RELATIVE IMPORTANCE OF DT-DIAPHORASE AND HYPOXIA IN THE BIOACTIVATION OF EO9 BY HUMAN LUNG-TUMOR CELL-LINES

Purpose: Although a number of bioreductive agents are substrates for purified DT-diaphorase the role of this enzyme in either activation or detoxification of these agents in the whole cell is unclear. The aim of this study was to determine the role of DT-diaphorase in the metabolic activation of EO9 under both aerobic and hypoxic conditions. Methods and Materials: A panel of lung cancer cell lines was used and drug sensitivity was determined by clonogenic or tetrazolium-dye-based assays. Activities of DT-diaphorase, cytochrome P450 and cytochrome b5 reductase were determined spectrophotometrically by following the reduction of cytochrome c. Results: Small-cell lung cancer cell lines showed a 600-fold range in DT-diaphorase activities but levels were much higher in three of the four non-small-cell lines. Activities of cytochromes P450 and b5 reductase were much lower than those of DT-diaphorase and showed much less variation between cell lines. There was no relationship between the activities of any of the enzymes and aerobic sensitivity to SR 4233, BCNU and cis-platin. Under aerobic conditions there was a clear correlation between DT-diaphorase activity and sensitivity to EO9. The small-cell lines were much more resistant to EO9 than the DT-diaphorase rich non-small-cell lines. A doxorubicin resistant variant of one of the small-cell lines (H69LX10) did not show cross resistance to EO9 but did show a small degree (3-fold) of cross resistance to SR 4233. Under hypoxic conditions, cell lines with high levels of DT-diaphorase showed only a small increase in sensitivity to EO9 (1.5-7 fold); cell lines with low levels of activity showed a 10-37-fold increase in sensitivity. Conclusion: These results suggest that under hypoxic conditions, EO9 is metabolized by 1-electron reducing enzymes to a toxic species. This reduction product is oxygen sensitive but a similar degree of activation is obtained under aerobic conditions in cell lines with high levels of 2-electron reducing DT-diaphorase.