Selective iNOS inhibition reduces renal damage induced by cisplatin

Cisplatin is a chemotherapeutic agent used in the treatment of several cancer tumors; however, nephrotoxicity has restricted its use. Reactive oxygen species and peroxynitrite, which is formed by the reaction between superoxide anion and nitric oxide (NO*), are implicated in cisplatin-induced nephrotoxicity. In contrast, both toxic and beneficial effects of NO* have been suggested in cisplatin-induced nephrotoxicity. Therefore, nowadays the role of NO* in this experimental model remains controversial. The aim of the present work was to elucidate the role of NO* in cisplatin-induced renal damage using N-[3-(aminomethyl)benzyl]acetamidine (1400W), a selective and irreversible inhibitor of iNOS. The mRNA levels of iNOS were increased in cisplatin-treated rats. The administration of 1400W reduced the cisplatin induced histological damage, renal dysfunction (increase in proteinuria and kidney injury molecule expression and decrease in creatinine clearance), tubulointerstitial infiltration, oxidative stress (increase in renal malondialdehyde and inmmunostaining for 4-hydroxy-2-nonenal) and nitrosative stress (immunostaining for 3-nitrotyrosine). In addition, the administration of 1400W was unable to modify systolic blood pressure in control rats. Our data demonstrate that selective iNOS inhibition reduces the cisplatin-induced nephrotoxicity and nitrosative stress which strongly suggest that in this experimental model (1) the NO* production is toxic and (2) iNOS is the main source of NO*.     

Protective effect of persimmon peel polyphenol against high glucose-induced oxidative stress in LLC-PK(1) cells.

The effect of persimmon peel polyphenol (PPP) on high glucose-induced oxidative stress was investigated using LLC-PK(1) cells, which is susceptible to oxidative stress. High-concentration glucose (30 mM) treatment induced LLC-PK(1) cell death, but high molecular-PPP (HMPPP) and low molecular-PPP (LMPPP), at concentrations of 5 or 10 microg/ml, significantly inhibited the high glucose-induced cytotoxicity. Furthermore, treatment with HMPPP or LMPPP dose-dependently reduced the intracellular reactive oxygen species level increased by 30 mM glucose. In addition, nitric oxide, superoxide and peroxynitrite levels were increased by 30 mM glucose treatment, but they were concentration-dependently inhibited by HMPPP or LMPPP treatment. High glucose levels induced the overexpressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins, but HMPPP or LMPPP treatment reduced the overexpressions of these proteins. HMPPP or LMPPP also inhibited the nuclear translocation of nuclear factor-kappa B (NF-kappaB) induced by 30 mM glucose in LLC-PK(1) cells. In particular, LMPPP exhibited stronger inhibitory activities on high glucose induced oxidative stress than HMPPP. These findings indicate the potential benefits of persimmon peel as a valuable source of antioxidants in the diabetic condition which will reduce the oxidative stress induced by hyperglycemia.     

Cardioprotective effects of a proanthocyanidin-rich fraction from Croton celtidifolius Baill: Focus on atherosclerosis

Proanthocyanidins are the most abundant polyphenols in human diets. Epidemiological studies have pointed to proanthocyanidins as promising molecules that could prevent the development of several coronary syndromes by inhibiting the atherogenic process. The present study was designed to investigate the antiatherogenic effects of a proanthocyanidin-rich fraction (PRF) obtained from Croton celtidifolius Baill (Euphorbiaceae) barks. In isolated human LDL, PRF caused a concentration-dependent inhibition of Cu²⁺-induced oxidative modifications, evidenced by the increasing of the lag phase of lipid peroxidation and decreasing in the oxidation rate (V_max), moreover, the protein moieties from LDL were protected against Cu²⁺-induced oxidation. In human umbilical vein endothelial cells (HUVECs), PRF reduced the ROS production stimulated by oxidized LDL. Herein, we demonstrate that oral treatment with PRF improved endothelium-dependent vasorelaxation in hypercholesterolemic LDL receptor knockout mice (LDLr^−/−), however, the fraction did not modify plasma lipids and atherosclerotic lesion size in this experimental model. Finally, our results showed for the first time that PRF prevent isolated LDL oxidation, decrease oxidative stress in endothelial cells and improve endothelial function in mice.     

Prooxidant action of knipholone anthrone: Copper dependent reactive oxygen species generation and DNA damage

Knipholone (KP) and knipholone anthrone (KA) are natural 4-phenylanthraquinone structural analogues with established differential biological activities including in vitro antioxidant and cytotoxic properties. By using DNA damage as an experimental model, the comparative Cu(II)-dependent prooxidant action of these two compounds were studied. In the presence of Cu(II) ions, the antioxidant KA (3.1–200 μM) but not KP (6–384 μM) caused a concentration-dependent pBR322 plasmid DNA strand scission. The DNA damage induced by KA could be abolished by reactive oxygen species scavengers, glutathione and catalase as well as EDTA and a specific Cu(I) chelator bathocuproine disulfonic acid. In addition to Cu(II) chelating activity, KA readily reduces Cu(II) to Cu(I). Copper-dependent generation of reactive oxygen species and the subsequent macromolecular damage may be involved in the antimicrobial and cytotoxic activity of KA.     

Antioxidant activities of the flaxseed lignan secoisolariciresinol diglucoside, its aglycone secoisolariciresinol and the mammalian lignans enterodiol and enterolactone in vitro.

The flaxseed lignan secoisolariciresinol diglucoside (SDG) and mammalian lignans enterodiol (ED) and enterolactone (EL) were previously shown to be effective antioxidants against DNA damage and lipid peroxidation. Others reported inhibition of activated cell chemiluminescence by supra-physiological concentrations of secoisolariciresinol (SECO), ED and EL. Thus, we evaluated the antioxidant efficacy of potential physiological concentrations of SDG, SECO, ED and EL against 1,1-diphenyl-2-picrylhydrazyl (DPPH()), and 2,2'-azo-bis(2-amidinopropane) dihydrochloride (AAPH)-initiated peroxyl radical plasmid DNA damage and phosphatidylcholine liposome lipid peroxidation. SDG and SECO were effective (p<0.01) antioxidants against DPPH() at 25-200muM; whereas, ED and EL were inactive. Efficacy of lignans and controls against AAPH peroxyl radical-induced DNA damage was: SDG>SECO=17alpha-estradiol>ED=EL>genistein>daidzein. Lignan efficacy against AAPH-induced liposome lipid peroxidation was: SDG>SECO=ED=EL. Plant lignan antioxidant activity was attributed to the 3-methoxy-4-hydroxyl substituents of SDG and SECO, versus the meta mono-phenol structures of ED and EL. Benzylic hydrogen abstraction and potential resonance stabilization of phenoxyl radicals in an aqueous environment likely contributed to the antioxidant activity of the mammalian lignans. These represent likely extra- and intracellular antioxidant activities of flax-derived lignans at concentrations potentially achievable in vivo.     

The role of reactive oxygen species in arsenite and monomethylarsonous acid-induced signal transduction in human bladder cells: acute studies

Arsenicals are known to induce ROS, which can lead to DNA damage, oxidative stress, and carcinogenesis. A human urothelial cell line, UROtsa, was used to study the effects of arsenicals on the human bladder. Arsenite [As(III)] and monomethylarsonous acid [MMA(III)] induce oxidative stress in UROtsa cells after exposure to concentrations as low as 1 microM and 50 nM, respectively. Previous research has implicated ROS as signaling molecules in the MAPK signaling pathway. As(III) and MMA(III) have been shown to increase phosphorylation of key proteins in the MAPK signaling cascade downstream of ErbB2. Both Src phosphorylation (p-Src) and cyclooxygenase-2 (COX-2) are induced after exposure to 50 nM MMA(III) and 1 microM As(III). These data suggest that ROS production is a plausible mechanism for the signaling alterations seen in UROtsa cells after acute arsenical exposure. To determine importance of ROS in the MAPK cascade and its downstream induction of p-Src and COX-2, specific ROS antioxidants (both enzymatic and non-enzymatic) were used concomitantly with arsenicals. COX-2 protein and mRNA was shown to be much more influenced by altering the levels of ROS in cells, particularly after MMA(III) treatment. The antioxidant enzyme superoxide dismutase (SOD) effectively blocked both As(III)-and MMA(III)- associated COX-2 induction. The generation of ROS and subsequent altered signaling did lead to changes in protein levels of SOD, which were detected after treatment with either 1 microM As(III) or 50 nM MMA(III). These data suggest that the generation of ROS by arsenicals may be a mechanism leading to the altered cellular signaling seen after low-level arsenical exposure.     

NADPH oxidase-mitochondria axis-derived ROS mediate arsenite-induced HIF-1α stabilization by inhibiting prolyl hydroxylases activity

Arsenic exposure has been shown to induce hypoxia inducible factor 1α (HIF-1α) accumulation, however the underlying mechanism remains unknown. In the present study, we tested the hypothesis that arsenic exposure triggered the interaction between NADPH oxidase and mitochondria to promote reactive oxygen species (ROS) production, which inactivate prolyl hydroxylases (PHDs) activity, leading to the stabilization of HIF-1α protein. Exposure of human immortalized liver cell line HL-7702 cells to arsenite induced HIF-1α accumulation in a dose-dependent manner, which was abolished by SOD mimetic MnTMPyP. Inhibition of NADPH oxidase with diphenyleneiodonium chloride (DPI) or inhibition of mitochondrial respiratory chain with rotenone significantly blocked arsenite-induced ROS production, and the mitochondria appeared to be the major source of ROS production. Arsenite treatment inhibited HIF-1α hydroxylation by prolyl hydroxylases (PHDs) and increased HIF-1α stabilization, but did not affect HIF-1α mRNA expression and Akt activation. Supplementation of ascorbate or Fe(II) completely abolished arsenite-induced PHDs inhibition and HIF-1α stabilization. In conclusion, these results define a unique mechanism of HIF-1α accumulation following arsenic exposure, that is, arsenic activates NADPH oxidase–mitochondria axis to produce ROS, which deplete intracellular ascorbate and Fe(II) to inactivate PHDs, leading to HIF-1α stabilization.     

Protective effect of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on copper-induced hydroxyl radical generation in the rat heart

The present study was examined the effect of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on Cu(II)-induced hydroxyl radical generation (OH) in the extracellular fluid of rat myocardium. Rats were anesthetized and sodium salicylate in Ringer's solution (0.5 nmol/microl/min) was infused through a microdialysis probe to detect the generation of OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the myocardium. When Cu(II) was infused through the microdialysis probe, Cu(II) increased in OH formation trapped as 2,3-DHBA in the dialysate. When fluvastatin (100 microM) was administered to Cu(II) (50 microM)-pretreated animals, the levels of 2,3-DHBA at 300 min after administration of fluvastatin significantly decreased. In cumulative dose dependent experiments, three concentrations of Cu(II), 10, 25 and 50 microM, were infused through the microdialysis probe in the rat myocardium. A positive linear correlation between Cu(II) and the formation of 2,3-DHBA (R(2)=0.980) was observed. However, when corresponding experiments were performed with fluvastatin (100 microM) pretreated animals, the level of 2,3-DHBA decreased. These results suggest that blocking LDL oxidation by fluvastatin may attenuate Cu(II)-induced OH formation in the rat heart.     

Investigating the potential neuroprotective effects of statins on DNA damage in mouse striatum

We investigated the protective effect of pravastatin, simvastatin and atorvastatin on striatal DNA damage as a potential method to conserve and protect the nigrostriatal neurons. C57Bl/J6 mice were treated with combinations of a statin and the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). DNA damage, DNA sensitivity to H(2)O(2)in vitro, and DNA repair capacity was measured using the single cell gel electrophoresis (comet) assay. MPTP treatment increased DNA damage in the striatum. Contrary to expectation, the statins studied here did not protect DNA against H(2)O(2) induced damage but did in fact cause DNA damage. Treatment with simvastatin showed a significant increase in levels of DNA damage (p0.0018) and DNA damage induced in vitro with H(2)O(2) was significantly increased by pravastatin (p0.0001). DNA repair and repair capacity was slightly increased by simvastatin, significantly increased by pravastatin (p=0.0093), but slightly decreased by atorvastatin. In the MPTP treated groups, pre-treated with statins, pravastatin (p0.0036) and simvastatin (p0.021), increased the level of DNA damage, while atorvastatin did not exhibit a significant effect. All three statins, administered prior to MPTP, offered slight protection against H(2)O(2) induced DNA damage and simvastatin and atorvastatin decreased the DNA repair capacity of the cells insignificantly. Treatment with statins, under the experimental conditions used, increased baseline levels of DNA damage, but DNA repair processes were left intact because the amount of repair also increased. Oxidative damage, however, largely exceeded the extent of DNA repair of mice treated with both the statins and MPTP.     

A novel marine compound xyloketal B protects against oxidized LDL-induced cell injury in vitro

Xyloketal B is a novel marine compound with unique chemical structure isolated from mangrove fungus Xylaria sp. (no. 2508). Pretreatment with xyloketal B (0.63-40 μM) significantly improved oxLDL (150 μg/ml)-induced injury in human umbilical vein endothelial cells (HUVECs) without either toxic or proliferative effects. Xyloketal B concentration-dependently attenuated oxLDL-induced ROS generation, peroxynitrite formation and decrease of Bcl-2 expression. In addition, xyloketal B significantly inhibited NADPH oxidase activity, as well as mRNA expression of gp91phox and p47phox. Furthermore, xyloketal B alone augmented the production of nitric oxide (NO). Collectively, these data indicate that xyloketal B protects against oxLDL-induced endothelial oxidative injury probably through inhibiting NADPH oxidase-derived ROS generation, promoting NO production and restoring Bcl-2 expression, making it a promising compound for further evaluation in the treatment of atherosclerosis.     

Protective effects of dieckol isolated from Ecklonia cava against high glucose-induced oxidative stress in human umbilical vein endothelial cells

The effect of dieckol, one of phlorotannin polyphenol compound purified from Ecklonia cava (E. cava) against high glucose-induced oxidative stress was investigated using human umbilical vein endothelial cells (HUVECs), which is susceptible to oxidative stress. High glucose (30 mM) treatment induced HUVECs cell death, but dieckol, at concentration 10 or 50 μg/ml, significantly inhibited the high glucose-induced cytotoxicity. Furthermore, treatment with dieckol dose-dependently decreased thiobarbituric acid reactive substances (TBARS), intracellular reactive oxygen species (ROS) generation and nitric oxide level increased by high glucose. In addition, high glucose levels induced the overexpressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and nuclear factor-kappa B (NF-kB) proteins in HUVECs, but dieckol treatment reduced the overexpressions of these proteins. These findings indicate that dieckol is a potential therapeutic agent that will reduce the damage caused by hyperglycemia-induced oxidative stress associated with diabetes.     

Mitochondria,reactive oxygen species and cadmium toxicity in the kidney

The heavy metal cadmium accumulates in kidney cells, particularly those of the proximal tubular epithelium, and the damage this causes is associated with development of chronic kidney disease. One of the causative mechanisms of chronic kidney disease is thought to be oxidative stress. Cadmium induces oxidative stress, but the molecular mechanisms involved in the cell damage from oxidative stress in cadmium-induced chronic kidney disease are not well understood. Mitochondrial damage is likely, given that dysfunctional mitochondria are central to the formation of excess reactive oxygen species (ROS), and are known key intracellular targets for cadmium. Normally, ROS are balanced by natural anti-oxidant enzymes. When mitochondria become dysfunctional, for example, through long term exposure to environmental toxicants like cadmium, they produce less cell energy and more ROS. The imbalance between these ROS and the natural anti-oxidants creates the condition of oxidative stress. The outcomes of mitochondrial injury are manyfold: injured mitochondria perpetuate oxidative stress; the loss of mitochondrial membrane potential causes release of cytochrome-c and activation of caspase pathways that lead to apoptotic deletion of renal cells; and attempts by cells to remove dysfunctional mitochondria through autophagy lead to “autophagic cell death” or apoptosis. Three pathways of mitochondrial regulation (upstream signalling pathways, direct mitochondrial targeting, and downstream cell death effector pathways) are therefore all promising targets for effective anti-oxidant treatment of cadmium toxicity in the kidney.     

Antioxidant and antigenotoxic activities in Acacia salicina extracts and its protective role against DNA strand scission induced by hydroxyl radical

The antioxidant potency of Acacia salicina extracts was investigated. Total antioxidant capacity was determined using an ABTS⁺ assay. Superoxide radical scavenging was measured using riboflavin-light-nitro blue tetrazolium (NBT) assay. In addition, the content of phenols, total flavonoids and sterols were measured in the tested extracts. The petroleum ether exhibited a potent scavenging activity toward ABTS radical cations. Whereas, chloroform extract showed the highest activity against superoxides radicals and was also able to protect pKS plasmid DNA against hydroxyl radicals induced DNA damages. The antimutagenicity of these extracts was assayed using the Ames assay against Salmonella typhimurium TA98 and S. typhimurium TA 1535 tester strains at different concentrations. These extracts decreased significantly the mutagenecity induced by sodium azide (SA) and 4-nitro-o-phenylenediamine (NOP). The antioxidant and antimutagenecity activities exhibited by A. salicina depended on the chemical composition of the tested extracts.     

Interactions of peroxynitrite with uric acid in the presence of ascorbate and thiols: implications for uncoupling endothelial nitric oxide synthase

It has been suggested that uric acid acts as a peroxynitrite scavenger although it may also stimulate lipid peroxidation. To gain insight into how uric acid may act as an antioxidant, we used electron spin resonance to study the reaction of uric acid and plasma antioxidants with ONOO-. Peroxynitrite reacted with typical plasma concentrations of urate 16-fold faster than with ascorbate and 3-fold faster than cysteine. Xanthine but not other purine-analogs also reacted with peroxynitrite. The reaction between ONOO- and urate produced a carbon-centered free radical, which was inhibited by either ascorbate or cysteine. Moreover, scavenging of ONOO- by urate was significantly increased in the presence of ascorbate and cysteine. An important effect of ONOO- is oxidation of tetrahydrobiopterin, leading to uncoupling of nitric oxide synthase. The protection of eNOS function by urate, ascorbate and thiols in ONOO(-)-treated bovine aortic endothelial cells (BAECs) was, therefore, investigated by measuring superoxide and NO using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine (CMH) and the NO-spin trap Fe[DETC]2. Peroxynitrite increased superoxide and decreased NO production by eNOS indicating eNOS uncoupling. Urate partially prevented this effect of ONOO- while treatment of BAECs with the combination of either urate with ascorbate or urate with cysteine completely prevented eNOS uncoupling caused by ONOO-. We conclude that the reducing and acidic properties of urate are important in effective scavenging of peroxynitrite and that cysteine and ascorbate markedly augment urate's antioxidant effect by reducing urate-derived radicals.     

New advances in molecular mechanisms and the prevention of adriamycin toxicity by antioxidant nutrients

Anthracyclines (doxorubicin, daunorubicin, epirubicin, and idarubicin) are currently the most effective group of anti-neoplastic drugs used in clinical practice. Of these, doxorubicin (also called adriamycin) is a key chemotherapeutic agent in cancer treatment, although its use is limited as a consequence of the chronic and acute toxicity associated with this drug. The molecular mechanisms of doxorubicin account for both the anti-cancer and the toxic side effects. Many antioxidants have been assayed, with positive or negative results, to prevent the toxicity of doxorubicin. The present review has two main goals: (1) to report the latest findings regarding the molecular mechanisms of doxorubicin toxicity; (2) to update our understanding of the role of natural antioxidants in preventive therapy against doxorubicin-induced toxicity. This review provides new evidence for the chemoprevention of doxorubicin toxicity, making use of natural antioxidants – in particular vitamin E, vitamin C, coenzyme Q, carotenoids, vitamin A, flavonoids, polyphenol, resveratrol, antioxidant from virgin olive oil and selenium – and offers new insights into the molecular mechanisms of doxorubicin toxicity with respect to DNA damage, free radicals and other parameters.     

Antioxidant, prooxidant and cytotoxic activity of hydroxylated resveratrol analogues: structure-activity relationship

Resveratrol (trans-3,4',5-trihydroxystilbene), a naturally occurring hydroxystilbene, is considered an essential antioxidative constituent of red wine possessing chemopreventive properties. However, resveratrol and even more its metabolite piceatannol were reported to have also cytostatic activities. In order to find out whether this is related to antioxidative properties of those compounds, we synthesized five other polyhydroxylated resveratrol analogues and studied structure-activity relationships between pro-/antioxidant properties and cytotoxicity. Radical scavenging experiments with O(2)(*-) (5,5-dimethyl-1-pyrroline-N-oxide/electron spin resonance (DMPO/ESR)) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (photometry) revealed that 3,3',4',5-tetrahydroxystilbene (IC(50): 2.69microM; k(9): 443000M(-1)s(-1)), 3,4,4',5-tetrahydroxystilbene (IC(50): 41.5microM; k(9): 882000M(-1)s(-1)) and 3,3',4,4',5,5'-hexahydroxystilbene (IC(50): 5.02microM), exerted a more than 6600-fold higher antiradical activity than resveratrol and its two other analogues. Furthermore, in HL-60 leukemic cells hydroxystilbenes with ortho-hydroxyl groups exhibited a more than three-fold higher cytostatic activity compared to hydroxystilbenes with other substitution patterns. Oxidation of ortho-hydroxystilbenes in a microsomal model system resulted in the existence of ortho-semiquinones, which were observed by ESR spectroscopy. Further experiments revealed that these intermediates undergo redox-cycling thereby consuming additional oxygen and forming cytotoxic oxygen radicals. In contrast to compounds with other substitution patterns hydroxystilbenes with one or two resorcinol groups (compounds 1 and 3) did not show an additional oxygen consumption or semiquinone formation. These findings suggest that the increased cytotoxicity of ortho-hydroxystilbenes is related to the presence of ortho-semiquinones formed during metabolism or autoxidation.     

BCNU-induced gR2 DEFECT mediates S-glutathionylation of Complex I and respiratory uncoupling in myocardium

A deficiency of mitochondrial glutathione reductase (or GR2) is capable of adversely affecting the reduction of GSSG and increasing mitochondrial oxidative stress. BCNU [1,3-bis (2-chloroethyl)-1-nitrosourea] is an anticancer agent and known inhibitor of cytosolic GR ex vivo and in vivo. Here we tested the hypothesis that a BCNU-induced GR2 defect contributes to mitochondrial dysfunction and subsequent impairment of heart function. Intraperitoneal administration of BCNU (40 mg/kg) specifically inhibited GR2 activity by 79.8 ± 2.7% in the mitochondria of rat heart. However, BCNU treatment modestly enhanced the activities of mitochondrial Complex I and other ETC components. The cardiac function of BCNU-treated rats was analyzed by echocardiography, revealing a systolic dysfunction associated with decreased ejection fraction, decreased cardiac output, and an increase in left ventricular internal dimension and left ventricular volume in systole. The respiratory control index of isolated mitochondria from the myocardium was moderately decreased after BCNU treatment, whereas NADH-linked uncoupling of oxygen consumption was significantly enhanced. Extracellular flux analysis to measure the fatty acid oxidation of myocytes indicated a 20% enhancement after BCNU treatment. When the mitochondria were immunoblotted with antibodies against GSH and UCP3, both protein S-glutathionylation of Complex I and expression of UCP3 were significantly up-regulated. Overexpression of SOD2 in the myocardium significantly reversed BCNU-induced GR2 inhibition and mitochondrial impairment. In conclusion, BCNU-mediated cardiotoxicity is characterized by the GR2 deficiency that negatively regulates heart function by impairing mitochondrial integrity, increasing oxidative stress with Complex I S-glutathionylation, and enhancing uncoupling of mitochondrial respiration.