Department of Entomology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061
Received September 29, 2000; accepted December 12, 2000
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ABSTRACT |
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Key Words: dopamine; dopamine uptake; dopamine release; respiration; cyclodiene; Parkinsonism.
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INTRODUCTION |
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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., 1993; Wong et al., 1991
), consumption of well water (Jimenez-Jimenez et al., 1992
; Koller et al., 1990
; Rybicki et al., 1993
; Wong et al., 1991
), and exposure to insecticides (Chapman et al., 1991
; Moses et al., 1993
; Semchuk et al., 1992
, 1993
; Siedler et al., 1996
; Tanner and Langston, 1990
). 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., 1999
), 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.
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MATERIAL AND METHODS |
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Mice and treatments.
All studies were performed on C57BL/6 retired breeder male mice (2835 g live weight, 79 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., 1999), where a single 20 mg/kg dose of MPTP was capable of depressing levels of striatal dopamine 4060% 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 TrisHCl, 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 0.033 µM dopamine for 2 min (100 µl final incubation volume). Aliquots of cortical membranes (90 µl) were similarly incubated in concentrations of 0.031 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 TrisHCl (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).
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RESULTS |
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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. 1A). 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. 1
, 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. 1B
).
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DISCUSSION |
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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., 1999). 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 (1820%), whereas 3 µM heptachlor treatment of [3H]5-HTloaded cortical synaptosomes caused no measurable release. Because heptachlor is metabolized to its corresponding and more toxic epoxide (Nakatsugawa and Morelli, 1976
), we tested it in identical release assays. Dose-response studies (Fig. 5
) 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., 1980
) or through neuroexcitation resulting from antagonism of GABAA receptor function (Bloomquist, 1993
), 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 (312 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. 3) 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 50100 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 312 mg/kg heptachlor appear to contradict the reduction in DAT observed in PD postmortems (Antonini et al., 1995; Innis et al., 1993
; Niznik et al., 1991
). 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. 2
). 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., 1991
). 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, 1974
), ring doves (Heinz et al., 1980
), and ducks (Sharma, 1973
) 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.
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ACKNOWLEDGMENTS |
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NOTES |
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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.
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