Different kinds of reactive oxygen and nitrogen species were detected in colon and breast tumors.

Several studies have shown the involvement of reactive oxygen species (ROS; O2*-, hypochlorite, hydroxyl radical, hydrogen peroxide) in carcinogenesis. With certain pathologies, nitric oxide (NO) is formed and can interact with superoxide radical (O2*-) resulting in the propagation of the highly reactive species, peroxynitrite. In order to study the molecular mechanisms underlying the ability of reactive oxygen and nitrogen species (RONS) to mediate carcinogenesis, we have measured ROS, NO, and peroxynitrite content of cancerous tissues obtained from colon and breast carcinoma cases by chemiluminescence technique. All ROS were significantly increased in cancerous colon tissues with hypochlorite making the most important contribution and suggesting the role of inflammatory cells. NO was also increased and the peroxynitrite concentration was higher in cancerous samples. For breast carcinoma cases, only O2*- was significantly increased. Hypochlorite was not detected excluding the contribution of inflammatory cells. NO concentrations were not significantly different, therefore, ROS might originate by change in the redox state of the tissue.

Effect of mitochondrial electron transport chain inhibitors on superoxide radical generation in rat hippocampal and striatal slices.

In this study, we have compared the generation of superoxide radical in rat hippocampal and striatal slices in the presence of specific mitochondrial electron transport chain (ETC) inhibitors (complexes I and III) under control and depolarization conditions [incubation in artificial cerebrospinal fluid (ACSF) or depolarizing ACSF (dACSF), respectively]. Superoxide radical generation was increased in both ACSF- and dACSF-incubated hippocampal and striatal slices when rotenone and antimycin A were added to the incubation medium. The increase in superoxide radical was dependent on the concentration of ETC inhibitors under control, but not depolarization conditions. Rotenone was found to be more effective than antimycin A in producing superoxide radical from hippocampal and striatal slices. Our results also showed that hippocampal slices were more sensitive to ETC inhibitors compared with striatal slices. Thus, different regions of the brain seem to differ in their capacity to generate free radicals and vulnerability to oxidative stress conditions. This difference should be considered in developing therapeutic modalities against oxidative stress-related disorders and neurodegeneration.

Oxygen radicals and nitric oxide in rat mesenteric ischaemia-reperfusion: modulation by L-arginine and NG-nitro-L-arginine methyl ester.

1. The aims of the present study were to detect changes in superoxide anion (O2.-), nitric oxide (NO) and other reactive oxygen species (ROS) directly by measurement of chemiluminescence (CL) and to investigate the role of L-arginine, a nitric oxide synthase (NOS) substrate, and NG-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, together with their molecular enantiomers D-arginine and D-NAME, in a rat mesenteric ischaemia-reperfusion (I/R) model. 2. Seventy-nine female Wistar albino rats were divided into eight groups. The first three groups underwent sham operation; group 1 was the control group, group 2 received L-arginine and group 3 received L-NAME. Ischaemia was produced in the remaining five groups by ligation of the superior mesenteric artery for 30 min followed by 60 min reperfusion. Group 4 rats were control I/R rats and groups 5-8 received either L-arginine, L-NAME, D-arginine or D-NAME, respectively. 3. Both luminol and lucigenin CL was significantly increased in I/R groups compared with sham-operated groups. L-Arginine significantly reduced CL measurements. D-Arginine was also protective, but not as much as L-arginine. Both L- and D-arginine had in vitro O2.- (-)scavenging potential, as tested by the xanthine-xanthine oxidase system. NG-Nitro-L-arginine methyl ester decreased lipid peroxidation values in addition to reducing CL measurements. Nitric oxide concentrations were significantly increased in I/R groups in comparison with sham-operated groups. Peroxynitrite formation was increased by I/R. Treatment with L-NAME was beneficial by reducing NO concentrations in the reperfused ileum. 4. In our I/R model, O2.-, NO and other ROS were increased. Although NOS inhibitors were effective in reducing oxidative damage, increasing NO concentrations with L-arginine was also beneficial, presumably due to the ability of L-arginine to inhibit phagocyte adherence and its radical scavenging potential. In fact, NO may have different effects in terms of tissue injury or protection depending on the concentration of oxygen and the haemodynamic state of the tissue.

Measurement of reactive oxygen species by chemiluminescence in diet-induced atherosclerosis: protective roles of vitamin E and probucol on different radical species.

We have investigated the effects of a high-cholesterol diet on the production of different reactive oxygen species in rabbit aortic rings and evaluated the protective effects of vitamin E and probucol in preventing peroxidative changes. Twenty-five male albino rabbits were divided into five groups. Control rabbits were fed a vitamin E-poor rabbit chow. Rabbits in the second group were given a vitamin E-poor diet supplemented with 2% cholesterol. Other groups received either 50 mg/kg vitamin E, 1% probucol, or both, in addition to 2% cholesterol for 4 weeks. Reactive oxygen species formation in aortic rings was measured by enhanced chemiluminescence using luminol and lucigenin. (The results were given as cpm/mg wet weight.) Further differentiation of radical species involved in luminol-enhanced chemiluminescence was performed using sodium azide and L-nitroarginine, a selective inhibitor of nitric oxide production. Our results indicated that cholesterol feeding increased lucigenin and luminol chemiluminescence, where the contribution of free radicals inhibited by sodium azide (radicals originating from endothelial cells or from phagocytes) were 53% and peroxynitrite 24%. Both vitamin E and probucol were effective as scavengers of free radicals, but the effect of vitamin E was more pronounced. In conclusion, the present study demonstrated excessive generation of reactive oxygen species within the atherosclerotic vessel. Peroxidative changes could be prevented by vitamin E and probucol treatment, but vitamin E seemed to be more efficient.

Early detection of peritonitis in continuous ambulatory peritoneal dialysis patients by use of chemiluminescence: evaluation of diagnostic accuracy by receiver-operating characteristic curve analysis.

Continuous ambulatory peritoneal dialysis (CAPD) is now a widely accepted treatment for end-stage renal disease. However, the high incidence of peritonitis is a major complication of CAPD. Polymorphonuclear leukocytes (PMNs) play a major role in antimicrobial response of the host. During phagocytosis, the PMNs undergo a striking increase in oxidative metabolism, known as the respiratory burst, and emit light as chemiluminescence (CL). CL is thus a sensitive measure of PMN oxidative potential and correlates well with antimicrobial activity. In view of the observation of increased susceptibility to infection in CAPD patients, we have studied lucigenin- and luminol-enhanced CL in peritoneal fluids of these patients and assessed the diagnostic accuracy of these tests by ROC curve analysis. ROC curves showed diagnostic accuracies for both tests that were superior to counts of PMNs in the dialysis fluid (P <0.001). At selected cutoff values of 150000 cpm/vial for lucigenin CL and 600000 cpm/vial for luminol CL, sensitivities were 100%. Specificities for lucigenin and luminol CL were 89% and 80%, respectively. Our results suggest that CL measurements can be used as an early marker for the presence of infection in CAPD patients.

Low dose MK-801 protects against iron-induced oxidative changes in a rat model of focal epilepsy.

We have used chemiluminescence measurements to examine the relationship between free radical formation and excitotoxicity in a post-traumatic epilepsy model. For this purpose, seven days after injecting iron in rat brain cortices, we measured luminol- and lucigenin-enhanced chemiluminescence in different brain regions (ipsilateral cortex, contralateral cortex, hypothalamus and hippocampus). In all brain regions (except contralateral cortices) both luminol- and lucigenin-enhanced chemiluminescence were increased in iron-injected group compared to saline-injected control group. These increases returned to control values in iron-injected rats pretreated with MK-801. Our results suggest that both free radicals and excitatory amino acids play important roles in the development of post-traumatic epilepsy and that MK-801 has protective effects against iron-induced chemiluminescence formation.

Effect of donor age on the susceptibility of erythrocytes and erythrocyte membranes to cumene hydroperoxide-induced oxidative stress.

A comparative study on erythrocytes and erythrocyte membranes of healthy elderly and young adults was carried out to understand how the antioxidant defense capacity is effected by aging. The levels of endogenous malondialdehyde and Ca(2+)-ATPase activity were taken as indices of oxidative damage. In addition, chemiluminescence measurements were performed on intact erythrocytes. The susceptibility of these parameters to in vitro cumene hydroperoxide, under low oxidant level that does not induce hemolysis, was also taken as an age-related indicator of the endogenous peroxidative potential of the erythrocytes. Our data showed that the content of malondialdehyde and Ca(2+)-ATPase activity did not change with age. Furthermore, the susceptibility of intact erythrocytes to oxidative stress did not change in the elderly group. However, under the same conditions erythrocyte membranes were more susceptible to oxidative damage in the elderly than young adults. Our results also showed that antioxidant defenses were overwhelmed in intact erythrocytes of the elderly at high concentrations of cumene hydroperoxide.

The functional assessment of autotransplanted splenic tissue by its capacity to remove oxidatively modified erythrocytes.

Free radicals and reactive oxygen species have been implicated in the pathogenesis of a variety of hematologic diseases and erythrocyte aging. Aged erythrocytes are removed from the circulation primarily by the spleen. In this study, we aimed to determine the functional effectiveness of autotransplanted splenic tissue by its capacity to remove oxidatively modified erythrocytes from the circulation. Our experimental model in rats includes splenectomy with autotransplantation of 80% of the excised splenic tissue into the omental pouch. In this model, free radical damage was estimated by different parameters of lipid peroxidation such as carbonyl content and thiobarbituric acid reactive substances (TBARS), together with Heinz body formation. Our results have shown that splenic autotransplantation was effective in removing oxidatively modified, aged erythrocytes from the circulation.

NMDA excitotoxicity and free radical generation in rat brain homogenates: application of a chemiluminescence assay.

NMDA, the specific agonist of glutamate gated ion channels permeable to calcium, is implicated as a causal factor in the pathogenesis of several neurobiological disorders such as stroke, seizures, ischemia, and chronic neurodegenerative diseases. On the other hand, evidence on the roles of oxidative mechanisms involved in NMDA-induced neurotoxicity is accumulating. In this study, we have used chemiluminescence measurements as an easy, rapid and sensitive assay to investigate the effects of NMDA and oxidative stress on brain cell vulnerability. Rat brain homogenates were incubated with increasing concentrations of glutamate and NMDA. Production of reactive oxygen species was followed by single photon emission measurements using the specific enhancers luminol and lucigenin. Increases in emission were observed at excitotoxic concentrations of glutamate and NMDA. Other parameters of oxidative stress such as diene conjugates, TBARS and carbonyl groups were also investigated. Our results indicated that chemiluminescence measurements may be used to study involvement of oxidative stress in neurotoxicity.

Involvement of free radicals in the cardioprotective effect of defibrotide.

Ischemia followed by reperfusion has deleterious effects on myocardial tissue and a wide range of drugs have been investigated to modulate these changes. Defibrotide (polydeoxyribonucleotides from bovine lung), a drug with antithrombotic and fibrinolytic activities, has also proven to be cardioprotective against myocardial ischemia/reperfusion damage. However, the mechanism of this protective effect has not been clarified yet. The aim of this study was to determine whether this effect is due to protection against free radical induced changes. The experimental model in rabbits includes coronary artery ligation for 60 min followed by a reperfusion period of 45 min. In this model, free radical damage was estimated by different parameters of lipid peroxidation such as diene conjugation, carbonyl content, and thiobarbituric acid reactive substances, together with protein oxidation determinations. The results demonstrate that defibrotide prevents free radical induced changes after myocardial ischemia/reperfusion.

BQ-123, a specific endothelin (ETA) receptor antagonist, prevents ischemia-reperfusion injury in kidney transplantation.

We studied the effects of the specific endothelin (ETA) receptor antagonist, BQ-123, on reperfusion injury in a rat model of kidney transplantation. First, Sprague-Dawley rats were divided into three groups: a sham nephrectomy (SNEPH), an autotransplantation (AUTO-Tx), and an allotransplantation (ALLO-Tx) group. In a fourth group, ALLO-Tx + BQ, allografts were flushed with 20 micrograms BQ-123 containing cold Ringer's lactate before transplantation. For the allograft groups, kidneys from white Wistar albino rats were transplanted into allogeneic Sprague Dawley recipients. Grafts were allowed 120 min of reperfusion after 40 min of cold ischemia. ET-1,2 plasma concentrations in the renal venous blood, and kidney tissue prostaglandin (PG) E2 and leukotriene (LT) B4 levels were studied. Diene conjugates (DC), hydroxyalkanals (HAA), hydroxyalkenals (HAE) and malondialdehyde (MDA) levels, as the products of lipid peroxidation, and protein carbonyls (PC) and protein sulphydryls (PS), as the parameters of protein oxidation, were also analyzed in the kidney tissue. Plasma ET concentrations increased significantly in the AUTO-Tx and ALLO-Tx groups (P < 0.05 and P < 0.01, respectively) but this increase was reversed in the ALLO-Tx + BQ group. None of the lipid peroxidation products except DCs (P < 0.05) increased in the AUTO-Tx group, whereas they all increased in the ALLO-Tx group (P < 0.01). Protein oxidation parameters also changed significantly (P < 0.01) in the ALLO-Tx group but did not in the AUTO-Tx group (P < 0.05). The differences in PGE2 and LTB4 levels were not significant. Histopathologic examination revealed prominent glomerular and tubular injury in the AUTO-Tx and ALLO-Tx groups but less in the ALLO-Tx + BQ group. In the last group, all parameters of lipid peroxidation (P < 0.001 for all) and PCs decreased, and PSs were preserved (P < 0.001 for both) when compared with the AUTO-Tx and ALLO-Tx groups. We conclude that BQ-123, in addition to inhibiting the binding of ET-1,2 to the ETA receptor, may also inhibit the release and/or synthesis of ET-1,2 and prevent reperfusion injury in kidney transplantation.

Evaluation of oxidant stress in chronic hemodialysis patients: use of different parameters.

Patients with chronic renal failure, particularly those undergoing regular dialysis treatment (RDT) are candidates for free radical damage. It is difficult to quantitate free radicals because of their short half-lives and reactive nature. Therefore, indirect methods measuring products of lipid peroxidation and protein oxidation are preferred. The present study displays a profile of lipid peroxidation and protein oxidation parameters, which are more sensitive and specific than the widely used method measuring thiobarbituric acid reactive substances (TBARS), adapted to the plasma and erythrocyte samples of RDT patients. We have observed increased levels of plasma and erythrocyte lipid peroxidation and also demonstrated increased protein oxidation in erythrocyte membranes of RDT patients.

Protective effects of cilazapril against free radical injury in myocardial ischaemia-reperfusion.

Cilazapril is a prodrug which is rapidly hydrolysed to the pharmacologically active cilazaprilat following absorption to the bloodstream. In clinical pharmacological studies, administration of cilazapril resulted in potent, reversible, selective and competitive angiotensin converting enzyme inhibition. In this study, we have examined the protective effect of cilazapril on a myocardial ischaemia-reperfusion model by using different parameters of lipid peroxidation and protein oxidation. We have observed increased levels of diene conjugates, carbonyls and malondialdehyde as well as protein carbonyls after ischaemia-reperfusion, whereas protein sulphydryl groups were decreased. Our results clearly demonstrate that cilazapril, a non-sulphydryl, long-acting angiotensin converting enzyme inhibitor, has free-radical-scavenging potential in a model comparable to the clinical situation observed in humans.

The effects of experimental splenic autotransplantation and imipenem-cilastatin treatment in postsplenectomy Pseudomonas aeruginosa sepsis.

Male Wistar albino rats were allocated to three groups-sham operated (n: 10), splenectomized (n: 20) and splenic autotransplanted (n: 10). Twelve weeks after operation, all were challenged with 1.8 x 10(8) cfu/ml Pseudomonas aeruginosa intranasally. Half of the splenectomized rats received imipenem-cilastatin after 2 h of bacterial challenge. Mortality was then observed for the next 12 days. All animals were autopsied and liver, kidney, spleen and lung specimens were processed for microbiological culture and histopathological examination. In 80% of autotransplanted rats, splenic tissue regeneration was histopathologically verified. Hemoglobin oxidation of erythrocytes increased in splenectomized rats and remained close to control levels in the autotransplanted group. No significant difference was detected between IgM levels of splenectomized and autotransplanted groups. Mortality rates were the same for all groups, except that splenectomized animals given antimicrobial therapy had increased survival rates. In conclusion, it is likely that the spleen has no role in protection against pulmonary sepsis and that only appropriate antimicrobial therapy can protect the splenectomized host from Pseudomonas sepsis.

Effect of antiepileptic drugs on erythrocyte osmotic fragility and lipid peroxidation.

Epileptic children receiving antiepileptics were studied to investigate the effect of carbamazepine and phenobarbital therapy on erythrocyte osmotic fragility and lipid peroxidation. Significant differences between the two groups were observed in erythrocyte osmotic fragility. In addition, there was a significant increase in erythrocyte malondialdehyde release in the epileptic group compared to controls. It is suggested that the use of antioxidants in addition to antiepileptic drugs may be beneficial.

Gastric lipid peroxidation, glutathione and calcium channel blockers in the stress-induced ulcer model in rats.

The antiulcer activity of verapamil and its analogues devapamil and gallopamil was studied. All three drugs reduced cold-restraint stress-induced ulcer development. Gallopamil almost abolished gastric ulcers. Verapamil prevented the increase in gastric lipid peroxidation (LP) due to stress. On the other hand, devapamil and gallopamil increased gastric lipid peroxidation and decreased glutathione levels. This effect may be attributed to the increase in oxygen supply due to possible effective vasodilation at gastric mucosa. The second part of this study revealed that stress-induced gastric ulcers in rats rapidly and spontaneously heal and disappear within 24 h. During recovery, gastric LP decreased and glutathione levels increased within 12 h after the withdrawal of stress, preceded by an initial reduction in glutathione. After 72 h, an unexplained increase in gastric LP and a decrease in glutathione were observed. Treatment with verapamil, devapamil and gallopamil promoted healing, gallopamil being again the most effective. Their effects on gastric LP and glutathione levels are in accordance with the results of pretreatment experiments. In conclusion, devapamil and gallopamil are effective antiulcer agents against stress-induced ulcers, but unlike verapamil, antioxidant activity does not seem likely to be among their mechanisms of action.

Captopril increases endothelin serum concentrations and preserves intestinal mucosa after mesenteric ischemia-reperfusion injury.

Endothelial cells modulate the tone of the underlying smooth muscle by generating endothelium-derived relaxing and constricting factors. Captopril (CPT), unlike other angiotensin-converting enzyme (ACE) inhibitors, contains a sulfhydryl (-SH) group and can act as a free radical scavenger. Iloprost (ILO) is a synthetic analogue of prostacyclin and mimics the effects of this compound. This study was designed to investigate the effect of ILO and CPT on the mechanism of endothelin (ET) release after mesenteric ischemia-reperfusion (I/R) injury in the rat. Sprague-Dawley rats were divided into five groups: sham-operated, control, ILO (25 micrograms/kg), CPT (10 micrograms/kg), and ILO + CPT. The superior mesenteric artery was occluded for 30 min and then allowed 90 min of reperfusion, except in the sham-operated group, and the corresponding agents were given to the treated groups prior to I/R injury. After I/R injury, portal venous blood was obtained for ET assay, and ileal tissue samples were also obtained for the determination of malondialdehyde (MDA), prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) and for histopathological examination. MDA levels were significantly lower in the CPT, ILO and, ILO + CPT groups than in the control group, indicating the inhibition of lipid peroxidation in all groups. ET levels increased in the control group, and this increase was reversed with ILO. In the CPT group, ET levels were significantly increased, and the addition of ILO did not affect this increase. Significant cytopreservative effect was achieved with ILO and CPT, the latter being more prominent histopathologically. CPT exerts a significant protective effect on the intestinal mucosa after I/R injury. This protection is accomplished by increased ET levels and seems to be unrelated to its inhibitory effect on lipid peroxidation and also unrelated to the arachidonic acid cascade.

Effect of verapamil and iloprost (ZK 36374) on endothelin release after mesenteric ischemia-reperfusion injury.

In this experimental study we studied the effect of verapamil and iloprost on endothelin release in ischemia/reperfusion (IR) injury of the rat intestine. Endothelin levels in the portal blood and malondialdehyde (MDA), PGE2, and LTC4 levels in the intestinal tissue were determined. The MDA levels increased in the control group and this increase was reversed with iloprost, verapamil and both. The change in the LTC4 levels was insignificant between the groups. Iloprost reduced PGE2 and endothelin release, but verapamil was not as effective and no synergistic effect was encountered. The increased PGE2/LTC4 ratio was also reversed in the experimental groups, verapamil being less effective. Endothelin release seems to be related to both PGE2 levels and the PGE2/LTC4 ratio after mesenteric IR injury.

Cumene hydroperoxide-induced chemiluminescence in human erythrocytes: effect of antioxidants and sulfhydryl compounds.

1. The time-course of cumene hydroperoxide-induced changes in lipid peroxidation, protein sulfhydryl groups and chemiluminescence intensity was determined in human erythrocytes. 2. Increase in lipid peroxidation was maximal within 60 min of incubation and was paralleled by a decrease in protein sulfhydryl groups and an increase in chemiluminescence formation. 3. A standard assay system was established to investigate the protective effects of antioxidants and scavenger compounds on cumene hydroperoxide-induced chemiluminescence formation. 4. Chain-breaking antioxidants (i.e. butylated hydroxytoluene) and sulfhydryl compounds (i.e. dithiothreitol) were able to suppress chemiluminescence formation. 5. Our results suggested that secondary free radicals, as well as sulfhydryl groups of proteins are involved in cumene hydroperoxide-induced chemiluminescence formation.