This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Quinones are important compounds used in antitumor therapy and to treat other human illnesses. The common feature of these compounds is that these are prone to be reduced chemically, photochemically or enzymatically in biological media to produce reactive intermediates and products. Upon reduction of these compounds, superoxide and nitrosyl production rates are enhanced in a way which depends on the quinone one-electron redox potential. In addition, alkylating quinones are also activated to covalently bind DMA upon reduction under hypoxic conditions. In our previous years we found that aziridinylquinones can be reduced by photosensitization, under anoxia, followed by DNA covalent binding. We also found that in the presence of a sacrificial electron donor such as hypoxanthine, an increase in both quinone reduction and covalent adducts to DNA were detected. Since electron donors could shift the photosensitized production of singlet oxygen to superoxide and since both superoxide and hydrogen peroxide can travel within tissues farther away than singlet oxygen to produce damage, we will deal here with a study on how the hydrophobicity/hydrophilicity of dyes and electron donors play interacting roles in the relative production of singlet oxygen and superoxide. Since many tumors overexpress folate receptors (FR), one way to reduce quinone toxicity to normal tissues, and increase toxic damage to tumor, is to deliver selectively quinones to FR-overexpressing tumors. The present research proposal is also aimed at exploring new potential pathways in quinone-enhanced toxicity initiated by quinone reduction using FR-targeted quinones. FRtargeted quinones will be synthesized and their abilities to catalyze the production of reactive oxygen species and DNA and protein alkylation will be determined.
Specific Aims : 1. To determine the effects of dyes and sacrificial electron donors hydrophobicities/hydrophilicities in lipid/buffer systems on the relative yields of superoxide and singlet oxygen. 2. To synthesize FR-targeted quinones and an FR-targeted etoposide quinone derivative. 3. To characterize the semiquinones and the rate of reactive oxygen species (ROS) production upon enzymatic and photosensitized reduction of FR-targeted quinones. 4. To determine if photosensitized FR-targeted aziridinylquinone-DNA covalent binding occurs, using red-absorbing dyes. 5. To determine if the FR-targeted etoposide quinone derivative binds to protein thiols and DNA bases. 6. To determine FR-targeted semiquinone and ROS production in HeLa and non-FRoverexpressing cells, using EPR spectroscopy, and whether or not the production of these species is related to FR binding. 7. To determine FR-targeted quinone derivative cytotoxic activity in HeLa and non-FR overexpressing cells.
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