Reactive Oxygen Species Scavenging Enzymes Research Articles

Transgenic plants: An insight into oxidative stress tolerance mechanisms

Environmental stresses considerably limit plant productivity. At the molecular level the negative effect of stress is often mediated by reactive oxygen species-initiated oxidative damage. Hence, it was hypothesised that increased tolerance to several environmental constraints could be achieved through enhanced tolerance to oxidative stress. In recent years much effort has been undertaken to improve oxidative stress tolerance by transforming plants with native or bacterial genes coding either for reactive oxygen species-scavenging enzymes or for enzymes modulating the cellular antioxidant capacity. This review deals with data on transgenic plants with altered antioxidant capacity and focuses on the new insight into the antioxidant defence mechanism given by this type of experimental model.

FSH induced stimulation of catalase activity in goat granulosa cells in vitro

Reactive oxygen species scavenging enzymes like catalase play diverse role in mammals. The presence of catalase in mammalian ovary is now well established. In the present investigation, changes in catalase activity in granulosa cells isolated from follicles at various stages of differentiation in response to FSH were studied. The follicles were dissected out from goat ovaries and classified as small (<3 mm), medium (3–6 mm) or large (>6 mm). Granulosa cells were isolated from categorized follicles. Results showed that there was a three-fold increase in catalase activity in granulosa cells from large follicles as compared to small and medium follicles. The catalase activity was stimulated significantly when granulosa cells were treated with FSH in vitro. The minimum effective dose that could stimulate catalase activity and estradiol secretion in case of granulosa cells from small and medium sized follicles was 100 ng/ml; for larger follicles, this value was 200 ng/ml. Concomitant to the increase in catalase activity, the estradiol secretion was significantly enhanced when cultured goat granulosa cells were treated with FSH. It was concluded that enzyme catalase may have a functional role in goat ovarian follicular development under endocrine regulation.

Abscisic acid, gibberellin and cell viability in cereal aleurone

The aleurone layer of cereals is a secretory tissue whose activity is regulated by abscisic acid (ABA) and gibberellins (GAs). Whereas GA triggers enzyme synthesis and secretion and initiates a program that culminates in cell death, ABA prevents enzyme production and cell death. Reactive oxygen species (ROS) are key players in regulating cell viability and GA sensitizes the aleurone cell to ROS. Sensitivity of GA-treated cells results in part from a reduction in steady-state amounts of mRNAs encoding enzymes that scavenge ROS. mRNAs encoding catalase, superoxide dismutase and ascorbate peroxidase are almost undetectable in aleurone layers 24 h after incubation in GA. For layers incubated in ABA, however, the amounts of these mRNAs increase. Western blotting and enzyme activity assays confirm that GA but not ABA reduced the amount and activity of ROS scavenging enzymes (Fath et al., 2001b). Substantial amounts of ROS are produced by enzymes engaged in lipid metabolism, and by the electron transport chain in the mitochondria. Aleurone layers contain abundant stores of triglycerides and ROS are produced as these lipids are rapidly converted to sugars. We hypothesize that the ROS produced in GA-treated aleurone cells bring about cell death by disrupting the plasma membrane. Aleurone cells incubated in ABA, on the other hand, are better able to maintain redox balance. ABA does not initiate rapid triglyceride metabolism, and the activities of ROS-scavenging enzymes remain high in ABA-treated cells. We conclude that GA initiates a metabolic cascade in aleurone cells that results in death from ROS. ABA maintains viability by keeping ROS under control.

Effects of thymic hormone on reactive oxygen species-scavengers and renal function in tacrolimus-induced nephrotoxicity

The effects of a thymic hormone (Facteur thymique serique; FTS) on renal reactive oxygen species-scavenging enzymes or substances in heminephrectomized rats with and without tacrolimus-induced nephrotoxicity were studied. Rats received both oral dose of tacrolimus (5 mg/kg/day) and subcutaneous administration of three dosages of FTS (5, 50, and 250 μg/kg/day) over 28 days (Group A). In Group B, they received three dosages of FTS alone (0.5, 5, and 50 μg/kg/day) or FTS 50 μg/kg/day with tacrolimus over 28 days. Each dose of FTS (Group A) partially elevated renal creatinine clearances. Tacrolimus enhanced renal glutathione reductase (GSH-R) activities and glutathione (GSH) and depressed catalase (CAT) activities. FTS increased GSH levels and GSH-R activities. Although FTS alone did not change CAT activities, CAT activities recovered as a result of concomitant use of FTS (Groups A and B). A significant positive correlation was found between CAT activity and creatinine clearance. These findings suggest that FTS is useful for the prevention of tacrolimus-induced nephrotoxicity, and that the increase of renal CAT activity in the defense mechanism of FTS might be important for cell protection against active oxygen species.

Enzymes that scavenge reactive oxygen species are down-regulated prior to gibberellic acid-induced programmed cell death in barley aleurone.

Gibberellins (GAs) initiate a series of events that culminate in programmed cell death, whereas abscisic acid (ABA) prevents this process. Reactive oxygen species (ROS) are key elements in aleurone programmed cell death. Incubation of barley (Hordeum vulgare) aleurone layers in H2O2 causes rapid death of all cells in GA- but not ABA-treated layers. Sensitivity to H2O2 in GA-treated aleurone cells results from a decreased ability to metabolize ROS. The amounts and activities of ROS scavenging enzymes, including catalase (CAT), ascorbate peroxidase, and superoxide dismutase are strongly down-regulated in aleurone layers treated with GA. CAT activity, protein, and Cat2 mRNA decline rapidly following exposure of aleurone layers to GA. In ABA-treated layers, on the other hand, the amount and activity of CAT and Cat2 mRNA increases. Incubation in ABA maintains high amounts of ascorbate peroxidase and superoxide dismutase, whereas GA brings about a rapid reduction in the amounts of these enzymes. These data imply that GA-treated cells loose their ability to scavenge ROS and that this loss ultimately results in oxidative damage and cell death. ABA-treated cells, on the other hand, maintain their ability to scavenge ROS and remain viable.

The role of nitric oxide in paraquat-induced cytotoxicity in the human A549 lung carcinoma cell line

Paraquat (PQ) is a well-known pneumotoxicant that exerts its toxic effect by elevating intracellular levels of superoxide. In addition, production of pro-inflammatory cytokines has possibly been linked to PQ-induced inflammatory processes through reactive oxygen species (ROSs) and nitric oxide (NO). However, the role of NO in PQ-induced cell injury has been controversial. To explore this problem, we examined the effect of NO on A549 cells by exposing them to the exogenous NO donor NOC18 or to cytokines; tumor necrosis factor-α, interleukin-1 β and interferon-γ, as well as PQ. Although the exogenous NO donor on its own had no effect on the release of lactate dehydrogenase (LDH), remarkable release was observed when the cells were exposed to high concentrations of NOC18 and PQ. This cellular damage caused by 1 mM NOC18 plus 0.2 mM PQ was ascertained by phase contrast microscopy. On the other hand, NO derived from 25–50 μM NOC18 added into the medium improved the MTT reduction activity of mitochondria, suggesting a beneficial effect of NO on the cells. Incubation of A549 cells with cytokines increased in inducible NO synthase (iNOS) expression and nitrite accumulation, resulting in LDH release. PQ further potentiated this release. The increase in nitrite levels could be completely prevented by NOS inhibitors, while the leakage of LDH was not attenuated by the inhibition of NO production with them. On the other hand, ROS scavenging enzymes, superoxide dismutase and catalase, inhibited the leakage of LDH, whereas they had no effect on the increase in the nitrite level. These results indicate that superoxide, not NO, played a key role in the cellular damage caused by PQ/cytokines. Our in vitro models demonstrate that NO has both beneficial and deleterious actions, depending on the concentrations produced and model system used.

Antioxidant therapy and streptozotocin-induced diabetes in pregnant rats.

The aim of our study was to analyse the effect of chronic hyperglycaemia on lipid peroxidation and scavenging enzyme activity in pregnant animals and their offspring supplemented and not supplemented with vitamin E - a natural antioxidant. Thirty pregnant female Wistar rats were used in our experiments. Diabetes was induced on day 7 of pregnancy using a single does of streptozotocin (40 mg/kg). Diabetic animals were divided into two equal groups: vitamin E supplemented and those fed with standard diet. Our controls consisted of 15 healthy rats. On day 1 after delivery homogenates of maternal liver and uterus as well as neonatal lungs and liver were prepared. Then the following parameters were measured: malondialdehyde (MDA) concentrations in the homogenates and blood serum, glutathione (GSH) levels, the activity of CuZn superoxide dismutase (SOD) and glutathione peroxidase (GPx) (Bioxytech, France). Statistical analysis was performed using Mann-Withney U test. The neonates of diabetic rats were smaller than those from healthy rats and serum glucose concentration was markedly higher in diabetic animals, both in mothers and neonates. MDA levels increased significantly, whereas GSH content and SOD as well as GPx activities were markedly diminished in diabetic pregnant rats and their offspring in comparison with the control group. In animals supplemented with tocopherol, MDA concentrations declined significantly, GSH contents and SOD activities were markedly elevated in almost all types of tissues studied, whereas glutathione peroxidase remained suppressed. Our results suggest that diabetic pregnant rats and their neonates are exposed to oxidative stress (OS), but vitamin E supplementation could in part reduce the imbalance between uncontrolled reactive oxygen species generation and scavenging enzyme activity, and may potentially serve as a useful prophylactic factor against OS development:

Peroxynitrite mediated damage and lowered superoxide tolerance in primary cortical glial cultures after induction of the inducible isoform of NOS.

The effect of the induction of i-NOS in primary glial cultures was studied with respect to the protein levels of reactive oxygen species (ROS) scavenging enzymes and the cytotoxicity of nitric oxide (.NO) formation at different levels of artificially generated superoxide. Stimulation of the cultures by bacterial lipopolysaccharides and gamma-interferon resulted in an induction of i-NOS exclusively in microglial cells. Among the ROS scavenging enzymes superoxide dismutase (Cu/Zn- and Mn-isoform), glutathione peroxidase and catalase only mitochondrial Mn-SOD was found to be upregulated in the course of i-NOS induction (Western blots). Although .NO formation did not affect cell viability at physiological levels of superoxide over a time period of 4 days, it caused an oxidative load particularly in microglial cells as observed by monitoring the oxidation of dichloro-dihydrofluorescein, an indicator for the formation of peroxynitrite and ROS. Elevated levels of superoxide, generated either intracellularly by paraquat or extracellularly via xanthine oxidase and hypoxanthine, resulted dose-dependently in a larger decline of cell viability in the .NO forming cultures compared to controls (release of lactate dehydrogenase, citrate synthase, stainability by propidium iodide, and tetramethylrhodamine). NOS-inhibitors reduced the degree of cell damage to that seen for control cultures, indicating an ONOO--/.NO mediated mechanism of cell damage. Our data support the concept that i-NOS catalyzed .NO-formation leads to an ONOO--mediated increased oxidative load. At physiological levels of superoxide and within a wide range of higher superoxide levels this nitrosative stress is well balanced in cultured glial cells by protective mechanisms.

SENSITIVITIES AND GENE-EXPRESSIONS OF ESCHERICHIA COLI MUTANTS DEFICIENT IN DNA REPAIR AND REACTIVE OXYGEN SPECIES SCAVENGING CAPACITY EXPOSED TO NATURAL SUNLIGHT

Sensitivities to sunlight of Escherichia coli mutants deficient in DNA repair capacities such as excision and recombination repair and their wild-type strain were compared. Higher sensitivities to sunlight were clearly observed in the mutant than wild-type strain, indicating that exposure to sunlight induces DNA damage which is repaired by DNA repair mechanisms. In order to assess the role of generation of reactive oxygen species (ROS) in DNA damage induced by exposure to natural sunlight, the kat-sod assay was performed using E. coli mutant strains deficient in ROS scavenging enzymes such as superoxide dismutase and/or catalase. Natural sunlight induced a significant generation of ROS, suggesting a possibility that sunlight induced ROS may be involved in DNA damage in bacterial cells.

DNA damage and intracellular generation of reactive oxygen species by allyl isothiocyanate inEscherichia coli

Allyl isothiocyanate (AITC) has been used as a food additive for paste-type wasabi (Japanese horseradish) and karashi (Japanese mustard) almost only in Japan, even though it has been reported to be carcinogenic and mutagenic. In the present paper, the capacities for DNA damage and the intracellular generation of reactive oxygen species (ROS) by AITC were examined by a ‘rec-lac assay’ and a ‘kat-sod assay’ by using E. coli mutants lacking DNA repair capacities and ROS scavenging enzymes, respectively. Both assays were developed by Nishioka et al. and consist of a streak test, a survival test and a β-galactosidase test. The results show strong DNA damaging capacity, and intracellular generation of ROS by AITC. These results indicate the possibility that intracellular ROS performs some role in DNA damage in bacterial cells treated with AITC. Copyright © 1999 John Wiley & Sons, Ltd.

Intracellular Generation of Reactive Oxygen Species and DNA Damage in Escherichia Coli Mutants Exposed to Electromagnetic Field and X-Ray

In order to examine intracellular generation of reactive oxygen species (ROS) and DNA damage in bacterial cells exposed to extremely low frequency magnetic field (ELFMF) at 400mT and 50Hz and X-ray, ‘kat-sod Assay’ and ‘rec-lac Assay’ were performed using E. coli mutants which were deficient in ROS scavenging enzymes, such as superoxide dismutase and/or catalase, and DNA repair capacities. Both assays were developed by Nishioka H. and Hayashi S. (1995) and Nunoshiba T. and Nishioka H. (1991) and consisted of survival test and β-galactosidase test. X-irradiation induced a clear intracellular generation of ROS and DNA damage, indicating a possibility that intracellular generated ROS may play a partial role in DNA damage by X-ray. On the other hand, no remarkable effects of ELFMF were seen in the cells using by ‘kat-sod Assay’ and ‘rec-lac Assay’, showing that ELFMF did not generate ROS intracellularly and damage DNA.

Phenoloxidase and cytotoxicity in the compound ascidian Botryllus schlosseri

The vacuoles of morula cells (MC) of the colonial ascidian Botryllus schlosseri contain phenoloxidase (PO). As the release of their vacuolar content at the border of incompatible contacting colonies is associated with the formation of necrotic masses which characterize the rejection reaction, the role of PO in Botryllus cytotoxicity was investigated. When hemocytes are incubated with blood plasma from incompatible (heterologous) colonies, MC degranulate and, after 60 min, the cytotoxicity index becomes significantly greater than that observed in controls incubated with autologous plasma. The rise in cell mortality is completely inhibited by the addition of PO inhibitors sodium benzoate, tropolone and phenylthiourea, and serine protease inhibitors phenylmethylsulfonyl fluoride, benzamidine, N-tosyl-L-phenylalanine chloromethyl ketone and N-tosyl-L-lysine chloromethyl ketone. The addition of either reducing agents L-cysteine and ascorbic acid or reactive oxygen species scavenger enzymes superoxide dismutase and catalase has a similar effect. Significant inhibition of cytotoxicity is also observed with the quinone scavenger, 3-methyl-2-benzothiazolinone hydrazone. In the presence of sodium benzoate and phenylthiourea, there is a significant reduction in the number, size and color intensity of necrotic masses along the contact border of incompatible colonies. A significant increase in superoxide anion production, completely inhibited by sodium benzoate, is observed when hemocytes are incubated with heterologous blood plasma. These results indicate that: (i) PO is the enzyme responsible for the cytotoxicity observed in both hemocyte cultures and rejection reactions; (ii) PO is present inside MC vacuoles as a proenzyme which is activated, upon release, by humoral proteases; (iii) cytotoxicity appears to be mainly due to oxidative stress generated by PO during oxidation of polyphenols to quinones without the involvement of other oxidases such as NADPH oxidase and peroxidase.

The effect of Photosystem II inhibitors DCMU and BNT on the high-light induced D1 turnover in two cyanobacterial strains Synechocystis PCC 6803 and Synechococcus PCC 7942

The effect of the Photosystem II (PSII) inhibitors dichlorophenyldimethylurea (DCMU) and bromonitrothymol (BNT) on the rate of the high-light induced D1 protein turnover was studied in whole cells of two cyanobacterial strains Synechocystis PCC 6803 and Synechococcus PCC 7942. In Synechocystis the D1 degradation was slowed down to a similar extent in the presence of either inhibitor compared with control cells. This slower degradation corresponded with the retardation of Photosystem II photoinactivation (PSIIPI) measured as a decline of PS II activity in the illuminated cells treated with chloramphenicol (CAP). The ongoing D1 synthesis in the presence of both PS II inhibitors was confirmed by unchanging PS II activity and the steady-state level of D1 during illumination in the absence of CAP. In Synechococcus cells both DCMU and BNT blocked the turnover of the 'low-light' D1 form (D1:1) but did not prevent the exchange of the 'high-light' form D1:2 for the D1:1 form. The similar effect of both herbicides on the D1 exchange was in contrast with their influence on the rate of PSIIPI. While DCMU had a pronounced protective effect, BNT significantly increased the rate of PS II photodamage. The fast BNT-induced decline of PS II activity was also observed in Synechocystis cells treated with azide, an inhibitor of reactive oxygen species scavenging enzymes. Therefore, we assume that the distinct sensitivity of the two cyanobacterial strains to BNT can be caused by different content and/or activity of these enzymes in each strain.

Effects of a single exposure to UVB radiation on the activities and protein levels of copper-zinc and manganese superoxide dismutase in cultured human keratinocytes.

Ultraviolet B irradiation has been believed to decrease or impair the activity of reactive oxygen species (ROS) scavenging enzymes such as superoxide dismutase (SOD) in the skin. It has been recently reported that two isozymes of SOD, namely copper-zinc SOD (Cu-Zn SOD) and manganese SOD (Mn SOD), exist in mammalian cells and that the two enzymes play different roles in living systems. The aim of this study was to investigate changes in SOD activities and protein levels in cultured human keratinocytes after acute UVB irradiation. In addition, the protein levels of Cu-Zn SOD and Mn SOD were quantified separately. A single exposure to UVB irradiation produced an increase in SOD activity and protein level that peaked immediately after UVB irradiation, after which a decline was observed, with subsequent recovery to baseline levels 24 h after irradiation. In individual assays of Mn SOD and Cu-Zn SOD, the amount of Mn SOD protein decreased and then gradually recovered 24 h after irradiation. In contrast, the amount of Cu-Zn SOD protein increased immediately after UVB irradiation, and then gradually declined. To evaluate the mechanisms of these changes, we examined the effects of the cytokines, interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha), which can be secreted from keratinocytes after UVB irradiation, on the SOD activity and protein levels in keratinocytes. Interleukin-1 alpha and TNF-alpha enhanced both the SOD activity and protein level of Mn SOD, while these cytokines had no effect on Cu-Zn SOD protein levels in cultured human keratinocytes after incubation for 24 h. Furthermore, when neutralizing antibodies against IL-1 alpha and TNF-alpha were added separately or together to the culture medium before UVB irradiation, the recovery of total SOD activity and Mn SOD protein level were markedly inhibited 24 h after irradiation. Our results suggest that significant increases in SOD activity and protein level occur as a cutaneous antioxidant defense mechanism that protects against the cytotoxicity as a result of UVB irradiation, and that this increase in SOD is attributed to Cu-Zn SOD. The Cu-Zn SOD and Mn SOD protein levels changed in a different manner after UVB irradiation. The former may participate in an early phase and the latter in a late phase defense mechanism directed against oxidant cytotoxicity through UVB irradiation. In addition, the recovery of Mn SOD to baseline levels 24 h after UVB irradiation seems to be mediated through cytokines such as IL-1 alpha and TNF-alpha, which are secreted from keratinocytes.

Role of Cytokines, Tyrosine Kinase, and Protein Kinase C on Production of Superoxide and Induction of Scavenging Enzymes in Human Leukocytes

We investigated the effects of proximal modulators of cytokines, tyrosine kinase (TK), and protein kinase C (PKC) on reactive oxygen species (ROS) generation and the induction of scavenging enzymes, superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-Px) of human neutrophils and lymphocytes, by using IL1-α, TNF-α, and IFN-γ and neutralizing antibodies to these cytokines. Inhibitors of TK (ST638 and herbimycin) or PKC (H-7, calphostin, and staurosporine) were also used. The results revealed that both O−2generation stimulated by five different agents (opsonized zymosan, A23187, PAF, PMA, and fMLP) and the inductions of all three scavenging enzymes were potentiated by priming with TNF-α. In contrast, both O−2generation and enzyme induction were attenuated by priming with IL1-α, with the exception of PMA-stimulated O−2generation. IFN-γ decreased O−2generation but increased scavenging enzyme induction. Antibodies to all three cytokines and all the TK and PKC inhibitors decreased O−2stimulated by most agents, but markedly enhanced O−2levels stimulated by PAF. Induction of all three enzymes was enhanced equally by low concentrations of each of the three anticytokine antibodies, while each of the TK or PKC inhibitors decreased induction of SOD and GSH-Px and increased catalase induction. These results suggest that both ROS generation and scavenging enzyme induction are controlled in complex ways by the actions of these three proximal mediators. This supports our hypothesis that disturbances in the regulation of early events of cell activation can lead to oxidative tissue injury.

Effects of antioxidants on human sperm preparationtechniques

The effect of two different sperm preparation techniques, Percoll gradient centrifugation and swim-up from a washed pellet were tested on the functional competence of the selected spermatozoa. Percoll gradient centrifugation brought about an improvement in sperm motility parameters such as curvilinear velocity and straight-line velocity, an increase in the rates of hyperactivation and the acrosome reaction and an increase in the percentage of motile spermatozoa after 24 h of incubation compared to the centrifugation, swim-up procedure. The effects of antioxidants such as dithiothreitol (DTT) or reduced glutathione (GSH), and reactive oxygen species-scavenging enzymes such as catalase or superoxide dismutase (SOD) during the stage of centrifugation before the swim-up procedure were also studied. Though all of these agents prevented the fall in sperm motility after 24 h incubation, only DTT and SOD improved the rates of both hyperactivation and the acrosome reaction. GSH also improved the acrosome reaction, whereas catalase was without significant effect on the rates of hyperactivation or the acrosome reaction. These results indicate that Percoll gradient centrifugation selects spermatozoa with better functional competence than does centrifugation swim-up. The damage caused by the centrifugation can be prevented by the addition of antioxidants, suggesting that the differences noted with the Percoll gradient method was due to better protection against peroxidative damage due to the centrifugation of unselected spermatozoa. However, the use of DTT is limited by virtue of the fact that this sulphydryl reducing agent leads to destabilization of the sperm chromatin. In contrast, GSH and SOD could have therapeutic potential.

The Effect of Paraquat on the Radiosensitivity of Melanoma Cells: the Role of Superoxide Dismutase &amp; CATALASE

The activities of reactive oxygen species scavenging enzymes, superoxide dismutases (SODs) and catalase (in cells of two melanomas (mouse B16 and human SK23) and in Chinese hamster ovary (CHO) cells were examined. Melanoma cells are relatively depleted in activities of superoxide dismutases and catalase as compared to CHO cells. Short equitoxic (500 microM for CHO and B16 cells and 5 microM for SK23 cells) paraquat treatment (15 min before the X-irradiation, 45 min in postirradiation period--the total time of treatment was 1 h) caused an increase in radiation resistance, measured as colony forming ability, in two of the three lines examined. It is proposed that PQ may exert its radioprotective effect by induction of antioxidant enzymes.

Changes in lipid peroxide levels and activity of reactive oxygen scavenging enzymes in skin, serum and liver following UVB irradiation in mice

The effect of acute UVB on the generation of reactive oxygen species (ROS) in the skin and the induction of ROS scavenging enzymes in situ was examined. Lipid peroxide levels and the activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px) and D-glucose-6-phosphate dehydrogenase (G-6-P-D) were determined in the skin, serum, and liver of ICR mice subjected to 1400 mJ/cm 2 of acute UVB irradiation. In irradiated skin, lipid peroxides were increased at 3 and 24 hr after irradiation, whereas the four ROS scavenging enzymes were generally decreased during the first 48 hr after irradiation. In the serum, lipid peroxides showed an increase at 3 hr, but enzyme activities remained negligible. In the liver, lipid peroxides showed similar behaviour to that in skin. GSH-Px activity in the liver was decreased during the first 24 hr, whereas G-6-P-D showed substantial fluctuation and SOD and catalase activities showed no change. These data are consistent with a model in which lipid peroxides generated in the UVB-irradiated lesions are transported to the liver and there metabolized by the scavenging enzymes induced in situ.