The molecular mechanism of protecting cells against oxidative stress by 2-selenium-bridged β-cyclodextrin with glutathione peroxidase activity

Ultraviolet B (UVB) induces apoptosis and lipid peroxidation of NIH3T3 cells by producing reactive oxygen species (ROS). Glutathione peroxidase (GPX) is one of the most important antioxidant enzymes in organism and it can scavenge ROS. 2-selenium-bridged β-cyclodextrin (2-SeCD) is a GPX mimic generated in our lab. Its GPX activity is 7.4 U/μmol, which is 7.5 times as much as that of ebselen. In this paper, we have established a cell damage system using UVB radiation. Using this system, we have determined antioxidant effect of 2-SeCD by comparison of malondialdehyde (MDA) and H₂O₂ contents in NIH3T3 cells before and after UVB radiation. Experimental results indicate that 2-SeCD can inhibit lipid peroxidation and protect the cells from the damage generated by UVB radiation. To evaluate the molecular mechanism of this protection, we determined the effect of 2-SeCD on the expression of p53 and Bcl-2 in NIH3T3 cells. The results showed that 2-SeCD inhibits the increase of p53 expression level and the decrease of expression of Bcl-2 induced by UVB radiation. Thus, we have concluded that protection of NIH3T3 cells against oxidative stress by 2-SeCD was carried out by regulation of the expression of Bcl-2 and p53.     

Naringin prevents ultraviolet‐B radiation‐induced oxidative damage and inflammation through activation of peroxisome proliferator‐activated receptor γ in mouse embryonic fibroblast (NIH‐3T3) cells

The present study, we investigate the preventive role of naringin, a dietary flavonoid, against ultraviolet‐B (UVB) radiation (280‐320 nm) induced oxidative damage and inflammatory responses in mouse embryonic fibroblast cell lines (NIH‐3T3). In this study, 20 mJ/cm ² of UVB radiation induces cell cytotoxicity, reactive oxygen species (ROS) generation, DNA damage, and antioxidants depletion in NIH‐3T3 cells. Treatment with naringin (60 µM) prior UVB exposure prevented the cell cytotoxicity, ROS generation, DNA damage, and antioxidants depletion in NIH‐3T3 cells. Furthermore, naringin prevents UVB‐induced mitogen‐activated protein kinase families and nuclear factor‐κB (NF‐κB)‐mediated activation of inflammatory factors, that is TNF‐α, IL‐6, IL‐10, and COX‐2 in NIH‐3T3 cells. Peroxisome proliferator‐activated receptor γ (PPARγ) is an anti‐inflammatory agent and it suppressed the UVB‐mediated oxidative and inflammatory responses. In this study, naringin activates PPARγ and prevents inflammatory biomarkers in NIH‐3T3 cells. Thus, naringin prevents UVB‐mediated inflammation and oxidative damage in NIH‐3T3 cells probably over controlling NF‐κB expression and activation of PPARγ.     

Human selenium-containing single-chain variable fragment with glutathione peroxidase activity protects NIH3T3 fibroblast against oxidative damage

Ultraviolet B (UVB medium wave, 280–315 nm) induces cellular oxidative damage and apoptosis by producing reactive oxygen species (ROS). Glutathione peroxidase functions as an antioxidant by catalyzing the reduction of hydrogen peroxide, the more important member of reactive oxygen species. A human selenium-containing single-chain variable fragment (se-scFv-B3) with glutathione peroxidase activity of 1288 U/μmol was generated and investigated for its antioxidant effects in UVB-induced oxidative damage model. In particular, cell viability, lipid peroxidation extent, cell apoptosis, the change of mitochondrial membrane potential, caspase-3 activity and the levels of intracellular reactive oxygen species were assayed. Human se-scFv-B3 protects NIH3T3 cells against ultraviolet B-induced oxidative damage and subsequent apoptosis by prevention of lipid peroxidation, inhibition of the collapse of mitochondrial membrane potential as well as the suppression of the caspase-3 activity and the level of intracellular ROS. It seems that antioxidant effects of human se-scFv-B3 are mainly associated with its capability to scavenge reactive oxygen species, which is similar to that of the natural glutathione peroxidase.     

RAF and antioxidants prevent cell death induction after growth factor abrogation through regulation of Bcl-2 proteins

We have shown previously that mitochondrial ROS production is essential to turn growth factor (GF) removal into cell death. Activated RAF, AKT, Bcl-2 and antioxidants protected equally well against ROS accumulation and subsequent death. Here we investigated whether protection by survival signaling and antioxidants utilizes shared or distinct targets. Using serum deprivation from NIH 3T3 fibroblasts and IL-3 withdrawal from promyeloid 32D cells, we showed that pro-survival signaling by activated RAF but not AKT prevented the decline in Mcl-1 following GF abrogation. GF starvation increased levels of Bim in both model systems, which was prevented by RAF in 32D cells but not in NIH 3T3 fibroblasts. RAF and AKT suppressed activation and mitochondrial translocation of BAX. Also, antioxidant treatment efficiently prevented BAX activation and death of 32D cells but showed little effect on its mitochondrial translocation. No significant impact of antioxidant treatment on Bim or Mcl-1 expression was observed. ROS produced during GF abrogation also did not alter the activity of intracellular signaling pathways, which have been implicated previously in cell killing by pro-oxidants. Together these data suggest Bcl-2 family proteins as convergence point for RAF and ROS in life and death decisions.     

Overexpression of human GPX1 modifies Bax to Bcl-2 apoptotic ratio in human endothelial cells

As they scavenge reactive oxygen species, antioxidants were studied for their ability to interfere with apoptotic processes. However, their mechanisms of action remain unclear. In this study, we measured the expression of two Bcl-2 family members, Bax and Bcl-2, in a human endothelial like cell-line overexpressing the organic hydroperoxide-scavenging enzyme glutathione peroxidase (GPX1), in the absence of any apoptotic/oxidant stimulus. ECV304 were stably transfected with the GPX1 cDNA and used for quantification of Bax (pro-apoptotic) and Bcl-2 (antiapoptotic) mRNA and protein levels, by quantitative RT-PCR and Western-blot. We found that, compared to control cells, cells from a clone showing a 13.2 fold increase in GPX1 activity had unchanged mRNA or protein Bcl-2 levels but expressed 42.6% and 46.1% less Bax mRNA and Bax protein respectively. Subsequently to Bax decrease, the Bax/Bcl-2 ratio, reflecting the apoptotic state of the cells, was also lower in cells overexpressing GPX1. Noticeably, the mRNA and the protein level of the cell-cycle protein p53, known to activate Bax expression, was unchanged. Our study showed that overexpressing an antioxidant gene such as GPX1 in endothelial cells is able to change the basal mRNA and protein Bax levels without affecting those of p53 and Bcl-2. This phenomenon could be useful to antiatherogenic therapies which use antioxidants with the aim of protecting the vascular wall against oxidative stress injury.     

Nitric oxide inhibits ultraviolet B-induced murine keratinocyte apoptosis by regulating apoptotic signaling cascades

Cytotoxic effects of nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) are considered to be one of the major causes of inflammatory diseases. On the other hand, protective effects of NO on toxic insults-induced cellular damage/apoptosis have been demonstrated recently. Ultraviolet B (UVB)-induced apoptosis of epidermal keratinocytes leads to skin inflammation and photoageing. However, it has not been elucidated what kind of effects NO has on UVB-induced keratinocyte apoptosis. Thus, in the present study, we investigated the problem and demonstrated that NO from NO donor suppressed UVB-induced apoptosis of murine keratinocytes. In addition, NO significantly suppressed activities of caspase 3, caspase 8 and caspase 9 that had been upregulated by UVB radiation. NO also suppressed p53 expression that had been upregulated by UVB radiation and upregulated Bcl-2 expression that had been downregulated by UVB radiation. These findings suggested that NO might suppress UVB-induced keratinocyte apoptosis by regulating apoptotic signaling cascades in p53, Bcl-2, caspase3, caspase 8 and caspase 9.     

The effects of UV radiation on the visual system of the crab Neohelice granulata: a protective role of melatonin

The first and main target-structure of ultraviolet (UV) radiation in animals is the body surface, including the skin and eyes. Here, we investigated cell damage in the visual system of the crab Neohelice granulata acclimated to constant light and exposed to UVA or UVB at 12:00 h for 30 min. The reactive oxygen species (ROS) production, antioxidant capacity against peroxyl radicals (ACAP), lipid peroxidation (LPO) damage, catalase (CAT) activity, and the melatonin immunohistochemical reactivity in the eyestalks were evaluated. The animals that received melatonin and were exposed to UVA and UVB radiation showed a decreased ROS concentration (p < 0.05).The ACAP test showed a decrease (p < 0.05) in their values when the animals received 2 pmol/crab of melatonin (physiological dose) before the exposure to UVA radiation. The animals exposed to UVB radiation after receiving the same dose of melatonin showed an increase (p < 0.05) in the ACAP test compared with the animals exposed to UVB radiation after receiving only crab physiological saline. The CAT activity increased (p < 0.05) in the animals that received melatonin and were exposed to UVA and UVB radiation. Animals exposed to UVA and UVB displayed an increase (p < 0.05) in the LPO levels, whereas animals treated with melatonin showed lower (p < 0.05) LPO levels when irradiated. The results indicate that the specific oxidative parameters altered by UV radiation can be modulated by a physiological dose of melatonin. Moreover, the melatonin regularly produced by virtually all eyestalk cells suggests that it may function to modulate the noxious effects of radiation, at least in the crab N. granulata.     

Effect of chlorophyllin against oxidative stress in splenic lymphocytes in vitro and in vivo

Chlorophyllin (CHL) has been examined as an antioxidant/radioprotector in splenic lymphocytes from BALB/c mice. CHL inhibited lipid peroxidation induced by 2,2'-azobis(2-propionimidinedihydrochloride) (AAPH) in lymphocytes in vitro. It also partially prevented radiation-induced suppression of mitogenic stimulation of lymphocytes in vitro. Generation of intracellular reactive oxygen species (ROS) by radiation or AAPH was measured as oxidation of dichlorodihydrofluorescein diacetate (H(2)DCF-DA) using flow cytometry. Addition of CHL to lymphocytes in vitro significantly inhibited the increase in intracellular ROS. Further, lymphocytes from mice treated with CHL (100-400 microg/gbw i. p.) showed varying levels of ROS depending on the dose and the time (24 to 72 h) after injection. The extent of radiation-induced apoptosis and suppression of concanavalin A (con A)-induced mitogenesis ex vivo corresponded with changes in ROS levels in CHL-administered mice. Antioxidant enzymes superoxide dismutase (SOD), catalase and glutathione peroxidase (GPX) were also estimated in lymphocytes from CHL-treated mice. CHL offered protection against whole body irradiation (WBI)-induced lipid peroxidation and apoptosis in lymphocytes at all the time points studied. These results demonstrate antioxidant effect of CHL in vivo.     

Dietary eicosapentaenoic acid prevents systemic immunosuppression in mice induced by UVB radiation.

Moison, R. M. W. and Beijersbergen van Henegouwen, G. M. J. Dietary Eicosapentaenoic Acid Prevents Systemic Immunosuppression in Mice Induced by UVB Radiation. Radiat. Res. 156, 36-44 (2001).Reactive oxygen species (ROS) contribute to the immunosuppression induced by UVB radiation. Omega-3 fatty acids in fish oil, e.g. eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), can modulate immunoresponsiveness, but because of their susceptibility to ROS-induced damage, they can also challenge the epidermal antioxidant defense system. The influence of dietary supplementation with different omega-3 fatty acids on systemic immunosuppression induced in mice by UVB radiation was studied using the contact hypersensitivity response to trinitrochlorobenzene. In an attempt to study the mechanisms involved, UVB-radiation-induced changes in epidermal antioxidant status were also studied. Mice received high-fat (25% w/w) diets enriched with either oleic acid (control diet), EPA, DHA, or EPA + DHA (MaxEPA). Immunosuppression induced by UVB radiation was 53% in mice fed the oleic acid diet and 69% in mice fed the DHA diet. In contrast, immunosuppression was only 4% and 24% in mice fed the EPA and MaxEPA diets, respectively. Increased lipid peroxidation and decreased vitamin E levels (P < 0.05) were found in unirradiated mice fed the MaxEPA and DHA diets. For all diets, exposure to UVB radiation increased lipid peroxidation (P < 0.05), but levels of glutathione (P < 0.05) and vitamin C (P > 0.05) decreased only in the mice given fish oil. UVB irradiation did not influence vitamin E levels. In conclusion, dietary EPA, but not DHA, protects against UVB-radiation-induced immunosuppression in mice. The degree of protection appears to be related to the amount of EPA incorporated and the ability of the epidermis to maintain an adequate antioxidant level after irradiation.     

Protective Effect of Carvacrol on Oxidative Stress and Cellular DNA Damage Induced by UVB Irradiation in Human Peripheral Lymphocytes

Exposure to ultraviolet B (UVB; 280‐320 nm) radiation induces the formation of reactive oxygen species (ROS) in the biological system. In this study, we examined the protective effect of carvacrol on UVB‐induced lipid peroxidation and oxidative DNA damage with reference to alterations in cellular an‐tioxidant status in human lymphocytes. A series of in vitro assays (hydroxyl radical, superoxide, nitric oxide, DPPH (2,2‐Diphenyl‐1‐picryl hydrazyl), and ABTS (2,2‐azino‐bis‐3‐ethylbenzothiazoline‐6‐sulfonic acid) radical scavenging assays) demonstrate antioxidant property of carvacrol in our study. UVB exposure significantly increased thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LHPs), % tail DNA and tail moment; decreased % cell viability and antioxidant status in UVB‐irradiated lymphocytes. Treatment with carvacrol 30 min prior to UVB‐exposure resulted in a significant decline of TBARS, LHP, % tail DNA, and tail moment and increased % cell viability as carvacrol concentration increased. UVB irradiated lymphocytes with carvacrol alone (at 10 μg/mL) gave no significant change in cell viability, TBARS, LHP, % tail DNA, and tail moment when compared with normal lymphocytes. On the basis of our results, we conclude that carvacrol, a dietary antioxidant, mediates its protective effect through modulation of UVB‐induced ROS.     

Aldose reductase in keratinocytes attenuates cellular apoptosis and senescence induced by UV radiation

Although aldose reductase (AR) has been implicated in the cellular response to oxidative stress, the role of AR in ultraviolet-B (UVB)-induced cellular injury has not been investigated. Here, we show that an increased expression of AR in human keratinocytes modulates UVB-induced apoptotic cell death and senescence. Overexpression of AR in HaCaT cells significantly attenuated UVB-induced cellular damage and apoptosis, with a decreased generation of reactive oxygen species (ROS) and aldehydes. Ablation of AR with small interfering RNA or inhibition of AR activity abolished these effects. We also show that increased AR activity suppressed UVB-induced activation of the p38 and c-Jun N-terminal kinases, but did not affect the extracellular signal-regulated kinase and phosphatidylinositol 3-kinase pathways. Similarly, UVB-induced translocation of Bax and Bcl-2 to mitochondria and cytosol, respectively, was markedly attenuated in cells overexpressing AR. Knockdown or inhibition of AR activity in primary cultured keratinocytes enhanced UVB-induced cellular senescence and increased the level of a cell-cycle regulatory protein, p53. Finally, cellular apoptosis induced by UVB radiation was significantly reduced in the epidermis of transgenic mice overexpressing human AR. These findings suggest that AR plays an important role in the cellular response to oxidative stress by sequestering ROS and reactive aldehydes generated in keratinocytes.     

UVB Irradiation Regulates ERK1/2- and p53-Dependent Thrombomodulin Expression in Human Keratinocytes

Thrombomodulin (TM) is highly expressed in endothelial cells and acts as a natural anticoagulation factor to maintain circulation homeostasis. TM is an interesting molecule with many physiological functions, including anti-inflammation, anti-thrombosis, and carcinogenesis inhibition. TM can also be detected on the spinous layer of epidermal keratinocytes. However, the role of epidermal TM is still under investigation. In this study, we investigated keratinocyte TM expression and regulation in response to sub-cytotoxic ultraviolet B (UVB) irradiation. Oxidative stress was assessed with DCF and the results revealed that UVB irradiation significantly and dose-dependently augmented reactive oxygen species (ROS) production in HaCaT cells. In addition, low-dose UVB irradiation decreased TM mRNA and protein levels. Blocking ROS production and ERK activation prevented UVB-induced TM down-regulation. The nuclear p53 accumulation and TM promoter binding was observed within 3 h after UVB exposure. Small interfering RNA-mediated p53 knockdown disrupted the UVB-mediated TM protein down-regulation. Our study demonstrates that UVB irradiation results in ROS accumulation and ERK activation, which causes the nuclear p53 accumulation and TM promoter binding to inhibit TM expression. This study provides novel evidence demonstrating that p53 serves as a key regulator of keratinocyte TM expression.     

The greater lethality of UVB radiation to cultured human cells is associated with the specific activation of a DNA damage-independent signaling pathway

UV radiation causes cell death through the activation of various intracellular signaling molecules in both DNA damage-dependent and -independent manners. The ability of middle-wavelength UV (UVB) radiation to form DNA photoproducts is less than that of short-wavelength UV (UVC) radiation; however, the differences between UVB and UVC radiation in the extent of DNA damage-independent signaling and its contribution to cell death have not been well characterized. When cells were irradiated with UVB or UVC radiation at doses that generated equivalent amounts of DNA photoproducts, UVB radiation induced more clonogenic cell death, apoptotic cells, mitochondrial cytochrome C release, and intracellular oxidative stress. Among the signaling molecules examined, levels of p53 phosphorylated at Ser-392 and p38 were higher in UVB-irradiated cells than in UVC-irradiated cells. Both phosphorylations were reduced by treating cells with an antioxidant. Furthermore, an inhibitor of p38 also blocked the phosphorylation of p53 at Ser-392. These results suggest that UVB radiation activates the p38 pathway through the generation of oxidative stress, which merges with the DNA p53 pathway by phosphorylation of p53 at ser392. This greater contribution of the DNA damage-independent pathway in UVB-irradiated cells may explain the greater lethality of UVB radiation.     

Up-Regulation and redistribution of Bax in ultraviolet B-irradiated melanocytes

Ultraviolet B (UVB) radiation may activate or deteriorate cultured human epidermal melanocytes, depending on the doses and culture conditions. It is also reported that cultured human epidermal melanocytes derived from different pigmentary phenotypes showed different responses to UVB radiation. In this study, we examined whether apoptosis of melanocytes can be induced by physiologic doses of UVB irradiation using cultured human epidermal melanocytes derived from oriental males of skin types III and IV. Propidium iodide staining for DNA condensation and flow cytometric analyses demonstrated the apoptotic cell death of melanocytes following UVB irradiation (0-30 mJ/cm2). The levels of p53, Bax, and Bcl-2, determined by immunoblotting, revealed a dose-dependent increase in p53 and Bax, but the level of Bcl-2 remained unchanged. Confocal microscopic examination showed that Bax moved from a diffuse to a punctate distribution after UVB irradiation. However, there were no changes in the pattern of distribution of Bcl-2. These data suggest that the high constitutional level of Bcl-2 may protect melanocytes from UVB-induced injury, and that apoptotic death of melanocytes may be induced by the elevation and redistribution of Bax.     

Suppression of transcription factor NF-kappaB activity by Bcl-2 protein in NIH3T3 cells: implication of a novel NF-kappaB p50-Bcl-2 complex for the anti-apoptotic function of Bcl-2

Bcl-2 can suppress apoptosis by controlling genes that encode proteins required for programmed cell death and by interference with peroxidative damage. Overexpression of Bcl-2 in NIH3T3 cells can prevent GSNO-induced (S-nitrosoglutathione-induced) apoptosis. The experimental results indicated that activation of NF-kappaB by GSNO is involved in inducing apoptosis. Surprisingly, we found that Bcl-2 delayed the release of IkB by formation of a Bcl-2-NF-kappaB complex (p50-p65-IkappaB) in the cytoplasm during cell apoptosis. Furthermore, a novel Bcl-2-p50 complex was found in the nucleus. These features were only observed in Bcl-2-transfected cells but not in the parental NIH3T3 cells. Overexpression of Bcl-2 suppressed the levels of c-myc, a target gene of NF-kappaB, and influenced the DNA-binding activity of NF-kappaB during GSNOinduced apoptosis. We suggest that the Bcl-2-p50 complex inhibits NF-kappaB DNA-binding activity by competing with the p65-p50 heterodimer for the DNA-binding site in the nucleus. Finally, it has been demonstrated that the anti-apoptotic potential of Bcl-2 may be attributed to its complexing with p50 in the nucleus that leads to blockage of nuclear gene expression.     

Insulin-like growth factor-binding protein-3 modulates expression of Bax and Bcl-2 and potentiates p53-independent radiation-induced apoptosis in human breast cancer cells

We report that transfection of insulin-like growth factor-binding protein-3 (IGFBP-3) cDNA in human breast cancer cell lines expressing either mutant p53 (T47D) or wild-type p53 (MCF-7) induces apoptosis. IGFBP-3 also increases the ratio of pro-apoptotic to anti-apoptotic members of the Bcl-2 family. In MCF-7, an increase in Bad and Bax protein expression and a decrease in Bcl-x(L) protein and Bcl-2 protein and mRNA were observed. In T47D, Bax and Bad proteins were up-regulated; Bcl-2 protein is undetectable in these cells. As T47D expresses mutant p53 protein, these modulations of pro-apoptotic proteins and induction of apoptosis are independent of p53. The effect of IGFBP-3 on the response of T47D to ionizing radiation (IR) was examined. These cells do not G(1) arrest in response to IR and are relatively radioresistant. Transfection of IGFBP-3 increased the radiosensitivity of T47D and increased IR-induced apoptosis but did not effect a rapid G(1) arrest. IR also caused a much greater increase in Bax protein in IGFBP-3 transfectants compared with vector controls. Thus, IGFBP-3 increases the expression of pro-apoptotic proteins and apoptosis both basally and in response to IR, suggesting it may be a p53-independent effector of apoptosis in breast cancer cells via its modulation of the Bax:Bcl-2 protein ratio.     

Possible involvement of glutathione and p53 in trichloroethylene- and perchloroethylene-induced lipid peroxidation and apoptosis in human lung cancer cells

Trichloroethylene (TCE) and perchloroethylene (PERC) are volatile organic compounds (VOCs) that are primarily inhaled through the respiratory system. The aim of this study was to elucidate the role of glutathione (GSH) and p53 in TCE- and PERC-induced lung toxicity. Human lung adenocarcinoma cells NCI-H460 (p53-wild-type) have constitutively lower levels of GSH than NCI-H1299 (p53-null) cells. The results showed that exposure to vapor TCE and PERC produced a dose-dependent and more pronounced accumulation of H(2)O(2) in p53-WT H460 than p53-null H1299 cells. The accumulation of H(2)O(2) was accompanied by severe cellular damage, as indicated by the significant increase of lipid peroxidation and apoptosis in p53-WT H460 cells, but not p53-null H1299 cells. Cotreatment of p53-WT H460 cells with free radical scavengers, such as D-mannitol, uric acid, and sodium selenite, significantly attenuated the TCE- or PERC-induced lipid peroxidation. In contrast, depletion of GSH in p53-null H1299 cells enhanced TCE- or PERC-induced lipid peroxidation. The levels of p53 and Bax proteins were elevated, while Bcl-2 protein was downregulated in TCE- or PERC-treated p53-WT H460 cells. Activity of caspase 3, the apoptotic executioner, was also significantly enhanced in TCE- or PERC-treated cells. These data suggest that, in human lung cancer cells, GSH plays a vital role in the protection of TCE- and PERC-induced oxidative stress and apoptosis, which may be mediated through a p53-dependent pathway.