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Dr. Luís Félix
i3S - Instituto de Inovação e Investigação em Saúde, Universidade do Porto, Portugal

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0 Oxidative Stress
0 Toxicology
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Journal article
Published: 18 July 2021 in Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology
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Previous embryonic fish data have shown caffeine to induce potential teratogenic and long-term neurodevelopmental outcomes through oxidative stress-mediated apoptosis. In this context, antioxidants may have the potential to counteract the caffeine-induced effects. Therefore, the present study aimed to investigate the potential protective role of 24-epibrassinolide (24-EPI), a natural brassinosteroid with proven antioxidant properties, against caffeine-induced teratogenic effects during early zebrafish development. Embryos (~2 h post-fertilization - hpf) were exposed to 0.5 mM caffeine, co-exposed to 24-EPI (0.01, 0.1 and 1 μM) and to 24-EPI alone (1 μM) for 96 h. During exposure, lethal and sublethal developmental parameters were evaluated. At the end of the exposure, biochemical evaluations were made, and 24 h after, different behavioural paradigms were assessed. An increased number of animals showing oedema and malformations were observed after caffeine exposure, while these were reduced after co-exposure to 24-EPI concentration, namely the tail curvature. The results showed oxidative stress and related parameters similar among treatments. Yet, caffeine exposure resulted in locomotor deficits (decreased speed and distance) and disrupted anxiety-like and avoidance responses. The co-exposure to caffeine and to the highest 24-EPI concentrations resulted in less pronounced behavioural deficits. Overall, there was an absence of effects in the embryo/larvae exposed solely to 24-EPI, while caffeine caused developmental and neurotoxic effects. Although further studies are needed, the results showed promising protective effects of the highest 24-EPI concentration tested against the toxicity induced by caffeine in zebrafish.

ACS Style

Luís Félix; Carolina Lobato-Freitas; Sandra M. Monteiro; Carlos Venâncio. 24-Epibrassinolide modulates the neurodevelopmental outcomes of high caffeine exposure in zebrafish (Danio rerio) embryos. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 2021, 249, 109143 .

AMA Style

Luís Félix, Carolina Lobato-Freitas, Sandra M. Monteiro, Carlos Venâncio. 24-Epibrassinolide modulates the neurodevelopmental outcomes of high caffeine exposure in zebrafish (Danio rerio) embryos. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology. 2021; 249 ():109143.

Chicago/Turabian Style

Luís Félix; Carolina Lobato-Freitas; Sandra M. Monteiro; Carlos Venâncio. 2021. "24-Epibrassinolide modulates the neurodevelopmental outcomes of high caffeine exposure in zebrafish (Danio rerio) embryos." Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 249, no. : 109143.

Journal article
Published: 21 May 2021 in Aquatic Toxicology
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Aluminium is a non-essential metal and potentially toxic to organisms whose environmental concentration increases due to pollution. In our previous studies, the behavioral changes induced by aluminium were already shown on zebrafish, a model organism widely used for ecotoxicology screening. To examine in depth the knowledge about the toxicity mechanism induced by this metal, zebrafish embryos, at 6 hpf, have been exposed to 50, 100 and 200 µM of AlCl3 for 72 h. Phenotypic alterations, apoptosis and oxidative stress responses have been assessed by evaluations of antioxidant defence and changes in metabolism at the end of treatment. The mRNA expression level of c-fos, appa and appb as marker genes of neural development and function were analyzed by qPCR for the highest used concentration. The data showed that aluminium significantly affected the development of zebrafish inducing morphological alterations and cell death. The oxidative state of larvae was altered, although the formation of reactive oxygen species and the levels of metallothioneins, and the activity of some antioxidant enzymes, decreased at the maximum concentration tested. In addition, at this concentration, the expression of the evaluated genes increased. The comprehensive information obtained gives a realistic snapshot of the aluminium toxicity and provides new information on the mechanism of action of this metal.

ACS Style

Teresa Capriello; Sandra M. Monteiro; Luis M. Félix; Aldo Donizetti; Vincenza Aliperti; Ida Ferrandino. Apoptosis, oxidative stress and genotoxicity in developing zebrafish after aluminium exposure. Aquatic Toxicology 2021, 236, 105872 .

AMA Style

Teresa Capriello, Sandra M. Monteiro, Luis M. Félix, Aldo Donizetti, Vincenza Aliperti, Ida Ferrandino. Apoptosis, oxidative stress and genotoxicity in developing zebrafish after aluminium exposure. Aquatic Toxicology. 2021; 236 ():105872.

Chicago/Turabian Style

Teresa Capriello; Sandra M. Monteiro; Luis M. Félix; Aldo Donizetti; Vincenza Aliperti; Ida Ferrandino. 2021. "Apoptosis, oxidative stress and genotoxicity in developing zebrafish after aluminium exposure." Aquatic Toxicology 236, no. : 105872.

Journal article
Published: 26 March 2021 in Aquatic Toxicology
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Microplastics (MPs, <5 mm) have been frequently detected in aquatic ecosystems, representing both health and ecological concerns. However data about the combined effects of MPs and other contaminants is still limited. This study aimed to evaluate the impact of MPs and the heavy metal copper (Cu) on zebrafish (Danio rerio) larvae development and behavior. Zebrafish embryos were subchronically exposed to MPs (2 mg/L), two sub-lethal concentrations of Cu (60 and 125 µg/L) and binary mixtures of MPs and Cu using the same concentrations, from 2-h post fertilization until 14 days post fertilization. Lethal and sub-lethal responses (mortality, hatching, body length) were evaluated during the embryogenesis period, and locomotor, avoidance, anxiety and shoaling behaviors, and acetylcholinesterase (AChE) activity were measured at 14 dpf. The results showed that survival of larvae was reduced in groups exposed to MPs, Cu and Cu+MPs. Regarding the behavioral patterns, the higher Cu concentration and mixtures decreased significantly the mean speed, the total distance traveled and the absolute turn angle, demonstrating an adverse effect on swimming competence of zebrafish larvae. Exposure to MPs and Cu, alone or combined, also affected avoidance behavior of zebrafish, with larvae not reacting to the aversive stimulus. There was a significant inhibition of AChE activity in larvae exposed to all experimental groups, compared to the control group. Moreover, a higher inhibition of AChE was noticed in larvae exposed to MPs and both Cu+MPs groups, comparatively to the Cu alone groups. Our findings demonstrate the adverse effects of MPs, alone or co-exposed with Cu, on fish early life stages behavior. This study highlights that MPs and heavy metals may have significant impacts on fish population fitness by disrupting locomotor and avoidance behaviors.

ACS Style

Dércia Santos; Ana Luzio; Carlos Matos; Juan Bellas; Sandra M. Monteiro; Luís Félix. Microplastics alone or co-exposed with copper induce neurotoxicity and behavioral alterations on zebrafish larvae after a subchronic exposure. Aquatic Toxicology 2021, 235, 105814 .

AMA Style

Dércia Santos, Ana Luzio, Carlos Matos, Juan Bellas, Sandra M. Monteiro, Luís Félix. Microplastics alone or co-exposed with copper induce neurotoxicity and behavioral alterations on zebrafish larvae after a subchronic exposure. Aquatic Toxicology. 2021; 235 ():105814.

Chicago/Turabian Style

Dércia Santos; Ana Luzio; Carlos Matos; Juan Bellas; Sandra M. Monteiro; Luís Félix. 2021. "Microplastics alone or co-exposed with copper induce neurotoxicity and behavioral alterations on zebrafish larvae after a subchronic exposure." Aquatic Toxicology 235, no. : 105814.

Journal article
Published: 16 March 2021 in Environmental Toxicology and Pharmacology
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Aluminium (Al) water pollution is an increasing environmental problem. Accordingly, this study aimed to find out more about its toxic effects on aquatic organisms. Adult zebrafish were exposed to 11 mg/L of Al and the behavioural responses and its correlation with brain oxidative stress, antioxidant-defences, changes in metabolism and neurotransmission were assessed at 10, 15 and 20 days of exposure. The behavioural and locomotory responses, suggest an increase in the anxiety state, especially observed in animals exposed to Al for 15 days. The reactive oxygen species increased in a time-dependent trend, while the oxidative damage varied over exposure time. The activity of antioxidant enzymes, as superoxide dismutase, glutathione peroxidase and glutathione S-transferases, and the metallothioneins levels increased after short-term exposures and tended to decrease or stabilize at longer times. The results contribute to understand the toxic mechanisms activated by Al highlighting correlations like behavioural disorders and oxidative state.

ACS Style

Teresa Capriello; Luis M. Félix; Sandra M. Monteiro; Dércia Santos; Rita Cofone; Ida Ferrandino. Exposure to aluminium causes behavioural alterations and oxidative stress in the brain of adult zebrafish. Environmental Toxicology and Pharmacology 2021, 85, 103636 .

AMA Style

Teresa Capriello, Luis M. Félix, Sandra M. Monteiro, Dércia Santos, Rita Cofone, Ida Ferrandino. Exposure to aluminium causes behavioural alterations and oxidative stress in the brain of adult zebrafish. Environmental Toxicology and Pharmacology. 2021; 85 ():103636.

Chicago/Turabian Style

Teresa Capriello; Luis M. Félix; Sandra M. Monteiro; Dércia Santos; Rita Cofone; Ida Ferrandino. 2021. "Exposure to aluminium causes behavioural alterations and oxidative stress in the brain of adult zebrafish." Environmental Toxicology and Pharmacology 85, no. : 103636.

Journal article
Published: 13 March 2021 in Chemosphere
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The evaluation of the interaction between microplastics (MPs) and heavy metals is of special importance for risk assessment. In this study, zebrafish (Danio rerio) were exposed to MPs (2 mg/L), two sub-lethal concentrations of copper (Cu, 60 and 125 μg/L) and their mixtures (Cu60 + MPs, Cu125 + MPs), from 2-h post-fertilization (hpf) until 14-days post-fertilization (dpf). Lethal and sublethal endpoints were evaluated, along with a set of biochemical and genetic biomarkers between 2 and 14 dpf. Exposure to MPs and Cu, single or combined, induced high mortality and oxidative stress in zebrafish larvae, with data showing that the antioxidant enzymes were inhibited at 6 dpf, increasing thereafter until 14 dpf, due to the accumulation of reactive oxygen species. MPs and Cu, single or combined, caused neurotoxicity in larvae by inhibiting acetylcholinesterase activity. There was an increased and significant effect of Cu + MPs groups on the evaluated biomarkers, concerning the corresponding Cu groups, suggesting that MPs may have a synergistic effect in relation to Cu. The Integrated Biomarker Response (IBR) evidenced that a higher degree of stress occurred at the larval period. Our findings highlight that MPs can act as a vector for heavy metals, therefore, influencing their bioavailability and toxicity in the organisms.

ACS Style

Dércia Santos; Luís Félix; Ana Luzio; Susana Parra; Juan Bellas; Sandra M. Monteiro. Single and combined acute and subchronic toxic effects of microplastics and copper in zebrafish (Danio rerio) early life stages. Chemosphere 2021, 277, 130262 .

AMA Style

Dércia Santos, Luís Félix, Ana Luzio, Susana Parra, Juan Bellas, Sandra M. Monteiro. Single and combined acute and subchronic toxic effects of microplastics and copper in zebrafish (Danio rerio) early life stages. Chemosphere. 2021; 277 ():130262.

Chicago/Turabian Style

Dércia Santos; Luís Félix; Ana Luzio; Susana Parra; Juan Bellas; Sandra M. Monteiro. 2021. "Single and combined acute and subchronic toxic effects of microplastics and copper in zebrafish (Danio rerio) early life stages." Chemosphere 277, no. : 130262.

Journal article
Published: 03 February 2021 in Water
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One of the most widespread aquatic organisms in the rivers and estuarine ecosystems, in the world, is Asian clam Corbicula fluminea. This clam, that can adapt to environmental changes, is an invasive species in several areas and it was adopted as a model for toxicity tests. This study evaluated the effects of the exposure to cadmium (Cd), to microplastics (MPs) and their mixtures on C. fluminea. The oxidative stress responses, lipid peroxidation (LPO), changes in the activity of energy-related enzymes and neurotoxicity were assessed on the gill, digestive gland and gonad. The results show that Cd, MPs and their mixtures cause oxidative stress, damage and neurotoxicity. The enzymes superoxide dismutase (SOD), glutathione S-transferase (GST), acetylcholinesterase (AChE) and the LPO levels could be chosen as biomarkers of Cd pollution. Exposure to MPs induced an increase in reduced/oxidized glutathione (GSH/GSSG) ratio and increased AChE activity. The combined exposure to Cd and MPs caused a synergetic effect in gill and gonad, while an antagonism response was recorded in the digestive gland. The results provide new insights for unveiling the biologic effects of heavy metal, microplastics and their mixtures on C. fluminea. Besides, we demonstrated that the Asian clam is a good bioindicator of microplastic pollution that can occur in aquatic environments.

ACS Style

Susana Parra; Simone Varandas; Dércia Santos; Luís Félix; Lisete Fernandes; Edna Cabecinha; Jesús Gago; Sandra Monteiro. Multi-Biomarker Responses of Asian Clam Corbicula fluminea (Bivalvia, Corbiculidea) to Cadmium and Microplastics Pollutants. Water 2021, 13, 394 .

AMA Style

Susana Parra, Simone Varandas, Dércia Santos, Luís Félix, Lisete Fernandes, Edna Cabecinha, Jesús Gago, Sandra Monteiro. Multi-Biomarker Responses of Asian Clam Corbicula fluminea (Bivalvia, Corbiculidea) to Cadmium and Microplastics Pollutants. Water. 2021; 13 (4):394.

Chicago/Turabian Style

Susana Parra; Simone Varandas; Dércia Santos; Luís Félix; Lisete Fernandes; Edna Cabecinha; Jesús Gago; Sandra Monteiro. 2021. "Multi-Biomarker Responses of Asian Clam Corbicula fluminea (Bivalvia, Corbiculidea) to Cadmium and Microplastics Pollutants." Water 13, no. 4: 394.

Journal article
Published: 25 January 2021 in Ecotoxicology and Environmental Safety
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Azoxystrobin is a broad-spectrum strobilurin fungicide for use on a wide range of crops available to end-users as formulated products. Due to its extensive application, it has been detected in aquatic ecosystems, raising concerns about its environmental impact, which is still poorly explored. The objective of this work was to study the effects of a commercial formulation of azoxystrobin in the zebrafish embryo model. Sublethal and lethal effects were monitored during the exposure period from 2 h post fertilisation (hpf) to 96 hpf after exposure to azoxystrobin concentrations (1, 10 and 100 μg L−1). The responses of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR)) as well as detoxifying enzymes (glutathione-s-transferase (GST) and carboxylesterase (CarE)) were evaluated at 96 hpf. Similarly, glutathione levels (reduced (GSH) and oxidised (GSSG) glutathione), neurotransmission (acetylcholinesterase (AChE)) and anaerobic respiration (lactate dehydrogenase (LDH)) -related enzymes were assayed. At 120 hpf, larvae from each group were used for behaviour analysis. Results from this study showed concentration-dependent teratogenic effects, particularly by increasing the number of malformations (yolk and eye), with a higher prevalence at the highest concentration. However, it was found that the lowest concentration induced a high generation of reactive oxygen species (ROS) and increased activity of SOD, GST, and CarE. In addition, GR and GSSG levels were decreased by the lowest concentration, suggesting an adaptive response to oxidative stress, which is also supported by the increased AChE activity and absence of behavioural changes. These findings advance the knowledge of the azoxystrobin developmental and environmental impacts, which may impose ecotoxicological risks to non-target species.

ACS Style

Raquel S.F. Vieira; Carlos A.S. Venâncio; Luís M. Félix. Embryonic zebrafish response to a commercial formulation of azoxystrobin at environmental concentrations. Ecotoxicology and Environmental Safety 2021, 211, 111920 .

AMA Style

Raquel S.F. Vieira, Carlos A.S. Venâncio, Luís M. Félix. Embryonic zebrafish response to a commercial formulation of azoxystrobin at environmental concentrations. Ecotoxicology and Environmental Safety. 2021; 211 ():111920.

Chicago/Turabian Style

Raquel S.F. Vieira; Carlos A.S. Venâncio; Luís M. Félix. 2021. "Embryonic zebrafish response to a commercial formulation of azoxystrobin at environmental concentrations." Ecotoxicology and Environmental Safety 211, no. : 111920.

Journal article
Published: 09 January 2021 in Toxics
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The improper use of synthetic fungicides has raised public concerns related to environmental pollution and animal health. Over the years, plant-derived antifungals have been investigated as safer alternatives, although little scientific evidence of its neurodevelopmental effects exist. The main objective of this study was to explore the effects of three alternative natural extracts (Equisetum arvense, Mimosa tenuiflora, Thymol) with antifungal properties during the early development of zebrafish by evaluating different teratogenic, oxidative stress and behavioural outcomes. Following the determination of the 96 h-LC50, exposure to sublethal concentrations showed the safety profile of both E. arvense and M. tenuiflora. However, following 96-h exposure to Thymol, increased lethality, pericardial oedema, yolk and eye deformations, and decreased body length were observed. The reduced and oxidized glutathione (GSH:GSSG) ratio was increased, and the glutathione-s-transferase activity in the group exposed to the highest Thymol concentration. Overall, these results support a more reducing environment associated with possible effects at the cellular proliferation level. In addition, the disruption of behavioural states (fear- and anxiety-like disorders) were noted, pointing to alterations in the c-Jun N-terminal kinase developmental signalling pathway, although further studies are required to explore this rationale. Notwithstanding, the results provide direct evidence of the teratogenic effects of Thymol, which might have consequences for non-target species.

ACS Style

Raquel Vieira; Carlos Venâncio; Luís Félix. Teratogenic, Oxidative Stress and Behavioural Outcomes of Three Fungicides of Natural Origin (Equisetum arvense, Mimosa tenuiflora, Thymol) on Zebrafish (Danio rerio). Toxics 2021, 9, 8 .

AMA Style

Raquel Vieira, Carlos Venâncio, Luís Félix. Teratogenic, Oxidative Stress and Behavioural Outcomes of Three Fungicides of Natural Origin (Equisetum arvense, Mimosa tenuiflora, Thymol) on Zebrafish (Danio rerio). Toxics. 2021; 9 (1):8.

Chicago/Turabian Style

Raquel Vieira; Carlos Venâncio; Luís Félix. 2021. "Teratogenic, Oxidative Stress and Behavioural Outcomes of Three Fungicides of Natural Origin (Equisetum arvense, Mimosa tenuiflora, Thymol) on Zebrafish (Danio rerio)." Toxics 9, no. 1: 8.

Journal article
Published: 25 December 2020 in Toxicology and Applied Pharmacology
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Tricaine methanesulfonate (MS-222) is a commonly used anaesthetic agent for immobilization of aquatic species. However, delayed development and malformations have been observed in 24 hpf (hours post-fertilization) zebrafish embryos after long-term immobilization. Still, no comprehensive study has been described regarding zebrafish exposure to MS-222 during the first hours of development, which are one of the most sensitive life stages to toxicants. Therefore, this research aimed to assess the toxicity of a 24 h exposure to MS-222 on zebrafish embryonic development. Based on the MS-222 LC50, early blastula stage embryos (~2 hpf) were exposed to 0, 12.5, 25 and 50 mg L−1 for 24 h and then allowed to develop up to 144 hpf. The chromatographic analysis showed that this anaesthetic agent bioaccumulates in 26 hpf zebrafish larvae in a concentration-dependent manner. In addition, increased mortalities and skeletal abnormalities were observed at 144 hpf, namely in the highest tested concentration. Yet, no craniofacial anomalies were observed either by alcian blue or calcein staining methods. Independently of the tested concentration, decreased speed and distance travelled were perceived in 144 hpf larvae. At the biochemical level, decreased in vivo reactive oxygen species (ROS) generation and apoptosis was observed. Additionally, catalase activity was increased at 26 hpf while results of mRNA expression showed a decreased gclc transcript content at the same time-point. Overall, data obtained highlight the toxicological risk of MS-222 and support ROS-mediated cell death signalling changes through the elevation of catalase activity as an adaptative or protective response.

ACS Style

Luís M. Félix; Ana Luzio; Luís Antunes; Ana M. Coimbra; Ana M. Valentim. Malformations and mortality in zebrafish early stages associated with elevated caspase activity after 24 h exposure to MS-222. Toxicology and Applied Pharmacology 2020, 412, 115385 .

AMA Style

Luís M. Félix, Ana Luzio, Luís Antunes, Ana M. Coimbra, Ana M. Valentim. Malformations and mortality in zebrafish early stages associated with elevated caspase activity after 24 h exposure to MS-222. Toxicology and Applied Pharmacology. 2020; 412 ():115385.

Chicago/Turabian Style

Luís M. Félix; Ana Luzio; Luís Antunes; Ana M. Coimbra; Ana M. Valentim. 2020. "Malformations and mortality in zebrafish early stages associated with elevated caspase activity after 24 h exposure to MS-222." Toxicology and Applied Pharmacology 412, no. : 115385.

Journal article
Published: 22 July 2020 in Chemosphere
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Data about the toxicological interactions of MPs and heavy metals in biota is limited, particularly in fish early life stages. This study aimed to evaluate the toxicological effects of MPs and copper (Cu), alone or combined, in zebrafish early life stages. Embryos were exposed from 2 until 96-h post-fertilization (hpf) to MPs (2 mg/L), three sub-lethal concentrations of Cu (15, 60 and 125 μg/L) and binary mixtures containing Cu and MPs (Cu15+MPs, Cu60+MPs, Cu125+MPs). Lethal and sub-lethal parameters, histopathological changes, biochemical biomarkers, gene expression and behavior were assessed. Our findings showed that Cu and Cu+MPs decreased embryos survival and hatching rate. Increased ROS levels were observed in larvae exposed to the two lowest Cu and Cu+MPs groups, suggesting an induction of oxidative stress. An increased CAT and GPx activities were observed in Cu and Cu+MPs, implying a response of the antioxidant defense system to overcome the metal and MPs stress. The sod1 expression was downregulated in all Cu groups and in the two highest Cu+MPs exposed groups. AChE was significantly inhibited in Cu and Cu+MPs groups, indicating neurotoxicity. A disruption of avoidance and social behaviors were also noticed in the Cu125 and Cu125+MPs exposed larvae. Evidences of Cu-toxicity modulation by MPs were observed in some endpoints. Overall, the findings of this study highlight that Cu alone or co-exposed with MPs lead to oxidative stress, neurotoxicity and ultimately behavioral alterations in early life stages of zebrafish, while MPs alone do not produce significant effects on zebrafish larvae.

ACS Style

Dércia Santos; Luís Félix; Ana Luzio; Susana Parra; Edna Cabecinha; Juan Bellas; Sandra M. Monteiro. Toxicological effects induced on early life stages of zebrafish (Danio rerio) after an acute exposure to microplastics alone or co-exposed with copper. Chemosphere 2020, 261, 127748 .

AMA Style

Dércia Santos, Luís Félix, Ana Luzio, Susana Parra, Edna Cabecinha, Juan Bellas, Sandra M. Monteiro. Toxicological effects induced on early life stages of zebrafish (Danio rerio) after an acute exposure to microplastics alone or co-exposed with copper. Chemosphere. 2020; 261 ():127748.

Chicago/Turabian Style

Dércia Santos; Luís Félix; Ana Luzio; Susana Parra; Edna Cabecinha; Juan Bellas; Sandra M. Monteiro. 2020. "Toxicological effects induced on early life stages of zebrafish (Danio rerio) after an acute exposure to microplastics alone or co-exposed with copper." Chemosphere 261, no. : 127748.

Journal article
Published: 12 July 2020 in Chemico-Biological Interactions
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Embryonic studies have demonstrated the neurotoxic, teratogenic, and neurobehavioral toxicity of ethanol (EtOH). Although multiple mechanisms may contribute to these effects, oxidative stress has been described as the major damage pathway. In this regard, natural antioxidants have the potential to counteract oxidative stress-induced cellular damage. Therefore, the present study aimed to investigate the potential protective role of 24-epibrassinolide (24-EPI), a natural brassinosteroid with proved antioxidant properties, in EtOH-induced teratogenic effects during early zebrafish development. Embryos (~2 h post-fertilization - hpf) were exposed to 1 % EtOH, co-exposed to 24-EPI (0.01, 0.1 and 1 μM) and to 24-EPI alone (1 μM) for 24 h. Following exposure, biochemical evaluations were made at 26 hpf, developmental analysis was made throughout the embryo-larval period, and behavioural responses were evaluated at 120 hpf. Exposure to 1 % EtOH caused an increase in the number of malformations, which were diminished by 24-EPI. In addition, EtOH induced an accumulation of GSSG and consequent reduction of GSH:GSSG ratio, indicating the involvement of oxidative mechanisms in the EtOH-induced effects. These were reverted by 24-EPI as proved by the GSSG levels and GSH:GSSG ratio that returned to control values. Furthermore, exposure to EtOH resulted in behavioural deficits at 120 hpf as observed by the disrupted response to an aversive stimulus, suggesting the involvement of neurotoxic mechanisms. 24-EPI restored the behavioural deficits observed in a dose-dependent manner. The absence of effects in the embryos exposed solely to 24-EPI showed its safety during the exposure period. In conclusion, EtOH caused developmental teratogenicity and behavioural toxicity by inducing glutathione changes, which were prevented by 24-EPI.

ACS Style

Paulo Fernandes; Sandra M. Monteiro; Carlos Venâncio; Luís Félix. 24-Epibrassinolide protects against ethanol-induced behavioural teratogenesis in zebrafish embryo. Chemico-Biological Interactions 2020, 328, 109193 .

AMA Style

Paulo Fernandes, Sandra M. Monteiro, Carlos Venâncio, Luís Félix. 24-Epibrassinolide protects against ethanol-induced behavioural teratogenesis in zebrafish embryo. Chemico-Biological Interactions. 2020; 328 ():109193.

Chicago/Turabian Style

Paulo Fernandes; Sandra M. Monteiro; Carlos Venâncio; Luís Félix. 2020. "24-Epibrassinolide protects against ethanol-induced behavioural teratogenesis in zebrafish embryo." Chemico-Biological Interactions 328, no. : 109193.

Journal article
Published: 23 June 2020 in Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology
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MS-222, the most widely used anaesthetic in fish, has been shown to induce embryotoxic effects in zebrafish. However, the underlying molecular effects are still elusive. This study aimed to investigate the effects of MS-222 exposure during early developmental stages by evaluating biochemical and molecular changes. Embryos were exposed to 50, 100 or 150 mg L−1 MS-222 for 20 min at one of three developmental stages (256-cell, 50% epiboly, or 1–4 somite stage) and oxidative-stress, cell proliferation and apoptosis-related parameters were determined at two time-points (8 and 26 hpf). Following exposure during the 256-cell stage, the biochemical redox balance was not affected. The genes associated with glutathione homeostasis (gstpi and gclc) were affected at 8 hpf, while genes associated with apoptosis (casp3a and casp6) and cellular proliferation (pcna) were found affected at 26 hpf. An inverted U-shaped response was observed at 8 hpf for catalase activity. After exposure at the 50% epiboly stage, the gclc gene associated with oxidative stress was found upregulated at 8 hpf, while gstpi was downregulated and casp6 was upregulated later on, coinciding with a decrease in glutathione peroxidase (GPx) activity and a non-monotonic elevation of protein carbonyls and casp3a. Additionally, MS-222 treated embryos showed a decrease in DCF-staining at 26 hpf. When exposure was performed at the 1–4 somite stage, a similar DCF-staining pattern was observed. The activity of GPx was also affected whereas RT-qPCR showed that caspase transcripts were dose-dependently increased (casp3a, casp6 and casp9). The pcna mRNA levels were also found to be upregulated while gclc was changed by MS-222. These results highlight the impact of MS-222 on zebrafish embryo development and its interference with the antioxidant, cell proliferation and cellular death systems by mechanisms still to be explained; however, the outcomes point to the Erk/Nrf2 signalling pathway as a target candidate.

ACS Style

Luís M.Félixab; Ana Luziob; Ana Santosc; Luís M.Antunesbd; Ana M.Coimbrabc; Ana M. Valentim. MS-222 induces biochemical and transcriptional changes related to oxidative stress, cell proliferation and apoptosis in zebrafish embryos. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 2020, 237, 108834 .

AMA Style

Luís M.Félixab, Ana Luziob, Ana Santosc, Luís M.Antunesbd, Ana M.Coimbrabc, Ana M. Valentim. MS-222 induces biochemical and transcriptional changes related to oxidative stress, cell proliferation and apoptosis in zebrafish embryos. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology. 2020; 237 ():108834.

Chicago/Turabian Style

Luís M.Félixab; Ana Luziob; Ana Santosc; Luís M.Antunesbd; Ana M.Coimbrabc; Ana M. Valentim. 2020. "MS-222 induces biochemical and transcriptional changes related to oxidative stress, cell proliferation and apoptosis in zebrafish embryos." Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 237, no. : 108834.

Journal article
Published: 17 June 2020 in Food Chemistry
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The polyphenol compositions of Thymus × citriodorus and Thymus vulgaris extracts as obtained by exhaustive hydroethanolic (HE) extraction and aqueous decoction (AD) were compared. In addition, their compositions and bioactivities were compared to those of Thymus pulegioides and Thymus mastichina, grown under the same edaphoclimatic conditions, and Thymus carnosus. Rosmarinic acid was the most abundant polyphenol followed by luteolin-hexuronide, salvianolic acids I and K. Cluster analysis suggests a similarity of the polyphenol composition of T. citriodorus and T. vulgaris. A significant antioxidant activity was observed and correlated with their polyphenol levels. The same being observed for the higher anti-proliferative activity/cytotoxicity of HE extracts on Caco-2 and HepG2 cells as compared to AD extracts. Significant association between the total phenolic compounds with the anti-proliferative activity, for both cell lines, was observed. These results support the importance of salvianolic acids levels in Thymus extracts and their in vitro anti-proliferative/cytotoxic activities.

ACS Style

Meriem Taghouti; Carlos Martins-Gomes; Luís M. Félix; Judith Schäfer; João A. Santos; Mirko Bunzel; Fernando M. Nunes; Amélia M. Silva. Polyphenol composition and biological activity of Thymus citriodorus and Thymus vulgaris: Comparison with endemic Iberian Thymus species. Food Chemistry 2020, 331, 127362 .

AMA Style

Meriem Taghouti, Carlos Martins-Gomes, Luís M. Félix, Judith Schäfer, João A. Santos, Mirko Bunzel, Fernando M. Nunes, Amélia M. Silva. Polyphenol composition and biological activity of Thymus citriodorus and Thymus vulgaris: Comparison with endemic Iberian Thymus species. Food Chemistry. 2020; 331 ():127362.

Chicago/Turabian Style

Meriem Taghouti; Carlos Martins-Gomes; Luís M. Félix; Judith Schäfer; João A. Santos; Mirko Bunzel; Fernando M. Nunes; Amélia M. Silva. 2020. "Polyphenol composition and biological activity of Thymus citriodorus and Thymus vulgaris: Comparison with endemic Iberian Thymus species." Food Chemistry 331, no. : 127362.

Research article
Published: 08 April 2020 in Environmental Science and Pollution Research
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The toxicological knowledge of mancozeb (MZ)-containing commercial formulations on non-target species is scarce and limited. Therefore, the objective of this work was to represent a realistic application scenario by evaluating the toxicity of environmental relevant and higher concentrations of a commercial formulation of MZ using zebrafish embryos. Following determination of the 96-h LC50 value, the embryos at the blastula stage (~ 2 h post-fertilisation, hpf) were exposed to 0.5, 5, and 50 μg L−1 of the active ingredient (~ 40× lower than the 96-h LC50). During the exposure period (96 h), lethal, sublethal, and teratogenic parameters, as well as behaviour analysis, at 120 hpf, were assayed. Biochemical parameters such as oxidative stress–linked enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR)), reactive oxygen species (ROS) levels, and glutathione levels (GSH and GSSG), as well as the activity of degradation (glutathione S-transferase (GST) and carboxylesterase (CarE)), neurotransmission (acetylcholinesterase (AChE)), and anaerobic respiration (lactate dehydrogenase (LDH))–related enzymes, were analysed at the end of the exposure period. Exposed embryos showed a marked decrease in the hatching rate and many malformations (cardiac and yolk sac oedema and spinal torsions), with a higher prevalence at the highest concentration. A dose-dependent decreased locomotor activity and a response to an aversive stimulus, as well as a light-dark transition decline, were observed at environmental relevant concentrations. Furthermore, the activities of SOD and GR increased while the activity of GST, AChE, and MDA contents decreased. Taken together, the involvement of mancozeb metabolites and the generation of ROS are suggested as responsible for the developmental phenotypes. While further studies are needed to fully support the hypothesis presented, the potential cumulative effects of mancozeb-containing formulations and its metabolites could represent an environmental risk which should not be disregarded.

ACS Style

Raquel Vieira; Carlos Venâncio; Luís M. Félix. Toxic effects of a mancozeb-containing commercial formulation at environmental relevant concentrations on zebrafish embryonic development. Environmental Science and Pollution Research 2020, 27, 21174 -21187.

AMA Style

Raquel Vieira, Carlos Venâncio, Luís M. Félix. Toxic effects of a mancozeb-containing commercial formulation at environmental relevant concentrations on zebrafish embryonic development. Environmental Science and Pollution Research. 2020; 27 (17):21174-21187.

Chicago/Turabian Style

Raquel Vieira; Carlos Venâncio; Luís M. Félix. 2020. "Toxic effects of a mancozeb-containing commercial formulation at environmental relevant concentrations on zebrafish embryonic development." Environmental Science and Pollution Research 27, no. 17: 21174-21187.

Journal article
Published: 04 April 2020 in Biomedicines
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The production of chestnut (Castanea sativa Miller) is mostly concentrated in Europe. Chestnut is recognized by its high content of antioxidants and phytosterols. This work aimed to evaluate the effects of dietary chestnut consumption over physiological variables of FVB/n mice. Eighteen FVB/n male 7-month-old mice were randomly divided into three experimental groups (n = 6): 1 (control group) fed a standard diet; 2 fed a diet supplemented with 0.55% (w/w) chestnut; and 3 supplemented with 1.1% (w/w) chestnut. Body weight, water, and food intake were recorded weekly. Following 35 days of supplementation, the mice were sacrificed for the collection of biological samples. Chestnut supplementation at 1.1% reduced abdominal adipose tissue. Lower serum cholesterol was also observed in animals supplemented with chestnut. There were no significant differences concerning the incidence of histological lesions nor in biochemical markers of hepatic damage and oxidative stress. These results suggest that chestnut supplementation may contribute to regulate adipose tissue deposition.

ACS Style

Pedro Rodrigues; Tiago Ferreira; Elisabete Gonçalves; Fernanda Seixas; Rui Miguel Gil Da Costa; Tânia Martins; Maria João Neuparth; Maria João Pires; Germano Augusto Bacin Lanzarin; Luís Félix; Carlos Venâncio; Isabel C.F.R. Ferreira; Margarida M.S.M. Bastos; Rui Medeiros; Isabel Gaivão; Eduardo Rosa; Paula A. Oliveira. Dietary Supplementation with Chestnut (Castanea sativa) Reduces Abdominal Adiposity in FVB/n Mice: A Preliminary Study. Biomedicines 2020, 8, 75 .

AMA Style

Pedro Rodrigues, Tiago Ferreira, Elisabete Gonçalves, Fernanda Seixas, Rui Miguel Gil Da Costa, Tânia Martins, Maria João Neuparth, Maria João Pires, Germano Augusto Bacin Lanzarin, Luís Félix, Carlos Venâncio, Isabel C.F.R. Ferreira, Margarida M.S.M. Bastos, Rui Medeiros, Isabel Gaivão, Eduardo Rosa, Paula A. Oliveira. Dietary Supplementation with Chestnut (Castanea sativa) Reduces Abdominal Adiposity in FVB/n Mice: A Preliminary Study. Biomedicines. 2020; 8 (4):75.

Chicago/Turabian Style

Pedro Rodrigues; Tiago Ferreira; Elisabete Gonçalves; Fernanda Seixas; Rui Miguel Gil Da Costa; Tânia Martins; Maria João Neuparth; Maria João Pires; Germano Augusto Bacin Lanzarin; Luís Félix; Carlos Venâncio; Isabel C.F.R. Ferreira; Margarida M.S.M. Bastos; Rui Medeiros; Isabel Gaivão; Eduardo Rosa; Paula A. Oliveira. 2020. "Dietary Supplementation with Chestnut (Castanea sativa) Reduces Abdominal Adiposity in FVB/n Mice: A Preliminary Study." Biomedicines 8, no. 4: 75.

Journal article
Published: 01 April 2020 in Chemosphere
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The use of herbicides with glyphosate as an active ingredient (a.i.) has increased dramatically in recent years, with its residues often being found in either soil or water. Nevertheless, concerns have arisen about its harmful side effects for both ecosystems and wildlife health. Therefore, the objective of this work was to assess the effects of a commercial formulation of glyphosate (RoundUp® UltraMax), at environmentally relevant concentrations on zebrafish embryos through a set of behavioural patterns. Zebrafish embryos were exposed to 0, 1, 2 and 5 μg a.i. mL−1 concentrations of the glyphosate formulation for 72 h (from 2.5 to 75 h post-fertilization (hpf)). After exposure, larvae were washed and allowed to develop until 144 hpf. At this point, the larvae behaviour was evaluated using a battery of tests to assess the general exploratory motility, escape-like responses, anxiety-related behaviours and social interactions. In addition, cortisol levels were assessed. No significant changes were observed relative to the exploratory behaviour in the standard open field. The anxiety-related behaviours were similar among groups, and no social interference was observed following exposure to these glyphosate concentrations. Likewise, cortisol levels remained similar among treatments. Still, the larvae exposed to 5 μg a.i. mL−1 did not react to the presence of an aversive stimulus, supporting glyphosate-induced changes in the sensory-motor coordination during development. In general, these results indicate a possible neurotoxic effect of this glyphosate-based formulation that should be further evaluated. In addition, the results obtained could impose a risk for wildlife sensitive species that should not be neglected.

ACS Style

Germano A.B. Lanzarin; Carlos Venâncio; Sandra Mariza Monteiro; Luís M. Félix. Behavioural toxicity of environmental relevant concentrations of a glyphosate commercial formulation - RoundUp® UltraMax - During zebrafish embryogenesis. Chemosphere 2020, 253, 126636 .

AMA Style

Germano A.B. Lanzarin, Carlos Venâncio, Sandra Mariza Monteiro, Luís M. Félix. Behavioural toxicity of environmental relevant concentrations of a glyphosate commercial formulation - RoundUp® UltraMax - During zebrafish embryogenesis. Chemosphere. 2020; 253 ():126636.

Chicago/Turabian Style

Germano A.B. Lanzarin; Carlos Venâncio; Sandra Mariza Monteiro; Luís M. Félix. 2020. "Behavioural toxicity of environmental relevant concentrations of a glyphosate commercial formulation - RoundUp® UltraMax - During zebrafish embryogenesis." Chemosphere 253, no. : 126636.

Review
Published: 21 April 2019 in Critical Reviews in Toxicology
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Over the years, the potential toxicity of anesthetics has raised serious concerns about its safe use during pregnancy. As evidence emerged from research in animal models, showing that some anesthetic drugs are potential teratogenic, the determination of the risk of exposures to anesthetic drugs at early life stages became mandatory. However, due to inaccessibility and ethical constrains related to experimental conditions, the use of early life stages in mammalian models is limited. In this regard, some animal and nonanimal models have been suggested to surpass mammalian use in experimentation. Among them, the zebrafish embryo test has been recognized as a promising alternative in toxicology research, as well as an inexpensive and practical test. Substantial information collected from developmental research following compounds exposure, has contributed to the application of zebrafish assays in research, although only a few studies have focused on the use of early life stages of zebrafish to evaluate the developmental effects of anesthetics. Based on the recent advances of science and technology, there is a clear potential for zebrafish early life stages to provide new insights into anesthetics teratogenicity. This review provides an overview of recent anesthesia research using zebrafish embryos, demonstrating its usefulness to the anesthesia field, discussing the recent findings on various aspects related to the effects of anesthetics during early life development and the strengths and limitations of this model system.

ACS Style

Luís Félix; Ana Maria Coimbra; Ana Maria Valentim; Luís Antunes. Review on the use of zebrafish embryos to study the effects of anesthetics during early development. Critical Reviews in Toxicology 2019, 49, 357 -370.

AMA Style

Luís Félix, Ana Maria Coimbra, Ana Maria Valentim, Luís Antunes. Review on the use of zebrafish embryos to study the effects of anesthetics during early development. Critical Reviews in Toxicology. 2019; 49 (4):357-370.

Chicago/Turabian Style

Luís Félix; Ana Maria Coimbra; Ana Maria Valentim; Luís Antunes. 2019. "Review on the use of zebrafish embryos to study the effects of anesthetics during early development." Critical Reviews in Toxicology 49, no. 4: 357-370.

Journal article
Published: 12 February 2019 in Chemosphere
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The use of herbicides with glyphosate as an active ingredient, the so-called glyphosate-based herbicides (GBH), has increased dramatically in recent years currently being the most widely used in the world. Therefore, glyphosate residues have been detected in water and soils near the application sites. Recent studies indicate that GBH may cause adverse effects on vertebrates although these have been attributed to the presence of adjuvants in the commercial formulations rather than to the sole compound. Accordingly, the objective of this work was to investigate the lethal and sub-lethal developmental effects, neurotoxic potential and oxidative stress responses of zebrafish embryos to Roundup® Ultramax (RU) exposure. Embryos were exposed during 72 h to 0, 2, 5, 8.5 μg a.i. mL-1 of RU. Increased mortality was observed in embryos exposed to concentrations above 8.5 μg a.i. mL-1 as well as increased number of malformations. Decreased heart rate and hatchability were also observed. By contrast, exposure to concentrations that do not evoke teratogenic outcomes induced a dose-dependent decrease of heart rate although not inducing significant developmental changes. However, histological changes were not observed in the larvae exposed to these concentrations. Moreover, the generation of reactive oxygen species, the antioxidant enzymes activities (SOD and CAT), the GST biotransformation activity, the glutathione levels (GSH and GSSG), the oxidative damage (MDA) and the acetylcholinesterase and lactate dehydrogenase were similar among groups following exposure. Overall, available evidence suggests a dose-dependent toxicological effect of this formulation at concentrations that are not routinely detected in the environment. However, additional studies should be performed to better understand the underlying molecular mechanisms in favor of this formulation.

ACS Style

Germano A. B. Lanzarin; Luís M. Félix; Dércia Santos; Carlos A. S. Venâncio; Sandra M. Monteiro. Dose-dependent effects of a glyphosate commercial formulation – Roundup® UltraMax - on the early zebrafish embryogenesis. Chemosphere 2019, 223, 514 -522.

AMA Style

Germano A. B. Lanzarin, Luís M. Félix, Dércia Santos, Carlos A. S. Venâncio, Sandra M. Monteiro. Dose-dependent effects of a glyphosate commercial formulation – Roundup® UltraMax - on the early zebrafish embryogenesis. Chemosphere. 2019; 223 ():514-522.

Chicago/Turabian Style

Germano A. B. Lanzarin; Luís M. Félix; Dércia Santos; Carlos A. S. Venâncio; Sandra M. Monteiro. 2019. "Dose-dependent effects of a glyphosate commercial formulation – Roundup® UltraMax - on the early zebrafish embryogenesis." Chemosphere 223, no. : 514-522.

Book chapter
Published: 20 September 2018 in Advances in Molecular Toxicology
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In developmental toxicity, the evaluation of early biomarkers may be predictive of later developmental outcomes. Over the last few years, new models have been proposed for predicting developmental effects through the analysis of different effect biomarkers which have been supported by the need to implement the 3Rs (replacement, refinement, and reduction) during laboratory experimentation. In this regard, the use of the early life stages of the zebrafish (Danio rerio) has become an accepted model for toxicological studies and an important tool in translational research. This chapter describes the most common and validated biomarkers used in biochemical and genetic approaches using this animal model and presents examples of many different types of biomarkers for many classes of toxicological compounds. Although this chapter outlines the potential of the proposed biomarkers and the early life stages of zebrafish for utilization as a part of biomonitoring systems, a number of experiments are required to examine and establish the utility of the selected biomarkers to predict the whole-organism and population-level responses.

ACS Style

Dércia Santos; Raquel Vieira; Ana Luzio; Luís Félix. Zebrafish Early Life Stages for Toxicological Screening: Insights From Molecular and Biochemical Markers. Advances in Molecular Toxicology 2018, 12, 151 -179.

AMA Style

Dércia Santos, Raquel Vieira, Ana Luzio, Luís Félix. Zebrafish Early Life Stages for Toxicological Screening: Insights From Molecular and Biochemical Markers. Advances in Molecular Toxicology. 2018; 12 ():151-179.

Chicago/Turabian Style

Dércia Santos; Raquel Vieira; Ana Luzio; Luís Félix. 2018. "Zebrafish Early Life Stages for Toxicological Screening: Insights From Molecular and Biochemical Markers." Advances in Molecular Toxicology 12, no. : 151-179.

Journal article
Published: 24 July 2018 in Reproductive Toxicology
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MS-222 has been widely used as an anaesthetic in fish, thus, raising the need to infer about its toxicological safety during development. In this study, MS-222 toxicity in zebrafish embryos was evaluated after a 20-min exposure at different stages of development. Embryos exposed during the 256-cell stage displayed an increase in mortality, associated with defective early developmental pathways. Following exposure during the 50% epiboly stage, an increase in mortality and abnormal cartilage development, as well as changes in noggin expression were observed. Locomotor deficits were detected and associated with changes in early signalling pathways through the involvement of noggin. When exposed at the 1–4 somites stage, zebrafish were phenotypically normal, although presenting changes in the expression pattern of developmental genes. These findings indicate a teratogenic impact, independent of sodium channels that should be taken into consideration when MS-222 toxicity is discussed.

ACS Style

Luís M. Félix; Ana Luzio; Maria Themudo; Luís Antunes; Manuela Matos; Ana M. Coimbra; Ana M. Valentim. MS-222 short exposure induces developmental and behavioural alterations in zebrafish embryos. Reproductive Toxicology 2018, 81, 122 -131.

AMA Style

Luís M. Félix, Ana Luzio, Maria Themudo, Luís Antunes, Manuela Matos, Ana M. Coimbra, Ana M. Valentim. MS-222 short exposure induces developmental and behavioural alterations in zebrafish embryos. Reproductive Toxicology. 2018; 81 ():122-131.

Chicago/Turabian Style

Luís M. Félix; Ana Luzio; Maria Themudo; Luís Antunes; Manuela Matos; Ana M. Coimbra; Ana M. Valentim. 2018. "MS-222 short exposure induces developmental and behavioural alterations in zebrafish embryos." Reproductive Toxicology 81, no. : 122-131.