Zinc at clinically-relevant concentrations potentiates the cytotoxicity of polysorbate 80, a non-ionic surfactant

Polysorbate 80, a non-ionic surfactant, is used in the formula of water-insoluble anticancer agents for intravenous application. In our recent studies, this surfactant decreased cellular thiol content and the chemicals decreasing cellular thiol content increased intracellular Zn²⁺ concentration. In this study using rat thymocytes, the effect of polysorbate 80 on FluoZin-3 fluorescence, an indicator for intracellular Zn²⁺, and the influence of ZnCl₂ on cytotoxicity of polysorbate 80 were examined in order to test the possibility that Zn²⁺ is involved in cytotoxic action of polysorbate 80. The surfactant at concentrations of 10 μg/ml or more significantly augmented FluoZin-3 fluorescent in a concentration-dependent manner, indicating an increase in intracellular Zn²⁺ concentration. The increase by polysorbate 80 was also observed after removing extracellular Zn²⁺, suggesting an intracellular Zn²⁺ release. The simultaneous application of polysorbate 80 (30 μg/ml) and ZnCl₂ (10–30 μM) significantly increased cell lethality. The simultaneous application of ZnCl₂ accelerated the process of cell death induced by polysorbate 80 and the combination increased oxidative stress. Results may indicate that the cytotoxicity of polysorbate 80 at clinical concentrations is modified by micromolar zinc. Although there is no clinical report that polysorbate 80 and zinc salt are simultaneously applied to human as far as our knowledge, it may be speculated that zinc induces some diverse actions in cancer treatment with water-insoluble anticancer agent including nanoparticle drug of which the solvent is polysorbate 80.     

Micromolar Zn2+ potentiates the cytotoxic action of submicromolar econazole in rat thymocytes: Possible disturbance of intracellular Ca2+ and Zn2+ homeostasis

Econazole, one of imidazole antifungals, has been reported to exhibit an inhibitory action on Mycobacterium tuberculosis and its multidrug-resistant strains under in vitro and ex vivo conditions. There is a chemotherapeutic potential of econazole against tuberculosis. We have revealed that Zn²⁺ at micromolar concentrations potentiates the cytotoxicity of imidazole antifungals by increasing membrane Zn²⁺ permeability. It is reminiscent of a possibility that econazole exhibits harmful action on human in the presence of Zn²⁺ at a physiological range when the agent is systemically administered. Because it is necessary to characterize the cytotoxic action of econazole in the presence of Zn²⁺, we have cytometrically examined the effects of econazole, ZnCl₂, and their combination on rat thymocytes. ZnCl₂ at concentrations ranging from 1 μM to 30 μM significantly increased the lethality induced by 10 μM econazole in a concentration-dependent manner. Econazole at a sublethal concentration of 1 μM significantly augmented the intensity of side scatter in the presence of micromolar ZnCl₂, suggesting the change in an intracellular circumstance by the combination of econazole and ZnCl₂. Econazole at 0.3 μM or more in the presence of ZnCl₂ increased the intensity of Fluo-3 fluorescence, an indicator for intracellular Ca²⁺. Furthermore, the intensity of FluoZin-3 fluorescence, an indicator for intracellular Zn²⁺, was also augmented by econazole at 0.1 μM or more in the presence of ZnCl₂. Results suggest that the combination of submicromolar econazole with micromolar ZnCl₂ may increase the intracellular concentration of Ca²⁺ and Zn²⁺, leading to disturbance of intracellular Ca²⁺ and Zn²⁺ homeostasis that triggers cytotoxic action.     

Low micromolar zinc exerts cytotoxic action under H2O2-induced oxidative stress: Excessive increase in intracellular Zn concentration

The ability of zinc to retard oxidative processes has been recognized for many years. However, zinc is cytotoxic under certain oxidative stress. In this study, we investigated the effect of H₂O₂ on intracellular Zn²⁺ concentration of rat thymocytes and its relation to the cytotoxicity. Experiments were cytometrically performed by the use of fluorescent probes, propidium iodide, FluoZin-3-AM, and 5-chloromethylfluorescein diacetate. ZnCl₂ potentiated cytotoxicity of H₂O₂ while TPEN, a chelator for intracellular Zn²⁺, attenuated it. Results suggested an involvement of intracellular Zn²⁺ in the cytotoxicity of H₂O₂. H₂O₂ at concentrations of 30 μM or more (up to 1000 μM) significantly increased intracellular Zn²⁺ concentration. There were two mechanisms. (1) H₂O₂ decreased cellular content of nonprotein thiols, possibly resulting in release of Zn²⁺ from thiols as cellular Zn²⁺ binding sites. (2) H₂O₂ increased membrane Zn²⁺ permeability because external ZnCl₂ application further elevated intracellular Zn²⁺ concentration. Micromolar H₂O₂ may induce excessive elevation of intracellular Zn²⁺ concentration that is harmful to cellular functions. However, the incubation with micromolar ZnCl₂ alone increased cellular content of nonprotein thiols, one of the factors protecting cells against oxidative stress. Though zinc is generally considered to be protective with its antioxidant property, this study reveals the toxic effect of zinc even in micromolar range under oxidative stress induced by H₂O₂.     

Biphenyl-induced cytotoxicity is mediated by an increase in intracellular Zn2+

Biphenyl is found both in natural and anthropogenic sources and is used as a fungistat in the packaging of citrus fruits. Acute exposure to high levels of biphenyl has been observed to cause skin irritation and toxic effects on the liver and kidneys. However, the mechanisms of cytotoxicity induced by biphenyl are not yet well understood. In the present study, the cytotoxicity of biphenyl was studied by flow cytometry with fluorescent probes. Biphenyl at 100?μM significantly increased cell lethality after 3?h in rat thymocytes. In addition, biphenyl at 100?μM or more elevated intracellular Zn²⁺ levels. N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), an intracellular and extracellular Zn²⁺ chelator, but not diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid (DTPA), a membrane-impermeable Zn²⁺ chelator, attenuated the biphenyl-induced increase in intracellular Zn²⁺ levels and cell death. These results suggested that biphenyl-induced cytotoxicity caused an increase in intracellular Zn²⁺ levels, which was dependent on internal Zn²⁺. Moreover, biphenyl led to an increase in sensitivity to oxidative stress, while TPEN inhibited this biphenyl-induced increase. Our findings revealed that biphenyl caused an increase in the intracellular free Zn²⁺ concentration, inducing cytotoxicity, cell death, and an increase in sensitivity to oxidative stress.     

Zn2+, derived from cell preparation,partly attenuates Ca2+-dependent cell death induced by A23187, calcium ionophore,in rat thymocytes

A23187, a calcium ionophore, is used to induce Ca²⁺-dependent cell death by increasing intracellular Ca²⁺ concentration ([Ca²⁺]_i) under in vitro condition. Since this ionophore also increases membrane permeability of metal divalent cations such as Zn²⁺ and Fe²⁺ rather than Ca²⁺, trace metal cations in cell suspension may affect Ca²⁺-dependent cell death induced by A23187. Therefore, the effects of chelators for divalent metal cations, EDTA and TPEN, on the A23187-induced cytotoxicity were cytometrically examined in rat thymocytes. The cytotoxicity of A23187 was attenuated by 1 mM EDTA while it was augmented by 50 μM EDTA and 10 μM TPEN. These changes were statistically significant. The A23187-induced increase in Fluo-3 fluorescence intensity, a parameter for [Ca²⁺]_i, was significantly reduced by 1 mM EDTA while it was not the case for 50 μM EDTA and 10 μM TPEN. The intensity of FluoZin-3 fluorescence, a parameter for [Zn²⁺]_i, increased by A23187 was respectively reduced by 50 μM EDTA and 10 μM TPEN. It is suggested that the attenuation of A23187-induced cytotoxicity by 1 mM EDTA is due to the chelation of extracellular Ca²⁺ and Zn²⁺ while the augmentation by 50 μM ETDA or 10 μM TPEN is due to the chelation of extracellular Zn²⁺. The Tyrode’s solution without thymocytes contained 32.4 nM of zinc while it was 216.9 nM in the cell suspension. In conclusion, trace Zn²⁺, derived from cell preparation, partly attenuates the Ca²⁺-dependent cell death induced by A23187.     

Increase in intracellular Zn concentration by thimerosal in rat thymocytes: Intracellular Zn release induced by oxidative stress

Thimerosal (TMR), an ethylmercury-containing preservative in pharmaceutical products, was recently reported to increase intracellular Zn²⁺ concentration. Therefore, some health concerns about the toxicity of TMR remain because of physiological and pathological roles of Zn²⁺. To reveal the property of TMR-induced increase in intracellular Zn²⁺ concentration, the effect of TMR on FluoZin-3 fluorescence, an indicator of intracellular Zn²⁺, of rat thymocytes was examined. TMR at concentrations ranging from 0.3 μM to 10 μM increased the intensity of FluoZin-3 fluorescence in a concentration-dependent manner under external Ca²⁺- and Zn²⁺-free condition. The threshold concentration was 0.3–1 μM. The increase in the intensity was significant when TMR concentration was 1 μM or more. N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a chelator for intracellular Zn²⁺, completely attenuated the TMR-induced augmentation of FluoZin-3 fluorescence. Hydrogen peroxide (H₂O₂) and N-ethylmaleimide, reducing cellular thiol content, significantly increased FluoZin-3 fluorescence intensity and decreased 5-chloromethylfluorescein (5-CMF) fluorescence intensity, an indicator for cellular thiol. The correlation coefficient between TMR-induced augmentation of FluoZin-3 fluorescence and attenuation of 5-CMF fluorescence was −0.882. TMR also attenuated the 5-CMF fluorescence in the presence of TPEN. Simultaneous application of H₂O₂ and TMR synergistically augmented the FluoZin-3 fluorescence. It is suggested that TMR increases intracellular Zn²⁺ concentration via decreasing cellular thiol content.     

Elevation of intracellular Zn level by nanomolar concentrations of triclocarban in rat thymocytes

It was recently reported that nanomolar concentrations of triclocarban, an antimicrobial agent, were detected in human blood after the use of soap containing triclocarban. Due to the widespread use of triclocarban in adult and infant personal care products, the report prompted us to study its cytotoxicity. The cytotoxicity of triclocarban was examined in rat thymocytes by using a cytometric technique with propidium iodide for examining cell lethality, FluoZin-3-AM for monitoring the intracellular Zn²⁺ level, and 5-chloromethylfluorescencein diacetate for estimating the cellular content of non-protein thiol. The incubation with triclocarban at nanomolar concentrations (50–500 nM) for 1 h did not affect cell lethality but significantly elevated the intracellular Zn²⁺ level. The elevation of the intracellular Zn²⁺ level by triclocarban was not significantly dependent on external Zn²⁺ level. There was a negative correlation (r = −0.9225) between the effect on the intracellular Zn²⁺ level and that on the cellular content of non-protein thiol. These results suggest that nanomolar concentrations of triclocarban decrease the cellular content of non-protein thiol, leading to intracellular Zn²⁺ release. Since zinc plays physiological roles in mammalian cells, the percutaneous absorption of triclocarban from soap may, therefore, affect some cellular functions.     

Ethylene bisdithiocarbamate pesticides Maneb and Mancozeb cause metal overload in human colon cells

Previous studies in our laboratory have shown that ethylene bisdithiocarbamate (EBDC) fungicides Maneb and Mancozeb are equipotent gastrointestinal toxicants that produce cell loss and metal accumulation within HT-29 and Caco2 colon cells. Nabam, MnCl₂, CuCl₂ and ZnCl₂ exposure produced no loss of viability up to 200 μM and increases in metal levels were noted but not to the same extent as Maneb and Mancozeb. EBDC exposure caused increases in copper levels (20–200 μM). Maneb and Mancozeb treatment also caused increases in manganese and zinc concentrations (20–200 μM). Nabam plus MnCl₂ and Nabam and MnCl₂ plus ZnCl₂ caused decreases in viability and increases in metal levels comparable to Maneb and Mancozeb. Decreases in the ratio of reduced glutathione to glutathione disulfide were observed with Maneb and Mancozeb (20–200 μM). Maneb and Mancozeb treatment results in intracellular metal accumulation leading to the oxidative stress. The metal moiety and the organic portion of EBDCs contribute to toxicity.     

Stimulation of Suicidal Erythrocyte Death by Ellipticine

Ellipticine is a potent antineoplastic alkaloid effective in part by triggering apoptosis. Mechanisms involved in ellipticine‐induced apoptosis include mitochondrial depolarization and DNA damage. Erythrocytes lack mitochondria and nuclei but may nevertheless enter suicidal death or eryptosis, which is characterized by cell shrinkage and phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase in cytosolic Ca²⁺ activity ([Ca²⁺]_i), ceramide formation and oxidative stress. This study tested whether ellipticine stimulates eryptosis. Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter (FSC), [Ca²⁺]_i from Fluo‐3 fluorescence, ceramide abundance from binding of specific antibodies and reactive oxygen species from 2′,7′‐dichlorodihydrofluorescein diacetate fluorescence. A 24‐hr exposure of human erythrocytes to ellipticine (5 μg/ml) significantly increased the percentage of annexin V binding cells, ceramide abundance and oxidative stress. Ellipticine did not significantly modify [Ca²⁺]_i, and the stimulation of annexin V binding by ellipticine (5 μg/ml) did not require the presence of extracellular Ca²⁺. Ellipticine (5 μg/ml) did not significantly modify FSC. Ionomycin (1 μM, 1 hr) decreased FSC, an effect slightly but significantly blunted by ellipticine (5 μg/ml). Ellipticine thus stimulates phosphatidylserine translocation in the erythrocyte cell membrane, an effect at least partially due to stimulation of oxidative stress and ceramide formation.     

Tri-n-butyltin increases intracellular Zn concentration by decreasing cellular thiol content in rat thymocytes

Effect of tri-n-butyltin (TBT), an environmental pollutant, on intracellular Zn²⁺ concentration was tested in rat thymocytes to reveal one of cytotoxic profiles of TBT at nanomolar concentrations using a flow cytometer and appropriate fluorescent probes. TBT at concentrations of 30 nM or more (up to 300 nM) significantly increased the intensity of FluoZin-3 fluorescence, an indicator for intracellular Zn²⁺ concentration, under external Ca²⁺- and Zn²⁺-free condition. Chelating intracellular Zn²⁺ completely attenuated the TBT-induced augmentation of FluoZin-3 fluorescence. Result suggests that nanomolar TBT releases Zn²⁺ from intracellular store site. Oxidative stress induced by hydrogen peroxide also increased the FluoZin-3 fluorescence intensity. The effects of TBT and hydrogen peroxide on the fluorescence were additive. TBT-induced changes in the fluorescence of FluoZin-3 and 5-chloromethylfluorescein, an indicator for cellular thiol content, were correlated with a coefficient of −0.962. Result suggests that the intracellular Zn²⁺ release by TBT is associated with TBT-induced reduction of cellular thiol content. However, chelating intracellular Zn²⁺ potentiated the cytotoxicity of TBT. Therefore, the TBT-induced increase in intracellular Zn²⁺ concentration may be a type of stress responses to protect the cells.     

Zinc-dependent and independent actions of hydroxyhydroquinone on rat thymic lymphocytes

Coffee contains hydroxyhydroquinone (HHQ). HHQ is one of the by-products released during bean roasting. Therefore, it is important to elucidate the bioactivity of HHQ to predict its beneficial or adverse effects on humans. We studied zinc-dependent and independent actions of commercially procured synthetic HHQ in rat thymocytes using flow cytometric techniques with propidium iodide, FluoZin-3-AM, 5-chloromethylfluorescein diacetate, and annexin V-FITC. HHQ at 1050?µM elevated intracellular Zn²⁺ levels by releasing intracellular Zn²⁺. HHQ at 10?µM increased cellular thiol content in a zinc-dependent manner. However, HHQ at 30–50?µM reduced cellular thiol content. Although the latter actions of HHQ (30–50?µM) were suggested to increase cell vulnerability to oxidative stress, HHQ at 0.3–100?µM significantly protected cells against oxidative stress induced by H₂O₂. The process of cell death induced by H₂O₂ was delayed by HHQ, although both H₂O₂ and HHQ increased the population of annexin V-positive living cells. However, HHQ at 10–30?µM promoted cell death induced by A23187, a calcium ionophore. HHQ at 10–30?µM exerted contrasting effects on cell death caused by oxidative stress and Ca²⁺ overload. Because HHQ is considered to possess diverse cellular actions, coffee with reduced amount of HHQ may be preferable to avoid potential adverse effects.     

Protective mechanism of quercetin and rutin on 2,2′-azobis(2-amidinopropane)dihydrochloride or Cu-induced oxidative stress in HepG2 cells

Protective effects of quercetin and rutin against oxidative stress were evaluated using in vitro and intracellular antioxidant assay. Quercetin showed higher peroxyl and hydroxyl radical-scavenging activity in a dose-dependent manner than did rutin in oxygen-radical absorbance capacity (ORAC). At 10 and 100 μM, quercetin had higher metal-chelating activity than rutin carrying rutinose at position C-3 and was also more efficient than rutin in reducing intracellular oxidative stress caused by peroxyl radicals and Cu²⁺. The protective activities of 10 and 100 μM quercetin against Cu²⁺-induced intracellular oxidation were 13.8% and 44.8%, respectively. Rutin showed no protective activity against Cu²⁺-induced oxidative stress. Quercetin showed significantly lower intracellular Cu²⁺-chelating activity than did 1,10-phenanthroline but offered greater protection from Cu²⁺-induced oxidative stress. Thus, quercetin may diffuse through the cell membrane more efficiently than rutin because quercetin does not carry rutinose, is hydrophilic, and reduces Cu²⁺-induced oxidative stress by scavenging radicals instead of chelating with metal ions.     

Involvement of oxidative stress-induced ERK/JNK activation in the Cu(2+)/pyrrolidine dithiocarbamate complex-triggered mitochondria-regulated apoptosis in pancreatic β-cells

Oxidative stress was demonstrated to promote the progression of diabetes mellitus (DM). It has been suggested that copper may play a specific role in the progression and pathogenesis of DM. Pyrrolidine dithiocarbamate (PDTC), a widely apply to the medicine, was known to be capable of enhancing copper accumulation. In this study, we investigated the effect of submicromolar-concentration Cu²⁺/PDTC complex on pancreatic β-cell damage and evaluated the role of oxidative stress in this effect. CuCl₂ (0.01-300 μM) did not affect the cell viability in β-cell line RIN-m5F cells. However, combination of CuCl₂ (0.5 μM) and PDTC (0.3 μM) markedly reduced RIN-m5F cell viability. Cu²⁺/PDTC complex could also increase the LPO and decrease the intracellular reduced GSH levels, and display several features of apoptosis signals including: increase in sub-G1 cell population, annexin-V binding, and caspase-3 activity, mitochondrial dysfunctions, and the activation of PARP and caspase cascades, which accompanied with the marked increase the intracellular Cu²⁺ levels. These apoptotic-related responses of Cu²⁺/PDTC complex-induced could be effectively prevented by antioxidant N-acetylcysteine (NAC). Furthermore, Cu²⁺/PDTC complex was capable of increasing the phosphorylations of ERK1/2 and JNK, and its upstream kinase MEK1/2 and MKK4, which could be reversed by NAC. Transfection with ERK2- and JNK-specific si-RNA and specific inhibitors SP600125 and PD98059 could inhibit ERK1/2 and JNK activation and attenuate MMP loss and caspase-3 activity induced by the Cu²⁺/PDTC complex. Taken together, these results are the first report to demonstrate that the Cu²⁺/PDTC complex triggers a mitochondria-regulated apoptosis via an oxidative stress-induced ERK/JNK activation-related pathway in pancreatic β-cells.     

Activation of the N‐methyl‐d‐aspartate receptor is involved in glyphosate‐induced renal proximal tubule cell apoptosis

Glyphosate‐based herbicides have been used worldwide for decades and have been suggested to induce nephrotoxicity, but the underlying mechanism is not yet clear. In this study, we treated a human renal proximal tubule cell line (HK‐2) with glyphosate for 24 hours at concentrations of 0, 20, 40 and 60 μm . Glyphosate was found to reduce cell viability and induce apoptosis and oxidative stress in a dose‐dependent manner. Because the chemical structures of glyphosate and those of its metabolite AMPA are similar to glycine and glutamate, which are agonists of the N‐methyl‐d ‐aspartate receptor (NMDAR), we investigated the potential role of the NMDAR pathway in mediating the proapoptotic effect of glyphosate on proximal tubule cells. We found that NMDAR1 expression, as well as intracellular Ca²⁺ ([Ca²⁺]_i) and reactive oxygen species (ROS) levels, increased after glyphosate treatment. Blocking NMDAR attenuated glyphosate‐induced upregulation of [Ca²⁺]_i and ROS levels as well as apoptosis. Meanwhile, inhibition of [Ca²⁺]_i reduced glyphosate‐induced ROS and apoptosis, and inhibition of ROS alleviated glyphosate‐induced apoptosis. In mice exposed to 400 mg/kg glyphosate, the urine low molecular weight protein levels started to increase from day 7. Upregulation of apoptosis and NMDAR1 expression in renal proximal tubule epithelium and an imbalance of oxidant and antioxidative products were observed. These results strongly suggest that activation of the NMDAR1 pathway, together with its downstream [Ca²⁺]_i and oxidative stress, is involved in glyphosate‐induced renal proximal tubule epithelium apoptosis.     

Toxicity of zinc oxide (ZnO) nanoparticles on human bronchial epithelial cells (BEAS-2B) is accentuated by oxidative stress

Although several studies reported that cytotoxic effects of various nanoparticles are partially due to induction of oxidative stress, it is unclear how oxidative state of the cell per se could influence its sensitivity to cytotoxic nanoparticles. This is of clinical significance because certain pathological conditions such as inflammation is associated with elevated oxidative stress and this may alter sensitivity of cells and tissues to cytotoxic nanoparticles. Hence, this study investigated how initial exposure of BEAS-2B human bronchial epithelial cells to oxidative stress influences subsequent response to cytotoxic challenge with zinc oxide (ZnO) nanoparticles (≈10 nm). Oxidative stress was induced by exposing BEAS-2B cells to 5 and 10 μM of H₂O₂ for 45 min in PBS (with Ca²⁺). Subsequently, the H₂O₂ solutions were washed off and the cells were exposed to varying concentrations (5–25 μg/ml) of ZnO nanoparticles in culture media for 24 h, followed by cell viability assessment with the WST-8 assay. The results demonstrated that initial transient exposure of cells to oxidative stress accentuated cytotoxicity of ZnO nanoparticles. In the negative control unexposed to H₂O₂, >99% of cells remained viable up to a ZnO nanoparticle concentration of 10 μg/ml, but displayed a steep decrease in viability above 10 μg/ml ZnO. By contrast, cells that were initially exposed to 5 and 10 μM of H₂O₂, displayed a sharp drop in viability even at concentrations below 10 μg/ml ZnO. At 10 μg/ml ZnO, cells initially exposed to 10 μM H₂O₂ displayed a viability of 40.6 ± 2.0%, which is significantly lower than the corresponding values of 72.8 ± 2.0% and 99.9 ± 1.1% obtained for initial exposure to 5 μM H₂O₂ and the negative control, respectively. Hence, initial exposure of BEAS-2B cells to oxidative stress sensitized their subsequent response to cytotoxic challenge with ZnO nanoparticles.     

Effects of 17 β-estradiol on lipopolysacharride-induced intracellular adhesion molecule-1 mRNA expression and Ca2+ homeostasis alteration in human endothelial cells

Recent evidence showed that 17 β-estradiol (E₂) decreased cytokine-induced expression of cell adhesion molecules (CAM). Changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) has been shown to be associated with CAM expression in endothelial cells. Here, the effects of E₂ (1 μM, 24 h) on the expression of intracellular adhesion molecule-1 (ICAM-1) and [Ca²⁺]_i were investigated in a lipopolysaccharide (LPS) (100 ng/mL, 18 h)-stimulated human endothelial cell line, EA.hy926, using real-time PCR and spectrofluorometry, respectively. PCR analysis revealed a significant increase in ICAM-1 expression in calcium ionophore A23187 (1 nM)- or LPS-stimulated cells. Pretreatment of cells with E₂ significantly inhibited LPS-induced ICAM-1 mRNA expression. [Ca²⁺]_i was monitored in Fura-2 AM-loaded cells in the presence and absence of extracellular Ca²⁺ with thapsigargin (TG, 1 μM), a sarco/endoplasmic reticulum ATPase inhibitor or ATP (100 μM). The extent of TG- or ATP-induced [Ca²⁺]_i increase was significantly higher in LPS-stimulated cells than in control cells. Pre-treatment of LPS-stimulated cells with E₂ limited the Ca²⁺ response to the same level as in control cells. Furthermore, ICI 182,780, an estrogen receptor antagonist, attenuated the inhibitory actions of E₂ on ICAM-1 mRNA expression and Ca²⁺ responses, suggesting that estrogen receptors mediate, at least in part, the effects of estrogen. These data suggest a potential underlying mechanism for the protective effect of E₂ against atherosclerosis.     

Air pollution-related metals induce differential cytokine responses in bronchial epithelial cells

Different transition metals have been shown to induce inflammatory responses in lung. We have compared eight different metal ions with regard to cytokine responses, cytotoxicity and signalling mechanisms in a human lung epithelial cell model (BEAS-2B). Among the metal ions tested, there were large differences with respect to pro-inflammatory potential. Exposure to Cd^2 +, Zn^2 + and As^3 + induced CXCL8 and IL-6 release at concentrations below 100 μM, and Mn^2 + and Ni^2 + at concentrations above 200 μM. In contrast, VO₄^3 −, Cu^2 + and Fe^2 + did not induce any significant increase of these cytokines. An expression array of 20 inflammatory relevant genes also showed a marked up-regulation of CXCL10, IL-10, IL-13 and CSF2 by one or more of the metal ions. The most potent metals, Cd^2 +, Zn^2 + and As^3 + induced highest levels of oxidative activity, and ROS appeared to be central in their CXCL8 and IL-6 responses. Activation of the MAPK p38 seemed to be a critical mediator. However, the NF-κB pathway appeared predominately to be involved only in Zn^2 +- and As^3 +-induced CXCL8 and IL-6 responses. Thus, the most potent metals Cd^2 +, Zn^2 + and As^3 + seemed to induce a similar pattern for the cytokine responses, and with some exceptions, via similar signalling mechanisms.     

Effects of the mycotoxin destruxin A on Locusta migratoria visceral muscles

Destruxins, a family of cyclic peptides, are produced by various species of entomopathogenic fungi. These peptides have been shown to influence calcium-dependent processes in insect cell lines and tissues, such as skeletal muscles. To better understand the mechanism of action of these peptide toxins on insect muscular tissues, we have evaluated the effects of destruxin A on the contractions of oviducts and hindgut of Locusta migratoria. In oviducts, destruxin A increased the frequency of spontaneous contractions and induced a dose-dependent tonic contraction; the EC₅₀ for lower lateral and upper lateral oviducts was 0.7 μM and 8.7 μM, respectively. In hindgut, destruxin A also caused an increase in the frequency of spontaneous contractions; the EC₅₀ was 3.2 μM. The action of destruxin A was abolished in Ca²⁺-free saline or when the Ca²⁺ channel blocker CoCl₂ was added to the incubation saline. Likewise, the presence of 50 μM nifedipine or 100 μM verapamil in the medium reduced the magnitude of destruxin A′s effect, particularly in hindgut. The depolarization of muscle membranes by 100 mM K⁺ saline prevented the action of destruxin A. Preincubation of lower lateral oviducts in the intracellular Ca²⁺ antagonist TMB-8 did not have any effect on destruxin A action; however, preincubation in the calmodulin inhibitor trifluoperazine greatly reduced the effect of destruxin A. Taken together, these results show that destruxin A has an excitatory effect on contractions of insect visceral muscles of L. migratoria. Destruxin A-induced contractions appear to be dependent on extracellular, but not on intracellularly-released Ca²⁺, which suggest that this peptide toxin might be acting on insect visceral muscle by facilitating an influx of extracellular Ca²⁺.