Abstract
Defective copper excretion from hepatocytes in Wilson's disease causes accumulation of copper ions with increased generation of reactive oxygen species via the Fenton-type reaction. Here we developed a nanoflow liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry coupled with the isotope-dilution method for the simultaneous quantification of oxidatively induced DNA modifications. This method enabled measurement, in microgram quantities of DNA, of four oxidative stress-induced lesions, including direct ROS-induced purine cyclonucleosides (cPus) and two exocyclic adducts induced by byproducts of lipid peroxidation, i.e. 1,N(6)-etheno-2'-deoxyadenosine (εdA) and 1,N(2)-etheno-2'-deoxyguanosine (εdG). Analysis of liver tissues of Long-Evans Cinnamon rats, which constitute an animal model of human Wilson's disease, and their healthy counterparts [i.e. Long-Evans Agouti rats] showed significantly higher levels of all four DNA lesions in Long-Evans Cinnamon than Long-Evans Agouti rats. Moreover, cPus were present at much higher levels than εdA and εdG lesions. In contrast, the level of 5-hydroxymethyl-2'-deoxycytidine (5-HmdC), an oxidation product of 5-methyl-2'-deoxycytidine (5-mdC), was markedly lower in the liver tissues of Long-Evans Cinnamon than Long-Evans Agouti rats, though no differences were observed for the levels of 5-mdC. In vitro biochemical assay showed that Cu(2+) ions could directly inhibit the activity of Tet enzymes. Together, these results suggest that aberrant copper accumulation may perturb genomic stability by elevating oxidatively induced DNA lesions, and by altering epigenetic pathways of gene regulation.
Highlights
From the ‡Environmental Toxicology Graduate Program and §Department of Chemistry, University of California, Riverside, California 92521; ¶Department of Medicine and ʈDepartment of Pathology, **Marion Bessin Liver Research Center, Diabetes Center, Cancer Center, and Ruth L. and David S
NanoLC-NSI/MS2 Method for Quantifying Oxidative Stressinduced DNA Lesions in Rat Tissues—Relatively large amounts of unmodified nucleosides and buffer salts are present in the enzymatic digestion mixture of tissue DNA, which can significantly interfere with the detection of low levels of DNA lesions
We employed off-line HPLC separation to enrich DNA lesions of interest from the digestion mixtures prior to NanoLC-NSI/MS2 analysis
Summary
From the ‡Environmental Toxicology Graduate Program and §Department of Chemistry, University of California, Riverside, California 92521; ¶Department of Medicine and ʈDepartment of Pathology, **Marion Bessin Liver Research Center, Diabetes Center, Cancer Center, and Ruth L. and David S. Oxidative DNA Modifications and Wilson’s Disease as iron or copper, may result in diseases In this vein, Wilson’s disease (WD) is an autosomal recessive genetic disorder arising from mutations in the P-type ATPase gene, ATP7B, and patients suffering from this disease are manifested by defective excretion of copper ions into bile [26, 27]. Wilson’s disease (WD) is an autosomal recessive genetic disorder arising from mutations in the P-type ATPase gene, ATP7B, and patients suffering from this disease are manifested by defective excretion of copper ions into bile [26, 27] This leads to accumulation of copper in the body with progressive damage in liver, brain and kidneys [26, 27]. The LEC rat serves as an excellent animal model for studying the pathophysiology of human Wilson’s disease
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