Caracterização e avaliação dos aspectos fitotóxicos e citogenotóxicos do organofosforado clorpirifós, por meio de ensaios in vivo e in vitro
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Data
2021-11-04
Autores
Costa, Mariana Santos
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Universidade Estadual Paulista (Unesp)
Resumo
Os agrotóxicos são usados no Brasil como a principal estratégia para combater e/ou prevenir pragas agrícolas. O crescente uso desses químicos tornou o país um dos líderes mundiais no consumo dessa substância. O uso contínuo e irregular dos agrotóxicos pode gerar efeitos adversos tanto aos ecossistemas como uma ameaça à saúde pública. Dentre os agrotóxicos disponíveis para utilização, se destacam os pertencentes a classe dos organofosforados, considerados um dos principais responsáveis por intoxicações e óbitos por agrotóxicos registrados no Brasil. O organofosforado clorpirifós, que é muito utilizado no Brasil, exerce sua ação tóxica pela inibição da enzima acetilcolinesterase, o que resulta em excesso de acetilcolina nas sinapses colinérgicas, promovendo sinais e efeitos diversos. Tendo em vista o potencial que esse químico tem em causar alterações citogenotóxicas e mutagênicas, esse trabalho objetivou avaliar diferentes concentrações de clorpirifós, para investigar o seu potencial citogenotóxico e mutagênico, por testes com bioindicadores vegetais e com células de hepatoma humano (C3A). Os bioindicadores vegetais utilizados neste estudo foram Lactuca sativa, para avaliação do potencial fitotóxico (ensaios de germinação e de inibição do crescimento de radícula e hipocótilo), Allium cepa, para as avaliação dos potenciais tóxico e cito genotóxico (ensaios de germinação, de índice mitótico e de aberrações cromossômicas - AC) e investigação do potencial mutagênico (ensaio do micronúcleo – MN, em células F1). Para os ensaios com organismos vegetais, foram testadas seis concentrações do inseticida Clorpirifós: C1: 10 mg mL-1 (concentração de uso na agricultura), C2: concentração de 5 mg mL-1; C3: concentração de 2,5 mg mL-1; C4: concentração de 1, 25 mg mL-1 ; C5: concentração de 0,625 mg mL-1; C6: concentração de 0,3125 mg mL-1. Os resultados com Allium cepa mostraram potencial fitotóxico para C1, C2 e C3. Já os ensaios de avaliação dos potenciais genotóxico e mutagênico, que foram avaliado apenas com C3, C4, C5 e C6, mostraram resultados estatisticamente diferentes do controle negativo para C3, C4, C5. Os ensaios com células HepG2 foram primeiramente realizados com as concentrações acima citadas (C1 a C6), mas, como todas as concentrações foram citotóxicas, pelo ensaio do MTT, houve a necessidade de diminuir as concentrações (C7: 0,15625 mg mL-1; C8: 0,078 mg mL-1; C9: 0,039 mg mL-1 e C10: 0,019 mg mL-1). Essas concentrações foram então utilizadas tanto no teste de citotoxicidade do MTT como nos demais ensaios colorimétricos (do Azul de Tripan e Resazurina). O teste do MTT mostrou que, somente C9 e C10 não foram citotóxicas. Quanto aos ensaios de resazurina, todas as concentrações foram citotóxicas. Pelo ensaio do azul de tripan, somente a concentração C7 foi tóxica. Adicionalmente aos ensaios realizados com os bioindicadores, foi feita uma ampla revisão bibliográfica em bases de dados científicas disponíveis, sobre o potencial poluidor dos organofosforados, o uso do inseticida clorpirifós na agricultura brasileira e os bioensaios mais utilizados e indicados para a avaliação da toxicidade desse grupo. Os dados desta pesquisa realizada na literatura científica foram compilados em um artigo de revisão, que também faz parte do conteúdo dessa dissertação.
Pesticides are used in Brazil as the main strategy to combat and/or prevent agricultural pests. The growing chemical mineral use has made the country one of the global leaders in the consumption of this substance. The continuous and irregular use of pesticides can generate adverse effects on ecosystems as well as a threat to public health. Among the pesticides available for use, those belonging to the class of organophosphates stand out, considered one of the main causes of poisoning and deaths caused by pesticides registered in Brazil. The organophosphate chlorpyrifos, which is widely used in Brazil, exerts its toxic action by inhibiting the enzyme acetylcholinesterase, which results in excess acetylcholine in cholinergic synapses, promoting different signs and effects. Considering the potential that this chemical has to cause cytogenotoxic and mutagenic changes, this work aimed to evaluate different options of chlorpyrifos, to investigate its cytogenotoxic and mutagenic potential, by testicles with plant bioindicators and with human hepatoma cells (C3A). The plant bioindicators used in this study were Lactuca sativa, for evaluation of phytotoxic potential (germination tests and inhibition of radicle and hypocotyl growth), Allium cepa, for the needs of toxic and cytogenotoxic potentials (germination tests, mitotic index and of chromosomal aberrations - AC) and investigation of the mutagenic potential (micronucleus assay - MN, in F1 cells). For tests with plant organisms, six requirements of the insecticide Chlorpyrifos were tested: C1: 1 mg mL-1 (concentration of use in agriculture), C2: concentration of 5 mg mL-1; C3: 2,5 mg mL-1 concentration; C4: 1,25 mg mL-1 concentration; C5: 0,625 mg mL-1, concentration C6: concentration of 0,3125 mg mL-1. The results with Allium cepa excellent phytotoxic potential for C1, C2 and C3. As for the assays for evaluating genotoxic and mutagenic potentials, which were evaluated only with C3, C4, C5 and C6, results were statistically different from the negative control for C3, C4, C5. Assays with HepG2 cells were first performed with the concentrations mentioned above (C1 to C6), but, as all concentrations were cytotoxic, by the MTT assay, it was necessary to decrease the concentrations (C7: 0.15625 mg mL-1; C8: 0.078 mg mL-1; C9: 0.039 mg mL-1 and C10: 0.019 mg mL-1). These concentrations were then used both in the MTT cytotoxicity test and in the other colorimetric assays (Trypan Blue and Resazurin). The MTT test showed that only C9 and C10 were not cytotoxic. As for for the resazurin assays, all concentrations were cytotoxic and according to the trypan blue assay, only the C7 concentration was toxic. In addition to the tests carried out with the bioindicators, an extensive literature review was carried out in available scientific databases, on the polluting potential of organophosphates, the use of the insecticide chlorpyrifos in Brazilian agriculture and the most used and indicated bioassays for the assessment of the toxicity of this group de The data from this research carried out in the scientific literature were compiled in a review article, which is also part of the content of this dissertation.
Pesticides are used in Brazil as the main strategy to combat and/or prevent agricultural pests. The growing chemical mineral use has made the country one of the global leaders in the consumption of this substance. The continuous and irregular use of pesticides can generate adverse effects on ecosystems as well as a threat to public health. Among the pesticides available for use, those belonging to the class of organophosphates stand out, considered one of the main causes of poisoning and deaths caused by pesticides registered in Brazil. The organophosphate chlorpyrifos, which is widely used in Brazil, exerts its toxic action by inhibiting the enzyme acetylcholinesterase, which results in excess acetylcholine in cholinergic synapses, promoting different signs and effects. Considering the potential that this chemical has to cause cytogenotoxic and mutagenic changes, this work aimed to evaluate different options of chlorpyrifos, to investigate its cytogenotoxic and mutagenic potential, by testicles with plant bioindicators and with human hepatoma cells (C3A). The plant bioindicators used in this study were Lactuca sativa, for evaluation of phytotoxic potential (germination tests and inhibition of radicle and hypocotyl growth), Allium cepa, for the needs of toxic and cytogenotoxic potentials (germination tests, mitotic index and of chromosomal aberrations - AC) and investigation of the mutagenic potential (micronucleus assay - MN, in F1 cells). For tests with plant organisms, six requirements of the insecticide Chlorpyrifos were tested: C1: 1 mg mL-1 (concentration of use in agriculture), C2: concentration of 5 mg mL-1; C3: 2,5 mg mL-1 concentration; C4: 1,25 mg mL-1 concentration; C5: 0,625 mg mL-1, concentration C6: concentration of 0,3125 mg mL-1. The results with Allium cepa excellent phytotoxic potential for C1, C2 and C3. As for the assays for evaluating genotoxic and mutagenic potentials, which were evaluated only with C3, C4, C5 and C6, results were statistically different from the negative control for C3, C4, C5. Assays with HepG2 cells were first performed with the concentrations mentioned above (C1 to C6), but, as all concentrations were cytotoxic, by the MTT assay, it was necessary to decrease the concentrations (C7: 0.15625 mg mL-1; C8: 0.078 mg mL-1; C9: 0.039 mg mL-1 and C10: 0.019 mg mL-1). These concentrations were then used both in the MTT cytotoxicity test and in the other colorimetric assays (Trypan Blue and Resazurin). The MTT test showed that only C9 and C10 were not cytotoxic. As for for the resazurin assays, all concentrations were cytotoxic and according to the trypan blue assay, only the C7 concentration was toxic. In addition to the tests carried out with the bioindicators, an extensive literature review was carried out in available scientific databases, on the polluting potential of organophosphates, the use of the insecticide chlorpyrifos in Brazilian agriculture and the most used and indicated bioassays for the assessment of the toxicity of this group de The data from this research carried out in the scientific literature were compiled in a review article, which is also part of the content of this dissertation.
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Palavras-chave
Toxicidade, Inseticida, Allium cepa, Lactuca sativa, Citotoxicidade, C3A, Toxicity, Allium cepa, Lactuca sativa, Cytotoxicity, C3A