The classic symptoms of Parkinson's Disease, which are related to motor disorders, are not the only symptoms to negatively affect the quality of life of patients. Several non-motor symptoms are reported as the first apparent symptoms of PD, including behavioral changes, such as anxiety and depression (Poewe et al. 2017). Another feature of PD is the complexity of treatment, which, to date, is only symptomatic, as there is still no effective treatment or drug that can stop or delay the neurodegeneration process of PD (Doyle and Croll 2022). Due to these factors, studies with animal models in which PD-like symptoms are reproduced and necessary to provide more tools capable of helping in the formulation of new treatments and drugs. In a recent study, it was observed that exposure of zebrafish embryos to rotenone can induce PD-like motor symptoms (Kalyn et al. 2020). However, there is a lack of insights into behavioral and embryonic developmental changes in zebrafish exposed to rotenone. In our study, we add information to improve the quality of this model. We observed new toxic effects such as delayed epiboly, morphometric alterations, and teratogenic defects. These important parameters can be used for toxicological evaluation in the screening of PD drugs. Furthermore, behavioral changes caused by exposure to rotenone were observed, which may be related to PD-like symptoms. These studied endpoints can be used in the screening of drugs to develop new PD treatments.
4.1. Toxicological parameters
The assessment of the percent epiboly of zebrafish embryos can be performed by fixing the eggs after 8 hpf, followed by microscopic photographic records for measurements of the plant, animal, and germinal ring poles, enabling the measurement of the advance of epiboly movement. Epiboly is the first morphogenesis event during zebrafish development and is essential for normal development, which occurs from the late blastula (4.3 hpf) to the bud stage (10 hpf) (Bruce and Heisenberg 2020). Zebrafish development is the stage of life most sensitive to chemical exposure, and to avoid this problem, the chorion acts as a protective barrier that has the potential to limit the effects of this exposure (Mandrell et al. 2012). However, in our results, it was observed that exposure to rotenone at concentrations between 10 and 20 µg/L in the first hours of life (2–8 hpf) can cause embryotoxicity, as seen in the delayed percent epiboly. In the literature, no evaluations of epiboly were found in zebrafish exposed to chemicals that have been used for PD-like models. This may be related to the fact that the initial exposure time for PD-like induction in zebrafish normally occurs at 24 hpf (Kalyn et al. 2020). In order to evaluate the susceptibility of zebrafish embryos to rotenone, the percent epiboly provided is adequate to confirm the influence on early stages of development. In addition, the percent epiboly can represent a rapidly obtained toxicological endpoint for use in the evaluation of protective effectiveness or treatment in the zebrafish PD-like model.
The exposure of zebrafish embryos to rotenone caused teratogenic defects and mortality during the entire test. The affected embryos belonged to the ROT10, ROT15, and ROT20 groups, the same groups that showed delayed epiboly, indicating that a delay in this stage of development can cause later adverse effects. This fact can be confirmed, since at 24 hpf the affected embryos of the ROT10, ROT15, and ROT20 groups presented developmental delay teratogenic effects, mainly related to the absence of tail detachment and incomplete ocular formation, absence of pigmentation, and pericardial edema. Similar results were found in the study carried out by Melo et al. (2015), in which the authors observed a lack of pigmentation and developmental delay in embryos at 24 hpf exposed to concentrations of 10, 20, 40, and 80 µg/L of rotenone since the beginning of fertilization. At the 48 hpf, the ROT10 group did not have a significantly higher rate of affected animals than the DMSO group. Furthermore, the teratogenic defect absence of pigmentation was not observed at 48 hpf or in a subsequent observation period, indicating that the teratogenic effects may be reversible and, because of the pigmentation recovery, the ROT10 group presented a reduced percentage of affected animals at 48 hpf. However, the most common teratogenic defects were developmental delay and pericardial edema, which are different from the results observed by Melo et al. (2015), who noticed the appearance of pericardial edema only at 72 hpf. At the periods of 72 and 96 hpf, the three groups with the highest concentrations of rotenone presented affected animals. However, unlike at 24 and 48 hpf, the main effect observed in the embryos was the proportionally increasing mortality in relation to the increase in rotenone concentrations. A teratogenic effect that began to be observed at 72 and 96 hpf was yolk sac edema, but developmental delay and pericardial edema effects continued to be observed in both periods. In the study carried out by Kalyn et al. (2020), in which zebrafish exposure to a 50 nM concentration of rotenone started at 72 hpf, the presence of cardiac edema was observed as a teratogenic effect. A teratogenic effect was also observed in our results at 72 hpf, with loss of chorion and larval hatching. Another study by Melo et al. (2015), observed the presence of cardiac malformations (edema). However, the details of the mechanisms of action of rotenone that influence cardiac malformations remain unknown.
Fish in embryonic and larval stages are known to be sensitive to xenobiotics (Çalışkan and Emekli-Alturfan 2021), as observed in our epiboly and affected animal analyses. However, the 5 µg/L concentration of rotenone did not provoke other adverse effects, besides morphometry, in the zebrafish embryos tested. In the study by Bretaud et al. (2004), in which the authors exposed zebrafish embryos at 24 hpf to concentrations of 5 and 10 µg/L, no teratogenic effects were observed at either concentration. In our results, exposure to 10 µg/L of rotenone alone was able to provoke teratogenic effects, while exposure to 5 µg/L had no observable negative effects.
Studies with adult zebrafish exposed to concentrations of 2 to 5 µg/L of rotenone for 28 days, reported induced locomotor changes and non-motor PD-like symptoms (Khotimah et al. 2015; Wang et al. 2017). The high sensitivity of adult zebrafish to rotenone can be explained by the long duration of exposure and the greater contact area of the gills, intestine, and skin absorption. In addition, rotenone easily crosses the blood-brain barrier and enters the central nervous system due to its lipophilic structure, penetrating directly into neurons, and accumulating mainly in mitochondria (Ünal et al. 2020). According to Melo et al. (2015), the limit between rotenone doses that cause effects and that are ineffective is quite narrow, something that was also observed in our study. Therefore, we measured the EC50 in order to find an ideal concentration to induce PD-like symptoms in zebrafish embryos, without causing high mortality rates, but with several observable and measurable effects on developmental parameters, behavior, and locomotor activity. In our study, an EC50 of 11.24 µg/L was observed for zebrafish embryos exposed to rotenone, a value similar to that observed by Melo et al. (2015) who reported an EC50 of 12.2 µg/L.
For morphometric analysis, the larvae at 6 dpf were euthanized immediately after the end of the behavioral tests, i.e., those fish that had obvious teratogenic effects were excluded. According to our data, rotenone was able to negatively affect the size of the head and body of the tested larvae. The morphometric evaluation was the only test that demonstrated toxicity at the lowest concentration of rotenone (5 µg/L - ROT5) in our work. Although no delay in the epiboly phase and changes in other parameters were observed, continuous exposure to rotenone in the ROT5 group delayed larval growth. This fact may be associated with the high sensitivity of bone tissues to rotenone, as reported by Heinz et al. (2017) when observing bone marrow depletion and bone atrophy in 7–8 week old rats exposed to rotenone. In general, there was a significant reduction in the head sizes of the larvae, caused by the ability of rotenone to decrease bone ossification.
Despite having smaller heads compared to the DMSO group, it was expected that in proportion the eye size of the larvae exposed to rotenone would also decrease; however, no reductions in the eye diameter were observed. The absence of changes in the size of the eye may be an indication of the results obtained in the OMR test, in which the larvae of all groups reacted normally to the visual stimuli applied. Therefore, the concentrations of rotenone and the form of exposure used in our study were not able to cause changes in the eye development of zebrafish. Additionally, the lack of neurotoxicity in the ROT5 and ROT10 groups, although rotenone can easily cross the blood-brain barrier (Ünal et al. 2020), may have been caused by the absorption of most of the rotenone concentration in bone tissue formation. Thus, it is possible to observe effects on the size of the larvae, but no behavioral changes. A similar result was observed in the work by Khotimah et al. (2021) in which the authors exposed zebrafish embryos at 3 dpf to a concentration of 12.5 µg/L of rotenone and observed a reduction in the body size of zebrafish larvae at 6 dpf. Based on the data obtained in our tests, the morphometric evaluation of the head and body of zebrafish larvae exposed to rotenone as a PD-like model can be a useful tool when performing tests to evaluate drugs that have a protective action against PD-like inducing agents.
4.2. Behavioral parameters
Behaviors that have evolutionary conservation and are exhibited by a wide variety of species, including rodents, fish, and humans (Basnet et al. 2019), which is the case with thigmotaxis, are important tools to assess anxiety-like patterns. Thigmotaxis is a specific behavior related to anxiety and represents one of the most used behavioral parameters in preclinical studies employing rodent models, in which animals are placed in a novel environment and, by default, strongly avoid the center of that environment while trying to move close to the edges (Schnörr et al. 2012). In our thigmotaxis tests, larvae were placed in an unfamiliar environment and those that showed apparent teratogenic defects, or destabilized swimming were not evaluated. It was observed that thigmotaxis was affected by the two highest concentrations of rotenone (15 and 20 µg/L). Previous studies that exposed zebrafish embryos and larvae to PD-like substances did not analyze anxiety-like behaviors, a non-motor symptom of PD. The study by Wang et al. (2017), evaluated anxiety-like behaviors in the PD-like model of adult zebrafish exposed to rotenone using a light/dark preference test to observe whether rotenone caused changes in standard behavior. The authors concluded that behavioral symptoms were associated with decreased levels of dopamine in the brains of fish treated with rotenone, something that may also have occurred in the larvae of our experiment. From the observation of these results, the use of zebrafish larvae exposed to rotenone as a model of induction of PD-like non-motor symptoms is valid. In the TS test, used to evaluate the response of larvae to physical stimuli and escape behavior, only the concentration of 20 µg/L of rotenone showed a reduction (of 14.55%) compared to the DMSO group. The reduction in sensitivity may be related to the neurotoxic action of rotenone, which interferes with the normal state of the central nervous system and may indicate a mild locomotion defect (Kalyn et al. 2020). Given the results of the TH and TS tests, behavioral changes were observed in larvae exposed to the highest concentrations of rotenone, even in animals that did not have apparent teratogenic effects.
The OMR test evaluates the visual and motor components of zebrafish larvae. Assessment of these components may be useful since many patients with PD present sensory dysfunctions, such as changes in visual perception (Weil et al. 2016). Rotenone is used as a chemical capable of causing retinal degeneration in rats (Sasaoka et al. 2020), but no studies evaluating the optomotor response in zebrafish exposed to rotenone were found in the literature. However, a study that exposed zebrafish larvae to 6-OHDA observed optomotor damage at 7 dpf (Benvenutti et al. 2018). Benvenutti et al. (2018) described that 6-OHDA, besides causing locomotor defects, was also involved in sensory impairment. In our study, all groups exposed to rotenone responded positively to the visual stimulus that was applied in larvae without apparent teratogenic effects, indicating that the concentrations of rotenone were not sufficient to affect locomotion and the visual capacity of the animals in this test, even in the groups that demonstrated alterations in the TH and TS tests.
The negative effects of rotenone in the early life stages of zebrafish as a model to study PD-like symptoms appeared in different ways according to the rotenone concentration. The lowest concentration used (ROT5), for example, only causes a decrease in the size of the head and body but did not present effects for the evaluation of PD-like symptoms. Rotenone exposure in the ROT10 group did not cause behavioral changes, although effects on percent epiboly, teratogenic defects, and morphometric reductions were observed. Thus, exposure to 10 µg/L of rotenone is not indicated for inducing behavioral changes in this animal model. However, 10 µg/L of rotenone can be used to provoke toxicological defects in zebrafish animal models for PD drug screening. The concentrations of 15 and 20 µg/L (ROT15 and ROT20, respectively) showed high toxicity of rotenone in zebrafish, and there were significant effects in all non-behavioral parameters compared to the DMSO group. Regarding behavior, the ROT15 group presented only reduced thigmotaxis behavior, while the ROT20 animals also showed reduced sensitivity to physical stimuli. However, the high mortality rate observed in these groups could make it difficult to carry out tests and reduces the survival of animals in drug tests. As mentioned above, the use of a concentration between 10 to 15 µg/L is recommended (Table 2), in which mortality is reduced, but there are significant observable effects. These rotenone concentrations will be suitable for evaluating the potential of new drugs to protect against rotenone-inducing defects or for treating the non-motor symptoms caused by rotenone exposure in our zebrafish embryo PD-like model.
The use of zebrafish exposed to rotenone as a PD-like model presents advantages and disadvantages according to the life stage. The zebrafish at an early stage of life in our test enables the observation of toxicity in embryonic and larval development (delayed epiboly, teratogenic, and morphometric defects) at concentrations of rotenone that did not cause high mortality (10 µg/L). The larval stage of zebrafish from 5 to 7 dpf allows the application of tests to evaluate several motor and non-motor qualitative endpoints on a large scale, such as anxiety-like behaviors, and sensitivity to physical stimuli, as seen in our study. However, the downside of exposure to rotenone in the early life stages of zebrafish is that there is a narrow limit of concentrations that cause or do not cause effects, making it difficult to standardize an ideal concentration able to cause developmental, motor, and non-motor effects of PD-like symptoms. Adult zebrafish are more sensitive to rotenone (Ünal et al. 2020), enabling induction of PD-like symptoms with lower concentrations of 2 to 5 µg/L, resulting in lower limits needed to cause the motor and non-motor effects and low mortality (Khotimah et al. 2015; Wang et al. 2017; Andrade et al. 2022). In addition, adult zebrafish have a wider variety of behavioral and locomotor patterns, allowing access to additional information compared to zebrafish larvae (Wang et al. 2017; Ilie et al. 2022). However, adult zebrafish as an animal model for PD-like induction with rotenone require a prolonged exposure of 28 days, and, due to the size of the animals, the sample size is reduced compared to tests using early stages of zebrafish. In view of this, the use of zebrafish in the early stages is recommended for screening for new drugs with a therapeutic effect on PD, while in the adult stage, a deeper evaluation of the protective and therapeutic effects of drugs is indicated.