OBJECTIVE

Steatohepatitis (SH) is associated with mitochondrial dysfunction and excessive production of superoxide, which can then be converted into H(2)O(2) by <em>SOD</em>2. Since mitochondrial GSH (mGSH) plays a critical role in H(2)O(2) reduction, we explored the interplay between superoxide, H(2)O(2), and mGSH in nutritional and genetic models of SH, which exhibit mGSH depletion.

METHODS

We used isolated mitochondria and primary hepatocytes, as well as in vivo SH models showing mGSH depletion to test the consequences of superoxide scavenging.

RESULTS

In isolated mitochondria and primary hepatocytes, superoxide scavenging by <em>SOD</em> mimetics or purified <em>SOD</em> decreased superoxide and peroxynitrite generation but increased H(2)O(2) following mGSH depletion, despite mitochondrial peroxiredoxin/thioredoxin defense. Selective mGSH depletion sensitized hepatocytes to cell death induced by <em>SOD</em> mimetics, and this was prevented by RIP1 kinase inhibition with necrostatin-1 or GSH repletion with GSH ethyl ester (GSHee). Mice fed the methionine-choline deficient (MCD) diet or MAT1A(-/-) mice exhibited reduced <em>SOD</em>2 activity; in vivo treatment with <em>SOD</em> mimetics increased liver damage, inflammation, and fibrosis, despite a decreased superoxide and <em>3</em>-nitrotyrosine immunoreactivity, effects that were ameliorated by mGSH replenishment with GSHee, but not NAC. As a proof-of-principle of the detrimental role of superoxide scavenging when mGSH was depleted transgenic mice overexpressing <em>SOD</em>2 exhibited enhanced susceptibility to MCD-mediated SH.

CONCLUSIONS

These findings underscore a critical role for mGSH in the therapeutic potential of superoxide scavenging in SH, and suggest that the combined approach of superoxide scavenging with mGSH replenishment may be important in SH.

The mechanisms of toxicity related to human hepatocellular carcinoma HepG2 cell exposures to cadmium telluride quantum dots (CdTe-QDs) were investigated. CdTe-QDs caused cytotoxicity in HepG2 cells in a dose- and time-dependent manner. Treated cells showed an increase in reactive oxygen species (ROS). Altered antioxidant levels were demonstrated by depletion of reduced glutathione (GSH), a decreased ratio of reduced glutathione to oxidized glutathione (GSH/GSSG) and an increased NF-E2-related Factor 2 (Nrf2) activation. Enzyme assays showed that superoxide dismutase (<em>SOD</em>) activity was elevated whereas catalase (CAT) and glutathione-S-transferase (GST) activities were depressed. Further analyses revealed that CdTe-QD exposure resulted in apoptosis, indicated by changes in levels of caspase-<em>3</em> activity, poly ADP-ribose polymerase (PARP) cleavage and phosphatidylserine externalization. Extrinsic apoptotic pathway markers such as Fas levels and caspase-8 activity increased as a result of CdTe-QD exposure. Involvement of the intrinsic/mitochondrial apoptotic pathway was indicated by decreased levels of B-cell lymphoma 2 (Bcl2) protein and mitochondrial cytochrome c, and by increased levels of mitochondrial Bcl-2-associated X protein (Bax) and cytosolic cytochrome c. Further, mitogen-activated protein kinases (MAPKs) such as c-Jun N-terminal kinases (JNK), extracellular signal-regulated kinases (Erk1/2), and p<em>3</em>8 were all activated. Our findings reveal that CdTe-QDs cause oxidative stress, interfere with antioxidant defenses and activate protein kinases, leading to apoptosis via both extrinsic and intrinsic pathways. Since the effects of CdTe-QDs on selected biomarkers were similar or greater compared to those of CdCl2 at equivalent concentrations of cadmium, the study suggests that the toxicity of CdTe-QDs arises from a combination of the effects of cadmium and ROS generated from the NPs.

Formaldehyde (FA) can cause severe central nervous system impairment. But, there are only a few studies about biochemical and histopathological changes of frontal cortex and hippocampal tissue caused by FA toxicity. The aim of our study was to investigate these changes occurring after chronic formaldehyde toxicity in frontal cortex and hippocampal tissues, and protective effect of Vitamin E (vit E) against oxidative damage. Eighteen rats were divided into three groups: (1) control, (2) treated with FA (FAt), and (<em>3</em>) treated with FA and vit E (FAt+vit E) groups. After the treatment, the animals were sacrificed and frontal cortex and hippocampal tissues were removed for biochemical and histopathological investigation. FA significantly increased tissue malondialdehyde (MDA) and protein carbonyl (PC) levels and also decreased superoxide dismutase (<em>SOD</em>) and catalase (CAT) enzyme activities in frontal cortex and hippocampal tissue compared to control. Vit E treatment decreased MDA and PC levels and prevented inhibition of <em>SOD</em> and CAT enzymes in the tissues. In the FAt group, the neurons of both tissues became extensively dark and degenerated with picnotic nuclei. The morphology of neurons in FAt+vit E group was protected well, but not as neurons of the control group. The number of neurons in frontal cortex and hippocampal tissue of FAt group was significantly less than both control and FAt+vit E groups. It was concluded that vit E treatment might be beneficial in preventing FA-induced oxidative frontal cortex and hippocampal tissue damage, therefore, shows potential for clinical use.

Pathological and physiological hypertrophy of the heart is associated with decreased expression of the Kv4.<em>3</em> transient outward current (Ito) channel. The downregulation of channel mRNA and protein, which may be proarrhythmic, is recapitulated with cultured neonatal rat ventricular myocytes treated with angiotensin II (Ang II). Here we show that the 4.9 kb <em>3</em>' untranslated region (<em>3</em>' UTR) of the Kv4.<em>3</em> channel transcript confers Ang II sensitivity to a promoter-reporter construct. In contrast, Kv4.2 and Kv1.5 <em>3</em>'-UTR sequences are insensitive to Ang II. Both Kv4.<em>3</em> <em>3</em>'-UTR reporter mRNA and activity are decreased in Ang II-treated cardiac myocytes, in accordance with a decrease in mRNA stability. This regulation is mediated by Ang II type 1 (AT1) receptors and abolished by NADPH oxidase inhibitors and dominant negative rac. The Ang II effect is also blocked by expression of superoxide dismutase (<em>SOD</em>), but not catalase, showing that superoxide is required. Dominant negative subunits, enzyme inhibitors and hydrogen peroxide experiments show that the apoptosis signal-regulating kinase 1 (ASK1)-p<em>3</em>8 kinase pathway mediates downstream signaling from NADPH oxidase. Mechanical stretch also downregulates Kv4.<em>3</em> <em>3</em>'-UTR reporter activity and this requires AT1 receptors and NADPH oxidase. Thus, activation of AT1 receptors by Ang II or stretch specifically destabilizes cardiac myocyte Kv4.<em>3</em> channel mRNA by activating NADPH oxidase. These results link long-term control of cardiac K+ channel gene expression to a physiological reactive oxygen species signaling pathway.

Vascular diseases are important clinical complications of diabetes. Advanced glycation end-products (AGE) are mediators of vascular dysfunction, but their effects on vascular smooth muscle cell (VSMC) ROS production are unclear. We studied the source and downstream targets of AGE-mediated ROS and reactive nitrogen species production in these cells. Significant increases in superoxide production in AGE-treated VSMC were measured using lucigenin (7650+/-4<em>3</em><em>3</em> vs 4485+/-424 LU/10(6) cells, p<0.001) or coelenterazine (277,907+/-71,295 vs 120,456+/-4140 LU/10(6) cells, p<0.05) and confirmed by ESR spectroscopy. These signals were blocked by the flavin-containing oxidase inhibitor diphenylene iodonium (DPI). AGE-stimulated NF-kappaB activity was abolished by DPI and the superoxide scavenger MnTBAP. AGE differentially regulated VSMC NADPH oxidase catalytic subunits, stimulating the transcription of Nox1 (201+/-12.7%, p<0.0001), while having no effect on Nox4. AGE also increased <em>3</em>-nitrotyrosine formation, which was inhibited by MnTBAP, DPI, or the NOS inhibitor L-NAME. Regarding the source of NO, AGE stimulated inducible nitric oxide synthase mRNA (1 vs 9.7+/-<em>3</em>.0, p=0.046), which was abolished by a NF-kappaB inhibitor, <em>SOD</em>, catalase, or siRNA against Nox1. This study establishes that AGE activate iNOS in VSMC through a ROS-sensitive, NF-kappaB-dependent mechanism involving ROS generation by a Nox1-based oxidase.

Prevention of cancer remains a primary need and new chemopreventive agents must be developed for this purpose. Towards this goal, a chemoprevention study was conducted to evaluate the activity of resveratrol (Res), a phytoalexin, as an inhibitor of colon carcinogenesis. Wistar male rats were divided into six groups, group 1 were control rats, group 2 were control rats that received Res (8 mg/kg body wt p.o. everyday), rats in groups <em>3</em>-6 were treated weekly with 1,2-dimethylhydrazine (DMH, 20 mg/kg body wt, s.c. x 15 times). In addition, groups 4, 5 and 6 received Res as in group 2. Modifying effects were assessed using aberrant crypt foci (ACF) and the extent of histopathological lesions as end point markers. At the end of <em>3</em>0 weeks, Res markedly reduced tumor incidence, the degree of histological lesions and also the size of tumors significantly (P < 0.05) as compared with the rats treated with unsupplemented DMH. The number of ACF consisting of more than six aberrant crypts per rat was observed in group 6 (6.2 +/- 1.4), group 5 (7.7 +/- 1.0) and group 4 (8.2 +/- 1.4) which were significantly lower than that of group <em>3</em> (22.<em>3</em> +/- 2.4) (P < 0.05). The most pronounced inhibition of ACF development was noted in rats fed Res for the entire period and also during the post-initiation period. Also, Res administration lowered the number of argyrophilic nucleolar organizing region-associated proteins (AgNORs) per nucleus in non-lesional colonic crypts, which reflects the cell proliferation activity. Oxidative imbalance in DMH-treatment was significantly (P < 0.01) modulated on Res supplementation as indicated by optimal concentration of thiobarbituric acid reactive substances (TBARS), superoxide dismutase (<em>SOD</em>), catalase (CAT) and reduced glutathione (GSH). The results of our study suggest Res to be an effective chemopreventive agent, which suppresses DMH-induced colon carcinogenesis at various stages.

The interaction of endothelium-derived relaxing factor (EDRF) and oxygen-derived free radicals may potentially play an important role in the pathophysiology of complications associated with diabetes. In the present study, we investigated spontaneous EDRF release in diabetic rat aorta that is unmasked by the addition of superoxide dismutase (<em>SOD</em>). <em>SOD</em> produced a significantly greater relaxation in diabetic aorta compared with control aorta using both aortic ring and bioassay preparations. This relaxation was unaltered by pretreatment with catalase or indomethacin. Removal of the endothelium or pretreatment with either NG-monomethyl-L-arginine or methylene blue eliminated <em>SOD</em>-induced relaxation in both control and diabetic rings. Measurement of antioxidant enzymes revealed an elevation in catalase in diabetic aorta, with no difference in the <em>SOD</em> or glutathione peroxidase activity. The increase in catalase activity suggests increased exposure of diabetic aorta to hydrogen peroxide. Pretreatment of rings with the catalase inhibitor, <em>3</em>-amino-1,2,4-triazole, attenuated the <em>SOD</em>-induced relaxation in diabetic aortic rings but had no effect in control aortic rings. In summary, our observations suggest that the diabetic rat aorta releases more spontaneous EDRF than control aorta; however, the activity of EDRF on vascular smooth muscle tone is masked by increased destruction by oxygen-derived free radicals.

Purple sweet potato color (PSPC), a class of naturally occurring anthocyanins, protects brain function against oxidative stress induced by D-galactose (D-gal) (Sigma-Aldrich, St. Louis, MO, USA). Our data showed that PSPC enhanced open-field activity, decreased step-through latency, and improved spatial learning and memory ability in D-gal-treated old mice by decreasing advanced glycation end-products' (AGEs) formation and the AGE receptor (RAGE) expression, and by elevating Cu,Zn-superoxide dismutase (Cu,Zn-<em>SOD</em>) (Sigma-Aldrich) and catalase (CAT) expression and activity. Cleavage of caspase-<em>3</em> and increased terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end-labeling (TUNEL)-positive cells in D-gal-treated old mice were inhibited by PSPC, which might be attributed to its antioxidant property. PSPC also suppressed the activation of c-Jun NH(2)-terminal kinase (JNK) and the release of cytochrome c from mitochondria that counteracted the onset of neuronal apoptosis in D-gal-treated old mice. Furthermore, it was demonstrated that phosphoinositide <em>3</em>-kinase (PI<em>3</em>K) activation was required for PSPC to promote the neuronal survival accompanied with phosphorylation and activation of Akt and p44/42 mitogen-activated protein kinase (MAPK) by using PI<em>3</em>K inhibitor LY294002 (Cell Signaling Technology, Inc., Beverly, MA, USA), implicating a neuronal survival mechanism. The present results suggest that neuronal survival promoted by PSPC may be a potentially effective method to enhance resistance of neurons to age-related disease.

Influence of 100 microM Ni on growth, Ni accumulation, [Formula: see text], H2O2 and lipid peroxides contents as well as the activities of superoxide dismutase (<em>SOD</em>), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POD) and glutathione peroxidase (GSH-Px) were studied in the leaves of wheat plants on the <em>3</em>rd, 6th and 9th days after treatment. Exposure of the plants to Ni for only <em>3</em> days led to almost 200-fold increase in this metal concentration in the leaf tissue but later the rate of Ni accumulation was much slower. Length and fresh weight of the leaves were substantially reduced, up to 25% and <em>3</em>9%, respectively at the end of experiment. Visible symptoms of Ni toxicity: chlorosis and necrosis were observed following the <em>3</em>rd day. Treatment with Ni resulted in the increase in [Formula: see text] and H2O2 contents in the leaves. Both showed their highest values, approximately 250% of those of the control, on the <em>3</em>rd day and then their levels decreased but still markedly exceeded the control values. <em>SOD</em> and CAT activities decreased significantly in response to Ni treatment, however a several-fold increase in APX and POD activities was found. No significant changes in lipid peroxides content were observed in the leaves after Ni application. The activity of GSH-Px showed a 29% induction on the <em>3</em>rd day. Our results indicated that despite prolonged increases in [Formula: see text] and H2O2 levels, oxidative damage, measured as the level of lipid peroxidation, did not occur in the leaves of Ni-treated wheat.

OBJECTIVE

To compare the levels of <em>3</em> oxidative stress markers (glutathione peroxidase [GPX], superoxide dismutase [<em>SOD</em>], and malondialdehyde [MDA]) and 2 antioxidants (vitamin C and lycopene) in healthy and pre-eclamptic pregnant women.

METHODS

Circulating levels of GPX, SOD, MDA, vitamin C and lycopene were measured in 50 healthy pregnant women and 50 women with pre-eclampsia (PE) (41 with mild PE and 9 with severe PE) attending the antenatal clinic or admitted to the maternity ward of the All-India Institute of Medical Sciences, New Delhi, India.

RESULTS

The levels of GPX, SOD and MDA were significantly higher in women with PE than in controls, and the increase was higher in women with severe PE (P<0.001 using analysis of variance and the Kruskal Wallis test). The levels of vitamin C and lycopene were significantly lower in women with PE than in controls, with a greater decrease in women with severe PE.

CONCLUSIONS

Increased levels of oxidative stress markers and decreased levels of antioxidants in pre-eclamptic women suggest that oxidative stress markers play a significant role in the pathophysiology of pre-eclampsia, and that supplemental dietary antioxidants may have a beneficial role in the prevention of pre-eclampsia in women at high-risk for this condition.

The toxicity of gentamicin (GEN) in the kidney seems to relate to the generation of reactive oxygen species (ROS). Caffeic acid phenethyl ester (CAPE) has been demonstrated to have antioxidant, free radical scavenger and anti-inflammatory effects. It has been proposed that antioxidant maintain the concentration of reduced glutathione (GSH) may restore the cellular defense mechanisms and block lipid peroxidation thus protect against the toxicity of wide variety of nephrotoxic chemicals. We investigated the effects of CAPE on GEN-induced changes in renal malondialdehyde (MDA), a lipid peroxidation product, nitric oxide (NO) generation, superoxide dismutase (<em>SOD</em>), catalase (CAT) activities, GSH content, blood urea nitrogen (BUN) and serum creatinine (Cr) levels. Morphological changes in the kidney were also examined. A total of <em>3</em>2 rats were equally divided into four groups which were: (1) control, (2) injected with intraperitoneally (i.p.) GEN, (<em>3</em>) injected with i.p. GEN+CAPE and (4) injected with i.p. CAPE. GEN administration to control rats increased renal MDA and NO generation but decreased <em>SOD</em> and CAT activities, and GSH content. CAPE administration with GEN injections caused significantly decreased MDA, NO generation and increased <em>SOD</em>, CAT activities and GSH content when compared with GEN alone. Serum level of BUN and Cr significantly increased as a result of nephrotoxicity. CAPE also, significantly decreased serum BUN and Cr levels. Morphological changes in the kidney due to GEN, including tubular necrosis, were evaluated qualitatively. In addition, CAPE reduced the degree of kidney tissue damage induced by GEN. Both biochemical findings and histopathological evidence showed that administration of CAPE reduced the GEN-induced kidney damage. Our results indicated that CAPE acts in the kidney as a potent scavenger of free radicals to prevent the toxic effects of GEN both at the biochemical and histological level. Thus, CAPE could be effectively combined with GEN treatment.

Arachidonic acid (AA), 5,8,11,14-eicosateraenoic acid is abundant, active and necessary in the human body. In the present study, we reported the neuroprotective effects and mechanism of arachidonic acid on hippocampal slices insulted by glutamate, NaN(<em>3</em>) or H(2)O(2)in vitro. Different types of models of brain injury in vitro were developed by 1mM glutamate, 10mM NaN(<em>3</em>) or 2mM H(2)O(2). After <em>3</em>0 min of preincubation with arachidonic acid or linoleic acid, hippocampal slices were subjected to glutamate, NaN(<em>3</em>) or H(2)O(2), then the tissue activities were evaluated by using the 2,<em>3</em>,5-triphenyltetrazolium chloride method. Endogenous antioxidant enzymes activities (<em>SOD</em>, GSH-PX and catalase) in hippocampal slices were evaluated during the course of incubation. MK886 (5 microM; a noncompetitive inhibitor of proliferator-activated receptor [PPAR]alpha), BADGE (bisphenol A diglycidyl ether; 100 microM; an antagonist of PPARgamma) and cycloheximide (CHX; <em>3</em>0 microM; an inhibitor of protein synthesis) were tested for their effects on the neuroprotection afforded by arachidonic acid. Population spikes were recorded in randomly selected hippocapal slices. Arachidonic acid (1-10 microM) dose dependently protected hippocampal slices from glutamate and H(2)O(2) injury (P<0.01), and arachidonic acid (10 microM) can significantly improve the activities of Cu/Zn-<em>SOD</em> in hippocampal slices after 1h incubation. In addition, 10 microM arachidonic acid significantly increased the activity of Mn-<em>SOD</em> and catalase, and decreased the activities of Cu/Zn-<em>SOD</em> to control value after <em>3</em>h incubation. These secondary changes of <em>SOD</em> during incubation can be reversed by indomethacine (10 microM; a nonspecific cyclooxygenase inhibitor) or AA 861 (20 microM; a 5-lipoxygenase inhibitor). Its neuroprotective effect was completely abolished by BADGE and CHX. These observations reveal that arachidonic acid can defense against oxidative stress by boosting the internal antioxidant system of hippocampal slices. Its neuroprotective effect may be mainly mediated by the activation of PPARgamma and synthesis of new protein in tissue.

Photodynamic therapy (PDT), a promising therapeutic modality for the management of solid tumors, is a two-phase treatment consisting of a photosensitizer and visible light. Increasing evidence indicates that tumor cells in regions exposed to sublethal doses of PDT can respond by rescue responses that lead to insufficient cell death. We decided to examine the role of superoxide dismutases (<em>SODs</em>) in the effectiveness of PDT and to investigate whether 2-methoxyestradiol (2-MeOE(2)), an inhibitor of <em>SODs</em>, is capable of potentiating the antitumor effects of this treatment regimen. In the initial experiment we observed that PDT induced the expression of Mn<em>SOD</em> but not Cu,Zn-<em>SOD</em> in cancer cells. Pretreatment of cancer cells with a cell-permeable <em>SOD</em> mimetic, Mn(II)-tetrakis(4-benzoic acid)porphyrin chloride, and transient transfection with the Mn<em>SOD</em> gene resulted in a decreased effectiveness of PDT. Inhibition of <em>SOD</em> activity in tumor cells by preincubation with 2-MeOE(2) produced synergistic antitumor effects when combined with PDT in <em>3</em> murine and 5 human tumor cell lines. The combination treatment was also effective in vivo producing retardation of the tumor growth and prolongation of the survival of tumor-bearing mice. We conclude that inhibition of Mn<em>SOD</em> activity by 2-MeOE(2) is an effective treatment modality capable of potentiating the antitumor effectiveness of PDT.

20-HETE, a metabolite of arachidonic acid, has been implicated as a mediator of free radical formation and tissue death following ischemia-reperfusion (IR) injury in the brain and heart. The present study examined the role of this pathway in a simulated IR renal injury model in vitro. Modified self-inactivating lentiviral vectors were generated to stably overexpress murine Cyp4a12 following transduction into LLC-PK(1) cells (LLC-Cyp4a12). We compared the survival of control and transduced LLC-PK(1) cells following 4 h of ATP depletion and 2 h of recovery in serum-free medium. ATP depletion-recovery of LLC-Cyp4a12 cells resulted in a significantly higher LDH release (P < 0.05) compared with LLC-enhanced green fluorescent protein (EGFP) cells. Treatment with the <em>SOD</em> mimetic MnTMPyP (100 microM) resulted in decreased cytotoxicity in LLC-Cyp4a12 cells. The selective 20-HETE inhibitor HET-0016 (10 microM) also inhibited cytotoxicity significantly (P < 0.05) in LLC-Cyp4a12 cells. Dihydroethidium fluorescence showed that superoxide levels were increased to the same degree in LLC-EGFP and LLC-Cyp4a12 cells after ATP depletion-recovery compared with control cells and that this increase was inhibited by MnTMPyP. There was a significant increase (P < 0.05) of caspase-<em>3</em> cleavage, an effector protease of the apoptotic pathway, in the LLC-Cyp4a12 vs. LLC-EGFP cells (P < 0.05). This was abolished in the presence of HET-0016 (P < 0.05) or MnTMPyP (P < 0.01). These results demonstrate that 20-HETE overexpression can significantly exacerbate the cellular damage that is associated with renal IR injury and that the programmed cell death is mediated by activation of caspase-<em>3</em> and is partially dependent on enhanced CYP4A generation of free radicals.

Reactive oxygen species are important mediators that exert a toxic effect during ischemia-reperfusion (I/R) injury of various organs. Sulforaphane is known to be an indirect antioxidant that acts by inducing Nrf2-dependent phase 2 enzymes. In this study, we investigated whether sulforaphane protects heart against I/R injury. Sprague-Dawley rats received sulforaphane (500microg/kg/day) or vehicle intraperitoneally for <em>3</em> days and global ischemia was performed using isolated perfused Langendorff hearts. Hearts were perfused with Krebs-bicarbonate buffer for 20min pre-ischemic period followed by a 20min global ischemia and 50min reperfusion. Treatment with sulforaphane inhibited an increase in the post-ischemic left ventricular end-diastolic pressure (LVEDP) and improved the post-ischemic left ventricular developed pressure (LVDP), +/-dP/dt, and coronary flow as compared with the untreated control hearts. Pretreatment with 5-hydroxydecanoic acid (5-HD), a mitochondrial K(ATP) channel blocker, for 10min before ischemia attenuated the improvement of LVEDP, LVDP, +/-dP/dt, and coronary flow induced by sulforaphane. Sulforaphane markedly decreased the infarcted size and attenuated the increased lactate dehydrogenase level in effluent during reperfusion. Pretreatment with 5-HD also blocked these protective effects of sulforaphane. Post-ischemia increased the concentration of atrial natriuretic peptide in coronary effluent, which attenuated by sulforaphane treatment. Decreases on Mn-superoxide dismutase (<em>SOD</em>), catalase, and heme oxygenase-1 levels by I/R were increased by sulforaphane treatment and pretreatment of 5-HD blocked the sulforaphane effects. Increases in Bax and caspase-<em>3</em> levels, and decrease in Bcl-2 level by I/R were attenuated by sulforaphane treatment. These results suggest that the protective effects of sulforaphane against I/R injury may be partly mediated through mitochondrial K(ATP) channels and antioxidant pathway.

Resveratrol (RSV) is a natural polyphenol that is known as a powerful chemopreventive and chemotherapeutic anticancer molecule. This study focused on the effects of RSV on the activities and expression levels of antioxidant enzymes in the cancer cells. Prostate cancer PC-<em>3</em> cells, hepatic cancer HepG2 cells, breast cancer MCF-7 cells and the non-cancerous HEK29<em>3</em>T kidney epithelial cells were treated with a wide range of RSV concentrations (10-100 μM) for 24-72 h. Cell growth was estimated by trypan blue staining, activities of the antioxidant enzymes were measured spectrophotometrically, expression levels of the antioxidant enzymes were quantified by digitalizing the protein band intensities on Western blots, and the percentage of apoptotic cells was determined by flow cytometry. Treatment with a low concentration of RSV (25 μM) significantly increased superoxide dismutase (<em>SOD</em>) activity in PC-<em>3</em>, HepG2 and MCF-7 cells, but not in HEK29<em>3</em>T cells. Catalase (CAT) activity was increased in HepG2 cells, but no effect was found on glutathione peroxidase (GPX) upon RSV treatment. RSV-induced <em>SOD</em>2 expression was observed in cancer cells, although the expression of <em>SOD</em>1, CAT and GPX1 was unaffected. Apoptosis increased upon RSV treatment of cancer cells, especially in PC-<em>3</em> and HepG2 cells. Together, our data demonstrated that RSV inhibits cancer cell growth with minimal effects on non-cancerous cells. We postulate that the disproportional up-regulation of <em>SOD</em>, CAT and GPX expression and enzymatic activity in cancer cells results in the mitochondrial accumulation of H2O2, which in turn induces cancer cell apoptosis.

The purpose of this study was to determine the effects and mechanisms of sCD40L on endothelial dysfunction in both human coronary artery endothelial cells (HCAECs) and porcine coronary artery rings. HCAECs treated with sCD40L showed significant reductions of endothelial nitric oxide synthase (eNOS) mRNA and protein levels, eNOS mRNA stability, eNOS enzyme activity, and cellular NO levels, whereas superoxide anion (O(2)(-)) production was significantly increased. sCD40L enhanced eNOS mRNA <em>3</em>'UTR binding to cytoplasmic molecules and induced a unique expression pattern of 95 microRNAs. sCD40L significantly decreased mitochondrial membrane potential, and catalase and <em>SOD</em> activities, whereas it increased NADPH oxidase (NOX) activity. sCD40L increased phosphorylation of MAPKs p<em>3</em>8 and ERK1/2 as well as IkappaBalpha and enhanced NF-kappaB nuclear translocation. In porcine coronary arteries, sCD40L significantly decreased endothelium-dependent vasorelaxation and eNOS mRNA levels, whereas it increased O(2)(-) levels. Antioxidant seleno-l-methionine; chemical inhibitors of p<em>3</em>8, ERK1/2, and mitochondrial complex II; as well as dominant negative mutant forms of IkappaBalpha and NOX4 effectively blocked sCD40L-induced eNOS down-regulation in HCAECs. Thus, sCD40L reduces eNOS levels, whereas it increases oxidative stress through the unique molecular mechanisms involving eNOS mRNA stability, <em>3</em>'UTR-binding molecules, microRNAs, mitochondrial function, ROS-related enzymes, p<em>3</em>8, ERK1/2, and NF-kappaB signal pathways in endothelial cells.

Reactive oxygen species (ROS) may be involved in the toxicity of chlorpyrifos-ethyl (CE) [O,O-diethyl-O-(<em>3</em>,5,6-trichloro-2-pyridyl)phosphorothioate]. We have, therefore, examined the in vivo effects of CE on the rat erythrocyte antioxidant system and evaluated the ameliorating effects of melatonin and a combination of vitamin E and vitamin C on the oxidative damage induced by CE. The experimental groups were: (1) control group, (2) CE-treated group (CE), (<em>3</em>) vitamin E plus vitamin C treatment group (Vit), (4) melatonin-treated group (Mel), (5) vitamin E plus vitamin C plus CE treatment group (Vit + CE), and (6) melatonin plus CE treatment group (Mel + CE). Vitamin E and vitamin C were administered intramuscularly once a day for 6 consecutive days at 150 and 200 mg/kg, respectively, in the Vit and Vit + CE groups. Melatonin was administered intramuscularly at 10 mg/kg per day for 6 consecutive days in the Mel and Mel + CE groups. At the end of the fifth day, the rats of CE, Vit + CE and Mel + CE groups were treated orally with the first of two equal doses of 41 mg/kg CE, the second oral dose being given 21 h later. Blood samples were taken 24 h after the first CE administration. Levels of thiobarbituric acid reactive substance (TBARS), antioxidant defence potential (AOP), and the activities of superoxide dismutase (<em>SOD</em>), glutathione peroxidase (GSH-Px), and catalase (CAT) were determined in erythrocytes. In comparison with the control group, oral administration of CE significantly (P < 0.05) stimulated TBARS activity while significantly (P < 0.05) inhibiting AOP and the activities of <em>SOD</em> and CAT. However, GSH-Px activity remained unchanged by CE treatment. Treatment with melatonin and vitamins E plus C significantly (P < 0.05) reduced the CE-induced increase of TBARS, and overcame the inhibitory effect of CE on <em>SOD</em> and CAT, but not on AOP. Melatonin treatment significantly (P < 0.05) increased only GSH-Px activity, irrespective of the effect of CE. These results suggest that CE treatment increases in vivo lipid peroxidation and decreases antioxidant defence by increasing oxidative stress in erythrocytes of rats, and melatonin and a combination of vitamin E and vitamin C can reduce this lipoperoxidative effect.

Recent studies have implicated a contribution of oxidative stress to the development of hypertension. Studies were performed to determine the effects of the superoxide dismutase (<em>SOD</em>) mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (Tempol) on vascular superoxide production and renal sympathetic nerve activity (RSNA) in anesthetized Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Compared with WKY rats (n=6), SHR showed a doubled vascular superoxide production, which was normalized by treatment with Tempol (<em>3</em> mmol/L, n=7). In WKY rats (n=6), Tempol (<em>3</em>0 mg/kg IV) significantly decreased mean arterial pressure (MAP) from 108+/-5 to 88+/-6 mm Hg and HR from <em>3</em>04+/-9 to 282+/-6 beats/min. In SHR (n=6), Tempol significantly decreased MAP from 166+/-4 to 12<em>3</em>+/-9 mm Hg and HR from <em>3</em>80+/-7 to <em>3</em>29+/-12 beats/min. Furthermore, Tempol significantly decreased RSNA in both WKY rats and SHR. On the basis of group comparisons, the percentage decreases in MAP (-28+/-4%), HR (-16+/-<em>3</em>%) and integrated RSNA (-6<em>3</em>+/-6%) in SHR were significantly greater than in WKY rats (-17+/-<em>3</em>%, -9+/-2%, and -<em>3</em>0+/-4%, respectively). In SHR, changes in integrated RSNA were highly correlated with changes in MAP (r=0.85, P<0.0001) during administration of Tempol (<em>3</em>, 10, and <em>3</em>0 mg/kg IV). In both WKY rats and SHR (n=4, respectively), intracerebroventricular injection of Tempol (<em>3</em>00 micro g/1 micro L) did not alter MAP, HR, or RSNA. Intravenous administration of a <em>SOD</em> inhibitor, diethyldithio-carbamic acid (<em>3</em>0 mg/kg), significantly increased MAP, HR, and integrated RSNA in both WKY rats and SHR (n=6, respectively). These results suggest that augmented superoxide production contributes to the development of hypertension through activation of the sympathetic nervous system.

The contribution of heme oxygenase HO-2, the primary source of bilirubin and carbon monoxide (CO) under physiological conditions, to the regulation of vascular function has remained largely unexplored. Using siRNA HO-2, we examined the effect of suppressed levels of HO-2 on vascular antioxidant and survival proteins. In vivo HO-2 siRNA treatment decreased the basal levels of EC-<em>SOD</em>, pAKT proteins (serine-47<em>3</em> and threonine-<em>3</em>08), without changing Akt protein expression. HO-2 siRNA treatment increased <em>3</em>-nitrotyrosine (<em>3</em>-NT) and apoptotic signaling kinase-1 (ASK-1) (P < 0.01). HO activity was decreased by the use of siRNA HO-2. We extended these studies to the mitochondria, examining for the presence of HO-1 and its role in the regulation of pro- and anti-apoptotic proteins. HO activity was increased by the administration of CoPP resulting in the translocation of HO-1 into the mitochondria, mainly to the inner face of the mitochondrial inner membrane. These findings suggest that HO-2 is critical in the maintenance of heme homeostasis and also the regulation of apoptosis by controlling levels of EC-<em>SOD</em>, Akt, <em>3</em>-NT, and ASK-1. In addition, localization of HO-1 in the mitochondrial compartment plays a critical role in mitochondria-mediated apoptosis.

Age-associated changes in hypothalamic catalase activity and level, and Cu/Zn superoxide dismutase (Cu/Zn <em>SOD</em>) activity were examined in Ames dwarf mice with growth hormone (GH) deficiency and prolonged lifespan, in PEPCK-hGH transgenic mice with overexpression of GH and reduced lifespan, and compared to values measured in normal controls. Hypothalami from young (<em>3</em>-4 months), middle-aged (9-10 months), and old (19-2<em>3</em> months) male mice were examined using spectrophotometric assay and Western blot. In dwarf mice, Cu/Zn <em>SOD</em> and catalase activities declined with age, and were higher than the corresponding normal values in young and middle-aged groups. Catalase levels also declined with age, but were similar to values in normal controls. In GH transgenic mice, age-associated decline of both catalase and Cu/Zn <em>SOD</em> occurred earlier than in normal animals. Catalase levels and activities in transgenic animals were similar to controls, whereas Cu/Zn <em>SOD</em> activity was higher in transgenics than in normal mice. The present results suggest that dwarf mice, during early life, have enhanced hypothalamic free radical defenses, which may contribute to their extended lifespan. However, from the present results in GH transgenic mice, it is impossible to conclude whether early decline of hypothalamic catalase and Cu/Zn <em>SOD</em> in these animals represents a correlate of accelerated aging, or contributes to their reduced lifespan.

Manganese superoxide dismutase (Mn-<em>SOD</em>) is a primary antioxidant enzyme whose expression is essential for life in oxygen. Mn-<em>SOD</em> has tumor suppressor activity in a wide variety of tumors and transformed cell systems. Our initial observations revealed that Mn-<em>SOD</em> expression was inversely correlated with expression of AP-2 transcription factors in normal human fibroblasts and their SV-40 transformed counterparts. Thus we hypothesized that AP-2 may down-regulate Mn-<em>SOD</em> expression. To examine the functional role of AP-2 on Mn-<em>SOD</em> promoter transactivation we cotransfected AP-2-deficient HepG2 cells with a human Mn-<em>SOD</em> promoter-reporter construct and expression vectors encoding each of the three known AP-2 family members. Our results indicated that AP-2 could significantly repress Mn-<em>SOD</em> promoter activity, and that this repression was both Mn-<em>SOD</em> promoter and AP-2-specific. The three AP-2 proteins appeared to play distinct roles in Mn-<em>SOD</em> gene regulation. Moreover, although all three AP-2 proteins could repress the Mn-<em>SOD</em> promoter, AP-2alpha and AP-2gamma were more active in this regard than AP-2beta. Transcriptional repression by AP-2 was not a general effect in this system, because another AP-2-responsive gene, c-erbB-<em>3</em>, was transactivated by AP-2. Repression of Mn-<em>SOD</em> by AP-2 was dependent on DNA binding, and expression of AP-2B, a dominant negative incapable of DNA binding, relieved the repression on Mn-<em>SOD</em> promoter and reactivated Mn-<em>SOD</em> expression in the AP-2 abundant SV40-transformed fibroblast cell line MRC-5VA. These results indicate that AP-2-mediated transcriptional repression contributes to the constitutively low expression of Mn-<em>SOD</em> in SV40-transformed fibroblasts and suggest a mechanism for Mn-<em>SOD</em> down-regulation in cancer.

The effects of 2-Methoxyestradiol (2ME)-induced apoptosis was examined in human leukemia cells (U9<em>3</em>7 and Jurkat) in relation to mitochondrial injury, oxidative damage, and perturbations in signaling pathways. 2ME induced apoptosis in these cells in a dose-dependent manner associated with release of mitochondrial proteins (cytochrome c, AIF), generation of reactive oxygen species (ROS), downregulation of Mcl-1 and XIAP, and inactivation (dephosphorylation) of Akt accompanied by activation of JNK. In these cells, enforced activation of Akt by a constitutively active myristolated Akt construct prevented 2ME-mediated mitochondrial injury, XIAP and Mcl-1 downregulation, JNK activation, and apoptosis, but not ROS generation. Conversely, 2ME lethality was potentiated by the phosphatidylinositol <em>3</em>-kinase (PI<em>3</em>K) inhibitor LY294002. Furthermore, in U9<em>3</em>7 cells, the hydrogen peroxide scavenger catalase and a superoxide dismutase (<em>SOD</em>) mimetic, TBAP, blocked these events, as well as Akt inactivation. Interruption of the JNK pathway by pharmacologic or genetic (e.g. siRNA) means attenuated 2ME-induced mitochondrial injury, XIAP and Mcl-1 downregulation, and apoptosis. Collectively, these findings suggest a hierarchical model of 2ME-related apoptosis induction in human leukemia cells in which 2ME-induced oxidative injury represents a primary event resulting in Akt inactivation, leading, in turn, to JNK activation, and culminating in XIAP and Mcl-1 downregulation, mitochondrial injury, and apoptosis. They also suggest that in human leukemia cells, the Akt pathway plays a critical role in mediating the response to oxidative stress induced by 2ME.

OBJECTIVE

The present study was designed to determine the possible pathway underlying the enhancement of apoptosis induced by the combined use of arsenic trioxide (As(2)O(<em>3</em>)) and ascorbic acid (AA).

METHODS

The level of intracellular reactive oxygen species (ROS) was detected by means of flow cytometry analysis with an oxidation-sensitive fluorescent probe (6-carboxy-2',7' dichlorodihydrofluorescein diacetate) uploading. The activity of glutathione (GSH), glutathione peroxidase (GPx), and superoxide dismutase (SOD) were detected by biochemical methods. The mitochondrial membrane potential was measured by flow cytometry analysis with rhodamine 12<em>3</em> staining. Bcl-2, Bax, and p17 subunit of caspase-<em>3</em> were analyzed using the Western blot method. The apoptosis rate was determined by flow cytometry with annexin-V/propidium iodide staining.

RESULTS

Compared with As(2)O(<em>3</em>) (2.0 micromol/L) treated alone, As(2)O(<em>3</em>) (2.0 micromol/L) in combination with AA (100 micromol/L) decreased intracellular GSH content from 101.<em>3</em>0+/-5.76 to 81.91+/-<em>3</em>.12 mg/g protein, and increased ROS level from 127.61+/-5.12 to 152.60+/-5.88, which was represented by the 2, 7-dichlorofluorescein intensity. The loss of mitochondria membrane potential was increased from 1269.97+/-<em>3</em>6.11 to 1540.52+/-52.6<em>3</em>, which was presented by fluorescence intensity. The p17 subunit of caspase-<em>3</em> expression was increased approximately 2-fold. However, SOD and GPx depletion and the ratio of Bcl-2 to Bax were equal to that of As2O<em>3</em> treated alone (P>0.05). When the ROS scavenger, N-acetyl-L-cysteine, was added to As(2)O(<em>3</em>) and AA combined treatment group, the apoptosis rate decreased from 15.60 %+/-1.14% to 9.48%+/-0.67%, and the ROS level decreased from 152.60+/-5.88 to 102.77+/-10.25.

CONCLUSIONS

AA potentiated As(2)O(<em>3</em>)-induced apoptosis through the oxidative pathway by increasing the ROS level. This may be the result of depleting intracellular GSH. It may influence the downstream cascade following ROS, including mitochondria depolarization and caspase-<em>3</em> activation. However, SOD and GPx depletion and the ratio of Bcl-2 to Bax influenced by As(2)O(<em>3</em>) was not found to be potentiated by AA.