Dispelling Dampness, Relieving Turbidity and Dredging Collaterals Decoction, Attenuates Potassium Oxonate-Induced Hyperuricemia in Rat Models.

Purpose: Dispelling dampness, relieving turbidity and dredging collaterals decoction (DED), is a traditional Chinese medicine used in the treatment of hyperuricemia. We aimed to explore the effect and mechanism of DED in the treatment of hyperuricemia. Methods: The effects of DED (9.48, 4.74, and 2.37 g/kg/d) on potassium oxonate (750 mg/kg/d)-induced hyperuricemia in rats were evaluated by serum uric acid (UA), creatinine (CRE), blood urea nitrogen (BUN), and renal pathological changes. Network pharmacology was used to identify the effective components and targets of DED, and the key targets and signaling pathways for its effects on hyperuricemia were screened. Molecular docking was used to predict the action of DED. H&E, immunohistochemistry, WB, and PCR were used to validate the network pharmacology results. Results: DED can effectively alleviate hyperuricemia, inhibit UA, CRE, BUN, and xanthine oxidase (XOD) activity, and reduce renal inflammatory cell infiltration and glomerular atrophy. The experiment identified 27 potential targets of DED for hyperuricemia, involving 9 components: wogonin, stigmasterol 3-O-beta-D-glucopyranoside, 3ß-acetoxyatractylone, beta-sitosterol, stigmasterol, diosgenin, naringenin, astilbin, and quercetin. DED can relieve hyperuricemia mainly by inhibiting RAGE, HMGB1, IL17R, and phospho-TAK1, and by regulating the AGE-RAGE and IL-17 signaling pathways. Conclusion: DED can alleviate hyperuricemia by inhibiting XOD activity and suppressing renal cell apoptosis and inflammation via the AGE-RAGE signaling pathway and IL-17 signaling pathway. This study provides a theoretical basis for the clinical application of DED.

Naringenin Prevents Inflammation, Apoptosis, and DNA Damage in Potassium Oxonate-Induced Hyperuricemia in Rat Liver Tissue: Roles of Cytochrome C, NF-κB, Caspase-3, and 8-Hydroxydeoxyguanosine.

Background: Hyperuricemia (HU) is a metabolic disease characterized by high uric acid levels in the blood. HU is a risk factor for diabetes, cardiovascular complications, metabolic syndrome, and chronic kidney disease. Purpose: The present study was performed to determine the effect of experimental HU on xanthine oxidase (XO), tumor necrosis factor-alpha (TNF-α), nuclear factor-kappa B (NF-κB), interleukin-17 (IL-17), cytochrome C, glutathione peroxidase (GPx), caspase-3, and 8-hydroxydeoxyguanosine (8-OHdG) levels in liver tissues of rats. Study Design: Thirty-five, male, Wistar albino-type rats were used for this study. Experimental groups were formed as follows: Group 1: control group; Group 2: potassium oxonate (PO) group; group 3: PO+NAR (naringenin; 2 weeks) group; and Group 4: PO (2 weeks)+NAR (2 weeks) group (total of 4 weeks). Methods: The first group was not given anything other than normal rat food and drinking water. In the second group, a 250 mg/kg intraperitoneal dose of PO was administered for 2 weeks. In the third group, 250 mg/kg intraperitoneal PO (application for 2 weeks) and 100 mg/kg NAR intraperitoneally 1 hr after each application were administered. In the fourth group, intraperitoneal PO administration was applied for 2 weeks, followed by intraperitoneal administration of NAR for 2 weeks (4 weeks in total). At the end of the experimental period, XO, TNF-α, NF-κB, IL-17, cytochrome C, GPx, caspase-3, and 8-OHdG levels were determined in liver tissues. Results: HU increased XO, TNF-α, NF-κB, IL-17, cytochrome C, caspase-3, and 8-OHdG levels in liver tissues. However, both 2 and 4 weeks of NAR supplementation decreased these values, and also NAR supplementation led to an increase in GPx levels in tissues. Conclusions: The results of the study show that increased inflammation, apoptosis, and DNA damage in experimental HU can be prevented by administration of NAR due to inhibition of cytochrome C, NF-κB, caspase-3, and 8-OHdG.

Acute and chronic modulation of placental chorionic plate artery reactivity by reactive oxygen species.

Control of vascular resistance and blood flow in the fetoplacental circulation is incompletely understood. Reactive oxygen species (ROS), physiological and pathophysiological regulators of vascular tone, are elevated in preeclampsia (PE), a disease of pregnancy characterized by increased fetoplacental vascular resistance. We tested the hypothesis that ROS modulate vascular reactivity in placental chorionic plate arteries. Wire myography was used to examine (1) the effects of acute exposure to ROS on arterial function in normal pregnancy and (2) the effects of maternal antioxidant supplementation on arterial reactivity in women at high risk for PE participating in the Vitamins in Pre-eclampsia (VIP) trial. ROS generated by xanthine plus xanthine oxidase enhanced basal tension, vasoconstriction in response to the thromboxane mimetic U46619, and relaxation in response to sodium nitroprusside. Hydrogen peroxide and peroxynitrite increased basal tone and relaxed preconstricted arteries (U44619), respectively. In women at risk for PE, chorionic plate artery constriction in response to U46619 was greater in the women receiving placebo compared to the women supplemented with the antioxidant vitamins C and E. ROS may regulate fetoplacental vascular resistance and blood flow in the short term, and chronic exposure to raised ROS could contribute to elevated fetoplacental vascular resistance in PE and fetal growth restriction (FGR).

Effect of docosahexaenoic acid on oxidative stress in placental trophoblast cells.

BACKGROUND: Docosahexaenoic acid (DHA) is an indispensable component of cell membranes that is required at high levels during pregnancy. STUDY DESIGN: We investigated the effects of DHA (0 to 100 microM) in the placenta on oxidative stress using a trophoblast cell line, BeWo. RESULTS: Oxidative stress levels (as evaluated using a fluorescent probe) and lipid peroxidation were significantly higher in cells preincubated with 100 microM of DHA. Oxidative DNA damage in cells preincubated with modest levels of DHA (1 or 10 microM) was significantly lower than for untreated cells or cells preincubated with 100 microM DHA. Survival rates of cells preincubated with modest levels of DHA after oxidative challenge (with H(2)O(2) or Xanthine plus Xanthine oxidase) were significantly higher than without DHA preincubation. CONCLUSION: Modest levels of DHA alleviate oxidative DNA damage whereas high levels of DHA accelerate lipid peroxidation. Thus, DHA supplementation during pregnancy may exert oxidative or antioxidant properties via different mechanisms and depending on dosage.

Effects of keishibukuryogan on vascular function in adjuvant-induced arthritis rats.

It is known that rheumatoid arthritis (RA) accelerates atherosclerosis. Further, the soluble form of vascular adhesion molecule-1 (VCAM-1) is known as a predictive marker of atherosclerosis in RA patients. We reported that keishibukuryogan, one of the Kampo formulas, improved articular symptoms and decreased soluble VCAM-1 in patients with RA. In adjuvant-induced arthritis (AIA) rats, an animal model of RA, it is known that endothelial function is injured by inflammation. So, we investigated the effect of keishibukuryogan on endothelial function in AIA rats. Lewis rats were divided into control, AIA control, and AIA with keishibukuryogan groups. The AIA with keishibukuryogan group was fed 3% keishibukuryogan contained in normal chow. On day 25 after injection of Mycobacterium butyricum, endothelium-dependent relaxation by acetylcholine in the AIA control group was suppressed, but it was improved in the AIA with keishibukuryogan group. The contractions by xanthine/xanthine oxidase in both AIA rats increased, but that in keishibukuryogan decreased compared to the AIA control group. Plasma levels of lipid peroxide increased in the AIA control group, but keishibukuryogan decreased these levels. Plasma levels of nitric oxide (NO) increased in both AIA groups. The expressions of endothelial NO synthase, inducible NO synthase and VCAM-1 of thoracic aorta were investigated by western blotting. These expressions increased in the AIA control group, but were restricted in the AIA with keishibukuryogan group. We considered that keishibukuryogan protected the endothelial function of AIA rats mainly by its anti-oxidative effect.

Estrogenic and antioxidant effects of a phytoestrogen daidzein on ovarian germ cells in embryonic chickens.

The estrogenic and antioxidant effects of the phytoestrogen daidzein (DAI) on germ cell proliferation were evaluated by a chicken ovarian germ-somatic cell coculture model. Ovarian cells were dispersed from 18-day-old embryos, cultured in serum-free McCoy's 5A medium and challenged with DAI alone or in combinations with estrogen receptor antagonist tamoxifen for 48 h. The number of germ cells was counted and the proliferating cells were identified by immunocytochemistry of proliferating cell nuclear antigen (PCNA). The labeling index (LI) was determined for germ cells. Results showed that DAI significantly increased the number of germ cells (P<0.05) and this stimulating effect was inhibited by tamoxifen in a dose-dependent manner. Furthermore, PCNA-LI of germ cells displayed similar changes with the number of germ cells. To estimate the antioxidant action of DAI, ovarian cells were exposed to the reactive oxygen species (ROS)-producing system hypoxanthine/xanthine oxidase (HX/XO). The changes of superoxide dismutase (SOD) activity and glutathione (GSH) level were measured for estimation of the antioxidant status. Ovarian cells were severely damaged by free radicals and this deteriorating effect could be prevented by DAI. Moreover, HX/XO-induced decrease in SOD activity and GSH level was restored by DAI (P<0.05). These results indicated that DAI promoted proliferation of cultured ovarian germ cells by estrogenic action and attenuated ROS-induced toxicity by antioxidant action in embryonic chickens.

Hypouricemic effects of acacetin and 4,5-o-dicaffeoylquinic acid methyl ester on serum uric acid levels in potassium oxonate-pretreated rats.

The effects of acacetin (1) and 4,5-O-dicaffeoylquinic acid methyl ester (2), compounds contained in the flowers of Chrysanthemum sinense SABINE, on the serum uric acid level were investigated using the rats pretreated with the uricase inhibitor potassium oxonate as an animal model for hyperuricemia. When administered per orally at doses of 20 and 50 mg/kg, 1 reduced the serum uric acid level by 49.9 and 63.9%, respectively and 2 reduced the level by 31.2 and 44.4%, respectively. On the other hand, when the same doses were given intraperitoneally, both of compounds also exhibited a dose-dependent and more marked reduction of the serum uric acid level (% reduction at 20 and 50 mg/kg were 63.0 and 95.1% in 1, respectively and 66.9 and 86.5% in 2, respectively). Furthermore, the compounds 1 and 2 inhibited the rat liver xanthine oxidase activity with IC(50) values of 2.22 muM and 5.27 muM, respectively. These results demonstrated the hypouricemic action of 1 and 2, which may be attributable to their xanthine oxidase inhibitory activity.

NAD(P)H oxidase inhibition attenuates neuronal chronotropic actions of angiotensin II.

It is well established that the central cardiovascular effects of angiotensin II (Ang II) involve superoxide production. However, the intracellular mechanism by which reactive oxygen species (ROS) signaling regulates neuronal Ang II actions remains to be elucidated. In the present study, we have used neuronal cells in primary cultures from the hypothalamus and brain stem areas to study the role of ROS on the cellular actions of Ang II. Ang II increases neuronal firing rate, an effect mediated by the AT(1) receptor subtype and involving inhibition of the delayed rectifier potassium current (I(Kv)). This increase in neuronal activity was associated with increases in NADPH oxidase activity and ROS levels within neurons, the latter evidenced by an increase in ethidium fluorescence. The increases in NADPH oxidase activity and ethidium fluorescence were blocked by either the AT(1) receptor antagonist losartan or by the selective NAD(P)H oxidase inhibitor gp91ds-tat. Extracellular application of the ROS scavenger, Tempol, attenuated the Ang II-induced increase in neuronal firing rate by 70%. In addition, gp91ds-tat treatment resulted in a 50% inhibition of Ang II-induced increase in firing rate. In contrast, the ROS generator Xanthine-Xanthine oxidase significantly increased neuronal firing rate. Finally, Ang II inhibited neuronal I(Kv,) and this inhibition was abolished by gp91ds-tat treatment. These observations demonstrate, for the first time, that Ang II regulates neuronal activity via a series of events that includes ROS generation and inhibition of I(Kv). This signaling seems to be a critical cellular event in central Ang II regulation of cardiovascular function.

Anti-angiogenic, antioxidant and xanthine oxidase inhibition activities of the mushroom Phellinus linteus.

Fruiting bodies of Phellinus linteus were extracted with 70% ethanol at room temperature. The Phellinus linteus extract (PL) showed strong anti-angiogenic activity, which was detected using the chick embryo chorioallantoic membrane (CAM) assay. The in vitro antioxidant activities of PL were evaluated using two different bioassays. PL was comparable to Vitamin C in scavenging the stable free radical 1,1-diphenyl-2-picrylhyrazyl (DPPH). It also inhibited lipid peroxidation (LPO) in a concentration-dependent manner. These results suggest that antioxidant and anti-angiogenic activities of Phellinus linteus would be partly responsible for its anti-tumor effect.

Protection of insulin-secreting INS-1 cells against oxidative stress through adenoviral-mediated glutathione peroxidase overexpression.

OBJECTIVES: A large fraction of an islet graft can be lost early following allotransplantation from various non specific mechanisms including oxidative stress. Overexpression of antioxidant enzymes could confer a beneficial effect on islets exposed to reactive oxygen and nitrogen species. We examined the viability of beta cells driven to overexpress glutathione peroxidase (GPx) and exposed to a superoxide donor (hypoxanthine/xanthine oxidase HX/XO) and a nitric oxide donor (3-morpholinosydnonimine SIN-1). METHODS: Cultured INS-1 rat-derived insulin-secreting cells were transfected by an E1-deleted adenovirus carrying GPx cDNA (AdGPx). Additional experiments were performed with an adenovector carrying Cu/Zn superoxide dismutase cDNA (AdSOD). Cellular viability was tested by the WST-1 colorimetric assay and functionality by static incubation. RESULTS: AdGPx increased GPx activity within 48 hours from 0 (untransfected cells) to 60 +/- 11 U/g (cells transfected at an MOI of 25: 1). GPx overexpression significantly reduced cytotoxicity induced by HX/XO from 10.81 +/- 1.41 to 5.42 +/- 2.62% at 10 mU/ml and from 61.19 +/- 4.17 to 52.9 +/- 4.39% at 20 mU/ml (p=0.0002, transfected cells vs control cells). Doses of SIN-1 from 600 to 1000 micromol/l resulted in cytotoxicity ranging from 17.66 +/- 3.48 to 45.97 +/- 6.48% in control cells and from 5.65 +/- 1.37 to 35.80 +/- 5.59% in AdGPx transfected cells (p=0.015). The combination of AdGPx and AdSOD did not exhibit any synergistic cytoprotective effect. Control cells exposed to a HX/XO stress exhibited a reduction in glucose-theophylline stimulated insulin secretion by half, while stressed GPx overexpressing-cells maintained the same insulin secretion level than non-stressed cells. CONCLUSIONS: Adenoviral-induced overexpression of GPx enhances the resistance of a rat beta cell line to both reactive oxygen (ROS) and reactive nitrogen species (RNS) cytotoxicity. Transposition of these findings to human islet transplantation with a clinically-relevant procedure deserves further investigations.

Effect of Rhus coriaria L. (Anacardiaceae) on superoxide radical scavenging and xanthine oxidase activity.

Rhus coriaria L. (Anacardiaceae) is a well-known spice in the Middle-East and grown in the Central and East Anatolia region of Turkey. A methanolic extract (water-soluble part constituents) of R. coriaria, was found to be an uncompetitive inhibitor of xanthine oxidase and scavenger of superoxide radical in vitro with IC50 values of 172.5 microg/mL and 232 microg/mL respectively. Superoxide radicals were generated either by an enzymatic or a non-enzymatic system, and scavenging ability was evaluated by the inhibition of nitroblue tetrazolium reduction. This study provides evidence that a crude extract of R. coriaria exhibits interesting antioxidant properties, expressed either by the capacity to scavenge superoxide radical or to uncompetitively inhibit xanthine oxidase.

Redox regulation of HIF-1alpha levels and HO-1 expression in renal medullary interstitial cells.

The present study hypothesized that superoxide (O2(-)*) importantly contributes to the regulation of hypoxia-inducible factor (HIF)-1alpha expression at posttranscriptional levels in renal medullary interstitial cells (RMICs) of rats. By Western blot analysis, it was found that incubation of RMICs with O2(-)* generators xanthine/xanthine oxidase and menadione significantly inhibited the hypoxia- or CoCl(2)-induced increase in HIF-1alpha levels and completely blocked the increase in HIF-1alpha levels induced by ubiquitin-proteasome inhibition with CBZ-LLL in the nuclear extracts from these cells. Under normoxic conditions, a cell-permeable O2(-)* dismutase (SOD) mimetic, 4-hydroxyl-tetramethylpiperidin-oxyl (TEMPOL) and PEG-SOD, significantly increased HIF-1alpha levels in RMICs. Two mechanistically different inhibitors of NAD(P)H oxidase, diphenyleneiodonium and apocynin, were also found to increase HIF-1alpha levels in these renal cells. Moreover, introduction of an anti-sense oligodeoxynucleotide specific to NAD(P)H oxidase subunit, p22(phox), into RMICs markedly increased HIF-1alpha levels. In contrast, the OH* scavenger tetramethylthiourea had no effect on the accumulation of HIF-1alpha in these renal cells. By Northern blot analysis, scavenging or dismutation of O2(-)* by TEMPOL and PEG-SOD was found to increase the mRNA levels of an HIF-1alpha-targeted gene, heme oxygenase-1. These results indicate that increased intracellular O2(-)* levels induce HIF-1alpha degradation independently of H(2)O(2) and OH* radicals in RMICs. NAD(P)H oxidase activity may importantly contribute to this posttranscriptional regulation of HIF-1alpha in these cells under physiological conditions.

Prevention of cellular ROS damage by isovitexin and related flavonoids.

The antioxidant properties of isovitexin and related flavonoids were studied. Isovitexin inhibited xanthine oxidase with an IC50 value of = 15.2 microM. The flavonoid analogues, apigenin, kaempferol, quercetin, myricetin, and genistein also inhibited xanthine oxidase with IC50 values of 0.58, 2.18, 1.09, 9.90, and 4.83 microM, respectively. Isovitexin protected DNA from the Fenton reaction-induced breakage in a dose-dependent manner with an IC50 value of 9.52 microM. Isovitexin also protected HL-60 cells from the ROS damage induced by the xanthine/xanthine oxidase reaction. Isovitexin exhibited the lowest cytotoxicity toward HL-60 cells (LD50 >400 microM) compared to the other flavonoids examined. In addition, excess hydrogen peroxide induced by cadmium in A2780 ovarian cells was significantly suppressed by isovitexin. These results suggest that isovitexin in rice may protect cells from oxidative stress.

Inhibition of xanthine and monoamine oxidases by stilbenoids from Veratrum taliense.

The bioassay guided refractionation of the methanol extract of roots and rhizomes of Veratrum taliense (Liliaceae) yielded five stilbenoids: veraphenol, resveratrol, piceid, isorhapontin, and mulberroside E, all inhibiting xanthine oxidase (XO, EC 1.2.3.2.) in vitro in a dose-dependent manner with IC50 values of 11.0, 96.7, 66.1, 70.0, and 78.4 microM, respectively. Veraphenol and mulberroside E were found to be mixed XO inhibitors with the Ki and Ki data of the former being 32.8 and 239.3 microM, and those of latter 32.5 and 13.8 microM, respectively. However, the inhibition on the enzyme by resveratrol, isorhapontin, and piceid was shown to be competitive with their Ki values of 9.7, 19.1, and 14.3 microM, respectively. Among the five stilbenoids, veraphenol and resveratrol were also revealed to inhibit competitively monoamine oxidase A (MAO, EC 1.4.3.4) with IC50 values at 38.0 and 26.6 microM, and Ki data 36.4 and 47.3 microM, respectively. However, none of the stilbenoids was inhibitory on MAO B in our assay. The structure-activity relationship examination showed that glycosylation of the stilbenoids could reduce the inhibition on XO and diminish the activity against MAO A, indicating that the free phenolic hydroxy group of the compounds was most likely essential for these bioactivities.

Polymorphism of the glutathione transferase subunit 3 in Sprague-Dawley rats involves a reactive cysteine residue.

Comparison of Hirosaki hairless rat (HHR) and Sprague-Dawley (SD) rat liver glutathione transferase (GST) subunits by HPLC revealed differences in subunit 3; a new peak was detected in HHR GSTs and this was tentatively named X. By chromatofocusing, the HHR GST form composed of peak X and SD rat GST 3-3 were eluted at pH 8.8 and 9.1 respectively. The former was more sensitive to the SH reagent N-ethylmaleimide (NEM) than the latter. GSSG treatment of peak X resulted in a shift of retention time (peak Y) by HPLC analysis. However, such conversion was not observed for the SD rat GST 3-3 following GSSG or dithiothreitol (DTT) treatment. Peak Y exhibited m/z values of 26091.9 and 26125.4 by matrix-assisted laser-desorption ionization-time-of-flight MS, higher than those of peak X by 304-307, equivalent to the molecular-mass value of GSH. On treatment with DTT, peak Y was converted into peak X, with release of a substance with HPLC-characteristics of GSH. This substance was confirmed to be GSH by liquid chromatography/MS. These results thus indicated peak Y to be a glutathionylated form of peak X. Quantification revealed the release of 4 nmol of GSH from 0.12 mg of the peak Y protein, corresponding to 4.8 nmol (M(r) 25000). The nucleotide sequence of HHR GST subunit 3 cDNA proved identical to that reported for pGTA/C44, possessing asparagine and cysteine as the 198th and 199th amino acid residues, respectively, corresponding to lysine and serine in subunit 3 of the SD rat. Thus peak X appeared to be the product of HHR GST subunit 3 cDNA. Treatment with N-(4-dimethylamino-3,5-dinitrophenyl)maleimide, a coloured analogue of NEM, followed by trypsin-treatment and sequencing of labelled peptides, identified the reactive cysteine residue of HHR GST subunit 3 to be located at position 199. Unlike SD rat GST 3-3, HHR GST 3-3 was not activated by treatment with xanthine and xanthine oxidase. These results suggest polymorphism of the rat GST subunit 3 gene with individual gene product variation in sensitivity to oxidative stress.

On the role of hydroxyl radical and the effect of tetrandrine on nuclear factor--kappaB activation by phorbol 12-myristate 13-acetate.

Nuclear factor kappaB (NF-kappaB) is considered to be an important target for therapeutic intervention because of its role in the regulation of proinflammatory and profibrotic mediators. The present study examined the role of hydroxyl (*OH) radical and the effect of tetrandrine, an alkaloid extracted from the Chinese medicinal herb Stephania tetrandra, on NF-kappaB activation by a tumor promoter, phorbol 12-myristate 13-acetate (PMA) in human lymphoid T cells (ie, Jurkat cells). Exogenous superoxide dismutase (SOD) enhanced the NF-kappaB activation by PMA, while catalase blocked it. Formate, a scavenger of *OH radical, also was inhibitory, as was deferoxamine, a metal chelator. These data suggest an important role of *OH radical in PMA-induced NF-kappaB activation. Incubation of the cells with tetrandrine prior to the stimulation of the cells was found to inhibit PMA-induced NF-kappaB activation. Tetrandrine activity was so potent that 50 microM of tetrandrine was sufficient to inhibit activation of NF-kappaB completely. Electron spin resonance (ESR) spin trapping was used to investigate the antioxidant action of tetrandrine using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap. Tetrandrine is an antioxidant for both *OH and superoxide (O2-)radicals. The reaction rate constant of tetrandrine with *OH is 1.4 x 10(10) M(-1)sec(-1), which is comparable with several well established antioxidants, such as ascorbate, glutathione, and cysteine. The Fenton reaction (Fe(II) + H2O2-->Fe(III) + *OH + OH-) and xanthine/xanthine oxidase were used as sources of *OH and O2- radicals. The free radical scavenging activity of tetrandrine is responsible for its inhibition of PMA-induced NF-kappaB activation.

The iron chelator pyridoxal isonicotinoyl hydrazone (PIH) protects plasmid pUC-18 DNA against *OH-mediated strand breaks.

Pyridoxal isonicotinoyl hydrazone (PIH) has previously been studied for use in iron chelation therapy in iron-overload diseases. It is an efficient in vitro antioxidant due to its Fe(III) complexing activity (Schulman, H. M., et al. Redox Report 1:373-378; 1995). Pathologies associated with iron-overload include hepatic and other cancers. Since oxidative alterations of DNA can be linked to the development of cancer, we decided to study whether PIH protects DNA against in vitro oxidative stress. We report here that pUC-18 plasmid DNA is damaged by *OH radicals generated from Fe(II) plus H2O2 or from Fe(II) plus hypoxanthine/xanthine oxidase. The DNA damage was quantified by determining the diminution of supercoiled DNA forms after oxidative attack using agar gel electrophoresis. Micromolar amounts of PIH (20-30 microM) were able to half-protect DNA from iron (1-7.5 microM)-mediated *OH formation. The antioxidant capacity of PIH was significantly higher than that of some of its analogs and desferrioxamine. PIH and some of its analogues could also inhibit the oxidative degradation of 2-deoxyribose caused by Fenton reagents. Since we observed that PIH enhances the Fe(II) autoxidation rate, measured by the ferrozine technique, PIH may limit *OH formation and consequently DNA damage by decreasing the amount of Fe(II) available to catalyze Fenton reactions.

Apoptosis of myelin-reactive T cells induced by reactive oxygen and nitrogen intermediates in vitro.

Apoptosis is a major mechanism of T cell elimination during ontogeny and tolerance induction as well as in autoimmunity. To assess the possible involvement of reactive oxygen and nitrogen intermediates (ROI and NO.) in T-cell apoptosis during autoimmune demyelination we investigated the effects of H2O2 and NO. in vitro on activated autoreactive CD4+ T cell lines capable of transferring experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune neuritis (EAN). For detection and quantitation of apoptotic cells, DNA fragmentation was assessed by in situ tailing with fluorescein-ddUTP and subsequent flow cytometric analysis. H2O2 applied directly to the cell cultures for 6 to 18 hr at concentrations of 10 to 300 microM and ROI released by combination of hypoxanthine and xanthine oxidase (HX/XO) caused apoptosis in a dose-dependent manner in 13-33% of T cells of neuritogenic and encephalitogenic T cell lines. Apoptosis induction could be suppressed by the H2O2-neutralizing enzyme catalase. NO. released by the penicillamine derivative SNAP induced apoptosis to a similar extent as ROI. Maximum values were 38% in an encephalitogenic V beta 8.2-T cell receptor-bearing T cell line and 26% in a neuritogenic T cell line. T cell lines with specificity to ovalbumin revealed slightly lower susceptibility to apoptosis induction by all three kinds of trigger, which is, however, most probably not due to the different antigen specificity, but rather a result of fewer in vitro restimulation cycles of these cells. In neuritogenic cells high-dose (100 units/ml) exogenous interleukin-2 (IL-2) prevents H2O2-induced apoptosis. In conclusion, macrophage-derived reactive oxygen and nitrogen intermediates have the potency to limit inflammatory demyelination by elimination of autoreactive and bystander T cells via apoptotic cell death, and IL-2 is a rescue factor.

Metallothionein and zinc as potential antioxidants in radical-induced lipid peroxidation in cultured hepatocytes.

Rat hepatocytes were isolated by a two-step collagenase perfusion technique and introduced to the hydroxyl radical (OH)-generating xanthine-xanthine oxidase-iron (X/XO/Fe) system. The amount of thiobarbituric acid reactive substances (TBA) and thiobarbituric acid bound malondialdehyde (TBA-MDA) were assayed in homogenates after different phases of cultivation. The effects on lipid peroxidation of supplemented metallothionein (MT) ranging from 25 to 75 microM and zinc ranging from 14.5 to 77.8 microM, as well as the effect of a Zn-pretreatment for 18 h were investigated. The addition of X/XO/Fe resulted in a 3 to 4-fold increase in the levels of TBA and TBA-MDA. These results show that X/XO/Fe initiated the lipid peroxidation in the hepatocyte cell system. High doses of supplemented MT inhibited the production of TBA and TBA-MDA. Neither Zn nor the Zn-pretreatment, which resulted in an increase of intracellular MT, had any effect on TBA and TBA-MDA levels. This study suggests that MT can act as an antioxidant in high concentrations via the cysteinyl groups of the protein. The postulated protective effects of Zn via its release from the oxidized MT can be ruled out.

The protective role of glutathione peroxidase in apoptosis induced by reactive oxygen species.

Selenium-dependent glutathione peroxidase (GPx) plays a protective role in oxidative stress-induced apoptosis. In this study, we demonstrated that MDBK cells, a bovine renal epithelial cell line, exhibited internucleosomal DNA fragmentation characteristic of apoptotic cell death under selenium-deficient conditions with lower doses of hydrogen peroxide (H2O2) than under selenium-supplemented ones. This was due to a decreased amount of GPx in the cells under selenium-deficient conditions, because other antioxidative enzyme activities were not affected by the selenium supplementation. Cumene hydroperoxide also induced DNA fragmentation in selenium-deficient cells but no ladder formation was observed. Flow cytometric analysis showed that selenium-deficient cells were less capable of scavenging intracellular peroxides after exposure to exogenous H2O2 than selenium-supplemented ones. In contrast, there was no difference in viability between selenium-supplemented and non-supplemented cells in cell survival after exposure to menadione, which activates the electron transport system and increases intracellular superoxide radicals. Clofibrate, a peroxisomal proliferator and an inducer of catalase (CAT), partially protected both Se-deficient and Se-supplemented cells from exogenous H202. We concluded that selenium-deficient cells were more easily brought to apoptotic cell death by peroxides, but not by superoxide radicals, than selenium-supplemented ones and that CAT could compensate for the depletion of GPx to a certain degree by scavenging H2O2.