engleski

DNA damage and morphological changes to symbiotic and free-living hydra caused by norflurazon and UVB radiation

Individuals of symbiotic green hydra (Hydra viridissima Pallas, 1766) and free-living brown hydra (Hydra oligactis Pallas, 1766) were treated with aqueous solutions of herbicide norflurazon (2x10-7, 2x10-6 mol/L) for 72 h and hydras were also exposed to simultaneous treatment to norflurazon and UVB radiation (λ = 254 nm, 2 min), along with control groups. The experiment was performed in triplicate. Migration and change of body shape (relaxed, contracted), as the evasion signs of deleterious effects, were observed by stereo microscope, and responses to mechanical stimuli were also noted. Comet assay was applied to measure the level of primary DNA damage of exposed green and brown hydras. Beside single and double strand breaks, it detects DNA base alkylation, oxidatively changed bases, DNA-adducts, covalently bound proteins to DNA, cross-linking and base dimers. Each hydra was put into a conical tube containing chilled homogenization buffer (75 mM NaCl, 24 mM Na2EDTA, pH 7.5, +4 °C) and its cells were separated mechanically. The obtained homogenate was mixed with the low melting point agarose, spread across the microscopic slide and left for the gel to polymerize. Preparations were treated with a series of buffers to remove the cell structures and isolate nucleoids. By immersing the slides into alkaline buffer, pH 13.0, DNA denatures rapidly. At the spots of primary damage, DNA strands broke, which led to the formation of relaxed loops and free DNA fragments. In electrophoresis, these loops and fragments are pulled from the nucleoid core toward the anode, and following fluorescent staining, under the microscope, they appear as comets. Comet preparations were analysed under an epifluorescent microscope equipped with CCD camera, and the level of damage was measured by image analysis software for the comet assay. Based on the intensity of fluorescence of the tail compared to the nucleoid, the percentage of DNA affected by primary damage was calculated. The treatment did induce a concentration related increase in primary DNA damage, but no significant difference between green and brown hydra was observed (4.40 % vs. 5.23 %, respectively ; negative controls 1.13 % and 1.17 %, respectively). On the other hand, in the presence of radiation, brown hydras showed significantly higher level of primary DNA damage already when exposed to the lowest concentration compared to the green ones (15.10 % and 3.77 %, respectively), proving the advantage of the symbiotic organism. The increase in damage was concentration dependent in either case.

hydra ; comet assay ; morphology ; norflurazon ; UVB radiation

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