Protective Effect of Aldo-keto Reductase 1B1 Against Neuronal Cell Damage Elicited by 4'-Fluoro-α-pyrrolidinononanophenone.

Chronic exposure to cathinone derivatives increases the risk of severe health hazards, whereas little is known about the detailed pathogenic mechanisms triggered by the derivatives. We have recently shown that treatment with α-pyrrolidinononanophenone (α-PNP, a highly lipophilic cathinone derivative possessing a long hydrocarbon main chain) provokes neuronal cell apoptosis and its 4'-fluorinated analog (F-α-PNP) potently augments the apoptotic effect. In this study, we found that neuronal SK-N-SH cell damage elicited by F-α-PNP treatment is augmented most potently by pre-incubation with an AKR1B1 inhibitor tolrestat, among specific inhibitors of four aldo-keto reductase (AKR) family members (1B1, 1C1, 1C2, and 1C3) expressed in the neuronal cells. In addition, forced overexpression of AKR1B1 remarkably lowered the cell sensitivity to F-α-PNP toxicity, clearly indicating that AKR1B1 protects from neurotoxicity of the derivative. Treatment of SK-N-SH cells with F-α-PNP resulted in a dose-dependent up-regulation of AKR1B1 expression and activation of its transcription factor NF-E2-related factor 2. Metabolic analyses using liquid chromatography/mass spectrometry/mass spectrometry revealed that AKR1B1 is hardly involved in the F-α-PNP metabolism. The F-α-PNP treatment resulted in production of reactive oxygen species and lipid peroxidation byproduct 4-hydroxy-2-nonenal (HNE) in the cells. The enhanced HNE level was reduced by overexpression of AKR1B1, which also lessened the cell damage elicited by HNE. These results suggest that the AKR1B1-mediated neuronal cell protection is due to detoxification of HNE formed by F-α-PNP treatment, but not to metabolism of the derivative.

Sibutramine facilitates apoptosis and contraction of aortic smooth muscle cells through elevating production of reactive oxygen species.

Sibutramine had been prescribed as an oral anorexiant that reduces dietary intake, but was withdrawn from the market due to frequent occurrence of severe cardiovascular events including hypertension. To elucidate the pathogenic mechanism of hypertension, we here investigated whether sibutramine facilitates damage and contraction of human aortic smooth muscle (HASM) cells or not. Treatment with sibutramine provoked HASM cell apoptosis, which was attributed to production of reactive oxygen species and mitochondria dysfunction. In addition, the drug treatment of the cell promoted calcium influx, phosphorylation of myosin light chain and contraction, which were abrogated by pretreating the cells with antioxidant and nitric oxide (NO) donor. Thus, the drug-evoked contraction is likely due to a preceding disturbance of balance between the drug-elicited reactive oxygen species production and exogenous NO supply. Compared to sibutramine, its N-desmethyl and N-didesmethyl metabolites exhibited much less toxicity and contraction against HASM cells, in which sibutramine was hardly demethylated. The low metabolic capacity of the cells may also be pertinent to the damage and contraction elicited by sibutramine. Taken together, our data suggest that sibutramine facilitates apoptosis and contraction of aortic smooth muscle cells through elevating production of reactive oxygen species and decreasing exogenous NO supply, leading to pathogenesis of hypertension.

Enhancement of Endothelial Barrier Permeability by Mitragynine.

Persistent inhalation of mitragynine (MG), a major alkaloid in the leaves of Mitragyna speciosa, causes various systemic adverse effects such as seizure, diarrhea and arthralgias, but its toxicity to endothelial cells and effects on barrier function of the cells are poorly understood. In this study, we compared toxicities of MG and mitraphylline, another constituent of the leaves, against human aortic endothelial (HAE), bronchial BEAS-2B, neuronal SK-N-SH, hepatic HepG2, kidney HEK293, gastric MKN45, colon DLD1, lung A549, breast MCF7 and prostate LNCaP cells, and found that MG, but not mitraphylline, shows higher toxicity to HAE cells compared to the other cells. Forty-eight-hours incubation of HAE cells with a high concentration of MG (60 µM) provoked apoptotic cell death, which was probably due to signaling through enhanced reactive oxygen species (ROS) generation and resultant caspase activation. Treatment of the cells with MG at sublethal concentrations less than 20 µM significantly lowered transendothelial electrical resistance and elevated paracellular permeability, without affecting the cell viability. In addition, the MG-elicited lowering of the resistance was abolished by a ROS inhibitor N-acetyl-L-cysteine and augmented by H2O2 and 9,10-phenanthrenequinone, which generates ROS through its redox cycle. These results suggest the contribution of ROS generation to the increase in endothelial barrier permeability.

α-Pyrrolidinononanophenone provokes apoptosis of neuronal cells through alterations in antioxidant properties.

In this study, we found that exposure to α-pyrrolidinononanophenone (α-PNP), a highly lipophilic synthetic cathinone, provokes apoptosis of human neuronal SK-N-SH cells. The drug sensitivity of the cells (50% lethal concentration of 12µM) was similar to those of aortic endothelial and smooth muscle cells, and was higher than those of cells derived from colon, liver, lung and kidney, suggesting that α-PNP overdose and abuse cause serious damage in central nervous and vascular systems. SK-N-SH cell treatment with lethal concentrations (20 and 50µM) of α-PNP facilitated the reactive oxygen species (ROS) production. The treatment also prompted elevation of Bax/Bcl-2 ratio, lowering of mitochondrial membrane potential, release of cytochrome-c into cytosol, and resultant activation of caspase-9 and caspase-3. The apoptotic events (caspase-3 activation and DNA fragmentation) were abolished by pretreatment with antioxidants, N-acetyl-l-cysteine and polyethyleneglycol-conjugated catalase. These results suggest that ROS production, mitochondrial dysfunction and caspase activation are potential events in the mechanism underlying the α-PNP-triggered neuronal cell apoptosis. Intriguingly, the α-PNP treatment of SK-N-SH cells was found to promote formation of 4-hydroxynonenal, a reactive aldehyde generated from lipid peroxidation. The α-PNP treatment also decreased cellular levels of total and reduced glutathiones, expression of γ-glutamylcysteine synthetase mRNA and glutathione reductase activity. Furthermore, the α-PNP treatment resulted in both decrease in proteasomal activities and increase in expression of autophagy-related factors, which were significantly prevented by pretreating with N-acetyl-l-cysteine. Therefore, the ROS formation by α-PNP treatment may be ascribable to the decrease in glutathione level through its consumption during 4-hydroxynonenal detoxification and dysfunction of both de novo synthesis and regeneration of glutathione, in addition to impairments in proteasomal and autophagic systems that degrade cellular oxidized components.

Sibutramine provokes apoptosis of aortic endothelial cells through altered production of reactive oxygen and nitrogen species.

Overdose administration of sibutramine, a serotonin-noradrenalin reuptake inhibitor, is considered to elicit severe side effects including hypertension, whose pathogenic mechanism remains unclear. Here, we found that 48-h incubation with >10µM sibutramine provokes apoptosis of human aortic endothelial (HAE) cells. Treatment with the lethal concentration of sibutramine facilitated production of reactive oxygen species (ROS), altered expression of endoplasmic reticulum stress response genes (heat shock protein 70 and C/EBP homologous protein), and inactivated 26S proteasome-based proteolysis. The treatment also decreased cellular level of nitric oxide (NO) through lowering of expression and activity of endothelial NO synthase. These results suggest that ROS production and depletion of NO are crucial events in the apoptotic mechanism and may be linked to the pathogenesis of vasoconstriction elicited by the drug. Compared to sibutramine, its metabolites (N-desmethylsibutramine and N-didesmethylsibutramine) were much less cytotoxic to HAE cells, which hardly metabolized sibutramine. In contrast, both the drug and metabolites showed low cytotoxicity to hepatic HepG2 cells with high metabolic potency and expression of cytochrome P450 (CYP) 3A4. The cytotoxicity of sibutramine to HepG2 and Chang Liver cells was remarkably augmented by inhibition and knockdown of CYP3A4. This study also suggests an inverse relationship between sibutramine cytotoxicity and CYP3A4-mediated metabolism into the N-desmethyl metabolites.

Simultaneous determination of antidepressants by non-aqueous capillary electrophoresis-time of flight mass spectrometry.

Simultaneous determination of 20 antidepressants in plasma samples was carried out by non-aqueous capillary electrophoresis with time of flight mass spectrometry via electrospray ionization, where a mixture of 60 mM ammonium acetate and 1M acetic acid in acetonitrile, and water, as well as methanol (100:1:0.5, v/v/v) was selected as the background electrolyte. By using time of flight mass spectrometry, accurate mass information was obtained and the background noise was dramatically decreased, thus causing a great improvement in qualitative ability. As for the plasma sample, solid phase extraction with Oasis HLB was used. The limits of detection and quantification were in the range of 0.5-1 and 1-5 ng/ml, respectively. The sensitivity of the present method was found better, i.e. approximately 10-60 folds compared to that using photo diode array detectors because the analyte peak could be clearly distinguished from the background derived from the plasma. The present method was found very useful and practical as regards to routine analysis of plasma samples.

Simultaneous determination of antidepressants by non-aqueous or quasi-non-aqueous capillary electrophoresis.

Simultaneous determinations of 20 antidepressants were carried out by non-aqueous capillary electrophoresis using a background electrolyte consisting of an organic solvent. A bubble cell fused silica capillary (112.5 cm x 50 microm i.d., 150 microm i.d. bubble) was used as an electrophoresis tube. The determination was carried out at 215 nm, while the detection wavelength between 190 and 500 nm was selected for qualitative analysis. When an acetonitrile solution alone was used as the background electrolyte, good separation was observed, but it was not sufficient to separate all tested analytes. It was found that better separation was acquired by adding a few other solvents into acetonitrile, such as water and methanol; the best separation was achieved with a mixture of acetonitrile containing 60 mM ammonium acetate and 1 M acetic acid/water/methanol (100:1:0.5, v/v/v). As for the plasma sample, liquid-liquid extraction and solid-phase extraction (SPE) were considered; as a result, SPE with Oasis HLB was found to be most suitable. The present method is very useful as regards to plasma samples.

On-line sample extraction and enrichment of non-steroidal anti-inflammatory drugs by pre-column in capillary liquid chromatography mass spectrometry.

A rapid and sensitive analytical method has been developed for the simultaneous determination of 16 non-steroidal anti-inflammatory drugs (NSAIDs) in human plasma by capillary liquid chromatography (LC) and quadrupole mass spectrometry with electrospray ionization operated in the negative ion mode. The sample clean-up and enrichment on a pre-column were accomplished on-line to improve the sensitivity. This method greatly reduced sample preparation time and sample volume compared with off-line sample extraction methods and conventional LC methods, respectively. The recoveries of NSAIDs from human plasma were 56.7-96.9%. The total analytical time for a single analytical run was approximately 15 min. The detection limits of NSAIDs were 0.001-0.075 microg ml(-1) using a selected ion monitoring mode.

Rapid and simultaneous determination of nonsteroidal anti-inflammatory drugs in human plasma by LC-MS with solid-phase extraction.

A rapid, selective and sensitive analytical method for the simultaneous determination of 16 nonsteroidal anti-inflammatory drugs (NSAIDs) in human plasma was carried out using Oasis HLB solid-phase extraction (SPE), followed by reversed-phase high-performance liquid chromatography and quadrupole mass spectrometry with electrospray ionization operated in the negative ion mode. The recoveries of NSAIDs from human plasma by SPE were greater than 76.7%. The use of a short column packed with small particles enabled rapid and simultaneous determination within 7 min. The detection limits for the NSAIDs were 0.01-0.9 mug/ml using the selected ion monitoring mode.

Direct determination of non-steroidal anti-inflammatory drugs by column-switching LC-MS.

A method for determination of 16 non-steroidal anti-inflammatory drugs (NSAIDs) in human plasma samples without time-consuming sample pre-treatments was developed. The system consisted of two pumps for mobile phase delivery, a six-port switching valve, a pre-column (Oasis HLB Cartridge Column), and a reversed phase analytical column (COSMOSIL 3C18-MS-II). The analytes were trapped on the precolumn and subsequently separated on the analytical column. The present method allowed on-line sample clean-up and enrichment, leading to improved sensitivity without any tedious sample preparation. The recoveries of NSAIDs from human plasma by column-switching were greater than 72.6%. The total analysis time for a single analytical run was approximately 11 min. The detection limits of NSAIDs were 0.0025 to 0.2 microg/mL using the selected ion monitoring mode.

Use of disposable GRC electrodes for the detection of phenol and chlorophenols in liquid chromatography.

In this study, a wall-jet flow cell with a GRC (graphite reinforced by carbon) electrode was designed for the amperometric detection of phenol and chlorophenols in liquid chromatography. The voltammetric responses of these analytes at the GRC electrodes are very similar to those at conventional glassy carbon electrodes. As the GRC electrodes were made of the same materials as commercially available mechanical pencil leads, they exhibit the advantages of low cost, simple surface renewability, lower residual current, and good electrode-to-electrode reproducibility, and thus can be used as disposable-type electrodes. Chromatographic separations of phenol, o-chlorophenol (o-CP), 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP), and pentachlorophenol (PCP) were achieved with an ODS column using a mobile phase containing a mixture of CH3CN and H20 (40:60) containing 25 mM L-(+)tartaric acid (pH = 4.5). Amperometric detections were based on the electrochemical oxidation of these compounds around +0.9 V vs. Ag/AgCl. Under the optimized conditions, linear calibrations were obtained in a range up to 100 microM for phenol, o-CP, 2,4-DCP, 2,4,6-TCP, and 200 microM for PCP, with the correlation coefficients r2 of 0.9992, 0.9997, 0.9986, 0.9992, and 0.9968, respectively. The chromatographic detection limits for the tested analytes were obtained at pmol levels.