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TOXICOLOGY

Deltamethrin, a Pyrethroid Insecticide, Is a Potent Inducer for the Activity-Dependent Gene Expression of Brain-Derived Neurotrophic Factor in Neurons

Toyama Medical and Pharmaceutical University, Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Toyama, Japan (L.I., M.Y., K.K., D.H., A.T., M.T.); and Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Tokyo, Japan (L.I., A.T., M.T.)

Received July 12, 2005; accepted September 13, 2005.

	Abstract

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The mRNA expression of brain-derived neurotrophic factor (BDNF) is controlled in an activity-dependent manner through Ca²⁺ influx into neurons. Pyrethroids are widely used insecticides of low acute toxicity in mammals, but their effects on sodium channels are known to lead to hyperexcitation in neuronal cells of insects. In this study, we found that deltamethrin, a type II pyrethroid insecticide, was highly effective in inducing BDNF expression in culture and in the rat brain. Addition of deltamethrin to rat cortical cells in culture markedly increased the expression of BDNF exon III–V mRNA and protein, dependent upon the neuronal activity accompanying the influx of Ca²⁺ into neurons and the Ca²⁺ influx-dependent phosphorylation of extracellular signal-regulated kinases 1/2. The elevated expression was maintained for at least 48 h, even after deltamethrin was withdrawn from the culture medium. Comparison of the effects of selected pyrethroids on the expression revealed that type II but not type I pyrethroids effectively induced BDNF mRNA expression. In addition, administration of deltamethrin to rats increased the level of BDNF protein in the cerebral cortex and hippocampus. These results indicate that deltamethrin is a potent inducer of BDNF expression in neurons and that it may induce neuronal hyperexcitation if it reaches the brain.

Pyrethroids are highly active synthetic derivatives of natural pyrethrins, toxins present in the flowers of Chrysanthemum cinerariaefolium. Pyrethroid insecticides are classified into two major groups on the basis of chemical structures (Miyamoto et al., 1995): type I pyrethroids are devoid of a cyano moiety at the -position (i.e., permethrin), whereas type II pyrethroids have an -cyano moiety (i.e., cypermethrin and deltamethrin). In rats, type I pyrethroids cause a type I poisoning syndrome called "T syndrome", which is characterized by hyperexcitation, tremors, and convulsions, whereas type II pyrethroids induce a type II choreoathetosis syndrome known as "CS syndrome", which produces hypersensitivity, salivation, choreoathetosis, and chronic seizures (Verschoyle and Aldridge, 1980; Soderlund et al., 2002).

Since the 1970s, pyrethroids have been widely used to control insect pests in agriculture and in the home. Although pyrethroids are reported to be rapidly metabolized in mammals and hence have low toxicity, it is still unclear whether or how pyrethroids affect the neuronal structure and function of the mammalian brain (Soderlund et al., 2002). Pyrethroids prolong the opening of the voltage-sensitive sodium channel (VSSC) and raise a prolonged sodium tail current in mammalian as well as invertebrate neurons (Vijverberg et al., 1982; Narahashi, 1985). Furthermore, it has been reported that pyrethroids act on not only VSSC but also other ion channels or neurotransmitter receptors, such as voltage-gated chloride channels (Burr and Ray, 2004) and GABA_A receptors (Crofton and Reiter, 1987). In contrast, we have already demonstrated that relatively high concentrations (more than 10 µM) of pyrethroid insecticides decreased the expression of c-fos and BDNF genes induced by membrane depolarization in mouse cerebellar granule cells (Imamura et al., 2000). In the present study, however, we found that, in contrast to the inhibitory effect of pyrethroids, administration of deltamethrin to rat cortical neuronal cells in culture markedly increased the BDNF mRNA expression in an activity-dependent manner at relatively low concentrations (10 nM–1 µM).

	Materials and Methods

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Chemicals. Technical-grade deltamethrin, (S)--cyano-3-phenoxybenzyl-(1R,3R)-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylate (DM), other pyrethroids, and corn oil were purchased from Wako Pure Chemicals (Tokyo, Japan). A stock solution of pyrethroids was prepared with dimethyl sulfoxide (Sigma-Aldrich. St. Louis, MO). At the concentrations used in the culture media, dimethyl sulfoxide did not have any effect on the results of the present experiment (data not shown).

Primary Culture of Rat Cortical Cells. A primary culture of rat cortical cells was prepared from the cerebral cortexes of 17-day-old Sprague-Dawley rat (Japan SLC, Hamamatsu, Japan) embryos as described previously (Tabuchi et al., 1998). The dissociated cells were suspended in DMEM (Invitrogen, Carlsbad, CA) containing 10% fetal calf serum, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin and seeded at 2.5 x 10⁶ cells in culture dishes (35 mm in diameter; Asahi Techno Glass, Tokyo, Japan) that had been coated with polyethylenimine (Sigma-Aldrich). The cells were grown for 3 days, and then the medium was replaced with serum-free DMEM containing 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin. Under such culture conditions, Kato-Negishi et al. (2004) have demonstrated that spontaneous Ca²⁺ oscillation can be synchronously induced among neurons, indicating that active synapses are formed in this culture. For the mRNA analysis or ELISA experiments, the cells were stimulated with DM or other drugs after 5 days in culture.

Preparation of Animals. Seven-week-old male Sprague-Dawley rats (Japan SLC) were used. Procedures for animal care were performed in accordance with Guidelines for the Care and Use of Laboratory Animals of Toyama Medical and Pharmaceutical University. The lowest observed effect level for motor-depressant effects of intraperitoneally administered DM using corn oil as a vehicle was 30 mg/kg (Crofton et al., 1995). Based on this, DM (25 mg/kg) was administered intraperitoneally in a corn oil suspension. In addition, kainate (15 mg/kg) was injected intraperitoneally as a positive control of BDNF inducer (Zafra et al., 1990; Metsis et al., 1993). We administered corn oil alone to the control rats. Three or four rats were used for the DM, kainate, and vehicle treatment, respectively. These rats were killed at different times after injection (5 h for DM and vehicle; 1 h for kainate), and their cerebral cortexes and hippocampi were used for measurements of BDNF mRNA and protein by real-time RT-PCR and ELISA, respectively, as described below.

RNA Isolation and Analysis. Total cellular RNA was extracted by the acid guanidine phenol-chloroform method. The isolation of RNA from cultured cells or rat tissues was described in detail previously (Imamura et al., 2000, 2002). In brief, total cellular RNA was isolated according to the ISOGEN protocol (Nippon Gene, Tokyo, Japan; Imamura et al., 2000, 2002) and quantified with a Beckman spectrophotometer (Beckman Coulter, Fullerton, CA). One microgram of RNA was used for reverse transcription with SuperScript II (Invitrogen). Quantitative RT-PCR was conducted in an ABI PRISM 7700 sequence detection system (Applied Biosystems, Foster City, CA) using 1x SYBR Green master mix (Applied Biosystems) containing 2 µl of cDNA solution and 0.5 µM primer pairs. For the amplification of rat BDNF, exon III–V cDNA, BDNF exon III sense (5'-TCGGCCACCAAAGACTC-3'), and BDNF exon V antisense (5'-GCCCATTCACGCTCTCCA-3') primers were used. For the amplification of rat c-fos cDNA, rat c-fos sense (5'-GTTTCAACGCGGACTACGAG-3') and rat c-fos antisense (5'-AGCGTATCTGTCAGCTCCCT-3') were used. For the internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was amplified using rat GAPDH sense (5'-TCCATGACAACTTTGGCATCGTGG-3') and rat GAPDH antisense (5'-GTTGCTGTTGAAGTCACAGGAGAC-3') primers. The mRNA expression levels were computed from the C_T value and normalized to the concentration of GAPDH mRNA as an internal standard.

Immunoassay for Measurement of BDNF. BDNF protein was measured with a conventional two-site enzyme immunoassay system (ELISA). All samples were prepared using the slightly modified method of Katoh-Semba et al. (1998). For animal experiments, after decapitation, cerebral cortexes and hippocampi were immediately frozen in liquid nitrogen, weighed, and stored at –80°C. Each tissue was homogenized with 10 volumes of a solution of 2 M guanidine hydrochloride in 0.1 M sodium phosphate buffer, pH 7.0, in the presence of 1 mM EDTA, 10 mM N-ethylmaleimide, 0.36 mM pepstatin A (Wako Pure Chemicals), and 1 mM phenylmethylsulfonyl fluoride. For the cultured cells, after stimulation with drugs, cells were washed with phosphate-buffered saline two times and extracted with 100 µl of the solution as described above. The homogenates and the cell extracts were sonicated before centrifugation at 46,000g for 30 min at 4°C. The supernatants were used for quantitation of BDNF. The BDNF Emax immunoassay system (Promega, Madison, WI) was used according to the manufacturer's protocol. Briefly, PRO-BIND flat-bottomed plates (96-well plates; Falcon; BD Biosciences Discovery Labware, Bedford, MA) were coated overnight at 4°C with 100 µl of anti-BDNF monoclonal antibody in 25 mM sodium bicarbonate buffer, pH 9.7. The nonspecific binding sites were blocked with block and sample buffer (Promega) for 1 h at 25°C. Fifty microliters of supernatant diluted with 50 µl of lysis solution or 100 µl of standard (recombinant human BDNF) was added to each well and incubated for 2 h at 25°C with shaking. The wells were rinsed, anti-human BDNF polyclonal antibody was added, and the plate was incubated for 2 h at 25°C with shaking. This was followed by the addition of anti-IgY horseradish peroxidase conjugate, and the plate was incubated for 1 h at 25°C with shaking. The wells were washed with 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% (v/v) Tween 20 five times after each step. The assay was developed by adding TMB One Solution (Promega) and incubating for 10 min at 25°C with shaking and stopped by the addition of 1 M phosphoric acid. The absorbance at 450 nm was then recorded on a microplate reader (model 550; Bio-Rad, Hercules, CA). Protein concentrations in tissue homogenates and cell lysates were determined using a protein assay kit (Bio-Rad) with bovine serum albumin (Sigma-Aldrich) as a standard.

Immunoblotting Analysis. Total cellular lysates were extracted from the cultured cells using whole cell extract buffer (20 mM HEPES, pH 7.7, 0.3 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.1% Triton X-100, 20 mM -glycerophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM dithiothreitol, and 1% protease inhibitor mixtures; Wako Pure Chemicals). After stimulation with drugs, cells were washed with phosphate-buffered saline two times and extracted with 100 µl of the solution as described above. The total cell extract was vortexed before centrifugation at 15,000g for 10 min at 4°C. The supernatant was denatured in 1x sample buffer (10 mM Tris-HCl, pH 6.8, 1% SDS, 1% -mercaptoethanol, and 20% glycerol) for 5 min at 95°C, and 15 µg of protein was separated on 10% SDS-polyacrylamide gel at 20 mA. Sample proteins were transferred onto an Immun-Blot PVDF membrane (Bio-Rad), and the membrane was blocked with 5% skim milk in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.1% Tween 20. The membrane was incubated with rabbit anti-active mitogen-activated protein kinase (1:1000; Promega) or rabbit anti-ERK1/2 (1:1000; Promega) overnight at 4°C and then with horseradish peroxidase-conjugated rabbit anti-IgG (1:10,000; GE Healthcare, Little Chalfont, Buckinghamshire, UK) for 1 h at room temperature. ERK/mitogen-activated protein kinase was detected with the ECL Western blotting detection reagents (GE Healthcare).

DNA Transfection and Luciferase Assay. DNA transfection was carried out over 4 days in culture. A plasmid DNA, pGL3-BDNF promoter III (pGL3-BDNFpIII), was prepared as described previously (Tabuchi et al., 2000), and an internal control vector, phRL-TK(–), was purchased from Promega. The procedure used for DNA transfection was calcium phosphate/DNA precipitation as described previously (Tabuchi et al., 1998). Briefly, the calcium phosphate/DNA precipitates were prepared by mixing 1 volume (67 µl) of plasmid DNA (5.3 µg; pGL3-BDNFpIII:phRL-TK(–) = 10:1) in a 250 mM CaCl₂ solution with an equal volume of 2x HEPES-buffered saline, and added to each 35-mm dish. The dish was washed three times with phosphate-buffered saline and replenished with fresh serum-free DMEM medium. After 2 days, the transfected cells were stimulated with pyrethroids, 25 mM KCl, or vehicle for 12 h, and cell lysates were extracted with passive lysis buffer (Promega) and dual (firefly and Renilla) luciferase activity was measured using a Dual-Luciferase Reporter assay system (Promega) with luminometer TD-20/20 (Promega).

Statistics. All data were expressed as the mean ± S.D. Statistical analyses were performed using one-way analysis of variance, followed by Tukey-Kramer procedure for multiple comparisons as post hoc analysis.

	Results

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Expression of BDNF Exon III–V mRNA and BDNF Protein Induced by Deltamethrin. We stimulated the cultured rat cortical cells with DM and monitored the expression of BDNF exon III–V and c-fos mRNAs by real-time RT-PCR. As shown in Fig. 1A, the addition of 1 µMDMtothe medium markedly induced the expression of BDNF exon III–V mRNA at 1 h of exposure with a maximum reached 3 h later. The maximal expression level induced by DM was about 150 times higher than that of the control. The elevated expression level was continuously detected until at least 72 h after starting the incubation. In addition, the increase in BDNF exon III–V mRNA expression was detected at doses from 10 nM to 10 µM DM (Fig. 1B). Since the maximum was obtained at 1 µM, we used 1 µM DM for further experiments. The expression of c-fos mRNA increased until 1 h after the exposure to DM but decreased after that time (Fig. 1A). To know whether BDNF protein synthesis is also induced by DM coupled with the increase in mRNA expression, we measured the expression levels of BDNF protein using an ELISA system. As shown in Fig. 1C, the amount of BDNF protein increased upon stimulation of cortical cells with DM for 3 h, and the elevated BDNF synthesis continued for at least 24 h.

View larger version (26K): [in this window] [in a new window]   Fig. 1. Expression of BDNF exon III–V mRNA and BDNF protein in rat cortical neurons induced by deltamethrin. A, time course of mRNA expression of BDNF exon III–V and c-fos. Deltamethrin (1 µM) was added, and the cells were further incubated for the periods indicated before total cellular RNA was extracted for measurement by a real-time RT-PCR. The ratio of mRNA expression to the control level at time 0 is shown. B, effect of deltamethrin concentration on BDNF exon III–V mRNA expression. Deltamethrin was added at the concentrations indicated and the cells further incubated for 3 h. The ratio of mRNA expression relative to the control is shown. C, effect of deltamethrin on BDNF protein expression. Deltamethrin (1 µM) was added, and the cells further incubated for 3 or 24 h. After preparing the cell lysates, BDNF protein was measured with ELISA and total protein concentration was determined using bovine serum albumin as a standard. The concentration of BDNF protein was normalized with the total amount of protein. The columns represent the mean ± S.D. from three independent experiments. *, p < 0.05; **, p < 0.01 compared with the control.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Effect of TTX on deltamethrin-induced BDNF exon III–V mRNA expression in rat cortical neurons. Cortical neuronal cells in culture were treated with or without 1 µM TTX for 30 min before the addition of 1 µM deltamethrin, 10 µM veratridine, or 100 ng/ml BDNF and incubated for another 3 h. The ratio of mRNA expression to the control level is shown. The columns represent the mean ± S.D. from three independent experiments. *, p < 0.05; **, p < 0.01 compared with the control. , p < 0.01 compared with 1 µM deltamethrin or 10 µM veratridine.

View larger version (35K): [in this window] [in a new window]   Fig. 3. Effect of deltamethrin on the cellular Ca2+ signaling cascade in rat cortical neurons. A, nicardipine and U0126 inhibited deltamethrin-induced BDNF exon III–V mRNA expression. Cortical neuronal cells in culture were treated with or without 5 µM nicardipine, 200 µM AP-V, 20 µM U0126, or 20 µM U0124 for 10 min before the addition of 1 µM deltamethrin, and the cells were incubated for another 3 h. The ratio of mRNA expression relative to the control is shown. The columns represent the mean ± S.D. from three independent experiments. **, p < 0.01 compared with the control. , p < 0.01 compared with 1 µM deltamethrin. B, effect of deltamethrin on phosphorylation of ERK1/2. Deltamethrin (1 µM) or BDNF (100 ng/ml) was added, and the cells further incubated for the periods indicated before total cellular protein was extracted for immunoblotting. C, nicardipine inhibited deltamethrin-induced phosphorylation of ERK1/2. Cells were treated with 5 µM nicardipine for 10 min before the addition of 1 µM deltamethrin or 50 mM KCl and then incubated for another 10 min before total cellular protein was extracted for immunoblotting.

Prolonged Expression of BDNF Exon III–V mRNA after Withdrawal of DM. To examine the effect of the transient treatment of cortical cells with DM on BDNF mRNA expression, we treated the cortical cells with 1 µM DM for 3 h and then changed the medium to fresh serum-free DMEM without DM three times, before continuing the incubation (washout). As shown in Fig. 4, the BDNF exon III–V mRNA expression level obtained by the treatment of washout was significantly higher than that of the control after 24 and 48 h. However, the samples with washout showed a lower level of BDNF mRNA expression than those obtained by further stimulation of the cortical cells with 1 µM DM (washout + DM). Thus, the withdrawal of DM from the culture medium after the transient stimulation with DM caused a gradual decrease in the expression of BDNF exon III–V mRNA, but further addition of DM recovered the expression.

View larger version (13K): [in this window] [in a new window]   Fig. 4. Effect of withdrawal of deltamethrin from the medium on BDNF exon III–V mRNA expression in rat cortical neurons. Cortical neuronal cells in culture were treated with 1 µM deltamethrin and further incubated for 3 h. Then, the cells were washed with fresh serum-free DMEM without deltamethrin three times and incubated for another 24 or 48 h before total cellular RNA was prepared for measuring the mRNA by real-time RT-PCR (washout). In addition, cortical cells were further treated with 1 µM deltamethrin after the washout and incubated for another 24 or 48 h (washout + DM). The ratio of mRNA expression to the control level is shown. The columns represent the mean ± S.D. from three independent experiments. *, p < 0.05; **, p < 0.01 compared with the control.

Type II but Not Type I Pyrethroids Induced BDNF Exon III–V mRNA Expression. Pyrethroids are divided into two groups according to chemical structure, and they show different pharmacological activities and poisoning syndromes (Ray and Forshaw, 2000). To investigate the effect of pyrethroids on BDNF exon III–V mRNA expression, we examined the effect of eight different pyrethroids in comparison with that obtained by membrane depolarization (Fig. 5A). Interestingly, the type II pyrethroids caused a significant increase in BDNF exon III–V mRNA expression, whereas the type I pyrethroids had no effect. Next, we examined the effect of pyrethroids on the activity of BDNF-pIII. As shown in Fig. 5B, the luciferase activity was increased by type II pyrethroids, both DM and cypermethrin, whereas the type I pyrethroids such as cis-permethrin did not increase the promoter activity.

View larger version (20K): [in this window] [in a new window]   Fig. 5. Effect of pyrethroids on BDNF exon III–V mRNA expression (A) and BDNF promoter III activity (B) in rat cortical neurons. A, type II pyrethroids induced BDNF exon III–V mRNA expression. Cortical neuronal cells in culture were treated with 25 mM KCl or 1 µM of eight kinds of pyrethroids and further incubated for 3 h before total cellular RNA was extracted for real-time RT-PCR. The ratio of mRNA expression to the control level is shown. B, deltamethrin and cypermethrin induced BDNF promoter III activity. Forty hours after the DNA transfection with BDNF-pIII, the cells were stimulated with depolarization (25 mM KCl) or pyrethroids (1 µM each), and the incubation was continued for 6 h before preparing the cellular extracts for the luciferase assay. The columns represent the mean ± S.D. from three independent experiments. *, p < 0.05; **, p < 0.01 compared with the control.

View larger version (18K): [in this window] [in a new window]   Fig. 6. Effect of deltamethrin treatment on expression of BDNF exon III–V mRNA and protein in rat cerebral cortex and hippocampus. Three or four rats were intraperitoneally administered with deltamethrin (25 mg/kg) or vehicle. These rats were killed 5 h after injection, and their cerebral cortexes and hippocampi were used for measuring BDNF protein (A) and BDNF exon III–V mRNA (B) expression. Kainate (15 mg/kg) was intraperitoneally administered to three rats. The rats were killed 1 h after injection and BDNF protein content in their cerebral cortexes and hippocampi was measured (A). The concentration of BDNF protein was normalized with the total amount of protein in tissue. The columns represent the mean ± S.D. from three or four independent rats. *, p < 0.05 compared with the control.

	Discussion

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In the present study, we clearly demonstrated that the exposure of cortical neurons to DM induced the expression of BDNF exon III–V mRNA and the synthesis of BDNF protein both in vitro and in vivo, which is controlled by neuronal activity accompanying the influx of Ca²⁺ into neurons (Zafra et al., 1990; Bading et al., 1993; Sano et al., 1996; West et al., 2001). The fact that nicardipine completely inhibited the activation of BDNF exon III–V mRNA expression induced by DM indicates that DM-induced BDNF gene expression is mainly mediated by the Ca²⁺ influx through L-VDCC (Fig. 3A). The VSSC is the primary target of pyrethroid insecticides (Soderlund, 1985); however, DM is thought to affect the sodium channel kinetics and to induce sodium tail currents. In support of this, DM- as well as veratridine-induced BDNF gene expression was inhibited by administration of TTX (Fig. 2). Thus, it is evident that activation of Na⁺ channels is involved in the DM-induced BDNF gene expression. In addition, DM induced the phosphorylation of ERK1/2, and the activation of BDNF mRNA expression was inhibited by U0126 but not by U0124 (Fig. 3A). Based on these observations, it seems highly possible that the membrane depolarization that can be evoked via the action of DM toward Na⁺ channels induces the Ca²⁺ influx through L-VDCC, resulting in an increase in BDNF exon III–V mRNA expression through the activation of the ERK/MAP kinase signaling pathway in neurons.

It has been reported that pyrethroids can bind to membrane lipid bilayers because of their high hydrophobicity and that they produce membrane depolarization in the rat synaptosome (Michelangeli et al., 1990; Eells et al., 1992). In addition, it has also been reported that DM acts as an inhibitor of calcineurin, Ca²⁺-dependent protein phosphatase 2B (Misonou et al., 2004), which may intensify the Ca²⁺ signal-dependent phosphorylation of protein kinases induced by DM. Besides these actions, some studies have demonstrated an antagonistic effect of pyrethroids on the GABA_A receptor complex (Abalis et al., 1986), which may result in an increase in the excitatory activity of neurons. Nevertheless, it is clear that the induction of BDNF mRNA expression by DM is essentially caused by the excitatory effect of pyrethroids on neurons through the action on Na⁺ channels because administration of TTX markedly abolished the induction (Fig. 2). Since type I pyrethroids also give rise to the sodium tail currents through their action against Na⁺ channels (Soderlund et al., 2002), however, it remains unclear why only the type II pyrethroids induced the BDNF exon III–V mRNA expression. Further investigation is needed to explore the mechanisms behind the activation of BDNF gene expression induced by DM. As for the relationship between the structure and function of pyrethroids, it is clear that the cyano moiety at the -position of pyrethroids plays an important role in inducing the expression because the type II pyrethroids have a cyano moiety and activate BDNF gene expression. It is still unknown, however, how the cyano moiety of type II pyrethroids is able to induce the BDNF gene expression.

In our previous report, we have demonstrated that cis- and trans-permethrin, type I pyrethroids, repressed the membrane depolarization at concentrations above 10 µM and resulted in a reduction of BDNF mRNA expression in parallel with that of Ca²⁺ influx in mouse cerebellar granule cells in culture (Imamura et al., 2000). We have already observed that the type II pyrethroids also reduced the Ca²⁺ influx into neurons at more than 10 µM (data not shown), a concentration that was less inducible for the BDNF exon III–V mRNA expression than the optimal concentration (1 µM) (Fig. 1B). This same inhibitory effect on calcium influx in mouse cerebellar granule cells has been reported by Hildebrand et al. (2004), in which high-voltage-activated calcium channels are blocked by allethrin, a type I pyrethroid. In the present study, however, we used cultures of rat cortical neuronal cells to examine the effect of DM on BDNF gene expression, in which active synapses are formed between neurons (Kato-Negishi et al., 2004). Therefore, it can be speculated that the induction of BDNF mRNA expression detected at relatively low concentrations (10 nM–1 µM) of DM could be related to the formation of active synapses; that is, the formation of active synapses in culture might be a prerequisite for the DM-induced BDNF gene expression.

Even after the withdrawal of DM from the medium upon the stimulation of cells for 3 h, the expression of BDNF exon III–V mRNA remained highly elevated for at least 48 h (Fig. 4). Since the promoter activity of BDNF gene promoter III was induced by DM (Fig. 5B) and the half-life of BDNF exon III–V mRNA was not changed in the presence of DM (data not shown), the continued mRNA expression should be supported at the transcriptional level. The prolonged expression observed with DM may be explained by the fact that type II pyrethroids remained in neuronal tissue after the washout and continued to exert their effects on sodium channel function, whereas the effect of type I pyrethroids was reversible (Song et al., 1996). Although there remains a possibility that the residual DM is still acting even after withdrawal from the medium, it is evident that the inducible effect of DM on BDNF gene expression tended to be maintained once the cortical cells were stimulated and additively induced by a fresh administration of DM after the washout (Fig. 4). From these findings, it seems likely that repeated contamination with DM might increase the risk of chronic exposure of the mammalian brain.

Administration of DM to the rat through a single peripheral injection revealed that the BDNF protein levels in the cerebral cortex and the hippocampus were significantly increased (Fig. 6A). In contrast, the BDNF exon III-V mRNA was significantly increased in the hippocampus but tended to be raised in the cerebral cortex by DM treatment (Fig. 6B). In either case, DM treatment increased the expression of BDNF mRNA and protein in the hippocampus. Irrespective of the fact that pyrethroids are easily eliminated from the bodies of mammals, many toxic effects have been reported with DM (Miyamoto et al., 1995). After a single oral administration of 26 mg/kg DM to rats, the maximal plasma concentration of DM was reported to be about 1 µM (Anadon et al., 1996). In cultures of rat cortical cells, 10 nM DM was enough to increase the BDNF exon III–V mRNA expression (Fig. 1B). Based on these results, it seems possible that chronic exposure of mammals to DM could initiate and continue the synthesis of BDNF in the mammalian brain even if the brain is exposed to a concentration of DM lower than 10 nM.

Koyama et al. (2004) reported that BDNF is necessary and sufficient to evoke the mossy fiber sprouting and the resultant network reorganization, which are induced in the epileptic rat hippocampus. In addition, there is evidence of epileptic activity in rats chronically exposed to cypermethrin (Condes-Lara et al., 1999). These results support the idea that an excessive synthesis and secretion of BDNF may induce neuronal hyperexcitation. Since BDNF plays a pivotal role in strengthening the synaptic structures (Tartaglia et al., 2001), it is possible that long-term exposure of animal brains to DM causes an aberrant formation of neuronal networks through a constitutively elevated expression of BDNF, resulting in an impairment of brain function. This possible dysfunction induced by DM is thought to be serious in the developing brain of mammals because the development of the brain after birth progresses in a neuronal activity-dependent manner, in which the expression of BDNF is critically important. Pyrethroids are widely used insecticides, but little is known about the consequences of long-term exposure in mammals. Further investigation is needed to declare the neurotoxicity of DM and other pyrethroid insecticides in the brain, in terms of aberrantly elevated BDNF gene expression by environmental disrupters.

	Footnotes

 
This study was supported by a grant-in-aid for Core Research for Evolutional Science and Technology from the Science and Technology Corporation of Japan and a grant-in-aid for scientific research on priority areas from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

doi:10.1124/jpet.105.092478.

ABBREVIATIONS: BDNF, brain-derived neurotrophic factor; L-VDCC, L-type voltage-dependent calcium channel; NMDA, N-methyl-D-aspartate; BDNF-pX, brain-derived neurotrophic factor promotor I, II, III, and IV; VSSC, voltage-sensitive sodium channel; DM, deltamethrin; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ERK, extracellular signal-regulated kinase; TTX, tetrodotoxin; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene; U0124, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadine.

Address correspondence to: Dr. Masaaki Tsuda, Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani 2630, Toyama 930-0194, Japan. E-mail: tsuda{at}ms.toyama-mpu.ac.jp

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