The mechanism of inhibition by xanthine of adenosine A1-receptor responses in rat hippocampus

We have recently observed that the free radical-generating mixture of xanthine and xanthine oxidase (X/XO) can suppress the inhibitory effects of adenosine on synaptic transmission in the hippocampus, but that this action can be mimicked by xanthine alone. We have now clarified the mechanism of these interactions by using the new, potent and highly selective inhibitor of xanthine oxidase, 1-(3-cyano-4-neopentyloxyphenyl)pyrazole-4-carboxylic acid (Y-700). Field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 region of rat hippocampal slices. X/XO induced a long-lasting increase of fEPSP slope and significantly reduced the presynaptic inhibitory effect of adenosine. Both these actions were prevented by Y-700 at a concentration of only 200nM. Similarly the superfusion of xanthine alone increased fEPSP slope and reduced sensitivity to adenosine but these effects were also prevented by Y-700. The results indicate that the antagonism of adenosine responses by X/XO or by xanthine alone are entirely attributable to the activity of the added or endogenous XO activity, probably generating free radicals, and are not likely to be caused by a direct antagonistic action at the xanthine-sensitive site on the adenosine receptor.     

超氧阴离子自由基对大鼠肝卵圆细胞胞内自由钙浓度的影响

采用激光共聚焦扫描显微镜,观察黄嘌呤黄嘌呤氧化酶反应系统（Ｘ／ＸＯ）生成不同浓度的超氧阴离子自由基（Ｏ２）致培养的大鼠肝卵圆细胞（ＷＢ细胞）胞质内钙浓度的变化,结果发现：仅小剂量的Ｏ２引起胞质内钙浓度升高,约３０Ｓ后达到峰值,６０ｓ后恢复正常。部分细胞观察到数次钙浓度间断升高、幅度逐渐下降的现象。超氧化物歧化酶（ＳＯＤ）可抑制此变化,过氧化氢酶（ＣＡＴ）无效果,细胞在无钙液中观察仍出现钙峰,但受     

氧自由基对人胚软骨细胞超微结构的影响

采用体外软骨细胞培养方法，观察由黄嘌呤－黄嘌呤氧化酶系产生的氧自由对人胚软骨细胞超微结构的影响，结果表明，该酶系产生的自由基对人胚软骨细胞膜及细胞器产生了明显的损伤作用，从而间接提示炎性关节疾病时活化中性白细胞、巨噬细胞释放的氧自由基可能是引起关节软骨损伤的原因之一。     

Chondrocyte apoptosis induced by nitric oxide.

Chondrocytes stimulated with IL-1 produce high levels of nitric oxide (NO), which inhibits proliferation induced by transforming growth factor-beta or serum. This study analyzes the role of NO and IL-1 in the induction of chondrocyte cell death. NO generated from sodium nitroprusside induced apoptosis in cultured chondrocytes as demonstrated by electron microscopy, 4',6-dianidino-2-phenylindole dihydrochloride staining, FACS analysis, and histochemical detection of DNA fragmentation. Similar results were obtained with two other NO donors, 3-morpholinosynonimide-hydrochloride and s-nitroso-N-acetyl-D-L-penicillamine. In contrast, oxygen radicals generated by hypoxanthine/xanthine oxidase caused necrosis but did not induce chondrocyte apoptosis. To analyze whether endogenously generated NO induces apoptosis, chondrocytes were stimulated with IL-1, but there was no evidence for apoptotic changes. Combinations of NO inducers such as IL-1, lipopolysaccharide, tumor necrosis factor, and interferon-gamma also failed to trigger apoptosis. IL-1-stimulated chondrocytes are known to produce oxygen radicals that react with NO to form products that can induce cell death in other systems. We thus tested IL-1 in combination with the oxygen radical scavengers N-acetyl cysteine, dimethyl sulfoxide, or 5,5'-dimetylpyrroline 1-oxide. Under these conditions IL-1 was able to induce apoptosis, which was inhibited in a dose-dependent manner by the NO synthase inhibitor N-monomethyl L-arginine. Conversely, endogenous oxygen radicals induced by inflammatory mediators caused necrosis under conditions in which the simultaneous production of NO was reduced. These results suggest that NO, but not oxygen radicals, is the primary inducer of apoptosis in human articular chondrocytes.     

The protective effect of allopurinol on cholestatic liver injury induced by bile duct ligation.

To determine whether oxygen free radicals are responsible for the pathogenesis of the cholestasis induced by ligation of common bile duct (CBD) variables which reflect the hepatic function in the serum, the amount of superoxide radical production, and xanthine oxidase(XO) activity were studied. The activity of serum alanine aminotransferase, bilirubin level in the serum and the amount of superoxide radical production were lower in a CBD ligation with allopurinol treated group than in a CBD ligation without allopurinol treated group. Abnormalities of the microscopic structures were reduced in a CBD ligation with allopurinol treated group than in a CBD ligation without allopurinol treated group. Allopurinol, an inhibitor of XO, prevented the hepatic damage induced by CBD ligation through the inhibition of XO. These experiments demonstrate that oxygen free radicals are responsible for the pathogenesis of the cholestatic liver.     

Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa

The objective of this study was to evaluate the effect of the generation of reactive oxygen species (ROS) on the integrity of the DNA of human spermatozoa, and to determine if pretreatment with antioxidants can reduce DNA damage. Samples were obtained from 47 men undergoing infertility investigation. ROS were generated in the samples by the addition of xanthine/xanthine oxidase (X/XO) with or without antioxidants. After incubation at timed intervals (0-2 h) with X/XO, the percentage of spermatozoa with DNA fragmentation was determined using the method of TdT-mediated DNA end-labelling (TUNEL). Time intervals were selected to mimic the clinical situation in which spermatozoa are held for a period of time after swim-up while the oocytes are prepared for ICSI. A significant increase in sperm DNA damage was evident when samples were incubated in the presence of ROS for intervals of 1 and 2 h, but not when incubated with ROS for <1 h (P = 0.0001). The addition of antioxidants significantly decreased the amount of DNA damage induced by ROS generation (P < 0.04). ROS can cause an increase in DNA fragmentation and pretreatment with antioxidants can reduce DNA damage.      

Modulation of monocyte chemokine production and nuclear factor kappa B activity by oxidants.

Reactive oxygen species can directly damage tissue. In this setting, amplification of tissue damage also occurs through infiltration of inflammatory cells either acutely or chronically. Several recent studies suggest that reactive oxygen species stimulate production of certain chemokines, which are potent chemoattractants for inflammatory cells. In the present study, we examined whether oxidants, generated by the combination of xanthine and xanthine oxidase (X/XO), alter chemokine production by monocytes and U937 cells. Our findings demonstrate that X/XO stimulates monocytes, but not U937 cells, to produce increased amounts of interleukin-8 (IL-8) and monocyte chemoattractant protein. This effect is attenuated by pretreatment with dimethylsulfoxide (DMSO), a scavenger of hydroxyl radicals, but is not affected by superoxide dismutase or catalase. In contrast, X/XO-induced cytotoxicity, evidenced by lactate dehydrogenase release, is mediated primarily by hydrogen peroxide, as catalase reverses this effect. Finally, exposure to X/XO causes an increase in nuclear factor kappa B (NF-kappaB), and this effect is attenuated by DMSO. These studies suggest that reactive oxygen species can induce production of molecules that amplify inflammation through attraction of inflammatory cells. It appears the hydroxyl radical is the principal oxidant species involved in stimulation of chemokine production.     

Response of fibroblast cultures from ataxia-telangiectasia patients to reactive oxygen species generated during inflammatory reactions

Cells from patients with ataxia-telangiectasia (AT) are more sensitive than cells from normal individuals to a number of compounds which induce DNA damage via oxygen-derived free radical attack. We tested the hypothesis that AT cells would show a sensitivity to reactive oxygen species (ROS) generated by activated inflammatory cells. AT cells were exposed to neutro-phils activated with 12-O-tetradecanoyl-phor-bol-13-acetate (TPA) or to xanthine/xanthine oxidase (X/XO), an enzyme system which generates superoxide and hydrogen peroxide. Induced micronuclei (MN) frequencies (corrected for spontaneous MN frequencies) were significantly higher in AT cell cultures than in cultures from normal individuals (comparison of MN frequencies of AT vs. normal cultures: for treatment with activated neutrophils, P = 0.003; for X/XO, P = 0.05). The comet assay was used to determine whether the elevated chromosomal damage in the treated AT cells was due to a difference in strand breakage or its rejoining. X/XO treatment was used in studies of single-stranded (SS) DNA breakage, and X-ray treatment for double-stranded (DS) DNA damage. AT and normal cells showed no significant differences in the initial levels of SS (P = 0.29) or DS (P = 0.91) DNA damage. Likewise, they exhibited similar rejoining kinetics (rejoining half-time for SS = 10 min, for DS = 30 min). These data support the involvement of the AT loci in determining a cell's ability to deal with oxidative stress, although the mechanism underlying this effect has yet to be resolved. The data also suggest that AT patients are at elevated risk of sustaining DNA damage in tissues undergoing inflammatory reactions. © 1994 Wiley-Liss, Inc.     

Polynitroxylated Starch/TPL Attenuates Cachexia and Increased Epithelial Permeability Associated with TNBS Colitis

Free radicals play an important role in the initiation and progression of inflammatory bowel disease (IBD). Therefore, the reduction or elimination of adverse oxidant effects can provide novel therapy for IBD. Here, the antioxidant capacity and protective effects of a new class of chemically modified hetastarch (polynitroxyl starch, or PNS) plus 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl (Tempol or TPL) (PNS/TPL) were assessed in a model of colitis. The superoxide scavenging capacity of PNS/TPL—that is, the inhibition of the reduction of cytochrome c in the presence of xanthine/xanthine oxidase (X/XO)—was evaluated in vitro. The effects of PNS/TPL on X/XO–induced neutrophil endothelial adhesion in vitro were investigated. Also, this study tested the protection produced by PNS/TPL in a mouse model of trinitrobenzene sulfonic acid (TNBS)–induced colitis. PNS/TPL was given intravenously immediately before (<30 min) and intraperitoneally at 24 and 72 hr after TNBS induction. The body weight and survival rate of the mice were checked daily. Colonic mucosal damage was assessed on the 7th day by measuring intestinal permeability to Evans blue (EB) in vivo. The ability of PNS to reoxidize bioreduced TPL was documented by whole-body electron paramagnetic resonance (EPR) detection. We found that PNS or TPL exhibits superoxide dismutase (SOD)–like activity, with approximately 2% of SOD activity occurring on a molar basis. The endothelial–neutrophil adherence induced by X/XO was significantly inhibited by PNS/TPL but not by TPL alone. PNS/TPL protected against cachexia and mortality, both usually induced by TNBS. Epithelial permeability was increased significantly in TNBS mice but was ameliorated by the administration of PNS/TPL. In conclusion, PNS/TPL may be beneficial in the treatment or prevention of IBD through its antioxidant effects, which inhibit oxidant-mediated leukocyte adhesion and injury to endothelial cells.     

Ontogenetic study of enteric xanthine oxidase in the chick embryos: focus on the late embryogenesis

Xanthine oxidase (XO) is a member of xanthine oxidoreductase (XOR) enzyme system that catalyses the terminal reactions of purine degradation to uric acid (UA). The synthesis of both antioxidant (i.e. UA) and numerous free radicals makes XO an important regulator of the cellular redox potential involved in normal foetal development. The aim of this study was to investigate ontogenetic changes of enteric XO activity and UA content during the last week of chick embryonic development. Fertilized eggs (n?=?180) of layer-breeder (Bovans White) and broiler-breeder hens (Ross 308) were incubated at 37.7 °C and 60 % relative humidity. Enteric XO activity (n?=?10 for each strain) was spectrophotometrically measured, based on the generation of UA using xanthine as substrate, at various incubation days namely D14, D16, D18, D20 and D21. Intestinal XO activity and UA content of both strains fluctuated and showed polynomial relationships with embryo age. The XO activity tended to be increased from D14 and reached its maximum level around D18 in both strains; however, its activity returned to minimal values at D21. Intestinal XO activity in broiler embryos was higher when compared to that of layer embryos at D18. The pattern of change in levels of intestinal UA were similar for the two strains with the highest UA levels being detected at D21. We have shown that the intestinal XO activity and UA content of chick embryos fluctuates during the last week of embryonic development.