Quinones represent an important class of naturally occurring compounds that exhibit diverse biological (re)activity. The quinones of polycyclic hydrocarbons are prevalent as environmental contaminants and are present in automobile exhaust, cigarette smoke and air particulates. In addition, many foodstuffs contain quinones and many anti-cancer drugs of clinical and research interest contain the quinone nucleus. Quinones may also be mutagenic and carcinogenic. The reactivity of quinones resides in their ability to undergo """"""""redox-cycling"""""""" and to thereby create an oxidative stress and/or to react directly with cellular nucleophiles such as protein and non-protein sulfhydryls. Glutathione (GSH) is the major non-protein sulfhydryl present in cells although there are relatively few studies on the addition of sulfur nucleophiles to quinones and especially of the biological consequences of these reactions. In contrast to the generally accepted role of GSH conjugation as a detoxication reaction, we have shown that conjugation of quinones with GSH results in the formation of potent, and selective, nephrotoxicants. Moreover, there is now substantial evidence indicating that quinone-thioethers exhibit a variety of toxicological activity. The ubiquitous nature of quinones and the high concentrations of GSH within cells virtually guarantees that human exposure to quinone-thiothers will occur. Since the molecular mechanisms(s) by which the quinone-thioethers produce their adverse effects are unknown the present application will address this deficiency in our knowledge. We have established a comprehensive understanding of the metabolic disposition of the quinone-thioethers, and especially of those biotransformations that regulate the inherent reactivity of these compounds. We now plan to integrate our knowledge on quinone-thioether metabolism to the cellular and molecular events that lead to toxicity. Our Preliminary Data indicate that macromolecular arylation plays a role in 2-Br-(diGSyl)HQ-mediated nephrotoxicity. Immunochemical techniques will be employed to identify macromolecular targets of 2-Br-(diGSyl)-HQ, and their role in the initiation and progression of cytotoxicity. In addition, the role of quinone-thioether catalyzed reactive oxygen generation will be investigated. Preliminary Data also implicate a role for H2O2 generation and DNA damage as initial events in 2-Br-(diGSyl)HQ- mediated cytotoxicity. We will therefore address the mechanism of 2-Br- (diGSyl)HQ-mediated DNA damage, and the role of hydrogen peroxide, iron redistribution and changes in Ca2+ and pH regulation in 2-Br-(diGSyl)HQ- mediated cytotoxicity. Damaged proteins and reactive oxygen species induce the transcription of a number of stress-related genes. Preliminary Data in this area indicate that 2-Br-(diGSyl)HQ regulates the expression of specific DNA-damage inducible genes and other acute stress response genes. Another aim of our proposed studies will address the mechanism of this regulation of gene expression and the toxicological consequences thereof. We believe we have a sound foundation from which to progress with the studies detailed in this application.
