Proteasome inhibitor MG132 reduces growth of As4.1 juxtaglomerular cells via caspase-independent apoptosis

The proteasome inhibitor MG132 has been shown to induce apoptotic cell death through the formation of reactive oxygen species (ROS). Here, we investigated the molecular mechanisms of MG132 in As4.1 juxtaglomerular cell death in relation to apoptosis and levels of ROS and glutathione (GSH). MG132 inhibited the growth of As4.1 cells with an IC₅₀ of approximately 0.3–0.4 μM at 48 h and induced cell death, accompanied by the loss of mitochondrial membrane potential (MMP; ∆Ψ_m), Bcl-2 decrease, activations of caspase-3 and caspase-8, and PARP cleavage. MG132 increased intracellular ROS levels and GSH-depleted cell numbers. However, caspase inhibitors, especially Z-VAD (pan-caspase inhibitor) intensified cell growth inhibition, cell death, MMP (∆Ψ_m) loss, and Bcl-2 decrease in MG132-treated As4.1 cells. Z-VAD also slightly intensified increases in ROS levels and GSH depletion in MG132-treated As4.1 cells. In conclusion, MG132 reduced the growth of As4.1 cells via caspase-independent apoptosis. The changes in ROS and GSH levels by MG132 and caspase inhibitors partially influenced the growth inhibition and death of As4.1 cells.     

Crambescidin 800, a pentacyclic guanidine alkaloid,protects a mouse hippocampal cell line against glutamate-induced oxidative stress

Oxidative stress is implicated in the pathogenesis of neuronal degenerative diseases. In our search for protective substances against glutamate-induced oxidative stress using mouse hippocampal cell line HT22, we have isolated crambescidin 800, a pentacyclic guanidine alkaloid, from the Indonesian marine sponge, Monanchora ungiculata. Crambescidin 800 (1) protected HT22 cells against glutamate-induced oxidative toxicity at 0.06 μM (EC₅₀) concentration. Crambescidin 800 (1) also protected HT22 and neuroblastoma cells from the oxidative stress induced by a hypoxic condition or nitric oxide (NO). Crambescidin 800 (1) showed no effect on the depletion of intracellular glutathione (GSH) of HT22 cells induced by glutamate, and production of reactive oxygen species (ROS) and activation of mitogen-activated protein kinase (MAPK) were inhibited only by the high concentration (1 μM) of 1.     

Inhibition of Ca2+ transport associated with cAMP-dependent protein phosphorylation in rat cardiac sarcoplasmic reticulum by triorganotins

Organotin compounds have been shown to interfere with cardiovascular system. We have studied the in vitro and in vivo effects of tributyltin bromide (TBT), triethyltin bromide (TET) and trimethyltin chloride (TMT) on the cardiac SR Ca²⁺ pump, as well as on protein phosphorylation of SR proteins, in order to understand the relative potency of these tin compounds. All the three tin compounds inhibited cardiac SR⁴⁵Ca uptake and Ca²⁺-ATPase in vitro in a concentration-dependent manner. The order of potency for Ca²⁺-ATPase as determined by IC₅₀, is TBT (2 M) > TET (63 M) > TMT (280 M). For⁴⁵Ca uptake, it followed the same order i.e., TBT (0.35 M) > TET (10 M) > TMT (440 M). In agreement with the in vitro results, both SR Ca²⁺-ATPase and⁴⁵Ca uptake were significantly inhibited in rats treated with these tin compounds, indicating that these tin compounds inhibit cardiac SR Ca²⁺ transport. cAMP significantly elevated (70–80%) the³²P-binding to SR proteins in vitro in the absence of any organotin. In the presence of organotins, cAMP-stimulated³²P-binding to proteins was significantly reduced, but the decrease was concentration dependent only at lower concentrations. The order of potency is TBT > TET > TMT. In agreement with in vitro studies, cAMP-dependent³²P bound to proteins was significantly reduced in rats treated with TBT, TET and TMT. SDS-polyacrylamide gel electrophoresis of the cardiac SR revealed at least 30 Coomassie blue stainable bands ranging from 9 to 120 kDa. Autoradiographs from samples incubated in the presence of cAMP indicated³²P incorporation in seven bands. Of these, the band corresponding to about 24 kDa molecular weight protein decreased in its intensity with the treatment of organotins. These results suggest that triorganotins may be affecting Ca²⁺ pumping mechanisms through the alteration of phosphorylation of specific proteins in rat cardiac SR.This work has been presented in part at the Annual meeting of Society of Toxicology, 1990 at Miami Beach, FL. The Toxicologist 10: 35 & 108 (1990).     

Inhibitory effect of 2-arylbenzofurans from the Mori Cortex Radicis (Moraceae) on oxygen glucose deprivation (OGD)-induced cell death of SH-SY5Y cells

Three known 2-arylbenzofurans, moracin P (1), moracin O (2) and mulberrofuran Q (3) were isolated from the MeOH extract of the Mori Cortex Radicis. These compounds 1–3 enhanced cell viability in dose-dependent manner against oxygen-glucose deprivation (OGD)-induced cell death in neuroblastoma SH-SY5Y cells, which was measured by MTT reduction assay. (EC₅₀ values of 10.4, 12.6, and 15.9 μM, respectively). In addition, the compounds 1–3 were examined for their inhibitory effect on OGD-induced ROS production by FACS analysis. We observed these compounds reduced ROS production in OGD-induced cell death (IC₅₀ values of 1.9, 0.3 and 12.1 μM, respectively). Consequently, reactive oxygen species (ROS) were overexpressed in OGD-induced cells and all three compounds reduced ROS induced by OGD in dosedependent manner. Taken together, compounds 1–3 might protect neuronal cell death against the oxidative stress induced by OGD, though further studies in vitro and in vivo models are necessary.     

Toxicity of β-blockers in a rat whole embryo culture: concentration-response relationships and tissue concentrations

Beta-adrenoceptor blockers are widely used drugs for the treatment of cardiovascular diseases. Since β-blockers cross the placenta, it is essential to consider possible adverse effects on the embryo. Six β-adrenoceptor blockers were tested at various concentrations (10 – 5000 μM) in a rat whole embryo culture. Although inducing a very similar pattern of dysmorphogenetic effects (incomplete flexure, disturbed development of the neural tube, the head, the heart and the tail bud), the compounds exhibited a wide range of embryotoxic potency. Estimation of the EC₅₀ (median-concentration producing dysmorphogenesis in 50% of the embryos) for the six compounds revealed differences of more than two orders of magnitude: propranolol 25 μM, alprenolol 30 μM, metoprolol 100 μM, pindolol 150 μM, acebutolol 500 μM, atenolol 4000 μM. Measurements of the concentrations of the various drugs in the cultured embryos at corresponding EC₅₀ levels showed differing values: metoprolol 4.5 μM, propranolol 5.2 μM, alprenolol 8.4 μM, pindolol 9.0 μM, acebutolol 12.5 μM and atenolol 77.0 μM. With regard to the EC₅₀ and the degree of substance transfer to the embryo it can be stated that propranolol and metoprolol show a much higher intrinsic potency to interfere with normal in vitro embryonic development than, e.g. atenolol. Received: 1 September 1993 / Accepted: 16 February 1994     

Arsenic induces mitochondria-dependent apoptosis by reactive oxygen species generation rather than glutathione depletion in Chang human hepatocytes

This study was conducted to evaluate the possible involvement of mitochondrial pathway in NaAsO₂-induced apoptosis and the role of reactive oxygen species (ROS) and reduced glutathione (GSH) in the apoptotic effect in Chang human hepatocytes. The MTT assay demonstrated that sodium arsenite (NaAsO₂) treatment for 24 h caused a dose-dependent decrease of cell viability. NaAsO₂ treatment (0–30 μM) was also found to induce phosphatidylserine externalization, a hallmark of apoptosis; to disrupt the mitochondrial membrane potential (Δψ _m ); to cause the release of cytochrome c into the cytosol, and to trigger cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP) in a dose-dependent manner. All these changes were accompanied with the enhanced generation of intracellular ROS and malondialdehyde (MDA). Increase of intracellular GSH also coincided unexpectedly. Moreover, the extracellular addition of N-acetyl-l-cysteine (NAC, 5 mM) effectively reduced the generation of ROS and MDA, and rescued the cells from NaAsO₂ induced apoptosis and related alteration of mitochondria. These data suggest that the arsenic-induced cell apoptosis occurs though the mitochondrial pathway, and is mostly dependent on generation of ROS rather than GSH depletion in Chang human hepatocytes.     

The anti-apoptotic effects of caspase inhibitors on propyl gallate-treated HeLa cells in relation to reactive oxygen species and glutathione levels

Propyl gallate (PG) as a synthetic antioxidant is widely used in processed food, cosmetics and medicinal preparations. Despite the assumed low toxicity of PG, it exerts a variety of effects on tissue and cell functions. In the present study, we evaluated the anti-apoptotic effects of caspase inhibitors on PG-treated human cervix adenocarcinoma HeLa cells in relation to the changes of reactive oxygen species (ROS) and glutathione (GSH) levels. PG induced apoptosis in a dose-dependent manner, as evidenced by sub-G1 cells and annexin V staining cells. Treatment with pan-caspase inhibitor, caspase-3 inhibitor, caspase-8 inhibitor or caspase-9 inhibitor significantly prevented apoptosis in PG-treated HeLa cells at 24 h. The intracellular ROS levels including O₂^•− were increased or decreased in PG-treated HeLa cells depending on the incubation times (1 or 24 h). PG depleted intracellular GSH content in HeLa cells at 24 h. Treatment with caspase inhibitor reduced ROS levels and significantly prevented GSH depletion in PG-treated HeLa cells at 24 h. In conclusion, PG induced apoptosis in HeLa cells. The anti-apoptotic effect of caspase inhibitor on PG-induced HeLa cell death was closely related to the reduction of ROS levels, especially mitochondrial O₂^•−, as well as to the inhibition of GSH depletion.     

Effects of glutathione and pH on the oxidation of biomarkers of cellular oxidative stress

 Cellular oxidative stress is associated with such pathological conditions as arteriosclerosis, inflammatory diseases and cancer. The oxidation of the biomarkers 2′,7′-dichlorofluorescin (DCFH), 2-deoxyribose, and lipid peroxidation are often used to assess the status of oxidative stress in cells and tissues. Since high levels of reduced glutathione (GSH) and acidic conditions have been associated with diminished chemical lethality, we evaluated the influence of these parameters on the cellular response to oxidative stress. We used a cultured hepatocyte line (ch/ch cells) that is susceptible to oxidative toxicity. A hydroxyl radical-generating system consisting of H₂O₂, ascorbate and iron produced a pH-dependent lethality, with complete cell killing at pH 7.4 and none at pH 6.8. Lethality correlated with the depletion of intracellular GSH, and with an increase in DNA fragmentation. The influence of GSH and pH was assessed for DCFH and 2-deoxyribose oxidation, and for lipid peroxidation. The oxidation of DCFH and 2-deoxyribose was inhibited by GSH, with about 4-fold greater inhibition efficacy at pH 6.8 than at pH 7.4 [IC₅₀ values (μM GSH) for pH 6.8 and 7.4, respectively: DCFH=7 and 30; 2-deoxyribose=125 and 490]. GSH did not affect lipid peroxidation at either pH, even at a high intracellular concentration of 10 mM. We conclude: 1) GSH is not inhibiting DCFH and 2-deoxyribose oxidation by simply quenching reactive oxygen (hydroxyl radical or perferryl oxygen), since GSH did not inhibit lipid peroxidation; 2) the protonated form GSH is more likely to be the inhibitory species rather than GS⁻, since even in the simple cell-free systems lower pH inhibited biomarker oxidation; and; 3) hydroxyl radical may not be the primary intracellular oxidant of DCFH, since intracellular GSH concentrations are typically 10- to 100-fold higher than the IC₅₀ values for GSH inhibiting reactive oxygen-mediated DCFH oxidation. Received: 24 October 1995/Accepted: 29 February 1996     

Endogenous bile acid disposition in rat and human sandwich-cultured hepatocytes

We hypothesized that flavonoid-induced glutathione (GSH) efflux through multi-drug resistance proteins (MRPs) and subsequent intracellular GSH depletion is a viable mechanism to sensitize cancer cells to chemotherapies. This concept was demonstrated using chrysin (5-25 μM) induced GSH efflux in human non-small cell lung cancer lines exposed to the chemotherapeutic agent, doxorubicin (DOX). Treatment with chrysin resulted in significant and sustained intracellular GSH depletion and the GSH enzyme network in the four cancer cell types was predictive of the severity of chrysin induced intracellular GSH depletion. Gene expression data indicated a positive correlation between basal MRP1, MRP3 and MRP5 expression and total GSH efflux before and after chrysin exposure. Co-treating the cells for 72 h with chrysin (5-30 μM) and DOX (0.025-3.0 μM) significantly enhanced the sensitivity of the cells to DOX as compared to 72-hour DOX alone treatment in all four cell lines. The maximum decrease in the IC₅₀ values of cells treated with DOX alone compared to co-treatment with chrysin and DOX was 43% in A549 cells, 47% in H157 and H1975 cells and 78% in H460 cells. Chrysin worked synergistically with DOX to induce cancer cell death. This approach could allow for use of lower concentrations and/or sensitize cancer cells to drugs that are typically resistant to therapy.     

Evaluation of toxicity indicators in rat primary astrocytes, C6 glioma and human 1321N1 astrocytoma cells: can gliotoxicity be distinguished from cytotoxicity?

A comparison was made of rat primary astrocytes, C6 glioma cells pre-treated with dibutyryl cyclic AMP, and the human astrocyte 132N1 cell line using a range of 40 compounds and the neutral red (NR) assay. The 40 chemicals included substances known to be toxic to astrocytes or neurons, to be generally cytotoxic or not thought to be toxic to nervous tissue. For those compounds which were toxic, changes in glial fibrillary acidic protein (GFAP) levels were measured in the primary and C6 cultures, and changes in vimentin and S-100 measured in the C6 cells. The number of compounds with EC₅₀ values <2000 μg/ml for the NR assay for the different cell cultures were as follows: primary astrocytes, 19; C6 cells, 15; and 1321N1 cells, 11. The log of the EC₅₀ values for the NR assay for the test compounds between the three cell types was not significantly different at the 5% level by paired Student's t-test. For the toxic substances the correlation coefficients of the EC₅₀ values between primary cells and the C6 or 1321N1 cells were r>0.5, and between the C6 and 1321N1 cells r>0.9. For GFAP there was a similar degree of correlation in EC₅₀ values between the different cell types. The GFAP, vimentin and S-100 levels showed similar EC₅₀ values for the toxicants, but were not as sensitive as the NR assay. The toxic substances caused altered morphology in the primary, C6 and 1321N1 cells, with increased branching of cell processes. The combined astrocyte systems identified 8 out of 9 substances reported to be toxic to astrocytes in vivo, together with substances which have general cytotoxic properties. A␣number of substances (including the 1 out of 9 reported gliotoxic substances), which may primarily affect neurons, which may affect nervous tissue after long-term exposure, or which are not thought to be toxic to nervous tissue, were not detected. The astrocyte systems positively identify gliotoxic and cytotoxic substances and will allow detailed mechanistic studies to be made on the different underlying mechanisms. Received: 26 January 1998 / Accepted: 3 February 1998     

Cytotoxicity of sphingoid marine compound analogs in mono- and multilayered solid tumor cell cultures

Summary A subset of four synthetic sphingoid marine compound analogs was chosen from a preliminary in vitro cytotoxicity study for further analysis. The selected analogs were initially screened in monolayer cultures for their anticancer potential against a panel of eight human tumor cell lines, ovarian, colon and lung cancer, squamous cell carcinoma and leukemia producing IC₅₀ values ranging from 1.5 to 6.9 μM. In a secondary screening, the sphingoid analogs were evaluated against multilayered postconfluent cultures of A2780 ovarian cancer and WiDr colon cancer cells. In this model, compounds 5 and 8 were the most active derivatives showing EC₅₀ values in the range 25–32 μM. The performance of 5 and 8 against both cell lines was not dependent on the cell culture model as shown with resistance factor values in the range 8–12. Cell cycle studies in HL60 leukemia cells showed an arrest in G ₀/G ₁ at a low drug concentration (3 μM) but accumulation in S phase at a high drug concentration (9 μM). It can be concluded that the analogs showed a cell line independent activity, with an apparent selectivity against cells grown in more physiological three-dimensional condition compared to standard anticancer drugs.     

Effect of selenium compounds on murine B16 melanoma cells and pigmented cloned pB16 cells

The effects of selenium compounds such as sodium selenite, sodium selenate, seleno-Dl-cystine and seleno-Dl-methionine (100 μM and 10 μM) on B16 and pigmented cloned pB16 murine melanoma cells were investigated in vitro. At the tested concentrations, B16 cells showed a greater sensitivity to the toxic effects of sodium selenite and seleno-Dl-cystine than pB16 cells, whereas no decrease of B16 and pB16 cell number was observed after incubation with sodium selenate or seleno-Dl-methionine. Glutathione (GSH) percentages were strongly decreased only by selenite and seleno-Dl-cystine; it was marked more in B16 than in pB16 cells. The pretreatment of B16 cells with a GSH depleting agent (10 μM buthionine-[S,R]-sulfoximine) did not significantly influence the cytotoxic effects of selenite and seleno-Dl-cystine. On both cell populations, GSH preincubation (50 μM) enhanced the cytotoxicity of selenite whereas the survival of seleno-Dl-cystine treated cells was increased. Glutathione peroxidase (GSH-Px) activity in B16 cells was more sensitive than in pB16 cells to the activating effect of selenite, and particularly of seleno-Dl-cystine; however, cell-free controls indicated that activation was mainly due to glutathione reductase. The rate of⁷⁵Se (as sodium selenite) uptake in both cell populations was maximal within the first hour of incubation, with a preferential accumulation in the cytosol; after 24 h of incubation, the amount of⁷⁵Se in cytosol and pellet was approximately the same. Gel filtration chromatography of lysed cells after incubation for 6 h with 10 μM⁷⁵Se-selenite showed that the radioactivity was eluted as two peaks corresponding to low (4–9 kDa) and high (280–320 kDa) molecular weights. Possible toxicological mechanisms are discussed at molecular level. For selenite, a major involvement of GSH is proposed, with production of selenodiglutathione and selenopersulfide, which should be directly responsible of the decrease in cell number, thiol oxidation and protein synthesis inhibition. For selenocystine, an active selenol species (Cy-Se⁻) is also hypothesized as being responsible for thiol oxidation and mutagenic effects. For both compounds oxygen active species could also be formed; however, a relevant role of GSH-Px was not apparent. The minor sensitivity of pB16 cells to the toxic effects of selenite and seleno-Dl-cystine could be explained by the smaller depletion of GSH induced by those compounds in pB16 cells, a minor formation of selenium active species, the larger amount present of the oxyradical scavenger melanin, the secretion of some mitogenic factor by pB16 cells and/or a greater resistance to autocrine cytotoxic factors.     

The role of glutathione in L-2-chloropropionic acid induced cerebellar granule cell necrosis in the rat

 The role of glutathione (GSH) in the neurotoxicity produced following a single oral dose of 750 mg/kg L-2-chloropropionic acid (L-CPA) has been investigated in rats. L-CPA-induced neurotoxicity was characterised by up to 80–90% loss in cerebellar granule cells and cerebellar oedema leading to locomotor dysfunction. Neurochemically, L-CPA-induced neurotoxicity produced a reduction in the concentration of aspartate and glutamate in the cerebellum and a reduction in the density of NMDA receptors in the cerebellar cortex, whilst there was an increase in cerebellar glycine, glutamine and GABA concentrations. Treatment of rats with buthionine sulfoximine (BSO) at 1 g/kg, i.p., an inhibitor of GSH synthesis, potentiated the toxicity of L-CPA, such that many of the neurochemical markers were significantly different from controls at earlier time points, compared to animals which had received L-CPA alone, and toxicity was also seen in the kidney of BSO plus L-CPA treated rats. In contrast, supplementing GSH concentrations by administration of the isopropyl ester of glutathione (ip-GSH) at 1 g/kg, s.c., was able to protect rats against L-CPA neurotoxicity and prevent many of the neurochemical changes. In order to assess whether the depletion of GSH in the rat cerebellum following L-CPA treatment was related to the delivery of cysteine or cystine, the accumulation of [¹⁴C] cystine into cerebellar slices was characterised and found to be energy dependent, Na⁺ independent and obey saturation kinetics with an apparent K_m of 77 μM and an apparent V_max of 450 nmol/g wet weight per h. The accumulation of cystine into cerebellar slices was non-competitively inhibited by the cysteine conjugate of L-CPA with an apparent K_i of approximately 60 μM, whilst glutamate only inhibited cystine accumulation at doses which were cytotoxic to cerebellar slices. Hence the depletion of GSH in the rat cerebellum, following L-CPA administration, may be due to a reduction in the delivery to the brain of cysteine or cystine, one of the components required for GSH synthesis, by the cysteine conjugate of L-CPA. Our studies show the pivotal role GSH plays in cerebellar granule cell necrosis induced by L-CPA in the rat, indicating that a marked and sustained reduction in cerebellar GSH content by L-CPA may leave granule cells vulnerable to cytotoxic free radical damage leading to cell death, possibly mediated through excitatory amino acids. Received: 24 October 1995/Accepted: 24 January 1996     

Gallic acid inhibits the growth of HeLa cervical cancer cells via apoptosis and/or necrosis

Gallic acid (GA) is widely distributed in various plants and foods, and its various biological effects have been reported. Here, we evaluated the effects of GA on HeLa cells in relation to cell growth inhibition and death. HeLa cell growth was diminished with an IC₅₀ of approximately 80 μM GA at 24 h whereas an IC₅₀ of GA in human umbilical vein endothelial cells (HUVEC) was approximately 400 μM. GA-induced apoptosis and/or necrosis in HeLa cells and HUVEC, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m). The percents of MMP (ΔΨ_m) loss cells and death cells were lower in HUVEC than HeLa cells. All the tested caspase inhibitors (pan-caspase, caspase-3, -8 or -9 inhibitor) significantly rescued HeLa cells from GA-induced cell death. GA increased reactive oxygen species (ROS) level and GSH (glutathione) depleted cell number in HeLa cells. Caspase inhibitors reduced GSH depleted cell number but not ROS level in GA-treated HeLa cells. In conclusion, GA inhibited the growth of HeLa cells and HUVEC via apoptosis and/or necrosis. The susceptibility of HeLa cells to GA was higher than that of HUVEC. GA-induced HeLa cell death was accompanied by ROS increase and GSH depletion.     

Neurotoxic effects of trimethyltin and triethyltin on human fetal neuron and astrocyte cultures: a comparative study with rat neuronal cultures and human cell lines

Trimethyltin (TMT) and triethyltin (TET) caused cell death in cultures of primary human neurons and astrocytes, rat neurons and human neuroblastoma cell lines. Human neurons and astrocytes showed a delayed response to TMT cytotoxicity. After 24h of TMT exposure, LC50 values were 148.1, 335.5 and 609.7 microM for SK-N-MC neuroblastoma cell line, neurons and astrocytes, respectively. Over 5 days of exposure, the cytotoxic potency of TMT increased about 70-fold in human cortical neurons. Rat hippocampal neurons were the most vulnerable cells to TMT cytotoxicity, exhibiting an LC50 value 30-fold lower (1.4 microM) than that of rat cerebellar granule cells (44.28 microM). With the exception of rat hippocampal neurons, TET was more potent than TMT in inducing cell death (LC50 values of 3.5-16.9 microM). Moreover, TET was more effective than TMT in increasing intracellular free Ca2+ concentration in human and rat neurons. This work shows that human fetal neuron and astrocyte cultures are a useful model for studying the neurotoxic effects of these environmental contaminants and, thus, predicting their impact on human health.     

Structural isomerization of synephrine influences its uptake and ensuing glutathione depletion in rat-isolated cardiomyocytes

Synephrine is a natural compound, frequently added to ephedra-free dietary supplements for weight-loss, due to its effects as a nonspecific adrenergic agonist. Though only p-synephrine has been documented in plants, the presence of m-synephrine has also been reported in weight-loss products. The use of synephrine in dietary supplements was accompanied by reports of adverse effects, especially at the cardiovascular level. It is well known that the imbalance in cardiac glutathione levels can increase the risk of cardiomyopathy. The present work aimed to study the role of organic cation-mediated transport of m- and p-synephrine and the possibility that p- and m-synephrine induce intracellular changes in glutathione levels in calcium-tolerant freshly isolated cardiomyocytes from adult rat. After a 3 h incubation with 1 mM p- or m-synephrine, the intracellular content of synephrine was measured by gas chromatography/ion trap-mass spectrometry (GC/IT-MS); cell viability and intracellular glutathione levels were also determined. To evaluate the potential protective effects of antioxidants against the adverse effects elicited by m-synephrine, cells were pre-incubated for 30 min with Tiron (100 μM) or N-acetyl-cysteine (NAC) (1 mM). To assess the influence of α₁-adrenoceptors activation in glutathione depletion, a study with prazosin (100 nM) was also performed. The results obtained provide evidence that organic cation transporters OCT3 and OCT1 play a major role in m- and p-synephrine-mediated transport into the cardiomyocytes. The importance of these transporters seems similar for both isomers, although p-synephrine enters more into the cardiomyocytes. Furthermore, only m-synephrine induced intracellular total glutathione (GSHt) and reduced glutathione (GSH) depletion. NAC and Tiron were able to counteract the m-synephrine-induced GSH and GSHt decrease. On the other hand, the incubation with prazosin was not able to change m-synephrine-induced glutathione depletion showing that this effect is independent of α₁-adrenoceptor stimulation. In conclusion, both positional isomers require OCT3 and OCT1-mediated transport to enter into the cardiomyocytes; however, the hydroxyl group in the p-position favours the OCT-mediated transport into cardiomyocytes. Furthermore, the structural isomerization of synephrine influences its toxicological profile since only m-synephrine caused GSH depletion.     

Mitochondrial transmembrane potential and free radical production in excitotoxic neurodegeneration

Excitotoxic cell death is involved in many forms of acute and chronic neurodegeneration. We induced excitotoxic cell death in cultured rat hippocampal neurons by brief exposure to two selective glutamate receptor agonists with different neurotoxic potencies, N-methyl-d-aspartate (NMDA) and kainate (KA). Digital video imaging was performed during exposure to the agonists to monitor free radical production and changes in mitochondrial transmembrane potential, Ψ_m. Brief exposure to NMDA (10min) induced significant cell death in the hippocampal neurons reaching a maximum at a concentration of 300μM (57.2±2.6% cell death; P<0.001). In parallel imaging experiments we found that exposure to NMDA (300μM, 10min) induced a significant increase in superoxide production monitored with the oxidation-sensitive probe, hydroethidine (increase of 280±33% above baseline; P<0.001). Rhodamine-123-based imaging revealed a loss of Ψ_m in 70.1±10.1% of the hippocampal neurons during the exposure to NMDA. In contrast to NMDA, brief exposure to KA (10min) produced limited neurotoxicity reaching a maximum at a concentration of 100μM (10.2±4.0% cell death; P<0.05). Exposure to KA (100μM, 10min) also caused a significant increase in superoxide production. This increase, however, was significantly less pronounced than that produced by NMDA (increase of 94±17% above baseline; P<0.001 compared to controls or NMDA-exposed cultures). Moreover, rhodamine-123-based imaging revealed that KA (100μM) caused a collapse of Ψ_m in only 13.5±1.4% of the hippocampal neurons. In conclusion, the present study demonstrates that early changes in intracellular superoxide production and Ψ_m relate to neuronal survival outcome in excitotoxic cell death. Received: 11 September 1997 / Accepted: 8 December 1997     

Comparison of hepatotoxicity caused by mono-, di- and tributyltin compounds in mice

 The in vivo induction of hepatotoxicity, as evaluated by the activity of ornithine carbamyl transferase in serum, was investigated in mice administered orally with the following three butyltin compounds: tributyltin chloride (TBTC), dibutyltin dichloride (DBTC) and monobutyltin trichloride (MBTC). The minimal concentrations of TBTC and DBTC that caused hepatotoxicity at 24 h after oral administration were 180 μmol and 60 μmol/kg, respectively, while MBTC did not induce liver injury even at 7000 μmol/kg. Additionally, when the administered doses were equivalent (180 μmol/kg), a time course (3–96 h) study revealed that the hepatotoxicity of TBTC and DBTC appeared at 24 and 12 h, respectively, but that MBTC showed no hepatotoxicity even at 96 h. The amounts of Sn excreted into urine for 4 days were 1.5 fold greater with TBTC than with DBTC treatment and were lowest in MBTC group. Similarly, the total liver Sn content was 2- to 5-fold greater in the TBTC group than in the DBTC group whereas the liver Sn content in the MBTC treatment showed the lowest value throughout the 3- to 96-h period. Thus, the non-hepatotoxicity of MBTC may be due either to low absorption through the digestive tract of mice or to the low levels of Sn in liver; however, the level of Sn in liver was not associated with the induction of hepatotoxicity by TBTC and DBTC. The analysis of metabolites of TBTC (180 μmol/kg) and DBTC (60 μmol/kg) at equivalent hepatotoxicity showed that the main tin compounds in the liver after the administration of TBTC were dibutyltin and monobutyltin as well as inorganic tin compounds, while most (>78%) of the total tin compounds in the liver of mice treated with DBTC was in the form of dibutyltin. In addition, the levels of monobutyltin and inorganic tin compounds in the livers of mice treated with TBTC were greater than those with DBTC, but the levels of dibutyltin did not differ significantly between TBTC and DBTC. The levels of lipid peroxidation (LPO) and hepatic glutathione (GSH) content in the liver showed a transitory increase after the administration of MBTC and TBTC, respectively. These results suggest that DBTC is more hepatotoxic than TBTC, and that dibutyltin inside the cells may be the main form of tin which is responsible for induction of hepatotoxicity following in vivo administration of TBTC and DBTC. The generation of free radical species, as evaluated by LPO and GSH levels, may not be associated with the hepatotoxicity caused by butyltin compounds. Received: 13 April 1994 / Accepted: 25 May 1994     

P2-Receptor-mediated inhibition of noradrenaline release in the rat pancreas

The aim of the study was to find out whether, and if so through which receptors, nucleotides modulate the release of noradrenaline in the rat pancreas. Segments of the pancreas were preincubated with [³H]-noradrenaline, superfused with medium containing desipramine (1μM) and yohimbine (1μM), and stimulated electrically, in most experiments by 60 pulses/1Hz. The adenosine A₁-receptor agonist N⁶-cyclopentyl-adenosine (CPA; EC₅₀ 32nM), the non-subtype-selective adenosine receptor agonists adenosine (EC₅₀ 15μM) and 5’-N-ethylcarboxamidoadenosine (NECA; EC₅₀ 135nM), and the nucleotides ATP (EC₅₀ 13μM), adenosine-5’-O-(3-thiotriphosphate) (ATPγS; EC₅₀ 19μM) and adenosine-5’-O-(2-thiodiphosphate) (ADPβS; EC₅₀ 16μM) decreased the evoked overflow of tritium. The adenosine A_2A-agonist 2-p-(2-carboxyethyl)-phenethylamino-5’-N-ethylcarboxamido-adenosine (CGS 21680) caused no change. The concentration-response curve of CPA was shifted to the right by the A₁-antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX 10nM; pK_d 9.1) but, like the concentration-response curve of adenosine, hardly affected by the P2-receptor antagonist cibacron blue 3GA (30μM). Combined administration of a high concentration of DPCPX (1μM) and 8-phenyltheophylline (10μM) abolished the effects of CPA and NECA. The concentration-response curves of ATP and ADPβS were shifted to the right by both DPCPX (10nM; pK_d 8.7 and 8.9, respectively) and cibacron blue 3GA (30μM; pK_d 5.0 and 5.2, respectively). The antagonist effects of DPCPX (10nM) and cibacron blue 3GA (30μM) against ATP were additive in a manner compatible with the blockade of two separate receptors for ATP. In the presence of the high concentration of DPCPX (1μM) and 8-phenyltheophylline (10μM), ATP and ADPβS still decreased evoked tritium overflow, and this decrease was attenuated by additional administration of cibacron blue 3GA (30μM). The P2-antagonists cibacron blue 3GA, reactive blue 2, reactive red 2, and to a limited extent also suramin and 8-(3,5-dinitro-phenylenecarbonylimino)-1,3,5-naphthalenetrisulphonate (XAMR0721), increased the evoked overflow of tritium by up to 114%. Pyridoxalphosphate-6-azophenyl-2’,4’-disulphonate (PPADS) caused no change. The results indicate that the postganglionic sympathetic axons of the rat pancreas possess A₁-adenosine and P2-receptors. Both receptors mediate an inhibition of noradrenaline release. The presynaptic P2-receptors are activated by an endogenous ligand, presumably ATP, during appropriate trains of action potentials. This is the first demonstration of presynaptic P2-receptors at postganglionic sympathetic neurons that are located in prevertebral ganglia. Received: 6 November 1997 / Accepted: 6 January 1998     

Suppression of acrylamide toxicity by carboxyfullerene in human neuroblastoma cells in vitro

In our previous study, we found that caspase-dependent apoptosis played a role in the genesis of toxicity of acrylamide in human neuroblastoma (SH-SY5Y) cells (Sumizawa and Igisu in Arch Toxicol 81:279–282, 2007). In the present experiment, we examined whether carboxyfullerene may suppress the cytotoxicity of acrylamide because carboxyfullerene has been reported to protect nerve cells from various pathologic processes including apoptosis. Carboxyfullerene lowered lactate dehydrogense leakage and elevated cell viability in SH-SY5Y cells exposed to acrylamide. It also lowered caspase-3 activities and cell population in the sub-G₁ phase induced by acrylamide. Nevertheless, carboxyfullerene enhanced cellular uptake of [¹⁴C]acrylamide. On the other hand, acrylamide markedly decreased glutathione (GSH)-content in cells and carboxyfullerene blocked the decrease. The toxicity of acrylamide was suppressed by adding GSH or GSH monoethyl ester, whereas it was not lowered by carboxyfullerene when GSH synthesis was inhibited by l-buthionine-(S,R)-sulfoximine. Thus, the cytotoxicity of acrylamide including apoptotic processes is closely related to GSH level in SH-SY5Y cells and carboxyfullerene suppresses the toxicity by maintaining GSH content. Neither tricarboxylic acids without fullerene moiety nor hydroxylated fullerene showed comparable effects of carboxyfullerene (60 μM) against 1–5 mM acrylamide, suggesting the importance of the three malonic acid groups at specific positions in a fullerene molecule for the effects.