Antioxidant Protective Genes

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An important effect of oxidative stress in the lungs is the upregulation of protective antioxidant genes. The antioxidant glutathione (GSH) is concentrated in epithelial lining fluid compared with plasma (101) and has an important protective role, together with its redox enzymes, in the airspaces and intracellularly in epithelial cells. Human studies have shown elevated levels of glutathione in epithelial lining fluid in chronic cigarette smokers compared with nonsmokers (26,101). However, this increase is not present immediately after acute cigarette smoking (26).

The discrepancy between glutathione levels in epithelial lining fluid in chronic and acute cigarette smokers has been investigated in animal models and in vitro using cultured epithelial cells (100,104,182). Exposure of airspace epithelial cells to CSC in vitro produces an initial decrease in intracel-lular GSH with a rebound increase after 12 hr (183). This effect in vitro is mimicked by a similar change in glutathione in rat lungs in vivo following intratracheal instillation of cigarette smoke condensate (100). The initial fall in lung and intracellular glutathione after treatment with cigarette smoke condensate was associated with a decrease in the activity of glutamylcysteine ligase (GCL) formerly known as y-GCS, the rate limiting enzyme for glutathione synthesis, with recovery of the activity by 24 hr (100,183). The increased levels of glutathione following cigarette smoke condensate exposure have been shown to be due to transcriptional upregulation of the gene for GSH synthesis GCL by components within cigarette smoke (Fig. 12) (183,184). Thus, oxida-tive stress, including that produced by cigarette smoking, causes upregulation of the gene involved in the synthesis of glutathione as a protective mechanism against oxidative


Figure 12 The effect of cigarette smoke exposure in A549 epithelial cells on y-GCS mRNA by RT-PCR.


Figure 12 The effect of cigarette smoke exposure in A549 epithelial cells on y-GCS mRNA by RT-PCR.

stress. These events are likely to account for the increased levels of glutathione seen in the epithelial lining fluid in chronic cigarette smokers (26,101). However, the injurious effects of cigarette smoke may occur repeatedly during and immediately after cigarette smoking when the lung is depleted of antioxidants, including glutathione (26).

The cytokine TNF, which is present as part of the airway inflammation in COPD (185), also decreases intracellular glutathione levels initially in epithelial cells by a mechanism involving the generation of intracellular oxidative stress, which is followed after 24 hr by a rebound increase in intracellular glutathione, as a result of AP-1 activation and an increased GCL mRNA expression (183,184). Corticosteroids have been used as anti-inflammatory agents in COPD, but there is still doubt over their effectiveness in reducing airway inflammation in COPD. Interestingly, dexamethasone also causes a decrease in intracellular glutathione in airspace epithelial cells, but no rebound increase compared with the effects of TNF (186). Moreover, the rebound increase in glutathione produced by TNF in epithelial cells is prevented by cotreatment with dexametha-sone (186). These effects may have relevance for the treatment of COPD patients with corticosteroids.

Gilks et al. (187) have shown that rats exposed to whole cigarette smoke had increased expression of a number of antioxidant genes in the bronchial epithelial cells for up to 14 days (187). While, mRNA of manganese superoxide dismutase (MnSOD) and metallothionein (MT) was increased at 1-2 days and returned to normal by 7 days, mRNA for glutathione peroxidase did not increase until 7 days of exposure, suggesting the importance of the glutathione redox system as a mechanism for chronic protection against the effects of cigarette smoke (187).

The oncogene cfos belongs to a family of growth and differentiation-related immediate early genes, the expression of which generally represents the first measurable response to a variety of chemical and physical stimuli (173). Studies in various cell lines have shown enhanced gene expression of the cfos in response to cigarette smoke condensate (170,188). These effects of cigarette smoke condensate can be mimicked by peroxynitrite and smoke-related aldehydes in concentrations that are present in cigarette smoke condensate (170). This effect can be enhanced by pretreatment of the cells with buthionine sulfoximine to decrease intracellular glutathione and can be prevented by treatment with the thiol antioxidant N-acetylcysteine (170). These studies emphasize the importance of intracellular levels of the antioxidant glu-tathione in regulating gene expression.

Ishii et al. (189) have shown that glutathione S-transferase P1 (GSTP1) acts as a protective enzyme against cigarette smoke in the airway cells. Similarly, Maestrelli et al. (190) have recently shown that HO-1 is induced in alveolar spaces of smokers suggesting that oxidative stress due to cigarette smoke may increase the gene expression of HO-1 leading to increased levels of exhaled CO. Cigarette smoke also induces heat-shock protein 70 (HSP70) in human monocytes and HO-1, which have been implicated in the regulation of cell injury and cell death and, in particular, modulation of apoptosis in human endothelial cells and monocytes (173,191). The induction of HSP70 may stabilize IkBa, possibly through the prevention of IkB kinase activation (192).

Thus, oxidative stress, including that produced by cigarette smoke, causes increased gene expression of both proinflammatory genes, by oxidant-mediated activation of NF-kB and also activation of protective genes, such as y-glutamylcysteine synthetase through other transcription factors (AP-1/ARE). A balance may therefore exist between pro- and anti-inflammatory gene expressions in response to cigarette smoke, which may be critical to whether cell injury is induced by cigarette smoking (Fig. 13). Such an imbalance of an array of redox-regulated antioxidant versus proinflammatory genes might, therefore, be associated with the susceptibility or tolerance to disease. Knowledge of the molecular mechanisms that regulate these events may open new therapeutic avenues in the treatment of COPD.

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