Mitochondrial dysfunction increases oxidative stress and decreases chronological life span in fission yeast

Background

Oxidative stress is a probable cause of aging and associated diseases. Reactive oxygen species (ROS) originate mainly from endogenous sources, namely the mitochondria.

Methodology/Principal Findings

We analyzed the effect of aerobic metabolism on oxidative damage in Schizosaccharomyces pombe by global mapping of those genes that are required for growth on both respiratory-proficient media and hydrogen-peroxide-containing fermentable media. Out of a collection of approximately 2700 haploid yeast deletion mutants, 51 were sensitive to both conditions and 19 of these were related to mitochondrial function. Twelve deletion mutants lacked components of the electron transport chain. The growth defects of these mutants can be alleviated by the addition of antioxidants, which points to intrinsic oxidative stress as the origin of the phenotypes observed. These respiration-deficient mutants display elevated steady-state levels of ROS, probably due to enhanced electron leakage from their defective transport chains, which compromises the viability of chronologically-aged cells.

Conclusion/Significance

Individual mitochondrial dysfunctions have often been described as the cause of diseases or aging, and our global characterization emphasizes the primacy of oxidative stress in the etiology of such processes.     

The emphysematous lung is abnormally sensitive to TRAIL-mediated apoptosis

Background

Alveolar apoptosis is increased in the emphysematous lung. However, mechanisms involved are not fully understood. Recently, we demonstrated that levels of TRAIL receptor 1 and 2, levels of p53, and Bax/Bcl-x_L ratio were elevated in the lung of subjects with emphysema, despite smoking cessation. Thus, we postulate that due to chronic pulmonary oxidative stress, the emphysematous lung would be abnormally sensitive to TRAIL-mediated apoptosis.

Methodology

A549 cells were exposed to rTRAIL, cigarette smoke extract, and/or H₂O₂ prior to caspase-3 activity measurement and annexin V staining assessment. In addition, freshly resected lung samples were obtained from non-emphysematous and emphysematous subjects and exposed ex vivo to rTRAIL for up to 18 hours. Lung samples were harvested and levels of active caspase-3 and caspase-8 were measured from tissue lysates.

Results

Both cigarette smoke extract and H₂O₂ were able to sensitize A549 cells to TRAIL-mediated apoptosis. Moreover, following exposure to rTRAIL, caspase-3 and -8 were activated in lung explants from emphysematous subjects while being decreased in lung explants from non-emphysematous subjects.

Significance of the study

Alveolar sensitivity to TRAIL-mediated apoptosis is strongly increased in the emphysematous lung due to the presence of oxidative stress. This might be a new mechanism leading to increased alveolar apoptosis and persistent alveolar destruction following smoking cessation.     

Detoxification of Multiple Heavy Metals by a Half-Molecule ABC Transporter,HMT-1, and Coelomocytes of Caenorhabditis elegans

Background

Developing methods for protecting organisms in metal-polluted environments is contingent upon our understanding of cellular detoxification mechanisms. In this regard, half-molecule ATP-binding cassette (ABC) transporters of the HMT-1 subfamily are required for cadmium (Cd) detoxification. HMTs have conserved structural architecture that distinguishes them from other ABC transporters and allows the identification of homologs in genomes of different species including humans. We recently discovered that HMT-1 from the simple, unicellular organism, Schizosaccharomyces pombe, SpHMT1, acts independently of phytochelatin synthase (PCS) and detoxifies Cd, but not other heavy metals. Whether HMTs from multicellular organisms confer tolerance only to Cd or also to other heavy metals is not known.

Methodology/Principal Findings

Using molecular genetics approaches and functional in vivo assays we showed that HMT-1 from a multicellular organism, Caenorhabditis elegans, functions distinctly from its S. pombe counterpart in that in addition to Cd it confers tolerance to arsenic (As) and copper (Cu) while acting independently of pcs-1. Further investigation of hmt-1 and pcs-1 revealed that these genes are expressed in different cell types, supporting the notion that hmt-1 and pcs-1 operate in distinct detoxification pathways. Interestingly, pcs-1 and hmt-1 are co-expressed in highly endocytic C. elegans cells with unknown function, the coelomocytes. By analyzing heavy metal and oxidative stress sensitivities of the coelomocyte-deficient C. elegans strain we discovered that coelomocytes are essential mainly for detoxification of heavy metals, but not of oxidative stress, a by-product of heavy metal toxicity.

Conclusions/Significance

We established that HMT-1 from the multicellular organism confers tolerance to multiple heavy metals and is expressed in liver-like cells, the coelomocytes, as well as head neurons and intestinal cells, which are cell types that are affected by heavy metal poisoning in humans. We also showed that coelomocytes are involved in detoxification of heavy metals. Therefore, the HMT-1-dependent detoxification pathway and coelomocytes of C. elegans emerge as novel models for studies of heavy metal-promoted diseases.     

Crosstalk between JNK and SUMO signaling pathways: deSUMOylation is protective against H2O2-induced cell injury

Background

Oxidative stress is a key feature in the pathogenesis of several neurological disorders. Following oxidative stress stimuli a wide range of pathways are activated and contribute to cellular death. The mechanism that couples c-Jun N-terminal kinase (JNK) signaling, a key pathway in stress conditions, to the small ubiquitin-related modifier (SUMO), an emerging protein in the field, is largely unknown.

Methodology/Principal Findings

With this study we investigated if SUMOylation participates in the regulation of JNK activation as well as cellular death in a model of H₂O₂ induced-oxidative stress. Our data show that H₂O₂ modulates JNK activation and induces cellular death in neuroblastoma SH-SY5Y cells. Inhibition of JNK''s action with the D-JNKI1 peptide rescued cells from death. Following H₂O₂, SUMO-1 over-expression increased phosphorylation of JNK and exacerbated cell death, although only in conditions of mild oxidative stress. Furthermore inhibition of SUMOylation, following transfection with SENP1, interfered with JNK activation and rescued cells from H₂O₂ induced death. Importantly, in our model, direct interaction between these proteins can occur.

Conclusions/Significance

Taken together our results show that SUMOylation may significantly contribute to modulation of JNK activation and contribute to cell death in oxidative stress conditions.     

Transcript and Protein Analysis Reveals Better Survival Skills of Monocyte-Derived Dendritic Cells Compared to Monocytes during Oxidative Stress

Background

Dendritic cells (DCs), professional antigen-presenting cells with the unique ability to initiate primary T-cell responses, are present in atherosclerotic lesions where they are exposed to oxidative stress that generates cytotoxic reactive oxygen species (ROS). A large body of evidence indicates that cell death is a major modulating factor of atherogenesis. We examined antioxidant defence systems of human monocyte-derived (mo)DCs and monocytes in response to oxidative stress.

Methods

Oxidative stress was induced by addition of tertiary-butylhydroperoxide (tert-BHP, 30 min). Cellular responses were evaluated using flow cytometry and confocal live cell imaging (both using 5-(and-6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate, CM-H₂DCFDA). Viability was assessed by the neutral red assay. Total RNA was extracted for a PCR profiler array. Five genes were selected for confirmation by Taqman gene expression assays, and by immunoblotting or immunohistochemistry for protein levels.

Results

Tert-BHP increased CM-H₂DCFDA fluorescence and caused cell death. Interestingly, all processes occurred more slowly in moDCs than in monocytes. The mRNA profiler array showed more than 2-fold differential expression of 32 oxidative stress–related genes in unstimulated moDCs, including peroxiredoxin-2 (PRDX2), an enzyme reducing hydrogen peroxide and lipid peroxides. PRDX2 upregulation was confirmed by Taqman assays, immunoblotting and immunohistochemistry. Silencing PRDX2 in moDCs by means of siRNA significantly increased CM-DCF fluorescence and cell death upon tert-BHP-stimulation.

Conclusions

Our results indicate that moDCs exhibit higher intracellular antioxidant capacities, making them better equipped to resist oxidative stress than monocytes. Upregulation of PRDX2 is involved in the neutralization of ROS in moDCs. Taken together, this points to better survival skills of DCs in oxidative stress environments, such as atherosclerotic plaques.     

Protective Antioxidant and Antiapoptotic Effects of ZnCl2 in Rat Pancreatic Islets Cultured in Low and High Glucose Concentrations

Aim/Hypothesis

Rat pancreatic islet cell apoptosis is minimal after prolonged culture in 10 mmol/l glucose (G10), largely increased in 5 mmol/l glucose (G5) and moderately increased in 30 mmol/l glucose (G30). This glucose-dependent asymmetric V-shaped profile is preceded by parallel changes in the mRNA levels of oxidative stress-response genes like Metallothionein 1a (Mt1a). In this study, we tested the effect of ZnCl₂, a potent inducer of Mt1a, on apoptosis, mitochondrial oxidative stress and alterations of glucose-induced insulin secretion (GSIS) induced by prolonged exposure to low and high vs. intermediate glucose concentrations.

Methods

Male Wistar rat islets were cultured in RPMI medium. Islet gene mRNA levels were measured by RTq-PCR. Apoptosis was quantified by measuring islet cytosolic histone-associated DNA fragments and the percentage of TUNEL-positive β-cells. Mitochondrial thiol oxidation was measured in rat islet cell clusters expressing “redox sensitive GFP” targeted to the mitochondria (mt-roGFP1). Insulin secretion was measured by RIA.

Results

As observed for Mt1a mRNA levels, β-cell apoptosis and loss of GSIS, culture in either G5 or G30 vs. G10 significantly increased mt-roGFP1 oxidation. While TPEN decreased Mt1a/2a mRNA induction by G5, addition of 50–100 µM ZnCl₂ to the culture medium strongly increased Mt1a/2a mRNA and protein levels, reduced early mt-roGFP oxidation and significantly decreased late β-cell apoptosis after prolonged culture in G5 or G30 vs. G10. It did not, however, prevent the loss of GSIS under these culture conditions.

Conclusion

ZnCl₂ reduces mitochondrial oxidative stress and improves rat β-cell survival during culture in the presence of low and high vs. intermediate glucose concentrations without improving their acute GSIS.     

Treatment of CoQ10 Deficient Fibroblasts with Ubiquinone,CoQ Analogs,and Vitamin C: Time- and Compound-Dependent Effects

Background

Coenzyme Q₁₀ (CoQ₁₀) and its analogs are used therapeutically by virtue of their functions as electron carriers, antioxidant compounds, or both. However, published studies suggest that different ubiquinone analogs may produce divergent effects on oxidative phosphorylation and oxidative stress.

Methodology/Principal Findings

To test these concepts, we have evaluated the effects of CoQ₁₀, coenzyme Q₂ (CoQ₂), idebenone, and vitamin C on bioenergetics and oxidative stress in human skin fibroblasts with primary CoQ₁₀ deficiency. A final concentration of 5 µM of each compound was chosen to approximate the plasma concentration of CoQ₁₀ of patients treated with oral ubiquinone. CoQ₁₀ supplementation for one week but not for 24 hours doubled ATP levels and ATP/ADP ratio in CoQ₁₀ deficient fibroblasts therein normalizing the bioenergetics status of the cells. Other compounds did not affect cellular bioenergetics. In COQ2 mutant fibroblasts, increased superoxide anion production and oxidative stress-induced cell death were normalized by all supplements.

Conclusions/Significance

These results indicate that: 1) pharmacokinetics of CoQ₁₀ in reaching the mitochondrial respiratory chain is delayed; 2) short-tail ubiquinone analogs cannot replace CoQ₁₀ in the mitochondrial respiratory chain under conditions of CoQ₁₀ deficiency; and 3) oxidative stress and cell death can be counteracted by administration of lipophilic or hydrophilic antioxidants. The results of our in vitro experiments suggest that primary CoQ₁₀ deficiencies should be treated with CoQ₁₀ supplementation but not with short-tail ubiquinone analogs, such as idebenone or CoQ₂. Complementary administration of antioxidants with high bioavailability should be considered if oxidative stress is present.     

Oxidative stress induces distinct physiological responses in the two Trebouxia phycobionts of the lichen Ramalina farinacea

Background and Aims

Most lichens form associations with Trebouxia phycobionts and some of them simultaneously include genetically different algal lineages. In other symbiotic systems involving algae (e.g. reef corals), the relative abundances of different endosymbiotic algal clades may change over time. This process seems to provide a mechanism allowing the organism to respond to environmental stress. A similar mechanism may operate in lichens with more than one algal lineage, likewise protecting them against environmental stresses. Here, the physiological responses to oxidative stress of two distinct Trebouxia phycobionts (provisionally named TR1 and TR9) that coexist within the lichen Ramalina farinacea were analysed.

Methods

Isolated phycobionts were exposed to oxidative stress through the reactive oxygen species propagator cumene hydroperoxide (CuHP). Photosynthetic pigments and proteins, photosynthesis (through modulated chlorophyll fluorescence), the antioxidant enzymes superoxide dismutase (SOD) and glutathione reductase (GR), and the stress-related protein HSP70 were analysed.

Key Results

Photosynthetic performance was severely impaired by CuHP in phycobionts, as indicated by decreases in the maximal PSII photochemical efficiency (F_v/F_m), the quantum efficiency of PSII (Φ_PSII) and the non-photochemical dissipation of energy (NPQ). However, the CuHP-dependent decay in photosynthesis was significantly more severe in TR1, which also showed a lower NPQ and a reduced ability to preserve chlorophyll a, carotenoids and D1 protein. Additionally, differences were observed in the capacities of the two phycobionts to modulate antioxidant activities and HPS70 levels when exposed to oxidative stress. In TR1, CuHP significantly diminished HSP70 and GR but did not change SOD activities. In contrast, in TR9 the levels of both antioxidant enzymes and those of HSP70 increased in response to CuHP.

Conclusions

The better physiological performance of TR9 under oxidative conditions may reflect its greater capacity to undertake key metabolic adjustments, including increased non-photochemical quenching, higher antioxidant protection and the induction of repair mechanisms.     

Sinapic Acid Prevents Hypertension and Cardiovascular Remodeling in Pharmacological Model of Nitric Oxide Inhibited Rats

Objectives

Hypertensive heart disease is a constellation of abnormalities that includes cardiac fibrosis in response to elevated blood pressure, systolic and diastolic dysfunction. The present study was undertaken to examine the effect of sinapic acid on high blood pressure and cardiovascular remodeling.

Methods

An experimental hypertensive animal model was induced by L-NAME intake on rats. Sinapic acid (SA) was orally administered at a dose of 10, 20 and 40 mg/kg body weight (b.w.). Blood pressure was measured by tail cuff plethysmography system. Cardiac and vascular function was evaluated by Langendorff isolated heart system and organ bath studies, respectively. Fibrotic remodeling of heart and aorta was assessed by histopathologic analyses. Oxidative stress was measured by biochemical assays. mRNA and protein expressions were assessed by RT-qPCR and western blot, respectively. In order to confirm the protective role of SA on endothelial cells through its antioxidant property, we have utilized the in vitro model of H₂O₂-induced oxidative stress in EA.hy926 endothelial cells.

Results

Rats with hypertension showed elevated blood pressure, declined myocardial performance associated with myocardial hypertrophy and fibrosis, diminished vascular response, nitric oxide (NO) metabolites level, elevated markers of oxidative stress (TBARS, LOOH), ACE activity, depleted antioxidant system (SOD, CAT, GPx, reduced GSH), aberrant expression of TGF-β, β-MHC, eNOS mRNAs and eNOS protein. Remarkably, SA attenuated high blood pressure, myocardial, vascular dysfunction, cardiac fibrosis, oxidative stress and ACE activity. Level of NO metabolites, antioxidant system, and altered gene expression were also repaired by SA treatment. Results of in vitro study showed that, SA protects endothelial cells from oxidative stress and enhance the production of NO in a concentration dependent manner.

Conclusions

Taken together, these results suggest that SA may have beneficial role in the treatment of hypertensive heart disease by attenuating fibrosis and oxidative stress through its antioxidant potential.     

Cytotoxicity Mechanism of Two Naphthoquinones (Menadione and Plumbagin) in Saccharomyces cerevisiae

Background

Quinones are compounds extensively used in studies of oxidative stress due to their role in plants as chemicals for defense. These compounds are of great interest for pharmacologists and scientists, in general, because several cancer chemotherapeutic agents contain the quinone nucleus. However, due to differences in structures and diverse pharmacological effects, the exact toxicity mechanisms exerted by quinones are far from elucidatation.

Methodology/Principal Findings

Using Saccharomyces cerevisiae, we evaluated the main mechanisms of toxicity of two naphthoquinones, menadione and plumbagin, by determining tolerance and oxidative stress biomarkers such as GSH and GSSG, lipid peroxidation levels, as well as aconitase activity. The importance of glutathione transferases (GST) in quinone detoxification was also addressed. The GSSG/GSH ratio showed that menadione seemed to exert its toxicity mainly through the generation of ROS while plumbagin acted as an electrophile reacting with GSH. However, the results showed that, even by different pathways, both drugs were capable of generating oxidative stress through their toxic effects. Our results showed that the control strain, BY4741, and the glutathione transferase deficient strains (gtt1Δ and gtt2Δ) were sensitive to both compounds. With respect to the role of GST isoforms in cellular protection against quinone toxicity, we observed that the Gtt2 deficient strain was unable to overcome lipid peroxidation, even after a plumbagin pre-treatment, indicating that this treatment did not improve tolerance when compared with the wild type strain. Cross-tolerance experiments confirmed distinct cytotoxicity mechanisms for these naphthoquinones since only a pre-treatment with menadione was able to induce acquisition of tolerance against stress with plumbagin.

Conclusions/Significance

These results suggest different responses to menadione and plumbagin which could be due to the fact that these compounds use different mechanisms to exert their toxicity. In addition, the Gtt2 isoform seemed to act as a general protective factor involved in quinone detoxification.     

Effects of unaccustomed downhill running on muscle damage,oxidative stress,and leukocyte apoptosis

[Purpose]

The purpose of this study was to investigate the effect of unaccustomed downhill running on muscle damage, oxidative stress, and leukocyte apoptosis.

[Methods]

Thirteen moderately trained male subjects performed three 40 min treadmill runs at ~70% VO_2max on separate days: a level run (L) followed by two downhill runs (DH1 and DH2). Blood samples were taken at rest (PRE) and immediately (POST), 2 h, 24 h, and 48 h after each run. Data were analyzed using 2-way repeated measures ANOVA with post hoc Tukey tests.

[Results]

Creatine kinase (CK) activity and oxidative stress level were significantly elevated at 24 h and 48 h following DH1 (P < 0.05). The level of oxidative stress at the POST measurement following DH1 and DH2 was greater than PRE. The rate of leukocyte apoptosis was significantly increased at the POST measurement following all three runs, and remained elevated for up to 48 h following DH1 (P < 0.01).

[Conclusion]

CK activity and oxidative stress were elevated following an acute bout of moderate intensity downhill running, resulting in a greater apoptotic response at 24 h and 48 h post-exercise in comparison with level grade running or a second downhill run. These elevations were blunted following DH2. Although the link between exercise-induced muscle damage and leukocyte apoptosis is currently unknown, the differential response to DH1 vs. L and DH2 indicates that it may be mediated by the elevation of oxidative stress.