An ESR contrast agent is transported to rat liver through organic anion transporter

Carboxy PROXYL is a useful extracellular paramagnetic contrast reagent in electron spin resonance (ESR) and magnetic resonance imaging (MRI). Active transfer of the probe was investigated using an in situ liver model in rats. Carboxy PROXYL, a nitroxyl spin probe, was perfused into in situ liver perfusion system from Wistar rats. Concentration of nitroxyl form of the spin probe in effluent increased gradually after introducing perfusate with the spin probe and reached a plateau. The disappearance of Carboxy PROXYL from the perfusate was 40%, which could not be explained with its partition coefficient. Administration of non-selective inhibitors of organic anion transporters, p-aminohippuric acid and penicillin G, inhibited competitively and in a dose dependent manner the transfer of Carboxy PROXYL into rat liver in situ, resulting in increases of Carboxy PROXYL in the effluent. The results demonstrate that there is an active transfer system of an ESR contrast reagent into in situ rat liver through organic anion transporters.     

The Effect of Xanthine/Xanthine Oxidase Generated Reactive Oxygen Species on Synaptic Transmission

The effect of reactive oxygen species generated by the interaction of xanthine and xanthine oxidase on synaptic transmission was examined at the squid giant synapse and the lobster neuromuscular junction. Exposure of these synaptic regions to xanthine/xanthine oxidase produced a significant depression in evoked release, with no change in either resting membrane properties or in the action potential. Addition of catalase to the xanthine/xanthine oxidase-containing media partially blocked the synaptic depression, indicating that H₂ O ₂ contributes to the synaptic changes induced by exposure to xanthine/xanthine oxidase. H₂ O ₂ applied directly to the perfusing media also produced a decrease in synaptic efficacy. The results demonstrate that reactive oxygen species, in general, depress evoked synaptic transmission.     

Cell Kinetic Evidence Suggests Elevated Oxidative Stress in Cultured Cells of Bloom's Syndrome

Bromodeoxyuridine/Hoechst flow cytometry was used to analyse disturbed cell proliferation of fibroblasts and lymphoblastoid cells from Bloom's syndrome (BS). Fibroblasts show poor activation, arrest in the G2 phase of the cell cycle along with a prolongation of the Gl phase. This pattern of perturbed cells proliferation is akin to that elicited in normal fibroblasts by 4-hydroxy-nonenal, a breakdown product of lipid peroxides. Treatment with vitamin E improved growth of BS fibroblasts more strongly than growth of normal fibroblasts. Lymphoblastoid cells from BS, to the contrary, experience only a minor arrest in the G2 phase after one round of bromodeoxyuridine incorporation, but are strongly inhibited during and after the second S phase. Thus, their cell cycle arrest is dependent upon BrdU incorporation, as has been found previously in normal cells exposed to elevated concentrations of oxygen or paraquat, a superoxide generating compound. These results suggest that BS cells may suffer from an elevated, endogenous generation of oxygen free radicals.     

Generation of superoxide and singlet oxygen from α-tocopherolquinone and analogues

Three potential routes to generation of reactive oxygen species (ROS) from α-tocopherolquinone (α-TQ) have been identified. The quinone of the water-soluble vitamin E analogue Trolox C (Trol-Q) is reduced by hydrated electron and isopropanol α-hydroxyalkyl radical, and the resulting semiquinone reacts with molecular oxygen to form superoxide with a second order rate constant of 1.3 × 10⁸ dm³/mol/s, illustrating the potential for redox cycling. Illumination (UV-A, 355 nm) of the quinone of 2,2,5,7,8-pentamethyl-6-hydroxychromanol (PMHC-Q) leads to a reactive short-lived (ca. 10^{? 6} s) triplet state, able to oxidise tryptophan with a second order rate constant greater than 10⁹ dm³/mol/s. The triplet states of these quinones sensitize singlet oxygen formation with quantum yields of about 0.8. Such potentially damaging reactions of α-TQ may in part account for the recent findings that high levels of dietary vitamin E supplementation lack any beneficial effect and may lead to slightly enhanced levels of overall mortality.     

In vitro Damage to Rat Lens by Lumazine and Xanthine Oxidase: Prevention by Superoxide Dismutase

Intact rat lenses incubated with lumazine and xanthine oxidase are physiologically damaged as evidenced by a decrease in the net accumulation of rubidium ions against a concentration gradient. Superoxide dismutase protected the tissue against this damage. These experiments, therefore, demonstrate the susceptibility of the lens tissue to O₂^?_? injury under ambient and nonphotochemical conditions, suggesting a possible implication of this radical in the tissue in vivo and eventual cataract formation. The lumazine/xanthine oxidase system which is known to cause oxygen reduction predominantly by the monovalent route, producing superoxide, appears quite suitable to evaluate the toxicity of O₂^?_? to the tissues in vitro.     

Hydroxylation of Phenol to Hydroquinone Catalyzed by A Human Myeloperoxidase-Superoxide Complex: Possible Implications In Benzene-Induced Myelotoxicity

Benzene, a known human rnyelotoxin and leukemogen is metabolized by liver cytochrome P-450 mono-oxygenase to phenol. Further hydroxylation of phenol by cytochrome P-450 monooxygenase results in the formation of mainly hydroquinone, which accumulates in the bone marrow. Bone marrow contains high levels of myeloperoxidase. Here we report that phenol hydroxylation to hydroquinone is also catalyzed by human myeloperoxidase in the presence of a superoxide anion radical generating system, hypoxanthine and xanthine oxidase. No hydroquinone formation was detected in the absence of myeloperoxidase. At low concentrations superoxide disniutase stimulated, but at high concentrations inhibited, the conversion of phenol to hydroquinone. The inhibitory effect at high superoxide dismutase concentrations indicates that the active hydroxylating species of myeloperoxidase is not derived from its interaction with hydrogen peroxide. Furthermore, catalase a hydrogen peroxide scavenger, was found to have no significant effect on hydroxylation of phenol to hydroquinone, supporting the lack of hydrogen peroxide involvement. Mannitol (a hydroxyl radical scavenger) was found to have no inhibitory effect, but histidine (a singlet oxygen scavenger) inhibited hydroquinone formation. Based on these results we postulate that a myeloperoxidase-superoxide complex spontaneously rearranges to generate singlet oxygen and that this singlet oxygen is responsible for phenol hydroxylation to hydroquinone. These results also suggest that myeloperoxidase dependent hydroquinone formation could play a role in the production and accumulation of hydroquinone in bone marrow, the target organ of benzene-induced myelotoxicity.     

Thioctic Acid and Dihydrolipoic Acid are Novel Antioxidants Which Interact With Reactive Oxygen Species

Thioctic acid (TA) and its reduced form dihydrolipoic acid (DHLA) have recently gained somc recognition as useful biological antioxidants. In particular, the ability of DHLA to inhibit lipid peroxidation has been reported. In the present study, the effects of TA and DHLA on reactive oxygen species (ROS) generated in the aqueous phase have been investigated. Xanthine plus xanthine oxidase-generated superoxide radicals (O₂), detected by electron spin resonance spectroscopy (ESR) using DMPO as a spin trap. were eliminated by DHLA but not by TA. The sulhydryl content of DHLA, measured using Ellman's reagent decreased subsequent to the incubation with xanthine plus xanthine oxidase confirming the interaction between DHLA and O₂^-. An increase of hydrogen peroxide concentration accompanied the reaction between DHLA and O₂x, suggesting the reduction of O₂^- by DHLA. Competition of O₂^- with epinephrine allowed us to estimate a second order kinetic constant of the reaction between O₂^- and DHLA, which was found to be a 3.3 × 10⁵ M^-1 s^-1. On the other hand, the DMPO signal of hydroxyl radicals (HO ·) generated by Fenton's reagent were eliminated by both TA and DHLA. Inhibition of the Fenton reaction by TA was confirmed by a chemiluminescence measurement using luminol as a probe for HO ·. There was no electron transfer from Fe²⁺ to TA or from DHLA to Fe^{3 +} detected by measuring the Fe²⁺ -phenanthroline complex. DHLA did not potentiate the DMPO signal of HO · indicating no prooxidant activity of DHLA. These results suggest that both TA and DHLA possess antioxidant properties. In particular. DHLA is very effective as shown by its dual capability by eliminating both O₂^-; and HO ·.     

Evidence for Superoxide Dismutase and Catalase in Mollicutes and Release of Reactive Oxygen Species

The presence of superoxide dismutase was demonstrated in 21 strains of mollicutes, including achuloplas-mas, mycoplasmas and ureaplasmas. Additionally, catalase activities were demonstrated in nearly 50% of the cell lysates. whereas no peroxide activities were detectable. The production of O₂-and H₂O₂ with glucose as substrate was demonstrated for 8 strains of 10 strains tested. Anaerobic mycoplasmas showed the highest amount of radical production, whereas superoxide dismutase and catalase activities were in the range of activities estimated for aerobic mollicutes. Some pathogenic strains additionally released compounds into the culture medium, which stimulated O₂-production by PMNs.     

Evaluation of the mechanisms involved in leucine-induced oxidative damage in cerebral cortex of young rats

Maple syrup urine disease (MSUD) is a metabolic disorder caused by the deficiency of the activity of the mitochondrial enzyme complex branched-chain l-2-keto acid dehydrogenase. The metabolic block results in tissue and body fluid accumulation of the branched-chain amino acids leucine (Leu), isoleucine and valine, as well as of their respective α-keto acids. Neurological sequelae are usually present in MSUD, but the pathophysiologic mechanisms of neurotoxicity are still poorly known. It was previously demonstrated that Leu elicits oxidative stress in rat brain. In the present study we investigated the possible mechanisms involved in Leu-induced oxidative damage. We observed a significant attenuation of Leu-elicited increase of thiobarbituric acid-reactive substances (TBA-RS) measurement when cortical homogenates were incubated in the presence of the free radical scavengers ascorbic acid plus trolox, dithiothreitol, glutathione, and superoxide dismutase, suggesting a probable involvement of superoxide and hydroxyl radicals in this effect. In contrast, the use of N^ω-nitro-l-arginine methyl ester or catalase (CAT) did not affect TBA-RS values. We also demonstrated an inhibitory effect of Leu on the activities of the antioxidant enzymes CAT and gluthathione peroxidase, as well as a significant reduction in the membrane-protein thiol content from mitochondrial enriched preparations. Furthermore, dichlorofluorescein levels were increased although not significantly by Leu. Taken together, our present data indicate that an unbalance between free radical formation and inhibition of critical enzyme activities may explain the mechanisms involved in the Leu-induced oxidative damage.