OBJECTIVE

To clarify the effect of superoxide dismutase (SOD) on vasospasm after subarachnoid hemorrhage (SAH), we investigated sequential changes in arterial diameter after SAH in transgenic mice overexpressing CuZn-SOD (SOD-1).

METHODS

SOD-transgenic mice and nontransgenic littermates (35 to 40 g) were subjected to SAH produced by endovascular perforation of left anterior cerebral artery. At 4 hours and 1, 3, 7, and 14 days after SAH, the mice were perfused with 10% formalin and consequently with a mixture of carbon black and 10% gelatin to cast all vessels. Vasospasm was evaluated by measuring the diameter of the left middle cerebral artery (MCA) with a microscope.

RESULTS

In nontransgenic mice, the diameter of the MCA on day 3 after SAH (110.5+/-20.5 microm [mean+/-SD]; n=16) was significantly reduced compared with that without SAH (138.5+/-14.5 microm; n=12) (P<0.01). Moreover, on day 3 after SAH, the diameter of the MCA in SOD-transgenic mice (127. 9+/-20.2 microm; n=20) was significantly larger than that in nontransgenic mice (110.5+/-20.5 microm; n=16) (P<0.05).

CONCLUSIONS

These results suggest that SOD is effective on the amelioration of vasospasm after SAH and that oxygen free radicals, particularly superoxide, play an important role in the pathogenesis of vasospasm after SAH.

The peroxidative activity of Cu,Zn-containing superoxide dismutase (Cu, Zn-<em>SOD</em>) was studied by using a chromogen, 2,2'-azinobis-(<em>3</em>-ethylbenzthiazoline-6-sulfonate) (ABTS) which reacts with .OH radicals to form ABTS+, and the spin traps, N-tert-butyl-alpha-phenylnitrone (PBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The formation of ABTS+. in this study required both active Cu,Zn-<em>SOD</em> and H2O2 and followed first order kinetics with respect to <em>SOD</em> and H2O2. However, it showed a binding isotherm with ABTS that yielded a dissociation constant of ABTS-enzyme as Kd = 7.1 +/- 0.5 microM. The Kd values for DMPO and PBN were obtained as 0.6<em>3</em> and 11 mM, respectively, by competition reactions. A radical scavenger, formate anion, inhibits the formation of ABTS+., whereas ethanol does not. The results together indicate that DMPO and anionic scavengers have easy access inside the positively charged active channel of Cu,Zn-<em>SOD</em> and are thus in a position to intercept the newly formed .OH radicals. PBN and ethanol, however, stay outside of the channel and are not able to compete with ABTS for .OH radicals. We trapped free radicals, which were produced in the presence of free radical scavengers, with PBN. The formation curve of PBN-hydroxyethyl radical adduct observed in the presence of ethanol indicated that the enzyme became inactivated in a relatively short period. In contrast, in the presence of anionic scavenger formate, formyl radicals were produced catalytically, and the enzyme activity was protected by the formate against H2O2 inactivation. Thus Cu,Zn-<em>SOD</em> behaves as an enzyme that catalyzes the formation of free radicals using anionic scavengers and H2O2 as substrates.

The teratogenicity of many xenobiotics is thought to depend at least in part upon their bioactivation by embryonic cytochromes P450, prostaglandin H synthase (PHS) and lipoxygenases (LPOs) to electrophilic and/or free radical reactive intermediates that covalently bind to or oxidize cellular macromolecules such as DNA, protein and lipid, resulting in in utero death or teratogenesis. Using as models the tobacco carcinogens benzo[a]pyrene (B[a]P) and 4-(methylnitrosamino)-1-(<em>3</em>-pyridyl)-1-butanone (NNK), the anticonvulsant drug phenytoin, structurally related anticonvulsants (e.g. mephenytoin, nirvanol, trimethadione, dimethadione) and the sedative drug thalidomide, we have examined the potential teratologic relevance of free radical-initiated, reactive oxygen species (ROS)-mediated oxidative molecular target damage, genotoxicity (micronucleus formation) and DNA repair in mouse and rabbit models in vivo and in embryo culture, and in vitro using purified enzymes or cultured rat skin fibroblasts. These teratogens were bioactivated by PHS and LPOs to free radical reactive intermediary metabolites, characterized by electron spin resonance spectrometry, that initiated ROS formation, including hydroxyl radicals, which were characterized by salicylate hydroxylation. ROS-initiated oxidation of DNA (8-hydroxy-2'-deoxyguanosine formation), protein (carbonyl formation), glutathione (GSH) and lipid (peroxidation), and embryotoxicity were shown for phenytoin, its major hydroxylated metabolite 5-(p-hydroxyphenyl)-5-phenylhydantoin [HPPH], thalidomide, B[a]P and NNK in vivo and/or in embryo culture, the latter indicating a teratologically critical role for embryonic, as distinct from maternal, processes. DNA oxidation and teratogenicity of phenytoin and thalidomide were reduced by PHS inhibitors. Oxidative macromolecular lesions and teratogenicity also were reduced by the free radical trapping agent phenylbutylnitrone (PBN), and the antioxidants caffeic acid and vitamin E. In embryo culture, addition of superoxide dismutase (<em>SOD</em>) to the medium enhanced embryonic <em>SOD</em> activity, and <em>SOD</em> or catalase blocked the oxidative lesions and embryotoxicity initiated by phenytoin and B[a]P, suggesting a major contribution of ROS, as distinct from covalent binding, to the teratologic mechanism. In in vivo studies, other antioxidative enzymes like GSH peroxidase, GSH reductase and glucose-6-phosphate dehydrogenase (G6PD) were similarly protective. Even untreated G6PD-deficient mice had enhanced embryopathies, indicating a teratological role for endogenous oxidative stress. In cultured fibroblasts, B[a]P, NNK, phenytoin and HPPH initiated DNA oxidation and micronucleus formation, which were inhibited by <em>SOD</em>. Oxidation of DNA may be particularly critical, since transgenic mice with +/- or -/- deficiencies in the p5<em>3</em> tumor suppressor gene, which facilitates DNA repair, are more susceptible to phenytoin and B[a]P teratogenicity. Even p5<em>3</em>-deficient mice treated only with normal saline showed enhanced embryopathies, suggesting the teratological importance of endogenous oxidative stress, as observed with G6PD deficiency. These results suggest that oxidative macromolecular damage may play a role in the teratologic mechanism of xenobiotics that are bioactivated to a reactive intermediate, as well in the mechanism of embryopathies occurring in the absence of xenobiotic exposure.

We tested the hypothesis that exercise training (Ex) attenuates hypercholesterolemia-induced impairment of endothelium-dependent relaxation (EDR) in male porcine coronary arteries [left anterior descending coronary arteries (LAD)] by increasing nitric oxide (NO) release [due to increased endothelial NO synthase (NOS) expression] and/or increased bioactivity of NO. Adult male pigs were fed a normal-fat (NF) or high-fat (HF) diet for 20-24 wk. Pigs were Ex or remained sedentary (Sed) for 16-20 wk, beginning after 4 wk on diet. Four groups of pigs were used: NF-Sed, NF-Ex, HF-Sed, and HF-Ex. HF enhanced LAD contractions induced by KCl, aggregating platelets (AP), and serotonin (5-HT). AP and 5-HT produced EDR after blockade of cyclooxygenase with indomethacin (Indo) and smooth-muscle 5-HT(2) receptors with ketanserin. HF impaired EDR induced by AP, 5-HT, and bradykinin. Results indicate a decreased contribution of NO to EDR in HF-Sed LADs, because the percentage of bradykinin-induced EDR inhibited by N(G)-nitro-L-arginine methyl ester was 27% in NF-Sed and <em>3</em>4% in NF-Ex but only 17% in HF-Sed. Also, N(G)-nitro-L-arginine methyl ester + Indo results indicate that release of an Indo-sensitive vasoconstrictor contributes to blunted EDR in HF-Sed LAD. Immunoblot and immunohistochemistry results indicate the following: 1) LAD endothelial NOS protein content was similar among groups; 2) HF decreased LAD superoxide dismutase (<em>SOD</em>) but increased caveolin-1 content; and <em>3</em>) Ex increased <em>SOD</em> content of HF LADs. We conclude that HF impairs EDR by impairing the contribution of NO released from NOS (due to decreased <em>SOD</em> and increased caveolin-1 protein content) and by production of an Indo-sensitive vasoconstrictor. Ex preserves EDR in HF LADs by decreasing the production of the constrictor and increasing NO-release by NOS and/or NO bioactivity and bioavailability.

A role mutations in the superoxide dismutase (<em>SOD</em>)-1 gene in the pathogenesis of amyotrophic lateral sclerosis (ALS) has been discussed. To investigate immunohistochemical alterations of <em>SOD</em> in the spinal cord affected with the disease, we examined <em>3</em> patients with <em>SOD</em>1 mutation-associated family with ALS, 20 patients with sporadic ALS and 10 control individuals. Lewy body-like hyaline inclusions (LBHIs) were seen in the anterior horn cells of all the familial patients and 10 of the 20 sporadic patients, while skein-like inclusions (SIs) and Bunina bodies (BBs) were present in the 20 sporadic patients but not in the familial patients. The primary antibodies used for immunostaining were rabbit antisera raised against human <em>SOD</em>1 and <em>SOD</em>2. The anti-<em>SOD</em>1 antibody reacted strongly with all LBHIs of each familial patient and with some LBHIs of each sporadic patient. The cytoplasm of morphologically intact and degenerated spinal cord neurons as well as spheroids seen in the cases examined was only weakly stained by the antibody to <em>SOD</em>1 or not at all. The reactive astrocytes displayed weak to moderate staining for <em>SOD</em>1. The anti-<em>SOD</em>2 antibody strongly immunolabeled the reactive astrocytes and microglia. LBHIs of both familial and sporadic ALS were negatively stained for <em>SOD</em>2. Spinal cord neurons and spheroids of each case exhibited no significant <em>SOD</em>2 immunoreactivity. Neither antibodies reacted with SIs nor BBs. These results indicate that <em>SOD</em>1 may be involved in the formative process of LBHIs especially in familial ALS but not always in that of SIs or BBs, and imply that <em>SOD</em>2 may have no connection with any of these ALS-related abnormal structures.

Our main objective was to determine whether reactive oxygen species (ROS), such as superoxide (O(2)(-)) and hydrogen peroxide (H(2)O(2)), contribute to altered pulmonary vascular responses in piglets with chronic hypoxia-induced pulmonary hypertension. Piglets were raised in either room air (control) or hypoxia for <em>3</em> days. The effect of the cell-permeable superoxide dismutase mimetic (<em>SOD</em>; M4040<em>3</em>) and/or PEG-catalase (PEG-CAT) on responses to acetylcholine (ACh) was measured in endothelium-intact and denuded pulmonary resistance arteries (PRAs; 90-to-<em>3</em>00-microm diameter). To determine whether NADPH oxidase is an enzymatic source of ROS, PRA responses to ACh were measured in the presence and absence of a NADPH oxidase inhibitor, apocynin (APO). A Western blot technique was used to assess expression of the NADPH oxidase subunit, p67phox. A lucigenin-derived chemiluminescence technique was used to measure ROS production stimulated by the NADPH oxidase substrate, NADPH. ACh responses, which were dilation in intact control arteries but constriction in both intact and denuded hypoxic arteries, were diminished by M4040<em>3</em>, PEG-CAT, the combination of M4040<em>3</em> plus PEG-CAT, as well as by APO. Although total amounts were not different, membrane-associated p67phox was greater in PRAs from hypoxic compared with control piglets. NADPH-stimulated lucigenin luminescence was nearly doubled in PRAs from hypoxic vs. control piglets. We conclude that ROS generated by NADPH oxidase contribute to the aberrant pulmonary arterial responses in piglets exposed to <em>3</em> days of hypoxia.

Angiotensin II and hypertension increase vascular oxidant stress. We examined how these might affect expression of the extracellular superoxide dismutase (ec<em>SOD</em>), a major form of vascular <em>SOD</em>. In mice, angiotensin II infusion (1.1 mg/kg for 7 days) increased systolic blood pressure from 107+/-<em>3</em> to 152+/-9 mm Hg and caused a <em>3</em>-fold increase in ec<em>SOD</em>, but there was no change in the cytosolic Cu/Zn <em>SOD</em> protein, as determined by Western blot analysis. This was associated with a similar increase in ec<em>SOD</em> mRNA as assessed by RNase protection assay and was prevented by losartan. Induction of ec<em>SOD</em> by angiotensin II was not due to hypertension alone, because hypertension caused by norepinephrine (5.6 mg. kg-1. d-1) had no effect on ec<em>SOD</em>. Similarly, exposure of mouse aortas to angiotensin II (100 nmol/L) in organoid culture increased ec<em>SOD</em> by approximately 2-fold. In the organoid culture, angiotensin II-induced upregulation of ec<em>SOD</em> was prevented by losartan (10 micromol/L) and PD985059 (<em>3</em>0 micromol/L), a specific inhibitor of p42/44 MAP kinase kinase. Angiotensin II activates the NADH/NADPH oxidase; however, diphenyleneiodonium chloride (10 micromol/L), an inhibitor of this oxidase, did not prevent p42/44 MAP kinase phosphorylation or ec<em>SOD</em> induction by angiotensin II. Finally, in human aortic smooth muscle cells, angiotensin II moderately increased transcriptional rate (as assessed by nuclear run-on analysis) but markedly increased ec<em>SOD</em> mRNA stability. Thus, angiotensin II increases ec<em>SOD</em> expression independent of hypertension, and this increase involves both an increase in ec<em>SOD</em> transcription and stabilization of ec<em>SOD</em> mRNA. This effect of angiotensin II on ec<em>SOD</em> expression may modulate the oxidative state of the vessel wall in pathological processes in which the renin-angiotensin system is activated.

Observations from nematodes to mammals indicate that insulin/insulin-like growth factor signaling (IIS) regulates lifespan. As in other organisms, IIS is conserved in mosquitoes and signaling occurs in multiple tissues. During bloodfeeding, mosquitoes ingest human insulin. This simple observation suggested that exogenous insulin could mimic the endogenous hormonal control of aging in mosquitoes, providing a new model to examine this phenomenon at the organismal and cellular levels. To this end, female Anopheles stephensi mosquitoes were maintained on diets containing human insulin provided daily in sucrose or three times weekly by artificial bloodmeal. Regardless of delivery route, mosquitoes provided with insulin at 1.7 x 10(-4) and 1.7 x 10(-<em>3</em>) micromol l(-1), doses 0.<em>3</em>-fold and <em>3</em>.0-fold higher than non-fasting blood levels, died at a faster rate than controls. In mammals, IIS induces the synthesis of reactive oxygen species and downregulates antioxidants, events that increase oxidative stress and that have been associated with reduced lifespan. Insulin treatment of mosquito cells in vitro induced hydrogen peroxide synthesis while dietary supplementation reduced total superoxide dismutase (<em>SOD</em>) activity and manganese <em>SOD</em> activity relative to controls. The effects of insulin on mortality were reversed when diets were supplemented with manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a cell-permeable <em>SOD</em> mimetic agent, suggesting that insulin-induced mortality was due to oxidative stress. In addition, dietary insulin activated Akt/protein kinase B and extracellular signal-regulated kinase (ERK) in the mosquito midgut, suggesting that, as observed in Caenorhabditis elegans, the midgut may act as a 'signaling center' for mosquito aging.

The influence of cytokines on extracellular superoxide dismutase (EC-<em>SOD</em>) expression by human dermal fibroblasts was investigated. The expression was markedly stimulated by interferon-gamma (IFN-gamma), was varying between fibroblast lines stimulated or depressed by interleukin-1 alpha (IL-1 alpha), was intermediately depressed by tumor necrosis factor-alpha (TNF-alpha), and markedly depressed by transforming growth factor-beta (TGF-beta). TNF-alpha, however, enhanced the stimulation by a high dose of IFN-gamma, whereas TGF-beta markedly depressed the stimulations given by IFN-gamma and IL-1 alpha. The ratio between the maximal stimulation and depression observed was around <em>3</em>0-fold. The responses were generally slow and developed over periods of several days. There were no effects of IFN-alpha, IL-2, IL-<em>3</em>, IL-4, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, human growth hormone, Escherichia coli lipopolysaccharide, leukotriene B4, prostaglandin E2, formylmethionylleucylphenylalanine, platelet-activating factor, and indomethacin. The cytokines influencing the EC-<em>SOD</em> expression are also known to influence superoxide production by leukocytes and other cell types, and the EC-<em>SOD</em> response pattern is roughly compatible with the notion that its function is to protect cells against extracellular superoxide radicals. The results show that EC-<em>SOD</em> is a participant in the complex inflammatory response orchestrated by cytokines. The CuZn-<em>SOD</em> activity of the fibroblasts was not influenced by any of the cytokines, whereas the Mn-<em>SOD</em> activity was depressed by TGF-beta. TNF-alpha, IL-1 alpha, and IFN-gamma stimulated the Mn-<em>SOD</em> activity, as previously known, and these responses were reduced by TGF-beta. The different responses of the three <em>SOD</em> isoenzymes illustrate their different physiological roles.

The expression of the two Cu,Zn superoxide dismutase (<em>SOD</em>) genes of tomato was followed in different organs and plant developmental stages at the transcript and enzymatic activity levels. The cDNA clones used as probes code for the chloroplast Cu,Zn <em>SOD</em> (clone T1) and the cytosolic Cu,Zn <em>SOD</em> (clone P<em>3</em>1). The two genes were found to display distinct expression patterns. While the T1 transcript was rare or absent from roots, stems and ripening fruits, the P<em>3</em>1 transcript was very abundant in these organs. Shoot tips, flower buds, seedlings and young leaves contained high levels of the two mRNAs. During leaf expansion, the levels of both transcripts diminish markedly. Despite the diminished presence of transcripts, <em>SOD</em> activity levels of the corresponding cytosolic and chloroplast isozymes accumulated and were sustained throughout leaf expansion. In non-photosynthetic organs, the <em>SOD</em>-<em>3</em> (cytosolic) isozyme contained most of the activity, while in the expanded leaf the <em>SOD</em>-1 (chloroplast) isozyme was more abundant. Light-regulated accumulation of both the P<em>3</em>1 transcript (1.7-fold) and the T1 transcript (<em>3</em>-fold) was observed upon light exposure of etiolated seedlings. However, only <em>SOD</em>-1 activity was observed to increase, after a lag of a few hours. The levels of both transcripts increased in response to paraquat and mechanical wounding. The level of the cytosolic transcript and the respective isozyme activity increased dramatically during prolonged drought stress while the chloroplast transcript remained unaffected. The expression of both genes was enhanced by spraying tomato plants with ethephon--a compound that releases ethylene. Our data show that the expression of Cu,Zn <em>SOD</em> genes in tomato is modulated in response to a variety of factors and suggest the importance of oxyradical toxicity as well as the role of <em>SOD</em> in the defence mechanism of plants exposed to stress.

BACKGROUND

Superoxide anion may contribute to erectile dysfunction (ED) in diabetes mellitus by reducing cavernosal nitric oxide (NO) bioavailability. The purpose of this study was to determine if gene transfer of extracellular superoxide dismutase (EC-SOD) can reduce superoxide anion formation and determine if this reactive oxygen species may contribute to diabetes-related ED in an experimental model of diabetes.

METHODS

Three groups of animals were utilized: (1) control; (2) streptozotocin (STZ)-diabetic rats [60 mg/kg intraperitoneally (ip)] intracavernosally injected with AdCMVbetagal (negative control); and (3) STZ-rats intracavernosally injected with AdCMVEC-SOD. Two months after ip injection of STZ, groups 2 and 3 were transfected with the adenoviruses and 2 days after transfection, all animals underwent cavernosal nerve stimulation (CNS) to assess erectile function. Confocal microscopy for superoxide anion and von Willebrand Factor (vWF) was performed in the STZ-diabetic rat. Superoxide anion production, total SOD activity, and cyclic guanosine monophosphate (cGMP) levels were measured in each experimental group of rats.

RESULTS

Confocal microscopy demonstrated superoxide in smooth muscle and endothelial cells of the STZ-rat cavernosum and colocalized with vWF in the endothelium. Higher superoxide anion levels and decreased cGMP levels were found in the penis of STZ-rats at a time when erectile function was reduced. Two days after administration of AdCMVEC-SOD, superoxide anion levels were significantly lower in the penis of STZ-rats. Total SOD activity and cavernosal cGMP was increased in the penis of EC-SOD-transfected rats. STZ-rats transfected with AdCMVEC-SOD had a peak intracavernosal pressure (ICP) and total ICP to CNS that was similar to control rats.

CONCLUSIONS

These data demonstrate that in vivo adenoviral gene transfer of EC-SOD can reduce corporal superoxide anion levels and raise cavernosal cGMP levels by increasing NO bioavailability thus restoring erectile function in the STZ-diabetic rat.

Superoxide dismutase (<em>SOD</em>) and catalase (CAT), enzymes that degrade superoxide anion and hydrogen peroxide, respectively, reduce size of infarction in anesthetized, open-chest dogs subjected to coronary occlusion followed by reperfusion. To evaluate potential protective effects of these enzymes in conscious animals, three groups of dogs were instrumented at sterile surgery with a hydraulic occluder on the left circumflex (LCX) coronary artery, sonomicrometers to measure regional wall thickness, and catheters to monitor arterial and left ventricular pressures. Ten to 14 days after surgery, the animals were sedated with morphine sulfate (0.5 mg/kg). The LCX artery was occluded for <em>3</em> hr by inflation of the hydraulic cuff. Infusions of <em>SOD</em> (n = 7), CAT (n = 6), or saline (control group, n = 7) were begun 15 min before reperfusion and lasted for 45 min of reperfusion. The doses of <em>SOD</em> and CAT were 5 mg/kg, dissolved in 60 ml of saline, and infused at a rate of 1 ml/min. Myocardial blood flow was measured with tracer-labeled microspheres (15 micron diameter) before occlusion, after 5 to 10 min of occlusion, after 150 min of occlusion, and 5 to 10 min after reperfusion. Size of infarction was measured 24 hr later by dual-perfusion staining with Evans blue and triphenyl tetrazolium. Size of infarction (expressed as a percentage of area at risk) did not differ significantly among the three groups: control, <em>3</em>2 +/- 17% (mean +/- SD); <em>SOD</em>, <em>3</em>8 +/- 17%; CAT, 27 +/- 17%. Hemodynamic parameters and myocardial blood flows (measured before infusion of any agents) were not significantly different among the three groups. Serum <em>SOD</em> levels in <em>SOD</em>-treated dogs were 19 +/- 2 micrograms/ml at the onset of reperfusion and 29 +/- <em>3</em> micrograms/ml at the end of the infusion. Blood assays collected after infusion showed a monoexponential decay of <em>SOD</em> levels with a half-life of 22 +/- 6 min. We conclude that myocardial protection by <em>SOD</em> or CAT is model dependent. In conscious dogs subjected to <em>3</em> hr of coronary occlusion followed by reperfusion, <em>SOD</em> and CAT failed to alter size of infarction.

BACKGROUND

Advanced endometrial cancer often shows resistance to clinical chemotherapy although potencies of anticancer drugs in vitro are promising. The disparity suggests that in vivo microenvironments are not recapitulated by in vitro models used for preclinical testing. However, spheroids replicate some important properties of tumours in vivo. Therefore, for the first time, we compared effects of doxorubicin and cisplatin on <em>3</em>D multicellular structures and 2D cell monolayers of endometrial cancer cells.

METHODS

<em>3</em>D multicellular structures were generated by culturing cancer cells on non-adherent surfaces; and for comparison cell monolayers were cultured on adherent culture plates. Ishikawa, RL95-2, and KLE cell lines were studied. Morphologies of <em>3</em>D multicellular structures were examined. After 48 hours treatment with anticancer drugs, apoptosis, proliferation, glucose metabolism and vascular endothelial growth factor (VEGF) were analysed. Immunostaining of PCNA, Glut-1, p-Erk1/2, <em>SOD</em>-1 and p-Akt1/2/<em>3</em> was also performed.

RESULTS

Distinct <em>3</em>D multicellular morphologies were formed by three different endometrial cancer cell lines. Doxorubicin induced less apoptosis in <em>3</em>D multicellular structures of high grade cancer cells (RL95-2 and KLE cell lines) than in cell monolayers. Parallel alterations in Erk1/2 phosphorylation and cell proliferation might suggest they were linked and again doxorubicin had less effect on <em>3</em>D multicellular structures than cell monolayers. On the other hand, there was no correlation between altered glucose metabolism and proliferation. The responses depended on cancer cell lines and were apparently not mediated by altered Glut-1 levels. The level of <em>SOD</em>-1 was high in <em>3</em>D cell cultures. The effects on VEGF secretion were various and cancer cell line dependent. Importantly, both doxorubicin and cisplatin had selective paradoxical stimulatory effects on VEGF secretion. The microenvironment within <em>3</em>D multicellular structures sustained Akt phosphorylation, consistent with it having a role in anchorage-independent pathways.

CONCLUSIONS

The cancer cells responded to microenvironments in a distinctive manner. <em>3</em>D multicellular structures exhibited greater resistance to the agents than 2D monolayers, and the differences between the culture formats were dependent on cancer cell lines. The effects of anticancer drugs on the intracellular mediators were not similar in <em>3</em>D and 2D cultures. Therefore, using <em>3</em>D cell models may have a significant impact on conclusions derived from screening drugs for endometrial carcinomas.

Many flavonoids extracted from nature plants have been reported to exert antidepressant-like effect in animal studies. The present study was designed to observe the effects of liquiritin, a flavone compound derived from Glycyrrhiza uralensis, on the behaviors of chronic variable stress induced depression model rats and to explore the possible association between its antidepressant-like effect and antioxidative activity by measuring erythrocyte superoxide dismutase (<em>SOD</em>) activity and plasma malondialdehyde (MDA) level of the experimental animals. With the exposure to stressor once daily for consecutive 5 weeks, liquiritin and a positive control drug fluoxetine were administered via gastric intubation to rats once daily for consecutive <em>3</em> weeks from the <em>3</em>rd week. The results showed that CVS reduced open-field activity and sucrose consumption significantly, but increased immobility time in forced swimming test. Treatment of liquiritin could effectively reverse alteration in immobility time and sucrose consumption but did not show significant effect on open-field activity. Moreover, liquiritin could increase <em>SOD</em> activity, inhibit lipid peroxidation, and lessen production of MDA, while fluoxetine did not. In conclusion, the present study demonstrated a potential antidepressant-like effect of liquiritin treatment on chronic variable stress induced depression model rats, which might be related to defense of liquiritin against oxidative stress.

Glutamate has toxic effects on a number of tissues, partly by inducing toxic (e.g., oxidative) stress, whereas adenosine can be protective. Since there is evidence that glutamate and adenosine receptors are present in bone, we set out to study whether oxidative stress, induced by hydrogen peroxide (H2O2), affected viability in the MC<em>3</em>T<em>3</em>-E1 osteoblast-like cell line and whether treatment with adenosine receptor ligands attenuated this. Hydrogen peroxide (100 microM to 5 mM) reduced the viability of the MC<em>3</em>T<em>3</em>-E1 cells, while catalase reversed this cell loss and itself had some mitogenic effect. Superoxide dismutase (<em>SOD</em>) increased the number of viable cells alone but failed to modify significantly the effect of H2O2 treatments. Glutamate (100 microM, 1 mM) and NMDA (10 microM), applied alone for up to 1 h, had a mitogenic effect (P < 0.05). Adenosine A1 and A2A receptor agonists and antagonists at low and high concentrations showed some mitogenic effects when added singly, but only high concentrations of the agonists showed significant protection against cell death resulting from H2O2 treatments. Contributions from both apoptotic and necrotic pathways were implicated in the H2O2-induced cell loss as was demonstrated by the use of the caspase-<em>3</em> inhibitor (Z-DEVD-fmk) and the PARP-1 inhibitor (DPQ). The results demonstrate that hydrogen peroxide was toxic to MC<em>3</em>T<em>3</em>-E1 cells, whereas glutamate was not and may even have a trophic influence. Adenosine and its receptors afforded some protection to osteoblasts against cellular death mediated partly by apoptosis and partly by necrosis.

Hexavalent chromium [Cr(VI)] is a well-known human carcinogen associated with the incidence of lung cancer. Although overproduction of reactive oxygen species (ROS) has been suggested to play a major role in its carcinogenicity, the mechanisms of Cr(VI)-induced ROS production remain unclear. In this study, we investigated the role of NADPH oxidase (NOX), one of the major sources of cellular ROS, in Cr(VI)-induced oxidative stress and carcinogenesis. We found that short-term exposure to Cr(VI) (2μM) resulted in a rapid increase in ROS generation in Beas-2B cells, and concomitantly increased NOX activity and expression of NOX members (NOX1-<em>3</em> and NOX5) and subunits (p22(phox), p47(phox), p40(phox), and p67(phox)). Cr(VI) also induced phosphorylation of p47(phox) and membrane translocation of p47(phox) and p67(phox), further confirming NOX activation. Knockdown of p47(phox) with a short hairpin RNA attenuated the ROS production induced by Cr(VI). Chronic exposure (up to <em>3</em> months) to low doses of Cr(VI) (0.125, 0.25, and 0.5μM) also promoted ROS generation and the expression of NOX subunits, such as p47(phox) and p67(phox), but inhibited the expression of main antioxidant enzymes, such as superoxidase dismutase (<em>SOD</em>) and glutathione peroxidase (GPx). Chronic Cr(VI) exposure resulted in transformation of Beas-2B cells, increasing cell proliferation, anchorage independent growth in soft agar, and forming aggressive tumors in nude mice. Stable knockdown of p47(phox) or overexpression of <em>SOD</em>1, <em>SOD</em>2, or catalase (CAT) eliminated Cr(VI)-induced malignant transformation. Our results suggest that NOX plays an important role in Cr(VI)-induced ROS generation and carcinogenesis.

Effective medical treatments of infertile men with idiopathic oligoasthenoteratospermia (OAT) have yet to be determined. This study considered two major aims: (i) to measure the changes in semen parameters, omega-<em>3</em> fatty acids (FA) compositions and anti-oxidant activity; (ii) to determine if the administration of omega-<em>3</em> FA affect semen quality in infertile men with OAT. Two hundred thirty-eight infertile men with idiopathic OAT were randomised to eicosapentaenoic (EPA) and docosahexaenoic acids (DHA), 1.84 g per day (EPAX 5500TG; Lysaker, Norway), or placebo for <em>3</em>2 weeks. The semen parameters were assessed according to WHO criteria, and the EPA and DHA concentrations were determined in red blood cells (RBCs), seminal plasma and sperm cells at baseline and <em>3</em>2-week treatment period. Of randomised subjects, 211 (88.7%) completed the full <em>3</em>2-week randomisation period. The anti-oxidant status of seminal plasma was also evaluated by measuring the superoxide dismutase (<em>SOD</em>) and catalase-like activity. In the total group of participants, all EPA and DHA levels in RBC, and seminal plasma, were statistically significantly correlated with those in spermatozoa (both P = 0.001). A significant improvement of sperm cell total count (from <em>3</em>8.7 ± 8.7 ' 10⁶ to 61.7 ± 11.2 ' 10⁶, P = 0.001) and sperm cell concentration (from 15.6 ± 4.1 ' 10⁶ per ml to 28.7 ± 4.4 ' 10⁶ per ml, P = 0.001) was observed in the omega-<em>3</em> group. A significant positive correlation was found between the EPA and DHA in seminal plasma and the semen parameters. Seminal plasma EPA and DHA concentrations were positively correlated with seminal plasma <em>SOD</em>-like and catalase-like activity (both P = 0.001). In seminal plasma, both <em>SOD</em>-like and catalase-like activity were positively correlated with sperm count, sperm motility, and sperm morphology. Oligoasthenoteratospermic men with low levels of EPA and DHA may benefit from omega-<em>3</em> FA supplementation. Further studies are warranted to shed more light on this important issue.

In this study, we analysed the implication of superoxide (O2-.) and nitric oxide (NO.) free radicals and their resulting product peroxynitrite (ONOO-) in the neuronal death induced by the activation of the glutamatergic receptor of the N-methyl-D-aspartate (NMDA) subtype using cultured cerebellar granule cells. The NOl donor SIN-1 (<em>3</em>-morpholinosydnonimine N-ethylcarbamide), at concentrations which produced a much higher guanylate cyclase activation (i.e. NO. concentration) than NMDA, was not neurotoxic and did not increase the NMDA-induced neuronal death. The absence of involvement of NO. in NMDA-induced neuronal death was confirmed by the ineffectiveness of L-NG-nitroarginine (L-Narg) as a neuroprotective compound. Electron paramagnetic resonance (EPR) experiments, using 5,5-dimethyl pyrroline 1-oxide (DMPO) as a spin trap, indicated that NMDA receptor stimulation led to the generation of O2-. from at least 15-<em>3</em>0 min. The generation of O2-. by xanthine (XA)-xanthine oxidase (XO) induced a neuronal death similar to that of NMDA. XA-XO-induced neuronal death was suppressed by addition of either superoxide dismutase (<em>SOD</em>) plus catalase (CAT), or DMPO in the incubation medium. In contrast, NMDA-induced neuronal death was widely blocked by DMPO and other spin trap compounds, but not by <em>SOD</em> +/- CAT. XA-XO-induced neuronal death was not potentiated by SIN-1 indicating that ONOO- is not more toxic than O2-. in our neuronal model.

Mice lacking Cu,Zn superoxide dismutase (<em>SOD</em>1) show accelerated, age-related loss of muscle mass. Lack of <em>SOD</em>1 may lead to increased superoxide, reduced nitric oxide (NO), and increased peroxynitrite, each of which could initiate muscle fiber loss. Single muscle fibers from flexor digitorum brevis of wild-type (WT) and Sod1(-/-) mice were loaded with NO-sensitive (4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, DAF-FM) and superoxide-sensitive (dihydroethidium, DHE) probes. Gastrocnemius muscles were analyzed for <em>SOD</em> enzymes, nitric oxide synthases (NOS), and <em>3</em>-nitrotyrosine (<em>3</em>-NT) content. A lack of <em>SOD</em>1 did not increase superoxide availability at rest because no increase in ethidium or 2-hydroxyethidium (2-HE) formation from DHE was seen in fibers from Sod1(-/-) mice compared with those from WT mice. Fibers from Sod1(-/-) mice had decreased NO availability (decreased DAF-FM fluorescence), increased <em>3</em>-NT in muscle proteins indicating increased peroxynitrite formation and increased content of peroxiredoxin V (a peroxynitrite reductase), compared with WT mice. Muscle fibers from Sod1(-/-) mice showed substantially reduced generation of superoxide in response to contractions compared with fibers from WT mice. Inhibition of NOS did not affect DHE oxidation in fibers from WT or Sod1(-/-) mice at rest or during contractions, but transgenic mice overexpressing nNOS showed increased DAF-FM fluorescence and reduced DHE oxidation in resting muscle fibers. It is concluded that formation of peroxynitrite in muscle fibers is a major effect of lack of <em>SOD</em>1 in Sod1(-/-) mice and may contribute to fiber loss in this model, and that NO regulates superoxide availability and peroxynitrite formation in muscle.

Two genes encoding manganese superoxide dismutase (<em>sod</em>-2 and <em>sod</em>-<em>3</em>) have been identified in the nematode Caenorhabditis elegans. Each gene is composed of five exons, and intron positions are identical; however, intron sizes and sequences are not the same. The predicted protein sequences are 86.<em>3</em>% homologous (91.8% conservative), and the cDNAs are only 75.2% homologous. Both deduced protein sequences contain the expected N-terminal mitochondrial transit peptides. Reverse transcriptase polymerase chain reaction analysis shows that both genes are expressed under normal growth conditions and that their RNA transcripts are trans-spliced to the SL-1 leader sequence. The latter result together with Northern blot analysis indicate that both genes have mono-cistronic transcripts. The <em>sod</em>-<em>3</em> gene was mapped to chromosome X, and the location of <em>sod</em>-2 was confirmed to be chromosome I. Polymerase chain reaction was used to amplify the cDNA regions encoding the predicted mature manganese superoxide dismutase proteins and each was cloned and expressed to high levels in Escherichia coli cells deficient in cytosolic superoxide dismutases. Both proteins were shown to be active in E. coli, providing similar protection against methyl viologen-induced oxidative stress. The expressed enzymes, which were not inhibited by hydrogen peroxide or cyanide, are dimeric, show quite different electrophoretic mobilities and isoelectric points, but exhibit comparable specific activities.

Menhaden fish oil (FO) containing n-<em>3</em> fatty acids dramatically extends the life span and delays the onset and progression of autoimmune disease in (NZBxNZW)F1 (B/W) female mice as compared to those fed corn oil (CO) rich in n-6 lipids. As an inefficient antioxidant defense system has been linked to autoimmune diseases, the present study was undertaken to determine whether the protective action of n-<em>3</em> lipids is mediated through their antioxidant defense system. Weanling B/W mice were fed a nutritionally adequate, semipurified diet containing CO or krill oil (KO) or FO at 10% level (w/w) ad libitum until the mice were 6.5 months old. All diets contained the same level of vitamin E (21.5 mg/100 g diet). We compared the effects of feeding n-6 and n-<em>3</em> lipids on survival, kidney disease, hepatic microsomal lipid composition, peroxidation, and on the activity and mRNA expression of the antioxidant enzymes catalase, glutathione peroxidase (GSH-Px) and superoxide dismutase (<em>SOD</em>) in 6.5-month-old B/W mice. The results showed that when compared to livers from CO-fed mice, livers from KO- and FO-fed mice showed: (i) significantly higher (P < 0.001) activities and expression of CAT, GSH-Px and <em>SOD</em>; (ii) significantly lower (P < 0.001) arachidonic acid (20:4n-6) and linoleic acid (18:2n-6) and higher (P < 0.001) eicosapentaenoic acid (20:5n-<em>3</em>) and docosahexaenoic acid (22:6n-<em>3</em>) levels in hepatic microsomes; and (iii) significantly lower (P < 0.001) estimated peroxidation indices and thiobarbituric acid reactive substances generation.(ABSTRACT TRUNCATED AT 250 WORDS)

[reaction: see text]. The riboflavin-catalyzed photooxidation of 2',<em>3</em>',5'-tri-O-acetyl-8-oxo-7,8-dihydroguanosine generates a radical intermediate that is competitively trapped by H(2)O, O2(-)(*), or O(2). The products of H(2)O trapping have been previously described as the spiroiminodihydantoin (pH>>or= 7) and iminoallantoin/guanidinohydantoin (pH < 7) nucleosides. Trapping by O2(-)(*) leads to the oxaluric acid (pH <or= 7) and imidazolone (pH>>or= 8.6) pathways (R' ', R' ' = H or 2,<em>3</em>,5-tri-O-Ac-ribofuranosyl). The pH-dependent role of superoxide was probed using Mn-<em>SOD</em> and compared to guanosine and 8-methoxyguanosine photooxidation.

This investigation studied the contribution of antioxidants in delaying healing in excision cutaneous wounds (8 mm) in diabetic, aged and immunocompromised animals. Skin levels of catalase, glutathione (GSH), ascorbic acid (AA) and vitamin E in streptozotocin-induced diabetic rat were lower as compared to nondiabetics. The 7-d wound tissue of diabetic rats showed an increased vitamin E level along with depleted GSH content. In aged rats (18 months old), higher levels of skin superoxide dismutase (<em>SOD</em>), glutathione peroxidase (Gpx) and thiobarbituric acid reactive substances (TBARS) and lower levels of catalase and GSH were found as compared to their values in young rats (<em>3</em>-4 months old). The levels of <em>SOD</em>, GPx, catalase, AA, GSH and vitamin E in 7-d wound tissue of aged rats were significantly lower in comparison to those in young rats. However, TBARS were elevated in these wound tissues. The non-wounded skin of immunocompromised (athymic) mice showed lower levels of <em>SOD</em>, catalase, and TBARS and higher GSH and GPx levels in comparison to those present in normal mouse skin. Surprisingly, the analysis of 7-d wound tissue showed higher levels of <em>SOD</em>, catalase, GPx, and GSH and lower TBARS level in athymic mice compared to the wound tissue of normal mice. Thus low levels of antioxidants accompanied by raised levels of markers of free radical damage play a significant role in delaying wound healing in aged rats. In diabetic rats reduced glutathione levels may have a contributory role in delaying the healing process. However, in immunocompromised mice the antioxidant status following injury showed an adapted response.

Skeletal muscle dysfunction contributes to poor exercise performance in patients with chronic obstructive pulmonary disease (COPD). Increased oxygen radicals and nitric oxide (NO) have been proposed as mechanisms. In this study, we assessed the levels of protein oxidation (carbonyl formation), lipid peroxidation (4-hydroxy-2-nonenal formation), catalase and Mn-superoxide dismutase (Mn-<em>SOD</em>) expressions, nitric oxide synthases (NOSs), and protein tyrosine nitration in quadriceps muscles of 12 patients with patients with COPD and 6 control subjects. Lipid peroxidation was elevated in muscles of patients with patients with COPD as compared with control subjects, but protein oxidation was not. Muscle Mn-<em>SOD</em> but not catalase protein expression was significantly higher (200%) in patients with patients with COPDas compared with control subjects. Expression of neuronal NOS and endothelial NOS isoforms did not differ between control subjects and patients with COPD, whereas no inducible NOS protein expression was detected in limb muscles of the two groups of subjects. In patients with COPD, neuronal NOS expression correlated negatively with the degree of the airway obstruction (%FEV1 predicted). <em>3</em>-Nitrotyrosine levels were significantly elevated in muscles of patients with COPDas compared with control subjects, and correlated positively with nNOS protein levels. These results indicate the development of both oxidative and nitrosative stresses in the quadriceps of patients with COPD, suggesting their involvement in muscle dysfunction.