Nitroreduction of Nitrated and C-9 Oxidized Fluorenes in Vitro

Widespread environmental pollution with mutagenic and carcinogenic nitrofluorenes contributes to human health risks. Since nitroreduction leads to activation of many nitro compounds, nitroreduction of the nitrofluorene (NF) derivatives by one- and two-electron reductants was examined. Rates of nitroreduction catalyzed by xanthine oxidase (XO)/hypoxanthine and measured via stimulation of acetylated cytochrome c reduction increased with the number of nitro groups and oxidation at C-9:  9-oxo-2,4,7-triNF > 9-oxo-2,7-diNF > 2,7-diNF > 9-oxo-2-NF = 2,5-diNF > 9-hydroxy-2-NF > 2-NF. Ascorbate catalyzed one-electron reduction to nitro anion radicals which reacted with molecular O<INF>2</INF> to yield superoxide. Rates of O<INF>2</INF> uptake with 9-oxo-2,4,7-triNF and 9-oxo-2,7-diNF were 63 and 0.17 times those, respectively, with equivalent concentrations of nitrofurazone, a classical substrate. Superoxide formation was indicated by the ~75% regeneration of O<INF>2</INF> upon addition of superoxide dismutase and catalase. 9-Oxo-2,4,7-triNF stimulated O<INF>2</INF> uptake in the presence of XO/NADH with typical Michaelis−Menten kinetics with an apparent K<INF>m</INF> of 0.476 ± 0.054 μM versus a K<INF>m</INF> of 6.18 ± 0.719 μM for nitrofurazone. HPLC analyses of products from reduction catalyzed by XO or diaphorase of Clostridium with NADH showed the following trends for the rates of amine formation from 9-oxo-2,7-diNF > 2,7-diNF; 9-oxo-2-NF > 9-hydroxy-2-NF > 2-NF; 2,7-diNF > 2-NF; and 9-oxo-2,7-diNF > 9-oxo-2-NF. Little or no amine was formed in 95% O<INF>2</INF>, suggesting O<INF>2</INF>-labile intermediates. The data herein suggest that oxidation at C-9 and multiple nitro groups increase the potential for nitroreduction of the nitrofluorenes in vivo which may lead to genotoxic effects.