HPLC Determination of Oxidative DNA Damage in Rat Liver Perfusates Influenced by Addition of Copper

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C. Mišľanováa,b, A. Kebisc, J. Príbojováa, and M. Valachovičováa

aFaculty of Nursing and Professional Health Studies, Slovak Medical University in Bratislava, bDepartment of Analytical Chemistry, Faculty of Natural Sciences, Comenius University, Bratislava, cFaculty of Public Health, Slovak Medical University in Bratislava

Keywords: electrochemical detection, HPLC, 8-oxodG, liver, DNA damage

 

HPLC determination of a background level of 8 oxodG and 2-dG in rat liver after addition of copper to perfusate was developed. The reversed phase analytical column Purospher® STAR C18e with 50 mmol L–1 phosphate buffer, pH 5.5 and methanol (92:8, v/v) mobile phase was applied for the analysis. The validation of the HPLC method according to linearity, accuracy and precision was carried out. Oxidative DNA damage (expressed as concentration ratio of 8-oxodG/106 2-dG) was determined by the simultaneous measuring of 2-dG with UV detection followed by coulochemical detection of 8 oxodG. The procedure using a model of liver damage caused by intoxication with copper and ischemia / reperfusion with addition of various concentrations of CuSO4 to the perfused rat livers was tested. The aim of this study was to decide whether the toxicity of copper in liver perfusates is related to protein oxidation and oxidative DNA damage. The high contribution to the DNA damage can be related to the physical liver manipulation during harvest and reperfusion as well as to artefacts induced during the sample preparation (time-consuming sample handling during DNA isolation and extraction). The obtained results pointed out that the DNA damage occurred already during liver handling even before application of CuSO4, whereby concentration of CuSO4 higher than 0.03 mmol L–1 caused a total liver damage, which led to a complete stop of the flow of the perfusate.

 

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