O'Malley Yunxia Q, Reszka Krzysztof J, Spitz Douglas R, Denning Gerene M, Britigan Bradley E
Department of Internal Medicine, Veterans Affairs Medical Center-Iowa City, Iowa 52242, USA.
Am J Physiol Lung Cell Mol Physiol. 2004 Jul;287(1):L94-103. doi: 10.1152/ajplung.00025.2004. Epub 2004 Mar 12.
Production of pyocyanin enhances Pseudomonas aeruginosa virulence. Many of pyocyanin's in vitro and in vivo cytotoxic effects on human cells appear to result from its ability to redox cycle. Pyocyanin directly accepts electrons from NADH or NADPH with subsequent electron transfer to oxygen, generating reactive oxygen species. Reduced glutathione (GSH) is an important cellular antioxidant, and it contributes to the regulation of redox-sensitive signaling systems. Using the human bronchial epithelial (HBE) and the A549 human type II alveolar epithelial cell lines, we tested the hypothesis that pyocyanin can deplete airway epithelial cells of GSH. Incubation of both cell types with pyocyanin led to a concentration-dependent loss of cellular GSH (up to 50%) and an increase in oxidized GSH (GSSG) in the HBE, but not A549 cells, at 24 h. An increase in total GSH, mostly as GSSG, was detected in the culture media, suggesting export of GSH or GSSG from the pyocyanin-exposed cells. Loss of GSH could be due to pyocyanin-induced H(2)O(2) formation. However, overexpression of catalase only partially prevented the pyocyanin-mediated decline in cellular GSH. Cell-free electron paramagnetic resonance studies revealed that pyocyanin directly oxidizes GSH, forming pyocyanin free radical and O(2)(-). Pyocyanin oxidized other thiol-containing compounds, cysteine and N-acetyl-cysteine, but not methionine. Thus GSH may enhance pyocyanin-induced cytotoxicity by functioning as an alternative source of reducing equivalents for pyocyanin redox cycling. Pyocyanin-mediated alterations in cellular GSH may alter epithelial cell functions by modulating redox sensitive signaling events.
绿脓菌素的产生会增强铜绿假单胞菌的毒力。绿脓菌素对人类细胞的许多体外和体内细胞毒性作用似乎源于其氧化还原循环的能力。绿脓菌素直接从NADH或NADPH接受电子，随后将电子转移到氧，产生活性氧。还原型谷胱甘肽（GSH）是一种重要的细胞抗氧化剂，它有助于调节对氧化还原敏感的信号系统。我们使用人支气管上皮（HBE）细胞系和A549人II型肺泡上皮细胞系，测试了绿脓菌素会耗尽气道上皮细胞中GSH的假设。在24小时时，用绿脓菌素孵育这两种细胞类型会导致细胞内GSH浓度依赖性损失（高达50%），并且HBE细胞中氧化型谷胱甘肽（GSSG）增加，但A549细胞中没有增加。在培养基中检测到总GSH增加，主要是GSSG，这表明GSH或GSSG从暴露于绿脓菌素的细胞中输出。GSH的损失可能是由于绿脓菌素诱导的H(2)O(2)形成。然而，过氧化氢酶的过表达仅部分阻止了绿脓菌素介导的细胞内GSH下降。无细胞电子顺磁共振研究表明，绿脓菌素直接氧化GSH，形成绿脓菌素自由基和O(2)(-)。绿脓菌素氧化其他含硫醇的化合物，半胱氨酸和N-乙酰半胱氨酸，但不氧化甲硫氨酸。因此，GSH可能通过作为绿脓菌素氧化还原循环的还原当量的替代来源来增强绿脓菌素诱导的细胞毒性。绿脓菌素介导的细胞内GSH改变可能通过调节对氧化还原敏感的信号事件来改变上皮细胞功能。
