O'Donnell V B, Spycher S, Azzi A
Institute of Biochemistry and Molecular Biology, University of Bern, Switzerland.
Biochem J. 1995 Aug 15;310 ( Pt 1)(Pt 1):133-41. doi: 10.1042/bj3100133.
Cellular signalling by the inflammatory cytokine tumour necrosis factor alpha (TNF alpha) has been suggested to involve generation of low levels of reactive oxygen species (ROS). Certain antioxidants and metal chelators can inhibit cytotoxicity and gene expression in response to TNF alpha in numerous cell types. However, neither the source nor function of TNF alpha-induced oxidant generation is known. Using specific inhibitors, we ruled out involvement of several oxidant-generating enzymes [cyclo-oxygenase (indomethacin), cytochrome P-450 (metyrapone), nitric oxide synthase (NG-methyl-L-arginine), NADPH oxidase (iodonium diphenyl), xanthine oxidase (allopurinol), ribonucleotide reductase (hydroxyurea)] in TNF alpha-mediated apoptosis of the murine fibrosarcoma line, L929. We also demonstrated no role for mitochondrial-derived radicals/respiratory chain in the lytic pathway using specific inhibitors/uncouplers (rotenone, KCN, carboxin, fluoroacetate, antimycin, malonate, carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and chloramphenicol-derived respiration-deficient cells. Significant ROS (H2O2, O2-.) generation was not observed in response to TNF alpha in L929 cells using four separate assays. Also, prevention of intracellular H2O2 removal by inhibition of catalase did not potentiate TNF alpha-mediated cell death. These data suggest that neither H2O2 nor O2-. plays a direct role in TNF alpha cytotoxicity. Finally, we suggest a central role for lipoxygenase in TNF alpha-mediated lysis. Three inhibitors of this radical-generating signalling pathway, including an arachidonate analogue (5,8,11,14-eicosatetraynoic acid), could protect cells against TNF alpha. The inhibitor nordihydroguaiaretic acid is also a radical scavenger, but it could not protect cells from ROS toxicity at concentrations that effectively prevented TNF alpha killing. Therefore protection by nordihydroguaiaretic acid cannot be due to scavenging of cytotoxic H2O or O2-.. The lipoxygenase product, (12S)-hydroxyeicosatetraenoic acid, was also significantly protective. As this analogue can act as a substrate for certain lipoxygenases, this effect may be due to prevention of generation of physiological products.
炎症细胞因子肿瘤坏死因子α（TNFα）的细胞信号传导被认为涉及低水平活性氧（ROS）的产生。某些抗氧化剂和金属螯合剂可抑制多种细胞类型中TNFα诱导的细胞毒性和基因表达。然而，TNFα诱导的氧化剂产生的来源和功能尚不清楚。我们使用特异性抑制剂排除了几种氧化剂生成酶[环氧化酶（吲哚美辛）、细胞色素P-450（甲吡酮）、一氧化氮合酶（NG-甲基-L-精氨酸）、NADPH氧化酶（二苯基碘鎓）、黄嘌呤氧化酶（别嘌呤醇）、核糖核苷酸还原酶（羟基脲）]参与小鼠纤维肉瘤细胞系L929的TNFα介导的凋亡。我们还使用特异性抑制剂/解偶联剂（鱼藤酮、KCN、羧基青霉素、氟乙酸、抗霉素、丙二酸、羰基氰对三氟甲氧基苯基腙）和氯霉素诱导的呼吸缺陷细胞证明线粒体衍生的自由基/呼吸链在裂解途径中不起作用。使用四种不同的测定方法，未观察到L929细胞中TNFα诱导的显著ROS（H2O2、O2-）产生。此外，通过抑制过氧化氢酶来阻止细胞内H2O2的清除并不能增强TNFα介导的细胞死亡。这些数据表明H2O2和O2-在TNFα细胞毒性中均不发挥直接作用。最后，我们认为脂氧合酶在TNFα介导裂解中起核心作用。该自由基生成信号通路的三种抑制剂，包括一种花生四烯酸类似物（5,8,11,14-二十碳四炔酸），可保护细胞免受TNFα的损伤。抑制剂去甲二氢愈创木酸也是一种自由基清除剂，但在有效阻止TNFα杀伤的浓度下，它不能保护细胞免受ROS毒性。因此，去甲二氢愈创木酸的保护作用不能归因于清除细胞毒性的H2O或O2-。脂氧合酶产物（12S）-羟基二十碳四烯酸也具有显著的保护作用。由于这种类似物可作为某些脂氧合酶的底物，这种作用可能是由于阻止了生理产物的产生。
