Cesar A. Lau-Cam

Comparison of the protective actions of N-acetylcysteine, hypotaurine and taurine against acetaminophen-induced hepatotoxicity in the rat

When used in overdoses, acetaminophen (APAP) is a common cause of morbidity and mortality in humans. At present, N-acetylcysteine (NAC) is the antidote of choice for acetaminophen overdoses. Prompt administration of NAC can prevent the deleterious actions of APAP in the liver. In view of the similarities in antioxidant effects demonstrated by NAC, hypotaurine (HYTAU) and taurine (TAU) in this and other our laboratories, the present study was undertaken to compare these compounds for the ability to attenuate plasma and liver biochemical changes associated with a toxic dose of APAP. For this purpose, fasted male Sprague-Dawley rats, 225-250 g in weight, were intraperitoneally treated with APAP (800 mg/kg), NAC, HYTAU or TAU (2.4 mM/kg) followed 30 min later by APAP, or 50% PEG 400 (the vehicle for APAP). At 6 hr after APAP administration, all animals were sacrificed by decapitation and their blood and livers collected. The plasma fractions were analyzed for indices of liver damage (alanine transaminase, aspartate transaminase, lactate dehydrogenase), levels of malondialdehyde (MDA), reduced (GSH) and oxidized (GSSG) glutathione, and activities of glutathione reductase (GR), glutathione S-transferase (GST) and γ-glutamylcisteinyl synthetase (GCS). Suitable liver homogenates were analyzed for the same biochemical parameters as the plasma but indices of liver damage. By itself, APAP increased MDA formation and had a significant lowering influence on the levels of GSH and GSSG, the GSH/GSSH ratio, and the activities of GR, GST and GCS both in the plasma and liver. In addition, APAP promoted the leakage of transaminases and lactate dehydrogenase from the liver into the plasma. Without exceptions, a pretreatment with a sulfur-containing compound led to a significant attenuation of the liver injury and the biochemical changes induced by APAP. Within a narrow range of potency differences, HYTAU appeared to be the most protective and TAU the least. The present results suggest that, irrespective of the differences in structural features and in vitro antioxidant properties that may exist among NAC, TAU and HYTAU, these compounds demonstrate equivalent patterns of protection and, to a certain extent, equipotent protective actions against the toxic actions of APAP in the liver when tested in equimolar doses and under the same conditions in an animal model.

The Effects of Taurine, Taurine Homologs and Hypotaurine on Cell and Membrane Antioxidative System Alterations Caused by Type 2 Diabetes in Rat Erythrocytes

This study compared taurine, aminomethanesulfonic acid, homotaurine and hypotaurine for the ability to modify indices of oxidative stress and membrane damage associated with type 2 diabetes. In the study, male Goto-Kakizaki and Wistar-Kyoto rats were allowed free access to a high fat and normal diet, respectively, for 9 weeks. At the end of week 8, half of the animals in each group received a daily intraperitoneal dose of a sulfur compound (0.612 M/kg) for 5 days and, 24 hr after the last treatment, blood samples were withdrawn by cardiac puncture to obtain plasma and erythrocyte fractions for biochemical analyses. Relative to control values, taurine and its congeners reduced membrane damage, the formation of intracellular malondialdehyde and oxidized glutathione, and the decreases in reduced glutathione and antioxidative enzyme activities in diabetic erythrocytes. Except for a few isolated instances, all test compounds were equiprotective.

Attenuating effect of taurine on lipopolysaccharide-induced acute lung injury in hamsters

This study has evaluated the ability of the semiessential amino acid taurine to attenuate lipopolysaccharide (LPS)-induced lung inflammation, oxidative stress and apoptosis in a small animal model. For this purpose, bacterial LPS (0.02mg in phosphate buffered saline (PBS) pH 7.4) was instilled intratracheally into female Golden Syrian hamsters, before or after a 3-day intraperitoneal treatment with a single dose (50mg/kg in PBS pH 7.4) of taurine. At 24h after the last treatment, lung tissue and bronchoalveolar lavage fluid (BALF) samples were collected. In comparison to samples from animals receiving only PBS pH 7.4, serving as controls, those of LPS-stimulated animals exhibited a higher count of both total leukocytes and neutrophils in the BALF, and increased incidence of apoptosis, depletion of intracellular glutathione and evidence of inflammation confined to the parenchyma in the lung. In addition, LPS caused cells in the BALF to exhibit a higher expression of tumor necrosis factor-1, a higher activity of caspase-3, marked lipid peroxidation, and altered activities of catalase, glutathione peroxidase and superoxide dismutase relative to control samples. In contrast, a treatment with taurine was found to significantly attenuate all of the cellular and biochemical alterations induced by LPS, more so when given before rather than after the endotoxin. The present results suggest that taurine possesses intrinsic antiinflammatory and antioxidant properties that may be of benefit against the deleterious actions of LPS in the lung.

Comparative study of the binding characteristics to and inhibitory potencies towards PARP and in vivo antidiabetogenic potencies of taurine, 3-aminobenzamide and nicotinamide

BackgroundPoly(ADP-ribose) is a NAD+-requiring, DNA-repairing, enzyme playing a central role in pancreatic β-cell death and in the development of endothelial dysfunction in humans and experimental animals. PARP activation is also relevant to the development of complications of diabetes. Hence, agents capable of inhibiting PARP may be useful in preventing the development of diabetes and in slowing down complications of diabetes.MethodsPARP inhibition was assessed with a colorimetric assay kit. Molecular docking studies on the active site of PARP were conducted using the crystalline structure of the enzyme available as Protein Data Bank Identification No. 1UK1. Type 2 diabetes was induced in male Sprague-Dawley rats with streptozotocin (STZ, 60 mg/kg, i.p.). The test compounds (3-aminobenzamide = 3-AB, nicotinamide = NIC, taurine = TAU) were given by the i.p. route 45 min before STZ at 2.4 mM/kg (all three compounds) or 1.2 and 3.6 mM/kg (only NIC and TAU). Blood samples were collected at 24 hr after STZ and processed for their plasma. The plasma samples were used to measure glucose, insulin, cholesterol, triglycerides, malondialdehyde, nitric oxide, and glutathione levels using reported methods.Results3-AB, NIC and TAU were able to inhibit PARP, with the inhibitory potency order being 3-AB>NIC>>TAU. Molecular docking studies at the active site of PARP showed 3-AB and NIC to interact with the binding site for the nicotinamide moiety of NAD+ and TAU to interact with the binding site for the adenine moiety of NAD+. While STZ-induced diabetes elevated all the experimental parameters examined and lowered the insulin output, a pretreatment with 3-AB, NIC or TAU reversed these trends to a significant extent. At a dose of 2.4 mm/kg, the protective effect decreased in the approximate order 3-AB>NIC≥TAU. The attenuating actions of both NIC and TAU were dose-related except for the plasma lipids since NIC was without a significant effect at all doses tested.ConclusionsAt equal molar doses, 3-AB was generally more potent than either TAU or NIC as an antidiabetogenic agent, but the differences were not as dramatic as would have been predicted from their differences in PARP inhibitory potencies. NIC and TAU demonstrated dose-related effects, which in the case of TAU were only evident at doses ≥2.4 mM/kg. The present results also suggest that in the case of NIC and TAU an increase in dose will enhance the magnitude of their attenuating actions on diabetes-related biochemical alterations to that achieved with a stronger PARP inhibitor such as 3-AB. Hence, dosing will play a critical role in clinical studies assessing the merits of NIC and TAU as diabetes-preventing agents.

Growth Inhibitory Action of Ebselen on Fluconazole-Resistant Candida albicans: Role of the Plasma Membrane H+-ATPase

PMA1 is a yeast gene that codes for the plasma membrane H(+)-ATPase, a protein commonly referred to as Pma1p. Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is a synthetic selenium-containing compound that has recently been shown to display antimicrobial activity owing to its ability to inhibit Pma1p. Ebselen is able to block the activity of Pma1p not only in opportunistic pathogens such as Cryptococcus neoformans and Candida albicans but also in nonpathogenic yeasts such as Saccharomyces cerevisiae. A series of in vitro studies aimed at evaluating the antifungal activity of ebselen were performed. At low concentrations (<10 microM), ebselen was fungistatic against three strains of S. cerevisiae (IC(50) approximately 3 microM) and one fluconazole-resistant strain of C. albicans (IC(50) approximately 6 microM), and at a high concentration (30 microM) it was fungicidal against C. albicans. Moreover, ebselen was found to inhibit medium acidification by the fluconazole-resistant strain of C. albicans in a concentration-dependent manner. In comparison to currently used antifungal agents represented by azole (itraconazole, ketoconazole, fluconazole) and polyene (amphotericin B) compounds, ebselen was at least 10-fold more potent than fluconazole but less active than the other compounds tested. The present results suggest that the growth inhibitory activity of ebselen toward fluconazole-resistant yeast cells is due, at least in part, to inhibition of Pma1p. Ebselen may also serve as a useful agent in the treatment of infections caused by fluconazole-resistant fungi.

Evaluation of the antifungal and plasma membrane H+-ATPase inhibitory action of ebselen and two ebselen analogs in S. cerevisiae cultures

The plasma membrane H+-ATPase pump (Pma1p) has been proposed as a viable target for antifungal drugs since this high capacity proton pump plays a critical role in the intracellular regulation of pH and in nutrient uptake of yeast and other fungi. In recent years, this and other laboratories have verified that the antifungal activity of 2-phenylbenzisoselenazol-3(2H)-one, an organoselenium compound commonly referred to as ebselen (1), stems, at least in part, from its inhibitory action on the fungal Pma1p. In the present study, the antifungal efficacy of 2-(3-pyridinyl)-benzisoselenazol-3(2H)-one (2) and 2-phenylbenzisoselenazol-3(2H)-one 1-oxide (3), two ebselen analogs, was evaluated using a strain of S. cerevisiae and compared against that of 1. In addition, the study also examined the inhibitory potential of these three compounds toward the Pma1p of S. cerevisiae. Based on mean IC50 values, the antifungal potency was found to decrease in the order 3 > 1 > 2. However, in terms of inhibitory action on Pma1p, the potency decreased in the order 1 > 3 > 2. The magnitude of these activities appears to be correlated with the corresponding log P values, with compound 2 being the most hydrophilic and the least active of the three.

Simplified reversed-phase HPLC method with spectrophotometric detection for the assay of verapamil in rat plasma

A high-performance liquid chromatographic (HPLC) method was developed for the assay of verapamil in rat plasma. After deproteinization of the plasma sample with an acetonitrile-perchloric acid (8:2) mixture containing dextromethorphan, the internal standard, an aliquot of the supernatant was directly analyzed on a cyanopropylsilane column with methanol-acetonitrile-triethylamine acetate buffer (10:30:60) as the mobile phase and detection at 235 mm. At a flow rate of 1.5 ml min-1, a complete analysis was completed in less than 6 min. The method was linear for verapamil concentrations in the range 0.5-10 micrograms ml-1 (r = 0.9999). Recoveries for the same drug concentrations from spiked rat plasma ranged from 85.6-93.0% (n = 8). The mean RSD values for intraday and interday assay reproducibility (n = 3) were, in both cases, less than 0.9%. The limit of detectability was about 0.1 microgram ml-1. The method was found useful to monitor the plasma levels of verapamil in rats that had received this drug by the nasal, oral and intravenous routes of administration.

1H-NMR Spectroscopic Assay Method for Metoclopramide Hydrochloride in Tablets and Injections

AbstractA simple, accurate and specific proton nuclear magnetic resonance (1H-NMR) spectroscopic method has been developed for the assay of metoclopramide hydrochloride in tablets and injections. In deuterium oxide, the analyte and acetamide, the internal standard, produced corresponding resonance signals at 1.35 ppm and 2.03 ppm of utility for quantitative purposes. The average ± SD recovery of drug from 10 synthetic formulations was 99.7 ± 0.7% of the added amount. The assay of commercial products by the proposed method resulted in average assay values of 100.43 (range = 98.8-100.8, n = 6)% of declared for tablets, and of 99.45 (range = 99.6-100.4, n = 4)% of declared for injections. These results were validated by a high performance liquid chromatographic (HPLC) method.

Evaluation of Resveratrol and Piceatannol Cytotoxicity in Macrophages, T Cells, and Skin Cells

Evaluation of Resveratrol and Piceatannol Cytotoxicity in Macrophages, T Cells, and Skin Cells The cytotoxicity of resveratrol and of piceatannol, a structural analog of resveratrol, was examined in cultured cells. Using a MTT-based assay, which measures the conversion of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) to a colored formazan product in living cells, resveratrol was found to inhibit the viability of transformed mouse macrophages, tumor-derived human T cells and human epidermoid carcinoma cells in a concentration-dependent manner, with the effect decreasing in the order: T cells (LC50 ~27 μmol L-1, 24 h; ~9 μmol L-1; 48h) > macrophages (LC50~29 μmol L-1, 24 h; 39 μmol L-1, 48 h) > skin cells (LC50 ~91 μmol L-1, 24 h; ~66 μmol L-1, 48 h). Paradoxically, a high concentration of resveratrol (50 μmol L-1) inhibited the proliferation of all three cell types, and a low concentration (5 μmol L-1) stimulated the proliferation of macrophages. The viability of macrophages was also decreased by piceatannol in a concentration-dependent manner. The stimulation of macrophages with zymosan lowered the cytotoxicity of both resveratrol and piceatannol. Scanning electron microscopy of cells treated with resveratrol revealed changes in cellular morphology that were consistent with toxicity. In macrophages and skin cells, resveratrol (50 μmol L-1) induced a time-dependent increase in reduced glutathione levels but did not alter the background levels of thiobarbituric acid-reactive substances. Taken together, the present data indicate that resveratrol is toxic to cultured macrophages, T cells and skin cells at concentrations ≥25 μmol L-1, and that the cytotoxicity occurs via a mechanism that does not involve oxidative stress. Furthermore, the degree of toxicity of both resveratrol and piceatannol towards macrophages depends on the activation status of these cells, with zymosan-activated cells appearing more resistant than nonstimulated cells. Evaluacija Citotoksičnosti Resveratrola I Piceatanola U Makrofazima, T-Stanicama I Stanicama Kože Citotoksičnost resveratrola i piceatanola, strukturnog analoga resveratrola, ispitivana je u uzgojenim stanicama. Primjenom MTT-testa koji mjeri pretvorbu 3-[4,5-dimetiltiazol-2-il]2,5-difenil-tetrazolijeva bromida (MTT) u obojeni formazan produkt u živim stanicama, nađeno je da resveratrol inhibira preživljavanje transformiranih makrofaga miša, ljudskih tumorskih T-stanica i humanih stanica epidermoidnog karcinoma u ovisnosti o koncentraciji, a učinak opada redom: T-stanice (LC50 ~27 μmol L-1, 24 h; ~9 μmol L-1; 48 h) > makrofazi (LC50 ~29 μmol L-1, 24 h; 39 μmol L-1, 48 h) > stanice kože (LC50 ~91 μmol L-1, 24 h; ~66 μmol L-1, 48 h). Paradoksalno, pri visokoj koncentraciji resveratrola (50 μmol L-1) inhibirana je proliferacija svih triju tipova stanica, a pri niskim koncentracijma (5 μmol L-1) stimulirana je proliferacija makrofaga. Preživljavanje makrofaga bilo je također smanjeno primjenom piceatanola ovisno o koncentraciji. Stimulacija makrofaga zimosanom smanjila je citotoksičnost i resveratrola i piceatanola. Skenirajuća elektronska mikroskopija stanica tretiranih resveratrolom pokazala je promjene u morfologiji stanica, što je bilo u skladu s toksičnosti. U makrofazima i stanicama kože resveratrol (50 μmol L-1) inducirao je porast smanjenja razina glutationa ovisan o vremenu, ali nije mijenjao osnovne razine reaktivnih spojeva tiobarbiturne kiseline. Gledajući skupno, prikazani rezultati indiciraju da je resveratrol toksičan za uzgojene makrofage, T-stanice i stanice kože pri koncentracijama ≥25 μmol L-1 i da se citotoksičnost zbiva mehanizmom koji ne uključuje oksidativni stres. Nadalje, stupanj toksičnosti resveratrola i piceatanola prema makrofagima ovisi o aktivacijskom statusu tih stanica, pri čemu su stanice aktivirane zimosanom rezistentnije od nestimuliranih stanica.

Protective action of taurine, given as a pretreatment or as a posttreatment, against endotoxin-induced acute lung inflammation in hamsters

To assess the effect of taurine on lipopolysaccharide (LPS)-induced lung inflammation, oxidative stress and apoptosis, female Golden Syrian hamsters were intratracheally instilled with bacterial LPS (0.02 mg in phosphate buffered saline (PBS) pH 7.4), before or after a 3-day intraperitoneal treatment with a single dose of taurine (50 mg/kg/day in PBS pH 7.4), and bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected at 24 hr after the last treatment. In comparison to BALF samples from animals receiving only PBS pH 7.4, and serving as controls, those of LPS-stimulated animals exhibited a higher count of both total leukocytes and neutrophils and increased expression of tumor necrosis factor receptor 1. In comparison to lungs from control animals, those from LPS-treated animals showed increased cellular apoptosis, lipid peroxidation, decreased glutathione levels, altered activities of antioxidant enzymes (catalase, glutathione peroxidase, superoxide dismutase) and focal inflammation confined to the parenchyma. A treatment with taurine was found to significantly attenuate all these alterations, with the protection being, in all instances, greater when given before rather than after LPS. The present results suggest that taurine is endowed with antiinflammatory and antioxidant properties that are protective in the lung against the deleterious actions of Gram negative bacterial endotoxin.