Long-term Objectives-To test the hypothesis that cellular two-electron transfer systems-involving DT-diaphorase catalysis and glutathione nucleophilic addition-play a central role in the bioreductive activation of quinonoid compounds and are associated with their cytotoxicity and carcinogenicity as well as with the activation of chemotherapeutic agents. This hypothesis appears tenable since [I] our recent results indicate that the two-electron transfer to quinones-as accomplished by DT-diaphorase and thiol nucleophilic addition-cannot be regarded as a detoxification system, but as a process accompanied in most instances by high production of oxygen radicals. [II] Thiol reactivity is a salient feature of many classes of therapeutic drugs, hence, it should be considered as matter of course when evaluating the metabolic pathways of quinonoid compounds. [III] The cellular role of DT-diaphorase-whose activity is enhanced in transformed cells and preneosplatic nodules-is still controversial and it has been implicated in both the resistance against certain toxins and the metabolic activation of carcinogens.
Specific aims - The aims of this research are directed to an understanding of the molecular mechanisms inherent in the two-electron bioreduction of quinones and their implications for quinone cytotoxicity. The proposed research represents a systematic and comprehensive approach to gather such information and it involves three major specific aims: [1] To determine the effect of substitution patterns on quinone reactivity and cytotoxicity within the framework of biological two-electron transfer systems. [2] To determine the role of DT-diaphorase in the initial reduction and subsequent redox transitions of quinone bioalkylating agents. [3] To determine the toxic effects of quinones on cultured cell lines in terms of [a] the significance of two-electron transfer processes in the activation of quinones, [b] the factors regulating cellular induction of DT-diaphorase and whether or not this induction is accompanied by a parallel induction of superoxide dismutase, and [c] how cells with an induced DT-diaphorase activity regulate quinone metabolism as a function of the physico-chemical properties of the quinonoid compound. Experimental design and methods - [1] Various compounds from the p-benzo- and 1,4-naphthoquinone series, selected on the basis of their substitution pattern, reduction potential, and capability to become reactive electrophiles, will be studied in relation to two-electron transfer processes: DT-diaphorase catalysis and thiol nucleophilic addition. [2] The inborn redox properties of p-benzoquinone and naphthoquinone bioalkylating agents will be assessed in experimental models involving the above-described two-electron transfer systems. [3] Quinone cytotoxicity will be evaluated with C3H/10T1/2 cells -a mouse embryofibroblast line suitable for these investigations- as a function of the induced levels of DT-diaphorase and superoxide dismutase and the chemical reactivity of the quinonoid compounds. Cytotoxicity assays will be performed by measuring reduction in plating efficiency of treated cells. Spector- and fluorometric methods will be used to measure enzyme activity, active oxygen species, glutathione, and glutathione disulfide. ESR with or without spin trapping technique will be used for the detection of oxygen-derived- and thiyl radicals, and semiquinones. HPLC methods with different detection modes will be used as follows: [a] electrochemical detection: determination quinones, hydroquinones, quinone epoxides, quinone-thioether derivatives, and hydroxyl radical. [b] UV detection: determination of glutathione disulfide and glutathione sulfonate.
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