Avaliação da fito- e genotoxicidade do chumbo em Lactuca sativa L.
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2014-12-11
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A contaminação dos ecossistemas com metais pesados (MP) é uma preocupação mundial. Dentre os vários MP poluentes, o chumbo (Pb) é um dos mais tóxicos, colocando em risco a saúde humana e dos animais. Este elemento não-essencial encontra-se difundido de forma generalizada no ambiente e é altamente fito e genotóxico.
A alface (Lactuca sativa L.) é uma hortícola muito importante na alimentação humana e encontra-se referenciada como espécie standard para testes de toxicidade e genotoxicidade. Neste estudo, avaliou-se a ação do Pb em concentrações legais para águas de rega em plantas de alface, ao nível da germinação, crescimento e integridade do DNA.
Expondo sementes de duas cultivares de alface, “Reine de Mai” (acumuladora de cádmio) e “Bola de Manteiga” (considerada sensível a MP), durante 7 dias às concentrações de 0, 0,05, 0,5, 5, 10 e 20 mg/L de Pb, verificou-se que a maioria das doses utilizadas não afetou de forma significativa a germinação, os comprimentos das partes radiculares e aéreas, e os pesos frescos das plântulas, levando, em certos casos, a um estímulo destes parâmetros, possivelmente devido a um papel como micronutriente do Pb. A velocidade de germinação e o vigor das sementes foram afetados tanto de forma positiva (normalmente, nas concentrações mais baixas) como negativa (nas concentrações mais elevadas). Estes resultados foram obtidos para ambas as cultivares, sendo que a “Bola de Manteiga” não se demonstrou sensível para estas concentrações de Pb, mas menos tolerante que a “Reine de Mai”.
Realizou-se também um ensaio de exposição ao Pb em hidroponia com plantas da cultivar “Reine de Mai”, com cerca de duas semanas, usando as concentrações de 0, 0,05, 0,5, 5 mg/L de Pb. Ao fim de 14 e 28 dias de exposição foram avaliados os comprimentos, e respetivos pesos frescos, das partes aéreas e radiculares. Os resultados apontam para um efeito hormético das concentrações de Pb testadas, e para uma possível ativação/ desenvolvimento de mecanismos de defesa contra o Pb.
Através do ensaio do cometa em células dos ápices radiculares de plantas expostas durante 28 dias ao Pb, verificou-se que com o aumento da concentração deste metal, maior é a quantidade de danos nos núcleos destas células. A concentração de 0,05 mg/L não se demonstrou tão tóxica como as restantes utilizadas.
De modo a avaliar melhor a genotoxicidade do Pb, aplicaram-se os marcadores moleculares ISSRs e EST-SSRs a amostras de folhas e raízes expostas a ambos os tempos de exposição. Os ISSRs demonstraram que com o aumento da concentração de Pb maior é a instabilidade genómica das amostras. Permitiram ainda verificar que nas raízes há uma maior instabilidade comparativamente às folhas, e que com o aumento do tempo de exposição a estabilidade genómica aumenta. Estes resultados confirmam os obtidos no ensaio de exposição, para um possível processo de tolerância/adaptação. Os EST-SSRs não demonstraram qualquer instabilidade entre as diversas amostras testadas.
Com este trabalho verificou-se que a alface é uma cultura apropriada para estudos de fito e genotoxicidade, e que os parâmetros avaliados são bons biomarcadores/bioindicadores da toxicidade do Pb.
The contamination of the ecosystems with heavy metals (HM) is a global concern. Among the various HM pollutants, lead (Pb) is one of the most toxic, presenting a big risk for human and animal health. This non-essential element, widely distributed in the environment, is highly phyto- and genotoxic. The lettuce (Lactuca sativa L.) constitutes an important vegetable for human nutrition and is referred as a standard species for toxicity and genotoxicity tests. The current study, reports on the effect of legal Pb concentrations on germination, growth and DNA integrity in this species. Seeds of two lettuce cultivars, namely “Reine de Mai” (cadmium accumulator) and “Bola de Manteiga” (considered HM sensitive), were exposed to a range of Pb concentrations (0, 0.05, 0.5, 5, 10 and 20 mg/L) for 7 days and the effect on several parameters (germination, roots and shoots length and fresh weights of the seedlings) were evaluated. The results demonstrate that most of the doses did not negatively affect these characteristics, and in some cases, it was even possible to observe a positive relation between the measured parameters and the Pb concentrations, possibily related with a role of Pb as micronutrient. Furthermore, the germination speed and seed vigour were affected both positively (usually at lower concentrations) and negatively (at higher concentrations). These results were obtained for both cultivars and the “Bola de Manteiga” demonstrated to not be a sensitive cultivar to these concentrations of Pb, but was less tolerant than “Reine de Mai”. Also, the present study reports a test for Pb exposure in hydroponic conditions with “Reine de Mai” plants, (with approximately two weeks old), using the following Pb concentration range: 0, 0.05, 0.5 and 5 mg/L. After 14 and 28 days of exposure, roots and shoots lengths, as well as fresh weights, were evaluated. The results point to a hormetic effect of the Pb concentrations, and a possible activation/development of defence mechanisms against Pb. Through a comet assay in apical root cells Pb-exposed for 28 days, it is shown an increase in DNA fragmentation correlates with increasing Pb concentrations. The concentration of 0.05 mg/L demonstrated that it is not as toxic as the others. In order to more closely assess the genotoxicity of Pb, a molecular markers-based analysis (ISSRs and EST-SSRs) was applied to leaves and roots samples previously exposed to both exposure times. The ISSRs demonstrated that an increasing in Pb concentration leads to a greater genomic instability. Furthermore, this analysis also allowed to verify that there is a higher instability in roots compared to leaves, and with increasing time of exposure there is an improvement of the genomic stability. These results are in accordance with those obtained in the exposure test for a possible process of tolerance/ adaptation. The EST-SSRs did not reveal instability between the various samples tested. This work presents the lettuce as a suitable crop for phyto- and genotoxic studies, as well as several parameters as reliable biomarkers / bioindicators of Pb toxicity.
The contamination of the ecosystems with heavy metals (HM) is a global concern. Among the various HM pollutants, lead (Pb) is one of the most toxic, presenting a big risk for human and animal health. This non-essential element, widely distributed in the environment, is highly phyto- and genotoxic. The lettuce (Lactuca sativa L.) constitutes an important vegetable for human nutrition and is referred as a standard species for toxicity and genotoxicity tests. The current study, reports on the effect of legal Pb concentrations on germination, growth and DNA integrity in this species. Seeds of two lettuce cultivars, namely “Reine de Mai” (cadmium accumulator) and “Bola de Manteiga” (considered HM sensitive), were exposed to a range of Pb concentrations (0, 0.05, 0.5, 5, 10 and 20 mg/L) for 7 days and the effect on several parameters (germination, roots and shoots length and fresh weights of the seedlings) were evaluated. The results demonstrate that most of the doses did not negatively affect these characteristics, and in some cases, it was even possible to observe a positive relation between the measured parameters and the Pb concentrations, possibily related with a role of Pb as micronutrient. Furthermore, the germination speed and seed vigour were affected both positively (usually at lower concentrations) and negatively (at higher concentrations). These results were obtained for both cultivars and the “Bola de Manteiga” demonstrated to not be a sensitive cultivar to these concentrations of Pb, but was less tolerant than “Reine de Mai”. Also, the present study reports a test for Pb exposure in hydroponic conditions with “Reine de Mai” plants, (with approximately two weeks old), using the following Pb concentration range: 0, 0.05, 0.5 and 5 mg/L. After 14 and 28 days of exposure, roots and shoots lengths, as well as fresh weights, were evaluated. The results point to a hormetic effect of the Pb concentrations, and a possible activation/development of defence mechanisms against Pb. Through a comet assay in apical root cells Pb-exposed for 28 days, it is shown an increase in DNA fragmentation correlates with increasing Pb concentrations. The concentration of 0.05 mg/L demonstrated that it is not as toxic as the others. In order to more closely assess the genotoxicity of Pb, a molecular markers-based analysis (ISSRs and EST-SSRs) was applied to leaves and roots samples previously exposed to both exposure times. The ISSRs demonstrated that an increasing in Pb concentration leads to a greater genomic instability. Furthermore, this analysis also allowed to verify that there is a higher instability in roots compared to leaves, and with increasing time of exposure there is an improvement of the genomic stability. These results are in accordance with those obtained in the exposure test for a possible process of tolerance/ adaptation. The EST-SSRs did not reveal instability between the various samples tested. This work presents the lettuce as a suitable crop for phyto- and genotoxic studies, as well as several parameters as reliable biomarkers / bioindicators of Pb toxicity.
Descrição
Dissertação de Mestrado em Genética Molecular Comparativa e Tecnológica
Palavras-chave
Lactuca sativa , Fitotoxicidade , Genotoxicidade , Marcadores moleculares , Ensaio cometa , Melhoramento de plantas