Estudo dos efeitos biológicos da poliamina putrescina em diferentes organismos-teste
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Data
2017-05-19
Autores
Pereira, Franco Dani Campos [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A qualidade das águas superficiais e subterrâneas tem sido ameaçada por diferentes fontes de contaminação ambiental. Os cemitérios têm se destacado neste quesito, principalmente por disponibilizar para o ambiente um líquido produzido pela decomposição cadavérica, denominado de necrochorume. O necrochorume é rico em substâncias químicas, dentre elas a poliamina putrescina, uma substância que desempenha importantes funções fisiológicas nos organismos. Porém, se essa substância se apresenta em concentrações maiores que as fisiológicas, pode desencadear problemas para os organismos expostos, pois, devido à sua alta bioatividade, passa a ser um contaminante perigoso para os seres vivos. Como esse tipo de contaminação tem sido pouco estudada ecotoxicologicamente, este trabalho é pioneiro nesta abordagem. Para a avaliação dos efeitos biológicos desencadeados pela exposição à diferentes concentrações de putrescina, foram realizados diversos ensaios com diferentes bioindicadores. Ensaios biológicos, in vitro e in vivo, foram desenvolvidos com os organismos testes Allium cepa, Salmonella typhimurium, cultura de células humanas (HepG2) e ratos Wistar. Em células meristemáticas de A. cepa foram avaliados o potencial genotóxico, por meio do teste de aberrações cromossômicas, e mutagênico, por meio da análise de micronúcleos. As células meristemáticas expostas a concentração de 23 mg/kg de putrescina apresentaram aumento significativo no índice de mutagenicidade e aumento na frequência de aderências cromossômicas, indicando uma ação aneugênica da substância. O potencial mutagênico da putrescina foi avaliado pelo teste de Ames, com as linhagens de S. typhimurium TA 98 e TA 100 (com e sem fração metabolizadora). Foi observada uma resposta positiva para as duas linhagens utilizadas, após ativação metabólica, mostrando que a metabolização da putrescina aumenta seu potencial de causar mutações no DNA. Foram desenvolvidos com células HepG2, testes de citotoxicidade (MTT e resazurina), genotoxicidade (cometa), de mutagenicidade (micronúcleo com bloqueio de citocinese). Também foram avaliados nestas células os padrões de expressão gênica para genes envolvidos com a via de metabolização de xenobióticos, na resposta a danos no reparo do DNA e estresse oxidativo. Os testes de citotoxicidade permitiram selecionar concentrações não citotóxicas para a realização dos demais experimentos com HepG2. Foram estabelecidas as concentrações de 46,3, 138,9, 231,5 e 324,1 mg/kg, correspondendo respectivamente a 10, 30, 50 e 70 % da DL50 estabelecida para ratazanas (463 mg/kg). No ensaio do cometa, todas as concentrações testadas foram significativas para o critério intensidade da cauda, enquanto que para os critérios comprimento da cauda e momento da cauda, apenas as concentrações de 10 e 50 % foram significativas. Houve aumento da frequência de MN para as concentrações de 10, 30 e 50 %, enquanto que a frequência de alterações nucleares (brotos e pontes) foram significativas somente para a maior concentração testada (70 %). A expressão de genes relacionados ao estresse oxidativo aumentaram, em relação ao grupo controle. Houve também aumento da expressão dos genes de reparo do DNA, principalmente para as concentrações de 10 e 50 %. Os genes relacionados ao metabolismo de xenobióticos tiveram maior expressão nas células expostas às maiores concentrações. Esses resultados indicam uma ação genotóxica e mutagênica da putrescina para células HepG2 e mostram que a substância foi capaz de modular os níveis de expressão gênica desses genes estudados. Os ratos Wistar foram expostos, por gavage, a três diferentes concentrações de putrescina (10, 30 e 50 % da DL50), durante 56 dias consecutivos. Foram avaliados parâmetros biométricos, hematológicos e bioquímicos, que forneceram informações sistêmica sobre o potencial tóxico da substância. Os animais expostos apresentaram parâmetros alterados para o peso de órgão como fígado, rim, pulmão; alterações nos índices hematológicos (aumento de células vermelhas, plaquetas e células do sistema imunológico); e alterações nos parâmetros bioquímicos, como distúrbios nos índices glicêmicos e colesterolêmicos. O potencial genotóxico da putrescina foi avaliado em células sanguíneas dos ratos, por meio do ensaio do cometa e do teste do micronúcleo em medula óssea. Nestes organismos, a putrescina se mostrou genotóxica para todos os grupos expostos, porém em maior intensidade no grupo exposto à menor concentração (46,3 mg/kg). Essa mesma concentração induziu um aumento significativo de eritrócitos policromáticos micronúcleados (EPCMN), confirmando o seu potencial genotóxico. Foram também avaliados parâmetros reprodutivos como contagem de espermatozoides nos testículos e nos epidídimos, expressão proteica de enzimas envolvidas na biossíntese de testosterona, alterações morfológicas, morfométricas e estereológicas nos testículos e morfológicas nos epidídimos, além de avaliação do estresse oxidativo nos testículos e nos epidídimos. Os resultados demonstraram forte ação da putrescina sobre os índices reprodutivos, como diminuição na produção espermática, diminuição na expressão de enzimas importantes para a biossíntese de testosterona (CYP11A1, 17β-HSD e StAR) e indução de estresse oxidativo e alterações morfológicas.
The quality of surface water and groundwater has been threatened by different sources of environmental contamination. The cemeteries have stood out in this question, mainly for making available to the environment a liquid produced by the cadaveric decomposition, denominated of necroslurry. Necroslurry is rich in chemicals, among them a polyamine called putrescine, a substance that plays important physiological functions in organisms. However, if this substance is present in higher concentrations than the physiological ones, it can cause problems for the exposed organisms, because, due to its high bioactivity, it becomes a contaminant dangerous to living beings. As this type of contamination has been little studied ecotoxicologically, this work is pioneering in this approach. For the evaluation of the biological effects triggered by the exposure to different concentrations of putrescine, several tests with different bioindicators were carried out. Biological assays, in vitro and in vivo, were developed with the test organisms Allium cepa, Salmonella typhimurium, culture of human cells (HepG2) and Wistar rats. The genotoxic potential were evaluted in meristematic cells of A. cepa by the chromosomal aberration test and the mutagenic potencial by the micronucleus analysis. The meristematic cells exposed to the concentration of 23 mg/kg of putrescine showed a significant increase in the index of mutagenicity and increase in the frequency of chromosomal adherence, indicating aneugenic action of the substance. The mutagenic potential of putrescine was evaluated by the Ames test, with S. typhimurium TA 98 and TA 100 strains (with and without metabolizing fraction). A positive response was observed for the two strains used after metabolic activation, showing that the metabolic process of putrescine increases its potential to cause mutations in the DNA. Cytotoxicity (MTT and resazurin), genotoxicity (comet) and, mutagencicity test (micronucleus with block of cytokinesis) were developed with HepG2 cells. We also evaluated the gene expression patterns of genes involved in the xenobiotic metabolism pathway in response to damage to DNA repair and oxidative stress. Cytotoxicity tests allowed the selection of non-cytotoxic concentrations, which were used in the other experiments with HepG2. Concentrations of 46.3, 138.9, 231.5 and 324.1 mg/kg, corresponding respectively to 10, 30, 50 and 70 % of the LD50 for rats (463 mg/kg) were established. From the comet assay it was observed that all the concentrations tested were significant for the tail intensity criterion, whereas for the tail length and tail moment criteria only the concentrations of 10 and 50 % were significant. There was an increase in the frequency of MN at concentrations of 10, 30 and 50 %, while the frequency of nuclear changes (buds and chromosomal bridges) were significant only at the highest concentration tested (70 %). The expression of genes related to oxidative stress increased in relation to the control group. There was also increased expression of DNA repair genes, especially at concentrations of 10 and 50 %. Genes related to xenobiotic metabolism had greater expression in the cells exposed to higher concentrations. These results indicate a genotoxic and mutagenic action of putrescine for HepG2 cells and show that the substance was able to modulate the levels of gene expression of these studied genes. Wistar rats were exposed by gavage to three different concentrations of putrescine (10, 30 and 50 % of LD50) for 56 consecutive days. Biometric, hematological and biochemical parameters were evaluated, which provided systemic information about the toxic potential of the substance. The exposed animals presented altered parameters for organ weight such as liver, kidney, lung; changes in hematological indices (increase of red blood cells, platelets and cells of the immune system); and alterations in biochemical parameters, such as disturbances in glycemic and cholesterolemic indexes. The genotoxic potential of putrescine was evaluated in the blood cells of the rats by the comet assay and the mutagenic potential by the micronucleus test in bone marrow. In these organisms, putrescine was genotoxic for all exposed groups, but more intense in the group exposed to the lowest concentration (46.3 mg/kg). This same concentration induced a significant increase of Micronucleated Polychromatic Erythrocytes (PCEMN), confirming its genotoxic potential. Reproductive parameters such as sperm counts in the testicles and epididymides, protein expression of enzymes involved in testosterone biosynthesis, morphological, morphometric and stereological alterations in the testicles and morphological aspects of the epididymides, as well as evaluation of oxidative stress in the testicles and epididymides were also evaluated. The results showed a strong action of putrescine on the reproductive indices, such as decrease in sperm production, decrease in the expression of enzymes important for testosterone biosynthesis (CYP11A1, 17β-HSD and StAR) and induction of oxidative stress and morphological changes. In view of all these evaluations, it was possible to aggregate information on the biological effects promoted by the exposure of different organisms to the different concentrations of putrescine evaluated. These results contribute to information concerning the toxicity of this substance and alert to its potential as an environmental contaminant.
The quality of surface water and groundwater has been threatened by different sources of environmental contamination. The cemeteries have stood out in this question, mainly for making available to the environment a liquid produced by the cadaveric decomposition, denominated of necroslurry. Necroslurry is rich in chemicals, among them a polyamine called putrescine, a substance that plays important physiological functions in organisms. However, if this substance is present in higher concentrations than the physiological ones, it can cause problems for the exposed organisms, because, due to its high bioactivity, it becomes a contaminant dangerous to living beings. As this type of contamination has been little studied ecotoxicologically, this work is pioneering in this approach. For the evaluation of the biological effects triggered by the exposure to different concentrations of putrescine, several tests with different bioindicators were carried out. Biological assays, in vitro and in vivo, were developed with the test organisms Allium cepa, Salmonella typhimurium, culture of human cells (HepG2) and Wistar rats. The genotoxic potential were evaluted in meristematic cells of A. cepa by the chromosomal aberration test and the mutagenic potencial by the micronucleus analysis. The meristematic cells exposed to the concentration of 23 mg/kg of putrescine showed a significant increase in the index of mutagenicity and increase in the frequency of chromosomal adherence, indicating aneugenic action of the substance. The mutagenic potential of putrescine was evaluated by the Ames test, with S. typhimurium TA 98 and TA 100 strains (with and without metabolizing fraction). A positive response was observed for the two strains used after metabolic activation, showing that the metabolic process of putrescine increases its potential to cause mutations in the DNA. Cytotoxicity (MTT and resazurin), genotoxicity (comet) and, mutagencicity test (micronucleus with block of cytokinesis) were developed with HepG2 cells. We also evaluated the gene expression patterns of genes involved in the xenobiotic metabolism pathway in response to damage to DNA repair and oxidative stress. Cytotoxicity tests allowed the selection of non-cytotoxic concentrations, which were used in the other experiments with HepG2. Concentrations of 46.3, 138.9, 231.5 and 324.1 mg/kg, corresponding respectively to 10, 30, 50 and 70 % of the LD50 for rats (463 mg/kg) were established. From the comet assay it was observed that all the concentrations tested were significant for the tail intensity criterion, whereas for the tail length and tail moment criteria only the concentrations of 10 and 50 % were significant. There was an increase in the frequency of MN at concentrations of 10, 30 and 50 %, while the frequency of nuclear changes (buds and chromosomal bridges) were significant only at the highest concentration tested (70 %). The expression of genes related to oxidative stress increased in relation to the control group. There was also increased expression of DNA repair genes, especially at concentrations of 10 and 50 %. Genes related to xenobiotic metabolism had greater expression in the cells exposed to higher concentrations. These results indicate a genotoxic and mutagenic action of putrescine for HepG2 cells and show that the substance was able to modulate the levels of gene expression of these studied genes. Wistar rats were exposed by gavage to three different concentrations of putrescine (10, 30 and 50 % of LD50) for 56 consecutive days. Biometric, hematological and biochemical parameters were evaluated, which provided systemic information about the toxic potential of the substance. The exposed animals presented altered parameters for organ weight such as liver, kidney, lung; changes in hematological indices (increase of red blood cells, platelets and cells of the immune system); and alterations in biochemical parameters, such as disturbances in glycemic and cholesterolemic indexes. The genotoxic potential of putrescine was evaluated in the blood cells of the rats by the comet assay and the mutagenic potential by the micronucleus test in bone marrow. In these organisms, putrescine was genotoxic for all exposed groups, but more intense in the group exposed to the lowest concentration (46.3 mg/kg). This same concentration induced a significant increase of Micronucleated Polychromatic Erythrocytes (PCEMN), confirming its genotoxic potential. Reproductive parameters such as sperm counts in the testicles and epididymides, protein expression of enzymes involved in testosterone biosynthesis, morphological, morphometric and stereological alterations in the testicles and morphological aspects of the epididymides, as well as evaluation of oxidative stress in the testicles and epididymides were also evaluated. The results showed a strong action of putrescine on the reproductive indices, such as decrease in sperm production, decrease in the expression of enzymes important for testosterone biosynthesis (CYP11A1, 17β-HSD and StAR) and induction of oxidative stress and morphological changes. In view of all these evaluations, it was possible to aggregate information on the biological effects promoted by the exposure of different organisms to the different concentrations of putrescine evaluated. These results contribute to information concerning the toxicity of this substance and alert to its potential as an environmental contaminant.
Descrição
Palavras-chave
Necrochorume, Cemitérios, Aderências cromossômicas, Teste de Ames, Ratos Wistar, Genotoxicidade, Mutagênicidade, Estresse oxidativo, Parâmetros reprodutivos, Esteroidogênese