafish larvae possess resemblance to patients with bi-allelic SDHB mutations with a Leigh-syndrome-like phenotype, resulting in severe progressive neurodegeneration and myopathy with the onset in infancy and poor prognosis [1,39]. The supplementation of low-dosage levels of Vitamin C was also shown to be effective in pre-clinical studies for neuropathy [40], and other anti-oxidants showed a beneficial effect for patients with mitochondrial disorders [41]. More research is required to investigate the therapeutic potential of low-dosage levels of Vitamin C for Leigh-syndrome-like patients.
To follow up the PPGL research, we will investigate whether adult heterozygous sdhb fish develop tumours in comparison to human SDHB mutations, which are at risk of developing PPGLs. If successful, this zebrafish tumour model can be complementarily used to test the potential of the most promising compounds identified in the larval drug seen for the effectiveness on tumour growth and to further unravel the mode of action behind its pathomechanism. Additionally, the onset of tumorigenesis and the prevention of tumour formation could be investigated in more detail.
In this study, we identified increased ROS levels in our homozygous sdhb larvae at baseline. Further, we validated the zebrafish larvae drug screen as tool to screen for therapeutic compounds and possible combination of compounds to target pathways involved in the tumorigenesis of SDHB-associated PPGLs. The most powerful advantage of this zebrafish model is its ability to screen many targets for possible new therapeutics in a cheap and cost-effective manner. This enables us to narrow down possible therapeutics to test for effectiveness in a more advanced tumour model. Patient-derived xenografts (PDXs) or cancer "Avatars" could attribute to personalized medicine in the future. In addition to mouse and rat PDXs [38,42], zebrafish Avatars are emerging as a cheaper and faster alternative [43,44] to hopefully accelerate personalised drug discovery for currently incurable metastatic SDHB-associated PPGLs.

Zebrafish Maintenance and Husbandry
Experimental procedures were conducted in accordance with institutional guidelines and National and European laws. Ethical approval of the experiments was granted by Radboud University's Institutional Animal Care and Use Committee (IACUC, application numbers RU-DEC 2015-0098 and RU-DEC 2020-0030). Wild-type adult Oregon AB* zebrafish (Danio Rerio) and heterozygous adult sdhb rmc200 mutants were used [22]. Eggs were obtained from natural spawning. Larvae were maintained and raised by standard methods [45].

ROS Measurements
ROS levels were assessed in 6 dpf zebrafish larvae using the 2 ,7 -dichlorodihydrofluorescein diacetate (CM-H2DCFDA) dye (Fisher Scientific). When oxidized, this non-fluorescent dye is converted into a fluorescent compound, 2 ,7 -dichlorofluorescein (DCF) [33]. The ROS levels were measured according to protocol [33]. In brief, each larva was individually placed in a well of a 96-well plate with 100 µL of an E3 embryo medium at 6 dpf. A working solution of H2DCFDA (500 µg/mL in dimethyl sulfoxide (DMSO, 14.1 M)/E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4)) was prepared, and 100 µL were added to each well. Then, the solutions were mixed for 20 s at 150 rpm and incubated for 3.5 h at 28 • C in the dark. After incubation, the plates were analysed with the use of a fluorescence microscope (EVOS M5000 Imaging System) for the low-dosage levels of Vitamin C and a fluorescence microscope (Leica MZFL-III) for the high-dosage levels of Vitamin C. The level of fluorescence was calculated with the use of ImageJ [34].

Vitamin C Treatments
Fertilised eggs originating from a heterozygous sdhb rmc200 incross were reared in petri dishes filled with E3 medium supplemented with 0.1% methylene blue (Sigma-Aldrich) and incubated at 28 • C with a day/night rhythm. At 2 dpf, the hatched larvae were put in a 48-wells plate containing 200 µL of medium with or without Vitamin C (A4544, Sigma-Aldrich) until 6 dpf. At day 5, the medium was replaced with E3 medium without or with appropriate concentrations of Vitamin C. All working solutions (20, 500, or 1000 mg·L −1 ) were freshly prepared in E3 medium, and the pH was adjusted using 0.5 M NaOH between 6.8 and 8.5 [46].

Lethality Score Analysis
Heterozygous sdhb rmc200 adult fish were crossed to collect eggs. The larvae were divided into two groups. An E3 medium was added for the control group, and a Vitamin C dosage (20, 500, or 1000 mg·L −1 ) was added from 2 dpf onwards. Larvae were either raised in petri dishes (max 60 larvae per dish) for low-dosage levels of Vitamin C experiments or transferred to 1 L tanks for high-dosage levels of Vitamin C experiments. Minimally, twice a day, the larvae were checked to collect death larvae. Death larvae were collect in 75 µL of lysis buffer (40 mM NaOH and 0.2 mM EDTA) and then genotyped. Every day, the medium was refreshed, and in the afternoon, the larvae were fed with Gemma micro 75 ZF for the low-dosage level Vitamin C experiments and with rotifers for the high-dosage level Vitamin C experiments.

Behavioural Assessment: Locomotion Assay
Using DanioVision (Noldus Information Technologies, Wageningen, The Netherlands), the locomotion of 6 dpf larvae was tracked. Each larva was individually placed in a well of a 48-well plate with 200 µL of an E3 embryo medium. The study was conducted at a constant 28 • C and 3000 lux. The short protocol (<5 min in total) to induce startle responses consisted of tapping stimuli with random intervals varying between 2 and 35 s. Afterward, the complete larval body was used for genotyping. Larvae were pooled based on genotype and data grouped per phenotype were exported to Microsoft Excel (version 1906). The max velocity was used as read-outs for startle response to detect possible differences between the three different genotypes and Vitamin C treatment.

Statistical Analysis
GraphPad Prism software (Version 5.03 for Windows, GraphPad Software, La Jolla, CA, USA) was used to generate scatter plots, calculate mean values, and perform statistical analyses. The Log-rank (Mantel-Cox) test was used for the survival curve analysis. The one-way ANOVA with Tukey's post hoc test was used for the ROS basal levels and startle response quantification. A two-tailed unpaired Student's t-test was used for ROS levels without or with Vitamin C treatment.

Conclusions
In this study, we showed that the sdhb zebrafish model possesses an unbalanced redox homeostasis, as indicated by elevated ROS levels at baseline. Further, we evaluated the utility of sdhb zebrafish larvae to test drugs for their therapeutic potential for SDHBassociated PPGLs. We demonstrated that high-dosage levels of Vitamin C shortened the lifespan of homozygous sdhb larvae. This zebrafish model could potentially be used for preclinical drug screening and the identification of new therapeutic targets.  Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.