Production of superoxides and nitric oxide generation in haemocytes of mussel Mytilus galloprovincialis (Lmk.) after exposure to cadmium: A possible involvement of Na/H exchanger in the induction of cadmium toxic effects

The present study investigates cadmium (Cd) ability to enhance superoxides (O₂⁻) and nitric oxide (NO) production (as nitrites) in haemocytes of mussel Mytilus galloprovincialis as well as the possible involvement of Na⁺/H⁺ exchanger (NHE) in the induction of NADPH oxidase and NO synthase activity. PMA, a well-known PKC-mediated NADPH oxidase as well as NO synthase stimulator was also used, in order to verify Cd effects on both O₂⁻ and NO generation. According to the results of the present study, micromolar concentrations of Cd (0.05, 5, 10 and 50 μM) seemed to enhance O₂⁻ and NO generation in haemocytes of mussels. Moreover, O₂⁻ and NO generation in haemocytes exposed to Cd could be enhanced by its ability to induce reactive oxygen species (ROS) but respiratory burst activation as well. Inhibition of NO synthase with 10 μM l-NAME, significantly attenuated Cd ability to enhance O₂⁻ production and diminished NO generation, thus leading to the suggestion that Cd toxic effects, started at concentration of 50 μM, could enhance NADPH oxidase and NO synthase stimulation in haemocytes of mussels. NHE seems to play a regulatory role in the induction of either O₂⁻ or NO generation in haemocytes exposed to the metal, since its inhibition with the use of 10 μM EIPA significantly decrease both O₂⁻ and NO production. The involvement of NHE in the induction of O₂⁻ and NO generation, probably via PKC-mediated NADPH oxidase and NO synthase activation, is likely to be crucial to haemocytes exposed to heavy metals, such as Cd.     

Function of nitric oxide and superoxide anion in the adventitious root development and antioxidant defence in <Emphasis Type="Italic">Panax ginseng</Emphasis>

The involvement of NO in O₂ ^·− generation, rootlet development and antioxidant defence were investigated in the adventitious root cultures of mountain ginseng. Treatments of NO producers (SNP, sodium nitroprusside; SNAP, S-nitroso-N-acetylpenicillamine; and sodium nitrite with ascorbic acid), and NO scavenger (PTIO, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxide) revealed that NO is involved in the induction of new rootlets. Severe decline in number of new rootlets compared to the control under PTIO treatment indicates that NO acts downstream of auxin action in the process. NO producers (SNP, SNAP and sodium nitrite with ascorbic acid) activated NADPH oxidase activity, resulting in greater O₂ ^·− generation and higher number of new rootlets in the adventitious root explants. Moreover, treatment of diphenyliodonium chloride, a NADPH oxidase inhibitor, individually or along with SNP, inhibited root growth, NADPH oxidase activity and O₂ ^·− anion generation. NO supply also enhanced the activities of antioxidant enzymes that are likely to be responsible for reducing H₂O₂ levels and lipid peroxidation as well as modulation of ascorbate and non-protein thiol concentrations in the adventitious roots. Our results suggest that NO-induced generation of O₂ ^·− by activating NADPH oxidase activity is related to adventitious root formation in mountain ginseng.     

Intraphagosomal peroxynitrite as a macrophage-derived cytotoxin against internalized Trypanosoma cruzi: consequences for oxidative killing and role of microbial peroxiredoxins in infectivity

Macrophage-derived radicals generated by the NADPH oxidase complex and inducible nitric-oxide synthase (iNOS) participate in cytotoxic mechanisms against microorganisms. Nitric oxide (^•NO) plays a central role in the control of acute infection by Trypanosoma cruzi, the causative agent of Chagas disease, and we have proposed that much of its action relies on macrophage-derived peroxynitrite (ONOO⁻ + ONOOH) formation, a strong oxidant arising from the reaction of ^•NO with superoxide radical (O₂^˙̄). Herein, we have shown that internalization of T. cruzi trypomastigotes by macrophages triggers the assembly of the NADPH oxidase complex to yield O₂^˙̄ during a 60–90-min period. This does not interfere with IFN-γ-dependent iNOS induction and a sustained ^•NO production (∼24 h). The major mechanism for infection control via reactive species formation occurred when ^•NO and O₂^˙̄ were produced simultaneously, generating intraphagosomal peroxynitrite levels compatible with microbial killing. Moreover, biochemical and ultrastructural analysis confirmed cellular oxidative damage and morphological disruption in internalized parasites. Overexpression of cytosolic tryparedoxin peroxidase in T. cruzi neutralized macrophage-derived peroxynitrite-dependent cytotoxicity to parasites and favored the infection in an animal model. Collectively, the data provide, for the first time, direct support for the action of peroxynitrite as an intraphagosomal cytotoxin against pathogens and the premise that microbial peroxiredoxins facilitate infectivity via decomposition of macrophage-derived peroxynitrite.     

Involvement of nitric oxide-induced NADPH oxidase in adventitious root growth and antioxidant defense in Panax ginseng

Nitric oxide (NO) affects the growth and development of plants and also affects plant responses to various stresses. Because NO induces root differentiation, we examined whether or not it is involved in increased ROS generation. Treatments with sodium nitroprusside (SNP), an NO donor, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), a specific NO scavenger, and Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME), an NO synthase (NOS) inhibitor, revealed that NO is involved in the adventitious root growth of mountain ginseng. Supply of an NO donor, SNP, activates NADPH oxidase activity, resulting in increased generation of O₂ ^·−, which subsequently induces growth of adventitious roots. Moreover, treatment with diphenyliodonium chloride (DPI), an NADPH oxidase inhibitor, individually or with SNP, inhibited root growth, NADPH oxidase activity, and O₂ ^·− anion generation. Supply of the NO donor, SNP, did not induce any notable isoforms of enzymes; it did, however, increase the activity of pre-existing bands of NADPH oxidase, superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, and glutathione reductase. Enhanced activity of antioxidant enzymes induced by SNP supply seems to be responsible for a low level of H₂O₂ in the adventitious roots of mountain ginseng. It was therefore concluded that NO-induced generation of O₂ ^·− by NADPH oxidase seems to have a role in adventitious root growth of mountain ginseng. The possible mechanism of NO involvement in O₂ ^·− generation through NADPH oxidase and subsequent root growth is discussed.     

Investigation of Cadmium-Induced Genotoxicity and Oxidative Stress Response in Indian Major Carp,Labeo rohita

We investigated genotoxicity and oxidative stress in the gills of Labeo rohita exposed to 33.6, 67.1, and 100.6 mg L^–1of cadmium chloride at 96 h. Genotoxicity was assessed using single cell gel electrophoresis whereas oxidative stress was monitored through lipid peroxidation induction and antioxidant response parameters, namely reduced glutathione (GSH), glutathione peroxidase, glutathione-S-transferase, superoxide dismutase, and catalase (CAT) activities. Significant (p < .05) effect of both concentration and time of exposure was observed on the extent of DNA damage in treated fish. Similarly, malondialdehyde content, level of GSH, and activities of antioxidant enzymes were significantly elevated in treated groups, except CAT. The increased DNA damage and lipid peroxidation (LPO) content along with fluctuation in antioxidant defense system in fish indicated the interaction of cadmium (Cd) with DNA repair processes and production of reactive oxygen species. Thus, Cd is liable for induction of LPO, alteration of antioxidant defenses, and DNA damage in gills of L. rohita.     

镉胁迫下紫花苜蓿幼苗内源一氧化氮和活性氧的生成

以"甘农三号"紫花苜蓿幼苗为材料,在水培条件下,研究了不同浓度镉(Cd)胁迫下紫花苜蓿根、茎和叶内源一氧化氮(NO)和活性氧(ROS)的生成机制以及根系活力的变化.结果表明:在0~2.0 mmol·L-1范围内,随着Cd浓度的增加,幼苗内NO含量呈现先升高后降低的趋势,最后可维持在略高或持平于对照的水平.幼苗内一氧化氮合成酶(NOS)活性、硝酸还原酶(NR)活性、亚硝酸根离子(NO2-)含量和类胡萝卜素(Car)含量的变化与NO含量变化规律相似却又不全相同.NOS和NR是影响幼苗茎中NO含量的主要因素,NOS、NO2-和NR则是影响叶中NO含量的主要因素,而根中NO含量主要与NOS活性和NO2-含量有较大相关性.随着Cd浓度的增加,幼苗内过氧化氢(H2 O2)含量、丙二醛(MDA)含量、超氧阴离子(O-2·)含量和相对电导率(REC)呈现显著升高趋势,说明高浓度的Cd处理会使ROS大量积累,细胞膜遭破坏,细胞质外流,进而引发膜脂过氧化.随着Cd浓度的增加,紫花苜蓿根系活力的变化为先升高后降低,指示了低浓度Cd处理会促进植物代谢,增强其生命力;而高浓度Cd会致使植株代谢受抑制,细胞受损害.NO和ROS的相关性不大,说明二者虽同为自由基,但它们产生和变化方式大有差别.     

Inhibition of cell membrane lipid peroxidation by cadmium- and zinc-metallothioneins

The effects of all-zinc metallothionein (Zn-metallothionein) and predominantly cadmium metallothionein (Cd/Zn-metallothionein) on free radical lipid peroxidation have been investigated, using erythrocyte ghosts as the test system. When treated with xanthine and xanthine oxidase, Zn-metallothionein and Cd/Zn-metallothionein underwent thiolate group oxidation and metal ion release that was catalase-inhibitable, but superoxide dismutase-non-inhibitable. Similar treatment in the presence of ghosts and added Fe(III) resulted in metallothioneen oxidation that was significantly inhibited by superoxide dismutase. Ghosts incubated with xanthine/xanthine oxidase/Fe(III) underwent H₂O₂- and O₂⁻-dependent lipid peroxidation, as measured by thiobarbituric acid reactivity. Neither type of metallothionein had any effect on xanthine oxidase activity, but both strongly inhibited lipid peroxidation when added to the membranes concurrently with xanthine/xanthine oxidase/iron. This inhibition was far greater and more sustained than that caused by dithiothreitol at a concentration equivalent to that of metallothionein thiolate. Significant protection was also afforded when ghosts plus Cd/Zn-metallothionein or Zn/metallothionein were preincubated with H₂O₂ and Fe(III), and then subjected to vigorous peroxidation by the addition of xanthine and xanthine oxidase. These results could be mimicked by using Cd(II) or Zn(II) alone. Previous studies suggested that Zn(II) inhibits xanthine/xanthine oxidase/iron-driven lipid peroxidation in ghosts by interfering with iron binding and redox cycling. Therefore, the primary determinant of metallothionein proteciion appears to be metal release and subsequent uptake by the membranes. These results have important implications concerning the antioxidant role of metallothionein, a protein known to be induced by various prooxidant conditions.     

Evidence for phosphatidylinositol-3-OH-kinase (PI3-kinase) involvement in Cd-mediated oxidative effects on hemocytes of mussels

This study investigated phosphatidylinositol-3-OH-kinase (PI3-kinase) involvement in the induction of cadmium-mediated oxidative effects on hemocytes of mussel Mytilus galloprovincialis. PI3-kinase was investigated with the use of wortmannin, a specific covalent inhibitor of PI3-kinase. Moreover, phorbol-myristate acetate (PMA), a well-known protein kinase C (PKC)-mediated NADPH oxidase and nitric oxide (NO) synthase stimulator, was also used for elucidating PI3-kinase involvement during the respiratory burst process in challenge hemocytes. According to the results, cells pre-treated with non-toxic concentrations of wortmannin (1 and/or 50 nM, as revealed by neutral red retention assay) for 15 min, showed a significant attenuation of cadmium ability (at concentration of 50 μM) to promote cell death, superoxide anion (O(2)(-)) production, NO generation and lipid peroxidation (in terms of malondialdehyde equivalents). On the other hand, wortmannin-treated cells showed a significant attenuation of PMA ability to induce NO generation but not O(2)(-) production. These findings reveal that PI3-kinase could lead to a PKC-independent induction of NO synthase activity in cells faced with pro-oxidants, such as cadmium, while its activation could be fundamental for the regulation of NAPDH oxidase activity, probably through a PKC-dependent signaling pathway.     

ROS generation and proline metabolism in calli of halophyte Nitraria tangutorum Bobr. to sodium nitroprusside treatment

Nitric oxide (NO) is a stress factor or a signal molecule involved in various plant physiological and developmental processes. In the present study, the generation of reactive oxygen species and the metabolism of proline due to different sodium nitroprusside (SNP, an NO donor) concentrations were investigated in callus from halophyte Nitraria tangutorum Bobr. Treatment with SNP led to significant increases of hydrogen peroxide (H₂O₂) content and cell viability but notable reductions in hydrogen radical level and lipid peroxidation degree, and superoxide onion (O₂ ^?) content also enhanced in 100 μM SNP-treated calli. Using a chemical inhibitor for plasma membrane (PM) NADPH oxidase diphenylene iodonium (DPI), we found low O₂ ^? generation in untreated and 25 μM SNP-treated calli, whereas in those treated with 100 μM SNP O₂ ^? level exhibited a very little alteration, comparable to the absence of DPI. These suggest a high activity of PM NADPH oxidase in untreated calli. H₂O₂ scavenging enzymes (catalase, peroxidase [POD] and ascorbate peroxidase) and H₂O₂ forming enzymes (superoxide dismutase [SOD], cell wall-POD and diamine oxidase [DAO]) stimulated significantly in calli treated with different SNP concentrations while glutathione reductase activity decreased. In addition, a reduction in proline content was observed in SNP-treated calli. Moreover, different SNP concentrations stimulated proline dehydrogenase (PDH) and ornithine δ-aminotransferase but inhibited r-glutamyl kinase (GK). In conclusion, our results suggest that the increasing H₂O₂ generation was associated with the stimulation of SOD, cell wall-POD and DAO, and that the reduction of proline content might be the consequence of increased PDH activity and decreased GK activity in N. tangutorum Bobr. calli under SNP treatment.     

24-Epibrassinolide alleviated zinc-induced oxidative stress in radish (Raphanus sativus L.) seedlings by enhancing antioxidative system

This article encompasses the results on the effects of 24-epibrassinolide (EBR) on the changes in reactive oxygen species (ROS) and activities of antioxidative enzymes in radish (Raphanus sativus L.) seedlings subjected to zinc (Zn) stress. Zn toxicity resulted in significant enhancement in the level of membrane lipid peroxidation, protein oxidation, contents of hydrogen peroxide (H₂O₂) and hydroxyl radical (^·OH), the production rate of superoxide radicals (O ₂ ^·? ) and the activities of lipoxygenase and NADPH oxidase in radish seedlings indicating the induction of oxidative stress. However, Zn-mediated enhancement in indices of oxidative stress was considerably decreased by EBR treatment. EBR application enhanced the activities of catalase, superoxide dismutase, guaiacol peroxidase, glutathione peroxidase, and peroxidase in radish seedlings under Zn stress. EBR treatment reduced the activity of ascorbic acid oxidase in Zn stressed seedlings. Further, EBR application also enhanced the free proline and phenol levels under Zn stress. From the results obtained in this study, it can be inferred that EBR application alleviated oxidative damage caused by over production of ROS through the up regulation of antioxidative capacity in Zn stressed radish seedlings.     

Evaluation of the antioxidative and genotoxic effects of sodium butyrate on breast cancer cells

Oncogenic stimulation shows a rise in reactive oxygen species (ROS), and ROS can eventually induce carcinogenesis by causing DNA damage. In this context, this study aims to evaluate some biochemical and genotoxic changes in the control of cell death caused by NaBu (Sodium butyrate). treatment in breast cancer cells. NaBu’s impact on cell proliferation was determined via WST-1 assay. The lipid peroxidation (MDA), reduced glutathione (GSH), Nitric Oxide (NO), hydrogen peroxide (H₂O₂), and superoxide dismutase (SOD) enzyme levels were determined biochemically. NaBu-induced genotoxic damage was estimated via single-cell gel electrophoresis (SCGE). NaBu reduced cell viability and potentially induced GSH, but decreased SOD enzyme activity and the level of MDA and NO decreased also H₂O₂ decreased at different times and NaBu concentrations. Higher NaBu concentrations amplified DNA damage in MCF-7 cells compared to the control group. NaBu shows anticancer and genotoxic effects, especially through antioxidant enzymes, one of the oxidative stress parameters in breast cancer. However, the anticancer and genotoxic effects of NaBu is changed in the oxidative stress parameters with time and treatment concentration of NaBu in MCF-7 cells. Furthermore, his oxidative stress-dependent effect changes need to be clarified by further evaluation with molecular and more biochemical parameters.     

Cadmium-induced early changes in O2 •−, H2O2 and antioxidative enzymes in soybean (Glycine max L.) leaves

Cadmium-induced initial changes in the production of reactive oxygen species (ROS) and antioxidant mechanism were investigated in soybean (Glycine max L. cv. Don Mario 4800 RR) leaves. Whole plants (WP) and plants without roots (PWR) were exposed to 0.0, 10.0 and 40.0 μM Cd for 0, 4, 6 and 24 h. Compared to PWR, a higher level of endogenous Cd in WP was associated with a lower oxidative stress measured in terms of lipid peroxidation. Furthermore, O₂ ^•− content decreased in the leaves of Cd-treated WP, whereas it increased in those of Cd-treated PWR. Although O₂ ^•− accumulation in PWR was associated with a decrease in superoxide dismutase (SOD) activity, O₂ ^•− diminution in WP leaves was not related to any increase in SOD activity. H₂O₂ content increased in the leaves of both Cd-treated WP and PWR, and it was concomitant with a corresponding decline in catalase (CAT) and ascorbate peroxidase (APX) activities. When diphenyl iodonium (DPI), an inhibitor of NADPH oxidase, was added, H₂O₂ content remained unchanged in Cd-treated WP, suggesting that NADPH oxidase does not participate in the early hours of Cd toxicity. Taken together, our results showed that early ROS evolution and oxidative damage were different in WP and PWR. This suggests that the response in soybean leaves during the early hours of Cd toxicity is probably modulated by the root.     

A fish oil-rich diet reduces vascular oxidative stress in apoE–/– mice

Abstract

Oxidative stress contributes to lipid peroxidation and decreases nitric oxide (NO) bioavailability in atherosclerosis. While long-chain (n-3) polyunsaturated fatty acids (PUFA) are easily oxidized in vitro, they improve endothelial function. Hence, this study postulates that long-chain (n-3) PUFA decrease atherogenic oxidative stress in vivo. To test this, apoE^–/– mice were fed a corn oil- or a fish oil (FO)-rich diet for 8, 14 or 20 weeks and parameters related to NO and superoxide (O₂^.–) plus markers of lipid peroxidation and protein oxidative damage in the aortic root were evaluated. The FO-rich diet increased NO production and endothelial NO synthase (NOS) expression and lowered inducible NOS, p22^phox expression and O₂^.–production after 14 and 20 weeks of diet. Protein lipoxidative damage (including 4-hydroxynonenal) was decreased after a long-term FO-diet. This supports the hypothesis that a FO-rich diet could counteract atherogenic oxidative stress, showing beneficial effects of long-chain (n-3) PUFA.     

Evaluation of genotoxic and oxidative stress response to dimethoate in freshwater fish Channa punctatus (Bloch)

Abstract

Dimethoate is one of the organophosphate insecticides widely used in agriculture throughout the world and is an inhibitor of cholinesterase in animals. The objective of the present study was to detect oxidative stress and DNA damage induced by dimethoate in the freshwater fish Channa punctatus (C. punctatus). The LC₅₀-96 h value of technical grade dimethoate was estimated at 19.10 μg L^-1 in a semi-static system and, on the basis of the LC₅₀ value, three concentrations were determined. The fish were exposed to these concentrations of dimethoate for 96 h and samplings were done at 24 and 96 h for assessment of oxidative stress and DNA damage. After exposure to dimethoate, the level of superoxide dismutase declined while lipid peroxidation, glutathione, induction of micronucleus and DNA damage were increased in C. punctatus as the concentration and exposure time increased. Thus our results suggest that dimethoate induces genotoxic effects which invariably accompanied and correlated with oxidative stress.     

Effects of exogenous nitric oxide on cadmium toxicity, element contents and antioxidative system in perennial ryegrass

The effects of sodium nitroprusside (SNP, a donor of NO) on cadmium (Cd) toxicity in ryegrass seedlings (Lolium perenne L.) were studied by investigating the symptoms, plant growth, chlorophyll content, lipid peroxidation, H⁺-ATPase enzyme and antioxidative enzymes. Addition of 100???M CdCl₂ caused serious chlorosis and inhibited the growth of ryegrass seedlings, and dramatically increased accumulation of Cd in both shoots and roots, furthermore, the absorption of macro and micronutrients were inhibited. Addition of 50, 100, 200???M SNP significantly decreased the transport of Cd from roots to shoots, alleviated the inhibition of K, Ca, Mg and Fe, Cu, Zn absorption induced by Cd, reduced the toxicity symptoms and promoted the plant growth. The accumulation of reactive oxygen species (ROS) significantly increased in ryegrass seedlings exposed to Cd, and resulted in the lipid peroxidation, which was indicated by accumulated concentration of thiobarbituric acid-reactive substances. Addition of 50, 100, 200???M SNP significantly decreased the level of ROS and lipid peroxidation. Activities of antioxidant enzymes also showed the same changes. Addition of 50, 100, 200???M SNP increased activities of superoxide dismutase, peroxidase, catalase and ascorbate peroxidase in ryegrass seedlings exposed to Cd. Addition of 100???M SNP had the most significant alleviating effect against Cd toxicity while the addition of 400???M SNP had no significant effect with Cd treatment.     

Phospholipase Dα1 and Phosphatidic Acid Regulate NADPH Oxidase Activity and Production of Reactive Oxygen Species in ABA-Mediated Stomatal Closure in Arabidopsis

We determined the role of Phospholipase Dα1 (PLDα1) and its lipid product phosphatidic acid (PA) in abscisic acid (ABA)-induced production of reactive oxygen species (ROS) in Arabidopsis thaliana guard cells. The pldα1 mutant failed to produce ROS in guard cells in response to ABA. ABA stimulated NADPH oxidase activity in wild-type guard cells but not in pldα1 cells, whereas PA stimulated NADPH oxidase activity in both genotypes. PA bound to recombinant Arabidopsis NADPH oxidase RbohD (respiratory burst oxidase homolog D) and RbohF. The PA binding motifs were identified, and mutation of the Arg residues 149, 150, 156, and 157 in RbohD resulted in the loss of PA binding and the loss of PA activation of RbohD. The rbohD mutant expressing non-PA-binding RbohD was compromised in ABA-mediated ROS production and stomatal closure. Furthermore, ABA-induced production of nitric oxide (NO) was impaired in pldα1 guard cells. Disruption of PA binding to ABI1 protein phosphatase 2C did not affect ABA-induced production of ROS or NO, but the PA–ABI1 interaction was required for stomatal closure induced by ABA, H₂O₂, or NO. Thus, PA is as a central lipid signaling molecule that links different components in the ABA signaling network in guard cells.     

Strobilanthes crispus attenuates renal carcinogen, iron nitrilotriacetate (Fe-NTA)-mediated oxidative damage of lipids and DNA

This study was aimed to evaluate the effect of Strobilanthes crispus extract for possible protection against lipid peroxidation and DNA damage induced by iron nitrilotriacetate (Fe-NTA) and hydrogen peroxide (H₂O₂). Fe-NTA is a potent nephrotoxic agent and induces acute and subacute renal proximal tubular necrosis by catalyzing the decomposition of H₂O₂-derived production of hydroxyl radicals, which are known to cause lipid peroxidation and DNA damage. Incubation of postmitochondrial supernatant and/or calf thymus DNA with H₂O₂ (40 mM) in the presence of Fe-NTA (0.1 mM) induces lipid peroxidation and DNA damage to about 2.3-fold and 2.9-fold, respectively, as compared to control (P < 0.05). In lipid peroxidation protection studies, S. crispus treatment showed a dose-dependent inhibition (45–53% inhibition, P < 0.05) of Fe-NTA and H₂O₂ induced lipid peroxidation. Similarly, in DNA damage protection studies, S. crispus treatment also showed a dose-dependent inhibition (18–30% inhibition, P < 0.05) of DNA damage. In addition, the protection was closely related to the content of phenolic compounds as evident by S. crispus extract showing the value of 124.48 mg/g total phenolics expressed as gallic acid equivalent (GAE, mg/g of extract). From these studies, it is concluded that S. crispus inhibits peroxidation of membrane lipids and DNA damage induced by Fe-NTA and H₂O₂ and possesses the potential to be used to treat or prevent degenerative diseases where oxidative stress is implicated.