Neurotoxicology and Teratology, Vol. 18, No. 1, pp. 77-81, 1996 Copyright 0 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0892-0362196 $15.00 + .oO zyxwvutsrq ELSEVIER 0892-0362(95)02024-l Effects of Inhaled on Locomotor SCOTT E. BOWEN 1 , 1,l zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIH -Trichloroethane Activity in Mice AND ROBERT L. BALSTER’ Department of Pharmacology & Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298-0613 Received 27 January 1995; Accepted 25 September 1995 BOWEN, S. E. AND R. L. BALSTER. Effects of inholed I, I,]-trichloroethane on locomotor activity in mice. NEUROTOXICOL TERATOL 18( 1) 77-81, 1996. -To quantify the motoric effects of an abused solvent, photocells were added to two exposure systems. The first system utilized a static exposure chamber that recirculated vapor-laden air. A second dynamic system allowed for removal of waste gases with replenishment of fresh air combined with test vapors. In the present studies, male mice were examined for effects on locomotor activity following 30-min inhalation exposures to several concentrations of 1,1, I-trichloroethane (TCE), a widely used and abused solvent. TCE produced significant increases in locomotor activity at intermediate concentrations. Minimally effective concentrations for activity-increasing effects in the dynamic and static systems were 1,250 ppm and 2,500 ppm, respectively. At higher concentrations, motor activity was decreased with the highest dynamic system concentration (10,000 ppm), resulting in 26% of baseline control values. Biphasic, motor activity increasing and decreasing effects of TCE as a function of exposure concentration may reflect the CNS-depressant drug-like effects of abused solvents. Solvents Inhalant abuse Locomotor Activity 1,1,1 -Trichloroethane tradictory. The present study focuses on the widely abused solvent l,l, I-trichloroethane (TCE). TCE (also known as methyl chloroform) is widely found in both household products and in the industrial setting. Although TCE has been shown to be detrimental to the ozone and is slowly being phased out of commercial use, it was one of the most widely used solvents because of its high solvency and inflammability characteristics. TCE was commonly used in a variety of commercial products including typewriter correction fluids, suede protectors, brake cleaners, and as a degreasing agent for metals (22). Due to the large volume usage, TCE was a solvent to which many individuals were frequently exposed via inhalation through product use or abuse (9,13). Animal studies of TCE have shown that this solvent produces behavioral effects characteristic of abused solvents such as toluene. TCE produces reversible, concentration-related drug-like effects on schedule-controlled behavior (2,16), produces impairment of coordinated motor performance (15), and, in combination with ethanol, enhancement of the motoric incoordination and lethal effects are observed (25). TCE also produces ethanol- (17,21) and pentobarbital-like (20) discriminative stimulus effects. Mice exposed chronically to TCE VOLUNTARY inhalation of organic solvents is a significant problem in society today (5,18). Because these compounds are highly lipophilic and volatile at room temperature, inhalation of these solvents results in a rapid uptake. This rapid uptake into the brain results in the production of central nervous system (CNS) effects, which include euphoria and intoxication (22). In high concentration exposures, these depressant effects may lead to death (1,15,25). The acute intoxication produced by solvents has been hypothesized to be the basis for why individuals abuse them (4). Support for this comes from previous investigations that have shown that some solvents share behavioral and pharmacological properties with abused CNS depressant drugs (6,17). As with barbiturates and alcohol, acute exposures to abused solvents such as toluene have been shown to increase locomotor activity at lower exposures and decrease behavior at higher exposures in both rats (8) and mice (11,24). If this biphasic effect of solvents on motor activity represents their depressant drug-like abuse potential, then other abused solvents should produce this profile of effects as well. Unfortunately, there is relatively little information on the effects of abused solvents other than toluene in this procedure, and some of that is con- ’ To whom requests for reprints should Mice be addressed. 77 BOWEN 78 AND BALSTER an additional flow regulator to achieve the final test concenshow withdrawal effects indicative of physical dependence, tration. Flow rates through the exposure chamber were mainwith evidence for cross dependence with abused depressant drugs and alcohol (7). In seeming contradiction to this profile tained at 10 l/min and TCE concentration was continuously of depressant drug-like effects seen with TCE, previous studmonitored during all exposures using a single wavelength monitoring infrared spectrometer (Miran lA, Foxboro Anaies of its effects on motor activity have failed to provide consistent evidence for a biphasic effect on locomotor activity. lytical, North Haven, CT). Any fluctuations from the desired Two studies in rats (1) and mice (11) failed to obtain clear concentration during the experiment were corrected through motor activity increasing effects, whereas in another study manual adjustment of the flow regulators. Both static and some evidence for activity increases was obtained (12). Theredynamic exposure systems were absent of bedding. While the fore, in the present report, two separate investigations of the dynamic system was without a fan, an attempt was made to effects of a wide range of TCE concentrations on motor activprovide similar white background noise and light levels as well ity of mice are described. The first study utilized static expoas isolate the chambers from the laboratory environment by sure chambers in which TCE was introduced into a closed placing both exposure systems under a fume hood. system and then completely volatilized to produce a chosen Motor Activity concentration. The second study utilized dynamic exposures in a differently configured exposure chamber. In this system, Motor activity was measured unobtrusively via two sets of TCE vapor was constantly generated and delivered to the exphotocells (Micro Switch, Freeport, IL) that bisected both the posure chamber. static and dynamic exposure chambers. Interruptions of these An additional interest of this project was to ascertain the photobeams resulted in an analog signal being delivered by the feasibility of a within-subject design in evaluating concentraphotocell, which in turn, triggered a counter. Mice were tion-effect curves for solvent exposure. A within-subject deplaced into the same exposure chamber in the same sequence sign would reduce the number of animals needed and possibly each day. Activity was monitored once daily (Monday-Friday) produce greater sensitivity by controlling for individual differfor 30 min for approximately 1 week prior to solvent expoences. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA sures. This resulted in stable day to day levels of activity that served as a baseline against which solvent effects could be METHOD determined. Subjects TCE Exposure Ten experimentally naive male mice (CFW, Charles River Mice were placed individually into the chamber (static or Co., Wilmington, MA) were used in the static chamber experidynamic) and exposed for 30 min to a randomized graded ment, and nine similar mice were used in the dynamic chamber concentration of TCE vapor. Subjects were tested daily, 5 experiment. Subjects were housed in groups of five in standays a week, with TCE exposures occurring on Tuesdays and dard mouse cages (18 x 29 x 13 cm) with wood chip bedding Fridays. The concentrations of TCE tested were as follows: in a room with controlled temperature (22-24OC) on a 12 L : static-500, 1,250, 2,500, 5,000, 7,500, 10,000, 12,500 ppm; 12 D cycle. The tail of each mouse was color coded with a dynamic-500, 1,250, 2,500, 5,000, 7,500, 10,ooO ppm. One permanent marker for individual identification. Testing was of the days between solvent exposure was arbitrarily selected done during the light cycle. Water was available ad lib in the as an air control test for purposes of data analysis. The order home cage and mice were maintained at 30 f 5 g during testof testing was randomized for each subject. Certified TCE ing by postsession feeding of rodent chow (Rodent Laboratory was purchased commercially (T 391-4, Fischer Scientific Co.). Chow, Ralston-Purina Co., St. Louis, MO). Static Exposure Chambers Vapor exposures were conducted in 29-l cylindrical jars (47 cm H x 35 cm diameter; total floor space = 962 cm’), which have been described previously (3). Briefly, vapor generation commenced when liquid solvent was injected through a port onto filter paper suspended below the sealed lid. A fan, mounted on the inside of the lid, was then turned on which volatilized and distributed the agent within the chamber. Nominal chamber concentrations did not vary by more than 10% from measured concentration, as determined by single wavelength monitoring infrared spectrometry (Miran lA, Foxboro Analytical, North Haven, CT). All vapor exposures were 30 min in duration. Dynamic Exposure Chambers Vapor exposures were conducted in a 20.8-l glass rectangular tank (41 L x 21 W x 27 H cm; total floor space = 861 cm2) fitted with a Teflon lined lid into which TCE vapor was continuously delivered in the air inflow. The methodology for this solvent exposure system has been described previously (7). Briefly, a flow regulator allowed filtered air to pass through a gas dispersion tube immersed in a 2-l flask containing TCE. This vapor-laden air was then combined with filtered air from Data Analysis Concentration-effect curves were analyzed using repeated measures analysis of variance (ANOVA) and Tukey post hoc comparisons (p < 0.05). Because the control performance of one mouse was several times greater than the mean activity of the group, motor activity for each exposure was expressed as a percentage of the control motor activity. These control activity levels were determined by averaging motor activity on three control air only test sessions for each animal prior to determination of the concentration effect curve so that each animal served as its own control. RESULTS All mice maintained steady baseline activity counts during both studies. The mean baseline counts/minute for the air only test session before solvent testing for the static and dynamic systems were 20.8 f 5.0 SE and 15.5 k 1.1 SE, respectively. Locomotor activity returned to baseline levels during air only sessions the day after solvent test sessions. Static Exposure As shown in the top panel of Fig. 1, TCE exposure produced concentration-dependent increases in locomotor activ- EFFECTS OF INHALED I, 1, I-TRICHLORETHANE STATIC 3001 EXPOSURE ON LOCOMOTOR 79 zyxwvutsrqponm ACTIVITY changes in locomotor activity, F(6, 48) = 14.56, p < 0.001. Motor activity was not significantly affected at the 500 ppm TCE exposure; however, the greatest significant increase in locomotor activity was observed at the next highest concentration tested of 1,250 ppm (p < 0.05). Although locomotor activity was about 130-140% of control at 2,500 and 5,000 ppm TCE, none of these concentrations proved to be significantly different from air control. The higher concentrations of 7,500 and 10,000 ppm reduced activity with the highest concentration producing a significant decrease (p < 0.05). At 10,000 ppm, motor activity was fourfold lower than that observed under the air control. Again, as in the static exposure system, no seizures or deaths were observed at any of the tested concentrations. DISCUSSlON Clear evidence was obtained for increases in mouse locomotor activity after TCE exposure in two separate experiDYNAMIC EXPOSURE ments using within-subject designs. Using 30-min static exposures, we observed statistically significant increases with 2,500, 5,000 and 7,500 ppm. Dynamic exposures produced a statistically significant increase at only 1,250 ppm. The biphasic nature of the concentration-effect curve, with activity increases at intermediate concentrations and decreases at high concentrations, was most evident using the dynamic exposure system. This biphasic effect of TCE on locomotor activity is similar to what is obtained with the abused solvent toluene (8,25) and to what is seen with abused depressant drugs and OJ . , . , , ethanol (14,23). Along with the other evidence showing deAl, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 25w 7500 12500 pressant drug-like effects of TCE in animal tests (7,15,17, 20,21), these results further support the suggestion that biphaTCE CONCENTRATION (PPM) sic effects on motor activity can be viewed as one aspect of a depressant profile of effects of abused solvents. FIG. 1. Concentration-response curves for effects of l,l, l-trichloroOne goal of the present study was to ascertain the feasibilethane (TCE) on mouse locomotor activity for the static exposure ity of a within-subject design for evaluating concentrationsystem (upper panel) and the dynamic exposure system (lower panel). Locomotor activity is shown as a percentage of air control. Error bars effect curves for vapor exposure. While direct comparisons represent one standard error of the mean. Exposures were 30 min in between within-subject and between-subject designs are not duration. possible in the present report, it is still interesting to note that the acute effects of TCE on locomotor activity were observed at concentrations in the same range as those described in previous behavioral studies in rodents using between-subject deity [ANOVA for TCE concentration, F(7, 63) = 17.21, p < signs. In fact, these results report the lowest minimally effecO.OOl]. Although increases were observed at the 500 and 1,250 tive concentration for TCE (1,250 ppm) reported for learned ppm TCE concentrations, none of these exposures were signifor unlearned behavior in the mouse. In a previous operant icantly different from the air control (p > 0.05). At 5,000 study investigating TCE exposure, the reported EC,, for reppm, locomotor activity was significantly increased almost sponse rate suppression under a fixed-ratio 20 schedule in mice threefold above air control. Significant increases were also exposed for 30 min was 2,727 ppm (16). In a later study by observed at 2,500 and 7,500 ppm. Although the higher TCE Moser and Balster (17), 1,800-3,600 ppm TCE exposures were concentrations of 10,000 ppm and 12,500 ppm resulted in a reported to increase responding under a fixed-interval schedreduction of the previously elevated behavior, this reduction ule in mice during 30-min exposures while higher exposures was not significantly different from air control. While no sysdecreased responding (E&, of 7,129 ppm). Thus, earlier retematic attempt was made to quantitate the time course of ports of schedute-controlled operant behavior have not been effects, subjective observations of the mice during the expoas sensitive as motor activity for detecting acute effects of sures revealed that as exposure concentrations increased, the TCE exposure. Earlier investigations have also shown that behavior of the mice changed during the exposures. Animals behavioral disruptions occur at much lower concentrations of would initially show elevations in motor activity with activity TCE than those that produce lethality (15,25). For example, levels decreasing considerably as the exposure progressed. No Moser and Balster (15) reported that performance on an inseizures or deaths were observed in any of the animals at any verted screen test of motor performance was concentration concentration, nor was there any evidence for residual effects dependently disrupted by 30-min exposures to TCE. For 30on subsequent control exposure sessions. min exposures, the calculated ECSo for TCE was 5,216 ppm while the LC,O value was four times higher at 20,616 ppm. Dynamic Exposure The evidence obtained for a biphasic effect of TCE on locomotor activity in mice is in contrast to previous reports in The lower panel of Fig. 1 shows that TCE exposure in the dynamic system again produced concentration-dependent mice and rats. Adams et al. (1) reported that rats exposed to BOWEN 80 various TCE exposures (600 to 30,000 ppm) with different durations did not display significant increases in locomotor activity. In another study, Kjellstrand et al. (11) reported that mice displayed no increases of motor activity at TCE exposures below 2000 ppm with only small (less than twofold) increases observed at the 2,000 ppm exposure. In a later investigation, Kjellstrand et al. (12) demonstrated that TCE exposures of 5,100 ppm for 40 min, 60 min, and 180 min resulted in stimulatory effects (twofold increase) with no observed decreases during exposures. The discrepancies between the present investigation and previous reports of TCE exposure may be attributable to the use of a within-subjects design vs. the previous between-subjects designs. The sensitivity of our model may be due to the fact that repeated exposures to TCE may have sensitized the animals to locomotor increasing effects, although a previous report of repeated TCE exposure would give little support for this hypothesis (16). Test conditions and baseline rate levels may have also played an important role in the differences among studies. In the present investigation, measurement of locomotion occurred during the light phase of the light/dark cycle vs. measures taken during the dark phase in the Kjellstrand et al. (11,12) investigations. Because baseline activity levels are lower during the light phase and higher during the dark phase, it may have been easier in our study to demonstrate increases in locomotion. These differences may also be due to the measurement of activity by photobeam breakage vs. Doppler radar in the Kjellstrand et al. (11,12) investigations, differences in mouse strain, or exposure chamber design. Both static and dynamic systems were used in the present study to compare TCE’s effects on spontaneous locomotor AND BALSTER behavior. It is clear from both panels in Fig. 1 that we were successful in implementing photobeam breakage in our enclosed exposure systems as a measurement of spontaneous motor activity. Both systems produced qualitatively similar results with TCE exposure producing a biphasic effect. In addition, observations of behavior during higher exposures revealed that the time course of effects for activity was also biphasic in nature. Activity was initially increased and, as TCE exposure continued, activity gradually decreased with cessation of activity occurring at the highest TCE exposure in the dynamic system. In conclusion, TCE produced biphasic effects on locomotor activity in mice under two different exposure and testing conditions using a within-subject design. These effects, like similar effects reported earlier for toluene, may reflect the CNS depressant drug-like profile of acute effects of abused solvents. Locomotor activity changes were also shown to be a sensitive measure of the acute behavioral effects of TCE, and should prove useful for further investigations of the role of concentration and exposure duration for this and other solvents. These results also indicate that within-group methods of solvent presentation are viable and may accentuate the locomotor effects in mice creating a reliable difference that may not be found using between-group designs. zyxwvutsrqponmlkjihgfedc ACKNOWLEDGEMENTS The authors wish to thank Josiah Hamilton and Mary Tokarz for their technical assistance during the execution and analysis of this study. Research supported by NIDA grant DA-031 12. S. E. Bowen is a postdoctoral fellow supported by NIEHS Training Grant ES-07087. REFERENCES 1. Adams, E. M.; Spencer, H. C.; Rowe, V. K.; Irish, D. D. 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