Increased Reactive Oxygen Species Activity Research Articles

Oxidative Damage to Mitochondria Enhanced by Ionising Radiation and Gold Nanoparticles in Cancer Cells.

Gold nanoparticles (AuNP) can increase the efficacy of radiation therapy by sensitising tumor cells to radiation damage. When used in combination with radiation, AuNPs enhance the rate of cell killing; hence, they may be of great value in radiotherapy. This study assessed the effects of radiation and AuNPs on mitochondrial reactive oxygen species (ROS) generation in cancer cells as an adjunct therapeutic target in addition to the DNA of the cell. Mitochondria are considered one of the primary sources of cellular ROS. High levels of ROS can result in an intracellular state of oxidative stress, leading to permanent cell damage. In this study, human melanoma and prostate cancer cell lines, with and without AuNPs, were irradiated with 6-Megavolt X-rays at doses of 0–8 Gy. Indicators of mitochondrial stress were quantified using two techniques, and were found to be significantly increased by the inclusion of AuNPs in both cell lines. Radiobiological damage to mitochondria was quantified via increased ROS activity. The ROS production by mitochondria in cells was enhanced by the inclusion of AuNPs, peaking at ~4 Gy and then decreasing at higher doses. This increased mitochondrial stress may lead to more effectively kill of AuNP-treated cells, further enhancing the applicability of functionally-guided nanoparticles.

Blast-Associated Shock Waves Result in Increased Brain Vascular Leakage and Elevated ROS Levels in a Rat Model of Traumatic Brain Injury.

Blast-associated shock wave-induced traumatic brain injury (bTBI) remains a persistent risk for armed forces worldwide, yet its detailed pathophysiology remains to be fully investigated. In this study, we have designed and characterized a laboratory-scale shock tube to develop a rodent model of bTBI. Our blast tube, driven by a mixture of oxygen and acetylene, effectively generates blast overpressures of 20–130 psi, with pressure-time profiles similar to those of free-field blast waves. We tested our shock tube for brain injury response to various blast wave conditions in rats. The results show that blast waves cause diffuse vascular brain damage, as determined using a sensitive optical imaging method based on the fluorescence signal of Evans Blue dye extravasation developed in our laboratory. Vascular leakage increased with increasing blast overpressures and mapping of the brain slices for optical signal intensity indicated nonhomogeneous damage to the cerebral vasculature. We confirmed vascular leakage due to disruption in the blood-brain barrier (BBB) integrity following blast exposure. Reactive oxygen species (ROS) levels in the brain also increased with increasing blast pressures and with time post-blast wave exposure. Immunohistochemical analysis of the brain sections analyzed at different time points post blast exposure demonstrated astrocytosis and cell apoptosis, confirming sustained neuronal injury response. The main advantages of our shock-tube design are minimal jet effect and no requirement for specialized equipment or facilities, and effectively generate blast-associated shock waves that are relevant to battle-field conditions. Overall data suggest that increased oxidative stress and BBB disruption could be the crucial factors in the propagation and spread of neuronal degeneration following blast injury. Further studies are required to determine the interplay between increased ROS activity and BBB disruption to develop effective therapeutic strategies that can prevent the resulting cascade of neurodegeneration.

Oral antioxidants supplementation for women with unexplained infertility undergoing ICSI/IVF: Randomized controlled trial

Objective: Good oocyte quality and maturity are important prerequisites for high fertilization and implantation rates in IVF/ICSI treatment cycles. Reactive oxygen species (ROS) are produced within ovarian follicles, especially during the ovulation process, and increased ROS activity may be a cause of impaired oocyte maturation and higher rate of failure of IVF/ICSI cycles. Study design: RCT evaluating the effect of antioxidant supplementation on ICSI/IVF outcomes. Two hundred and eighteen women with unexplained subfertility undergoing IVF/ICSI were randomized into two groups. The study group (n = 112) received daily oral antioxidants in the form of multivitamins and minerals (amino acid chelated) while the control group (n = 106) did not. Main outcomes were number of mature metaphase II (MII) oocytes and clinical pregnancy rate. Results: There were no significant changes between the groups as regards age, BMI, basal FSH, number of mature (MII) oocytes (12.7 ± 9.4 vs. 13.2 ± 8.6, P = 0.7) and clinical pregnancy rate per woman randomized (38% vs. 34%; [OR = 1.2; 95% CI, 0.70–2.11]. Conclusion: Oral antioxidants in the form of a combination of multivitamins and minerals (amino acid chelated) did not improve oocyte quality and pregnancy rates in women with unexplained infertility undergoing IVF/ICSI treatment.

Basal and T3-induced ROS Production in Lymphocyte Mitochondria is Increased in Type 2 Diabetic Patients

Mitochondrial function, including production of reactive oxygen species (ROS), is important in the pathogenesis of diabetes and its complications. Thyroid hormones are major regulator of these processes. Hence, the aim of this study was to examine the thyroid hormone regulation of ROS production in human lymphocytes in patients with diabetes mellitus type 2 (T2DM). Lymphocytes from 10 controls and 10 persons with T2DM were examined. Mitochondrial membrane potential (MMP) was examined by flow cytometry after staining with MitoTracker Green (MTG). Similarly ROS was measured following staining with carboxy-H₂DCFDA. MMP was increased in T2DM patients and T₃ stimulation increased MMP in controls [1398 a.u. (979-4094) vs. 2156 a.u. (1611-15189), p=0.04, median and quartiles] as well as in T2DM patients [9167 a.u. (7387-11746) vs. 20274 a.u. (17183-27839 p=0.004, median and quartiles]. Basal ROS concentration was increased in lymphocytes from T2DM and T₃ significantly stimulated ROS concentration in controls [3691 a.u. (2584-6396) vs. 5650 a.u. (3001-7802) p=0.013, median and quartiles] and in T2DM patients [19271 a.u. (6288-25282) vs. 23178 a.u. (10004-28857) p=0.013, median and quartiles]. The ratio of ROS production related to MMP was significantly higher in T2DM, unstimulated as well as T₃-stimulated in T2DM. Unstimulated and T₃ stimulated ROS production and MMP were higher in lymphocytes from diabetic patients. An altered balance between ROS production and MMP, favoring ROS production in T2DM patients, was found suggesting that an increased mitochondrial sensitivity for T₃ may be a significant factor responsible for increased ROS activity in diabetic patients.

Expression of oxidative stress in metastatic retinoblastomaa comparative study

To compare oxidative stress between primary retinoblastoma and retinoblastoma with distant metastasis. Forty consecutive patients presented with primary retinoblastoma and the same number of patients presented with distant metastasis, attending the outpatient department of our hospital between August 2002 and April 2005. All the patients with retinoblastoma underwent a standard metastasis workup and were subsequently categorized into two groups (without metastasis and with metastasis).Venous blood samples were drawn from each patient. After proper centrifugation, serum was collected and antioxidant enzymes and reactive oxygen species (ROS) were assayed. Serum collected from the patients was subjected to biochemical assay of the antioxidant enzymes (superoxide dismutase, catalase and peroxidise) and ROS to determine any difference in enzyme activity between the two groups. Antioxidant levels were found to be less in the metastasis group as compared to the primary intraocular retinoblastoma group(p less than 0.05).Mean ROS activity was found to be increased in metastatic group (p less than 0.05). The decreased antioxidant enzymes level along with increased ROS activity in patients with metastatic retinoblastoma reflect increased oxidative stress as compared to primary intraocular retinoblastoma patients.

Impaired Endothelial Function in Ovariectomised Rats: Effects of Tempol

Menopause causes endothelial dysfunction. We investigated the hypothesis that reactive oxygen species (ROS) mediate the adverse effects of ovariectomy (OVX) on microvessels. Mesenteric arterioles (MAs) were isolated from rats 4–6 weeks after sham‐operation (S) or OVX alone, S or OVX rats were given vehicle or tempol (2 mmol · l−1) throughout (n=6/group) to measure endothelium derived relaxation factor (EDRF) and endothelium dependent contracting factor (EDCF) using a wire myograph. NO and ROS were quantitated by DAF‐FM and tempo‐9AC‐fluorescence using RatioMasterTM. OVX rats had increased mean blood pressure (MBP) (114±7 vs 88±6.5 mmHg, P<0.05). Their MAs had increased medium:lumen ratio (4.44±0.38 vs 1.54±0.29, P<0.01) and EDRF (20±4 vs 41±4%, P<0.001) and decreased NO activity (0.37±0.1 vs 0.7±0.1 Units, P<0.01). However, OVX rats developed a new EDCF response (20±6 vs 5±5%, P<0.05) accompanied by increased ROS activity (0.22±0.09 vs 0.10±0.01 units; P<0.01). Tempol prevented significant changes in BP, vascular remodeling, EDRF and NO activity, and prevented any EDCF or ROS responses in OVX rats. In conclusion, OVX causes hypertension and microvascular remodeling and redirects endothelial responses from relaxation to contraction accompanied by reduced vascular NO and enhanced ROS. All of these effects are dependent on ROS and could, therefore, be targeted with effective antioxidant therapy.

Physical injury stimulates aerobic methane emissions from terrestrial plants

Abstract. Physical injury is common in terrestrial plants as a result of grazing, harvesting, trampling, and extreme weather events. Previous studies demonstrated enhanced emission of non-microbial CH4 under aerobic conditions from plant tissues when they were exposed to increasing UV radiation and temperature. Since physical injury is also a form of environmental stress, we sought to determine whether it would also affect CH4 emissions from plants. Physical injury (cutting) stimulated CH4 emission from fresh twigs of Artemisia species under aerobic conditions. More cutting resulted in more CH4 emissions. Hypoxia also enhanced CH4 emission from both uncut and cut Artemisia frigida twigs. Physical injury typically results in cell wall degradation, which may either stimulate formation of reactive oxygen species (ROS) or decrease scavenging of them. Increased ROS activity might explain increased CH4 emission in response to physical injury and other forms of stress. There were significant differences in CH4 emissions among 10 species of Artemisia, with some species emitting no detectable CH4 under any circumstances. Consequently, CH4 emissions may be species-dependent and therefore difficult to estimate in nature based on total plant biomass. Our results and those of previous studies suggest that a variety of environmental stresses stimulate CH4 emission from a wide variety of plant species. Global change processes, including climate change, depletion of stratospheric ozone, increasing ground-level ozone, spread of plant pests, and land-use changes, could cause more stress in plants on a global scale, potentially stimulating more CH4 emission globally.

Caffeic acid phenethyl ester preferentially enhanced radiosensitizing and increased oxidative stress in medulloblastoma cell line

Caffeic acid phenethyl ester (CAPE), an active component of propolis, was recently reported to have radiosensitizing effects on medulloblastoma (MB) cells. However, the mechanisms of radiosensitivity involved in medulloblastoma cells are still unclear. The specific aim of this study was to investigate the role of CAPE-induced oxidative stress to influence of radiosensitivity and anti-proliferative effects in medulloblastoma cells. Medulloblastoma (Daoy) cells were treated with CAPE in different concentrations and assessed for cell viability. The following were also evaluated: migratory ability, reduced glutathione (GSH) level, reactive oxygen species (ROS) level, nuclear factor-kappaB (NF-kappaB) activity, and apoptosis in CAPE alone, radiation alone, or radiation combined with CAPE in Daoy cells. The results indicated that CAPE inhibited the growth of Daoy cells. CAPE treatment in Daoy cells could effectively decrease glutathione reductase and significantly increase glutathione peroxidase. Radiation-activated NF-kappaB was reversed by CAPE pretreatment. Finally, the result of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay showed that CAPE treatment can enhance radiation-induced apoptosis in Daoy cells. Our study demonstrated the anti-proliferative and radiosensitizing effects of CAPE on MB cells, which may be achievable through depleting GSH, increased ROS activity, and inhibiting NF-kappaB activity.

Upregulation of Endothelial and Inducible Nitric Oxide Synthase Expression by Reactive Oxygen Species

The effect of reactive oxygen species (ROS) on nitric oxide synthase (NOS) expression remains uncertain. This study explored the effect of increased ROS activity on NOS expression in vitro in human coronary artery endothelial cells (HCAECs) grown in culture and in intact animals. Endothelial NOS (eNOS) expression and nuclear factor kappaB (NFkappaB) activation were determined in HCAECs grown in culture and exposed to oxidative stress with xanthine-xanthine oxidase (X-XO) generated superoxide, H(2)O(2), or glutathione depletion with buthionine sulfoximine (BSO) for 24 h. In parallel experiments, cells were treated with a nitric oxide (NO) scavenger (hemoglobin), and with an NO donor S-nitroso-N-acetyl penicillamine (SNAP)]. In addition, eNOS and inducible NOS (iNOS) expressions were determined in rats treated with either BSO or vehicle for 48 h. Increases in ROS activity, achieved by exogenous superoxide and H(2)O(2) or by glutathione depletion, upregulated the expression of eNOS at both transcriptional and translational levels in HCAECs. Similar effects were seen with the non-radical NO scavenger, hemoglobin. The upregulatory action of hemoglobin on eNOS messenger RNA (mRNA) and protein expressions was overcome by the NO donor, SNAP, thereby suggesting that there is a negative feedback regulation of eNOS by NO. Nuclear translocation of NFkappaB (p65) was noted within 5 min of exposure to H(2)O(2) and at least 15 min after exposure to superoxide or BSO. Induction of oxidative stress by glutathione depletion led to upregulation of renal and aorta eNOS and iNOS in live animals. An increase in ROS activity upregulates NOS expression in vitro in HCAECs grown in culture, and also in vivo in animals. This effect appears to be, in part, mediated by limiting the availability of NO, thereby exerting a negative feedback influence on NOS expression through activation of NFkappaB.

Serum Lipid Peroxidation and Antioxidant Potential Levels in Hyperemesis Gravidarum

Hyperemesis gravidarum (HEG) is a severe form of nausea and vomiting with unknown etiology. Recent studies have suggested that there might be an etiologic role for HELICOBACTER PYLORI (HP) in HEG. HP is a Gram-negative bacterium that colonizes the gastric mucosa, increases the generation of reactive oxygen species (ROS), and decreases plasma antioxidants such as ascorbic acid. The aim of this study was to investigate plasma ROS activities and antioxidant status, and their relationship with HP infection, in HEG womens. Twenty-five HEG women, 20 gestational age- and gravida-matched healthy pregnant controls, and 15 nonpregnant women were examined for specific immunoglobulin G (IgG) against HP, serum malondialdehyde (MDA) as a measure of lipid peroxidation, and activity of antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CT). Compared with controls, the seropositivity of HP for IgG was significantly higher ( P < 0.01) in pregnancies with HEG. When three study groups (including both HP + and HP- patients) were compared with each other with respect to the study parameters, MDA concentration increased significantly in pregnant women when compared with control group (nonpregnant women; P < 0.01), as well as in HEG compared with healthy pregnant women ( P < 0.01). The SOD, GSH-Px, and CT activity were increased in the group of normal pregnant women versus the nonpregnant group ( P < 0.01), but decreased significantly in the group of HEG pregnant women ( P < 0.01). Serum MDA levels and SOD, GSH-Px, and CT activity were not affected by the seropositivity of HP for IgG in either group ( P > 0.05). The results of the present study suggest that HEG is an oxidative stress condition, as reflected by the increased ROS activity and decreased antioxidant status, regardless of HP infection.

Extracellular ATP in Plants. Visualization, Localization, and Analysis of Physiological Significance in Growth and Signaling

Extracellular ATP (eATP) in animals is well documented and known to play an important role in cellular signaling (e.g. at the nerve synapse). The existence of eATP has been postulated in plants; however, there is no definitive experimental evidence for its presence or an explanation as to how such a polar molecule could exit the plant cell and what physiological role it may play in plant growth and development. The presence of eATP in plants (Medicago truncatula) was detected by constructing a novel reporter; i.e. fusing a cellulose-binding domain peptide to the ATP-requiring enzyme luciferase. Application of this reporter to plant roots allowed visualization of eATP in the presence of the substrate luciferin. Luciferase activity could be detected in the interstitial spaces between plant epidermal cells and predominantly at the regions of actively growing cells. The levels of eATP were closely correlated with regions of active growth and cell expansion. Pharmacological compounds known to alter cytoplasmic calcium levels revealed that ATP release is a calcium-dependent process and may occur through vesicular fusion, an important step in the polar growth of actively growing root hairs. Reactive oxygen species (ROS) activity at the root hair tip is not only essential for root hair growth, but also dependent on the cytoplasmic calcium levels. Whereas application of exogenous ATP and a chitin mixture increased ROS activity in root hairs, no changes were observed in response to adenosine, AMP, ADP, and nonhydrolyzable ATP (betagammameATP). However, application of exogenous potato (Solanum tuberosum) apyrase (ATPase) decreased ROS activity, suggesting that cytoplasmic calcium gradients and ROS activity are closely associated with eATP release.

GSTM1 and GSTT1 null genotypes as possible heritable factors of rosacea

Rosacea might be related to an increased activity of reactive oxygen species (ROS) and deficient function of the antioxidant system. Glutathione S-transferases (GSTs) play a primer role in cellular defense against electrophilic chemical species and radical oxygen species. We hypothesized that increased ROS activity or decreased antioxidant potential, possibly induced by GST gene polymorphism, might have a pathogenic role in rosacea. The study group consisted of 45 patients with rosacea and 100 control subjects. DNA samples were isolated from blood samples using high pure polymerase chain reaction (PCR) Template preparation Kit. The GSTM1, GSTT1, and P1 polymorphisms were detected using a real-time PCR and fluorescence resonance energy transfer with a Light-Cycler Instrument. Associations between specific genotypes and the development of rosacea were examined using logistic regression analyses to calculate odds ratios (OR) and 95% confidence intervals (CI). GSTM1 and GSTT1 null genotypes were found to be statistically different from control (P=0.005, P=0.009, respectively), and associated with an increased risk of rosacea (OR [95% CI]: 2.84 [1.37-5.89]; OR [95% CI]: 2.68 [1.27-5.67], respectively). There was a statistically significant relationship between both null combination of the GSTM1 and GSTT1 genotype polymorphisms and rosacea (P=0.003, OR [95% CI]: 4.18 [1.57-11.13]). There were no statistically significant differences between patient and control groups for the GSTP1 Ile/Ile, Ile/Val, and Val/Val genotypes (P>0.05). We demonstrated a significant association between the GSTT1 and/or GSTM1 null genotypes and rosacea. However, the potential role of GSTs as markers of susceptibility to rosacea needs further studies in larger patient groups.

Plasma reactive oxygen species activity and antioxidant potential levels in rosacea patients: correlation with seropositivity to Helicobacter pylori.

Recent studies have suggested that there might be an etiologic role for Helicobacter pylori (HP) in rosacea. HP is a Gram-negative bacterium that colonizes the gastric mucosa, increases the generation of reactive oxygen species (ROS), and decreases plasma antioxidants such as ascorbic acid. To investigate plasma ROS activities and antioxidant status, and their relationship with HP infection, in rosacea patients. Twenty-nine rosacea patients and 20 age- and sex-matched healthy controls were examined for specific immunoglobulin G (IgG) and IgM against HP, plasma malondialdehyde (MDA), and antioxidant potential (AOP) levels. Compared with controls, the seropositivity of HP for IgM was significantly higher (P = 0.03) and the seropositivity of HP for IgG was significantly lower (P = 0.0001) in patients with rosacea. Plasma MDA levels were higher (P = 0.0001) and AOP levels were lower (P = 0.019) in patients than in controls, regardless of the severity of the disease. Plasma MDA and AOP levels were not affected by the seropositivity of HP for IgM and/or IgG in either group. Our results suggest that rosacea is an oxidative stress condition, as reflected by the increased ROS activity and decreased AOP, regardless of HP infection.

Stimulation of Na,K-ATPase by low potassium requires reactive oxygen species.

The signaling pathway that transduces the stimulatory effect of low K+ on the biosynthesis of Na,K-ATPase remains largely unknown. The present study was undertaken to examine whether reactive oxygen species (ROS) mediated the effect of low K+ in Madin-Darby canine kidney (MDCK) cells. Low K+ increased ROS activity in a time- and dose-dependent manner, and this effect was abrogated by catalase and N-acetylcysteine (NAC). To determine the role of ROS in low-K+-induced gene expression, the cells were first stably transfected with expression constructs in which the reporter gene chloramphenicol acetyl transferase (CAT) was under the control of the avian Na,K-ATPase alpha-subunit 1.9 kb and 900-bp 5'-flanking regions that have a negative regulatory element. Low K+ increased the CAT expression in both constructs. Catalase or NAC inhibited the effect of low K+. To determine whether the increased CAT activity was mediated through releasing the repressive effect or a direct stimulation of the promoter, the cells were transfected with a CAT expression construct directed by a 96-bp promoter fragment that has no negative regulatory element. Low K+ also augmented the CAT activity expressed by this construct. More importantly, both catalase and NAC abolished the effect of low K+. Moreover, catalase and NAC also inhibited low-K+-induced increases in the Na,K-ATPase alpha1- and beta1-subunit protein abundance and ouabain binding sites. The antioxidants had no significant effect on the basal levels of CAT activity, protein abundance, or ouabain binding sites. In conclusion, low K+ enhances the Na,K-ATPase gene expression by a direct stimulation of the promoter activity, and ROS mediate this stimulation and also low-K+-induced increases in the Na,K-ATPase protein contents and cell surface molecules.

ANTIOXIDANT THERAPY POTENTIATES ANTIHYPERTENSIVE ACTION OF INSULIN IN DIABETIC RATS

Background: Poorly controlled longstanding diabetes frequently results in sustained hypertension (HTN) which plays a major role in the pathogenesis of diabetic nephropathy. In addition, hyperglycemia, per se, causes a reversible rise in blood pressure (BP) and increases production of reactive oxygen species (ROS). Increased ROS activity may raise BP by promoting inactivation of nitric oxide (NO) and/or nonenzymatic generation of vasoconstrictive prostaglandins from peroxidation of arachidonic acid. Therefore, we hypothesized that antioxidant therapy may enhance the BP-lowering effect of glycemia control with insulin replacement in diabetes. Methods: Male Sprague-Dawley rats were rendered diabetic by streptozotocin administration and randomized to untreated, antioxidant-treated (vitamin E-fortified food, tocopherol 5000 U/kg chow and vitamin C-fortified H2O, 1000 mg/L), insulin-treated and insulin plus antioxidant-treated groups. Normal rats fed a regular diet or antioxidant-fortified diet served as controls and monitored for 4 weeks. Results: The diabetic animals showed marked hyperglycemia, HTN, proteinuria, depressed tissue glutathione level and elevated plasma lipid peroxidation product, malondialdehyde (MDA) denoting increased ROS activity. Insulin therapy alone resulted in significant, but incomplete reduction in BP and plasma MDA but not proteinuria. Antioxidant therapy alone had no effect on the measured parameters in either the diabetic or control animals. However, combined insulin and antioxidant therapies normalized BP, plasma MDA and urinary protein in the diabetic animals. As expected, uncontrolled diabetes resulted in glomerular hyperfiltration which was partially reversed by insulin therapy, but was unaffected by antioxidant therapy. Conclusion: Uncontrolled hyperglycemia in the early phase of diabetes was associated with elevated plasma MDA, HTN and proteinuria. Insulin therapy alone resulted in significant but incomplete reduction of plasma MDA and BP. Antioxidant therapy which was ineffective when given alone, normalized plasma MDA, BP and reduced urinary protein excretion when combined with insulin treatment.

Association of Renal Injury with Increased Oxygen Free Radical Activity and Altered Nitric Oxide Metabolism in Chronic Experimental Hemosiderosis

Chronic iron (Fe) overload is associated with a marked increase in renal tissue iron content and injury. It is estimated that 10% of the American population carry the gene for hemochromatosis and 1% actually suffer from iron overload. The mechanism of iron overload-associated renal damage has not been fully elucidated. Iron can accelerate lipid peroxidation leading to organelle membrane dysfunction and subsequent cell injury/death. Iron-catalyzed generation of reactive oxygen species (ROS) is responsible for initiating the peroxidatic reaction. We investigated the possible association of oxidative stress and its impact on nitric oxide (NO) metabolism in iron-overload-associated renal injury. Rats were randomized into Fe-loaded (given 0.5 g elemental iron/kg body weight as iron dextran; IV), Fe-depleted (given an iron-free diet for 20 weeks), and control groups. Renal histology, tissue expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS), renal tissue expression of nitrotyrosine, plasma, and renal tissue lipid peroxidation product, malondialdehyde (MDA), and plasma and urinary NO metabolites (NOx) were examined. Iron overload was associated with mild proteinuria, tissue iron deposition together with significant glomerulosclerosis, tubular atrophy, and interstitial fibrosis. Rare focal glomerulosclerosis and tubulointerstitial changes were noted in normal controls. No renal lesions were observed in Fe-depleted rats. Iron deposits were seen in glomeruli, proximal tubules, and interstitium. The iron staining in the distal tubules was negligible. Both plasma and renal tissue MDA and renal tissue nitrotyrosine were increased significantly in Fe-loaded rats compared with control rats. In contrast, Fe-depleted animals showed a marked reduction in plasma and renal tissue MDA and nitrotyrosine together with significant elevation of urinary NOx excretion. In addition, iron-overload was associated with up-regulation of renal eNOS and iNOS expressions when compared with the control and Fe-depleted rats that showed comparable values. In conclusion, chronic iron overload resulted in iron deposition in the glomeruli and proximal tubules with various renal lesions and evidence of increased ROS activity, enhanced ROS-mediated inactivation, and sequestration of NO and compensatory up-regulation of renal eNOS and iNOS expressions. However, iron depletion was associated with reduced MDA and tissue nitrotyrosine abundance, increased urinary NOx excretion, normal nitric oxide synthase (NOS) expression, and absence of renal injury. These findings point to the possible role of ROS in chronic iron overload-induced renal injury.

Increased nitric oxide inactivation by reactive oxygen species in lead-induced hypertension

We have recently found evidence for increased reactive oxygen species (ROS) in rats with lead-induced hypertension. We hypothesized that increased ROS activity may contribute to hypertension by enhancing inactivation of nitric oxide (NO) in this model. Rats were treated for 12 weeks with either lead acetate (100 p.p.m.) alone (Pb group) or lead acetate plus vitamin E-fortified food (5000 U/kg rat chow, Pb + E group). The control animals were fed either regular rat chow or a vitamin E-fortified diet. Blood pressure, creatinine clearance, and urinary excretion of stable NO metabolites (NOx) were monitored, and plasma and tissue abundance of nitrotyrosine, which is the footprint of NO oxidation by ROS, were determined. The Pb group showed a marked rise in blood pressure, a significant increase in plasma and kidney, heart, liver, and brain nitrotyrosine abundance, and a substantial fall in urinary NOx excretion. Concomitant administration of high-dose vitamin E in the Pb + E group ameliorated hypertension and normalized both urinary NOx excretion and tissue nitrotyrosine without altering tissue lead content. However, vitamin E supplementation had no discernible effect on either blood pressure or nitrotyrosine abundance in the normal controls. These findings point to enhanced ROS-mediated inactivation and sequestration of NO, which can potentially contribute to hypertension, tissue damage, and reduced urinary NOx excretion in rats with lead-induced hypertension. The beneficial effects of high-dose vitamin E on blood pressure, tissue nitrotyrosine burden, and urinary NOx excretion support the role of increased ROS activity in the pathogenesis of these abnormalities in this model.