Oxidative Stress-mediated Mechanism Research Articles

Effect of Doxazosin on Oxidative Stress-Related Proteins in Benign Prostatic Hyperplasia

Background and Objectives: Oxidative stress can induce cell mutations or proliferation which then can progress to carcinogenesis or remodeling. The same oxidative stress-mediated mechanism could participate in prostate cell proliferation and remodeling present in benign prostatic hyperplasia (BPH). Doxazosin induces prostate epithelial and stromal cell apoptosis through production of transforming growth factor-β (TGF-β), but cellular mechanisms are not completely clarified. In 10 prostate samples from BPH untreated patients who underwent TUR, we have assessed the gene and protein expression of: p22^phox (subunit of NAD(P)H oxidase essential for O₂^– production); heme oxygenase-1 (HO-1) (induced by oxidative stress and antiapoptotic); TGF-β (inhibitor of prostatic epithelial and stromal cell growth); the in vitro effect of doxazosin on expression of these markers. Methods: RT-PCR and Western blot with specific primers and antibodies. p22^phox, HO-1 and TGF-β were quantified by the ratio between their PCR and Western blot products and GAPDH. Results: Doxazosin significantly reduced p22^phox gene and protein expression (0.61 ± 0.04 vs. 0.36 ± 0.04 d.u., p < 0.0002; 0.85 ± 0.03 vs. 0.47 ± 0.03, p < 0.0001, respectively). Doxazosin concentration dependently reduced HO-1 gene and protein expression (0.57 ± 0.07 vs. 0.49 ± 0.06 d.u. (1 µM) p < 0.04, vs. 0.22 ± 0.08 (10 µM) p < 0.0001; 0.78 ± 0.04 vs. 0.44 ± 0.1 (10 µM) p < 0.003 respectively) and increased TGF-β protein expression (0.58 ± 0.05 vs. 0.74 ± 0.16 (1 µM) n.s. vs. 0.81 ± 0.07 (10 µM) p < 0.01). Conclusions: Induction of oxidative stress-related proteins seems to be involved in the prostate cell proliferation and remodeling present in BPH. Doxazosin may reduce oxidative stress through reduction of p22^phox. Surprisingly, HO-1, which is induced and protected by oxidative stress, is also reduced by doxazosin. HO-1 is a potent antiapoptotic factor and downregulator of TGF-β. From the results of this preliminary study it could be proposed that the proapoptotic effect of doxazosin could be mediated, at least in part, through the contemporary inhibition of HO-1.

Soluble transition metals in welding fumes cause inflammation via activation of NF-κB and AP-1

We previously reported that the molecular pro-inflammatory effects of welding fumes in vitro were caused by soluble transition metals via an oxidative stress-mediated mechanism. Herein, we tested the hypothesis that transition metals in welding fume drive the in vivo inflammatory response caused by welding fume. Rats were instilled with either whole, soluble extract or washed welding fume particulates or soluble extracts pre-treated with a transition metal chelator. Markers of pulmonary inflammation were measured in the bronchoalveolar lavage fluid (BALF) and nuclear translocation of transcription factor was assessed in BAL cells by electrophoretic mobility shift assay. Instillation of either whole or soluble fractions of welding fume caused a significant influx of inflammatory cells and other markers of inflammation in the BALF 24 h later. MIP-2 protein in BALF and nuclear translocation of NF-κB and AP-1 were significantly greater following instillation of whole and soluble fractions than in saline-instilled lungs. Chelation of the soluble fraction, to remove transition metals, abolished the ability to cause inflammation, MIP-2 increase or transcription factor translocation to the nucleus. Instillation of washed particulates alone caused no significant change in any end-point compared to saline. This study demonstrates that soluble transition metals present in welding fumes cause inflammation via activation of the redox-sensitive transcription factors NF-κB and AP-1 and confirms the validity of utilising in vitro models to assess inflammatory responses to such particles.

CD40 ligand enhances monocyte tissue factor expression and thrombin generation via oxidative stress in patients with hypercholesterolemia

We tested the hypothesis that CD40 ligand (CD40L) induces a prothrombotic state by enhancing oxidative stress. Patients with hypercholesterolemia show an ongoing prothrombotic state, but the underlying mechanism is still unclear. Circulating levels of the soluble form of CD40L (sCD40L), prothrombin fragment (F1+2, a marker of thrombin generation), and 8-hydroxy-2'-deoxyguanosine (8-OHdG, a marker of oxidative stress) were measured in 40 patients with hypercholesterolemia and in 20 age- and gender-matched healthy subjects. Patients with hypercholesterolemia showed significantly higher levels of sCD40L (p <0.005), 8-OHdG (p <0.005), and prothrombin F1+2 (p <0.005), as compared with control subjects. Soluble CD40L significantly correlated with 8-OHdG (r=0.85, p <0.0001) and prothrombin F1+2 (r=0.83, p <0.0001); a significant correlation between 8-OHdG and prothrombin F1+2 was also observed (r=0.64, p <0.0001). An in vitro study demonstrated that CD40L-stimulated monocytes from patients with hypercholesterolemia expressed more tissue factor (TF) and prothrombin F1+2 than monocytes from controls; co-incubation of monocytes with either an inhibitor of NADPH oxidase or an inhibitor of phosphatidylinositol-3-kinase significantly reduced CD40L-mediated clotting activation. A marked inhibition of CD40L-mediated clotting activation was also observed in two male patients with hereditary deficiency of gp91 phox, the central core of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Finally, we demonstrated that CD40L-mediated clotting activation was significantly inhibited by vitamin C, a known antioxidant. This study indicates that in patients with hypercholesterolemia, CD40L over-expresses TF and increases the thrombin generation rate by an oxidative stress-mediated mechanism that requires the activation of NADPH oxidase.

Activation of paraquat in the earthworm Allolobophora chlorotica is mediated by NAD(P)H-cytochrome c reductase activities

This study reports that earthworms, Allolobophora chlorotica, are capable of biotransforming paraquat, a toxic herbicide, resulting in the formation of reactive oxygen species (ROS). We found that in earthworms the reduction of paraquat is mediated by NADPH- and NADH-cytochrome c reductase activities. The formation of superoxide anion ( O 2 −) from the incubation of paraquat with the earthworm extracts was demonstrated by using both Cypridina luciferin analog (CLA) chemiluminescence and the SOD-inhibitable cytochrome c reduction reaction. In addition, in vivo exposure of earthworms to paraquat in solution (24 and 48 h) was performed to investigate whether or not the herbicide affects the levels of the NAD(P)H-cytochrome c reductase activities. Although in vitro NADPH-cytochrome c reductase reduces paraquat more easily than the NADH-dependent activity, after the in vivo exposure an increase of NADH-cytochrome c reductase activity(s) by 12% compared to control values was observed, whereas NADPH-cytochrome c reductase activity was not affected. Xanthine oxidase (XO) is an enzyme implicated in paraquat toxicity, however, no XO was detected in earthworm extracts nor hypoxanthine was a source of electrons for the herbicide reduction. For comparative reasons menadione, a redox cycling quinone, was also incubated with the earthworm extracts. It was found that the incubation of menadione with earthworm extracts formed about two times more ( O 2 −) than with paraquat. It is concluded that the exposure of paraquat to earthworms could elicit radical formation and consequently toxic effects via oxidative stress-mediated mechanisms. The reduction of paraquat by the reductases leads to the formation of paraquat radical, which reacts with molecular oxygen, accounting for the formation of superoxide anion. Further studies are required to conclude that the observed increase of NADH-cytochrome c reductase activity(s) should be used as a biomarker for paraquat exposure in earthworms.

Activation of paraquat in the earthworm Allolobophora chlorotica is mediated by NAD(P)H-cytochrome c reductase activities

This study reports that earthworms, Allolobophora chlorotica, are capable of biotransforming paraquat, a toxic herbicide, resulting in the formation of reactive oxygen species (ROS). We found that in earthworms the reduction of paraquat is mediated by NADPH- and NADH-cytochrome c reductase activities. The formation of superoxide anion (O2−) from the incubation of paraquat with the earthworm extracts was demonstrated by using both Cypridina luciferin analog (CLA) chemiluminescence and the SOD-inhibitable cytochrome c reduction reaction. In addition, in vivo exposure of earthworms to paraquat in solution (24 and 48 h) was performed to investigate whether or not the herbicide affects the levels of the NAD(P)H-cytochrome c reductase activities. Although in vitro NADPH-cytochrome c reductase reduces paraquat more easily than the NADH-dependent activity, after the in vivo exposure an increase of NADH-cytochrome c reductase activity(s) by 12% compared to control values was observed, whereas NADPH-cytochrome c reductase activity was not affected. Xanthine oxidase (XO) is an enzyme implicated in paraquat toxicity, however, no XO was detected in earthworm extracts nor hypoxanthine was a source of electrons for the herbicide reduction. For comparative reasons menadione, a redox cycling quinone, was also incubated with the earthworm extracts. It was found that the incubation of menadione with earthworm extracts formed about two times more (O2−) than with paraquat. It is concluded that the exposure of paraquat to earthworms could elicit radical formation and consequently toxic effects via oxidative stress-mediated mechanisms. The reduction of paraquat by the reductases leads to the formation of paraquat radical, which reacts with molecular oxygen, accounting for the formation of superoxide anion. Further studies are required to conclude that the observed increase of NADH-cytochrome c reductase activity(s) should be used as a biomarker for paraquat exposure in earthworms.

Very long chain fatty acids activate NADPH oxidase in human dermal fibroblasts

Very long chain fatty acids (VLCFAs) are exclusively oxidized in peroxisomes and their levels are significantly increased in tissues of patients with peroxisomal disorders. Although the biochemical indicators of peroxisomal dysfunction, such as elevated VLCFAs, are well known, the mechanisms of pathogenesis of peroxisomal diseases are unclear. In this study we have examined the effect of VLCFAs on NADPH oxidase (NOX), a complex enzyme system responsible for the production of superoxide anions, in order to understand the oxidative stress-mediated mechanisms involved in pathology of peroxisomal disorders. Varying concentrations (2.5 to 10 microg ml(-1)) of VLCFAs, lignoceric acid and cerotic acid, significantly (p < 0.001) increased the enzymic activity of NOX in cultures of human dermal fibroblasts. VLCFAs did not affect the expression of gp91phox or p22phox whereas the mRNA and protein levels of p47phox were significantly (two or three-fold) increased following treatment of fibroblasts with lignoceric acid or cerotic acid. VLCFAs also caused a significant (p < 0.01) increase in lipid peroxidation in dermal fibroblasts which could be markedly reversed by treatment with apocyanin (10 mM) or superoxide dismutase (SOD, 25 U ml(-1)). With these results, we report for the first time that VLCFAs enhance NOX activity and superoxide anion-mediated lipid peroxidation in cultured dermal fibroblasts. This study proposes a mechanism that may be taking place in vivo during peroxisomal dysfunction and that leads to oxidative stress-mediated pathogenesis.

Vitamin E reduces progression of atherosclerosis in low-density lipoprotein receptor-deficient mice with established vascular lesions.

A growing body of evidence from animal studies supports the hypothesis that oxidative stress-mediated mechanisms play a central role in early atherogenesis. In contrast, clinical trials with antioxidant vitamins have not produced consistent results in humans with established atherosclerosis. Low-density lipoprotein receptor-deficient mice (LDLR KO) were fed a high-fat diet for 3 months to induce atheroma. At this time, 1 group of mice was euthanized for examination of atherosclerosis, and 2 other groups were randomized to receive high-fat diet either alone or supplemented with vitamin E for 3 additional months. At the end of the study, LDLR KO on a vitamin E-supplemented fat diet had decreased 8,12-iso-isoprostane (iP)F(2alpha)-VI and monocyte chemoattractant protein-1 levels, but increased nitric oxide levels compared with mice on placebo. No difference in lipid levels was observed between the 2 groups. Compared with baseline, placebo group had progression of atherosclerosis. In contrast, vitamin E-treated animals showed a significant reduction in progression of atherosclerosis. These results demonstrate that in LDLR KO, vitamin E supplementation reduces progression of established atherosclerosis by suppressing oxidative and inflammatory reactions and increasing nitric oxide levels.

CI-1010 induced opening of the mitochondrial permeability transition pore precedes oxidative stress and apoptosis in SY5Y neuroblastoma cells

The hetero-bifunctional nitroimidazole radiosensitizer CI-1010, R-α-{[(2-bromoethyl)-amino]methyl}-2-nitro-1H-imidazole-1-ethanol monohydrobromide, causes selective irreversible apoptotic loss of retinal photoreceptor cells in vivo. The human neuroblastoma cell line, SH-SY5Y, was used as a neuronotypic model of CI-1010-mediated retinal degeneration. Exposure to CI-1010 for 24 h induced apoptosis in neuroblastoma cells, as determined by histopathological and ultrastructural analysis and by TUNEL technique. CI-1010 causes a dose-dependent decrease in cell viability in SY5Y cells, as measured by the reduction of MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Superoxide dismutase reduced loss of cell viability following CI-1010 treatment suggesting an oxidative stress-mediated mechanism of toxicity. The effects of CI-1010 on mitochondrial membrane potential and intracellular levels of reactive oxygen species were assessed in live SY5Y cells by confocal microscopy using the fluorescent dyes, tetramethylrhodamine methyl ester and 5,6-carboxy-2′,7′-dihydrodichlorofluorescein diacetate. CI-1010 caused a rapid depolarization of mitochondria in SY5Y cells followed by an increase in ROS. Both CI-1010-induced mitochondrial depolarization and subsequent increases in ROS were prevented by pretreatment with either the permeability transition pore inhibitor, cyclosporin A (CsA), and by the antioxidant, α-tocopherol. However, CsA and α-tocopherol were unable to prevent apoptosis in CI-1010-treated cells, suggesting the influence of additional mechanism(s) of CI-1010-induced toxicity. This study evaluates intracellular oxidative stress associated with pore opening prior to apoptosis and provides evidence in support of a mitochondrial mechanism of CI-1010-induced neuronal cell death.

Role of antioxidants in paraquat toxicity

Paraquat, a quarternary nitrogen herbicide, is a highly toxic compound for humans and animals and many cases of acute poisoning and death have been reported over the past few decades. The mechanisms of paraquat toxicity involve: the generation of the superoxide anion, which can lead to the formation of more toxic reactive oxygen species, such as hydrogen peroxide and hydroxyl radical; and the oxidation of the cellular NADPH, the major source of reducing equivalents for the intracellular reduction of paraquat, which results in the disruption of important NADPH-requiring biochemical processes. The major cause of death in paraquat poisoning is respiratory failure due to an oxidative insult to the alveolar epithelium with subsequent obliterating fibrosis. Management of paraquat poisoning has remained mostly supportive and has been directed towards the modification of the toxicokinetics of the poison. Currently, there are no true pharmacological antagonists for paraquat and there are no chelating agents capable of binding the poison in the blood or other tissues. Recognizing the fact that paraquat induces its toxic effects via oxidative stress-mediated mechanisms, innovations in the management of paraquat poisoning are directed towards the use of antioxidants. In this review, the status of antioxidants in ameliorating or treating the toxic effects produced by paraquat is presented.

α-Tocopherol liposomes alleviate LPS-induced hepatotoxicities

Gram-negative bacteria, in part through lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNFα), activate phagocytes to generate reactive oxygen species (ROS), which have been known to play a key role in the pathogenesis of liver injury. Accordingly, we hypothesized that the susceptibility of the liver to ROS should be reduced by augmenting its antioxidant status. Adult male Sprague-Dawley rats were pretreated with α-tocopherol liposomes (20 mg α-tocopherol/kg body weight, i.v.), plain liposomes or saline. 24 h after liposomal treatment, rats were injected intravenously with LPS (1 mg/kg, Escherichia coli: 0111:B4) and killed 2 h later. Livers of saline-pretreated animals challenged with LPS were damaged as demonstrated by increases in plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities. The hepatic injury appeared to be associated with oxidative stress-mediated mechanisms as evidenced by increases in lipid peroxidation and decreases in glutathione concentration in the liver, both indices of oxidative stress. Also, LPS injection resulted in increases in plasma TNFα and thromboxane B2 (TXB2) levels, as well as increases in hepatic myeloperoxidase (MPO) activity and chloramine concentration, suggestive of activation of the inflammatory response. Pretreatment of rats with plain liposomes, 24 h prior to LPS challenge, failed to protect against the LPS-induced liver injury. Although pretreatment of animals with α-tocopherol liposomes was not effective in preventing the LPS-induced inflammatory response, it conferred a partial protection against the LPS-induced changes in plasma AST and ALT activities as well as in hepatic levels of lipid peroxidation, glutathione and chloramine concentrations. These data appear to suggest that augmentation of the hepatic antioxidant status is effective in alleviating the LPS-induced liver injury.

Nitrofurantoin-induced pulmonary toxicity: In vivo evidence for oxidative stress-mediated mechanisms

The present study was carried out to examine whether nitrofurantoin-induced pulmonary toxicity in normal rats was mediated via oxidant stress mechanisms. The relative importance of the cellular antioxidant enzymes in nitrofurantoin toxicity was also assessed. For this, the pulmonary toxicity induced by nitrofurantoin in rats was evaluated at various time intervals after a single subcutaneous injection. Data from this study showed that nitrofurantoin (200 mg/kg, s.c.) resulted in transient but measurable lung damage as evidenced by the increases in wet lung weight/body weight ratio and decreases in lung angiotensin converting enzyme activity. A transient decrease in GSH concentrations with a concurrent increase in GSSG concentrations as well as an increase in lipid peroxidation levels (measured by the formation of diene conjugates and thiobarbituric acid reactants) were also evident in lungs of nitrofurantoin-treated rats. In addition, nitrofurantoin did not alter the pulmonary Superoxide dismutase and glutathione peroxidase activities, but it did produce transient decreases in catalase and glutathione reductase activities. These data indicate that impairment of the ability of the lung to detoxify reactive oxygen species may play an important role in the development of nitrofurantoin-induced pulmonary toxicity. The results of the present study suggest that nitrofurantoin can damage the lungs of rats, probably through oxidative stress-mediated mechanisms. Also, our data have provided in vivo evidence for substantiating lipid peroxidation as a possible cause of lung damage.