Micronuclei and comet assay in adult Cnesterodon decemmaculatus as indicators of genotoxic effect in environmental samples

OSSANA, N A; SOLONESKI S; FERRARI, L

Buenos Aires

Congreso; SETAC Latin America 11th Biennial Meeting; 2015

The aim was to assess the genotoxic impact of the water from theReconquista river (high basin) with and without a pollution pulse aggregate(Cadmium-Cd). Samples for the assays were taken at Ing. Roggero dam in thespring of 2013 and fall of 2014, and the physico-chemical profile wasdetermined. Assays during 96 h, with controlled temperature and photoperiod (22ºC,16L:8O), 7-day acclimation in moderately hard water (MHW) with ad libitumfeeding. 200 adult C. decemmaculatus from the culture were used; weight107,10 ± 4,26 mg and length 24,89 ± 0,27 mm (median ± ESM)and were distributed to five groups: [1] river water (Rg), [2] river water + 2ppm Cd (RgCd), [3] MHW + 2 ppm Cd (Cd) (positive control metal), [4] MHW + 5ppmcyclophosphamide (CP) (positive control genotoxicity) and [5] MWH (negativecontrol-CN). The assay was carried out by duplicate (n=10/replicate) with amedium renovation at 48 h and continued aeration. At the end of experiment,animals where anaesthetized in cold and blood was drawn. For MN, slides preparationswere fixed with methanol and stained with Giemsa. A count was performed in 1500blood cells and the frequency of micronuclei (MN) and nuclear aberrations (NAs)(notches, nuclear buds and double nuclei) was quantified. For the Cometassay (EC) performed during the fall, one drop of blood was collected ineppendorf with PBS and centrifuged, and the pellet was resuspended withagarose. The electrophoresis was done in pH 13 buffer at 25 mV and 250 mA. Slideswas stained with DAPI and damage was classified for 100 cells (class 0 to IV)and the genomic damage index (IDG) was calculated. The statistic assessment wasperformed using ANOVA or Kruskal Wallis. In the first assay, animals exposed to CP and Cd suffered a significantincrease in MN frequency with respect to CN. ANs in all treatments increasedsignificantly with respect to CN. In the next assay, there was a significantincrease in MNs from animals exposed to CP, Cd and RgCd and no differences wereobserved in ANs. Regarding EC, a significant increase was observed in alltreatments compared to CN in IDG. These results allow us to conclude that,under our experimental conditions, the water sample from Reconquista river ispotentially inductive of genomic instability in blood cells from C.decenmaculatus, which is increased by the aggregate of a polluting pulse ofCd.