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.
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