Caracterização da secreção cutânea do anfíbio Trachycephalus nigromaculatus e investigação da atividade antimicrobiana

Trachycephalus nigromaculatus is a helmeted treefrog belonging to the Hylidae family, distinguished by its cranial apparatus adapted for phragmosis, a unique defense strategy. Its skin is notable for the abundance of poison glands, playing a crucial role in resistance against predators and pathogens in its natural habitat. However, the chemical composition of the venom and the effects of exposure to cutaneous secretion in humans and animals remain poorly understood. This study aimed to characterize the cutaneous secretion, assess its toxic effects, and explore the antimicrobial activity of Trachycephalus nigromaculatus secretion. We employed chromatographic analyses, SDS-PAGE gel electrophoresis, and mass spectrometry to identify the fractions of cutaneous secretion. The cell-inflammatory-epithelial interaction was examined through intravital microscopy, while the impact on cell viability was evaluated using the MTT assay. Antimicrobial activity was tested in vitro against S. aureus, S. coli, and C. albicans. Results revealed a complex mixture in T. nigromaculatus cutaneous secretion, with proteins ranging from 8.5 to 77 kDa. Noteworthy components include hyaluronidase, peroxiredoxin-6, and the toxin anntoxin. MALDI- TOF analysis indicated an abundant presence of peptides. Low concentrations (1μg/mL) of the secretion exhibited significant cytotoxic activity (76%) against VERO cells. In intravital microscopy, the secretion (3 μg and 30 μg) displayed relevant toxic activity, causing loss of elasticity, edema, infiltration, and hemorrhagic points, preventing the experiment's continuation after 2 hours of injection. Within 24 hours, signs of necrosis and infiltration were evident, accompanied by an increase in adhered and migrated cells. These effects were observed within just 5 minutes of topical application. Inhibition of microbial growth was observed in crude secretion against S. coli, S. aureus, and C. albicans. At least half of the fractions tested by HPLC exhibited relevant activity. Antimicrobial activity was particularly effective against gram-positive bacteria (S. aureus) and gram-negative bacteria (S. coli), demonstrating notable antifungal action against C. albicans. These results are significant as they represent the first description of the antimicrobial activity of T. nigromaculatus cutaneous secretion, highlighting substantial inhibition against fungi. Furthermore, this study provides the first detailed description of the biochemical content of cutaneous secretion, revealing a unique composition of compounds inducing notable toxic effects both in vivo and in vitro. These findings, coupled with the distinctive cranial apparatus of this species, reinforce its efficacy as an efficient defense mechanism.

Trachycephalus nigromaculatus é uma perereca-de-capacete da família Hylidae, destacada pelo seu aparelho craniano adaptado para a fragmose, uma estratégia de defesa peculiar. Sua pele é notória pela abundância de glândulas de veneno, desempenhando um papel crucial na resistência contra predadores e patógenos em seu habitat natural. No entanto, a composição química do veneno e os efeitos da exposição à secreção cutânea em humanos e animais permanecem pouco compreendidos. Este estudo objetivou caracterizar a secreção cutânea, avaliar seus efeitos tóxicos e explorar a atividade antimicrobiana da secreção de Trachycephalus nigromaculatus. Empregamos análises cromatográficas, eletroforese em gel SDS- PAGE e espectrometria de massa para identificar as frações da secreção cutânea. A interação célula-inflamatória-epitelial foi examinada por microscopia intravital, enquanto o impacto na viabilidade celular foi avaliado pelo ensaio MTT. A atividade antimicrobiana foi testada in vitro contra S. aureus, S. coli e C. albicans. Os resultados revelaram uma mistura complexa na secreção cutânea de T. nigromaculatus, com proteínas variando de 8,5 a 77 kDa. Destacam-se hialuronidase, peroxirredoxina-6 e a toxina anntoxina. A análise MALDI-TOF indicou uma presença abundante de peptídeos. Concentrações baixas (1μg/mL) da secreção apresentaram notável atividade citotóxica (76%) contra células VERO. Na microscopia intravital, a secreção (3 μg e 30 μg) exibiu atividade tóxica relevante, causando perda de elasticidade, edema, infiltração e pontos hemorrágicos, impedindo a continuidade após 2h de injeção. Em 24 horas, sinais de necrose e infiltração foram evidentes, juntamente com um aumento na contagem de células aderidas e migradas. Esses efeitos foram observados após apenas 5 minutos de aplicação tópica. Observou-se inibição do crescimento microbiano na secreção bruta para S. coli, S. aureus e C. albicans. Pelo menos metade das frações testadas por HPLC exibiram atividade relevante. A atividade antimicrobiana foi especialmente eficaz contra bactérias gram-positivas (S. aureus) e gram-negativas (S. coli), além de apresentar notável ação antifúngica contra C. albicans. Esses resultados são significativos, pois representam a primeira descrição da atividade antimicrobiana da secreção cutânea de T. nigromaculatus, destacando uma inibição importante contra fungos. Além disso, este estudo oferece a primeira descrição detalhada do conteúdo bioquímico da secreção cutânea, revelando uma composição singular de compostos que induzem efeitos tóxicos notáveis tanto in vivo quanto in vitro. Essas descobertas, aliadas ao distintivo aparelho ósseo na cabeça desta espécie, reforçam sua eficácia como uma eficiente arma de defesa.

Recapitulation of Retinal Damage in Zebrafish Larvae Infected with Zika Virus.

Zebrafish are increasingly being utilized as a model to investigate infectious diseases and to advance the understanding of pathogen-host interactions. Here, we take advantage of the zebrafish to recapitulate congenital ZIKV infection and, for the first time, demonstrate that it can be used to model infection and reinfection and monitor anti-viral and inflammatory immune responses, as well as brain growth and eye abnormalities during embryonic development. By injecting a Brazilian strain of ZIKV into the yolk sac of one-cell stage embryos, we confirmed that, after 72 h, ZIKV successfully infected larvae, and the physical condition of the virus-infected hosts included gross morphological changes in surviving embryos (84%), with a reduction in larval head size and retinal damage characterized by increased thickness of the lens and inner nuclear layer. Changes in locomotor activity and the inability to perceive visual stimuli are a result of changes in retinal morphology caused by ZIKV. Furthermore, we demonstrated the ability of ZIKV to replicate in zebrafish larvae and infect new healthy larvae, impairing their visual and neurological functions. These data reinforce the deleterious activity of ZIKV in the brain and visual structures and establish the zebrafish as a model to study the molecular mechanisms involved in the pathology of the virus.

TnP Peptide Suppresses Experimental Autoimmune Encephalomyelitis (EAE) in a Preclinical Mouse Model.

TnP is a family of patented synthetic peptides which is in a preclinical development stage with valuable potential therapeutic indication for multiple sclerosis (MS), an autoimmune demyelinating disease of the central nervous system (CNS). The use of a preclinical animal model, such as experimental autoimmune encephalomyelitis (EAE) has deepened our knowledge of the immunomodulatory functions of TnP as a drug. We have shown that TnP possesses a disease suppressive function in EAE, ameliorating disease severity by 40% and suppressing the accumulation of T helper (Th)1- and Th17-producing lymphocytes (by 55% and 60%, respectively) in CNS along with activated microglia/macrophages populations (by 33% and 50%, respectively), and also conferred a protective effect anticipating the remyelination process to day 66 compared to day 83 of untreated cuprizone-mice. Here we expanded our knowledge about its effects compared with current first-line disease-modifying therapies (DMT). We demonstrated that prophylactic treatment with TnP generated similar protection to betaseron (30%) or was more effective than glatiramer (44% versus 6%) or fingolimod (50% versus 19%) against the development of clinical symptoms. Although TnP controlled the leukocyte infiltration (87% versus 82%) into demyelinated areas of the spinal cord in the same way as betaseron and fingolimod, it was more effective (72% to 78% decrease) in the long-term control of neuronal degeneration compared to them. Also, when compared to glatiramer, TnP was more efficient in reversing leukocytes infiltration into the spinal cord (55% versus 24%), as well as induced a higher percentage of regulatory cells in spleen (2.9-fold versus 2.3-fold increase over vehicle-treated EAE mice) an in the spinal cord (8-fold versus 6-fold increase over vehicle-treated EAE mice). This specialized TnP profile for inducing immune tolerance and neuronal regeneration has significant therapeutic potential for the treatment of MS and other autoimmune diseases.

Impact of Pesticides on Human Health in the Last Six Years in Brazil.

Every year, Brazil intensifies its activity in agriculture and, as a result, it has become one of the biggest consumers of pesticides in the world. The high rate of these substances raises environmental and human health concerns. Therefore, we collected papers from PubMed, Scopus, Scielo, and Web of Science databases, from 2015 to 2021. After a blind selection using the software Rayyan QCRI by two authors, 51 studies were included. Researchers from the South and the Southeast Brazilian regions contributed to most publications, from areas that concentrate agricultural commodity complexes. Among the pesticides described in the studies, insecticides, herbicides, and fungicides were the most frequent. The articles reported multiple toxic effects, particularly in rural workers. The results obtained can be used to direct policies to reduce the use of pesticides, and to protect the health of the population.

Natterin-like depletion by CRISPR/Cas9 impairs zebrafish (Danio rerio) embryonic development.

BACKGROUND: The Natterin protein family was first discovered in the venom of the medically significant fish Thalassophryne nattereri, and over the last decade natterin-like genes have been identified in various organisms, notably performing immune-related functions. Previous findings support natterin-like genes as effector defense molecules able to activate multiprotein complexes driving the host innate immune response, notably due to the pore-forming function of the aerolysin superfamily members. Herein, employing a combination of the CRISPR/Cas9 depletion system, phenotype-based screening, and morphometric methods, we evaluated the role of one family member, LOC795232, in the embryonic development of zebrafish since it might be implicated in multiple roles and characterization of the null mutant is central for analysis of gene activity. RESULTS: Multiple sequence alignment revealed that the candidate natterin-like has the highest similarity to zebrafish aep1, a putative and better characterized fish-specific defense molecule from the same family. Compared to other species, zebrafish have many natterin-like copies. Whole-mount in situ hybridization confirmed the knockout and mutant embryos exhibited epiboly delay, growth retardation, yolk sac and heart edema, absent or diminished swim bladder, spinal defects, small eyes and head, heart dysfunction, and behavioral impairment. As previously demonstrated, ribonucleoproteins composed of Cas9 and duplex guide RNAs are effective at inducing mutations in the F0 zebrafish. CONCLUSIONS: The considerably high natterin-like copies in zebrafish compared to other species might be due to the teleost-specific whole genome duplication and followed by subfunctionalization or neofunctionalization. In the present work, we described some of the natterin-like features in the zebrafish development and infer that natterin-like proteins potentially contribute to the embryonary development and immune response.

Recapitulation of retinal damage in zebrafish larvae infected with Zika virus

Zebrafish are increasingly being utilized as a model to investigate infectious diseases and to advance the understanding of pathogen–host interactions. Here, we take advantage of the zebrafish to recapitulate congenital ZIKV infection and, for the first time, demonstrate that it can be used to model infection and reinfection and monitor anti-viral and inflammatory immune responses, as well as brain growth and eye abnormalities during embryonic development. By injecting a Brazilian strain of ZIKV into the yolk sac of one-cell stage embryos, we confirmed that, after 72 h, ZIKV successfully infected larvae, and the physical condition of the virus-infected hosts included gross morphological changes in surviving embryos (84%), with a reduction in larval head size and retinal damage characterized by increased thickness of the lens and inner nuclear layer. Changes in locomotor activity and the inability to perceive visual stimuli are a result of changes in retinal morphology caused by ZIKV. Furthermore, we demonstrated the ability of ZIKV to replicate in zebrafish larvae and infect new healthy larvae, impairing their visual and neurological functions. These data reinforce the deleterious activity of ZIKV in the brain and visual structures and establish the zebrafish as a model to study the molecular mechanisms involved in the pathology of the virus.

TnP peptide suppresses experimental autoimmune Encephalomyelitis (EAE) in a preclinical mouse model

TnP is a family of patented synthetic peptides which is in a preclinical development stage with valuable potential therapeutic indication for multiple sclerosis (MS), an autoimmune demyelinating disease of the central nervous system (CNS). The use of a preclinical animal model, such as experimental autoimmune encephalomyelitis (EAE) has deepened our knowledge of the immunomodulatory functions of TnP as a drug. We have shown that TnP possesses a disease suppressive function in EAE, ameliorating disease severity by 40% and suppressing the accumulation of T helper (Th)1- and Th17-producing lymphocytes (by 55% and 60%, respectively) in CNS along with activated microglia/macrophages populations (by 33% and 50%, respectively), and also conferred a protective effect anticipating the remyelination process to day 66 compared to day 83 of untreated cuprizone-mice. Here we expanded our knowledge about its effects compared with current first-line disease-modifying therapies (DMT). We demonstrated that prophylactic treatment with TnP generated similar protection to betaseron (30%) or was more effective than glatiramer (44% versus 6%) or fingolimod (50% versus 19%) against the development of clinical symptoms. Although TnP controlled the leukocyte infiltration (87% versus 82%) into demyelinated areas of the spinal cord in the same way as betaseron and fingolimod, it was more effective (72% to 78% decrease) in the long-term control of neuronal degeneration compared to them. Also, when compared to glatiramer, TnP was more efficient in reversing leukocytes infiltration into the spinal cord (55% versus 24%), as well as induced a higher percentage of regulatory cells in spleen (2.9-fold versus 2.3-fold increase over vehicle-treated EAE mice) an in the spinal cord (8-fold versus 6-fold increase over vehicle-treated EAE mice). This specialized TnP profile for inducing immune tolerance and neuronal regeneration has significant therapeutic potential for the treatment of MS and other autoimmune diseases.

Impact of pesticides on human health in the last six years in Brazil

Every year, Brazil intensifies its activity in agriculture and, as a result, it has become one of the biggest consumers of pesticides in the world. The high rate of these substances raises environmental and human health concerns. Therefore, we collected papers from PubMed, Scopus, Scielo, and Web of Science databases, from 2015 to 2021. After a blind selection using the software Rayyan QCRI by two authors, 51 studies were included. Researchers from the South and the Southeast Brazilian regions contributed to most publications, from areas that concentrate agricultural commodity complexes. Among the pesticides described in the studies, insecticides, herbicides, and fungicides were the most frequent. The articles reported multiple toxic effects, particularly in rural workers. The results obtained can be used to direct policies to reduce the use of pesticides, and to protect the health of the population.

Natterin-like depletion by CRISPR/Cas9 impairs zebrafish (Danio rerio) embryonic development

Background The Natterin protein family was first discovered in the venom of the medically significant fish Thalassophryne nattereri, and over the last decade natterin-like genes have been identified in various organisms, notably performing immune-related functions. Previous findings support natterin-like genes as effector defense molecules able to activate multiprotein complexes driving the host innate immune response, notably due to the pore-forming function of the aerolysin superfamily members. Herein, employing a combination of the CRISPR/Cas9 depletion system, phenotype-based screening, and morphometric methods, we evaluated the role of one family member, LOC795232, in the embryonic development of zebrafish since it might be implicated in multiple roles and characterization of the null mutant is central for analysis of gene activity. Results Multiple sequence alignment revealed that the candidate natterin-like has the highest similarity to zebrafish aep1, a putative and better characterized fish-specific defense molecule from the same family. Compared to other species, zebrafish have many natterin-like copies. Whole-mount in situ hybridization confirmed the knockout and mutant embryos exhibited epiboly delay, growth retardation, yolk sac and heart edema, absent or diminished swim bladder, spinal defects, small eyes and head, heart dysfunction, and behavioral impairment. As previously demonstrated, ribonucleoproteins composed of Cas9 and duplex guide RNAs are effective at inducing mutations in the F0 zebrafish. Conclusions The considerably high natterin-like copies in zebrafish compared to other species might be due to the teleost-specific whole genome duplication and followed by subfunctionalization or neofunctionalization. In the present work, we described some of the natterin-like features in the zebrafish development and infer that natterin-like proteins potentially contribute to the embryonary development and immune response.

The Natterin Proteins Diversity: A Review on Phylogeny, Structure, and Immune Function.

Since the first record of the five founder members of the group of Natterin proteins in the venom of the medically significant fish Thalassophryne nattereri, new sequences have been identified in other species. In this work, we performed a detailed screening using available genome databases across a wide range of species to identify sequence members of the Natterin group, sequence similarities, conserved domains, and evolutionary relationships. The high-throughput tools have enabled us to dramatically expand the number of members within this group of proteins, which has a remote origin (around 400 million years ago) and is spread across Eukarya organisms, even in plants and primitive Agnathans jawless fish. Overall, the survey resulted in 331 species presenting Natterin-like proteins, mainly fish, and 859 putative genes. Besides fish, the groups with more species included in our analysis were insects and birds. The number and variety of annotations increased the knowledge of the obtained sequences in detail, such as the conserved motif AGIP in the pore-forming loop involved in the transmembrane barrel insertion, allowing us to classify them as important constituents of the innate immune defense system as effector molecules activating immune cells by interacting with conserved intracellular signaling mechanisms in the hosts.

Heat-Labile Toxin from Enterotoxigenic Escherichia coli Causes Systemic Impairment in Zebrafish Model.

Heat-labile toxin I (LT-I), produced by strains of enterotoxigenic Escherichia coli (ETEC), causes profuse watery diarrhea in humans. Different in vitro and in vivo models have already elucidated the mechanism of action of this toxin; however, their use does not always allow for more specific studies on how the LT-I toxin acts in systemic tracts and intestinal cell lines. In the present work, zebrafish (Danio rerio) and human intestinal cells (Caco-2) were used as models to study the toxin LT-I. Caco-2 cells were used, in the 62nd passage, at different cell concentrations. LT-I was conjugated to FITC to visualize its transport in cells, as well as microinjected into the caudal vein of zebrafish larvae, in order to investigate its effects on survival, systemic traffic, and morphological formation. The internalization of LT-I was visualized in 3 × 104 Caco-2 cells, being associated with the cell membrane and nucleus. The systemic traffic of LT-I in zebrafish larvae showed its presence in the cardiac cavity, yolk, and regions of the intestine, as demonstrated by cardiac edema (100%), the absence of a swimming bladder (100%), and yolk edema (80%), in addition to growth limitation in the larvae, compared to the control group. There was a reduction in heart rate during the assessment of larval survival kinetics, demonstrating the cardiotoxic effect of LT-I. Thus, in this study, we provide essential new depictions of the features of LT-I.

Molecular Characterization and Functional Analysis of the Nattectin-like Toxin from the Venomous Fish Thalassophryne maculosa.

TmC4-47.2 is a toxin with myotoxic activity found in the venom of Thalassophryne maculosa, a venomous fish commonly found in Latin America whose envenomation produces an injury characterized by delayed neutrophil migration, production of major pro-inflammatory cytokines, and necrosis at the wound site, as well as a specific systemic immune response. However, there are few studies on the protein structure and functions associated with it. Here, the toxin was identified from the crude venom by chromatography and protein purification systems. TmC4-47.2 shows high homology with the Nattectin from Thalassophryne nattereri venom, with 6 cysteines and QPD domain for binding to galactose. We confirm its hemagglutinating and microbicide abilities independent of carbohydrate binding, supporting its classification as a nattectin-like lectin. After performing the characterization of TmC4-47.2, we verified its ability to induce an increase in the rolling and adherence of leukocytes in cremaster post-capillary venules dependent on the α5ß1 integrin. Finally, we could observe the inflammatory activity of TmC4-47.2 through the production of IL-6 and eotaxin in the peritoneal cavity with sustained recruitment of eosinophils and neutrophils up to 24 h. Together, our study characterized a nattectin-like protein from T. maculosa, pointing to its role as a molecule involved in the carbohydrate-independent agglutination response and modulation of eosinophilic and neutrophilic inflammation.

Early preclinical screening using zebrafish (Danio rerio) reveals the safety of the candidate anti-inflammatory therapeutic agent TnP.

The patented anti-inflammatory peptide TnP had its effectiveness recently confirmed in vivo in a murine model of multiple sclerosis and asthma. In this work, the safety of the TnP was evaluated in investigative toxicology tests using zebrafish (Danio rerio) as a model. We conducted the OECD #236 test to investigate effects of the TnP on the survival, hatching performance, and morphological formation of zebrafish embryos. After determining these endpoints, morphometric analysis termination of locomotion eartbeat rate in zebrafish larvae were evaluated to identify adverse effects such as neurotoxicity and cardiotoxicity. The results highlight a wide therapeutic index for TnP with non-lethal and safe doses rom 1 nM to 10 µM, without causing neurotoxicity or cardiotoxic effect. The low frequencyf abnormalities by TnP was associated with high safety of the molecule and the developing embryo's ability to process and eliminate it. TnP crossed the blood-brain barrier without disturbing the normal architecture of forebrain, midbrain and hindbrain. Our data reinforce the importance of zebrafish as an accurate investigative toxicology model to assess acute toxicity as well as cardiotoxicity and neurotoxicity of molecules in the preclinical phase of development.

Molecular characterization and functional analysis of the nattectin-like toxin from the venomous fish thalassophryne maculosa

TmC4-47.2 is a toxin with myotoxic activity found in the venom of Thalassophryne maculosa, a venomous fish commonly found in Latin America whose envenomation produces an injury characterized by delayed neutrophil migration, production of major pro-inflammatory cytokines, and necrosis at the wound site, as well as a specific systemic immune response. However, there are few studies on the protein structure and functions associated with it. Here, the toxin was identified from the crude venom by chromatography and protein purification systems. TmC4-47.2 shows high homology with the Nattectin from Thalassophryne nattereri venom, with 6 cysteines and QPD domain for binding to galactose. We confirm its hemagglutinating and microbicide abilities independent of carbohydrate binding, supporting its classification as a nattectin-like lectin. After performing the characterization of TmC4-47.2, we verified its ability to induce an increase in the rolling and adherence of leukocytes in cremaster post-capillary venules dependent on the α5β1 integrin. Finally, we could observe the inflammatory activity of TmC4-47.2 through the production of IL-6 and eotaxin in the peritoneal cavity with sustained recruitment of eosinophils and neutrophils up to 24 h. Together, our study characterized a nattectin-like protein from T. maculosa, pointing to its role as a molecule involved in the carbohydrate-independent agglutination response and modulation of eosinophilic and neutrophilic inflammation.

The natterin proteins diversity: a review on phylogeny, structure, and immune function

Since the first record of the five founder members of the group of Natterin proteins in the venom of the medically significant fish Thalassophryne nattereri, new sequences have been identified in other species. In this work, we performed a detailed screening using available genome databases across a wide range of species to identify sequence members of the Natterin group, sequence similarities, conserved domains, and evolutionary relationships. The high-throughput tools have enabled us to dramatically expand the number of members within this group of proteins, which has a remote origin (around 400 million years ago) and is spread across Eukarya organisms, even in plants and primitive Agnathans jawless fish. Overall, the survey resulted in 331 species presenting Natterin-like proteins, mainly fish, and 859 putative genes. Besides fish, the groups with more species included in our analysis were insects and birds. The number and variety of annotations increased the knowledge of the obtained sequences in detail, such as the conserved motif AGIP in the pore-forming loop involved in the transmembrane barrel insertion, allowing us to classify them as important constituents of the innate immune defense system as effector molecules activating immune cells by interacting with conserved intracellular signaling mechanisms in the hosts.

Heat-Labile toxin from enterotoxigenic Escherichia coli causes systemic impairment in Zebrafish model

Heat-labile toxin I (LT-I), produced by strains of enterotoxigenic Escherichia coli (ETEC), causes profuse watery diarrhea in humans. Different in vitro and in vivo models have already elucidated the mechanism of action of this toxin; however, their use does not always allow for more specific studies on how the LT-I toxin acts in systemic tracts and intestinal cell lines. In the present work, zebrafish (Danio rerio) and human intestinal cells (Caco-2) were used as models to study the toxin LT-I. Caco-2 cells were used, in the 62nd passage, at different cell concentrations. LT-I was conjugated to FITC to visualize its transport in cells, as well as microinjected into the caudal vein of zebrafish larvae, in order to investigate its effects on survival, systemic traffic, and morphological formation. The internalization of LT-I was visualized in 3 × 104 Caco-2 cells, being associated with the cell membrane and nucleus. The systemic traffic of LT-I in zebrafish larvae showed its presence in the cardiac cavity, yolk, and regions of the intestine, as demonstrated by cardiac edema (100%), the absence of a swimming bladder (100%), and yolk edema (80%), in addition to growth limitation in the larvae, compared to the control group. There was a reduction in heart rate during the assessment of larval survival kinetics, demonstrating the cardiotoxic effect of LT-I. Thus, in this study, we provide essential new depictions of the features of LT-I.

Early preclinical screening using zebrafish (Danio rerio) reveals the safety of the candidate anti-inflammatory therapeutic agent TnP

The patented anti-inflammatory peptide TnP had its effectiveness recently confirmed in vivo in a murine model of multiple sclerosis and asthma. In this work, the safety of the TnP was evaluated in investigative toxicology tests using zebrafish (Danio rerio) as a model. We conducted the OECD #236 test to investigate effects of the TnP on the survival, hatching performance, and morphological formation of zebrafish embryos. After determining these endpoints, morphometric analysis termination of locomotion eartbeat rate in zebrafish larvae were evaluated to identify adverse effects such as neurotoxicity and cardiotoxicity. The results highlight a wide therapeutic index for TnP with non-lethal and safe doses rom 1 nM to 10 μM, without causing neurotoxicity or cardiotoxic effect. The low frequencyf abnormalities by TnP was associated with high safety of the molecule and the developing embryo's ability to process and eliminate it. TnP crossed the blood-brain barrier without disturbing the normal architecture of forebrain, midbrain and hindbrain. Our data reinforce the importance of zebrafish as an accurate investigative toxicology model to assess acute toxicity as well as cardiotoxicity and neurotoxicity of molecules in the preclinical phase of development.