Hydroxyl Radical Production by Mouse Epidermal Cell Lines in the Presence of Quinone Anti-Cancer Compounds

The effect of quinonr anti-cancer compounds, 3,6-diaziridinyl-2,5-bis(carboethoxyamino)1,4-benzoquinone (diaziquone or AZQ) and 3,6-dihydro-2,5-bis(aziridmyl)-l,4-benzoquinone (DZQ), on the production of hydroxyl radical (OH) in a series of mouse epidermal cell lines was examined using a new, highly sensitive method that employs a fluorescamine-derivatized nitroxide probe. Cell lines that were examined included the mouse epidermal cell line, JB6 clone 41, and JB6 cells transfected with the human Cu-Zn superoxide dismutase (SOD) genes (SOD3 and SOD15) and human catalase (CAT) genes (CAT13 and CAT10). Bioreduction of the nitroxide probe by these cell lines was insignificant at the cell densities employed in these experiments, and thus did not interfere with the ʹOH measurements. In the presence of low concentrations of AZQ or DZQ (20-200 muM), ʹOH production rates were highest in the JB6 cells, intermediate in the SOD-transfected cells, and lowest in the CAT-transfected cells, illustrating that both superoxide and hydrogen peroxide are involved in the production of ʹOH in these systems. Further experiments in which the addition of exogenous SOD and CAT was employed, as well as measurements of probe incorporation into the cells, indicated that this probe can cross cell membranes and detect ʹOH generated intracellularly. In the presence of 100 muM diethylenetriaminepentaacetic acid (DTPA), the rate of ʹOH production in the presence of 100 muM AZQ is ~4.7 x 10^4 molecules s^-1 cell^-1; as much as 45% of this production appears to originate within the cells. This new method should be broadly applicable to the rapid screening of compounds or treatments thought to induce oxidative stress in mammalian cells.