Searching for tryptase in the RBL‐2H3 mast cell model: Preparation for comparative mast cell toxicology studies with zebrafish

Mast cells comprise a physiologically and toxicologically important cell type that is ubiquitous among species and tissues. Mast cells undergo degranulation, in which characteristic intracellular granules fuse with the plasma membrane and release many bioactive substances, such as enzymes β‐hexosaminidase and tryptase. Activity of mast cells in the toxicology model organism, zebrafish, has been monitored via tryptase release and cleavage of substrate N‐α‐benzoyl‐dl ‐Arg‐p‐nitroanilide (BAPNA). An extensively used in vitro mast cell model for studying toxicant mechanisms is the RBL‐2H3 cell line. However, instead of tryptase, granule contents such as β‐hexosaminidase have usually been employed as RBL‐2H3 degranulation markers. To align RBL‐2H3 cell toxicological studies to in vivo mast cell studies using zebrafish, we aimed to develop an RBL‐2H3 tryptase assay. Unexpectedly, we discovered that tryptase release from RBL‐2H3 cells is not detectable, using BAPNA substrate, despite optimized assay that can detect as little as 1 ng tryptase. Additional studies performed with another substrate, tosyl‐Gly‐Pro‐Lys‐pNA, and with an enzyme‐linked immunosorbent assay, revealed a lack of tryptase protein released from stimulated RBL‐2H3 cells. Furthermore, none of the eight rat tryptase genes (Tpsb2, Tpsab1, Tpsg1, Prss34, Gzmk, Gzma, Prss29, Prss41) is expressed in RBL‐2H3 cells, even though all are found in RBL‐2H3 genomic DNA and even though β‐hexosaminidase mRNA is constitutively expressed. Therefore, mast cell researchers should utilize β‐hexosaminidase or another reliable marker for RBL‐2H3 degranulation studies, not tryptase. Comparative toxicity testing in RBL‐2H3 cells in vitro and in zebrafish mast cells in vivo will require use of a degranulation reporter different from tryptase.     

Properties of hydrogen peroxide-induced histamine release from rat mast cells

Incubation of rat peritoneal mast cells with hydrogen peroxide results in a marked release of histamine. Maximal release is observed with 0.05–0.1 mM H₂O₂, but higher concentrations of H₂O₂ instead suppresses the release. Histamine release starts after about 2 min of lag time and reaches a plateau in about 10 min. Hydrogen peroxide-induced release does not exceed 50–60 per cent of total histamine if the incubations are prolonged or additional H₂O₂ is given at 10 min. This would be explained by the fact that H₂O₂ causes impairment of the histamine releasing system of mast cells simultaneously with the release of histamine. Hydrogen peroxide-induced release is not due to nonspecific lysis of the cells because lactate dehydrogenase, a cytoplasmic enzyme, is not liberated during the reaction. The reaction requires the presence of Ca²⁺, is enhanced by D₂O and suppressed by colchicine. It is not, however, affected by dibutyryl cAMP or dibutyryl cGMP. No significant alteration of intracellular levels of cyclic AMP and cyclic GMP is observed during the incubation of mast cells with 0.1 mM H₂O₂. These results indicate that microtubular functions would be involved in the releasing reaction although they are not under the control of cyclic nucleotides. Microscopic observation shows that H₂O₂-induced release is accompanied by degranulation.     

The catalase activity of Nα‐acetyl‐microperoxidase‐8

Abstract: N ^α ‐Acetylated microperoxidase‐8 (Ac‐MP‐8) is a water soluble, ferric heme model for peroxidases. We report here that Ac‐MP‐8 catalyzes catalase‐type reaction in addition to peroxidase‐type and cytochrome P450‐type reactions. The catalase activity of Ac‐MP‐8 was determined by the Clark oxygen electrode, which measures the production of oxygen in solution. The K_m and k_cat of the decomposition of hydrogen peroxide (H₂O₂) catalyzed by Ac‐MP‐8 are 40.9 mm and 4.1 per s, respectively. The specificity constant (k_cat/K_m) of Ac‐MP‐8 in catalase‐type reaction of H₂O₂ is 100.2,/m /s, which is 5‐ to 12‐ and 50‐ to 100‐fold less than those of MPs in cytochrome P450‐type reaction of aniline/H₂O₂ and peroxidase‐type reaction of o‐methoxyphenol/H₂O₂, respectively. These results indicate that Ac‐MP‐8 can catalyze three different types of reactions, and the relative catalytic specificities of Ac‐MP‐8 with a histidyl ligand exhibit the following orders: peroxidase‐type > cytochrome P450‐type > catalase‐type reactions. Comparisons of the enzyme activities of Ac‐MP‐8 suggest that the fifth ligands of hemoproteins influence the ratio of the three types of reactions.     

Improved Antioxidant Effect of Idebenone-Loaded Polyethyl-2-Cyanoacrylate Nanocapsules Tested on Human Fibroblasts

Purpose. The protective antioxidant role of idebenone both as free drug and drug-loaded Tween 80-coated polyethyl-2-cyanoacrylate (PECA) nanocapsules is reported. The relationship between oxidative damage and apoptotic or nonapoptotic cell death is evaluated in vitro. Methods. Idebenone-loaded nanocapsules were prepared with the interfacial polymerization method in the presence of Tween 80. Human nonimmortalized fibroblasts, under different stress conditions, either 0.5 mM diethylmaleate (DEM) for 60 min or 0.1 mM H₂O₂ for 30 min, were used as the experimental in vitromodel. The production of reactive oxygen species, the cell viability, and the nuclear DNA damage were evaluated. The presence of apoptotic damage was evaluated both by the determination of caspase-3-like protein activity and by Promega's fluorescent apoptotic detection system. Results. DEM and H₂O₂ affected the cultured cells in different ways. DEM induced a moderate cellular insult, which was efficaciously antagonized by idebenone-loaded PECA nanocapsules. H₂O₂ elicited severe damage to nuclear DNA, which was reduced by idebenone-loaded PECA nanocapsules. The free drug was less effective than idebenone-loaded nanocapsules. Conclusions. The findings reported here demonstrate that an improved antioxidant effect was obtained with a low idebenone concentration (0.5 M) when the drug was entrapped within Tween 80-coated PECA nanocapsules.     

Evaluation of endogenous urinary biomarkers for indirect detection of urine adulteration attempts by five different chemical adulterants in mass spectrometry methods

Reliable detection of urine adulteration attempts to circumvent positive drug testing represents a critical step for laboratories in abstinence control settings. An ideal workflow for high‐throughput testing would involve simultaneous detection of adulteration attempts in the same run with drug detection. Monitoring of degraded or oxidized endogenous urinary compounds as indirect markers has been previously evaluated for that purpose exemplified for the adulterant potassium nitrite (KNO₂). Fifteen, previously identified endogenous markers should now be evaluated for their general applicability to detect adulteration attempts for the adulterants hypochlorite‐based bleach (NaOCl), peroxidase and peroxide (H₂O₂), pyridinium chlorochromate (PCC), and iodine (I₂). Initial experiments revealed similar results for the tested adulterants regarding degradation of indolylacryloylglycine (IAG), uric acid (UA), or UA derivatives. 5‐Hydroxyisourate (HIU), the oxidation product of UA, was however only formed by KNO₂, PCC, and H₂O₂. Amino acids showed larger adulterant‐dependent differences. All reactions were shown to be influenced by the adulterant concentration and the urinary pH with large variances depending on compound and adulterant. Except for HIU/PCC, all markers were stable within +/? 30% variation for all adulterants at ?20°C. Receiver operating characteristics indicated best sensitivity and specificity over all adulterants for IAG (specificity 0.9, sensitivity 1.0) and UA (specificity 1.0, sensitivity 0.9). HIU gave best results for KNO₂, PCC, and H₂O₂ and N‐acetylneuraminic acid for PCC and H₂O₂, respectively. When integrating a limited number of targets into existing screening methods, monitoring of UA, IAG, N‐acetylneuraminic acid, and HIU is recommended.     

Mast Cell Tryptase is Increased in the Nasal and Bronchoalveolar Lavage Fluids of Humans after Ozone Exposure

Abstract

Human mast cell tryptase, a marker for mast cell degranulation, was immunologically measured in nasal lavage fluids (NALF) and bronchoalveolar lavage fluids (BALF) from subjects exposed to 0.4 ppm ozone (O₃) for 2 hr with intermittent exercise. Tryptase antigen was significantly elevated in the NALF immediately postexposure to O₃ (p - .0008) in comparison to samples taken immediately after exposure to air. Additionally, an examination of data from five subjects, serving as their own controls, revealed elevated tryptase levels immediately postexposure (p - .015) and 18 hr after exposure (p - .026), in comparison to corresponding samples obtained with air exposure. An examination of BALF samples (n - 9) from these same subjects 18 hr after exposure showed that tryptase was significantly (p - .01) elevated. These data suggest that O₃ exposure results in mast cell degranulation and that mast cell-derived mediators may contribute to the physiological effects noted with O₃ inhalation.     

3‐butyl‐6‐fluoro‐1 (3H)‐isobenzofuranone,a derivative of dl‐n‐butylphthalide,attenuates hydrogen peroxide‐induced damage in PC12 cells

The anti‐cerebral ischemia agent, dl‐3‐n‐butylphthalide (NBP), is effective in models of vascular dementia in animals. The present study investigates the protective effect of 3‐butyl‐6‐fluoro‐1 (3H)‐isobenzofuranone (6‐F‐NBP), a fluoro derivative of dl‐nbutylphthalide, in hydrogen peroxide (H₂O₂)‐induced damage in PC12 cells. Exposure of PC12 cells to H₂O₂ for 24 h led to decreased cell survival, glutathione peroxidase (GSH‐PX), and mitochondrial membrane potential (MMP). In contrast, malondialdehyde (MDA) production, nitric oxide synthase (NOS) activity, nitric oxide (NO) formation, and intracellular reactive oxygen species (ROS) were increased, as was intracellular accumulation of [Ca²⁺]_i. However, pretreatment with 6‐F‐NBP markedly reversed the changes induced by H₂O₂, exhibiting a protective effect against H₂O₂‐induced cytotoxicity in PC12 cells. The compound may have therapeutic potential in the treatment of cerebral ischemia by inhibiting the oxidative damage. Drug Dev Res 72: 259–264, 2011. © 2010 Wiley‐Liss, Inc.     

Effects on Lipid Peroxidation and Antioxidative Enzymes of Euonymus alatus in Cultured Rat Hepatocytes

Abstract: The Euonymus alatus (Thunb.) Sieb. has long been used as a crude drug. In this paper, we investigate the effects of E. alatus on cultured hepatocyte cell system and lipid peroxidation in hydrogen peroxide (H₂O₂) treatment conditions. The study covers the physiological activity (the antioxidative activity and the nitrite‐scavenging effect) of E. alatus. H₂O₂ that can produce intracellular free radical was used for inducer of the peroxidation of cellular lipids. Treatment of E. alatus attenuated in cell killing enhanced by increasing concentrations of H₂O₂. The increased malondialdehyde level induced by H₂O₂ treatment was reduced by pre‐treatment of E. alatus. Furthermore, addition of E. alatus in cell culture medium significantly reduced cell killing and content of intracellular antioxidants. Changes in nitrite‐scavenging effect of E. alatus at various concentrations (5–25 mg/ml) and various pH levels (pH 1.2, 4.2 and 6.0) were also observed. The present study was also done to investigate the effects of E. alatus on cultured hepatocyte cell system, H₂O₂‐induced cytotoxicity and antioxidative enzyme activities, including catalase, superoxide dismutase, glutathione peroxidase and glutathione S‐transferase in H₂O₂ treatment conditions. E. alatus treatment had significant protective or elevating activities on these antioxidative enzyme activities compared to a normal group. The results indicate that E. alatus provides a strong antioxidant protection of cells against H₂O₂‐induced oxidative stress.     

Hydrogen peroxide increases the activity of γ-glutamylcysteine synthetase in cultured Chinese hamster V79 cells

Hydrogen peroxide (H₂O₂) caused a rapid and a concentration-dependent increase in the activity of γ-glutamylcysteine synthetase (γ-GCS) in cultured Chinese hamster V79 cells. The increase in the activity was transient and declined rapidly during post-treatment incubation. Inhibition of protein synthesis by cycloheximide, chelation of divalent iron byo-phenanthroline, and scavenging of free radicals by butyl-4-hydroxyanisole failed to suppress the increase in activity of γ-GCS caused by H₂O₂. However, catalase completely inhibited the increase in the activity of the enzyme. H₂O₂ did not change the level of total glutathione (GSH+GSSG) but is oxidized GSH. The increased in levels of GSSG caused by H₂O₂ was enhanced byo-phenanthroline. These results suggest that the increase in activity of γ-GCS caused by H₂O₂ is not an inducible phenomenon, nor it is attributable to the action of free radicals generated by an iron-catalyzed Fenton reaction. Furthermore, the changes in levels of GSH and GSSG caused by H₂O₂ appear not to be responsible for the increase in activity of γ-GCS caused by the hydroperoxide. However, chemical reduction of the enzyme, the activity of which had been increased by H₂O₂, resulted in a decrease, in the activity, suggesting the involvement of oxidation of the enzyme in the increased activity of γ-GCS caused by H₂O₂. The results also suggest that the activity of γ-GCS in cultured V79 cells can be regulated by the cellular oxidation-reduction state.     

Hetastarch reduces neuronal cell death caused by oxidative stress

We investigated the effect of hetastarch, used for the treatment of acute ischemic stroke, on neuronal cell damage by oxidative stress, a main pathogenic mechanism in ischemic stroke. Neuronally differentiated PC12 cells (nPC12 cells) were treated with varying concentrations of hetastarch and hydrogen peroxide (H₂O₂), and their viability was measured with a 3,(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay and trypan blue staining. The effect of hetastarch on free radical production by H₂O₂ was evaluated using the fluorescent probe 2′,′‐dichlorodihydrofluorescein diacetate (DCFH‐DA) and by quantifying the amount of 2,5‐ and 2,3‐dihydroxybenzoic acid (DHBA). Additionally, the expression levels of BAX, Bid, Bcl‐2, Bcl‐xL, cytosolic cytochrome c, and cleaved caspase‐3 were examined using Western blot analysis. Following exposure to 100 µM H₂O₂, the viability of nPC12 cells significantly decreased; however, cell viability increased with hetastarch treatment. Free radical production related to H₂O₂ exposure was significantly reduced after 100 µM hetastarch treatment. The expression levels of BAX, Bid, cytosolic cytochrome c, and activated caspase‐3 were reduced, whereas Bcl‐2 and Bcl‐xL levels increased in H₂O₂‐injured nPC12 cells treated with 100 µM hetastarch, as compared with nPC12 cells that were treated with only 100 µM H₂O₂. These results demonstrate that hetastarch can reduce oxidative stress‐induced neuronal cell death. Drug Dev Res 73: 35–42, 2012. © 2011 Wiley Periodicals, Inc.     

Synthesis of N‐methylmorpholine N‐(17O‐oxide) and N‐methylmorpholine 15N‐(17O‐oxide)

N‐Methylmorpholine N‐(¹⁷O‐oxide) and N‐methylmorpholine ¹⁵N‐(¹⁷O‐oxide) were prepared from N‐methylmorpholine and ¹⁵N‐methylmorpholine by oxidation with H₂ ¹⁷O₂. The facile one‐pot procedure provided yields of 82 and 76%, respectively. The labeled hydrogen peroxide was obtained by electrolysis of H₂ ¹⁷O followed by autoxidation of 2‐ethylanthraquinol with the molecular oxygen ¹⁷O₂ generated. The compounds serve for mechanistic studies into gold nanoparticle generation in NMMO solution. Copyright © 2009 John Wiley & Sons, Ltd.     

A Possible Mechanism of Naproxen‐Induced Lipid Peroxidation in Rat Liver Microsomes

Abstract: Previous papers from our laboratory report that naproxen and salicylic acid induced lipid peroxidation in rat liver microsomes, however, the mechanism is still unclear. In the present paper, ferrous iron release, nicotinamide‐adenine dinucleotide phosphate reduced form (NADPH) oxidation and hydrogen peroxide (H₂O₂) formation have been measured to find out which mechanisms are involved in naproxen‐ and salicylic acid‐induced lipid peroxidation. While the increase of ferrous iron release was observed with high concentrations of naproxen, salicylic acid did not stimulate ferrous iron release. Neither of these drugs stimulated NADPH oxidation and H₂O₂ formation. However hexobarbital and perfluorohexane, known as uncouplers of cytochrome P450, stimulated microsomal NADPH oxidation, O₂ consumption, H₂O₂ formation and water (H₂O) formation involving four‐electron oxidase reaction. These results suggest that ferrous iron release contributes to naproxen‐induced microsomal lipid peroxidation and that naproxen and salicylic acid are not uncouplers of cytochrome P450. Apparently H₂O₂ does not play an important role in naproxen‐ and salicylic acid‐induced microsomal lipid peroxidation.     

Non‐IgE‐mediated hypersensitivity induced by multivitamins containing Tween‐80

Multivitamins have been widely used for years. Adverse reactions, especially hypersensitivity, to multivitamins are becoming noteworthy. However, the classification of hypersensitivity is confusing, and the trigger is unknown. Multivitamins consist of two vials labelled vial 1 containing Tween‐80 and vial 2. Multivitamins without Tween‐80 were used as a contrast. Behaviouristics, histamine, IgE, and blood pressure of beagle dogs and guinea‐pigs were investigated by observation, ELISA and sphygmomanometer, and degranulation and apoptotic of RBL‐2H3 cells were assayed by spectrophotometry and flow cytometry. The results showed that dogs suffered from multiorgan anaphylactoid symptoms, and dramatically decreased blood pressure, and high plasma concentrations of histamine after the first administration of multivitamins and multivitamins vial 1, which contains Tween‐80, compared to the control, multivitamins vial 2 or multivitamins without Tween‐80. In anaphylaxis assay, guinea‐pigs did not display any anaphylaxis symptoms and there were no changes in plasma histamine and IgE concentrations in the multivitamins and multivitamins vial 1 groups or in the multivitamins vial 2 and multivitamins without Tween‐80 groups except ovalbumin. Compared to the control, the release of β‐hexosaminidase and histamine, and the apoptosis of non‐antigen‐sensitized RBL‐2H3 cells significantly increased in the Tween‐80 and multivitamins and multivitamins vial 1 groups in a concentration‐dependent manner. However, there was no alteration in multivitamins vial 2 and multivitamins without Tween‐80 groups. The results indicate that the hypersensitivity induced by multivitamins may be anaphylactoid reaction, but not anaphylaxis. Multivitamin‐induced release of inflammatory factors is triggered by Tween‐80 through a non‐IgE‐mediated pathway.     

The versatility of polysorbate 80 (tween 80) as an ionophore

A number of experiments were performed to illustrate the unusual versatility of Polysorbate 80 (Tween 80) as an ionophore. New ions shown to be transported by it from and to water layers through a model membrane (CH₂Cl₂) include H₃O⁺, Li⁺, Pb²⁺, Co²⁺, piperidinium ion, guanidinium ion, and Paraquat, while two complex ions resisted transport under the conditions used. “Reverse” transport of lipophilic guests (azobenzene, azulene, ferrocene) from and to organic solvents through water was also promoted by Tween 80, but C₆₀ was not carried. Three water molecules were transported per molecule of KSCN by the Tween.     

Developmental exposure to decabrominated diphenyl ether (BDE‐209): Effects on sperm oxidative stress and chromatin dna damage in mouse offspring

Polybrominated diphenyl ethers (PBDEs) are used as brominated flame retardants and have been found in human milk in recent years. This study investigates whether prenatal exposure to decabrominated diphenyl ether (BDE‐209) induces sperm dysfunction in male offspring. Pregnant CD‐1 mice were gavaged once daily with corn oil (control), 10, 500, and 1500 mg kg^?1 body weight of BDE‐209 from day 0 of gestation to day 17. The outcomes of male reproductive parameters were assessed on postnatal day 71. Anogenital distance, sperm‐head abnormalities, and testicular histopathology were significantly affected in male offspring prenatally exposed to 1500 mg kg^?1. Significant increases in the tendency for sperm DNA denaturation (αT) induction and the DNA fragmentation index (DFI) were found in those exposed to 10, 500, and 1500 mg kg^?1 (P < 0.05). We observed a significant increase of sperm hydrogen peroxide (H₂O₂) generation in the 10 and 1500 mg/kg/day groups compared to the control group (P < 0.05). Although our findings suggested that the mechanisms underlying BDE‐209‐induced sperm DNA damage and H₂O₂ generation might not be represented as a dose‐response relationship, we found that the greater the excess production of sperm H₂O₂, the greater the sperm αT (r = 0.65, P = 0.0155) and DFI (r = 0.53, P = 0.002). In conclusion, developmental exposure to BDE‐209 induced sperm‐head abnormality, oxidative stress, chromatin DNA damage, and testicular histopathological changes. These findings suggest that BDE‐209‐induced male reproductive effects might involve the formation of sperm H₂O₂ which attacks nucleic acids via H₂O₂ generation. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2013.     

Synthesis of deuterium‐labeled 3‐O‐methyldopa and 4‐O‐methyldopa

Concise methods for the synthesis of 4‐hydroxy‐3‐[²H₃]‐methoxyphenylalanine (3‐O‐[²H₃]‐methydopa) and 3‐hydroxy‐4‐[²H₃]‐methoxyphenylalanine (4‐O‐[²H₃]‐methydopa) are described. The 3‐O‐[²H₃]‐methydopa is a valuable internal standard for the tandem MS quantification of 3‐O‐methyldopa, a metabolite of value in the diagnosis of aromatic l‐amino acid decarboxylase (AADC) deficiency. Copyright © 2003 John Wiley & Sons, Ltd.     

Preparation and characterization of poly(methyl methacrylate)—iron (III) oxide microparticles using a modified solvent evaporation method

At present, there is widespread interest in developing new, biocompatible microparticles made from polymers such as poly(methyl methacrylate) (PMMA) that could have applications ranging from diagnostic imaging to drug delivery. In these experiments, there were two primary objectives: (1) to stabilize a suspension of iron (III) oxide (α-Fe₂O₃; mean diameter?=?100?nm) nanoparticles in a solution of PMMA by using an emulsifier and different mixtures of two miscible solvents; and (2) to fabricate PMMA-α-Fe₂O₃ microparticles by using an oil-in-water (o/w) solvent evaporation method. By accomplishing the first objective, it was hypothesized that the encapsulation efficiency of α-Fe₂O₃ within PMMA microparticles would improve and induce the clustering of α-Fe₂O₃ along the circumferential edges of the microparticles. Of the seven emulsifiers tested, Tween 80 was selected primarily for its hydrophilicity and its ability to produce a stable α-Fe₂O₃ dispersion. As a result, 22 batches of microspheres (11 with Tween 80 and 11 without) were made and the solvent (dichloromethane) to co-solvent (ethyl acetate) ratios were varied. Particles made with solvent mixtures of >50% ethyl acetate (<50% dichloromethane) were more likely to be hollow and had larger mean volumetric particle diameters (>5 microns) than particles made with mixtures containing >50% dichloromethane. Particles made with Tween 80 were larger, more porous, and had α-Fe₂O₃ aligned along the circumferential edges of the particles. The use of solvent mixtures did not improve the encapsulation efficiency of α-Fe₂O₃ but the use of ethyl acetate helped to induce the clustering of α-Fe₂O₃ along the peripheries of the microparticles.     

Oxidant‐Induced Cell Death in Renal Epithelial Cells: Differential Effects of Inorganic and Organic Hydroperoxides

Abstract: This study was undertaken in order to examine the roles of lipid peroxidation and poly (ADP‐ribose) polymerase (PARP) activation in oxidant‐induced renal cell death. Opossum kidney cell cultures were used as the renal epithelial cell model, and an inorganic hydroperoxide H₂O₂ and an organic hydroperoxide t‐butylhydroperoxide were employed as model oxidants. Cell death by both oxidants could be prevented by thiols (dithiothreitol and glutathione), iron chelators (deferoxamine and phenanthroline), and hydroxyl radical scavengers (dimethylthiourea and pyruvate). Phenolic antioxidants N,N′‐diphenyl‐p‐phenylenediamine (DPPD) and butylated hydroxyanisole had no effect on the H₂O₂‐induced cell death. However, the t‐butylhydroperoxide‐induced cell death was effectively prevented by these antioxidants. The PARP inhibitor 3‐aminobenzamide prevented the cell death induced by H₂O₂, but not cell death by t‐butylhydroperoxide. The PARP activity was increased in cells exposed to H₂O₂ but not t‐butylhydroperoxide. Unlike in opossum kidney cells, in rabbit renal cortical slices both oxidants H₂O₂ and t‐butylhydroperoxide induced cell death through a lipid peroxidation‐dependent and PARP‐independent mechanism. Effects of DPPD and 3‐aminobenzamide on H₂O₂‐induced cell death in primary cultured rabbit proximal tubular cells were similar to those in opossum kidney cells. These results indicate that 1) the H₂O₂‐induced cell death in cultured renal epithelial cells is associated with PARP activation but not lipid peroxidation, whereas the t‐butylhydroperoxide‐induced cell death is mediated by lipid peroxidation, and 2) the role of lipid peroxidation in H₂O₂ cytotoxicity may be different between freshly isolated renal tubular cells and cultured renal epithelial cells.     

Inhibitory effects of Zataria multiflora essential oil and its main components on nitric oxide and hydrogen peroxide production in lipopolysaccharide-stimulated macrophages

Objectives Zataria multiflora is an aromatic plant that is used in flavouring and preserving foods and also used as an antispasmodic, anaesthetic and antinociceptive agent. In this study, the effects of Z. multiflora essential oil on nitric oxide (NO) and hydrogen peroxide (H₂O₂) production in lipopolysaccharide (LPS)‐stimulated macrophages was investigated. Methods Z. multiflora essential oil was extracted by water‐distillation, analysed by GC‐MS and then the effect of the essential oil on NO and H₂O₂ production was investigated. Key findings Carvacrol (52%), thymol (16%) and p‐cymene (10%) were the main components of the oil. The IC50 (concentration providing 50% inhibition) for reactive oxygen scavenging was estimated to be 5.7, 3 and 4.2 µg/ml for the essential oil, thymol and carvacrol, respectively, while the corresponding IC50 values for reactive nitrogen scavenging were estimated to be 8.6, 4.7 and 6.6 µg/ml. Z. multiflora essential oil, thymol, and carvacrol significantly reduced NO and H₂O₂ production as well as NO synthase and NADH oxidase activity in LPS‐stimulated murine macrophages while p‐cymene did not show any antioxidant activity. Conclusions Z. multiflora essential oil has the potential to be used in the therapy of oxidative damage.     

Synthesis of deuterium‐labelled (−)‐galanthamine

The synthesis of deuterium‐labelled galanthamine is reported. 6‐[²H₃]methoxy‐N‐[²H₃]methyl‐(?)‐galanthamine was obtained in seven steps from galanthamine. The synthesis was carried out by selective O‐ and N‐demethylations. The [²H₃]‐N‐methyl and [²H₃]‐O‐methyl‐groups were introduced by selective aminoreduction and O‐methylation. Copyright © 2008 John Wiley & Sons, Ltd.