Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae)

Abstract

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies.

Toxic, cytotoxic, and genotoxic effects of a glyphosate formulation (Roundup®SL–Cosmoflux®411F) in the direct‐developing frogEleutherodactylus johnstonei

“…Neonates of E. johnstonei were highly sensitive (100% mortality) to application rates lower than those used during Colombian aerial spraying program (3.74 kg a.e./ha). This finding highlights the need for herbicide toxicity studies in other amphibian species, Valencia et al, 2011). c Percentage of damaged cells 5 sum of comet types 2, 3, and 4 (after Palus et al, 1999).…”

“…e NC, negative control was a solution of 0.9% NaCl. Valencia et al, 2011). c Percentage of damaged cells 5 Sum of comet types 2, 3, and 4 (after Palus et al, 1999).…”

Detecting genomic damages in the frog Dendropsophus minutus: preserved versus perturbed areas

“…These features make them excellent bioindicators of environmental quality (Ralph and Petras 1997;Gauthier et al 2003;Frenzilli et al 2009;Yin et al 2009;Burlibasa and Gavrila 2011;Valencia et al 2011;Gonçalves et al 2013).…”

Amphibian sperm DNA fragmentation

“…Their highly porous skin coupled with a dependence on water for breeding means they often react adversely to changes in the environment before that of higher order vertebrates Burlibasa and Gavrila 2011). Accordingly, amphibians have been described as a sentinel species for modelling the effects of climate change and environmental damage (Kloas and Lutz 2006;Burlibasa and Gavrila 2011;Valencia et al 2011).…”

“…The integrity of sperm DNA is now recognised as a potential indicator of fertility and is essential for successful syngamy and embryonic development (D'Occhio et al 2007;Gosálvez et al 2015). There have been extremely limited studies that have investigated DNA fragmentation in amphibians, and the majority of these have focused on the effects of environmental toxicity on somatic cells (Wang and Jia 2009;Marselli et al 2010;Valencia et al 2011). To date, only two studies exist reporting DNA fragmentation in amphibian spermatozoa (in African clawed frog; Xenopus laevis; Pollock et al 2015 and in Western clawed frog; Xenopus tropicalis; Morrow et al 2014; poster presented at the World Congress of Reproductive Biology).…”