Coenzyme Q 10 Depletion is Comparatively Less Detrimental to Human Cultured Skin Fibroblasts than Respiratory Chain Complex Deficiencies

The oxidative stress possibly resulting from an inherited respiratory chain (RC) deficiency was investigated in a series of human cultured skin fibroblasts presenting either ubiquinone depletion or isolated defect of the various RC complexes. Taken as an index for superoxide overproduction, a significant induction of superoxide dismutase activity was observed in complex V-deficient fibroblasts harboring the NARP-mutation in the ATPase 6 gene. Superoxide dismutase induction was also noticed, albeit to a lesser extent, in complex II-deficient fibroblasts with a mutation in the nuclear gene encoding the flavoprotein subunit of the succinate dehydrogenase. No sign of oxidative stress could be found in ubiquinone-depleted fibroblasts. In all cases but complex IV-defect, increased oxidative stress was associated with increased cell death. In glucose-rich medium, apoptosis appeared as the main cell death process associated with all types of RC defect. However, similar to the great variations in oxidative stress associated with the various types of RC defect, we found that apoptotic features differed noticeably between defects. No indication of increased cell death was found in ubiquinone-depleted fibroblasts.     

Lack of protection of prior heat shock against UV-induced oxidative stress in human skin fibroblasts

The effect of prior hyperthermia on UV-induced oxidative stress was studied in human skin fibroblasts. UV radiation alone induced an increased release of superoxide anions and increased lipid peroxidation in skin fibroblasts accompanied by a rise in catalase and superoxide dismutase activities. Hyperthermia was found to induce a significant rise in the cell content of heat-shock proteins, HSP60 and HSP70, but this treatment prior to UV radiation did not influence any indicators of oxidative stress in the fibroblasts. In contrast, the combination of heat shock prior to UV-exposure reduced fibroblast cell viability compared with UV radiation-exposure alone.     

Extracellular superoxide dismutase is a major antioxidant in human fibroblasts and slows telomere shortening

There is good evidence that telomere shortening acts as a biological clock in human fibroblasts, limiting the number of population doublings a culture can achieve. Oxidative stress also limits the growth potential of human cells, and recent data show that the effect of mild oxidative stress is mediated by a stress-related increased rate of telomere shortening. Thus, fibroblast strains have donor-specific antioxidant defense, telomere shortening rate, and growth potential. We used low-density gene expression array analysis of fibroblast strains with different antioxidant potentials and telomere shortening rates to identify gene products responsible for these differences. Extracellular superoxide dismutase was identified as the strongest candidate, a correlation that was confirmed by Northern blotting. Over-expression of this gene in human fibroblasts with low antioxidant capacity increased total cellular superoxide dismutase activity, decreased the intracellular peroxide content, slowed the telomere shortening rate, and elongated the life span of these cells under normoxia and hyperoxia. These results identify extracellular superoxide dismutase as an important antioxidant gene product in human fibroblasts, confirm the causal role of oxidative stress for telomere shortening, and strongly suggest that the senescence-like arrest under mild oxidative stress is telomere-driven.     

Lack of protection of prior heat shock against UV-induced oxidative stress in human skin fibroblasts

Abstract

The effect of prior hyperthermia on UV-induced oxidative stress was studied in human skin fibroblasts. UV radiation alone induced an increased release of superoxide anions and increased lipid peroxidation in skin fibroblasts accompanied by a rise in catalase and superoxide dismutase activities. Hyperthermia was found to induce a significant rise in the cell content of heat-shock proteins, HSP60 and HSP70, but this treatment prior to UV radiation did not influence any indicators of oxidative stress in the fibroblasts. In contrast, the combination of heat shock prior to UV-exposure reduced fibroblast cell viability compared with UV radiation-exposure alone.     

Effect of antioxidant supplementation on the adaptive response of human skin fibroblasts to UV-induced oxidative stress

The effect of supplementation with substances having antioxidant properties on the adaptive responses of human skin fibroblasts to UV-induced oxidative stress was studied in vitro. UVR was found to induce a substantial oxidative stress in fibroblasts, resulting in an increased release of superoxide anions and an increase in lipid peroxidation (shown by an elevated malonaldehyde content). Sub-lethal doses of UVR were also found to induce adaptive responses in the fibroblast antioxidant defences, with a transient rise in catalase and superoxide dismutase activities followed by a slower, large increase in cellular glutathione content. Supplementation of the fibroblasts with the antioxidants, Trolox (a water soluble analogue of alpha-tocopherol), ascorbic acid or beta-carotene, had differential effects on these responses. Trolox supplementation reduced the UVR-induced cellular oxidative stress and adaptive response in a predictable concentration-dependent manner. This was in contrast to ascorbic acid which increased superoxide release from fibroblasts. At low doses, ascorbate supplements also reduced the magnitude of the adaptive increases in catalase and superoxide dismutase activities and increase in glutathione content. Beta-carotene had a similar effect to ascorbic acid, reducing the extent of the adaptations to UVR at lower doses while simultaneously increasing superoxide release and malonaldehyde content. These in vitro data indicate that only the vitamin E analogue suppressed UVR-induced oxidative stress in a predictable manner and suggest that common dietary antioxidants may not be equally effective in reducing the potential deleterious effects of UVR-induced oxidative stress in skin.     

Effect of antioxidant supplementation on the adaptive response of human skin fibroblasts to UV-induced oxidative stress

Abstract

The effect of supplementation with substances having antioxidant properties on the adaptive responses of human skin fibroblasts to UV-induced oxidative stress was studied in vitro. UVR was found to induce a substantial oxidative stress in fibroblasts, resulting in an increased release of superoxide anions and an increase in lipid peroxidation (shown by an elevated malonaldehyde content). Sub-lethal doses of UVR were also found to induce adaptive responses in the fibroblast antioxidant defences, with a transient rise in catalase and superoxide dismutase activities followed by a slower, large increase in cellular glutathione content. Supplementation of the fibroblasts with the antioxidants, Trolox (a water soluble analogue of α-tocopherol), ascorbic acid or β-carotene, had differential effects on these responses. Trolox supplementation reduced the UVR-induced cellular oxidative stress and adaptive response in a predictable concentration-dependant manner. This was in contrast to ascorbic acid which increased superoxide release from fibroblasts. At low doses, ascorbate supplements also reduced the magnitude of the adaptive increases in catalase and superoxide dismutase activities and increase in glutathione content. β-Carotene had a similar effect to ascorbic acid, reducing the extent of the adaptations to UVR at lower doses while simultaneously increasing superoxide release and malonaldehyde content. These in vitro data indicate that only the vitamin E analogue suppressed UVR-induced oxidative stress in a predictable manner and suggest that common dietary antioxidants may not be equally effective in reducing the potential deleterious effects of UVR-induced oxidative stress in skin.     

Exposure to polychlorinated biphenyls enhances lipid peroxidation in human normal peritoneal and adhesion fibroblasts: A potential role for myeloperoxidase

Nitric oxide, superoxide, and lipid peroxidation (LPO) produced under oxidative stress may contribute to the development of postoperative adhesions. The objective of this study was to determine the effects of polychlorinated biphenyls (PCBs) on LPO, superoxide dismutase, myeloperoxidase (MPO), and nitrite/nitrate in human normal peritoneal and adhesion fibroblasts. PCB treatment reduced inducible nitric oxide synthase (iNOS) expression as well as levels of nitrite/nitrate in both cell lines. Although there was no difference in iNOS expression between the two cell lines, adhesion fibroblasts manifested lower basal levels of MPO compared to normal peritoneal fibroblasts. There was a reduction in MPO expression and its activity in response to PCB treatment in normal peritoneal fibroblasts; however, this effect was minimal in adhesion fibroblasts. Moreover, adhesion fibroblasts manifested higher levels of LPO compared to normal peritoneal fibroblasts, whereas PCB treatment increased LPO levels in both cell types. We conclude that PCBs promote the development of the adhesion phenotype by generating an oxidative stress environment. This is evident by lower iNOS, MPO, and nitrite/nitrate and a simultaneous increase in LPO. Loss of MPO activity, possibly through a mechanism involving MPO heme depletion and free iron release, is yet another source of oxidative stress.     

Role of Oxidative Damage in Friedreich's Ataxia

Plasma malondialdehyde (MDA) levels were raised in Friedreich's ataxia (FRDA) patients. These levels correlated with increasing age and disease duration, suggesting lipid peroxidation increased with disease progression. Using fibroblasts from FRDA patients we observed that GSH levels and aconitase activities were normal, suggesting their antioxidant status was unchanged. When exposed to various agents to increase free radical generation we observed that intracellular superoxide generation induced by paraquat caused enhanced oxidative damage. This correlated with the size of the GAA1 expansion, suggesting decreased frataxin levels may render the cells more vulnerable to mild oxidative stress. More severe oxidative stress induced by hydrogen peroxide caused increased cell death in FRDA fibroblasts but was not significantly different from control cells. We propose that abnormal respiratory chain function and iron accumulation may lead to a progressive increase in oxidative damage, but increased sensitivity to free radicals may not require detectable respiratory chain dysfunction.     

Manganese superoxide dismutase induction by iron is impaired in Friedreich ataxia cells.

Iron-mediated oxidative stress has been implicated in the pathology of the neurodegenerative disease Friedreich ataxia (FRDA). Here, we show that normal upregulation of the stress defense protein manganese superoxide dismutase (MnSOD) fails to occur in FRDA fibroblasts exposed to iron. This impaired induction was observed at iron levels in which increased activation of the redox-sensitive factor NF-kappaB was absent. Furthermore, MnSOD induction could only be partially suppressed by antioxidants. We conclude that an NF-kappaB-independent pathway that may not require free radical signaling is responsible for the reduction of MnSOD induction. This impairment could constitute both a novel defense mechanism against iron-mediated oxidative stress in cells with mitochondrial iron overload and conversely, an alternative source of free radicals that could contribute to the disease pathology.     

Supplemental ascorbate or alpha-tocopherol induces cell death in Cu-deficient HL-60 cells

Cytochrome c oxidase (CCO) is the Cu-dependent, terminal respiratory complex of the mitochondrial electron transport chain. Inhibition of CCO can promote oxidative stress by increasing mitochondrial production of reactive oxygen species (ROS). Because mitochondria have an important role in apoptosis as both a target and source for ROS, enhanced ROS production resulting from inhibition of CCO by Cu deficiency may trigger apoptosis. The present study focuses on the mitochondrial effects of N,N'-bis(2-aminoethyl)-1,3-propanedi-amine (TET), which inhibits CCO by causing cellular Cu deficiency, and the antioxidants ascorbate and alpha-tocopherol in a human promyelocytic leukemia cell line (HL-60). The following effects were observed: (i) TET reduced both cell growth and viability only in the presence of ascorbate or alpha-tocopherol; (ii) TET reduced CCO activity and increased mitochondrial ROS production as indicated by increased expression of Mn super-oxide dismutase, but the induction of Mn superoxide dismutase was not affected by ascorbate or alpha-tocopherol; (iii) TET acted independently of ascorbate or alpha-tocopherol in disrupting mitochondrial membrane potential; (iv) TET did not increase caspase-8 activity in the absence of ascorbate or alpha-tocopherol; and (v) TET did not increase transfer of cytochrome c from mitochondria to the cytosol unless alpha-tocopherol was present. These findings indicate that reduction in CCO activity by TET-induced Cu deficiency increased oxidative stress in HL-60 cells sufficiently to disrupt the electrochemical gradient of the inner mitochondrial membrane but did not trigger cell death. Also, ascorbate and alpha-tocopherol did not alleviate oxidative stress but may have become pro-oxidants, adding to the oxidant burden sufficiently to trigger cell death in TET-treated cells.     

Reduction of DNA fragmentation and hydroxyl radical production by hyaluronic acid and chondroitin-4-sulphate in iron plus ascorbate-induced oxidative stress in fibroblast cultures

Glycosaminoglycans (GAGs), components of extracellular matrix, are thought to play important roles in cell proliferation and differentiation in the repair process of injured tissue. Oxidative stress is one of the most frequent causes of tissue and cell injury and the consequent lipid peroxidation is the main manifestation of free radical damage. It has been found to play an important role in the evolution of cell death. Since several reports have shown that hyaluronic acid (HYA) and chondroitin-4-sulphate (C4S) are able to inhibit lipid peroxidation during oxidative stress, We investigated the antioxidant capacity of these GAGs in reducing oxidative damage in fibroblast cultures.

Free radicals production was induced by the oxidizing system employing iron (Fe₂₊) plus ascorbate. We evaluated cell death, membrane lipid peroxidation, DNA damage, protein oxidation, hydroxyl radical (OH_•) generation and endogenous antioxidant depletion in human skin fibroblast cultures.

The exposition of fibroblasts to FeSO₄ and ascorbate caused inhibition of cell growth and cell death, increased OH_• production determined by the aromatic trap method; furthermore it caused DNA strand breaks and protein oxidation as shown by the DNA fragments analysis and protein carbonyl content, respectively. Moreover, it enhanced lipid peroxidation evaluated by the analysis of conjugated dienes (CD) and decreased antioxidant defenses assayed by means of measurement of superoxide dismutase (SOD) and catalase (CAT) activities.

When fibroblasts were treated with two different doses of HYA or C4S a protective effect, following oxidative stress induction, was shown. In fact these GAGs were able to limit cell death, reduced DNA fragmentation and protein oxidation, decreased OH_• generation, inhibited lipid peroxidation and improved antioxidant defenses.

Our results confirm the antioxidant activity of HYA and C4S and this could represent a useful step in the understanding of the exact role played by GAGs in living organisms.     

Differential metabolic consequences of fumarate hydratase and respiratory chain defects

Defects of the oxidative ATP production pathway lead to an amazing variety of disease phenotypes, ranging from childhood encephalomyopathies to hereditary tumor formation. A key enzyme of tricarboxylic cycle, fumarate hydratase (FH), is involved in encephalopathies, but also in leiomyoma formation, and occasionally also in various types of cancer. MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) and NARP (neuropathy ataxia retinitis pigmentosa) are progressive neurological disorders, caused by mitochondrial DNA mutations and respiratory chain (RC) deficiency. These diseases lead to disability and premature death, but not to tumorigenesis. We studied the cellular consequences of FH and RC deficiencies, aiming to identify general responses to energy metabolism defect and those specific for FH-deficiency, suggestively connected to tumorigenesis. Unlike in RC deficiency, the FH-deficient diploid human fibroblasts showed no signs of oxidative stress, but had a reduced redox state with high glutathione levels. The cytoplasmic FH isoform, previously described, but with an unknown function, was completely lacking in all FH-deficient lines. Fumarate was increased in two of our FH-lines, but accumulation of HIF-1alpha was not detected. Glycolysis was induced in both MELAS and in FH-deficiency. Accumulation of fumarate in primary fibroblasts did not activate a hypoxia response, suggesting that hypoxia activation due to fumarate accumulation may be a tissue-specific response. The lack of cytoplasmic form of FH and the reduced redox environment were typical for all FH-mutant lines, and their role in FH-related tumorigenesis requires further attention.     

Oxidative stress-dependent p66Shc phosphorylation in skin fibroblasts of children with mitochondrial disorders

p66Shc, the growth factor adaptor protein, can have a substantial impact on mitochondrial metabolism through regulation of cellular response to oxidative stress. We investigated relationships between the extent of p66Shc phosphorylation at Ser36, mitochondrial dysfunctions and an antioxidant defense reactions in fibroblasts derived from five patients with various mitochondrial disorders (two with mitochondrial DNA mutations and three with methylglutaconic aciduria and genetic defects localized, most probably, in nuclear genes). We found that in all these fibroblasts, the extent of p66Shc phosphorylation at Ser36 was significantly increased. This correlated with a substantially decreased level of mitochondrial superoxide dismutase (SOD2) in these cells. This suggest that SOD2 is under control of the Ser36 phosphorylation status of p66Shc protein. As a consequence, an intracellular oxidative stress and accumulation of damages caused by oxygen free radicals are observed in the cells.     

Cytotoxicity of a mitochondriotropic quercetin derivative: Mechanisms

The mitochondriotropic compound 7-O-(4-triphenylphosphoniumbutyl)quercetin iodide (Q-7BTPI) in the μM concentration range caused necrotic death of cultured cells by acting as a prooxidant, with generation of superoxide anion in the mitochondria. Externally added membrane-permeating superoxide dismutase or catalase largely prevented death. Rescue by permeant catalase indicates that the toxicant is H(2)O(2), or reactive species derived from it. Rescue by permeant dismutase suggests the possibility of a chain mechanism of H(2)O(2) production, in which dismutation of superoxide constitutes a termination step. Oxidative stress was due to the presence of free phenolic hydroxyls and to accumulation in mitochondria, since the analogous mitochondriotropic per-O-methylated compound -3,3',4',5-tetra-O-methyl,7-O-(4-triphenylphosphoniumbutyl) quercetin iodide (QTM-7BTPI)-or Quercetin itself induced no or little superoxide production and cell death. Q-7BTPI did not cause a significant perturbation of the mitochondrial transmembrane potential or of respiration in cells. On the other hand its presence led to inhibition of glutathione peroxidase, an effect expected to accentuate oxidative stress by interfering with the elimination of H(2)O(2). An exogenous permeable glutathione precursor determined a strong increase of cellular glutathione levels but did not rescue the cells. Death induction was selective for fast-growing C-26 tumoral cells and mouse embryonic fibroblasts (MEFs) while sparing slow-growing MEFs. This suggests a possible use of Q-7BTPI as a chemotherapeutic agent.     

X-linked inhibitor of apoptosis protein increases mitochondrial antioxidants through NF-kappaB activation

X chromosome-linked inhibitor of apoptosis protein is an endogenous inhibitor of caspases and is an important regulator of cell death. XIAP can also influence cell signaling, but downstream proteins affected are largely unknown. We show here using neuronal PC6.3 cells that XIAP increases the levels of antioxidants, particularly superoxide dismutase-2 that is localized to mitochondria. Studies using reporter constructs and NF-κB Rel-A deficient mouse embryonic fibroblasts showed that NF-κB signaling is required for the induction of Sod2 by XIAP. XIAP also reduced oxidative stress in the PC6.3 cells as shown by decreased production of reactive oxygen species. These findings disclose a novel role for XIAP in control of oxidative stress and mitochondrial antioxidants that may contribute to cell protection after various injuries.     

Nuclear-mitochondrial cross-talk in global myocardial ischemia. A time-course analysis

There is an ongoing concern regarding the biocompatibility of nanoparticles with sizes less than 100 nm as compared to larger particles of the same nominal substance. In this study, we investigated the toxic properties of magnetite stabilized with polyacrylate sodium. The magnetite was characterized by X-ray powder diffraction analysis, and the mean particle diameter was calculated using the Scherrer formula and was found to be 9.3 nm. In this study, we treated lung epithelial cells with different concentrations of magnetite and investigated their effects on oxidative stress and cell proliferation. Our data showed an inhibition of cell proliferation in magnetite-treated cells with a significant dose-dependent activation and induction of reactive oxygen species. Also, we observed a depletion of antioxidants, glutathione, and superoxide dismutase, respectively, as compared with control cells. In addition, apoptotic-related protease/enzyme such as caspase-3 and -8 activities, were increased in a dose-dependent manner with corresponding increased levels of DNA fragmentation in magnetite-treated cells compared to than control cells. Together, the present study reveals that magnetite exposure induces oxidative stress and depletes antioxidant levels in the cells to stimulate apoptotic pathway for cell death.     

Quiescent fibroblasts are protected from proteasome inhibition-mediated toxicity

Proteasome inhibition is used as a treatment strategy for multiple types of cancers. Although proteasome inhibition can induce apoptotic cell death in actively proliferating cells, it is less effective in quiescent cells. In this study, we used primary human fibroblasts as a model system to explore the link between the proliferative state of a cell and proteasome inhibition-mediated cell death. We found that proliferating and quiescent fibroblasts have strikingly different responses to MG132, a proteasome inhibitor; proliferating cells rapidly apoptosed, whereas quiescent cells maintained viability. Moreover, MG132 treatment of proliferating fibroblasts led to increased superoxide anion levels, juxtanuclear accumulation of ubiquitin- and p62/SQSTM1-positive protein aggregates, and apoptotic cell death, whereas MG132-treated quiescent cells displayed fewer juxtanuclear protein aggregates, less apoptosis, and higher levels of mitochondrial superoxide dismutase. In both cell states, reducing reactive oxygen species with N-acetylcysteine lessened protein aggregation and decreased apoptosis, suggesting that protein aggregation promotes apoptosis. In contrast, increasing cellular superoxide levels with 2-methoxyestradiol treatment or inhibition of autophagy/lysosomal pathways with bafilomycin A1 sensitized serum-starved quiescent cells to MG132-induced apoptosis. Thus, antioxidant defenses and the autophagy/lysosomal pathway protect serum-starved quiescent fibroblasts from proteasome inhibition-induced cytotoxicity.     

Changes in antioxidant gene expression and induction of oxidative stress in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) under Al stress

Aluminium toxicity has been recognized as a primary growth-limiting factor in acid soil, resulting in a decrease in plant growth and production. In this experiment we have studied the induction of oxidative stress and changes in antioxidant gene expression in pea (var. ALASKA) under aluminium (Al) stress. We have found that Al treatment affected the growth of pea plant and induced oxidative stress with a change in antioxidant gene expression profile. While the expression of glutathione-s-transferase (GST) and catalase (CAT) was more in root, cytosolic Ascorbate peroxidase (cAPX) expression increased in shoot under aluminium stress. Copper- Zinc Superoxide dismutase (Cu-Zn SOD) gene expression was higher after 24 h but decreased after 48 h along with elevated expression of manganese superoxide dismutase (MnSOD) and iron-superoxide dismutase (FeSOD) at higher aluminium contentrations after 24 and 48 h. Aluminium stress elevated hydrogen peroxide (H₂O₂) level and affected the growth. The proline content did not change significantly, whereas glutathione content increased with a decreased ascorbate content under Al stress. The present study indicates that aluminium treatment affected the antioxidant gene expression and induced oxidative stress in pea plant.