Insulin secretion defects of human type 2 diabetic islets are corrected in vitro by a new reactive oxygen species scavenger.

Oxidative stress is a putative mechanism leading to beta-cell damage in type 2 diabetes. We studied isolated human pancreatic islets from type 2 diabetic and non-diabetic subjects, matched for age and body mass index. Evidence of increased oxidative stress in diabetic islets was demonstrated by measuring nitrotyrosine concentration and by electron paramagnetic resonance. This was accompanied by reduced glucose-stimulated insulin secretion, as compared to non-diabetic islets (Stimulation Index, SI: 0.9 +/- 0.2 vs. 2.0 +/- 0.4, P<0.01), and by altered expression of insulin (approximately -60%), catalase (approximately +90%) and glutathione peroxidase (approximately +140%). When type 2 diabetic islets were pre-exposed for 24 h to the new antioxidant bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate di-hydrochloride, nitrotyrosine levels, glucose-stimulated insulin secretion (SI: 1.6+/-0.5) and gene expressions improved/normalized. These results support the concept that oxidative stress may play a role in type 2 diabetes beta-cell dysfunction; furthermore, it is proposed that therapy with antioxidants could be an interesting adjunctive pharmacological approach to the treatment of type 2 diabetes.

Inhibition of autoxidation of egg yolk phosphatidylcholine in homogenous solution and in liposomes by oxidized ubiquinone.

The aim of this study was to obtain a quantification of the antioxidant activity of ubiquinone. To this purpose the oxidation of egg yolk phosphatidylcholine both in solvent and in liposomes initiated by an azocompound has been studied either in the absence or in the presence of ubiquinone-3, using alpha-tocopherol as a reference antioxidant. The two experimental systems gave similar results. In the presence of ubiquinone-3 the oxidation rate was reduced with respect to control experiments but was faster than that in the presence of alpha-tocopherol. The amount of ubiquinone required to decrease the autoxidation rate was so high as to prevent detection of the induction period. The stoichiometric factor was greater than 2 and the rate constant of inhibition was two orders of magnitude lower than that of alpha-tocopherol. It is concluded that high concentrations of ubiquinone are required to exhibit significant antioxidant activity. A possible mechanism compatible with the stoichiometric factor larger than 2 for the inhibiting effect of ubiquinone is also suggested.

Measurement of oxidative stress by EPR radical-probe technique.

An EPR method for the measurement of the oxidative stress status in biological systems is described. The method is based on the X-band EPR detection of a persistent nitroxide generated under physiological or pseudo-physiological conditions by oxidation of a highly lipophylic hydroxylamine probe. The probe employed is bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)-decandioate which is administrated as hydrochloride salt. This probe is able to give a fast reaction with the majority of radical species involved in the oxidative stress. Furthermore, it crosses cell membranes and distributes in a biological environment without the need to alter or destroy compartmentation. The method is therefore suitable for quantitative measurements of ROS and can be applied to human tissues in real clinical settings. It has been successfully employed in systems of growing complexity and interest, ranging from subcellular fractions to whole animals and human liver.

Medium effects on the antioxidant activity of dipyridamole.

Very strong medium effects have been observed when testing the antioxidant activity of dipyridamole (DP) in different media such as benzene, tert-butanol, methanol solutions and egg yolk lecithin unilamellar and multilamellar vesicles. Actually, dipyridamole behaves as a very poor antioxidant in benzene while its ability to inhibit the lipid peroxidation reaction increases with increasing solvent polarity, being the highest in lipid vesicles. This behavior can not be rationalized on the basis of the classical chain breaking mechanism which operates in the case of phenolic and amine antioxidants and involving the transfer of a hydrogen atom to peroxyl radicals. An explanation is instead given in terms of an electron transfer reaction which leads to the oxidation of DP by the chain carrying peroxyl radical to give the dipyridamole cation radical, DP+*, and the peroxyl anion LOO-, and whose rate constant is expected to increase in strongly polar media. EPR and electrochemical data supporting this interpretation have been collected.

Measurement of oxidative stress in human liver by EPR spin-probe technique.

A method for the measurement of reactive oxygen species (ROS) in human hepatic tissue has been developed. The method is based on the EPR detection of the nitroxide radical produced by reaction of the hydroxylamine spin-probe bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate with ROS generated under pseudo-physiologic conditions in fine needle biopsies of healthy (10 controls) and diseased (22 patients) human liver. Measures of malonaldehyde in 9 liver biopsies (3 controls and 6 patients) have also been obtained by high pressure liquid chromatography and values parallel those obtained by the spin-probe technique. The amount of ROS found in healthy human liver (median = 1.8 x 10(-11) mol/mg) was significantly lower than values found in liver affected by hepatitis B (median=5.8 x 10(-10) mol/mg; p < 0.02) or by hepatitis C (median = 2.7 x 10(-9) mol/mg; p < 0.003) as well as compared to some other non-viral liver diseases (NVLD): autoimmune hepatitis, primary biliary cirrhosis, primary schlerosing cholangitis (median = 9.8 x 10(-9) mol/mg; p < 0.005). NVLD also showed significantly higher ROS levels compared to hepatitis B (p < 0.04) and hepatitis C (p < 0.04). The mechanism, potentiality and limitations of our method are discussed.

On the hepatotoxicity of 1,1,2,2-tetrachloroethane.

Intoxication of male and female mice with a single dose (300 or 600 mg/kg) of 1,1,2,2-tetrachloroethane (TTCE) resulted in significant decreases in cytochrome P-450 (to 58-73% of the control) and NADPH-cytochrome (P-450) c-reductase (to 29-35% of the control) in hepatic microsomes. This was accompanied by an alteration of mixed function monooxygenases stemming from the marked reduction (to 20-64% of the control) of several oxidative activities to selected substrates towards different P-450 isozymes (classes IA1, IA2, IIB1, IIE1 and IIIA). As phase II markers, epoxide hydrolase (approximately 35% loss), UDP-glucuronosyl transferase (approximately 42% loss) and to a lesser extent glutathione S-transferase (approximately 17% loss) were all affected. Also, the activity of delta-aminolevulinic (ALA) synthetase was decreased (approximately 57% of the control). On the contrary, heme oxygenase activity was increased (up to 35%) at the maximal dose tested. The decrease of P-450-function may be explained in terms of an alteration in the rate of heme biosynthesis and degradation, provoking a loss of heme content (approximately 33%) as well as of the direct inactivation of both P-450 and reductase. Because of increasing evidence on the involvement of free radical intermediates in the case of toxicity of haloalkanes, electron spin resonance spectroscopy (ESR) spin-trapping in vivo techniques were used to characterize the possible free radical species involved in the observed liver damage. The results obtained with the spin-trap N-benzylidene-2-methylpropylamine N-oxide (phenyl t-butylnitrone, PBN) provide evidence for the formation and trapping of the CHCl2CHCl free radicals. The detection of conjugated diene signals by means of second-derivative spectrophotometry, have enabled us to show that in vivo lipid peroxidation may be one of the main mechanisms responsible for TTCE hepatotoxicity.

The nature of prooxidant activity of vitamin C.

It was recently reported that vitamin C (500 mg/day for 6 weeks) administered as a dietary supplement to healthy humans exhibits a prooxidant, as well as an antioxidant effect in vivo. Here we show that high intakes of vitamin C (500 mg/kg b.w. for 4 days) in the rat are able to markedly induce hepatic cytochrome P4502E1-linked monooxygenases, measured as p-nitrophenol hydroxylase activity and corroborated by means of Western blot analyses. Furthermore, using Electron Paramagnetic Resonance Spectroscopy (EPR) coupled to a spin-trapping technique, we have also found that this induction generates large amounts of the anion radical superoxide (O2-). Therefore we can conclude that the adverse prooxidant outcomes (i.e. oxidative DNA damage) associated to vitamin C supplementation, being associated to a typical reversible boosting effect (i.e. enzymatic induction), may be easily controlled by a discontinuous supply. However, since the induced P4502E1 isoforms by vitamin C are responsible for ethanol metabolism to highly reactive radicals, care should be taken even in moderate drinkers.

The antioxidant activity of ubiquinol-3 in homogeneous solution and in liposomes.

With a view to determining the antioxidant effectiveness of ubiquinol, the autoxidation of egg phosphatidylcholine initiated by an azocompound was studied both in homogeneous solution and in liposomes, either in the presence or in the absence of ubiquinol-3. The results show that ubiquinol behaves as a chain-breaking antioxidant by trapping lipid peroxyl radicals, its inhibition rate constant being about one half of that of alpha-tocopherol in both systems under investigation. In organic solvents the stoichiometric factor was found approx. 2 and in liposomes approx. 0.5, i.e. one fourth of that of alpha-tocopherol. We suggest that the lower value found in model membranes is due to autoxidation of the quinol itself by a radical chain reaction taking place at the polar interface. Ubiquinol-3 exhibits a sparing effect toward alpha-tocopherol, both in liposomes and in tert-butanol. It is suggested, on a thermodynamic basis, that the regeneration of vitamin E from the corresponding radical is more likely to occur by reaction with the ubisemiquinone rather than with the ubiquinol. Although these results, obtained in in vitro systems, can not be directly extrapolated to an in vivo system, they may be useful to clarify the antioxidant role of ubiquinol in biomembranes.

Captan impairs CYP-catalyzed drug metabolism in the mouse.

To investigate whether the fungicide captan impairs CYP-catalyzed drug metabolism in murine liver, kidney and lung, the modulation of the regio- and stereo-selective hydroxylation of testosterone, including 6beta-(CYP3A), 6alpha-(CYP2A1 and CYP2B1) and 16alpha-(CYP2B9) oxidations was studied. Specific substrates as probes for different CYP isoforms such as p-nitrophenol (CYP2E1), pentoxyresorufin (CYP2B1), ethoxyresorufin (CYP1A1), aminopyrine (CYP3A), phenacetin and methoxyresorufin (CYP1A2), and ethoxycoumarin (mixed) were also considered. Daily doses of captan (7.5 or 15 mg/kg b.w., i.p.) were administered to different groups of Swiss Albino CD1 mice of both sexes for 1 or 3 consecutive days. While a single dose of this fungicide did not affect CYP-machinery, repeated treatment significantly impaired the microsomal metabolism; in the liver, for example, a general inactivating effect was observed, with the sole exception of testosterone 2alpha-hydroxylase activity which was induced up to 8.6-fold in males. In vitro studies showed that the mechanism-based inhibition was related to captan metabolites rather than the parental compound. In the kidney, both CYP3A- and CYP1A2-linked monooxygenases were significantly induced (2-fold) by this pesticide. Accelerated phenacetin and methoxyresorufin metabolism (CYP1A2) was also observed in the lung. Data on CYP3A (kidney) and CYP1A2 (kidney and lung) induction were corroborated by Western immunoblotting using rabbit polyclonal anti-CYP3A1/2 and CYP1A1/2 antibodies. By means of electron spin resonance (EPR) spectrometry coupled to a spin-trapping technique, it was found that the recorded induction generates a large amounts of the anion radical superoxide (O*2-) either in kidney or lung microsomes. These findings suggest that alterations in CYP-associated activities by captan exposure may result in impaired (endogenous) metabolism as well as of coadministered drugs with significant implications for their disposition. The adverse outcomes associated to CYP changes (e.g. cotoxicity, comutagenicity and promotion) may also have harmful consequences.

Paramagnetic resonance in detecting carcinogenic risk from cytochrome P450 overexpression.

BACKGROUND: Despite the increasing interest in the role of oxygen radicals on human degenerative disorders including cancer, oxidative stress status is not yet measurable in vivo, largely precluding clinical application. Limited semi-quantitative assays of damage to broad classes of biomolecules such as lipids, proteins, and DNA are currently available. The detection of radicals in humans by a whole-body electron paramagnetic resonance (EPR) technique has not yet been developed, although this possibility has long fascinated free radical investigators. METHODS: While the EPR spin trapping procedure can be used to detect carbon centered or hydroxyl radical in human tissues, the most common spin traps are much less useful for capturing the superoxide anion (O2). To overcome these limitations, we propose a whole-body-harvest approach that utilizes a highly lipophilic spin scavenger that when injected in the animal is capable of trapping the O2 generated in vivo throughout the body with formation of a stable nitroxide measurable by EPR in the urine. A process known to generate the O2 is the induction of certain cytochrome P450 (CYP) isozymes by drugs or environmental pollutants. RESULTS: We report: 1) a correlation between the induction of each CYP gene family and the O2 yield; 2) support to an observation reported previously that the tumor promoting ability of CYP inducers is mainly mediated by the O2; and 3) the description of a method for nitroxide mediated O2 detection in vivo. CONCLUSION: These findings could open the way for using electron spin resonance in diagnostic practice.

Antioxidant mechanism of flavonoids. Solvent effect on rate constant for chain-breaking reaction of quercetin and epicatechin in autoxidation of methyl linoleate.

The rate of oxygen depletion, as measured by electron spin resonance spectroscopy (oximetry using a spin probe), in a homogeneous solution of peroxidating methyl linoleate (initiated by an azo initiator) in the presence or absence of antioxidants was converted to second-order rate constants for the inhibiting reaction of quercetin and epicatechin. In the non-hydrogen-bonding solvent chlorobenzene at 50 degrees C, k(inh) had values of 4.3 x 10(5) M(-)(1) s(-)(1) for quercetin and 4.2 x 10(5) M(-)(1) s(-)(1) for epicatechin, respectively. In the hydrogen-accepting "water-like" solvent tert-butyl alcohol, the values were 2.1 x 10(4) and 1.7 x 10(4) M(-)(1) s(-)(1), respectively. The solvent effect (factor of 20) is more significant than for alpha-tocopherol (factor of 4), and the two flavonoids have efficiencies comparable to that of alpha-tocopherol in scavenging peroxyl radicals in the nonpolar solvent but not in the hydrogen-bonding solvent.

Critical re-evaluation of the O-H bond dissociation enthalpy in phenol.

The gas-phase O-H bond dissociation enthalpy, BDE, in phenol provides an essential benchmark for calibrating the O-H BDEs of other phenols, data which aids our understanding of the reactivities of phenols, such as their relevant antioxidant activities. In a recent review, the O-H BDE for phenol was presented as 90 +/- 3 kcal mol(-1) (Acc. Chem. Res. 2003, 36, 255-263). Due to the large margin of error, such a parameter cannot be used for dynamic interpretations nor can it be used as an anchor point in the development of more advanced computational models. We have reevaluated the existing experimental gas-phase data (thermolyses and ion chemistry). The large errors and variations in thermodynamic parameters associated with the gas-phase ion chemistry methods produce inconsistent results, but the thermolytic data has afforded a value of 87.0 +/- 0.5 kcal mol(-1). Next, the effect of solvent has been carefully scrutinized in four liquid-phase methods for measuring the O-H BDE in phenol: photoacoustic calorimetry, one-electron potential measurements, an electrochemical cycle, and radical equilibrium electron paramagnetic resonance (REqEPR). The enthalpic effect due to solvation, by, e.g., water, could be rigorously accounted for by means of an empirical model and the difference in hydrogen bond interactions of the solvent with phenol and the phenoxyl radical. For the REqEPR method, a second correction is required since the calibration standard, the O-H BDE in 2,4,6-tri-tert-butylphenol, had to be revised. From the gas-phase thermolysis data and three liquid-phase techniques (excluding the electrochemical cycle method), the present analysis yields a gas-phase BDE of 86.7 +/- 0.7 kcal mol(-1). The O-H BDE was also estimated by state-of-the-art computational approaches (G3, CBS-APNO, and CBS-QB3) providing a range from 86.4 to 87.7 kcal mol(-1). We therefore recommend that in the future, and until further refinement is possible, the gas-phase O-H BDE in phenol should be presented as 86.7 +/- 0.7 kcal mol(-1).

Quantitative evaluation of oxidative stress status on peripheral blood in beta-thalassaemic patients by means of electron paramagnetic resonance spectroscopy.

High oxidative stress status (OSS) is known to be one of the most important factors determining cell injury and consequent organ damage in thalassaemic patients with secondary iron overload. Using an innovative hydroxylamine 'radical probe' capable of efficiently trapping majority of oxygen-radicals including superoxide we measured, by electron paramagnetic resonance (EPR) spectroscopy, OSS in peripheral blood of 38 thalassaemic patients compared with sex-/age-matched healthy controls. Thalassaemic patients showed sixfold higher EPR values of OSS than controls. Significantly higher EPR values of OSS were observed in those with a severe phenotype (thalassaemia major, transfusion-dependent) with respect to mild phenotype (sickle-cell/beta-thalassaemia, not transfusion-dependent) or thalassaemia intermedia. In patients with thalassaemia major, EPR values of OSS were positively correlated with serum ferritin and with alanine aminotransferase levels. In patients with sickle cell/beta-thalassaemia, there was no correlation between EPR value of OSS and all parameters considered. The type of chelating therapy (desferrioxamine or deferiprone) did not have an effect on EPR value of OSS. In conclusion, EPR 'radical probe' seems to be a valid innovative method to determine total OSS in patients affected by thalassaemia and might be used for evaluating new strategies of chelation, new chelators, or the efficacy of antioxidant formula.

Thermochemical and kinetic studies of a bisphenol antioxidant.

The results of a thermodynamic and kinetic investigation on the homolytic reactivity of 3,3'-di-tert-butyl-5,5'-dimethyl(1,1'-biphenyl)-2,2'-diol (1) are reported. EPR studies of the equilibration between 1, 2,4,6-trimethylphenol, and the corresponding radicals obtained by abstraction of a hydroxylic hydrogen allowed us to determine the OH bond dissociation energy (BDE) of investigated bisphenol as 83.10 kcal/mol. This value is considerably larger than that reported for the structurally related 2,6-di-tert-butyl-4-methylphenol (BHT), i.e., 81.02 kcal/mol. Absolute rate constants for the reaction of 1 with alkyl, alkoxyl, and peroxyl radicals, at or nearly to room temperature, were also determined by competition kinetics in the first two cases and by autoxidation studies under controlled conditions in the last one. The experimental data indicate that this bisphenol is a moderately efficient antioxidant and polymerization inhibitor.

Induction of cytochrome P450 enzymes and over-generation of oxygen radicals in beta-carotene supplemented rats.

Effects of beta-carotene (betaCT) on microsomal CYP-linked monooxygenases were investigated using both the regio- and stereo-selective hydroxylation of testosterone (as multibiomarker) and highly specific substrates as probes of various isoenzymes. CYP-catalyzed reactions were studied in the liver, kidney, lung and intestine of Sprague-Dawley rats of both sexes supplemented with 250 or 500 mg/kg body wt betaCT (per os) in a single or repeated (daily for 5 days) fashion. Generalized boosting effects (2-15-fold increases) were observed in the various tissues for carcinogen metabolizing enzymes associated with CYP1A1/2, CYP3A1/2, CYP2E1, CYP2B1/2 and CYP2C11. Induction of the most affected CYPs was corroborated by western blot linked to densitometric analyses. Measurement of reactive oxygen species (ROS) produced by subcellular preparations from either control or betaCT supplemented rats was performed by EPR detection of the nitroxide radical yielded by the reaction with ROS of the hydroxylamine spin probe bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate. Marked ROS over-generation associated with CYP induction (up to 33-fold increase in the liver) was recorded in the various organs (liver > lung > intestine > kidney). CYP and ROS induction are substantially in keeping with the concentration of betaCT accumulated in the various tissues, the liver being the most affected organ. These findings are consistent with the concept that betaCT is a pro-oxidant and potentially co-carcinogenic pro-vitamin, and may help explain why, in large quantities, it can have harmful effects in humans.