Stimulation of Catecholamine Synthesis by Environmental Estrogenic Pollutants
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内分泌学杂志 2005年第1期
Departments of Pharmacology (N.Y., Y.T., S.U., M.T., M.L., K.T.) and Psychiatry (K.U.), University of Occupational and Environmental Health, School of Medicine, Kitakyushu 807-8555, Japan
Address all correspondence and requests for reprints to: Nobuyuki Yanagihara, Ph.D., Department of Pharmacology, University of Occupational and Environmental Health, School of Medicine, 1-1, Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. E-mail: yanagin@med.uoeh-u.ac.jp.
Abstract
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Environmental estrogenic pollutants are compounds that have been shown to have estrogenic effects on fetal development and reproductive systems. Less attention, however, has been paid to their influence on neuronal functions. We report here the effects of estrogenic pollutants on catecholamine synthesis in bovine adrenal medullary cells used as a model system of noradrenergic neurons. Treatment of cultured bovine adrenal medullary cells with p-nonylphenol and bisphenol A at 10 nM for 3 d stimulated [14C]catecholamine synthesis from [14C]tyrosine and tyrosine hydroxylase activity, an effect that was not inhibited by ICI 182,780, an antagonist of estrogen receptors. Significant effects of p-nonylphenol on [14C]catecholamine synthesis were observed at 0.1 nM, which is 45 times lower than that of the international regulatory standard (4.5 nM), and the maximum effects were around 10–100 nM. The concentrations (0.1–10 nM) used in the present study are similar to the range observed in rivers in the United States or Europe. On the other hand, short-term treatment of cells with 10 nM p-nonylphenol for 10 min also activated tyrosine hydroxylase, which was suppressed by U0126, an inhibitor of MAPK kinase. Furthermore, treatment of cells with p-nonylphenol for 5 min increased the phospho-p44/42MAPK in a concentration-dependent (1–1000 nM) manner, whereas p-nonylphenol (100 nM, 2 d) enhanced both levels of non-phospho- and phospho-p44/42MAPK. These findings suggest that short-term and long-term treatment of cells with estrogenic pollutants at environmental concentrations stimulates catecholamine synthesis and MAPK through an estrogen receptor-independent pathway.
Introduction
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NATURAL ESTROGENS PLAY important roles in cell differentiation and proliferation, homeostasis, and the female reproductive system. The long-term genomic actions of estrogens are mediated by the binding of estrogens to their cytoplasmic/nuclear receptors (1, 2). In addition to estrogens, there exist environmental estrogenic pollutants or endocrine-disrupting chemicals that mimic the actions of estrogen; these include a variety of nonsteroidal substances with diverse chemical structures (3). Estrogenic pollutants bind to estrogen receptors (4) and may induce estrogen-dependent gene expression in wildlife and humans. Furthermore, as Ruehlmann et al. (5) have reported, environmental estrogenic pollutants acutely inhibit L-type Ca2+ channels in vascular smooth muscle cells and evoke a rapid relaxation of the coronary vasculatures. Therefore, it is also possible that estrogenic pollutants affect neuronal functions through modification of neurotransmission via genomic or nongenomic pathways.
Adrenal medullary cells are derived from the embryonic neural crest and share many physiological and pharmacological properties with postganglionic sympathetic neurons. Stimulation of adrenal medullary cells by acetylcholine released from splanchnic nerves stimulates catecholamine synthesis associated with an increase in activity of tyrosine hydroxylase, which catalyzes the conversion of tyrosine to L-3,4-dihydroxyphenylalanine (DOPA) (6). Tyrosine hydroxylase is regulated by two different mechanisms: short-term regulation by allosteric activation (7) and long-term regulation by enzyme induction (8). Because of similarity to that of the sympathetic neurons or brain noradrenergic neurons, the adrenal medullary cells have provided a convenient model system for studying the regulation of tyrosine hydroxylase in these neurons (9, 10).
p-Nonylphenol and bisphenol A are industrial compounds that have generated the most concern on the part of regulatory agencies and researchers as production of these chemicals has become widespread in the world. Bisphenol A is a monomer found in polycarbonate plastics and a constituent of epoxy and polystyrene resins employed extensively in the food-packing industry and in dentistry (11, 12). Alkylphenol ethoxylates are among the most widely used groups of surfactants. Approximately 500,000 tons of alkylphenol ethoxylates (nonylphenol ethoxylates encompass 80% of the world market) are produced annually worldwide (13). These compounds are aerobically and anaerobically degraded to alkylphenols. p-Nonylphenol is the main metabolite of alkylphenol polyethoxylates, which are widely used for surfactants in products such as detergents, paints, petroleum recovery chemicals, and so on. Although there is ongoing scientific debate concerning the potential threat of these estrogenic pollutants to animal and human reproductive health (3, 4), little information, however, is available to us about the effects of estrogenic pollutants on neuronal functions. Recently, we reported that estrogenic pollutants, including bisphenol A, inhibit the function of norepinephrine transporters in bovine adrenal medullary cells (14). We speculated that these pollutants might pose a potential threat to human mental health, because the norepinephrine transporter is known to be largely responsible for efficient termination of noradrenergic neurotransmission in the brain. In the present study, we examined the effects of estrogenic pollutants on catecholamine synthesis in cultured bovine adrenal medullary cells and found an increase in catecholamine synthesis and MAPK activity induced by estrogenic pollutants at environmental concentrations.
Materials and Methods
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Reagents
Oxygenated Krebs-Ringer phosphate (KRP) buffer was used throughout. Its composition is as follows (in mM): 154 NaCl, 5.6 KCl, 1.1 MgSO4, 2.2 CaCl2, 0.85 NaH2PO4, 2.15 Na2HPO4, and 10 glucose (adjusted to pH 7.4). Materials were obtained from the following sources: Eagle’s MEM was from Nissui Pharmaceutical (Tokyo, Japan); collagenase was from Nitta Zerachin (Osaka, Japan); calf serum, acetylcholine, 17?-estradiol, bisphenol A and p-nonylphenol were from Nacalai Tesque (Kyoto, Japan); MAPK kinase inhibitor U0126 was from Promega (Madison, WI); PhosphoPlus p44/42 MAPK (Thr 202/Tyr 204) antibody kit was from BioLabs (Berverly, MA); L-[1-14C]tyrosine (54.45 mBq/mmol) was from Perkin-Elmer Life Sciences (Boston, MA); and L-[U-14C]tyrosine (460 mBq/mmol) and L-[3-14C]DOPA (6.8 mBq/mmol) were from Amersham Biosciences (Buckinghamshire, UK).
Isolation and primary culture of bovine adrenal medullary cells
From bovine adrenal glands, the medullary cells were isolated by collagenase digestion according to the previously described method (9, 15). The cells were plated at a density of 4 x 106 cells per dish (Falcon 35 mm, Becton Dickinson Labware, Franklin Lakes, NJ) and maintained in monolayer culture in Eagle’s MEM containing 10% calf serum and antibiotics at 37 C under 5% CO2/95% air. The cells were used for experiments after being cultured between 2 and 7 d.
Long-term and short-term treatments of cells with estrogenic pollutants
Cultured cells were pretreated with or without estrogenic pollutants for 1–7 d. After treatment, the cells were used for experiments of [14C]catecholamine synthesis, tyrosine hydroxylase activity, and MAPK. In some experiments, after cells were treated for 5 or 10 min with estrogenic pollutants, tyrosine hydroxylase activity and MAPK were measured.
[14C]Catecholamine synthesis from [14C]tyrosine or [14C]DOPA
After treatment of cells with estrogenic pollutants for the indicated periods, the cells were incubated with 20 μM L-[U-14C]tyrosine (1 μCi) and L-[3-14C]DOPA (0.25 μCi) in 1.0 ml of KRP buffer at 37 C for 20 and 15 min, respectively. After aspiration of the reaction medium, the cells were harvested in 2.5 ml of 0.4 M perchloric acid and left standing for more than 30 min on ice to extract the radioactive catecholamines. The precipitated protein was removed by centrifugation at 1600 x g for 10 min at 4 C, and 14C-labeled catechol compounds in the supernatant were separated further into the fractions containing [14C]dopamine and [14C]epinephrine plus [14C]norepinephrine by ion exchange chromatography on Duolite C-25 columns (H+ type, 0.4 x 7.0 cm) (16). The 14C-labeled catecholamines separated were counted in a toluene base scintillator using a liquid scintillation counter (Aloka LSC-3500E, Aloka Co., Ltd., Tokyo, Japan). [14C]Catecholamine synthesis was expressed as the sum of the [14C]catecholamines (epinephrine, norepinephrine, and dopamine), because the ratio of [14C]epinephrine plus [14C]norepinephrine/[14C]dopamine was not significantly changed by stimulants.
Tyrosine hydroxylase activity
After treatment of cells (106 cells per well, 24 wells; Falcon) with 10 nM estrogenic pollutants for 3 d, the cells were exposed to 250 μl of the KRP buffer supplemented with 18 μM L-[1-14C]tyrosine (0.2 μCi) for 10 min at 37 C. In some experiments of short-term treatment with estrogenic pollutants, cells were pretreated with or without estrogenic pollutants for 10 min, and then tyrosine hydroxylase activity was measured. Upon addition of L-[1-14C]tyrosine, each well was immediately sealed with an acrylic tube capped with a rubber stopper and fitted with a small plastic cup containing 200 μl of NCS-II tissue solubilizer (Amersham Biosciences) to absorb 14CO2 released by the cells (17).
Western blot analysis of MAPK
The cells were washed three times with 1.0 ml of KRP buffer and were incubated in KRP buffer at 37 C for 30 min. Then, the cells were stimulated with or without p-nonylphenol (1–1000 nM) for 5 min. They were harvested with 100 μl of a buffer containing 20 mM Tris-HCl (pH 7.5), 5 mM EGTA, 60 mM ?-glycerophosphate, 1 mM NaVO3, 0.5% Triton X-100, 6 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 20 μg/ml aprotinin. The cell suspension was homogenized and centrifuged at 15,000 x g for 30 min. The supernatants were analyzed by SDS-PAGE, transferred to an Immobilon-P membrane (Millipore, Bedford, MA), and subjected to immunodetection using an anti-phospho- or anti-nonphospho-p44/42 MAPK (Thr 202/Tyr 204) antibody kit (17). To visualize the proteins, the membrane was exposed to x-ray film and analyzed by Mac BAS (version 2.52, Fuji Photo Film Co. Ltd., Tokyo, Japan).
Statistics
Data are presented as the mean ± SEM. The statistical evaluation of the data was performed by ANOVA. When a significant F-value was found by ANOVA, Dunnett’s or Scheffé’s test for multiple comparisons was used to identify differences among the groups. Values were considered statistically significant when P < 0.05. Statistical analyses were performed using StatView for Macintosh version 5.0J software (Abacus Concept Inc., Berkeley, CA).
Results
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Stimulation of catecholamine synthesis by environmental estrogenic pollutants
Treatment of cultured bovine adrenal medullary cells with 17?-estradiol and estrogenic pollutants such as bisphenol A and p-nonylphenol at 10 nM for 3 d stimulated [14C]cat-echolamine synthesis from [14C]tyrosine by 22, 45, and 72% over the control, respectively (Fig. 1). p-Nonylphenol (10 nM) continued to stimulate [14C]catecholamine synthesis during incubation for 1–7 d (Fig. 2A). The significant increase in [14C]catecholamine synthesis induced by p-nonylphenol was observed at concentrations as low as 0.1 nM with a maximum increase around 10–100 nM (Fig. 2B). To resolve whether estrogenic pollutants exert their effect on [14C]catecholamine synthesis through estrogen receptors, we used ICI182,780, a pure antagonist of classical estrogen receptors (18). ICI182,780 (100 nM), by itself, significantly increased [14C]catecholamine synthesis. This antagonist abolished [14C]catecholamine synthesis induced by 17?-estradiol, whereas the stimulatory effect of p-nonylphenol was not inhibited by ICI182,780 (Table 1). To ascertain which step in the biosynthesis of catecholamines was stimulated by p-nonylphenol, [14C]DOPA was used as a substrate instead of [14C]tyrosine (Fig. 3). In this case, however, p-nonylphenol (10 or 100 nM, 3 d) did not increase [14C]catecholamine synthesis from [14C]DOPA, indicating that stimulation of catecholamine synthesis induced by p-nonylphenol must occur predominantly before the DOPA decarboxylase step.
FIG. 1. Effects of various environmental estrogenic pollutants on [14C]catecholamine synthesis from [14C]tyrosine in cultured bovine adrenal medullary cells. Cultured cells (4 x 106 cells per dish) were pretreated with 17?-estradiol, bisphenol A, and p-nonylphenol at 10 nM for 3 d. The cells were then incubated for 20 min at 37 C in 1.0 ml KRP buffer containing L-[U-14C]tyrosine (20 μM, 1 μCi) without estrogenic pollutants. The 14C-labeled catecholamines formed are shown as the total [14C]catecholamines. Data are expressed as the mean ± SEM of four separate experiments carried out in triplicate. *, P < 0.05, compared with control.
FIG. 2. Effects of p-nonylphenol on [14C]catecholamine synthesis in adrenal medullary cells. Cells were treated with or without 10 nM p-nonylphenol for the indicated period (A) or with various concentrations of p-nonylphenol (0–100 nM) for 3 d (B). After treatment, the cells were incubated for 20 min in 1.0 ml KRP buffer containing L-[U-14C]tyrosine. [14C]Catecholamines formed in the cells were measured and are shown as the total [14C]catecholamines. Data are expressed as the mean ± SEM of four separate experiments carried out in triplicate. *, P < 0.05, compared with d 0 (A) or with 0 nM p-nonylphenol (B); **, P < 0.05, compared with 0.1 or 1.0 nM p-nonylphenol (B).
TABLE 1. Effects of ICI182,780, an antagonist of estrogen receptors on [14C]catecholamine synthesis
FIG. 3. Effect of p-nonylphenol on [14C]catecholamine synthesis from [14C]DOPA in the cells. After treatment with or without p-nonylphenol (10 or 100 nM) for 3 d, cells were incubated with [14C]DOPA (20 μM, 0.25 μCi) for 15 min. [14C]Catecholamines formed in the cells were measured. Data are expressed as the mean ± SEM of three separate experiments carried out in triplicate.
Stimulation of tyrosine hydroxylase activity induced by p-nonylphenol through a nongenomic pathway
Treatment of cells with bisphenol A and p-nonylphenol at 10 nM for 3 d increased the activity of tyrosine hydroxylase by 22 and 40% over the control, respectively (Table 2). To examine whether the stimulation of tyrosine hydroxylase activity induced by p-nonylphenol is mediated by newly synthesized proteins of the enzyme, we measured the protein level of tyrosine hydroxylase after treatment of cells with 10 nM p-nonylphenol for 2 d. p-Nonylphenol (10 nM) did not affect the amount of tyrosine hydroxylase protein, whereas dibutyryl cAMP (a derivative of cAMP) (1 mM, 2 d) significantly increased the tyrosine hydroxylase protein (Fig. 4). These results suggest that tyrosine hydroxylase activity increased by p-nonylphenol is not because of the newly synthesized protein of the enzyme. Indeed, neither 0.2 ng/ml actinomycin D, an inhibitor of DNA-dependent RNA polymerase, nor 0.2 ng/ml cycloheximide, an inhibitor of ribosomal protein synthesis, abolished the stimulatory effect of p-nonylphenol (10 nM, 2 d) on tyrosine hydroxylase activity (dpm/106 cells·10 min: control, 1570 ± 80; p-nonylphenol, 2240 ± 170; actinomycin D, 560 ± 170; actinomycin D plus p-nonylphenol, 930 ± 290; cycloheximide, 610 ± 70; cycloheximide plus p-nonylphenol, 1050 ± 80).
TABLE 2. Effects of various estrogenic pollutants on tyrosine hydroxylase activity in adrenal medullary cells
FIG. 4. Effects of p-nonylphenol and dibutyryl cyclic AMP on tyrosine hydroxylase protein in the cells. After treatment of cells without (lane 1) or with 100 nM p-nonylphenol (lane 2) and 1 mM dibutyryl cAMP (DB-cAMP) (lane 3) for 2 d, the protein level of tyrosine hydroxylase was measured by Western blotting (A) and by a densitometric analysis (B). Data are expressed as mean ± SEM of three to four separate experiments carried out in triplicate. *, P < 0.05, compared with control.
Acute activation of tyrosine hydroxylase by p-nonylphenol
We then examined the effect of short-term treatment with p-nonylphenol on tyrosine hydroxylase activity in cells. Incubation of adrenal medullary cells with p-nonylphenol (10 nM) and acetylcholine, a physiological secretagogue (0.3 mM) (19), for 10 min caused a significant increase in tyrosine hydroxylase activity by 36 and 527% over the control, respectively (Fig. 5A). Concurrent incubation of cells with p-nonylphenol and acetylcholine at maximal concentrations augmented the stimulatory effect of acetylcholine alone by 806% over the control. Tyrosine hydroxylase is reported to be phosphorylated and activated by several multiple protein kinases (7) such as MAPK (20). Then, we examined the effect of U0126, an inhibitor of MAPK kinase, on the tyrosine hydroxylase activity induced by p-nonylphenol. Treatment of cells with U0126 (0.5 μM) for 10 min suppressed the acute effect of p-nonylphenol on tyrosine hydroxylase activity (Fig. 5B).
FIG. 5. Effects of short-term treatment with p-nonylphenol and/or acetylcholine (A) and U0126 (B) on tyrosine hydroxylase activity in the cells. Cells were preincubated in 250 μl of KRP buffer with or without 10 nM p-nonylphenol and/or 0.3 mM acetylcholine (ACh) (A) and 10 nM p-nonylphenol and/or 0.5 nM U0126 (B) for 10 min and then incubated for 10 min more in the presence of L-[1-14C]tyrosine (18 μM, 0.2 μCi). Tyrosine hydroxylase activity was measured. Data are the mean ± SEM of three to four separate experiments carried out in triplicate. *, P < 0.05, compared with control (A and B); **, P < 0.01, compared with control (A); ***, P < 0.05, compared with ACh alone (A).
Effect of short-term or long-term treatment with p-nonylphenol on MAPK activity
Recent reviews (18, 21) showed that estrogens acutely activate several cellular signals such as MAPK or phosphoinositide 3-kinase in vascular endothelial cells. To study the signal transduction of tyrosine hydroxylase activation induced by p-nonylphenol, we examined the effect of p-nonylphenol on p44/42 MAPK activity in adrenal medullary cells. Incubation of cells with p-nonylphenol for 5 min significantly increased phospho-p44/42 MAPK in a concentration-dependent manner (1–1000 nM) (Fig. 6). ICI182,780 (1 μM), by itself, increased MAPK phosphorylation but did not inhibit p44/42 MAPK phosphorylation induced by p-nonylphenol (100 nM) (Fig. 7). Furthermore, long-term treatment of cells with p-nonylphenol at 10 nM for 2 d also increased non-phospho- and phospho-p44/42 MAPK (Fig. 8).
FIG. 6. Effects of various concentrations of p-nonylphenol on phospho-p44/42 MAPK in cells. The cells were incubated with various concentrations (0–1000 nM) of p-nonylphenol for 5 min and then harvested with 100 μl of a lysis buffer. The cell suspension was homogenized and centrifuged at 15,000 x g for 30 min. The supernatants were analyzed by SDS-PAGE and Western blotting and subjected to the immunodetection. Top, Phospho- and non-phospho-p44/42 MAPK; bottom, data are the mean ± SEM of three to four separate experiments carried out in duplicate and expressed as a percentage of control (0 nM p-nonylphenol). *, P < 0.05, compared with 0 nM p-nonylphenol.
FIG. 7. Effect of ICI182,780, an antagonist of nuclear estrogen receptors, on phospho-p44/42 MAPK. After treatment with or without ICI182,780 (1 μM) for 30 min, the cells were incubated with or without p-nonylphenol (100 nM) and/or ICI182,780 (1 μM) for 5 min. Top, Phospho- and non-phospho-p44/42 MAPK; bottom, data are the mean ± SEM of five separate experiments carried out in duplicate and expressed as a percentage of control (0 nM p-nonylphenol). White and gray columns show p44 MAPK and p42 MAPK, respectively. *, P < 0.05, compared with control.
FIG. 8. Effect of long-term treatment with p-nonylphenol on p44/42 MAPK. After pretreatment of cells with or without 100 nM for 2 d, phospho- and non-phospho-p44/42 MAPK were measured. Top, Phospho- and non-phospho-p44/42 MAPK; bottom, data are the mean ± SEM of four separate experiments carried out in duplicate and expressed as a percentage of control. A and B, Phospho- (A) and non-phospho- (B) p44/42 MAPK; white and gray columns show p44 MAPK and p42 MAPK, respectively. *, P < 0.05, compared with control.
??Discussion
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In the present study, we demonstrated that treatment (3 d) of adrenal medullary cells with 17?-estradiol and environmental estrogenic pollutants such as bisphenol A and p-nonylphenol stimulates the synthesis of [14C]catecholamines from [14C]tyrosine. When [14C] DOPA was used as a substrate, p-nonylphenol failed to stimulate [14C]catecholamine synthesis, suggesting that stimulation of catecholamine synthesis induced by p-nonylphenol must occur upstream from the DOPA decarboxylase step, and probably at the tyrosine hydroxylase step. Indeed, treatment of cells with estrogenic pollutants for 3 d increased tyrosine hydroxylase activity in the cells. Based on these findings, it is likely that estrogenic pollutants activate tyrosine hydroxylase activity, which, in turn, stimulates catecholamine synthesis in adrenal medullary cells. To our knowledge, this is the first direct evidence to show the stimulatory effect of environmental estrogenic pollutants on catecholamine synthesis.
Nongenomic activation of tyrosine hydroxylase by estrogenic pollutants
The mechanism by which estrogenic pollutants increase tyrosine hydroxylase activity was then examined. ICI182,780, an antagonist of estrogen receptors (18), did not inhibit the stimulatory effect of p-nonylphenol but nullified the stimulatory effect of 17?-estradiol on [14C]catecholamine synthesis, suggesting that classical estrogen receptors are involved in catecholamine synthesis induced by 17?-estradiol but not by p-nonylphenol. Treatment of the cells with 10 nM p-nonylphenol for 2 d failed to increase the protein level of tyrosine hydroxylase. Furthermore, the stimulatory effects of p-nonylphenol on tyrosine hydroxylase activity were not inhibited by treatment with protein synthesis inhibitors, actinomycin D, and cycloheximide. These findings suggest that p-nonylphenol stimulates tyrosine hydroxylase activity and catecholamine synthesis in a nongenomic manner. Indeed, short-term treatment of cells with p-nonylphenol (10 nM) for 10 min induced an activation of tyrosine hydroxylase. Concomitant treatment of cells with acetylcholine (0.3 mM) and p-nonylphenol (10 nM) further enhanced the stimulatory effect of acetylcholine alone. The present finding suggests that estrogenic pollutants accelerate catecholamine synthesis produced by stress or emotional excitation, which induces the stimulation of splanchnic nerves and subsequently the adrenal medulla.
Stimulation of p44/42MAPK induced by p-nonylphenol
Recent studies reported that estrogens exert their functions via activation of several protein kinases such as MAPK and phosphoinositide 3-kinase through cell membrane estrogen receptors (18, 21). In the present study, treatment of cells with p-nonylphenol activated MAPK in a concentration-dependent (1–1000 nM) manner. Therefore, it is likely that p-nonylphenol stimulates the MAPK signaling pathway in bovine adrenal medullary cells. This is consistent with previous data that 17?-estradiol or p-nonylphenol increased MAPK phosphorylation in the pituitary tumor cell line GH3/B6/F10 (22) or breast cancer cells (23). The activation of MAPK induced by p-nonylphenol was not inhibited by ICI182,780, suggesting a classical estrogen receptor-independent pathway, as observed in catecholamine synthesis. On the other hand, tyrosine hydroxylase is acutely regulated by various factors (7, 24), such as enzyme phosphorylation. A number of in vitro and in situ experiments have shown that tyrosine hydroxylase can be phosphorylated by multiple protein kinases (7, 24), including MAPK (20). In the present study, U0126, an inhibitor of MAPK kinase, suppressed the stimulatory effect of p-nonylphenol on tyrosine hydroxylase activity, suggesting that acute treatment of cells with p-nonylphenol stimulates tyrosine hydroxylase, at least in part, through activation of MAPK.
Physiological significance of the stimulatory effects of estrogenic pollutants on catecholamine synthesis
The environmental distribution of p-nonylphenol has been documented by a number of studies in the world. For example, in metropolitan Tokyo in Japan, p-nonylphenol concentrations in river water samples were reported to range from 0.051–1.08 μg/liter (0.23–4.9 nM) (25, 26). These concentrations are less than those observed for some European rivers and are similar to the range observed in rivers in the United States. Because some regulators in the United States note that effluent p-nonylphenol concentrations greater than 10 μg/liter (45 nM) could result in detrimental environmental effects, emerging international regulatory concentrations are approximately 1 μg/liter (4.5 nM) (13). Nonetheless, plasma concentrations of p-nonylphenol in healthy humans were recently reported at a range from 0.5–1.0 ng/ml (2.3–4.5 nM) (27). In the present study, p-nonylphenol significantly stimulated [14C]catecholamine synthesis even at 0.1 nM and maximally at 10 nM. It is noteworthy that 0.1 nM of p-nonylphenol concentration is 45 times lower than that of the international regulatory standard and that the concentrations (0.1–10 nM) used in the present study are plausible environmental concentrations or possibly circulating concentrations in human blood.
On the other hand, serum concentrations of bisphenol A also were reported at 0.64 ng/ml (2.8 nM) and 1.5 ng/ml (6.5 nM) in normal women and men, respectively (28). Furthermore, because bisphenol A is usually used as dental sealants and composite fillings, it can be detected in saliva of patients with treated teeth (29). Lipid-soluble environmental pollutants such as organochloride insecticides and other estrogenic pollutants accumulate to high concentrations in various tissues, including the brain in humans (30) and rats (31). Indeed, Sun et al., (32) reported that bisphenol A is capable of penetrating the blood-brain barrier of the rat. Furthermore, several lines of evidence have demonstrated that prenatal and neonatal exposure to bisphenol A enhances central dopamine D1 receptor-dependent neurotransmissions, resulting in the supersensitivity of methamphetamine-induced pharmacological actions related to psychological dependence on psychostimulants in mice (33). Such exposure also induces aggressive behaviors in pubescent male mice (34).
Presently, there is no direct evidence that the disturbing actions of estrogenic pollutants on catecholamine synthesis and MAPK function are responsible for any deleterious effects on human health. We, however, are very concerned that the widespread use and persistence of estrogenic pollutants and their degradations might pose a potential threat to human health, because brain catecholamines such as norepinephrine or dopamine play important roles in neuronal functions of the midbrain and brain stem, a center of emotional behaviors (35). Future studies are required to determine whether environmental estrogenic pollutants exert these effects in the brain.
In summary, we demonstrated for the first time that environmental concentrations of estrogenic pollutants activate MAPK and stimulate catecholamine synthesis in adrenal medullary cells.
Acknowledgments
We thank Dr. Ryosuke Shinmi, Dr. Tomo Nakao, and Dr. Motohide Gotoh for their helpful experimental assistance.
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Address all correspondence and requests for reprints to: Nobuyuki Yanagihara, Ph.D., Department of Pharmacology, University of Occupational and Environmental Health, School of Medicine, 1-1, Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. E-mail: yanagin@med.uoeh-u.ac.jp.
Abstract
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Environmental estrogenic pollutants are compounds that have been shown to have estrogenic effects on fetal development and reproductive systems. Less attention, however, has been paid to their influence on neuronal functions. We report here the effects of estrogenic pollutants on catecholamine synthesis in bovine adrenal medullary cells used as a model system of noradrenergic neurons. Treatment of cultured bovine adrenal medullary cells with p-nonylphenol and bisphenol A at 10 nM for 3 d stimulated [14C]catecholamine synthesis from [14C]tyrosine and tyrosine hydroxylase activity, an effect that was not inhibited by ICI 182,780, an antagonist of estrogen receptors. Significant effects of p-nonylphenol on [14C]catecholamine synthesis were observed at 0.1 nM, which is 45 times lower than that of the international regulatory standard (4.5 nM), and the maximum effects were around 10–100 nM. The concentrations (0.1–10 nM) used in the present study are similar to the range observed in rivers in the United States or Europe. On the other hand, short-term treatment of cells with 10 nM p-nonylphenol for 10 min also activated tyrosine hydroxylase, which was suppressed by U0126, an inhibitor of MAPK kinase. Furthermore, treatment of cells with p-nonylphenol for 5 min increased the phospho-p44/42MAPK in a concentration-dependent (1–1000 nM) manner, whereas p-nonylphenol (100 nM, 2 d) enhanced both levels of non-phospho- and phospho-p44/42MAPK. These findings suggest that short-term and long-term treatment of cells with estrogenic pollutants at environmental concentrations stimulates catecholamine synthesis and MAPK through an estrogen receptor-independent pathway.
Introduction
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NATURAL ESTROGENS PLAY important roles in cell differentiation and proliferation, homeostasis, and the female reproductive system. The long-term genomic actions of estrogens are mediated by the binding of estrogens to their cytoplasmic/nuclear receptors (1, 2). In addition to estrogens, there exist environmental estrogenic pollutants or endocrine-disrupting chemicals that mimic the actions of estrogen; these include a variety of nonsteroidal substances with diverse chemical structures (3). Estrogenic pollutants bind to estrogen receptors (4) and may induce estrogen-dependent gene expression in wildlife and humans. Furthermore, as Ruehlmann et al. (5) have reported, environmental estrogenic pollutants acutely inhibit L-type Ca2+ channels in vascular smooth muscle cells and evoke a rapid relaxation of the coronary vasculatures. Therefore, it is also possible that estrogenic pollutants affect neuronal functions through modification of neurotransmission via genomic or nongenomic pathways.
Adrenal medullary cells are derived from the embryonic neural crest and share many physiological and pharmacological properties with postganglionic sympathetic neurons. Stimulation of adrenal medullary cells by acetylcholine released from splanchnic nerves stimulates catecholamine synthesis associated with an increase in activity of tyrosine hydroxylase, which catalyzes the conversion of tyrosine to L-3,4-dihydroxyphenylalanine (DOPA) (6). Tyrosine hydroxylase is regulated by two different mechanisms: short-term regulation by allosteric activation (7) and long-term regulation by enzyme induction (8). Because of similarity to that of the sympathetic neurons or brain noradrenergic neurons, the adrenal medullary cells have provided a convenient model system for studying the regulation of tyrosine hydroxylase in these neurons (9, 10).
p-Nonylphenol and bisphenol A are industrial compounds that have generated the most concern on the part of regulatory agencies and researchers as production of these chemicals has become widespread in the world. Bisphenol A is a monomer found in polycarbonate plastics and a constituent of epoxy and polystyrene resins employed extensively in the food-packing industry and in dentistry (11, 12). Alkylphenol ethoxylates are among the most widely used groups of surfactants. Approximately 500,000 tons of alkylphenol ethoxylates (nonylphenol ethoxylates encompass 80% of the world market) are produced annually worldwide (13). These compounds are aerobically and anaerobically degraded to alkylphenols. p-Nonylphenol is the main metabolite of alkylphenol polyethoxylates, which are widely used for surfactants in products such as detergents, paints, petroleum recovery chemicals, and so on. Although there is ongoing scientific debate concerning the potential threat of these estrogenic pollutants to animal and human reproductive health (3, 4), little information, however, is available to us about the effects of estrogenic pollutants on neuronal functions. Recently, we reported that estrogenic pollutants, including bisphenol A, inhibit the function of norepinephrine transporters in bovine adrenal medullary cells (14). We speculated that these pollutants might pose a potential threat to human mental health, because the norepinephrine transporter is known to be largely responsible for efficient termination of noradrenergic neurotransmission in the brain. In the present study, we examined the effects of estrogenic pollutants on catecholamine synthesis in cultured bovine adrenal medullary cells and found an increase in catecholamine synthesis and MAPK activity induced by estrogenic pollutants at environmental concentrations.
Materials and Methods
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Reagents
Oxygenated Krebs-Ringer phosphate (KRP) buffer was used throughout. Its composition is as follows (in mM): 154 NaCl, 5.6 KCl, 1.1 MgSO4, 2.2 CaCl2, 0.85 NaH2PO4, 2.15 Na2HPO4, and 10 glucose (adjusted to pH 7.4). Materials were obtained from the following sources: Eagle’s MEM was from Nissui Pharmaceutical (Tokyo, Japan); collagenase was from Nitta Zerachin (Osaka, Japan); calf serum, acetylcholine, 17?-estradiol, bisphenol A and p-nonylphenol were from Nacalai Tesque (Kyoto, Japan); MAPK kinase inhibitor U0126 was from Promega (Madison, WI); PhosphoPlus p44/42 MAPK (Thr 202/Tyr 204) antibody kit was from BioLabs (Berverly, MA); L-[1-14C]tyrosine (54.45 mBq/mmol) was from Perkin-Elmer Life Sciences (Boston, MA); and L-[U-14C]tyrosine (460 mBq/mmol) and L-[3-14C]DOPA (6.8 mBq/mmol) were from Amersham Biosciences (Buckinghamshire, UK).
Isolation and primary culture of bovine adrenal medullary cells
From bovine adrenal glands, the medullary cells were isolated by collagenase digestion according to the previously described method (9, 15). The cells were plated at a density of 4 x 106 cells per dish (Falcon 35 mm, Becton Dickinson Labware, Franklin Lakes, NJ) and maintained in monolayer culture in Eagle’s MEM containing 10% calf serum and antibiotics at 37 C under 5% CO2/95% air. The cells were used for experiments after being cultured between 2 and 7 d.
Long-term and short-term treatments of cells with estrogenic pollutants
Cultured cells were pretreated with or without estrogenic pollutants for 1–7 d. After treatment, the cells were used for experiments of [14C]catecholamine synthesis, tyrosine hydroxylase activity, and MAPK. In some experiments, after cells were treated for 5 or 10 min with estrogenic pollutants, tyrosine hydroxylase activity and MAPK were measured.
[14C]Catecholamine synthesis from [14C]tyrosine or [14C]DOPA
After treatment of cells with estrogenic pollutants for the indicated periods, the cells were incubated with 20 μM L-[U-14C]tyrosine (1 μCi) and L-[3-14C]DOPA (0.25 μCi) in 1.0 ml of KRP buffer at 37 C for 20 and 15 min, respectively. After aspiration of the reaction medium, the cells were harvested in 2.5 ml of 0.4 M perchloric acid and left standing for more than 30 min on ice to extract the radioactive catecholamines. The precipitated protein was removed by centrifugation at 1600 x g for 10 min at 4 C, and 14C-labeled catechol compounds in the supernatant were separated further into the fractions containing [14C]dopamine and [14C]epinephrine plus [14C]norepinephrine by ion exchange chromatography on Duolite C-25 columns (H+ type, 0.4 x 7.0 cm) (16). The 14C-labeled catecholamines separated were counted in a toluene base scintillator using a liquid scintillation counter (Aloka LSC-3500E, Aloka Co., Ltd., Tokyo, Japan). [14C]Catecholamine synthesis was expressed as the sum of the [14C]catecholamines (epinephrine, norepinephrine, and dopamine), because the ratio of [14C]epinephrine plus [14C]norepinephrine/[14C]dopamine was not significantly changed by stimulants.
Tyrosine hydroxylase activity
After treatment of cells (106 cells per well, 24 wells; Falcon) with 10 nM estrogenic pollutants for 3 d, the cells were exposed to 250 μl of the KRP buffer supplemented with 18 μM L-[1-14C]tyrosine (0.2 μCi) for 10 min at 37 C. In some experiments of short-term treatment with estrogenic pollutants, cells were pretreated with or without estrogenic pollutants for 10 min, and then tyrosine hydroxylase activity was measured. Upon addition of L-[1-14C]tyrosine, each well was immediately sealed with an acrylic tube capped with a rubber stopper and fitted with a small plastic cup containing 200 μl of NCS-II tissue solubilizer (Amersham Biosciences) to absorb 14CO2 released by the cells (17).
Western blot analysis of MAPK
The cells were washed three times with 1.0 ml of KRP buffer and were incubated in KRP buffer at 37 C for 30 min. Then, the cells were stimulated with or without p-nonylphenol (1–1000 nM) for 5 min. They were harvested with 100 μl of a buffer containing 20 mM Tris-HCl (pH 7.5), 5 mM EGTA, 60 mM ?-glycerophosphate, 1 mM NaVO3, 0.5% Triton X-100, 6 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 20 μg/ml aprotinin. The cell suspension was homogenized and centrifuged at 15,000 x g for 30 min. The supernatants were analyzed by SDS-PAGE, transferred to an Immobilon-P membrane (Millipore, Bedford, MA), and subjected to immunodetection using an anti-phospho- or anti-nonphospho-p44/42 MAPK (Thr 202/Tyr 204) antibody kit (17). To visualize the proteins, the membrane was exposed to x-ray film and analyzed by Mac BAS (version 2.52, Fuji Photo Film Co. Ltd., Tokyo, Japan).
Statistics
Data are presented as the mean ± SEM. The statistical evaluation of the data was performed by ANOVA. When a significant F-value was found by ANOVA, Dunnett’s or Scheffé’s test for multiple comparisons was used to identify differences among the groups. Values were considered statistically significant when P < 0.05. Statistical analyses were performed using StatView for Macintosh version 5.0J software (Abacus Concept Inc., Berkeley, CA).
Results
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Stimulation of catecholamine synthesis by environmental estrogenic pollutants
Treatment of cultured bovine adrenal medullary cells with 17?-estradiol and estrogenic pollutants such as bisphenol A and p-nonylphenol at 10 nM for 3 d stimulated [14C]cat-echolamine synthesis from [14C]tyrosine by 22, 45, and 72% over the control, respectively (Fig. 1). p-Nonylphenol (10 nM) continued to stimulate [14C]catecholamine synthesis during incubation for 1–7 d (Fig. 2A). The significant increase in [14C]catecholamine synthesis induced by p-nonylphenol was observed at concentrations as low as 0.1 nM with a maximum increase around 10–100 nM (Fig. 2B). To resolve whether estrogenic pollutants exert their effect on [14C]catecholamine synthesis through estrogen receptors, we used ICI182,780, a pure antagonist of classical estrogen receptors (18). ICI182,780 (100 nM), by itself, significantly increased [14C]catecholamine synthesis. This antagonist abolished [14C]catecholamine synthesis induced by 17?-estradiol, whereas the stimulatory effect of p-nonylphenol was not inhibited by ICI182,780 (Table 1). To ascertain which step in the biosynthesis of catecholamines was stimulated by p-nonylphenol, [14C]DOPA was used as a substrate instead of [14C]tyrosine (Fig. 3). In this case, however, p-nonylphenol (10 or 100 nM, 3 d) did not increase [14C]catecholamine synthesis from [14C]DOPA, indicating that stimulation of catecholamine synthesis induced by p-nonylphenol must occur predominantly before the DOPA decarboxylase step.
FIG. 1. Effects of various environmental estrogenic pollutants on [14C]catecholamine synthesis from [14C]tyrosine in cultured bovine adrenal medullary cells. Cultured cells (4 x 106 cells per dish) were pretreated with 17?-estradiol, bisphenol A, and p-nonylphenol at 10 nM for 3 d. The cells were then incubated for 20 min at 37 C in 1.0 ml KRP buffer containing L-[U-14C]tyrosine (20 μM, 1 μCi) without estrogenic pollutants. The 14C-labeled catecholamines formed are shown as the total [14C]catecholamines. Data are expressed as the mean ± SEM of four separate experiments carried out in triplicate. *, P < 0.05, compared with control.
FIG. 2. Effects of p-nonylphenol on [14C]catecholamine synthesis in adrenal medullary cells. Cells were treated with or without 10 nM p-nonylphenol for the indicated period (A) or with various concentrations of p-nonylphenol (0–100 nM) for 3 d (B). After treatment, the cells were incubated for 20 min in 1.0 ml KRP buffer containing L-[U-14C]tyrosine. [14C]Catecholamines formed in the cells were measured and are shown as the total [14C]catecholamines. Data are expressed as the mean ± SEM of four separate experiments carried out in triplicate. *, P < 0.05, compared with d 0 (A) or with 0 nM p-nonylphenol (B); **, P < 0.05, compared with 0.1 or 1.0 nM p-nonylphenol (B).
TABLE 1. Effects of ICI182,780, an antagonist of estrogen receptors on [14C]catecholamine synthesis
FIG. 3. Effect of p-nonylphenol on [14C]catecholamine synthesis from [14C]DOPA in the cells. After treatment with or without p-nonylphenol (10 or 100 nM) for 3 d, cells were incubated with [14C]DOPA (20 μM, 0.25 μCi) for 15 min. [14C]Catecholamines formed in the cells were measured. Data are expressed as the mean ± SEM of three separate experiments carried out in triplicate.
Stimulation of tyrosine hydroxylase activity induced by p-nonylphenol through a nongenomic pathway
Treatment of cells with bisphenol A and p-nonylphenol at 10 nM for 3 d increased the activity of tyrosine hydroxylase by 22 and 40% over the control, respectively (Table 2). To examine whether the stimulation of tyrosine hydroxylase activity induced by p-nonylphenol is mediated by newly synthesized proteins of the enzyme, we measured the protein level of tyrosine hydroxylase after treatment of cells with 10 nM p-nonylphenol for 2 d. p-Nonylphenol (10 nM) did not affect the amount of tyrosine hydroxylase protein, whereas dibutyryl cAMP (a derivative of cAMP) (1 mM, 2 d) significantly increased the tyrosine hydroxylase protein (Fig. 4). These results suggest that tyrosine hydroxylase activity increased by p-nonylphenol is not because of the newly synthesized protein of the enzyme. Indeed, neither 0.2 ng/ml actinomycin D, an inhibitor of DNA-dependent RNA polymerase, nor 0.2 ng/ml cycloheximide, an inhibitor of ribosomal protein synthesis, abolished the stimulatory effect of p-nonylphenol (10 nM, 2 d) on tyrosine hydroxylase activity (dpm/106 cells·10 min: control, 1570 ± 80; p-nonylphenol, 2240 ± 170; actinomycin D, 560 ± 170; actinomycin D plus p-nonylphenol, 930 ± 290; cycloheximide, 610 ± 70; cycloheximide plus p-nonylphenol, 1050 ± 80).
TABLE 2. Effects of various estrogenic pollutants on tyrosine hydroxylase activity in adrenal medullary cells
FIG. 4. Effects of p-nonylphenol and dibutyryl cyclic AMP on tyrosine hydroxylase protein in the cells. After treatment of cells without (lane 1) or with 100 nM p-nonylphenol (lane 2) and 1 mM dibutyryl cAMP (DB-cAMP) (lane 3) for 2 d, the protein level of tyrosine hydroxylase was measured by Western blotting (A) and by a densitometric analysis (B). Data are expressed as mean ± SEM of three to four separate experiments carried out in triplicate. *, P < 0.05, compared with control.
Acute activation of tyrosine hydroxylase by p-nonylphenol
We then examined the effect of short-term treatment with p-nonylphenol on tyrosine hydroxylase activity in cells. Incubation of adrenal medullary cells with p-nonylphenol (10 nM) and acetylcholine, a physiological secretagogue (0.3 mM) (19), for 10 min caused a significant increase in tyrosine hydroxylase activity by 36 and 527% over the control, respectively (Fig. 5A). Concurrent incubation of cells with p-nonylphenol and acetylcholine at maximal concentrations augmented the stimulatory effect of acetylcholine alone by 806% over the control. Tyrosine hydroxylase is reported to be phosphorylated and activated by several multiple protein kinases (7) such as MAPK (20). Then, we examined the effect of U0126, an inhibitor of MAPK kinase, on the tyrosine hydroxylase activity induced by p-nonylphenol. Treatment of cells with U0126 (0.5 μM) for 10 min suppressed the acute effect of p-nonylphenol on tyrosine hydroxylase activity (Fig. 5B).
FIG. 5. Effects of short-term treatment with p-nonylphenol and/or acetylcholine (A) and U0126 (B) on tyrosine hydroxylase activity in the cells. Cells were preincubated in 250 μl of KRP buffer with or without 10 nM p-nonylphenol and/or 0.3 mM acetylcholine (ACh) (A) and 10 nM p-nonylphenol and/or 0.5 nM U0126 (B) for 10 min and then incubated for 10 min more in the presence of L-[1-14C]tyrosine (18 μM, 0.2 μCi). Tyrosine hydroxylase activity was measured. Data are the mean ± SEM of three to four separate experiments carried out in triplicate. *, P < 0.05, compared with control (A and B); **, P < 0.01, compared with control (A); ***, P < 0.05, compared with ACh alone (A).
Effect of short-term or long-term treatment with p-nonylphenol on MAPK activity
Recent reviews (18, 21) showed that estrogens acutely activate several cellular signals such as MAPK or phosphoinositide 3-kinase in vascular endothelial cells. To study the signal transduction of tyrosine hydroxylase activation induced by p-nonylphenol, we examined the effect of p-nonylphenol on p44/42 MAPK activity in adrenal medullary cells. Incubation of cells with p-nonylphenol for 5 min significantly increased phospho-p44/42 MAPK in a concentration-dependent manner (1–1000 nM) (Fig. 6). ICI182,780 (1 μM), by itself, increased MAPK phosphorylation but did not inhibit p44/42 MAPK phosphorylation induced by p-nonylphenol (100 nM) (Fig. 7). Furthermore, long-term treatment of cells with p-nonylphenol at 10 nM for 2 d also increased non-phospho- and phospho-p44/42 MAPK (Fig. 8).
FIG. 6. Effects of various concentrations of p-nonylphenol on phospho-p44/42 MAPK in cells. The cells were incubated with various concentrations (0–1000 nM) of p-nonylphenol for 5 min and then harvested with 100 μl of a lysis buffer. The cell suspension was homogenized and centrifuged at 15,000 x g for 30 min. The supernatants were analyzed by SDS-PAGE and Western blotting and subjected to the immunodetection. Top, Phospho- and non-phospho-p44/42 MAPK; bottom, data are the mean ± SEM of three to four separate experiments carried out in duplicate and expressed as a percentage of control (0 nM p-nonylphenol). *, P < 0.05, compared with 0 nM p-nonylphenol.
FIG. 7. Effect of ICI182,780, an antagonist of nuclear estrogen receptors, on phospho-p44/42 MAPK. After treatment with or without ICI182,780 (1 μM) for 30 min, the cells were incubated with or without p-nonylphenol (100 nM) and/or ICI182,780 (1 μM) for 5 min. Top, Phospho- and non-phospho-p44/42 MAPK; bottom, data are the mean ± SEM of five separate experiments carried out in duplicate and expressed as a percentage of control (0 nM p-nonylphenol). White and gray columns show p44 MAPK and p42 MAPK, respectively. *, P < 0.05, compared with control.
FIG. 8. Effect of long-term treatment with p-nonylphenol on p44/42 MAPK. After pretreatment of cells with or without 100 nM for 2 d, phospho- and non-phospho-p44/42 MAPK were measured. Top, Phospho- and non-phospho-p44/42 MAPK; bottom, data are the mean ± SEM of four separate experiments carried out in duplicate and expressed as a percentage of control. A and B, Phospho- (A) and non-phospho- (B) p44/42 MAPK; white and gray columns show p44 MAPK and p42 MAPK, respectively. *, P < 0.05, compared with control.
??Discussion
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In the present study, we demonstrated that treatment (3 d) of adrenal medullary cells with 17?-estradiol and environmental estrogenic pollutants such as bisphenol A and p-nonylphenol stimulates the synthesis of [14C]catecholamines from [14C]tyrosine. When [14C] DOPA was used as a substrate, p-nonylphenol failed to stimulate [14C]catecholamine synthesis, suggesting that stimulation of catecholamine synthesis induced by p-nonylphenol must occur upstream from the DOPA decarboxylase step, and probably at the tyrosine hydroxylase step. Indeed, treatment of cells with estrogenic pollutants for 3 d increased tyrosine hydroxylase activity in the cells. Based on these findings, it is likely that estrogenic pollutants activate tyrosine hydroxylase activity, which, in turn, stimulates catecholamine synthesis in adrenal medullary cells. To our knowledge, this is the first direct evidence to show the stimulatory effect of environmental estrogenic pollutants on catecholamine synthesis.
Nongenomic activation of tyrosine hydroxylase by estrogenic pollutants
The mechanism by which estrogenic pollutants increase tyrosine hydroxylase activity was then examined. ICI182,780, an antagonist of estrogen receptors (18), did not inhibit the stimulatory effect of p-nonylphenol but nullified the stimulatory effect of 17?-estradiol on [14C]catecholamine synthesis, suggesting that classical estrogen receptors are involved in catecholamine synthesis induced by 17?-estradiol but not by p-nonylphenol. Treatment of the cells with 10 nM p-nonylphenol for 2 d failed to increase the protein level of tyrosine hydroxylase. Furthermore, the stimulatory effects of p-nonylphenol on tyrosine hydroxylase activity were not inhibited by treatment with protein synthesis inhibitors, actinomycin D, and cycloheximide. These findings suggest that p-nonylphenol stimulates tyrosine hydroxylase activity and catecholamine synthesis in a nongenomic manner. Indeed, short-term treatment of cells with p-nonylphenol (10 nM) for 10 min induced an activation of tyrosine hydroxylase. Concomitant treatment of cells with acetylcholine (0.3 mM) and p-nonylphenol (10 nM) further enhanced the stimulatory effect of acetylcholine alone. The present finding suggests that estrogenic pollutants accelerate catecholamine synthesis produced by stress or emotional excitation, which induces the stimulation of splanchnic nerves and subsequently the adrenal medulla.
Stimulation of p44/42MAPK induced by p-nonylphenol
Recent studies reported that estrogens exert their functions via activation of several protein kinases such as MAPK and phosphoinositide 3-kinase through cell membrane estrogen receptors (18, 21). In the present study, treatment of cells with p-nonylphenol activated MAPK in a concentration-dependent (1–1000 nM) manner. Therefore, it is likely that p-nonylphenol stimulates the MAPK signaling pathway in bovine adrenal medullary cells. This is consistent with previous data that 17?-estradiol or p-nonylphenol increased MAPK phosphorylation in the pituitary tumor cell line GH3/B6/F10 (22) or breast cancer cells (23). The activation of MAPK induced by p-nonylphenol was not inhibited by ICI182,780, suggesting a classical estrogen receptor-independent pathway, as observed in catecholamine synthesis. On the other hand, tyrosine hydroxylase is acutely regulated by various factors (7, 24), such as enzyme phosphorylation. A number of in vitro and in situ experiments have shown that tyrosine hydroxylase can be phosphorylated by multiple protein kinases (7, 24), including MAPK (20). In the present study, U0126, an inhibitor of MAPK kinase, suppressed the stimulatory effect of p-nonylphenol on tyrosine hydroxylase activity, suggesting that acute treatment of cells with p-nonylphenol stimulates tyrosine hydroxylase, at least in part, through activation of MAPK.
Physiological significance of the stimulatory effects of estrogenic pollutants on catecholamine synthesis
The environmental distribution of p-nonylphenol has been documented by a number of studies in the world. For example, in metropolitan Tokyo in Japan, p-nonylphenol concentrations in river water samples were reported to range from 0.051–1.08 μg/liter (0.23–4.9 nM) (25, 26). These concentrations are less than those observed for some European rivers and are similar to the range observed in rivers in the United States. Because some regulators in the United States note that effluent p-nonylphenol concentrations greater than 10 μg/liter (45 nM) could result in detrimental environmental effects, emerging international regulatory concentrations are approximately 1 μg/liter (4.5 nM) (13). Nonetheless, plasma concentrations of p-nonylphenol in healthy humans were recently reported at a range from 0.5–1.0 ng/ml (2.3–4.5 nM) (27). In the present study, p-nonylphenol significantly stimulated [14C]catecholamine synthesis even at 0.1 nM and maximally at 10 nM. It is noteworthy that 0.1 nM of p-nonylphenol concentration is 45 times lower than that of the international regulatory standard and that the concentrations (0.1–10 nM) used in the present study are plausible environmental concentrations or possibly circulating concentrations in human blood.
On the other hand, serum concentrations of bisphenol A also were reported at 0.64 ng/ml (2.8 nM) and 1.5 ng/ml (6.5 nM) in normal women and men, respectively (28). Furthermore, because bisphenol A is usually used as dental sealants and composite fillings, it can be detected in saliva of patients with treated teeth (29). Lipid-soluble environmental pollutants such as organochloride insecticides and other estrogenic pollutants accumulate to high concentrations in various tissues, including the brain in humans (30) and rats (31). Indeed, Sun et al., (32) reported that bisphenol A is capable of penetrating the blood-brain barrier of the rat. Furthermore, several lines of evidence have demonstrated that prenatal and neonatal exposure to bisphenol A enhances central dopamine D1 receptor-dependent neurotransmissions, resulting in the supersensitivity of methamphetamine-induced pharmacological actions related to psychological dependence on psychostimulants in mice (33). Such exposure also induces aggressive behaviors in pubescent male mice (34).
Presently, there is no direct evidence that the disturbing actions of estrogenic pollutants on catecholamine synthesis and MAPK function are responsible for any deleterious effects on human health. We, however, are very concerned that the widespread use and persistence of estrogenic pollutants and their degradations might pose a potential threat to human health, because brain catecholamines such as norepinephrine or dopamine play important roles in neuronal functions of the midbrain and brain stem, a center of emotional behaviors (35). Future studies are required to determine whether environmental estrogenic pollutants exert these effects in the brain.
In summary, we demonstrated for the first time that environmental concentrations of estrogenic pollutants activate MAPK and stimulate catecholamine synthesis in adrenal medullary cells.
Acknowledgments
We thank Dr. Ryosuke Shinmi, Dr. Tomo Nakao, and Dr. Motohide Gotoh for their helpful experimental assistance.
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