Metabolismo do malondialdeído em peixes: implicações na avaliação da peroxidação lipídica como biomarcador de contaminação aquática
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2016-03-29
Autores
Garcia, Danielly Pereira [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Dentre os efeitos negativos da exposição a poluentes nos animais aquáticos podemos destacar o estresse oxidativo, uma produção exacerbada de espécies reativas de oxigênio, consequentemente, uma cascata de eventos bioquímicos denominados peroxidação lipídica (PL) ocorrem tendo como principal produto o malondialdeído (MDA). Assim, ainda que o aumento dos níveis de MDA esteja relacionado a intoxicação por poluentes, alguns trabalhos têm mostrado uma diminuição do MDA frente a essa exposição, mesmo com alterações em enzimas antioxidantes. Deste modo, este trabalho teve por objetivo avaliar se possíveis decréscimos nos níveis de MDA em peixes Astyanax altiparanae expostos a contaminantes ambientais pode ter relação com aumentos na atividade de defesas antioxidantes, aumentos na atividade da ALDH ou excreção do MDA na água. Assim, os lambaris foram expostos a misturas de metais (cádmio e cobre) nas concentrações 40 e 100 μg/L e a biodiesel B5 nas concentrações de 0,001, 0,01 e 0,1 mL/L para verificar a relação entre MDA e as enzimas antioxidantes, em amostras de brânquia e fígado. Um segundo experimento, injetando MDA nos espécimes foi realizado, nestes organismos, foram injetados intraperitonealmente doses de 10 mg/kg e 100 mg/kg de MDA com coleta do material biológico após 5 dias para avaliar o efeito do MDA no fígado e na brânquia do lambari. Um terceiro experimento foi realizado para avaliar a metabolização do MDA em brânquia e fígado e sangue com coleta de material biológico e água nos tempos zero (logo após receber a dose), 1 e 12 horas. Nestes experimentos a metabolização do MDA foi observada pela atividade da enzima aldeído desidrogenase (ALDH) e as enzimas antioxidantes catalase (CAT), glutationa peroxidase (GPx), glicose – 6- fosfato- desidrogenase (G6PDH) e a de biotransformação glutationa S-transferase (GST), peróxidos lipídicos (FOX), níveis de glutationa total (GSH-t) e ensaio do MDA. Por fim foi realizado um experimento in vitro para avaliar o efeito do MDA nas enzimas (CAT, GPx, G6PDH, GST e ALDH). Nossos resultados mostraram que o MDA tem vias de metabolização e que o aumento das enzimas antioxidantes não é responsável por sua diminuição. Sugerimos que o B5 foi capaz de induzir a atividade da ALDH e esta diminuiu a quantidade de MDA nos dois grupos de maior concentração de B5. O MDA não foi capaz de induzir a atividade da ALDH, entretanto observamos uma forte correlação entre o MDA e a ALDH. O experimento in vitro mostrou que em concentrações elevadas o MDA pode até mesmo inibir a atividade da ALDH, sugerindo que esta enzima atue na manutenção dos níveis basais deste aldeído. Por fim, o trabalho mostrou que o lambari foi capaz de eliminar o MDA em excesso por meio da excreção, fortalecendo nossa hipótese de que o organismo dos peixes utiliza vias de excreção e da ALDH para eliminar o excesso deste aldeído toxico produzido na PL por estressores ambientais
Among the adverse effects of exposure to pollutants in aquatic animals, we can highlight oxidative stress, which can be caused due to an exacerbated production of reactive oxygen species along with a cascade of biochemical events. During this cascade, one of oxidative consequences we can observe is called lipid peroxidation (LPO) with its main product malondialdehyde (MDA). Thus, although the increase in MDA levels is related to intoxication by pollutants, some studies have shown a decrease in MDA levels in aquatic organisms exposed to pollutants, even with significant improvment in antioxidant enzymes. Thus, this study aimed to assess the relationship among the reduced MDA levels in Astyanax altiparanae fish exposed to environmental contaminants, and increased antioxidant defenses and aldehyde dehydrogenase (ALDH) activity, or excretion of MDA in the water. In this way, the fishes were exposed to mixtures of metals (cadmium or copper) at concentrations of 40 g/L and 100 g/L and to B5 biodiesel in concentrations of 0.001 mL/L, 0.01 mL/L and 0.1 mL/L in order to verify the relation between MDA and antioxidant enzymes in the liver, and gill samples. We performed a second experiment by injecting MDA in the fish specimens. We intraperitoneally injected doses of 10 mg/kg and 100 mg/kg of MDA. After 5 days, we collected the liver and gill in order to evaluate the effect of MDA in the fishes. A third experiment was conducted to investigate MDA metabolization in blood, gill and liver samples, through collection of biological material and water at zero time (shortly after receiving the same intraperitoneal injections as in the second experiment), 1 and 12 hours after the injections. In these experiments, the MDA metabolization was monitored by the activity of ALDH and by the antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GPx), glucose-6-phosphate dehydrogenase (G6PDH) and glutathione-S-transferase biotransformation (GST), lipid peroxides by ferrous ion oxidation xylenol orange (FOX) assay, the total glutathione (GSH-t) and MDA assay. Finally, an experiment was conducted in vitro to evaluate the effect of enzymes on MDA (CAT, GPx, G6PDH, GST and ALDH). Our results showed that the MDA levels have metabolizing pathways and increased antioxidant enzymes are not responsible for their decrease. We suggest that the B5 was able to induce the ALDH activity, Moreover, ALDH was related to reduced amount of MDA in both groups of higher B5 concentration. The MDA was not able to induce the activity of ALDH, despite we found a strong correlation between the MDA levels and ALDH activity. In the in vitro experiment, we observed that high concentrations of MDA could even inhibit the activity of ALDH, suggesting that this enzyme acts in the maintenance of basal levels of this aldehyde. Finally, the study showed that the Astyanax altiparanae have been able to eliminate the MDA excess by excretion. This fact reinforces our hypothesis that the fish uses excretion routes and ALDH to eliminate excess of this toxic aldehyde produced during LPO cased by stressors environmental.
Among the adverse effects of exposure to pollutants in aquatic animals, we can highlight oxidative stress, which can be caused due to an exacerbated production of reactive oxygen species along with a cascade of biochemical events. During this cascade, one of oxidative consequences we can observe is called lipid peroxidation (LPO) with its main product malondialdehyde (MDA). Thus, although the increase in MDA levels is related to intoxication by pollutants, some studies have shown a decrease in MDA levels in aquatic organisms exposed to pollutants, even with significant improvment in antioxidant enzymes. Thus, this study aimed to assess the relationship among the reduced MDA levels in Astyanax altiparanae fish exposed to environmental contaminants, and increased antioxidant defenses and aldehyde dehydrogenase (ALDH) activity, or excretion of MDA in the water. In this way, the fishes were exposed to mixtures of metals (cadmium or copper) at concentrations of 40 g/L and 100 g/L and to B5 biodiesel in concentrations of 0.001 mL/L, 0.01 mL/L and 0.1 mL/L in order to verify the relation between MDA and antioxidant enzymes in the liver, and gill samples. We performed a second experiment by injecting MDA in the fish specimens. We intraperitoneally injected doses of 10 mg/kg and 100 mg/kg of MDA. After 5 days, we collected the liver and gill in order to evaluate the effect of MDA in the fishes. A third experiment was conducted to investigate MDA metabolization in blood, gill and liver samples, through collection of biological material and water at zero time (shortly after receiving the same intraperitoneal injections as in the second experiment), 1 and 12 hours after the injections. In these experiments, the MDA metabolization was monitored by the activity of ALDH and by the antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GPx), glucose-6-phosphate dehydrogenase (G6PDH) and glutathione-S-transferase biotransformation (GST), lipid peroxides by ferrous ion oxidation xylenol orange (FOX) assay, the total glutathione (GSH-t) and MDA assay. Finally, an experiment was conducted in vitro to evaluate the effect of enzymes on MDA (CAT, GPx, G6PDH, GST and ALDH). Our results showed that the MDA levels have metabolizing pathways and increased antioxidant enzymes are not responsible for their decrease. We suggest that the B5 was able to induce the ALDH activity, Moreover, ALDH was related to reduced amount of MDA in both groups of higher B5 concentration. The MDA was not able to induce the activity of ALDH, despite we found a strong correlation between the MDA levels and ALDH activity. In the in vitro experiment, we observed that high concentrations of MDA could even inhibit the activity of ALDH, suggesting that this enzyme acts in the maintenance of basal levels of this aldehyde. Finally, the study showed that the Astyanax altiparanae have been able to eliminate the MDA excess by excretion. This fact reinforces our hypothesis that the fish uses excretion routes and ALDH to eliminate excess of this toxic aldehyde produced during LPO cased by stressors environmental.
Descrição
Palavras-chave
Malondialdeído, Aldeído desidrogenase, Stress oxidativo, Astyanax sp., Malondialdehyde, Aldehyde dehydrogenase, Oxidative stress