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Abstract

Bifenthrin, a commonly used pyrethroid pesticide, evokes various toxicological effects in different models. However, the effect of bifenthrin on cytosolic-free Ca²⁺ level ([Ca²⁺] _i ) and cytotoxicity in human prostate cancer cells is unclear. This study examined whether bifenthrin altered Ca²⁺ homeostasis and cell viability in PC3 human prostate cancer cells. [Ca²⁺] _i in suspended cells were measured using the fluorescent Ca²⁺-sensitive dye fura-2. Cell viability was examined by 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] water soluble tetrazolium-1 assay. Bifenthrin (100–400 μM) concentration-dependently induced [Ca²⁺] _i rises. Ca²⁺ removal reduced the signal by approximately 30%. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) abolished bifenthrin-evoked [Ca²⁺] _i rises. Conversely, treatment with bifenthrin abolished BHQ-evoked [Ca²⁺] _i rises. Inhibition of phospholipase C (PLC) with U73122 significantly inhibited bifenthrin-induced [Ca²⁺] _i rises. Mn²⁺ has been shown to enter cells through similar mechanisms as Ca²⁺ but quenches fura-2 fluorescence at all excitation wavelengths. Bifenthrin (400 μM)-induced Mn²⁺ influx implicates that Ca²⁺ entry occurred. Bifenthrin-induced Ca²⁺ entry was inhibited by 30% by protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate) and inhibitor (GF109203X) and three inhibitors of store-operated Ca²⁺ channels: nifedipine, econazole, and SKF96365. Bifenthrin at 175–275 μM decreased cell viability, which was not reversed by pretreatment with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetra acetic acid-acetoxymethyl ester. Together, in PC3 cells, bifenthrin-induced [Ca²⁺] _i rises by evoking PLC-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ entry. Bifenthrin also caused Ca²⁺-independent cell death.

Keywords

Bifenthrin cytotoxicity endoplasmic reticulum prostate cancer cells

Introduction

Synthetic pyrethroid insecticides were introduced into widespread use for the control of insects and disease vectors for decades. Pyrethroids account for approximately one-fourth of the worldwide insecticide market, and the use of these compounds is increasing.¹ This usage results in an increased potential for human exposure, including exposure to pregnant women, infants, and children.² Based on both their chemical structures and biologic responses to acute exposure, pyrethroids are classified into two groups: type I and type II. Type I pyrethroids lack a cyano group at the α carbon of the 3-phenoxybenzyl alcohol moiety and produce hyperexcitation, tremors, and convulsions, whereas type II pyrethroids have a cyano group at the α carbon and produce hypersensitivity, choreoathetosis, salivation, and seizures.³ Therefore, it is important to explore the toxic action of pyrethroid pesticides in human.

Bifenthrin, a type I pyrethroid pesticide, has been shown to possess different in vitro effects. Bifenthrin caused neurite retraction in the absence of cell death.⁴ In differentiating PC12 cells, bifenthrin was reported to inhibit neurite outgrowth.⁵ Furthermore, bifenthrin activated homotypic aggregation in human T-cell lines.⁶ It is reported that bifenthrin induced apoptosis through mitogen-activated protein kinase signaling pathway in HepG2 cells⁷ and evoked oxidative stress in human erythrocytes.⁸ At the channel level, bifenthrin altered Ca²⁺ oscillations and cortical neuron development independent of Na⁺ channel activity.⁹ Bifenthrin was reported to induce stimulation of Ca²⁺ influx in neocortical neurons¹⁰ and alter activity of Na⁺ channels expressed in rat cerebral cortical neurons.¹¹ However, the effect of bifenthrin on Ca²⁺ homeostasis in human prostate cancer cells has not been explored.

An alteration in [Ca²⁺] _i is a key regulator of many cellular processes such as fertilization, protein activation, gene expression, proliferation, plasticity, apoptosis, secretion, contraction, and so on.¹² To allow a precise regulation of [Ca²⁺] _i and various signaling pathways, cells possess many mechanisms to control [Ca²⁺] _i both globally and at the subcellular level.¹³ Among these are many members of the superfamily of G-protein-coupled receptors, which are characterized by the presence of seven transmembrane domains.¹⁴ Typically, these receptors are able to activate phospholipase C (PLC) leading to Ca²⁺ release from intracellular stores, which subsequently evoked Ca²⁺ entry across the plasma membrane. Many intracellular molecules can also regulate Ca²⁺ signal, such as protein kinase C (PKC)¹⁵ and cAMP.¹⁶

A previous study has shown that bifenthrin caused prostate dysfunction and increased risk of the antiandrogenic activity in rats.¹⁷ Therefore, the risk of exposing to bifenthrin in human prostate should be cautioned. This study was aimed to explore the effect of bifenthrin on Ca²⁺ homeostasis and cell viability and to explore their relationship. The PC3 human prostate cancer cells were used because it produces measurable [Ca²⁺] _i rises upon pharmacological stimulation. It has been shown that in this cell, [Ca²⁺] _i rises and death can be evoked by stimulation with chemicals, such as timolol,¹⁸ carvacrol,¹⁹ and thymol.²⁰ To understand the physiological significance of this Ca²⁺ signal, it is important to elucidate the mechanisms underlying the signal. Fura-2 was used as a Ca²⁺-sensitive dye to measure [Ca²⁺] _i . The [Ca²⁺] _i rises were characterized, the concentration–response plots were established, and the mechanisms underlying bifenthrin-evoked Ca²⁺ entry and Ca²⁺ release were examined. The effect of bifenthrin on cell viability was also explored.

Materials and methods

Chemicals

The reagents for cell culture were from Gibco^® (Gaithersburg, Maryland, USA). Aminopolycarboxylic acid/acetoxy methyl (fura-2/AM) and 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetra acetic acid/acetoxy methyl (BAPTA/AM) were from Molecular Probes^® (Eugene, Oregon, USA). Bifenthrin (Figure 1(a)) and other reagents were from Sigma-Aldrich^® (St Louis, Missouri, USA) unless otherwise indicated. The purity (>98%) of bifenthrin was determined by high performance liquid chromatography densitometry.

Figure 1.

Effect of bifenthrin (a) on [Ca²⁺] _i in fura-2-loaded PC3 cells. (b) Bifenthrin was added at 25 s. The concentration of bifenthrin was indicated. The experiments were performed in Ca²⁺-containing medium. Y-axis is the [Ca²⁺] _i induced by bifenthrin in Ca²⁺-containing medium. (c) Effect of bifenthrin on [Ca²⁺] _i in the absence of extracellular Ca²⁺. Bifenthrin was added at 25 s in Ca²⁺-free medium. Y-axis is the [Ca²⁺] _i rise induced by bifenthrin in Ca²⁺-free medium. (d) Concentration–response plots of bifenthrin-induced [Ca²⁺] _i rises in the presence or absence of extracellular Ca²⁺. Y-axis is the percentage of the net (baseline subtracted) area under the curve (25–250 s) of the [Ca²⁺] _i rises induced by 400 μM bifenthrin 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

PC3 cells obtained from Bioresource Collection and Research Center (Taiwan) were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U ml⁻¹ penicillin, and 100 μg ml⁻¹ streptomycin.

Solutions used in [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 contained similar chemicals as Ca²⁺-containing medium except that CaCl₂ was replaced with 0.3 mM ethylene glycol tetra acetic acid (EGTA) and 2 mM MgCl₂. Bifenthrin was dissolved in dimethyl sulfoxide (DMSO) as a 0.1 M stock solution. The other chemicals were dissolved in water, ethanol, or DMSO. The concentration of organic solvents in the experimental solutions did not exceed 0.1% and did not affect viability or basal [Ca²⁺] _i .

[Ca²⁺] _i measurements

[Ca²⁺] _i was measured as previously described.^18
–20 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. 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 1 ml of medium and 0.5 million cells. Fluorescence was monitored with a Shimadzu 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. During the recording, reagents were added to the cuvette by pausing the recording for 2 s to open and close the cuvette-containing chamber. For calibration of [Ca²⁺] _i , after completion of the experiments, the detergent Triton X-100 (0.1%) and CaCl₂ (5 mM) were added to the cuvette to obtain the maximal fura-2 fluorescence. Then, the Ca²⁺ chelator EGTA (10 mM) was 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.²¹ Manganese ion (Mn²⁺) quenching of fura-2 fluorescence was performed in Ca²⁺-containing medium containing 50 μM MnCl₂. MnCl₂ was added to cell suspension in the cuvette 30 s before the fluorescence recoding started. Data were recorded at excitation signal at 360 nm (Ca²⁺-insensitive) and emission signal at 510 nm at 1-s intervals as described previously.²²

Cell viability analyses

Viability was assessed as previously described.^18
–20 The measurement of viability was based on the ability of cells to cleave tetrazolium salts by dehydrogenases. An increase in the amount of developed color correlated with the number of live cells. Assays were performed according to 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 bifenthrin. The fluorescent cell viability detecting reagent 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 bifenthrin treatment, and cells were incubated for 30 min in a humidified atmosphere. The cells were incubated with/without bifenthrin for 24 h. The absorbance of samples (A₄₅₀) was determined using an enzyme-linked immunosorbent assay (ELISA) reader. In experiments using BAPTA/AM to chelate cytosolic Ca²⁺, cells were treated with 5 μM BAPTA/AM for 1 h prior to incubation with bifenthrin. The cells were washed once with Ca²⁺-containing medium and incubated with/without bifenthrin for 24 h. The absorbance of samples (A₄₅₀) was determined using an ELISA reader. Absolute optical density was normalized to the absorbance of unstimulated cells in each plate and expressed as a percentage of the control value.

Statistics

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

Results

Effect of bifenthrin on [Ca²⁺] _i in PC3 cells

Figure 1(b) shows that the basal [Ca²⁺] _i level was 52 ± 2 nM (n = 3). At 100–400 μM, bifenthrin induced concentration-dependent rises in [Ca²⁺] _i . At a concentration of 400 μM, bifenthrin induced [Ca²⁺] _i rises of 90 ± 2 nM (n = 3). This signal was followed by a slow decay within 200 s. The Ca²⁺ response saturated at 400 μM bifenthrin because 600 μM bifenthrin did not evoke greater responses (not shown). In Ca²⁺-free medium, bifenthrin also induced concentration-dependent rises in [Ca²⁺] _i at 100–400 μM. At 400 μM, bifenthrin induced rises in [Ca²⁺] _i of 41 ± 2 nM (n = 3; Figure 1(c)). Figure 1(d) shows the concentration–response relationship. Ca²⁺ removal reduced the Ca²⁺ signal by approximately 30%. The EC₅₀ value was 140 ± 3 μM in Ca²⁺-containing or 100 ± 3 μM in Ca²⁺-free medium, respectively, by fitting to a Hill equation (p < 0.05). Hill equation is Y = min + (max − min)/1 + (X/EC₅₀)^−Hillslope. The X value for the curve point is midway between the max and min parameters. Hillslope characterizes the slope of the curve at its midpoint. Large values result in a steep curve whereas small values a shallow curve. By definition, EC₅₀ value is the effective concentration that induces 50% of maximum response. In our study, EC₅₀ of 140 μM means that at 140 μM, bifenthrin induced 50% of maximum response in Ca²⁺-containing medium. Thus, this value can give us an idea of the effective concentration range of an agent. The p < 0.05 is relative to the basal [Ca²⁺] _i level (without bifenthrin).

The source of bifenthrin-induced Ca²⁺ release in PC3 cells

Because bifenthrin-induced Ca²⁺ response saturated at 400 μM, in the following experiments, the response induced by 400 μM bifenthrin was used as control. Experiments were conducted in Ca²⁺-free medium to exclude the involvement of Ca²⁺ influx. Figure 2(a) shows that the addition of 50 μM BHQ, a selective inhibitor of Ca²⁺ ATPases pump,²³ induced [Ca²⁺] _i rises of 51 ± 2 nM (n = 3). Bifenthrin (400 μM) added afterward at 500 s failed to induce [Ca²⁺] _i rises. Figure 2(b) shows that after 400 μM bifenthrin-induced [Ca²⁺] _i rises, the addition of 50 μM BHQ at 500 s failed to induce [Ca²⁺] _i rises (n = 3). The data suggest that the endoplasmic reticulum played a dominant role in bifenthrin-induced Ca²⁺ release from intracellular stores.

Figure 2.

Effect of BHQ on bifenthrin-induced Ca²⁺ release in PC3 cells. (a) and (b) BHQ (50 μM) and bifenthrin (400 μM) were added at time points indicated. Experiments were performed in Ca²⁺-free medium. Data are mean ± SD of three independent experiments. Ca²⁺: calcium ion; BHQ: 2,5-di(tert-butyl)-1,4-benzohydroquinone; SD: standard deviation.

A role of PLC in bifenthrin-induced [Ca²⁺] _i rises in PC3 cells

U73122,²⁴ a PLC inhibitor, was applied to explore if the activation of PLC was required for bifenthrin-induced Ca²⁺ release. Figure 3(a) shows that adenosine triphosphate (ATP; 10 μM) induced [Ca²⁺] _i rises of 51 ± 2 nM (n = 3). ATP is a PLC-dependent agonist of [Ca²⁺] _i rises in most cell types.²⁵ Figure 3(b) shows that incubation with 2 μM U73122 did not change basal [Ca²⁺] _i but abolished ATP-induced [Ca²⁺] _i rises. This suggests that U73122 effectively suppressed PLC activity. The data also show that incubation with 2 μM U73122 nearly abolished 400 μM bifenthrin-induced [Ca²⁺] _i rises (n = 3). As a negative control of U73122 effect, the PLC-insensitive structural analog of U73122, U73343, was used to test its effect on ATP-induced Ca²⁺ signal. Our results suggest that U73343 (2 μM) failed to affect ATP-caused [Ca²⁺] _i rises (not shown). These data implicate that bifenthrin-evoked Ca²⁺ release from the endoplasmic reticulum depended on PLC activity.

Figure 3.

Effect of U73122 on bifenthrin-induced Ca²⁺ release in PC3 cells. Experiments were performed in Ca²⁺-free medium. (a) ATP (10 μM) was added at 25 s. (b) First column is 400 μM bifenthrin-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 200 s abolished ATP-induced [Ca²⁺] _i rises (*p < 0.05 compared to first column). Fifth column shows that U73122 (incubation for 200 s) and ATP (incubation for 50 s) pretreatment inhibited 400 μM bifenthrin-induced [Ca²⁺] _i rises. Data are mean ± SD of three independent experiments. Ca²⁺: calcium ion; ATP: adenosine triphosphate; SD: standard deviation.

Bifenthrin-induced Mn²⁺ influx in PC3 cells

Experiments were performed to confirm that bifenthrin-evoked [Ca²⁺] _i rises involved Ca²⁺ influx. Figure 4 shows that 400 μM bifenthrin evoked an instant decrease in the 360 nm excitation signal that reached a value of 130 ± 4 arbitrary units at 250 s (n = 3). The data implicate that bifenthrin-caused [Ca²⁺] _i rises involved Ca²⁺ influx.

Figure 4.

Effect of bifenthrin on Ca²⁺ influx by measuring Mn²⁺ quenching of fura-2 fluorescence in PC3 cells. 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 bifenthrin. Trace b: bifenthrin (400 μM) was added as indicated. Data are mean ± SD of three independent experiments. Ca²⁺: calcium ion; Mn²⁺: manganese ion; MnCl₂: manganese chloride; SD: standard deviation.

Regulation of bifenthrin-induced [Ca²⁺] _i rises in PC3 cells

Phorbol 12-myristate 13 acetate (PMA; 1 nM; PKC activator), GF109203X (2 μM; a PKC inhibitor), econzaole (0.5 μM), nifedipine (1 μM), or SKF96365 (5 μM) was applied 1 min before bifenthrin (400 μM), in Ca²⁺-containing medium, then [Ca²⁺] _i changes were measured. All these five chemicals inhibited bifenthrin-induced [Ca²⁺] _i rises by approximately 30% (p < 0.05; n = 3; Table 1). Furthermore, we have performed Ca²⁺ adding back experiments. Cells were first incubated in Ca²⁺-free medium, then bifenthrin was added at 25 s to induce a [Ca²⁺] _i rise. At the time point of 500 s, 3 mM Ca²⁺ was added back to the suspension. This immediately induced a [Ca²⁺] _i rise which was taken as control. In this experiment, each of the inhibitors was added 30 s before 3 mM Ca²⁺. Nifedipine, econazole, SKF96365, PMA, and GF109203X all significantly inhibited Ca²⁺-induced [Ca²⁺] _i rises by 30% (n = 3; not shown). Therefore, bifenthrin-induced Ca²⁺ influx appears to be partly mediated by PKC-regulated store-operated Ca²⁺ entry.

Table 1.

Effect of Ca²⁺ channel modulators on bifenthrin-induced [Ca²⁺] _i rises in PC3 cells.

Treatment
Control	100 ± 2
+PMA	72 ± 1^b
+GF109203X	72 ± 2^b
+econazole	72 ± 3^b
+nifedipine	74 ± 2^b
+SKF96365	69 ± 4^b

Ca²⁺: calcium ion; PMA: phorbol 12-myristate 13 acetate; SD: standard deviation.

^aIn modulator-treated group, the regulator was added 1 min before terfenadine (1000 μM). The concentration was 2 μM for GF109203X, 10 nM for PMA, 0.5 μM for econazole, 1 μM for nifedipine, and 5 μM for SKF96365. Data are presented as the percentage of control that is the area under the curve (25–200 s) of 400 μM bifenthrin-evoked [Ca²⁺] _i rises in Ca²⁺-containing medium and are mean ± SD of three independent experiments.

^bp < 0.05 compared to control.

Effect of bifenthrin on viability of PC3 cells

Tetrazolium assay was performed after cells were treated with 0–275 μM bifenthrin for 24 h. In the presence of 175–275 μM bifenthrin, cell viability decreased in a concentration-dependent manner (n = 3; Figure 5). The EC₅₀ value was 212 ± 3 μM by fitting to a Hill equation (p < 0.05). The intracellular Ca²⁺ chelator BAPTA/AM²⁶ was applied to prevent [Ca²⁺] _i rises during bifenthrin pretreatment, to explore the role of Ca²⁺ in bifenthrin-induced cell death. Figure 5 also shows that 5 μM BAPTA/AM loading did not change the control value of cell viability. Bifenthrin (400 μM) did not evoke [Ca²⁺] _i rises in BAPTA/AM-treated cells in both Ca²⁺-containing and Ca²⁺-free solutions (not shown). This suggests that BAPTA loading for 25 h still effectively chelated cytosolic Ca²⁺. In the presence of 175–275 μM bifenthrin, BAPTA loading failed to reverse bifenthrin-induced cell death. Collectively, the results imply that bifenthrin-evoked death of PC3 cells was dissociated from preceding rises in [Ca²⁺] _i .

Figure 5.

Effect of bifenthrin on cell viability in PC3 cells. Cells were treated with 0–275 μM bifenthrin for 24 h, and the cell viability assay was performed. Data are mean ± SD of three independent experiments. Each treatment had six replicates (wells). Data are expressed as percentage of control response that is the increase in cell numbers in bifenthrin-free groups. Control had 11,158 ± 222 cells/well before experiments and had 13,965 ± 211 cells/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 bifenthrin in Ca²⁺-containing medium. Ca²⁺: calcium ion; BAPTA/AM: 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra acetic acid/acetoxy methyl; SD: standard deviation.

Discussion

Bifenthrin is one of the most potent pyrethroids with LD₅₀ values of 53–70 mg kg⁻¹ in rats after oral gavage.²⁷ Bifenthrin, as low as 10 mg kg⁻¹, produces type I pyrethroid-like episodes of whole-body shakes, strong tremorigenic responses, hyperthermia, vocalizations, and prostration.²⁷ This dose range corresponds to a brain concentration of bifenthrin of 600 ng g⁻¹ of brain tissue.²⁸ In vivo, a multilevel evaluation of potential liver injury of bifenthrin has been performed.²⁹ In addition to toxicological effects of bifentrhin on brain or liver, the increased risk of the antiandrogenic activity and the reduction of prostate weight were shown in bifenthrin-treated rats.¹⁷ Therefore, our study explored the toxic effect of bifentrhin on human prostate cancer cells.

Previous studies showed that bifentrhin affected Ca²⁺ signaling in cortical neuron development.⁹ However, whether bifentrhin affects Ca²⁺ homeostasis and physiology in PC3 cells is unclear. This study explored the effect of bifenthrin on Ca²⁺ signaling and viability in PC3 cells. Our study shows that bifenthrin concentration-dependently increased [Ca²⁺] _i in PC3 cells. The Ca²⁺ signal was composed of Ca²⁺ entry and Ca²⁺ release because the signal was reduced by 30% by removing extracellular Ca²⁺.

Among all the organelles, the endoplasmic reticulum has been shown to be the main Ca²⁺ store in most cell types.³⁰ Thus, the role of the endoplasmic reticulum in bifenthrin-evoked Ca²⁺ release in PC3 cells was explored. The BHQ-sensitive endoplasmic reticulum store appears to be the pivotal Ca²⁺ store. Regarding the mechanism, one possibility was that bifenthrin acts similarly to BHQ by inhibiting the endoplasmic reticulum Ca²⁺-ATP pump.³¹ Many pathways regulate the release of Ca²⁺ from intracellular stores. Among them, PLC is one of the pivotal proteins.³² Accordingly, the role of PLC activation in bifenthrin-evoked Ca²⁺ release was examined. The results demonstrate that the Ca²⁺ release mainly depended on PLC activation, because the release was significantly inhibited when PLC activity was suppressed.

The mechanism of bifenthrin-induced Ca²⁺ influx was explored. Because Mn²⁺ enters cells through similar mechanisms as Ca²⁺ but quenches fura-2 fluorescence at all excitation wavelengths,³³ quenching of fura-2 fluorescence excited at the Ca²⁺-insensitive excitation wavelength of 360 nm by Mn²⁺ implicates Ca²⁺ influx. In PC3 cells, the main pathway of Ca²⁺ influx is the store-operated Ca²⁺ entry.¹⁸ The main internal Ca²⁺ store is the endoplasmic reticulum Ca²⁺ store.³⁴ Since Ca²⁺ signaling is so important, cells have complex mechanisms to regulate Ca²⁺ influx and release. Previous studies have shown that different compounds such as timolol or carvacrol induced Ca²⁺ entry through store-operated Ca²⁺ channels in PC3 cells.^18,19 Our findings show that bifenthrin-evoked [Ca²⁺] _i rises were inhibited by 30% by econazole, nifedipine, and SKF96365. These three compounds have been used to inhibit store-operated Ca²⁺ entry.^35
–37 Therefore, bifenthrin appears to cause Ca²⁺ entry via store-operated Ca²⁺ entry which is induced by depletion of intracellular Ca²⁺ stores.³⁸

The activity of many protein kinases is known to associate with Ca²⁺ homeostasis.^39,40 Our data show that bifenthrin-evoked [Ca²⁺] _i rises were inhibited by enhancing or inhibiting PKC activity. This may suggest that a normally maintained PKC activity was necessary for a full response of 400 μM bifenthrin-induced [Ca²⁺] _i rises. Literature also shows that PKC plays a key role in the activation of store-operated Ca²⁺ entry in models such as myotubes.⁴¹ Because 30% of bifenthrin-induced [Ca²⁺] _i rises were via Ca²⁺ influx, this influx appears to be totally contributed by PKC-sensitive store-operated Ca²⁺ entry.

Evidence accumulates that abnormal rises in [Ca²⁺] _i may lead to changes in cell viability.⁴² Thus, the effect of bifenthrin on viability of PC3 cells was explored. Ca²⁺ and cell viability may have an interactive relationship.^43,14 Our data show that bifenthrin-induced Ca²⁺-independent cell death in a concentration-dependent fashion. Although bifenthrin-evoked [Ca²⁺] _i rises were not cytotoxic, they may affect other Ca²⁺-related cellular responses in PC3 cells.^43,14

The literature shows that the plasma level of bifenthrin in human patients may reach 20 μM.^44,29 In patients with liver or kidney dysfunction or healthy persons exposing to higher doses, this level may go much higher. Our findings show that at a concentration of 175 μM, bifenthrin evoked death of 30% of PC3 cells. Thus, the clinical relevance of our data may not be excluded.

Together, the data implicate that bifenthrin caused Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ entry and also Ca²⁺ release from the endoplasmic reticulum in a PLC-dependent fashion. Furthermore, bifenthrin caused Ca²⁺-independent cell death. Since [Ca²⁺] _i rises play a triggering or modulatory role in numerous cellular phenomena, the effect of bifenthrin on [Ca²⁺] _i and cell viability should be taken into account in other in vitro study. There are some limitations in this study. First, the evaluation of the toxicological effect of bifenthrin was restricted to one human prostate cancer cell line, PC3, and thus other human prostate cancer (e.g., LNCaP or/and DU145) cells could also be employed to examine the toxicological effect. Second, the toxicity of bifenthrin to prostate function was not evaluated in an animal model. Because an in vitro study cannot perfectly mimic an in vivo exposure, in the future, our research will expand to the in vivo toxicity of bifenthrin.

Footnotes

Author contribution

J-MC, W-ZL, and W-CL are 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 no financial support for the research, authorship, and/or publication of this article: This work was supported by PS105013, 106-001, and 107-001 to J-MC and VGHKS 107-169 to C-RJ.

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Ca 2+ movement and cytotoxicity induced by the pyrethroid pesticide bifenthrin in human prostate cancer cells

Abstract

Keywords

Introduction

Materials and methods

Chemicals

Cell culture

Solutions used in [Ca2+] i measurements

[Ca2+] i measurements

Cell viability analyses

Statistics

Results

Effect of bifenthrin on [Ca2+] i in PC3 cells

The source of bifenthrin-induced Ca2+ release in PC3 cells

A role of PLC in bifenthrin-induced [Ca2+] i rises in PC3 cells

Bifenthrin-induced Mn2+ influx in PC3 cells

Regulation of bifenthrin-induced [Ca2+] i rises in PC3 cells

Effect of bifenthrin on viability of PC3 cells

Discussion

Footnotes

Author contribution

Declaration of conflicting interests

Funding

References

Solutions used in [Ca²⁺] _i measurements

[Ca²⁺] _i measurements

Effect of bifenthrin on [Ca²⁺] _i in PC3 cells

The source of bifenthrin-induced Ca²⁺ release in PC3 cells

A role of PLC in bifenthrin-induced [Ca²⁺] _i rises in PC3 cells

Bifenthrin-induced Mn²⁺ influx in PC3 cells

Regulation of bifenthrin-induced [Ca²⁺] _i rises in PC3 cells