Neuroprotective Effect of Decalepis hamiltonii in Paraquat-Induced Neurotoxicity in Drosophila melanogaster: Biochemical and Behavioral Evidences

In this paper, we have demonstrated for the first time, the antioxidant and neuroprotective effects of Decalepis hamiltonii (Dh) root extract against paraquat (PQ)-induced oxidative stress and neurotoxicity in Drosophila melanogaster. Exposure of adult D. melanogaster (Oregon K) to PQ induced oxidative stress as evidenced by glutathione depletion, lipid peroxidation and enhanced activities of antioxidant enzymes such as catalase, superoxide dismutase as well as elevated levels of acetylcholine esterase. Pretreatment of flies by feeding with Dh extract (0.1, 0.5 %) for 14 days boosted the activities of antioxidant enzymes and prevented the PQ-induced oxidative stress. Dietary feeding of Dh extract prior to PQ exposure showed a lower incidence of mortality and enhanced motor activities of flies in a negative geotaxis assay; both suggesting the neuroprotective potential of Dh. Based on the results, we contemplate that the roots of Dh might prevent and ameliorate the human diseases caused by oxidative stress. The neuroprotective action of Dh can be attributed to the antioxidant constituents while the precise mechanism of its action needs further investigations.     

Attenuation of Rotenone-Induced Mitochondrial Oxidative Damage and Neurotoxicty in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> Supplemented with Creatine

Creatine (Cr), an ergogenic nutritional supplement is demonstrated to possess bioenergetic, antiexcitotoxic and antioxidant properties. This study investigated the neuroprotective effects of Cr against rotenone induced oxidative stress, mortality and neurotoxicty in Drosophila melanogaster. We found significant diminution in the endogenous levels of oxidative markers in whole body homogenates of flies exposed to Cr (2–10 mM). Cr supplementation resulted in reduced mortality in flies exposed to rotenone (500 μM) and better performance in a negative geotaxis assay. Further Cr (10 mM) markedly offset rotenone induced mitochondrial oxidative stress, completely restored the GSH levels, nitric oxide levels, activity of Mn-SOD and dopamine depletion. In an oxidative stress bioassay, flies given Cr prophylaxis exhibited marked resistance to paraquat exposure. These data allow us to hypothesize that the neuroprotective action of Cr in Drosophila may be related to its direct antioxidant activity and ability to abrogate rotenone induced mitochondrial oxidative stress.     

Differential susceptibility of a few members of the nasuta-albomicans complex of Drosophila to paraquat-induced lethality and oxidative stress

The evolution of karyotypically stabilized short-lived (SL) and long-lived (LL) cytoraces in the laboratory have been established and validated through our previous lifespan studies. In the present investigation, we examined the possible reason(s) for the differential longevity among selected members of SL and LL cytoraces, employing the well known paraquat (PQ) resistance bioassay. Exposure of these races to varying concentrations of PQ revealed relatively higher resistance among LL cytoraces than SL cytoraces, as evident by the lower incidence of mortality. Biochemical analysis for endogenous markers of oxidative stress revealed that LL-2 cytorace exhibited lower reactive oxygen species (ROS) and lipid peroxidation (LPO) levels, higher activity levels of superoxide dismutase (SOD), and coupled with higher levels of reduced glutathione (GSH) compared with the levels found in SL-2 cytorace. These findings suggest that the higher susceptibility of SL cytoraces to PQ challenge may be, at least in part, related to the higher endogenous levels of oxidative stress markers. Although the precise mechanisms responsible for the longer longevity among LL cytoraces of the nasuta-albomicans complex of Drosophila merits further investigation, our data suggest that the relatively longer lifespan may be related to the status of endogenous markers that renders them more resistant towards oxidative-stress-mediated lethality, as evident in the PQ assay.     

Role of oxidative stress in Drosophila aging.

We review the role that oxidative damage plays in regulating the lifespan of the fruit fly, Drosophila melanogaster. Results from our laboratory show that the lifespan of Drosophila is inversely correlated to its metabolic rate. The consumption of oxygen by adult insects is related to the rate of damage induced by oxygen radicals, which are purported to be generated as by-products of respiration. Moreover, products of activated oxygen species such as hydrogen peroxide and lipofuscin are higher in animals kept under conditions of increased metabolic rate. In order to understand the in vivo relationship between oxidative damage and the production of the superoxide radical, we generated transgenic strains of Drosophila melanogaster that synthesize excess levels of enzymatically active superoxide dismutase. This was accomplished by P-element transformation of Drosophila melanogaster with the bovine cDNA for CuZn superoxide dismutase, an enzyme that catalyzes the dismutation of the superoxide radical to hydrogen peroxide and water. Adult flies that express the bovine SOD in addition to native Drosophila SOD are more resistant to oxidative stresses and have a slight but significant increase in their mean lifespan. Thus, resistance to oxidative stress and lifespan of Drosophila can be manipulated by molecular genetic intervention. In addition, we have examined the ability of adult flies to respond to various environmental stresses during senescence. Resistance to oxidative stress, such as that induced by heat shock, is drastically reduced in senescent flies. This loss of resistance is correlated with the increase in protein damage generated in old flies by thermal stress and by the insufficient protection from cellular defense systems which includes the heat shock proteins as well as the oxygen radical scavenging enzymes. Collectively, results from our laboratory demonstrate that oxidative damage plays a role in governing the lifespan of Drosophila during normal metabolism and under conditions of environmental stress.     

Effects of redox cycling compounds on glutathione content and activity of glutathione-related enzymes in rainbow trout liver

In fish, as in other aerobic organisms, glutathione and glutathione-related enzymes are important components in the defences against oxidative stress. To study if hepatic glutathione levels and/or activities of glutathione-related enzymes can act as indicators of oxidative stress in fish, we injected rainbow trout (Oncorhynchus mykiss) intraperitoneally with paraquat (PQ), menadione (MD), naphthazarin (DHNQ), or beta-naphthoflavone (beta-NF), all known to cause a rise in reactive oxygen species (ROS). After 2 and 5 days of exposure, we measured the activities of hepatic glutathione peroxidase (GPox), glutathione S-transferase (GST), gamma-glutamylcysteine synthetase (GCS), and glutathione reductase (GR). We also measured total glutathione (tGSH) and oxidised glutathione (GSSG) in the liver of fish treated with PQ and MD. All chemicals caused an increase in GR activity after 5 days, which ranged from 160% in fish treated with beta-NF to 1,500% in fish treated with PQ. All chemicals except beta-NF caused moderate elevation in GST activity; GPox activity was lower in fish treated with DHNQ and MD, while GCS activity increased twofold in the fish treated with DHNQ, without being affected by beta-NF, PQ or MD. After 5 days of treatment with PQ or MD, tGSH content was elevated. Our findings demonstrated that of the parameters included in the study, GR activity was the most responsive to treatment with redox cycling compounds, indicating that GR activity is a promising biomarker of such compounds and possibly indicating oxidative stress in rainbow trout.     

Compartment-specific protection of iron-sulfur proteins by superoxide dismutase

Iron and oxygen are essential but potentially toxic constituents of most organisms, and their transport is meticulously regulated both at the cellular and systemic levels. Compartmentalization may be a homeostatic mechanism for isolating these biological reactants in cells. To investigate this hypothesis, we have undertaken a genetic analysis of the interaction between iron and oxygen metabolism in Drosophila. We show that Drosophila iron regulatory protein-1 (IRP1) registers cytosolic iron and oxidative stress through its labile iron sulfur cluster by switching between cytosolic aconitase and RNA-binding functions. IRP1 is strongly activated by silencing and genetic mutation of the cytosolic superoxide dismutase (Sod1), but is unaffected by silencing of mitochondrial Sod2. Conversely, mitochondrial aconitase activity is relatively insensitive to loss of Sod1 function, but drops dramatically if Sod2 activity is impaired. This strongly suggests that the mitochondrial boundary limits the range of superoxide reactivity in vivo. We also find that exposure of adults to paraquat converts cytosolic aconitase to IRP1 but has no affect on mitochondrial aconitase, indicating that paraquat generates superoxide in the cytosol but not in mitochondria. Accordingly, we find that transgene-mediated overexpression of Sod2 neither enhances paraquat resistance in Sod1+ flies nor compensates for lack of SOD1 activity in Sod1-null mutants. We conclude that in vivo, superoxide is confined to the subcellular compartment in which it is formed, and that the mitochondrial and cytosolic SODs provide independent protection to compartment-specific protein iron-sulfur clusters against attack by superoxide generated under oxidative stress within those compartments.     

Spermine and putrescine enhance oxidative stress tolerance in maize leaves

The protective effects of spermine (SPM) and putrescine (PUT) against paraquat (PQ), a herbicide in agriculture and oxidative stress inducer, were investigated in the leaves of maize. Maize leaves were pretreated to SPM and PUT at concentrations of 0.2 and 1 mM and treated with PQ afterwards. Pretreatment with 1 mM of SPM and PUT significantly prevented the losses in chlorophyll and carotenoid levels induced by PQ. Ascorbic acid content in the leaves pretreated with both polyamines was found to be higher than those of the leaves pretreated with water. Also, pretreatment with SPM and PUT was determined to have some effects on the activities of superoxide dismutase (SOD) and peroxidase (POD). 1 mM of SPM increased SOD activity, but PUT has no significant effect on SOD activity. On the other hand, POD activity was recorded to increase slightly in response to both concentrations of SPM and 1 mM of PUT. The results showed that such polyamine pretreated plants may become more tolerant to oxidative stress due to increases in the antioxidative enzymes and antioxidants.     

Mechanism underlying the antioxidant activity of taurine: prevention of mitochondrial oxidant production

An important function of the β-amino acid, taurine, is the regulation of oxidative stress. However, taurine is neither a classical scavenger nor a regulator of the antioxidative defenses, leaving uncertain the mechanism underlying the antioxidant activity of taurine. In the present study, the taurine antagonist and taurine transport inhibitor, β-alanine, was used to examine the mechanism underlying the antioxidant activity of taurine. Exposure of isolated cardiomyocytes to medium containing β-alanine for a period of 48?h led to a 45% decrease in taurine content and an increase in mitochondrial oxidative stress, as evidenced by enhanced superoxide generation, the inactivation of the oxidant sensitive enzyme, aconitase, and the oxidation of glutathione. Associated with the increase in oxidative stress was a decline in electron transport activity, with the activities of respiratory chain complexes I and III declining 50–65% and oxygen consumption falling 30%. A reduction in respiratory chain activity coupled with an increase in oxidative stress is commonly caused by the development of a bottleneck in electron transport that leads to the diversion of electrons from the respiratory chain to the acceptor oxygen forming in the process superoxide. Because β-alanine exposure significantly reduces the levels of respiratory chain complex subunits, ND5 and ND6, the bottleneck in electron transport appears to be caused by impaired synthesis of key subunits of the electron transport chain complexes. Co-administration of taurine with β-alanine largely prevents the mitochondrial effects of β-alanine, but treatment of the cells with 5?mM taurine in the absence of β-alanine has no effect on the mitochondria, likely because taurine treatment has little effect on cellular taurine levels. Thus, taurine serves as a regulator of mitochondrial protein synthesis, thereby enhancing electron transport chain activity and protecting the mitochondria against excessive superoxide generation.     

Paraquat pre-treatment increases activities of antioxidant enzymes and reduces lipid peroxidation in salt-stressed cucumber leaves

To investigate whether paraquat (PQ) is involved in regulation of antioxidant enzymes and lipid peroxidation under short-term salt stress, and to elucidate the physiological mechanism of salt stress mitigated by PQ, a cucumber cultivar (cv. Chunguang no. 2) was exposed to 100 mM NaCl for 48 h after pre-treatment with 10 μM PQ for 1 h. When compared to the control, salt stress increased the levels of malonaldehyde (MDA), superoxide radical (O₂^·−) and hydrogen peroxide (H₂O₂) and the activities of antioxidant enzymes, such as superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2) in the cucumber leaves. Under salt conditions, PQ pre-treatment prevented oxidative stress as observed by the decreases in MDA, H₂O₂ and O₂^·− that correlated with the increase in antioxidant defenses. We propose that, at low concentrations, the PQ pre-treatment can reduce the salt-induced oxidative damage by increasing the antioxidative mechanisms in cucumber plants.     

Mitochondrial defects and oxidative damage in patients with peripheral arterial disease

Abnormal mitochondrial function is present in patients with peripheral arterial disease and may contribute to its clinical manifestations. However, the specific biochemical mitochondrial defects and their association with increased oxidative stress have not been fully characterized. Gastrocnemius muscle was obtained from peripheral arterial disease patients (n = 25) and age-matched controls (n = 16) and mitochondrial parameters were measured. Complexes I through IV of the electron transport chain were individually evaluated to assess for isolated defects. Muscle was also evaluated for protein and lipid oxidative changes by measuring the levels of protein carbonyls, lipid hydroperoxides, and total 4-hydroxy-2-nonenal binding and for the activities of the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase. Mitochondrial electron transport chain complexes I, III, and IV in arterial disease patients demonstrated significant reductions in enzymatic activities and mitochondrial respiration compared to controls. Oxidative stress biomarker analysis demonstrated significantly increased levels of protein carbonyls, lipid hydroperoxides, and 4-hydroxy-2-nonenal compared to control muscle. Antioxidant enzyme activities were altered, with a significant decrease in superoxide dismutase activity and significant increases in catalase and glutathione peroxidase. Peripheral arterial disease is associated with abnormal mitochondrial function and evidence of significant oxidative stress.     

Mitochondrial superoxide plays a crucial role in the development of mitochondrial dysfunction during high glucose exposure in rat renal proximal tubular cells

Diabetic nephropathy is the leading cause of end-stage renal disease in the United States. Despite several studies indicating a role for mitochondrial oxidative stress and mitochondrial dysfunction in the development of diabetic complications, the precise mechanisms underlying renal mitochondrial dysfunction and renal cell injury remain unclear. The hypothesis of the current study was that high-glucose-mediated generation of mitochondrial superoxide is a key early event that leads to mitochondrial injury in renal proximal tubular cells. To ascertain the role of mitochondrial superoxide we have tested whether overexpression of the primary mitochondrial antioxidant, manganese superoxide dismutase (MnSOD), protects against hyperglycemia-induced renal injury using normal rat renal proximal tubular cells (NRK). NRK cells were exposed to high glucose (25 mM) and the changes in the mitochondrial membrane potential, ATP levels, and superoxide generation and the loss of cell viability were measured at 24 and 48 h after high glucose exposure. Our results indicate that high glucose first induced superoxide generation and hyperpolarization in the mitochondria, followed by a secondary event, which involved a decline in ATP levels, partial Complex III inactivation, and loss of cell viability. These high-glucose-induced changes were completely prevented by overexpression of MnSOD in NRK cells. However, MnSOD activity was not changed after high glucose exposure in vitro or during the early stages of diabetes using the streptozotocin rat model. These findings show for the first time that hyperglycemic induction of superoxide production within the mitochondria initiates specific mitochondrial injury (i.e., Complex III) via a mechanism independent of MnSOD inactivation.     

Biomarkers of oxidative stress in the fungal strain Humicola lutea under copper exposure

The fungal strain Humicola lutea 103 was used as a model organism to examine the relationship between copper toxicity and oxidative stress in low eukaryotes such as filamentous fungi. Spores or submerged cultures were treated with different copper concentrations and the oxidative stress-inducing agent paraquat (PQ). Oxidative stress biomarkers such as reactive oxygen species (ROS), cyanide-resistant respiration, protein carbonyls, reserve carbohydrates, and antioxidant defence were identified in cells treated or not treated with either copper ions or PQ. Copper inhibited the growth and conidiospore formation of H. lutea 103 in a concentration-dependent manner. This treatment also resulted in increased superoxide anion radical formation. Copper stress was furthermore accompanied by transient accumulation of trehalose and glycogen, as well as increased protein carbonyl content. Compared to control cultures, copper-treated mycelia demonstrated a marked increase in the activity of protective enzymes (superoxide dismutase, catalase, and glucose-6-phosphate dehydrogenase). These increased antioxidant enzyme activities were blocked by inhibitors of protein synthesis, suggesting that de novo enzyme formation was involved. Biomarker response to the heavy metal was similar to treatment with known ROS generators such as PQ. The observed hyper-oxidative status and increased oxidative damage suggest a relationship between acute metal treatment and oxidative stress in fungal cells.     

Forkhead box O3 protects the heart against paraquat‐induced aging‐associated phenotypes by upregulating the expression of antioxidant enzymes

Paraquat (PQ) promotes cell senescence in brain tissue, which contributes to Parkinson's disease. Furthermore, PQ induces heart failure and oxidative damage, but it remains unknown whether and how PQ induces cardiac aging. Here, we demonstrate that PQ induces phenotypes associated with senescence of cardiomyocyte cell lines and results in cardiac aging‐associated phenotypes including cardiac remodeling and dysfunction in vivo. Moreover, PQ inhibits the activation of Forkhead box O3 (FoxO3), an important longevity factor, both in vitro and in vivo. We found that PQ‐induced senescence phenotypes, including proliferation inhibition, apoptosis, senescence‐associated β‐galactosidase activity, and p16^INK4a expression, were significantly enhanced by FoxO3 deficiency in cardiomyocytes. Notably, PQ‐induced cardiac remolding, apoptosis, oxidative damage, and p16^INK4a expression in hearts were exacerbated by FoxO3 deficiency. In addition, both in vitro deficiency and in vivo deficiency of FoxO3 greatly suppressed the activation of antioxidant enzymes including catalase (CAT) and superoxide dismutase 2 (SOD2) in the presence of PQ, which was accompanied by attenuation in cardiac function. The direct in vivo binding of FoxO3 to the promoters of the Cat and Sod2 genes in the heart was verified by chromatin immunoprecipitation (ChIP). Functionally, overexpression of Cat or Sod2 alleviated the PQ‐induced senescence phenotypes in FoxO3‐deficient cardiomyocyte cell lines. Overexpression of FoxO3 and CAT in hearts greatly suppressed the PQ‐induced heart injury and phenotypes associated with aging. Collectively, these results suggest that FoxO3 protects the heart against an aging‐associated decline in cardiac function in mice exposed to PQ, at least in part by upregulating the expression of antioxidant enzymes and suppressing oxidative stress.     

Oxidative Stress in Severely Obese Persons Is Greater in Those With Insulin Resistance

The postprandial state seems to have a direct influence on oxidative status and insulin resistance. We determined the effect of an increase in plasma triglycerides after a high‐fat meal on oxidative stress in severely obese patients with differing degrees of insulin resistance. The study was undertaken in 60 severely obese persons who received a 60‐g fat overload with a commercial preparation. Measurements were made of insulin resistance, the plasma activity of various antioxidant enzymes, the total antioxidant capacity (TAC) and the plasma concentration of thiobarbituric acid reactive substances (TBARS). The patients with greater insulin resistance had a lower plasma superoxide dismutase (SOD) activity (P < 0.05) and a greater glutathione peroxidase (GSH‐Px) activity (P < 0.05). The high‐fat meal caused a significant reduction in SOD activity and an increase in the plasma concentration of TBARS in all the patients. Only the patients with lower insulin resistance experienced a significant increase in plasma catalase activity (2.22 ± 1.02 vs. 2.93 ± 1.22 nmol/min/ml, P < 0.01), remaining stable in the patients with greater insulin resistance. These latter patients had a reduction in plasma TAC (6.92 ± 1.93 vs. 6.29 ± 1.80 mmol/l, P < 0.01). In conclusion, our results show a close association between the degree of insulin resistance and markers of oxidative stress, both before and after a high‐fat meal. The postprandial state causes an important increase in oxidative stress, especially in severely obese persons with greater insulin resistance. However, we are unable to determine from this study whether there is first an increase in oxidative stress or in insulin resistance.     

Depletion of reduction potential and key energy generation metabolic enzymes underlies tellurite toxicity in Deinococcus radiodurans

Oxidative stress resistant Deinococcus radiodurans surprisingly exhibited moderate sensitivity to tellurite induced oxidative stress (LD₅₀ = 40 μM tellurite, 40 min exposure). The organism reduced 70% of 40 μM potassium tellurite within 5 h. Tellurite exposure significantly modulated cellular redox status. The level of ROS and protein carbonyl contents increased while the cellular reduction potential substantially decreased following tellurite exposure. Cellular thiols levels initially increased (within 30 min) of tellurite exposure but decreased at later time points. At proteome level, tellurite resistance proteins (TerB and TerD), tellurite reducing enzymes (pyruvate dehydrogense subunits E1 and E3), ROS detoxification enzymes (superoxide dismutase and thioredoxin reductase), and protein folding chaperones (DnaK, EF‐Ts, and PPIase) displayed increased abundance in tellurite‐stressed cells. However, remarkably decreased levels of key metabolic enzymes (aconitase, transketolase, 3‐hydroxy acyl‐CoA dehydrogenase, acyl‐CoA dehydrogenase, electron transfer flavoprotein alpha, and beta) involved in carbon and energy metabolism were observed upon tellurite stress. The results demonstrate that depletion of reduction potential in intensive tellurite reduction with impaired energy metabolism lead to tellurite toxicity in D. radiodurans.     

Effect of cadmium on antioxidant enzyme activities and lipid peroxidation in the gills of the clam Ruditapes decussatus

Metals are known to influence the oxidative status of marine organisms, and antioxidant enzymes have been often proposed as biomarkers of effect. The clam Ruditapes decussatus is a well-known metal bioindicator. In this species cadmium (Cd) induces metallothionein (MT) synthesis only after 7 days of exposure. Before MT synthesis is induced, the other mechanisms capable of handling the excess of Cd are unknown. In order to identify some of these mechanisms, variations in antioxidant systems (superoxide dismutase, catalase, selenium-dependent glutathione peroxidase and non-selenium-dependent glutathione peroxidase), malondialdehyde (MDA) and MT were studied in the gills of R. decussatus exposed to different Cd concentrations (4, 40 and 100 gl^-1) for 28 days. These parameters, together with total proteins and Cd concentrations, were measured in the gills of the clams over different periods of exposure. Results indicate that Cd accumulation increased linearly in the gills of R. decussatus with the increase in Cd concentration. This increase induces an imbalance in the oxygen metabolism during the first days of Cd exposure. An increase in cytosolic superoxide dismutase (SOD) activity and a decrease in mitochondrial SOD activity was observed at the same time as or after a decrease in cytosolic and mitochondrial catalase activity and of selenium-dependent and non-selenium-dependent glutathione peroxidase activity. After 14 days of exposure, Cd no longer affect these enzymes but there was elevation of other cellular activities, such as MDA and MT production. MT bound excess Cd present in the cell. These variations in these parameters suggest their potential use as biomarkers of effects such as oxidative stress resulting from Cd contamination in molluscs.     

The activities of antioxidant enzymes in response to oxidative stresses and hormones in paraquat-tolerant Rehmannia glutinosa plants

All members of R. glutinosa show the unique characteristic of intrinsic tolerance to paraquat (PQ). Antioxidant enzymes have been proposed to be the primary mechanism of PQ resistance in several plant species. Therefore, the antioxidant enzyme systems of R. glutinosa were evaluated by comparatively analyzing cellular antioxidant enzyme levels, and their responses of oxidative stresses and hormones. The levels of ascorbate peroxidase (APX), glutathione reductase (GR), non-specific peroxidase (POX), and superoxide dismutase (SOD) were 7.3-, 4.9-, 2.7- and 1.6-fold higher in PQ-tolerant R. glutinosa than in PQ-susceptible soybeans. However, the activity of catalase (CAT) was about 12-fold higher in the soybeans. The activities of antioxidant enzymes reduced after PQ treatment in the two species, with the exception of POX and SOD in R. glutinosa, which increased by about 40 %. Interestingly, the activities of APX, SOD and POX in R. glutinosa, relative to those in soybeans, were further increased by 49, 67 and 93 % after PQ treatment. The considerably higher intrinsic levels, and increases in the relative activities of antioxidant enzymes in R. glutinosa under oxidative stress support the possible role of these enzymes in the PQ tolerance of R. glutinosa. However, the relatively lower levels of SOD versus PQ tolerance, and the mixed responses of antioxidant enzymes to stresses and hormones, suggest a possible alternative mechanism(s) for PQ tolerance in R. glutinosa.     

Protective role of exogenous spermidine against paraquat toxicity in radish chloroplasts

When radish chloroplasts were pretreated with 1 mM spermidine (Spd) and then exposed to 30 M paraquat (PQ), they improved their tolerance to subsequent PQ-induced oxidative damages. That included the decreases in the contents of chlorophyll, protein, and ascorbate, as well as the increases in malondialdehyde (MDA) and H₂O₂ levels. Analysis of antioxidant enzymes showed that Spd pretreatment effectively prevented the PQ-induced decreases in the total activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX). In contrast, the normally enhanced activities of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) in PQ-treated chloroplasts were reversed by Spd pretreatment In a native gel assay, the Cu/ZnSOD isozyme, which disappeared under the PQ alone treatment, was significantly recovered when tissues were pretreated with Spd. The dominant APX4 isozyme activity, which was preferentially decreased in response to PQ alone treatment, was also strongly reactivated by earlier Spd exposure. Therefore, we suggest that Spd could play a substantial role in protecting the radish chloroplasts from PQ stress. Furthermore, the enhancement of the Cu/ZnSOD and APX4 isozymes by Spd pretreatment seems to be responsible for prevention of the PQ-induced decreases in the total activities of SOD and APX, thereby providing a tolerance to PQ toxicity.