Tert-butyl Hydroperoxide Treatment Research Articles

Hydroxysafflor yellow A attenuates oxidative stress injury-induced apoptosis in the nucleus pulposus cell line and regulates extracellular matrix balance via CA XII.

Intervertebral disc degeneration (IVDD) is the main cause of lower back pain. Oxidative stress injury and degradation of the extracellular matrix (ECM) are important factors causing IVDD, while hydroxysafflor yellow A (HSYA) has significant anti-oxidative stress and anti-apoptotic effects. The present study aimed to investigate the protective role of HSYA in IVDD using nucleus pulposus (NP) cells. A Cell Counting Kit-8 assay was used to detect cell viability following HSYA and tert-Butyl hydroperoxide (TBHP) treatment. Cellular reactive oxygen species levels and the level of apoptosis were measured using flow cytometry. The concentration of superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT) and glutathione peroxidase GSH-Px were detected using ELISA. DAPI staining was performed for nuclear morphology analysis, while western blot analysis was used to detect apoptotic- and ECM-related protein expression levels. Bioinformatics analysis was used to predict the binding site between HSYA and carbonic anhydrase 12 (CA12; CA XII). NP cells were transfected withsmall interference RNA (siRNA) for CA XII downregulation. Following TBHP treatment, the level of ROS increased significantly, and the concentrations of SOD, CAT and GSH-Px were decreased. In addition, the apoptosis level of the NP cell line significantly increased following TBHP treatment. Furthermore, the expression levels of ECM-related proteins, collagen II and aggrecan were significantly decreased, and the protein expression level of MMP-13 was significantly increased. HSYA (10 µM) could effectively alleviate the effects of TBHP on NP cell apoptosis, oxidative stress damage and the expression level of ECM-related proteins. A binding site was found between HSYA and CA XII. In addition, CA XII-siRNA significantly reduced the increase in the expression level of collagen II and aggrecan proteins and decrease in the expression level of MMP-13 induced by HSYA in the NP cell line. In conclusion, HSYA could attenuate oxidative stress injury and apoptosis induced by TBHP in the NP cell line, and could improve the regulation of ECM balance.

Involvement of oxidative stress-induced annulus fibrosus cell and nucleus pulposus cell ferroptosis in intervertebral disc degeneration pathogenesis.

Ferroptosis is a necrotic form of regulated cell death that was associated with lipid peroxidation and free iron‐mediated Fenton reactions. It has been reported that iron deficiency had been implicated in the pathogenesis of intervertebral disc degeneration (IVDD) by activating apoptosis. However, the role of ferroptosis in the process of IVDD has not been illuminated. Here, we demonstrate the involvement of ferroptosis in IVDD pathogenesis. Our in vitro models show the changes in protein levels of ferroptosis marker and enhanced lipid peroxidation level during oxidative stress. Safranin O staining, hematoxylin‐eosin staining, and immunohistochemical were used to assess the IVDD after 8 weeks of surgical procedure in vivo. Treatment with ferrostatin‐1, deferoxamine, and RSL3 demonstrate the role of ferroptosis in tert‐butyl hydroperoxide (TBHP)‐treated annulus fibrosus cells (AFCs) and nucleus pulposus cells (NPCs). Ferritinophagy, nuclear receptor coactivator 4 (NCOA4)‐mediated ferritin selective autophagy, is originated during the process of ferroptosis in response to TBHP treatment. Knockdown and overexpression NCOA4 further prove TBHP may induce ferroptosis of AFCs and NPCs in an autophagy‐dependent way. These findings support a role for oxidative stress‐induced ferroptosis in the pathogenesis of IVDD.

Catalytic oxidative desulfurization of model and real diesel over a molybdenum anchored metal-organic framework

In this work, we report the catalytic performance of a MoO2Cl2 anchored metal-organic framework, [email protected], in the oxidative desulfurization (ODS) process on a continuous fixed-bed reactor. [email protected] was examined as a catalyst in the oxidation of model oils containing dibenzothiophene (DBT), benzothiophene (BT) or 4,6-dimethyldibenzothiophene (4,6-DMDBT). In a 50-h ODS reaction of DBT at 70 °C, with tert-butyl hydroperoxide (TBHP) as the oxidant, a constant high DBT conversion of nearly 80% was observed along with a high oxidant utility of over 85%, where DBT was converted rapidly into DBT sulfone. XRD and ICP analysis show that the structure of [email protected] was well preserved, with no significant leaching of Mo species. In addition, [email protected] can also oxidize a hydrogenated diesel oil, and 74% sulfur removal efficiency was achieved, with an extraction recovery of 92.5% after acetonitrile extraction. Spectroscopic characterization confirmed the formation of a molybdenum peroxide complex from MoO2Cl2 under TBHP treatment, a plausible catalytic process was proposed based on the experiment evidence as well as FT-IR and FT-FIR characterization results.

Suppression of AMPK/aak-2 by NRF2/SKN-1 down-regulates autophagy during prolonged oxidative stress.

NF-E2–related factor 2 (NRF2) transcription factor has a fundamental role in cell homeostasis maintenance as one of the master regulators of oxidative and electrophilic stress responses. Previous studies have shown that a regulatory connection exists between NRF2 and autophagy during reactive oxygen species–generated oxidative stress. The aim of the present study was to investigate how autophagy is turned off during prolonged oxidative stress, to avoid overeating and destruction of essential cellular components. AMPK is a key cellular energy sensor highly conserved in eukaryotic organisms, and it has an essential role in autophagy activation at various stress events. Here the role of human AMPK and its Caenorhabditis elegans counterpart AAK-2 was explored upon oxidative stress. We investigated the regulatory connection between NRF2 and AMPK during oxidative stress induced by tert-butyl hydroperoxide (TBHP) in HEK293T cells and C. elegans. Putative conserved NRF2/protein skinhead-1 binding sites were found in AMPK/aak-2 genes by in silico analysis and were later confirmed experimentally by using EMSA. After addition of TBHP, NRF2 and AMPK showed a quick activation; AMPK was later down-regulated, however, while NRF2 level remained high. Autophagosome formation and Unc-51–like autophagy activating kinase 1 phosphorylation were initially stimulated, but they returned to basal values after 4 h of TBHP treatment. The silencing of NRF2 resulted in a constant activation of AMPK leading to hyperactivation of autophagy during oxidative stress. We observed the same effects in C. elegans demonstrating the conservation of this self-defense mechanism to save cells from hyperactivated autophagy upon prolonged oxidative stress. We conclude that NRF2 negatively regulates autophagy through delayed down-regulation of the expression of AMPK upon prolonged oxidative stress. This regulatory connection between NRF2 and AMPK may have an important role in understanding how autophagy is regulated in chronic human morbidities characterized by oxidative stress, such as neurodegenerative diseases, certain cancer types, and in metabolic diseases.—Kosztelnik, M., Kurucz, A., Papp, D., Jones, E., Sigmond, T., Barna, J., Traka, M. H., Lorincz, T., Szarka, A., Banhegyi, G., Vellai, T., Korcsmaros, T., Kapuy, O. Suppression of AMPK/aak-2 by NRF2/SKN-1 down-regulates autophagy during prolonged oxidative stress.

Abstract 022: Proteasome Inhibition Prevents Renal Inflammation and Hypertension by Abrogation of Dendritic Cell Autoantigen Presentation

Reactive oxygen species (ROS) have been shown to be important mediators of hypertension. Isolevuglandins (isoLG) are oxidation products of fatty acids that adduct to lysine residues of proteins. Our prior data suggest that isoLG-adducted proteins are presented by dendritic cells (DCs) and activate T-cells resulting in hypertension. Identification of the source and mechanism of presentation of isoLG adducts is critical for the development of therapies targeting this process. Antigens formed intracellularly are processed by the proteasome and presented to CD8 + T cells in MHC class I (MHC-I). We hypothesize that within DCs, isoLG-adducted peptides are processed by the proteasome and presented in MHC-I. Treatment of mouse DCs with the oxidant tert-butyl hydroperoxide (TBHP) increased levels of isoLG-adducts on the DC surface, as detected by flow cytometry, from 1.3 x 10 3 to 11.4 x 10 3 /10 6 DCs (N = 9, P < 0.01). Co-treatment of mouse DCs with TBHP and proteasome inhibitors (PIs) bortezomib (BTZ) or MG132 attenuated surface isoLG-adduct levels when compared to TBHP alone (2.7 x 10 3 or 3.5 x 10 3 vs 11.4 x 10 3 /10 6 DCs, N = 9, P < 0.001). Moreover, using flow cytometry-based fluorescence resonance energy transfer, we found that isoLG-adducted peptides and MHC-I interact at the intermolecular level on mouse DCs, suggesting that IsoLG-modified peptides are presented within MHC-I. Co-treatment of these cells with TBHP and BTZ attenuated this interaction compared with TBHP treatment alone (2.9 x 10 3 vs 32.7 x 10 3 /10 6 DCs, N = 9, P < 0.001). Next, mice were treated with angiotensin II (ang II) with or without co-treatment with BTZ. Co-treated animals exhibited reduced mean systolic blood pressure (161.7 ± 7.2 vs 122.9 ± 17.9 mmHg, N = 4-5, P < 0.05) and a reduction in renal macrophages (13.4 x 10 3 vs 6.57 x 10 3 DCs/Kidney, N = 4-5, P < 0.001) and DCs (129.4 x 10 3 vs 79.9 x 10 3 DCs/Kidney, N = 4-5, P < 0.05). These studies provide evidence that the proteasome processes isoLG adducts into peptides that are presented within MHC -I, and that proteasome inhibitors can not only block this process, but in doing so, can reduce renal inflammation and lower blood pressure. Efforts to inhibit processing and presentation of isoLG-modified peptides may therefore have therapeutic benefit in hypertension.

Hippocampal microvasculature changes in association with oxidative stress in Alzheimer's disease.

Vascular endothelial dysfunction is a primary phenotype of aging, and microvascular (MV) lesion is mainly associated with Alzheimer's disease (AD). Here we have studied the correlation of MV wall thickness and CA1 pyramidal neuronal pathology in autopsy-confirmed AD brains. Both hyaline (h-MV) and increased cell number (c-MV) associated MV wall thickening was found in age-matched control (AC) hippocampus without significant change in Aβ level (Braak stages 0-III). AC neurons neighboring the h-MV showed lower levels of oxidative DNA/RNA damage and Aβ precursor protein (APP), while the neurons around c-MV showed higher oxidative DNA/RNA damage with increased APP expression. Neurons in AC hippocampus without MV wall thickening (thin wall) showed increased DNA/RNA damage and APP levels compared to AC cases with h-MV and c-MV walls. In the AD hippocampus neurons neighboring h-MV walls showed increased levels of Aβ and decreased number of dendritic spines (at Braak stages IV-VI). C-MV neighboring neurons in the AD cases showed higher levels of DNA/RNA damage with increased APP at stages II - III, followed by lower levels of oxidative DNA/RNA damage, decreased APP and increased Aβ levels with loss of dendritic spines at stages IV-VI. Prolonged treatment of primary human fetal hippocampal neurons with tert-butyl hydroperoxide (TBHP) induced oxidative DNA damage with a sustained increase in APP. Aβ increased rapidly and then decreased overtime. Short-term TBHP treated neurons showed lower levels of superoxide (O2• -) without significant DNA damage. Short-term TBHP treatment induced a gradual decrease in APP but an increase in Aβ levels over time. In conclusion this study indicates that AD hippocampus at Braak stages II-III are characterized by strong oxidative DNA/RNA damage with increased APP in neurons associated with c-MV, while stages IV-VI are characterized by a slow increase in Aβ in neurons neighboring both h-MV and c-MV.

142 - Alterations of Endogenous Coenzyme Q10 Levels in Human Cells after the Induction of Lipid Peroxidation by an Organic Peroxide

Coenzyme Q10 (CoQ10) is an essential electron carrier in the mitochondrial electron transport chain and also an endogenously synthesized lipid-soluble antioxidant. We have previously demonstrated that hydrogen peroxide increased CoQ10 levels, but disruption of mitochondrial membrane potential by the chemical uncoupler FCCP suppressed CoQ10 levels in the human 143B cells. Mor bioeover, we have also shown that the COQ5 protein, which is required for CoQ biosynthesis in yeast, is essential for maintaining CoQ10 levels in 143B cells. In this study, we investigated how the condition of enhanced lipid peroxidation affected levels of the reduced form (ubiquinol-10) and oxidized form (ubiquinone-10) of CoQ10. The organic peroxide tert-butyl hydroperoxide (TBHP) was used to induce lipid peroxidation in 143B cells. Levels of esterified F2-isoprostanes (F2-IsoPs) in cells, detected by the gas chromatography/mass spectrometry method, were confirmed to be elevated by a low, medium, or high dose of TBHP as early as after 4 h of treatment. The levels remained augmented after 18 h of TBHP treatment but the degree of the increase was less. Only the medium-dose and high-dose TBHP treatments enhanced total CoQ10 levels after 18 h or 24 h of treatment, but the high-dose TBHP treatment also enhanced ubiquinol-10 levels after 4 h of treatment. Transient knockdown of COQ5, which decreased CoQ10 levels, suppressed the increase of both ubiquinol-10 and ubiquinone-10 levels after 24-h TBHP treatment. These results demonstrate for the first time that specific augmentation of lipid peroxidation could up-regulate formation of endogenous CoQ10 levels and ubiquinol-10 could be the initially generated form.

Dynamic Interplay between the Transcriptome and Methylome in Response to Oxidative and Alkylating Stress.

In recent years, it has been shown that free radicals not only react directly with DNA but also regulate epigenetic processes such as DNA methylation, which may be relevant within the context of, for example, tumorigenesis. However, how these free radicals impact the epigenome remains unclear. We therefore investigated whether methyl and hydroxyl radicals, formed by tert-butyl hydroperoxide (TBH), change temporal DNA methylation patterns and how this interferes with genome-wide gene expression. At three time points, TBH-induced radicals in HepG2 cells were identified by electron spin resonance spectroscopy. Total 5-methylcytosine (5mC) levels were determined by liquid chromatography and tandem mass spectrometry and genome-wide changes in 5mC and gene expression by microarrays. Induced methylome changes rather represent an adaptive response to the oxidative stress-related reactions observed in the transcriptome. More specifically, we found that methyl radicals did not induce DNA methylation directly. An initial oxidative and alkylating stress-related response of the transcriptome during the early phase of TBH treatment was followed by an epigenetic response associated with cell survival signaling. Also, we identified genes of which the expression seems directly regulated by DNA methylation. This work suggests an important role of the methylome in counter-regulating primary oxidative and alkylating stress responses in the transcriptome to restore normal cell function. Altogether, the methylome may play an important role in counter-regulating primary oxidative and alkylating stress responses in the transcriptome presumably to restore normal cell function.

Extra virgin olive oil phenolic extracts counteract the pro-oxidant effect of dietary oxidized lipids in human intestinal cells

The phenolic fraction of extra virgin olive oil (EVOO) concentrates before absorption in the intestinal lumen, where it may contribute to the modulation of enterocytes response to oxidative and inflammatory stimuli. We evaluated the ability of two monovarietal EVOOs phenolic extracts, Bosana and Nera di Gonnos/Tonda di Cagliari, typical and widespread varieties in Sardinia (Italy), to counteract in enterocytes like Caco-2 cells the pro-oxidant action of oxidized lipids, tert-butyl hydroperoxide (TBH) or a mixture of oxysterols of dietary origin. We confirmed that TBH treatment causes a significant increase of ROS production, GSH depletion, increase of MDA, fatty acids hydroperoxides and 7-ketocholesterol, and showed first evidence of oxidative imbalance and cell damage due to oxysterols exposure. Preincubation of cells with the phenolic extracts significantly attenuated oxidative modifications. Bosana extract showed the highest concentration of total phenols, mainly hydroxytyrosol and tyrosol, and was the most active in presence of TBH, where the free radical scavenging activity of these simple phenols seems to be a determining factor. The two extracts were equally effective, in spite of the different composition, in presence of oxysterols, where ROS production probably occurs according to different and more complex mechanisms.

Absence of Peroxiredoxin 6 Amplifies the Effect of Oxidant Stress on Mobility and SCSA/CMA3 Defined Chromatin Quality and Impairs Fertilizing Ability of Mouse Spermatozoa.

Oxidative stress, the imbalance between reactive oxygen species production and antioxidant defenses, is associated with male infertility. Peroxiredoxins (PRDXs) are antioxidant enzymes with a wide distribution in spermatozoa. PRDX6 is highly abundant and located in all subcellular compartments of the spermatozoon. Infertile men have lower levels of sperm PRDX6 associated with low sperm motility and high DNA damage. In order to better understand the role of PRDX6 in male reproduction, the aim of this study was to elucidate the impact of the lack of PRDX6 on male mouse fertility. Spermatozoa lacking PRDX6 showed significantly increased levels of cellular oxidative damage evidenced by high levels of lipid peroxidation, 8-hydroxy-deoxyguanosine (DNA oxidation), and protein oxidation (S-glutathionylation and carbonylation), lower sperm chromatin quality (high DNA fragmentation and low DNA compaction, due to low levels of protamination and a high percentage of free thiols), along with decreased sperm motility and impairment of capacitation as compared with wild-type (WT) spermatozoa. These manifestations of damage are exacerbated by tert-butyl hydroperoxide treatment in vivo. While WT males partially recovered the quality of their spermatozoa (in terms of motility and sperm DNA integrity), Prdx6−/− males showed higher levels of sperm damage (lower motility and chromatin integrity) 6 mo after the end of treatment. In conclusion, Prdx6−/− males are more vulnerable to oxidative stress than WT males, resulting in impairment of sperm quality and ability to fertilize the oocyte, compatible with the subfertility phenotype observed in these knockout mice.

Chronic oxidative stress upregulates Drusen-related protein expression in adult human RPE stem cell-derived RPE cells: A novel culture model for dry AMD

The goal of this study was to examine changes in the expression of transcripts and proteins associated with drusen in Age-related Macular Degeneration (AMD) after exposing human retinal pigment epithelium (hRPE) cells to chronic oxidative stress. Primary adult human RPE cells were isolated from cadaveric donor eyes. The subpopulation of RPE stem cells (RPESCs) was activated, expanded, and then differentiated into RPE progeny. Confluent cultures of RPESC-derived hRPE and ARPE-19 cells were exposed to a regimen of tert-butylhydroperoxide (TBHP) for 1-5 days. After treatment, gene expression was measured by quantitative PCR (qPCR), protein expression was assessed by immunocytochemistry and transepithelial resistance and cell toxicity were measured. hRPE cells exposed to a regimen of TBHP for 5 days upregulate expression of several molecules identified in drusen, including molecular chaperones and pro-angiogenic factors. 5-day TBHP treatment was significantly more effective than 1-day treatment at eliciting these effects. The extent of hRPE response to 5-day treatment varied significantly between individual donors, nevertheless, 6 transcripts were reliably significantly upregulated. ARPE-19 cells treated with the same 5-day stress regime did not show the same pattern of response and did not upregulate this group of transcripts. RPESC-derived hRPE cells change significantly when exposed to repeated oxidative stress conditions, upregulating expression of several drusen-related proteins and transcripts. This is consistent with the hypothesis that hRPE cells are competent to be a source of proteins found in drusen deposits. Our results suggest that donor-specific genetic and environmental factors influence the RPE stress response. ARPE-19 cells appear to be less representative of AMD-like changes than RPESC-derived hRPE. This adult stem cell-based system using chronic TBHP treatment of hRPE represents a novel in vitro model useful for the study of drusen formation and dry AMD pathophysiology.

Protective effects of oligomeric and polymeric procyanidin fractions from defatted grape seeds on tert-butyl hydroperoxide-induced oxidative damage in HepG2 cells

Oligomeric and polymeric procyanidins have been reported to possess different antioxidant capacities. However, the intracellular antioxidant mechanisms of oligomeric and polymeric procyanidins are still poorly understood. In this study, we evaluated the cytoprotective effects of the oligomeric procyanidin fraction (OPF) and the polymeric procyanidin fraction (PPF) from grape seeds against the oxidative damage induced by tert-butyl hydroperoxide (TBHP) in HepG2 cells. The levels of cellular reactive oxygen species (ROS), cellular lipid peroxidation, glutathione (GSH), and antioxidant enzyme activities were measured as biomarkers of cellular oxidative status. HepG2 cells were treated with different concentrations of procyanidin samples (0–20μg/ml) for 6h prior to treatment with TBHP for 3h. The incubation of HepG2 cells with TBHP led to an approximately 60% decrease in cell viability. However, pretreatment of the cells with the samples, at 5–20μg/ml, rescued cell viability in a dose-dependent manner. Cellular generation of ROS, formation of malondialdehyde (MDA), and depletion of GSH were reduced by OPF and PPF. Moreover, TBHP treatment increased the activities of glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD). However, a 6h pretreatment with each of the samples at 20μg/ml significantly decreased the activities of these enzymes. These results clearly showed that treatment with OPF and PPF protected against oxidative damage by modulating ROS production, GSH levels, MDA generation, and antioxidant enzyme activities in HepG2 cells.

Wine extracts from Sardinian grape varieties attenuate membrane oxidative damage in Caco-2 cell monolayers

One of the most important sites of polyphenol action seems to be in the gastrointestinal system before absorption. We investigated the ability of three wine phenolic extracts, obtained from grape varieties grown in Sardinia, Cannonau (red), Vermentino and Malvasia (white), to exert an antioxidant action against tert-butyl hydroperoxide (TBH)-induced oxidative damage to Caco-2 cell monolayers as a model system of the human intestine. TBH treatment caused the disruption of epithelial integrity, measured as transepithelial electrical resistance, and markers of the peroxidation process of membrane lipids, MDA, fatty acid hydroperoxides and 7-ketocholesterol. All wine extracts were able to counteract the oxidising action of TBH and, in spite of the differences in phenolic composition, exerted a comparable activity. Our findings point out a direct antioxidant action of the wine extracts on enterocytes exposed to oxidising species and further support the opinion that total phenolic content is not essential for antioxidant activity.

Corilagin prevents tert-butyl hydroperoxide-induced oxidative stress injury in cultured N9 murine microglia cells

Oxidative stress plays an important role in neurodegenerative diseases. Reactive oxygen species (ROS)-mediated stress in microglia in vivo could result in cellular injuries and preferentially induces neuronal injury. Corilagin, a novel member of the phenolic tannin family, has been shown to possess antioxidant properties. In this study, we investigated the effects of corilagin on tert-butyl hydroperoxide (TBHP)-induced injury in cultured N9 murine microglial cells and the underlying mechanisms by a methyltetrazolium assay and oxidative damage assay. We found that exposure of N9 cells to TBHP induced cytotoxicity as demonstrated by cell shrinkage, loss of cell viability, increased lactate dehydrogenase (LDH) leakage, and increased intracellular levels of ROS. By contrast, TBHP reduced both superoxide dismutase activity and total cell anti-oxidation capacity, but glutathione was not reduced. Moreover, TBHP treatment was associated with the loss of mitochondrial membrane potential, and it induced cell apoptosis through the mitochondrial-mediated pathway involving the down-regulation of Bcl-2 expression and up-regulation of the Bax/Bcl-2 ratio. Interestingly, pre-treatment with corilagin reversed these reactions. These data collectively indicated that corilagin could attenuate TBHP-induced oxidative stress injury in microglial cells, and its protective effects may be ascribed to its antioxidant and antiapoptotic properties. Our findings suggest that corilagin should be a potential candidate for the treatment of oxidative stress-induced neurodegenerative diseases.

Protection against Oxidative Stress-induced Hepatic Injury by Intracellular Type II Platelet-activating Factor Acetylhydrolase by Metabolism of Oxidized Phospholipids in Vivo

Membrane phospholipids are susceptible to oxidation, which is involved in various pathological processes such as inflammation, atherogenesis, neurodegeneration, and aging. One enzyme that may help to remove oxidized phospholipids from cells is intracellular type II platelet-activating factor acetylhydrolase (PAF-AH (II)), which hydrolyzes oxidatively fragmented fatty acyl chains attached to phospholipids. Overexpression of PAF-AH (II) in cells or tissues was previously shown to suppress oxidative stress-induced cell death. In this study we investigated the functions of PAF-AH (II) by generating PAF-AH (II)-deficient (Pafah2(-/-)) mice. PAF-AH (II) was predominantly expressed in epithelial cells such as kidney proximal and distal tubules, intestinal column epithelium, and hepatocytes. Although PAF-AH activity was almost abolished in the liver and kidney of Pafah2(-/-) mice, Pafah2(-/-) mice developed normally and were phenotypically indistinguishable from wild-type mice. However, mouse embryonic fibroblasts derived from Pafah2(-/-) mice were more sensitive to tert-butylhydroperoxide treatment than those derived from wild-type mice. When carbon tetrachloride (CCl(4)) was injected into mice, Pafah2(-/-) mice showed a delay in hepatic injury recovery. Moreover, after CCl(4) administration, liver levels of the esterified form of 8-iso-PGF(2alpha), a known in vitro substrate of PAF-AH (II), were higher in Pafah2(-/-) mice than in wild-type mice. These results indicate that PAF-AH (II) is involved in the metabolism of esterified 8-isoprostaglandin F(2alpha) and protects tissue from oxidative stress-induced injury.

Cytotoxic and genotoxic effects of tert-butyl hydroperoxide on Chinese hamster B14 cells

The organic hydroperoxide, tert-butyl hydroperoxide (t-BHP), is a useful model compound to study mechanisms of oxidative cell injury. In the present work, we examined the features of the interactions of this oxidant with Chinese hamster B14 cells. The aim of our study was to reveal a possible role of structural modifications in membranes and loss of DNA integrity in t-BHP-induced cell injury and death. The tert-butyl hydroperoxide treatment (100–1000μM, 37°C for 1h) did not decrease cell viability (as measured by cell-specific functional activity with an MTT test), but completely prevented cell growth. We observed intracellular reduced glutathione (GSH) oxidation and total glutathione (GSH+GSSG) depletion, a slight increase in the level of lipid-peroxidation products, an enhancement of membrane fluidity, intracellular potassium leakage and a significant decrease of membrane potential. At oxidant concentrations of 100–1500μM, a significant damage to DNA integrity was observed as revealed by the Comet assay. The inhibition of cell proliferation (cell-growth arrest) may be explained by genotoxicity of t-BHP, by disturbance of the cellular redox-equilibrium (GSH oxidation) and by structural membrane modifications, which result in ion-non-selective pore formation. The disturbance in passive membrane permeability and the DNA damage may be the most dramatic cell impairments induced by t-BHP treatment. The presence of another oxidant, hypochlorous acid (HOCl), completely prevented t-BHP-induced DNA strand breaks, perhaps due to extracellular oxidation of t-BHP by HOCl.

Effect of Aminoethylcysteine Ketimine Decarboxylated Dimer, a Natural Sulfur Compound Present in Human Plasma, on Tert-Butyl Hydroperoxide-induced Oxidative Stress in Human Monocytic U937 Cells

Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD) is a natural sulphur compound present in human plasma and urine and in mammalian brain. Recently, it has been detected in many common dietary vegetables. The aim of the present study was to evaluate the ability of AECK-DD to affect cellular response of U937 human monocytic cells to tert-butyl hydroperoxide-induced oxidative stress. AECK-DD was incorporated into cells, as confirmed by GC-MS analyses, without any cytotoxic effect. A 24 h treatment with 50 and 250 μM AECK-DD resulted in the incorporation of 0.10±0.01 and 0.47±0.08 ng AECK-DD × 106 cells, respectively. U937 cells pretreated with AECK-DD (in the range 4–100 μM) showed an increased resistance to tert-butyl hydroperoxide-induced necrotic death, as revealed by a higher percent of survival measured at all incubation times with respect to control cells. Moreover, the protective effect exhibited by AECK-DD is significantly stronger with respect to that obtained with other common antioxidants (N-acetyl cysteine and trolox) and comparable, although somewhat higher, to that of vitamin E. This effect seems to be due to the ability of AECK-DD to reduce glutathione depletion and to inhibit lipid peroxidation during tert-butyl hydroperoxide treatment. It can be concluded that AECK-DD protects cultured human monocytic cells against tert-butyl hydroperoxide-induced oxidative stress and subsequent cell death, likely through an antioxidant action inside the cell. Due to its presence in both human plasma and urine, AECK-DD may play a role in the modulation of oxidative processes in vivo.

The effect of posttranslational modifications on the in vitro activity of recombinant human thyroid-stimulating hormone.

Posttranslational modification can influence the biologic activity of recombinant proteins. The effects of beta-subunit C-terminal truncation, oligosaccharide heterogeneity, and chemical oxidation on the in vitro activity of recombinant human thyroid-stimulating hormone (rhTSH) were investigated. beta-Subunit C-terminal truncation up to residue 113 did not effect the in vitro activity of the hormone. The relationship between the heterogeneity of oligosaccharide structures on rhTSH and specific activity of the glycoprotein hormone was also examined. Oligosaccharide profiles were generated for preparations of rhTSH containing similar sialic acid levels. A weak correlation was observed between relative levels of monosialylated biantennary, bisialylated biantennary, and trisialylated triantennary oligosaccharide species and in vitro activity of the recombinant hormone (p < 0.05). To examine the effect of chemically induced methionine oxidation on the activity of rhTSH, the hormone was treated with tert-butyl hydroperoxide and then characterized. Using peptide mapping and mass spectrometry, the degree of oxidation of the five methionine residues within rhTSH was measured. Met-71 in the alpha-subunit was the most susceptible to oxidation whereas Met-9 in the beta-subunit was the most resistant. Also, after tert-butyl hydroperoxide treatment, levels of oxidation of Met-32 in the beta-subunit, and Met-29 and Met-47 in the alpha-subunit were less than half of that observed for Met-71. The in vitro activity of rhTSH initially declined with increasing oxidation; however, the loss in activity plateaued at approximately 50% of the control sample activity. In summary, despite the possible effects that posttranslational modifications may have on the bioactivity of a protein, a limited degree of variation in bioactivity was observed for the rhTSH preparations described in this study.

Green tea extract decreases muscle necrosis in mdx mice and protects against reactive oxygen species

Duchenne muscular dystrophy is a severe X-linked congenital disorder characterized by lethal muscle wasting caused by the absence of the structural protein dystrophin. Because generation of reactive oxygen species appears to play an important role in the pathogenesis of this disease, we tested whether antioxidant green tea extract could diminish muscle necrosis in the mdx mouse dystrophy model. A diet supplemented with 0.01% or 0.05% green tea extract was fed to dams and neonates for 4 wk beginning on the day of birth. Muscle necrosis and regeneration were determined in stained cryosections of soleus and elongator digitorum longus muscles. Radical scavenging by green tea extract was determined in differentiated cultured C2C12 cells treated with tert-butylhydroperoxide, with the use of 2',7'-dichlorofluorescin diacetate as a radical detector. This feeding regimen significantly and dose-dependently reduced necrosis in the fast-twitch muscle elongator digitorum longus but at the doses tested had no effect on the slow-twitch soleus muscle. Green tea extract concentration-dependently decreased oxidative stress induced by tert-butylhydroperoxide treatment of cultured mouse C2C12 myotubes. The lower effective dose tested in mdx mice corresponds to approximately equal to 1.4 L (7 cups) green tea/d in humans. Green tea extract may improve muscle health by reducing or delaying necrosis in mdx mice by an antioxidant mechanism.

Different effects of tert-butylhydroperoxide-induced peroxynitrite-dependent and -independent DNA single-strand breakage on PC12 cell poly(ADP-ribose) polymerase activity.

The short-chain lipid hydroperoxide analogue tert-butylhydroperoxide induces peroxynitrite-dependent and -independent DNA single strand breakage in PC12 cells. U937 cells that do not express constitutive nitric oxide synthase respond to tert-butylhydroperoxide treatment with peroxynitrite-independent DNA cleavage. Under experimental conditions leading to equivalent strand break frequencies, the analysis of poly(ADP-ribose) polymerase activity showed an increase in PC12 cells but not in U937 cells. The enhanced poly(ADP-ribose) polymerase activity observed in PC12 cells was paralleled by a significant decline in NAD+ content and both events were prevented by treatments suppressing formation of peroxynitrite. Although DNA breaks were rejoined at similar rates in the two cell lines, an inhibitor of poly(ADP-ribose) polymerase delayed DNA repair in PC12 cells but had hardly any effect in U937 cells. The results obtained using the latter cell type were confirmed with an additional cell line (Chinese hamster ovary cells) that does not express nitric oxide synthase. Collectively, our data suggest that tert-butylhydroperoxide-induced peroxynitrite-independent DNA strand scission is far less effective than the DNA cleavage generated by endogenous peroxynitrite in stimulating the activity of poly(ADP-ribose) polymerase.