Peter S. Gilmour

Oxidative stress and calcium signaling in the adverse effects of environmental particles (PM10)

This review focuses on the potential role that oxidative stress plays in the adverse effects of PM(10). The central hypothesis is that the ability of PM(10) to cause oxidative stress underlies the association between increased exposure to PM(10) and both exacerbations of lung disease and lung cancer. Pulmonary inflammation may also underlie the cardiovascular effects seen following increased PM(10), although the mechanisms of the cardiovascular effects of PM(10) are not well understood. PM(10) is a complex mix of various particle types and several of the components of PM(10) are likely to be involved in the induction of oxidative stress. The most likely of these are transition metals, ultrafine particle surfaces, and organic compounds. In support of this hypothesis, oxidative stress arising from PM(10) has been shown to activate a number of redox-responsive signaling pathways in lung target cells. These pathways are involved in expression of genes that play a role in responses relevant to inflammation and pathological change, including MAPKs, NF-kappaB, AP-1, and histone acetylation. Oxidative stress from particles is also likely to play an important role in the carcinogenic effects associated with PM(10) and hydroxyl radicals from PM(10) cause DNA damage in vitro.

Oxidative stress and TNF-alpha induce histone acetylation and NF-kappaB/AP-1 activation in alveolar epithelial cells: potential mechanism in gene transcription in lung inflammation.

Oxidants and inflammatory mediators such as tumour necrosis factor-alpha (TNF-alpha) activate nuclear factor kappa B (NF-kappaB) and activator protein-1 (AP-1) transcription factors, and enhance the expression of both pro-inflammatory and protective antioxidant genes. Remodelling of chromatin within the nucleus, controlled by the degree of acetylation/deacetylation of histone residues on the histone core around which DNA is coiled, is important in allowing access for transcription factor DNA binding and hence gene transcription. Unwinding of DNA is important in allowing access for transcription factor DNA binding and hence gene transcription. Nuclear histone acetylation is a reversible process, and is regulated by a group of acetyltransferases (HATs) which promote acetylation, and deacetylases (HDACs) which promote deacetylation. The aim of this study was to determine whether oxidative stress and the pro-inflammatory mediator, TNF-alpha, altered histone acetylation/deacetylation and the activation of NF-kappaB and AP-1, leading to the release ofthe pro-inflammatory cytokine IL-8 in human alveolar epithelial cells (A549). Hydrogen peroxide (H2O2) (100 microM) and TNF-alpha (10 ng/ml) imposed oxidative stress in A549 cells as shown by depletion of the antioxidant reduced glutathione (GSH) concomitant with increased levels of oxidised glutathione (GSSG). Treatment of A549 cells with H2O2, TNF-alpha and the HDAC inhibitor, trichostatin A, TSA (100 ng/ml) significantly increased acetylation of histone proteins shown by immunostaining of cells and increased HAT activity, compared to the untreated cells. H2O2, and TNF-a, and TSA all increased NF-kappaB and AP-1 DNA binding to their consensus sites assessed by the electrophoretic mobility shift assay. TSA treatment potentiated the increased AP-1 and NF-KB binding, produced by H2O2 or TNF-alpha treatments in A549 cells. Both H2O2 and TNF-alpha significantly increased IL-8 release, which was further enhanced by pre-treatment of A549 cells with TSA compared to the individual treatments. This study shows that the oxidant H2O2 and the pro-inflammatory mediator, TNF-a induce histone acetylation which is associated with decreased GSH levels and increased AP-1 and NF-kappaB activation leading to enhanced proinflammatory IL-8 release in alveolar epithelial cells. This indicates a mechanism for the pro-inflammatory effects of oxidative stress.

Oxidant-mediated lung epithelial cell tolerance: the role of intracellular glutathione and nuclear factor-kappaB

The airway epithelium is injured by oxidants inhaled as atmospheric pollutants or produced during inflammatory responses. We studied the effect of modulating the antioxidant intracellular glutathione, both using thiol compounds and by the adaptive effect of hyperoxia, on oxidant-induced injury and activation of the nuclear factor-kappaB (NF-kappaB) in two cell lines: the human bronchial (16HBE) and type II alveolar epithelial cells (A549). The thiol antioxidants glutathione (GSH) and glutathione monoethyl ester (GSH-MEE) [2 mM] increased GSH levels (nmol/mg protein) in A549 cells (GSH 383 +/- 26 and GSH-MEE 336 +/- 23 vs control 171 +/- 13, P < 0.001) and in 16HBE cells (GSH 405 +/- 33, GSH-MEE 362 +/- 37 vs control 198 +/- 12, P < 0.001, N = 3). Treatment of hyperoxia (95% oxygen) also increased GSH levels between 4 and 24 hr exposure compared with control (P < 0.01). Hydrogen peroxide (H(2)O(2)) (0.01 mM) induced NF-kappaB activation, whereas hyperoxia exposure did not affect NF-kappaB activation in either cell line. Pretreatment with dl-buthionine (SR)-sulfoximine, which decreased intracellular glutathione, increased NF-kappaB binding induced by H(2)O(2) and increased lactate dehydrogenase (LDH) release (P < 0.001). Pretreatment with the thiol compounds and hyperoxia totally inhibited H(2)O(2)-induced NF-kappaB binding and cell injury as measured by LDH release. These data indicate the importance of intracellular glutathione and inhibition of NF-kappaB in both protection/tolerance against oxidant-induced epithelial cell injury, and NF-kappaB activation in response to oxidative stress which may be important in lung inflammation. Thus, increasing intracellular glutathione may be of therapeutic relevance if able to modulate NF-kappaB activation and hence attenuate inflammation.

Ergothioneine inhibits oxidative stress- and TNF-α-induced NF-κB activation and interleukin-8 release in alveolar epithelial cells

Oxidants and inflammatory mediators such as tumour necrosis factor-alpha (TNF-alpha) activate transcription factors such as NF-kappa B. Interleukin-8 (IL-8) is a ubiquitous inflammatory chemokine that mediates a multitude of inflammatory events in the lung. Ergothioneine is a naturally occurring thiol compound, which possesses antioxidant property. The aim of this study was to determine whether ergothioneine can inhibit the hydrogen peroxide (H(2)O(2))- and TNF-alpha-mediated activation of NF-kappa B and the release of IL-8 in human alveolar epithelial cells (A549). Treatment of A549 cells with H(2)O(2) (100 microM) and TNF-alpha (10 ng/ml) significantly increased NF-kappa B activation using a reporter assay. Ergothioneine inhibited both H(2)O(2)- and TNF-alpha-mediated activation of NF-kappa B. Both H(2)O(2) and TNF-alpha significantly increased IL-8 release, which was inhibited by pre-treatment of A549 cells with ergothioneine compared to the control untreated cells. Ergothioneine also abolished the transcriptional activation of IL-8 in an IL-8-chloramphenicol acetyltransferase (CAT) reporter system, transfected into A549 cells. This indicates a molecular mechanism for the anti-inflammatory effects of ergothioneine.

Cigarette Smoke-Induced Oxidative Stress and TGF-β1 Increase p21waf1/cip1 Expression in Alveolar Epithelial Cells

Abstract: Sustained oxidative stress caused by cigarette smoking induces a chronic inflammatory response, resulting in the destruction of the alveolar cell wall characteristic of emphysema. The loss of tissue may involve the progressive depletion of epithelial cells through inhibition of proliferation leading to cell death. The cell cycle regulator p21waf1/cip1 acts as a G1/S and G2/M phase checkpoint regulator. We hypothesize that cigarette smoke‐induced oxidative stress and transforming growth factor beta 1 (TGF‐β1) may inhibit cellular proliferation by p21waf1/cip1 in type II alveolar epithelial cells (A549). A significant increase was observed in p21waf1/cip1 mRNA expression in A549 cells by cigarette smoke condensate, H2O2, and TGF‐β1. In conclusion, cigarette smoke‐induced oxidative stress and TGF‐β1 modulate expression of the cell cycle inhibitor p21waf1/cip1. This may be important in the growth arrest and cell survival of alveolar type II cells in the G1 phase.

Cardiovascular responses in unrestrained WKY rats to inhaled ultrafine carbon particles

Based on epidemiologic observations, the issue of adverse health effects of inhaled ultrafine particles (UFP) is currently under intensive discussion. We therefore examined cardiovascular effects of UFP in a controlled animal exposure on young, healthy WKY rats. Short-term exposure (24 h) to carbon UFPs (38 nm, 180 μg m−3), generated by spark discharging, induced a mild but consistent increase in heart rate (18 bpm, 4.8%), which was associated with a significant decrease in heart-rate variability during particle inhalation. The timing and the transient character of these responses point to a particle induced alteration of cardiac autonomic balance, mediated by a pulmonary receptor activation. After 24 h of inhalation exposure, bronchoalveolar lavage revealed significant but low-grade pulmonary inflammation (clean air 1.9% vs. UFPs 6.9% polymorphonuclear cells) and on histopathology sporadic accumulation of particle-laden macrophages was found in the alveolar region. There was no evidence of an inflammation-mediated increase in blood coagulability, as UFP inhalation did not induce any significant changes in plasma fibrinogen or factor VIIa levels and there were no prothrombotic changes in the lung or the heart at both the protein and mRNA level. Histological analysis revealed no signs of cardiac inflammation or cardiomyopathy. This study therefore provides toxicological evidence for UFP-associated pulmonary and cardiac effects in healthy rats. Our findings suggest that the observed changes are mediated by an altered sympatho-vagal balance in response to UFP inhalation, but do not support the concept of an inflammation-mediated prothrombotic state by UFP.

The antioxidant cocktail effective microorganism X (EM-X) inhibits oxidant-induced interleukin-8 release and the peroxidation of phospholipids in vitro

The antioxidant beverage EM-X is derived from the ferment of unpolished rice, papaya, and sea-weeds with effective microorganisms. Oxidative stress enhances the expression of proinflammatory genes, causing the release of the chemokine interleukin-8 (IL-8), which mediates a multitude of inflammatory events. Human alveolar epithelial cells (A549) were treated with H(2)O(2) (100 microM) or TNF-alpha (10ng/ml) alone or with the addition of EM-X (100 microl/ml), incubated for 20h, and the release of IL-8, measured using ELISA. EM-X inhibited the release of IL-8 at the transcriptional level in A549 cells. EM-X also decreased the iron/ascorbate dependent peroxidation of ox-brain phospholipids in a concentration dependent manner. A TEAC value of 0.10+/-0.05mM was obtained for EM-X, indicating antioxidant potential. We suggest that the anti-inflammatory and antioxidant properties of EM-X are dependent on the flavonoid contents of the beverage.

Systemic Effect of Particulate Air Pollution.

The association in epidemiological studies between particulate air pollution (PM10) and increased mortality and morbidity from cardiovascular disease is well established. However, the mechanism(s) by which PM10 produces these effects is unknown. We have developed a hypothesis that ultrafine components of PM10 cause lung inflammation by creating local lung oxidative stress, which activates transcription factors such as NF-kB and hence the transcription of genes for inflammatory mediators. In a series of studies in vivo in the rat and using cultured airspace epithelial cells in vitro, we have tested this hypothesis using environmental PM10 and fine (CB 260 nm in diameter) and ultrafine (UfCB, 14 nm in diameter) carbon particles, since carbon is a major component of PM10 We have shown that compared with CB, which produces trivial effects, UFCB and PM10 produce local lung inflammation, increased epithelial permeability, and evidence of oxidative stress. We have also shown that PM10 also activates NF-kB in airspace epithelial cells. We also hypothesized that the local lung inflammation produced by PM10, may result in systemic effects, in particular systemic oxidative stress and enhanced blood coagulation, which may have a role in the adverse cardiovascular effects induced by PM10. To test this hypothesis we measured oxidative stress and changes in coagulation factors in plasma following the inhalation of UFCB, CB, or instillation ofPM,10 in the rat. Immediately after 7 h inhalation of UFCB (1000 (g/m3) there was a significant decrease in the antioxidant capacity in rat plasma, which fell further 16 and 48 h after cessation of the inhalation. A similar fall in plasma antioxidant capacity was shown 6 h after instillation of PM10 (125 tig). In contrast, there were no significant changes in antioxidant capacity in rats after 7 h of inhalation of CB at similar concentrations. The levels of factor VII in plasma, which is a key factor in the intrinsic pathway of coagulation cascade, increased at time points from 6 h to 7 days after inhalation of UFCB, but did not change after CB exposure. There were no changes in plasma fibrinogen or other coagulation factors after inhalation of particles. Thus, inhalation of ultrafine carbon black particles and instillation of PMio in rats decreases plasma antioxidant capacity as an indication of systemic oxidative stress. Ultrafine carbon black particles also cause increased factor VII levels in the plasma, a known risk factor for adverse cardiovascular events. These studies help to explain the relationship between the levels of particulate air pollutants and cardiovascular morbidity/mortality.