Suppression by phthalates of the calcium signaling of human nicotinic acetylcholine receptors in human neuroblastoma SH-SY5Y cells

Suppression by phthalates of the calcium signaling of human nicotinic acetylcholine receptors in human neuroblastoma SH-SY5Y cells

Toxicology 200 (2004) 113–121 Suppression by phthalates of the calcium signaling of human nicotinic acetylcholine receptors in human neuroblastoma SH...

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Toxicology 200 (2004) 113–121

Suppression by phthalates of the calcium signaling of human nicotinic acetylcholine receptors in human neuroblastoma SH-SY5Y cells Lu Kaun-Yu, Tseng Fu-Wei, Wu Chia-Jung, Liu Pei-Shan∗ Department of Microbiology, Soochow University, Shihlin, Taipei, Taiwan, ROC Received 18 November 2003; received in revised form 15 December 2003; accepted 12 March 2004 Available online 18 May 2004

Abstract Phthalates are widely used in industry and cause public concern since they have genomic estrogenic-like effects via estrogen receptors. We previously found that some phthalates have nongenomic effects, exerting inhibitory effects on the functional activities of nicotinic acetylcholine receptors (nAChRs) in bovine chromaffin cells. In this study, we investigated the effects of eight phthalates on the calcium signaling of human nAChR by using human neuroblastoma SH-SY5Y cells. All eight phthalates, with different potency, have inhibitory roles on the calcium signaling coupled with human nAChR, but not muscarinic acetylcholine receptors (mAChRs). For inhibition of human nAChR, the strongest to weakest potencies were observed as di-n-pentyl phthalate (DPP) → butyl benzyl phthalate (BBP) → di-n-butyl phthalate (DBP) → dicyclohexyl phthalate (DCHP) → di-n-hexyl phthalate (DHP) → di-(2-ethyl hexyl) phthalate (DEHP) → di-n-propyl phthalate (DPrP) → diethyl phthalate (DEP). The potencies of phthalates were associated with their structures such that the most effective ones had dialkyl group carbon numbers of C4 or C5, with shorter or longer numbers resulting in decreased potency. At as low as 0.1 ␮M, DPP, DBP, BBP, DCHP and DHP significantly inhibited the calcium signaling of human nAChR. The IC50 of phthalates on human nAChR, ranging from 0.32 to 7.96 ␮M, were 10–50 lower than those for bovine nAChR. We suggest that some phthalates effectively inhibit the calcium signaling of human nAChR, and these nongenomic effects are cause for concern. © 2004 Published by Elsevier Ireland Ltd. Keywords: Phthalates; Human nicotinic acetylcholine receptors; Muscarinic acetylcholine receptors; Calcium signaling; Bovine adrenal chromaffin cells

1. Introduction Phthalates, extensively used in consumer products and medical devices, have been described as the ∗ Corresponding author. Tel.: +886-2-28819471x6857; fax: +886-2-28831193. E-mail address: [email protected] (L. Pei-Shan).

most abundant man-made environmental pollutants (Schulz, 1989). Phthalates are known to act on some cellular receptors to mediate their cytotoxicity. Some phthalates and their metabolites have the potential to cause carcinogenic hazards that might be related to their character to cause peroxisome proliferation through activating peroxisome proliferators-activated receptors and binding to peroxisome proliferators

0300-483X/$ – see front matter © 2004 Published by Elsevier Ireland Ltd. doi:10.1016/j.tox.2004.03.018

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response elements (Willhite, 2001; Hurst and Waxman, 2003). In addition to this, some phthalates compete with estradiol for binding to estrogen receptors and induce estrogen receptor-mediated responses (Harris et al., 1997; Zacharewski et al., 1998). Due to these cellular disturbances and their wide-distribution, phthalates attract public concern. Steroid hormones have, in addition to their traditional genomic effects, nongenomic immediate effects on ion channels in the plasma membrane. Phthalates also have these two effects. We previously found that some phthalates have inhibitory effects on the calcium signaling coupled with nicotinic acetylcholine receptor (nAChRs) in bovine adrenal chromaffin cells (Liu et al., 2003; Liu and Lin, 2002). Nicotinic acetylcholine receptors, belonging to the family of ligand-gated cation channels, are important sites of excitatory neurotransmissions in both vertebrate and invertebrate animals. nAChR, widely distributed in the central and peripheral nervous systems, neuromuscular junctions, and adrenal glands, mediates cholinergic neurotransmissions. nAChR apparently plays a pivotal role in a number of functional processes including learning and memory and is implicated in several central nervous system (CNS) disorders, including Alzheimer’s disease, Parkinson’s disease, schizophrenia and epilepsy (Paterson and Nordberg, 2000). nAChR also modulates the level of blood catecholamine, heart rate and blood pressure (Khan et al., 2001). Alarmingly, nAChR is also the target of some insecticides and other environmental toxicants, including phthalates (Eldefrawi et al., 1985; Sattelle et al., 1989; Liu and Lin, 2002). Various nicotinic receptor subtypes are composed of unique combinations of homologous subunits encoded by at least 16 distinct genes (␣1 –␣9 , ␤1 –␤4 , ␥, ␦ and ⑀) (Heinemann et al., 1991). nAChRs exist widely from invertebrates to vertebrates, insects to humans, and variations even exist in homologous subunits across species (Lindstrom et al., 1995). These diversities afford a large potential for nAChRs to possess different cation conducting properties and pharmacological heterogeneity. Only a small difference in C terminus leads to the enhancement of estradiol in human nAChR ␣4␤2 but not in rats (Paradiso et al., 2001). Considering the existence of these variations in nAChR and the public interest in phthalates, we investigated the effects of phthalates on human nAChRs in

Fig. 1. Chemical structures of the examined phthalates. Dialkyl phthalates we used are shown in the upper left. The alkyl groups are linear from C2 to C6: di-n-ethyl phthalate (DEP); di-n-propyl phthalate (DPrP); di-n-butyl phthalate (DBP); di-n-pentyl phthalate (DPP), di-n-hexyl phthalate (DHP). Butyl benzyl phthalate (BBP) (98%) shown in upper right. Lower show dicyclohexyl phthalate (DCHP) and di-(2-ethyl hexyl) phthalate (DHEP) (99%).

human neuroblastoma SH-SH5Y cells and compared their effects in bovine chromaffin cells. Eight phthalates, named diethyl phthalate (DEP), di-n-propyl phthalate (DPrP), butyl benzyl phthalate (BBP), di-n-butyl phthalate (DBP), di-n-pentyl phthalate (DPP), dicyclohexyl phthalate (DCHP), di-n-hexyl phthalate (DHP), di-(2-ethyl hexyl) phthalate (DEHP) are investigated in this study (Fig. 1). Chemically, these phthalates are structure-related. All eight phthalates are commonly found in the environment (Yuan et al., 2002). DEHP and DBP were considered most relevant as they occur in the highest concentrations in the Dutch (Belfroid, 1998) and Taiwan environments (Yuan et al., 2002).

2. Material and methods 2.1. Chemicals 1,1-Dimethyl-4-phenyl-piperazinium iodide (DMPP), carbachol, epibatidine, methacholine, digitonin, deoxyribonuclease I and EGTA were all obtained from Sigma Chemical Co. NaCl, KCl, and other salts were obtained from Merck Chemical Co. Fura-2

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acetoxymethyl ester was obtained from Molecular Probes. Collagenase was obtained from Worthington Biochemical Co. Culture media were obtained from Gibico Co. Phthalates (98% pure), including DEP, DPrP, DBP, DPP, DHP, BBP, DCHP, DEHP were purchased from the Tokyo Kasei Kogyo company. 2.2. Cell culture The human neuroblastoma SH-SY5Y cells, obtained from ATCC (CRL-2266), were cultured in a minimally essential medium and F12 medium (1:1), supplemented with 10% fetal bovine serum and 100 U pencillin/streptomycin, and grown in a 5% CO2 humidified incubator at 37 ◦ C (Lambert et al., 1989). The medium was changed every 3–4 days and subcultured every 7 days. The confluent cells were collected to process the [Ca2+ ]c measurements. 2.3. Cell isolation Bovine adrenal chromaffin cells were isolated with the aid of collagenase as described by Wilson (1987) with slight modifications (Liu et al., 1994). Adrenal glands were perfused with a perfusion buffer (145 mM NaCl, 5.4 mM KCl, 1.0 mM NaH2 PO4 , 11.2 mM glucose, 15 mM HEPES, pH 7.4) and then digested with a collagenase solution (0.2% collagenase and 0.002% deoxyribonuclease I in perfusion buffer). The medulla was separated from the cortex, cut into small pieces, and digested with the collagenase solution at 37 ◦ C. Freshly isolated cells were used for cytosolic free Ca2+ concentration ([Ca2+ ]c ) measurements. 2.4. [Ca2+ ]c measurements Human neuroblastoma SH-SY5Y cells and bovine chromaffin cells were collected and loaded with fura-2 by incubation (5 × 106 cells/ml) with 10 ␮M fura-2 acetoxymethyl ester at 37 ◦ C for 30 min. Cells were then washed twice with a loading buffer (150 mM NaCl, 5 mM KCl, 2.2 mM CaCl2 , 1 mM MgCl2 , 5 mM glucose, 10 mM HEPES, pH 7.4). All the experiments were proceeded in the loading buffer with calcium or without calcium (CaCl2 was withdrawn and 0.25 mM EGTA was added). The fluorescent measurements were taken using a dual-excitation fluorometer (SPEX, CM system); fluorescence was

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measured at 340, 380 nm excitation and 505 nm emission. Cytosolic Ca2+ concentration ([Ca2+ ]c ) was calculated using a fluorescence ratio of 340–380 nm (Grynkiewicz et al., 1985). Rmax was achieved by adding 0.01% digitonin to the cuvette at the end of experiments; excess EGTA was subsequently added to obtain Rmin . A Kd of 224 nM Ca2+ for fura-2 was used. Phthalates, at concentrations from 0.1 ␮M to 1 mM, were added 100 s before the stimulation; examples were carbachol, epibatidine and the like. Data were presented as a mean + S.D. that were calculated from five individual experiments by using different cell batches and duplicates in each individual experiment. The representative Ca2+ curve data was the closest one to the mean of the five experiments.

3. Results We used carbachol, an analog of acetylcholine, to stimulate acetylcholine receptors in both bovine chromaffin cells and human neuroblastoma SH-SY5Y cells. Carbachol was capable of inducing a transient [Ca2+ ]c rise in both cells. DBP suppressed the carbachol-induced [Ca2+ ]c rise in both cells with different potencies. The inhibition of DBP, at 20 ␮M, was much larger in bovine chromaffin cells than in human SH-SY5Y cells (Fig. 2A and B). Both nAChR and muscarinic acetylcholine (mAChR) receptors—both acetylcholine receptors— differ in their signaling pathways: nAChRs act as ligand-gated ion channels, while some of mAChR receptors couple with G protein to activate phospholipase C to produce inositol 1,4,5-trisphosphate, which releases intracellular stored Ca2+ . Bovine adrenal chromaffin cells only possess a small number of mAChRs and we only found a small rise of [Ca 2+ ] coupled with mAChR in a few batches of prepac rations. Fortunately, mAChR is dominantly present in human SH-SY5Y cells (Kukkonen et al., 1992); thus we used these cells to observe the influence of phthalates on mAChR. We used two strategies to distinguish mAChR and nAChR to find the blockage target of DBP. Firstly, we used carbachol to stimulate cells in a Ca2+ -free buffer to exclude Ca2+ influx via nAChR and found the suppression capability of DBP was counteracted with the depletion of the extracellular Ca2+ (Fig. 2C). Secondly, we observed the effects

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Fig. 2. Effects of DBP on carbachol-induced [Ca2+ ]c changes in bovine adrenal chromaffin cells and human SH-SY5Y cells. Bovine adrenal chromaffin cells (panel A) and human SH-SY5Y cells (panel B) were stimulated with 0.3 mM carbachol (↑Car) with (curve b) or without (curve a) 20 ␮M DBP in loading buffer (with Ca2+ ). In panel C, human SH-SY5Y cells were used to carry similar experiments in a Ca2+ free buffer and 0.25 mM EGTA (E↑) was added at first to deplete the extracellular Ca2+ and 2.5 mM Ca2+ (↑Ca) was re-introduced at the end of the observation. In panel D, human SH-SY5Y cells were treated with (curve b) or without (curve a) 20 ␮M DBP and stimulated with 0.3 mM methacholine (↑Mch).

of DBP on [Ca2+ ]c rise induced by methacholine, a selective agonist of mAChRs. Similar results were obtained in the presence or absence of DBP, so DBP had no effects on methacholine-induced [Ca2+ ]c rise (Fig. 2D). Epibatidine, a selective ligand, was used to stimulate nAChRs in both cells (Fig. 3). DBP, at 1 ␮M, significantly suppressed the epibatidine-induced [Ca2+ ]c rise (only 20% remained) in human SH-SY5Y cells, but only slightly suppressed the epibatidine-induced [Ca2+ ]c rise in bovine chromaffin cells. DBP was also found suppressing the [Ca2+ ]c rise induced by other selective nAChRs ligands, namely DMPP or nicotine, in both cells (data not shown). In the presence of 1 ␮M DBP, its suppression could not be counteracted by

Fig. 3. Effects of DBP on [Ca2+ ]c changes induced by epibatidine in bovine adrenal chromaffin cells and human SH-SY5Y cells. Bovine adrenal chromaffin cells (panel A) and human SH-SY5Y cells (panel B) were stimulated with 10 ␮M epibatidine (↑EPI) in the presence (curve b) or absence (curve a) 1 ␮M DBP in loading buffer.

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increasing the concentration of epibatidine (data not shown); thus, DBP might address a non-competitive inhibition on nAChR. Under the stimulation of all the selective nicotinic ligands we used, the inhibition of DBP was much larger in human SH-SY5Y cells than that in bovine chromaffin cells. The lack of suppression effects of DBP on mAChR might count the contradictory potency on [Ca2+ ]c rise induced by carbachol and epibatidine in these two cells. In order to understand the suppressive capability of other phthalates and the relationship between suppression and the chemical structure of phthalates, eight phthalates (Fig. 1) with different carbon number alkyl groups, C2–C8, were investigated: DEP (C2), DPrP (C3), DBP (C4), DPP (C5), DHP (C6), DCHP (cyclic alkyl C6 ring), DEHP (one main C6 alkyl chain as hexyl and one ethyl group attached to the C2 position of the main hexyl chain). BBP contains one alkyl chain as butyl and one benzyl group attached to the methyl group. In both human SH-SY5Y cells and bovine chromaffin cells, the suppression potency of phthalates from low to high was listed as: DPP ≥ BBP > DBP > DCHP ≥ DHP > DPrP > DEHP > DEP (Fig. 4). The relationship between the potency and the carbon number of the dialkyl group of phthalates was bell shaped. The most potent one was DPP in the C5 alkyl group, and the potency decreased with the shorter or longer carbon numbers of alkyl group. Fig. 5 shows the dose-dependence of the phthalates on the epibatidine-induced [Ca2+ ]c rise in human SH-SY5Y cells and bovine chromaffin cells, respectively. All phthalates suppressed the epibatidine-induced [Ca2+ ]c rise in human SH-SY5Y cells at lower concentrations than in bovine chromaffin cells (Fig. 5). 17␣-Estradiol showed a similar phenomenon. In human SH-SY5Y cells, all the phthalates that we investigated for IC50 values were

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Fig. 4. Inhibitory potency of eight phthalates on epibatidineinduced [Ca2+ ]c rise in bovine adrenal chromaffin cells and human SH-SY5Y cells. Data present as the inhibitory percentages of phthalates on epibatidine-induced [Ca2+ ]c rise. The phthalates are listed from C2 to C8 of their alkyl group. At the concentration of 1 ␮M, phthalates were used to inhibit the epibatidine-induced [Ca2+ ]c rise in human SH-SY5Y cells (solid bars). At the concentration of 10 ␮M, phthalates were used to inhibit the epibatidine-induced [Ca2+ ]c rise in human bovine adrenal chromaffin cells (open bars).

lower than 10 ␮M except DEHP; its IC50 value was not able to approach this level because its maximal suppression was less than 50% (Table 1). The IC50 of phthalates was about 10–50-folds higher in bovine adrenal chromaffin cells than that in human SH-SY-5Y cells. All eight phthalates we investigated showed significant suppression at 1 ␮M in human SH-SY5Y cells (P < 0.01). Eight phthalates, not including DEP and DEHP, had lower IC50 when compared with 17 ␣-estradiol. Five phthalates, namely DBP, BBP, DPP, DCHP, DHP, had IC50 10-folds lower than 17␣-estradiol in human SH-SY5Y cells.

Table 1 The IC50 value of phthalates in bovine chromaffin cells and human SH-SY5Y cells Phthalates

Chromaffin cell SH-SY5Y cell

IC50 (␮M) DEP

DPrP

DBP

BBP

DPP

DCHP

DHP

17␣-E2

n.d. 7.96

37.4 3.10

4.89 0.61

4.30 0.38

3.94 0.32

14.7 0.54

31.6 0.56

36.9 6.50

n.d.: not detectable. The 100% was the [Ca2+ ]c rise induced by epibatidine subtracted the basal [Ca2+ ]c .

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Fig. 5. Concentration-dependent effects of eight phthalates on epibatidine-induced [Ca2+ ]c rise in bovine adrenal chromaffin cells and human SH-SY5Y cells. (A) DEP; (B) DPrP; (C) DBP; (D) BBP; (E) DPP; (F) DCHP; (G) DHP; (H) 17 ␣-estradiol. Bovine adrenal chromaffin cells (䊊) and human SH-SY5Y cells (䊉) were stimulated with 10 ␮M epibatidine in the presence of phthalates at various concentrations. Data are presented as percentages of control response SEM, where a 100% response was the difference between the transient following the addition of 0 and 10 ␮M epibatidine. All the results are from three to five experiments using different batch cells and duplicate in each experiment.

4. Discussion In this study, we demonstrated that the phthalates were capable of addressing suppression of the [Ca2+ ]c rise coupled with human nAChR but not the suppression of human mAChR. Our evidence includes the results that: (a) DBP blocked the [Ca2+ ]c rise induced by agonists of nicotinic receptors, including carbachol, epibatidine, nicotine and DMPP; (b) DBP did not affect the Ca2+ release from the intracellular Ca2+ pools induced by carbachol in the absence of extracellular Ca2+ ; (c) DBP did not affect the [Ca2+ ]c rise induced by methacholine, an agonist of muscarinic receptors. The interference of the phthalates with the

functional activities in human nAChRs evidenced in this study proceeded our previous finding of their actions on bovine nAChRs (Liu et al., 2003). Moreover, the potency of the phthalates was 10–50 times higher in human SH-SY5Y cells than in bovine chromaffin cells. The interference of phthalates with the human neuroendocrine system is a cause of concern. Two possibilities arise to explain the higher suppression potency of the phthalates with regard to human nAChR than to bovine. One possibility arises from the fact that different species have nAChRs with different amino acid sequences and stereo protein structures. These differences might influence phthalates with different potencies on nAChR from different species.

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This suggestion is supported by Paradiso’s study that small differences at the C terminus of rat and human ␣4 nAChR lead to differing regulations of estradiol (Paradiso et al., 2001). Bovine and human nAChR have different amino acids even in the same nAChR isotype, thus leading to differing potency of phthalate suppression. In ␣3 subunit of nAChR, there is 91% homology between human and bovine (Kawamata and Shimohama, 2002; Maneu et al., 2002). In ␣7 subunit of nAChR, there is 88% homology between human and bovine (Peng et al., 1994; Maneu et al., 2002). The other possibility arises from the different subtypes of nAChR in the two cells we used in this study. The functional activities of ␣7 nAChR were absent because ␣-bungarotoxin could not inhibit the Rb influx in human SH-SY5Y cells (Lukas et al., 1993) although their mRNA can be detected (Gould et al., 1992). In our lab, the inhibition of ␣-bungarotoxin, up to 300 nM, on the [Ca2+ ]c rise induced by epibatidine could not be found in human SHSY5Y cells (data not shown). However, ␣7 nAChR is present and has functional activities in bovine adrenal chromaffin cells (Garcia-Guzman et al., 1995; Criado et al., 1992). ␣3 and ␣5 nAChR are present and have their functional activities in both human SHSY5Y cells (Lukas et al., 1993; Ke and Lukas, 1996) and bovine chromaffin cells (Tachikawa et al., 2001). ␣7 nAChR is known to possess large Ca2+ permeability and exhibits rapid desensitization, while ␣3 and ␣5 nAChR are permeable for Na+ and represent a slow desensitizing receptor (Lopez et al., 1998). Based on the different potency of phthalates between two cells, we suggest that phthalates are more selective for ␣3 and ␣5 nAChR and have smaller effects on ␣7 nAChR. The allosteric protein nAChR possesses several transitional states that regulate its functional activities. We found that the inhibition of DBP on nAChR could not be counteracted by the increase of ligand concentrations in human SHSY5Y cells, and we predicted that the inhibition of phthalates might be a noncompetitive blockage. In addition to acetylcholine binding sites, there are several regulating sites of nAChRs including two noncompetitive inhibitory binding sites with different affinities and steroid compounds, such as estrogen, that predictably address at a hydrophobic interaction (low affinity binding) site (Arias, 1998a,b). Paradiso et al. (2001) found two binding sites on ␣4␤2 nAChR for steroids; one was C terminus for

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potentiating its responses and the other was a membrane spanning helix for inhibiting its responses. Since phthalates are lipophilic and are reported to have an estrogen-like character, we suggested that their inhibitions might be acting at the interface between the membrane and receptor membrane domain. We found a bell shaped relationship between the potency of phthalates involving human and bovine nAChR and their dialkyl structures. The hydrophobicity of phthalates will increase following an increase in the carbon number of the dialkyl group. We therefore suggest that the lower potency of the phthalates with a small dialkyl group—i.e., the propyl [C3] and ethyl group [C2]—may be due to their lower hydrophobicity. Phthalates with longer alkyl chains were less potent because the longer alkyl chain causes steric hindrances in fitting the noncompetitive binding site. Based on these two structural characteristics, the chart of the potency of phthalates at nAChR was bell shaped in relation to their alkyl group carbon number. The bell structure potency relationship is similar to the structural features of dialkyl phthalates associated with estrogen receptor binding activities, with the butyl group (C4) exhibiting maximum effect (Nakai et al., 1999). We suggest that the steroid binding site at nAChRs and estrogen receptors might share some similarities, such as hydrophobicity tendency and steric hindrances. Phthalates contamination is common in our worldwide habitats. Levels of DBP in foods range from 50 to 500 ␮g/kg (0.2–2 ␮M) in the United States (ATSDR, 1991). A 1987 study in the United Kingdom estimated that the average intake of DBP from food packaged in cellulose film was 230 ␮g/day (1 ␮mol/day) (Ministry of Agriculture, 1987). Up to 14 mg DBP/kg (50 ␮M DBP) was found in chocolate bars and potato snacks wrapped in printed polypropylene films (Ministry of Agriculture, 1990). Moreover, DEHP plasma concentration in four children after a parental nutrition session was 6.9 ␮g/ml (17.1 ␮M) (Kambia et al., 2001). In this study, all the phthalates we used significantly suppressed nAChR at 1 ␮M, a level which is possibly occurring in our habitats, especially with regard to foods. Liubchenko et al. (1997) reported that workers engaged in the production of plastic materials were diagnosed as showing latent signs of neurotoxicity. Rosendale mentioned in 1999 that almost all cats having bitten toys containing DBP exhibit various

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neurologic signs. These reports support our finding that phthalates might interfere with neurotransmission, and our data show interferences are more dominant in human specimens. In humans, multiple nicotinic receptor subtypes are present in the central and peripheral nervous systems; they are associated with memory and emotion in central nervous system and mediate control of muscle contraction and secretion in the peripheral nervous system. Our studies show the possibility of phthalates acting on nAChR in the nervous system, and are therefore cause of concern.

Acknowledgements We wish to thank Miss Cynthia Wei and Dr. Hamilton for help editing this manuscript. This study was supported by grants from the National Science Council, Taiwan, ROC (NSC922311B031-002).

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