No effect of creatine supplementation on oxidative stress and cardiovascular parameters in spontaneously hypertensive rats

Background: Exacerbated oxidative stress is thought to be a mediator of arterial hypertension. It has been postulated that creatine (Cr) could act as an antioxidant agent preventing increased oxidative stress. The aim of this study was to investigate the effects of nine weeks of Cr or placebo supplementation on oxidative stress and cardiovascular parameters in spontaneously hypertensive rats (SHR). Findings: Lipid hydroperoxidation, one important oxidative stress marker, remained unchanged in the coronary artery (Cr: 12.6 +/- 1.5 vs. Pl: 12.2 +/- 1.7 nmol.mg(-1); p = 0.87), heart (Cr: 11.5 +/- 1.8 vs. Pl: 14.6 +/- 1.1 nmol.mg(-1); p = 0.15), plasma (Cr: 67.7 +/- 9.1 vs. Pl: 56.0 +/- 3.2 nmol.mg(-1); p = 0.19), plantaris (Cr: 10.0 +/- 0.8 vs. Pl: 9.0 +/- 0.8 nmol.mg(-1); p = 0.40), and EDL muscle (Cr: 14.9 +/- 1.4 vs. Pl: 17.2 +/- 1.5 nmol.mg(-1); p = 0.30). Additionally, Cr supplementation affected neither arterial blood pressure nor heart structure in SHR (p > 0.05). Conclusions: Using a well-known experimental model of systemic arterial hypertension, this study did not confirm the possible therapeutic effects of Cr supplementation on oxidative stress and cardiovascular dysfunction associated with arterial hypertension.

Phenolic compounds from Rosemary (Rosmarinus officinalis L.) attenuate oxidative stress and reduce blood cholesterol concentrations in diet-induced hypercholesterolemic rats

Abstract Background Phenolic compounds combine antioxidant and hypocholesterolemic activities and, consequently, are expected to prevent or minimize cardiometabolic risk. Methods To evaluate the effect of an aqueous extract (AQ) and non-esterified phenolic fraction (NEPF) from rosemary on oxidative stress in diet-induced hypercholesterolemia, 48 male 4-week old Wistar rats were divided into 6 groups: 1 chow diet group (C) and 5 hypercholesterolemic diet groups, with 1 receiving water (HC), 2 receiving AQ at concentrations of 7 and 140 mg/kg body weight (AQ70 and AQ140, respectively), and 2 receiving NEPF at concentrations of 7 and 14 mg/kg body weight (NEPF7 and NEPF14, respectively) by gavage for 4 weeks. Results In vitro, both AQ and NEPF had remarkable antioxidant activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH●) assay, which was similar to BHT. In vivo, the group that received AQ at 70 mg/kg body weight had lower serum total cholesterol (−39.8%), non-HDL-c (−44.4%) and thiobarbituric acid reactive substance (TBARS) levels (−37.7%) compared with the HC group. NEPF (7 and 14 mg/kg) reduced the tissue TBARS levels and increased the activity of tissular antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase). Neither AQ nor NEPF was able to ameliorate the alterations in the hypercholesterolemic diet-induced fatty acid composition in the liver. Conclusions These data suggest that phenolic compounds from rosemary ameliorate the antioxidant defense in different tissues and attenuate oxidative stress in diet-induced hypercholesterolemic rats, whereas the serum lipid profile was improved only in rats that received the aqueous extract.

Oxidative Stress and Friedreich’s Ataxia

Friedreich’s Ataxia (FRDA) is a neurodegenerative disorder caused by a deficiency of the protein frataxin and characterized by oxidative stress. The first aim of my research project was to analyze the effects of tocotrienol in FRDA patients. Patients received for 2 months a low dose of tocotrienol. A number of biochemical parameters related to oxidative stress were studied. We consistently showed that taking for 2 months a low dose of tocotrienol led to the decrease of oxidative stress indexes in FRDA patients. Also, this study provides a suitable model to investigate the efficacy of natural compounds to counteract the oxidative stress in FRDA. Furthermore, we investigated whether the tocotrienol was able to modulate the expression of the frataxin isoforms (FXN-1, FXN -2, FXN-3) in FRDA patients. We demonstrated that tocotrienol leads to a specific and significant increase of FXN-3 expression. As no structural and functional details were available for FNX-2 and FXN-3, 3D-models were built. FXN-1, the canonical isoform, was then docked on the human iron-sulphur complex and functional interactions were computed; when FXN-1 was replaced by FXN-2 or FNX-3, we found that the interactions were maintained, thus suggesting a possible biological role for both isoforms. The second aim of my research project was to investigate the role of a single nucleotide polymorphism (SNP) in the protein Sirtuin 6 in FRDA patients. In fact, it was known that those who harbour a SNP (Asn46/Ser46) in the gene enconding Sirt6 show a better outcome those individuals who are homozygous for the Asn 46 allele. We found that fibroblasts and iPSC-derived neurons from FRDA patients harboring the SNP (Asn46/Ser46) have a reduced amount of Sirt6 protein compared to cells from individuals who are homozygous for the prevalent Asn allele. Our studies provide new information on the role of Sirtuins in FRDA pathogenesis.

Oxidative stress and inflammation response after nanoparticle exposure: differences between human lung cell monocultures and an advanced three-dimensional model of the human epithelial airways

Combustion-derived and manufactured nanoparticles (NPs) are known to provoke oxidative stress and inflammatory responses in human lung cells; therefore, they play an important role during the development of adverse health effects. As the lungs are composed of more than 40 different cell types, it is of particular interest to perform toxicological studies with co-cultures systems, rather than with monocultures of only one cell type, to gain a better understanding of complex cellular reactions upon exposure to toxic substances. Monocultures of A549 human epithelial lung cells, human monocyte-derived macrophages and monocyte-derived dendritic cells (MDDCs) as well as triple cell co-cultures consisting of all three cell types were exposed to combustion-derived NPs (diesel exhaust particles) and to manufactured NPs (titanium dioxide and single-walled carbon nanotubes). The penetration of particles into cells was analysed by transmission electron microscopy. The amount of intracellular reactive oxygen species (ROS), the total antioxidant capacity (TAC) and the production of tumour necrosis factor (TNF)-alpha and interleukin (IL)-8 were quantified. The results of the monocultures were summed with an adjustment for the number of each single cell type in the triple cell co-culture. All three particle types were found in all cell and culture types. The production of ROS was induced by all particle types in all cell cultures except in monocultures of MDDCs. The TAC and the (pro-)inflammatory reactions were not statistically significantly increased by particle exposure in any of the cell cultures. Interestingly, in the triple cell co-cultures, the TAC and IL-8 concentrations were lower and the TNF-alpha concentrations were higher than the expected values calculated from the monocultures. The interplay of different lung cell types seems to substantially modulate the oxidative stress and the inflammatory responses after NP exposure.

Oxidative stress is involved in heat-induced cell death in Saccharomyces cerevisiae.

The cause for death after lethal heat shock is not well understood. A shift from low to intermediate temperature causes the induction of heat-shock proteins in most organisms. However, except for HSP104, a convincing involvement of heat-shock proteins in the development of stress resistance has not been established in Saccharomyces cerevisiae. This paper shows that oxidative stress and antioxidant enzymes play a major role in heat-induced cell death in yeast. Mutants deleted for the antioxidant genes catalase, superoxide dismutase, and cytochrome c peroxidase were more sensitive to the lethal effect of heat than isogenic wild-type cells. Overexpression of catalase and superoxide dismutase genes caused an increase in thermotolerance. Anaerobic conditions caused a 500- to 20,000-fold increase in thermotolerance. The thermotolerance of cells in anaerobic conditions was immediately abolished upon oxygen exposure. HSP104 is not responsible for the increased resistance of anaerobically grown cells. The thermotolerance of anaerobically grown cells is not due to expression of heat-shock proteins. By using an oxidation-dependent fluorescent molecular probe a 2- to 3-fold increase in fluorescence was found upon heating. Thus, we conclude that oxidative stress is involved in heat-induced cell death.

Oxidative stress and its haemodynamic consequences in chronic kidney disease

Chronic kidney disease (CKD) is associated with increased cardiovascular risk in comparison with the general population. This can be observed even in the early stages of CKD, and rises in proportion to the degree of renal impairment. Not only is cardiovascular disease (CVD) more prevalent in CKD, but its nature differs too, with an excess of morbidity and mortality associated with congestive cardiac failure, arrhythmia and sudden death, as well as the accelerated atherosclerosis which is also observed. Conventional cardiovascular risk factors such as hypertension, dyslipidaemia, obesity, glycaemia and smoking, are highly prevalent amongst patients with CKD, although in many of these examples the interaction between risk factor and disease differs from that which exists in normal renal function. Nevertheless, the extent of CVD cannot be fully explained by these conventional risk factors, and non-conventional factors specific to CKD are now recognised to contribute to the burden of CVD. Oxidative stress is a state characterised by excessive production of reactive oxygen species (ROS) and other radical species, a reduction in the capacity of antioxidant systems, and disturbance in normal redox homeostasis with depletion of protective vascular signalling molecules such as nitric oxide (NO). This results in oxidative damage to macromolecules such as lipids, proteins and DNA which can alter their functionality. Moreover, many enzymes are sensitive to redox regulation such that oxidative modification to cysteine thiol groups results in activation of signalling cascades which result in adverse cardiovascular effects such as vascular and endothelial dysfunction. Endothelial dysfunction and oxidative stress are present in association with many conventional cardiovascular risk factors, and can be observed even prior to the development of overt, clinical, vascular pathology, suggesting that these phenomena represent the earliest stages of CVD. In the presence of CKD, there is increased ROS production due to upregulated NADPH oxidase (NOX), increase in a circulating asymmetric dimethylarginine (ADMA), uncoupling of endothelial nitric oxide synthase (eNOS) as well as other mechanisms. There is also depletion in exogenous antioxidants such as ascorbic acid and tocopherol, and a reduction in activity of endogenous antioxidant systems regulated by the master gene regulator Nrf-2. In previous studies, circulating markers of oxidative stress have been shown to be increased in CKD, together with a reduction in endothelial function in a stepwise fashion relating to the severity of renal impairment. Not only is CVD linked to oxidative stress, but the progression of CKD itself is also in part dependent on redox sensitive mechanisms. For example, administration of the ROS scavenger tempol attenuates renal injury and reduces renal fibrosis seen on biopsy in a mouse model of CKD, whilst conversely, supplementation with the NOS inhibitor L-NAME causes proteinuria and renal impairment. Previous human studies examining the effect of antioxidant administration on vascular and renal function have been conflicting however. The work contained in this thesis therefore examines the effect of antioxidant administration on vascular and endothelial function in CKD. Firstly, 30 patients with CKD stages 3 – 5, and 20 matched hypertensive controls were recruited. Participants with CKD had lower ascorbic acid, higher TAP and ADMA, together with higher augmentation index and pulse wave velocity. There was no difference in baseline flow mediated dilatation (FMD) between groups. Intravenous ascorbic acid increased TAP and O2-, and reduced central BP and augmentation index in both groups, and lowered ADMA in the CKD group only. No effect on FMD was observed. The effects of ascorbic acid on kidney function was then investigated, however this was hindered by the inherent drawbacks of existing methods of non-invasively measuring kidney function. Arterial spin labelling MRI is an emerging imaging technique which allows measurement of renal perfusion without administration of an exogenous contrast agent. The technique relies upon application of an inversion pulse to blood within the vasculature proximal to the kidneys, which magnetically labels protons allowing measurement upon transit to the kidney. At the outset of this project local experience using ASL MRI was limited and there ensued a prolonged pre-clinical phase of testing with the aim of optimising imaging strategy. A study was then designed to investigate the repeatability of ASL MRI in a group of 12 healthy volunteers with normal renal function. The measured T1 longitudinal relaxation times and ASL MRI perfusion values were in keeping with those found in the literature; T1 time was 1376 ms in the cortex and 1491 ms in the whole kidney ROI, whilst perfusion was 321 mL/min/100g in the cortex, and 228 mL/min/100g in the whole kidney ROI. There was good reproducibility demonstrated on Bland Altman analysis, with a CVws was 9.2% for cortical perfusion and 7.1% for whole kidney perfusion. Subsequently, in a study of 17 patients with CKD and 24 healthy volunteers, the effects of ascorbic acid on renal perfusion was investigated. Although no change in renal perfusion was found following ascorbic acid, it was found that ASL MRI demonstrated significant differences between those with normal renal function and participants with CKD stages 3 – 5, with increased cortical and whole kidney T1, and reduced cortical and whole kidney perfusion. Interestingly, absolute perfusion showed a weak but significant correlation with progression of kidney disease over the preceding year. Ascorbic acid was therefore shown to have a significant effect on vascular biology both in CKD and in those with normal renal function, and to reduce ADMA only in patients with CKD. ASL MRI has shown promise as a non-invasive investigation of renal function and as a biomarker to identify individuals at high risk of progressive renal impairment.

Cuminum cyminum Linn (Apiaceae) extract attenuates MPTP-induced oxidative stress and behavioral impairments in mouse model of Parkinson’s disease

Purpose: To evaluate the protective effects of Cuminum cyminum Linn (Apiaceae, CCY) against 1- methyl-4 phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced oxidative stress and behavioral impairments in mouse model of Parkinson’s disease (PD). Methods: MPTP-intoxicated mice model of PD was used for evaluating the effect of CCY extract on behavioral deficits through rota rod, passive avoidance and open field tasks. The effect of CCY extract on oxidative stress levels were assessed by estimating enzyme status, including superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation(LPO) in brain tissues of MPTP-induced mice. Results: MPTP (25 mg/kg, i.p.)-treated mice resulted in a significant (p < 0.001) behavioral deficit in locomotor behavior (from 56.24 ± 1.21 to 27.64 ± 0.94) and cognitive functions (from 298 ± 3.68 s to 207.28 ± 4.12 s) compared with their respective control groups. Administration of CCY extract (100, 200 and 300 mg/kg, p.o.) for three weeks significantly and dose-dependently improved (p < 0.001 at 300 mg/kg) locomotor and cognitive deficits in MPTP-treated mice. CCY treatment also significantly (p < 0.001 at 300 mg/kg) inhibited MPTP-induced decrease in antioxidant enzyme levels (superoxide dismutase and catalase) and lipid peroxides in mice brain tissues. Conclusion: CCY extract exhibits strong protection against MPTP-induced behavioral deficit through enhancement of antioxidant defense mechanisms. Therefore, CCY may be developed as a therapeutic strategy in the treatment of neurodegeneration seen in PD.

A Mycobacterial Phosphoribosyltransferase Promotes Bacillary Survival by Inhibiting Oxidative Stress and Autophagy Pathways in Macrophages and Zebrafish

Mycobacterium tuberculosis employs various strategies to modulate host immune responses to facilitate its persistence in macrophages. The M. tuberculosis cell wall contains numerous glycoproteins with unknown roles in pathogenesis. Here, by using Concanavalin A and LC-MS analysis, we identified a novel mannosylated glycoprotein phosphoribosyltransferase, encoded by Rv3242c from M. tuberculosis cell walls. Homology modeling, bioinformatic analyses, and an assay of phosphoribosyltransferase activity in Mycobacterium smegmatis expressing recombinant Rv3242c (MsmRv3242c) confirmed the mass spectrometry data. Using Mycobacterium marinum-zebrafish and the surrogate MsmRv3242c infection models, we proved that phosphoribosyltransferase is involved in mycobacterial virulence. Histological and infection assays showed that the M. marinum mimG mutant, an Rv3242c orthologue in a pathogenic M. marinum strain, was strongly attenuated in adult zebrafish and also survived less in macrophages. In contrast, infection with wild type and the complemented Delta mimG: Rv3242c M. marinum strains showed prominent pathological features, such as severe emaciation, skin lesions, hemorrhaging, and more zebrafish death. Similarly, recombinant Msm Rv3242c bacteria showed increased invasion in non-phagocytic epithelial cells and longer intracellular survival in macrophages as compared with wild type and vector control M. smegmatis strains. Further mechanistic studies revealed that the Rv3242c- and mimG-mediated enhancement of intramacrophagic survival was due to inhibition of autophagy, reactive oxygen species, and reduced activities of superoxide dismutase and catalase enzymes. Infection with MsmRv3242c also activated the MAPK pathway, NF-kappa B, and inflammatory cytokines. In summary, we show that a novel mycobacterial mannosylated phosphoribosyltransferase acts as a virulence and immunomodulatory factor, suggesting that it may constitute a novel target for antimycobacterial drugs.

Induction of oxidative stress and apoptosis by PFOS and PFOA in primary cultured hepatocytes of freshwater tilapia (Oreochromis niloticus)

Perfluorinated organic compounds (PFOCs) are emerging persistent organic pollutants (POPs) widely present in the environment, wildlife and human. We studied the cellular toxicology of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) on oxidative stress and induction of apoptosis in primary cultured hepatocytes of freshwater tilapia (Oreochromis niloticus). Cultured hepatocytes were exposed to PFOS or PFOA (0, 1, 5, 15 and 30 mg L-1) for 24 h, and a dose-dependent decrease in cell viability was determined using trypan blue exclusion method. Significant induction of reactive oxygen species (ROS) accompanied by increases in activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) were found, while activities of glutathione peroxidase (GPx) and glutathione-S-transferase (GST) were decreased. Glutathione (GSH) content was reduced following treatment of PFOA and PFOS. A dose-dependent increase in the lipid peroxidation (LPO) level (measured as maleic dialdehyde, MDA) was observed only in the PFOA exposure groups, whereas LPO remained unchanged in the PFOS exposure groups. Furthermore, a significant activation of caspase-3, -8, -9 activities was evident in both PFOS and PFOA exposure groups. Typical DNA fragmentation (DNA laddering) was further characterized by agarose gel electrophoresis. The overall results demonstrated that PFOS and PFOA are able to produce oxidative stress and induce apoptosis with involvement of caspases in primary cultured tilapia hepatocytes. (c) 2007 Elsevier B.V. All rights reserved.

pkn22 (alr2502) encoding a putative Ser/Thr kinase in the cyanobacterium Anabaena sp PCC 7120 is induced by both iron starvation and oxidative stress and regulates the expression of isiA

In cyanobacteria, the isiA gene is required for cell adaptation to oxidative damage caused by the absence of iron. We show here that a putative Ser/Thr kinase gene, pkn22 (alr2052), is activated by iron deficiency and oxidative damage in Anabaena sp. PCC 7120. A pkn22 insertion mutant is unable to grow when iron is limiting. pkn22 regulates the expression of isiA (encoding CP43') but not of isiB (encoding flavodoxin) and psbC (CP43). Fluorescence measurement at 77 K reveals the absence of the typical signature of CP43' associated with photosystem I in the mutant under iron-limiting conditions. We propose that Pkn22 is required for the function of isiA/CP43' and constitutes a regulatory element necessary for stress response. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Identification and analysis of a Scophthalmus maximus ferritin that is regulated at transcription level by oxidative stress and bacterial infection

Ferritins are conserved Iron storage proteins that exist in most living organisms and play an essential role in Iron homeostasis. In this study, we reported the identification and analysis a ferritin M subunit, SmFerM, from turbot Scophthalmus maximus. The full length cDNA of SmFerM contains a 5'-untranslated region (UTR) of 232 bp, an open reading frame (ORF) of 531 bp, and a 3'-UTR of 196 bp The ORF encodes a putative protein of 176 amino acids, which shares extensive sequence identities with the M terrains of several fish species. In silico analysis identified in SmFerM both the ferroxidase center of mammalian H ferritins and the iron nucleation site of mammalian L ferritins. Quantitative real time reverse transcriptase-PCR analysis indicated that SmFerM expression was highest in muscle and lowest in heart and responded positively to experimental challenges with bacterial pathogens and poly(I center dot C) Exposure of cultured turbot hepatocytes to treatment of stress inducers (iron, copper, and H2O2) significantly upregulated the expression of SmFerM in a dose dependent manner. Iron chelating analysis showed that recombinant SmFerM purified from Escherichia coli exhibited apparent iron binding activity. These results suggest that SmFerM is a functional M ferritin and is likely to play a role in iron sequestration and protection against oxidative stress and microbial infection (C) 2010 Elsevier Inc All rights reserved

Reduced Nitro-oxidative Stress and Neural Cell Death Suggests a Protective Role for Microglial Cells in TNFα−/− Mice in Ischemic Retinopathy

Purpose. Neovascularization occurs in response to tissue ischemia and growth factor stimulation. In ischemic retinopathies, however, new vessels fail to restore the hypoxic tissue; instead, they infiltrate the transparent vitreous. In a model of oxygen-induced retinopathy (OIR), TNFa and iNOS, upregulated in response to tissue ischemia, are cytotoxic and inhibit vascular repair. The aim of this study was to investigate the mechanism for this effect.

Methods. Wild-type C57/BL6 (WT) and TNFa-/- mice were subjected to OIR by exposure to 75% oxygen (postnatal days 7–12). The retinas were removed during the hypoxic phase of the model. Retinal cell death was determined by TUNEL staining, and the microglial cells were quantified after Z-series capture with a confocal microscope. In situ peroxynitrite and superoxide were measured by using the fluorescent dyes DCF and DHE. iNOS, nitrotyrosine, and arginase were analyzed by real-time PCR, Western blot analysis, and activity determined by radiolabeled arginine conversion. Astrocyte coverage was examined after GFAP immunostaining.

Results. The TNFa-/- animals displayed a significant reduction in TUNEL-positive apoptotic cells in the inner nuclear layer of the avascular retina compared with that in the WT control mice. The reduction coincided with enhanced astrocytic survival and an increase in microglial cells actively engaged in phagocytosing apoptotic debris that displayed low ROS, RNS, and NO production and high arginase activity.

Conclusions. Collectively, the results suggest that improved vascular recovery in the absence of TNFa is associated with enhanced astrocyte survival and that both phenomena are dependent on preservation of microglial cells that display an anti-inflammatory phenotype during the early ischemic phase of OIR.