Guardiola Abellán, Francisco Antonio

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Guardiola Abellán, Francisco Antonio

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Guardiola Abellán, Francisco Antonio

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Universidad de Murcia. Departamento de Biología Celular e Histología

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Open Access
In vitro effects of a natural marine algae polysaccharide (λ-carrageenin) on seabream erythrocytes, tumour cell lines and marine bacterial pathogens
Campos-Sánchez, José Carlos; Esteban Abad, María Ángeles; Guardiola Abellán, Francisco Antonio; Biología Celular e Histología
Carrageenin is a high molecular weight sulphated mucopolysaccharide used to induce experimental inflammation in mammals. In addition, it possesses a wide variety of properties that have not yet been studied in fish. This study evaluated the hemagglutinating, hemolytic, cytotoxic, and antibacterial activities of λ-carrageenin. The results showed that λ-carrageenin has hemagglutinating and hemolytic activities on gilthead seabream erythrocytes, which were dose and time-dependent during the first 6 hours of incubation. No significant effects on the haemolytic activity of erythrocytes were observed after incubation for 12 or 24 h with λ-carrageenin. The PLHC-1 cell line showed significant increases in cytotoxic activity after 6 or 12 hours of incubation compared with control cells, and the highest doses of λ-carrageenin caused cytotoxicity in PLHC-1 cells after 24 h of incubation. The morphology of PLHC1 cells incubated with the highest doses of λ-carrageenin for 12 or 24 h showed obvious cell death changes compared with control cells. Interestingly, no significant variations in cytotoxic activity were observed in SAF-1 or HeLa cell lines after incubation with λ-carrageenin. λ-carrageenin showed significant dose-dependent bactericidal activity against Photobacterium damselae but had no significant effect on the bactericidal activity of Vibrio harveyi, V. anguillarum, and Tenacibaculum maritimum. The study suggests that λ-carrageenin has potential applications in aquaculture and aquatic pharmaceutical industries as a hemagglutinating, hemolytic, and antibacterial agent.
Open Access
Uncovering microplastics contamination in canned seafood
(2024) Guardiola Abellán, Francisco Antonio; Biología Celular e Histología
Open Access
Use of carrageenan in the screening of natural anti-inflammatory molecules in fish: in vitro effects of Chiliadenus glutinosus extracts
Campos-Sánchez, Jose Carlos; Esteban Abad, María Ángeles; Guardiola Abellán, Francisco Antonio; Biología Celular e Histología
Carrageenan, a high molecular weight sulphated polysaccharide, has been used in the screening of anti-inflammatory molecules in mammals. In this sense, Chiliadenus glutinosus (L.) Fourr., an endemic species of plant has been reported in the Spanish popular medicine due to their anti-inflammatory properties in humans. However, their properties have poorly been studied in fish. In this assay, different concentrations (0, 0.001, 0.01, 0.125, 0.25, 0.5 and 1 mg mL-1) of aqueous, ethanolic and methanolic extracts of this plant were elaborated, and the antioxidant, bactericidal (against four marine fish pathogen) and cytotoxic activity, as well as several immunological parameters in head-kidney leucocytes (HKLs) of gilthead seabream (Sparus aurata) were evaluated in a first experiment to determinate the extract with the best properties. Our results showed a dose-dependent antioxidant activity of each plant extract increasing with the concentration, a species- and extract-specific bactericidal activity, as well as a cytotoxic activity against the PLHC1 tumour cell line by the ethanolic and methanolic extracts. However, taking into account the cytotoxic effects of these two extracts on HKLs and the strong immunosuppressive effects on the immunological parameters assayed (peroxidase, respiratory burst activity and phagocytic capacity), a second experiment was developed by using only adjusted doses of the aqueous extract (0, 0.25 and 0.5 mg mL-1), in which immune parameters, cell morphology and inflammatory-related genes were studied in HKLs stimulated with λ-carrageenan. The present results offer a detailed view of the biological effects of C. glutinosus extracts, which could be interesting to the aquaculture sector due to their possible anti-inflammatory effects.
Open Access
Effect of dietary fenugreek seeds on the antioxidant status of gilthead seabream (Sparus aurata L.)
(Springer, 2024-10-17) Jiménez Monreal, Antonia María; Guardiola Abellán, Francisco Antonio; Murcia Tomás, María Antonia; Ceballos Francisco, Diana; Esteban Abad, María Ángeles; MartÍnez Tomé, Magdalena; Tecnología de Los Alimentos, Nutrición y Bromatología
The goal of this work was to determine the effects of dietary supplementation with fenugreek seeds on the oxidative status of gilthead seabream. Fish were fed four different diets for 8 weeks: three groups of fish were fed diets containing 1%, 5% or 10% (w/w) fenugreek seeds, while the control group was fed a diet without fenugreek. At the end of the experiment, the expression gene of the main antioxidant enzymes (superoxide dismutase, catalase and glutathione reductase) was measured in the fish liver. Supplementation with fenugreek seeds at 10% significantly increased the growth of fish while different effects on gene expression were detected. An increase in H2O2 and OH⋅ scavenging ability and oxidative stability was observed in fish fed fenugreek-supplemented diets. In the present study, an 8-week supplementation of the diet of gilthead seabream specimens with fenugreek seeds resulted in an improvement of fish growth and antioxidant status (ROS scavenging, catalase and oil oxidative stability). These results afford a new perspective on the use of spices with medicinal properties as a supplement in fish feed in order to improve their growth and antioxidant status.
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Role of mucosal immune response and histopathological study in European eel (Anguilla anguilla L.) intraperitoneal challenged by Vibrio anguillarum or Tenacibaculum soleae
(Elsevier, 2021-05-18) Conforto, Enmanuelle; Vilchez Gómez, Luciano; Parrinello, Daniela; Giovanna,Parisi, María; Esteban Abad, María Ángeles; Cammarata, Matteo; Guardiola Abellán, Francisco Antonio; Biología Celular e Histología; Facultad de Biología
The external mucus layer that covers fish skin contains numerous immune substances scarcely studied that act as the first line of defence against a broad spectrum of pathogens. This study aimed to characterize and describe for the first time several humoral immune defence parameters in the skin mucus of the European eel (Anguilla anguilla) after intraperitoneal injection with Vibrio anguillarum or Tenacibaculum soleae. This study evaluated several immune-related enzymes and bactericidal activity against fish pathogenic bacteria in the skin mucus of European eels at 24, 48, and 72 h post-challenge. The results demonstrated that European eel skin mucus showed significant increments in peroxidase and lysozyme activity at 48 and 72 h after V. anguillarum challenge, compared to other experimental groups. In the case of antiprotease activity, an increase was observed at 24 h in the skin mucus of fish challenged with V. anguillarum compared to unchallenged fish, while this activity was undetected at 48 and 72 h. In contrast, protease activity had decreased at 48 and 72 h in the skin mucus of fish challenged with V. anguillarum compared to the unchallenged group. Regarding bactericidal activity, a high growth capacity of T. soleae was observed in the skin mucus of all experimental groups. Interestingly, the skin mucus from fish challenged with V. anguillarum exhibited increased bactericidal activity against this bacterium at 48 h, compared to unchallenged fish. Finally, severe histopathological alterations were observed in the gills and liver at the end of the trial (72 h), whereas the skin showed only an overspread presence of goblet cells in the challenged fish compared to unchallenged fish. The present results may give new insights into the mucosal immune system of this primitive species with potential applications in aquaculture.
Open Access
Erythrocyte phagocytosis in gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax).
(2024-11-04) Campos Sánchez, Jose Carlos; Meseguer, José; Esteban Abad, María Ángeles; Guardiola Abellán, Francisco Antonio; Biología Celular e Histología
Open Access
Inmunotoxicología producida por metales pesados y caracterización del moco de piel en peces= Heavy metal immunotoxicology and skin mucus in fish
(2014-09-02) Guardiola Abellán, Francisco Antonio; Guardiola Abellán, Francisco Antonio; Meseguer Peñalver, José; Cuesta Peñafiel, Alberto; Esteban Abad, María Ángeles; Facultad de Biología
Durante la presente Tesis Doctoral se han estudiado los efectos inmunotoxicológicos de la exposición mediante baño a arsénico, cadmio y mercurio en la dorada (Sparus aurata L.), la cual es una especie que posee la mayor tasa de producción en la acuicultura mediterránea. Por otra parte, también se han estudiado los parámetros inmunológicos y físico-químicos del moco de la piel de cinco especies de peces teleósteos marinos. En el caso de la dorada los parámetros inmunológicos en el moco se compararon con los presentes en el suero. Por último, se evaluaron los efectos de estos metales pesados en la inmunidad de la mucosa de la piel de la dorada. Por otra parte, los peces ocupan una posición filogenética clave en la evolución de los vertebrados representando el primer grupo animal que tiene un sistema inmunitario innato y adaptativo bien estructurado, en este sentido, el estudio de la inmunología de este grupo de vertebrados tiene un doble interés científico y filogenético básico. En primer lugar, hemos evaluado los efectos de la exposición mediante baño a concentraciones sub-letales de arsénico (As), cadmio (Cd) y mercurio (Hg) en la dorada, con especial énfasis en la respuesta inmunitaria innata. En el caso del As, se observaron alteraciones histológicas en el hígado, así como acumulación de este metal en este órgano después de 30 días de exposición. Los parámetros inmunitarios humorales (niveles de IgM, actividades del complemento y la peroxidasa) no se vieron afectados, mientras que los parámetros innatos celulares del riñón cefálico (peroxidasa, explosión respiratoria y las actividades fagocíticas en los leucocitos) se incrementaron significativamente después de 10 días de exposición en comparación con los peces control. Del mismo modo, con respecto a la exposición a Cd, se observaron alteraciones morfológicas progresivas en el hígado y en el páncreas exocrino que se correlacionaron con la acumulación hepática de Cd, la reducción en la actividad del complemento en el suero y la explosión respiratoria en los leucocitos en un grado significativo después de 10 y 30 días de exposición. La actividad de la peroxidasa en el suero y la fagocitosis de los leucocitos aumentaron en diferentes momentos del muestreo, mientras que los niveles de IgM en el suero y la actividad de la peroxidasa en los leucocitos resultaron inalterados. Finalmente, se confirmaron los efectos toxicológicos del Hg porque los ejemplares de dorada expuestos mediante baño mostraron un aumento de las enzimas antioxidantes en el hígado después de 2 días, alteraciones histopatológicas en el hígado y la piel, así como la sobre-expresión de genes relacionados con el metabolismo de xenobióticos, el estrés celular y la apoptosis en la piel. Además, las actividades del complemento y la peroxidasa en el suero se incrementaron, así como la peroxidasa, la explosión respitaroria y las actividades fagocíticas en los leucocitos de riñón cefálico que se incrementaron en diferentes tiempos de exposición. En segundo lugar, se identificaron y caracterizaron los diferentes mecanismos de defensa humorales constitutivos de la mucosa de la piel de la dorada (Sparus aurata) y se compararon con los presentes en el suero. Por lo tanto, se demostró que el moco de la piel de la dorada contiene niveles más bajos de IgM, niveles similares de lisozima, fosfatasa alcalina y proteasas, y más altos en actividades como la esterasa, peroxidasa y antiproteasa que en el suero. Asimismo, el moco de la piel reveló una fuerte actividad bactericida contra bacterias patógenas de peces testadas en comparación con la actividad encontrada en el suero, mientras que las bacterias no patógenas pueden incluso crecer mejor en la presencia de moco. Además, se evaluaron los mecanismos de defensa humoral y parámetros físico-químicos en el moco de la piel de cinco especies de teleósteos marinos. Se observaron varias correlaciones entre los parámetros como el pH, la conductividad y el potencial redox, así como, entre la densidad y la osmolaridad. Centrándose en la respuesta inmunitaria, los resultados mostraron que mientras que algunas de las actividades inmunitarias fueron muy similares en los peces estudiados, otras, tales como las actividades de la proteasa, antiproteasa, fosfatasa alcalina, esterasa y peroxidasa variaron dependiendo de las especies de peces. Por último, se investigó si los parámetros de la inmunidad innata de las mucosas de la piel determinados en la dorada se ven afectados o no por los metales pesados ensayados anteriormente. De este modo, se detectaron algunos cambios en la composición del moco y en las funciones inmunitarias después de la exposición a estos metales. En general, el perfil de carbohidratos sufrió pequeñas variaciones y la mayoría de las actividades enzimáticas se incrementaron después de la exposición. Curiosamente, el Hg provocó los incrementos más importantes en la mucosa de la piel, mientras que los perfiles de proteínas obtenidos por SDS-PAGE y HPLC mostraron poca variación en el moco de la dorada después de la exposición a estos metales. En conclusión, estos resultados podrían ser útiles para una mejor comprensión de los efectos de la exposición a metales pesados en el sistema inmunitario de la dorada y como podrían afectar a la biología marina, la acuicultura o a los consumidores humanos. Por otra parte, el estudio de la función y el comportamiento de la inmunidad de las mucosas en la piel, como un componente clave del sistema inmunitario innato, podría ayudar a comprender la resistencia de los peces, así como la presencia y distribución de los patógenos y la magnitud de las infecciones y el uso de los parámetros como biomarcadores en la toxicología de los peces, los cuales serían los aspectos de mayor importancia para la acuicultura. During the present PhD Thesis, we have studied the immunotoxicological effects of waterborne exposure to arsenic, cadmium and mercury in the gilthead seabream (Sparus aurata L.), which is a species with the highest rate of production in Mediterranean aquaculture. Furthermore, the immunological and physico-chemical parameters of the skin mucus from five marine teleost fish species have been also studied. In the case of gilthead seabream the immunological parameters in mucus were compare with those present in serum. Finally, the effects of these heavy metals in the skin mucosal immunity of gilthead seabream were evaluated. Moreover, fish occupy a key phylogenetic position in the evolution of vertebrates representing the first animal group that has a well structured innate and adaptive immune system; in this sense, the study of the immunology of this group of vertebrates has a dual basic scientific and phylogenetic interest. Firstly, we have evaluated the effects of waterborne exposure to sub-lethal concentrations of arsenic (As), cadmium (Cd) and mercury (Hg) in the teleost fish gilthead seabream, with special emphasis in the innate immune response. In the case of As, we observed histological alterations in the liver as well as As-accumulation in this organ after 30 days of exposure. The humoral immune parameters (seric IgM, complement and peroxidase activities) were not affected while the cellular innate parameters of head-kidney (leucocyte peroxidase, respiratory burst and phagocytic activities) were significantly increased after 10 days of exposition compared to the control fish. Similarly, regarding Cd exposure, it was observed progressive deleterious morphological alterations in liver and exocrine pancreas that correlated with the hepatic Cd-accumulation, reduction in the serum complement activity and leucocyte respiratory burst to a significant extent after 10 and 30 days of exposure. The serum peroxidase activity and leucocyte phagocytosis were increased at different sampling times while serum IgM levels and leucocyte peroxidase activity resulted unaltered. Finally, toxicological effects were confirmed for Hg because waterborne-exposed seabream specimens showed increased liver antioxidant enzymes after 2 days, histopathological alterations in the liver and skin as well as up-regulation of the expression of genes related to xenobiotic metabolism, cellular stress and apoptosis in the skin. Further, serum complement and peroxidase activities were increased, as well as head-kidney leucocyte peroxidase, respiratory burst and phagocytic activities were increased in different exposure time. Secondly, different constitutive humoral defence mechanisms of the skin mucus of gilthead seabream (Sparus aurata) were identified and characterized and compared with those present in the serum. Thus, it was demonstrated that gilthead seabream skin mucus contains lower levels of IgM, similar levels of lysozyme, alkaline phosphatase and proteases, and higher esterase, peroxidase and antiprotease activities than serum. Skin mucus revealed stronger bactericidal activity against tested fish pathogen bacteria compared to the serum activity, whilst non-pathogen bacteria can even grow better in the presence of mucus. In addition, humoral defence mechanisms and physico-chemical parameters in the skin mucus of five species of teleosts were evaluated. Several correlations were observed between parameters as pH, conductivity and redox potential, as well as, density and osmolarity. Focusing on the immunological response, results showed that while some immune activities were very similar in the studied fish other such as protease, antiprotease, alkaline phosphatase, esterase and peroxidase activities varied depending on the fish species. Finally, we investigated whether the skin mucus innate immune parameters determined in the gilthead seabream are affected or not by the heavy metals assayed above. Thus, some changes in the mucus composition and immune functions after heavy metal exposure were detected. Overall, carbohydrate profile suffered little changes and most of the enzymatic activities were increased after exposition. Interestingly, Hg evoked the most important increments in the skin mucus and protein profiles obtained by SDS-PAGE and HPLC showed little variations in the seabream mucus after exposure to heavy metals. In conclusions, these results could be useful for better understanding the effects of exposition to heavy metals in the seabream immune system and as could interfere with fish biology, aquaculture management or human consumers. Moreover, the study of the role and behaviour of the mucosal immunity in skin, as a key component of the innate immune system, could help to understand the fish resistance as well as the presence and distribution of pathogens and magnitude of infections and use of parameters as biomarkers in fish toxicology, which would be aspects of major importance for the aquaculture industry.
Open Access
In vitro effects of Origanum vulgare leaf extracts on gilthead seabream (Sparus aurata L.) leucocytes, cytotoxic, bactericidal and antioxidant activities
(2018) García Beltrán, José María; Espinosa Ruíz, Cristóbal; Esteban Abad, María Ángeles; Guardiola Abellán, Francisco Antonio; Biología Celular e Histología
Origanum vulgare is a well-known medicinal plant that has been used since ancient times as an additive in foods and cosmetic preparations. The possible application of O. vulgare extracts in fish was assessed by using gilthead seabream (Sparus aurata) as a marine fish model due to its importance in aquaculture. The in vitro effects of aqueous and ethanolic leaf extracts of O. vulgare were tested in order to observe any immunostimulant, cytotoxic, bactericidal or antioxidant properties. The results showed that medium or high concentration of aqueous extracts and low concentrations of ethanolic extract, increased head kidney leucocyte activities as well as the number of SAF-1 cells. However, moderate to high concentrations of ethanolic extracts decreased both leucocyte activities and the number of viable SAF-1 cells, suggesting some possible toxic effect towards them. Only the highest concentration of the aqueous extract and medium to high concentrations of the ethanolic extracts showed cytotoxic activity against the tumor PLHC-1 cell line. Bactericidal activity was only detected against Vibrio harveyi, V. anguillarum and Photobacterium damselae when using the highest concentration of aqueous extract and moderate to high concentrations of ethanolic extract. Finally, both plant extracts presented antioxidant activity particularly the aqueous extract. Overall, the results suggest that both extracts (when used at the appropriate concentration) have immunostimulant, cytotoxic, bactericidal and antioxidant properties, making O. vulgare an interesting candidate for incorporation as additive in functional diets for farmed fish.
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Molecular oxidative stress markers in olive ridley turtles (Lepidochelys olivacea) and their relation to metal concentrations in wild populations
(Elsevier, 2017-10-23) Cortés-Gómez, Adriana A.; Morcillo, Patricia; Espinosa, Cristóbal; Girondot, Marc; Cuesta Peñafiel, Alberto; Esteban Abad, María Ángeles; Guardiola Abellán, Francisco Antonio; Romero García, Diego; Ciencias Sociosanitarias
Due to their longevity and extensive migration areas, marine turtles are able to accumulate diverse contaminants over many years and as a consequence they represent an interesting bioindicator species for marine ecosystem pollution. Metals provoke toxicological effects in many aquatic animal species, but marine turtles have been under-investigated in this area. Thus, we have determined the presence of certain inorganic elements (As, Cd, Cu, Ni, Pb, Se and Zn) in olive ridley turtles (Lepidochelys olivacea) and related them to metallothionein (MT), superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) transcription and/or enzymatic activities. Gene expression of sod, cat and gr was found to be higher in blood than liver or kidney but most of the significant relationships were found in liver, not only for gene expression but also for enzyme activities. This must be related to the role the liver has as the first filter organ. Several positive relationships ofsod, cat and gr gene expression in the different tissues were found in this population, as well as very high Cd concentrations. This could mean that these turtles are adapting to the metals-production of ROS and damage through a high transcription of these antioxidants. Multiple positive relationships with GR seem to be part of its compensatory effect due to the decrease of SOD production against the high and chronic exposure to certain xenobiotics. CAT, on the other hand, seems not to be used much, and glutathione detoxification of H2O2 may be more important in this species. Finally, despite the very high Cd concentrations found in this population, no significant relationship was found in any tissue with metallothionein gene expression. These results, along with very high Cd concentrations and a negative relationship with Cu, lead us to consider some kind of disruption in mt gene expression in these turtles.
Open Access
Evaluation of immune-related parameters in acellular and cellular fractions in male and female of Atlantic blue crabs (Callinectes sapidus)
(Elsevier, 2025-05-25) Gálvez, Ricardo; Guardiola Abellán, Francisco Antonio; Biología Celular e Histología; Facultad de Biología
Brachyurans are relatively understudied compared to other decapods, with numerous fields remaining to be explored, including the immune response. Therefore, the aim of this study was to characterize several immune-related enzymes and activities of the Atlantic blue crab (Callinectes sapidus) in cell-free haemolymph (CFH) and in the haemocyte lysate supernatant (HLS), considering the gender factor and the suitability of measuring each activity in CFH or HLS. Data were expressed by millilitres of haemolymph or milligrams of protein to identify which of these units would be more robust and informative. For this, haemolymph of each gender was collected, and centrifuged to obtain CFH samples, whilst the cells were sonicated to get the HLS. Our results revealed that the protein concentration was higher in CFH compared to HLS. Considering milligrams of protein as units, phenoloxidase and esterase activities were lower in CFH regarding to HLS in both genders. Similarly, the lysozyme and phosphatase activities were lower in CFH compared to HLS but only in females. The values of lysozyme activity were higher in HLS of females compared to males. Studying millilitre of haemolymph as units, lysozyme activity exhibited higher activity in CFH compared to HLS in both genders, whilst phenoloxidase and esterase activities were higher in HLS, showing variations only in males. Interestingly, alkaline phosphatase activity was undetected in CFH male. Any variation was observed between the activities measured in both genders. Protease and antiprotease activities were higher and lower in CFH compared to HLS values, respectively, whilst any variation was observed between genders. Bactericidal activity against V. harveyi, V. alginolyticus and V. anguillarum exhibited stronger values in HLS than in CFH, independently of gender, as well as against P. damselae in females. Considering gender factor, the bactericidal activity against V. alginolyticus, V. anguillarum and P. damselae was higher in HLS of females than in males. However, the bactericidal activity against V. alginolyticus and P. damselae was higher in CFH of males compared to females. Our results could contribute to gender selection in future brachyurans studies considering the variability between males and females due to their distinct biology. Additionally, we provide data regarding the baseline values of several immune-related activities, establishing the most appropriate units from the biological point of view.