Institute of Food Safety and Nutrition, Danish Veterinary and Food Administration, Mørkhøj Bygade 19, Dk-2860 Søborg, Denmark
Received September 26, 2002; accepted October 25, 2002
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ABSTRACT |
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Key Words: endocrine disrupters; combination effects; isobole method; vinclozolin; procymidone; antiandrogen; rats; reporter gene assay.
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INTRODUCTION |
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Many compounds have been shown to be antiandrogenic in vitro and in animal studies (Gray et al., 1999), but most of these compounds are weak agonists or antagonists of the steroid receptors in vitro and efficient only at concentrations in the µM range, affinities that are at least 1000-fold lower than those of the natural steroid hormones. Based on the effect of single compounds, it is difficult to explain the possible impact on human health. Very little is known on the human exposure to these compounds, but it is anticipated that humans are exposed to a mixture of many different endocrine disrupters from different sources, each present at low levels (Wilkinson et al., 2000
), that may cause an accumulated effect. Thus, it is highly relevant to get more knowledge on potential combination effects of similarly acting compounds and investigate this issue thoroughly in diverse model systems.
One work by Arnold et al.(1997) that showed a marked synergistic effect of estrogenic compounds on the activation of the estrogen receptor was discussed thoroughly both scientifically and legislationally until it was retracted as the results could not be reproduced (McLachlan, 1997
). Some studies on combinatorial effects have only tested one dose level of each compound alone and in combination. In these studies it was predicted that the combination effects followed effect summation at all concentration levels, thus a linear dose-response curve with a plot passing through the origin was assumed. Linear dose-response curves can be found in the low dose range, e.g., for carcinogens, but are seldom found for endocrine disrupters that often show sigmoidal curves with differing slopes or differing maximal effects when compared (Kortenkamp and Alternburger, 1998
). As the linearity was not demonstrated in most of the combination studies mentioned above, they were found to be inconclusive when evaluated thoroughly (Kortenkamp and Altenburger, 1999
). In most studies, additivity and thus no interaction of the combination of compounds was observed, but a thorough evaluation of the results showed that synergism had been overlooked in a few cases (Kortenkamp and Altenburger, 1998
).
Concentration-response analyses of 17ß-estradiol, bisphenol A, and o,p'-DDT as single compounds and in combination showed additivity in a yeast reporter gene assay for estrogenic effects (Rajapakse et al., 2001). The best fitted regression lines for the observed effects of the mixtures were congruent with the curves for the predicted additive effect. Following the same isobole analyses of concentration-response curves, the combination of o,p'-DDT, genistein, 4-nonylphenol, and 4-n-octylphenol was analyzed in a yeast estrogen screen (Payne et al., 2000
). By calculated predictions and concentration-response curves they found the combination of the estrogenic compounds to be additive. Payne et al.(2001)
studied o,p'-DDT, p,p'-DDE, ß-hexachlorocyclohexane, and p,p'-DDT on the induction of cell proliferation in MCF-7 cells. The combined effect was found additive by this approach. Recently, Rajapakse et al.(2002)
published a study showing that a combination of 11 xenobiotics, present at concentrations that were inactive by themselves, had marked but still additive effect on the potency of 17ß-estradiol in a yeast reporter gene assay with the human estrogen receptor
.
Most of the published studies concerning mixtures of endocrine disrupters have looked at the effect of combination of the compounds in vitro. Interactions with the receptor in vitro are often found to be additive (Payne et al., 2000) but whether this reflects the situation in vivo is still an open question. Adding to the complexity, it has been shown that combination effects can vary with different endpoints within the same experimental design. Compounds working synergistically on the molecular level can elicit additivity on more complex endpoints such as cell death (Grimme et al., 1996
).
In this study, the combination of two similarly acting fungicides was investigated in vitro and in vivo with endpoints at the molecular as well as the organ level. Dose-response curves of both fungicides known to elicit antiandrogenic effects and mixtures of these were analyzed in an in vitro androgen reporter gene assay (Vinggaard et al., 1999) as well as in the Hershberger assay involving young castrated male rats (Hershberger et al., 1953
). The Hershberger assay was supplemented with hormone analyses as well as with quantitative analyses of androgen-dependent gene expression as previously described (Nellemann et al., 2001
) in order to elucidate the combination effects at different levels of molecular complexity. Few studies of endocrine disrupters so far have included evaluation of neurochemical parameters. This study includes analyses of noradrenaline (NA), dopamine (DA), 5-hydroxytryptamine (5-HT), and acethylcholine esterase (AChE) as indicators of effects on respective parts of the CNS.
Following analyses of the dose-response curves for the single fungicides and assuming additivity, the models of concentration addition were used to predict the relationships for combination of the two with a variety of fixed mixture ratios.
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MATERIALS AND METHODS |
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AR reporter gene assay.
Effects on androgen receptor activity were tested in a previously developed reporter gene assay (Vinggaard et al., 1999). Chinese Hamster Ovary cells (CHO K1, 7000 cells per well) were transfected with 75 ng cDNA/well of the expression vector pSVAR0 and the MMTV-LUC reporter plasmid (both provided by Dr. Albert Brinkmann, Erasmus University, Rotterdam) in a ratio of 1:100 using 300 nl of the transfection reagent FuGene (Boehringer Mannheim, Germany). The chemicals to be tested were added with or without 0.01 nM of the AR agonist R1881 (NEN, Boston, MA). Hydroxyflutamide was included in every experiment as a positive control. Luciferase activity was measured directly using a BioOrbit Galaxy luminometer by automatically injection of 40 µl substrate containing 1 mM luciferin (Amersham Int., Buckinghamshire, UK) and 1 mM ATP (Boehringer Mannheim, Germany) in lysis buffer and the chemiluminiscense generated from each well was measured. The observations were normalized to correct for interassay variation. The procedure is described in detail in Andersen et al.(2002)
.
Animal Experiments
Test species.
Castrated male Wistar rats were acquired from M&B (Eiby, Denmark). All rats were castrated at the age of four weeks, 14 days prior to study start. All animals were delivered one week prior to study start and upon arrival rats were housed in Bayer Makrolon type 3118 cages (Type: 80-III-420-H-MAK, Techniplast), three per cage with Tapvai bedding. They were fed Syn 8.IT (a diet known to be free of phytoestrogens) and were provided with acidified tap water ad libitum. Animal rooms were maintained on a 12-h light/dark cycle, a temperature of 22 ± 1°C and a relative humidity of 55 ± 5%. Rats were weighed and divided by randomization into treatment groups. During testing rats were weighed daily and visually inspected for health effects twice a day.
Testing of vinclozolin and procymidone in castrated animals.
In Experiment 1 all groups of male castrated Wistar rats (seven groups of six animals each) were administered 0.5 mg/kg/day testosterone propionate sc. One group served as negative control while the second group serving as positive control was administered 20 mg/kg flutamide sc. The rest of the groups received 10, 25, 50, 100, or 200 mg/kg of vinclozolin orally.
In Experiment 2 all groups of male castrated Wistar rats (seven groups of six animals each) were administered 0.5 mg/kg/day testosterone propionate sc. One group served as control while the second group serving as positive control was administered 20 mg/kg flutamide sc. The rest of the groups received 10, 25, 50, 100, or 200 mg/kg of procymidone orally.
In Experiment 3 all groups of male castrated Wistar rats (nine groups of six animals each) were administered 0.5 mg/kg/day testosterone propionate sc. One group served as control while the second group serving as positive control was administered 20 mg/kg flutamide sc. The following five groups were orally administered 10, 25, 50, 100, or 200 mg/kg of vinclozolin and procymidone in a 1:1 combination. The 10 mg/kg group received 5 mg/kg vinclozolin and 5 mg/kg procymidone and so forth for the other groups. The two last groups received 100 mg/kg vinclozolin or 100 mg/kg procymidone alone.
For all three experiments all compounds were dissolved in peanut oil. Sterile oil (The Royal Veterinary Agriculture Pharmacy, Copenhagen, Denmark) was used for testosterone propionate and flutamide solutions. All compounds were administered in a dosing volume of 2 ml/kg body weight and the dosing period was seven days for all animals. The testosterone dose was always given a few minutes after the test compound and the last dosing was performed in the morning on the day of killing the animals. Body weights were recorded and animals were euthanized using CO2/O2 followed by decapitation. All the animals from each group underwent a thorough autopsy. The ventral prostate, seminal vesicles, levator ani/bulbocavernosus muscle, bulbourethral glands, pituitary, liver, and paired kidneys were dissected and weighed. The ventral prostates were stored in 0.5 ml RNAlater (Ambion) at 20°C until gene expression analysis. Blood was collected in plain glass tubes and serum was prepared and stored at 80°C until measurement of hormones.
Hormone Analyses
Rat LH and FSH concentrations were analyzed in serum using the technique of time-resolved fluorescence (Delfia, Wallac) by Pirjo Pakarinen, Turku University, Finland. Rat FSH immunoreactivity was determined by a two-site immunofluorometric assay (IFMA; van Casteren et al., 2000). A monoclonal mouse antibody against human recombinant FSHß (Mab ahFSHß FG 5020, N.V. Organon, Oss, The Netherlands) was used as a capture antibody coated to the walls of 96-well plates (Maxisorp 4-73709A, Nunc, Denmark). As signal antibody biotin-labeled rabbit polyclonal antibody against human recombinant FSH
(R-ahFSH-Biotin R93-2705, N.V. Organon) was used. Finally, europium-labeled streptavidin was bound to the biotin-labeled antibody. Time-resolved fluorescence evoked by a europium label was used for signal detection with Wallac Victor2 1420 Multilabel Counter (PerkinElmer Life Sciences, Wallac Finland Oy, Turku, Finland). The standard used was a NIDDK standard FSH-RP-2 obtained from the National Hormone and Pituitary Program, NIH, Rockville, MD. Buffers and washing solutions used throughout the assay were Delfia® reagents obtained from Wallac. Rat LH was measured using the time-resolved fluorimetric assay (IFMA, Delfia, Wallac OY, Turku, Finland) as described by Haavisto et al.(1993)
. The standard rLH RP-3 was kindly provided by NIDDK, NIH (Baltimore, MD).
Gene Expression Analyses
RNA isolation and cDNA production.
Ventral prostates were homogenized in RLT buffer (RNeasy Mini-kit, QIAGEN) by an UltraTurrax® rotor-stator homogenizer. Subsequent extraction of total RNA was performed using the RNeasy Mini-kit (QIAGEN) following the manufacturers instructions. The quantity and quality of the purified RNA was evaluated by spectrophotometry. cDNA was produced from 0.51.0 µg of total RNA using Omniscript Reverse Transcriptase kit and the manufacturers instructions (QIAGEN).
Real-time RT-PCR.
Real-time RT-PCR with online detection of the PCR reaction based on fluorescence monitoring (LightCycler, Roche) was employed. We used hybridization probes (TIB MolBiol, Berlin, Germany) to monitor the amount of specific target sequence produced. Quantitative results were obtained by the cycle threshold value where a signal rises above background level. Expression of the genes coding for TRPM-2 and PBP C3 was compared to the steady expression of 18S rRNA. PCR was performed with 5 mM MgCl2 for 18S rRNA and with 4 mM MgCl2 for TRPM-2 and PBP C3, respectively. 0.5 µM primers (5'-ACGAACCAGAGCGAAAGCAT-3', 5'-GGACATCTAAGGGCATCACAGAC-3') and 0.2 µM probes (FL530: 5'-TCGGAACTACGACGGTATCTGATCGTC-3', LC640: 5'-CGAACCTCCGACTTTCGTTCTTGAT-3') for 18S rRNA, 0.3 and 0.2 µM of primers (5'-TTGCTGCTATGCCAGTGGTT-3', 5'-CCTCCATCATCACGCTAACATT-3') and probes (FL530: 5'-AGGCTGTGAAGCAATTCAAGCAGTGT-3', LC640: 5'-TTCTAGATCAGACCGACAAGACTCTGGAAA-3'), respectively, for PBP C3. For TRPM-2 0.7 µM primers (5'-CTGACCCAGCAGTACAACGA-3', 5'- TGACACGAGAGGGGACTTCT-3') and 0.3 µM probes (FL530: 5'-TAACCTCACACAGGGCGATGACCA-3'; LC640: 5'-ACCTTCGGGTCTCCACAGTGACAAC-3') were employed.
The PCR program followed the manufacturers instructions (LightCycler-DNA Master Hybridization Probes, Roche) except that Taq start antibody (Clontech; 0.16 µl per 20 µl reaction) was incubated with the DNA Master Hybridization Probes mix at room temperature for 5 min prior to addition of the rest of the components. Gene expressions were analyzed at least three times for each animal in the same cDNA preparation.
Neurochemistry
Preparation of brain samples.
The whole brain was isolated, weighed, and homogenized in 8 ml ice-cold 0.32M sucrose at 04°C with an UltraTurrax® at maximum speed. Samples of homogenate were taken and processed for protein, enzyme, and neurotransmitter analyses.
Protein analysis.
A sample of homogenate was added to 0.05% M NaOH, and quantified for protein content by the bicinchoninic acid (BCA) method (BCA Protein Assay Reagent®, Pierce Cat. No. 23225) using bovine serum albumin as the standard.
Determinations of enzyme activities.
Homogenate was added to equal volumes of 2% Triton X-100. After 1 h at ambient temperature, samples were centrifuged at 10,000 x g for 10 min. Supernatants were used for determination of acetylcholinesterase (AChE) and buturylcholinesterase (BuChE) activities at 37°C (Lam et al., 1996).
Neurotransmitter analyses.
Immediately after homogenization, one part of a 0.5 M perchloric acid was added to two parts of brain homogenate and centrifuged at 10,000 x g for 10 min. One aliquot of perchloric acid supernatant (Lam et al., 1996) was used directly for the determination of 5-HT. Another aliquot was used for NA and DA determinations after "conventional" purification on aluminium oxide (Anton and Sayre, 1962
). NA, DA, and 5-HT were determined by HPLC with electrochemical detection (Lam et al., 1996
).
Isobole method.
In order to calculate a function describing the predicted effect of a combination of the compounds, additivity was assumed. For every effect level we calculated the equation: dV/DV + dP/DP = 1 where dV and dP are the doses of vinclozolin and procymidone in a mixture that produces a specified effect and DV and DP are the doses of the single compounds that on their own elicit the same effect. Synergism is indicated if data points lie below the addivity isobole such that the dV/DV + dP/DP < 1. Antagonism is proposed when dV/DV + dP/DP > 1. The concentrations of the single compounds resulting in a given effect were calculated from the regression line giving the best fit of the concentration-response curve for each fungicide. For the in vitro studies and for the weight of the reproductive organs, the best fit was found to be a four-parameter logistic curve fit (F = min + (max min)/(1 + (x/EC50)Hillslope)). For the hormone levels a curve fit following a hyperbola modified fit (y = a (b/(1 + cx)d)) and for the gene expression levels an exponential growth Stirling model (y = yo + a(ebx 1)/b) were chosen. The in vivo results in Experiments 1 and 2 were normalized using the data from the 100 mg/kg groups of the single compounds in Experiment 3 by adjusting the data in the 100 mg/kg vinclozolin and 100 mg/kg procymidone group in Experiments 1 and 2, respectively.
Statistical analyses.
All data was analyzed by comparing the group treated with testosterone (control) and the groups treated with testosterone and flutamide or testosterone and vinclozolin or procymidone alone or in combination. A t-test was performed to compare the flutamide-treated animals with the controls. A one-way ANOVA was performed excluding the flutamide group. Pair-wise comparisons between test and control group were made with Dunnetts test (SigmaPlot ver. 7.0, Statistical Solutions). If the data did not show normal distribution and homogeneity of variance, a nonparametric test (Kruskal-Wallis) was performed followed by Dunns method (SigmaPlot ver. 7.0, Statistical Solutions). Significance was judged at p < 0.05. IC50 values of the in vitro data were determined using a four-parameter logistic function (SigmaPlot ver. 7.0, Statistical Solutions).
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RESULTS |
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DISCUSSION |
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Initially, the combination effects of procymidone and vinclozolin were tested in an AR reporter gene assay. This is to our knowledge the first combination experiment on endocrine disrupters performed on AR in vitro. The assay is very useful for this purpose because it allows dose-response curves of the compounds alone and in several mixture ratios to be tested simultaneously. The results obtained showed that the isobole coefficients were 0.84 and 1.09 for effect levels of IC25 and IC50, respectively. In general, all results indicated unambiguously that the blocking effect on AR did not deviate from additivity.
In vitro, procymidone showed a lower inhibitory effect than vinclozolin with an IC50 of 0.6 µM compared to 0.1 µM for vinclozolin, whereas procymidone in vivo showed effects significantly different from the control rats at the same or lower concentrations than for vinclozolin with respect to most of the reproductive organ weights and LH analyses. This is in contrast to findings in an in utero study in Long-Evans rats treated from gestational day 14 to postnatal day 3 by Ostby et al.(1999) where procymidone had antiandrogenic effects in vivo at 25 mg/kg, but vinclozolin was about 2.5-fold more potent. In intact adult male rats, Hosokawa et al. (1993a
,b
) found that administration of 6000 ppm procymidone in the diet, corresponding to approximately 300 mg/kg, administered for two weeks resulted in little effect on the reproductive tract. The current study shows that the potencies of androgen receptor antagonists in vitro do not always predict the potency of the compounds in vivo. One reason for the strong potency of procymidone in vivo might be that metabolites of procymidone act as antiandrogens. In addition, the study shows that the Hershberger assay using young castrated male rats is a sensitive assay for evaluating the antiandrogenic effects of both procymidone and vinclozolin.
The two pesticides were administered orally because the metabolites of vinclozolin, M1 and M2, are the active androgen receptor inhibitors and because human exposure is primarily oral (Ashby and Lefevre, 2000; Kelce et al., 1997
). Even the lowest dose of 10 mg/kg procymidone resulted in lowered weight of seminal vesicles, levator ani/bulbocavernosus muscle, and the bulbourethral glands. For vinclozolin a dose of 10 mg/kg reduced the weight of ventral prostates. The NOEL found in intact animals is 12.5 mg/kg for procymidone (Ostby et al., 1999
) and 12 mg/kg vinclozolin in Wistar rats (Hellwig et al., 2000
). This indicates that the Hershberger assay is a sensitive assay, valuable for screening of endocrine disrupters.
The increases in LH and FSH indicate that both vinclozolin and procymidone antagonized central androgen receptors blocking the negative feedback mechanism of testosterone on the hypothalamus/pituitary level. The level of LH was significantly increased with as low as 25 mg/kg procymidone, 100 mg/kg vinclozolin, and 25 mg/kg combination of the two fungicides. FSH was increased by 25 mg/kg vinclozolin or procymidone alone or by 10 mg/kg vinclozolin and procymidone in combination. Vinclozolin and procymidone induced increases in both LH and FSH levels of a magnitude corresponding to the effect of flutamide. Hosokawa et al. (a,b) found LH to be elevated after administration of of 6000 ppm procymidone to intact male rats for 14 days, but not after administration of 2000 ppm, corresponding to approximately 300 mg/kg and 100 mg/kg procymidone, respectively. Kelce et al.(1997) found LH to be increased by administration of 200 mg/kg vinclozolin for five days in a Hershberger assay.
As examples of androgen-responsive gene expression, the expression of prostate binding protein polypeptide C3 (PBP C3; Bossyns et al., 1986) and testosterone-repressed prostatic message 2 (TRPM-2; Montpetit et al., 1986
) in the ventral prostate of the rat was chosen. PBP constitutes approximately 50% of the secreted protein from the normal rat prostate and is thereby the most abundant androgen-regulated protein in this tissue. The androgen regulation of PBP C3 is presumably caused by an androgen-responsive element in the first intron (Vercaeren et al., 1996
). TRPM-2 is not normally expressed in the rat ventral prostate but is induced by castration (Leger et al., 1987
) and has been suggested to be involved as a survival factor from apoptosis (Viard et al., 1999
). Gene expression studies often result in significant effects prior to other endpoints and can be a very sensitive endpoint for endocrine disrupters acting on the androgen receptor (Nellemann et al., 2001
; Vinggaard et al., 2002
). In the study described here, the gene expression evaluations are less sensitive than the weights of reproductive organs or than hormone levels. The relative level of TRPM-2 showed significant difference to the testosterone group with administration of 100 mg/kg vinclozolin and procymidone in combination and for 100 mg/kg procymidone alone but still with an effect much lower than for the flutamide group. The much higher effect of flutamide is also found for the relative expression of PBP C3 where only the administration of 100 mg/kg vinclozolin and procymidone in combination or the 100 mg/kg procymidone alone resulted in a significant change. Thus, the effects of the two fungicides at the highest doses either alone or in combination approach the effect of flutamide on both organ and hormone levels but not at the gene expression level. In a Hershberger assay with sc administration of 50 mg/kg vinclozolin for seven days, we found that evaluation of gene expression of the two genes TRPM-2 and PBP C3 was more sensitive than hormone levels (Nellemann et al., 2001
). Kelce et al.(1997)
found vinclozolin to induce TRPM-2 expression and inhibit PBP C3 expression at a dose of 200 mg/kg for 5 days in 120-day-old Sprague-Dawley rats. As the TRPM-2 mRNA level has an optimum after four to five days of treatment with an antiandrogen, the seven-day period might be too long to measure any effect at lower doses of the compounds.
The levels of different neurotransmitters were included in these experiments. These studies showed significant changes for 5-HT, but no effects for the levels of NA, DA, AChE, or BuChE. This indicates that the noradrenergic, dopaminergic, and cholinergic parts of the CNS are not markedly affected by administration of the two fungicides. 5-HT is known to regulate luteinizing hormone-releasing hormone (LHRH) in the female rat, and a feedback to the level of 5-HT could be the cause of our finding (Moguilevsky and Wuttke, 2001). No such effect has previously been reported to be induced by endocrine disrupters and thus this aspect deserves further investigation. However, flutamide treatment did not induce any significant increase in the level of 5-HT in the current study. In a related study, no differences between castrated rats and castrated rats supplemented with testosterone were observed on the level of 5-HT. In that study flutamide did not have any effect on 5-HT either (unpublished data).
The classical isobole method is a practical approach for evaluating potential interaction of compounds. The concept is to compare doses that produce effects of equal strength (isoboles). One of the advantages of the isobole method is that it can compare compounds irrespective of the shape of their dose-response curves (Kortenkamp and Altenburger, 1998). A combined effect can be additive, synergistic, or antagonistic depending on whether the observed effect is the expected, a stronger, or a weaker one (Berembaum, 1989
). Analysis using the isobole method demands a design involving dose-response curves for each compound alone and in combination. This gives the possibility to compare doses of compounds that produce the same specified effect. By studying combination effects of many different endocrine disrupters a better evaluation of the presence or absence of interaction between similarly and differently acting chemicals may be obtained, thereby achieving a better understanding of the effects of total human exposure to endocrine disrupters.
As combination experiments require high reproducibility of the biologic responses and as in vivo experiments show greater variation than do in vitro tests, some of this variation was corrected for by harmonizing the data between experiments by using the 100 mg/kg vinclozolin and the 100 mg/kg procymidone groups in Experiment 3 to harmonize Experiments 1 and 2, respectively, before isobole analyses were performed. Even though the isobole formulas of weights of ventral prostates and seminal vesicles showed values well below one known to reflect synergism, the observed inhibitory effects were additive both in vitro and in vivo when statistics was employed and 95% confidence belts added. In Gray et al.(2001) a study involving combinations of 25100 mg/kg of vinclozolin and procymidone is mentioned. In agreement with results from our study, the combination was found to be additive for effect on weights of ventral prostate and levator ani/bulbocavernosus muscle for lower doses whereas the effect in their study was less than additive for higher doses probably because the single compounds showed maximal inhibition of the effect of testosterone on their own.
In conclusion, additivity of combination of vinclozolin and procymidone was found both in vitro and in vivo and for all three endpoints tested, organ weights, hormone levels, and gene expression levels in a Hersbherger assay. However, it cannot be excluded that interactions of the two fungicides can be found on endpoints investigated in developmental studies, an issue that deserves further attention in future studies.
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ACKNOWLEDGMENTS |
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NOTES |
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