Estudo do potencial citotóxico e genotóxico do geraniol em células mononucleares do sangue periférico e de hepatocarcinoma humano HepG2
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Data
2017-02-20
Autores
Queiroz, Thaís Bernardes de [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Os monoterpenos são compostos terpênicos não-nutritivos, conhecidos por possuírem atividade protetora contra doenças. São produzidos pelas plantas como metabólitos secundários, sendo os compostos de maior frequência nos óleos essenciais. Presente no óleo essencial de diversas plantas aromáticas, o geraniol – monoterpeno alcoólico acíclico – é uma das moléculas mais comumente utilizadas pelas indústrias de sabor e fragrância. Além disso, inúmeras pesquisas o apontam como um composto promissor contra o câncer, o que torna imprescindível avaliar os riscos genéticos de seu uso frequente pela população. Diante do exposto, o presente estudo objetivou avaliar o potencial citotóxico, genotóxico e mutagênico do geraniol em células mononucleares do sangue periférico humano (células não-metabolizadoras) e em células de hepatocarcinoma humano HepG2 (células metabolizadoras), através, respectivamente, do teste do MTT, do ensaio cometa, e do teste do micronúcleo com bloqueio da citocinese. Para as células mononucleares, 4 concentrações (100, 50, 25 e 10 µg/mL) obtiveram viabilidade ≥ 80% nos ensaios de citotoxicidade, enquanto que para HepG2 foram 3 concentrações (5, 2,5 e 1,25 µg/mL), as quais foram empregadas nos testes. Para verificação de quebras em cadeia simples e duplas do DNA, foi realizado o ensaio do cometa alcalino, expondo os dois tipos celulares aos tratamentos com as concentrações de geraniol por um período de 4 horas, em triplicata, e 100 cometas por lâmina/concentração foram contados visualmente, classificando-os em: dano 0, 1, 2 ou 3, de acordo com a extensão da cauda. Já para o teste do MN, os linfócitos foram tratados por 28 horas – em duplicata –, enquanto que 24 horas de tratamentos foram empregadas para as células HepG2 – em triplicata. 1000 células binucleadas foram analisadas por lâmina/concentração, verificando a presença de micronúcleos, pontes nucleoplasmáticas e brotos nucleares. Os resultados obtidos apontaram que o geraniol, sem e após sua metabolização por enzimas hepáticas, não apresentou efeito genotóxico nas concentrações estudadas para ambos os tipos celulares. No entanto, foi constatada uma significativa redução, de maneira dose-dependente, na viabilidade de células HepG2 após exposição ao geraniol, reforçando os dados já existentes na literatura quanto à sua potente ação contra células tumorais.
Monoterpenes are non-nutritive terpenic compounds, known for their protective action against deseases. Produced by plants as secondary metabolites, they are the most common compounds in essential oils. Present in the essential oil of many aromatic plants, the geraniol – acyclic monoterpene alcoholic – is one of the most frequently used molecules by the flavour and fragrance industries. Furthermore, several research has shown it to be a promising compound against cancer, which makes it crucial to assess the genetic risks of its frequent use by the population. In light of this, this study aimed at evaluating the cytotoxic, genotoxic and mutagenic potential of the geraniol in peripheral blood mononuclear cells (non-metabolising cells) and in human hepatoma cell line HepG2 (metabolising cells), by means of the MTT test, the comet assay, the cytokinesis-block micronucleus assay, respectively. For mononuclear cells, 4 concentrations (100, 50, 25, 10 µg/mL) resulted in viability ≥80% in the cytotoxicity assay, while 3 concentrations for HepG2 (5, 2,5, 1,25 µg/mL) showed the same results, all of them being applied to the assays. To verify the single and double strand breaks, the alkaline comet assay was carried out, exposing both cell types to the treatment with geraniol concentrations for a period of 4 hours, in triplicate, and 100 comets per slide/concentrations were visually counted, being labelled as: damage 0, 1, 2, 3, according to the tail extent. Regarding the micronucleus test, the lymphocytes were treated for 28 hours – in duplicate – while 24 hours of treatment were applied to the HepG2 cells – in triplicate. 1000 binucleated cells were counted per slides/concentrations, verifying the presence of micronucleus, nucleoplasmic bridges and nuclear buds. The results obtained showed that geraniol, without and after being metabolised by liver enzymes, did not present genotoxic effect in the concentrations studied for both cell types. However, it was verified a significant dose-dependent reduction in the viability of HepG2 cells after exposure to geraniol, corroborating the data found in literature as to its potent action against tumoral cells.
Monoterpenes are non-nutritive terpenic compounds, known for their protective action against deseases. Produced by plants as secondary metabolites, they are the most common compounds in essential oils. Present in the essential oil of many aromatic plants, the geraniol – acyclic monoterpene alcoholic – is one of the most frequently used molecules by the flavour and fragrance industries. Furthermore, several research has shown it to be a promising compound against cancer, which makes it crucial to assess the genetic risks of its frequent use by the population. In light of this, this study aimed at evaluating the cytotoxic, genotoxic and mutagenic potential of the geraniol in peripheral blood mononuclear cells (non-metabolising cells) and in human hepatoma cell line HepG2 (metabolising cells), by means of the MTT test, the comet assay, the cytokinesis-block micronucleus assay, respectively. For mononuclear cells, 4 concentrations (100, 50, 25, 10 µg/mL) resulted in viability ≥80% in the cytotoxicity assay, while 3 concentrations for HepG2 (5, 2,5, 1,25 µg/mL) showed the same results, all of them being applied to the assays. To verify the single and double strand breaks, the alkaline comet assay was carried out, exposing both cell types to the treatment with geraniol concentrations for a period of 4 hours, in triplicate, and 100 comets per slide/concentrations were visually counted, being labelled as: damage 0, 1, 2, 3, according to the tail extent. Regarding the micronucleus test, the lymphocytes were treated for 28 hours – in duplicate – while 24 hours of treatment were applied to the HepG2 cells – in triplicate. 1000 binucleated cells were counted per slides/concentrations, verifying the presence of micronucleus, nucleoplasmic bridges and nuclear buds. The results obtained showed that geraniol, without and after being metabolised by liver enzymes, did not present genotoxic effect in the concentrations studied for both cell types. However, it was verified a significant dose-dependent reduction in the viability of HepG2 cells after exposure to geraniol, corroborating the data found in literature as to its potent action against tumoral cells.
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Palavras-chave
Monoterpeno, Geraniol, MTT, Ensaio do cometa, Teste do micronúcleo