Dopamine Transporter Binding in the Rat Striatum Is Increased by Gestational, Perinatal, and Adolescent Exposure to Heptachlor

Sherry Purkerson-Parker*,{dagger}, Katherine L. McDaniel{dagger} and Virginia C. Moser{dagger},1

* The University of North Carolina, Curriculum in Toxicology, Chapel Hill, North Carolina 27599; and {dagger} Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711

Received May 1, 2001; accepted August 23, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Heptachlor is a persistent cyclodiene pesticide that affects GABAergic function. Recent reports indicate that heptachlor exposure also alters dopamine transporter (DAT) expression and function in adult mice. The aim of this study was to determine whether gestational, perinatal, and/or adolescent heptachlor exposure in rats altered dopamine-receptor and DAT binding. Adolescent exposure to dieldrin was included to evaluate the generality of the findings. Sprague-Dawley rats received doses (po) ranging from 0 to 8.4 mg/kg/day of heptachlor, or dieldrin, 3 mg/kg/day, during different developmental periods. There were dose-related decreases in maternal weight gain and pup survival, as well as delayed righting reflex, at heptachlor doses >=3 mg/kg/day. There were no changes in striatal dopamine receptor-D1 ([3H]SCH-23390) and -D2 ([3H]spiperone) binding in preweanling pups exposed perinatally to heptachlor, and no differences in the response of adult rats to the motor activity-increasing effects of d-amphetamine. However, there were significant (27–64%) increases in striatal DAT binding of [3H]mazindol in preweanling rats exposed only gestationally. In rats exposed perinatally and/or during adolescence, there were also increases (34–65%) in striatal DAT binding at postnatal days (PND) 22, 43, and 128. Adolescent exposure to dieldrin also increased DAT binding. In other rats exposed perinatally and throughout adolescence, even the lowest dose of heptachlor 0.3 mg/kg/d increased DAT binding on PND 130. The DAT affinity for mazindol was unchanged in heptachlor-exposed striata. In vitrobinding studies indicated that heptachlor (>=10 µM) displaced mazindol binding. Thus, gestational, perinatal, and/or adolescent exposure to heptachlor produced an increase in DAT binding as early as PND 10, and this change persisted into adulthood.

Key Words: heptachlor; dieldrin; dopamine transporter; neurotoxicity; developmental; rats.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Heptachlor is a cyclodiene organochloride that was used as a pesticide for more than 30 years until its ban in the mid-1980s. Heptachlor and its metabolite, heptachlor epoxide, have persisted in the environment for years (Fendick et al., 1990Go). Incidents of high pesticide exposure have been reported; for example; in 1981–82, heptachlor epoxide contaminated a considerable part of Hawaii's milk supply (Smith, 1982Go). This has led to concern that adverse health effects might have resulted from consumption of contaminated dairy products, especially in those exposed in utero and/or as children. However, little is known regarding the impact of these exposures, or how heptachlor and heptachlor epoxide might influence the development of the central nervous system. One recent study showed that developing rats exposed to heptachlor exhibited developmental delays and alterations in adult cognitive function (Moser et al., 2001Go). Since heptachlor and other cyclodienes are persistent and bioaccumulate, additional studies are needed to identify potential long-term health effects of these exposures.

A large etiologic study of World War II veteran twins over the age of 65 failed to indicate any genetic component to Parkinson's disease (PD) when the disease begins after age 50 (Tanner et al., 1999Go). These findings suggested that environmental factors may play a role in this disease, and recent epidemiological studies indicate that pesticide exposure is a risk factor for PD (Priyadarshi et al., 2000Go). For example, one study showed a significant increase in mortality from PD in California counties with extensive agricultural pesticide use (Ritz and Yu, 2000Go). In other studies, analysis of postmortem human brain tissue showed a significantly greater concentration of dieldrin, another cyclodiene pesticide, in PD brains than in either control or Alzheimer diseased brains (Corrigan et al., 2000Go; Fleming et al., 1994Go). Thus, there is a growing concern that exposure to pesticides, and particularly to cyclodienes, may play a role in the onset of PD (Priyadarshi et al., 2000Go).

The dopamine transporter (DAT) normally functions to allow rapid uptake of dopamine (DA) and therefore is important in regulating DA actions (Fischman et al., 1998Go; Giros and Caron, 1993Go). DAT binding decreases with age and is additionally decreased due to neuronal loss in PD (Fischman et al., 1998Go; Frost et al., 1993Go; Madras et al., 1998Go; Seibyl et al., 1997Go). The DAT may also play an important role in neuronal development, appearing first at gestational day (GD) 14. In DAT knockout mice, D1 and D2 DA receptors and preproenkephalin A mRNA levels were decreased from GD 14 on, while dynorphin mRNA levels were increased from GD 17 on (Fauchey et al., 2000Go). Thus, changes in DAT number and DA levels may affect neuronal development early on, and may play a role in the etiology of PD later in life.

A direct link between heptachlor and the dopaminergic system has been reported (Kirby et al., 2001Go; Miller et al., 1999bGo). Striatal DA uptake was increased in adult mice given injections of heptachlor (6–12 mg/kg, 3 times over 2 weeks, intraperitoneally), as measured by the rate of [3H]-dopamine uptake as well as Western-blot analyses of striatal synaptosomes using monoclonal DAT antibodies. These data led to the question of whether repeated exposure to lower doses of heptachlor would produce similar effects (1) in rats, and (2) following developmental exposure. Several ongoing behavioral studies allowed access to brain tissues with which to examine these questions. The hypothesis was that developmental exposure to heptachlor would alter DAT binding, and, as a consequence, would affect DA function as evidenced by changes in receptor density. Thus, ligand binding to DA receptors and the DAT were examined at various times during and after exposure.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Pregnant female Sprague-Dawley rats were obtained from Charles River Laboratories, Inc. (Raleigh, NC). All litters were culled (4 males, 4 females) on postnatal day (PND) 7, and weaned on PND 21. Rats were housed on hardwood chips (pregnant dams) or heat-treated pine shavings (postweanling and adult rats). They were given free access to food (Purina Rodent Chow 5001, or 5008 for pregnant dams) and tap water, and maintained at 72 ± 2°F, 50 ± 10% humidity, and a 12-h light cycle.

Four separate groups of animals (summarized in Table 1Go) were dosed with heptachlor by oral gavage (2 ml/kg for preweanling rats, 1 ml/kg for all others). In the perinatal study (Group 1), pregnant dams (10/dose; 0, 4.2, or 8.4 mg/kg/day) were dosed (5 days/week) from mid-gestation through one week after delivery, and thereafter pups were directly dosed until weaning. In Group 2, pregnant dams (9/dose; 0, 4.2, or 8.4 mg/kg/day) were dosed (5 days/week) only during gestation. In a perinatal/adolescent study (Group 3), pregnant dams (10/heptachlor dose [0.3, 3 mg/kg/day], 20 controls) were dosed as in Group 1, except that the pups were dosed until PND 42. To examine effects of early adolescent exposure, half of the control litters (which had been dosed with vehicle until PND 21) were dosed with 3 mg/kg/day on PND 21–42. In another perinatal/adolescent study (Group 4), pregnant dams (5/dose; 0, 0.3, 1, or 3 mg/kg/day) were dosed (7 days/week) until lactational day 7, and all pups were dosed on PND 7–42.


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TABLE 1 Description of Dosing Paradigms for Different Heptachlor Studies
 
Yet another group was dosed (7 days/week) on PND 21–42 with corn oil or dieldrin 5 mg/kg/day (1 ml/kg); this dose was lowered to 3 mg/kg/day after 3 days, due to unexpected toxicity.

Behavioral testing.
All pups in all litters of the first 3 groups were evaluated for the development of the righting reflex. Each pup was placed on its back, and the amount of time required for the pup to right itself (all 4 paws flat on the surface) was measured using a stopwatch, with a maximum of 30 s. This testing took place on PND 2–3 in Groups 1 and 2, and PND 2–5 in Group 3.

Ontogeny of motor activity was measured on PND 13, 17, and 21 in Groups 1 and 2, in 30-min sessions using photocell-based devices shaped like a figure-8 (Reiter et al., 1975Go). For Group 1, testing took place before administering each day's dose.

To evaluate the functional integrity of the dopaminergic system, adult rats from the perinatal study were challenged with a series of d-amphetamine doses (0, 0.3, 1, and 3 mg/kg, ip) and tested during 1-h sessions in the figure-8 activity chambers. Each rat received each dose in a semi-random order; testing took place on Tuesdays and Fridays over 2 weeks.

Chemicals.
Heptachlor (99% purity, Radian Corp., Austin, TX) and dieldrin (98.5% purity, Chem Service, Westchester, PA) were dissolved in corn oil for animal dosing. For in vitro binding studies, heptachlor was dissolved in 95% ethanol and diluted in assay buffer. Solutions were attempted with heptachlor epoxide (99% purity, Radian Corp.) in either 95% ethanol or 100% DMSO, followed by buffer dilution; however, the epoxide was not sufficiently soluble under these conditions. Mazindol, butaclamol, nomifensine, ketanserin, and d-amphetamine were obtained from Sigma Chemical (St. Louis, MO). [3H]Mazindol (specific activity 21.0 Ci/mmol), [3H]SCH-23390 (75.5 Ci/mmol), and [3H]spiperone (16.5 Ci/mmol) were obtained from New England Nuclear (Boston, MA). All other chemicals were obtained from commercial sources and were of the highest available grade.

Binding studies.
To obtain tissues for binding studies, rats were euthanized as follows, on: (1) PND 10, and PND 22 in the perinatal and gestational studies, (2) PND 22, PND 43, and PND 128 in the first perinatal/adolescent study, (3) PND 130 in the second perinatal/adolescent study, and (4) PND 43 in the dieldrin study. The brains were removed and striata were quickly dissected and stored at –80°C until use in binding assays. For all binding assays, n = 4–5/sex/dose, each representing a separate litter. Male and female tissues were assayed on separate days and therefore analyzed separately.

The procedures for DA-receptor binding using [3H]SCH-23390 (D1 receptors) and [3H]spiperone (D2) followed previously published methods (Madras et al., 1988Go; Xu et al., 1991Go). Briefly, striata were homogenized in ice-cold buffer (50 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, and 5 mM MgCl2, pH 7.4), centrifuged twice at 30,000 x g for 10 min, and resuspended in assay buffer (50 mM Tris-HCl buffer plus 40 nM ketanserin to block serotonergic receptors). This homogenate was incubated with a saturating concentration of either [3H]SCH-23390 (4 nM), or [3H]spiperone (2 nM), ± 10 µM butaclamol (to define nonspecific binding).

The procedures for DAT binding using [3H]mazindol followed the methods of Javitch et al. (1984). Striata were homogenized and centrifuged as above in ice-cold assay buffer (50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, pH 7.4). Resuspended membrane homogenates were incubated with 30 nM [3H]mazindol ± unlabeled mazindol (10 µM). In this study, cold mazindol (10 µM) was used to determine nonspecific [3H]mazindol binding, as done in the studies on which we based our procedures (e.g., Javitch et al., 1984Go; Moody et al., 1996Go; Shimizu and Prasad, 1991Go). In other studies in this laboratory, however, the use of another DAT inhibitor (nomifensine 10 µM) revealed the same level of nonspecific binding compared to mazindol (within 1%).

For competition binding assays, membrane homogenates were incubated with 4 nM [3H]mazindol and unlabeled mazindol (0.1 nM to 10 µM, 11 concentrations). In heptachlor binding assays, 30 nM [3H]mazindol was incubated with 0–50 µM (6 concentrations) heptachlor ± 10 µM mazindol. Unfortunately, heptachlor epoxide was not completely soluble when diluted in the assay buffer, and thus the direct effects of heptachlor epoxide could not be determined.

All binding assays were conducted in triplicate. Tissues were incubated for 1 h at 0°C (DAT binding) or 1 h at 37°C (DA receptor binding). Incubation was terminated by the addition of ice-cold assay buffer and rapid filtration through 0.3% polyethylenimine pre-treated glass fiber filters in a cell harvester apparatus (Brandel, U.S.). Following 2 more rinses with ice-cold buffer, remaining radioactivity was measured by liquid scintillation spectrometry at a counting efficiency of 45%. Protein was measured by the bicinchoninic acid (BCA) method (kit, Sigma Chemical, St. Louis, MO).

Data analysis.
Dam and pup weight, proportion of pups righting within 5 s, and motor activity counts were analyzed by a general linear model (GLM; SAS, 1990). For saturation and competition data, KD and Bmax values were determined by nonlinear regression (GraphPad Prism 3.0, San Diego, CA). Binding data were analyzed using unpaired 2-tailed t-tests or 1-way ANOVAs (with more than 2 treatment groups) followed by post hoc group comparisons using Dunnett's t-test. In all cases, resulting probability values < 0.05 were considered significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Developmental and Growth Indices
Heptachlor was maternally toxic in the perinatal and gestational studies, resulting in depression (6–7%) of maternal weight gain at the end of gestation (GD 18–21; dose by gestation day, p = 0.0001). Maternal weight gain was decreased in the perinatal study by both doses (4.2 and 8.4 mg/kg/day), and only by the high dose in the gestational study. In addition, the high dose of 8.4 mg/kg/day reduced pup survival and caused some maternal convulsions. With continued dosing in the perinatal study, the health of the high-dose dams was compromised and the rats were euthanized. In the perinatal/adolescent studies, 3 mg/kg/day and lower had no influence on maternal health. In all groups receiving 4.2 mg/kg/day or less, litter size, survival, and pup growth parameters throughout the study were not affected.

Behavioral Tests
The proportion of rats reaching criteria (righting within 5 s) was lowered across days in female pups dosed with 4.2 mg/kg/day in the gestational but not the perinatal study (data not shown). Figure 1Go shows that righting at 3 mg/kg/day was also slowed in both sexes (dose p = 0.0026 and p = 0.0122 for females and males, respectively) in the Group-3 litters.



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FIG. 1. Righting reflex ontogeny (Group 3, righting < 5 s) from PND 2 to 5 during heptachlor exposure (0, 0.3, or 3.0 mg/kg/day). Data shown are mean proportion of litters reaching criterion for each sex in each dose group on each test day.

 
Motor activity in control rats increased approximately 4-fold from PND 13 to 17, and remained nearly the same (within 10%) on PND 21. There were no treatment-related differences in the pattern (ontogeny) or levels of activity (data not shown).

Adult rats showed an increase in activity (~20%) at 1 mg/kg amphetamine, and the highest dose (3 mg/kg) decreased activity by 50%. Again, there were no differences in the heptachlor-treated rats on this dose response (data not shown).

D1 and D2 Binding
In saturation binding experiments of untreated PND 10 rat striata, the Bmax and KD values for [3H]SCH-23390 were 13.4 fmol/mg protein and 0.59 nM (r2 = 0.978), respectively. The Bmax and KD values for [3H]spiperone were 10.7 fmol/mg protein and 0.68 nM (r2 = 0.859), respectively. In PND 22 rat striata, the Bmax and KD values for [3H]SCH-23390 were 60.5 fmol/mg protein and 0.73 nM (r2 = 0.998), respectively. The Bmax and KD values for [3H]spiperone were 31.9 fmol/mg protein and 0.4 nM (r2 = 0.915), respectively. As presented in Table 2Go, there was no significant difference in D1 and D2 receptor binding in controls compared to heptachlor-exposed striata (Group 1) in either age or sex.


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TABLE 2 [3H]SCH-23390 and [3H]Spiperone Binding to Striatal D1 and D2 Receptors (fmol/mg protein) in Rats Perinatally Exposed to Heptachlor (Group 1)
 
DAT Binding
Saturation [3H]mazindol binding assays were performed in control adult striata. The binding data indicated one binding site with KD = 15.7 nM and Bmax = 2.74 pmol/mg protein (r2 = 0.993). Studies have shown that the KD for mazindol does not vary during development, while the Bmax increases from PND 0 to PND 21, levels off between PND 21 and 28, and declines sometime after PND 145 (Broaddus and Bennett, 1990Go; Shimizu and Prasad, 1991Go; Stadlin et al., 1994Go). A concentration of 30 nM [3H]mazindol, which is about twice the KD and labels about 60–65% of the total binding sites in adult striatum, was used in further studies. Nonspecific binding was approximately 10% of total binding.

Striatal [3H]mazindol binding in gestationally-exposed (Group 2) rats is shown in Figure 2Go. At PND 10, there was a dose-dependent increase in [3H]mazindol binding in male rats only (dose p = 0.0008). Since 8.4 mg/kg/d was a toxic dose of heptachlor, only rats dosed with 4.2 mg/kg/day were retained until PND 22. In both sexes, at PND 22, there was a significant increase in binding (p = 0.03 and p = 0.02 for females and males, respectively).



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FIG. 2. Effect of gestational exposure (GD 10 to 21) to heptachlor 4.2 mg/kg/day on striatal [3H]mazindol binding (pmol/mg protein at 30 nM mazindol) in males and females at PND10 and 22. Data shown are mean ± SEM (n = 4/sex/dose). Numbers above bars indicate percent change from control values. *Significant difference (p < 0.05) from controls.

 
Figure 3Go shows [3H]mazindol binding at PND 22, 43, and 128 in perinatal/adolescent-exposed (Group 3) rats. The data show that binding was increased significantly at PND 22 in both males and females exposed to 3.0 mg/kg/day of heptachlor (females p = 0.006; males p = 0.0015). At PND 43, there was an overall increase in binding in males only, including those exposed only from PND 21 to 42 (p = 0.018). At this age, the apparent increase in females did not attain statistical significance (p = 0.08). In adult rats at PND 128, there was a significant increase in binding in both sexes (females p = 0.005; males p = 0.011). Group comparisons revealed that both dose groups were significantly different from controls in females, but in males, only the group dosed with 3.0 mg/kg/day throughout the dosing period was significantly different from controls.



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FIG. 3. Effect of perinatal (GD 10 to PND 42) and adolescent (PND 21 to PND 42) exposure to heptachlor 3 mg/kg/day on striatal [3H]mazindol binding (pmol/mg protein at 30 nM mazindol) in males and females on PNDs 22, 43, and 128. Numbers above bars indicate percent change from control values. Data shown are mean ± SEM (n = 4/sex/dose). *Significant difference (p < 0.05) from controls.

 
The data for Group 4 (exposed GD 10 through PND 42) males and females on PND 130 are shown in Figure 4Go. There was an overall increase in DAT binding (females p = 0.002; males p = 0.0006). Group comparisons revealed a significant increase at even the lowest dose administered (0.3 mg/kg/day).



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FIG. 4. Effect of perinatal/adolescent (GD 10 to PND 42) exposure to heptachlor (0.3, 1, and 3 mg/kg/day) on striatal [3H]mazindol binding on PND 130. Numbers above bars indicate percent change from control values. Data shown are mean ± SEM (n = 4/sex/dose). *Significant difference (p < 0.05) from controls.

 
In rats dosed with dieldrin during adolescence (3.0 mg/kg/day), DAT binding was increased by 23% in females, and by 20% in males (data not shown).

Competition binding assays between labeled and unlabeled mazindol in control and heptachlor-exposed brains (perinatal/adolescent, PND 43) produced control KI values of 16.4 and 14.9 nM in females and males, respectively. In heptachlor-exposed striata (3 mg/kg/day from GD 10 to PND 42), KI values were 16.7 and 15.2 nM in females and males, respectively. Thus, there was no significant difference in the affinity of mazindol for the DAT in control compared to heptachlor-exposed striata.

To examine any direct effect of heptachlor on DAT binding, adult male untreated striata were incubated with [3H]mazindol and increasing concentrations of heptachlor. Nonspecific binding was not changed by the addition of heptachlor. Heptachlor (10 µM and above) displaced [3H]mazindol binding, reaching at least 50% inhibition at 50 µM (highest concentration tested).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Heptachlor doses of 3 mg/kg/day and less were neither maternally nor developmentally toxic. However, 3 mg/kg/day and higher produced a delay in the righting reflex ontogeny, but only in perinatally dosed rats. This finding is consistent with an earlier study (Moser et al., 2001Go). The ontogeny of motor activity was not altered. A slightly higher dose of 4.2 mg/kg/day decreased maternal weight gain in 1 (Group 1) of 2 studies. The highest dose, 8.4 mg/kg/day, produced maternal and developmental toxicity: data from those pups were only included to show dose response.

DAT binding was increased by all heptachlor treatments, including those that were ineffective on other endpoints. Even the lowest dose of 0.3 mg/kg/d significantly increased DAT binding, i.e., a no-effect level could not be determined. These doses produced neurobehavioral and cognitive deficits in the adult offspring as well as decreased GABAA receptor binding immediately after dosing (Moser et al., 2001Go). In addition, a similar increase in DAT binding was shown following adolescent exposure to another cyclodiene, dieldrin. There is evidence that some pesticides, e.g., triadimefon (Walker and Mailman, 1996Go) and rotenone (Ferrante et al., 1997Go), affect the dopaminergic system by binding to the DAT. The present in vitro results show that heptachlor may be added to this list.

Cyclodienes affect GABAergic neurotransmission (Abalis et al., 1986Go; Cole and Casida, 1986Go; Gant et al., 1987Go; Lawrence and Casida, 1984Go), and are cytotoxic to primary cultures of dopaminergic neurons (Sanchez-Ramos et al., 1998Go). In this study, D1 and D2 DA-receptor binding at PND 10 and 21 was unchanged in rats exposed perinatally to heptachlor (Group 1). DA-receptor binding was also not altered (data not shown) in 1-year-old rats from a previous heptachlor study (Moser et al., 2001Go). DA-receptor expression does not fully reflect potential changes in dopaminergic function, and changes in receptor levels may not necessarily be expected since DAT expression does not correspond to DA levels in young rats (Shimizu and Prasad, 1991Go). As further evidence of a lack of correlation between DAT and DA-receptor binding, the behavioral response to d-amphetamine was not different in heptachlor-treated rats compared to controls, indicating a normal response of the dopaminergic system.

In the present study, striatal DAT binding was increased in both males and females, even in rats exposed only during gestation (Group 2) or only during adolescence (Group 3). Thus, it appears that heptachlor exposure at any time during neuronal development increases DAT binding. In addition, this increase persisted into adulthood, as long as 3 months after the end of dosing. Since there was no difference in the affinity (KD) of mazindol for the DAT in heptachlor-exposed striata, these changes in binding reflect increases in Bmax. It is possible that these changes could persist into old age, and thus would represent a long-term consequence of very early exposure.

One possible mechanism by which heptachlor affects DAT levels is by directly binding to the protein. Developmental exposure to DAT inhibitors, such as cocaine, has been shown to increase DAT binding (e.g., Fang and Rønnekleiv, 1999Go), but results of adult exposures have been less clear (Letchworth et al., 1999Go). However, it seems unlikely that the weak heptachlor effect to decrease DAT binding could alone result in robust increases in DAT expression. This mechanism may also lack physiological relevance since the predominant form of heptachlor in tissues is heptachlor epoxide. Unfortunately, in the present study, the binding assays with the epoxide were inconclusive.

Alternatively, it is possible that heptachlor alters DAT expression through its potent binding to the GABAA receptor. Heptachlor and heptachlor epoxide displace binding of 35S-TBPS (t-butylbicyclophosphorothionate) to the GABAA receptor in rat brain, with IC50s of 400 and 70 nM, respectively (Abalis et al., 1985Go). Since GABA is a trophic factor for the development of the monoaminergic system (Lauder et al., 1998Go), blockade of the GABAA receptor during development could result in alterations in the dopaminergic system, including long-term increases in DAT levels. Arguing against that hypothesis, however, is the finding that adolescent (PND 21–42) exposure also altered DAT binding; at that age neuronal proliferation is essentially complete. On the other hand, blockade of the GABAA receptor in the striatum has been shown to increase DA release in adult rats (Gong et al., 1998Go), which could lead to a compensatory increase in DAT levels.

Many toxic substances may be substrates for the DAT (Miller et al., 1999aGo). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces PD-like effects in human and non-human primates (Burns et al., 1983Go). The active metabolite of MPTP, 1-methyl-4-phenylpyridinium (MPP+), is actively transported into presynaptic nerve terminals through the DAT, and is cytotoxic to dopaminergic cells (Javitch et al., 1985Go; Pifl et al., 1993Go; Santiago et al., 1996Go). MPTP is not neurotoxic in mice lacking the DAT (Bezard et al., 1999Go; Gainetdinov et al., 1997Go), and overexpression of the DAT in mice results in a 50% greater loss of dopaminergic neurons following a course of MPTP (Donovan et al., 1999Go). Consequently, the DAT may be an important site by which some environmental neurotoxicants enter and subsequently damage dopaminergic nerve terminals.

The present finding of increased DAT binding concurs with, and extends, similar results of heptachlor exposure in adult mice (Kirby et al., 2001Go; Miller et al., 1999bGo). Several doses of heptachlor, 3 to 4 times higher than in these studies, were shown to cause upregulation of DAT in adult mice (measured by immunoreactivity), and increased synaptic uptake of dopamine ex vivo, but not in vitro in cells expressing the DAT. An increased susceptibility to MPTP neurotoxicity in heptachlor-treated mice has also been reported (Garcia et al., 2000Go). Therefore, by increasing DAT levels, heptachlor and dieldrin may increase the susceptibility of dopaminergic neurons to pesticides and other neurotoxicants. These findings support the idea that cyclodienes may be involved in a link between pesticide exposure and Parkinson's disease (Corrigan et al., 2000Go; Fleming et al., 1994Go).

In summary, gestational, perinatal, and adolescent exposure to heptachlor and dieldrin increased DAT levels, both during and for months after exposure. This is the first evidence of such prolonged neurochemical alteration of early heptachlor exposure at doses that produce no overt toxicity. In addition, a direct effect of heptachlor on the DAT was demonstrated in vitro, albeit at a high concentration. Such a persistent change may be cause for concern. One hypothesis is that these heptachlor-induced alterations in the dopaminergic system during infancy and childhood could increase the risk of developing Parkinson's disease later in life by increasing the vulnerability of dopaminergic neurons to other neurotoxicants. Further studies in aged rats, which are exposed to cyclodienes early in life, may help to address these questions.


    ACKNOWLEDGMENTS
 
This study has been funded in part by the U.S. Environmental Protection Agency. The authors gratefully acknowledge the excellent technical assistance of Ms. P. M. Phillips. We also thank Dr. S. Padilla for expert advice and use of equipment in her laboratory, and Drs. H. Tilson and T. Shafer for review of an earlier version of this manuscript. Portions of this research were funded by a grant from the Hawaii Heptachlor Research and Education Foundation to NIEHS. S.P.-P. was funded by the EPA/UNC Toxicology Research Program, Training Agreement CT902908, with the Curriculum in Toxicology, University of North Carolina at Chapel Hill.


    NOTES
 
1 To whom correspondence should be addressed at NTD (MD-74B), U.S. EPA, RTP, NC 27711. Fax: (919) 541-4849. E-mail: moser.ginger{at}epa.gov. Back

This paper has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Abalis, I. M., Eldefrawi, M. E., and Eldefrawi, A. T. (1985). High affinity stereospecific binding of cyclodiene insecticides and {gamma}-hexachlorocyclohexane for {gamma}-aminobutyric receptors of rat brain. Pestic. Biochem. Physiol. 24, 95–102.[ISI]

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