Embryotoxic Effects of Pesticides in Zebrafish (Danio rerio): Diflubenzuron, Pyriproxyfen, and Its Mixtures.

Diflubenzuron (DFB) and pyriproxyfen (PPF) are larvicides used in crops to control insect plagues. However, these pesticides are known to impact non-target organisms like fish and mammals. Here, we aimed at assessing the embryotoxicity of purified DFB, PPF, and their mixtures in a non-target organism-zebrafish. Zebrafish embryos were exposed to different concentrations for 120 h: 0.025, 0.125, 0.25, 1.25, 2.5, and 10 mg/L of purified PPF and purified DFB, while we used 0.025 mg/L PPF + 10 mg/L DFB (Mix A), 0.125 mg/L PPF + 10 mg/L DFB (Mix B), and 0.25 mg/L PPF + 10 mg/L DFB (Mix C) for the mixtures of PPF + DFB. We observed mortality, teratogenicity, and cardiotoxicity. For the neurotoxicity tests and evaluation of reactive oxygen species (ROS) levels in the brain, embryos were exposed for 120 h to 0.379 and 0.754 mg/L of PPF and 0.025 and 0.125 mg/L of DFB. We established the LC50 for PPF as 3.79 mg/L, while the LC50 for DFB was not determinable. Survival and hatching were affected by PPF concentrations above 0.125 mg/L, DFB concentrations above 1.25 mg/L, and the lower pesticide mixtures. PPF exposure and mixtures induced different types of malformations, while a higher number of malformations were observed for the mixtures, suggesting a potentiating effect. Pesticides diminished avoidance responses and increased the levels of ROS across all concentrations, indicating neurotoxicity. Our findings underscore the detrimental impact of PPF and DFB exposure, spanning from biochemistry to morphology. There is a critical need to reconsider the global use of these pesticides and transition to more ecologically friendly forms of pest control, raising an alarm regarding repercussions on human and animal health and well-being.

COMPARATIVE EVALUATION OF SKIN SUTURE IN RATS WITH POLYGLYCAPRONE 25 AND NYLON.

Currently, the market offers a wide variety of suture threads, made of materials with different structural and chemical properties. Among many other characteristics, they vary in origin, absorption or degradation, and structure. From this variety, the clinical doubt arises as to which material provides the patient with the best healing quality. Objective: This study aims to comparatively evaluate two different types of suture threads-Monocryl® (polyglycaprone 25) and Ethilon® (nylon)-regarding their ability to aid in tissue regeneration by a histological and immunohistochemical analysis of the skin of rats sutured with the aforementioned materials. Methods: This basic experimental study used 12 adult Wistar rats, randomly divided into three groups with four animals each and subjected to four longitudinal incisions under anesthesia. Each group corresponded to a postsurgical evaluation date (one, seven, and 14 days). Results: At 14 postoperative days, the studied groups had no histological difference. However, the use of nylon thread showed greater evidence of earlier fibrotic union. Conclusion: This study found no histological difference in healing 14 days after surgery among the techniques and the types of suture threads. Level of Evidence II, Therapeutic Studies.

Atualmente, encontra-se disponível no mercado uma grande variedade de fios de sutura, compostos de materiais com diferentes propriedades estruturais e químicas, que variam quanto à origem, absorção ou degradação e estrutura, entre outras características. A partir dessa disponibilidade, emerge a dúvida clínica quanto ao material que propicia a melhor qualidade de cicatrização ao paciente. Objetivo: Avaliar comparativamente dois tipos de fios - Monocryl® (poliglicaprone 25) e Ethilon® (nylon) - quanto à sua capacidade de auxílio na regeneração tecidual, por meio da análise histológica e imuno-histoquímica da pele de ratos submetidos a suturas com esses materiais. Métodos: Neste estudo básico experimental, foram utilizados 12 ratos adultos da linhagem Wistar, randomicamente divididos em três grupos com quatros animais cada, que foram submetidos a quatro incisões longitudinais sob anestesia. Cada grupo correspondeu a uma data de avaliação pós-cirúrgica (1, 7 e 14 dias). Resultados: Passados 14 dias após a operação, não houve diferença histológica em relação aos grupos estudados. No entanto, o uso de fio de nylon apresentou evidência de união fibrótica mais precoce. Conclusão: Não há diferença histológica de cicatrização após 14 dias pós-operatórios entre as técnicas e os tipos de fio de sutura. Nível de Evidência II, Estudos Terapêuticos.

Multilevel Toxicity Evaluations of Polyethylene Microplastics in Zebrafish (Danio rerio).

Microplastics in freshwater environments pose a serious threat to living beings. Polyethylene microplastics (PE-MP) are the type most used around the world as microbeads in personal care products, and they have been found in aquatic organisms. The behavior and toxicity of fluorescent PE-MP spheres with an average diameter of 58.9 µm were studied in adult, juvenile and embryo zebrafish (Danio rerio). The adults were studied for genotoxicity, cytotoxicity, histology and biochemical markers. Juveniles underwent a follow-up in the gastrointestinal (GI) tract with histologic observations, and embryos were studied for embryotoxicity with the FET-test. In adults, micronucleus test and comet assays found neither genotoxicity after acute exposure for 96 h at concentrations of 0.0, 12.5, 50 and 100 mg.L-1, nor cytotoxicity through the nuclear abnormalities test. Acetylcholinesterase (AChE), Glutathione-S-Transferase (GST) and Lactate Dehydrogenase (LDH) activities were measured in adults exposed for 96 h. The AChE and GST activities were significantly changed, while no changes occurred for LDH. In conclusion, these PE-MP spheres did not cause serious toxic effects in zebrafish because there was no internalization. The observed biochemical changes in AChE and GST may be associated with GI microbiological dysbiosis, previously reported. The PE-MP spheres in the intestine of juveniles remained present for 12-15 days on average after the post-exposure clearance study, showing a slow depuration. The histological analysis, in adults, found no internalization of these microbeads, with complete depuration. The PE-MP spheres did not cross the chorion barrier, showing no embryotoxic effects after exposures at 0.0, 6.25, 12.5, 50.0 or 100.0 mg.L-1 for 96 h.

Comparative evaluation of skin suture in rats with polyglycaprone 25 and nylon / Avaliação comparativa da sutura de pele com poliglicaprone 25 e nylon em ratos

ABSTRACT Currently, the market offers a wide variety of suture threads, made of materials with different structural and chemical properties. Among many other characteristics, they vary in origin, absorption or degradation, and structure. From this variety, the clinical doubt arises as to which material provides the patient with the best healing quality. Objective: This study aims to comparatively evaluate two different types of suture threads-Monocryl® (polyglycaprone 25) and Ethilon® (nylon)-regarding their ability to aid in tissue regeneration by a histological and immunohistochemical analysis of the skin of rats sutured with the aforementioned materials. Methods: This basic experimental study used 12 adult Wistar rats, randomly divided into three groups with four animals each and subjected to four longitudinal incisions under anesthesia. Each group corresponded to a postsurgical evaluation date (one, seven, and 14 days). Results: At 14 postoperative days, the studied groups had no histological difference. However, the use of nylon thread showed greater evidence of earlier fibrotic union. Conclusion: This study found no histological difference in healing 14 days after surgery among the techniques and the types of suture threads. Level of Evidence II, Therapeutic Studies.

RESUMO Atualmente, encontra-se disponível no mercado uma grande variedade de fios de sutura, compostos de materiais com diferentes propriedades estruturais e químicas, que variam quanto à origem, absorção ou degradação e estrutura, entre outras características. A partir dessa disponibilidade, emerge a dúvida clínica quanto ao material que propicia a melhor qualidade de cicatrização ao paciente. Objetivo: Avaliar comparativamente dois tipos de fios - Monocryl ® (poliglicaprone 25) e Ethilon ® (nylon) - quanto à sua capacidade de auxílio na regeneração tecidual, por meio da análise histológica e imuno-histoquímica da pele de ratos submetidos a suturas com esses materiais. Métodos: Neste estudo básico experimental, foram utilizados 12 ratos adultos da linhagem Wistar, randomicamente divididos em três grupos com quatros animais cada, que foram submetidos a quatro incisões longitudinais sob anestesia. Cada grupo correspondeu a uma data de avaliação pós-cirúrgica (1, 7 e 14 dias). Resultados: Passados 14 dias após a operação, não houve diferença histológica em relação aos grupos estudados. No entanto, o uso de fio de nylon apresentou evidência de união fibrótica mais precoce. Conclusão: Não há diferença histológica de cicatrização após 14 dias pós-operatórios entre as técnicas e os tipos de fio de sutura. Nível de Evidência II, Estudos Terapêuticos.

Histological and Immunohistochemical Analysis of the Effects of Topical Melatonin Treatment Associated with Collagen Sponge and rhBMP-2 Protein on Bone Remodeling.

Extensive bone defect healing is an important health issue not yet completely resolved. Different alternative treatments have been proposed but, in face of a critical bone defect, it is still very difficult to reach a complete regeneration, with the new-formed bone presenting all morphological and physiological characteristics of a normal, preinjury bone. Topical melatonin use has shown as a promising adjuvant for bone regeneration due to its positive effects on bone metabolism. Thus, to search for new, safe, biological techniques that promote bone repair and favor defect healing, we hypothesized that there is a synergistic effect of melatonin treatment associated with rhBMP-2 to guide bone regeneration. This study aimed to investigate bone repair effects of topical melatonin administration in different concentrations (1, 10, and 100 µg), associated or not with rhBMP-2. Surgical-induced bone defect healing was qualitatively evaluated through histopathological analysis by light microscopy. Additionally, quantitative stereology was performed in immunohistochemistry-prepared tissue to identify angiogenic, osteogenic, and osteoclastogenic factors. Quantification data were compared between groups by the ANOVA/Tukey test and differences were considered significant when p < 0.05. Our results showed that the presence of the scaffold in the bone defect hindered the process of bone repair because in the group treated with "blood clot + scaffold" the results of bone formation and immunolabeling were reduced in comparison with all other groups (treated with melatonin alone or in association with rhBMP-2). Statistical analysis revealed a significant difference between the control group (bone defect + blood clot), and groups treated with different concentrations of melatonin in association with rhBMP-2, indicating a positive effect of the association for bone repair. This treatment is promising once it becomes a new safe alternative technique for the clinical treatment of fractures, bone defects, and bone grafts. Our results support the hypothesis of the safe use of the association of melatonin and rhBMP-2 and have established a safe and effective dose for this experimental treatment.

Treatment of Muscle Injury with Stem Cells - Experimental Study in Rabbits.

Objective Histological and macroscopic evaluation of the healing process of acute lesions of the femoral rectus muscle using stem cells derived from adipose tissue-derived stem cells (ADSCs). Method An experimental study was conducted with 18 hind legs of New Zealand rabbits, which were divided into three study groups according to the intervention to be performed. In group I, no surgical procedure was performed; in group II-SHAN, the experimental lesion was performed without any additional intervention protocol; in group III-Intervention, the addition of ADSCs was performed in the same topography of the experimental lesion. After the proposed period, 2 weeks, the material was collected and submitted to macroscopic and histological evaluation. Results The quantitative analysis showed that the addition of ADSCs is related to the reduction of inflammatory cells in the 2-week evaluation (164.2 cells in group II - SHAN to 89.62 cells in group III - ADSC). The qualitative analysis of the slides with Picrosirius red, noticed an increase in orange/yellow fibers in group III - ADSC, which evidences a final healing process. The macroscopic evaluation found no difference between the groups. Conclusion The use of ADSCs in the treatment of acute muscle injury presented histological advantages when compared to their non-use.

Treatment of Muscle Injury with Stem Cells - Experimental Study in Rabbits / Tratamento da lesão muscular com células-tronco - Estudo experimental em coelhos

Abstract Objective Histological and macroscopic evaluation of the healing process of acute lesions of the femoral rectus muscle using stem cells derived from adipose tissue-derived stem cells (ADSCs). Method An experimental study was conducted with 18 hind legs of New Zealand rabbits, which were divided into three study groups according to the intervention to be performed. In group I, no surgical procedure was performed; in group II—SHAN, the experimental lesion was performed without any additional intervention protocol; in group III—Intervention, the addition of ADSCs was performed in the same topography of the experimental lesion. After the proposed period, 2 weeks, the material was collected and submitted to macroscopic and histological evaluation. Results The quantitative analysis showed that the addition of ADSCs is related to the reduction of inflammatory cells in the 2-week evaluation (164.2 cells in group II - SHAN to 89.62 cells in group III - ADSC). The qualitative analysis of the slides with Picrosirius red, noticed an increase in orange/yellow fibers in group III - ADSC, which evidences a final healing process. The macroscopic evaluation found no difference between the groups. Conclusion The use of ADSCs in the treatment of acute muscle injury presented histological advantages when compared to their non-use.

Resumo Objetivo Avaliação histológica e macroscópica do processo de cicatrização das lesões agudas do músculo reto femoral, com utilização de células-tronco derivadas de tecido adiposo (ADSCs, na sigla em inglês). Método Foi realizado um estudo experimental com 18 patas traseiras de coelhos Nova Zelândia, que foram divididos em três nos grupos de estudo de acordo com a intervenção a ser realizada. No grupo I não foi realizado procedimento cirúrgico; no grupo II - SHAN foi realizado a lesão experimental sem nenhum protocolo de intervenção adicional; e no grupo III - Intervenção foi realizado a adição de ADSCs na mesma topografia onde foi realizada a lesão experimental. Após o período proposto, 2 semanas, o material foi coletado, submetido a avaliação macroscópica e histológica. Resultados A análise quantitativa demonstrou que a adição de ADSCs está relacionada com a diminuição de células inflamatórias na avaliação com 2 semanas (164,2 células no grupo II - SHAN para 89,62 células no grupo III - ADSC). A análise qualitativa das lâminas coradas com Picrosírius red demonstrou um aumento das fibras de cor laranja/amarela no grupo III - ADSC, o que evidencia um processo final de cicatrização. A avaliação macroscópica não encontrou diferença entre os grupos. Conclusão A utilização de ADSCs no tratamento de lesão muscular aguda apresentou vantagens histológicas quando comparada a sua não utilização.

Microcystin-LR at sublethal concentrations induce rapid morphology of liver and muscle tissues in the fish species Astyanax altiparanae (Lambari).

The process of eutrophication and consequent proliferation of cyanobacteria in rivers and lakes leads to increasing numbers of harmful algal blooms and higher concentration of toxic metabolites in freshwater bodies. Microcystin is a toxic metabolite produced by cyanobacteria that is frequently detected and can pose health risks to important freshwater species including fish. The aim of the present study was to evaluate the effects of microcystin-LR on the morphology of Astyanax altiparanae's liver and muscle. One hundred (n = 100) Astyanax altiparanae were divided into 5 groups (n = 20) with 24 h and 96 h of microcystin exposition at two doses of 0.5 and 1.0 µg/L. Differences were observed in the microcystin treatment with respect to histopathological analyses including cytoplastic degradation, displacement, and increase in nuclei volume and area of hepatocytes. Hyperemia and dilation of blood capillaries were seen in the liver. There were also observable changes in the size of muscle fibers and muscle inflammation. Our results demonstrate that microcystins can impact the integrity of both tissues even at sublethal concentrations. Low doses of microcystins are therefore sufficient to intoxicate fish livers and muscle tissues.

An ultrastructural approach for gill responses after pollutants exposure / Un enfoque ultraestructural de las respuestas branquiales después de la exposición a contaminantes

SUMMARY: Morphological and physiological responses of gills exposed to environmental contaminants are widely described in the literature. Among the primary findings are the lamellar fusion, hyperplasia, aneurysms, proliferation of mitochondria-rich cells and mucous cells. This work has as main objectives, to show the main changes caused by a polluted lake in gills, and understand how each gill's cell type responds to pollutants' exposure. To those individuals of the species, Astyanax altiparanae were exposed to water from an urban lake, which receives such contaminants from various sources. The gills were analyzed under a TEM. Our results showed that prior to these changes, commonly found, only the mucous cells did not show structural changes. Among the main results, we observed the emergence of Rodlet cells in the group exposed to contaminants, as well as severe damage to the mitochondria-rich cells. The latter result was extremely relevant for demonstrating that the proliferation of this cell type, widely described as an alternative ionic regulation, may in some cases be completely inefficient because structurally these cells are no longer able to perform their basic metabolic functions.

RESUMEN: Las respuestas morfológicas y fisiológicas de las branquias expuestas a contaminantes ambientales se describen ampliamente en la literatura. Entre los hallazgos principales se encuentran la fusión laminar, la hiperplasia, los aneurismas, la proliferación de células ricas en mitocondrias y las células mucosas. Este trabajo tiene como objetivo principal, mostrar los principales cambios en las branquias, causados por un lago contaminado y comprender cómo responde el tipo de célula de las branquias a la exposición de los contaminantes. Individuos de la especie Astyanax altiparanae fueron expuestos al agua de un lago urbano, receptor de contaminantes de diversas fuentes. Las branquias se analizaron bajo un TEM. Nuestros resultados mostraron que antes de estos cambios, comúnmente encontrados, solo las células mucosas no mostraban cambios estructurales. Entre los resultados principales, observamos la aparición de células Rodlet en el grupo expuesto a contaminantes, así como el daño severo a las células ricas en mitocondrias. Este último resultado fue extremadamente relevante para demostrar que la proliferación de este tipo de células, ampliamente descrito como una regulación iónica alternativa, en algunos casos puede ser completamente ineficiente porque estructuralmente estas células ya no son capaces de realizar sus funciones metabólicas básicas.

Effects of biodegradable detergents in the accumulation of lipofuscin (age pigment) in gill and liver of two neotropical fish species / Efectos de detergentes biodegradables en la acumulación de lipofuscina (pigmento de envejecimiento) en branquias e hígado de dos espécies de peces neotropicales

Currently there is a growing concern, in both population and governments, to identify the effects of substances commonly disposed of into rivers and lakes, on aquatic fauna and flora. Thus the objective of the present study was to verify effects of biodegradable detergents and water from an urban lake on gills and liver of two neotropical fish species of great economic importance, Astyanax altiparanae and Prochilodus lineatus. In order to do so, lipofuscin, also called the ageing pigment, was used as bioindicator. After one and five months of experiment both tissues accumulated this pigment. These data are discussed from physiological points of view, related with lipid peroxidation and mitochondrial damage.

Existe una preocupación creciente de la población y los gobiernos para identificar los efectos de substancias comúnmente arrojadas en ríos y lagos, sobre la fauna y flora acuática. El objetivo fue verificar los efectos de detergentes biodegradables y agua de un lago urbano sobre las branquias e hígado de dos especies de peces neo-tropicales de gran importancia económica, Astyanax altiparanae y Prochilodus lineatus. Analizamos los pigmentos de lipofuscina, también llamado pigmento de envejecimiento, el que fue utilizado como biomarcador. Después de uno y cinco meses de experimento, ambos tejidos acumulados con el pigmento fueron analizados. Los datos fueron discutidos desde el punto de vista fisiológico, relacionado con la peroxidación lipídica y daño mitocondrial.

Effects of exposition to polluted environments on blood cells of the fish Prochilodus lineatus.

In this study, we investigated the effects of contaminated water on the blood parameters of the fish Prochilodus lineatus exposed during 7 and 20 days. Blood was collected with heparinized syringes, and blood smears were prepared and stained with Leishman stain. Slides were examined and photographed using a Leica light microscope. Total white blood cell counts and differential counts of thrombocytes and leukocytes were obtained for statistical analysis. The group exposed to water samples from Lago Azul exhibited an increase in the number of leukocytes and in the total number of white blood cells, suggesting that the chemical contaminants in this environment were acting similarly to antigens in this fish species, causing the proliferation of defense cells. In the group exposed to detergent during 20 days, the number of thrombocytes decreased. These results suggest that the variations in the number of leukocytes were indicators of environmental pollution and that biodegradable detergents may, after a certain time of exposure, affect vital functions in fish, such as coagulation and prevention of infections, which directly involves thrombocytes.

Histology, histochemistry and stereology of the adipose fin of Prochilodus lineatus.

The adipose fin is small, nonpared, and usually located medially between the dorsal and caudal fin. Its taxonomic occurrence is very restrict; thus, it represents an important trace for taxon distinction. As it does not play a known vital physiological roll and it is easily removed, it is commonly used in marking and recapture studies. The present study characterizes the adipose fin of Prochilodus lineatus, as it is poorly explored by the literature. The adipose fin consists basically of a loose connective core, covered by a stratified epithelium supported by collagen fibers. At the epithelium, pigmented cells and alarm substance cells are found. Despite the name, adipocytes or lipid droplets are not observed on the structure of the fin.

Histochemical technique for the detection of chloride cells in fish.

Chloride cells are responsible for ionic exchanges in the fish gill. These cells have been widely studied, considering its importance in vital functions of the gill, and because they proliferate when exposed to unfavorable environments. One of the main characteristic of these cells is an acidic cytoplasm, which has been used for identification through histochemical techniques with dyes such as Toluidine Blue and Hematoxylin and Eosin. However, these techniques can be problematic, since epithelial cells can, in certain situations, acquire acidic characteristics similar to those of chloride cells, thus staining in a similar way. Among other functions, chloride cells play a role in calcium uptake from the environment, and therefore have a high concentration of this element. Based on this information, this study aims at developing a specific protocol for the identification of chloride cells. With this purpose, the Von Kossa method specific for calcium was used combined with Hematoxylin counterstaining. Chloride cells had cytoplasm slightly stained with Hematoxylin and the presence of dark stained granules dispersed in the cytoplasm resulted from the Von Kossa reaction due to the calcium present in these cells. This was not found in any other gill cell. Thus, the technique used in this study was specific and efficient to identify chloride cells in fish gills.