Neurotoxicity of the Organochlorine Insecticide Heptachlor to Murine Striatal Dopaminergic Pathways

Michael L. Kirby,1, Rebecca L. Barlow and Jeffrey R. Bloomquist,2

Department of Entomology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061

Received September 29, 2000; accepted December 12, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Changes in biochemical status of nerve terminals in the corpus striatum, one of the primary brain regions affected in Parkinson's disease, were studied in groups of C57BL/6 mice treated by ip injection three times over a 2-week period with 3–100 mg/kg heptachlor. On average, the maximal rate of striatal dopamine uptake increased > 2-fold in mice treated at doses of 6 mg/kg heptachlor and 1.7-fold at 12 mg/kg heptachlor. Increases in maximal rate of striatal dopamine uptake were attributed to induction of the dopamine transporter (DAT) and a compensatory response to elevated synaptic levels of dopamine. Significant increase in Vmax of striatal DAT was not observed at doses > 12 mg/kg, which suggested that toxic effects of heptachlor epoxide may be responsible for loss of maximal dopamine uptake observed at higher doses of heptachlor. In support of this conclusion, polarigraphic measurements of basal synaptosomal respiration rates from mice treated with doses of heptachlor > 25 mg/kg indicated marked, dose-dependent depression of basal tissue respiration. At doses of 6 and 12 mg/kg heptachlor, which increased expression of striatal DAT, uptake of 5-hydroxytryptamine into cortical synaptosomes was unaffected. Thus, striatal dopaminergic nerve terminals were found to be differentially sensitive to heptachlor. This reduced sensitivity of serotonergic pathways was mirrored in the greater potency of heptachlor epoxide to cause release of dopamine from preloaded striatal synaptosomes in vitro compared to release of serotonin from cortical membranes. These results suggest that heptachlor, and perhaps other organochlorine insecticides, exert selective effects on striatal dopaminergic neurons and may play a role in the etiology of idiopathic Parkinson's disease.

Key Words: dopamine; dopamine uptake; dopamine release; respiration; cyclodiene; Parkinsonism.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human exposure to organochlorine insecticides has been, and continues to be, an important issue in human health. Although these insecticides are now restricted from use in the United States, the resistance of these compounds to environmental degradation has raised concerns regarding their documented ability for bioaccumulation and potential public health impact (Matsumura, 1985Go). A major incidence of human exposure to organochlorines occurred in 1982, when the Hawaii State Department of Health reported that the entire milk supply of Oahu was contaminated with heptachlor epoxide (Baker et al., 1991Go; Smith, 1982Go), which entered the food chain via "green chop" dairy cattle fodder derived from heptachlor-treated pineapple tops. Concentrations of heptachlor epoxide, the bioactivation product of heptachlor (Nakatsugawa and Morelli, 1976Go), in milk averaged 1.2 µg/g in a U.S. Environmental Protection Agency (U.S. EPA) study, 12 times greater than the intervention threshold set by the U.S. Food and Drug Administration (FDA). Despite public concerns, no statistically significant increase in birth defects or low birth weight children was noted (Baker et al., 1991Go). However, toxicokinetic models of milk-drinking children, assuming an age of 2 years and exposure to milk containing 1.0 µg heptachlor epoxide/g milk fat, indicate that serum concentrations of heptachlor epoxide may have remained above average in the exposed individuals for upwards of 8 or more years (Baker et al., 1991Go). Although the doses to which the residents of Oahu were exposed are below the estimated NOEL (No Observable Effect Level) in chronic feeding studies in dogs (U.S. EPA, 1982Go), the long-term, lifetime effects upon children following the consumption of relatively large doses of heptachlor epoxide over a brief period are entirely unknown.

Numerous epidemiological studies of Parkinson's disease (PD), a human neurodegenerative disease that affects nigrostriatal dopaminergic neurons, have demonstrated a strong association between the incidence of PD and factors that increase the likelihood of exposure to insecticides. These factors include rural living and agricultural work (Rybicki et al., 1993Go; Wong et al., 1991Go), consumption of well water (Jimenez-Jimenez et al., 1992Go; Koller et al., 1990Go; Rybicki et al., 1993Go; Wong et al., 1991Go), and exposure to insecticides (Chapman et al., 1991Go; Moses et al., 1993Go; Semchuk et al., 1992Go, 1993Go; Siedler et al., 1996Go; Tanner and Langston, 1990Go). A recent study containing a cohort of over 19,000 sets of identical twins failed to find any genetic linkage underlying the presence of PD (Tanner et al., 1999Go), which strengthens a role for an environmental factor in the development of this disease.

In support of epidemiological studies of PD based on survey data, Fleming et al. (1994) observed a strong correlation between the incidence of PD and the presence of brain residues of the cyclodiene insecticide dieldrin, which has a chemical structure similar to that of heptachlor. In cases where dieldrin was detectable, tissue samples taken from corpus callosum of postmortem PD patients averaged 6.0 ppb, and those taken from cortex averaged 16.6 ppb. Of the 20 PD cases and 21 control samples examined, tissue samples taken from PD patients were more than twice as likely to contain detectable levels of dieldrin than were controls (p = 0.045). These findings were confirmed by Corrigan et al. (1998), who found significantly higher levels of dieldrin (p = 0.005) in the caudate nucleus of Parkinson's disease patients, compared to controls.

In the present study, mice were treated with heptachlor to determine the existence of any neurochemical effects consistent with the development of PD, or any selective action of heptachlor or heptachlor epoxide upon nigrostriatal neurons. Selective effects of cyclodienes on striatal neurochemistry might indicate a potential future health problem related to development of PD in persons exposed to organochlorines, such as those in Hawaii in 1982.


    MATERIAL AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Analytical grade heptachlor and heptachlor epoxide were obtained from Chem Serv, Inc. (West Chester, PA). Pargyline was a gift from Neal Castagnoli, Jr. (Dept. of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA). Bovine serum albumin (fraction V), Coomassie Brilliant Blue G250, phosphoric acid, and methoxytriglycol were purchased from Sigma Chemical Co. (St. Louis, MO). [3H]Dopamine (20.3 Ci/mmol) and [3H]5-hydroxytryptamine (5-HT, 17.3 Ci/mmol) were purchased from New England Nuclear (E.I. DuPont et Nemours, Wilmington, DE).

Mice and treatments.
All studies were performed on C57BL/6 retired breeder male mice (28–35 g live weight, 7–9 months of age), obtained from either Harlan Sprague Dawley, Indianapolis, IN, or Harlan Sprague Dawley, Dublin, VA. C57BL/6 mice were assigned randomly to treatment groups having at least five mice each. Mice were arranged in weight categories that were evenly distributed among treatment groups to ensure no significant differences in mean weight and kurtosis. C57BL/6 mice were treated by ip injection either with vehicle or with vehicle containing toxicant. A treatment paradigm of three doses over a 2-week period was used based on previous studies (Bloomquist et al., 1999Go), where a single 20 mg/kg dose of MPTP was capable of depressing levels of striatal dopamine 40–60% at 2 weeks posttreatment. Animals were injected three times with 10 µl methoxytriglycol alone or methoxytriglycol containing heptachlor. Doses of heptachlor ranged from 3 to 100 mg/kg.

Striatal synaptosome respiration.
Respiration assays were modified from the method of Gleitz et al. (1993), and both tissue preparation and respiration measurements were performed using a blind procedure, to ensure no observer bias in dissection or analysis. Treatment groups were subsampled by removing striata from single (100 mg/kg group) or pairs (all other groups) of C57BL/6 mice treated with heptachlor. Striata were dissected into physiological sucrose (pH 7.4) and homogenized in a motor-driven Potter-Elvenheijm homogenizer. Following a 1000 x g (15 min, 4°C) centrifugation, supernatants were recentrifuged at 10,000 x g (15 min, 4°C). Pellets were resuspended at two brain equivalents of striatal tissue per milliliter fresh Krebs-Henseleit buffer (140 mM NaCl, 5 mM KCl, 1.3 mM MgSO4, 5 mM NaHCO3, 1 mM NaHPO4, 10 mM HEPES, 1.2 mM CaCl2, 10 mM glucose; pH 7.4). Membranes were then dispensed into a stirred, temperature-controlled chamber and allowed to equilibrate with air for 5 min at 37°C. A 5-min initial estimation of the respiration rate of membranes prepared from each treatment group was recorded with a Clark-type polarigraphic electrode in a sealed system (YSI Inc., Yellow Springs, OH) using a MacLab® chart recording unit (sampling rate; 4 samples/s). Slopes corrected for buffer-dependent electrode voltage drift were analyzed from raw data by linear regression using the least squares method. Electrode voltage was transformed to oxygen consumption rates by an estimated maximal buffer oxygen saturation value of 5.02 µl O2/ml buffer at 37°C (data provided by YSI Inc., Yellow Springs, OH). Data were expressed by striatal equivalent and a per-milligram-protein basis, as different amounts of tissue were used in the samples. Mean rates of oxygen consumption were compared by one-way ANOVA, followed by Student-Newman-Keuls means separation test, if a statistically significant main effect of treatment was observed (p < 0.05). Membrane protein was measured by the method of Bradford (1976) using a bovine serum albumin standard.

Neurotransmitter uptake.
For measurements of the kinetic properties of striatal dopamine or cortical 5-HT transport, striatal dissections of C57BL/6 mice were pooled by treatment group and homogenized in physiological sucrose (0.32 M sucrose, 4.2 mM HEPES; pH 7.4). In studies of 5-HT uptake kinetics, cortical dissections of C57BL/6 mice were similarly prepared, pooled by treatment group, and homogenized in physiological sucrose. Uptake of 5-HT was performed on animals treated with 6 mg/kg or 12 mg/kg heptachlor as a means of comparing the response of serotonergic terminals with dopaminergic nigrostriatal terminals. Homogenates were centrifuged as described above to obtain synaptosomes. The resulting pellets were washed once with Na+-free incubation buffer (pH 7.4; 0.02% L-ascorbic acid, 50 µM pargyline, 50 mM Tris–HCl, 125 mM Choline-Cl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM sucrose) and resuspended at 300 µl/striatal equivalent of synaptosomes (defined as a brain equivalent of striatal tissue) in either 125 mM NaCl-containing medium or medium with equimolar choline-Cl substitution. Aliquots of striatal membranes (90 µl) were warmed for 1 min at 37°C, then incubated with 0.03–3 µM dopamine for 2 min (100 µl final incubation volume). Aliquots of cortical membranes (90 µl) were similarly incubated in concentrations of 0.03–1 µM 5-HT. For dopamine concentrations greater than 30 nM, 50 nM [3H]dopamine (1:1, acetic acid:methanol) was used as a tracer with unlabeled dopamine to maintain the final solvent concentration at or below 0.1%. The commercial source of [3H]5-HT was dissolved in aqueous buffer, so no solvent limitation of tritiated label existed in these assays. Incubations were stopped by dilution with 3 ml ice-cold wash buffer [50 mM Tris–HCl (pH 7.4), 125 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM sucrose], and vacuum filtered through glass microfiber filters (Whatman GF/B). The filters were then washed three times with 3 ml ice-cold wash buffer. Filters were air dried and placed in scintillation vials with cocktail (Scintiverse E, Fisher Scientific, Raleigh, NC), and uptake was determined by liquid scintillation spectrometry. Membrane protein was measured by the method of Bradford (1976) using a bovine serum albumin standard. Uptake rates were determined in triplicate incubations with and without Na+ (equimolar choline-Cl substitution) in order to correct for low affinity transport by the method of Krueger (1990). Uptake parameters for each treatment group were determined by nonlinear regression to isotherm plots, with statistical comparisons by one-way ANOVA (Prism 2.0, GraphPad Software, San Diego, CA). The dopamine assay was then repeated on additional groups of mice given the same doses of heptachlor. Statistical comparisons of average Vmax and Km values were compared using one-way ANOVA, followed by Student-Newman-Keuls means separation test, if a statistically significant main effect of treatment was observed (p < 0.05).

Neurotransmitter release.
Release studies were performed using a modification of the above preparatory procedure of synaptosomes from untreated C57 mice. The 10,000 x g pellets were pooled and resuspended in incubation buffer containing either 200 nM [3H]dopamine (striatal synaptosomes) or 115 nM [3H]5-HT (cortical synaptosomes). Membranes were incubated for 5 min at 37°C, then centrifuged at 10000 x g for 10 min. Labeled pellets were resuspended in incubation buffer and incubated with heptachlor epoxide dissolved in DMSO (0.1% solvent) for 10 min at 37°C. Dilution, filtration, washing, and scintillation counting of incubates were the same as described above for uptake experiments, except that the wash buffer was at 37°C. For dose-response studies, data points were replicated three times in synaptosomes prepared on different days and were analyzed by four-parameter nonlinear regression, with EC50 (effective concentration giving 50% release) and maximal release determinations compared by T- test (Prism 2.0, GraphPad Software, San Diego, CA).

Striatal neurotransmitter content.
Striatal dissections from each C57BL/6 mouse were prepared according to the method of Hall et al. (1992). Striata were homogenized in 5% TCA with 10 ng 3,4-dihydroxybenzylamine/mg wet weight of striatal tissue as an internal standard and stored at –70°C until analyzed. Samples were thawed and centrifuged to pellet membranes, and the supernatant was analyzed for dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) content by HPLC through a C18 column. Separated fractions were analyzed by electrochemical detection and compared against a 3,4-dihydroxybenzylamine standard. Comparison of mean picomoles per milligram wet striatal weight for dopamine and DOPAC concentrations by treatment group was performed by one-way ANOVA and Student-Newman-Keuls means separation test, if a statistically significant main effect of treatment was observed (p < 0.05).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
No outward signs of intoxication or death were observed in mice given <= 25 mg/kg heptachlor. However, some mice treated at doses of 50 mg/kg or 100 mg/kg heptachlor showed hyperexcitability and became convulsive, which resulted in the death of some individuals. This amounted to 4% mortality at 50 mg/kg and 30% mortality at 100 mg/kg, although the mortality was not consistent in all the trials, which were performed on different cohorts of mice.

Basal respiration rates of striatal tissues from heptachlor-treated C57BL/6 mice in the high-dose regime were significantly diminished in a dose-dependent manner when expressed by nmol O2/min/striatal equivalent (Fig. 1AGo). Treatments of 25 mg/kg and 50 mg/kg heptachlor resulted in 27% and 37% decreases in striatal respiration rates compared with controls, whereas treatment with 100 mg/kg heptachlor caused a 92% decrease in respiration. A significant and concordant decrease in total striatal protein was also measured (Fig. 1Go, inset). When respiration was expressed by nmol O2/min/mg protein, respiration rates of tissues from mice treated with 25 or 50 mg/kg heptachlor were not significantly different from striatal tissue respiration rates of control animals, whereas tissue respiration rates of mice treated with 100 mg/kg heptachlor were diminished 50% compared to controls (Fig. 1BGo).



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FIG. 1. Basal striatal synaptosome respiration rates in heptachlor-treated C57BL/6 mice. Bars represent the mean rates (± SE) of three striatal homogenates from each treatment group. Rates are presented as oxygen consumed per striatal equivalent [(A) ANOVA for main treatment effect: p < 0.0001] and per milligram protein [(B) ANOVA for main treatment effect: p = 0.0195]. Figure inset represents synaptosomal protein concentrations measured for each heptachlor treatment group (ANOVA for main treatment effect: p = 0.0002). In all cases, letters above bars are the result of a Student-Newman-Keuls means separation test. Bars with different letters are significantly different (p < 0.05).

 
Rates of apparent Vmax for striatal dopamine uptake in heptachlor-treated C57BL/6 mice were increased in a treatment-dependent manner. Figure 2Go is a representative dopamine uptake kinetics plot from one experiment on three treatment groups. Dopamine uptake data showed the expected Michaelis-Menten type kinetics and was fit to isotherms typically having r2 values > 0.9. In this particular experiment, 6 mg/kg heptachlor gave a larger increase in maximal transport than 12 mg/kg, and both were significantly elevated compared to controls. A slightly higher Km in treated mice was also observed (Fig. 2Go). More extensive dose-response studies (Figs. 3A and 3BGo) found that increases in Vmax had a threshold dose of 3 mg/kg, were maximally enhanced at 6 mg/kg, and still significantly above control levels at 12 mg/kg heptachlor (Fig. 3AGo). Estimated induction maxima for Vmax of striatal dopamine transporter (DAT) was 216.8% (216.2–217.4%) of control (mean, with 95% confidence interval). Changes in apparent Km did not match the increases in Vmax in a dose-dependent way and displayed a variable response to heptachlor treatment (Fig. 3BGo). Treatments of 3 and 50 mg/kg heptachlor produced 45% increases in apparent Km of dopamine transport, but effects at other doses were not statistically significant.



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FIG. 2. A representative dopamine uptake kinetics plot from one experiment in heptachlor-treated C57BL/6 mice. Data points represent means of three incubations from pooled homogenates. Absence of bars indicates that the standard error resides within the size of the symbol. Data were analyzed by nonlinear regression and results are summarized in the inset table. ANOVA for main treatment effects were Vmax, p < 0.0001; Km, p = 0.0287. In the table, letters beside kinetic values (95% confidence limits) are the results of a Student-Newman-Keuls means separation test, and values labeled by a different letter are significantly different (p < 0.05).

 


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FIG. 3. Dose-dependent changes in dopamine uptake kinetics in heptachlor-treated C57BL/6 mice. Bars are normalized to matched controls set equal to 100 and represent means (± SE) of two experiments, on groups containing 5–7 mice each, performed on different days. (A) Vmax (pmol/min/mg protein), presented as percent of control. (B) Km (nM), presented as percent of control. Asterisks above bars are the results of a t-test using the raw data and represent means that are significantly different from controls (p < 0.05).

 
Changes in cortical serotonin uptake were measured in an experiment using 6 mg/kg and 12 mg/kg heptachlor-treated C57BL/6 mice (Fig. 4Go), the doses that in previous experiments produced the greatest increases in Vmax for dopamine uptake. Measurement of Vmax found no significant change from control in mice treated with 6 mg/kg heptachlor. There was a 20.8% reduction in calculated maximal rates of 5-HT uptake for 12 mg/kg heptachlor-treated C57BL/6 mice, but this reduction was essentially attributable to a decrease in uptake at 1 µM 5-HT, as the other points on the curve (Fig. 4Go) overlapped closely with those of the other treatment groups. No significant change in Km was measured for 5-HT uptake in either group of heptachlor-treated mice.



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FIG. 4. Uptake kinetics of 5-HT in heptachlor-treated C57BL/6 mice. Data points represent means of three incubations from pooled homogenates. Absence of bars indicates that the standard error resides within the size of the symbol. Kinetic parameters were estimated by nonlinear regression. The table summarizes ANOVA of kinetic values (main treatment effects: Vmax, p = 0.004; Km, nonsignificant). Adjacent letters are the results of a Student-Newman-Keuls means separation test, where values (with 95% confidence limits) labeled by a different letter are significantly different (p < 0.05).

 
In order to compare the effects of heptachlor epoxide on dopaminergic and serotonergic nerve terminals, in vitro transmitter release assays were performed (Fig. 5Go). The EC50 for dopamine release by heptachlor epoxide was 2.6 µM (1.0–6.4, 95% C.L.) and for 5-HT release was 15.3 µM (15.0–15.5). This difference in potency was about 6-fold and was statistically significant (p < 0.02, T-test).



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FIG. 5. Release of preloaded dopamine and serotonin from striatal and cortical synaptosomes, respectively prepared from untreated C57 mice. Symbols represent means with bars signifying the SE. Kinetic parameters of potency and maximal release are given in the text.

 
Measurements of striatal dopamine and DOPAC concentrations did not indicate any statistically significant reduction of these compounds following heptachlor treatment when given at 25 mg/kg (data not shown).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Changes in the capacity of striatal dopamine transport is a sensitive and specific indicator of striatal dopaminergic alterations resulting from heptachlor treatment. Dopamine transport in C57BL/6 retired breeder mice was enhanced as much as 2-fold at 6 mg/kg heptachlor, which is about 4% of the reported LD50 in albino mice (145 mg/kg by ip injection; Cole and Casida, 1986). Recently published collaborative studies (Miller et al., 1999Go) in C57BL/6 mice treated with heptachlor demonstrated that the increase in Vmax for striatal dopamine uptake measured here was the result of a concordant increase in dopamine transporter expression as determined by DAT antibody-labeled Western blots. Another insecticide that produces a documented upregulation of dopamine transport is the pyrethroid deltamethrin, which increases uptake by 70% at 6 mg/kg using the same treatment paradigm we used here for heptachlor (Kirby et al., 1999Go). The effect of these insecticides is greater than that of cocaine, which causes a 50% upregulation of dopamine transport in mice treated intermittently for 5 days by ip injection (Miller et al., 1993Go). Thus, our data suggest that dopamine transporter uptake kinetics or expression is a sensitive index of subclinical toxicant insult and should be investigated further as a biomarker of environmental toxicant exposure, since expression of DAT was affected at low doses of toxicant.

Whereas increases in dopamine uptake were measured in striatum, pronounced changes in maximal rates of 5-hydroxytryptamine (5-HT) uptake, another biogenic amine, were not detected in the cortex. The lack of change in 5-HT uptake suggests less effect on cortical serotonergic pathways, as compounds that either block uptake (fluoxetine; Hrdina and Vu, 1993) or inhibit synthesis of 5-HT (p-chlorphenylalanine; Rattray et al., 1996) have been shown to affect the expression of 5-HT transporter in rat cortex.

We assume that changes in dopamine transporter expression were compensatory for heptachlor-dependent increases in dopamine release, in vivo. We have recently shown that heptachlor releases a variety of neurotransmitters from preloaded synaptosomes (Bloomquist et al., 1999Go). In screening studies, 3 µM heptachlor caused a complete release of preloaded [3H]dopamine (96% release) from striatal synaptosomes. Similar treatments of [3H]glutamate- and [3H]GABA-loaded striatal synaptosomes produced only marginal release of transmitter (18–20%), whereas 3 µM heptachlor treatment of [3H]5-HT–loaded cortical synaptosomes caused no measurable release. Because heptachlor is metabolized to its corresponding and more toxic epoxide (Nakatsugawa and Morelli, 1976Go), we tested it in identical release assays. Dose-response studies (Fig. 5Go) confirmed that striatal dopaminergic terminals were about 6-fold more sensitive to heptachlor epoxide than cortical serotonergic terminals. Augmentation of dopamine release by cyclodienes in vivo could occur directly from altered calcium ion regulation (Yamaguchi et al., 1980Go) or through neuroexcitation resulting from antagonism of GABAA receptor function (Bloomquist, 1993Go), although this latter mechanism is unlikely to underlie the release we observed in vitro.

Observed changes in neurotransmitter uptake kinetics at lower doses appear to forecast more pronounced and severe alterations in cellular function that occur at higher doses of heptachlor. Pronounced depression of striatal tissue respiration occurred at doses of heptachlor at least twice as high as those used to produce induction of the dopamine transporter. Moreover, inhibition of respiration was measured at doses that reversed the increase in Vmax for dopamine transport measured at lower doses of heptachlor (3–12 mg/kg), thus suggesting that diminished transporter function may relate to cell damage. Cell damage is also suggested in the reduced amount of synaptosomal protein that could be isolated as the dose of heptachlor was increased. Therefore, disturbances in dopamine uptake are apparently more sensitive biomarkers of neurochemical imbalance than measurements of respiratory efficiency, although the latter is probably better correlated with cytotoxicity. In a study by Sanchez-Ramos (1998) on cultured mesencephalic neurons, dieldrin caused cytotoxicity in dopaminergic neurons more so than GABAergic neurons, albeit at relatively high concentrations: the EC50 for killing TH-positive cells was 12 µM. It is also possible, however, that the reduced dopamine uptake observed at heptachlor doses > 12 mg/kg (Fig. 3Go) represents the release effect of heptachlor epoxide, reflected in a limited ability of the terminals to effectively sequester the dopamine once it entered the terminals via the DAT.

Cyclodienes at concentrations of 50–100 µM are reported to inhibit state 3 (ADP-stimulated) respiration rates of free liver mitochondria in the presence of succinate as an energy substrate, whereas respiration rates are relatively unaffected by cyclodienes when using downstream salvage pathway substrates for the electron transport chain (heptachlor: Ogata et al., 1989; Meguro et al., 1990; endosulfan: Dubey et al., 1984; Mishra, 1994). Although depressed basal tissue respiration rates in cyclodiene-treated animals were observed here, experiments conducted in our laboratory with fresh striatal synaptosomes from untreated mice failed to produce significant respiratory inhibition at concentrations of heptachlor up to 100 µM (data not shown). Thus, we conclude that the decreased respiration observed in synaptosomes was probably a secondary effect, and that the high concentrations of heptachlor epoxide required to inhibit respiration in vitro make it unlikely to be a significant contributor to neurotoxicity in vivo.

The marked sensitivity of dopaminergic terminals in the striatum to toxicant-evoked release of neurotransmitter, as well as the specific upregulation of dopamine, but not 5-HT transport, supports an association between cyclodiene exposure, effects on striatal dopamine neurochemistry, and environmental parkinsonism. Such an association was originally suggested by the postmortem pathology studies of Fleming et al. (1994) and Corrigan et al. (1998). However, we do not know whether the neurochemical effects observed are permanent or are temporary changes resulting from the last heptachlor treatment. Moreover, several inconsistencies remain that may be related to the incipient nature of heptachlor effects on the striatum in the short duration, 2-week treatment regime that we used. Increased dopamine uptake kinetics in mice treated with 3–12 mg/kg heptachlor appear to contradict the reduction in DAT observed in PD postmortems (Antonini et al., 1995Go; Innis et al., 1993Go; Niznik et al., 1991Go). PD is also characterized by a loss of striatal dopamine, which was not observed when heptachlor was given at a dose of 25 mg/kg, even though this dose reduced respiration and synaptosomal protein content (Fig. 2Go). It has been shown in aged mice that 68% of the dopaminergic neurons are naturally lost and that there is a 103% increase in dopamine synthesis in the remaining cells to counteract this effect (Tatton et al., 1991Go). Such plasticity may explain the lack of significant reduction in dopamine levels following heptachlor treatment. Thus, longer exposures to heptachlor may be required to produce depletion of dopamine, bradykinesia, and the reduction in DAT observed in PD. Depletion of brain dopamine in rats (Wagner and Greene, 1974Go), ring doves (Heinz et al., 1980Go), and ducks (Sharma, 1973Go) have been observed in longer duration feeding studies with the related compound dieldrin, and additional work will be required to define exposure conditions that produce similar results with heptachlor.


    ACKNOWLEDGMENTS
 
We thank Kay Castagnoli for assistance with the HPLC analysis. This work was supported by the Hawaii Heptachlor Research and Education Foundation, grant #HHHERP 94–01 and USDA Hatch project #6122040 to J.R.B.


    NOTES
 
1 Current address: The Parkinson's and Movement Disorder Institute, Long Beach Memorial Medical Center, Long Beach, CA 90806. Back

2 To whom correspondence should be addressed at Neurotoxicology Laboratory, Dept. of Entomology, 216 Price Hall, Blacksburg, VA 24061-0319. Fax: (540) 231-9131. E-mail: jbquist{at}vt.edu. Back


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
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