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Hydroxyl Radical Production by Mouse Epidermal Cell Lines in the Presence of Quinone Anti-Cancer Compounds
journal contribution
posted on 1999-10-01, 00:00 authored by Beibei Li, Peter L. Gutierrez, Paul Amstad, Neil V. BloughThe effect of quinone anti-cancer compounds, 3,6-diaziridinyl-2,5-bis(carboethoxyamino)-1,4-benzoquinone (diaziquone or AZQ) and 3,6-dihydro-2,5-bis(aziridinyl)-1,4-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 •OH measurements. In the presence of low
concentrations of AZQ or DZQ (20−200 μM), •OH 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 •OH
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 •OH generated intracellularly. In the presence of
100 μM diethylenetriaminepentaacetic acid (DTPA), the rate of •OH production in the presence
of 100 μM AZQ is ∼4.7 × 104 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.