Zebrafish in the spotlight: expanding Frontiers in toxicology and drug discovery

Abstract

Zebrafish (Danio rerio) has emerged as a valuable model organism in toxicology and drug discovery research. This article provides an overview of the significant contributions of zebrafish to advancing our understanding of toxicology and drug development. Zebrafish offers several advantages, including high fecundity, transparent embryos, and genetic tractability, making it an ideal system for studying drug toxicity and efficacy. The review article highlights key areas where zebrafish has made substantial contributions, such as assessing chemical toxicity, understanding drug metabolism and pharmacokinetics, and identifying novel therapeutic compounds. Furthermore, zebrafish-based assays and screening platforms have been developed to evaluate drug candidates and identify potential toxic effects and researchers have used zebrafish-based tests to study the therapeutic properties of natural compounds. The unique capabilities of zebrafish with its physiological and genetic similarities to humans, have propelled it to the forefront of toxicology and drug discovery, expanding the frontiers of research in these fields.

Graphical Abstract

Graphical Abstract.

Introduction: Zebrafish as a model organism in toxicology and drug discovery

Zebrafish (D. rerio) has gained significant recognition as a powerful model organism in various fields of research, including toxicology and drug discovery. Originally native to freshwater habitats in Southeast Asia, zebrafish possess a number of advantageous characteristics that make them well-suited for studying the effects of toxins and investigating potential drug candidates.¹ One of the key reasons zebrafish have become popular in toxicology research is their high genetic and physiological similarity to humans.² The zebrafish model originated in the 1980s when Streisinger pioneered methods for generating clonal populations via gynogenesis, enabling large-scale genetic screens.³ By the 1990s, the model gained prominence in developmental biology due to its optically clear embryos, which permit real-time visualization of organogenesis-a feature unattainable in murine models.⁴ The completion of the zebrafish genome project in 2013 further highlighted its genetic homology to humans, particularly in disease-relevant pathways.⁵

They share a remarkable degree of genetic conservation, with approximately 70% of human genes having zebrafish counterparts. This similarity allows researchers to study the impact of toxins and drugs on zebrafish with the expectation that the results can be translated to humans, providing valuable insights into potential human health effects.⁶ Zebrafish also offer unique benefits due to their transparent embryos, which enable direct visualization of organ development and physiological processes in real-time. This transparency makes it easier to study the effects of toxic substances on different organ systems during embryonic development.⁷ Researchers can observe the impact on various organs, such as the heart, liver, and central nervous system, providing valuable insights into toxic mechanisms and potential drug interactions.⁸ Another advantage of zebrafish is their relatively low cost and rapid reproduction rate. They are highly prolific, producing hundreds of transparent embryos that develop rapidly, reaching adulthood within a few months. In visualizing organ development, tracking fluorescently labeled toxins, or evaluating real-time morphological changes.⁹ This allows researchers to conduct high-throughput screening of compounds, testing large numbers of potential toxins or drugs simultaneously, and obtaining quick results. The cost-effectiveness and efficiency of zebrafish-based experiments make them particularly attractive for screening and early-stage drug discovery.¹⁰ The zebrafish possess a remarkable regenerative capacity, with the ability to regenerate damaged tissues and organs, including the heart and spinal cord. This regenerative ability makes zebrafish an excellent model for studying the effects of toxic insults on tissue repair and regeneration.¹¹ It also provides an opportunity to investigate potential therapeutic approaches for promoting tissue regeneration in humans.¹² The zebrafish models have been instrumental in drug discovery, helping identify potential drug candidates, determining their efficacy, and investigating their mechanisms of action. This review contemplated the latest findings on the use of Zebrafish as a model in Toxicology and drug discovery during the period of (2019–2024). The relevant data has been collected from databases including PubMed, Google Scholar, ScienceDirect, and Web of Knowledge.

Zebrafish in assessing chemical toxicity: Advantages and applications

Zebrafish (D. rerio) is a useful model organism for chemical toxicity testing due to its unique features and flexible applications. Many genes associated with toxicological responses in humans have counterparts in zebrafish, enabling researchers to investigate the effects of chemicals on similar biological pathways. Zebrafish have a high reproductive capacity, producing a large number of embryos that develop rapidly.¹³ Zebrafish embryos are transparent, allowing for real-time visualization of developmental processes and toxicological effects. This transparency enables researchers to directly observe the impacts of chemicals on organ development, tissue morphology, and physiological functions.¹⁴ It also facilitates the use of imaging techniques to study dynamic processes in living organisms. Zebrafish embryos provide a platform to investigate the organ-specific toxicity of chemicals.¹⁵ By labelling specific tissues or organs with fluorescent markers, researchers can monitor the effects of chemicals on targeted systems, such as the cardiovascular, nervous, or digestive systems. This approach helps identify potential toxicological effects on specific organs and understand underlying mechanisms.¹⁶ Zebrafish exhibit a wide range of behaviors, including locomotion, feeding, and social interactions. Monitoring behavioural changes in response to chemical exposure can provide insights into neurological and neuromuscular toxicity.¹⁷ Automated tracking systems can quantify behavioural alterations, enabling the assessment of chemical impacts on locomotor activity, anxiety-like behaviors, learning, and memory.¹⁸ Zebrafish offer advantages for studying the absorption, distribution, metabolism, and excretion (ADME) of chemicals. Their small size and transparent embryos allow researchers to visualize the distribution of fluorescently labelled chemicals in real-time.¹⁹ This information aids in understanding the bioavailability and tissue-specific accumulation of toxicants, helping to predict potential toxicity and elucidate mechanisms of action. Zebrafish provide an excellent model for investigating the mechanisms underlying chemical toxicity.²⁰ This approach helps unravel the complex interactions between chemicals and biological systems, shedding light on toxicological mechanisms.²¹ Zebrafish help measure environmental contaminant toxicity. Their susceptibility to heavy metals, herbicides, and endocrine disruptors makes them excellent for evaluating their effects on aquatic environments. Zebrafish can measure pollutant ecological impact and environmental remediation effectiveness.

Understanding drug metabolism and pharmacokinetics using zebrafish

Zebrafish (D. rerio) has emerged as a valuable model organism for studying drug metabolism and pharmacokinetics (DMPK). DMPK is a crucial aspect of drug development and involves the investigation of how drugs are metabolized, distributed, and eliminated within the body.²² Zebrafish embryos are permeable to small molecules, allowing for the assessment of drug absorption and distribution. Researchers can expose zebrafish embryos to drugs via various routes (e.g. immersion, injection) and evaluate their distribution within the organism. This information helps understand the bioavailability of drugs and their ability to reach target tissues.²³ Zebrafish have many metabolic enzymes resembling humans. These enzymes are essential for drug metabolism. Phase I and Phase II drug metabolism can be studied in zebrafish. Metabolite analysis helps researchers identify drug metabolism enzymes and assess metabolic activation or detoxification. Phase I metabolism primarily involves oxidation, reduction, and hydrolysis reactions that introduce or expose functional groups on xenobiotics. In zebrafish, aside from CYPs, several other enzymes contribute to this phase such as Flavin-containing monooxygenases (FMOs), Alcohol dehydrogenases (ADHs), Aldehyde dehydrogenases (ALDHs).¹⁹ Phase II enzymes conjugate polar groups to xenobiotics, increasing their solubility for excretion. Zebrafish exhibit early expression and activity of major Phase II enzymes such as UDP-glucuronosyltransferases (UGTs), Sulfotransferases (SULTs), Glutathione-S-transferases (GSTs).²⁴ Zebrafish have a diverse repertoire of cytochrome P450 (CYP) enzymes, which are responsible for the metabolism of many drugs in humans. Zebrafish CYP enzymes show similarities in structure and function to human CYP enzymes, making them useful for studying drug metabolism.²⁵ Pharmacokinetic models of drug absorption, distribution, metabolism, and excretion (ADME) can be created using zebrafish. In addition to CYPs, enzymes like FMOs, ADHs, UGTs, SULTs, and GSTs significantly contribute to xenobiotic biotransformation.²⁶ By measuring drug concentrations at different time points, researchers can derive important pharmacokinetic parameters, such as half-life, clearance, and volume of distribution. These models help predict drug behavior in humans and guide the optimization of drug dosing regimens.²⁷ Zebrafish models provide a platform to investigate drug–drug interactions and evaluate the possibility for such interactions in humans. Researchers can concurrently administer various medications to zebrafish and assess their impact on each other’s pharmacokinetics. This information aids in identifying potential drug–drug interactions that may influence efficacy or elevate the risk of unwanted consequences.^18,28 Zebrafish can offer information about the potential toxicity of pharmaceuticals and their metabolites. Examining the toxicokinetics of pharmaceuticals enables the elucidation of the correlation between drug exposure and adverse effects, facilitating the assessment of medication safety in individuals.²⁹ Many embryos can be exposed to different chemicals, and automated imaging devices can track drug metabolism.²² This enables quick screening of drug candidates or detection of molecules with unacceptable pharmacokinetic characteristics.³⁰ While zebrafish have proven to be valuable models for studying various aspects of toxicology, and drug discovery, there are certain limitations compared to mammalian systems, especially concerning translating pharmacokinetics data to humans.¹ For example, zebrafish absorb medications through their skin and gills, which may not match human gastrointestinal absorption. Bioavailability and systemic exposure may vary between species due to this variance.^9,31 Despite zebrafish possessing conserved metabolic pathways, gene duplications can confuse the comprehension of drug metabolism. The complicated nature of human metabolism, affected by aspects like gut microbiota and enzyme polymorphisms, may not be entirely represented in zebrafish models.³²

Zebrafish (D. rerio) models have proven to be highly useful for studying organ toxicity and developmental disorders. Zebrafish embryos provide a platform to study the toxicity of various substances on organ systems. Researchers can expose zebrafish embryos to toxicants, such as environmental pollutants or pharmaceutical compounds, and evaluate their effects on organ development and function.³³ The transparency of zebrafish embryos allows for real-time visualization of organ systems, enabling the direct observation of toxicological impacts on organs such as the heart, liver, kidney, brain, and others. These studies aid in understanding the toxic mechanisms and potential long-term consequences on organ function.^34,35 Zebrafish have a well-developed cardiovascular system, which makes them an excellent model for studying cardiotoxicity. Researchers can evaluate the effects of various drugs or chemicals on cardiac development, heart rate, cardiac morphology, and function. Zebrafish models have been instrumental in identifying potential cardiotoxic effects of drugs, elucidating underlying mechanisms, and assessing the risk of cardiac side effects in humans.³⁶ Zebrafish possess a sophisticated nervous system, making them valuable for studying neurotoxicity. Researchers can investigate the effects of neurotoxic substances on neuronal development, behavior, and neurological disorders.³⁷ Zebrafish models have been used to study the impacts of pesticides, heavy metals, and other chemicals on neurodevelopmental disorders, such as autism spectrum disorders and cognitive impairments. These studies provide insights into the neurotoxic mechanisms and potential therapeutic interventions.³⁸ Zebrafish liver shares many similarities with the human liver, making it a suitable model for studying hepatotoxicity. Researchers can assess the effects of drugs, environmental toxins, or industrial chemicals on liver development, hepatocyte function, and liver disease. Zebrafish models have been used to investigate drug-induced liver injury, hepatotoxic mechanisms, and potential therapeutic strategies for liver diseases.³⁹ These zebrafish models enable the investigation of disease mechanisms, identification of potential therapeutic targets, and testing of novel drug candidates.

Zebrafish-based assays for drug screening and efficacy testing

Zebrafish (D. rerio) have become a popular model organism for drug screening and efficacy testing. Zebrafish are well-suited for phenotypic drug screening, where compounds are tested for their ability to induce specific phenotypic changes or rescue disease-related phenotypes.⁴⁰ Researchers can expose zebrafish embryos to compounds and monitor changes in morphology, organ development, behaviour, or disease-related phenotypes. This approach helps identify potential drug candidates with desired effects.⁴¹ Zebrafish exhibit a range of complex behaviours that can be measured to assess the efficacy of drugs. Behavioural assays can include locomotor activity, feeding behaviour, social interactions, anxiety-like behaviour, or learning and memory. Researchers can expose zebrafish to drugs and quantify changes in behaviour using automated tracking systems. This approach provides insights into the effects of drugs on the nervous system and behaviour-related disorders.⁴² Zebrafish possess a well-developed cardiovascular system that shares similarities with humans. Drug screening assays targeting cardiovascular diseases can involve assessing heart rate, blood flow, cardiac morphology, or the function of specific cardiac chambers. Researchers can use zebrafish embryos or adult zebrafish to evaluate the effects of drugs on cardiovascular parameters. These assays help identify potential drug candidates for cardiovascular disorders.⁴³ Zebrafish models are also valuable for evaluating drug toxicity. Researchers can expose zebrafish embryos or adults to compounds and assess their effects on survival, development, organ function, or behavior. By monitoring toxicity in zebrafish, researchers can identify potential adverse effects or toxic mechanisms of drugs. This information aids in predicting drug safety and optimizing therapeutic regimens.⁴⁴ Zebrafish can be genetically manipulated or exposed to specific chemicals to develop disease models. Zebrafish disease models have been developed for a wide range of conditions, including cancer, cardiovascular diseases, neurological disorders, and genetic diseases. These models allow for the screening of potential drug candidates and investigation of their mechanisms of action.^45,46 Zebrafish can be used to study drug delivery strategies and pharmacokinetics. Researchers can incorporate fluorescently labelled drugs into zebrafish embryos or administer drugs directly to adult zebrafish. This allows for visualization of drug distribution, absorption, metabolism, and excretion. Zebrafish-based assays can be adapted to high-throughput screening platforms, allowing for the rapid screening of large compound libraries. Automated imaging and analysis systems can be used to assess multiple parameters simultaneously, enabling the screening of thousands of compounds for desired effects or therapeutic potential.⁴⁷ Zebrafish assays provide insights into drug effects on various systems and behaviours, toxicity assessment, disease modeling, and drug delivery optimization. These assays contribute to the identification of potential drug candidates and the development of effective therapeutic interventions.⁴⁸

Bioactive peptides have gained significant attention due to their potential therapeutic applications across various medical conditions, including diabetes, oncology, and rare diseases.^49,50 Their ability to target specific biological pathways makes them promising candidates for drug development. Zebrafish models are increasingly used in drug discovery for their ability to screen drug candidates in vivo.⁵¹ Incorporating bioactive peptides into these models could provide insights into their safety and efficacy profiles. Zebrafish can help assess the toxicological effects of bioactive peptides, which is crucial for advancing them as drug candidates. A recent study explores the therapeutic potential of a bioactive peptide named HPAEDR using hyperlipidemic zebrafish larvae to assess the peptide’s effects on lipid metabolism and atherosclerosis. Zebrafish models are valuable for studying cardiovascular diseases.⁵² Another study evaluates how the supplementation of a short antimicrobial peptide affects the growth of zebrafish. Antimicrobial peptides are known to enhance growth by improving gut health and reducing pathogenic bacteria. It is suggested that short antimicrobial peptides can be valuable dietary supplements for zebrafish, potentially improving their health and resilience against pathogens.⁵³

Identification of novel therapeutic compounds through zebrafish research

Zebrafish (D. rerio) research has proven to be a fruitful avenue for the identification of novel therapeutic compounds. Leveraging the unique attributes of zebrafish, researchers have made significant discoveries that have the potential to translate into new therapeutic strategies. Zebrafish phenotypic screening has emerged as a powerful approach for identifying compounds with therapeutic potential.⁵⁴ By subjecting zebrafish embryos or larvae to chemical libraries, researchers can observe changes in phenotypes associated with specific diseases or disorders. This unbiased screening method allows for the discovery of compounds that modulate disease-related phenotypes, providing leads for further development.⁵⁵ Zebrafish can be utilized to model various human diseases, including cancer, cardiovascular diseases, neurodegenerative disorders, and genetic diseases. By introducing genetic mutations or using chemical inducers, researchers can recreate disease conditions in zebrafish. This enables the evaluation of potential therapeutic compounds in a relevant disease context, facilitating the identification of novel drug candidates or repurposing existing drugs.⁵⁶ Zebrafish research can aid in the identification of novel drug targets. By manipulating gene expression in zebrafish embryos or larvae, researchers can study the effects of specific genes on disease-related phenotypes. This approach can uncover previously unrecognized genes or pathways involved in disease progression. Target identification through zebrafish research provides opportunities for the development of targeted therapies.⁴⁶ Zebrafish models can be instrumental in drug repurposing efforts. Existing drugs that have been approved for one indication can be tested in zebrafish models of different diseases. This approach capitalizes on the ability of zebrafish to model diverse disease conditions and provides a platform for identifying alternative therapeutic uses for known compounds. Drug repurposing saves time and resources by bypassing the lengthy process of new drug development.^26,37 Zebrafish models allow for the exploration of drug combinations to enhance therapeutic efficacy. Researchers can test the effects of combining different compounds or therapies in zebrafish models of diseases. This approach can identify synergistic effects, improve treatment outcomes, and overcome drug resistance. Zebrafish-based drug combination studies provide valuable insights into the optimal use of therapeutic compounds⁵⁷  Fig. 1.

Fig. 1.

Overall mechanism and role of zebrafish in the research field regarding various aspects of biology, drug discovery and toxicity.

Zebrafish research can help identify compounds with low toxicity and high safety profiles. By assessing the effects of compounds on zebrafish development, organ function, or behavior, researchers can identify potential toxicities at an early stage of drug discovery. This enables the prioritization of compounds with better safety profiles, reducing the risk of adverse effects during subsequent development stages.^29,58 Zebrafish research holds potential for personalized medicine approaches. Zebrafish embryos or larvae can be derived from patient samples, allowing for the investigation of individualized drug responses. This approach offers the opportunity to identify compounds that are effective for specific patient populations or tailored to particular genetic backgrounds.⁵⁹ Through phenotypic screening, disease modeling, target identification, drug repurposing, drug combination studies, toxicity prediction, and personalized medicine approaches, zebrafish research contributes to the discovery and development of innovative therapeutic strategies.

Zebrafish as a tool for investigating neurotoxicity and behavioural effects

Zebrafish (D. rerio) have emerged as a valuable model organism for investigating neurotoxicity and studying the behavioural effects of chemicals. Their genetic similarity to humans, well-characterized nervous system, and behavioural repertoire makes them an excellent tool for understanding the impact of toxic substances on the nervous system and behaviour.⁶⁰ Zebrafish embryos and larvae undergo rapid and well-defined neurodevelopment, allowing researchers to study the effects of toxic substances on early brain development. By exposing zebrafish embryos to neurotoxic chemicals, researchers can assess their impact on neuronal proliferation, migration, differentiation, and circuit formation.⁶¹ This approach helps identify critical windows of vulnerability and elucidate the underlying mechanisms of neurotoxicity. Zebrafish exhibit a wide range of complex behaviors, providing opportunities to study the effects of neurotoxicants on behavior. Researchers can assess behavioural endpoints such as locomotor activity, anxiety-like behavior, learning and memory, social interactions, aggression, and feeding behavior.⁶² By exposing zebrafish to neurotoxic substances and monitoring changes in behaviour, researchers can identify alterations in neurological function and evaluate the potential behavioural effects of toxicants.⁶³ The dopaminergic system plays a crucial role in regulating behaviour, reward, and movement. Zebrafish possess a highly conserved dopaminergic system, making them a suitable model for studying dopaminergic function and its modulation by neurotoxicants.⁶⁴ Researchers can investigate the effects of chemicals on dopamine synthesis, release, reuptake, and receptor signalling using zebrafish models. This approach provides insights into the neurotoxic mechanisms underlying dopamine-related disorders.⁶⁵ Zebrafish offer the advantage of studying the effects of neurotoxicants on other neurotransmitter systems, such as glutamate, serotonin, GABA, and acetylcholine.⁶⁶ Researchers can assess changes in neurotransmitter levels, receptor expression, or synaptic function using zebrafish models.⁶⁷ This helps understand the impacts of neurotoxicants on neurotransmission and their potential contribution to behavioural alterations. Zebrafish models allow for high-throughput behavioural screening of chemicals or drug libraries.⁶⁸ Automated tracking systems can monitor zebrafish behaviour, enabling the assessment of large numbers of individuals simultaneously. This approach facilitates the screening of compounds for potential neurotoxic effects or behavioural alterations, saving time and resources in the early stages of toxicological studies or drug discovery.⁶⁹ Zebrafish are amenable to genetic and pharmacological manipulations, allowing researchers to investigate the specific mechanisms underlying neurotoxicity and behavioural effects. Researchers can selectively manipulate genes or administer drugs to target specific neurochemical pathways or molecular targets.⁷⁰ This approach helps identify key factors involved in neurotoxicity and behavior and offers opportunities for the development of targeted therapeutic interventions. Plants provide a vast reservoir of natural compounds with possessing a wide range of pharmacological properties.^71–77 Recent studies on natural products utilized the Zebrafish model to investigate their biological effects are illustrated in Table 1, Fig. 2.

Table 1.

Recent research utilized the zebrafish model to investigate the anxiolytic-like effects of plant extracts of natural plant extracts, isolated compounds, and essential oils.

Natural compound/Extarct	Dose	Model	Main Pharmacological Activity Effect on Zebrafish	References
Sweroside from Schenkia spicata	12.79, 8.35, and 13.95 nM	Novel tank-diving test (NTT), Y-maze, and novel object recognition (NOR) tests.	Increased time spent at the surface Increased locomotion in the Y-maze by increasing the total distance traveled. Reduced amnesic effects in NOR. Significant effect on anxiety and cognitive impairment in the zebrafish model.	1
Baicalein 5,6-dimethyl from Alnus rugosa	1, 3, 5 μg/mL	Novel tank-diving test (NTT), Y-maze, and novel object recognition (NOR) tests.	Increased time spent at the surface Increased locomotion in the Y-maze by increasing the total distance traveled. Reduced amnesic effects in NOR. It provided substantial defense against neuronal oxidative stress in the brain tissue of zebrafish treated with Scopolamine.	2
Rhoifolin from Chorisia crispiflora	1, 3, 5 μg/mL	Novel tank-diving test (NTT), Y-maze, and novel object recognition (NOR) tests.	Increased time spent at the surface Increased locomotion in the Y-maze by increasing the total distance travelled. Reduced amnesic effects in NOR. Reduce anxiety, memory problems, and oxidative stress in the brain and control cholinergic function by reducing AChE activity.	3
Citrus reticulata Leaves essential oil	25, 150, 300 μL/L	Novel tank-diving test (NTT), Y-maze, and novel object recognition (NOR) tests.	Increased time spent at the surface Increased locomotion in the Y-maze by increasing the total distance travelled. Reduced amnesic effects in NOR.	4
Coriandrum sativum var. microcarpum essential oil	25, 150, 300 μL/L	Novel tank-diving test (NTT), Y-maze, and novel object recognition (NOR) tests.	Increased time spent at the surface Increased locomotion in the Y-maze by increasing the total distance travelled. Reduced amnesic effects in NOR.	5
Glaucosciadium cordifolium essential oil	25, 150, μL/L	Novel tank-diving test (NTT), Y-maze, and novel object recognition (NOR) tests.	Increased time spent at the surface Increased locomotion in the Y-maze by increasing the total distance travelled. Reduced amnesic effects in NOR.	6
Lantana camara leaves extract	10, 30, 100 mg/kg	Y-maze and T-maze	Increased time spent in the novel arm. Also there was a rise in the amount of time spent in the food-containing arm in the T-maze.	7
Momordica cochinchinensis (Gac fruit) extract	200 mg/kg	T-maze test and an inhibitory avoidance test	increased time spent in the green arm and latency time lower AChE activity	8

Fig. 2.

Schematic presentation of the anxiolytic-like effects of natural plant products that can be evaluated in the zebrafish model induced with scopolamine.

Advances in zebrafish genetic engineering for targeted drug discovery

Zebrafish (D. rerio) genetic engineering has witnessed significant advances, enabling targeted drug discovery efforts. These advancements have expanded the scope and capabilities of zebrafish models, facilitating the identification and validation of drug targets, elucidation of drug mechanisms, and screening of potential therapeutics.⁷⁸ The advent of CRISPR/Cas9 technology has revolutionized zebrafish genetic engineering. CRISPR/Cas9 allows for precise and efficient modification of specific genes in zebrafish, including knockout, knock-in, and point mutation generation. This technique enables the creation of zebrafish models with specific genetic alterations associated with human diseases or drug targets. In zebrafish-based research. Technologies such as 3D imaging, real-time biosensors, and AI-driven behavioral analytics are increasingly influencing how zebrafish are used in high-throughput drug screening and mechanistic toxicology.⁷⁹ By introducing disease-related mutations or modifying drug target genes, researchers can study the effects of genetic changes on disease mechanisms and drug responses. Zebrafish models can be engineered to express transgenes or knockdown specific genes in a tissue-specific manner.⁸⁰ Promoters with specific tissue expression patterns can be utilized to drive the expression of transgenes or RNA interference (RNAi) constructs selectively. This allows researchers to investigate the role of specific genes in particular tissues or cell types relevant to drug discovery, such as the liver, heart, or central nervous system.⁸¹ Optogenetics and chemogenetics techniques have been successfully applied in zebrafish to manipulate neuronal activity with precise spatial and temporal control. Light or small molecules can be used to activate or inhibit specific neurons or neuronal circuits in zebrafish models. These techniques enable the study of neuronal function, neural circuits involved in disease pathology, and the evaluation of potential drug targets for neurological disorders.^82,83 Zebrafish transgenic reporter lines allow for the visualization of specific cellular or molecular events. Fluorescent protein markers can be linked to specific genes or promoters, enabling the visualization of gene expression patterns, cellular processes, or specific cell types. These reporter lines facilitate the identification of drug targets, assessment of drug responses, and real-time monitoring of cellular and molecular events during drug discovery studies.⁸⁴ Zebrafish models are valuable for validating drug targets and assessing their therapeutic potential. This approach helps confirm the relevance of drug targets and provides insights into potential drug mechanisms and side effects. Zebrafish models also allow for in vivo testing of potential therapeutics against specific targets, aiding in the validation and optimization of drug candidates.⁸⁵

Omics technologies (genomics, proteomics, and metabolomics) have revolutionized the study of treatment effects in zebrafish, enabling systematic analyses of molecular responses and drug mechanisms. Plasma proteomics using LC–MS/MS and SWATH identified 959 proteins in zebrafish plasma, enabling biomarker discovery for disease and toxicity.⁸⁶ Thermal Proteome Profiling (TPP) in zebrafish embryos revealed napabucasin’s effects on aldehyde dehydrogenases (ALDHs), linking protein stability changes to retinoic acid pathway activation.⁸⁷ Organ-specific proteomic analyses (e.g. brain, gills) have mapped tissue responses to environmental toxins and therapeutics.^86,88 Regarding metabolomics in zebrafish studies a multidimensional workflow combining mass spectrometry imaging and isotope tracing mapped hydroxychloroquine (HCQ) distribution in adult zebrafish, showing organ-specific accumulation and metabolic alterations as elevated pyruvate carboxylase activity in the brain.⁸⁹ Untargeted metabolomics revealed HCQ-induced shifts in pathways like N-acetylaspartate synthesis, highlighting off-target effects. Furthermore, metabolomic profiling supports drug discovery by correlating pharmacokinetics with pharmacodynamics in whole organisms.⁸⁹ Summary of Key Gene Targets in Zebrafish Toxicology and Pharmacology Studies is illustrated in Table 2.

Table 2.

Summary of key gene targets in zebrafish toxicology and pharmacology studies.

Gene Target	Function	Relevance to Toxicology/Pharmacology Studies	Reference
aldh2.1	Acetaldehyde detoxification	Knockout causes increased acetaldehyde, impaired glucose metabolism, and microvascular retinal damage; model for studying retinopathy	9
aldh1a2	Retinoic acid synthesis	Expressed in hematopoietic sites; potential target for modulating hematopoietic development	10
aldh16a1	Aldehyde metabolism	Expressed in hematopoietic sites; potential role in blood cell development	10
aldh3a1	4-HNE detoxification	Mutations disrupt pancreas function, leading to hyperglycemia and retinal vessel alterations	9
akr1a1a	Acrolein detoxification	Mutations lead to insulin resistance and diabetic retinopathy/nephropathy	9
gck (glucokinase)	Glucose phosphorylation	Expression inhibited by acetaldehyde; links xenobiotic metabolism to glucose homeostasis	9
g6pc (glucose-6-phosphatase)	Glucose release from G6P	Expression inhibited by acetaldehyde; contributes to metabolic dysregulation	9
pcxa (pyruvate carboxylase)	Gluconeogenesis	Activity elevated by hydroxychloroquine in brain; biomarker of metabolic toxicity	11
JNK	Stress-activated kinase	Imbalance mediates acetaldehyde-induced pathological angiogenesis	9
p38 MAPK	Stress-activated kinase	Imbalance contributes to abnormal vessel formation in response to toxicants	9
STAT3	Transcription factor	Indirect target of napabucasin; involved in developmental processes and stress response	12
Pora	Oxidoreductase	Direct target of napabucasin; mediates effects on STAT3 signaling	12
scl, lmo2	HSPC transcription factors	Expression increased by ALDH inhibition; markers for hematopoietic effects	10
gata1, α/β-eHb	Erythroid development	Expression increased by ALDH inhibition; markers for erythropoietic effects	10
spi1	Myeloid development	Expression increased by ALDH inhibition; a marker for myelopoietic effects	10

Translational potential: Human toxicology and drug development

Zebrafish holds significant translational potential as a bridge between basic research in toxicology and drug development and its application in human health. The unique characteristics and genetic similarity to humans make zebrafish an invaluable tool for advancing our understanding of human toxicology, disease mechanisms, and the development of potential therapeutics.⁹⁰ Their genetic similarity allows researchers to study the effects of toxicants or drugs on zebrafish with the expectation that the results can be translated to humans.⁹¹ Zebrafish models provide insights into the biological mechanisms underlying human diseases and drug responses, aiding in the identification of potential therapeutic targets and evaluation of drug efficacy.^92,93 Zebrafish possess conserved organ systems and molecular pathways relevant to human physiology and disease. Their cardiovascular, nervous, immune, and metabolic systems exhibit similarities to humans, allowing for the study of disease processes and toxicological effects in a biologically relevant context.⁹⁴ The conservation of these key organ systems and pathways enables the translation of findings from zebrafish research to human health. Zebrafish models have proven valuable in drug discovery efforts.⁹⁵ Their high-throughput capabilities, rapid development, and transparent embryos facilitate the screening of compound libraries, identification of potential drug candidates, and evaluation of compound efficacy and toxicity.⁹⁶ The results obtained from zebrafish studies can guide the selection and optimization of drug candidates for further development in human trials.⁹⁷ Zebrafish models provide a platform for assessing the toxicity of chemicals and pharmaceutical compounds. By exposing zebrafish to toxicants, researchers can evaluate the effects on organ systems, developmental processes, and behavior.⁹⁸ Zebrafish-based toxicity studies aid in predicting potential adverse effects in humans, providing valuable information for safety evaluation during drug development. Zebrafish models also allow for the screening of compound libraries to identify potential toxicants, reducing the likelihood of human exposure to harmful substances.⁹⁹ Zebrafish models play a crucial role in validating therapeutic targets.¹⁰⁰ This information helps confirm the relevance of drug targets and provides insights into potential drug mechanisms and side effects. The validation of therapeutic targets in zebrafish models increases the likelihood of successful translation to human therapeutics.⁴² Zebrafish models offer opportunities for personalized medicine approaches. Zebrafish embryos or larvae derived from patient samples allow for the investigation of individualized drug responses based on specific genetic backgrounds. This approach aids in predicting drug efficacy and optimizing therapeutic regimens for specific patient populations.¹⁰¹ Zebrafish models also contribute to the development of precision medicine by enabling the testing of potential drug combinations, optimizing dosing regimens, and evaluating drug responses in a patient-specific context.¹⁰² Zebrafish models facilitate the identification of potential therapeutic targets, drug discovery, safety evaluation, and personalized medicine approaches. The translational potential of zebrafish research contributes to the advancement of human toxicology, disease understanding, and the development of effective therapeutics.¹⁸

Potential drawbacks of using zebrafish in drug discovery and toxicology studies.

The use of zebrafish as a model organism in biomedical research, particularly for drug discovery and toxicology, comes with several limitations and drawbacks relative to mammalian systems.¹⁰³ While zebrafish have all major organ systems, their complexity is reduced compared to mammals. This simplification can limit the model’s ability to replicate human disease states and responses to drugs accurately.^31,104 Moreover, zebrafish have different anatomical structures than mammals. Their aquatic breathing mechanism makes studying human respiration medicines difficult. Zebrafish have distinct heart and liver functions than mammals, which may affect drug metabolism and toxicity.^9,31Standardized guidelines for zebrafish maintenance and experimental settings are notably absent. This variability may result in uneven outcomes among investigations, complicating the reproduction of findings or the comparison of data from other laboratories.¹⁰⁵ Zebrafish exhibit sensitivity to environmental variables (e.g. temperature, pH), potentially influencing experimental results. In the absence of tight control over these variables, outcomes may fluctuate considerably, complicating data analysis.¹⁰⁶ Zebrafish have distinct benefits for high-throughput screening and preliminary drug development; yet, their limitations compared to mammalian systems require critical evaluation when interpreting results and applying discoveries to human contexts. Ongoing efforts to standardize methods and integrate zebrafish research with mammalian models may augment their applicability in biological research.

Conclusion

In summary, zebrafish function as a significant intermediary between fundamental toxicology research and drug development, owing to their genetic resemblance to humans, transparent embryos facilitating visualization, rapid reproductive cycle, cost-efficiency, regenerative abilities, and relevance to human health. Zebrafish models reveal human disease mechanisms, therapeutic targets, and toxicological consequences due to their genetic similarities, conserved organ systems, and high throughput. They aid in drug discovery and safety assessment. The future perspectives are related to the combination of zebrafish with gene editing technologies like CRISPR/Cas9 will enhance the utility of zebrafish models by enabling precise genetic modifications, leading to more sophisticated disease models and drug screening assays. Also, advanced imaging techniques will likely enhance their utility in drug discovery. This integration can facilitate the study of specific disease mechanisms and the effects of drugs at single-cell resolution within a living organism.Future advancements in technology and methodology will likely expand their application, making them an integral part of modern biomedical research.

Author contribution

S.P. and S.V.T.: conceptualization, data curation, investigation, resources, writing—original draft preparation, S.D., G.K. AND A.C. resources, investigation and data curation, writing—review and editing; S.P. and S.H.A.: writing—original draft preparation, visualization; M.E.S: visualization, validation, supervision. The authors confirm that no paper mill and artificial intelligence was used.

Funding

Not Applicable.

Data availability

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Ethical approval

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Consent to participate

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Consent for publication

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Competing interests

The authors declare no competing interests.