Application of Zebrafish in Mitochondrial Dysfunction

Abstract

This chapter provides an overview of the zebrafish (Danio rerio) as a model organism for studies of mitochondrial dysfunction. Zebrafish possess a genetic similarity with humans and have conserved mitochondrial genomes, rendering them a valuable research tool for examining the intricate mechanisms that govern mitochondrial processes at diverse developmental stages. The chapter explores several methods for evaluating mitochondrial health and function. Examples include in vitro cell culture and in vivo analysis in embryos, larvae, and adults. The chapter discusses the use of zebrafish models in toxicological research to investigate mitochondrial reactions to environmental stressors and xenobiotics. The importance of implementing standardized protocols, validating marker, integrating different omics data, and using in vivo and in vitro approaches to advance mitochondrial research will be highlighted. In summary, zebrafish are suitable for analyzing both mitochondrial function and dysfunction, as well as their impact on human health.

1. Introduction

Mitochondria are indispensable organelles for eukaryotic organisms, enabling aerobic metabolism, thus consuming oxygene and oxidizing molecules, such as sugars, to produce adenosine triphosphate (ATP), the energy currency used by cells [1, 2]. In addition, mitochondria actively participate in the control of cell homeostasis and in signaling processes, including cell injury [3], the intrinsic apoptosis pathway, and mitophagy [2, 4].

Given their crucial importance, exposure to toxic agents that can affect mitochondria is a factor that has been explored in toxicological analyses of chemical substances and compounds [5, 6]. Damage to mitochondria, often of a structural nature [3], triggered by exposure to toxic agents, can lead to inhibition of oxidative phosphorylation, production of reactive oxygen species, alterations in the mitochondrial permeability transition, mitochondrial DNA damage, and inhibition of beta-oxidation of fatty acids [7, 8, 9] . The adverse effects derived from this damage and dysfunction are wide-ranging, including hepatotoxicity [8], cellular aging, senescence, genomic instability, inflammation [10], as well as metabolic, neurodegenerative, and cardiac disorders [9, 11] and hereditary diseases that affect the mitochondrial network and cell metabolism [11].

For regulatory purposes and to understand the toxicity of compounds, the definition of mitotoxicity is relevant and can help in the weight of evidence and in defining the safe use of substances, for which various experimental models and organisms are used. Various organisms are currently used to assess mitotoxicity. Among these model organisms, the Zebrafish (Danio rerio) has stood out worldwide [12, 13].

The advantages of using this model are related to its genetic, mitochondrial, and xenobiotic metabolism similarities when compared to humans [12, 13, 14, 15], as well as aspects related to scientific research. In addition, zebrafish are a model applied to One Health, which is based on linking human, animal, and environmental health [16]. This makes it possible, by examining possible mitochondrial dysfunction in zebrafish, for researchers to obtain information about the potential implications for human health, for the fish themselves, and for their ecosystems [16].

In view of the growing application of zebrafish in mitochondrial dysfunction analysis, the object of this chapter is to present the principles of mitochondrial function and dysfunction, the application of the model organism, and the main techniques used in this context.

2. Mitochondria: function and dysfunction

Occupying up to 20% of the cytoplasmic volume, mitochondria are important ubiquitous organelles for the evolution of complex animals, since they carry out the complete metabolism of sugars, making the production of ATP 15 times greater than anaerobic glycolysis. Their shape is similar to that of small bacteria, and because they have their own genome (circular DNA), produce tRNAs (RNA transporters), and have ribosomes, it is the most widely accepted theory that their origin is from an endosymbiotic relationship between a primitive eukaryote and aerobic bacteria [1, 2, 8].

Mitochondria have two different membranes that surround specific compartments. The outer membrane contains pores that allow small molecules and ions to pass but prevent larger molecules like proteins. Located between the outer and inner membranes is the intermembrane space. The inner membrane defines the mitochondrial matrix and is highly impermeable to small molecules, including H+ ions. The mitochondrial cristae, invaginations in the inner membrane, greatly increase its surface area and house the machinery responsible for energy conversion. Here we find the complex of electron transport proteins of the respiratory chain (CTE) (complex I–V), ADP-ATP translocase, ATP synthase, and other membrane transporters. The mitochondrial matrix contains important processes, including the Krebs cycle, which converts pyruvate and fatty acids into NADH. In this space, we find the pyruvate dehydrogenase complex and various enzymes related to these processes. In addition, the matrix houses mitochondrial DNA and ribosomes, which are essential for mitochondrial function and replication [1, 2].

Mitochondrial DNA has undergone substantial changes during evolution, so most of the genes underwent migration to the nucleus through gene transfer. However, current mitochondria still contain smaller genes that encode hydrophobic proteins forming the electron transport chain (ETC) in addition to genes related to the mitochondrial genetic system like ribosomal proteins. Some important mitochondrial genes include rns and rnl, which encode ribosomal RNA, cob (cytochrome b), and cox1 and cox3, which encode two subunits of cytochrome oxidase [1, 2, 17].

As dynamic and plastic organelles, mitochondria move within cells via microtubules of the cytoskeleton, change shape, and are able to divide and fuse. They possess the ability to change their shape and undergo division and fusion, collectively known as mitochondrial dynamics. These processes involve mitochondrial fission and fusion. These processes enable the regulation of mitochondrial number and shape according to cell requirements while preserving the integrity of mitochondrial compartments [1, 18].

The essential function of mitochondria is related to the energy conversion machinery located in the inner membrane. This process is known as chemiosmotic coupling, in which ATP synthesis is directly linked to transport processes across the membrane. In addition, in the complex context of cellular homeostasis, mitochondria interact with each other, including the endoplasmic reticulum, during fusion and lipid trafficking, and with lysosomes during the process of mitophagy [1, 2, 19].

The presence of mitochondrial permeability transition pores (mPTP) is a characteristic feature of mitochondrial permeability to compounds, especially with respect to passage through the outer membrane. The mPTPs maintain mitochondrial homeostasis and are composed of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocase (ANT), and cyclophilin D (Cyp-D) [20].

The mitochondrial matrix houses crucial enzymes that facilitate conversion of pyruvate and fatty acids into acetyl-CoA. Subsequently, acetyl-CoA is oxidized in the citric acid cycle to produce NADH, which provides high-energy electrons for the respiratory chain. In the inner membrane, the respiratory chain utilizes these electrons to transport protons out of the matrix, thereby creating an electrochemical gradient. In this process, NAD-linked dehydrogenases contribute electrons by oxidizing substrates like a-ketoglutarate and malate. These electrons are passed to the respiratory chain that reduces oxygen to create water [1, 2].

The respiratory chain is composed of three primary enzyme complexes that transfer electrons through protein carriers. In each complex, energy is released for the pumping of protons. Technical term abbreviations will be explained on first use. The sequence of electron flow is as follows: NADH → Complex I (NADH dehydrogenase) → Ubiquinone → Complex III (Cytochrome c reductase) → Cytochrome c → Complex IV (Cytochrome c oxidase) → Molecular oxygen (O₂). NADH transfers its reducing equivalents to ubiquinone, which then guides the electrons through complexes III and IV. In this phase, oxygen undergoes a reduction and becomes water. Observe that the language is objective, clear, and avoids complexity [1, 2].

The electrochemical gradient promotes the proton-motive force, propelling hydrogen ions back into the mitochondrial matrix. ATP synthase utilizes this force to produce ATP while serving as “rotational catalysis” coupled with the proton flow. Along with ATP production, the electrochemical gradient enables the active transport of metabolites, efficiently converting ADP into ATP. This sustains elevated cellular levels of ATP, furnishing energy for a diverse array of cellular processes [1, 2]. Figure 1 illustrates the processes of CTE and OXPHOS and the mitochondrial structures.

Figure 1.

Mitochondria: Structure and oxidative phosphorylation.

The oxygen reduction pathway processes naturally generate ROS, including superoxide (^•O₂⁻), hydrogen peroxide (H₂O₂), and hydroxyl radicals (−OH). These compounds necessitate speedy inactivation to avoid reactivity and potential destruction of various cellular structures. Mitochondria contain antioxidant enzyme complexes such as superoxide dismutase and glutathione peroxidase to neutralize ROS and prevent damage. On the other hand, low levels of ROS play a role in signaling and coordinating mitochondrial oxidative phosphorylation with other metabolic pathways [2, 8, 21].

Furthermore, mitochondria perform various metabolic functions, such as redox buffering in the cytosol and calcium buffering, as well as play a critical role in biosynthesizing heme groups and membranes, the urea cycle, and metabolic adaptation [1, 19]. The intricate nature of mitochondria underscores their diverse significance in cellular metabolism, and consequently, any injury to these organelles by toxic agents can disrupt cell homeostasis in numerous ways.

One of the important mechanisms of action of toxic agents in mitochondria is mediated by oxidative stress caused by excessive production of ROS that exceeds the capacity of the antioxidant system. ROS can impact different cellular and mitochondrial structures, and they are genotoxic while interfering with protein structures. Excess ROS production can result from mtDNA damage and protein synthesis alteration, activating apoptotic pathways [8, 22, 23].

Another mechanism involves the mPTP induction, which leads to the release of pro-apoptotic proteins into the cytosol, initiating cell death. The cascade may or may not be mediated by the caspase. The primary proteins involved in this context are cytochrome C (Cyt C), apoptosis induction factor (AIF), and endonuclease G (EndoG), which are prevalent in the intermembrane space. Various mechanisms facilitate the permeabilization of the outer mitochondrial membrane, including BAX/BAK, which produce pores, the activation of BH3-only proteins, and others that contribute to mitochondria-mediated intrinsic cell death. Disruption of calcium homeostasis or increased production of mtROS results in the disruption of both the inner and outer mitochondrial membranes. This causes a decrease in membrane potential, a reduction in ATP production, and ultimately a cellular demise through apoptosis or necrosis [8, 24, 25].

The induction of mutations in mtDNA by toxic agents may represent a molecular initiating event (MIE) of the mitotoxicity of these agents, as this induction can result in the abnormal synthesis of proteins involved in oxidative phosphorylation. mtDNA is especially susceptible to damage due to its proximity to CTE, which generates mtROs, and its limited repair mechanism. Consequently, mtDNA is prone to various types of damage, including strand breaks and base deamination, among others. These modifications can impact the function of CTE complexes, heighten the generation of reactive oxygen species (ROS), and ultimately initiate phenomena such as mitophagy, apoptosis mediated by mitochondria, or necrosis [8, 26, 27, 28].

The primary mechanism behind the mitotoxic effects of toxic agents is the inhibition of oxidative phosphorylation. These chemicals can impact mitochondrial respiration in multiple ways, such as by uncoupling it, inhibiting CTE complexes, or blocking ATP synthesis directly. Uncouplers transport H+ ions, changing the membrane potential, lowering ATP production, increasing oxygen consumption, and releasing energy as heat. Inhibition of ATP synthase and CTE results in ATP depletion, which disrupts calcium homeostasis. This leads to cytoplasmic calcium overload, activates proteases, and causes cell dysfunction or death. The decrease in ATP generation linked to this process is related to various pathologies, aging, and adverse medication effects. Moreover, blocking CTE raises the production of superoxide radicals and mtROS, which leads to cellular impairment [8, 29].

Ultimately, toxic substances could target fatty acid oxidation (FAO) in mitochondria. Inhibiting this process can occur directly through interaction with beta-oxidation enzymes or indirectly by depleting the coenzymes necessary for the electron transport chain (ETC). This inhibition of FAO oxidation can result in the uncoupling of oxidative phosphorylation, leading to potential consequences and apoptosis through the intrinsic mitochondrial pathway. Extended and severe reduction of mitochondrial beta-oxidation is associated with steatosis causing liver failure, coma, and mortality [8, 30].

3. Zebrafish as a model for mitochondrial toxicology

The fish species Danio rerio, popularly called zebrafish, is a small, blue, and gray striped fish native to the south of the Asian continent, first described by the Scottish physician Francis Hamilton in India [31]. Its typical habitat consists of shallow, slow-moving streams and, in particular, still pools that form along streams during the monsoon, a very rainy season when the waters become turbid. Having co-occurred with humans for thousands of years, zebrafish are also found in rice paddies, drainage ditches, storage tanks, and the like, although they certainly also suffer from the effects of pollution and habitat loss [32]. In terms of their natural diet, these fish are omnivorous, consuming larvae and adult insects, as well as small crustaceans and other zooplankton, while also consuming algae, plant materials, and various detritus [33]. Many studies have been carried out since their description, in relation to their diet, habitat, and cultivation, involving fish farming, and they have also been much investigated in relation to their genetics, mainly by George Streisinger (University of Oregon, USA), who made a significant contribution in the area of developmental biology and the use of zebrafish (D. rerio) as a model organism [34, 35, 36].

Since then, zebrafish have also been recognized as a useful organism for research into human diseases. Among the model organisms most commonly used in research, fish are already in second place in many countries, just behind rodents [37]. The zebrafish is widely used worldwide for a number of reasons. It is a low-cost model organism to keep in the laboratory; it is easy to cultivate and maintain because of its small size; it has a high reproduction rate, which allows several experiments to be carried out at the same time; fertilization is external, making it possible to collect eggs for studies such as toxicological tests, and its embryos are transparent, which allows the entire embryonic development to be visualized, observing its organs, heartbeat, and movement of the bloodstream; it is within the 3Rs (Replacement, Reduction, and Refinement), mainly because of the tests carried out with embryos up to 120 hpf, which is considered an in vitro test, and, above all, because it shares common organs and 71.4% of the same genes as humans and 84% similarity with disease genes [14, 38, 39].

The genetic similarity leads to the use of zebrafish as a model to study genetic functions and disorders in mammals, such as metabolic syndromes [40]. In the last two decades, zebrafish have been shown to be useful for discovering and studying toxicities in a wide range of organs and systems. Teratogenicity, cardiotoxicity, hepatotoxicity, nephrotoxicity, neurotoxicity, gastrointestinal toxicity, myotoxicity, and carcinogenicity have all been successfully explored in zebrafish [14]. We can see that there is a great demand for research in various areas of toxicology using this model organism, and in addition to these, mitotoxicity is also gaining ground today.

We can say that mitotoxicity is a new term, used in scientific articles since 2011, when we searched scientific platforms such as PubMed, with 147 scientific publications in total and only 21 review articles. Parallels between human and zebrafish mitochondria extend to their composition and genetic organization, encompassing genes for rRNAs, tRNAs, and proteins integral to oxidative phosphorylation [12, 13, 41, 42]. Consequently, zebrafish serve as an apt model for scrutinizing mitochondrial function, enriching the arena of comparative genomics and providing insights into genetic pathways and key molecular processes among vertebrates [43, 44]. Zebrafish can be used to study mitotoxicity at different stages of development and also in cultured cells from embryos, larvae, or adult tissues (Figure 2).

Figure 2.

The zebrafish model organism, stages of development, and the possibility of cell culture.

Xenobiotics, such as pollutants and drugs, target multiple macromolecules (DNA, proteins, lipids), including mitochondrial components, suggesting that their toxicity mechanisms may also be related to mitochondrial dysfunction [45, 46, 47, 48]. Scientific publications evaluating mitochondrial dysfunction in zebrafish have grown exponentially since 1994, according to data from the PubMed platform. Most of these studies are for the evaluation of xenobiotics, which are often substances that we come into contact with on a daily basis [49, 50, 51, 52, 53, 54]. The use of zebrafish as a model organism, in addition to all the advantages already described in this topic, is also of great importance for assessing mitotoxicity, and according to Azevedo et al. [12], it is because of the contribution of this small teleost that our knowledge of the intricate patterns linking mitochondrial function to the balance between health and disease is now more easily acquired. In the next topic, we will look more precisely and in detail at existing studies involving mitotoxicity and zebrafish [12, 42].

4. Approaches for mitochondrial analysis in zebrafish

Zebrafish enable researchers to investigate mitochondrial function and dysfunction using a range of methodological approaches. Established methodologies can be adapted, and new experimental procedures can be developed using various experimental models with the zebrafish organism. The structure is clear, with a logical progression and causal connections between statements. Analyses can be performed on primary and secondary cell cultures, embryos up to 48 hours after fertilization, larvae from 72 hours after fertilization, as well as juveniles and adults. Tissue extracts from adult zebrafish are utilized for the isolation of mitochondria, which allows for various analyses to investigate mitochondrial functions and potential dysfunctions.

This method is widely employed in mitochondrial toxicology research. Previous studies have focused on comprehending the similarity of isolated zebrafish mitochondria. In this context, certain studies have achieved the isolation of zebrafish mitochondria via methodologies, including differential centrifugation. This technique employs a series of centrifugation steps to divide cellular components by size and density and has successfully resulted in the isolation of mitochondria from zebrafish embryos. Another method for organelle isolation is density gradient centrifugation. This approach involves layering samples onto a density gradient medium, then separating organelles through centrifugation based on density fluctuations. Mitochondria can also be isolated utilizing specific commercial kits [55, 56, 57].

Microscopy and flow cytometry are important tools in mitochondrial toxicology studies, permitting visualization and analysis of mitochondrial morphology and dynamics and quantification of mitochondrial function and integrity at the single cell level [58]. The most commonly used microscopy techniques in this field include epifluorescence, confocal, and electron microscopy.

Fluorescence microscopy serves as an auxiliary technique for various experimental procedures, like immunofluorescence. A particular application of this method involves analyzing real-time and in vivo mitochondrial transport and dynamics in zebrafish larvae. The two-photon laser axotomy technique was utilized to axotomize M axons while concurrently recording the regeneration process and mitochondrial transport. In this context, it was observed that axons that have a greater ability to regenerate maintain higher levels of mitochondrial mobility [59].

Confocal microscopy can analyze the effects of toxic agents on mitochondrial fusion and fission processes, providing data on the regulation of mitochondrial segregation [58]. In addition, electron microscopy can be used to explore mitochondrial ultrastructure, with a focus on zebrafish cells. This approach allows for precise imaging of mitochondrial cristae morphology, size, shape, and architecture, providing essential insights into mitochondrial function and identifying any abnormalities associated with dysfunction [60].

Additionally, flow cytometry has been utilized to quantitatively measure mitochondrial function and integrity in individual cells, as well as to assess mitochondrial content and mass in zebrafish cells. This method enables the examination of mitochondrial biogenesis and regeneration, making it a useful tool for assessing the impact of harmful substances on mitochondrial metabolism and energy generation [61].

Combined with microscopy and flow cytometry, and in addition to blotting techniques, the use of fluorescent dyes constitutes a particularly sensitive method for detecting various structures and homeostasis conditions. In this context, immunofluorescence and immunoblotting techniques are noteworthy, as they allow for the evaluation of specific proteins within mitochondrial complexes. Immunoblotting analyses concentrate on the expression and localization of proteins that relate to mitochondrial function. For instance, the levels of mitochondrial apoptotic proteins in zebrafish testes following exposure to carbon ion radiation were evaluated using immunoblotting [62].

In immunofluorescence of cell cultures, MitoTracker is a commonly employed fluorescent dye. This dye enables the visualization and quantification of mitochondria, as it infiltrates the mitochondria depending on the mitochondrial membrane potential and continues binding even after this potential dissipates [63]. In another study, various fluorescent dyes or fluorescent reporters were employed to analyze neuronal mitochondria in vivo, in Zebrafish embryos and larvae, following the procedure in the study. To this end, Mandal et al. refined techniques for visualizing mitochondria and analyzing their life, health, and function using parameters including movement, location, size, and function [64].

The evaluation of co-localization of mitochondria in relation to other cellular components using fluorescent dyes and immunostaining is pertinent for understanding mitochondrial dynamics. Ito et al. employed immunostaining with an anti-raldh2 antibody to investigate the localization of raldh2 in zebrafish and medaka subsequent to cardiac injury. Periostin-b from zebrafish and periostin-b from medaka, which are markers of fibrillogenesis, were detected at the wound site where collagen deposition occurred, implying their co-localization with mitochondria [65].

To evaluate mitochondrial health and the potential mitotoxic effect of a compound, measuring mitochondrial membrane potential (ΔΨM) is a fundamental analysis. Fluorescent dyes are frequently employed for this purpose. For instance, researchers developed a novel cyanine dye, ZMJ214, for live zebrafish larvae to detect mitochondrial membrane potential [66]. In addition, zebrafish embryos were subjected to another fluorescent dye; JC-10 is a lipophilic cationic dye that indicates mitochondrial depolarization [67].

Assessing ROS production is another parameter used to determine mitochondrial toxicity, and zebrafish can be used as a model organism to identify changes in ROS levels. Fluorescent probes, such 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) and diamino fluorophore 4-amino-5-methylamino-2′,7′difluorofluorescein (DAF-FM), are commonly used because they can be internalized by cells and oxidized by ROS to generate fluorescent signals [68]. Finally, another frequent assessment is the analysis by biochemical assays of the activity of antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), which are responsible for scavenging ROS [68, 69].

In studies that concentrate on mitochondrial genetics, the main techniques used are mtDNA knockout and knockdown. Zhou et al. developed a tissue-specific knockout approach, which is an exemplar of this method. The main objective of the study was to explore essential developmental genes related to mitochondrial function in zebrafish [70]. Furthermore, the manipulation of genes responsible for generating or scavenging ROS can be employed to examine the impact of altered ROS concentrations on zebrafish physiology and development [71]. Moreover, transcriptome and proteome analyses can provide information on the molecular mechanisms underlying ROS production and its effects on zebrafish [72].

Oxygen Consumption rate (OCR) measurement provides valuable information on the mitochondrial function and bioenergetics of zebrafish. It enables the quantification of oxygen consumption rate (OCR) measurement, a direct measure of mitochondrial respiration and ATP synthesis [73]. The measurement of OCR using respirometry instruments permits the quantification of mitochondrial respiration in zebrafish. OCR measures the metabolic activity of organisms and provides important insights into mitochondrial function and bioenergetics. It has been utilized to explore the impact of temperature on the bioenergetics of zebrafish embryos and to evaluate how environmental stressors affect mitochondrial function [74]. OCR measurements can be utilized to evaluate the metabolic activity of zebrafish in response to different interventions or treatments [75, 76].

One method of determining OCR in zebrafish is by employing plate-based respirometry with the use of an extracellular flux analyzer like the Seahorse XF96 [74, 77]. This approach enables the evaluation of OCR in multi-well plates, providing a high-throughput method for measuring mitochondrial respiration in zebrafish embryos and larvae. The Seahorse analyzer employs Islet Capture microplates where a single embryo is situated in each well, permitting the measurement of bioenergetic parameters such as oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) [74].

Another innovative method involves utilizing the high-resolution Oroboros O2k Oxygraph instrument and microfluidic devices, which are combined with light modulation systems for precise measurement. This technology enables the calculation of OCR through the measurement of oxygen decrease over time. This method is supported by various studies. Using mitochondrial modulators that act at different stages of respiratory chain and oxidative phosphorylation, technology allows for the assessment of various mitochondrial components and complexes [78, 79]. This method enables precise and sensitive evaluation of mitochondrial oxygen consumption, enabling measurements of mitochondrial respiratory capacity, integrity, and energy metabolism [80].

4.1 Evaluation and enhancement of existing methodologies

Numerous techniques are employed to investigate mitochondria using zebrafish, as previously mentioned. However, in order to provide further insights into their applications, the developmental stages utilized, as well as the advantages and disadvantages associated with each, are elaborated. Below, Table 1 is presented.

Technique	Model	Application	Advantage	Limitation
Immunoblotting and immunofluorescence techniques using fluorescent dyes	C [63, 81]; E [64]; L [82]; A [83]	Visualization and quantification of mitochondrial proteins and respiratory chain components [84]	Quantitative results and small sample research [85]	Requires specific antibodies; Not so fast and sensitive [86, 87]
Genetic approaches	C, E and A [88]	Investigate effects of mitochondrial gene absence or reduction [88]	Efficient and accurate tool for targeted genetic manipulation [89]	Potential for nonspecific side effects [89]
Assessment of Mitochondrial Membrane Potential (ΔΨM)	C [90]	Measure the electrical potential across the mitochondrial membrane.; Evaluate mitochondrial functional integrity [91]	Direct indicator of mitochondrial health [91]	Does not provide insights into other parameters.; Limits in terms of high throughput performance and the need for multiple washing steps [92]
Assessment of Reactive Oxygen Species (ROS)	C [93]; E [94]; L [95]	Monitor mitochondrial oxidative stress [96]	Detection of oxidative damage [96]	Analytical and coordination limitations as well as the interference of concomitant ozone [97]
Assessment of mitochondrial co-localization	C [98]; E [99]; L [100]	Investigate mitochondrial interactions with other cellular structures [101]	Efficiency and prediction when analyzing and quantifying mitochondrial movements [102]	Challenges in image enhancement, trajectory tracking, and quantitative analysis [103]
Oxygen consumption rate (OCR) measurement techniques	C [104]; E [105]; L [106]	Comprehensive understanding of mitochondrial metabolism [79, 107]	Direct and quantitative measurement; Live analysis and real-time monitoring; Possibility of using mitochondrial modulators [79, 107, 108]	Requires specialized equipment.; The cost of equipment and in some cases analysis well plates.; Robustness of statistical inference [109].

Table 1.

Methods for assessing mitochondrial function in different experimental models of zebrafish (Danio rerio).

An overview of techniques employed for investigating mitochondrial function within diverse experimental models of zebrafish is presented. The experimental models considered include zebrafish cell culture (C), embryos up to 48 hours postfertilization (E), larvae from hatching to 1 month (L), and adults utilizing tissue grafts and isolated mitochondria (A). For each technique, a description of its specific application, associated advantages, and limitations is provided.

Within this context, it becomes evident that there is substantial room for advancement in the realm of mitochondrial studies using the zebrafish model organism. Below, we delve into several potential areas that warrant exploration and development.

• Standardization and reproducibility: many of the mentioned studies have employed a wide array of techniques, reagents, and equipment to assess mitochondrial function. The standardization of experimental protocols and reagents is paramount to ensuring result reproducibility across various laboratories and studies. This is particularly crucial in the realm of toxicological analyses, where assay reliability and reproducibility hold pivotal importance. In this context, the goal is for the collected data to constitute a substantial body of evidence, informing regulatory agencies’ decision-making processes [33].

• Validation of markers: despite the utilization of numerous fluorescent markers and probes to visualize mitochondrial function, validating these markers in terms of specificity and sensitivity is imperative. Moreover, employing multiple markers to address distinct facets of mitochondrial function can provide a more comprehensive understanding. Another avenue in this regard involves research and development focused on creating novel markers for analyzing new mitochondrial characteristics and functionalities while simultaneously reducing costs and enhancing analysis robustness [34].

• Real-time quantification of mitochondrial function: the adaptation of assay protocols that leverage extracellular flux analysis and oxygen concentration detection is pivotal for advancing the field of these analyses using the in vivo and in vitro models facilitated by zebrafish. Considering the potential to employ methodologies with enhanced cost-effectiveness, in contrast to established evaluations with substantial costs, holds promise. This approach aligns with the broader goal of accessibility and affordability in mitochondrial function assessments, in addition to leveraging the advantages offered by Zebrafish models.

• Integration of multi-omics data: mitochondrial function is influenced by a myriad of genetic and metabolic factors. The integration of data derived from multi-omic approaches, including genomics, transcriptomics, proteomics, and metabolomics, has the potential to offer a more comprehensive understanding of the mechanisms underpinning mitochondrial function and the mitochondrial action mechanisms of xenobiotics throughout the various developmental stages of zebrafish. This holistic approach facilitates unraveling the intricate interplay of molecular events driving mitochondrial responses and contributes to a more nuanced comprehension of the impact of external agents on mitochondrial dynamics within the zebrafish model system [35].

• Integration of in vivo and in vitro studies: while numerous studies are centered on in vivo systems, particularly focusing on embryos and larvae, supplementation with in vitro studies employing zebrafish cell cultures can yield invaluable insights into mitochondrial function under more controlled conditions. This complementary approach enhances our understanding of mitochondrial dynamics, allowing us to discern intricacies that might be obscured by the complex milieu of whole organisms. It also facilitates the dissection of specific cellular responses and contributes to a more comprehensive appreciation of the molecular underpinnings of mitochondrial processes in the context of zebrafish models [36].

5. Application of zebrafish in mitochondrial toxicology analysis

Most of the studies that began to study mitochondrial dysfunction in zebrafish were aimed at understanding human diseases, as in the work of Van Raamsdonk et al. [110], Guo et al. [111], and Bretaud et al. [112], who used this model mainly to analyze neurological diseases. According to the dates of these publications, we can say that the evaluation of mitochondrial alterations using D. rerio has been recent. One of the first studies to be published on mitotoxicity using zebrafish as a model organism was by Oulmi and Braunback [113], who at the time were microinjecting D. rerio embryos to evaluate an organochlorine compound (4-chloroaniline) used to make dyes, insecticides, and various industrial products. In this work, for the toxicological analysis, the researchers evaluated the size, shape, and function of the mitochondria in relation to the quantity of other organelles in the liver and kidney, concluding that, as well as the zebrafish being a suitable model for microinjection tests, it is also suitable in terms of histological and cytological methods for diagnosing the potential dangers of environmental chemical products [113].

We can observe that over the years, mitochondrial assessment has evolved, and new methodologies and techniques have been improved, as in the work of Mendelsohn et al., who investigated the role of energy detection in the response of the zebrafish embryo to anoxia, testing the arrest of development in mitochondrial inhibitors, through oxygen consumption measured by respirometer equipment [114]. Since then, several studies have evaluated mitochondrial dysfunction, mainly by analyzing mitochondrial oxygen consumption for mitochondrial toxicity, such as the work of Bourdineaud et al. [115], evaluating environmental pollutants; the work of Geffroy et al. [116] and Ladhar et al. [117], evaluating nanoparticles, already entering the area of nanotoxicology; the work of Bestman et al. [118], also evaluating an organic compound for the manufacture of pesticides; and Cowie et al. stating mitochondrial dysfunction of zebrafish caused by the pesticide Dieldrina [119]. Other studies have also evaluated pesticides using mitochondrial dysfunction as a method of toxicological analysis [49, 53, 54, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133].

In the literature, there are still few studies related to the toxicity of xenobiotics and mitochondrial dysfunction in zebrafish, especially in relation to mitochondrial oxygen consumption. Most of the existing publications have evaluated human diseases and drugs. Therefore, more studies are needed in the area of mitochondrial dysfunction and xenobiotic toxicity in zebrafish because of the importance of this analysis complementing conventional analyses and because of the help this analysis provides in understanding the mechanisms of action of substances.

6. Concluding remarks

This chapter discusses the function and dysfunction of mitochondria and how the zebrafish model organism can be utilized in various stages of development as well as in cell cultures. Zebrafish have proved to be a crucial tool for examining the intricate nature of mitochondrial function in the presence of xenobiotics.

Moreover, the model’s distinctive features render it a valuable tool for studies on mitotoxicity. Due to its close genetic similarities to humans and shared mitochondrial structure, combined with the zebrafish’s capacity for genetic manipulation, researchers are able to observe firsthand mitochondrial dynamics in action. Additionally, the zebrafish’s capability to monitor mitochondrial respiration directly in vivo and in real-time serves as a valuable tool for thoroughly examining the effects of xenobiotic exposure on mitochondrial function. This approach offers valuable insights into the underlying mechanisms of mitochondrial dysfunction.

In summary, the research focused on analyzing mitochondria in zebrafish shows great potential. By illuminating the potential effects of xenobiotics targeting mitochondria, this research reveals complex molecular responses.

Conflict of interest

The authors declare that they have no conflict of interest.