Erythropoietin protects retinal pigment epithelial cells from oxidative damage

Oxidative damage from reactive oxygen species (ROS) has been implicated in many diseases, including age-related macular degeneration, in which the retinal pigment epithelium (RPE) is considered a primary target. The aim of this study was to determine whether erythropoietin (EPO) protects cultured human RPE cells against oxidative damage and to identify the pathways that may mediate protection. EPO (1 IU/ml) significantly increased the viability of oxidant-treated RPE cells, decreased the release of the inflammatory cytokines tumor necrosis factor-α and interleukin-1β, recovered the RPE cells' barrier integrity disrupted by oxidative stress, prevented oxidant-induced cell DNA fragmentation and membrane phosphatidylserine exposure, and also reduced the levels of oxidant-induced intracellular ROS and restored cellular antioxidant potential, total antioxidant capacity, glutathione peroxidase, and superoxide dismutase and decreased malondialdehyde, the end product of lipid peroxidation. EPO inhibited caspase-3-like activity. Protection by EPO was partly dependent on the activation of Akt1 and the maintenance of the mitochondrial membrane potential. No enhanced or synergistic protection was observed during application of Z-DEVD-FMK (caspase-3 inhibitor) combined with EPO compared with cultures exposed to EPO and H₂O₂ alone. Together, these results suggest that EPO could protect against oxidative injury-induced cell death and mitochondrial dysfunction in RPE cells through modulation of Akt1 phosphorylation, mitochondrial membrane potential, and cysteine protease activity.     

Celecoxib mitigates cigarette smoke induced oxidative stress in mice

Cigarette smoke (CS) is a rich source of radicals, predisposing the cell to oxidative stress resulting in inflammation. Chronic inflammation is a recognized risk factor for carcinogenesis. Cyclooxygenase-2 (COX-2) is a mediator of inflammatory pathway and may, therefore, contribute to carcinogenesis. There are several reports that suggest the association between CS and COX-2 associated risk to cancer. In the present study, we examined the role of celecoxib (a selective COX-2 inhibitor) in modulating the oxidative stress caused by CS inhalation in mice. CS exposure for a period of 10 weeks caused oxidative stress in the pulmonary and hepatic tissues, as evident from the increase in lipid peroxidation levels (LPO) and decrease in reduced glutathione (GSH) levels. Celecoxib (125 mg/kg body weight for 8 weeks) administration to CS inhaling mice reduced the oxidative stress by decreasing the LPO levels and enhancing the GSH levels in comparison to the CS-exposed group. CS exposure repressed the enzymatic antioxidant defense system, as evident from the decrease in catalase (CAT) and superoxide dismutase (SOD) activities. Co-adminstration of celecoxib considerably reversed the changes in the enzymatic antioxidant defense system. Histopathological studies of lungs showed that CS exposure induced alveolar wall destruction and air space enlargement. In co-treated group, the alveolar septa were thicker than normal with apparent infiltration of inflammatory cells. In CS-exposed group, hepatic tissue exhibited vacuolization and macrophage infiltration. Co-treatment with celecoxib restored the normal histoarchitechture in hepatic tissues of CS inhaling mice. Thus, the present study demonstrated that celecoxib adminstration reduced the oxidative stress-mediated risk to carcinogenesis, due to its ability to boost the antioxidant defense system.     

Zinc protects against oxidative damage in cultured human retinal pigment epithelial cells

This study was undertaken to determine whether bioavailable zinc can influence the effects of oxidative stress on cultured human retinal pigment epithelial (RPE) cells. RPE cells were maintained for 7 d in culture medium containing 14 microM total zinc, or in medium containing 0.55 microM total zinc. After 1 week, MTT assays were performed to determine the relative cytotoxicity of H2O2 or paraquat on RPE cells. Conjugated dienes and thiobarbituric acid reactive substances (TBARS) were measured in RPE cells treated with 0, 0.5 mM H2O2, 10 microM FeSO4 + 0.5 mM H2O2 or 10 microM FeSO4 + xanthine/xanthine oxidase for 24 h or paraquat for 7 d. Oxidized proteins were determined by the formation of carbonyl residues. The antioxidants metallothionein, catalase, superoxide dismutase, and glutathione peroxidase were also measured. The MTT assays showed that zinc protected cultured RPE from the toxicity of H2O2 and paraquat. RPE cells in 0.55 microM zinc medium contained higher levels of TBARS, conjugated dienes and protein carbonyls due to the oxidative stresses, compared to cells in 14 microM zinc. Catalase and MT content were reduced in cells cultured in 0.55 microM zinc medium and were reduced additionally when treated with above stresses. Superoxide dismutase activity increased in 0.55 microM zinc medium in response to these stresses. Our results show RPE cells cultured in zinc-reduced medium are more susceptible to oxidative insult.     

STAT3 activation inhibits human bronchial epithelial cell apoptosis in response to cigarette smoke exposure

We have previously reported that cigarette smoke can induce DNA damage in human lung cells without leading to apoptosis or necrosis. In this study, we report that STAT3 is required for the survival of human bronchial epithelial cells (HBECs) following cigarette smoke-induced DNA damage. Cigarette smoke extract (CSE) exposure increases STAT3 phosphorylation (Tyr 705) and DNA binding activity in HBECs. CSE also stimulates IL-6 release and mRNA expression. Anti-IL-6 neutralizing antibody partially blocks STAT3 activation and renders the cells sensitive to CSE-induced DNA damage. Suppression of STAT3 by siRNA results in severe DNA damage and cell death in response to CSE exposure. These findings suggest that STAT3 mediates HBEC survival in response to CSE-induced DNA damage, at least in part, through the IL-6/STAT3 signaling pathway.     

Hepatic growth factor (HGF) inhibits cigarette smoke extract induced apoptosis in human bronchial epithelial cells

Low concentrations of cigarette smoke induced DNA damage and repair without leading to apoptosis in human bronchial epithelial cells. Higher concentrations of cigarette smoke, however, could induce either apoptosis or necrosis. The current study demonstrated that 15% cigarette smoke extract (CSE) induced apoptosis as evidenced by DNA content profiling (17.8 ± 2.1% vs 10.2 ± 1.6% of control, p < 0.05), LIVE/DEAD staining (60.2 ± 2.1% viable cells in CSE-treated vs 86.5 ± 2.3% in control cells, p < 0.05), and COMET assay (24.3 ± 0.6% of Apoptotic Index in the cells treated with CSE vs 4.7 ± 0.6% of control, P < 0.05). Hepatocyte growth factor (HGF) significantly blocked the cigarette smoke-induced apoptosis as shown by DNA profiling (10.8 ± 1.5% of CSE + HGF, p < 0.05), LIVE/DEAD staining (78.5 ± 1.2% in CSE + HGF treated cells, p < 0.05), and COMET assay (Apoptotic Index: 10.0 ± 0.8% in CSE + HGF treated cells, P < 0.05). This protective effect of HGF on CSE-induced apoptosis was abolished by PI3K inhibitors, wortmannin and LY294002, and by introduction of the dominant negative AKT into the cells. Furthermore, CSE plus HGF could induce phosphorylation of AKT Thr 308 and the pro-apoptotic protein, BAD. These results suggest that HGF modulates cell survival in response to cigarette smoke exposure through the PI3K/AKT signaling pathway.     

Vitamin C prevents cigarette smoke induced oxidative damage of proteins and increased proteolysis

Aqueous extract of cigarette smoke (CS) contains some stable oxidants, which oxidize human plasma proteins, bovine serum albumin, amino acid homopolymers, and also cause extensive oxidative degradation of microsomal proteins. Similar observations are made when the aqueous extract of cigarette smoke is replaced by whole phase CS solution or whole phase cigarette smoke. CS-induced microsomal protein degradation is a two step process: (i) oxidation of proteins by the oxidants present in the CS and (ii) rapid proteolytic degradation of the oxidized proteins by proteases present in the microsomes. Using aqueous extract of CS equivalent to that produced from one-twentieth of a cigarette, the observed initial and postcigarette smoke treated values of different parameters of oxidative damage per milligram of microsomal proteins are respectively: 0.24 and 1.74 nmoles for carbonyl formation, 125.4 and 62.8 fluorescence units for tryptophan loss, 10.2 and 33.4 fluorescence units for bityrosine formation, and 58.3 and 12.2 nmoles for loss of protein thiols. When compared with sodium dodecyl sulphate polyacrylamide gel electrophoresis profiles of untreated microsomal proteins, the extent of microsomal protein degradation after treatment with whole phase CS solution or aqueous extract of CS is above 90%. Ascorbate (100 microM) almost completely prevents cigarette smoke-induced protein oxidation and thereby protects the microsomes from subsequent proteolytic degradation. Glutathione is partially effective, but other antioxidants including superoxide dismutase, catalase, vitamin E, probucol, beta-carotene, mannitol, thiourea, and histidine are ineffective. The gas phase cigarette smoke contains unstable reactive oxygen species such as superoxide (O2*-) and hydrogen peroxide (H2O2) that can cause substantial oxidation of pure protein like albumin but is unable to produce significant oxidative damage of microsomal proteins. Gas phase cigarette smoke-induced albumin oxidation is not only inhibited by ascorbate and glutathione but also by superoxide dismutase, catalase and mannitol. The stable oxidants in the cigarette smoke are not present in the tobacco and are apparently produced by the interaction of O2*-/H2O2/OH* of the gas phase with some components of the tar phase during/following the burning of tobacco.     

Cilomilast counteracts the effects of cigarette smoke in airway epithelial cells

Cigarette smoke extracts (CSE) alter TLR4 expression and activation in bronchial epithelial cells. Cilomilast, a phosphodiesterase-4 inhibitor, inhibits cigarette smoke-induced neutrophilia.This study was aimed to explore whether cilomilast, in a human bronchial epithelial cell line (16-HBE), counteracted CSE effects. In particular, TLR4 expression, IP-10 and IL-8 release, lymphocyte and neutrophil chemotactic activity and ERK and IkBa phosphorylation in CSE and LPS-stimulated 16-HBE were assessed.CSE increased TLR4 expression, reduced IP-10 release and lymphocyte chemotactic activity and increased IL-8 release and neutrophil chemotactic activity. Cilomilast reduced TLR4 expression, IL-8 release and neutrophil chemotactic activity as well as it increased IP-10 release and lymphocyte chemotactic activity. All these cilomilast mediated effects were associated with a reduced ERK1/2 and with an increased IkBa phosphorylation.In conclusion, the present study provides compelling evidences that cilomilast may be considered a possible valid therapeutic option in controlling inflammatory processes present in smokers.     

Hypoxia controls iron metabolism and glutamate secretion in retinal pigmented epithelial cells

Background

Blood-barrier systems are essential in controlling iron levels in organs such as the brain and eye, both of which experience hypoxia in pathological conditions. While hypoxia's effects on numerous iron regulatory and storage proteins have been studied, little is known about how hypoxia affects iron metabolism. Iron also controls glutamate production and secretion; therefore the effects of hypoxia on iron metabolism and glutamate secretion were studied in polarized retinal pigmented epithelial (RPE) cells.

Methods

Primary canine RPE were cultured in Millicells to create polarized cell cultures. Iron uptake and efflux were measured in hypoxic and normoxic conditions. RPE were loaded with ⁵⁹Fe-transferrin. Glutamate concentrations in the cell conditioned media were also measured.

Results

Hypoxia induced a large increase in iron efflux from RPE in the basolateral direction. Glutamate secretion occurred mainly in the basolateral direction which is away from the retina and out of the eye in vivo. Glutamate secretion was doubled under hypoxic conditions.

Conclusions

Hypoxia is known to induce oxidative damage. The current results show that iron, a key catalyst of free radical generation, is removed from RPE under hypoxic conditions which may help protect RPE from oxidative stress. Results obtained here indicate the importance of using polarized tight junctional cells as more physiologically relevant models for blood-barrier-like systems.

General significance

While the effects of hypoxia on iron efflux and glutamate secretion may be protective for RPE cells and retina, increased glutamate secretion in the brain could cause some of the damaging neurological effects seen in stroke.     

Exposure of mice to cigarette smoke and/or light causes DNA alterations in heart and aorta

Cigarette smoke (CS) is a major risk factor for cardiovascular diseases, cancer, and other chronic degenerative diseases. UV-containing light is the most ubiquitous DNA-damaging agent existing in nature, but its possible role in cardiovascular diseases had never been suspected before, although it is known that mortality for cardiovascular diseases is increased during periods with high temperature and solar irradiation. We evaluated whether exposure of Swiss CD-1 mice to environmental CS (ECS) and UV-C-covered halogen quartz lamps, either individually or in combination, can cause DNA damage in heart and aorta cells. Nucleotide alterations were evaluated by (32)P postlabeling methods and by HPLC-electrochemical detection. The whole-body exposure of mice to ECS considerably increased the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and of bulky DNA adducts in both heart and aorta. Surprisingly, even exposure to a light that simulated solar irradiation induced oxidatively generated damage in both tissues. The genotoxic effects of UV light in internal organs is tentatively amenable to formation of unidentified long-lived mutagenic products in the skin of irradiated mice. Nucleotide alterations were even more pronounced when the mice were exposed to smoke and/or light during the first 5weeks of life rather than during adulthood for an equivalent period of time. Although the pathogenetic meaning is uncertain, DNA damage in heart and aorta may tentatively be related to cardiomyopathies and to the atherogenesis process, respectively.     

Sodium butyrate ameliorates lipopolysaccharide-induced cow mammary epithelial cells from oxidative stress damage and apoptosis

This study investigated the molecular mechanism by which sodium butyrate (NaB) causes oxidative stress damage induced by lipopolysaccharide (LPS) on cow mammary epithelial cells (MAC-T). We found that NaB significantly increased the activities of antioxidant enzymes, including superoxide dismutase, glutathione peroxidase, catalase, peroxidase, and total antioxidant capacity and decreased the reactive oxygen species production in LPS-induced MAC-T cells. NaB attenuated protein damage and reduced apoptosis in LPS-induced MAC-T cells. The messenger RNA (mRNA) levels of caspase-3, caspase-9, and Bax decreased, while the Bcl-2 mRNA level increased in LPS-induced MAC-T cells treated with NaB. Our results showed that NaB treatment increased the phosphoinositide 3-kinase (PI3K) and phospho-AKT (P-AKT) protein levels, whereas it decreased the Bax, caspase-3, and caspase-9 protein levels in LPS-induced MAC-T cells. However, the increase in PI3K and P-AKT protein levels and the decrease in Bax, caspase-3, and caspase-9 protein levels induced by NaB treatment were reversed when the cells were pretreated with LY294002 (PI3K inhibitor). These results indicate that NaB ameliorates LPS-induced oxidative damage by increasing antioxidative enzyme activities and ameliorating protein damage in MAC-T cells. In addition, NaB decreased apoptosis by inhibiting caspase-3, caspase-9, and Bax protein levels, and this action was mainly achieved via activation of the PI3K/AKT signaling pathways in LPS-induced MAC-T cells. These results provide substantial information for NaB as a chemical supplement to treat oxidative stress and its related diseases in ruminants.     

Effects of sham air and cigarette smoke on A549 lung cells: implications for iron-mediated oxidative damage

Inhalation of airborne pollution particles that contain iron can result in a variety of detrimental changes to lung cells and tissues. The lung iron burden can be substantially increased by exposure to cigarette smoke, and cigarette smoke contains iron particulates, as well as several environmental toxins, that could influence intracellular iron status. We are interested in the effects of environmental contaminants on intracellular iron metabolism. We initiated our studies using lung A549 type II epithelial cells as a model, and we evaluated the effects of iron dose and smoke treatment on several parameters of intracellular iron metabolism. We show that iron at a physiological dose stimulates ferritin synthesis without altering the transferrin receptor (TfR) mRNA levels of these cells. This is mediated primarily by a reduction of iron regulatory protein 2. Higher doses of iron reduce iron regulatory protein-1 binding activity and are accompanied by a reduction in TfR mRNA. Thus, for A549 cells, different mechanisms influencing IRP-IRE interaction allow ferritin translation in the presence of TfR mRNA to provide for iron needs and yet prevent excessive iron uptake. More importantly, we report that smoke treatment diminishes ferritin levels and increases TfR mRNA of A549 cells. Ferritin serves as a cytoprotective agent against oxidative stress. These data suggest that exposure of lung cells to low levels of smoke as are present in environmental pollutants could result in reduced cytoprotection by ferritin at a time when iron uptake is sustained, thus enhancing the possibility of lung damage by iron-mediated oxidative stress.     

Thymoquinone protects human retinal pigment epithelial cells against hydrogen peroxide induced oxidative stress and apoptosis

Oxidative stress in retinal pigment epithelium (RPE) cells may contribute to the progression of age-related macular degeneration. Thymoquinone (TQ), an active component derived from Nigella sativa, possesses antioxidative effect. However, the role of TQ in RPE cells under oxidative stress condition remains unclear. The present study aimed to examine the protective effect of TQ against hydrogen peroxide (H₂O₂)-induced oxidative stress in human RPE cells. Our results showed that TQ improved the cell viability and apoptosis in H₂O₂-induced ARPE cells. We also found that the levels of reactive oxygen species and malondialdehyde induced by H₂O₂ were reduced after the pretreatment of TQ. In addition, the inhibitory effect of H₂O₂ on the glutathione (GSH) level and superoxide dismutase activity was markedly attenuated by TQ pretreatment. Moreover, TQ enhanced the activation of Nrf2/heme oxygenase 1 (HO-1) signaling pathway in H₂O₂-induced ARPE cells. Knockdown of Nrf2 abolished the protective effect of TQ on H₂O₂-induced oxidative damage. These results suggested that TQ protected ARPE cells from H₂O₂-induced oxidative stress and apoptosis via the Nrf2/HO-1 signaling pathway.     

Beraprost sodium attenuates cigarette smoke extract-induced apoptosis in vascular endothelial cells

Apoptosis is now widely recognized as an important part of chronic obstructive pulmonary disease (COPD) pathogenesis. Our previous study demonstrated that a prostacyclin (PGI₂) analogue (beraprost sodium, BPS) prevented cigarette smoke extract (CSE) induced apoptosis of the pulmonary endothelium in rats. So we determined to clarify the apoptosis of vascular endothelial cells in COPD patient and the role of prostacyclin in the protection against apoptosis in vascular endothelial cells induced by CSE. Surgical specimens were obtained from 12 patients with COPD and 10 controls, and the level of apoptosis, prostacyclin synthase (PGI₂S) expression and 6-keto-PGF1α (a stable metabolite of PGI₂) were detected. The apoptotic index (AI), caspase-3 activity, expression of caspase-3 and 6-keto-PGF1α were examined in human umbilical vein endothelial cells (HUVECs) under exposure to varied concentrations of CSE for 24?h as well as under exposure to 2.5?% CSE for varied durations. Then, HUVECs under 2.5?% CSE were exposed to varied concentrations of BPS for 24?h and observed the alteration and the level of cAMP. Increased AI, decreased expression of PGI₂S and 6-keto-PGF1α, were found in the lungs of patients with COPD compared with controls. Moreover, CSE induced apoptosis in means of both dose-dependent and time-dependent manners, and reduced the level of 6-keto-PGF1α in HUVECs. And with the treatment of BPS, an enhanced level of cAMP and decreased apoptosis were detected. The deficiency of PGI₂ critically contributes to the COPD-associated endothelial dysfunction and apoptosis. And BPS protects against the apoptosis in the vascular endothelial cells induced by CSE.     

Increased sister-chromatid exchange in bone-marrow cells of mice exposed to whole cigarette smoke

Male Wistar rats were fed diets of varying selenium content in order to obtain selenium-deficient and selenium-supplemented rats. After 5-6 weeks on the respective diet, the rats were used to investigate how selenium influences the effect of dimethylnitrosamine (DMN) on some liver enzymes and related reactions. The selenium-dependent glutathione peroxidase activity in postmicrosomal supernatant from liver was about 1% in selenium-deficient rats as compared to selenium-supplemented rats or rats fed a standard diet. The highest DMN-demethylase activity was observed in postmitochondrial supernatant from selenium-deficient rat liver, and the lowest in selenium-supplemented rats. No dietary effect was observed on hepatic microsomal cytochrome P450 levels. C-Oxygenation of N,N-dimethylaniline (DMA) was not affected by the selenium level. On the other hand, selenium deficiency seemed to reduce N-oxygenation of DMA. The mutagenicity of DMN in Chinese hamster V79 cells after metabolic activation by the isolated perfused rat liver, was approximately doubled when selenium-deficient livers were used as compared to selenium-supplemented livers and livers from rats fed a standard diet. A negative correlation between DMA-N-oxygenation and mutagenicity from DMN was observed, whereas no correlation between DMA-C-oxygenation and mutagenicity from DMN was found.     

Chemoprevention of genotoxic damage in lung cells of rats exposed to cigarette smoke]

Male Sprague-Dawley rats were exposed to cigarette smoke (CS) according to a complex protocol which lasted 40 days. Some of the groups were pre-treated with N-acetyl-L-cysteine (NAC) by gavage (1 g/kg b.w.) 5 h before each exposure. Bronchoalveolar lavage was performed in each animal at the end of each exposure period in order to recover pulmonary alveolar macrophages (PAM). Cells were identified and counted under the microscope, and the number of micronucleated (MN) and binucleated (BN) PAM was registered. The results showed an increase in the number of MN PAM, which was already evident after 8 days of CS exposure; this increase remained constant after 28 and 40 days of exposure. A significant decrease in the number of MN PAM was observed in the animals pre-treated with NAC. BN PAM were significantly increased after 28 and 40 days of exposure; again, a slight yet not significant decrease was detected in NAC-pre-treated animals. On the whole, this study demonstrates that CS is clearly clastogenic to alveolar macrophages and that NAC can efficiently prevent this cytogenetic damage.