Citometria de fluxo como metodologia para análise de citogenotoxidade em Allium cepa l.: uma abordagem comparativa com a citogenética

Abstract

Allium cepa L. assay has been routinely used for the evaluation of ecotoxicology and prospecting of biological effects. The most common strategy associated with this test is the analysis of cytogenetic parameters such as mitotic index and percentage of chromosomal abnormalities. The use of flow cytometry for cytogenotoxicity and mutagenesis analysis in A. cepa is still an unexplored aspect. The present work aims to establish parameters from flow cytometry for cytogenotoxicity analysis in the Allium cepa test using with mutagen, sodium chloride. A. cepa bulbs were exposed in distilled water for a minimum of 24 hours and then exposed to the concentrations of sodium chloride (0, 31.25, 62.5, 125, 250, and 500mM) for 2, 4, 8, 12 and 24 hours. The sodium chloride was used because it is known in the literature as an inducer of cell death in plants. The parameters considered in flow cytometry were: fluorescence intensity, FSC (nuclear diameter) and SSC (nuclear complexity). In the cytogenetic analysis, the parameters evaluated were: mitotic index (MI) and chromosomal alterations. Comet assay was carried out in treatments exposed for 24 hours to the investigation of DNA fragmentation. Control cells were characterized by nuclear size, either by cytogenetic as by flow cytometry. A decrease in the percentage of dividing cells was observed in a dose-time dependent. Likewise, the results of flow cytometry showed significant decrease in the percentage of cells in G1, S and S + G2. In the treatments with mitotic index equal or close to zero features of cell death were observed as nuclei condensation (decrease of the FSC and fluorescence intensity) and fragmentation of genetic material (increased sub-G1 and coefficient of variation); this fragmentation was also viewed by comet assay. The results of analysis of internal complexity (SSC) were inversely proportional to the FSC data indicating that chromatin condensation occurs irregularly in the nuclear membrane. From the results, you can identify those parameters that flow cytometry can be used safely in assessing cell death in A. cepa, considering the characteristics of the nucleus condensation and DNA fragmentation. Interestingly, in all analyzes the NaCl concentration 62,5mM stood out with a high percentage of c-metaphase and some polyploid metaphase. This same concentration was tested for ability to induce c-metaphases lettuce and tomato cells and the results suggest the potential sodium chloride as antimitotic agent.