Effects Of Oxygen Free Radicals Research Articles

A threshold concept for cancer therapy

Oxygen free radicals have been implicated in many disease processes, including aging and carcinogenesis, and have been associated with a variety of complications resulting from the treatment of cancer. As a result, the treatment of free radical-induced disease with antioxidants or free radical scavengers has become an important therapeutic modality. Ironically, these same oxygen free radicals also play a critical role in anti-cancer therapies. The use of antioxidants such as superoxide dismutase (SOD), in this setting, has been found to decrease the efficacy of anti-tumor therapies, which depend on free radical generation for their action. In addition, increased antioxidant activity can often be utilized by the tumor cell to favor increased growth. Therefore, the appropriate application of oxygen free radicals and antioxidants seems to be critically important in designing proper strategies for both prevention and treatment of malignant disorders. This review will summarize free radical and antioxidant regimens that have been employed to date, examine some of the problems associated with these regimens, introduce the ‘threshold concept’ explaining the dual effects of oxygen free radicals and antioxidants, and discuss a novel hypothesis regarding therapy that could potentially improve outcome in cancer patients.

Effect of Oxygen Free Radicals on Myosin in Muscle Fibres. A DSC and EPR study

Differential scanning calorimetry (DSC) and electron paramagnetic resonance spectroscopy (EPR, both conventional and saturation transfer EPR) were used to study the motional dynamics and segmental flexibility of myosin in muscle fibres in the presence of free radical generating system. Muscle fibre bundles isolated from psoas muscle of rabbit were spin-labelled with maleimide-and isothiocyanate-based probe molecules at the reactive sulfhydryl sites (Cys-707) of the motor domain. In the presence of hydroxyl free radicals the spectral intensity of the maleimide probe molecules decreased with time following a single exponential curve. MgADP and MgATP plus orthovanadate that produce flexibility changes in the multisubunit structure of myosin enhanced the reduction of the attached nitroxide molecules in free radical generating system. The analysis of the EPR spectra of spin-labelled and oriented fibres showed that the narrow distribution of spin labels changed in the presence of hydroxyl free radicals. Spectrum analysis by computer subtraction showed that short irradiation by UV light resulted in the enhancement of the ordered population at the expense of the disordered population. This suggests a transition of myosin heads from weak-binding state into strong-binding state. DSC measurements performed on calf cardiac myosin resulted in two main transitions at 49.4 and 54.1°C, respectively. Addition of MgADP produced a decrease of the 49.4°C transition, whereas a shift towards higher temperature was detected at the 54.1°C transition. It shows that there is an inter-site communication between the domains of the myosin. Hydroxyl free radicals induced further shifts of the transition temperatures and affected the width of the heat absorption curves.

Hyperglycemia Exacerbates the Effect of Ischemia-Reperfusion on Peripheral Nerve in Rat

Ischemia-reperfusion is known to induce generation of oxygen free radicals (OFRs), thereby resulting in tissue damage. To clarify the pathogenesis of diabetic neuropathy, we examined whether hyperglycemia exacerbates the effects of ischemia-reperfusion on peripheral nerves. Using light microscopy. characteristic pathological changes seen in myelin and axons of nerve fibers were classified into two types: “loose myelin” and “dark axon,” respectively. After a 2-hour ischemia and 24-hour reperfusion of sciatic nerves of rats, the concentrations of lipid peroxide and “loose myelin” in the nerves were significantly increased in the diabetic rats, but not in the nondiabetic rats. Thereafter, “dark axon” was significantly increased in both diabetic and nondiabetic rats. “Loose myelin” may be increased as a result of the effects of OFRs produced by ischemia-reperfusion, which are exacerbated by hyperglycemia.

Effects of free radicals on ciliary movement in the human nasal epithelial cells

Objective: There have been few reports on the effects of free oxygen radicals on ciliary mobility of nasal respiratory epithelial cells. The aim of this study was to determine the effects of free radicals and antioxidants on human nasal epithelial cells (HNECs) using video-computerized analysis. Methods: Human nasal epithelial cells were obtained from the nasal cavity of normal volunteers. Ciliary beat frequency (CBF) was calculated as the mean value of ten randomly selected cells. The proportion of the area with normal CBF (above 8 Hz) was calculated from 10 randomly selected sites per specimen. Free radicals were produced by xanthine–xanthine oxidase enzymatic system. The generation of free radicals was confirmed by chemoilluminometer. CBF and the proportion of the area with normal CBF were measured at every 5 min for 30 min after the addition of enzyme. For the evaluation of the antioxidant effects on free radical-mediated ciliary slowing in HNECs, cells were incubated in superoxide dismutase solution (300 unit/ml) for 30 min and 3-aminobenzamide (5 mM). Results: Superoxide produced by 0.4 mM xanthine and 400 miliunit/ml xanthine oxidase decreased CBF (7.71±1.91 Hz). A total of 2 min later, ciliary slowing was evident (3.87±1.10 Hz). Regarding the changes in proportion of epithelial area that showed normal CBF experimental group showed a significant decrease in percentage of epithelial area with normal CBF over time. Superoxide dismutase prevented ciliary slowing (8.76±0.99 Hz). Moreover, 3-aminobenzamide, an inhibitor of the DNA repair enzyme poly-ADP ribose polymerase, prevented inhibition of CBF (8.32±0.61 Hz). Conclusions: These results suggest that oxygen-mediated damage to DNA may be the mechanism of the deterioration effects of oxygen radicals on the ciliated respiratory nasal epithelium.

Oxidation of gonadotrophin (PMSG) by oxygen free radicals alters its structure and hormonal activity.

The effect of oxygen free radicals produced by the Fenton reaction was used to induce oxidation and other structural changes in pregnant mare serum gonadotrophin (PMSG). Modifications in the spectrophotometric scan, an increase in exposed carbonyl groups, and the ability to reduce nitroblue tetrazolium, was achieved by the oxidized hormone when compared to the control PMSG. PMSG loses its biological activity when coming in contact with the free-radical generating system. This lack of activity is manifested as a loss of ovulation and a decrease in the weight of the ovaries and uterus. It was demonstrated that oxygen free radicals can induce structural and biological changes in the gonadotrophin.

Effects of hydroxyl radicals on KATP channels in guinea-pig ventricular myocytes.

We studied the effects of oxygen free radicals on the ATP-sensitive potassium channel (KATP channel) of guinea-pig ventricular myocytes. Single KATP channel currents were recorded from inside-out patches in the presence of symmetrical K+ concentrations (140 mM in both bath and pipette solutions). Reaction of xanthine oxidase (0.1 U/ml) on hypoxanthine (0.5 mM) produced superoxide anions (.O2-) and hydrogen peroxide (H2O2). Exposure of the patch membrane to.O2- and H2O2 increased the opening of KATP channels, but this activation was prevented by adding 1 microM glibenclamide to the bath solution. In the presence of ferric iron (Fe3+: 0.1 mM), the same procedure produced hydroxyl radicals (.OH) via the iron-catalysed Haber-Weiss reaction.OH also activated KATP channels; however, this activation could not be prevented by, even very high concentrations of glibenclamide (10 microM). These different effects of glibenclamide suggest that the mode of action of these oxygen free radicals on KATP channels is different and that.OH is more potent than.O2-/H2O2 in activating KATP channels in the heart.

Lung hyaluronan and water content in preterm and term rabbit pups exposed to oxygen or air.

Rabbit pups were delivered by cesarean section 1 or 2 d before term, or vaginally around term, and then reared in room air or exposed to intermittent or continuous hyperoxia (> 85%) for up to 9 d. Pups were killed at different ages, and lung hyaluronan (HA; microgram/g of dry lung weight) and lung water content, measured as wet/dry lung weight, were determined. Compared with the day of birth, the lung HA concentration did not change significantly on succeeding days in pups kept in air delivered 2 d (-2 d) or 1 d (-1 d) before term, whereas the water content decreased significantly. Continuous exposure to hyperoxia resulted in a significantly raised lung HA concentration 6 d postterm in both -2 d and -1 d pups, and intermittent exposure to hyperoxia resulted in a significantly raised HA concentration 6 d postterm in -1 d pups, compared with the groups exposed to room air. These increases were accompanied by significantly elevated wet/dry lung weight ratios. Microscopic examination revealed significantly increased HA staining scores in alveoli, arterioles, and bronchioli in both hyperoxia-exposed groups of -2 d pups 6 d postterm, and nonsignificantly higher scores in -1 d and vaginally delivered pups of comparable age, compared with the scores at birth. The results indicate that oxygen exposure neonatally may result in an increase in lung HA accompanied by an increase in lung water content. The increase in lung HA concentration in our study may be an effect of oxygen free radicals or of oxygen-induced stimulation of inflammatory mediators.

Effect of hydrogen peroxide on permeability of the main pancreatic duct and morphology of the pancreas

Background: The direct effect of free oxygen radicals, if any, on the morphology of the pancreas has never been studied in vivo. This study was designed to evaluate the effects of hydrogen peroxide (H 2O 2) on permeability of the main pancreatic duct (MPD) and morphology of pancreas in cats when administered intraductally or intraarterially. Methods: Thirty-six mongrel cats were randomly allocated into three groups, and all groups were divided into two subgroups. In group I and III, MPD was perfused with either standard perfusate (group IA and IIIA) or H 2O 2 at a concentration of 150 μM (group IB and IIIB) for 3 hours. In group II, the splenic artery was infused either with 0.9% sodium chloride (group IIA) or H 2O 2 (group IIB) for 3 hours. After 3 hours, in group I and II, MPD was perfused with 99mTc labelled dextran, and the percentage of the dextran permeated from the MPD into the portal vein was calculated for the evaluation of the pancreatic duct permeability. Then, tissue samples were obtained for the examination of early histopathological changes in pancreas. In group III, following ductal perfusion studies, the cats were allowed to recover. After 24 hours animals were killed, and samples were taken for the examination of late histological changes in pancreas. In all groups, an inflammatory score was created for each animal based on the pathological changes in pancreas: edema, leukocyte infiltration, parenchymal necrosis, and hemorrhage. Results: Group I: All cats developed acute edematous pancreatitis with significantly higher inflammatory scores than controls ( P <0.01). Desquamation of the single layer of columnar epithelium that normally lined the duct and leukocyte infiltration around the MPD duct were found. Pancreatic duct permeability was found to be increased significantly ( P <0.01). Group II: There were no statistical differences in inflammatory scores and pancreatic duct permeability between experimental and control groups ( P >0.05). Group III: All animals developed gross acute edematous pancreatitis after 3 hours of intraductal H 2O 2 perfusion. Histopathological changes at 24 hours were much more pronounced in group IIIB than in group IIIA including focal necrosis and hydropic degeneration of acinar cells. Conclusion: This study has shown that intraductal H 2O 2 perfusion induced acute edematous pancreatitis with marked histopathological changes and increased pancreatic duct permeability in cats. Intraarterial H 2O 2 infusion, however, has no effect on the permeability of the MPD and morphology of pancreas in our model.

The effect of oxygen free radicals on myocardial nuclear calcium transport in rat

The 1H and 13C NMR spectra of the chiral NADH models 3 and 4 were studied both in the presence or absence of Mg2+ ions. These two compounds have a chiral carboxamide part derived from (S)-phenylalaninol but contrary to 3, compound 4 has the ability to free-rotate about the C3CO bond. Careful analysis of the spectral results and comparison with some other studies recently published in the literature allowed the following assumptions: 1-the 1,4-dihydropyridine structure is planar both in the absence or presence of magnesium ions. 2-in the case of the rigid model 3, the CO is out of the plane of the dihydropyridine structure. 3-with Mg 2+ the chiral amino alcohol moiety adopts a quasi-cyclic conformation in the case of 4 but not in the case of 3. The observed enantioselectivities are explained by the mean of ternary complexes between the model, Mg2+ and the substrate.

Effect of oxygen free radicals on myosin in muscle fibres

Experiments were performed on glycerol-extracted muscle fibres prepared from psoas muscle of rabbit in the presence of hydroxyl free radical generating system. Short irradiation of spin-labelled muscle fibres by UV light showed the interaction of probe molecules with oxygen free radicals. The intensity of the EPR signal from maleimide or isothiocyanate spin labels attached to the essential thiol groups decreased following irradiation. Oxygen free radicals affected the rate constant of the transition AM.ADP.V i→AM.ADP in the ATP hydrolysis cycle. It was found that the essential –SH groups of myosin were involved in the oxidation of sulphydryls by Ce(IV). Ce(IV) complexed to nitrilotriacetic acid in the presence of spin trap produced long-lived free radicals located partly on SH-1 sulphydryls.

Role of antioxidants in gastric mucosal damage induced by indomethacin in rats.

Reactive oxygen species play a role in the formation of gastric lesions induced by nonsteroidal antiinflammatory drugs. The present study was undertaken to determine whether endogenous antioxidants in gastric mucosa can protect it against the damaging effects of oxygen free radicals. This study examine oxygen free radical production (superoxide anions and hydrogen peroxide), gastric mucosa antioxidant defense mechanisms (glutathione, catalase, superoxide dismutase), the lesion-inducing effects of the generated oxygen free radicals (vascular permeability, lipid peroxidation) and gastric ulceration in rats treated orally with indomethacin at 10 mg/kg at 2 and 6 h after drug administration. Two hours after administration of the antiinflammatory drug, there was a sharp increase in production of oxygen free radicals in the gastric mucosa with no alteration in other parameters examined. Six hours after indomethacin administration the production of oxygen free radicals returned to basal levels, but there was a high degree of gastric ulceration and a significant increase in lipid peroxidation and vascular permeability together with decreases of 45% in glutathione concentration and 30% in catalase relative to the control group. These results suggest that like plasma, the gastric mucosa has an antioxidant capacity and only when this capacity is exhausted are the lesive effects of the oxygen free radicals manifested.

O-183 - Effects of oxygen free radicals on Ca2 + mobilization in human nasal epithelium

O-183 - Effects of oxygen free radicals on Ca2 + mobilization in human nasal epithelium

Differential induction of superoxide dismutase in core and penumbra regions after transient focal ischemia in the rat neocortex

Oxygen free radicals are postulated to participate in the pathogenesis of ischemic brain injury. The present study investigated the response of the endogenous antioxidant enzyme, superoxide dismutase (SOD), in a model of transient focal ischemia in the rat neocortex. SOD activity was increased significantly in the penumbra region at 6–24 h postischemia, while no significant changes in SOD activity were observed in either the core region or striatum. These results indicate that endogenous antioxidant activity is differentially affected by the intensity of ischemic challenge and suggest that the regional effects of oxygen free radicals may vary substantially following ischemia-reperfusion.

H 2O 2-derived free radicals treated fibronectin substratum reduces the bone nodule formation of rat calvarial osteoblast

Fibronectin (FN) is involved in various cellular activities such as adhesion, proliferation and migration as a substratum. Since the metabolic turnover of FN is much slower than other cellular components, it may be affected by the oxygen free radicals produced in the aging process. However, the effect of oxygen free radicals on FN as substratum in bone formation has not been well characterized. The objective of this study was to examine the effect on the bone forming activity of osteoblasts using an oxygen free radical treated FN substratum in vitro (H 2O 2–Cu 2+system). SDS-PAGE, Western blotting and immuno-blotting analysis revealed that FN was degradated and/or modified by H 2O 2–Cu 2+ (·OH) treatment. Bone nodule formation per well was examined for total number, total area and area per nodule, which data were then compared between non-coated and FN-coated, and between FN-coated and ·OH treated FN-coated. Bone nodule formation in the FN-coated was significantly greater than in the non-coated. Furthermore, bone nodule formation in ·OH treated FN-coated was significantly less than that of FN-coated. These findings suggested that FN plays important roles in osteoblast activity and that FN substratum damaged by the oxygen free radicals produced by the aging process may cause decline of bone nodule formation through inhibition of the proliferation, differentiation and calcification processes.

Puberty influences expression of phospholipid hydroperoxide glutathione peroxidase (GPX4) in rat testis: probable hypophysis regulation of the enzyme in male reproductive tract.

Mammalian spermatozoa are unusually rich in polyunsaturated fatty acids, a property that predisposes them to the deleterious effects of oxygen free radicals. Mouse and human spermatozoa utilize glutathione peroxidase, (GPX), to inactivate oxygen free radicals. In the GPX super-family there is the enzyme phospholipid hydroperoxide glutathione peroxidase (GPX4) that specifically protects membrane phospholipids against peroxidation. GPX4 is present, primarily, in testis where its enzymatic activity seems to be present only after puberty. In order to clarify this question we utilized total RNA from rat testis, liver and lung to carry out cDNA synthesis and the following RT-PCR amplification of cDNA products by using specific primers of rat liver sequence. RT-PCR products of the expected size for GPX4 (525 bp) were obtained from the three tissues. At last, these fragments were submitted to sequencing analysis. Here we demonstrate that the sequence analysis of rat testis GPX4 coding region is identical to that of rat liver and lung; however puberty influences the expression pattern of rat testis GPX4. In fact Northern blot analysis of total RNA from normal and pre-puberal hypophysectomized rats demonstrates the absence of a specific GPX4 mRNA in total RNA from pre-puberal hypophysectomized rat testis; on the other hand this specific transcript is present in both normal rat testis and liver and in pre-puberal hypophysectomized rat liver. Expression pattern of GPX4 is very low in lung both in post-puberal and pre-puberal hypophysectomized rats. Therefore hypophysis could regulate GPX4 transcript in rat testis.

ENDOTHELIAL EICOSANOID METABOLISM AND SIGNAL TRANSDUCTION DURING EXPOSURE TO OXYGEN RADICALS INJURY

Several physiological agonists that induce elevation of cytosolic free calcium (Ca 2+)-levels act via receptor coupled G-proteins, involving activation of phospholipase C (PLC) and hydrolysis of phosphatidylinositol 4,5-bisphosphate. Activation of the inositol signal transduction pathway that precedes Ca 2+ ion mobilization is a well accepted signaling pathway in endothelial cell eicosanoid synthesis. This study was designed to examine possible involvement of phosphoinositides in the effects of oxygen free radicals on Ca 2+ liberation and eicosanoid synthesis in human umbilical venous endothelial cells (HUVEC). Hydrogen peroxide (H 2O 2) was chosen as oxygen radicals generating agent. Stimulation of HUVEC with H 2O 2 (0.1 mmol/l) led to significant rises in inositol phosphate and diacylglycerol (DAG) levels within 300 seconds and an inhibition of Ca 2+ release from internal stores. Eicosanoid formation was detectable despite unchanged levels of cytosolic free Ca 2+ and no detectable activation of membrane associated phospholipase A 2 (PLA 2). This suggests that eicosanoid formation may be mediated through the activation of a Ca 2+ independent, cytosolic 40 kDa PLA 2 isoenzyme and that DAG could serve as an alternative source for arachidonic acid and seems to sensitize a cytosolic PLA 2. © 1997 Elsevier Science Ltd

Effects of free oxygen radicals on Ca 2+ release mechanisms in the sarcoplasmic reticulum of scallop ( Pecten jacobaeus) adductor muscle

In vitro oxyradical effects on SR Ca 2+ regulation were studied by using a SR-containing cell-free preparation from scallop ( Pecten jacobaeus) adductor muscle. Ca 2+ variations were fluorimetrically detected after incubation with Fluo-3 in the presence of ATP. Exposure to Fe 3+/ascorbate produced dose-dependent Ca 2+ release from SR vesicles, eventually leading to massive Ca 2+ loss. Exposure to hypoxanthine/xanthine oxidase also caused Ca 2+ release but at a much slower rate. Pre-incubations with catalase or with the hydroxyl radical scavenger KMBA led to a significant decrease in the Fe 3+/ascorbate-induced Ca 2+ release rate and to a delay of massive Ca 2+ loss. Pre-incubations with GSH or DTT strongly reduced the Ca 2+ release caused by Fe 3+/ascorbate and, moreover, they prevented massive Ca 2+ loss from SR vesicles. Addition of GSH or DTT after Fe 3+/ascorbate promptly reduced the Ca 2+ release rate and delayed massive Ca 2+ release. Pre-incubation with the SR Ca 2+ channel blocker ruthenium red strongly reduced the Ca 2+ release caused by Fe 3+/ascorbate, and also prevented massive Ca 2+ loss. In the presence of ruthenium red, Fe 3+/ascorbate treatments followed by Ca 2+ addition revealed that Ca 2+ uptake inhibition was slower than Ca 2+ release. Taken together, data showed that free radicals and, in particular, hydroxyl radicals, affected the scallop SR Ca 2+ regulation. This mainly occurred through Ca 2+ channel opening, most likely triggered by sulfhydryl oxidation, which eventually led to massive Ca 2+ release from SR vesicles. The demonstration of a specific effect of oxyradicals on SR Ca 2+ channels is in line with their possible involvement in cell signaling.

Role of macrophage overactivation in the development of acute pancreatic injury in rats

An increase in systemic inflammatory mediators from stimulated leucocytes and macrophages has been noted during acute pancreatitis. The role of cytolytic inflammatory macrophages and potential mechanisms in the development of acute pancreatic injury and endothelial barrier dysfunction are less well defined. Rats were challenged by an intraperitoneal injection of cytolytic or non-cytolytic inflammatory macrophage stimulators at various concentrations. The effects of oxygen free radicals, prostaglandin and extracellular calcium influx on macrophage-associated pancreatic endothelial compromise, measured by pancreatic intravascular plasma volume, pancreatic interstitial fluid volume, and the pancreatic extravascular human serum albumin distribution volume, were explored. Zymosan-induced overactivation of cytolytic inflammatory macrophages resulted in the development of acute pancreatic endothelial dysfunction in a dose- and time-dependent pattern. An increase in pancreatic water content and interstitial fluid volume was observed following a higher dose (0.5 mg/g) of concanavalin A without alteration in plasma lipase level, while thioglycollate medium did not compromise pancreatic endothelial barrier function. Oxygen free radicals, but also prostaglandins and extracellular calcium influx, seemed to be involved in macrophage overactivation-induced pancreatic injury. Overactivation of cytolytic macrophages plays a role in the pathogenesis of pancreatic injury by initiating the development of endothelial barrier dysfunction. Multiple inflammatory mediators from overactivated macrophages act as intercellular signals between macrophages and the endothelium during acute pancreatic injury.

The effect of free oxygen radicals on fracture healing in rats

Thirty male rats were used to study the effect of free oxygen radicals on fracture healing. The rats were divided into two groups and the right forelimbs of the rats were broken by light manual compression. One hour before the fracture the following treatments were given to groups of 15 animals; saline 5 ml/kg i.p. (control group) or zyomosan 100 mg/kg i.p. to induce free oxygen radicals through stimulation of NADPH oxidase in polymorphonuclear leucocytes. Treatment were stopped on the fifth post-fracture day. All rats were sacrificed with high dose ether on day 22, and histological sections of the radius and ulna were examined without knowledge of the treatment regiment. The administration of zymosan impaired the fracture healing and therefore free oxygen radicals appear to play an important role on fracture healing.

Increasing vasoconstrictor response to ergonovine with oxidative injury in canine coronary artery.

The effects of oxygen free radicals on coronary vasoreactivity remain unknown. To examine whether oxygen free radicals increase coronary arterial tone and sensitivity to vasoconstrictor stimulation in closed-chest dogs. Oxygen radicals were generated by the reaction of xanthine plus xanthine oxidase (XXO) and effects of these substances on the left coronary artery (the percentage diameter change) and on the constrictor effect of ergonovine were examined in vivo in 19 anesthetized, closed-chest dogs by selective coronary angiography. The effects of XXO solution and ergonovine were assessed in a cumulative fashion using 100, 200, and 300 ml XXO and 50, 100, 150, and 200 micrograms ergonovine, in 5 (group I) and 6 dogs (group II), respectively. The effects of XXO on the constrictor responses elicited by 50 micrograms ergonovine were examined in eight dogs (group III). Changes in the vascular endothelium were examined by postmortem electron microscopic examination. Oxidative injury alone produced slight constriction of the coronary artery, but the change was not significant. However, ergonovine-induced vasoconstriction was enhanced after administration of 100 and 200 ml (cumulative amount) XXO solution (P < 0.05, group II versus group III). The enhancement was no longer observed after administration of 300 ml (cumulative amount) XXO solution. Scanning and transmission electron microscopies revealed the formation of blebs and ulceration in the coronary endothelium after administration of XXO solution. These results suggest that oxygen radicals can enhance the ergonovine-induced coronary vasoconstriction in a concentration-dependent manner. There seems to be a critical level of oxygen radicals for the production of the effect.