The effect of bicarbonate anion (HCO(<em>3</em>)(-)) on the peroxidase activity of copper, zinc superoxide dismutase (<em>SOD</em>1) was investigated using three structurally different probes: 5, 5'-dimethyl-1-pyrroline N-oxide (DMPO), tyrosine, and 2, 2'-azino-bis-[<em>3</em>-ethylbenzothiazoline]-6-sulfonic acid (ABTS). Results indicate that HCO(<em>3</em>)(-) enhanced <em>SOD</em>/H(2)O(2)-dependent (i) hydroxylation of DMPO to DMPO-OH as measured by electron spin resonance, (ii) oxidation and nitration of tyrosine to dityrosine, nitrotyrosine, and nitrodityrosine as measured by high pressure liquid chromatography, and (iii) oxidation of ABTS to the ABTS cation radical as measured by UV-visible spectroscopy. Using oxygen-17-labeled water, it was determined that the oxygen atom present in the DMPO-OH adduct originated from H(2)O and not from H(2)O(2). This result proves that neither free hydroxyl radical nor enzyme-bound hydroxyl radical was involved in the hydroxylation of DMPO. We postulate that HCO(<em>3</em>)(-) enhances <em>SOD</em>1 peroxidase activity via formation of a putative carbonate radical anion. This new and different perspective on HCO(<em>3</em>)(-)-mediated oxidative reactions of <em>SOD</em>1 may help us understand the free radical mechanism of <em>SOD</em>1 and related mutants linked to amyotrophic lateral sclerosis.

An experimental chemical N-[2-(2-oxo-1-imidazolidinyl)ethyl]-N'-phenylurea (EDU), is an effective protectant against acute and chronic foliar injury due to ozone (0(<em>3</em>)) when sprayed on intact leaves or supplied to the plants through soil application. An 0(<em>3</em>)-sensitive snap bean cultivar (Phaseolus vulgaris L. ;Bush Blue Lake 290') was systemically treated with EDU (0, 25, 50, and 100 milligrams per 15-centimeter diameter pot) to determine if EDU-induced or activated protective oxyradical and peroxyl scavenging enzymes. EDU-enhanced tolerance to O(<em>3</em>) injury always correlated with increases in superoxide dismutase (<em>SOD</em>) and catalase activities in the leaves. Peroxidase levels correlated more closely with foliar injury. Greater <em>SOD</em> levels in young leves compared to older leaves were associated with lower ozone sensitivities in these tissues.Polyacrylamide slab gel electrophoresis separations and specific determinations of <em>SOD</em> activity showed that EDU-treated plants possessed markedly greater <em>SOD</em> activity than non-treated plants. Tolerant plant tissues may have enhanced enzyme scavenging capabilities for the protection against toxic oxyradicals. Experimental confirmation for the oxyradical theory for O(<em>3</em>) phytotoxicity and <em>SOD</em> involvement in the detoxification process are presented.

OBJECTIVE

Subarachnoid hemolysate (SAH) has been associated with oxidative brain injury, cell death, and apoptosis. We hypothesized that over-expression of CuZn-superoxide dismutase (CuZn-SOD) would protect against injury after SAH, whereas reduction of its expression would exacerbate injury.

METHODS

Saline (n=16) or hemolysate (n=50) was injected into transgenic mice overexpressing CuZn-<em>SOD</em> (<em>SOD</em>1-Tg), CuZn-<em>SOD</em> heterozygous knockout mutants (<em>SOD</em>1+/-), and wild-type littermates (Wt). Mice were killed at 24 hours. Stress gene induction was evaluated by immunocytochemistry and Western blotting for hemeoxygenase-1 and heat shock protein 70. Apoptosis was evaluated by 3'-OH nick end-labeling and DNA gel electrophoresis. Cell death was quantified through histological assessment after cresyl violet staining.

RESULTS

Histological assessment demonstrated neocortical cell death in regions adjacent to the blood injection. Overall cell death was reduced 43% in <em>SOD</em>1-Tg mutants (n=6) compared with Wt littermates (n=6; P<0.02). In contrast, cell death was increased >40% in <em>SOD</em>1+/- mutants (n=6; P<0.05). Both hemeoxygenase-1 and heat shock protein 70 were induced after SAH. Apoptosis was also present after SAH, as evidenced by 3'-OH end-labeling and DNA laddering. However, the degree of stress gene induction and apoptosis did not vary between Wt, <em>SOD</em>1-Tg, and <em>SOD</em>1+/- mice.

CONCLUSIONS

The extent of CuZn-SOD expression in the cytosol correlates with cell death after exposure to SAH in a manner separate from apoptosis. Overexpression of CuZn-SOD may potentially be an avenue for therapeutic intervention.

We recently showed that intraischemic moderate hypothermia (<em>3</em>0 degrees C) reduces ischemic damage through the Akt pathway after permanent distal middle cerebral artery occlusion in rats. The only Akt pathway component preserved by hypothermia is phosphorylated phosphatase and tensin homolog deleted on chromosome 10 (p-PTEN), which suggests that p-PTEN may have a central role in neuroprotection. Reactive oxygen species (ROS) are critically involved in mediating ischemic damage after stroke by interacting with signaling molecules, including Akt, PTEN, and delta-protein kinase C (PKC). We investigated the protective mechanisms of moderate hypothermia on these signaling proteins after transient focal ischemia in rats. Early moderate hypothermia (<em>3</em> h) was administered 15 mins before reperfusion, and delayed moderate hypothermia (<em>3</em> h) was applied 15 mins after reperfusion. Our results indicate that early hypothermia reduced infarction, whereas delayed hypothermia did not. However, both early and delayed hypothermia maintained levels of Mn-<em>SOD</em> (superoxide dismutase) and phosphorylated Akt and blocked delta-PKC cleavage, suggesting that these factors may not be critical to the protection of hypothermia. Nevertheless, early hypothermia preserved p-PTEN levels after reperfusion, whereas delayed hypothermia did not. Furthermore, ROS inhibition maintained levels of p-PTEN after stroke. Together, these findings suggest that phosphorylation levels of PTEN are closely associated with the protective effect of early hypothermia against stroke.

We investigated the effects of high cholesterol diet in the absence and presence of vitamin E on the activity of antioxidant enzymes [superoxide dismutase (<em>SOD</em>), catalase, glutathione peroxidase (GSH-Px)] in rabbits. The animals were divided into 4 groups each comprising of 10 rabbits. Group I, regular rabbit chow diet; Group II, regular rabbit chow diet with added vitamin E; Group III, high cholesterol diet; and Group IV, high cholesterol diet+vitamin E. Antioxidant enzymes of blood were measured in each group before and after 1, 2, <em>3</em>, and 4 months on the experimental diets. The aorta was removed at the end of the protocol for measurement of antioxidant enzymes. There was a decrease in activity of <em>SOD</em> and GSH-Px and an increase in activity of catalase in blood of Group III. Vitamin E produced a decrease in blood <em>SOD</em>, catalase and GSH-Px activity in Group II and prevented the decrease in <em>SOD</em> and GSH-Px activity in Group IV but did not affect the changes in the catalase activity. <em>SOD</em>, catalase and GSH-Px activity of aortae from Group III increased significantly, while catalase activity increased and GSH-Px activity decreased in those from Group II. Vitamin E prevented the cholesterol-induced rise in catalase and GSH-Px activity in aorta but did not prevent the rise in <em>SOD</em> activity. These results suggest that the activity of antioxidant enzymes in blood is affected differently from that in aortic tissue. There appears to be a mutually supportive interaction among the antioxidant enzymes which provide defense against oxidant injury. The protective effects of vitamin E against hypercholesterolemic atherosclerosis may not be due to changes in the antioxidant enzymes but may be mainly mediated through its chain-breaking antioxidant activity.

The aims of this study are to investigate the contribution effect of oxidative stress in MK-801-induced experimental psychosis model, and to show that prevention of oxidative stress may improve prognosis. Because oxidative damage has been suggested in the neuropathophysiology of schizophrenia, the possible protecting agents against lipid peroxidation are potential target for the studies in this field. For this purpose, Wistar Albino rats were divided into three groups: the first group was used as control, MK-801 was given to the rats in the second group and MK-801+omega-<em>3</em> essential fatty acids (EFA) was given to the third group. MK-801 was given intraperitoneally at the dose of 0.5mg/(kgday) once a day for 5 days in experimental psychosis group. In the second group, 0.8g/(kgday), omega-<em>3</em> FA (eicosapentaenoic acid, 18%, docosahexaenoic acid, 12%) was given to the rats while exposed MK-801. In control group, saline was given intraperitoneally at the same time. After 7 days, rats were killed by decapitation. Prefrontal brain area was removed for histological and biochemical analyses. As a result, malondialdehyde (MDA), as an indicator of lipid peroxidation, protein carbonyl (PC), as an indicator of protein oxidation, nitric oxide (NO) levels and superoxide dismutase (<em>SOD</em>), glutathione peroxidase (GSH-Px) activities as antioxidant enzymes, and xanthine oxidase (XO) and adenosine deaminase (AD) activities as an indicator of DNA oxidation was found to be increased significantly in prefrontal cortex (PFC) of MK-801 group (P<0.0001) compared to control group. In omega-<em>3</em> FA treated rats, prefrontal tissue MDA, PC and NO levels as well as <em>SOD</em>, GSH-Px, XO, and AD enzyme activities were significantly decreased when compared to MK-801 groups (P<0.0001) whereas catalase (CAT) enzyme activity was not changed. Moreover, in the light of microscopic examination of MK-801 groups, a great number of apoptotic cells were observed. omega-<em>3</em> FA supplementation decreased the apoptotic cell count in PFC. The results of this study revealed that oxidative stress and apoptotic changes in PFC may play an important role in the pathogenesis of MK-801-induced neuronal toxicity. This experimental study also provides some evidences for the protective effects of omega-<em>3</em> FA on MK-801-induced changes in PFC of rats.

OBJECTIVE

The clinical usefulness of doxorubicin (adriamycin, ADR) is restricted by the risk of developing congestive heart failure. Probucol has been reported to completely prevent ADR cardiomyopathy without interfering with its antitumor effects. The current study investigated the effects of ADR and probucol on antioxidant enzyme gene expression during adriamycin-induced cardiomyopathy in a rat model.

METHODS

The mRNA abundance by Northern and immunoreactive protein levels by Western blotting of myocardial antioxidant enzymes, glutathione peroxidase (GSHPx), manganese superoxide dismutase (MnSOD) and catalase (CAT) were examined in relation to the enzyme activities in hemodynamically assessed control and treated animals.

RESULTS

At <em>3</em> weeks post-treatment duration, ADR caused heart failure which was prevented by probucol. Mn<em>SOD</em> mRNA abundance as well as protein levels were depressed by ADR treatment by 45% and 20%, respectively, and this change was prevented by probucol. However, the mRNA and protein levels of GSHPx and CAT were not significantly changed by ADR or probucol. ADR had no effect on <em>SOD</em> activity but this enzyme activity was increased by probucol and probucol plus ADR. GSHPx enzyme activity was decreased and oxidative stress as indicated by TBARS was increased by ADR and these changes were also modulated by probucol.

CONCLUSIONS

An increase in oxidative stress, GSHPx inactivation and MnSOD downregulation during ADR cardiomyopathy were prevented by probucol treatment.

The identification of the pathogenic mechanism of selective motor neuron (MN) death in amyotrophic lateral sclerosis (ALS) may lead to the development of new therapies to halt or slow the disease course. A novel, MN-specific, Fas-mediated programmed cell death (PCD) pathway has been reported in MNs which involves the activation of p<em>3</em>8 MAP kinase (phospho-p<em>3</em>8) and neuronal nitric oxide synthase (nNOS). PCD was found to be exacerbated in MNs expressing ALS-linked superoxide dismutase (<em>SOD</em>) mutations. Because this MN-specific pathway was investigated in vitro, we performed an in vivo study to evaluate its potential involvement in MN loss in the lumbar region of spinal cord of mutant <em>SOD</em> transgenic (G9<em>3</em>A) mice. Compared to nontransgenic littermates, we found significant increases in the numbers of immunopositive ventral horn MNs of G9<em>3</em>A mice as young as 60 days of age for several constituents of this putative PCD pathway, including phospho-p<em>3</em>8, nNOS, phospho-ASK1 MAP kinase kinase, and active caspase-<em>3</em>. This study provides in vivo evidence of an MN-specific PCD pathway that may be a pathogenic mechanism of ALS and may be activated very early in the disease process, well before clinical symptoms are evident (200 days). These findings suggest that early diagnosis and therapeutic intervention may be critical for the successful treatment of the disease. These enzymes may provide new markers for earlier diagnosis of ALS and new molecular targets for therapeutic intervention.

1. Tardive dyskinesia (TD), a syndrome of potentially irreversible, involuntary hyperkinetic disorder occurring in 20 - 40% of the patient population undergoing chronic neuroleptic treatment is a major limitation of neuroleptic therapy. 2. Oxidative stress and products of lipid peroxidation are implicated in the pathophysiology of various neurological disorders including tardive dyskinesia. <em>3</em>. Chronic treatment with neuroleptics leads to the development of abnormal oral movements in rats known as vacuous chewing movements (VCMs). Vacuous chewing movements in rats are widely accepted as an animal model of tardive dyskinesia. 4. All the antipsychotics were administered i.p. once daily for 21 days, whereas carvedilol (also i.p.) was administered twice daily. Rats chronically treated with haloperidol (1.0 mg kg(-1)) or chlorpromazine (5 mg kg(-1)) but not clozapine (2 mg kg(-1)) significantly developed vacuous chewing movements and tongue protrusions. Carvedilol dose dependently (0.5 - 2 mg kg(-1)) reduced the haloperidol or chlorpromazine-induced vacuous chewing movements and tongue protrusions. 5. Biochemical analysis revealed that chronic haloperidol or chlorpromazine but not clozapine treatment significantly induced lipid peroxidation and decreased the glutathione (GSH) levels in the forebrains of rats. Chronic haloperidol or chlorpromazine but not clozapine treated rats showed decreased forebrain levels of antioxidant defence enzymes, superoxide dismutase (<em>SOD</em>) and catalase. 6. Co-administration of carvedilol (0.5-2 mg kg(-1)) significantly reduced the lipid peroxidation and restored the decreased glutathione levels by chronic haloperidol or chlorpromazine treatment. Co-administration of carvedilol (1-2 mg kg(-1)) significantly reversed the haloperidol or chlorpromazine-induced decrease in forebrain <em>SOD</em> and catalase levels in rats. However, lower dose of carvedilol (0.5 mg kg(-1)) failed to reverse chronic haloperidol or chlorpromazine-induced decrease in forebrain <em>SOD</em> and catalase levels. 7. The major findings of the present study suggest that oxidative stress might play a significant role in neuroleptic-induced orofacial dyskinesia. In conclusion, carvedilol could be a useful drug for the treatment of neuroleptic-induced orofacial dyskinesia.

It is widely accepted that human islet amyloid polypeptide (hIAPP) aggregation plays an important role in the loss of insulin-producing pancreatic beta cells. hIAPP-induced cytotoxicity is mediated by generation of reactive oxygen species (ROS). Phycocyanin (PC) is a natural compound from blue-green algae that is widely used as food supplement. Currently, little is known about the effects of PC on beta cells with the presence of hIAPP. The aim of this study was to investigate the in vitro protective effects of PC on INS-1E rat insulinoma beta cells against hIAPP-induced cell death, as well as the underlying mechanisms. Our results showed that hIAPP-induced cell death with apoptotic characteristics including growth inhibition, chromatin condensation and DNA fragmentation. However, cytotoxicity of hIAPP was significantly attenuated by co-incubation of the cells with PC. The results of Western blotting showed that activation of caspase-<em>3</em> and cleavage of poly (ADP-ribose) polymerase (PARP) in hIAPP-treated cells was blocked by PC. Moreover, PC significantly prevented the hIAPP-induced overproduction of intracellular ROS and malondialdehyde (MDA), as well as changes in activities of superoxide dismutase (<em>SOD</em>) and glutathione peroxidase (GSH-Px) enzymes. Furthermore, hIAPP triggered the activation of mitogen-activated protein kinases (MAPKs), and these effects were effectively suppressed by PC. Taken together, our results suggest that PC protects INS-1E pancreatic beta cells against hIAPP-induced apoptotic cell death through attenuating oxidative stress and modulating c-Jun N-terminal kinase (JNK) and p<em>3</em>8 pathways.

BACKGROUND

Epidemiologic studies have shown an inverse correlation between acute coronary events and high intake of dietary vitamin E. Recent clinical studies, however, failed to show any beneficial effects of alpha-tocopherol on cardiovascular events. Absence of tocopherols other than alpha-tocopherol in the clinical studies may account for the conflicting results.

OBJECTIVE

This study compared the effect of a mixed tocopherol preparation rich in gamma-tocopherol with that of alpha-tocopherol on platelet aggregation in humans and addressed the potential mechanisms of the effect.

METHODS

Forty-six subjects were randomly divided into <em>3</em> groups: alpha-tocopherol, mixed tocopherols, and control. ADP and phorbol 12-myristate 1<em>3</em>-acetate-induced platelet aggregation, nitric oxide (NO) release, activation of endothelial constitutive nitric-oxide synthase (ecNOS; EC 1.14.1<em>3</em>.<em>3</em>9) and of protein kinase C (PKC), and ecNOS, superoxide dismutase (<em>SOD</em>; EC 1.15.1.1), and PKC protein content in platelets were measured before and after 8 wk of administration of tocopherols.

RESULTS

ADP-induced platelet aggregation decreased significantly in the mixed tocopherol group but not in the alpha-tocopherol and control groups. NO release, ecNOS activation, and SOD protein content in platelets increased in the tocopherol-treated groups. PKC activation in platelets was markedly decreased in the tocopherol-treated groups. Mixed tocopherols were more potent than alpha-tocopherol alone in modulating NO release and ecNOS activation but not SOD protein content or PKC activation.

CONCLUSIONS

Mixed tocopherols were more potent in preventing platelet aggregation than was alpha-tocopherol alone. Effects of mixed tocopherols were associated with increased NO release, ecNOS activation, and SOD protein content in platelets, which may contribute to the effect on platelet aggregation.

OBJECTIVE

The oxygen toxicity risk of hyperbaric oxygen (HBO) treatment has long been of interest. However, there are no comprehensive articles describing the relationship between HBO protocols and oxidative parameters used clinically. The purpose of this study was to determine the effects of various HBO pressure modalities on the oxidative values of rat lung, brain, and erythrocytes.

METHODS

A total of 64 male Sprague-Dawley rats was randomly divided into 7 groups. Group A was used as a control. Groups C to G were subjected to 100% oxygen at a pressure of 1, 1.5, 2, 2.5, and <em>3</em> ATA (atmosphere absolute), respectively, for 2 h. Group B was exposed to normal atmospheric air at <em>3</em> ATA for the same duration. The rat's lung, brain, and blood were taken immediately after the exposure and thiobarbituric acid reactive substances (TBARS) and superoxide dismutase (<em>SOD</em>) levels were determined.

RESULTS

Both TBARS levels and <em>SOD</em> activity increased concordantly with the pressure increase. Although a statistically significant change in TBARS levels started from 100% oxygen exposure at 1 ATA (normobaric), <em>SOD</em> activity was affected after 2 ATA. A significant correlation exists between exposure pressure and the aforementioned parameters. Ambient air exposure at <em>3</em> ATA did not affect any parameters besides the brain TBARS levels.

CONCLUSIONS

It is clear that HBO exposure causes oxidative stress. The main reason for this effect seems to be exposure to pure oxygen, since pure high pressure has no significant effect on the aforementioned parameters. However, clinicians should use as low pressures as possible since all oxidative parameters appear to be directly proportional to the extent of HBO exposure.

We reported previously that a single tryptophan residue, Trp<em>3</em>2, in human Cu,Zn-superoxide dismutase is specifically modified by peroxynitrite-CO2 [Yamakura et al. (2001) Biochim. Biophys. Acta 1548, <em>3</em>8-46]. In this study, we modified Cu,Zn-superoxide dismutase by using a combination of myeloperoxidase, hydrogen peroxide, and nitrite. The modified enzyme showed no loss of copper and zinc, and 15% less enzymatic activity. Trp<em>3</em>2 was the only significant amino acid lost. After trypsin digestion of the modified <em>SOD</em> with peroxynitrite-CO2 and the myeloperoxidase system, six newly appearing peptides containing tryptophan derivatives were observed on microLC-ESI-Q-TOF mass analyses and HPLC with a photodiode-array detector. The derivatives of the tryptophan residue exhibiting mass increases of 4, 16 (2 peaks), <em>3</em>2, 45 (major), and 45 Da (minor) were identified as kynurenine, oxindole-<em>3</em>-alanine and its derivatives, dihydroxytryptophan, 6-nitrotryptophan and 5-nitrotryptophan, respectively. We further identified 6-nitrotryptophan from the 1H-NMR spectrum for the pronase-digested product and calculated the yield of 6-nitrotryptophan as being about <em>3</em>0% for each of the modification methods. The tryptophan residue in the modified human Cu,Zn-superoxide dismutase gave the same spectra for the products including 6-nitrotryptophan as the major nitrated product with the two different modification systems.

Haemophilus influenzae type b, a causative agent of bacterial sepsis and meningitis in young children, contains a single superoxide dismutase (<em>SOD</em>), a cytoplasmic Mn<em>SOD</em>. To study the role of this enzyme, a chromosomal sodA::lacZ mutant (M-2) was constructed. M-2 had an increased sensitivity towards oxygen and the redox-active agent paraquat. A <em>3</em>.4-fold increase in sodA-lacZ expression was found in M-2 grown with oxygen supply rates between <em>3</em> and <em>3</em>6 mmol of O2/liter/h. In similar experiments with the wild type, assaying SodA activity, a <em>3</em>.1-fold increase was found. Both the wild type and M-2 grew best at the lowest oxygen supply rate tested, consistent with the notion that H. influenzae prefers a more anaerobic environment. In the infant rat model of infection, the ability of M-2 to colonize the nasopharynx was found to be impaired, but its ability to cause invasive disease was unaffected. This suggests that after invasion, the growth disadvantage imposed by a SodA- phenotype is not limiting.

Reactive oxygen intermediates have been implicated in lung injury induced by inhaled irritants. The present studies used mice overexpressing Cu/Zn-superoxide dismutase (<em>SOD</em>+/+) to analyze their role in ozone-induced lung inflammation and cytotoxicity. Treatment of wild-type mice with ozone (0.8 ppm, <em>3</em> h) resulted in increased bronchoalveolar lavage fluid protein, which was maximal after 24-48 h. Significant increases in lung macrophages and 4-hydroxyalkenals were also observed. In contrast, bronchoalveolar lavage fluid protein and macrophage content and 4-hydroxyalkenals were at control levels in ozone-treated <em>SOD</em>+/+ mice. There was also no evidence of peroxynitrite-mediated lung damage, demonstrating that <em>SOD</em>+/+ mice are resistant to ozone toxicity. Whereas alveolar macrophages from wild-type mice produced increased amounts of nitric oxide and expressed more inducible nitric oxide synthase, phospholipase A(2), and tumor necrosis factor-alpha after ozone inhalation, this was not evident in cells from <em>SOD</em>+/+ mice. Ozone-induced decreases in interleukin-10 were also not observed. In wild-type mice, ozone inhalation resulted in activation of nuclear factor-kappaB, which regulates proinflammatory gene activity. This response was significantly reduced in <em>SOD</em>+/+ mice. These data demonstrate that antioxidant enzymes play a critical role in ozone-induced tissue injury and in inflammatory mediator production.

1. Endotoxaemia causes an enhanced formation of reactive oxygen species (ROS) which contribute to the multiple organ dysfunction syndrome (MODS) in septic shock. Here we investigate (i) the effects of endotoxin on the expression of two isoforms of superoxide dismutase (<em>SOD</em>), namely Cu/Zn-<em>SOD</em> (cytosol) and Mn-<em>SOD</em> (mitochondria) in the rat kidney, and (ii) the effects of the radical scavenger tempol on the MODS caused by lipopolysaccharide (LPS, E. coli, 6 mg kg(-1) i.v.) in the rat. 2. Endotoxaemia resulted in a rapid, but transient, decline in the expression of both mRNA and protein of Cu/Zn-<em>SOD</em> as well as an increase in the expression of the mRNA of Mn-<em>SOD</em> in the kidney. Endotoxaemia for 6 h also caused hypotension, acute renal dysfunction, hepatocellular injury, pancreatic injury and an increase in the plasma levels of nitrite/nitrate. <em>3</em>. Pretreatment of rats with tempol (100 mg kg(-1) i.v. bolus injection, 15 min prior to LPS followed by an infusion of <em>3</em>0 mg kg(-1) i.v., n=9) did not affect the circulatory failure, but attenuated the renal dysfunction and the hepatocellular injury/dysfunction caused by LPS. Tempol did not affect the rise in nitrite/nitrate caused by endotoxin. 4. These results imply that an enhanced formation of ROS (including superoxide anions) in conjunction with inadequate defences against such ROS contributes to the injury and dysfunction of the kidney and the liver in endotoxic shock.

Many fungi of the genus Cercospora produce a light-induced, photoactivated polyketide toxin called cercosporin. In the presence of light an excited form (triplet state) of the toxin molecule is produced which, depending on the reducing potential of the environment, reacts with molecular oxygen to produce singlet oxygen and/or superoxide radicals. In this paper a system is presented for analysis of antioxidant defense gene response using purified cercosporin under conditions demonstrated to favor superoxide formation. Under the assay conditions employed, changes in total catalase activity, as well as individual isozyme protein levels generally mirrored the changes observed in corresponding steady-state RNA levels in response to applied cercosporin. In contrast, while transcript accumulation for most maize superoxide dismutases increased dramatically, both total superoxide dismutase activity and individual isozyme protein levels remained constant in all toxin treatments. In one case, the analyses indicated that there are two distinct transcripts that hybridize with a gene-specific probe for Sod<em>3</em>. These two transcripts responded differentially to applied toxin (levels of the larger transcript increased while the smaller decreased), whereas corresponding steady-state levels for the <em>SOD</em>-<em>3</em> isozyme proteins remained constant. This suggests that protein turnover might play a role in the response of these <em>SODs</em> to activated oxygen species.

The comparative pharmacology of endothelium-derived relaxing factor (EDRF) and nitric oxide (NO) was studied on isolated strips of rabbit aorta. Both compounds were equally unstable. The relaxations of the bioassay tissues induced by EDRF released by bradykinin (<em>3</em>-100 nM) and by NO (4-1<em>3</em>4 nM) were indistinguishable. The stability of both compounds was increased to a similar extent by infusions of superoxide dismutase (<em>SOD</em>; 15 U.ml-1) or of cytochrome c (40 microM). The relaxations induced by EDRF and NO were inhibited to similar extents by infusions of Fe2+, hydroquinone and pyrogallol, an effect attenuated by a concomitant infusion of <em>SOD</em> or cytochrome c. The relaxations induced by both compounds were also inhibited by haemoglobin, however, this effect was unaltered by concomitant infusion of <em>SOD</em>. These data indicate that EDRF and NO have identical biological activity, stability and susceptibility to inactivation by superoxide anions and haemoglobin, providing further confirmation that EDRF is NO.

Mutant strains of the yeast Saccharomyces cerevisiae which lack functional Cu,Zn superoxide dismutase (<em>SOD</em>-1) do not grow aerobically unless supplemented with methionine. The molecular basis of this O2-dependent auxotrophy in one of the mutants, Dscd1-1C, has been investigated. Sulfate supported anaerobic but not aerobic mutant growth. On the other hand, cysteine and homocysteine supported aerobic growth while serine, O-acetylserine, and homoserine did not, indicating that the interconversion of cysteine and methionine (and homocysteine) was not impaired. Thiosulfate (S2O<em>3</em>(2-] and sulfide (S2-) also supported aerobic growth; the activities of thiosulfate reductase and sulfhydrylase in the aerobic mutant strain were at wild-type levels. Although the levels of SO4(2-) and adenosine-5'-sulfate (the first intermediate in the SO4(2-) assimilation pathway) were elevated in the aerobically incubated mutant strain, this condition could be attributed to a decrease in protein synthesis caused by the de facto sulfur starvation and not to a block in the pathway. Therefore, the activation of SO4(2-) (to form <em>3</em>'-phosphoadenosine-5'-phosphosulfate) appeared to be O2 tolerant. Sulfite reductase activity and substrate concentrations [( NADPH] and [SO<em>3</em>(2-)]) were not significantly different in aerobically grown mutant cultures and anaerobic cultures, indicating that <em>SOD</em>-1- mutant strains could reductively assimilate sulfur oxides. However, the mutant strain exhibited an O2-dependent sensitivity to SO<em>3</em>(2-) concentrations of less than 50 microM not exhibited by any <em>SOD</em>-1+ strain or by <em>SOD</em>-1- strains supplemented with a cytosolic O2(-)-scavenging activity. This result suggests that the aerobic reductive assimilation of SO4(2-) at the level of SO<em>3</em>(2-) may generate a cytotoxic compound(s) which persists in <em>SOD</em>-(1-) yeast strains.

On the basis that ozone (O<em>3</em>) can upregulate cellular antioxidant enzymes, a morphological, biochemical and functional renal study was performed in rats undergoing a prolonged treatment with O<em>3</em> before renal ischaemia. Rats were divided into four groups: (1) control, a medial abdominal incision was performed to expose the kidneys; (2) ischaemia, in animals undergoing a bilateral renal ischaemia (<em>3</em>0 min), with subsequent reperfusion (<em>3</em> h); (<em>3</em>) O<em>3</em> + ischaemia, as group 2, but with previous treatment with O<em>3</em> (0.5 mg/kg per day given in 2.5 ml O2) via rectal administration for 15 treatments; (4) O2 + ischaemia, as group <em>3</em>, but using oxygen (O2) alone. Biochemical parameters as fructosamine level, phospholipase A, and superoxide dismutases (<em>SOD</em>) activities, as well as renal plasma flow (RPF) and glomerular filtration rate (GFR), were measured by means of plasma clearance of p-amino-hippurate and inulin, respectively. In comparison with groups 1 and <em>3</em>, the RPF and GFR were significantly decreased in groups 2 and 4. Interestingly, renal homogenates of the latter groups yielded significantly higher values of phospholipase A activity and fructosamine level in comparison with either the control (1) and the O<em>3</em> (<em>3</em>) treated groups. Moreover renal <em>SOD</em> activity showed a significant increase in group <em>3</em> without significant differences among groups 1, 2 and 4. Morphological alterations of the kidney were present in 100%, 88% and <em>3</em>0% of the animals in groups 2, 4 and <em>3</em>, respectively. It is proposed that the O<em>3</em> protective effect can be ascribed to the substantial possibility of upregulating the antioxidant defence system capable of counteracting the damaging effect of ischaemia. These findings suggest that, whenever possible, ozone preconditioning may represent a prophylactic approach for minimizing renal damage before transplantation.

This study was carried out to investigate the protective role of taurine (2-aminoethanesulphonicacid) against morphine-induced neurotoxicity in C6 cells. It was found that taurine significantly increased the viability of C6 cells treated by morphine, showing the neuroprotective role against morphine-induced neurotoxicity. However, such neuroprotective effect of taurine could not be blocked by bicuculline, an antagonist of gamma-amino butyrate (GABA) receptor. To determine the oxidative damage induced by morphine, the superoxide dismutase (<em>SOD</em>), catalase (CAT), and glutathione peroxidase (GPx) were measured in C6 cells. The decreased activities of <em>SOD</em>, CAT, and GPx in C6 cells were observed after morphine treatment for 48 h. However, taurine administration effectively ameliorated morphine-induced oxidative insult. To estimate anti-apoptosis effect of taurine, flow cytometry analysis as well as detection for caspase-<em>3</em> and Bcl-2 expressions was performed after morphine exposure for 48 h. It was found that Bcl-2 expression was down regulated by morphine, whereas taurine could reverse morphine-induced decrease in Bcl-2 expression. Taurine showed no effect on caspase-<em>3</em> expression. Collectively, the results show that taurine possesses the capability to ameliorate morphine-induced oxidative insult and apoptosis in C6 cells, probably due to its antioxidant activity rather than activation of GABA receptors.

Flavonoids exist extensively in plants and Chinese herbs, and several biological effects of flavonoids have been demonstrated. The antitumor effects in colorectal carcinoma cells (HT29, COLO205, and COLO<em>3</em>20HSR) of eight flavanones including flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone, 7-OH flavanone, naringenin, nargin, and taxifolin were investigated. Results of the MTT assay indicate that 2'-OH flavanone showed the most potent cytotoxic effect on these three cells, and cell death induced by 2'-OH flavanone was via the occurrence of DNA ladders, apoptotic bodies, and hypodiploid cells, all characteristics of apoptosis. Induction of caspase <em>3</em> protein processing and enzyme activity associated with cleavage of poly(ADP-ribose) polymerase (PARP) was identified in 2'-OH flavanone-treated cells, and a peptidyl inhibitor (Ac-DEVD-FMK) of caspase <em>3</em> attenuated the cytotoxicity of 2'-OH flavanone in COLO205 and HT-29 cells. Elevation of p21 (but not p5<em>3</em>) and a decrease in Mcl-1 protein were found in 2'-OH flavanone-treated COLO205 and HT-29 cells. Elevation of intracellular reactive oxygen species (ROS) was detected in 2'-OH flavanone-treated cells by the 2',7'-dichlorodihydrofluorescein diacetate (DCHF-DA) assay, and ROS scavengers including 4,5-dihydro-1,<em>3</em>-benzene disulfonic acid (tiron), catalase, superoxide dismutase (<em>SOD</em>), and pyrrolidine dithiocarbamate (PDTC) suppressed the 2'-OH flavanone-induced cytotoxic effect. Subcutaneous injection of COLO205 induced tumor formation in nude mice, and 2'-OH flavanone showed a significant inhibitory effect on tumor formation. The appearance of apoptotic cells with H&E staining, and an increase in p21, but not p5<em>3</em>, protein by immunohistochemistry were observed in tumor tissues under 2'-OH flavanone treatment. Primary tumor cells (COLO205-X) derived from a tumor specimen elicited by COLO205 were established, and 2'-OH flavanone showed an significant apoptotic effect in COLO205-X cells in accordance with the appearance of DNA ladders, caspase <em>3</em> protein processing, PARP protein cleavage, and increasing p21 protein. These results revealed in vitro, ex vivo, and in vivo antitumor activities of 2'-OH flavanone via apoptosis induction, and indicates that 2'-OH flavanone is an active compound worthy of development for cancer chemotherapy.

Seedlings of sweet orange (Citrus sinensis) were fertilized for 14 weeks with boron (B)-free or B-sufficient (2.5 or 10 microM H(<em>3</em>)BO(<em>3</em>)) nutrient solution every other day. Boron deficiency resulted in an overall inhibition of plant growth, with a reduction in root, stem and leaf dry weight (DW). Boron-starved leaves showed decreased CO(2) assimilation and stomatal conductance, but increased intercellular CO(2) concentrations. Activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), NADP-glyceraldehyde-<em>3</em>-phosphate dehydrogenase (NADP-GAPDH) and stromal fructose-1,6-bisphosphatase (FBPase) were lower in B-deficient leaves than in controls. Contents of glucose, fructose and starch were increased in B-deficient leaves while sucrose was decreased. Boron-deficient leaves displayed higher or similar superoxide dismutase (<em>SOD</em>), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR) and glutathione reductase (GR) activities, while dehydroascorbate reductase (DHAR) and catalase (CAT) activities were lower. Expressed on a leaf area or protein basis, B-deficient leaves showed a higher ascorbate (AsA) concentration, but a similar AsA concentration on a DW basis. For reduced glutathione (GSH), we found a similar GSH concentration on a leaf area or protein basis and an even lower content on a DW basis. Superoxide anion (O(2)(-)) generation, malondialdehyde (MDA) concentration and electrolyte leakage were higher in B-deficient than in control leaves. In conclusion, CO(2) assimilation may be feedback-regulated by the excessive accumulation of starch and hexoses in B-deficient leaves via direct interference with chloroplast function and/or indirect repression of photosynthetic enzymes. Although B-deficient leaves remain high in activity of antioxidant enzymes, their antioxidant system as a whole does not provide sufficient protection from oxidative damage.

Huntington's disease is a progressive neurodegenerative disorder that gradually reduces memory, cognitive skills and normal movements of affected individuals. Systemic administration of <em>3</em>-Nitropropionic acid induces selective striatal lesions in rodents and non-human primates. Therefore, the present study has been designed to elucidate the comparative mechanistic profile of gabapentin, lamotrigine and their interactions with GABAergic modulators against <em>3</em>-Nitropropionic acid induced neurotoxicity. Systemic <em>3</em>-Nitropropionic acid (10 mg/kg) administration for 14 days significantly reduced body weight, locomotor activity, grip strength, oxidative defense (LPO, nitrite, <em>SOD</em> and catalase) and impaired mitochondrial complex enzyme (I, II, IV and MTT assay) activities in the striatum. <em>3</em>-Nitropropionic acid treatment also increased TNF-α level in the striatum. Gabapentin (50 and 100 mg/kg) and lamotrigine (10, 20 and 40 mg/kg) treatments significantly restored behavioural, oxidative defense and mitochondrial complex enzyme activities and proinflammatory markers (TNF-α) as compared to <em>3</em>-Nitropropionic acid treated group. Systemic picrotoxin (1 mg/kg) pretreatment with sub effective dose of gabapentin (50 mg/kg) or lamotrigine (20mg/kg) significantly attenuated their protective effect. Further, GABA (50 mg/kg) and/or muscimol (0.05 mg/kg) pretreatment with sub effective dose gabapentin (50 mg/kg) and lamotrigine (20 mg/kg) significantly potentiated their protective effects which were significant as compared to their effect alone. The results of the present study suggest that a GABAergic mechanism is involved in the protective effect of gabapentin and lamotrigine against <em>3</em>-Nitropropionic acid induced neurotoxicity.