Tuttle S W, Hazard L, Koch C J, Mitchell J B, Coleman C N, Biaglow J E
University of Pennsylvania School of Medicine, Philadelphia 19104.
Int J Radiat Oncol Biol Phys. 1994 May 15;29(2):357-62. doi: 10.1016/0360-3016(94)90289-5.
Measurement of pentose cycle (PC) activity is shown to be a noninvasive means for monitoring the reduction of SR-4233 in whole cells. Comparing these measurements to the actual measurements of drug loss under aerobic and hypoxic conditions helps to define the mechanism for the associated aerobic toxicity.
SR-4233 is activated to a toxic species by bioreductive metabolism. NADPH is required for the activation of the drug by purified enzymes, cell homogenates and whole cells. In vivo the NADPH:NADP+ ratio is maintained by the oxidation of glucose via the oxidative limb of the pentose cycle. By measuring radiolabeled 14CO2 released as a product of this oxidation one can get an accurate measurement of the rate of drug metabolism in whole cells. These results are compared to measurements of drug consumption under aerobic and hypoxic conditions using an HPLC assay.
SR-4233 stimulates pentose cycle activity to a greater extent in air then under hypoxia, however, in the presence of added catalase, pentose cycle activity is stimulated to a similar extent under both conditions. The higher levels of PC activity observed in air are due to the production of hydrogen peroxide by the nitroxide free radical undergoing futile redox cycling. The contribution of H2O2 to the observed aerobic cytotoxicity of SR-4233 is minimal however, since toxicity is only slightly reduced in the presence of exogenous catalase and antioxidants such as vitamin E. The level of PC stimulation by SR-4233 suggests that the rate of electron addition to the drug is independent of O2 concentration. The loss of drug from the incubation medium, i.e., conversion to a stable intermediate species, occurs approximately five times faster under nitrogen than in air for A549 cells. It is the rate of drug loss from the cell and not the rate of reduction which best correlates with the observed aerobic and hypoxic toxicity.
Toxicity in air and in nitrogen is directly related to the rate of drug reduction, i.e., at equivalent levels of drug loss we observe equal levels of cytotoxicity.
戊糖循环（PC）活性的测定被证明是监测全细胞中SR - 4233还原的一种非侵入性方法。将这些测量结果与有氧和缺氧条件下药物损失的实际测量结果进行比较，有助于确定相关有氧毒性的机制。
SR - 4233通过生物还原代谢被激活为有毒物质。纯化酶、细胞匀浆和全细胞激活药物需要NADPH。在体内，NADPH:NADP⁺比值通过戊糖循环的氧化分支对葡萄糖的氧化来维持。通过测量作为该氧化产物释放的放射性标记的¹⁴CO₂，可以准确测量全细胞中药物代谢的速率。将这些结果与使用高效液相色谱法测定的有氧和缺氧条件下药物消耗的测量结果进行比较。
与缺氧条件相比，SR - 4233在空气中对戊糖循环活性的刺激程度更大，然而，在添加过氧化氢酶的情况下，两种条件下戊糖循环活性的刺激程度相似。在空气中观察到的较高水平的PC活性是由于氮氧自由基进行无效氧化还原循环产生过氧化氢所致。然而，H₂O₂对观察到的SR - 4233有氧细胞毒性的贡献最小，因为在外源过氧化氢酶和抗氧化剂如维生素E存在的情况下，毒性仅略有降低。SR - 4233对PC的刺激水平表明药物电子添加速率与O₂浓度无关。对于A549细胞，在氮气中孵育培养基中药物的损失，即转化为稳定中间物种的速度，比在空气中快约五倍。与观察到的有氧和缺氧毒性最相关的是药物从细胞中的损失速率，而不是还原速率。
空气中和氮气中的毒性与药物还原速率直接相关，即，在等效的药物损失水平下，我们观察到相等水平的细胞毒性。
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