Biphasic Effects of 1,1,1-Trichloroethane on the Locomotor Activity of Mice: Relationship to Blood and Brain Solvent Concentrations

D. Alan Warren*,1, Scott E. Bowen{dagger}, W. B. Jennings*, Cham E. Dallas* and Robert L. Balster{dagger}

* Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, Georgia 30602–2352; and {dagger} Department of Pharmacology and Toxicology, Medical College of Virginia, Richmond, Virginia 23298–0813

Received January 15, 2000; accepted April 12, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite the central nervous system (CNS) being a target of virtually all solvents, few solvents have been thoroughly studied for their effects on unlearned animal behaviors. Of the solvents that have been studied, little is known about the relationship of exposure concentration to behavioral effect, and quantitative data relating the toxicologically important target organ (i.e., brain) dose to behavioral effect are almost non-existent. To examine the concentration- and time-dependency of effects of 1,1,1-trichloroethane (TRI) on behavior, male albino Swiss-Webster mice were exposed to TRI (500–14,000 ppm) in static inhalation chambers for 30 min, during which locomotor activity was measured. Separate mice were exposed to the same concentrations under identical conditions for 6, 12, 18, 24, and 30 min, to determine blood and brain concentrations versus time profiles for TRI. This allowed for the relationships between blood and brain concentrations of TRI and locomotor activity to be discerned. The lowest TRI concentrations studied (500–2000 ppm) had no statistically significant effect on activity, intermediate concentrations (4000–8000 ppm) increased activity immediately to levels that remained constant over time, and higher concentrations (10,000–14,000 ppm) produced biphasic effects, i.e., increases in activity followed by decreases. 1,1,1-Trichloroethane concentrations in blood and brain approached steady-state equilibria very rapidly, demonstrated linear kinetics, and increased in direct proportion to one another. Locomotor activity increased monophasically ({approx}3.5-fold) as solvent concentrations increased from approximately 50–150 µg/g brain and µg/ml blood. As concentrations exceeded the upper limit of this range, the activity level declined and eventually fell below the control activity level at approximately 250 µg/g brain and µg/ml blood. Regression analyses indicated that blood and brain concentrations during exposure were strongly correlated with locomotor activity, as were measures of internal dose integrated over time. The broad exposure range employed demonstrated that TRI, like some classical CNS depressants, is capable of producing biphasic effects on behavior, supporting the hypothesis that selected solvents are members of the general class of CNS depressant drugs. By relating internal dose measures of TRI to locomotor activity, our understanding of the effects observed and their predictive value may be enhanced.

Key Words: 1,1,1-trichloroethane; methyl chloroform; locomotor activity; biphasic effects; internal dose; toxicokinetics..


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The extent of solvent exposure by inhalation ranges from low concentrations produced by the proper use of commercial products to dangerously high concentrations encountered during recreational solvent abuse and accidents or misuse in industry (Gerr and Letz, 1998Go; NIDA, 1996). Following high-level exposure, a general depression of the central nervous system (CNS) may occur, with effects ranging from impaired cognition and motor skills to death. Acute solvent exposures to human volunteers have impaired performance in tests of manual dexterity, eye-hand coordination, perceptual speed, and reaction time and have produced lightheadedness and imbalance (Gamberale and Hultengren, 1973Go; Mackay et al., 1987Go; Stewart et al., 1961Go; Torkelson et al., 1958Go). Exposure limits have been established to protect workers from such effects (ACGIH, 1991), but the majority of these are based on a limited number of poorly documented responses to occupational exposures, often with little or no experimental confirmation from animal studies. In addition, few animal studies have evaluated the toxicologic properties of solvents under exposure conditions typical of solvent abuse (Balster, 1987Go; Bruckner and Peterson, 1981Go).

With the exception of the prototypical aromatic hydrocarbon, toluene, effects of 1,1,1-trichloroethane (TRI) on animal behavior are better characterized than those of other industrial solvents. 1,1,1-Trichloroethane has produced changes in rates of food-reinforced lever-pressing of rats and mice (Balster et al., 1982Go; Moser and Balster, 1986Go; Warren et al., 1998Go), altered performance on a match-to-sample discrimination task in baboons (Geller et al., 1982Go), and impaired the ability of rats and mice to avoid shock by lever-pressing (Mullin and Krivanek, 1982Go) and remain atop an inverted screen (Moser and Balster, 1985Go). The results of these and other animal studies with TRI are consistent with the view that the profile of acute effects of this vapor resembles the effects of classical CNS depressant drugs, which typically produce a biphasic increasing and decreasing effect on locomotor activity in rodents (Evans and Balster, 1991Go; Wood and Colotla, 1990Go). However, studies of the effects of TRI on locomotor activity have produced conflicting results. Two studies in rats and mice failed to find evidence for motor activity increasing effects (Adams et al., 1950Go; Kjellstrand et al., 1985Go), whereas modest increases but no biphasic effects were seen in another study (Kjellstrand et al., 1990Go). In a recent study that examined a wide range of TRI concentrations, biphasic concentration-effect curves were obtained for motor activity as measured after 30-min exposure sessions (Bowen and Balster, 1996Go). One goal of the present study was to utilize one of the exposure protocols in this recent report to examine more closely both the concentration- and time-dependency of effects of TRI on mouse locomotor behavior.

Because of interspecies differences in TRI pharmacokinetics, the extrapolation of potential human risk from animal toxicity data without some knowledge of the corresponding target organ dose or a dose surrogate may involve uncertainty. This uncertainty can be reduced by data that describe, for example, the blood and brain concentrations of a solvent and their relationships to corresponding levels of behavioral toxicity. Unfortunately, few studies have generated data of this type. Therefore, the other major goal of the present study was to measure the time course for blood and brain concentrations of TRI under exposure conditions identical to those used for the behavioral studies. This enabled the relationship between locomotor activity and blood and brain solvent concentrations to be described for the first time. By relating internal dose measures to locomotor activity, our understanding of the effects observed and their predictive value may be enhanced.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
1,1,1-Trichloroethane of 97%+ purity was obtained from Aldrich Chemical Co., Inc. (Milwaukee, WI) and Fisher Scientific (Fair Lawn, NJ). Burdick and Jackson Brand, High Purity Solvent isooctane was obtained from Baxter Healthcare Corp. (Muskegon, MI).

Animals.
Male, albino Swiss-Webster (CFW®) mice (25–35 g) were obtained from Charles River Breeding Laboratories in Raleigh, NC. Mice were housed singly in polypropylene cages with hardwood chip bedding (Sani-Chips, Montville, NJ) and stainless steel wire lids in temperature- (23 ± 3°C) and humidity- (45 ± 10%) controlled rooms with 12-h light-dark cycles (light: 0700–1900 h). Mice were acclimated for at least 7 days prior to use, and allowed food (Agway Prolab RMH 3000, Syracuse, NY) and tap water ad libitum. All mice used in this study were chemically naïve.

Exposure apparatus.
Inhalation exposures were conducted in several identical static chambers, which have been described previously (Moser and Balster, 1986Go). The chambers were 29-l chromatography jars (Pyrex® 6942) with acrylic covers. A fan was projected into each chamber above a wire basket that supported a piece of filter paper. A mouse was placed in the bottom of the chamber, the top put in place, and a calculated amount of solvent injected onto the filter paper, which volatilized with the aid of the fan to produce the desired vapor concentration. Vapor concentrations reached target levels in 1–3 min and remained stable for the duration of exposure. Concentrations within the chambers were continuously monitored with a Miran 1A infrared spectrometer (The Foxboro Co., East Bridgewater, MA) during locomotor activity measurements, and by gas chromatographic analyses of air samples serially extracted with a 1.0 ml gas-tight syringe and a 12-in. needle during blood and brain concentration determinations. The extracted air samples were directly injected into a Shimadzu GC-14A gas chromatograph (GC) equipped with an electron capture detector (ECD), and TRI concentrations were calculated from standard curves based on atmospheres of known concentration prepared in 9-l glass jars. Chromatographic analyses were conducted using a stainless-steel column (182 x 0.317 cm) packed with 3% OV-17 (100–120 mesh) (Alltech Associates, Inc., Deerfield, IL). The GC operating conditions were injection port temperature, 150°C; column temperature, 80°C; ECD temperature, 360°C; flow rate for argon:methane (95:5) carrier gas, 60 ml/min.

Locomotor activity measurements.
Five mice per TRI concentration were adapted to the static exposure chambers for 30 min on the day prior to solvent exposure. Locomotor activity was measured during solvent exposure, beginning immediately upon vapor generation. Mice were free to move about inside the chambers and locomotor activity was measured via 2 sets of photocells that bisected each chromatography jar 2.5 cm above the base. During the 30-min exposure periods, locomotor activity was defined as the total number of photocell breaks. Locomotor activity was recorded in 1-min bins in order to maximize the sensitivity to detect temporal changes. All experiments were conducted during the light phase of the light-dark cycle and at the same time each day for all exposure groups.

Blood and brain sampling.
Blood and brain solvent concentrations were determined in mice during exposure to the same concentrations at which locomotor activity was measured. As in the behavioral study, mice received one 30-min chamber adaptation period on the day prior to solvent exposure. At 6, 12, 18, 24, and 30 min after the start of exposure, a mouse was removed from the exposure chamber and sacrificed by CO2 asphyxiation. Blood (0.2–0.5 ml) was withdrawn from the inferior vena cava with a 1-ml tuberculin syringe and a 25-gauge needle and whole brains were collected ({approx}0.4 g) within 1–2 min of sacrifice. Blood and brain samples were immediately placed into chilled scintillation vials containing 8 ml isooctane and 2 ml 0.9% saline. Four mice were sacrificed at each time point during exposure to each TRI concentration, except at 10,000 and 14,000 ppm, where only 2 mice were sacrificed at each time point. As with the measurement of locomotor activity, one mouse at a time was placed in an exposure chamber.

Blood and brain analysis.
Blood and brain samples were analyzed for TRI content by a method originally described by Chen et al. (1993). Briefly, blood and brain samples were homogenized as quickly as possible (5–10 s) with an Ultra-Turrax® homogenizer (Tekmar Co., Cincinnati, OH) to minimize volatilization of TRI. Then the samples were vortex-mixed for 30 s and the homogenates centrifuged at 2500 x g for 10 min at 4°C in the capped scintillation vials. An aliquot of the isooctane layer (5–10 ml) was either transferred directly to an 8-ml headspace vial or first diluted with isooctane. The vials were capped immediately with Teflon®-lined latex rubber septa in aluminum seals and crimped tightly. Analyses of TRI were made with a Sigma Model 300 GC (Perkin-Elmer Co., Norwalk, CT) equipped with an HS6 headspace sampler (headspace sampler temperature, 70°C) and an ECD, under the same chromatographic conditions previously described. 1,1,1-Trichloroethane concentrations were calculated from daily-prepared standard curves made by diluting various amounts of TRI in isooctane and corrected for the percent recovery characteristic of blood and brain samples. The percent recovery of TRI from blood (95.5%) and brain (94.3%) samples was previously determined by You et al. (1994) by injecting freshly harvested blood and brain samples with 4 µl of a solution containing 1 mg TRI per 1 ml isooctane and analyzing the samples as previously described. The limit of detection for TRI was {approx}0.5 ng in 8 ml of air.

Data analysis.
Locomotor activity during exposure to each TRI concentration was plotted relative to control activity (Fig. 1Go). A boundary zone encompassing the 95% confidence interval (±2 SD) for the control activity mean is represented by the shaded region in Figure 1Go. Repeated measures analysis of variance (RMANOVA) on total session activity counts was used to compare control activity to activity during TRI exposures. Bonferroni multiple comparisons (Johnson and Wickern, 1988Go) were then performed to ascertain those concentrations at which activity differed from control. Statistical analysis was conducted with GB-STAT for MS Windows Version 5.0.



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FIG. 1. Locomotor activity during exposure to a subset of nine TRI concentrations, relative to control activity. Locomotor activity was recorded in 1-min bins. Each data point represents the mean of 5 mice. The boundary zone encompassing the 95% confidence interval (± 2, SD) for mean control activity is represented by the shaded region.

 
Using data from all 9 exposure groups, mean blood and brain TRI concentrations measured at comparable times and equivalent exposure levels were plotted against one another (Fig. 2Go). The resulting scatter plot was subjected to least-squares linear regression analysis and the degree of correlation determined by comparing tr values based on sample data with values in a t-distribution table (Gad and Weil, 1986Go). The same mean blood and brain solvent concentrations were also plotted against locomotor activity (number of counts during TRI exposure minus the number of counts under control conditions) as measured at comparable times and equivalent exposure levels (Fig. 3Go). For example, the TRI concentration in blood after 12 min of exposure to 4000 ppm was plotted against the number of locomotor activity counts (relative to control) that occurred between the 11th and 12th min of exposure to 4000 ppm. Additionally, within each exposure concentration we plotted the total number of locomotor activity counts (relative to control) over the entire 30-min exposure period against the maximum blood and brain concentrations of TRI achieved during exposure (Cmax), as well as measures of time integrals of internal dose (areas under the blood and brain concentration vs. time curves [AUC0–30]) calculated by the trapezoidal rule (Rowland and Tozer, 1980Go). Maximum blood and brain concentrations (Cmax) were obtained by visual inspection of pharmacokinetic data. SigmaPlot Scientific Graphing Software (V. 2.01, Jandel Corporation) was used to fit the data with 1st (Fig. 2Go) and 2nd order (Figs. 3 and 4GoGo) regression lines. The minimum level of significance was set at p <= 0.05 for all statistical tests.



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FIG. 2. Scatter plot relating blood and brain concentrations of TRI as measured at comparable times and exposure levels. Each data point (circle) represents the mean blood and brain concentration of 2 (10,000 and 14,000 ppm) or 4 (500, 1000500, 2000, 4000, 6000, 8000, and 12,000 ppm) mice after 6, 12, 18, 24, or 30 min of exposure to one of nine TRI concentrations. The equation of the 1st order regression line: y = 0.954x + 5.095.

 


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FIG. 3. Scatter plots relating locomotor activity and (a) blood and (b) brain concentrations of TRI. Each data point represents the mean blood or brain concentration of 2 (10,000 and 14,000 ppm) or 4 (500, 1000500, 2000, 4000, 6000, 8000, and 12,000 ppm) mice after 6, 12, 18, 24, or 30 min of exposure to 500 (circle), 1000 (square), 2000 ({uparrow} triangle), 4000 ({downarrow} triangle), 6000 (diamond), 8000 (hexagon), 10,000 (circle +), 12,000 (square +) or 14,000 ppm TRI (triangle +), as well as the mean locomotor activity (relative to air control) of 5 mice at comparable times and equivalent exposure levels. The equations and correlation coefficients for the 2nd order regression lines are y = –6.755 + 0.485x – 0.0017x2 (blood; r = 0.81); y = –4.443 + 0.412x – 0.0014x2 (brain; r = 0.80).

 


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FIG. 4. Scatter plots relating locomotor activity and internal measures of TRI exposure. Each data point represents an internal measure of TRI exposure (blood Cmax, brain Cmax, blood AUC, brain AUC), as well as the total locomotor activity count (relative to air control) during 30-min exposures to 500 ppm (white circle, black circle), 1000 ppm (white square, black square), 2000 ppm (white {uparrow} triangle, black {uparrow} triangle), 4000 ppm (white {downarrow} triangle, black {downarrow} triangle), 6000 ppm (white diamond, black diamond), 8000 ppm (white hexagon, black hexagon), 10,000 ppm (white circle +, black circle +), 12,000 ppm (white square +, black square +) or 14,000 ppm TRI (white triange +, black triangle +). Cmax values are the maximum blood and brain concentrations of TRI achieved during 30-min exposures; AUC values are the areas under the blood and brain concentration vs. time curves from 0 to 30 min. The equations and correlation coefficients for the 2nd order regression lines are: y = –188.323 + 11.063x – 0.0333x2 (blood Cmax, r = 0.94); y = –131.496 + 9.197x – 0.0247x2 (brain Cmax, r = 0.95); y = –176.204 + 0.455x – 0.0000565x2 (blood AUC, r = 0.95); y = –157.226 + 0.439x – 0.0000558x2 (brain AUC, r = 0.93).

 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Locomotor activity during exposure to a subset of the nine TRI concentrations is shown, relative to control activity in Figure 1Go. The results clearly demonstrate that inhalation of TRI produces concentration-dependent biphasic stimulant and depressant effects on the locomotor activity of mice (F(9,40) = 11.18, p < 0.0001). Locomotor activity during exposures to 500–2000 ppm did not statistically differ from control activity (p > 0.05), which remained at a constant level for 30 min. Exposure to 4000 ppm did increase activity (p < 0.01), as did all concentrations in excess of this lowest-observed-effect-level. Exposures from 4000–8000 ppm elevated activity in the first minute of exposure, and this increased activity was sustained at roughly the same level for the remainder of the exposure. Higher concentrations (10,000, 12,000, and 14,000 ppm) had a biphasic effect on locomotor activity. At these concentrations, activity initially increased monophasically with the time to peak activity level being dependent upon the inhaled concentration. With continued exposure, activity decreased at a rate that was also concentration-dependent, and fell below the control level during the latter part of the exposure to 14,000 ppm. Only at 10,000, 12,000, and 14,000 ppm did any mice cease activity continuously for greater than 1 min. Ataxia was occasionally observed at the 2 highest concentrations. No seizures or fatalities occurred in any of the exposed animals. Virtually identical locomotor activity results were obtained in a study that employed a within-subjects design in which 10 mice were exposed to the same TRI concentrations as those used herein, in an ascending order, and to only one concentration per test day (unpublished observation).

The concentrations of TRI in blood and brain as a function of the degree and duration of exposure are shown in Table 1Go, and the Cmax and AUC values based on these data are shown in Table 2Go. As expected from the abrupt changes in locomotor activity upon the initiation of exposure, TRI was rapidly absorbed from the lung as evidenced by its substantial presence in blood and brain after just 6 min of exposure. 1,1,1-Trichloroethane concentrations in blood and brain approached steady-state equilibria very rapidly, as blood and brain concentrations at 6 min averaged 77% of those at 30 min, and those at 12 min rarely differed from those at 30 min. With a few isolated exceptions, TRI demonstrated linear kinetics over the broad exposure range as blood and brain concentrations and AUC and Cmax values were roughly proportional to the inhaled TRI concentrations.


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TABLE 1 Blood and Brain Concentrations of 1,1,1-Trichloroethane during Inhalation Exposure
 

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TABLE 2 Pharmacokinetic Parameters during Inhalation Exposure
 
As expected for a well-perfused and lipid-rich organ such as the brain, its pattern of TRI accumulation was very similar to that of the blood. The scatter plot relating mean blood and brain solvent concentrations (Fig. 2Go) clearly illustrates that brain levels of TRI increase in direct proportion to blood levels ({approx}1:1), making blood and brain solvent concentrations highly correlated (r = 0.97, df = 43, t = 24.89, p < 0.001). These findings strongly suggest that the blood concentration of TRI may serve as an effective surrogate for the target tissue (brain) concentration, making it a reasonable dose-metric to relate to locomotor activity.

The scatter plots relating mean blood and brain solvent concentrations to locomotor activity (Fig. 3Go) illustrates a monophasic increase in activity as solvent concentrations increase to approximately 150 µg/g brain and µg/ml blood. At higher solvent concentrations, activity decreases before eventually falling below the control level at approximately 250 µg/g brain and µg/ml blood. Based on the no-observed-effect-level of 2000 ppm and the lowest-observed-effect-level of 4000 ppm, the estimated threshold concentration for locomotor activity effects under our experimental conditions is approximately 50 µg/g brain or µg/ml blood. The biphasic relationships between blood and brain concentrations and locomotor activity were well described by second-order regression curves having correlation coefficients of 0.81 and 0.80, respectively.

While the scatter plots in Figure 3Go demonstrate that blood and brain TRI concentrations reasonably predict locomotor activity at various points in time during exposure, those in Figure 4Go suggest that other measures of internal dose (i.e., blood and brain Cmax and blood and brain AUC) are strongly correlated, and are thus highly predictive of the locomotor activity response when viewed in the aggregate, i.e., over the entire 30-min exposure period. Similar to those for blood and brain TRI concentrations, biphasic relationships between blood and brain Cmax or AUC and locomotor activity were well described by second-order regression curves having correlation coefficients ranging from 0.93 to 0.95.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Consistent with results shown for inhaled toluene and intraperitoneally administered ethanol (Hinman, 1987Go; Middaugh et al., 1992Go; Wood and Colotla, 1990Go), the results of this study indicate that inhaled TRI produces concentration- and time-dependent biphasic effects on locomotor activity. The lowest concentrations studied (500–2000 ppm) had no statistically significant effect on activity. Intermediate concentrations (4000–8000 ppm) increased activity immediately to levels that remained constant over time. Higher concentrations (10,000–14,000 ppm) produced biphasic effects, i.e., increases in activity followed by decreases.

In an earlier report by Kjellstrand et al. (1985), groups of 5 mice were repeatedly exposed to either TRI (890, 1300, 2000, or 4000 ppm), methylene chloride, perchloroethylene, toluene, or trichloroethylene for 1 h. The authors described the activity pattern for TRI as "...simple, consisting of an increase and a decrease closely related to the increase and decrease of the chamber concentration." They also noted that TRI was less effective at increasing activity than the other solvents, as its effectiveness was limited to the two highest concentrations tested. In an unpublished study sponsored by Dow Chemical Co., Midland, MI, Albee et al. (1990) also observed an increase in the motor activity of rats exposed to 4000 ppm TRI for 6 h per day for 4 days (ATSDR, 1994Go). Evidence of a biphasic effect for TRI comes from a study of schedule-controlled operant behavior in which both increases and decreases in the rate of lever-pressing for milk delivery were observed following inhalation exposure (Moser and Balster, 1986Go). This result supports a later report by Bowen and Balster (1996) in which intermediate TRI concentrations (2,500–7,500 ppm) were shown to increase locomotor activity, with the higher concentrations (> 10,000 ppm) producing decrements in locomotor activity.

Biphasic effects have also been documented in humans for TRI, as well as for other volatile, lipophilic agents such as ethyl alcohol, halothane and xylene (Kalant, 1978Go; Roth, 1979Go; Seppalainen et al., 1981Go). Body sway was decreased and reaction time improved in volunteers exposed to 200 ppm TRI, while both deteriorated at 400 ppm (Arlien-Soborg, 1992Go). 1,1,1-Trichloroethane has also been reported to have a biphasic effect on human equilibrium (Savolainen et al., 1982Go). In addition, exposure to 175 and 350 ppm TRI actually enhanced performance of volunteers in a test of distractibility of attention and concentration (Mackay et al., 1987Go).

Since most biphasic effects of solvents in humans clearly involve the CNS, there is little doubt that they are related to the accumulation of agents in the brain. Many behavioral effects in laboratory animals, however, while generally considered to indicate specific CNS effects, could be produced by actions on other organ systems or the sensory-stimulative properties of the agents themselves (Tilson and Mitchell, 1984Go). Recognizing this, Kjellstrand et al. (1985) exposed mice to an intense odorant stimulus (cologne) that was without effect on the locomotor activity of mice, indicating that solvent effects on activity were unlikely due to odor. This is further supported by observations of increased activity when toluene and ethanol are administered by a route other than inhalation (Middaugh et al., 1992Go; Wood and Colotla, 1990Go). Since the lowest-observed-adverse-effect-level for acute TRI exposure in any rodent organ system other than the CNS is 8000 ppm (ATSDR, 1994Go), it is also unlikely that effects on other organ systems are responsible for TRI-induced activity changes. Additionally, solvent-induced toxicities other than the classical behavioral syndrome are usually metabolite mediated (Andrews and Snyder, 1991Go), and TRI is metabolized to a very limited degree (Schumann et al., 1982aGo,bGo). Moreover, increases in activity, as opposed to decreases, are difficult to interpret as the mere disruption of activity by a nonspecific stressor such as irritation. Rather, activity increases would appear to reflect a biologically relevant event in the CNS, such as activation of the hypothalamo-pituitary-adrenal axis or the release of higher center control (Glowa, 1990Go). Therefore, we believe the biphasic effect observed in the present study was dependent on the deposition of TRI in the brain.

Hinman (1987) reported that toluene's biphasic effect on locomotor activity was consistent with the hypothesis that such behavior is dependent on the level of toluene in the CNS. He concluded that locomotor activity is increased at low CNS levels, while at higher CNS levels the hyperactivity is attenuated. Since Hinman's conclusion was based on a comparison of his locomotor activity data to time-courses of blood and brain concentrations determined by other investigators (Bruckner and Peterson, 1981Go; Benignus et al., 1981Go), no attempt was made to quantitatively relate internal solvent concentrations to locomotor activity. On the basis of the present study, however, we can conclude that locomotor activity counts increase roughly 3.5-fold as TRI concentrations increase from approximately 50–150 µg/g brain and µg/ml blood. As concentrations exceed the upper limit of this range, activity levels decline until they reach control level at approximately 250 µg/g brain and µg/ml blood. As expected for a well-perfused organ with a tissue:blood partition coefficient near unity (0.80) (Reitz et al., 1987Go), brain TRI concentrations seemingly paralleled those of the blood. As a result, blood TRI concentrations also appear to be a reliable index of the locomotor activity level, agreeing with similar conclusions made in correlative studies of other behaviors with toluene and TRI (Bruckner and Peterson, 1981Go; Warren et al., 1998Go).

The finding that increases in locomotor activity were observed in the first minute of exposure is not surprising, since TRI has been shown to be rapidly absorbed by the respiratory system. Dallas et al. (1989) have reported that arterial blood levels of TRI were quite high in rats within 2 min of exposure to 50 or 500 ppm. Furthermore, TRI was detected in the arterial blood of men within 10 s of exposure to 250 or 350 ppm (Astrand et al., 1973Go). Therefore, TRI would be expected to rapidly and extensively accumulate in the well-perfused and lipid-rich mouse brain. At intermediate exposure concentrations (4000–8000 ppm), locomotor activity changed very little over time, as did blood and brain concentrations of TRI, following a brief period of rapid uptake and distribution. At higher exposure levels (>= 10,000 ppm), activity steadily increased for a period of time that was inversely proportional to exposure concentration, then declined at a rate proportional to exposure concentration. The fact that locomotor activity only increases over time at the highest exposure concentrations suggests that the rate of TRI uptake by the brain may be the driving force behind this behavior, rather than absolute concentration. The rate of uptake has previously been implicated as a factor in m-xylene-induced body sway (Riihimaki and Savolainen, 1980Go) and diazepam-induced impairment of psychomotor skills (Linnoila and Mattila, 1973Go). Furthermore, the time course of solvent deposition in the brain suggests that the locomotor activity decrease seen at higher exposure concentrations occurs only after a critical target tissue concentration is reached, which we estimate to be 150 µg/g. If this were true, it would explain the failure of some previous investigations to show a biphasic response pattern, as concentrations used were not likely to result in brain levels sufficient to elicit such a response.

Attempts such as the present study to relate solvent pharmacokinetics to a behavioral effect have been few, with the exception of those studies with ethanol (Hurst and Bagley, 1972Go; Jones and Vega, 1972Go; Radlow and Hurst, 1985Go; Sidell and Pless, 1971Go). In two such studies, blood and brain toluene concentrations in mice were highly correlated with performance in tests of reflexes and unconditioned behaviors (Bruckner and Peterson, 1981Go), as were blood and brain TRI concentrations in rats to the rate of schedule-controlled operant responding for milk delivery (Warren et al., 1998Go). Also, Kishi et al. (1988, 1993) have reported relationships between shock avoidance performance decrements in rats and blood and brain levels of trichloroethylene and toluene. In studies of controlled human exposures, blood levels of m-xylene and TRI were measured and related to impaired body balance, eye-tracking deficits and altered reaction times (Mackay et al., 1987Go; Riihimaki and Savolainen, 1980Go). The present study is thought to be the first to relate internal dose measures of an industrial solvent to locomotor activity.

Given the diversity of neural influences on locomotor activity and the ubiquitous distribution of solvents within the brain (Ameno et al., 1992Go; Gospe and Calaban, 1988Go; Rafales, 1986Go), studies of solvent effects on locomotor activity usually do not allow for a determination of mechanisms within the CNS. The value of locomotor activity may instead lie in its economy and sensitivity to solvent-induced changes. For example, the lowest behaviorally-active toluene concentration to date in animals increases locomotor activity (Wood and Colotla, 1990Go), and the lowest-observed-effect-level in the present study is similar to that necessary to produce changes in schedule-controlled operant behavior under some reinforcement schedules (Balster et al., 1982Go; Moser and Balster, 1986Go). Therefore, as was demonstrated in the present study, locomotor activity may be useful for the routine determination of target tissue dose-response relationships that will enable behavioral modifications observed in animals to be extrapolated to humans with a greater degree of certainty. Progress in the conduct of such interspecies extrapolations ultimately hinges upon knowledge of the dose-metric that is most closely related to the behavioral change observed and the relative sensitivity of laboratory animals and humans to quantitatively equivalent dose-metrics. At present, little information is available on either. This is unfortunate as demonstrated by the routine application of Haber's Rule (i.e., all equal products of concentration and time [C x T] result in the same toxicity, regardless of the values of concentration and time) to the extrapolation of risks under circumstances common to solvent exposure (i.e., high level, short duration). The use of Haber's Rule under such circumstances has been seriously questioned (Atherley, 1985Go; Jarabek, 1995Go) and experimentally demonstrated not to be universally valid (Bushnell, 1997Go). Data from the present study bear this out, in that the locomotor activity level is substantially different after 20 min of exposure at 2000 ppm and 5 min of exposure at 8000 ppm, despite the products of concentration and time being equal (Fig. 1Go). The present experimental effort is of value in that it describes in a quantitative manner the relationship of internal dose-metrics to an easily measurable and universal behavior, locomotion. Equally important in moving toward a more scientifically defensible means of extrapolating behavioral endpoints in toxicology are efforts such as that of Benignus et al. (1998) who used PBPK modeling to make cross species comparisons of internal dose and effect relationships by examining arterial blood toluene concentration as it relates to avoidance behavior in rats and choice reaction time in humans.


    ACKNOWLEDGMENTS
 
This research was sponsored by AFOSR grant 910356 to C.E.D., NIDA grant DA-03112 to R.L.B., and NIEHS grant ES-07087 to R.L.B.


    NOTES
 
1 To whom correspondence should be addressed at TERRA, Inc., 1234 Timberlane Road, Tallahassee, FL 32312. Fax: (850) 309-1336. E-mail: awarren{at}terra1.com. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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