Endothelial protection from reperfusion injury by ischemic preconditioning and diazoxide involves a SOD-like anti-O2- mechanism.

Cardiac ischemia/reperfusion leads to coronary endothelial dysfunction, mediated by superoxide anion (O2-), but not hydroxyl radical (*OH). Ischemic preconditioning and mitochondrial ATP-dependent potassium channel opener (diazoxide) protect endothelium in the mechanism involving attenuation of O2- burst at reperfusion. We hypothesize that the endothelial protection involves upregulation of myocardial anty-O2- defense. Langendorff-perfused guinea-pig hearts were subjected to global ischemia/reperfusion (IR) or were preconditioned prior to IR with three cycles of ischemia/reperfusion (IPC) or infusion/washout of 0.5 microM diazoxide. Coronary flow responses to acetylcholine were measures of endothelium-dependent vascular function. Myocardial outflow of O2- and of *OH during reperfusion and myocardial activities of superoxide dismutase (SOD) and catalase were measured. IR impaired acetylcholine response and augmented cardiac O2- and *OH outflow. IPC, diazoxide, and SOD (150 IU/ml) attenuated O2- outflow, increased *OH outflow and protected endothelium. There were no differences in Cu/Zn-SOD, Mn-SOD and catalase activities between sham-perfused and IR hearts and only catalase activity was increased in the IPC hearts. We speculate that: (i) IPC and diazoxide endothelial protection involves activation of some SOD-like anti-O2- mechanism resulting in attenuation of O2- burst and increase in *OH burst, (ii) improved SOD activity might have not been detected because it was confined to a small, although functionally important, enzyme fraction, like that bound to the endothelial glycocalyx.     

Increased microsomal interaction with iron and oxygen radical generation after chronic acetone treatment

In vivo administration of acetone influences a variety of reactions catalyzed by rat liver microsomes. The effect of chronic treatment with acetone (1% acetone in the water for 10-12 days) on interaction with iron and subsequent oxygen radical generation by liver microsomes was evaluated. Microsomes from the acetone-treated rats displayed elevated rates of H2O2 generation, an increase in iron-dependent lipid peroxidation, and enhanced chemiluminescence upon the addition of t-butylhydroperoxide. The ferric EDTA-catalyzed production of formaldehyde from DMSO or of ethylene from 2-keto-4-thiomethylbutyrate was increased 2-fold after acetone treatment. This increase in hydroxyl radical generation was accompanied by a corresponding increase in NADPH utilization and was sensitive to inhibition by catalase and a competitive scavenger, ethanol, but not to superoxide dismutase. In vitro addition of acetone to microsomes had no effect on oxygen radical generation. Associated with the chronic acetone treatment was a 2-fold increase in the microsomal content of cytochrome P-450 and in the activity of NADPH-cytochrome-P-450 reductase. It appears that increased oxygen radical generation by microsomes after chronic acetone treatment reflects the increase in the major enzyme components which comprise the mixed-function oxidase system.     

Hydroxyl radical production by free and DNA-bound aminoquinone antibiotics and its role in DNA degradation. Electron spin resonance detection of hydroxyl radicals by spin trapping.

The reduced antitumor antibiotic mitomycin C in aqueous solution exposed to air gives a 36-line electron spin resonance spectrum of the semiquinone identified by computer simulation. Incubation of this radical with the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) gives the PBN.OH nitroxide radical identified by independent generation. This nitroxide radical is also formed from similar treatment of a DNA to which mitomycin C is covalently attached. Incubation of the semiquinone from mitomycin C, mitomycin B, or streptonigrin (SN) with catalase or with superoxide dismutase inhibits the generation of OH, implying the intermediacy of H2O2 and O2 in its formation. The formation of the spin-trapped nitroxide radical is similarly inhibited by EDTA, suggesting the intermediacy of trace metal ions in the generation of hydroxyl radicals from SN. The results are consistent with the generation by the aminoquinone antibiotics in vivo of OH. already implicated in the degradation of DNA.     

Oxygen radical generation by microsomes: role of iron and implications for alcohol metabolism and toxicity

Experiments were carried out to evaluate whether the molecular mechanism for ethanol oxidation by microsomes, a minor pathway of alcohol metabolism, involved generation of hydroxyl radical (.OH). Microsomes oxidized chemical .OH scavengers (KMB, DMSO, t-butyl alcohol, benzoate) by a reaction sensitive to catalase, but not SOD. Iron was required for microsomal .OH generation in view of the potent inhibition by desferrioxamine; however, the chelated form of iron was important. Microsomal .OH production was effectively stimulated by ferric EDTA or ferric DTPA, but poorly increased with ferric ATP, ferric citrate, or ferric ammonium sulfate. By contrast, the latter ferric complexes effectively increased microsomal chemiluminescence and lipid peroxidation, whereas ferric EDTA and ferric DTPA were inhibitory. Under conditions that minimize .OH production (absence of EDTA, iron) ethanol was oxidized by a cytochrome P-450-dependent process independent of reactive oxygen intermediates. Under conditions that promote microsomal .OH production, the oxidation of ethanol by .OH becomes more significant in contributing to the overall oxidation of ethanol by microsomes. Experiments with inhibitors and reconstituted systems containing P-450 and NADPH-P-450 reductase indicated that the reductase is the critical enzyme locus for interacting with iron and catalyzing production of reactive oxygen species. Microsomes isolated from rats chronically fed ethanol catalyzed oxidation of .OH scavengers, light emission, and inactivation of added metabolic enzymes at elevated rates, and displayed an increase in ethanol oxidation by a .OH-dependent and a P-450-dependent pathway. It is possible that enhanced generation of reactive oxygen intermediates by microsomes may contribute to the hepatotoxic effects of ethanol.     

Hydroxyl Radical Generation in the Nadh/Microsomal Reduction of vanadate

ESR spin trapping measurements demonstrate generation of hydroxyl (.OH) radical from reduction of vanadate by rat liver microsomes/NADH without exogenous H₂O₂. Catalase decreases the .OH signal while increasing a vanadium(4+) signal. Addition of superoxide dismutase (SOD) or measurements under an argon atmosphere show decreased .OH radical production. The results suggest that during the one-electron vanadate reduction process by microsomes/NADH, molecular oxygen is reduced to H₂O₂, which then reacts with vanadium (4+) to generate .OH radical via a Fenton-like mechanism.     

DNA strand scission by enzymatically reduced mitomycin C: evidence for participation of the hydroxyl radical in the DNA damage

Phage DNA, as well as plasmid and mammalian DNA's, were exposed to a superoxide and hydroxyl radical-generating system containing NADPH-cytochrome P-450 reductase and mitomycin C, both with and without added Fe3+-ADP, in phosphate buffer at pH 7.5. The generation of superoxide (O2-.) and hydroxyl (.OH) radicals in the system was demonstrated by using ESR spectrometry with N-tert -butyl-alpha-phenylnitrone (PBN) as a spin trapping agent. Only the lambda DNA isolated after exposure to the O2-./.OH-generating system containing many lower molecular weight DNA fragments indicating DNA strand breaks. This breakage was completely inhibited by a .OH radical scavenger (sodium benzoate) and by catalase, but only slightly by superoxide dismutase. Thyroid and plasmid DNA's were both cleaved when exposed to the O2-./.OH-generating systems. It is suggested that the mechanism of DNA scission by mitomycin C described here closely resembles that induced by the anthracycline drugs.     

Nadph-Cytochrome-P450 Reductase Promotes Hydroxyl Radical Production by the Iron Complex of ADR-925, the Hydrolysis Product of ICRF-187 (Dexrazoxane)

ICRF-187 (dexrazoxane) is currently in clinical trials as a cardioprotective agent for the prevention of doxorubicin-induced cardiotoxicity. ICRF-187 likely acts through its strongly metal ion-binding rings-opened hydrolysis product ADR-925 by removing iron from its complex with doxorubicin or by chelating free iron. The ability of NADPH-cytochrome-P450 reductase to promote hydroxyl radical formation by iron complexes of ADR-925 and EDTA was compared by EPR spin trapping. The iron-EDTA complex produced hydroxyl radicals at six times the rate that the iron-ADR-925 complex did. The aerobic oxidation of ferrous complexes of ADR-925, its tetraacid analog, EDTA and DTPA was followed spectropho-tometrically. The iron(II)-ADR-925 complex was aerobically oxidized 700 times slower than was the EDTA complex. It is concluded that even though ADR-925 does not completely eliminate iron-based hydroxyl radical production, it likely protects by preventing site-specific hydroxyl radical damage by the iron-doxorubicin complex.     

Calmodulin participation in oxygen radical-induced cardiac sarcoplasmic reticulum calcium uptake reduction

The effect of scavengers of oxygen radicals on canine cardiac sarcoplasmic reticulum (SR) Ca2+ uptake velocity was investigated at pH 6.4, the intracellular pH of the ischemic myocardium. With the generation of oxygen radicals from a xanthine-xanthine oxidase reaction, there was a significant depression of SR Ca2+ uptake velocity. Xanthine alone or xanthine plus denatured xanthine oxidase had no effect on this system. Superoxide dismutase (SOD), a scavenger of .O2-, or denatured SOD had no effect on the depression of Ca2+ uptake velocity induced by the xanthine-xanthine oxidase reaction. However, catalase, which can impair hydroxyl radical (.OH) formation by destroying the precursor H2O2, significantly inhibited the effect of the xanthine-xanthine oxidase reaction. This effect of catalase was enhanced by SOD, but not by denatured SOD. Dimethyl sulfoxide (Me2SO), a known .OH scavenger, completely inhibited the effect of the xanthine-xanthine oxidase reaction. The observed effect of oxygen radicals and radical scavengers was not seen in the calmodulin-depleted SR vesicles. Addition of exogenous calmodulin, however, reproduced the effect of oxygen radicals and the scavengers. The effect of oxygen radicals was enhanced by the calmodulin antagonists (compounds 48/80 and W-7) at concentrations which showed no effect alone on Ca2+ uptake velocity. Taken together, these findings strongly suggest that .OH, but not .O2-, is involved in a mechanism that may cause SR dysfunction, and that the effect of oxygen radicals is calmodulin dependent.     

Resistance of paraquat and adriamycin in human breast tumor cells: role of free radical formation

Generation and enhanced detoxification of toxic free radicals by glutathione peroxidase and glutathione transferase in human breast tumor cells have been suggested to play an important role in toxicity and in resistance to adriamycin. We have examined the biochemical basis of paraquat-induced free radical formation and the mechanism of resistance to this agent in human breast tumor cell lines. We have also compared the similarities and differences between adriamycin and paraquat in their mode of free radical formation and tumor cell kill. Anaerobic incubation of paraquat resulted in the formation of the paraquat cation radical in both the sensitive and resistant cells which increased with time and was enhanced by NADPH addition. Our studies show that while both adriamycin and paraquat form hydroxyl radicals (.OH) in these cell lines, adriamycin was 2-3 fold better at reducing oxygen. The formation of .OH was inhibited by exogenously added superoxide dismutase and catalase, indicating the involvement of both superoxide anion radical and hydrogen peroxide. In the adriamycin-resistant cell line, less .OH was formed by each of these drugs. While the .OH appeared to be formed outside by both adriamycin and paraquat in the drug-sensitive cells, experiments using chromium oxalate as a spin-broadening agent suggest that the drug-induced .OH formation in the resistant cells is an intracellular event. The adriamycin-resistant cell line was also cross-resistant to paraquat, suggesting a common mechanism of toxicity for both drugs. However, adriamycin was significantly more toxic (4000-times) to the sensitive cells suggesting that either other mechanisms or site-specific free radical formation are also important in biochemical mechanisms of adriamycin toxicity.     

The role of hydroxyl radicals in tetrachlorohydroquinone induced DNA strand break formation in PM2 DNA and human fibroblasts

Tetrachlorohydroquinone (TCHQ), which has previously been identified as a metabolite of pentachlorophenol, induces DNA strand breaks in isolated DNA and in human fibroblasts. Strand break formation in PM2 DNA is prevented by the addition of catalase and the hydroxyl radical scavengers DMSO, ethanol and mannitol, whereas addition of SOD reduced SSB only slightly. Oxygen radicals are formed by the autoxidation of TCHQ to the tetrachlorosemiquinone radical. Desferrioxamine (0.2 mM) completely abolished strand break formation, whereas the metal chelator DETAPAC (1 mM) reduced SSB by only 8.5%. The formation of the semiquinone radical at physiological conditions is shown by ESR spectroscopy. Exposure of human fibroblasts to TCHQ also leads to DNA single strand breaks measured by the alkaline elution assay. These were reduced by addition of 5% DMSO. This indicates that at least part of the strand break formation in human cells is also due to the action of hydroxyl radicals.     

Differential contractile actions of reactive oxygen species on rat aorta: selective activation of ATP receptor by H2O2

This study aims to examine the effects of different reactive oxygen species (ROS) on the resting tension of endothelium-denuded rat aortic rings. In these preparations, H2O2 (30 microM) induced a fast and transient contraction, which could be abolished by pretreatment of catalase (800 U/ml), but not affected by superoxide anion scavenger, superoxide dismutase (SOD; 150 U/ml) or the hydroxyl free radical scavenger, DMSO/mannitol (each 3 mM). In contrast, pyrogallol, a putative superoxide anion donor, induced a biphasic contraction, which could be abolished by SOD, but not by catalase or DMSO/mannitol. Unlike H2O2 and pyrogallol, Vitamin C(VitC)/Fe2+ (each 100 microM), a commonly used hydroxyl radical-generating system, triggered a tonic contraction which could be prevented by DMSO/mannitol, but not by SOD or catalase. Interestingly, H2O2-induced contraction could be concentration-dependently (10-100 microM) inhibited by suramin and reactive blue-2 (RB-2), two widely used ATP receptor antagonists. On the other hand, suramin or RB-2, at concentration up to 100 microM, affected neither pyrogallol nor VitC/Fe2+-induced contraction. In conclusion, we showed for the first time that different ROS could contract rat aorta with different mechanisms of action, and H2O2 elicits a transient contraction probably as a result of the ATP receptor activation.     

Downregulation of NADPH oxidase, antioxidant enzymes, and inflammatory markers in the heart of streptozotocin-induced diabetic rats by N-acetyl-L-cysteine

We investigated the effect of N-acetyl-l-cysteine (NAC) on the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, antioxidant enzymes, and inflammatory markers in diabetic rat hearts. Metabolic parameters, free 15-F(2t)-isoprostane level, protein expression of NADPH oxidase, superoxide dismutase (SOD), heme oxygenase (HO-1), interleukin-6 (IL-6), and cyclooxygenase-2 (COX-2) were analyzed in control and streptozotocin-induced diabetic rats treated with or without NAC in drinking water for 8 wk. The cardiac protein expression of p67(phox) and p22(phox) was increased in diabetic rats, accompanied by increased NADPH-dependent superoxide production. As a compensatory response to the increased NADPH oxidase, the protein expression of Cu-Zn-SOD and HO-1 and the total SOD activity were also increased in diabetic rat hearts. Consequently, cardiac free 15-F(2t)-isoprostane, an index of oxidative stress, was increased in diabetic rats, indicating that the production of reactive oxygen species becomes excessive in diabetic rat hearts. Cardiac inflammatory markers IL-6 and COX-2 were also increased in diabetic rats. NAC treatment prevented the increased expression of p22(phox) and translocation of p67(phox) to the membrane in diabetic rat hearts. Subsequently, the levels of cardiac free 15-F(2t)-isoprostane, HO-1, Cu-Zn-SOD, total SOD, IL-6, and COX-2 in diabetic rats were decreased by NAC. Consequently, cardiac hypertrophy was attenuated in diabetic rats treated with NAC. The protective effects of NAC on diabetic rat hearts may be attributable to its protection of hearts against oxidative damage induced by the increased NADPH oxidase and to its reduction in cardiac inflammatory mediators IL-6 and COX-2.     

The iron chelator Dp44mT does not protect myocytes against doxorubicin

The iron chelating agent Dp44mT (di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone) and the clinically approved cardioprotective agent dexrazoxane (ICRF-187) were compared for their ability to protect neonatal rat cardiac myocytes from doxorubicin-induced damage. Doxorubicin is thought to induce oxidative stress on the heart muscle through iron-mediated oxygen radical damage. While dexrazoxane was able to protect myocytes from doxorubicin-induced lactate dehydrogenase release, in contrast Dp44mT synergistically increased doxorubicin-induced damage. This occurred in spite of the fact that Dp44mT quickly and efficiently removed iron(III) from its complex with doxorubicin and that Dp44mT also rapidly entered myocytes and displaced iron from a fluorescence-quenched trapped intracellular iron-calcein complex. Electron paramagnetic resonance spin trapping was used to show that iron complexes of Dp44mT were not able to generate hydroxyl radicals, suggesting that its cytotoxicity was not due to reactive oxygen species formation. In conclusion Dp44mT is unlikely to be useful as an anthracycline cardioprotective agent.     

Microsomal interactions between iron, paraquat, and menadione: effect on hydroxyl radical production and alcohol oxidation

The addition of menadione or paraquat to rat liver microsomes resulted in about a threefold increase in the production of hydroxyl radical (.OH) as reflected by the increased oxidation of 2-keto-4-thiomethylbutyric acid (KMBA) to ethylene. This increase was not sensitive to superoxide dismutase but was blocked by catalase. The increase occurred in the absence of added iron and was not affected by the potent iron chelating agent, desferrioxamine, which suggests the possibility that .OH was produced from an interaction between H2O2 and the paraquat or menadione radical. Menadione and paraquat were especially effective in stimulating the oxidation of KMBA in the presence of certain iron chelates such as ferric-ADP, -ATP, or -EDTA, but not ferric-desferrioxamine, -citrate, or -histidine, or unchelated iron. In fact, ferric-ADP or -ATP only stimulated .OH production in the presence of menadione or paraquat. In the presence of ferric-EDTA, the greater than additive increase of .OH production was sensitive to catalase, but not to superoxide dismutase, suggesting the possibility of reduction of ferric-EDTA by paraquat or menadione radical. The interactions with ferric adenine nucleotides may increase the catalytic effectiveness of menadione or paraquat in producing potent oxidants such as the hydroxyl radical, and thus play a role in the toxicity associated with these agents. Paraquat and menadione had little effect on the overall oxidation of ethanol by microsomes. Microsomal drug metabolism was decreased by menadione or paraquat. As a consequence, the effect of these agents on the microsomal oxidation of ethanol was complex since it appeared that paraquat and menadione stimulated the oxidation of ethanol by a .OH-dependent mechanism, but inhibited the oxidation of ethanol by a cytochrome P-450-dependent oxidation pathway. Experiments with carbon monoxide, ferric-EDTA, and 2-butanol plus catalase tended to verify that microsomal oxidation of alcohols was increased by a .OH-dependent pathway when menadione or paraquat were added to microsomes.     

The in vitro metabolites of 2,4,6-trichlorophenol and their DNA strand breaking properties

The carcinogenic compound 2,4,6-trichlorophenol (2,4,6-TCP) was incubated with rat liver S-9 fraction. Three metabolites were identified: 2,6-dichloro-1,4-hydroquinone (DHQ), and two isomers of hydroxypentachlorodiphenyl ether (OH-Cl5-DPE). The latter are probably products of microsomal .OH radical attack on the trichlorophenol molecule forming phenoxy free radicals. These would undergo dimerizations with other molecules present in solution. The 2,6-dichloro-1,4-semiquinone free radical was identified by ESR spectroscopy. It is formed at physiological conditions in phosphate buffer at pH 7.2 and 7.8, with a more intensive signal at the more alkaline pH. The formation is probably due to the autoxidation of the corresponding hydroquinone. Incubation of a mixture of metabolites with PM2 DNA at pH 7.2 resulted in single strand breaks. Addition of catalase and dimethylsulfoxide (DMSO) inhibited the DNA strand scission. It was concluded that reactive oxygen species (ROS), produced during the formation of the semiquinone radical, were responsible for the observed DNA damage. The significance of the ROS and the semiquinone free radical is discussed in view of the reported tumorgenicity of 2,4,6-TCP in rats and mice.     

Relative importance of cellular uptake and reactive oxygen species for the toxicity of alloxan and dialuric acid to insulin-producing cells

The diabetogenic agent alloxan is selectively accumulated in insulin-producing cells through uptake via the GLUT2 glucose transporter in the plasma membrane. In the presence of intracellular thiols, especially glutathione, alloxan generates "reactive oxygen species" (ROS) in a cyclic reaction between this substance and its reduction product, dialuric acid. The cytotoxic action of alloxan is initiated by free radicals formed in this redox reaction. Autoxidation of dialuric acid generates superoxide radicals (O(2)(*-)) and hydrogen peroxide (H(2)O(2)), and finally hydroxyl radicals ((*)OH). Thus, while superoxide dismutase (SOD) only reduced the toxicity, catalase, in particular in the presence of SOD, provided complete protection of insulin-producing cells against the cytotoxic action of alloxan and dialuric acid due to H(2)O(2) destruction and the prevention of hydroxyl radical ((*)OH) formation, indicating that it is the hydroxyl radical ((*)OH) which is the ROS ultimately responsible for cell death. After selective accumulation in pancreatic beta cells, which are weakly protected against oxidative stress, the cytotoxic glucose analogue alloxan destroys these insulin-producing cells and causes a state of insulin-dependent diabetes mellitus through ROS-mediated toxicity in rodents and in other animal species, which express this glucose transporter isoform in their beta cells.     

Transformed target cell-derived superoxide anions drive apoptosis induction by myeloperoxidase

Myeloperoxidase induces apoptosis in src- or raxs-transformed fibroblasts, but not in parental nontransformed fibroblasts. This selectivity seems to be based on superoxide anion production by transformed cells, a recently described characteristic feature of transformed cells. Myeloperoxidase-mediated apoptosis induction is inhibited by SOD, catalase, 4-aminobenzoyl hydrazide, taurine and DMSO. This pattern of inhibition allows us to conclude that transformed cell derived superoxide anions dismutate to hydrogen peroxide, which fosters HOCl formation by myeloperoxidase. Hydrogen peroxide formation thereby is the rate-limiting step and depends on the cell density. In a second step, HOCl interacts with superoxide anions to yield the highly reactive apoptosis inducing hydroxyl radical. This conclusion was verified through selective apoptosis induction in transformed cells by direct addition of HOCl, which was also inhibited by SOD and DMSO. Our findings demonstrate a specific interplay between target cell derived superoxide anions and MPO during selective apoptosis induction.     

Activation of rat liver microsomal glutathione S-transferase by gallic acid

The effect of phenolic antioxidants on the rat liver microsomal glutathione S-transferase (MGST1) was investigated in vitro. When microsomes were incubated with various polyphenolic antioxidants, gallic acid (3,4,5-trihydroxybenzoic acid) markedly increased MGST1 activity and the increase was prevented in the presence of superoxide dismutase (SOD) or catalase. The MGST1 activity increased by gallic acid was decreased by further incubation with sodium arsenite, a sulfenic acid reducing agent, but was not with dithiothreitol, a disulfide bond reducing agent. The incubation of microsomes with gallic acid in the presence of the NADPH generating system which generates reactive oxygen species (ROS) through cytochrome P-450 system increased the MGST1activity in spite of scavenging the ROS and the increase was also depressed by SOD/catalase. The increase of MGST1 activity by gallic acid was prevented by co-incubation with a stable radical, 1,1-diphenyl-2-picrylhydrazyl or ferric chloride. These results suggest that the gallic acid acts as a pro-oxidant and activates MGST1 through oxidative modification of the enzyme.     

Generation of free radicals induced by nifurtimox in mammalian tissues

Nifurtimox is reduced by rat liver microsomes to a nitro anion-free radical as indicated by ESR spectroscopy. This subcellular fraction gives a steady state radical concentration which is proportional to the square root of the protein concentration, suggesting that the nifurtimox anion radical is a necessary intermediate in the reduction and that the radical decays through a nonenzymatic second order process. The steady state concentration of the anion radical in the microsomal system is not decreased by superoxide dismutase or catalase, thus indicating that neither the superoxide anion nor hydrogen peroxide is an intermediary in the generation of the anion radical. The steady state concentration of the anion radical in the microsomal system is also not altered in the presence of metyrapone or CO and is decreased in the presence of NADP+ and p-chloromercuribenzoate. This observation suggests that the formation of nifurtimox anion radical is mediated through NADPH-cytochrome P-450 (c) reductase and not by the cytochrome P-450 system. In accordance with this interpretation, a model system consisting of NADPH and FMN-reduced nifurtimox to the nitro anion-free radical. Nifurtimox anion radical generation is significantly stimulated by rat brain and testes homogenates. The enhanced free radical formation may be the basic cause of nifurtimox toxicity in mammals.     

Ciprofloxacin-induces free radical production in rat cerebral microsomes

In the presence of ciprofloxacin (CPFX), free radical adduct formation was demonstrated in rat cerebral microsomes using a spin trap α-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone by electron spin resonance spectroscopy. Active microsomes, dihydronicotinamide-adenine dinucleotide phosphate, and ciprofloxacin were necessary for the formation of a spin trap/radical adduct. Adduct formation increased dose-dependently at 0.5–1?mM CPFX concentration for 180?min, and 0.3–1 mM concentration level for 240?min. The addition of SKF 525A, ZnCl₂ or desferrioxamine to the incubation system caused complete inhibition of the radical formation. However, pretreatment of microsomal system with superoxide dismutase (SOD) did not induce any protective effect. Induction of lipid peroxidation, and depletion of thiol levels by CPFX were also shown in the system. These results strongly suggested that CPFX produces free radical(s) in the cerebral microsomes of rats.