Analysis of 8-oxo-2`-deoxyguanosine in human bronchoalveolar dells by immunoaffinity liquid chromatography/mass spectrometry

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Dipti Mangal, dipti@mail.med.upenn.edu1, Seon Hwa Lee, seonhwa@spirit.gcrc.upenn.edu2, Jong Heum Park, hmpark@mail.med.upenn.edu3, Trevor M. Penning, penning@pharm.med.upenn.edu4, and Ian A Blair, ian@spirit.gcrc.upenn.edu2. (1) Department of Pharmacology, Center for Cancer Pharmacology, University of Pennsylvania, 421, Curie Blvd, 846, Biomedical Research Building, Philadelphia, PA 19104, (2) Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, University of Pennsylvania, Biomedical Research Building, 421 Curie Boulevard, Philadelphia, PA 19104, (3) Center of Excellence in Environmental Toxicology, Department of Pharmacology, University of Pennsylvania School of Medicine, 135 John Morgan building, 3620 Hamilton Walk, Philadelphia, PA 19104-6084, (4) Deparment of Pharmacology, Univeristy of Pennsylvania, 130c John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104-6084
8-Oxo-2'-deoxyguanosine(8-oxo-dGuo) is an important pro-mutagenic DNA lesion resulting from oxidative damage. However, accurate quantification of 8-oxo-dGuo is difficult because artifactual formation occurs during the isolation and hydrolysis of DNA from cells and tissue samples. To avoid this problem, a cold guanidine thiocyanate non-phenolic method was used to extract DNA from cells. In addition, Desferal was used in Chelex-treated buffers to remove transition metal ions and prevent artifactual 8-oxo-dGuo formation through Fenton Chemistry Immunoaffinity purification removed interfering substances that co-eluted with 8-oxo-dGuo during subsequent chromatography and stable isotope dilution liquid chromatography multiple reaction monitoring/mass spectrometry (LC-MRM/MS) provided maximal specificity and sensitivity. Human bronchoalveolar cells were treated with potassium bromate, methyl methane sulfonate and polycyclic aromatic hydrocarbon metabolites in order to induce 8-oxo-dGuo formation. A linear dose response curve to potassium bromate was observed by LC-MRM/MS and there was an excellent correlation with the Comet assay when coupled to hOGG1. Supported by NIH P01CA 092537, P30ES013508, and R01CA039504
 

Poster Session and Awards
6:00 PM-10:00 PM, Tuesday, August 21, 2007 BCEC -- 204 A/B, Poster

Division of Chemical Toxicology

The 234th ACS National Meeting, Boston, MA, August 19-23, 2007