Desenvolvimento de um biomarcador molecular baseado no comprimento de telômeros em peixes da espécie Rhamdia quelen (ordem siluriforme)

Abstract

In recent decades, the National Council for Control of Animal Experimentation (CONCEA) has aimed to establish the 3Rs guideline: Replace, Reduce, and Refine. In this context, there is a need to adapt ecotoxicological tests to new bioindicators, with the adoption of fish being prioritized in new research. A field that has been gaining prominence in recent years is the measurement of telomeres, which are structures present at the ends of chromosomes and are composed of non-coding DNA, acting in DNA protection and regulation of cell senescence, delaying chromosome shortening and the eventual loss of coding gene regions at each cell division. In this scenario, this research aims to develop a standardized protocol for measuring telomeres, capable of analyzing oxidative stress caused by pollutants in neotropical fish of the species Rhamdia quelen, popularly known as Jundiá. For this research, five individuals from the control group of a bioassay of exposure to textile effluent at the UTFPR fish farm - Campus Dois Vizinhos were acquired. Muscle samples were collected from the individuals and stored at -80'C until the next DNA extraction and amplification step. DNA amplification was performed by two techniques, the first, called endpoint PCR, characterized as a qualitative technique that was used to provide initial parameters for amplifying the telomeric region, since this technique is more accessible than the second technique used in this research, called qPCR, also known as quantitative PCR. Through qPCR, it is possible to effectively measure the relative size of telomeres based on the measurement of an endogenous gene, here used the GAPDH gene. The standardization was successfully obtained for both telomeres and the GAPDH gene, with a mean cycle threshold (Ct) of 15.251 and 29.773, respectively. Using the average Cts it was possible to calculate the value of Act, which provides the difference in the amount of molecules, in cycles, of the target region and the endogenous gene. The difference between the telomeric region of Rhamdia quelen and the endogenous GAPDH gene is 14.522, that is, 14.5 more cycles are needed to start the amplification signal of the GAPDH gene. By obtaining this value, we can conclude that, in future ecotoxicological tests, values below 14.522 may mean changes in telomeres as a result of oxidative stress caused by one or more pollutants present in the environment to which the bioindicator is exposed.