Mechanism of cytotoxic action of perfluorinated acids. III. Disturbance in Ca²+ homeostasis

The global distribution of perfluorinated acids (PFAs) in industry and in household is well known. Their increasing environmental occurrence and biomagnification in the living organisms have drawn growing interests in efforts to describe precisely the mechanisms of action in vitro and in vivo.Our previous investigations widely described lipophilicity-dependent cytotoxicity of PFAs as well as the effect of perfluorination of carbon chain on depolarization of plasma membrane potential, acidification or mitochondrial dysfunctions. In this study we presented in dose- and time-dependent manner the impact of PFAs on calcium homeostasis in HCT116 cells. Comparative analysis of cytosolic [Ca²⁺]_c and mitochondrial calcium [Ca²⁺]_m carried out by flow cytometry revealed distinct uptake of calcium into mitochondria in correlation to increasing lipophilicity of PFAs. Massive accumulation of [Ca²⁺]_m was not accompanied by equivalent loss of [Ca²⁺]_c. Indeed, moderate changes of [Ca²⁺]_c were observed after incubation with 400 μM PFDoDA reaching 29.83% and 49.17% decrease at 4th and 72nd hour, respectively. At the same time, mitochondrial calcium uptake increased from 2- to more than 4-fold comparing with non-treated cells. Incubation with non-fluorinated decanoic acid (DA) did not cause any changes in calcium homeostasis.Presented data show that PFAs-induced perturbations in calcium distribution seem to be a missing link related to mitochondria dysfunction playing a crucial role in determination of apoptotic cell death. Complete scheme for the mechanism of cytotoxic action of PFAs has been included.     

A decrease of intracellular ATP is compensated by increased respiration and acidification at sub-lethal parathion concentrations in murine embryonic neuronal cells: measurements in metabolic cell-culture chips

We present a label-free in vitro method for testing the toxic potentials of chemical substances using primary neuronal cells. The cells were prepared from 16-day-old NMRI mouse embryos and cultured on silicon chips (www.bionas.de) under the influence of different parathion concentrations with sensors for respiration (Clark-type oxygen electrodes), acidification (pH-ISFETs) and cell adhesion (interdigitated electrode structures, IDES). After 12 days in vitro, the sensor readouts were simultaneously recorded for 350 min in the presence of parathion applying a serial 1:3 dilution. The parathion-dependent data was fitted by logistic functions. IC₅₀ values of approximately 105 μM, 65 μM, and 54 μM were found for respiration, acidification, and adhesion, respectively. An IC₅₀ value of approximately 36 μM was determined from the intracellular ATP-levels of cells, which were detected by an ATP-luminescence assay using micro-well plates. While the intracellular ATP level and cell adhesion showed no deviation from a simple logistic decay, increases of approximately 29% in the respiration and 15% in the acidification rates above the control values were found at low parathion concentrations, indicating hormesis. These increases could be fitted by a modified logistic function. We believe that the label-free, continuous, multi-parametric monitoring of cell-metabolic processes may have applications in systems-biology and biomedical research, as well as in environmental monitoring. The parallel characterization of IC₅₀ values and hormetic effects may provide new insights into the metabolic mechanisms of toxic challenges to the cell.     

A search for hepatoprotective activity of aqueous extract of Rhus coriaria L. against oxidative stress cytotoxicity

The protective effects of different concentrations of aqueous extract of Rhus coriaria L. fruit (75 and 100 μg/ml) and also gallic acid (100 μM) as one of its main components were examined against oxidative stress toxicity induced by cumene hydroperoxide (CHP) in isolated rat hepatocytes. Both extract concentrations and gallic acid (100 μM) significantly (P < 0.05) protected the hepatocyte against all oxidative stress markers including cell lysis, ROS generation, lipid peroxidation, glutathione depletion, mitochondrial membrane potential decrease, lysosomal membrane oxidative damage and cellular proteolysis. Aqueous extracts of Rhus coriaria L. (75 and 100 μg/ml) were more effective than gallic acid (100 μM) in protecting hepatocytes against CHP induced lipid peroxidation (P < 0.05). On the other hand gallic acid (100 μM) acted more effective than aqueous extracts of Rhus coriaria L. (75 and 100 μg/ml) at preventing hepatocyte membrane lysis (P < 0.05). In addition H₂O₂ scavenging effect of both extract concentrations (75 and 100 μg/ml) were determined in hepatocytes and compared with gallic acid (100 μM). Gallic acid (100 μM) was more effective than aqueous extracts of Rhus coriaria L. (75 and 100 μg/ml) at H₂O₂ scavenging activity (P < 0.05).     

Mechanism of cytotoxic action of perfluorinated acids: II. Disruption of mitochondrial bioenergetics

PFAs and derivatives due to perfect technological properties are broadly applied in industry and consumer goods, and in consequence widely disseminated, environmentally bioaccumulative and found at ppb level in human serum.Earlier we revealed that in vitro cytotoxicity increases with chain length (CF₆-CF₁₄). The compounds dissipate plasma membrane potential and acidify of cytosol. Here we determine whether there is an association between the protonophoric uncoupling of respiration and disruption of bioenergetics caused by CF₆-CF₁₂ on HCT116 cell apoptosis.Again the effects were stronger for longer molecules. Incubation of cells with CF₁₀ stimulated time-dependent generation of reactive oxygen species, opening of mitochondrial permeability transition (MPT) pore, release of cytochrome c, activation of caspases and depletion of intracellular level of ATP occurring in intrinsic pathway of apoptosis. Incubation with decanoic acid (DA) did not lead to mitochondrial dysfunctions neither to cell cycle disturbances. Synchronized removal of the phosphorylated state of Akt, ERK1/2 and PKCδ/θ kinases by CF₁₀ suggests presence of concerted action to uninhibit Bad protein activation and a cascade of intrinsic pathway of apoptosis. Blocking MPT pore by cyclosporin A (CsA) led to a reduction of mitochondrial potential dissipation (mtΔΨ). Such cells neither showed cytochrome c release nor the downstream activation of caspase-9 and caspase-3. Our results confirm that mitochondria play a crucial role in perfluorochemicals induced apoptosis by releasing apoptotic signals through MPT pore.     

Perchlorate transport and inhibition of the sodium iodide symporter measured with the yellow fluorescent protein variant YFP-H148Q/I152L

Perchlorate is an environmental contaminant that impairs thyroid function by interacting with the sodium iodide symporter (NIS), the transporter responsible for iodide uptake in the thyroid gland. Perchlorate is well known as a competitive inhibitor of iodide transport by NIS, and recent evidence demonstrates that NIS can also transport perchlorate. In this study, we evaluated the yellow fluorescent protein (YFP) variant YFP-H148Q/I152L, as a genetically encodable biosensor of intracellular perchlorate concentration monitored by real-time fluorescence microscopy. Fluorescence of recombinant YFP-H148Q/I152L was suppressed by perchlorate and iodide with similar affinities of 1.2 mM and 1.6 mM, respectively. Perchlorate suppressed YFP-H148Q/I152L fluorescence in FRTL-5 thyroid cells and NIS-expressing COS-7 cells, but had no effect on COS-7 cells lacking NIS. Fluorescence changes in FRTL-5 cells were Na⁺-dependent, consistent with the Na⁺-dependence of NIS activity. Perchlorate uptake in FRTL-5 cells resulted in 10-fold lower intracellular concentrations than iodide uptake, and was characterized by a higher affinity (K_m 4.6 μM for perchlorate and 34.8 μM for iodide) and lower maximal velocity (V_max 6.8 μM/s for perchlorate and 39.5 μM/s for iodide). Perchlorate also prevented iodide-induced changes in YFP-H148Q/I152L fluorescence in FRTL-5 cells, with half-maximal inhibition occurring at 1.1-1.6 μM. In conclusion, YFP-H148Q/I152L detects perchlorate accumulation by thyroid and other NIS-expressing cells, and reveals differences in the kinetics of perchlorate versus iodide transport by NIS.     

Cytotoxic activity of sanguinarine and dihydrosanguinarine in human promyelocytic leukemia HL-60 cells

The benzo[c]phenanthridine alkaloid sanguinarine has been studied for its antiproliferative activity in many cell types. Almost nothing however, is known about the cytotoxic effects of dihydrosanguinarine, a metabolite of sanguinarine. We compared the cytotoxicity of sanguinarine and dihydrosanguinarine in human leukemia HL-60 cells. Sanguinarine produced a dose-dependent decline in cell viability with IC₅₀ (inhibitor concentration required for 50% inhibition of cell viability) of 0.9 μM as determined by MTT assay after 4 h exposure. Dihydrosanguinarine showed much less cytotoxicity than sanguinarine: at the highest concentration tested (20 μM) and 24 h exposure, dihydrosanguinarine decreased viability only to 52%. Cytotoxic effects of both alkaloids were accompanied by activation of the intrinsic apoptotic pathway since we observed the dissipation of mitochondrial membrane potential, induction of caspase-9 and -3 activities, the appearance of sub-G₁ DNA and loss of plasma membrane asymmetry. This aside, sanguinarine also increased the activity of caspase-8. As shown by flow cytometry using annexin V/propidium iodide staining, 0.5 μM sanguinarine induced apoptosis while 1–4 μM sanguinarine caused necrotic cell death. In contrast, dihydrosanguinarine at concentrations from 5 μM induced primarily necrosis, whereas apoptosis occurred at 10 μM and above. We conclude that both alkaloids may cause, depending on the alkaloid concentration, both necrosis and apoptosis of HL-60 cells.     

Electrophysiological characterisation of the actions of kynurenic acid at ligand-gated ion channels

To better understand the effects of the tryptophan metabolite kynurenic acid (kynA) in the brain, we characterised its actions at five ligand-gated ion channels: NMDA, AMPA, GABA_A, glycine and α7 nicotinic acetylcholine receptors. Using whole-cell patch-clamp recordings, we found that kynA was a more potent antagonist at human NR1a/NR2A compared with NR1a/NR2B receptors (IC₅₀: 158 μM and 681 μM, respectively; in 30 μM glycine). KynA inhibited AMPA-evoked currents to a similar degree in cultured hippocampal neurons and a human GluR2(flip/unedited) cell line (IC₅₀: 433 and 596 μM, respectively) and at higher concentrations, kynA also inhibited the strychnine-sensitive glycine receptor (∼35% inhibition by 3 mM kynA). Interestingly, kynA inhibited the peak amplitude (IC₅₀: 2.9 mM for 10 μM GABA) and slowed the decay kinetics of GABA-evoked currents in cultured neurons. In contrast, we found that kynA (1-3 mM) had no effect on ACh-evoked, methyllycaconitine (MLA)-sensitive currents in a human α7 nicotinic receptor (nAChR) cell line, rat hippocampal neurons in primary culture or CA1 stratum radiatum interneurons in rat brain slices. However, DMSO (>1%) did inhibit α7 nAChR-mediated currents. In conclusion, kynA is an antagonist at NMDA, AMPA and glycine receptors and a modulator of GABA_A receptors, but we find no evidence for any effect of kynA at the α7 nAChR.     

Acute oral toxicity in mice of a new palytoxin analog: 42-Hydroxy-palytoxin

The acute oral toxicity of a new palytoxin congener, 42-hydroxy-palytoxin (42-OH-PLTX), was investigated in female CD-1 mice. The toxin (300-1697 μg/kg), administered by gavage, induced scratching, jumping, respiratory distress, cyanosis, paralysis and death of mice, with an LD₅₀ of 651 μg/kg (95% confidence limits: 384-1018 μg/kg) within 24 h. Hematoclinical analyses showed increased plasma levels of lactate dehydrogenase and aspartate-aminotransferase at doses of 600 μg/kg and above, as well as of alanine-aminotransferase, creatine phosphokinase and potassium ions at ≥848 μg/kg. Histology revealed inflammatory lesions in the non-glandular area of the stomach of mice that survived up to 24 h after gavage (424-1200 μg/kg). Although no histological alterations were seen in skeletal and cardiac muscles, changes in some plasma biomarkers (creatine phosphokinase, lactate dehydrogenase) suggested involvement of these tissues in 42-OH-PLTX oral toxicity, in agreement with epidemiological data on seafood poisonings ascribed to palytoxins. Complete recovery of the tissue and hematological changes was observed two weeks post-exposure.Furthermore, 42-OH-PLTX induced in vitro delayed erythrocyte hemolysis at concentrations similar to those of PLTX (EC₅₀ = 7.6 and 13.2 × 10⁻¹² M, respectively). This hemolysis could be completely neutralized by a monoclonal anti-PLTX antibody. The in vivo data, together with the in vitro data recorded for 42-OH-PLTX, seem to indicate Na⁺/K⁺-ATPase as one of the key cellular targets of this toxin.     

Determination of dynamic doxorubicin-EC50 value in an automated high-content workstation for cellular assays

To overcome the problems of endpoint tests routinely required for EC₅₀ determination, we utilized a novel automated high-content workstation and calculated a time-resolved EC₅₀ value of MCF-7 mamma carcinoma cells treated with a pharmacologic agent. Measuring parameters were the cellular oxygen consumption and the extracellular acidification. These parameters were detected in real-time and label free with optochemical sensor spots in a modified 24-well sensor plate. In particular, the objective was to compare the measuring data of the workstation with a classical standard resazurin cell assay and to transfer the benefit of continuously recorded metabolic data of the workstation to practical time-resolved information about the effect of the applied active reagent (doxorubicin). MCF-7 cells were treated with a broad range of doxorubicin concentrations (100 μM to 1 nM) over 24 h and cellular activities were investigated with both, the resazurin assay and the automated workstation. Twenty-four hours after treatment, the resazurin assay showed an EC₅₀ value (6.3 μM) which was about one decade above the value obtained from oxygen consumption rate (0.37 μM) and extracellular acidification rate (0.72 μM), measured with the workstation. Presumably, the differences are due to the different metabolic nature and regulation behind these measuring parameters. By polynomial fitting of continuously recorded metabolic data, we were able to point out a dynamic, time-resolved EC₅₀ characteristic for different time points. The workstation is a powerful tool to record in vitro kinetic data of pharmacologic effects in vital cells in an automated experimental run.     

Effects of AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide], a novel inhibitor of human microsomal prostaglandin E synthase-1, on prostanoid biosynthesis in human monocytes in vitro

Inhibitors of microsomal prostaglandin (PG) E synthase-1 (mPGES-1) are being developed for the relief of pain. Redirection of the PGH₂ substrate to other PG synthases, found both in vitro and in vivo, in mPGES-1 knockout mice, may influence their efficacy and safety. We characterized the contribution of mPGES-1 to PGH₂ metabolism in lipopolysaccharide (LPS)-stimulated isolated human monocytes and whole blood by studying the synthesis of prostanoids [PGE₂, thromboxane (TX)B₂, PGF_2α and 6-keto-PGF_1α] and expression of cyclooxygenase (COX)-isozymes and down-stream synthases in the presence of pharmacological inhibition by the novel mPGES-1 inhibitor AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide]. AF3442 caused a concentration-dependent inhibition of PGE₂ in human recombinant mPGES-1 with an IC₅₀ of 0.06 μM. In LPS-stimulated monocytes, AF3442 caused a concentration-dependent reduction of PGE₂ biosynthesis with an IC₅₀ of 0.41 μM. At 1 μM, AF3442 caused maximal selective inhibitory effect of PGE₂ biosynthesis by 61 ± 3.3% (mean ± SEM, P < 0.01 versus DMSO vehicle) without significantly affecting other prostanoids (i.e. TXB₂, PGF_2α and 6-keto-PGF_1α). In LPS-stimulated whole blood, AF3442 inhibited in a concentration-dependent fashion inducible PGE₂ biosynthesis with an IC₅₀ of 29 μM. A statistically significant inhibition of mPGES-1 activity was detected at 10 and 100 μM (38 ± 14%, P < 0.05, and 69 ± 5%, P < 0.01, respectively). Up to 100 μM, the other prostanoids were not significantly affected. In conclusion, AF3442 is a selective mPGES-1 inhibitor which reduced monocyte PGE₂ generation also in the presence of plasma proteins. Pharmacological inhibition of mPGES-1 did not translate into redirection of PGH₂ metabolism towards other terminal PG synthases in monocytes. The functional relevance of this observation deserves to be investigated in vivo.     

Citrinin-induced fluidization of the plasma membrane of the fission yeast Schizosaccharomyces pombe

Citrinin (CTN) is a toxic fungal metabolite that is a hazardous contaminant of foods and feeds. In the present study, its acute toxicity and effects on the plasma membrane of Schizosaccharomyces pombe were investigated. The minimum inhibitory concentration of CTN against the yeast cells proved to be 500 μM. Treatment with 0, 250, 500 or 1000 μM CTN for 60 min resulted in a 0%, 2%, 21% or 100% decrease, respectively, in the survival rate of the cell population. Treatment of cells with 0, 100, 500 or 1000 μM CTN for 20 min induced decrease in the phase-transition temperature of the 5-doxylstearic acid-labeled plasma membrane to 16.51, 16.04, 14.18 or 13.98 °C, respectively as measured by electron paramagnetic resonance spectroscopy. This perturbation was accompanied by the efflux of essential K⁺ from the cells. The existence of an interaction between CTN and glutathione was detected for the first time by spectrofluorometry. Our observations may suggest a direct interaction of CTN with the free sulfhydryl groups of the integral proteins of the plasma membrane, leading to dose-dependent membrane fluidization. The change in fluidity disturbed the ionic homeostasis, contributing to the death of the cells, which is a novel aspect of CTN cytotoxicity.     

A peripherally acting,selective T-type calcium channel blocker,ABT-639, effectively reduces nociceptive and neuropathic pain in rats

Activation of T-type Ca²⁺ channels contributes to nociceptive signaling by facilitating action potential bursting and modulation of membrane potentials during periods of neuronal hyperexcitability. The role of T-type Ca²⁺ channels in chronic pain is supported by gene knockdown studies showing that decreased Ca_v3.2 channel expression results in the loss of low voltage-activated (LVA) currents in dorsal root ganglion (DRG) neurons and attenuation of neuropathic pain in the chronic constriction injury (CCI) model. ABT-639 is a novel, peripherally acting, selective T-type Ca²⁺ channel blocker. ABT-639 blocks recombinant human T-type (Ca_v3.2) Ca²⁺ channels in a voltage-dependent fashion (IC₅₀ = 2 μM) and attenuates LVA currents in rat DRG neurons (IC₅₀ = 8 μM). ABT-639 was significantly less active at other Ca²⁺ channels (e.g. Ca_v1.2 and Ca_v2.2) (IC₅₀ > 30 μM). ABT-639 has high oral bioavailability (%F = 73), low protein binding (88.9%) and a low brain:plasma ratio (0.05:1) in rodents. Following oral administration ABT-639 produced dose-dependent antinociception in a rat model of knee joint pain (ED₅₀ = 2 mg/kg, p.o.). ABT-639 (10–100 mg/kg, p.o.) also increased tactile allodynia thresholds in multiple models of neuropathic pain (e.g. spinal nerve ligation, CCI, and vincristine-induced, and capsaicin secondary hypersensitivity). ABT-639 did not attenuate hyperalgesia in inflammatory pain models induced by complete Freund's adjuvant or carrageenan. At higher doses (e.g. 100 - 300 mg/kg) ABT-639 did not significantly alter hemodynamic or psychomotor function. The antinociceptive profile of ABT-639 provides novel insights into the role of peripheral T-type (Ca_v3.2) channels in chronic pain states.     

In vitro inhibitory effects of components from Salvia miltiorrhiza on catalytic activity of three human Arachidonic acid ω-hydroxylases

CYP4 enzymes are involved in the metabolism of xenobiotics and endogenous molecules. 20-Hydroxyeicosatetraenoic acid (20-HETE), the arachidonic acid (AA) ω-hydroxylation metabolite catalyzed by CYP4A/4F enzymes, is implicated in various biological functions. The goal of this investigation is to examine the inhibitory effects of components from Salvia miltiorrhiza(Danshen) on AA ω-hydroxylation using recombinant CYP4A11, CYP4F2, CYP4F3B, and microsomal systems. Tanshinone IIA had noncompetitive inhibition on CYP4F3B (K_i = 4.98 μM). Cryptotanshinone (K_i = 6.87 μM) and tanshinone I (K_i = 0.42 μM) had mixed-type inhibition on CYP4A11. Dihydrotanshinone I had mixed-type inhibition on CYP4A11 (K_i = 0.09 μM), and noncompetitive inhibition on CYP4F2 (K_i = 4.25 μM) and CYP4F3B (K_i = 3.08 μM). Salvianolic acid A had competitive inhibition on CYP4A11 (K_i = 19.37 μM), and noncompetitive inhibition on CYP4F2 (K_i = 15.28 μM) and CYP4F3B (K_i = 6.45 μM). Salvianolic acid C had noncompetitive inhibition on CYP4F2 (K_i = 5.70 μM) and CYP4F3B (K_i = 18.64 μM). In human kidney, human liver or rat heart microsomes, 20-HETE formation was significantly inhibited (P < 0.05) by dihydrotanshinone I (5 and 20 μM) and salvianolic acid A (20 and 50 μM). Given that low plasma concentrations of Danshen components after oral administration, Danshen preparations may not play pharmacological roles by inhibiting AA ω-hydroxylases; however, as Danshen components may reach high concentration in human intestine, drugs that have an important pre-systemic metabolism by these CYP4A/4F enzymes should avoid being co-administered with Danshen preparations.     

A Ca2+ chelator ameliorates chromium (VI)-induced hepatocyte L-02 injury via down-regulation of voltage-Dependent anion channel 1 (VDAC1) expression

Hexavalent chromium could result in cell malfunctions. Intracellular Ca²⁺ ([Ca²⁺]_i) content and VDAC1 expression are both important features related to cell survial. This study aimed to explore the mechanism of cell injury induced by Cr(VI) and tentatively offer clues to repairing this cell damage using [Ca²⁺]_i and VDAC1. L-02 hepatocytes were treated with Cr(VI)/BAPTA, and the levels of [Ca²⁺]_i and cell injury associated with Cr(VI) were determined in addition to the effect of BAPTA. The expression of VDAC1 in Cr(VI)-induced cells was evaluated. The results showed a dose-dependent elevation of the level of VDAC1 and the mRNA level of the VDAC1 biogenesis-related gene Sam50. BAPTA could ameliorate less severe damage induced by 4 μM Cr(VI) via reducing VDAC1 and Sam50. Additionally, cell injury caused by less than 4 μM Cr(VI) could be ameliorated by VDAC1 knockdown. Taken together, the findings of this study suggest that inhibition of intracellular Ca^2± overload could protect cells from damage and that VDAC1 plays a considerable role in Cr(VI)-induced liver injury.     

Contribution of NO,ATP-sensitive K+ channels and prostaglandins to adenosine receptor agonists-induced relaxation of the rat tail artery

The mechanism of relaxation in the rat tail artery induced by the adenosine A1 receptor-selective agonist N⁶-cyclohexyladenosine (CHA, 10 nM–300 μM) and the adenosine A1/A_2a receptor agonist 5’-N-ethylcarboxamidoadenosine (NECA, 10 nM–300 μM) has been characterized. To do this, we used α₁-receptor agonist phenylephrine to evoke contraction (10 μM), and inhibitors of nitric oxide synthase (L-NAME, 10 μM), ATP-sensitive K⁺ channels (glibenclamide, 10 μM) and prostaglandin synthesis (indomethacin, 10 μM). CHA and NECA induced relaxation of rat-tail artery by 80% and 70% in a concentration-dependent manner, respectively. The relaxation effect of NECA was completely abolished in the presence of L-NAME, while glibenclamide and indomethacin prevented CHA-induced relaxation of the rat tail artery by approximately 25% and 40%, respectively. Our results indicate that nonspecific effects such nitric oxide and prostaglandins release or the activation of potassium channels significantly contributed to the effects of CHA and NECA.     

Tellurium tetrachloride and diphenyl ditelluride cause cytotoxicity in rat hippocampal astrocytes

Tellurium tetrachloride (TeCl₄) and diphenyl ditelluride (DPDT) cytotoxicity, was investigated in rat astrocytes. Concentrations of 0.24-250 μM (24 h) were tested for viability using MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and trypan blue exclusion. MTT showed significant decreases at all concentrations tested for both compounds. Significant decreases in viability were seen in 1.95-250 μM of DPDT and 0.97-250 μM of TeCl₄ with trypan blue exclusion. The LC₅₀ for both compounds was 62.5 μM. Light and scanning microscopy confirm toxicity observed at higher concentrations. Thiobarbituric acid reactive substances (TBARs) assay, TUNEL, cytochrome c and caspase release were carried out. No significant increase in TBARS with either agent was observed (15.625-62.5 μM). TUNEL and cytochrome c assays demonstrated apoptosis in TeCl₄ treated cells (31.25-125 μM). Non-apoptotic cells were observed in DPDT treated cells. Studies of caspase 3/7 and caspase 9 indicated increased activity in TeCl₄ but not in DPDT treated cells. Optical Emission Spectroscopy of DPDT and TeCl₄ treated cells demonstrated significant accumulation of elemental tellurium in all treatment groups (31.25-125 μM). We conclude that DPDT and TeCl₄ are cytotoxic to astrocytes. TeCl₄ treated cells die via the intrinsic apoptotic pathway. Accumulation of tellurium occurs with both compounds, but results in different mechanisms of cell death.     

Manganese accumulation in membrane fractions of primary astrocytes is associated with decreased γ-aminobutyric acid (GABA) uptake,and is exacerbated by oleic acid and palmitate

Manganese (Mn) exposure interferes with GABA uptake; however, the effects of Mn on GABA transport proteins (GATs) have not been identified. We sought to characterize how Mn impairs GAT function in primary rat astrocytes. Astrocytes exposed to Mn (500 μM) had significantly reduced ³H-GABA uptake despite no change in membrane or cytosolic GAT3 protein levels. Co-treatment with 100 μM oleic or palmitic acids (both known to be elevated in Mn neurotoxicity), exacerbated the Mn-induced decline in ³H-GABA uptake. Mn accumulation in the membrane fraction of astrocytes was enhanced with fatty acid administration, and was negatively correlated with ³H-GABA uptake. Furthermore, control cells exposed to Mn only during the experimental uptake had significantly reduced ³H-GABA uptake, and the addition of GABA (50 μM) blunted cytosolic Mn accumulation. These data indicate that reduced GAT function in astrocytes is influenced by Mn and fatty acids accumulating at or interacting with the plasma membrane.     

Application of novel Ni(II) complex and ZrO2 nanoparticle as mediators for electrocatalytic determination of N-acetylcysteine in drug samples

The electrooxidation of N-acetylcysteine (N-AC) was studied by a novel Ni(II) complex modified ZrO₂ nanoparticle carbon paste electrode [Ni(II)/ZrO₂/NPs/CPE] using voltammetric methods. The results showed that Ni(II)/ZrO₂/NPs/CPE had high electrocatalytic activity for the electrooxidation of N-AC in aqueous buffer solution (pH = 7.0). The electrocatalytic oxidation peak currents increase linearly with N-AC concentrations over the concentration ranges of 0.05–600μM using square wave voltammetric methods. The detection limit for N-AC was equal to 0.009μM. The catalytic reaction rate constant, k_h, was calculated (7.01 × 10² M⁻¹ s⁻¹) using the chronoamperometry method. Finally, Ni(II)/ZrO₂/NPs/CPE was also examined as an ultrasensitive electrochemical sensor for the determination of N-AC in real samples such as tablet and urine.     

Chemical investigation of Hyptis suaveolens seed,a potential antihyperuricemic nutraceutical,with assistance of HPLC-SPE-NMR

The seed of Hyptis suaveolens, commonly known as wild flour ball (san fen yuan) in Taiwan, serves as a main refreshing drink substance in several regions. This study investigated firstly its secondary metabolites, leading to the isolation of five major caffeoylquinic acid derivatives (1–5) from the ethanol extract. In addition, ten minors, including three caffeoylquinic acid derivatives (12–14), were characterized via assistance of HPLC-SPE-NMR. Of these isolates, sodium 4,5-dicaffeoylquinate (2) and methyl 3,5-dicaffeoylquinate (4) showed moderate inhibitory activity against xanthine oxidase with the respective IC₅₀ values of 69.4 μM and 92.1 μM (c.f. allopurinol IC₅₀ 28.4 μM). Quantitative HPLC analysis of the EtOH extract indicates the content of sodium 3,5-dicaffeoylquinate (1) and sodium 4,5-dicaffeoylquinate (2) to be 0.1% and 0.08% (w/w, dry seed), respectively. This study not only discloses the bioactive constituents, but also demonstrates the potential of H. suaveolens seed as an antihyperuricemic nutraceutical.     

Inhibitory effects of non-steroidal anti-inflammatory drugs on human liver microsomal morphine glucuronidation: Implications for drug-drug interaction liability

The inhibitory effects of fifteen NSAIDs from six structurally distinct classes on human liver microsomal morphine glucuronidation were investigated. K_i values of selected NSAIDs were generated and employed to assess DDI liability in vivo. Potent inhibition was observed for mefenamic acid and tolfenamic acid; respective IC₅₀ values for morphine 3- and 6-glucuronidation were 9.2 and 13.5 μM, and 5.3 and 8.3 μM. Diclofenac and celecoxib showed moderate inhibition with IC₅₀ values of 78 and 52 μM, and 83 and 214 μM, respectively. Estimated IC₅₀ values for the other NSAIDs screened were >100 μM. Mefenamic acid, diclofenac, and S-naproxen competitively inhibited morphine 3- and 6-glucuronidation, with the K_i values of 11 and 12 μM, 110 and 76 μM, and 319 and 650 μM, respectively. Using the static mechanistic IVIVE approach, an approximate 40% increase in the AUC of morphine was predicted when co-administered with mefenamic acid, whereas the increase was <10% with diclofenac and S-naproxen. PBPK modeling predicted <15% increases in the morphine AUC from diclofenac and S-naproxen inhibition in virtual healthy and cirrhotic subjects. The data suggest that potential clinically significant DDIs arising from NSAID inhibition of morphine glucuronidation is unlikely, with the possible exception of some fenamates.