Investigation of DNA damage in Rana macrocnemis (amphibia: anura) during hibernation

INVESTIGATION OF DNA DAMAGE IN RANA MACROCNEMIS (AMPHIBIA: ANURA) DURING HIBERNATION Deoxyribonucleic acid (DNA) is inherently susceptible to damage from a multitude of endogenous and exogenous factors, which can compromise its integrity and, consequently, vital cellular functions. Genotoxic insults, including reactive oxygen species (ROS) generated as metabolic byproducts, ionizing radiation, environmental toxins, and thermal fluctuations, can induce a spectrum of lesions such as strand breaks, base modifications, and other structural aberrations. To quantitatively evaluate DNA damage, this study employed the Single Cell Gel Electrophoresis technique, widely known as the Comet Assay. This method was selected for its high sensitivity, technical practicality, and cost-effectiveness. Peripheral blood samples were collected from a cohort of 15 individuals of the anuran species Rana macrocnemis. Subsequently, the samples were embedded in a layer of low-melting-point agarose on microscope slides. Cell membranes and nuclear components were solubilized using a standard alkaline lysis buffer to liberate the DNA. Following lysis, the slides were subjected to electrophoresis under high pH conditions. This process causes negatively charged fragmented DNA to migrate away from the nuclear core (the "head") towards the anode, forming a characteristic "comet tail." The resulting nucleoids were stained with a fluorescent DNA-binding dye and visualized via fluorescence microscopy. DNA damage was quantified using four standard parameters: Comet Length (CL), Tail Length (TL), the percentage of DNA in the tail (%Tail DNA), and Olive Tail Moment (OTM), which integrates both the tail length and the distribution of DNA within it. In contrast to the physiological metrics, the Comet Assay revealed a profound genotoxic effect of hibernation. Post-hibernation analysis demonstrated statistically highly significant increases (P <0.001) in all measured DNA damage parameters: CL, TL, %Tail DNA, and OTM. The validity of the assay was confirmed by the positive control group, which exhibited the maximum level of DNA damage, thereby verifying the protocol's efficacy in detecting genotoxicity. The study also documented physiological changes associated with the pre-hibernation phase in R. macrocnemis. Individuals exhibited marked behavioral quiescence, adduction of the limbs towards the body, and a pronounced spherical body posture. A comparative analysis of morphometric data (body mass and snout-vent length) before and after the hibernation period indicated a trend toward reduction; however, these differences were not statistically significant (P> 0.05). The findings indicate that while hibernation in R. macrocnemis results in only minimal and non-significant changes in gross physiological parameters, it imposes significant molecular stress, culminating in substantial DNA fragmentation. This stress impact is likely attributable to metabolic suppression and a concomitant surge in the production of reactive oxygen species (ROS), particularly during the energetically demanding arousal phases. These results underscore that hibernation is not solely an adaptive energy-conserving state but also represents a period of considerable oxidative and genotoxic challenge. The study elucidates the association between hibernation physiology and molecular stress biomarkers in amphibians, providing critical insights into the overwintering strategies and subcellular vulnerability of R. macrocnemis inhabiting high-altitude ecosystems in Turkey.