This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Project 3 is developing methods to quantify oxidative modifications in DNA, proteins and metabolites. This year we have focused on introducing 14C isotope labels into biological molecules in order to characterize pathways and biomarkers of relevance to human disease. This labeling is done by either dosing cells or animals with a labeled building block such as an amino acid for incorporation into proteins or a nucleoside for synthesis of DNA. The presence of an oxidized nucleoside, 8-oxodG, in DNA acts a indicator of oxidative stress and it is more sensitive to further oxidation compared to the normal nucleosides G, C, A and T. For the DNA oxidation studies, we synthesized a 14C labeled 8-oxodG nucleoside. We dosed growing MCF-7 human breast cancer cells with the [14C]8-oxodG compound and monitored uptake of 14C into the newly synthesized DNA. Growth of these labeled cells in the presence of the hormone 17-?-estradiol (a naturally occurring carcinogen that plays a role in breast cell proliferation) caused further oxidation of the labeled 8-oxodG in the cellular DNA to form several novel mutagenic products. This result indicates that oxidative stress in breast cancer cells induced by hormones can induce the formation of novel types of mutagenic DNA damage that may play a strong role in breast cancer initiation and progression. Additionally, radiocarbon from the labeled 8-oxodG was incorporated into RNA, which indicates that 8-oxodG in the nucleotide pool may be a source for protein truncation or altered function as a result of the oxidized nucleotide interfering with transcription and possibly translation. Collaborators on this project include Jeffrey Gregg at the UC Davis Cancer Center and Cynthia Burrows at the University of Utah Department of Chemistry. This work has generated two manuscripts that were submitted for publication. One is currently under review for publication in the Nuclear Instrumentation Methods, Physics Research B and for the other, peer review is pendingfor publication in Nature. Protein oxidation is relevant to diseases such as arteriosclerosis. The hypothesis that chemical species generated by white blood cells react with DNA, protein and other biomolecules to generate a host of cytotoxic and genotoxic products. One type of oxidative damage arises from myeloperoxidase in activated macrophages that produce hypochlorous acid (HOCl), the active ingredient in bleach, which chlorinates tyrosine residues in proteins. Such tyrosine chlorination events can inactivate the proteins and enzymes. For example, ApoA1, a component of low density lipoprotein known as the good cholesterol , contains a tyrosine residue that when chlorinated, completely abrogates the ability of the protein to bind cholesterol in the blood stream. We are developing a method to quantitate chlorinated tyrosine in proteins in which tyrosine residues are labeled with 14C-containing acetate groups. A change in retention time during chromatography can allow separation of labeled chlorotyrosine-containing peptide fragments from the pristine parent peptides. Last year we hired post-doctoral fellow Dr. Janna Mundt to carryout these studies. She has successfully recapitulated the production of chlorinated peptides and developed a postlabeling protocol that was successful for a model peptide. Once the postlabeling method has been demonstrated on ApoA1 derived from clinical samples (from our collaborator Jay Heinecke at the University of Washington Medical Center) a manuscript will be submitted for publication.
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