Ensaio cometa no modelo vegetal Lactuca sativa: uma abordagem metodológica

Abstract

The comet assay is the most frequently used test for assessing DNA breaks. This assay presents relevant contribution to a number of areas, especially, genetic and environmental toxicology. The DNA breaks can occur in its single and double strands triggered by different mechanisms as an effect of physical, chemical or biological agents, producing aneugenic and clastogenic effects. The comet assay can be applied to various organisms, tissues and cell types. In plant models, this assay is recognized as a useful test which provides information about damage caused by genotoxic and mutagenic compounds. Its adaptation to plant models dates from 1991. At the beginning, plant models such as Nicotiana tabacum, Vicia fava and Allium cepa were commonly employed in the comet assay. Adjusting comet assay to new organisms, tissues and cell types requires standardization, optimization and calibration, which is assessed by means of DNA damage inducers, for example, Methyl methanesulfonate (MMS) and hydrogen peroxide (H2O2). Additionally, some parameters, such as nucleoid obtention, denaturation time, lysis solution efficiency and voltage level during electrophoretic run are relevant in order to achieve an effective performance with reliable results. Among a number of plant models, Lactuca sativa stands out for presenting easy cultivation, high seed yield and fast initial growth. Thus, this study aims to increase comet assay reproducibility and reliability in the Lactuca sativa plant model, through the optimization of experimental steps allowing a higher count of sampled nucleoids. Moreover, the obtention of comets with adequate shapes during electrophoretic migration was also the aim of the present study. Thus, a more reliable semi-automated scoring method, reduced evaluation time as well as faster assessment of results can be achieved. The methodological information obtained during optimization of essential steps in the comet assay applied to Lactuca sativa, presented here, will bring relevant directions that can be used to reduce time overall, number of sample repetitions and costs related to methodological performance.