Müller T, Gebel S
INBIFO Institut für Biologische Forschung, Köln, Germany.
Carcinogenesis. 1994 Jan;15(1):67-72. doi: 10.1093/carcin/15.1.67.
A dose-dependent and transiently elevated expression of a cytoplasmic 32 kDa protein was observed in Swiss albino 3T3 fibroblasts exposed to mainstream cigarette smoke (CS) trapped in phosphate-buffered saline solutions (smoke-bubbled PBS). The protein was identified as heme oxygenase (HO) (heme, hydrogen donor:oxygen oxidoreductase, EC 1.14.99.3) by Western blotting using an anti-rodent HO-specific antibody. Kinetic investigations revealed that HO protein and its mRNA were detectable in smoke-bubbled PBS-treated cells between 1 and 24 h after exposure to 0.03 puffs (approximately 1 cm3) CS per ml medium. As a result of transcriptional activation, a nearly 50-fold increase in the amount of HO mRNA was determined after 8 h exposure compared to control levels. Since literature data indicate that there is a link between glutathione depletion and HO expression, the same was assumed for cells exposed to smoke-bubbled PBS, as a decrease of more than 60% in glutathione levels was observed after the exposure. This was further supported by the observation that no elevated amounts of HO mRNA appeared in smoke-bubbled PBS-treated cells when cysteine was exogenously added. However, although these effects may be attributable to the formation of hydroxyl radicals (which have been shown to induce HO and to deplete glutathione levels and which appear in aqueous smoke-containing solutions via the iron-catalysed Fenton reaction) neither catalase nor the iron cation chelating agent o-phenanthroline were able to suppress or even to reduce HO expression in smoke-bubbled PBS-treated cells. On the contrary, at comparable concentrations both compounds were found to be potent inhibitors of smoke-dependent DNA strand breaks. Hence, reactive species other than Fenton reaction-derived hydroxyl radicals are responsible for the effects observed in the present study.
在暴露于困于磷酸盐缓冲盐溶液（烟熏气泡PBS）中的主流香烟烟雾（CS）的瑞士白化病3T3成纤维细胞中，观察到一种细胞质32 kDa蛋白的剂量依赖性和瞬时升高表达。通过使用抗啮齿动物HO特异性抗体的蛋白质印迹法，该蛋白被鉴定为血红素加氧酶（HO）（血红素，氢供体：氧氧化还原酶，EC 1.14.99.3）。动力学研究表明，在每毫升培养基暴露于0.03口（约1立方厘米）CS后1至24小时内，在烟熏气泡PBS处理的细胞中可检测到HO蛋白及其mRNA。由于转录激活，与对照水平相比，暴露8小时后HO mRNA的量增加了近50倍。由于文献数据表明谷胱甘肽消耗与HO表达之间存在联系，因此对于暴露于烟熏气泡PBS的细胞也作了同样的假设，因为暴露后观察到谷胱甘肽水平下降了60%以上。当外源添加半胱氨酸时，烟熏气泡PBS处理的细胞中未出现HO mRNA量升高的现象，这进一步支持了上述假设。然而，尽管这些效应可能归因于羟基自由基的形成（已证明其可诱导HO并消耗谷胱甘肽水平，且通过铁催化的芬顿反应出现在含烟雾的水溶液中），但过氧化氢酶和铁阳离子螯合剂邻菲罗啉均不能抑制甚至降低烟熏气泡PBS处理细胞中的HO表达。相反，在可比浓度下，发现这两种化合物都是烟雾依赖性DNA链断裂的有效抑制剂。因此，本研究中观察到的效应是由芬顿反应衍生的羟基自由基以外的活性物质引起的。
