Radical Oxygen Species Generation Research Articles

Anti- and Pro-Apoptotic Bcl2 Proteins Distribution and Metabolic Profile in Human Aorta Endothelial Cells Before and After Hyp-PDT

Deregulation of apoptosis can contribute to diverse pathologic processes. Understanding its regulatory mechanisms in endothelial cells (ECs) has great importance for the development of novel therapy strategies for cancer and cardiovascular pathologies. An oxidative stress with the generation of radical oxygen species (ROS) is a common mechanism causing ECs' dysfunction and apoptosis. The generation of ROS can be triggered by various stimuli including photodynamic therapy (PDT). The molecular mechanisms underlying PDT, and specifically Hypericin PDT (HypPDT), are not completely understood, although it has been shown that the sub-cellular Hyp localization and distribution determine which signaling pathway will lead to cell death. Cell responses to HypPDT are highly dependent on the Hyp intracellular localization and accumulation. The mechanisms by which ROS cause or regulate ECs apoptosis typically include receptor activation, caspase activation, Bcl-2 family proteins, and mitochondrial dysfunction. We were particularly interested in Bcl-2 family proteins and investigated their role in apoptosis of ECs triggered by HypPDT. In the present work, we show that the presence of Hyp itself has an effect on the distribution of Bcl2 family members. Presence of Hyp triggers translocation of Bax into mitochondria, and translocation of Bax and Bcl2 into nuclei in ECs. Further, HypPDT results in oxidative stress via mitochondrial superoxide production and primarily in necrotic type of death in HAEC cells. HAEC cell respiratory control is similar in control cells and in cells after 24 hrs incubation with Hyp without irradiation, suggesting that even though Hyp presence resulted in an increased number of apoptotic cells, the mitochondria function was not affected. However, HypPDT resulted in diminished OCR, indicating that HypPDT oxidative stress abolished mitochondria function in intact cells.

Fo Shou San, an Ancient Chinese Herbal Decoction, Protects Endothelial Function through Increasing Endothelial Nitric Oxide Synthase Activity

Fo Shou San (FSS) is an ancient herbal decoction comprised of Chuanxiong Rhizoma (CR; Chuanxiong) and Angelicae Sinensis Radix (ASR; Danggui) in a ratio of 2∶3. Previous studies indicate that FSS promotes blood circulation and dissipates blood stasis, thus which is being used widely to treat vascular diseases. Here, we aim to determine the cellular mechanism for the vascular benefit of FSS. The treatment of FSS reversed homocysteine-induced impairment of acetylcholine (ACh)-evoked endothelium-dependent relaxation in aortic rings, isolated from rats. Like radical oxygen species (ROS) scavenger tempol, FSS attenuated homocysteine-stimulated ROS generation in cultured human umbilical vein endothelial cells (HUVECs), and it also stimulated the production of nitric oxide (NO) as measured by fluorescence dye and biochemical assay. In addition, the phosphorylation levels of both Akt kinase and endothelial NO synthases (eNOS) were markedly increased by FSS treatment, which was abolished by an Akt inhibitor triciribine. Likewise, triciribine reversed FSS-induced NO production in HUVECs. Finally, FSS elevated intracellular Ca2+ levels in HUVECs, and the Ca2+ chelator BAPTA-AM inhibited the FSS-stimulated eNOS phosphorylation. The present results show that this ancient herbal decoction benefits endothelial function through increased activity of Akt kinase and eNOS; this effect is causally via a rise of intracellular Ca2+ and a reduction of ROS.

Phycocyanin Induces Apoptosis and Enhances the Effect of Topotecan on Prostate Cell Line LNCaP

C-phycocyanin (C-PC) from Spirulina has been previously shown to have anticancer properties. Here, we report on anticancer activity of C-PC that was isolated from the novel cyanobacterium Limnothrix sp. 37-2-1. C-PC from this organism exhibited anticancer properties in our in vitro systems; however, the required doses were well above the range of anticancer drugs normally used. Therefore, we conducted several experiments to test whether lower-than-usual doses of the anticancer drug topotecan (TPT) can offer the same level of cytotoxic effects as normal doses when combined with C-PC. For this purpose, cytotoxicities of C-PC and TPT were tested using the LNCaP (prostate cancer) cells. We found that when only 10% of a typical dose of TPT was combined with C-PC, the cancer cells were killed at a higher rate than when TPT was used alone at full dose. Similarly, we were also able to detect an increased level of radical oxygen species (ROS) generation as well as an increase in activities of caspase-9 and caspase-3 when these two compounds were used in combination. Taken together, our findings suggest that combining C-PC from Limnothrix sp. with the lower dose of TPT can induce apoptosis through generation of ROS and activation of caspases. In that respect, we suggest that C-PC can potentially improve the efficacy of the currently available anticancer drug, and therefore diminish its harsh side effects in the patient.

Resveratrol-loaded solid lipid nanoparticles versus nanostructured lipid carriers: evaluation of antioxidant potential for dermal applications

BackgroundExcessive generation of radical oxygen species (ROS) is a contributor to skin pathologies. Resveratrol (RSV) is a potent antioxidant. Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) can ensure close contact and increase the amount of drug absorbed into the skin. In this study, RSV was loaded into SLN and NLC for dermal applications.MethodsNanoparticles were prepared by high shear homogenization using Compritol 888ATO, Myglyol, Poloxamer188, and Tween80. Particle size (PS), polydispersity index (PI), zeta potential (ZP), drug entrapment efficiency (EE), and production yield were determined. Differential scanning calorimetry (DSC) analysis and morphological transmission electron microscopy (TEM) examination were conducted. RSV concentration was optimized with cytotoxicity studies, and net intracellular accumulation of ROS was monitored with cytofluorimetry. The amount of RSV was determined from different layers of rat abdominal skin.ResultsPS of uniform RSV-SLN and RSV-NLC were determined as 287.2 nm ± 5.1 and 110.5 nm ± 1.3, respectively. ZP was −15.3 mV ± 0.4 and −13.8 mV ± 0.1 in the same order. The drug EE was 18% higher in NLC systems. TEM studies showed that the drug in the shell model was relevant for SLN, and that the melting point of the lipid in NLC was slightly lower. Concentrations below 50 μM were determined as suitable RSV concentrations for both SLN and NLC in cell culture studies. RSV-NLC showed less fluorescence, indicating less ROS production in cytofluorometric studies. Ex vivo skin studies revealed that NLC are more efficient in carrying RSV to the epidermis.ConclusionThis study suggests that both of the lipid nanoparticles had antioxidant properties at a concentration of 50 μM. When the two systems were compared, NLC penetrated deeper into the skin. RSV-loaded NLC with smaller PS and higher drug loading appears to be superior to SLN for dermal applications.

Antioxidants halt axonal degeneration in a mouse model of X‐adrenoleukodystrophy

ObjectiveAxonal degeneration is a main contributor to disability in progressive neurodegenerative diseases in which oxidative stress is often identified as a pathogenic factor. We aim to demonstrate that antioxidants are able to improve axonal degeneration and locomotor deficits in a mouse model of X-adrenoleukodystrophy (X-ALD).MethodsX-ALD is a lethal disease caused by loss of function of the ABCD1 peroxisomal transporter of very long chain fatty acids (VLCFA). The mouse model for X-ALD exhibits a late onset neurological phenotype with locomotor disability and axonal degeneration in spinal cord resembling the most common phenotype of the disease, adrenomyeloneuropathy (X-AMN). Recently, we identified oxidative damage as an early event in life, and the excess of VLCFA as a generator of radical oxygen species (ROS) and oxidative damage to proteins in X-ALD.ResultsHere, we prove the capability of the antioxidants N-acetyl-cysteine, α-lipoic acid, and α-tocopherol to scavenge VLCFA-dependent ROS generation in vitro. Furthermore, in a preclinical setting, the cocktail of the 3 compounds reversed: (1) oxidative stress and lesions to proteins, (2) immunohistological signs of axonal degeneration, and (3) locomotor impairment in bar cross and treadmill tests.InterpretationWe have established a direct link between oxidative stress and axonal damage in a mouse model of neurodegenerative disease. This conceptual proof of oxidative stress as a major disease-driving factor in X-AMN warrants translation into clinical trials for X-AMN, and invites assessment of antioxidant strategies in axonopathies in which oxidative damage might be a contributing factor. Ann Neurol 2011;

Combination anti-CD74 (milatuzumab) and anti-CD20 (rituximab) monoclonal antibody therapy has in vitro and in vivo activity in mantle cell lymphoma

AbstractMantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a median survival of 3 years despite chemoimmunotherapy. Rituximab, a chimeric anti–CD20 monoclonal antibody (mAb), has shown only modest activity as single agent in MCL. The humanized mAb milatuzumab targets CD74, an integral membrane protein linked with promotion of B-cell growth and survival, and has shown preclinical activity against B-cell malignancies. Because rituximab and milatuzumab target distinct antigens and potentially signal through different pathways, we explored a preclinical combination strategy in MCL. Treatment of MCL cell lines and primary tumor cells with immobilized milatuzumab and rituximab resulted in rapid cell death, radical oxygen species generation, and loss of mitochondrial membrane potential. Cytoskeletal distrupting agents significantly reduced formation of CD20/CD74 aggregates, cell adhesion, and cell death, highlighting the importance of actin microfilaments in rituximab/milatuzumab–mediated cell death. Cell death was independent of caspase activation, Bcl-2 family proteins or modulation of autophagy. Maximal inhibition of p65 nuclear translocation was observed with combination treatment, indicating disruption of the NF-κB pathway. Significant in vivo therapeutic activity of combination rituximab and milatuzumab was demonstrated in a preclinical model of MCL. These data support clinical evaluation of combination milatuzumab and rituximab therapy in MCL.

Vesicles Generated during Storage of Red Blood Cells Enhance the Generation of Radical Oxygen Speciesin Activated Neutrophils

Erythrocytes are known to shed vesicles in vivo, under various conditions in vitro, and, with impact for transfusion medicine, during storage of red blood cell concentrates (Vsto vesicles). Vsto vesicles of blood transfusions have been shown to deliver glycosylphosphatidylinositol-linked proteins to recipient erythrocytes, to display prothrombotic activity, and to have an inhibitory effect on macrophages. The interaction of Vsto vesicles with and their effect on neutrophilic granulocytes has not yet been studied in detail. Fluorescentlylabeled Vsto and calcium-induced vesicles were preparedin order to study the uptake of labeled vesicular components by neutrophils as compared to the process of phagocytosis of zymosan using flow cytometry and confocal microscopy. The activating effect of Vsto vesicles on neutrophils was addressed by a luminometric assay for stimulated radical oxygen species (ROS) generation. Coincubation of vesicles and neutrophils results in a transfer of vesicular components to the cells. This uptake is different from a phagocytotic process and is enhanced upon interference with the cellular actin cytoskeleton. Preincubation of neutrophils with Vsto vesicles results in an enhanced ROS generation by neutrophils, which is further increased upon fMLP stimulation and during zymosan phagocytosis. The activating effect of Vsto vesicles on neutrophils might be due to the specific accumulation of lysophospholipidsin Vsto vesicles and should be considered as a possible contributor to the pathogenesis of transfusion-related acute lung injury.

The L-Arginine–Asymmetric Dimethylarginine Ratio Is Strongly Related to the Severity of Chronic Heart Failure. No Effects of Exercise Training

Background The aim of this study was to relate levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, L-arginine, the substrate for NO generation, and radical oxygen species (ROS) formation to severity of chronic heart failure. The effect of 4 months’ group-based exercise training was further investigated. Method and Results Eighty patients, aged 45-85 years with New York Heart Association (NYHA) functional class II-IIIb, all on optimal medical treatment, were included. A 6-minute walking test and a bicycle exercise test were performed, and fasting blood samples were collected for determination of N-terminal pro–brain natriuretic peptide (NT-proBNP), L-arginine, ADMA, and ROS generation in circulating leukocytes. ADMA levels were significantly higher in patients in NYHA functional class III versus II ( P = .024), and the L-arginine–ADMA ratio was significantly lower ( P = .005). After adjustment for covariates, L-arginine–ADMA ratio was associated with 6-minute walking distance ( P = .004), exercise capacity ( P = .026), and inversely with NT-proBNP ( P = .015). Stimulated levels of peroxynitrite on monocytes were inversely related to left ventricular ejection fraction ( P = .005). No effect of 4 weeks’ exercise training on the measured variables was obtained. Conclusions The strong relationship seen between L-arginine–ADMA ratio, ROS formation in leukocytes, and severity of chronic heart failure contributes to increased knowledge of endothelial dysfunction related to the NO pathway in such patients.

Bradykinin and adenosine receptors mediate desflurane induced postconditioning in human myocardium: role of reactive oxygen species

BackgroundDesflurane during early reperfusion has been shown to postcondition human myocardium, in vitro. We investigated the role of adenosine and bradykinin receptors, and generation of radical oxygen species in desflurane-induced postconditioning in human myocardium.MethodsWe recorded isometric contraction of human right atrial trabeculae hanged in an oxygenated Tyrode's solution (34 degrees Celsius, stimulation frequency 1 Hz). After a 30-min hypoxic period, desflurane 6% was administered during the first 5 min of reoxygenation. Desflurane was administered alone or with pretreatment of N-mercaptopropionylglycine, a reactive oxygen species scavenger, 8-(p-Sulfophenyl)theophylline, an adenosine receptor antagonist, HOE140, a selective B2 bradykinin receptor antagonist. In separate groups, adenosine and bradykinin were administered during the first minutes of reoxygenation alone or in presence of N-mercaptopropionylglycine. The force of contraction of trabeculae was recorded continuously. Developed force at the end of a 60-min reoxygenation period was compared (mean ± standard deviation) between the groups by a variance analysis and post hoc test.ResultsDesflurane 6% (84 ± 6% of baseline) enhanced the recovery of force after 60-min of reoxygenation as compared to control group (51 ± 8% of baseline, P < 0.0001). N-mercaptopropionylglycine (54 ± 3% of baseline), 8-(p-Sulfophenyl)theophylline (62 ± 9% of baseline), HOE140 (58 ± 6% of baseline) abolished desflurane-induced postconditioning. Adenosine (80 ± 9% of baseline) and bradykinin (83 ± 4% of baseline) induced postconditioning (P < 0.0001 vs control), N-mercaptopropionylglycine abolished the beneficial effects of adenosine and bradykinin (54 ± 8 and 58 ± 5% of baseline, respectively).ConclusionsIn vitro, desflurane-induced postconditioning depends on reactive oxygen species production, activation of adenosine and bradykinin B2 receptors. And, the cardioprotective effect of adenosine and bradykinin administered at the beginning of reoxygenation, was mediated, at least in part, through ROS production.

Photodamage to Multidrug‐resistant Gram‐positive and Gram‐negative Bacteria by 870 nm/930 nm Light Potentiates Erythromycin, Tetracycline and Ciprofloxacin

We have previously shown that 870 nm/930 nm wavelengths cause photodamage at physiologic temperatures in methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli via generation of endogenous radical oxygen species (ROS) and decreased plasma membrane potentials (Delta Psi p). We tested MRSA (Strain HSJ216) in vitro with sublethal 870 nm/930 nm laser energy and subinhibitory concentrations of erythromycin, tetracycline, penicillin, rifampin and trimethoprim to surmise whether photodamage could potentiate these antimicrobials. We also tested patient isolates of fluoroquinolone-resistant MRSA and E. coli with subinhibitory concentrations of ciprofloxacin. In MRSA (Strain HSJ216) we observed 97% potentiation (a 1.5 log(10) CFU decrease) with erythromycin and tetracycline. In patient isolates of E. coli, we observed 100% potentiation (>3 log(10) CFU decrease) in all irradiated samples with ciprofloxacin. To assess whether staphyloxanthin pigment conferred protection against the generated ROS, we created an isogenic carotenoid-deficient mutant of S. aureus that was significantly less tolerant of 870 nm/930 nm exposure than the wild type strain (P < 0.0001). We suggest that antibiotic potentiation results from a photobiological attenuation of ATP-dependent macromolecular synthetic pathways, similar to that observed with daptomycin, via disruption of Delta Psi p and endogenous generation of ROS. With erythromycin, tetracycline and ciprofloxacin, attenuation of energy-dependent efflux systems is also a possibility.

Dysregulation of very long chain acyl-CoA dehydrogenase coupled with lipid peroxidation

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease of unknown etiology. We previously revealed increased oxidative stress and high expression of antioxidant proteins in culture cell lines established from lesional lung tissues with IPF (Kabuyama Y, Oshima K, Kitamura T, Homma M, Yamaki J, Munakata M, Homma Y. Genes Cells 12: 1235-1244, 2007). In this study, we show that IPF cells contain high levels of free cholesterol and its peroxidized form as compared with normal TIG7 lung fibroblasts, suggesting that radical oxygen species (ROS) are generated within specific organelles. To understand the molecular basis underlying the generation of ROS in IPF cells, we performed proteomic analysis of mitochondrial proteins from TIG and IPF cells. This analysis shows that the phosphorylation of Ser586 of very long chain acyl-CoA dehydrogenase (VLCAD) is significantly reduced in IPF cells. Similar results are obtained from immunoblotting with anti-pS586 antibody. Kinase activity toward a peptide containing Ser586 from IPF cells is significantly lower than that from TIG cells. Furthermore, a phosphorylation-negative mutant (S586A) VLCAD shows reduced electron transfer activity and a strong dominant-negative effect on fatty acid beta-oxidation. The ectopic expression of the S586A mutant induced human embryonic kidney (HEK) 293 cells to produce significantly high amounts of oxidized lipids and hydrogen peroxide. HEK293 cells expressing the S586A mutant exhibit a reduction in cell growth and an enhancement in apoptosis. These results suggest a novel regulatory mechanism for homeostatic VLCAD activity, whose dysregulation might be involved in the production of oxidative stress and in the pathogenesis of IPF.

Transient Glutathione Depletion before Irradiation triggers Apoptosis in Head and Neck Squamous Cell Carcinoma

Glutathione, a major intra-cellular antioxidant, has been reported to play a fundamental role in the resistance of some cancer cells to radiotherapy so that this molecule can be considered as a potential clinical target for their radio-sensitization. The radio-resistant head and neck squamous carcinomas cell line SQ20B was shown to display a high endogenous level of reduced glutathione, up to 8-fold when compared to its radiosensitive counterpart SCC61 cell line. This result led us to consider the endogenous reduced glutathione as a key factor involved in the resistance of SQ20B cells to ionizing radiation. The purpose of this study was to experiment the incidence of a short and transient depletion of intracellular glutathione before irradiation of SQ20B cells. We therefore made use of the combination of dimethylfumarate (DMF), a glutathione-depleting agent in association with buthionine sulfoximine (BSO), a specific inhibitor of glutamate-cysteine ligase, the first enzyme of the glutathione biosynthesis pathway. In preliminary experiments, we have determined both the efficiency in lowering the endogenous glutathione level and the potential cyto-toxicity of these molecules in un-irradiated SQ20B cells. Although no toxicity was evidenced, a 95 % decrease of intracellular glutathione was obtained under our experimental conditions. This treatment combined to irradiation led to the triggering of apoptosis of the radioresistant SQ20B cell line which significantly increased from 48 hours to longer times, as evidenced by an enhancement of 50 % of the activity of caspases and of the number of cells in the sub-G1 phase, up to 55 % 96h after irradiation. Triggering of apoptosis in SQ210B cells was found to involve the c-Jun N-terminal Kinase (JNK) pathway which was phosphorylated 2 h after irradiation. This activation of JNK was dependent upon the generation of radical oxygen species which resulted in the dissociation of thioredoxin-ASK 1 complex (MAPKKK of JNK pathway). The phosphorylated form of JNK led to the activation and translocation of Bax to mitochondria which further resulted in the alteration of the organelle, as evidenced by the loss of the mitochondrial transmembrane potential (ΔΨm) and the increase of the secondary radical species generated through the mitochondrial respiratory chain. Taken altogether, our results demonstrate that a transient glutathione depletion before irradiation can trigger apoptosis of radio-resistant SQ20B cells through the activation of a JNK-dependent pathway. Moreover, the absence of a significant toxicity of this pharmacological treatment is of fundamental importance for further in vivo studies.

NO with no NOS in ischemic heart

See article by Martin et al. [5] (pages 46–55) in this issue. The seminal observation by Furchgott and Zawadzki [1] of a paradoxical vasoconstrictive effect of acetylcholine in arteries deprived of endothelium paved the way for the rapid discovery of nitric oxide (NO) and of the mechanisms of NO generation by a specific family of isoforms known as NO synthases (NOS). The role of NO in the endothelium was characterized as a predominant mechanism of vasodilation through the production of cyclic GMP (cGMP) in vascular smooth muscle cells. The rapid progress of this research in endothelial cells automatically led to the question whether the cardiac cell itself could be a source of NO. It was found that the three characterized isoforms of NOS are expressed in cardiac myocytes [2]. However, the role of NO in the myocardium, and the regulation of its production, seems much more complex than what was described before in the endothelium, and includes both cGMP-dependent and -independent mechanisms. Myocardial NO is involved in cellular processes as diverse as cardiac contractility, Ca2+ handling, mitochondrial respiration, substrate metabolism, generation of radical oxygen species, and ischemic preconditioning among others (see Fig. 1). In addition, the mechanisms of action of NO are diverse and sometimes contradictory. For example, depending on the experimental setting, blocking cardiac NOS can be either beneficial or deleterious for the ischemic heart [3,4]. Part of this complexity comes from the fact that NO production in the heart is performed by the three different isoforms (endothelial, inducible and neuronal) that differ in their properties, … *Corresponding author. Tel.: +1 973 972 3926; fax: +1 973 972 7489. Email address: deprech{at}umdnj.edu

Granzyme B induction signalling pathway in acute myeloid leukemia cell lines stimulated by Tumor Necrosis Factor alpha and Fas Ligand

Acute myeloid leukemia (AML) cell lines treated by genotoxic agents or by Tumor Necrosis Factor alpha (TNFα) acquire potent cytotoxicity towards myeloid cells through activation of granzyme B (GrB)/perforin (PFN) system. Here we first extend this observation to another death receptor activator, Fas Ligand (FasL). Moreover, we analyzed GrB induction signalling pathway in TNFα- and FasL-stimulated AML cells. The effects of TNFα and FasL on GrB expression were specifically mediated by p38MAPK (Mitogen-activated-protein-kinase) activation. Otherwise, TNFα and FasL stimulation led to radical oxygen species (ROS) generation and ASK1 (Apoptosis-signal-regulating-kinase-1) activation. Endogenous activation of ASK1 by either H2O2 or thioredoxin (Trx) reductase inhibition had the same effects as TNFα and FasL on GrB up regulation. Altogether, our results suggest that TNFα- and FasL-stimulated AML cell lytic induction is regulated by a signalling pathway involving sequentially, ROS generation, Trx oxidation, ASK1 activation, p38MAPK stimulation and GrB induction at mRNA and protein levels.

Homogeneous versus heterogeneous catalytic reactions to eliminate organics from waste water using H2O2

Homogeneous (Cu2+ ions) and heterogeneous (Cu2+-pillared clay) Fenton-like catalysts have been compared in the conversion of p-coumaric acid. The performances of the two classes of catalysts are similar for an analogous amount of copper, but there are some relevant differences in terms of (i) the presence of an induction time, (ii) the turnover frequency, (iii) the efficiency in the use of H2O2, (iv) the initial attack of p-coumaric acid (hydroxylation on the aromatic ring or oxidative attack on the double bond of the lateral chain), and (v) the effect of dissolved oxygen on the removal of total organic carbon (TOC). These differences were interpreted in terms of reaction network of generation of radical oxygen species and of organics conversion. The possible formation of a surface peroxo adduct coordinated to a copper binulcear site was also evidenced for the solid heterogeneous catalyst.

Insulin generates free radicals in human fibroblasts ex vivo by a protein kinase C-dependent mechanism, which is inhibited by pravastatin

Insulin can generate oxygen free radicals. Statins, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, exert a powerful antioxidant effect. The present study aimed to clarify the mechanisms through which insulin generates free radicals and to assess whether pravastatin modulates such effects. In cultured skin fibroblasts from human volunteers exposed to high insulin concentration, either in the presence or in the absence of pravastatin, insulin induced translocation of the p47 phox subunit of NAD(P)H oxidase from the cytosol to the membrane and generation of radical oxygen species through a PKC δ-dependent mechanism. The insulin-induced translocation of p47 phox was PKC δ dependent and attenuated by pravastatin, but independent of the activation of Akt and Rac1. Insulin-induced Akt phosphorylation was increased by pravastatin and ERK1/2 phosphorylation attenuated. The present study demonstrates a novel mechanism by which insulin stimulates the generation of free radicals in human fibroblasts, ex vivo. It involves phosphatidylinositol 3-kinase, PKC δ, and p47 phox translocation and promotes ERK1/2 phosphorylation. Pravastatin inhibited radical oxygen species production by inhibiting PKC δ. These observations offer a robust explanation for the positive effects of pravastatin treatment in patients with insulin resistance syndrome.

High Accumulation of Platinum-DNA Adducts in Strial Marginal Cells of the Cochlea Is an Early Event in Cisplatin but Not Carboplatin Ototoxicity

Ototoxicity is a typical dose-limiting side effect of cancer chemotherapy with cisplatin but much less so with carboplatin. To elucidate the underlying molecular pathological mechanisms, we have measured the formation and persistence of drug-induced DNA adducts in the nuclei of inner ear cells of guinea pigs after short-term exposure to either cisplatin or carboplatin using immunofluorescence staining and quantitative image analysis. After application of carboplatin, all cells of the cochlea exhibited a similar burden of guanine-guanine intrastrand cross-links in DNA. In contrast, we observed a pronounced 3- to 5-fold accumulation of this cytotoxic adduct exclusively in the marginal cells of the stria vascularis between 8 and 48 h after treatment with cisplatin. In the kidney, the other critical target tissue of cisplatin toxicity, a similar high preferential formation of cytotoxic DNA adducts was measured in the tubular epithelial cells but not in other renal cell types. As for the ear, this excessive formation of DNA damage in a particular cell type was seen in animals treated with cisplatin but not those treated with carboplatin. Because cisplatin ototoxicity is often attributed to oxidative stress mediated by the generation of radical oxygen species (ROS), we have measured in parallel the levels of the lead DNA oxidation product 8-oxoguanine (8-oxoG) in cochlear cryosections. Compared with basal levels in untreated control cochleas, no additional formation of 8-oxoG was detectable up to 48 h after cisplatin treatment in the DNA of either inner-ear cell type. This suggests that the generation of ROS may be a secondary event in cisplatin ototoxicity.

Development of a human mitochondria-focused cDNA microarray (hMitChip) and validation in skeletal muscle cells: implications for pharmaco- and mitogenomics

Mitochondrial research has influenced our understanding of human evolution, physiology and pathophysiology. Mitochondria, intracellular organelles widely known as 'energy factories' of the cell, also play fundamental roles in intermediary metabolism, steroid hormone and heme biosyntheses, calcium signaling, generation of radical oxygen species, and apoptosis. Mitochondria possess a distinct DNA (mitochondrial DNA); yet, the vast majority of mitochondrial proteins are encoded by the nuclear DNA. Mitochondria-related genetic defects have been described in a variety of mostly rare, often fatal, primary mitochondrial disorders; furthermore, they are increasingly reported in association with many common morbid conditions, such as cancer, obesity, diabetes and neurodegenerative disorders, although their role remains unclear. This study describes the creation of a human mitochondria-focused cDNA microarray (hMitChip) and its validation in human skeletal muscle cells treated with glucocorticoids. We suggest that hMitChip is a reliable and novel tool that will prove useful for systematically studying the contribution of mitochondrial genomics to human health and disease.

Ethanol-mediated stimulation of HCV replication in vitro: Role of MAP Kinase activity and generation of radical oxygen species

Aims: In chronic hepatitis C the natural course of the disease is more aggressive in alcoholics and leads earlier to liver cirrhosis. Furthermore, the sustained virological reponse upon interferon therapy is diminished in alcoholics.

Effect of acute and chronic psychostimulant drugs on redox status, AP-1 activation and pro-enkephalin mRNA in the human astrocyte-like U373 MG cells

In order to approach the astroglial implication of addictive and neurotoxic processes associated with psychostimulant drug abuse, the effects of amphetamine or cocaine (1–100 μM) on redox status, AP-1 transcription factor and pro-enkephalin, an AP-1 target gene, were investigated in the human astrocyte-like U373 MG cells. We demonstrated an early increase in the generation of radical oxygen species and in the formation of 4-hydroxynonenal-adducts reflecting the pro-oxidant action of both substances. After 1 h or 96 h of treatment, Fos and Jun protein levels were altered and the DNA-binding activity of AP-1 was increased in response to both substances. Using supershift experiments, we observed that the composition of AP-1 dimer differed according to the substance and the duration of treatment. FRA-2 protein represented the main component of the chronic amphetamine- or cocaine-activated complexes, which suggests its relevance in the long-term effects of psychostimulant drugs. Concomitantly, the pro-enkephalin gene was differently regulated by either 6 h or 96 h of treatment. Because astrocytes interact extensively with the neurons in the brain, our data led us to conclude that oxidation-regulated AP-1 target genes may represent one of the molecular mechanisms underlying neuronal adaptation associated with psychostimulant dependence.