Role of alkoxyl radicals on the fluorescein-based ORAC (Oxygen Radical Absorbance Capacity) assay.

During the last decades the ORAC (Oxygen Radical Absorbance Capacity) assay has been widely employed to evaluate the in vitro antioxidant capacity of polyphenol-rich fruits, vegetables and beverages. The method employs fluorescein (FLH) as target molecule and AAPH (2,2'-azo-bis(2-amidinopropane)dihydrochloride) as the source of peroxyl radicals (ROO•). The protection of FLH, afforded by antioxidants (XH), is often characterized by kinetic profiles with clear lag times (LT), which are directly associated with the stoichiometry (n) of the XH-ROO• reaction. However, even for simple phenolic compounds, the LT measured imply large n values (defined as the number of ROO• moles trapped by each antioxidant molecule) which cannot be explained by a simple reaction mechanism. Nonetheless, they can be explained when considering the formation of alkoxyl radicals (RO•) from the recombination of two AAPH-derived ROO•. In the present work, we provide kinetic data showing that, in the zero order kinetic limit of FLH consumption, there is a low reaction rate incompatible with total trapping of ROO•. Thus, the consumption of FLH should be mostly related to its reaction with RO•. In addition, we present data regarding the assumption that in competitive measurements, the LT is due to efficient trapping of the ROO• by the added phenols, leading to high n values (1.7 to 23) for mono and polyphenols. These values are not in agreement with kinetic studies of the antioxidant consumption mediated by the presence of AAPH carried out by HPLC-DAD technique, which imply a competition by RO•. The results suggest that the use of FLH as probe at low concentrations give, for several antioxidants, ORAC values mainly related to their reaction towards RO• radicals instead of primary ROO•radicals.

Mechanism of pyrogallol red oxidation induced by free radicals and reactive oxidant species. A kinetic and spectroelectrochemistry study.

Pyrogallol red (PGR) presents high reactivity toward reactive (radical and nonradical) species (RS). This property of PGR, together with its characteristic spectroscopic absorption in the visible region, has allowed developing methodologies aimed at evaluating the antioxidant capacity of foods, beverages, and human fluids. These methods are based on the evaluation of the consumption of PGR induced by RS and its inhibition by antioxidants. However, at present, there are no reports regarding the degradation mechanism of PGR, limiting the extrapolation to how antioxidants behave in different systems comprising different RS. In the present study, we evaluate the kinetics of PGR consumption promoted by different RS (peroxyl radicals, peroxynitrite, nitrogen dioxide, and hypochlorite) using spectroscopic techniques and detection of product by HPLC mass spectrometry. The same pattern of oxidation and spectroscopic properties of the products is observed, independently of the RS employed. Mass analysis indicates the formation of only one product identified as a quinone derivative, excluding the formation of peroxides or hydroperoxides and/or chlorinated compounds, in agreement with FOX's assays and oxygen consumption experiments. Cyclic voltammetry, carried out at different pH's, shows an irreversible oxidation of PGR, indicating the initial formation of a phenoxy radical and a second charge transfer reaction generating an ortho-quinone derivative. Spectroelectrochemical oxidation of PGR shows oxidation products with identical UV-visible absorption properties to those observed in RS-induced oxidation.

Pyrogallol red oxidation induced by superoxide radicals: application to evaluate redox cycling of nitro compounds.

The bleaching of the pyrogallol red (PGR) dye mediated by superoxide anion radicals (O(2)(-)) generated from the xanthine/xanthine oxidase system (X/XO) was studied by UV-visible spectrophotometry. The absorption band (at 540 nm) of PGR quickly decreased in the presence of X/XO, implying an efficient reaction of O(2)(-) with PGR. The process was unaffected by catalase (CAT), but completely abolished by superoxide dismutase (SOD). A mechanism of the reaction involving the consumption of one PGR molecule by two O(2)(-) to generate one molecule of H(2)O(2) is proposed. PGR was used as a probe to estimate the rate of O(2)(-) generation in redox cycling reactions of a series of nitro compounds mediated by rat liver microsomes. The consumption of PGR induced by the redox cycling of nitrofurantoin was totally eliminated by the addition of SOD but unaffected by CAT. The initial rate of consumption of PGR mediated by the redox cycling of others nitro derivatives follows the order: furazolidindione > nitrofurantoin > nifurtimox > benznidazole > chloramphenicol. We concluded that PGR can be used as a probe to estimate the release of O(2)(-) from enzymatic systems or from the redox cycling of nitro compounds.

Reactivity of 1,4-dihydropyridines toward SIN-1-derived peroxynitrite.

PURPOSE: To study the reactivity of C4-substituted 1,4-dihydropyridines (1,4-DHP), with either secondary or tertiary nitrogen in the dihydropyridine ring, toward SIN-1-derived peroxynitrite in aqueous media at pH 7.4. METHODS: Reactivity was followed by changes in the absorptivity of the UV-Vis bands corresponding to 1,4-DHP. Gas Chromatography/ Mass Spectrometer (GC-MS) and Electron Paramagnetic Resonance (EPR) spin trap techniques were used to characterize the final product and the intermediates of the reaction, respectively. RESULTS: 1,4-DHPs significantly reacted toward peroxynitrite at varied rates, according to the calculated kinetic rate constants. By EPR spectroscopy, a carbon-centered radical from the 1,4-DHP was intercepted with N-tert-butylamine-alpha-phenylnitrone (PBN), as the intermediate for the reaction with peroxynitrite. Likewise, the oxidized derivative (i.e., the pyridine) was identified as the final product of the reaction by GC-MS. By using the technique of deuterium kinetic isotope effect, the participation of the hydrogen of the 1-position on the 1,4-DHP ring was shown not to be the rate-limiting step of the reaction. CONCLUSIONS: The direct participation of the 1,4-DHP derivatives in the quenching of SIN-1-derived peroxynitrite has been demonstrated. Kinetic rate constant of tested 1,4-DHP toward peroxynitrite showed a direct relationship with the oxidation peak potential values; that is, compounds reacting faster were more easily oxidized.

A novel alkaloid antioxidant, Boldine and synthetic antioxidant, reduced form of RU486, inhibit the oxidation of LDL in-vitro and atherosclerosis in vivo in LDLR(-/-) mice.

A corollary to the oxidation hypothesis of atherosclerosis is that the consumption of antioxidants is beneficial. However, the literature is divided in support of this conclusion. In this study, Boldine, an alkaloid of Peumus boldus and reduced form of RU486, was tested for their antioxidant potency both in, in vitro oxidation system and in mouse models. Boldine decreased the ex-vivo oxidation of low-density lipoprotein (LDL). Two different in vivo studies were performed to study the effect of these compounds on the atherosclerotic lesion formation in LDLR(-/-) mice. In study I, three groups of LDLR(-/-) mice (N = 12 each) were fed an atherogenic diet. Group 1 was given vehicle and group 2 and 3 were given 1mg of Boldine or Red RU per day for 12 weeks. In study II, two groups of LDLR(-/-) mice N = 10 each) were fed an atherogenic diet. Group 1 was given vehicle and group 2 was given 5mg of Boldine per day. The results indicated that there was a decrease in lesion formation reaching a 40% reduction due to Boldine and 45% reduction by Red RU compared to controls. The in vivo tolerance of Boldine in humans (has been used as an herbal medicine in other diseases) should make it an attractive alternative to Vitamin E.

Chronic exposure of HepG2 cells to excess copper results in depletion of glutathione and induction of metallothionein.

Metallothionein (MT) and reduced glutathione (GSH) play an important role in the intracellular handling of copper by preventing the generation and favouring the removal of copper-derived free radicals. The present study addressed the changes in MT and GSH that follow chronic (2 or 5 weeks) exposure of human hepatoblastoma cells (HepG2) to excess copper. Copper treatment (100 microM, 2 weeks) led to a 28-fold elevation in intracellular copper. Concomitantly, cells exhibited a seven-fold increase in total MT and an increment in its saturation with copper from 45 to 86%. Around 38% of copper in the cytosolic fraction could be accounted for by MT. GSH equivalents were substantially lowered (to 37% of basal levels) in treated cells, with only part of it being accounted for by an increase in GSSG. Copper-treatment induced no changes in catalase or GSH-peroxidase activities but it was associated with a small reduction in SOD (20%) and GSH-reductase (26%) activities. Copper-loaded cells did not differ from controls in their basal oxidative tone; however, when exposed to tert-butylhydroperoxide they exhibited a markedly greater susceptibility to undergo both oxidative stress and cell lysis. It is proposed that chronic exposure of HepG2 cells to excess copper is accompanied by "adaptive changes" in GSH and MT metabolism that would render cells substantially more susceptibility to undergo oxidative stress-related cytotoxicity.

Effects of copper ions on the free radical-scavenging properties of reduced gluthathione: implications of a complex formation.

The interaction between glutathione (GSH) and copper ions was investigated in vitro to determine whether such interaction could affect the free-radical scavenging properties of the tripeptide. To this end, the bleaching (decrease in OD734 nm) of a coloured solution containing the stable free-radical cation ABTS+, (which results from the addition of thiols to such a solution) was employed as an in vitro indication of the ability of the tripeptide to scavenge free radicals. While GSH bleached concentration-dependently (1.0-7.5 gM) the ABTS+-containing solution, its prior incubation (5 microM) in the presence of Cu+1 or Cu+2 ions (1-7.5 M) led to a metal concentration-dependent decrease of the bleaching capacity. At a ratio equal to one (5 microM each), the bleaching capacity of the copper plus GSH mixture was 50% of that seen for GSH alone. Further additions of copper (reaching ratios up to 2) did not result in greater decreases in the GSH-bleaching capacity. Noteworthy at the ratio of onewas that the copper plus GSH solutions maintained their bleaching capacity despite the lack of any DTNB-reactivity, i. e., the complete absence of thiols in the mixture. Mixtures of increasing concentrations of a fixed ratio (equal to 2) of copper plus GSH, which were found not to exhibit any DTNB-reactivity, showed a linear and concentration-dependent increase in bleaching capacity. The bleaching capacity remained unaltered when TRIEN, EDTA or histidine were added to pre-incubated (1:1) mixtures of copper plus GSH. However, the incubation of copper with TRIEN or EDTA (but not histidine) prior to GSH addition, totally prevented the loss of the original GSH-bleaching capacity. The present data supports the formation of a copper-glutathione complex which is stable to the presence of some copper-chelators, lacks all thiol reactivity, but fully conserves the free-radical scavenging properties of GSH.

Free-radical-induced inactivation of lysozyme and carbonyl residue generation in protein are not necessarily associated.

The 2,2'-azobis(2-amidinopropane) (AAPH)-induced inactivation and oxidative modification of lysozyme, as determined by the loss of tryptophan-associated fluorescence (TAF) and the increase in dinitrophenylhydrazine-reactive carbonyl groups (CO), were studied in the absence and in the presence of antioxidants. AAPH induced a progressive inactivation of the enzyme and a parallel decrease of its TAF. Both changes were closely correlated (R2 = 0.97); however, the inactivation was only partially associated with an increase in CO. The latter reached maximal values at times half those needed to attain maximal losses in both lysozyme activity and TAF. A stoichiometric comparison reveals that whereas over 74% of the enzyme molecules had lost their activity, only 5% exhibited an increment in CO. CO formation was affected differentially by boldine and trolox. Both antioxidants fully protected against the early inactivation and loss of TAF; however, the increase in CO was completely unaffected by trolox. Exposure of lysozyme to Fe3+/ascorbate induced no loss of activity or TAF, but it led to an accumulation of CO similar to that induced by AAPH. Results indicate that CO formation and lysozyme inactivation are two mechanistically dissociable events and that changes in the former parameter can perfectly occur in the absence of changes in the latter.

Protective effects of boldine against free radical-induced erythrocyte lysis.

Boldine, an aporphine alkaloid extracted from the leaves and bark of boldo (Peumus boldus Mol.), has been shown to exhibit strong free-radical scavenger and antioxidant properties. Here, we report the in vitro ability of boldine to protect intact red cells against the haemolytic damage induced by the free radical initiator 2, 2'-azobis-(2-amidinopropane) (AAPH). Boldine concentration-dependently prevented the AAPH-induced leakage of haemoglobin into the extracellular medium. Substantial and similar cyto-protective effects of boldine were observed whether the antioxidant was added 1 h prior to, or simultaneously with, the azo-compound. The delayed addition of boldine, by 1 h relative to AAPH, diminished but did not abolish its cytoprotective effect. However, negligible effects of boldine were observed after its addition to erythrocytes previously incubated with AAPH for 2 h. The data presented demonstrate that, in addition to its well-established antioxidant effects, boldine also displays time-dependently strong cytoprotective properties against chemically induced haemolytic damage.

Biological disposition of boldine: in vitro and in vivo studies.

Boldine is a natural compound with well-established free radical scavenger and hepatoprotective properties. The further exploration of its actual therapeutic potential as an antioxidant is, however, partially limited by the absence of knowledge on its pharmacokinetics. In the present studies, we provide information on the in vitro and in vivo biological disposition of boldine. The addition of 200 microM boldine to an isolated rat hepatocyte suspension was followed by a time-dependent (0-60 min) disappearance of boldine from the extracellular medium. This decline was associated with an early (first 2 min) and swift accumulation (1600 microM) of boldine within the cells. Although the intracellular concentration of boldine diminished, boldine was always found to occur within the cells at concentrations substantially higher than those initially added to the preparation. Boldine was also concentration-dependently removed from the extracellular medium by isolated rat livers portally perfused with the antioxidant. In vivo studies, conducted in rats, revealed that following either its oral or its intravenous administration, plasma boldine concentrations declined rapidly and according to an apparently first order type of kinetics. After its oral administration (50 or 75 mg/kg), boldine was rapidly (within 30 min) absorbed and preferentially concentrated in the liver, with substantially lower concentrations being found in the brain and heart. Maximal hepatic concentrations of boldine were found to be equal to or greater than those needed to afford antioxidant and hepatoprotective effects in vitro.

Biochemical and behavioral effects of boldine and glaucine on dopamine systems.

The aporphine alkaloids boldine and glaucine have been reported to show "neuroleptic-like" actions in mice, suggesting that they may act as dopamine antagonists. We have found that in vitro boldine displaces specific striatal [3H]-SCH 23390 binding with IC50 = 0.4 microM and [3H]-raclopride binding with IC50 = 0.5 microM, while the affinities of glaucine at the same sites are an order of magnitude lower. In vivo, however, 40 mg/kg boldine (i.p.) did not modify specific striatal [3H]-raclopride binding and only decreased [3H]-SCH 23390 binding by 25%. On the other hand, 40 mg/kg glaucine (i.p.) displaced both radioligands by about 50%. Behaviors (climbing, sniffing, grooming) elicited in mice by apomorphine (0.75 mg/kg s.c.) were not modified by boldine at doses up to 40 mg/kg (i.p.) but were almost completely abolished by 40 mg/kg glaucine (i.p.). In the apomorphine-induced (0.1 mg/kg s.c.) rat yawning and penile erection model, boldine and glaucine appeared to be similarly effective, inhibiting both behaviors by more than 50% at 40 mg/kg (i.p.). Boldine and glaucine, injected i.p. at doses up to 40 mg/kg, were poor modifiers of dopamine metabolism in mouse and rat striatum. These data suggest that boldine does not display effective central dopaminergic antagonist activities in vivo in spite of its good binding affinity at D1- and D2-like receptors, and that glaucine, although less effective in vitro, does appear to exhibit some antidopaminergic properties in vivo.

Copper in infant nutrition: safety of World Health Organization provisional guideline value for copper content of drinking water.

BACKGROUND: Copper is an essential nutrient for humans. Recently, a limit of 31.48 micromol/l (2 mg/l) was proposed by the World Health Organization as the provisional guideline value for copper content of drinking water. The objective of the study was to determine the tolerance of chronic exposure to drinking water with low or high copper content in infants. METHODS: Healthy infants (n = 128) were randomly assigned to receive drinking water with less than 1.57 micromol/l (<0.1 mg/l) (n = 48) or 31.48 micromol/l (2 mg/l) of copper (n = 80) from 3 to 12 months of age. At 6, 9, and 12 months of age, serum concentrations of copper, ceruloplasmin, and superoxide dismutase; erythrocyte metallothionein; bilirubin; transaminases; and gamma-glutamyl transferase were measured. RESULTS: Small differences in biochemical indexes of copper nutrition were observed between the groups, but there was no evidence of adverse or toxic effects. These findings may be explained by an adaptive response to the higher copper intake, limiting copper absorption, and increasing biliary secretion, as well as by an increase in copper storage. It is also possible that the sensitivity of the biochemical indicators employed to detect differences in copper status is limited. CONCLUSION: No acute or chronic adverse consequences of consuming water with copper content of 31.48 micromol/l (2 mg/l) were detected in infants during the first year of life. The results support the safety of the World Health Organization's provisional guideline value for copper in drinking water during infancy.

Protective effect of boldine in experimental colitis.

The cytoprotective and anti-inflammatory effects of boldine in an experimental model of acute colitis are reported. The administration of boldine to animals with colitis induced by the intrarectal administration of acetic acid, was found to protect against colonic damage as expressed by major reductions in the extent of cell death, tissue disorganization, and edema. Boldine also reduced the colonic neutrophil infiltration, as measured by the myeloperoxidase activity, but it did not significantly affect tissue lipoperoxides. Boldine was found to preserve the colonic fluid transport, a function otherwise markedly affected in the tissue of acid-treated animals. Results presented here provide experimental evidence supporting new cytoprotective and anti-inflammatory properties of boldine.

DMPS-arsenic challenge test. I: Increased urinary excretion of monomethylarsonic acid in humans given dimercaptopropane sulfonate.

The purpose of the present study was to evaluate in a novel manner the arsenic exposure of humans living in two towns in Northeastern Chile. Residents of one town drink water containing 593 microg As/l. Those in the control town drink water containing 21 microg As/l. Our hypothesis was that the administration of the chelating agent, 2,3-dimercaptopropane-1-sulfonic acid, Na salt (DMPS, DIMAVAL) would increase the urinary excretion of arsenic, alter the urinary profile of arsenic species and thus result in a better indication of the body load of arsenic and a better biomarker for arsenic exposure. The method used to evaluate these subjects was to give them 300 mg DMPS by mouth, after an overnight fast, and collect urine at specified time periods. The urine samples were analyzed for inorganic arsenic, monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) and total arsenic by hydride generation and atomic absorption spectrophotometry. The results indicated that: 1) During the 2-hr period after DMPS administration, MMA represented 42%, inorganic As, 20 to 22% and DMA, 37 to 38% of the total urinary arsenic. The usual range of the MMA percentage in human urine has been 10 to 20%. The % MMA increased almost equally for both the arsenic-exposed and control subjects. 2) The exposed subjects had a greater urinary excretion of total arsenic, before and after DMPS administration, than the control subjects. 3) Although buccal cells were obtained only from a few subjects, the prevalence of mononucleated buccal cells, an indication of genotoxicity, was 5-fold greater for those who consumed drinking water with the higher arsenic content than among control subjects. Our conclusions are that 1) DMPS has a highly specific effect in humans on MMA metabolism and/or urinary excretion; 2) the human body stores substantial amounts of arsenic; and 3) the urinary arsenic concentration after DMPS administration may be more indicative of the body burden of arsenic because it was greater than that found before DMPS was given.

Loss of the metal binding properties of metallothionein induced by hydrogen peroxide and free radicals.

The relationship between the metal-binding properties of metallothionein (MT) and its ability to interact with peroxides and free radicals was explored in vitro. The binding of 109Cd to MT and the thiol density of the protein were determined after incubation of a purified Zn/Cd-metallothionein preparation with either hydrogen peroxide alone, or with a number of free radical generating systems. Exposure of MT to H2O2, whether in the presence or absence of Fe2+, resulted in the progressive loss of the thiol residues of the protein and led to a parallel decrease of its 109Cd-binding capacity. These changes correlated with r values of 0.999 (P = 0.001) and 0.998 (P = 0.001), in the absence and presence of iron, respectively. The effects of H2O2, alone or plus Fe2+, on MT were completely prevented by catalase, but totally unaffected by superoxide dismutase or desferrioxamine. Exposure of MT to xanthine/xanthine oxidase also led to thiol oxidation and to a concomitant loss of the Cd-binding properties. In this system, both changes correlated with an r of 0.993 (P = 0.001) and were completely inhibited by superoxide dismutase. Exposure of MT to the peroxyl radical generator, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), resulted in the progressive loss of its the metal-binding properties and its thiol residues, both changes correlating with an r of 0.986 (P = 0.002). The ability of MT to bind 109Cd, lost as a result of its prior exposure to either H2O2 alone, H2O2 plus Fe2+, xanthine/xanthine oxidase, or to AAPH was, in all cases, completely recovered after incubation of the modified protein with dithiothreitol. These results indicate that H2O2 alone, and/or the oxygen-derived species, superoxide anion and peroxyl radicals, can all directly interact in vitro with MT to modify the protein oxidatively, and suggest that, under in vivo conditions, these species may be implicated as modifying factors of the metal-binding capacity of metallothionein.

Liver copper storage and transport during development: implications for cytotoxicity.

Copper is an essential trace element for many biological processes. Its functions range from influencing specific gene expression to serving as a cofactor or prosthetic group for several enzymes. Intakes of copper at doses that exceed physiologic demands are normally met with efficient homeostatic mechanisms. Ceruloplasmin, albumin, and transcuprein, and to a lesser extent certain amino acids, are major copper-transporting constituents in circulating plasma. After its hepatic uptake, copper may be stored within hepatocytes, secreted into plasma, or excreted in bile. The biliary route represents the major excretory pathway of copper and largely accounts for its hepatic turnover. Copper retained by hepatocytes is mostly bound to specific metal-binding proteins, primarily metallothionein, or incorporated into several cuproenzymes. Copper incorporation into metallothionein and certain cuproproteins appears to require prior binding of copper to glutathione, thus defining a relation between copper metabolism and the intracellular availability of glutathione. Hepatic metallothionein concentrations can be modulated by dietary copper; changes in metallothionein and in copper status are significant throughout development. Binding of copper to metallothionein provides a temporary storage for cytoplasmic copper, preventing it from occurring as (potentially toxic) free ionic metal. In its unbound form, copper can generate hydroxyl radicals. Because metallothionein exhibits a high reactivity toward these radicals, it is increasingly recognized to play a protective role against copper-induced cytotoxicity. We discuss some of the possible toxicologic implications that may arise from changes in hepatic copper and metallothionein status during development.

Cytoprotective and antioxidant effects of boldine on tert-butyl hydroperoxide-induced damage to isolated hepatocytes.

Boldine, an aporphine alkaloid, was recently shown by us to exhibit potent antioxidant properties. We report here that boldine concentration-dependently inhibited the peroxidative (accumulation of thiobarbituric acid reactive substances) and lytic damage (trypan blue exclusion and lactate dehydrogenase leakage) to isolated rat hepatocytes induced by tert-butyl hydroperoxide (TBOOH). Boldine (200 micromol/L) fully cytoprotected and completely prevented the peroxidation induced by TBOOH at concentrations equal to or lower than 0.87 mmol/L. However, at a peroxide concentration of 0.91 mmol/L, although boldine completely inhibited lipid peroxidation it largely failed to afford cytoprotection against TBOOH. TBOOH alone (0.83 mmol/L) caused an early (within 60 s) sudden decline of reduced glutathione (by 50%) and an equivalent increase in the levels of oxidized glutathione. Neither of these effects was prevented by the simultaneous addition of a cytoprotective and antioxidant concentration of boldine (200 micromol/L). The delayed addition of boldine to the suspension (after 10 or 20 min), while effectively blocking any further increase in thiobarbituric acid reactive substances, totally failed to prevent the peroxide-induced loss in cell viability. Conversely, preincubation of the hepatocytes with boldine for 150 min (at which time no boldine could be detected in either intra- or extracellular spaces) prevented lipid peroxidation and was as effective in protecting the cells against the damage caused by the subsequent addition of TBOOH as the simultaneous addition of boldine and TBOOH to hepatocytes preincubated for 150 min under control conditions.

The inhibitory effects of boldine, glaucine, and probucol on TPA-induced down regulation of gap junction function. Relationships to intracellular peroxides, protein kinase C translocation, and connexin 43 phosphorylation.

The naturally occurring antioxidant boldine and its di-methoxy analogue glucine, as well as the drug antioxidant probucol, all inhibit TPA-induced downregulation of gap junctional intercellular communication in WB-F344 rat liver epithelial cells in dose-dependent manners. The compounds were essentially 100% inhibitory to the effect of TPA (10 nM) at 50 microM each. Analysis of the mechanism of the antitumor promotive action of these agents in vitro revealed that boldine and probucol (both at 10 microM) totally inhibited the TPA-induced accumulation of intracellular oxidants. Additionally, boldine, glaucine, and probucol, each at 50 microM, inhibited TPA-induced translocation of protein kinase C (PKC) to the particulate fraction of the cells, with concomitant inhibition of TPA-induced hyperphosphorylation of gap junctional connexin 43 (cx43) and TPA-induced internalisation of cx43 protein from the plasma membrane of the cells. None of the compounds inhibited the binding of (3H)-PDBu to TPA-specific binding sites in the cells. The results indicate that antioxidant molecules, irrespective of structure, possess common antitumor promotive potential in this model of gap junctional intercellular communication. The data also indicate that the compounds may interfere with the promotive function of TPA, at least in part, by the destruction of oxidants within the cells. Xanthine oxidase was excluded as a major source of such intracellular oxidants because allopurinol (50 microM) did not significantly affect either the accumulation of oxidants in the cells or the downregulation of gap junctional communication in response to TPA. Taken together, these data also suggest that TPA-induced oxidants play a role in the translocation of PKC to cellular membranes and it is at this level where the antioxidants may interfere in TPA-induced downregulation of gap junctional function.

[Effect of a dry boldo extract on oro-cecal intestinal transit in healthy volunteers]. / Effecto de un extracto seco de boldo sobre el tránsito intestinal oro-cecal en voluntarios sanos.

BACKGROUND: Boldo (Peumus boldus Molina) is a widely used medicinal plant. However, its physiological effects are not well known. Recent studies in animals showed that certain components of boldo relax smooth muscle and prolong intestinal transit. AIM: To assess the effects of a dry boldo extract on oro cecal transit time in normal humans. SUBJECTS AND METHODS: Twelve volunteers received 2.5 g of a dry boldo extract or a placebo (glucose) during two successive periods of four days. On the fourth day, 20 g of lactulose were administered and breath hydrogen was collected every 15 min. Oro cecal transit time was defined as the time in which breath hydrogen increased by 20 ppm over the fasting level. RESULTS: Oro cecal transit time was larger after dry boldo extract administration, compared to placebo (112.5 +/- 15.4 and 87 +/- 11.8 min respectively, paired t p < 0.05). CONCLUSIONS: Dry boldo extract prolongs oro cecal transit time, a possible explanation for its medicinal use.

[Pharmacokinetic implications associated to the use of drugs as racemates or pure enantiomers]. / Implicancias farmacocinéticas asociadas al uso de fármacos como racematos o enantiómeros puros.

This article critically reviews the recent specialized literature concerning the influence of the stereochemical nature of quiral drugs on the pharmacokinetic processes and its pharmacological implications. Evidence is presented indicating that as a function of the type of enantiomer administered, profound differences in the pharmacokinetic profiles, e.g. absorption, distribution, biotransformation and elimination can occur. As a consequence of the enantioselective nature of the drug-organism interaction, major differences in the therapeutic responses can be envisaged depending on whether the drug is administered as a pure enantiomer or as a racemic mixture.