Radiogenic free radicals in apatite: The influence of fluoride and hydroxide

Fluor-hydroxyapatites of varying fluoride and hydroxide contents were irradiated by soft x-rays at ?188°C and examined by means of electron spin resonance spectroscopy. Variation in fluoride-hydroxide contents affected radical yield and resulted in the observation of two kinds of oxygen anion resonances: a doublet due to hydrogen hyperfine splitting and a singlet. At higher temperatures a spin transfer process became operative which enhanced the oxygen anion radical population. Increasing fluoride content was paralleled by decreasingg-values for the oxygen anion resonances, possibly attributable to increased delocalization of the unpaired spin.     

Radiogenic free radicals as molecular probes in bone

Summary Exposure of bone mineral to X-rays generates free radicals. These are usually very labile, but can be stabilized at liquid nitrogen temperatures for study by electron spin resonance spectroscopy. The free radicals thus detected in the present study included one with resonances arising from an electron excess center and 2 species with electron-deficit centers: a phosphate anion radical and a radical associated with carbonate. Each of these radicals seemed to be located chiefly at the mineral surface and was sensitive to the surface environment. Presence of an organic phase, as in whole bone, modified free radical production in a manner that suggests interference with the formation of electron deficit centers. Comparison with other synthetic minerals suggests that precipitated carbonateapatites are good models for bone mineral.     

Selective impairment of endothelium-dependent relaxation by sevoflurane: oxygen free radicals participation.

To determine whether sevoflurane alters endothelium-mediated vasodilation of vascular smooth muscle, isolated ring preparations of canine mesenteric arteries were suspended for isometric tension recordings in modified Krebs-Ringer bicarbonate solution at 37 degrees C. Following contraction with norepinephrine, cumulative concentration-response curves were generated using endothelium-dependent vasodilators (acetylcholine, bradykinin, and calcium ionophore A23187) or nitroglycerin. The relaxation produced by acetylcholine, bradykinin, or A23187 was impaired by sevoflurane (2.3 and 4.6 vol%); sevoflurane did not affect relaxation caused by nitroglycerin, which, in these vessels, acts by an endothelium-independent mechanism. Under the same experimental conditions as those used for the concentration-response relationship, electron spin resonance spin-trapping with 5,5-dimethyl-1-pyrroline N-oxide verified generation of hydroxyl radical from the sevoflurane-delivered bathing media; the generation of hydroxyl radical was inhibited by superoxide dismutase, a scavenger of superoxide anion radical, or by the powerful iron chelator deferoxamine. Furthermore, sevoflurane-induced impairment of the relaxation caused by the endothelium-dependent vasodilators used was significantly decreased by superoxide dismutase. These results indicate that superoxide anion radical and/or closely related species of oxygen free radicals, possibly hydroxyl radical, are involved in the observed effect of sevoflurane. We propose that sevoflurane selectively impairs endothelium-dependent relaxation in canine mesenteric arteries by an oxygen free radical mechanism, mainly due to inactivation of endothelium-derived relaxing factor.     

Direct Assessments of the Antioxidant Effects of Propofol Medium Chain Triglyceride/Long Chain Triglyceride on the Brain of Stroke-prone Spontaneously Hypertensive Rats Using Electron Spin Resonance Spectroscopy

Background: Antioxidant anesthetics such as propofol (2,6-diisopropylphenol) directly inhibit lipid peroxidation via the generation of reactive oxygen species. Currently, there are no other studies regarding the direct effects of propofol medium chain triglyceride/long chain triglyceride (MCT/LCT) on reactive oxygen species generation or in experimental models of reactive oxygen species-induced oxidative stress in the brain.

Methods: The authors investigated the effects of propofol MCT/LCT on reactive oxygen species (hydroxyl radical or superoxide) by electron spin resonance spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide. The effects of propofol MCT/LCT on oxidative stress in the brain of Wistar-Kyoto rats or stroke-prone spontaneously hypertensive rats were investigated by using an in vivo L-band electron spin resonance system to monitor the decay rate of 3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl as a nitroxyl spin probe.

Results: These studies provided direct evidence that propofol MCT/LCT inhibited hydroxyl radical generation, but not superoxide generation. Regarding the hydroxyl radical from the Fenton system, it is likely to be due to the scavenging effects of vehicle. Anesthesia with propofol MCT/LCT reduced the degree of the high oxidative stress in the brain of stroke-prone spontaneously hypertensive rats.     

Systemic Free Radical Activation Is a Major Event Involved In Myocardial Oxidative Stress Related to Cardiopulmonary Bypass

Background: Cardiopulmonary bypass (CPB) can induce deleterious effects that could be triggered in part by radical oxygen species; however, their involvement in the course of surgery has been elusive. The aim of this study was to evaluate the time course and origin of radical oxygen species release, myocardial or not, in patients undergoing coronary artery surgery involving CPB.

Methods: Blood samples were taken from periphery and coronary sinus of patients during CPB, and oxidative stress was evaluated by direct and indirect approaches. Direct detection of alkyl and alkoxyl radicals was assessed by electron spin resonance spectroscopy associated with the spin-trapping technique using [alpha]-phenyl-N-tert-butylnitrone.

Results: The authors showed that the spin adduct concentration was not influenced by anesthesia and pre-CPB surgery. A rapid systemic increase of plasma spin adduct concentration occurred after starting CPB, and it stayed at a high concentration until the end of CPB. At the beginning of reperfusion period, radical oxygen species release was accelerated in the coronary sinus; however, it was not significant. A positive correlation was found between [alpha]-phenyl-N-tert-butylnitrone adduct concentrations and (1) the duration of CPB and (2) concentration of postoperative creatine phosphokinase of muscle band (CPK MB). Plasma vitamin E and C, ascorbyl radical, uric acid, thiol, plasma antioxidant status, and thiobarbituric acid reacting substances were also measured but did not give relevant indications, except for uric acid, which seemed to be consumed by the heart during reperfusion.     

Oxidant Mechanisms in Gentamicin Nephrotoxicity

Acute renal failure is a major complication of aminoglycoside antibiotics, which are widely used in the treatment of gram-negative infections. Sequential reduction of oxygen along the univalent pathway leads to the generation of superoxide anion, hydrogen peroxide, hydroxyl radical, and water. A large body of in vitro and in vivo evidence indicates that these partially reduced oxygen metabolites are important mediators of gentamicin nephrotoxicity. Gentamicin has been shown to enhance the generation of superoxide anion and hydrogen peroxide by renal cortical mitochondria. The interaction between superoxide anion and hydrogen peroxide in the presence of metal catalyst can lead to the generation of hydroxyl radical. Gentamicin has been shown to lead to release of iron from renal cortical mitochondria and to enhance generation of hvdroxyl radical. These in vitro observations have been supported by in vivo studies in which scavengers of reactive oxygen metabolites and iron chelators have shown to be protective in gentamicin induced acute renal failure. There is evidence to suggest that studies may have broader implication in being relevant to other aminglycosides including streptomycin and being applicable to other major toxicity of aminoglycoside such as ototoxicity.     

Oxidant mechanisms in gentamicin nephrotoxicity.

Acute renal failure is a major complication of aminoglycoside antibiotics, which are widely used in the treatment of gram-negative infections. Sequential reduction of oxygen along the univalent pathway leads to the generation of superoxide anion, hydrogen peroxide, hydroxyl radical, and water. A large body of in vitro and in vivo evidence indicates that these partially reduced oxygen metabolites are important mediators of gentamicin nephrotoxicity. Gentamicin has been shown to enhance the generation of superoxide anion and hydrogen peroxide by renal cortical mitochondria. The interaction between superoxide anion and hydrogen peroxide in the presence of metal catalyst can lead to the generation of hydroxyl radical. Gentamicin has been shown to lead to release of iron from renal cortical mitochondria and to enhance generation of hydroxyl radical. These in vitro observations have been supported by in vivo studies in which scavengers of reactive oxygen metabolites and iron chelators have shown to be protective in gentamicin induced acute renal failure. There is evidence to suggest that studies may have broader implication in being relevant to other aminoglycosides including streptomycin and being applicable to other major toxicity of aminoglycoside such as ototoxicity.     

Systemic free radical activation is a major event involved in myocardial oxidative stress related to cardiopulmonary bypass.

BACKGROUND: Cardiopulmonary bypass (CPB) can induce deleterious effects that could be triggered in part by radical oxygen species; however, their involvement in the course of surgery has been elusive. The aim of this study was to evaluate the time course and origin of radical oxygen species release, myocardial or not, in patients undergoing coronary artery surgery involving CPB. METHODS: Blood samples were taken from periphery and coronary sinus of patients during CPB, and oxidative stress was evaluated by direct and indirect approaches. Direct detection of alkyl and alkoxyl radicals was assessed by electron spin resonance spectroscopy associated with the spin-trapping technique using alpha-phenyl-N-tert-butylnitrone. RESULTS: The authors showed that the spin adduct concentration was not influenced by anesthesia and pre-CPB surgery. A rapid systemic increase of plasma spin adduct concentration occurred after starting CPB, and it stayed at a high concentration until the end of CPB. At the beginning of reperfusion period, radical oxygen species release was accelerated in the coronary sinus; however, it was not significant. A positive correlation was found between alpha-phenyl-N-tert-butylnitrone adduct concentrations and (1) the duration of CPB and (2) concentration of postoperative creatine phosphokinase of muscle band (CPK MB). Plasma vitamin E and C, ascorbyl radical, uric acid, thiol, plasma antioxidant status, and thiobarbituric acid reacting substances were also measured but did not give relevant indications, except for uric acid, which seemed to be consumed by the heart during reperfusion. CONCLUSION: The results indicate that a systemic production of free radicals occurs during CPB that may overwhelm the production related to reperfusion of the ischemic heart. This systemic oxidative stress is likely to participate in secondary myocardial damage.     

硅胶纤维囊的自由基检测

以Wistar大鼠为实验动物,在背部植入硅胶囊,术后5周,应用电子自旋共振(ESR)技术对纤维囊内的自由基进行检测,结果发现纤维囊形成过程中有大量氧自由基产生。注入自由基清除剂-超氧化物歧化酶(SOD)对氧自由基的产生有明显的清除效应。讨论了氧自由基与纤维囊胶原合成的关系。     

氧自由基与人体纤维囊形成

经动物实验研究，已证明硅橡胶囊植入体内形成的纤维囊与氧自由基有关，但在人体内的研究尚少。采用电子自旋共振（ｅｌｅｃｔｒｏｎｓｐｉｎｒｅｓｏｎａｎｃｅ，ＥＳＲ）技术，对７枚人体硅胶囊隆乳术后形成的纤维囊进行了检测，同时对纤维囊的组织病理学特征进行了观察。结果发现，纤维囊内有大量氧自由基产生，其病理改变主要为大量炎性细胞浸润和胶原沉积。表明人体内纤维囊的形成与氧自由基有关。应用氧自由基清除剂，可使增厚的纤维囊变薄     

Analysis of paramagnetic centers in X-ray-irradiated enamel,bone, and carbonate-containing hydroxyapatite by electron spin resonance spectroscopy

Summary Carbonate-containing hydroxyapatite, enamel, and bone were irradiated by an X-ray and investigated between 77° and 350°K by means of electron spin resonance (ESR) spectroscopy. The ESR spectrum of enamel irradiated at 77°K in vacuum and observed at the same temperature was almost the same as that of the carbonate-containing hydroxyapatite. The temperature dependence of signal intensities confirms a spin-energy exchange between the mineral and organic constituents in bone, but in enamel no or very little spinenergy exchange between the mineral and organic constituents. Considerable similarity among the ESR spectra of enamel, bone, and carbonate-containing apatite was obtained after X-ray irradiation in air at 300°K with both an X-band and a Q-band ESR spectrometer. The Q-band spectrum can be interpreted in terms of two paramagnetic species. One is identified as a CO ₃ ³⁻ anion radical which has an axial symmetry withg factors of 2.0029 and 1.9972. The other species is found to be centered atg=2.0019.     

Fluoride absorption: The influence of gastric acidity

Summary The influence of gastric acidity on the absorption of intragastrically administered fluoride was investigated in rats. Intact animals were pretreated with atropine or cimetidine to reduce gastric acid secretion or were given fluoride in NaHCO₃ to reduce the acidity of the gastric contents. Compared with pentagastrin-treated animals or animals that received fluoride in 0.1 N HCl, their rate of fluoride absorption was markedly reduced as judged by lower plasma fluoride concentrations and areas under the time-plasma concentration curves, especially during the first hour after dosing. In crossover studies with the stomachs isolatedin situ, fluoride absorption was at least 50% faster from a pH 2.1 buffer compared with its absorption from a pH 7.1 buffer. The findings are consistent with the hypothesis that fluoride is absorbed from the gastric lumen principally as the undissociated molecule, HF. The results may contribute to a more complete understanding of acute fluoride toxicity, the development of dental fluorosis and, perhaps, the use of fluoride in the treatment of osteoporosis.     

In vitro study of the antioxidant properties of nimesulide and 4-OH nimesulide: effects on HRP- and luminol-dependent chemiluminescence produced by human chondrocytes

OBJECTIVES: Reactive oxygen species (ROS) are now recognized to play an important role in the pathogenesis of rheumatic diseases and constitute an interesting therapeutic target for drugs. This in vitro study was designed to evaluate the antioxidant properties of nimesulide (NIM), a nonsteroidal antiinflammatory drug of the sulfonanilide class, and its main metabolite 4-OH nimesulide (4-OHNIM). METHODS: The scavenging effects of NIM and 4-OH NIM on hydroxyl radical ((.)OH) and superoxide anions (O(minusd)(2)) were investigated by electron spin resonance (ESR), using 5, 5-dimethylpyrroline-N-oxide (DMPO) as the spin trap agent. The quenching properties of these drugs on hypochlorite anion was studied by luminol enhanced chemiluminescence. Finally, the effects of NIM and 4-OHNIM on the reactive oxygen species production by human articular chondrocytes were recorded by HRP and luminol-enhanced chemiluminescence. RESULTS: By this method it has been demonstrated that NIM and 4-OHNIM, at concentrations ranging from 10 to 100 microM, are potent scavengers of(.)OH whereas only 4-OHNIM was capable to scavenge O(minusd)(2). Chemiluminescence generated by HOCl was also significantly and dose-dependently inhibited by both NIM and 4-OHNIM. Nevertheless, at each concentration tested, the inhibitory effect of 4-OHNIM was significantly more marked, even at the highest concentration (100 microM). Furthermore, when chondrocytes were pre-incubated for 48-96 h with NIM or 4-OHNIM, the luminol- and HRP-dependent CL produced by the cells was significantly inhibited in a dose-dependent manner. CONCLUSIONS: NIM and 4-OHNIM may protect cartilage against oxidative stress, not only by scavenging ROS but also by inhibiting their production by chondrocytes.     

Differences in production of melanin radicals by 694 nm ruby laser and UVA radiation

BACKGROUND AND OBJECTIVES: The 694 nm ruby laser is used clinically for hair removal and the mechanism is predominantly photo thermal via melanin targeting. We investigated 694 nm laser-irradiation of human hair, and laser-irradiation of synthetic dopa melanin to establish whether photolysis and oxygen radical production is also contributory, and which may have side effects. STUDY DESIGN/MATERIALS AND METHODS: Ultraviolet-A (UVA) irradiation of melanin was used as a positive control for radical production. Laser- and UVA-irradiated hair samples, and synthetic dopa melanin in media of different viscosity, were analyzed using electron spin resonance spectroscopy, and compared. The spin trap 5,5-dimethyl- 1-pyrroline N-oxide (DMPO) was used to probe laser-irradiated dopa melanin for superoxide radical production. RESULTS: Comparable to UVA, laser-irradiation of hair increased the signal-intensity of the intrinsic melanin radical. UVA-induced radicals decay rapidly; however, laser-induced radicals decayed slowly and did not fully revert to original levels after 24 hours. Laser-induced radicals were increasingly stable with viscosity of the medium. Superoxide radicals were detected using DMPO in UVA- but not laser-irradiated synthetic dopa-melanin at pH 4.5. CONCLUSIONS: Laser-irradiation of melanin does not result in oxygen radical formation; however, a paramagnetic species, long-lived in rigid media, is detected which is worth further investigation.     

Effect of pentoxifylline on superoxide anion production by human sperm

The in-vitro effect of pentoxifylline, a methylxanthine derivative, on superoxide anion generation by sperm was studied in 27 men with superoxide anion production ranging from 0.57 to 13.8 nmoles 02-. per 10(6) sperm. Superoxide anion release stimulated by phorbol myristate acetate, a potent activator of oxygen radical generation, was reduced by 29-72% following the addition of 10 mM pentoxifylline. The inhibitory effect of this millimolar concentration of the drug did not depend on the initial superoxide production in the presence of phorbol myristate acetate. These results point to a potential use for pentoxifylline in the treatment of male infertility in men with an increased superoxide anion generation capacity.     

Radiation induced free radicals as molecular probes in synthetic apatites

Summary Free radicals generated in synthetic apatitic calcium phosphates by X-ray radiation were investigated by electron spin resonance (ESR) spectroscopy. Among the species stable enough at –188° C to be identified were hydrogen atoms, phosphate radicals, and oxygen anion radicals. The ESR spectra were markedly dependent on the specific surface of the mineral. Oxygen radicals dominated the spectra of low specific surface samples while phosphate radicals were the predominant species at higher specific surfaces. Our studies suggest that the oxygen radicals are more stable in the bulk of the crystal while the hydrogen atoms and the phosphate radicals are stabilized at or near the crystal surface. It was concluded that the surface species are potentially capable of serving as probes of biologically relevant mineral-organic interfaces.     

Formation of ascorbate radicals as a measure of oxidative stress: an in vitro electron spin resonance-study using 2,2-Azobis (2-amidinopropane) dihydrochloride as a radical generator

Reactive oxygen species (ROS) are continuously formed in biological systems. Any increase in radical production or decrease in the defense against ROS induces oxidative stress. This imbalance between ROS formation and ROS detoxification is believed to be involved in a variety of pathogenic processes, including ischemia-reperfusion injury. Various markers indicating oxidative stress has been used in experimental and clinical studies. One of them is ascorbate free radical (AFR), electron spin resonance intensity of which correlates with the severity of radical formation. We investigated the impact of alkyl peroxyl radicals produced by 2,2-Azobis (2-amidinopropane) dihydrochloride decomposition on the magnitude of the AFR signal. Our data confirmed the principal applicability of AFR as a nontoxic marker of radical generation.     

The influence of fluoride on proteoglycan structure using a rat odontoblast in vitro system

Summary Using an in vitro rat incisor odontoblast system, the effect of fluoride on proteoglycans was investigated at both the metabolic and structural level. Incisors were removed from 4-week-old rats, split longitudinally, and the pulps removed. Teeth were incubated at 37°C, 5% CO₂ in Eagle's Minimum Essential Medium containing ³⁵S-sulfate for 7 hours in the presence of 0 mM, 3 mM, or 6 mM sodium fluoride. Teeth were demineralized in EDTA, proteoglycan was extracted from the residue with 4 M guanidinium chloride, and further purified by anion exchange chromatography. Uptake of radiolabel was monitored by liquid scintillation counting. The resultant products were examined by cellulose acetate electrophoresis, SDS-PAGE, chondroitinase digestion, and amino acid analysis. Differential effects of fluoride were observed in both metabolism and biochemical characterization of proteoglycans following incubation at the two concentrations. Fluoride decreased uptake of the radiolabel but led to an accumulation of glycosaminoglycan within the proteoglycan of the matrix. Chondroitin sulfate was the predominant glycosaminoglycan identified, with the additional presence of dermatan sulfate and heparan sulfate identified. Dermatan sulfate levels increased in 3 mM-treated teeth. Fluoride-treated proteoglycans had a reduced molecular weight (200–90K to 180–79K); this reduction is primarily a result of smaller glycosaminoglycan chains, with limited reduction in the size of the core protein of 6 mM-treated teeth occurring. Such alterations in the biochemical metabolism and hence structure and function of proteoglycan may be implicated in the hypomineralization seen in fluorosis.     

The effect of cimetidine on anesthetic metabolism and toxicity

Because the H2-receptor antagonist cimetidine has been shown to inhibit drug metabolism, the effects of cimetidine on anesthetic metabolism and toxicity were investigated in a rat model. Cimetidine decreased inorganic plasma fluoride production after methoxyflurane administration both in 21% oxygen (P less than 0.001) and in 100% oxygen (P less than 0.001). Phenobarbital produces an increased fluoride formation after methoxyflurane anesthesia, and this fluoride formation is also reduced by cimetidine (P less than 0.005). There was no significant difference between the plasma fluoride levels in rats anesthetized with halothane or enflurane. Although cimetidine inhibited the in vivo defluorination of methoxyflurane, fluoride levels were still within the nephrotoxic range, and cimetidine is not likely to play a role as part of a preanesthetic regimen that would permit the increased clinical use of methoxyflurane. Cimetidine also inhibited the oxidative metabolism of halothane; cimetidine decreased (P less than 0.05) trifluoroacetic acid concentrations after halothane anesthesia in 21% oxygen and in 100% oxygen and decreased (P less than 0.05) bromide concentrations after halothane anesthesia in 100% oxygen. Trifluoroacetic acid levels were less (P less than 0.02) after halothane anesthesia in 14% oxygen as compared with 100% oxygen, indicating a reduction in oxidative metabolism under hypoxic conditions. However, bromide concentrations were maximal after halothane anesthesia in 21% oxygen, and significantly (P less than 0.001) less after halothane anesthesia in 14% and 100% oxygen. Bromide production, therefore, seems to be inhibited by both hypoxia and hyperoxia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluoride,parathyroid hormone and calcitonin: Effects on metabolic processes involved in bone resorption

The effects of fluoride, parathyroid hormone and calcitonin on acid production and bone phosphatases were investigated using half-calvaria of 5-day-old mice in a stationary culture system. Fluoride was incorporated into the bones before culture by administering fluoride to the mothers and pups. Parathyroid hormone (0.2 U/ml) stimulated the production of both lactic and citric acids, while calcitonin (4.0 mU/ml) did not influence acid production, relative to control cultures. Half-calvaria with high and low fluoride contents produced similar quantities of both acids, and responded similarly to parathyroid hormone. The alkaline phosphatase activity of freshly-dissected half-calvaria was not influenced by their fluoride content, but acid phosphatase activity was 72% higher in bones with high fluoride contents than in bones with low fluoride contents. Parathyroid hormone stimulated acid phosphatase to a greater extent in low fluoride bones than in high fluoride bones. Calcitonin stimulated both alkaline and acid phosphatase activities, with a greater increase in acid phosphatase activity than in alkaline phosphatase activity. Fluoride did not appear to modify the influence of calcitonin on phosphatase activity. Since fluoride did not inhibit acid production or acid phosphatase activity, the inhibition of bone resorption by fluoride can best be explained by its effect on bone mineral solubility.Supported by Grant No. DE-01850, National Institute of Dental Research, Bethesda, Maryland 20014.