Roles of radicals in cancer research: Potential therapeutic agents and probes for studying carcinogenesis.

Unpaired electrons (radicals) have been implicated in numerous disease processes. They have also been proven useful in the study of intra- and intercellular processes. This work describes two approaches in the study of radicals. The first project involves the synthesis of a spin probe utilizing a stable nitroxide radical to probe DNA structures; the second includes studies of aryl radicals derived from arenediazonium ions found in common foods and other products. Formation of novel DNA structures such as triplex DNA has been associated with inhibited gene transcription and DNA replication. Electron Spin Resonance (ESR) has been used to study spin-labeled nucleic acids from the small oligonucleotide to the cellular level. We have improved the method for synthesis of a suitable nitroxide probe used to study the dynamics of double-stranded (ds) DNA, and we have incorporated a thymidine analog bearing this spin probe in a 15-mer triplex-forming oligonucleotide (TFO). The TFO was annealed to a ds DNA molecule, and formation of triplex DNA was verified by ESR. Isotropic and anisotropic simulations of the ESR data have been utilized to quantify the association of this TFO with its target sequence. The correlation of the simulation with the experimental data demonstrate an equilibrium favoring the association of the TFO with ds DNA. Arylhydrazines carcinogenesis remains poorly understood, although many arylhydrazines are carcinogenic. Arylhydrazines are metabolized to arenediazonium ions which yield C8-arylpurine adducts in calf thymus (ct) and cellular DNA. C8-Arylguanine adducts likely form from direct aryl radical addition to the C8 position of guanine. We demonstrated that C8-aryladenine is likely formed via an intermediate aryltriazene. ESR spin-trapping studies demonstrated the intermediacy of aryl radicals during decomposition of both N6-aryltriazene purines and arenediazonium treated ct DNA. The formation of these adducts results insignificant depurination. These metabolic processes also produce reactive oxygen species implicated in AP-1 activation. JB6 cells stably transfected with the AP-1 luciferase reporter plasmid were treated with a series of arenediazonium ions. Using luminometer analysis, ESR and Western blot analysis, the effect of arenediazonium ion on AP-1 activation and signal transduction was studied