Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder, characterized by the degeneration of upper and lower motor neurons (MNs). Central nervous system features include a loss of Betz cells and other pyramidal cells from sensorimotor cortex. The intrinsic mechanism underlying this selective motor neuron loss has not been identified. A recent in vitro study has provided evidence of a novel programmed cell death (PCD) pathway that is unique to spinal cord MNs and is exacerbated by superoxide dismutase (<em>SOD</em>) mutations. This PCD pathway is triggered through the Fas receptor and involves the apoptosis signal-regulating kinase 1 (ASK1), the p<em>3</em>8 MAP kinase, and the neuronal form of nitric oxide synthase (nNOS). Previously, we found significant increases in the numbers of ventral horn MNs immunopositive for these enzymes in the spinal cords of mutant <em>SOD</em> transgenic (G9<em>3</em>A) mice as early as 60 days of age, suggesting that this pathway may be active in vivo. Since the upper MNs of ALS patients and G9<em>3</em>A mice are also known to degenerate, the purpose of the present study was to investigate the possible activation of this PCD pathway in the MNs of the sensorimotor cortex of G9<em>3</em>A transgenic mice. Compared to non-transgenic littermates, the G9<em>3</em>A mice showed significant increases in the numbers of MNs immunopositive for the active (phosphorylated) forms of ASK1, p<em>3</em>8, MKK<em>3</em>/6 (the known activator of p<em>3</em>8), and also active caspase-<em>3</em>, as early as 60 days of age. Another stress-activated protein kinase, c-Jun N-terminal kinase (JNK), commonly activated in other neurodegenerative disorders such as Alzheimer's disease, showed no increases in G9<em>3</em>A mice at any age. These results suggest that, not only has a PCD pathway been activated in the cortical MNs, but one that may be unique to ALS. Moreover, these findings suggest that earlier diagnosis and therapeutic intervention may be possible for successful treatment of ALS. Consequently, these enzymes may provide the biochemical markers to enable earlier diagnosis of ALS and molecular targets for the development of new therapeutic compounds.

The effectiveness of bovine superoxide dismutase (<em>SOD</em>) in the prevention of bronchopulmonary dysplasia was evaluated in a prospective double-blind controlled study in 45 neonates (mean gestational age 28.7 weeks, mean weight 1154 gm) with severe respiratory distress syndrome. All were ventilator dependent with FiO2 greater than or equal to 0.7 at 24 hours of age. Either bovine <em>SOD</em> (0.25 mg/kg) or saline solution was administered subcutaneously every 12 hours according to random selection until patients could be maintained in room air without ventilatory or continuous positive airway pressure (CPAP) support. <em>SOD</em> levels were detected in all patients given treatment. Mean peak values at 4 hours after dose ranged from 0.15 micrograms/ml (dose 1) to 0.45 micrograms/ml (dose 10). The drug was well tolerated, and no side effects were detected. Among the <em>3</em>1 survivors (<em>SOD</em> 14, placebo 17) radiologic evidence of BPD was significantly less in patients given <em>SOD</em> (<em>3</em>/14 vs 12/17, P = 0.008). Clinical signs of bronchopulmonary dysplasia (wheezing, pneumonia) were less in patients given <em>SOD</em> (<em>3</em>/14 vs 11/17, P = 0.019). Patients given <em>SOD</em> required fewer days of CPAP (P less than 0.00<em>3</em>). There were no differences in days of O2 therapy, intermittent positive pressure breathing, or incidence and severity of patent ductus arteriosus or intraventricular hemorrhage. This preliminary study suggests that <em>SOD</em> may be helpful in reducing the severity of bronchopulmonary dysplasia in infants with respiratory distress syndrome.

Physiological function of reactive oxygen species (ROS) has been known since a long, but recently toxic effects of ROS on spermatozoa have gained much importance in male infertility. Mitochondrial DNA (mtDNA) is believed to be both source and target of ROS. mtDNA unlike nuclear DNA is not compactly packed and hence more susceptible to oxidative stress (OS) than nuclear DNA. In the present study, the role of OS in mitochondrial genome changes was studied in men with idiopathic infertility. The study included <em>3</em><em>3</em> infertile oligo-asthenozoospermic (OA) men and <em>3</em>0 fertile controls. Semen analyses were performed and OS was measured by estimating the level of malondialdehye (MDA) in the seminal plasma and ROS in the sperm. Sperm mtDNA was sequenced by standard PCR-DNA sequencing protocol for ATPase and nicotinamide adenine dinucleotide dehydrogenase (ND) groups of genes. Sperm count and progressive motility were found to be significantly lower in infertile group than the fertile controls. Semen MDA and ROS levels of infertile group were significantly higher (p<0.0001), when compared to the control group. However, catalase and glutathione peroxidase (GPx) levels were significantly lower in infertile group, compared to controls, but no significant difference in superoxide dismutase (<em>SOD</em>) activity was observed between control and cases. This might be due to higher expression of <em>SOD</em> alone in order to overcome OS in the semen. mtDNA analysis showed significant and high frequency of nucleotide changes in the ATPase (6 and 8), ND (2, <em>3</em>, 4 and 5) genes of infertile cases compared to the controls. Hence excess ROS and low antioxidant levels in the semen might cause mtDNA mutations and vice versa in OA men that might impair the fertilizing capacity of spermatozoa. Thus, it is important to understand the etiology of mitochondrial genome mutations in idiopathic OA cases for better diagnostic and prognostic value in infertility treatment/assisted reproductive technique.

BACKGROUND

Subjects of the Japan Collaborate Cohort Study (JACC Study) gave peripheral blood samples collected between 1988 and 1990. We conducted to investigate whether levels of serum components measured after 9 years of frozen storage are stable or not.

METHODS

To assess the degradation of frozen serum components in the JACC Study, we compared levels of various components (IGF-I, IGF-II, IGFBP-<em>3</em>, TGF-beta 1, sFas, and total <em>SOD</em> activity) between fresh and stored sera collected from other inhabitants. Serum levels of constituents were measured by immunoradiometric assay (IGF-I, IGF-II and IGFBP-<em>3</em>), quantitative enzyme immunoassay (TGF-beta1), enzyme-linked immuno-adsorbent assay (sFas), and an improved nitrite method (<em>SOD</em> activity).

RESULTS

The coefficients of variation for intra- and inter-assay precisions of the measurements were less than 9%. Levels of IGF-I, IGF-II, IGFBP-<em>3</em>, TGF-beta 1 and sFas in sera after storage for 9 years at--80 degrees C were similar to those of fresh sera newly collected from inhabitants. The distributions of serum IGF-I, IGF-II, IGFBP-<em>3</em>, TGF-beta 1, sFas and <em>SOD</em> activity for specimens collected from different individuals tended to be similar to those of serum levels for frozen specimens collected from different individuals and stored for 9 years.

CONCLUSIONS

There was no statistically significant difference in distribution of measured values of IGF-I, IGF-II, IGFBP-<em>3</em>, TGF-beta 1, and sFas between newly collected sera and frozen specimens stored for 9 years. Thus, measurements of these serum constituents of specimens stored for the JACC Study can be reliably used in nested case-control study.

Oxidative stress caused by amyloid β-peptide (Aβ) may play an important role in the pathogenesis of Alzheimer disease (AD). Aβ is known to be directly responsible for the production of reactive oxygen species (ROS) and induction of apoptosis. Tanshinone IIA (Tan IIA) is extracted from a traditional herbal medicine Salvia miltiorrhiza BUNGE, which has been shown to protect against oxidative stress and cell death. In this study, we investigated the neuroprotective effect of Tan IIA against Aβ₂₅₋₃₅-induced cell death in cultured cortical neurons. Exposure of cortical neurons to <em>3</em>0μM Aβ₂₅₋₃₅ caused a significant viability loss, cell apoptosis and decreased activities of superoxide dismutase (<em>SOD</em>) and glutathione peroxidase (GSH-Px) as well as increased levels of malondialdehyde (MDA) production. In parallel, Aβ₂₅₋₃₅ significant increased the intracellular ROS elevation and decreased mitochondrial membrane potential (MMP). However, pretreatment of the cells with Tan IIA prior to Aβ₂₅₋₃₅ exposure suppressed these Aβ₂₅₋₃₅-induced cellular events noticeably. In addition, Tan IIA reduced the Aβ₂₅₋₃₅-induced increase of caspase-<em>3</em> activity, and reduced cytochrome c translocation into the cytosol from mitochondria. Furthermore, Tan IIA also ameliorated the Aβ₂₅₋₃₅-induced Bcl-2/Bax ratio reduction in cortical neurons. Taken together, these data indicate that Tan IIA protected cultured cortical neurons against Aβ₂₅₋₃₅-induced neurotoxicity through its antioxidative potential. Our results strongly suggest that Tan IIA may be effective in treating AD associated with oxidative stress.

Recent studies have suggested that DNA methylation is implicated in age-related changes in gene expression as well as in cognition. DNA methyltransferase <em>3</em>a (Dnmt<em>3</em>a), which catalyzes DNA methylation, is essential for memory formation and underlying changes in neuronal and synaptic plasticity. Because caloric restriction (CR) and upregulation of antioxidants have been suggested as strategies to attenuate age-related alterations in the brain, we hypothesized that both a diet restricted in calories and transgenic overexpression of normal human Cu/Zn superoxide dismutase 1 (<em>SOD</em>) attenuate age-related changes in Dnmt<em>3</em>a in the aging mouse hippocampus. For this purpose, we performed qualitative and quantitative analyses of Dnmt<em>3</em>a-immunoreactivity (IR) for the hippocampal dentate gyrus (DG), CA<em>3</em> and CA1-2 regions in 12- and 24-month-old mice from 4 groups, i.e. (1) wild-type (WT) mice on a control diet (WT-CD), (2) <em>SOD</em>-CD mice, (<em>3</em>) WT mice on CR (WT-CR), and (4) <em>SOD</em>-CR. Qualitative analyses revealed two types of Dnmt<em>3</em>a immunoreactive cells: type I cells--present throughout all hippocampal cell layers showing moderate levels of nuclear Dnmt<em>3</em>a-IR, and type II cells--a subpopulation of hippocampal cells showing very intense nuclear Dnmt<em>3</em>a-IR, and colocalization with Bromodeoxyuridine. Quantitative analyses indicated that the age-related increase in Dnmt<em>3</em>a-IR within the CA<em>3</em> and CA1-2 in type I cells was attenuated by CR, but not by <em>SOD</em> overexpression. In contrast, the density of type II Dnmt<em>3</em>a immunoreactive cells showed an age-related reduction, without significant effects of both CR and <em>SOD</em>. These changes in Dnmt<em>3</em>a levels in the mouse hippocampus may have a significant impact on gene expression and associated cognitive functioning.

We have compared the active sites of Escherichia coli Fe-substituted (Mn)superoxide dismutase [Fe-sub-(Mn)<em>SOD</em>] and Fe-<em>SOD</em> to elucidate the basis for the inactivity of Fe-sub-(Mn)<em>SOD</em>, despite its apparent similarity to Fe-<em>SOD</em>. The active site of (reduced) Fe2+-sub-(Mn)<em>SOD</em> is qualitatively similar to that of native Fe2+-<em>SOD</em>, indicating similar active site structures and coordination environments for Fe2+. Its nativelike pK is indicative of nativelike local electrostatics, and consistent with Fe2+-sub-(Mn)<em>SOD</em>'s retention of ability to reduce O2*- [Vance and Miller (1998) J. Am. Chem. Soc. 120(<em>3</em>), 461-467]. The active site of (oxidized) Fe<em>3</em>+-sub-(Mn)<em>SOD</em> differs from that of Fe<em>3</em>+-<em>SOD</em> with respect to the EPR signals produced at both neutral and high pH, indicating different coordination environments for Fe<em>3</em>+. Although Fe<em>3</em>+-sub-(Mn)<em>SOD</em> binds the small anions N<em>3</em>- and F-, the KD for N<em>3</em>- is tighter than that of Fe<em>3</em>+-<em>SOD</em>, suggesting that the (Mn)<em>SOD</em> protein favors anion binding more than does the (Fe)<em>SOD</em> protein. The EPR spectral consequences of binding F- are reminiscent of those observed upon binding the first F- to Fe<em>3</em>+-<em>SOD</em>, but the EPR spectrum obtained upon binding N<em>3</em>- is different, consistent with crystallographic observation of a different binding mode for N<em>3</em>- in Thermus thermophilus Mn-<em>SOD</em> than Fe-<em>SOD</em> [Lah, M., et al. (1995) Biochemistry <em>3</em>4, 1646-1660]. We find a pK of 8.5 to be associated with dramatic changes in the EPR spectrum. In addition, we confirm the pK between 6 and 7 that has previously been reported based on changes in the optical signal and N<em>3</em>- binding [Yamakura, F., et al. (1995) Eur. J. Biochem. 227, 700-706]. However, this latter pK appears to be associated with much subtler changes in the EPR spectrum. The non-native pKs observed in Fe<em>3</em>+-sub-(Mn)<em>SOD</em> and the differences in the Fe<em>3</em>+ coordination indicated by the EPR spectra are consistent with Fe<em>3</em>+-sub-(Mn)<em>SOD</em>'s inability to oxidize O2*- and suggest that its low E degrees is due to perturbation of the oxidized state.

Stigmasterol, isolated from the bark of Butea monosperma was evaluated for its thyroid hormone and glucose regulatory efficacy in mice. Its administration at 2.6 mg/kg/d for 20 days reduced serum triiodothyronine (T(<em>3</em>)), thyroxin (T(4)) and glucose concentrations as well as the activity of hepatic glucose-6-phophatase (G-6-Pase) with a concomitant increase in insulin indicating its thyroid inhibiting and hypoglycemic properties. A decrease in the hepatic lipid peroxidation (LPO) and an increase in the activities of catalase (CAT), superoxide dismutase (<em>SOD</em>) and glutathione (GSH) suggested its antioxidative potential. The highest concentration tested (5.2 mg/kg) evoked pro-oxidative activity.

OBJECTIVE

Defective autophagy has been proposed as an important event in a growing number of autoimmune and inflammatory diseases such as rheumatoid arthritis and lupus. However, the precise role of mechanistic target of rapamycin (mTOR)-dependent autophagy and its underlying regulatory mechanisms in the intestinal epithelium in response to inflammation and oxidative stress remain poorly understood.

METHODS

The levels of p-mTOR, LC<em>3</em>B, p62 and autophagy in mice and LPS-treated cells were examined by immunoblotting, immunohistochemistry, confocal microscopy and transmission electron microscopy (TEM). We evaluated the expression of IL-1β, IL-8, TNF-α, MDA, <em>SOD</em> and T-AOC by quantitative real time-polymerase chain reaction (qRT-PCR) and commercially available kits after silencing of mTOR and ATG5. In vivo modulation of mTOR and autophagy was achieved by using AZD8055, rapamycin and <em>3</em>-methyladenine. Finally, to verify the involvement of TLR4 signalling and the NF-κB pathway in cells and active ulcerative colitis (UC) patients, immunofluorescence, qRT-PCR, immunoblotting and TEM were performed to determine TLR4 signalling relevance to autophagy and inflammation.

RESULTS

The mTOR-dependent autophagic flux impairment in a murine model of colitis, human intestinal epithelial cells and active UC patients is probably regulated by TLR4-MyD88-MAPK signalling and the NF-κB pathway. Silencing mTOR remarkably attenuated, whereas inhibiting ATG5 aggravated, LPS-induced inflammation and oxidative injury. Pharmacological administration of mTOR inhibitors and autophagy stimulators markedly ameliorated experimental colitis and oxidative stress in vivo.

CONCLUSIONS

Our findings not only shed light on the regulatory mechanism of mTOR-dependent autophagy, but also provided potential therapeutic targets for intestinal inflammatory diseases such as refractory inflammatory bowel disease.

Paraquat (PQ)-induced pulmonary toxicity is characterized by initial development of pulmonary edema, infiltration of inflammatory cells, and damage to the alveolar epithelium, which may progress to severe fibrosis. However, the exact role of PQ in the progression of the pathogenesis has not been clearly established. To understand the mechanism of PQ in pulmonary toxicity, we developed an animal model of PQ-induced lung injury by intranasal instillation of PQ solution using C57Black/6J mice. Twenty microliters of PQ solution (0.01, 0.01, and 0.04 mg/mouse) was applied through the nares, and the same amount of vehicle was applied in control mice. The pathological progression of lung pathology in our mouse model was very similar to that of patients suffering from PQ poisoning. The lungs of some animals exposed to PQ showed acute fulmination, resulting in death from 5 days post-exposure, but others showed a more protracted injury, resulting in typical pulmonary fibrosis at <em>3</em> weeks. Using this PQ-poisoned mouse model, we examined the gene expression at the initial destructive phase (within 5 days) that fibrosis has not completely developed. We prepared RNAs after 6h, 24h, and 5 days and examined the changes of the expression levels for 45 selected genes. The genes showing >2-fold increase at 6h or a time-dependent decrease during this experimental period may be the early markers for the destructive phase. These genes are Mt1, Mt2, Hmox1, Gcl, GR, IL-6, IL-1<em>3</em>, Txn1, Fas, FasL, Lpin2, Mmp1a, Mmp12, Sfp-B, Sfp-D, CAT, EC-<em>SOD</em>, GST, and Pltp. On the other hand, the genes involved in the development of fibrosis, such as procollagen, Fn1, Eln, SMA, and Mmp9, Timp1 were significantly increased on day 5, not at 6h nor at 24h, after PQ treatment (the late marker). The genes showing a significant increase (Mmp<em>3</em> and Mmp8) or decrease (VEGFA) at 24h and 5 days and not at 6h may be also the late markers. These changes in gene expression, which are equalled to functional activities of proteins, will be the targets for future studies focused on the development on PQ-induced pulmonary damage.

Oxidative stress and excitotoxicity have been implicated in selective striatal vulnerability caused by the mitochondrial toxin, <em>3</em>-nitropropionic acid (<em>3</em>-NP), which may simulate Huntington's disease in animals and humans. The detailed mechanism of the role of superoxide in striatal vulnerability induced by <em>3</em>-NP is still unknown. The authors investigated oxidative cellular injury and DNA fragmentation after systemic <em>3</em>-NP injection in wild-type (Wt) mice and mutant mice with a deficiency in manganese superoxide dismutase (Mn<em>SOD</em>; Sod2 -/+). Furthermore, they investigated the effects of decortication after <em>3</em>-NP treatment in Sod2 -/+ mice, and copper/zinc <em>SOD</em> (CuZn<em>SOD</em>) treatment in recently developed Sod2 -/+ mice that overexpress CuZn<em>SOD</em> (<em>SOD</em>1 +/- / Sod2 -/+ mice). Oxidized hydroethidine, 8-hydroxyguanosine immunoreactivity, and nitrotyrosine immunoreactivity were increased in the Sod2 -/+ mice compared with the Wt mice after <em>3</em>-NP treatment (P < 0.001). Decortication completely abolished oxidative striatal damage after <em>3</em>-NP treatment in the Sod2 -/+ mice. Increased CuZn<em>SOD</em> attenuated DNA fragmentation and striatal lesion volume after <em>3</em>-NP treatment in the Sod2 -/+ mice (P < 0.001). These data suggest that production of superoxide may be a critical step to excitotoxicity and subsequent DNA fragmentation in selective striatal vulnerability after <em>3</em>-NP treatment.

The LYS7 gene in Saccharomyces cerevisiae encodes a protein (yCCS) that delivers copper to the active site of copper-zinc superoxide dismutase (CuZn-SOD, a product of the SODSODSOD) although sod1Delta mutants show increased sensitivity to extracellular zinc, the lys7Delta strain is as resistant as wild type. To restore the SOD catalytic activity but not the zinc-binding capability of the SODSOD from Bacillus stearothermophilus in the cytoplasm of sod1Delta yeast. Paraquat resistance was restored to wild-type levels, but zinc was not. Conversely, expression of a mutant CuZn-SOD that binds zinc but has no SOD activity (H46C) restored zinc resistance but not paraquat resistance. Taken together, these results strongly suggest that CuZn-SOD, in addition to its antioxidant properties, plays a role in zinc homeostasis.

1. Pre-eclampsia is associated with elevated proinflammatory cytokine levels and endothelial dysfunction. This study examined the effect of two cytokines, tumour necrosis factor-alpha (TNF) and interleukin-1beta (IL-1) on endothelium-dependent relaxation in response to acetylcholine (ACH), bradykinin (BK) and histamine (HIS) in rat mesenteric small arteries in vitro. 2. Rat mesenteric arteries were mounted in an isometric myograph. Tone was induced with phenylephrine (PE) or a depolarizing solution containing 80 mM KCl (K(80)). Relaxation was measured in response to ACH, BK, HIS and sodium nitroprusside (SNP), an endothelium-independent relaxant. Inhibition of NO synthase by a combination of N(omega)-monomethyl-L-arginine (L-NMMA) and N(omega)-nitro-L-arginine methyl ester (L-NAME) significantly inhibited relaxation in response to ACH and BK. Addition of an inhibitor of cyclooxygenase, indomethacin, had no additional effect when added to L-NMMA and L-NAME. Inhibition of endothelium-derived hyperpolarizing factor (EDHF) by K(80) partially reduced responses to ACH and BK. Inhibition of HIS-induced relaxation was more marked with K(80). L-NMMA and L-NAME largely abolished the remaining relaxation to ACH, BK and HIS in arteries contracted with K(80). <em>3</em>. Preincubation with TNF for <em>3</em>0 min caused an inhibition of relaxation in response to ACH and BK in arteries contracted with PE. Responses to HIS and SNP were not affected by TNF under these conditions. TNF also inhibited ACH-induced relaxation in arteries contracted with K(80). IL-1 had no effect on responses to ACH and the combination of TNF and IL-1 was not more effective than TNF alone. 4. The inhibitory effect of TNF on ACH-induced relaxation was abolished by coincubation with superoxide dismutase (<em>SOD</em>) and was not seen if NO synthase was inhibited by L-NMMA and L-NAME. 5. TNF inhibits the NO-dependent component of endothelium-dependent relaxation in response to ACH and BK, but does not inhibit the EDHF-dependent component. This effect may be attributable to the ability of TNF to increase levels of superoxide anions (O(2)(-)) and the ability of O(2)(-) to inactivate NO. This mechanism could contribute to the endothelial dysfunction seen in situations where TNF is elevated, such as pre-eclampsia.

Forty-eight cattle were used in 4 experiments; 6-week-old calves in experiments 1-<em>3</em> (n = 24) and 10-month-old heifers in experiment 4 (n = 24). In experiments 1-<em>3</em>, 7 groups of <em>3</em> calves each were inoculated SC with 5 strains of Brucella abortus: virulent strain 2<em>3</em>08 (2 groups), vaccine strain 19 (2 groups), and mutant strains RB51. 19 delta <em>3</em>1K, and 19 delta <em>SOD</em>. Sera and lymph node tissues were examined at 2-week intervals for evidence of infection. At postinoculation (PI) week 12, 2 calves in each group were given dexamethasone for 5 days. Calves were then euthanatized and lymphoid tissue, spleen, liver, and bone marrow were examined for evidence of B abortus. Calves given strain 2<em>3</em>08 had large numbers of bacteria in their lymph nodes, marked granulomatous lymphadenitis in the deep cortex, and loss of lymphoid cells in superficial cortical areas. In addition, they had high serum antibody titers at PI week 16. Calves given strain 19, or genetic mutants derived from strain 19, cleared bacteria from lymph nodes more rapidly, had less lymphoid destruction, and developed antibody titers that did not persist for 16 weeks. The RB51 strain (rough) was cleared most rapidly from lymphoid tissues and induced serum antibody responses only to the core of the lipopolysaccharide molecule. Treatment of calves with dexamethasone did not cause B abortus to reappear in tissues of any calves, nor did serum antibody titers increase.(ABSTRACT TRUNCATED AT 250 WORDS)

Background. The nephroprotective effect of vitamins E and C or losartan against cisplatin (CP)- induced nephrotoxicity when they are accompanied by estrogen was investigated. Methods. The ovariectomized rats received estradiol valerate for two weeks. At the end of the first week, a single dose of CP (7 mg/kg, IP) was also administered, and they received placebo (group 1), vitamin E (group 2), vitamin C (group <em>3</em>), or losartan (group 4) every day during the second week, and they were compared with another three control groups. Results. CP alone increased the serum levels of blood urea nitrogen (BUN), creatinine (Cr), and kidney tissue damage score (KTDS), significantly (P < 0.05), however at the presence of estradiol and CP, vitamin C, vitamin E, or losartan not only did not decrease these parameters, but also increased them significantly (P < 0.05). The serum level of superoxidase dismutase (<em>SOD</em>) was reduced by CP (P < 0.05), but it was increased when estradiol or estradiol plus vitamin C or losartan were added (P < 0.05). Conclusion. The particular pharmacological dose of estrogen used in this study abolish the nephroprotective effects vitamins C and E or losartan against CP-induced nephrotoxicity.

We examined whether xanthine oxidoreductase (XOR), a hypoxia-inducible enzyme capable of generating reactive oxygen species, is involved in the onset of angiotensin (Ang) II-induced vascular dysfunction in double-transgenic rats (dTGR) harboring human renin and human angiotensinogen genes. In 7-week-old hypertensive dTGR, the endothelium-mediated relaxation of noradrenaline (NA)-precontracted renal arterial rings to acetylcholine (ACh) in vitro was markedly impaired compared with Sprague Dawley rats. Preincubation with superoxide dismutase (<em>SOD</em>) improved the endothelium-dependent vascular relaxation, indicating that in dTGR, endothelial dysfunction is associated with increased superoxide formation. Preincubation with the XOR inhibitor oxypurinol also improved endothelium-dependent vascular relaxation. The endothelium-independent relaxation to sodium nitroprusside was similar in both strains. In dTGR, serum 8-isoprostaglandin F(2alpha), a vasoconstrictor and antinatriuretic arachidonic acid metabolite produced by oxidative stress, was increased by 100%, and the activity of XOR in the kidney was increased by 40%. Urinary nitrate plus nitrite (NO(x)) excretion, a marker of total body NO generation, was decreased by 85%. Contractile responses of renal arteries to Ang II, endothelin-1 (ET-1), and NA were decreased in dTGR, suggesting hypertension-associated generalized changes in the vascular function rather than a receptor-specific desensitization. Valsartan (<em>3</em>0 mg/kg PO for <em>3</em> weeks) normalized blood pressure, endothelial dysfunction, and the contractile responses to ET-1 and NA. Valsartan also normalized serum 8-isoprostaglandin F(2alpha) levels, renal XOR activity, and, to a degree, NO(x) excretion. Thus, overproduction of Ang II in dTGR induces pronounced endothelial dysfunction, whereas the sensitivity of vascular smooth muscle cells to nitric oxide is unaltered. Ang II-induced endothelial dysfunction is associated with increased oxidative stress and vascular xanthine oxidase activity.

Oxidative damage of biomolecules increases with age and is postulated to be a major causal factor of various neurodegenerative disorders. Consequently, the concept of neuroprotection by antioxidants has been developed. Recently we have shown that the behaviour of young senescent-accelerated OXYS rats is similar to the behaviour of old Wistar animals. To determine the role of oxidative stress in this phenomenon we investigated age-related changes in protein carbonyls (PrC), lipid peroxides (LP), reduced glutathione (GSH), alpha-tocopherol (TP) and <em>SOD</em> activity in the brain of OXYS and Wistar rats. We also studied the effect of long-term supplementation with bilberry extract (2g/kg of diet) and Vitamin E (140 mg/kg of diet) on oxidative stress markers and on learning in passive avoidance test. In both rat strains LP, PrC and TP increased with age and at 24 months PrC was significantly higher (p<0.0001) in OXYS rats. At <em>3</em> months GSH was higher and <em>SOD</em> activity was lower in OXYS rats than in Wistar rats. <em>SOD</em> activity decreased with age in OXYS whereas increased in Wistar rats. Cognitive impairments in OXYS rats were manifested earlier than significant differences in the level of brain oxidative stress markers between two strains. By contrast, differences in antioxidant systems of Wistar and OXYS rats were registered at <em>3</em> months. Antioxidants attenuated cognitive deficits in OXYS rats, providing evidence for therapeutic role of antioxidants. Nevertheless, the exact mechanisms of neuroprotective effects of antioxidants in vivo and the real impact of oxidative stress on the development of cognitive impairments in OXYS rats still needs to be further investigated.

Mitochondrial integrity and antioxidative enzyme activity are two of the determinants of intracellular reactive oxygen species (ROS) accumulation probably underlying the aging mechanism. In this study, epigallocatechin-<em>3</em>-gallate (EGCG) was examined for its antiaging effect on human diploid fibroblasts (HDF). EGCG was evaluated for its cytotoxicity, and LC50 values were 78.0 and 84.4 microM for young and old HDF, respectively. HDF treated with EGCG at 25 and 50 microM for 24 h considerably increased catalase, superoxide dismutase (<em>SOD</em>)1, <em>SOD</em>2, and glutathione peroxidase gene expressions and their enzyme activities, thus protecting HDF against H2O2-induced oxidative damage, accompanied with decreased intracellular ROS accumulation and well-maintained mitochondrial potential. Moreover, HDF treated with EGCG at 12.5 microM for long term showed less intracellular ROS with higher mitochondrial potential, more intact mitochondrial DNA, much elevated antioxidative enzyme efficiency, and more juvenile cell status compared to those of the untreated group. Taken together, in this study we investigated the effects of EGCG in the regulation of mitochondrial integrity and antioxidative enzyme activity of HDF, suggesting that EGCG can be considered one of the possible antiaging reagents in the future.

Pre-eclampsia is a hypertensive disorder of pregnancy in which enzymatic antioxidant defenses fail and tissues are injured. This prospective case-control study evaluated whether pre-eclamptic women and their newborns show higher degrees of oxidative stress than normal pregnancies and sought to determine if this stress is related to clinical severity. Forty-four pre-eclamptic and thirty healthy pregnant women attending two hospitals in Valparaíso, Chile, were studied. The following plasmatic variables of antioxidant capacity were evaluated: glutathione peroxidase activity (GPx), total antioxidant capacity measured by oxidation of ABTS substrate (2,2'-azino-bis (<em>3</em>-ethylbenzthiazoline-6-sulfonic acid), and superoxide dismutase activity (<em>SOD</em>). malondialdehide (MDA) was measured to evaluate lipoperoxidation. The evaluation was performed at diagnosis of pre-eclampsia, delivery, <em>3</em>0 days and 120 days post delivery. Newborns were studied at delivery through umbilical cord blood samples. Our analysis shows that antioxidant enzyme activity (<em>SOD</em>, GPx, ABTS) was significantly decreased, while lipoperoxidation (MDA) was increased in both pre-eclamptic groups compared to normal pregnant women (p<0.01). Statistically significant difference was found between mild and severe pre-eclamptic groups (p<0.01), for all biochemical markers studied. Therefore, the clinical severity of this pathology is closely related to the degree of oxidative stress.

Cadmium may induce oxidative damage in different tissues by enhancing peroxidation of membrane lipids and altering the antioxidant system of the cells. The peroxidative damage to the cell membrane may cause injury to cellular components due to the interaction of metal ions with the cell organelles. The treatment with Cd (0.4 mg/kg body wt, ip) significantly increased lipid peroxidation (LPO) in heart within <em>3</em> h of the Cd injection, while the increase in kidney and liver followed 6 to 12 h after Cd intoxication. The antioxidant enzymes and other antioxidants provide protection to the cells against oxidative damage. The superoxide dismutase (<em>SOD</em>) activity increased in heart, kidney and liver within 24 h of Cd intoxication. The CAT activity increased significantly in heart 9 h after Cd injection; however, no significant change in CAT activity was observed in kidney and liver tissues. The GSH content and the activity of GR decreased in heart, kidney and liver 72 h after Cd administration, which has been suggested to be the cause for increased LPO in the tissues. The hexose monophosphate (HMP) shunt enzymes generate NADPH required for the activity of GR which may affect the GSH content in the tissues. The generalised decrease in glucose 6-phosphate dehydrogenase (G6PDH) and 6 phospho gluconate dehydrogenase (6PGDH) at 9 h followed by an increase in these enzymes in tissues 72 h after Cd intoxication suggest that the production of NADPH by the HMP shunt is required to reduce the oxidative damage. The results show that Cd induced LPO in the tissues and the condition was partially counteracted by the antioxidant system.

This study examined vascular function and the role of superoxide in mice that chronically express human renin (R+) and human angiotensinogen (A+). Responses of aortas from R+/A+ mice and from their normotensive littermates (RA- mice) were examined in vitro. Endothelium-dependent relaxation to acetylcholine was impaired in vessels from R+/A+ mice (e.g., maximal relaxation to 100 microM acetylcholine was 45 +/- 5% and 65 +/- <em>3</em>% in R+/A+ and RA- mice, respectively; P < 0.05). Relaxation was also impaired to the endothelium-independent dilators authentic nitric oxide and nitroprusside in vessels from R+/A+ mice. Maximal vasorelaxation to the endothelium-independent, non-nitric oxide dilator papaverine was similar in R+/A+ and RA- mice. Incubation of vessels from R+/A+ mice with Tiron (1 mM), a superoxide scavenger, improved relaxation to acetylcholine, nitric oxide, and nitroprusside. In contrast, incubation with diethyldithiocarbamate (1 mM), an inhibitor of copper-containing <em>SODs</em>, reduced acetylcholine- and nitroprusside-induced relaxation in vessels from both R+/A+ and RA- mice. Basal superoxide levels, measured with lucigenin-enhanced chemiluminescence (5 microM lucigenin) and hydroethidine-based fluorescent confocal microscopy, were higher in vessels from R+/A+ mice and were Tiron and polyethylene glycol-<em>SOD</em> sensitive. These results suggest that increased superoxide contributes to impaired nitric oxide-mediated relaxation in this genetic model of chronic angiotensin II-dependent hypertension.

OBJECTIVE

We evaluated the effect of rebamipide (2-(4-chlorobenzoylamino)-<em>3</em>-[2(1H)-quinolinon-4-yl] propionic acid), a novel anti-ulcer drug, on indomethacin-induced small intestinal lesions in rats.

METHODS

The animals were administered indomethacin (10 mg/kg, s.c.), and they were killed 24 h later. Rebamipide (<em>3</em>0-<em>3</em>00 mg/kg) was administered p.o. twice, <em>3</em>0 min before, and 6 h after indomethacin.

RESULTS

Indomethacin caused hemorrhagic lesions in the rat small intestine, accompanied by an increase in enterobacterial translocation, inducible nitric oxide synthase (iNOS) and myeloperoxidase (MPO) activities, as well as thiobarbituric acid (TBA) reactants, and these changes were significantly prevented by the supplementation with 16,16-dimethyl prostaglandin E2 (dmPGE2; 10 microg/kg, i.v.) or the pretreatment of animals with the antibiotic ampicillin. Treatment of the animals with rebamipide dose-dependently prevented the development of intestinal lesions, and this effect was mimicked by i.v. administration of superoxide dismutase (SOD: <em>3</em>000 U/kg) + catalase (CAT: 5000 U/kg). The protection by rebamipide was accompanied by a significant suppression of the increase in both MPO and iNOS activities, and a complete inhibition of the increase in TBA reactants, while SOD + CAT significantly inhibited the increase of MPO activity and TBA reactants, but not iNOS activity. The bacterial translocation following indomethacin was also significantly decreased by either rebamipide or SOD + CAT.

CONCLUSIONS

These results confirmed the importance of enterobacteria and iNOS/NO in the pathogenesis of indomethacin-induced small intestinal lesions, and suggested that rebamipide prevents the development of these lesions, probably by its radical scavenging action.

The activities of the endogenous O2- and H2O2 scavenging enzymes, superoxide dismutase (<em>SOD</em>), glutathionine peroxidase (GP), and catalase, were measured in lysates of the intracellular parasite, Toxoplasma gondii, and in various macrophage populations. During 72 h of cultivation in standard medium alone, the catalase activity of in vivo-activated toxoplasma-immune macrophages (IM) and immune-boosted macrophages (IB) progressively increased by eight- to ninefold, and correlated with the previously observed parallel decline in these cells' antitoxoplasma activity and capacity to release H2O2. <em>SOD</em> and GP activities either remained constant or decreased during this <em>3</em>-d period. Lymphokine exposure, which preserved the antitoxoplasma activity and oxidative capacity of 48- and 72-h cultures of IB and IM cells, blunted the rise in catalase levels and had no effect on <em>SOD</em> or GP. Inhibition of IB and IM macrophage catalase by aminotriazole maintained toxoplasmastatic activity otherwise lost after 48 h of cultivation. In addition, IB and IM cells from acatalasemic mice contained 20- to <em>3</em>0-fold less catalase, and showed comparatively little decline in either H2O2 release or antitoxoplasma activity during 72 h in culture. In vitro-(lymphokine) activated resident macrophages from normal mice had the highest levels of <em>SOD</em>, GP, and catalase, and these cells failed to kill or inhibit T. gondii despite enhanced extracellular release of O2- and H2O2. Toxoplasmas were also found to contain all three enzymatic scavengers. Aminotriazole inhibition of lymphokine-activated cells' catalase or of toxoplasma catalase was effective in inducing these macrophages to display antitoxoplasma activity. Moreover, and in contrast to normocatalasemic resident cells, those from acatalesemic mice were readily induced by lymphokine to inhibit the replication of untreated virulent toxoplasmas. These results suggest that endogenous O2- and H2O2 scavenging enzymes, which function within both T. gondii and activated macrophages as host cell antioxidant protective mechanisms, may reduce the effectiveness of phagocyte antimicrobial activity. Thus, the presence of <em>SOD</em>, GP, and especially catalase within both target and effector cell may be important determinants of macrophage oxygen-dependent processes.

Cytochrome c catalyzed the oxidation of various electron donors in the presence of hydrogen peroxide (H2O2), including 2-2'-azino-bis(<em>3</em>-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 4-aminoantipyrine (4-AP), and luminol. With ferrocytochrome c, oxidation reactions were preceded by a lag phase corresponding to the H2O2-mediated oxidation of cytochrome c to the ferric state; no lag phase was observed with ferricytochrome c. However, brief preincubation of ferricytochrome c with H2O2 increased its catalytic activity prior to progressive inactivation and degradation. Superoxide (O2-) and hydroxyl radical (.OH) were not involved in this catalytic activity, since it was not sensitive to superoxide dismutase (<em>SOD</em>) or mannitol. Free iron released from the heme did not play a role in the oxidative reactions as concluded from the lack of effect of diethylenetriaminepentaacetic acid. Uric acid and tryptophan inhibited the oxidation of ABTS, stimulation of luminol chemiluminescence, and inactivation of cytochrome c. Our results are consistent with an initial activation of cytochrome c by H2O2 to a catalytically more active species in which a high oxidation state of an oxo-heme complex mediates the oxidative reactions. The lack of <em>SOD</em> effect on cytochrome c-catalyzed, H2O2-dependent luminol chemiluminescence supports a mechanism of chemiexcitation whereby a luminol endoperoxide is formed by direct reaction of H2O2 with an oxidized luminol molecule, either luminol radical or luminol diazoquinone.