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
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.
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.
[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.
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.
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).
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).
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.
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.
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)
Mn2+-ethylenebis- DTC (µm)
Incubation time (s)
Mean ± SEM
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.
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.
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.
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.
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.
This work supported by the Department of Defense DAMD17-98-1-8628 (EKR); NIH ES11839 (EKR); ES01247, ES05905, ES05017 (DACS); and ES06484 (NB).