Adaptation and application of the Comet Assay to genotoxicity assessments in elasmobranchs

The Comet Assay (single-cell gel electrophoresis) is a widely employed genotoxicity biomarker in aquatic ecotoxicology. Its application to elasmobranchs, however, remains extremely limited, with only a single study reported to date. Given the ecological importance and conservation vulnerability of this group, an alkaline Comet Assay protocol was adapted and applied to six tissues (blood, liver, gills, gonads, brain, and kidneys) obtained from four elasmobranch species (Rhizoprionodon porosus, Rhizoprionodon lalandii, Hypanus berthalutzae, and Zapteryx brevirostris). A total of 22 individuals opportunistically obtained from artisanal fisheries along the coast of Paraná, southern Brazil, were analyzed. The protocol was optimized by adjusting blood volume during slide preparation and lysis conditions to account for the distinctive hematological and physiological elasmobranch characteristics, resulting in a standardized method applicable across all tissues and species. Significant differences in DNA damage were detected among tissues in all species (R. lalandii: p = 3.67 × 10^-6; R. porosus: p = 4.18 × 10^-3; H. berthalutzae: p = 5.86 × 10^-3; Z. brevirostris: p = 1.39 × 10^-5). The liver consistently exhibited the highest levels of DNA damage, consistent with its central role in xenobiotic metabolism and contaminant bioaccumulation. Sharks also exhibited greater DNA damage in blood, liver, and gills than batoids, suggesting differences in contaminant exposure and/or physiological susceptibility associated with habitat use and trophic ecology. These findings identify the liver as the most responsive tissue for detecting genotoxic damage in elasmobranchs and establish a standardized Comet Assay protocol for this group, expanding its application as a biomonitoring tool for assessing sublethal environmental contamination effects.