Evaluación de la genotoxicidad inducida por agentes mutagénicos mediante el ensayo cometa

Abstract

Mutagenic agents are the main causes of DNA damage, precursors of mutations causing errors in cell functions, for this reason the modified comet test protocol is sought to be standardized. Additionally, the repair enzymes FPG and ENDO III are used to identify the type of oxidative damage in the cell when induced by agents such as: hydrogen peroxide (H2O2), ultraviolet light (UV) and potassium bromate (KBrO3). The work consists of the isolation of lymphocytes from peripheral blood with the use of a density gradient, Ficoll. A cell lysis is carried out that allows the release of the DNA, followed by an alkalinization process and an electrophoresis of individual cells allowing the migration by charge of the genetic material. Subsequently, staining with the fluorescent DNA dye, DAPI, is performed and visualized in a fluorescence microscope with the corresponding filter, the formation of a tail or comet is identified by means of a mercury lamp at a wavelength of 590 nm, with the purpose of evaluating the degree of existing damage to the DNA. Negative, positive and enzyme-treated controls are used. The recorded values indicate the percentage of damage existing in the cells. The images are analyzed in automatic comet scoring software. In the present research work, the DNA damage caused by the various mutagenic agents was evaluated using the comet assay in human peripheral blood mononuclear cells. Said technique is a method of high sensitivity and specificity for the analysis of xenobiotics. Through the comet assay analysis with ENDO III and FPG enzyme, the results show that the induction of DNA damage to the mutagenic studied is mainly oxidative. The most harmful agent being potassium bromate (KBrO3), followed by ultraviolet light (UV) and the least damage, hydrogen peroxide (H2O2).