DT-DIAPHORASE PROTECTS CELLS FROM THE HYPOXIC CYTOTOXICITY OF INDOLOQUINONE EO9

Aerobic sensitivity to indoloquinone EO9 has been shown to correlate w ith cellular levels of the two-electron reducing enzyme DT-diaphorase. However, little is known about the relative roles of one- and two-ele ctron reducing enzymes in the hypoxic cytotoxicity of EO9. We have cha racterised a panel of 23 human tumour cell lines for both bioreductive enzyme activities and aerobic sensitivity to EO9. Eight cell lines we re then selected for a comparison of aerobic and hypoxic sensitivities . Activities of DT-diaphorase showed a wide range (> 10,000-fold), whi le activities of the one-electron reducing cytochrome b5 and cytochrom e P450 reductases were generally lower and showed only a 15- and 25-fo ld range respectively. The aerobic cytotoxicity of EO9 was clearly rel ated to the cellular levels of DT-diaphorase (r = 0.87), with higher l evels giving increased sensitivity, but not to the levels of one-elect ron reducing enzymes. In contrast, there was no relationship between s ensitivity to BCNU, cisplatin or the bioreductive agent SR 4233 (tirap azamine) and activities of any of these reducing enzymes. Under hypoxi c conditions sensitivity to EO9 was markedly increased in cell lines w ith low levels of DT-diaphorase activity, while cell lines with high l evels show only a small increase in sensitivity. This is reflected by a clear correlation (r = 0.98) between cellular DT-diaphorase activity and the ratio of aerobic to hypoxic sensitivity to EO9. However, we h ave now for the first time demonstrated an inverse correlation (r = 0. 93) between the cellular activity of DT-diaphorase and hypoxic sensiti vity to EO9, that is sensitivity decreases with increasing DT-diaphora se activity. Moreover, this correlation was lost when cells were expos ed to drug in the presence of dicoumarol, supporting an involvement of DT-diaphorase in this relationship. These observations question the p reviously straightforward role for DT-diaphorase in the metabolic acti vation of EO9. Whereas DT-diaphorase is associated with increased toxi city in air, it appears to reduce the cytotoxicity of EO9 in hypoxic c onditions. This suggests either that the one-electron reduction produc t of EO9 metabolism, the semiquinone, is more toxic than the two-elect ron reduction product, the hydroquinone, or that the hydroquinone is n ot cytotoxic and aerobic toxicity is due to the transient appearance o f the semiquinone upon back oxidation of the hydroquinone.