Increased synaptosomal dopamine content and brain concentration of paraquat produced by selective dithiocarbamates

Authors


Address correspondence and reprint requests to Eric K. Richfield, MD, PhD, Department of Pathology and Laboratory Medicine, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA. E-mail: Eric_Richfield@urmc.rochester.edu

Abstract

Exposure to pesticides may be a risk factor for Parkinson's disease based on epidemiologic data in humans, animal models and in vitro studies. Different dithiocarbamate pesticides potentiate the toxicity of both 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and paraquat in mouse models of Parkinsonism by an unknown mechanism. This study examined the effects of commercially used dithiocarbamates on [3H]dopamine transport in striatal synaptosomal vesicles and on the concentration of [14C]paraquat in vivo in mice. Different ethylenebis-dithiocarbamates and diethyl-dithiocarbamate increased dopamine accumulation in synaptosomes, whereas dimethyl-dithiocarbamate and methyl-dithiocarbamate did not. Increased dopamine accumulation in synaptosomes was dose dependent and was related to the carbon backbone of these molecules. The dithiocarbamates that increased accumulation of dopamine did not alter the influx of dopamine, but rather delayed the efflux out of synaptosomes. These same dithiocarbamates also increased the tissue content of [14C]paraquat in vivo by a mechanism that appeared to be distinct from the dopamine transporter. There was a consistent relationship between the dithiocarbamates that increased synaptosomal accumulation of dopamine and tissue content of paraquat, with those previously demonstrated to enhance paraquat toxicity in vivo. These results suggest that selective dithiocarbamates may alter the kinetics of different endogenous and exogenous compounds to enhance their neurotoxicity.

Abbreviations used
AUC

area under the curve

DA

dopamine

DAT

dopamine transporter

DTC

dithiocarbamate

MPP+

1-methyl-4-phenylpyridium ion

MPTP

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

PAT

polyamine transporter

PDP

Parkinson's disease phenotype

PQ

paraquat

SNpc

substantia nigra pars compacta

Recent data suggest that environmental factors play a more prominent role than genetic factors in the etiology of the Parkinson's disease phenotype (PDP) (Tanner et al. 1999; de la Fuente-Fernández and Calne 2002). Exposure to pesticides (herbicides, insecticides and fungicides) may be one environmental risk factor for the PDP as suggested by epidemiological data in humans (Semchuk et al. 1991, 1992; Tanner 1989; Poirier et al. 1991; Meco et al. 1994), animal models (Brooks et al. 1999; Thiruchelvam et al. 2000a, 2000b; Di Monte et al. 2001) and in vitro biochemical studies (Yang and Sun 1998; Woolley et al. 1989). The dithiocarbamates (DTCs) are a class of pesticides that have been used extensively worldwide for the past 40–50 years. Estimates of use range from 25 to 35 000 metric tons worldwide as of 1988 (International Programme on Chemical Safety 2001). A variety of neurotoxicant effects related to DTCs have been documented including Parkinsonism, although the mechanism(s) of neurotoxicity remain unclear (Meco et al. 1994; Maroni et al. 2000). Originally studied as an inhibitor of the antioxidant enzyme superoxide dismutase, diethyl-DTC was shown to potentiate the effect of the human PDP-inducing neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice (Corsini et al. 1985; Irwin et al. 1987; Takahashi et al. 1989; Yurek et al. 1989; Miller et al. 1991; Bachurin et al. 1996; Vaccari et al. 1996; Walters et al. 1999; McGrew et al. 2000). Although the mechanism for potentiation of MPTP toxicity of substantia nigra pars compacta neurons (SNpc) by DTCs has not been clearly defined, it must relate to basic concepts of chemical mixture toxicology (Cassee et al. 1998). As originally defined by Bliss (1939) these include: (1) simple similar action in which the different agents contribute to toxicity in proportion to their dose acting via a similar mechanism of toxicity; (2) simple dissimilar action in which the agents do not affect each others' toxic effects and the mechanism and site of toxicity differ between the agents; and (3) interactions result if agents modify the magnitude or nature of the toxic effect of each other. These interactions may relate to toxicokinetics (uptake, distribution, metabolism or excretion) or toxicodynamics (effect on specific organ or protein target). Understanding the mechanism of action of DTCs in neurotoxicant mouse models of the PDP will be important for determining whether DTCs play a similar role in humans and what the mechanism of potentiation might be.

Paraquat (PQ) (N,N′-dimethyl-4,4′-bipyridylium), another commonly used pesticide (Wesseling et al. 2001), has often been implicated as a human risk factor for the PDP based on epidemiologic studies (Sanchez-Ramos et al. 1987; Hertzman et al. 1990; Liou et al. 1997) and because of its structural similarity to MPTP (Woolley et al. 1989; Snyder and D'Amato 1985). Results of animal studies examining the effect of PQ on the nigrostriatal system have been inconsistent, with several studies failing to show a neurotoxic effect (Perry et al. 1986; Endo et al. 1988; Woolley et al. 1989; Widdowson et al. 1996). However, recent data on the effects of PQ alone or in combination with DTCs, using alternative outcome measures at different ages, have been more supportive of a potential role for PQ in the PDP in mice (Di Monte et al. 2001; Thiruchelvam et al. 2000a, 2000b, 2002, submitted). Furthermore, because DTCs and PQ are used in geographically overlapping areas, and because these agrichemicals may persist in the environment and as food residues, it is important to understand how exposure to combinations of these chemicals may contribute to the development of the PDP in humans.

The mechanism by which DTCs enhance the toxicity of both MPTP and PQ in mice is not known. One protein that may link the effects of MPTP and PQ is the dopamine (DA) transporter (DAT). The DAT is considered to be the primary route of access to nigrostriatal terminals for both 1-methyl-4-phenylpyridium ion (MPP+), the active metabolite of MPTP, and PQ based on their structural similarity, although uptake of PQ through the DAT has not been demonstrated. Another known route for brain uptake of PQ is via a polyamine transporter(s) (PAT) which has been demonstrated in lung and brain (Smith et al. 1982) to transport different polyamines including PQ into cells in an energy-dependent and saturable manner (Rose et al. 1976; Smith and Wyatt 1981; Gilad and Gilad 1991). The present study sought to determine whether DTCs had an effect on the DAT as a common link between these two mouse PDP-inducing neurotoxicants.

Experimental procedures

Materials

[3H]DA (specific activity 28–31.6 Ci/mmol) was purchased from NEN Life Science Products, Inc (Boston, MA, USA) and [methyl-14C]PQ dichloride (specific activity 110 mCi/mmol) was purchased from Amersham-Pharmacia Biotech (Arlington Heights, IL, USA) or received as a gift from Syngenta (Macclesfield, Cheshire, UK). Cocaine, amphetamine, DA, pargyline, diethyl-DTC, PQ, and all cations and buffers were obtained from Sigma (St Louis, MO, USA). Nomifensine was obtained from Hoechst-Roussel Pharmaceuticals Inc. (Somerville, NJ, USA). The pesticides Mn2+-ethylenebis-DTC (maneb), Na+-ethylenebis-DTC (nabam), Zn2+-ethylenebis-DTC (zineb), Zn2+-dimethyl-DTC (ziram), methyl-DTC (vapam) and triadimefon were obtained from Chem Service (West Chester, PA, USA).

Tissue procurement and processing

Mice were acquired and cared for in accordance with guidelines published in the National Institutes of Health Guide for Care and Use of Laboratory Animals. Protocols for use of mice were approved by the University of Rochester's Animal Care and Use Committee Guidelines. Adult C57Bl/6J mice were killed by cervical dislocation followed by harvesting of the brain. The dorsal striatum was rapidly dissected on ice, weighed and immediately homogenized as described previously (Richfield 1992). Briefly, tissue was homogenized in 20 volumes of ice-cold 0.32 m sucrose, 50 mm Tris–HEPES (pH 7.4), using a Teflon glass homogenizer (clearance, 0.15 mm), and using 14 up-and-down rotating strokes by hand. The homogenate was then centrifuged at 900 g for 10 min The supernatant was then centrifuged at 17 000 g for 20 min. The pellet was resuspended in 20 volumes of ice-cold incubation buffer and mixed by vortexing. The incubation buffer consisted of 10 mm Tris–HEPES, pH 7.4 (145 mm NaCl, 5 mm KCl, 1 mm MgCl2, 1 mm CaCl2, 10 mm glucose, 0.25 mm ascorbate and 15 µm pargyline) except as noted for ionic studies on the efflux of [3H]DA.

DA uptake and efflux

[3H]DA was used at a concentration of 10 nm for all experiments. Other drugs and ions were prepared in incubation buffer (except as noted below) and kept on ice before addition to the reaction mixture. The reaction mixture was kept on ice until a 5-min prewarming step at 37°C followed by incubation at 37°C. Uptake studies were initiated by the addition of the tissue. Uptake was terminated by the addition of 3 mL ice-cold normal saline and tubes were placed on ice until filtration (except for efflux studies).

For efflux studies, synaptosomes were prefilled with [3H]DA for 12–14 min and then diluted 10-fold by the addition of incubation buffer containing no [3H]DA. All efflux studies were conducted at 37° except one experiment conducted at 4°C. In some studies, the efflux buffer also contained additional compounds (DA, norepinephrine, amphetamine, cocaine or nomifensine) or an altered ionic condition (no Na+). When Na+ was removed from the efflux buffer, it was replaced by an equimolar concentration of choline. After variable efflux times, the tubes were filtered. Some efflux studies were done in the absence or presence of either Mn2+-ethylenebis-DTC or cocaine in the buffer. In these studies, synaptosomes were prefilled in the absence or presence of a compound and then placed in warm incubation either with or without the same compound. After variable efflux times, the tubes were filtered. In saturation experiments, the Km and Vmax for DA were calculated after incubating with a range of concentrations of unlabeled DA (1–100 nm) in the presence or absence of a single concentration of Mn2+-ethylenebis-DTC (0.5 µm) for two durations of influx.

The synaptosomes were rapidly filtered (Whatman GF/C filter paper) using a 48 probe harvester (Brandel Inc., Gaithersburg, MD, USA) and washed twice with 3 mL cold saline. Filters were counted using a liquid scintillation counter and 5 mL Ecoscint H (National Diagnostics, Atlanta, GA, USA) cocktail. Counting efficiency was corrected for each sample. Total uptake occurring at 37°C was corrected by subtracting uptake occurring at 4°C; the temperature-specific uptake always exceeded 90%. This correction for non-specific uptake was similar if a saturating dose of a DA uptake blocker was used instead of a 4°C blank. Uptake was proportional to tissue concentration (data not shown) and was time dependent. Protein was measured using the Bio-Rad protein assay (Bio-Rad, Richmond, CA, USA) or the NanoOrange protein assay (Molecular Probes, Eugene, OR, USA).

Eight different agrichemicals (Fig. 1a), two cations (Mn2+ and Zn2+) and cocaine (a known DAT blocker) were used at final concentrations ranging from 0.05 to 500 µm, depending on the agent. Triadimefon (a known DAT blocker) was dissolved in ethanol (10%) then diluted in incubation buffer.

Figure 1.

(a) Structure of selected DTCs. The structure of the four major classes of DTCs is illustrated. Molecules are color-coded by structural class and correspond to results in (b). The DTC backbone and complexed cations are shown. (b) Effect of agrichemicals, cations and cocaine (all at 0.5 µm) on DA uptake. Values represent mean ± SEM percentage of DA uptake in the presence of added compounds compared with DA uptake in the absence of these compounds (n = 3–4 independent experiments). Values do not reflect the maximal effect or the lowest effective concentration of the different compounds, but demonstrate the relative effect of each compound at the same concentration (0.5 µm). All compounds used were tested at both higher and lower concentrations to generate a dose–response curve for each agent (shown in Fig. 2 for two compounds). Concentrations tested were 0.05–10 µm (cocaine), 0.1–50 µm (PQ, Zn2+-dimethyl-DTC, zinc, manganese, triadimefon, Zn2+-ethylenebis-DTC), 0.1–100 µm (Mn2+-ethylenebis-DTC, Na+-ethylenebis-DTC) or 0.1–500 µm (diethyl-DTC). Some agents at higher concentrations reduced DA accumulation, but no other agent increased accumulation. *p < 0.05 versus control. Mn2+-ethylenebis-DTC, Na+-ethylenebis-DTC and diethyl-DTC all significantly increased DA synaptosomal content. Zinc, triadimefon and cocaine all significantly decreased DA synaptosomal content. Manganese, Zn2+-ethylenebis-DTC, Zn2+-dimethyl-DTC, methyl-DTC and PQ showed no significant effect. The control uptake was 23.72 ± 2.2 pmol per mg protein.

In vivo PQ measurements

Two sets of experiments were done. First, a time course comparing the uptake of [methyl-14C]PQ alone or with Mn2+-ethylenebis-DTC at different time points was analyzed. Subsequently, the effect of different DTCs was examined only at the 1-h time point. Male 2–3-month-old C57BL/6J (B6) mice were injected i.p. with 100 µCi [methyl-14C]PQ dissolved in saline. In other mice, Mn2+-ethylenebis-DTC (30 mg/kg) dissolved in saline was administered in a separate i.p. injection immediately before injection of [methyl-14C]PQ. For the time course study, mice (n = 4) were killed at four different time points (0.5, 1, 6 and 12 h) after injection. At each time point, blood was sampled from orbital bleeds. Following this, mice were anesthetized with 100 mg/kg pentobarbital, perfused with heparinized saline to remove blood from organs, and finally organs were harvested, including brain, lung, liver, kidney and heart. The brain was dissected and samples from four regions (striatum, midbrain, cerebellum and frontal cortex) were obtained and weighed. Samples from the other organs were blocked and weighed. All tissue samples were added to 1 mL Biosol (National Diagnostics) to dissolve tissue overnight or until clear at 50°C in an oven followed by the addition of 10 mL Bioscint (National Diagnostics) for liquid scintillation counting. Values reported are the mean ± SEM from four separate mice for each condition and time point. The effect of two additional DTCs, methyl-DTC (30 and 100 mg/kg) and diethyl-DTC (100 and 150 mg/kg) were examined in the same manner after 60 min.

Data and statistical analysis

For the DA uptake studies, data were averaged from three to five independent experiments. For in vivo[methyl-14C]PQ studies, three to four animals were used for each condition. Unpaired Student's t-tests were used to compare the effects of a compound with the effect in the absence of any compound. A two-factor anova was used to analyze the in vivo and in vitro time course study with treatment and time point as between-group factors. A one-factor anova was used to analyze the effect of different DTCs on [methyl-14C]PQ uptake. Significant main effects of treatment or interactions were followed by selected post-hoc analyses. The area under the curve (AUC) was calculated in SigmaPlot® 2000 (SPSS, Chicago, IL, USA) using a macro and the trapezoidal rule for equal or unequally spaced × values.

Results

In vitro DA uptake studies

Initial uptake studies were performed using a 10-nm concentration of [3H]DA and a 6-min incubation period. We tested a variety of DTC pesticides, two other pesticides (PQ and triadimefon), several cations and cocaine at varying concentrations (50 nm to 500 µm). The ethylenebis-DTCs, Mn2+-ethylenebis-DTC and Na+-ethylenebis-DTC, demonstrated a similar increase in synaptosomal DA content of about 18% at 500 nm (Fig. 1b). Zn2+-ethylenebis-DTC did not produce a significant increase in DA content (Fig. 1b). However, the zinc cation alone resulted in a significant decrease (∼15%) in DA content (as previously reported; Richfield 1992) and probably prevented an increase from the parent ethylenebis-DTC compound. Diethyl-DTC also produced in a significant increase in DA content (∼24%). The dimethyl-DTC (Zn2+-dimethyl-DTC) and methyl-DTC did not significantly increase synaptosomal DA content. The pesticide PQ did not alter the DA content at any concentration tested (0.1–50 µm; shown at 0.5 µm in Fig. 1b). The pesticide triadimefon, a known inhibitor of DA uptake (Walker and Mailman 1996), produced a significant dose-dependent decrease in uptake (0.1–50 µm), as did the classic DA uptake inhibitor cocaine (0.05–10 µm).

Mn2+-ethylenebis-DTC resulted in a dose-dependent increase in synaptosomal content of DA, with a maximal increase of about 30% at a concentration of 5 µm(Fig. 2). Significant increases in DA content were seen at concentrations ranging from 500 nm to 50 µm. No effect was seen at lower concentrations and a reduction in DA content was seen at 100 µm. Similar dose-dependent effects were seen with Na+-ethylenebis-DTC and diethyl-DTC (data not shown). No significant increase was seen with incubation of the complexed cation Mn2+ at similar concentrations (Fig. 2).

Figure 2.

Effect of different concentrations of Mn2+-ethylenebis-DTC or Mn2+ on synaptosomal DA content. Values represent mean ± SEM percentage of DA uptake in the presence of Mn2+-ethylenebis-DTC or Mn2+ compared with DA uptake in the absence of the compound (n = 3–5 independent experiments). Mn2+-ethylenebis-DTC showed a dose-dependent effect on synaptosomal DA content. Concentrations of Mn2+-ethylenebis-DTC that produced a significant increase ranged from 0.5 to 50 µm (p < 0.05). Mn2+-ethylenebis-DTC significantly decreased DA content at 100 µm (p < 0.05) and had no significant effect at the lowest concentration tested (0.1 µm). Mn2+ showed no significant effect on DA uptake from 0.5 to 50 µm (p > 0.05), but had an inhibitory effect at the lowest concentration tested (p < 0.05). The control uptake was 28.35 ± 2.4 pmol per mg protein.

We next performed more detailed kinetic studies of DA accumulation in the absence or presence of Mn2+- ethylenebis-DTC (1 µm; Figs 3a and b). Uptake was time dependent with or without Mn2+-ethylenebis-DTC (Fig. 3a). The initial rate of DA uptake was not affected by the presence of Mn2+-ethylenebis-DTC. Uptake was rapid and reached equilibrium after 10–14 min. Accumulation over time was greater in the presence of Mn2+-ethylenebis-DTC and reflected the net effect of influx and efflux. We then measured efflux of [3H]DA from prefilled synaptosomes (Fig. 3b). [3H]DA efflux was rapid in the absence of Mn2+- ethylenebis-DTC and was nearly complete after 20 min. However, in the presence of Mn2+-ethylenebis-DTC, [3H]DA efflux was slower, and was only about 50% complete after 20 min. The AUC was increased in the presence of Mn2+-ethylenebis-DTC (145% greater).

Figure 3.

Influx and efflux of synaptosomal DA. (a) DA uptake was rapid in the absence (○) or presence (•) of Mn2+-ethylenebis-DTC (1 µm) and typically reached a steady state after 10–14 min. (b) In the absence of Mn2+-ethylenebis-DTC (○), DA efflux was rapid and exponential, falling to near zero after 20 min. In the presence of Mn2+-ethylenebis-DTC (1 µm; •), efflux was dramatically slowed and was only about 50% complete after 20 min. Values are mean ± SEM of three to five independent experiments. Inserts (a′ and b′) represent values normalized to a common 100% maximal value to allow comparison of the shape of the curves. anova was performed separately for accumulation and efflux. There was a significant effect of time on both accumulation and efflux (both p < 0.001). There was also a significant effect of treatment with Mn2+-ethylenebis-DTC on both accumulation (p = 0.05) and efflux (p < 0.001). The effect of Mn2+-ethylenebis-DTC on synaptosomal DA accumulation (a) only became evident at later time points when both influx and efflux had reached equilibrium. However, as shown in the efflux curve (b), this effect was evident at all time points following prefilling.

Efflux studies were also performed in the absence or presence of cocaine (0.5 µm) to determine if cocaine blockade of the DAT might alter DA efflux (Fig. 4). We preloaded synaptosomes with [3H]DA for 13–14 min in the absence or presence of cocaine (0.5 µm) and then initiated efflux in solutions containing either 0.05 or 0.5 µm cocaine. The rate of DA efflux was similar in all conditions, suggesting that DA efflux was not modulated by cocaine at the DAT under these conditions.

Figure 4.

(a) Effect of cocaine on efflux of synaptosomal DA. Cocaine (500 nm) was included in various combinations during DA influx and efflux; both influx and efflux (•), influx only (○), efflux only (▪) or neither (□). Solid lines denote the presence of cocaine in efflux buffer, dotted lines indicate an absence of cocaine in efflux buffer. Cocaine reduced DA influx as expected when included in the influx buffer (○•). However, cocaine had no effect on efflux. Insert (a′) shows values normalized to a common 100% maximal value to allow comparison of the shape of the curves. The degree of efflux was approximately 30% complete under all conditions, suggesting that cocaine did not alter efflux in this preparation and was similar to the degree of efflux demonstrated in Fig. 3 (different x-axis) in the absence of Mn2+-ethylenebis-DTC. Values are mean ± SEM.

To gain insight into the [3H]DA efflux mechanism, studies were carried out under a variety of conditions (Figs 5a and b). [3H]DA efflux was temperature dependent and there was significantly greater efflux at 37°C than 4°C (p < 0.05) (Fig. 5a). We tested for trans-stimulation or trans-inhibition of [3H]DA efflux by adding compounds to the efflux buffer. DA, norepinephrine and amphetamine all significantly increased [3H]DA efflux (Fig. 5b) suggesting trans-stimulation of efflux. A sodium-free efflux buffer also increased [3H]DA efflux. In contrast, cocaine, nomifensine and Mn2+-ethylenebis-DTC did not alter [3H]DA efflux, suggesting that trans-inhibition of efflux did not occur.

Figure 5.

Effect of varying conditions on [3H]DA efflux of prefilled synaptosomes. Values are mean ± SEM from three to five independent experiments. (a) Following a 10-fold dilution into efflux buffer, we measured the amount of efflux from prefilled synaptosomes after 2 min at two temperatures. The amount of efflux was significantly greater at 37°C than 4°C (*p < 0.05). (b) Efflux as a result of dilution of [3H]DA was compared under several conditions with reagents added or ionic changes made to the standard efflux buffer at 37°C after 2 min. The presence of excess unlabeled DA, norepinephrine and amphetamine all significantly increased the percent of efflux (*p < 0.05). Efflux was also stimulated by a Na+-free efflux buffer. Cocaine, nomifensine and Mn2+-ethylenebis-DTC when present only in the efflux buffer did not alter efflux. The efflux at 37°C was 0.82 ± 0.14 pmol per mg protein.

We sought to determine if Mn2+-ethylenebis-DTC would alter the Km or Vmax for [3H]DA influx via the DAT after two durations of incubation (Table 1). After a short incubation (30 s, before efflux is likely to be significant), saturation studies in the absence or presence of Mn2+- ethylenebis-DTC resulted in similar Km values (mean ± SEM 311 ± 122 and 271 ± 184 nm respectively; p > 0.05) and Vmax (166.5 ± 62.9 and 161.7 ± 100.5 pmol per min per mg protein respectively; p > 0.05). After a longer incubation (6 min, when influx and efflux are both operative), saturation studies in the absence or presence of Mn2+-ethylenebis-DTC resulted in similar apparent Km values (42 ± 4 and 47 ± 7 nm respectively; p > 0.05), but significantly different Vmax values (7.7 ± 0.3 and 10.2 ± 0.7 pmol per min per mg protein respectively; p < 0.05). The higher Vmax in the presence of Mn2+-ethylenebis-DTC was consistent with blockade of efflux without altering the affinity of the DAT for DA. We also tested whether Mn2+-ethylenebis-DTC might need additional time to exert an effect on influx. After preincubating tissue for 6 min with either Mn2+-ethylenebis-DTC or cocaine, we compared the amount of influx with that of a control with no added drug. Cocaine resulted in significantly decreased influx (50.5 ± 9.5%; p ≤ 0.005), whereas Mn2+-ethylenebis-DTC had no effect (101.8 ± 2.4%; p = 0.89) compared with control.

Table 1.  Parameters derived from saturation experiments performed at two different time points (30 s or 6 min) in the absence or presence of Mn2+-ethylenebis-DTC (0.5 or 5 µm)
ParameterMn2+-ethylenebis- DTC (µm)Incubation time (s)n Mean ± SEM
  1. Mn2+-ethylenebis-DTC was used at a higher concentration for the 30-s incubation to maximize the likelihood of detecting an effect based on data demonstrated in Fig. 3. Varying concentrations of DA (1–100 nm) were incubated with synaptosomes for either 30 s or 6 min. *p < 0.05 versus no Mn2+-ethylenebis-DTC. The effect of Mn2+-ethylenebis-DTC on the Vmax at the longer incubation time reflects the net effect of influx and efflux.

Km (nm)0303311 ± 122
Km (nm)5303271 ± 184
Vmax (pmol per mg protein per min)0303166.5 ± 62.9
Vmax (pmol per mg protein per min)5303161.7 ± 100.5
Km (nm)0360542 ± 4
Km (nm)0.5360547 ± 7
Vmax (pmol per mg protein per min)036057.7 ± 0.3
Vmax (pmol per mg protein per min)0.5360510.2 ± 0.7*

In vivo[14C]PQ studies

Several DTCs have been shown to enhance the neurotoxicity of both MPTP and PQ in mice (Corsini et al. 1985; Irwin et al. 1987; Takahashi et al. 1989; Yurek et al. 1989; Miller et al. 1991; Bachurin et al. 1996; Vaccari et al. 1996; Walters et al. 1999; McGrew et al. 2000; Thiruchelvam et al. 2000a,b, 2002). In addition, one study has demonstrated a toxicokinetic effect on MPP+, with diethyl-DTC increasing the concentration and duration of MPP+ in the brain (Irwin et al. 1987). We sought to determine if a similar effect might occur with PQ. [14C]PQ was rapidly transported from peritoneum into blood and then into organs with significantly greater uptake into peripheral organs compared with the brain (Figs 6a–d). Levels generally peaked around 1 h. The simultaneous administration of Mn2+-ethylenebis-DTC resulted in significantly higher concentrations in all organs between 1 and 12 h compared with [14C]PQ alone (Figs 6 and 7). Mn2+-ethylenebis-DTC also enhanced organ concentrations of [14C]PQ by 50–350% between 1 and 6 h. The AUC between 0.5 and 12 h for each organ and brain region was increased in the presence of Mn2+-ethylenebis-DTC (midbrain 94%, cerebral cortex 105%, striatum 78%, cerebellum 94%, serum 127%, lung 205%, kidney 168%, heart 143% and liver 248%) compared with [14C]PQ alone. Organ concentrations were still raised at 12 h after injection in the presence of Mn2+-ethylenebis-DTC (Fig. 7a). By 12 h, most organs had eliminated the [14C]PQ, with brain and lung being the slowest to eliminate it in the presence of Mn2+-ethylenebis-DTC (Fig. 7b). A similar significant increase in the presence of diethyl-DTC (150 mg/kg) was observed at 1 h in all organs examined (Fig. 8). A lower dose of diethyl-DTC (100 mg/kg) produced a significant increase in some, but not all, organs. Methyl-DTC (30 and 100 mg/kg) did not significantly augment [14C]PQ concentration in any organ. Co-administration of dimethyl-DTC was toxic to mice at the lowest dose tested (30 mg/kg) and no results were obtained.

Figure 6.

Mean ± SEM [14C]PQ in various organs and brain regions over time following i.p. injection. Organs and tissues were harvested at various times (0.5–12 h) after injection of [14C]PQ (100 µCi) alone (filled symbols) or with Mn2+-ethylenebis(EB)-DTC (30 mg/kg; open symbols). Concentrations in brain were much lower than those in other organs or serum. Values peaked at 30 min in serum or around 1 h for other organs. Values in all organs and brain were increased in the presence of Mn2+-ethylenebis-DTC between 1 and 12 h. In brain and lung the augmentation by Mn2+-ethylenebis-DTC was greater than that in other organs, most noticeably at 6 and 12 h. A repeated-measures anova, with time point and treatment as independent variables and regions as dependent variables revealed a significant effect of time point (0.5, 1, 6 or 12 h; F3,168 = 48.9, p < 0.0001), treatment ([14C]PQ alone or [14C]PQ + MB; F1,168 = 15.2, p = 0.0008), and interaction of time point and treatment for the different regions analyzed (F24,168 = 4.9, p < 0.0001). Consequent two-factorial anova, with time point and treatment as between-factor variables and region as within-factor variable revealed significant interaction of time point and treatment for all the independent regions analyzed (p < 0.05).

Figure 7.

Mean ± SEM ratios of [14C]PQ concentration in different organs or regions of brain. (a) The change in [14C]PQ concentration in the presence of Mn2+-ethylenebis-DTC compared with its absence. After 30 min all organs displayed increased concentrations with the greatest effect between 1 and 6 h. Increases due to Mn2+-ethylenebis-DTC were still present after 12 h. (b) After 12 h, the effect of Mn2+-ethylenebis-DTC was similar in brain and lung, and exceeded that in other organs compared with values after 1 h. anova revealed a significant effect of region (F8,27 = 30.2, p < 0.0001), with subsequent post-hoc analysis showing striatum, midbrain, cortex, cerebellum and lung significantly different from serum, liver, kidney, and heart (p < 0.001).

Figure 8.

Mean ± SEM changes in [14C]PQ concentration in the striatum effected by two additional DTCs. Mice (n = 3 or 4) per condition were treated with [14C]PQ as described previously combined with methyl-DTC (30 and 100 mg/kg) or diethyl-DTC (100 and 150 mg/kg). Tissue was harvested after 1 h. Dimethyl-DTC was too toxic to use in combination. Effects similar to those in the striatum were seen in all other regions of brain and other organs. A dose effect was seen with diethyl-DTC, but only the 150 mg/kg dose produced a significant change in [14C]PQ concentration (*p = 0.005). Values obtained with Mn2+-ethylenebis-DTC are shown for comparison (*p = 0.002). The tissue concentrations in the striatum were as follows: [14C]PQ alone, 0.073 + 0.011 and 0.068 + 0.003 pmol per mg wet-weight tissue; [14C]PQ plus Mn2+-ethylenebis-DTC (30 mg/kg), 0.14 ± 0.012 pmol per mg wet-weight tissue; [14C]PQ plus methyl-DTC (30 and 100 mg/kg) 0.062 ± 0.009 and 0.073 ± 0.019 pmol per mg wet-weight tissue; [14C]PQ plus diethyl-DTC (100 and 150 mg/kg), 0.083 ± 0.017 and 0.125 ± 0.017 pmol per mg wet-weight tissue.

Discussion

Our data provide novel information about specific environmental agents, PQ and DTCs, implicated in the PDP in humans. A potent effect of certain DTCs on the kinetics of [3H]DA synaptosomal accumulation in vitro and tissue concentrations of systemic [14C]PQ in vivo was identified. Several ethylenebis-DTCs and diethyl-DTC had these effects, whereas methyl-DTC and dimethyl-DTC, which have a very similar chemical structure, did not. The DTCs with these kinetic effects have also been demonstrated to enhance MPTP and PQ neurotoxicity in vivo (Corsini et al. 1985; Irwin et al. 1987; Takahashi et al. 1989; Miller et al. 1991; Walters et al. 1999; McGrew et al. 2000; Thiruchelvam et al. 2000a,b), whereas DTCs without such effects either do not enhance neurotoxicity or have not been tested (McGrew et al. 2000). These findings have implications for these compounds in contributing to the PDP independently and in combination.

The ability of selective DTCs to increase synaptosomal [3H]DA accumulation in vitro was seen at concentrations as low as 500 nm, suggesting high sensitivity. Furthermore, the effect occurred after a short 6-min incubation period in vitro, suggesting a rapid onset. This effect of DTC was not seen after a 30-s incubation (with or without a 6-min preincubation) when the initial rate of DA uptake can best be measured, implying that the effect did not occur directly on the DAT itself. The action on synaptosomes lasted for at least 20 min in vitro, suggesting a prolonged effect. At longer incubation times (greater than 30 s), synaptosomal DA content reflects the net effects of both influx and efflux (see Schoemaker and Nickolson 1982); as efflux is slowed, synaptosomal DA content will increase to a new equilibrium. Based on further kinetic experiments, this effect was found to be due to a reduction in DA efflux. The apparent Km values (low nanomolar) of the DAT for DA at the two time points were near the range found by others using similar species and protocols. For example, Javitch et al. (1985) reported apparent Km values of 74 and 150 nm, and Chiba et al. (1985) reported an apparent Km of 180 nm. The difference in values (Km and Vmax) found between 30 s and 6 min reflects differences in the initial rate into an empty synaptosome and the complicated rates produced by competing effects between influx and efflux of a partially filled synaptosome. Differences between laboratories probably reflect differences in the time point used for measurement and the desire to measure an initial rate or a complicated rate reflecting both influx and efflux. The present study demonstrated a significant effect of selective DTCs on DA content (Vmax) of synaptosomes at 6 min.

Efflux or release of DA has been studied in striatal synaptosomal homogenates and tissue slices (Raiteri et al. 1979; Schoemaker and Nickolson 1982; Schoemaker and Nickolson 1983; Diliberto et al. 1988). Efflux has been shown to be both Ca2+ mediated (exocytic) and Ca2+ independent (non-exocytic) (Diliberto et al. 1988; Lonart and Zigmond 1991). Ca2+- independent efflux can be mediated in part by reverse transport through the DAT and by mechanisms that are independent of the DAT (Raiteri et al. 1979; Schoemaker and Nickolson 1982; Diliberto et al. 1988; Lonart and Zigmond 1991) depending on the type of preparation and conditions for efflux. However, the lack of a specific blocker and the little attention devoted to alternative efflux pathways has hampered the identification of the transporter(s) responsible for DAT-independent efflux.

There are several possible mechanisms for the effect of selective DTCs on inhibition of DA efflux. First, inhibition may involve the DAT, but only from the intrasynaptosomal side and only affecting efflux. We demonstrated that the initial rate of DA uptake was not altered by any DTC. We found that efflux was inhibited only when the selective DTCs were present during influx and efflux, allowing sufficient time for membrane penetration into the synaptosome. The lack of effect by a specific DTC present only in the efflux buffer suggests that an effect on the exterior portion of the DAT did not occur. Efflux of DA through the DAT was unlikely in our preparation as neither cocaine nor nomifensine altered efflux. A second possibility is that inhibition of efflux may be due to an increase in vesicular uptake, reducing the amount of cytoplasmic DA available for efflux. This possibility is remote given that compounds increasing transporter function have not been identified, efflux was rapid and complete from our synaptosomal preparation, and efflux followed Michaelis–Menton kinetics as demonstrated by others (Schoemaker and Nickolson 1983). Third, an alternative efflux transporter might be involved that is inhibited by selective DTCs. The presence of DAT-independent efflux reported by others makes this a possibility. However, a specific striatal DA efflux transporter has yet to be identified. Efflux transporters are common in humans and other species, and have wide tissue expression, and diverse substrates (see Taylor 2002). There are three large families of efflux transporters present in brain and other organs (Taylor 2002), each having several members: the multidrug resistance transporters (Ambudkar et al. 1999; Holland and Blight 1999; Leslie et al. 2001), the monocarboxcylic acid transporters and the organic ion transporters. Numerous members of these transporters are expressed in brain with differing distributions and substrate specificities (Ambudkar et al. 1999; Leslie et al. 2001). The two most interesting families of efflux transporters, in relationship to the findings presented here, are the organic cation transporter family (OCT1–3) and the organic cation/carnitine family (OCTN1–3). Several transporters in these families have been shown to efflux DA, MPTP and MPP+, and to be expressed on neurons (Zhang et al. 1997; Busch et al. 1998; Wu et al. 1998). Multiple members of these families are expressed in brain, liver, lung, kidney and heart, regions examined in vivo in this study (see Taylor 2002). Given the structural similarity of PQ to both MPTP and MPP+, and the broad substrate specificity of these transporters, efflux of PQ is a reasonable expectation, despite the selectivity of the DAT for DA and MPP+, but not PQ, as shown in this study. The fact that PQ is not metabolized in vivo suggests that it must be removed from cells by an active transport process. Finally, the specific DTCs might act by altering a non-transporter protein that modifies the DAT or another transporter. For example, the DAT and other transporters have been shown to be modified by protein kinase C and protein tyrosine kinases (Doolen and Zahniser 2001), making the involvement of a non-transporter protein a possible mechanism for the observed effects.

The DTCs shown to alter the kinetics of DA in vitro were also effective in altering the tissue concentration of [14C]PQ in vivo and were previously shown to alter striatal DA in vivo (Thiruchelvam et al. 2000a). Mice dosed acutely or repeatedly with Mn2+-ethylenebis-DTC have been shown to exhibit reduced locomotor activity and increased striatal levels of DA and metabolites (Thiruchelvam et al. 2000a; 2000b). Tissue concentrations of [14C]PQ in different organs were increased to variable levels by the simultaneous administration of selective DTCs. The duration of this effect varied in different organs; most prolonged levels were seen in the lung and brain. A common mechanism whereby selective DTCs might alter the kinetics of both [3H]DA in vitro in synaptosomes and [14C]PQ and DA in vivo is via direct inhibition of an efflux transporter that transports both compounds out of cells. Direct action on a protein involved in transport seems likely given the rapid nature of the effect, as opposed to a slower mechanism such as altered transcription or translation. Direct complexing of [14C]PQ with a selective DTC to increase transport is possible, although this effect was not seen in vitro as the selective DTCs did not alter influx of [3H]DA into synaptosomes.

The DTCs shown to alter the kinetics of both DA in vitro and PQ in vivo have also been shown to enhance the nigrostriatal neurotoxicity of both MPTP and PQ in vivo in mouse models (Corsini et al. 1985; Irwin et al. 1987; Takahashi et al. 1989; Miller et al. 1991; Walters et al. 1999; McGrew et al. 2000; Thiruchelvam et al. 2000a,b). Additionally, the toxicokinetics of MPP+ were increased by diethyl-DTC in vivo. The enhanced neurotoxicity of both MPTP and PQ in vivo may relate to a toxicokinetic effect of the DTCs on the neurotoxicants, a kinetic effect on DA or both. Although other mechanisms have been proposed or studied (such as the effect on mitochondria; Bachurin et al. 1996), none can support the clear correlation we have identified between different neurotoxicants (MPTP and PQ) and different DTCs. Other non-commercial DTCs exist that either do (dipropyl-DTC, diisopropyl-DTC and dicyclohexyl-DTC) or do not (ethylcyclohexyl-DTC and piperidinyl-DTC) potentiate systemic MPTP toxicity and could be tested to extend this correlation (Bachurin et al. 1996), but are not readily available and are not implicated as environmental risk factors for the PDP. The results obtained by combining certain other agents with MPTP/MPP+in vivo to enhance neurotoxicity further support the importance of a toxicokinetic mechanism (Irwin et al. 1987; Zuddas et al. 1989a,b; Lau et al. 1990).

Different lines of evidence, including epidemiologic studies in human (Hertzman et al. 1990; Sanchez-Ramos et al. 1987), animal models (Brooks et al. 1999; Thiruchelvam et al. 2000a, 2000b; McCormack et al. 2002) and in vitro studies (Yang and Sun 1998), have implicated PQ in the PDP. It has long been speculated, based on the similar structure shared by PQ and MPP+, that PQ might enter SNpc neurons via the DAT (Snyder and D'Amato 1985; Woolley et al. 1989). However, evidence from this study suggests that this is not the case. In contrast to the previously reported effect of MPP+ (Altar et al. 1986; Kitayama et al. 1998), PQ did not compete with [3H]DA for transport via the DAT in vitro at any concentration tested. Also, the concentration of [14C]PQ was not increased in the striatum compared with other brain regions at any time point between 0.5 and 12 h after injection, as would be expected if transport occurred only or preferentially via the DAT. In fact, the striatum had the lowest concentration with or without Mn2+-ethylenebis-DTC at every time point compared with other brain regions examined.

PQ and several other polyamines (spermine, spermidine, putrescene and cadaverine) have been demonstrated to accumulate in brain slices and specifically alveolar type I and II epithelial cells of the lung via a PAT that is energy dependent and saturable (Rose et al. 1976; Drew et al. 1979; Smith and Wyatt 1981; Groves et al. 1995; Chan et al. 1996; Yang and Sun 1998). The specific transporter has yet to be identified. PATs are part of a large superfamily of transporters, the amino acid/polyamine/organocation (APC) superfamily (see Jack et al. 2000). PATs have been identified in brain (Harman and Shaw 1981; Gilad and Gilad 1991; Dot et al. 2000), but their cellular location in brain remains unclear. PATs have been identified on glia in vitro (Dot et al. 2000). If PQ is transported via a PAT into cells in the brain, our data suggest widespread brain uptake without selectivity for the nigrostriatal system. However, SNpc neurons have been demonstrated to be selectively vulnerable to PQ in mice (Thiruchelvam et al. 2000a; 2000b; McCormack et al. 2002). PQ has the property of undergoing a single electron reduction from the cation to form a free radical that is stable in the absence of oxygen (Yang and Sun 1998; Witschi et al. 1977; Smith et al. 1978; Keeling and Smith 1982; Smith 1988; Adam et al. 1990), and dopaminergic neurons may be most highly sensitive to this oxidative stress.

Our data suggest a possible mechanism for the potentiation of PQ-induced neurotoxicity by DTCs in models of the PDP. We hypothesize that DTCs alter the toxicokinetics of exogenous neurotoxicants and endogenous DA. This effect might occur via inhibition of an efflux transporter, although alternative mechanisms relating to toxicokinetics are possible. In this model, systemic neurotoxicants are taken up into nigrostriatal terminals or dopaminergic cell bodies by the DAT (MPP+) or by a PAT (PQ). Effective DTCs alter their toxicokinetics to increase the effective concentration of those neurotoxicants and DA, and thus enhance their toxicity by increasing the concentration of each and prolonging their intracellular exposure, yielding an increased cellular AUC. PQ and DA both contribute to the generation of reactive oxygen species, which will ultimately reach a threshold for irreversible cellular damage. Others have identified and discussed the importance of toxicokinetics or biodisposition in the neurotoxicity of MPTP (Irwin et al. 1987; Johannessen et al. 1985). Effective DTCs and other agents are capable of converting a non-toxic dose to a toxic dose through alterations in their toxicokinetics (Walters et al. 1999; Thiruchelvam et al. 2000b). Features of this model are testable and will shed light on mechanisms of neurotoxicants speculated to play a role in the PDP in humans.

Acknowledgements

This work supported by the Department of Defense DAMD17-98-1-8628 (EKR); NIH ES11839 (EKR); ES01247, ES05905, ES05017 (DACS); and ES06484 (NB).

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