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

Laura Carolina Valencia,Martha Patricia Ramírez-Pinilla,Jorge Luis Fuentes,Adriana García

doi:10.1590/s1415-47572011005000048

Laura Carolina Valencia, Martha Patricia Ramírez-Pinilla + Show 2 more

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https://doi.org/10.1590/s1415-47572011005000048

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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.

Highlights

Amphibians are useful biological indicators because their sensitive skins and occurrence in aquatic and terrestrial habitats makes them vulnerable to environmental change (Blaustein and Wake, 1990; Lips, 1998)
Bleomycin (BML), dimethyl sulfoxide (DMSO), phosphotungstic acid, molecular grade and low gel temperature (LGT) agaroses, 4-nitroquinoline-1-oxide (4-NQO), silver nitrate and sodium N-lauryl sarcosine were obtained from Sigma-Aldrich Co
Since naked nuclei could not be obtained from E. johnstonei blood cells by the standard Comet procedure (Singh et al, 1988), we compared the ability of alkaline, enzymatic and alkaline/enzymatic treatments to produce these nuclei, as assessed by the DNA diffusion assay (Singh, 2000a)

Summary

Introduction

Amphibians are useful biological indicators because their sensitive skins and occurrence in aquatic and terrestrial habitats makes them vulnerable to environmental change (Blaustein and Wake, 1990; Lips, 1998). Eleutherodactylus johnstonei (Anura: Eleutherodactylidae) is a direct-developing frog (Hedges et al, 2008) from the Lesser Antilles with recently established populations in several Caribbean islands (Kaiser, 1997), as well as in Central and South America (Ortega et al, 2001; Kaiser et al., 2002). As a consequence of its widespread distribution and certain life history features (reproductive flexibility and high environmental adaptability) (Ortega et al, 2005), E. jonhstonei has been considered a successful invasive species (Rödder, 2009). These attributes suggest that this species could be a useful model for evaluating the genotoxicological impact of environmental xenobiotics such as pesticides

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