ToxSci Advance Access originally published online on April 9, 2007
Toxicological Sciences 2007 98(1):159-166; doi:10.1093/toxsci/kfm080
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Published by Oxford University Press 2007.
Evaluating the NMDA-Glutamate Receptor as a Site of Action for Toluene, In Vivo
* Neurotoxicology Division, MD B105-05, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, 27711
Pre-pharmacy Program, Campbell University, Buies Creek, North Carolina, 27506
Experimental Toxicology Division
Human Studies Division; National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, 27711
1 To whom correspondence should be addressed. Fax: (919) 541-4849. E-mail: boyes.william{at}epa.gov.
Received January 31, 2007; accepted March 31, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Acute exposure to toluene and other volatile organic solvents results in neurotoxicity characterized by nervous system depression, cognitive and motor impairment, and alterations in visual function. In vitro, toluene disrupts the function of N-methyl-D-aspartate (NMDA)-glutamate receptors, indicating that effects on NMDA receptor function may contribute to toluene neurotoxicity. NMDA-glutamate receptors are widely present in the visual system and contribute to pattern-elicited visual-evoked potentials (VEPs) in rodents, a measure that is altered by toluene exposure. The present study tested the hypothesis that effects on NMDA receptors contribute to toluene-induced alterations in pattern-elicited VEPs. Prior to examining the effects of NMDA receptor agonists and antagonists on toluene-exposed animals, a dose-range study was conducted to determine the optimal dose for NMDA (agonist) and MK801 (antagonist). Dose levels of 2.5 mg/kg NMDA and 0.1 mg/kg MK801 were selected from these initial studies. In the second study, Long-Evans rats were exposed to toluene by inhalation, and VEPs were measured during toluene exposure in the presence or absence of NMDA or MK801. Pattern-elicited VEPs were collected by exposing rats to a sinusoidal pattern modulated at a temporal frequency of 4.55 Hz. Following collection of baseline VEPs, rats were injected with either saline, NMDA (2.5 mg/kg, ip), or MK801 (0.1 mg/kg, ip) and 10 min later were exposed to air or toluene (2000 ppm). VEP amplitudes were calculated for 1x (F1) and 2x stimulus frequency (F2). The F2 amplitude was reduced by approximately 60, 60, and 50% in the toluene-exposed groups (TOL): SALINE/TOL (n = 11), NMDA/TOL (2.5 mg/kg; n = 13), and NMDA/TOL (10 mg/kg, n = 11), respectively. Thus, NMDA (2.5 and 10 mg/kg) did not significantly affect toluene-mediated F2 amplitude effects. Administration of 0.1 mg/kg MK801 prior to toluene exposure blocked the F2 amplitude decreases caused by toluene (n = 9). However, when 0.1 mg/kg MK801 was administered 20 min after the onset of toluene exposure, toluene-mediated F2 amplitude decreases persisted despite the challenge by MK801. These data support the hypothesis that acute actions of toluene on pattern-elicited VEPs involve NMDA receptors.
Key Words: NMDA receptor; toluene; visual-evoked potentials.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Toluene, a volatile organic solvent, is a major constituent in a number of commercial products such as cleaning fluids, paints, and glues. Inhalation is the typical route of human exposure to toluene, although it can be absorbed dermally or upon ingestion. Following acute exposure, toluene produces acute neurotoxic effects such as cognitive and visual impairments and motor coordination deficits (for review see Bushnell and Crofton, 1996
; Evans and Balster, 1991). The mechanisms underlying the neurotoxicity of toluene and other volatile organic compounds (VOCs) have not been elucidated clearly.
In vitro, toluene and other VOCs perturb function of ion channels found in the central nervous system. Specifically, studies have shown that toluene inhibits excitatory ion channels including the N-methyl-D-aspartate (NMDA) glutamate (Cruz et al., 1998, 2000) and nicotinic acetylcholine receptors (nAChRs) (Bale et al., 2002, 2005a) and potentiates the function of inhibitory ion channels such as the gamma-aminobutyric acid receptor type AA (Beckstead et al., 2000), glycine (Beckstead et al., 2000, 2001), and serotonin (Lopreato et al., 2003) receptors. In addition, toluene and other VOCs disrupt voltage-gated calcium channels (Shafer et al., 2005; Tillar et al., 2002) and ATP-gated ion channels (Woodward et al., 2004). These studies have demonstrated specific neurological targets for toluene and VOCs, resulting in the hypothesis that interactions of these compounds with ion channels contribute to their acute effects in vivo. However, the roles and relative importance of these ion channel targets in acute VOC neurotoxicity are presently unknown.
The current study examined whether the NMDA-glutamate receptor is an important target for toluene-mediated physiological modifications in vivo. The steady-state pattern-elicited visual-evoked potential (VEP) was used to assess the physiological effects of inhaled toluene. This potential, elicited by a sinusoidal on-off pattern, can be considered to involve a linear response component that corresponds to the frequency of stimulus presentation (F1) and a nonlinear response component at double the stimulus frequency (F2). These components of the VEP provide information regarding linear and nonlinear response properties of the visual system (Regan, 1989). Toluene decreases the F2 amplitude of the steady-state VEP (Boyes et al., submitted).
Glutamate is the primary excitatory neurotransmitter in the afferent visual pathway and is responsible for transmitting signals from photoreceptors to bipolar cells, bipolar cells to retinal ganglion cells, and retinal ganglion cells to thalamocortical projections. NMDA receptors are involved in transmission of responses from bipolar to ganglion cells which eventually send the responses to the visual cortex (Chen and Diamond, 2002; Cohen and Miller, 1994). Chronic disruption of NMDA receptor function during ocular development causes alterations in retinal and visual cortical signaling (Colonnese and Constantine-Patton, 2001). NMDA receptors have been demonstrated to be involved in the production of VEPs. MK801, an NMDA receptor antagonist, significantly alters the flash-evoked potential (Hetzler and Burkard, 1999). Recently, we demonstrated that NMDA (10 mg/kg) significantly increased the F1 amplitude and decreased the F2 amplitude in pattern-elicited steady-state VEPs (Bale et al., 2005b). Therefore, we hypothesized that if NMDA receptors are critical targets for the effects of toluene on the VEP, administration of a specific NMDA agonist or antagonist would alter toluene's effect on the steady-state VEP.
To test this hypothesis in an intact organism, VEP recordings were made following administration of an agonist (NMDA) or an antagonist (MK801) to Long-Evans rats also inhaling 2000 ppm toluene. Two studies were conducted to evaluate the hypothesis. The first study examined the effects of NMDA and MK801 alone on VEP amplitude. The results from this study were used to select doses of these compounds to test in an interaction with toluene by measuring F1 and F2 VEP amplitudes. Time points for testing were selected on the basis of previous experiments evaluating the effects acute of toluene exposure on VEPs (Boyes et al., submitted).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals.
Eighty-day-old male Long-Evans rats (350–450 g) were obtained from Charles River Laboratories (Raleigh, NC), housed individually in polycarbonate cages and provided ad libitum access to tap water and rat chow (PMI #5001, LabDiet). Animals were acclimated for a least 1 week prior to surgery. The rats were housed in an Association for Assessment and Accreditation of Laboratory Animal Care approved facility on a 12:12-h light/dark cycle (lights on from 6:00 A.M.–6:00 P.M.) at 22 ± 2°C with a relative humidity of 50 ± 10%. All aspects of the care and treatment of laboratory animals were approved by the institutional laboratory animal health care and use committee and were in compliance with applicable Federal guidelines for laboratory animal experimentation.
Electrode implantation.
Electrodes constructed from Nichrome wires soldered to stainless steel screws (00–90 x 1/16) were implanted into the rat skull, epidurally, as previously described (Boyes et al., 2003). Briefly, rats were anesthetized with sodium pentobarbital (50 mg/kg, ip), and recording electrodes were implanted in the following locations: 1 mm anterior to lambda and 4 mm left of the midline overlying the primary visual cortex, and 2 mm anterior to bregma and 2 mm left and right to the midline for the ground and reference electrodes. After implantation, the electrodes were connected to a ninepin connector (WirePro model 223-1609; Resource Electronics, Raleigh, NC), the entire assembly was encased in acrylic, and the wound was sutured. Approximately 1 week was allowed for recovery before VEP testing.
Visual stimuli.
The visual stimulus pattern was a vertical grating with a mean sinusoidal spatial luminance profile of 10 cd/m3, spatial frequency of 0.16 cpd, and visual contrast of 60% and was temporally modulated in an on-off fashion with a 4.55-Hz sinusoid. A value of 0.16 cpd was selected because it is approximately at the peak of the contrast sensitivity function of the pigmented rat (Boyes, 1994; Boyes et al., 2003; Silveira et al., 1987). A value of 60% was selected because it fell well within the linear portion of the video monitor luminance response function and yielded a strong evoked potential. Visual stimuli were generated from a computer-based system described in detail in Hamm et al. (2000) and were presented on a monitor (ViewSonic 15, model 1564M; Walnut, CA) located approximately 15 cm from the rat's eyes. Briefly, stimulus patterns were provided to the memory of a super-VGA graphics display card. The "green" video card output signal was then processed with analog circuitry, and analog multipliers were used to set the contrast, apply the temporal modulation, and set the overall luminance. Sixteen-bit D/A converters were used to generate the percent contrast and luminance control signals.
Toluene inhalation exposure.
Details of this exposure and setup are presented in Boyes et al. (2003). Briefly, a syringe pump (Model 22; Harvard Apparatus, South Natick, MA) was used to inject liquid toluene into the air stream of a J-tube design inhalation system (McGee et al., 1994). The J-tube was heated to 80°C and volatilized the liquid toluene. Vaporized toluene was presented to the animals in a head-only exposure chamber. Toluene concentrations were monitored online with a long path-length dispersive infrared spectrophotometer (Miran, 1A; Foxboro Co., East Bridgewater, MA).
Test compounds.
All administered drugs were purchased from Sigma-Aldrich (St Louis, MO). The drugs evaluated were NMDA and MK801 (dizocilpine). MK801 was dissolved in saline, and NMDA was dissolved in an equimolar concentration of NaOH (1:1 molar ratio of NMDA and NaOH) in saline. Drugs were administered ip at a volume of 1.0 ml/kg body weight. Doses of MK801 were 0.1, 0.33, and 0.7 mg/kg, and doses of NMDA were 2.5 and 10 mg/kg. Dose ranges, routes, and preparations for each compound were determined from selected studies (NMDA-Bale et al., 2005b; Knapp et al., 2001; Willmore et al., 2001a,b; MK801-Willmore et al., 2001a,b; Hetzler and Burkhard, 1999; toluene-Boyes et al., submitted).
VEP testing.
For testing, rats (85–99 days) remained awake and were restrained using a plastic cone (Braintree Scientific, Braintree, MA) with portions removed to uncover the rat's eyes, nose, and ears. The rats were placed in a head-only exposure chamber with interior dimensions of 10 x 10 x 17 cm (width x depth x height) that contained a glass face enabling the stimulus screen to be viewed. Prior to the start of the testing session, rats were acclimated to the restraining device and head-only exposure chamber to allow their behavior to stabilize. VEPs were measured in 5-s epochs, amplified and band-pass filtered (1–100 Hz). A total of 25 epochs were averaged for each time point. Averaged evoked potentials were then submitted for spectral analysis using a computer-based system described in detail in Hamm et al. (2000). The spectral amplitude at the stimulus rate (F1) and twice the stimulus rate (F2) were recorded as dependent variables. Further details of the VEP testing apparatus and protocol are provided elsewhere (Boyes et al., submitted).
Procedures for drug dosing and toluene exposure.
For the SALINE/AIR, NMDA/AIR, MK801/AIR, SALINE/TOL, NMDA/TOL, and MK801/TOL groups, rats were injected (ip) with vehicle or one of the test compounds after obtaining baseline VEP measurements. A second VEP measurement was collected 10 min after injection and then inhalation exposure began to either 0 (clean air) or 2000 ppm toluene. Remaining VEP measurements were made at 16, 30, 40, and 52 min, after injection.
For the experiment where TOL exposure preceded MK801 treatment (see "Results" section), the following procedure was used. After the baseline VEP measurement was made, another VEP measurement was made 10 min later, right before toluene (2000 ppm) exposure started. VEP measurements were taken at 6 and 20 min after the onset of toluene exposure (corresponds to the 16- and 30-min postinjection time points of the other groups). Immediately after collection of the 20-min toluene exposure VEP, rats were injected (ip) with MK801 (0.1 mg/kg), and two more VEP measurements were made at 30 and 42 min after start of toluene exposure (corresponds to the 40- and 52-min postinjection time point of the other groups). Toluene exposure was stopped for all groups immediately after the last VEP waveform was collected.
Statistical analysis of VEP measurements.
Results were analyzed using a mixed-model two-factor ANOVA (PROC MIXED: SAS, Cary, NC) with treatment as a between-group factor and time as a repeated measure. The mixed model permits both dependent and independent comparisons. Data from vehicle-treated animals run concurrently with the drug and toluene-treated rats were combined to form a single SALINE/AIR control group (n = 10). The F1 and F2 values from NMDA/AIR and MK801/AIR treatment groups (all doses) were compared individually to the SALINE/AIR control group. The NMDA/TOL, MK801/TOL, and TOL/MK801 groups were compared to SALINE/TOL and SALINE/AIR groups for time and treatment interactions. The alpha value for the two-way ANOVA depended on the number groups with two measures (F1 and F2). For example, if four groups were compared in the analysis with two measures each, 
' = (0.05)/(4 x 2) = 0.00625. If the interaction was significant, a step-down analysis (post hoc Tukey test) was performed and step-downs were evaluated at = 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All rats remained awake and kept their eyes open throughout the testing session. Saline (ip) did not change the F1 or F2 amplitude with respect to the baseline measurement. The data from all the vehicle control rats (SALINE/AIR) were combined and are repeated for comparison to the other treatment groups in Figures 1, 2, and 4. The baseline VEP measurements indicated that the rats typically had a strong F2 response in comparison to the F1 response. With the exception of 0.7 mg/kg MK801, none of the administered compounds produced any significant changes in body temperature during the testing duration (data not shown).
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Effect of NMDA and MK801 on VEPs in Air-Exposed Animals
NMDA caused a dose-dependent increase in F1 amplitude and either increased or decreased F2 amplitude, depending on dose. Animals dosed with 2.5 mg/kg NMDA did not exhibit profound VEP waveform changes (Fig. 1A). Overall, treatment with 2.5 mg/kg NMDA significantly increased F1 and F2 amplitudes (Figs. 1B and 1C; n = 10, p < 0.005, two-way ANOVA). Within individual animals, the VEP waveforms in animals dosed with 10 mg/kg NMDA were noticeably different from the baseline VEP measurements (not shown). At 16 min after injection, F1 amplitude was increased by 250% in comparison to the SALINE/AIR group (p < 0.05, two-way ANOVA). As demonstrated previously (Bale et al., 2005b
), 10 mg/kg NMDA significantly decreased F2 amplitude through the 52-min testing session (n = 10, p < 0.001, two-way ANOVA).
VEP waveforms from animals dosed with 0.1 and 0.33 mg/kg MK801 were not significantly different from their baselines (Fig. 2A). The two lower doses of MK801 (0.1 and 0.33 mg/kg) did not significantly change F1 or F2 amplitude (Figs. 2B and 2C). The highest administered dose, 0.7 mg/kg, significantly decreased F2 amplitude and increased F1 amplitude during the 52-min testing period; this effect may be due to drug-induced hypothermia in these animals. A similar hypothermic effect in rats dosed with high levels of MK801 (3 mg/kg, ip) on flash-evoked potentials has been documented (Hetzler and Burkhard, 1999). As a result of this effect and other adverse behavioral effects, such as ataxia, observed at this dose, only five animals were administered 0.7 mg/kg MK801.
Effect of NMDA and MK801 on Toluene-Induced Changes in VEPs
The next series of experiments was designed to determine the role of the NMDA receptor in toluene-induced changes in VEP by determining the extent to which NMDA, an agonist, or MK801, an antagonist, of this receptor altered the toluene response. Toluene, 2000 ppm, was selected as the exposure concentration based on a previous study (Boyes et al., submitted) evaluating effects of this compound on pattern-elicited VEPs measured under conditions identical to the present study. This toluene concentration produces a significant decrease in F2 amplitude during an hour-long exposure period. The results presented above demonstrate that both NMDA and MK801 (at 0.7 mg/kg) reach the central nervous system and exert effects within 1 h.
Based on effects of NMDA and MK801 on F1 and F2 amplitudes from the air-exposed animals, 2.5 mg/kg NMDA and 0.1 mg/kg MK801 were utilized in coadministration studies with toluene. These doses of NMDA and MK801 did not alter body temperature or produce any measured adverse effects except changes in F2 amplitude (2.5 mg/kg NMDA only). Additionally, 2.5 mg/kg NMDA significantly increases F2 amplitude, whereas toluene decreases F2 amplitude (Boyes et al., in preparation). Although 0.33 mg/kg of MK801 did not significantly affect F1 or F2 VEP amplitudes, in a pilot study animals receiving 0.33 mg/kg MK801 and 2000 ppm toluene experienced lower body temperatures, which may affect the VEP (Boyes et al., 1985; Hetzle et al., 1988).
Toluene concentrations measured in the chamber during VEP testing were 2052 ± 60 ppm (mean ± SD). The measured VEP waveforms decreased over time with toluene exposure (Fig. 3A). After injection of saline, toluene at 2000 ppm significantly decreased F2 amplitude (n = 11, p < 0.001, two-way ANOVA) across the 52-min test session without changing F1 amplitude, relative to baseline (Figs. 3B and 3C). When animals were treated with 2.5 mg/kg NMDA prior to toluene (2000 ppm) exposure, the F2 amplitude was not significantly changed with respect to the saline/toluene-exposed animals. Similarly, the effects of NMDA with toluene on F1 amplitude were not different from those of NMDA alone (n = 13, p < 0.001, two-way ANOVA). Thus, NMDA-mediated F1 amplitude increases were not affected by toluene exposure.
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Since the SALINE/TOL and MK801 (0.1–0.7 mg/kg)/AIR groups did not significantly alter F1 amplitudes, it was not surprising that no F1 effects were observed in animals treated with 0.1 mg/kg MK801 followed by toluene. The toluene-mediated decrease in F2 amplitude was significantly blocked by the MK801 pretreatment (n = 9, p < 0.001, two-way ANOVA, Fig. 3C). The measured F2 amplitude in the MK801/TOL group was not significantly different from the air-saline animals (compare to Figs. 1 and 2).
Based on the observed interaction between MK801 and toluene on the F2 amplitude, it was hypothesized that MK801 was either precluding toluene from interacting with the NMDA receptor or that toluene acts at a different site "upstream" of the NMDA receptor to produce its effect. To test this hypothesis, the treatment order was reversed. A new group of animals was exposed to toluene first and then challenged with 0.1 mg/kg MK801. Animals were challenged with 0.1 mg/kg MK801 20 min after the onset of toluene exposure, and VEP measurements were made at the same time points as the other groups in order to compare the data from previous treatment groups. When 0.1 mg/kg MK801 was administered after toluene inhalation, VEP waveform amplitudes decreased after the onset of toluene exposure, and the signal remained depressed until the end of the exposure session (Fig. 4A). Spectrally transformed VEPs revealed that the F2 amplitude remained depressed, similar to the SALINE/TOL animal group (Fig. 4B). This treatment group did not exhibit changes in F1 amplitude (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results of the present study are consistent with the involvement of NMDA receptors in toluene-mediated changes in the steady-state pattern-elicited VEP. Pretreatment of animals with the NMDA receptor antagonist MK801 was sufficient to prevent decreases in F2 amplitude induced by toluene. However, if exposure to toluene occured prior to MK801 treatment, this drug was ineffective at reversing the toluene-induced decrease in F2 amplitude. The agonist NMDA was without effect on toluene alterations in VEPs, even though by itself it increased F1 and decreased F2 amplitude. Thus, NMDA receptors contributed to pattern-evoked visual potentials, and actions of toluene, at least in part, appeared to be mediated by effects on this receptor.
When 2.5 mg/kg NMDA was administered prior to the onset of toluene exposure, F2 amplitude decreases mediated by toluene were not suppressed. This result was unexpected because toluene inhibits the NMDA receptor in vitro (Cruz et al., 1998) it was hypothesized that NMDA would counteract the effects of toluene on the VEP. However, as noted above, higher NMDA doses decrease F2 amplitude, similar to the effect of TOL exposure on this parameter. Thus, even in the absence of coexposure to the two compounds (TOL and NMDA), effects of NMDA were opposite of what would be predicted based on in vitro experiments, if direct antagonism of NMDA receptors mediates TOL effects on pattern-evoked VEPs.
MK801, like toluene, inhibits the NMDA receptor, in vitro. Thus, it was predicted that MK801 would reduce F2 amplitude, like toluene. MK801 administered alone, did not significantly alter F1 or F2 amplitude at doses that were without effect on temperature regulation. When 0.1 mg/kg MK801 was administered 10 min prior to the onset of toluene exposure, toluene-induced F2 amplitude decreases were blocked. There was a slight but insignificant increase in the F2 amplitude prior to the onset of toluene exposure. This effect was not observed in animals treated with MK801 alone. However, the significant effect of MK801 blocking toluene-mediated F2 amplitude decreases indicates that there is an interaction between MK801 and toluene. Additionally, this result suggests one of two possible mechanisms; either toluene precludes MK801 interaction with the NMDA receptor or toluene acts at a site proximal to the NMDA receptor in the formation of the VEP response.
If MK801 and toluene act at distinct sites, then MK801 treatment after toluene exposure should also attenuate F2 amplitude decreases mediated by toluene. However, F2 amplitude remained decreased in animals treated with MK801 after toluene exposure, similar to toluene-only exposed animals. Thus, this result suggests that toluene acts directly on the NMDA-glutamate receptor in vivo.
Although toluene and MK801 may both act directly on the NMDA-glutamate receptor, this does not necessarily indicate that the two compounds share a common binding site. It is widely known that MK801 binds the NMDA receptor in the ion channel pore. To date, potential locations on the NMDA receptor for the actions of toluene and other VOCs have not been determined. However, there is indirect evidence that the active site for toluene is not in the channel pore. Like toluene, ethanol inhibits NMDA receptor function in vitro. One study isolated an ethanol-sensitive amino acid (phenylalanine in the 639 position) on the NR1 subunit of the NMDA receptor (Ronald et al., 2001) that when mutated to an alanine results in decreased sensitivity of the receptor to ethanol. Similarly, unpublished studies conducted with toluene indicate that NMDA receptors containing the phenylalanine mutation in the 639 position of the NR1 subunit was also less sensitive to toluene (Bale and Woodward, personal observations). The 639 phenylalanine is located in a nonpore-facing domain of the NMDA receptor. These findings indicate that toluene-sensitive regions on the NMDA receptor may be in nonpore-facing areas and may not overlap with the MK801 binding site on the NMDA receptor. One possibility that may contribute to the in vivo interactions between MK801 and toluene at NMDA receptors is that once a compound binds, the receptor may undergo a conformational change that precludes the other compound from binding. Alternatively, other toluene-sensitive sites may overlap with the MK801 binding site, which would also result in functional antagonism.
The results of the present study suggest that the NMDA-glutamate receptor is one of the sites where toluene acts to alter VEPs. However, in a previous study by Shelton and Balster (2004), C57BL/6J and DBA/2J mice were evaluated to determine if toluene would fully substitute for MK801 in mice trained to discriminate MK801. It was found that there was little substitution between the two drugs, and the authors concluded that inhibition of the NMDA receptor may not be the primary action underlying the observed toluene-induced behavioral effects. Furthermore, Wiley et al. (2003) demonstrated that administration of NMDA to toluene-exposed mice did not affect the severity of seizures in the withdrawal period in comparison to the air control group. Although neither study indicates a direct interaction between toluene and the NMDA receptor, the studies were conducted in a different animal model from the present study and using behavioral rather than physiological measures. It could be possible that in the behavioral measures tested by Shelton and Balster (2004) and Wiley et al. (2003), NMDA receptor inhibition by toluene may not be a primary factor in contributing to these behaviors.
In vitro studies have illustrated that, in addition to NMDA receptors, toluene modulates many other receptors and ion channels, including nAChRs. A recent study demonstrated that nicotine, an agonist to the nAChR, attenuated some ethanol-mediated effects on flash-evoked potentials (Hetzler and Martin, 2006). Like toluene, ethanol inhibits homomeric 7 nAChRs (Yu et al., 1996). Furthermore, ethanol and toluene both produce changes in evoked potentials (ethanol—Hetzler and Bednarek, 2001; Hetzler et al., 1981; toluene—Boyes et al., submitted). Based on the ethanol-nicotine interactions in the flash-evoked potential and relative similarities in molecular sites of action for ethanol and toluene, in vitro, it is hypothesized that the nAChR may also be involved in toluene-mediated changes in the steady-state VEP. However, minimal effects in steady-state VEPs were noted with nicotine administration to rats (Bale et al., 2005b). In comparison, several treatment-related changes in flash-evoked potentials FEPs were observed in rats dosed with nicotine (Hetzler and Theinpeng, 2004). If other receptor systems such as the nAChRs are involved, it would partially explain why administration of NMDA alone did not block toluene-mediated F2 amplitude decreases. Selective targeting of toluene onto other sites could be a compensatory mechanism when the NMDA receptor cannot be inhibited.
In summary, toluene appears to interact with the NMDA receptor in reducing the amplitude of steady-state pattern-elicited VEPs in Long-Evans rats. MK801 administered prior to toluene exposure blocked toluene-mediated F2 amplitude decreases, and when administered after toluene exposure, did not change F2 inhibition in comparison to toluene-only exposure group. Although an interaction was demonstrated between the NMDA receptor and toluene in this physiological measure, it is still unclear if this molecular site is the primary site of action for producing the toluene-mediated F2 amplitude decreases. Additional studies, similar to the present study, need to be conducted in order to elucidate further and understand the major molecular sites of action for toluene and other VOCs.
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NOTES |
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2 Present address: Product Safety and Dermal Clinical Research, Colgate-Palmolive Company, Piscataway, NJ 08854.
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ACKNOWLEDGMENTS |
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The authors acknowledge and appreciate the outstanding technical assistance of Ms Marty Carroll and Mr Mark Bercegeay in the conduct of the present studies. In addition, we appreciate the useful comments and suggestions of Dr Andrew Geller (USEPA) and Dr Bruce Hetzler (Lawrence University, Appleton, WI) on an earlier version of this manuscript. The information in this document has been funded wholly by the U.S. Environmental Protection Agency. Preliminary data were presented previously at the 22nd International Neurotoxicology Meeting in Research Triangle Park, NC and at the 45th Society of Toxicology Meeting in San Diego, CA. This document has been reviewed by the National Health and Environmental Effects Research Laboratory and is approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
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