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Abstract

Cyfluthrin is a pyrethroid insecticide and common household pesticide. The effect of cyfluthrin on Ca²⁺-related physiology in human osteosarcoma is unclear. This study investigated the effect of cyfluthrin on cytosolic-free Ca²⁺ concentrations ([Ca²⁺]_i) and viability in MG63 human osteosarcoma cells. Cyfluthrin concentration-dependently induced [Ca²⁺]_i rises. Cyfluthrin-induced Ca²⁺ entry was confirmed by the Mn²⁺-induced quench of fura-2 fluorescence. Cyfluthrin at concentrations of 10–100 μM induced [Ca²⁺]_i rises. Ca²⁺ removal reduced the signal by approximately 50%. Cyfluthrin (100 μM) induced Mn²⁺ influx suggesting Ca²⁺ entry. Cyfluthrin-induced Ca²⁺ entry was inhibited 50% by protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate) and inhibitor (GF109203X) and also by three inhibitors of store-operated Ca²⁺ channels: nifedipine, econazole, and SKF96365. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin (TG) completely inhibited cyfluthrin-evoked [Ca²⁺]_i rises. Conversely, treatment with cyfluthrin abolished TG-evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with 1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dion abolished cyfluthrin-induced [Ca²⁺]_i rises. Cyfluthrin at 25–65 μM decreased cell viability, which was not reversed by pretreatment with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid–acetoxymethyl ester. Together, in MG63 cells, cyfluthrin induced [Ca²⁺]_i rises by evoking PLC-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ entry. Cyfluthrin also caused Ca²⁺-independent cell death.

Keywords

cyfluthrin endoplasmic reticulum human osteosarcoma cells phospholipase C

Introduction

Cyfluthrin belongs to a huge family of pesticides called pyrethroids¹ and is commonly used in household.² Its many forms of isomers are sold commercially. Most pyrethroids including cyfluthrin are considered to be highly toxic to animals and humans.³ Therefore, it is important to explore the mechanisms underlying the effect of cyfluthrin on toxicologic research in cancer models. Regarding toxicity, cyfluthrin has been shown to have a genotoxic potential in human lymphocytes and rats⁴ and several different cancer cell lines such as SH-SY5Y human neuroblastoma cells, HepG2 human hepatoma cells, and Caco-2 human colon cancer cells.⁵ In terms of Ca²⁺ signaling, cyfluthrin has been reported to stimulate Ca²⁺ influx in neocortical neurons,⁶ to alter N-type Ca(v)2.2 Ca²⁺ channel in neurons,⁷ and to influence Ca²⁺- adenosine triphosphatase activity of rat brain synaptosomes and leukocyte membranes.⁸ However, the effect of cyfluthrin on Ca²⁺ signaling and its related mechanism has not been explored in human osteosarcoma cells.

Ca²⁺ ions have a crucial role in cellular homeostasis.⁹ An increase in intracellular free Ca²⁺ level ([Ca²⁺]_i) can regulate many cellular responses, including proliferation, apoptosis, gene expression, protein folding, fertilization, channel gating, secretion, contraction, plasticity, enzyme activation, and so on.^10,11 Ca²⁺ homeostasis is also clinically relevant. Alzheimer’s disease,¹² cardiac disease,¹³ and cancer^10,11 have been shown to involve abnormal cellular Ca²⁺ movement. In cell models, [Ca²⁺]_i may rise as a result of Ca²⁺ influx from extracellular medium or be released from internal organelles like the endoplasmic reticulum.^10,11 Ca²⁺ could enter cells through various Ca²⁺ channels and receptors on the plasma membrane.^10,11 Literature shows that in MG63 human osteosarcoma cells, store-operated Ca²⁺ entry plays a key role in Ca²⁺ influx.¹⁴ Phospholipase C (PLC)-related pathways play a pivotal role in Ca²⁺ release in many cell types. Activation of PLC leads to an increase in cytosolic levels of inositol trisphosphate (IP₃) which stimulate Ca²⁺ release from the endoplasmic reticulum via the IP₃ receptors.¹⁵ Due to the importance of a Ca²⁺ signal, cells have different mechanisms to tightly control the signal. Elucidation of the pathways underlying a stimulus-induced Ca²⁺ signal is pivotal for understanding the effect of the stimulus on the physiology of the cell.

Since previous researches have shown that in MG63 cells,¹⁶ [Ca²⁺]_i rises can be evoked in response to the stimulation of external compounds such as niflumic acid,¹⁷ amitriptyline,¹⁸ and methoxsalen¹⁹. The goal of this study was to investigate the underlying pathways of this Ca²⁺ movement. To this end, fura-2 was applied as a Ca²⁺-sensitive dye to measure [Ca²⁺]_i. The mechanisms of cyfluthrin-induced [Ca²⁺]_i rises were explored. Furthermore, the cytotoxic effect of cyfluthrin and its relationship to Ca²⁺ were also examined.

Materials and methods

Chemicals

Cyfluthrin (Figure 1(a)) and other reagents were purchased from Sigma-Aldrich® (St Louis, Missouri, USA) unless otherwise indicated. The chemicals for cell culture were purchased from Gibco® (Gaithersburg, Maryland, USA). Aminopolycarboxylic acid/acetoxy methyl (fura-2/AM) and 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxy methyl (BAPTA/AM) were purchased from Molecular Probes® (Eugene, Oregon, USA).

Figure 1.

Effect of cyfluthrin (a) on [Ca²⁺]_i in fura-2-loaded MG63 cells. (b) Cyfluthrin was added at 25 s. The concentration of cyfluthrin was indicated. The experiments were performed in Ca²⁺-containing medium. (c) Effect of cyfluthrin on [Ca²⁺]_i in the absence of extracellular Ca²⁺. Cyfluthrin was added at 25 s in Ca²⁺-free medium. (d) Concentration–response plots of cyfluthrin-induced [Ca²⁺]_i rises in the presence or absence of extracellular Ca²⁺. The y axis is the percentage of the net (baseline subtracted) area under the curve (25–250 s) of the [Ca²⁺]_i rises induced by 100 μM cyfluthrin in Ca²⁺-containing medium. Data are mean ± SD of three independent experiments. *p < 0.05: compared to open circles. Ca²⁺: calcium ion; SD: standard deviation.

Cell culture

MG63 human osteosarcoma cells purchased from Bioresource Collection and Research Center (Taiwan) were cultured in minimum essential medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U ml⁻¹ penicillin, and 100 µg ml⁻¹ streptomycin.

Solutions for [Ca²⁺]_i measurements

Ca²⁺-containing medium (pH 7.4) had 140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 5 mM glucose. Ca²⁺-free medium had similar chemicals as Ca²⁺-containing medium except that CaCl₂ was substituted with 0.3 mM ethylene glycol tetraacetic acid (EGTA) and 2 mM MgCl₂. Cyfluthrin was dissolved in ethanol as a 2 M stock solution. The other chemicals were dissolved in water, ethanol, or dimethyl sulfoxide. The concentration of organic solvents in the experimental solutions was below 0.1% and did not affect basal [Ca²⁺]_i or viability.

[Ca²⁺]_i measurements

Confluent cells grown on 6 cm dishes were trypsinized and made into a suspension in culture medium at a concentration of 10⁶ cells per milliliter. Cell viability was determined by trypan blue exclusion (adding 0.2% trypan blue to 0.1 ml cell suspension). The viability was greater than 95% after the treatment. Cells were subsequently loaded with 2 μM fura-2/AM for 30 min at 25°C in the same medium. After loading, cells were washed with Ca²⁺-containing medium twice and were made into a suspension in Ca²⁺-containing medium at a concentration of 10⁷ cells per milliliter. Fura-2 fluorescence measurements were performed in a water-jacketed cuvette (25°C) with continuous stirring; the cuvette contained 0.5 million cells suspended in 1 ml of medium. Fluorescence was monitored with a Shimadzu (Kyoto, Japan) RF-5301PC spectrofluorophotometer immediately after 0.1 ml cell suspension was added to 0.9 ml Ca²⁺-containing or Ca²⁺-free medium, by recording excitation signals at 340 and 380 nm and emission signal at 510 nm at 1-s intervals. For calibration of [Ca²⁺]_i, after completion of the experiments, Triton X-100 (0.1%) and CaCl₂ (5 mM) were added to the cuvette to obtain the maximal fura-2 fluorescence. The Ca²⁺ chelator EGTA (10 mM) was subsequently added to chelate Ca²⁺ in the cuvette to obtain the minimal fura-2 fluorescence. Control experiments showed that cells bathed in a cuvette had a viability of 95% after 20 min of fluorescence measurements. [Ca²⁺]_i was calculated as previously described.²⁰

Manganese ions (Mn²⁺) and Ca²⁺ use the same movement pathways in cells, thus Mn²⁺ influx implicates Ca²⁺ entry. MnCl₂ (50 μM) was added to cell suspension in the cuvette 30 s before fura-2 fluorescence recording began to determine cyfluthrin-induced Ca²⁺ influx. Data were recorded at excitation signal at 360 nm (Ca²⁺-insensitive) and emission signal at 510 nm at 1-s intervals as described previously.²¹

Cell viability assays

The assessment of cell viability was based on the rationale that cells can cleave tetrazolium salts by dehydrogenases. Increases in the intensity of color correlated with the live cell number. Assays were conducted according to the manufacturer’s instructions (Roche Molecular Biochemical, Indianapolis, Indiana, USA). Cells were seeded in 96-well plates at a concentration of 10⁴ cells per well in culture medium for 24 h in the presence of cyfluthrin. The cell viability detecting tetrazolium dye 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] (WST-1; 10 µl pure solution) was added to samples after cyfluthrin incubation, and cells were incubated for 30 min in a humidified atmosphere. In experiments using BAPTA/AM to chelate cytosolic Ca²⁺, cells were incubated with 5 μM BAPTA/AM for 1 h before incubation with cyfluthrin. The cells were washed once with Ca²⁺-containing medium and incubated with/without cyfluthrin for 24 h. The absorbance of samples (A₄₅₀) was determined using an enzyme-linked immunosorbent assay reader. Absolute optical density was normalized to the absorbance of untreated cells in each plate and expressed as a percentage of the control.

Statistics

Data are shown as mean standard deviation of three independent experiments. Data were analyzed by one-way analysis of variances using the Statistical Analysis System (SAS®, SAS Institute Inc., Cary, North Carolina, USA). Multiple comparisons between group means were performed by post hoc analysis using the Tukey’s honestly significantly difference procedure. A p value of less than 0.05 represented significance.

Results

Cyfluthrin-induced [Ca²⁺]_i movement in MG63 cells

The basal [Ca²⁺]_i level was 52 ± 1 nM (Figure 1(b)). Cyfluthrin evoked [Ca²⁺]_i rises in a concentration-dependent manner at concentrations between μM 10 and 100 μM in Ca²⁺-containing medium. At a concentration of 100 μM, cyfluthrin evoked [Ca²⁺]_i rises that attained a net increase of 51 ± 2 nM. In the absence of extracellular Ca²⁺, 10–100 μM cyfluthrin induced concentration-dependent [Ca²⁺]_i rises (Figure 1(c)). The Ca²⁺ response saturated at 100 μM cyfluthrin because 200 μM cyfluthrin did not cause greater responses (Figure 1(b) and (c)). The concentration–response relationships of cyfluthrin-caused [Ca²⁺]_i rises were depicted in Figure 1(d). The half-maximal effective concentration value was 50 ± 4 μM in Ca²⁺-containing medium or 48 ± 2 μM in Ca²⁺-free medium, respectively, by fitting to a Hill equation. Ca²⁺ removal reduced the Ca²⁺ signal by approximately 50%.

Cyfluthrin-induced Mn²⁺ influx suggests Ca²⁺ entry in MG63 cells

Mn²⁺ and Ca²⁺ share the same routes of movement in cells but the former quenches fura-2 fluorescence at all excitation wavelengths,²¹ while fura-2 binding to Ca²⁺ amplifies the fura-2 fluorescence except at the Ca²⁺-insensitive excitation wavelength of 360 nm. Figure 2 shows that 100 μM cyfluthrin evoked an instant decrease in the 360 nm excitation signal that reached a maximum value of 51 ± 1 arbitrary units at 75 s. The Mn²⁺ quenching results imply that Ca²⁺ entry contributed to cyfluthrin-induced Ca²⁺ signal.

Figure 2.

Effect of cyfluthrin on Ca²⁺ influx by measuring Mn²⁺ quenching of fura-2 fluorescence. Experiments were performed in Ca²⁺-containing medium. MnCl₂ (50 μM) was added to cells 1 min before fluorescence measurements. The y axis is fluorescence intensity (in arbitrary units) measured at the Ca²⁺-insensitive excitation wavelength of 360 nm and the emission wavelength of 510 nm. Trace a: control, without cyfluthrin. Trace b: cyfluthrin (100 μM) was added as indicated. Data are mean ± SD of three independent experiments. Ca²⁺: calcium ion; SD: standard deviation; Mn²⁺: manganese ion.

Regulation of cyfluthrin-induced [Ca²⁺]_i rises in MG63 cells

In Ca²⁺-containing medium, three store-operated Ca²⁺ entry inhibitors: nifedipine (1 μM), econazole (0.5 μM), and SKF96365 (5 μM); phorbol 12-myristate 13-acetate (1 nM; a protein kinase C (PKC) activator); and GF109203X (2 μM; a PKC inhibitor) were added 1 min before cyfluthrin (100 μM). All the chemicals inhibited cyfluthrin-induced [Ca²⁺]_i rises by approximately 50% (Figure 3).

Figure 3.

Effect of Ca²⁺ channel modulators on cyfluthrin-induced [Ca²⁺]_i rises. In blocker- or modulator-treated groups, the reagent was added 1 min before cyfluthrin (100 μM). The concentration was 1 μM for nifedipine, 0.5 μM for econazole, 5 μM for SKF96365, 10 nM for PMA, and 2 μM for GF109203X. Data are expressed as the percentage of control (first column, 100%) that is the area under the curve (25–200 s) of 100 μM cyfluthrin-induced [Ca²⁺]_i rises in Ca²⁺-containing medium and are mean ± SD of three independent experiments. *p < 0.05 compared to the first column. Ca²⁺: calcium ion; PMA: phorbol 12-myristate 13-acetate; SD: standard deviation.

The role of the endoplasmic reticulum in cyfluthrin-evoked Ca²⁺ release in MG63 cells

In MG63 cells, the endoplasmic reticulum was thought to play a key role in releasing store-operated Ca²⁺.^17
–19 To exclude the contribution of Ca²⁺ entry, the experiments were performed in Ca²⁺-free medium. Figure 4(a) shows that addition of 1 μM thapsigargin (TG), an endoplasmic reticulum Ca²⁺ pump inhibitor,²² evoked [Ca²⁺]_i rises of 22 ± 2 nM. Subsequently added 100 μM cyfluthrin did not induce [Ca²⁺]_i rises. Conversely, Figure 4(b) shows that the addition of TG failed to release more Ca²⁺ after cyfluthrin-induced [Ca²⁺]_i rises. It appears that the endoplasmic reticulum played a pivotal role in cyfluthrin-evoked Ca²⁺ release from organelles.

Figure 4.

Effect of TG on cyfluthrin-induced Ca²⁺ release. ((a) and (b)) TG (1 μM) and cyfluthrin (100 μM) were added at time points indicated. Experiments were performed in Ca²⁺-free medium. Data are mean ± SD of three independent experiments. TG: thapsigargin; Ca²⁺: calcium ion; SD: standard deviation.

PLC played a role in cyfluthrin-evoked [Ca²⁺]_i rises in MG63 cells

PLC-dependent activation of IP₃ receptors results in the release of Ca²⁺ from the endoplasmic reticulum.¹¹ Thus, the role of this pathway in the cyfluthrin-induced release of Ca²⁺ was examined. 1-[6-[((17β)-3-Methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dion (U73122)²³ is a commonly used PLC inhibitor that is applied to examine if the activation of PLC was necessary for cyfluthrin-induced Ca²⁺ release. First, Figure 5(a) shows that adenosine triphosphate (ATP; 5 μM) was found to evoke [Ca²⁺]_i rises of 23 ± 2 nM. ATP was chosen in our experiments because it is a well-established PLC-dependent stimulus of [Ca²⁺]_i rises in most known cells.¹² U73122 was used to test its inhibitory effect on ATP-induced [Ca²⁺]_i rises. Figure 5(a) and (b) shows that incubation with U73122 (2 μM) did not alter basal [Ca²⁺]_i but completely inhibited ATP-induced [Ca²⁺]_i rises. This suggests that U73122 effectively suppressed PLC activity. Subsequently, the data show that incubation with 2 μM U73122 did not alter basal [Ca²⁺]_i but abolished 100 μM cyfluthrin-induced [Ca²⁺]_i rises. Although U73122 is generally deemed as a selective inhibitor of PLC, the possibility that U73122 acted by PLC-independent action needs to be excluded. Thus, U73343 was used as a negative control. U73343 is structurally very similar to U73122 while it lacks inhibitory effect on PLC. Our results show that U73343 (2 μM) failed to alter ATP-induced [Ca²⁺]_i rises (Figure 5(b)). Therefore, this most likely suggests that U73122 selectively inhibited PLC activity in cyfluthrin-induced [Ca²⁺]_i rises.

Figure 5.

Effect of U73122 on cyfluthrin-induced Ca²⁺ release. Experiments were performed in Ca²⁺-free medium. Experiments were performed in Ca²⁺-free medium. (a) ATP (5 μM) was added at 25 s. (b) First column is 100 μM cyfluthrin-induced [Ca²⁺]_i rises. Second column shows that 2 μM U73122 did not alter basal [Ca²⁺]_i. Third column shows ATP-induced [Ca²⁺]_i rises. Fourth column shows that U73122 pretreatment for 30 s abolished ATP-induced [Ca²⁺]_i rises (*p < 0.05 compared to the first column). Fifth column shows that U73122 (incubation for 30 s) and ATP (incubation for 30 s) pretreatment abolished 100 μM cyfluthrin-induced [Ca²⁺]_i rises. Data are mean ± SD of three independent experiments. Ca²⁺: calcium ion; SD: standard deviation; ATP: adenosine triphosphate; U73122: 1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dion.

Cyfluthrin decreased cell viability in MG63 cells

Cyfluthrin (0–65 μM) was added to cells for 24 h, and tetrazolium assay was performed. Cyfluthrin caused a decrease in viability in a concentration-dependent manner between 25 μM and 65 μM (Figure 6). Because cyfluthrin significantly induced [Ca²⁺]_i rises, the next question was whether cyfluthrin-caused cytotoxicity was induced by preceding [Ca²⁺]_i rises. The intracellular Ca²⁺ chelator BAPTA/AM (5 μM)²⁴ was applied to prevent [Ca²⁺]_i rises during cyfluthrin incubation. At a concentration of 100 μM, cyfluthrin did not evoke [Ca²⁺]_i rises in BAPTA/AM-treated cells (not shown). Figure 6 shows that 5 μM BAPTA/AM incubation did not change the viability of control cell. In the presence of 25–65 μM cyfluthrin, BAPTA/AM loading did not reverse cyfluthrin-induced cell death. Thus, the findings implicate that cyfluthrin-evoked cytotoxicity was not caused by [Ca²⁺]_i rises.

Figure 6.

Effect of cyfluthrin on cell viability. Cells were treated with 0–60 μM cyfluthrin for 24 h, and the cell viability assay was performed. Data are mean ± SD of three separate experiments. Each treatment had six replicates (wells). Data are expressed as a percentage of control response that is the increase in cell numbers in cyfluthrin-free groups. Control had 10,555 ± 112 cells per well before experiments, and had 13,125 ± 211 cells per well after incubation for 24 h. *p < 0.05 compared to control. In each group, the Ca²⁺ chelator BAPTA/AM (5 μM) was added to fura-2-loaded cells followed by treatment with cyfluthrin in Ca²⁺-containing medium. Ca²⁺: calcium ion; BAPTA/AM: 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester); SD: standard deviation.

Discussion

Ca²⁺ signaling plays a key role in numerous cellular responses. Cyfluthrin has been shown to trigger Ca²⁺ entry in neocortical neurons,⁶ to alter N-type Ca²⁺ channel in neurons,⁷ and to alter Ca²⁺-ATP pump activity of rat brain synaptosomes and leukocyte membranes.⁸ However, the effect of cyfluthrin on human osteosarcoma cells is unclear. The present study shows that cyfluthrin induced [Ca²⁺]_i rises in MG63 cells by depleting Ca²⁺ stores and inducing Ca²⁺ entry. Removal of extracellular Ca²⁺ inhibited 50% of cyfluthrin-induced Ca²⁺ entry throughout the measurement of 200 s, suggesting that cyfluthrin induced both Ca²⁺ entry and Ca²⁺ release. The Mn²⁺ quenching results also implicate that Ca²⁺ entry contributed to cyfluthrin-induced Ca²⁺ signal.

The pathway of cyfluthrin-induced Ca²⁺ entry was examined. In MG63 cells, store-operated Ca²⁺ channels have been shown to play a crucial role in [Ca²⁺]_i rises triggered by different compounds such as niflumic acid, amitriptyline, and methoxsalen.^17
–19 Although there are so far no selective inhibitors for this Ca²⁺ entry, nifedipine, econazole, and SKF96365 were commonly applied for this purpose.^25
–27 Our results suggest that all of these three chemicals inhibited cyfluthrin-induced [Ca²⁺]_i rises. Therefore cyfluthrin-induced Ca²⁺ entry appears to involve store-operated Ca²⁺ pathway.

The changes in the activity of protein kinases are well established to be able to alter Ca²⁺ signaling.^28,29 Our data show that cyfluthrin-evoked [Ca²⁺]_i rises were inhibited by 50% by enhancing or inhibiting PKC activity. It appears that cyfluthrin can only trigger full Ca²⁺ responses when PKC activity is at a normal status. The relationship between PKC and Ca²⁺ movement has been reported. For example, Na⁺-Ca²⁺ exchanger 3 was reported to be differentially regulated by PKC and protein kinase A.³⁰ Calretinin was shown to participate in regulating steroidogenesis by PLC-Ca²⁺-PKC pathway in Leydig cells.³¹

Regarding the Ca²⁺ stores involved in cyfluthrin-evoked Ca²⁺ release, the TG-sensitive endoplasmic reticulum store seemed to be the dominant one. TG was originally purified from the Mediterranean plant Thapsia garganica. The biological activity of TG was due to its high affinity for the sarco/endoplasmic reticulum Ca²⁺ ATPase. Binding of TG to the pump leads to collapse of the Ca²⁺ homeostasis by releasing the stored Ca²⁺ in the endoplasmic reticulum and eventually cell death.³² The data further show that PLC-dependent mechanisms contributed to the Ca²⁺ release. The IP₃ receptor is highly expressed in most cells and mediates conspicuous Ca²⁺ release from the endoplasmic reticulum. Receptor stimulation activates the G protein-PLC pathway, which leads to IP₃-induced Ca²⁺ release and subsequent cellular responses.³³

Cell viability could be altered in a Ca²⁺-dependent or -independent manner. Previous studies have shown that rapid disruption of cellular integrity of zinc-treated astroglia was regulated by Ca²⁺-dependent mechanisms.³⁴ In contrast, sensitization to autoimmune hepatitis in Ca²⁺-independent phospholipase A₂-null mice led to duodenal villous atrophy with apoptosis.³⁵ Our data show that cyfluthrin induced Ca²⁺-independent cell death in a concentration-dependent manner in MG63 cells. Literature shows that the plasma level of cyfluthrin in human patients may reach 10 μM.^36,37 In patients with liver or kidney dysfunction or healthy persons taking higher doses this level may go much higher. Our findings show that at a concentration of 45 μM cyfluthrin evoked death of 50% of MG63 cells. Thus, the clinical relevance of our data may not be excluded.

In sum, our data show that cyfluthrin evoked [Ca²⁺]_i rises via inducing Ca²⁺ influx through a PKC-sensitive store-operated Ca²⁺ entry and also via Ca²⁺ release from the endoplasmic reticulum in a PLC-dependent fashion. In the physiological level, cyfluthrin also induced Ca²⁺-dissociated cell death. Thus, caution should be exercised when using cyfluthrin for other in vitro research in MG63 human osteosarcoma cells.

Footnotes

Author contribution

W-ZL and Y-CL contributed equally to this work.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by VGHKS107-149 to Y-CL.

ORCID iD

C-R Jan

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Action of the insecticide cyfluthrin on Ca 2+ signal transduction and cytotoxicity in human osteosarcoma cells

Abstract

Keywords

Introduction

Materials and methods

Chemicals

Cell culture

Solutions for [Ca2+]i measurements

[Ca2+]i measurements

Cell viability assays

Statistics

Results

Cyfluthrin-induced [Ca2+]i movement in MG63 cells

Cyfluthrin-induced Mn2+ influx suggests Ca2+ entry in MG63 cells

Regulation of cyfluthrin-induced [Ca2+]i rises in MG63 cells

The role of the endoplasmic reticulum in cyfluthrin-evoked Ca2+ release in MG63 cells

PLC played a role in cyfluthrin-evoked [Ca2+]i rises in MG63 cells

Cyfluthrin decreased cell viability in MG63 cells

Discussion

Footnotes

Author contribution

Declaration of conflicting interests

Funding

ORCID iD

References

Solutions for [Ca²⁺]_i measurements

[Ca²⁺]_i measurements

Cyfluthrin-induced [Ca²⁺]_i movement in MG63 cells

Cyfluthrin-induced Mn²⁺ influx suggests Ca²⁺ entry in MG63 cells

Regulation of cyfluthrin-induced [Ca²⁺]_i rises in MG63 cells

The role of the endoplasmic reticulum in cyfluthrin-evoked Ca²⁺ release in MG63 cells

PLC played a role in cyfluthrin-evoked [Ca²⁺]_i rises in MG63 cells