Estimation of genomic DNA damage in populations of the marsh frog (Pelophylax ridibundus Pallas, 1771) of the Belgorod agglomeration by DNA comet assay

Anthropogenic pollution of water bodies is a serious problem for the sustainability of aquatic ecosystems. The main sources of pollution of water bodies are wastewater, washouts from agricultural fertilizers, pesticides and roads. Many of highly persistent pollutants are dangerous mutagens and carcinogens. The disruption they cause to the DNA and RNA structure and properties of organisms can have long-term consequences and can affect the livelihood of future generations. Among existing DNA damage estimation methods, DNA comet assay is the most widespread due to its accuracy and reliability. In our study, the marsh frog (Pelophylax ridibundus Pallas, 1771) was chosen as an object of study. This species is adapted to life under anthropogenic influence and actively inhabits transformed biotopes. The aim of our work was to estimate the level of DNA damage by DNA comet assay of P. ridibundus inhabiting Belgorod city and its suburbs. We studied a total of 6 marsh frog populations, with collection sites differing in the degree of anthropogenic pressure (See Table 1 and Fig. 1), and we selected 5 individuals of P. ridibundus from each locality. The experimental animals were handled in accordance with international ethical standards. Animal liver was used as the material for the study and analysis was performed on the day of collection. Tissue was homogenized in phosphate-salt buffer (pH 7.5) containing 20 mM EDTA-Na2 and 10% dimethyl sulfoxide (DMSO) at +4 °C. To precipitate large fragments, tubes were incubated for 5 min at room temperature before transferring 1.5 ml of the top layer to microcentrifuge tubes and centrifuged in a 5415 R Eppendorf microcentrifuge (Germany) at 1000 g for 10 min at +4 °C. The supernatant was removed and the cell precipitate was diluted in 1 ml of phosphate salt buffer cooled to +4 °C. To obtain microarrays, 60 pl of cell suspension was mixed with 240 pl of 1% fusible agarose solution (t<42 °C). The lysis was performed for at least one hour at +4 °C (lysis buffer: 10mM Tris-HCl (pH 10) 2.5M NaCl, 100 mM EDTA-Na2 and 1% Triton X-100 with 10% DMSO). Electrophoresis was performed in a darkened room in Tris-EDTA-borate electrode buffer (TBE) for 20 min at a voltage of 1V/cm. At the end of electrophoresis, the preparations were fixed with 70% ethanol for 15 min. The preparations were stained with SYBR Creen I dye, with subsequent analysis using a luminescent trinocular microscope MIKMED-2 of Lomo (Russia), with at least 100 cells analyzed from each preparation, which were divided into 5 types; cells in the state of apoptosis were counted separately (Durnev, et al., 2006). The degree of DNA damage was estimated using the Kruskel-Wallis criterion (“DNA comet index”) (Struwe et al., 2007; Filippov, 2014). To determine the statistical significance of differences between the studied populations we used one-way analysis of variance (ANOVA); statistically significant differences were considered atp < 0.05. According to the data obtained, in all studied populations, the mean value of the DNA damage index (DDI) did not exceed the first threshold of DNA degradation (See Table 2). This may indicate, on the one hand, the absence of high concentrations of genotoxic pollutants here (which is highly doubtful) and, on the other hand, the efficient operation of the reparative system of marsh frog. Nevertheless, the calculated DDI values made it possible to construct a gradational scale of DNA destruction level in the studied groups of frogs depending on the degree of pollution and proximity of residential and industrial buildings (See Table 2-4). Various chemical compounds from industrial production, particularly nitrogen compounds, are known to have genotoxic effects. Due to the lack of possibility to perform chemical analysis of water to identify concentrations of genotoxic pollutants, we analysed data on surface water pollution in some of the investigated locations from open literature sources. As a result of this analysis, we revealed the dynamics of surface water pollution. All investigated points by the mean value of the statistical DDI surpassed reliably the least polluted site “Sevrukovo”, which is located in the green zone and at a considerable distance from urbanized areas (See Table 2-4). High values of DDI were fixed in a zone of industrial and multi-storey buildings, localities “Seversky Donets” (DDI = 0.454) and “Veselka” (DDI = 0.416). Exceeding maximum permissible concentration of nitrogen compounds is observed in the above mentioned rivers. Noteworthy is the fact that the highest value of the DDI (0.521) was recorded in the estuary of the Razumnaya River remote from the industrial part of the city (“Razumnoye”). It should be noted that in this locality there is the greatest number of damaged cells, including apoptosis (See Table 2, Fig. 2 and 3). The highest DDI value is due to the high level of pollutants, since the predominant part of the river is occupied by “non-sewered” private low-rise buildings, as well as arable land. At the same time in the upper part of the river (“Sevryukovo”) due to the lack of obvious pollutants and the intensive current contributing to self-purification of the river, we recorded the minimum value of the DDI (0.057) and the absence of cells in the state of apoptosis. Thus, the present study revealed an increase in the degree of DNA destruction in populations of marsh frogs in the impacted areas of the city of Belgorod and its surroundings. At the same time, increased levels of toxic substances may contribute to a decrease in the viability of individuals of both analyzed species and other hydrobiont species, which will negatively affect the biodiversity of the region as a whole. The paper contains 3 Figures, 4 Tables and 42 References. Abbreviations: DDI - DNA damage index (comet assay)]; MPC - Maximum permissible concentration [ПДК In Russian]. The Authors declare no conflict of interest.

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