Oxygen-derived free radical damage in organ preservation: activity of superoxide dismutase and xanthine oxidase

It has been suggested that the generation of oxygen-derived free radicals during the reperfusion of injured tissue causes organ dysfunction. The mechanism apparently involves the xanthine oxidase (XOD) enzyme system, which becomes activated during reperfusion and generates oxygen-derived free radicals faster than their dismutation by naturally occurring superoxide dismutase (SOD). In this study, we measured the XOD and SOD activities of liver and kidney tissue in species used for organ preservation studies (rats, dogs, and human beings). We also measured the effect that up to 5 days of hypothermic preservation has on the activity of SOD and XOD in canine kidneys and expressed activities as the ratio of SOD to XOD; large ratios suggest lower susceptibility to oxygen-derived free radical damage. The SOD:XOD ratios for rat tissue were consistently lower (kidney = 2.7 X 10(4), liver = 8.9 X 10(3)) than for canine tissue (kidney = 7.0 X 10(5), liver = 4.1 X 10(4)) and human tissue (kidney = 1.1 X 10(6), liver = 6.4 X 10(5)). Canine kidneys perfused for 3 days showed no change in SOD:XOD ratio. After 5 days of perfusion, the SOD:XOD ratio decreased by 50% but was still quite large (5 X 10(5]. This high SOD:XOD ratio in canine and human tissues suggests that they may be less sensitive to oxygen-derived free radical damage than rat tissues.     

Catalase, superoxide dismutase, and glutathione peroxidase activities in various rat tissues after carbon tetrachloride intoxication

Background. The aim of this study was to determine the possible relationship between the activity of three different antioxidant enzymes — peroxidase superoxide dismutase, catalase, and glutathione peroxidase — and carbon tetrachloride-induced injury. Methods. Male Wistar rats weighing 200–250g were used in the experiments. Rats of the experimental groups were given carbon tetrachloride 0.5ml/kg i.p. in olive oil (5mmol/kg body mass) for 1 or 3 days. Control group rats were injected with olive oil only for the same period. Brain, liver, kidney, and heart supernatants were used for measurement of superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX) activities. Results. No statistically significant changes in SOD and GPX activities were observed in the liver after CCl₄ administration, but catalase activity was significantly increased after 24h and remained at that level during the course of the study. In the brain, SOD and catalase activities decreased after 24h of experiment, but GPX activity statistically significantly increased at all time points studied. Increased activities of SOD, catalase, and GPX were found in heart after CCl₄ intoxication. The CCl₄ injection in our experiment caused a reduction of SOD and catalase activities and increased GPX activity in the kidney. Conclusions. The results suggest that change in antioxidant enzyme activities may be relevant to the ability of the liver and other investigated organs to cope with oxidative stress during CCl₄ poisoning.     

The effect of xanthine oxidase inhibitor on hindlimb ischemia-induced thromboxane A2 release

Our previous studies demonstrated that the bilateral hindlimb ischemia/reperfusion stimulates thromboxane A2 (TXA2) production. The present study tests the role of xanthine oxidase-derived oxygen free radicals in mediating this event. In twelve anesthetized dogs, the abdominal aorta and the inferior vena cava were clamped for 150 min, declamped and reperfused for 30 min. Two groups were studied: untreated control group and pretreated group with xanthine oxidase inhibitor, allopurinol 100 mg.kg-1 orally 24 hr before clamping plus 25 mg.kg-1 intravenously 15 min before clamping. In the control group, plasma TXB2 levels increased markedly after reperfusion. On the other hand, prior treatment with allopurinol attenuated the increase in plasma TXB2 levels at 30 min after reperfusion. This model revealed partial ischemia, because the femoral arterial blood flow was approximately 15% of baseline during clamping. However, the present study suggests that ischemia/reperfusion stimulates TXA2 production, which may be partly affected by hypoxanthine-xanthine oxidase-derived oxygen free radicals and may be an important mechanism responsible for reperfusion injury.     

Suppressive effect of superoxide dismutase on adriamycin nephropathy.

A single intravenous injection of adriamycin (ADR) results in marked proteinuria and glomerular morphological changes that are similar to minimal-change disease in humans. We examined the effect of superoxide dismutase (SOD) on ADR-induced proteinuria. ADR in a dose of 7.5 mg/kg body weight significantly increased urinary protein by day 14; proteinuria rapidly increased thereafter. Concurrent administration of SOD (50 mg/kg) over 30 min prior to and 30 min following ADR injection markedly reduced proteinuria. Twenty-one days after the treatment with SOD, the amount of urinary protein was 108.6 +/- 43.1 mg/24 h in the experimental animals, while it was 221.6 +/- 102.9 mg/24 h in the ADR control group (p less than 0.05). There were also less severe glomerular morphologic changes in the SOD group versus ADR controls. The protective effects of SOD provide indirect evidence that oxygen free radicals are important mediators of ADR-induced proteinuria.     

Homocysteine induced impairment of nitric oxide-dependent vasorelaxation is reversible by the superoxide dismutase mimetic TEMPOL.

BACKGROUND: Elevated plasma homocysteine concentrations in renal patients are associated with accelerated cardiovascular disease. The mechanism(s) by which homocysteine acts remains unclear however, evidence implicates a role involving endothelial dysfunction. METHODS: Rat femoral arteries after acute or 4-h pre-incubation with racemic D,L-homocysteine (100 microM) were mounted on a myograph, pre-constricted with phenylephrine (10 microM) and responses to acetylcholine-dependent vasorelaxation examined. The incubations were repeated in the presence of indomethacin (10 microM), omega-nitro-L-arginine methyl ester (100 microM), L-arginine (100 microM), tetrahydrobiopterin (1 microM), catalase (1200 U/ml), ebselen, a peroxynitrite chelator (20 microM) and TEMPOL, a superoxide dismutase mimetic (1 mM). Results are shown as means+/-standard error, expressed as per cent relaxation to acetylcholine added (nmol/l). RESULTS: Increasing concentrations of homocysteine had no affect when added directly to basally relaxed or pre-constricted freshly isolated vessels. However, 4-h pre-incubation with or without homocysteine significantly shifted the acetylcholine EC(50) (EC(50) was defined as the concentration of acetylcholine that caused relaxation of the phenylephrine contracted tissue by 50%), control((4 h)) = 74.7 nmol/l+/-10.5 vs 100 microM D,L-homocysteine((4 h)) = 159.9 nmol/l+/-20.6; P<0.05) without affecting maximal relaxation. Response to endothelial independent relaxation was unaffected. Indomethacin, indomethacin and omega-nitro-L-arginine methyl ester, l-arginine and tetrahydrobiopterin, catalase and ebselen had no effect on the EC(50) in homocysteine-exposed arteries. However, TEMPOL normalized vasorelaxation in homocysteine-treated arteries (75.2 nmol/l+/-14.6) but had no effect on the 4-h control group. Moreover, washing TEMPOL from the treated vessels restored endothelial dysfunction in D,L-homocysteine-treated vessels (163.9 nmol/l+/-34.1). CONCLUSIONS: We conclude that homocysteine causes endothelial dysfunction by up-regulating a potential superoxide generating system resulting in reduced nitric oxide bio-availability.     

Morphologic changes induced by short-term ischemia in the rat testis are not affected by treatment with superoxide dismutase and catalase

Short-term testicular ischemia was induced unilaterally by ligating the testicular artery for 60 or 100 minutes in adult rats. After 7 days, the rats were fixed by vascular perfusion. The effect of ischemia and reperfusion was quantified using morphometric techniques. Occlusion of the testicular artery for 60 and 100 minutes resulted in a mild and moderate damage, respectively, to the seminiferous tubules. The contralateral control testis was not affected. To study the role of oxygen radicals in ischemia-reperfusion-induced testicular damage, rats were treated with superoxide dismutase and catalase. These radical scavengers did not influence the extent of testicular damage.     

Oxidative stress and dysregulation of superoxide dismutase and NADPH oxidase in renal insufficiency

BACKGROUND: Chronic renal failure (CRF) is associated with oxidative stress, the mechanism of which remains uncertain. Superoxide is the primary oxygen free radical produced in the body, NAD(P)H oxidase is the major source of superoxide production and superoxide dismutase (SOD) is responsible for removal of superoxide. We hypothesized that CRF-induced oxidative stress may be due to increased production and/or decreased dismutation of superoxide. METHODS: Immunodetectable superoxide dismutase isoforms (Cu Zn SOD and Mn SOD), as well as, NAD(P)H oxidase (gp91 phox subunit) proteins and xanthine oxidase (XO) activity were determined in the kidney and liver of CRF (5/6 nephrectomized) and sham-operated control rats. Subgroups of animals were treated with SOD-mimetic drug, tempol and blood pressure and urinary nitric oxide metabolites (NOx) were monitored. RESULTS: The CRF group showed marked down-regulations of CuZn SOD and Mn SOD and significant up-regulation of gp91 phox in the liver and kidney, which are among the metabolically most active tissues. In contrast, XO activity was depressed in both tissues. Arterial pressure and nitrotyrosine abundance were elevated while urinary NOx excretion was depressed, pointing to increased NO inactivation by superoxide and decreased NO availability in CRF animals. Administration of SOD-mimetic agent, tempol, for one week, ameliorated hypertension, reduced nitrotyrosine abundance and increased urinary NOx excretion in the CRF animals. CONCLUSIONS: CRF is associated with depressed SOD and elevated NAD(P)H oxidase expression, which can contribute to oxidative stress by increasing superoxide. This is evidenced by favorable response to administration of SOD-mimetic drug, tempol, and increased nitrotyrosine that is the footprint of NO interaction with superoxide.     

Oxygen free radical induced damage in kidneys subjected to warm ischemia and reperfusion. Protective effect of superoxide dismutase.

Superoxide anion free radical (O2-.) has been implicated in the pathogenesis of tissue injury consequent to ischemia/reperfusion in several different organs, including heart and bowel. Superoxide dismutase (SOD), an enzyme free radical scavenger specific for O2-., has been used successfully to protect these organs from structural damage during reoxygenation of ischemic tissue. It has been suggested that the catalytic action of xanthine oxidase in injured tissue is an important source of O2-. during reoxygenation. In order to evaluate the potential of SOD to protect against kidney damage resulting from transient ischemia followed by reperfusion with oxygenated blood, a model of warm renal ischemia was studied. LBNF1 rats underwent right nephrectomy and occlusion of the left renal artery for 45 minutes. Survival in the group of ischemic untreated rats (N = 30) was 56% at 7 days and serum creatinine was greatly elevated (p less than 0.01) in rats remaining alive over the full 7-day period. In strong contrast to these results, all of the animals treated with SOD before reperfusion (N = 18) were alive after 7 days similar to sham operated control rats (N = 8). Serum creatinine in the SOD treated rats was significantly elevated only to postoperative day 3 and thereafter returned to normal. Rats treated with inactive SOD (N = 4) or SOD before ischemia (N = 4) had decreased survival rates compared to ischemic untreated animals and prolonged elevation of serum creatinine. When the ischemia time was extended to 60 minutes, only 19% of the untreated animals (N = 16) survived at 7 days whereas nearly 60% of the SOD-treated animals survived (N = 19). Serum creatinine was greatly elevated during the full 7-day observation period in all surviving rats in the untreated ischemic group, whereas serum creatinine returned to normal (p less than 0.05) after 4 days in the surviving rats treated with SOD. To test whether the action of xanthine oxidase contributed to the kidney damage after reoxygenation, 45 min. ischemic rat kidneys were treated with allopurinol. All of the animals treated with allopurinol (N = 12) were alive at 7 days. Serum creatinine values returned to normal after the episode of ischemia and reperfusion but more slowly than after SOD treatment. Histologic evaluation of kidney tissue taken from animals after ischemia alone showed extensive renal tubular damage, which was essentially absent in kidneys from SOD-treated animals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Study on chronic renal injuries induced by carbon tetrachloride: selective inhibition of the nephrotoxicity by irradiation.

Carbon tetrachloride (CCl4) was intraperitoneally injected into Balb/c mice 4 times at biweekly intervals, and the morphological changes of the liver and kidney were examined during 12 weeks after the last injection. The renal injuries progressed in spite of cessation of CCl4 treatment; microcysts with tubular-cell degeneration were manifest on day 42 after the last injection of CCl4. At the end of the experiment, however, interstitial fibrosis with inflammatory cell infiltration was much more prominent. Glomerular changes with IgG deposits also developed following the tubulointerstitial changes. The CCl4 treatment induced liver damage as well, but it promptly subsided without formation of cirrhosis. The CCl4 nephrotoxicity was completely inhibited by whole body irradiation (200 rad) exposed at each injection of CCl4. In contrast, the hepatic damage was not changed by irradiation. These results seem to indicate etiologic independence of renal and hepatic events induced by CCl4 treatment. It is also suggested that chronic CCl4 nephrotoxicity is mediated, at least in part, by radiosensitive responses of the mice themselves.     

Salt intake, oxidative stress, and renal expression of NADPH oxidase and superoxide dismutase

The hypothesis that a high salt (HS) intake increases oxidative stress was investigated and was related to renal cortical expression of NAD(P)H oxidase and superoxide dismutase (SOD). 8-Isoprostane PGF(2alpha) and malonyldialdehyde were measured in groups (n = 6 to 8) of conscious rats during low-salt, normal-salt, or HS diets. NADPH- and NADH-stimulated superoxide anion (O(2)(.-)) generation was assessed by chemiluminescence, and expression of NAD(P)H oxidase and SOD were assessed with real-time PCR. Excretion of 8-isoprostane and malonyldialdehyde increased incrementally two- to threefold with salt intake (P < 0.001), whereas prostaglandin E(2) was unchanged. Renal cortical NADH- and NADPH-stimulable O(2)(.-) generation increased (P < 0.05) 30 to 40% with salt intake. Compared with low-salt diet, HS significantly (P < 0.005) increased renal cortical mRNA expression of gp91(phox) and p47(phox) and decreased expression of intracellular CuZn (IC)-SOD and mitochondrial (Mn)-SOD. Despite suppression of the renin-angiotensin system, salt loading enhances oxidative stress. This is accompanied by increased renal cortical NADH and NADPH oxidase activity and increased expression of gp91(phox) and p47(phox) and decreased IC- and Mn-SOD. Thus, salt intake enhances generation of O(2)(.-) accompanied by enhanced renal expression and activity of NAD(P)H oxidase with diminished renal expression of IC- and Mn-SOD.     

The beneficial effect of superoxide dismutase on the rat liver graft

We performed orthotopic liver transplantation in male Wistar rats and investigated the effect of superoxide dismutase (SOD) on the liver graft. Animals were divided into the four following experimental groups. Group I was an untreated control group, group II received oxygen, group III received SOD and group IV received both oxygen and SOD. The dose of SOD was 3 mg/kg which was injected intravenously into both donors and recipients during the operation. Oxygen was given through an oxygen inhaler to both donors and recipients during the operation. The preservation time of the liver graft ranged from 4 hours and 41 minutes to 5 hours and 40 minutes. The survival after liver transplantation was compared among groups I, II, III and IV. Group IV showed a significantly higher survival rate than groups I and II by two weeks after liver grafting, but there was no statistical difference in the survival rates between groups III and IV. These results indicate the beneficial effect of SOD on the rat liver graft and may implicate oxygen free radicals in the pathogenesis of ischemia/reperfusion injury in liver grafts.     

The beneficial effect of superoxide dismutase on the rat liver graft

We performed orthotopic liver transplantation in male Wistar rats and investigated the effect of superoxide dismutase (SOD) on the liver graft. Animals were divided into the four following experimental groups. Group I was an untreated control group, group II received oxygen, group III received SOD and group IV received both oxygen and SOD. The dose of SOD was 3 mg/kg which was injected intravenously into both donors and recipients during the operation. Oxygen was given through an oxygen inhaler to both donors and recipients during the operation. The preservation time of the liver graft ranged from 4 hours and 41 minutes to 5 hours and 40 minutes. The survival after liver transplantation was compared among groups I, II, III and IV. Group IV showed a significantly higher survival rate than groups I and II by two weeks after liver grafting, but there was no statistical difference in the survival rates between groups III and IV. These results indicate the beneficial effect of SOD on the rat liver graft and may implicate oxygen free radicals in the pathogenesis of ischemia/reperfusion injury in liver grafts.     

The chemistry of the inhibition of blood xanthine oxidase by -SH reagents.

In this study it was shown that the rat blood serum xanthine oxidase (X.O.) is differently inhibited by -SH reagents and seems that the blood serum enzyme has two types of -SH groups, one reacting relatively rapidly and unrelated to the enzyme activity and the other reacting slowly to produce inactivation. The results presented suggest that there are only few fundamental relationship between the different -SH reagents used and the inhibition of the enzyme activity in the blood serum. With mercurials it was shown that the most reactive -SH groups of the rat blood serum are not related to the X.O. activity, but when sufficient number are reacted the enzyme is structurally altered so that inhibition appears. With oxidants such as iodine the inhibition of the X.O. activity of rat blood serum seems to be not related specifically with the oxidation of -SH groups.     

Protective effect of Silybum marianum and Taraxacum officinale extracts against oxidative kidney injuries induced by carbon tetrachloride in rats

The protective effect of the extracts of the plants Silybum marianum and Taraxacum officinale by carbon tetrachloride (CCl₄) was researched. Sixty-six female Wistar albino rats were divided into six groups: Control, Silybum marianum, Taraxacum officinale, CCl₄, Silybum marianum+ CCl₄, Taraxacum officinale+CCl₄. The Silybum marianum and Taraxacum officinale extracts were administered as 100?mg/kg/day by gavage. The CCl₄ was administered as 1.5?mL/kg (i.p.). At the end of the trial period, in the serums obtained from the animals, in the CCl₄ group it was found that the MDA level increased in the kidney tissue samples as well as in the ALP and GGT enzyme activities. It was also found that the GSH level and the GST enzyme activities decreased (p<.05). The microscopic evaluations showed that the CCl₄ caused a serious hydropic degeneration, coagulation necrosis, and mono-nuclear cell infiltration in the kidney cell. In the animals where CCl₄ and Silybum marianum and Taraxacum officinale extracts were applied together, it was found that the serum ALP and GGT enzyme activities decreased and that the MDA level decreased in the kidney tissue, and that the GSH level and GST enzyme activities increased. It was observed that the histopathological changes caused by the CCl₄ toxicity were corrected by applying the extracts. Eventually, it was determined that the Silybum marianum was more effective. Silybum marianum and Taraxacum officinale extracts which were used against histopathological changes in the kidney caused by toxication showed a corrective effect, which were supported by biochemical parameters.     

Reduction by superoxide dismutase of leukocyte-endothelial adherence after liver transplantation.

One hour after orthotopic rat liver transplantation, hepatic microcirculation and adhesion characteristics of leukocytes to the endothelial wall were studied with intravital microscopy. Cold storage for 1 and 8 hours in Euro-Collins solution resulted in reduction of perfused sinusoids to 83% and 48% and a decrease of leukocyte velocity from 417 microns/sec in controls to 311 microns/sec and 269 microns/sec, respectively. Additionally, the number of permanently adherent leukocytes rose from 4% in controls to 33% and 43% after cold storage for 1 and 8 hours. Intravenous injection of the free radical scavenger human recombinant superoxide dismutase (40 mg/kg) during reperfusion resulted in marked improvement of the hepatic microcirculation after both 1 and 8 hours of cold storage (91% and 69% perfused sinusoids; 420 microns/sec and 350 microns/sec leukocyte velocity; p less than 0.05). Furthermore, the percentage of permanently adherent leukocytes decreased to 13% and 28% after 1 and 8 hours of cold ischemia, respectively. These results support the hypothesis that oxygen-derived free radicals contribute to leukocyte adherence and microcirculatory failure after cold liver ischemia and transplantation. Thus, application of oxygen free radical scavengers during liver transplantation procedures might be useful to improve graft function.     

Protective effect of allopurinol and superoxide dismutase in renal isografts in cyclosporin A-treated rats.

Acute tubular necrosis (ATN) after renal transplantation is related to the duration of warm and cold ischemia and leads to temporary or permanent impairment of graft function. An increased incidence of ATN has been reported since the introduction of cyclosporin A. Kidney damage resulting from hypothermic storage is generated in part during reperfusion rather than during ischemia itself. Potential mediators of the reperfusion injury are oxygen-derived free radicals. Therefore, the influence of two oxygen radical antagonists, allopurinol and superoxide dismutase, was evaluated in syngeneic rat kidney transplantation with and without concurrent administration of cyclosporin A. At 15 h cold ischemia, 28-day survival increased from 8% (no treatment) to 22% (superoxide dismutase), 33% (superoxide dismutase and allopurinol), and 73% (allopurinol). Cyclosporin A cotreatment (10 mg/kg over 14 days) resulted in survival rates of 0%, 25%, 17%, and 50% for the respective treatment groups. The results of serum creatinine values and morphological evaluation of biopsies paralleled the survival rates. Cyclosporin A nephrotoxicity was evidenced by significant serum creatinine elevations throughout the 28-day period of observation. In conclusion, allopurinol significantly protects syngeneic rat kidney transplants against a critical duration of cold ischemia. Under the conditions of this experiment, allopurinol was clearly superior to superoxide dismutase treatment. Cyclosporin A nephrotoxicity was, however, not ablated by the oxygen radical antagonists employed.