Toxicoiosy, 31 (1984) 143-150
Elsevier Scientific Publishers Ireland Ltd. EFFECTS OF TOLUENE AMINES IN THE RAT*
INHAI~TION
THOMAS WAYNE
NASI-P, J O S E P H
M. R E A °, J. F R A N K V. K E S S L E R ~
ON BRAIN BIOGENIC
E. Z A B I K b, G O R D O N
S. B O R N .... and
"School of Health Sciences and bDepartment of Pharmacology and Toxicology, Purdue University, West Lafayette, IN 47907 (U.S.A.)
(Received May 26th, 1983) (Accepted December 12th, 1983)
T h e effects of toluene e x p o s u r e on the biogenic a m i n e c o n c e n t r a t i o n s in t h e c e n t r a l n e r v o u s s y s t e m w e r e i n v e s t i g a t e d in the rat. T o l u e n e w a s a d m i n i s t e r e d via i n h a l a t i o n to g r o u p s of r a t s at c o n c e n t r a t i o n s of 0, 100, 300, or 1000 ppm. A f t e r an 8-h c o n t i n u o u s exposure, a n i m a l s w e r e sacrificed a n d whole b r a i n c o n c e n t r a t i o n s of d o p a m i n e (DA), n o r e p i n e p h r i n e (NE), a n d 5 - h y d r o x y t r y p t a m i n e (5-HT) w e r e d e t e r m i n e d . T h e d a t a indicated a significant i n c r e a s e in whole b r a i n c o n c e n t r a t i o n s of DA following the 100-ppm exposure. A regional a n a l y s i s of DA, NE, a n d 5 - H T c o n c e n t r a t i o n s in r a t s exposed to 1000 p p m of t o l u e n e for 8-h indicated a significant i n c r e a s e in DA c o n c e n t r a t i o n in t h e s t r i a t u m . A significant i n c r e a s e in N E c o n c e n t r a t i o n s w a s detected in the m e d u l l a a n d m i d b r a i n while 5 - H T c o n c e n t r a t i o n s w e r e significantly i n c r e a s e d in the c e r e b e l l u m , m e d u l l a , a n d s t r i a t u m . T h e s e r e s u l t s indicate t h a t t o l u e n e action r e s u l t s in e l e v a t e d c o n c e n t r a t i o n s of b e h a v i o r a l l y significant neurotransmitters. K e y w o r d s : T o l u e n e ; D o p a m i n e ; N o r e p i n e p h r i n e ; 5 - H y d r o x y t r y p t a m i n e ; Brain;
Inhalation INTRODUCTION T o l u e n e h a s been s h o w n to be r e l a t i v e l y non-toxic, even u n d e r e x t r e m e e x p o s u r e conditions [1-9l. R e c e n t r e v i e w s h a v e s u g g e s t e d t h a t t h e C N S m a y be the m o s t s e n s i t i v e t a r g e t t i s s u e in t e r m s of o b s e r v e d toxicity [5 9]. "A preliminary account of this research was presented at the joint meeting of the Society of Toxicology and American Society of Pharmacology and Experimental Therapeutics in Louisville, KY, U.S.A., August, 1982. "'To whom correspondence should be addressed. Abbreviations: DA, dopamine; N~, norepinephrine; 5-HT, 5-hydroxytryptamine; CNS, central nervous system. 0300-483X/84/$03.00 O 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
143
Accordingly, the primary effect of acute exposure to high concentrations (>5000 ppm) of toluene in humans has been summarized as being depression of the C N S [5--9].One study has indicated an increase in the concentrations of brain catecholamines in the rat at concentrations of toluene less than 1000ppm [10]. Equally important, studies of operant behavior following acute exposure to concentrations of toluene less than 1500ppm have indicated increased rates of responding on Sidman avoidance [11], fixed consecutive number [12], fixed interval [13], fixed interval-fixed ratio [14], and differential reinforcement at low rate schedules [15]. In contrast, a study has shown the absence of neurostructural changes in the rat brain following toluene exposure at concentrations less than 1500ppm [16]. Hence, the absence of neurostructural changes, the increase in catecholamines, and the increase in response rate following acute exposure to toluene at relatively low concentrations may suggest CNS toxicity limited to behaviorally significant neurochemistry. Behavioral effects have been reported in studies involving h u m a n s [17-19]. No precise molecular mechanism of toluene action on the CNS has been determined, although interference with the structure and function of ¢al.[lllar membranes dependent upon thermodynamic activity has been suggested for alkylbenzenes [5]. Since DA, NE, and 5-HT serve as neurotransmitters in discrete regions of the CNS [20,21], their relevance to maintaining normal physiological function has been investigated [22]. Alterations in the brain concentrations of these amines have been correlated with abnormal behavior and mood [23-25]. If the suggested interference with membranes does occur, then one possible consequence may be alteration of biogenic amine concentration regulation mechanism. Thus, the intent of the present investigation was to delineate the effects of acute, one-time exposure to toluene for 8 h at concentrations of 100, 300, and 1000 ppm on concentrations of DA, NE, and 5-HT in whole brain tissue and in discrete brain regions of the rat. The toluene exposure concentration range in this investigation would most-prvbably be relevant to common use conditions such as occupational exposures, rather than deliberate inhalation conditions. METHODS
-Subjects Adult, male Sprague-Dawley rats, weighing 220-240 g were obtained from Harlan Sprague-Dawley, Inc., Indianapolis, IN. Following their arrival, they were housed in an environmentally controlled room with a 14:10 light/dark cycle for at least 10 days to allow for acclimation before the start of the experiment. They received W a y n e Lab Blox ~ and tap water ad libitum. All inhalation studies were conducted between 8 a.m. and 6 p.m.
Exposure to toluene Inhalation exposure was conducted in two 20-liter cylindrical glass chromatographic jars. No more than 4 animals were placed in one chamber at 144
a time. W h e n more than 4 animals were used in the experimental design, 2 chambers were operated in parallel. Airflow through each chamber was 7 liters/min. Temperature inside the chamber remained within 1°C of ambient room temperature. Inhalation exposure target concentrations were 100, 300, or 1000 p p m of toluene. Toluene-laden atmospheres were generated by a syringe injection system in which toluene was vaporized by a turbulent flow of air. To facilitate vaporization of any aerosolized solvent, a flask containing 0.4-ram glass beads heated to 50°C was placed immediately after the syringe injection port. Toluene concentration inside the inhalation chamber was regulated by adjusting the infusion rate of a fine grade, variable speed syringe p u m p (Valley Scientific,Medway, MA). Entrance and exit ports were located at the geometric center of the top and base of the cylindrical chamber, positioned horizontally. Previous experimentation with the chamber has indicated concentration homogeneity. Toluene concentrations were monitored continuously by reading absorbance on an infrared spectrophotometer (Foxboro Analytical, South Norwalk, CT) at 13.7/zm. Standard curves were prepared prior to each exposure by the use of a closed-loop calibration system (Foxboro Analytical). Interpolated values from standard curves of liquid volume versus absorbance were used to adjust the syringe injection speed to obtain the absorbance corresponding to the target exposure level (_+10%). The toluene exposures were conducted continuously for 8 h. In the whole brain biogenic amine study, groups of 8 animals were exposed to 0, 100, 300, or 1000 p p m in a random sequence. The 0-ppm concentration represented an air only control. Animals tended to position themselves towards the exit port of the chamber; however, this was variable throughout the day. In a regional brain study, 6 animals were exposed to 1000 ppm, and another 6 served as controls. Upon completion of the 8°h exposure, individual rats were removed from the chamber and sacrificedby decapitation. The brain tissues were removed, rinsed in cold 0.9% saline, blotted dry, and immediately stored at -20°C until the time of assay. In the regional brain study, the brains were dissected into individual parts [26]. DA, NE, and 5-HT concentrations were measured spectrofluorometrically [27].
Data analyses The data were first analyzed for homogeneity of variance. A one-way analysis of variance ( A N O V A ) was then run to test for treatment effects.The A N O V A was run separately for each amine in the whole brain study and for each amine and each brain part in the regional study. RESULTS T h e effects of t o l u e n e e x p o s u r e on whole b r a i n biogenic a m i n e concent r a t i o n s a r e p r e s e n t e d in T a b l e I. While t h e r e w a s a c o n c e n t r a t i o n - d e p e n d e n t 145
TABLE I EFFECTS OF INHALATION OF TOLUENE FOR 8H ON WHOLE BRAIN LEVELS OF DOPAMINE (DA), NOREPINEPI-[RINE (NE), AND 5-HYDROXYTR~AMINE(5-HT) Concentration (ppm}
0 100 300 1000
Brain levels(ng/g)" DA
NE
5-HT
1106 ± 244 (4) 1195~ 76 1269 ± 145 1386 = 99b
3t4 ± 48 282± 8 316 ~-31 332 "- 23
545 -*47 571-~39 588 "- 58 607 r 80
'Mean ~-S.D., n : 8, except where indicated. bSignificantly different from 0 ppm (P < 0.05).
t e n d e n c y for i n c r e a s e d D A c o n c e n t r a t i o n s w i t h i n c r e a s i n g c o n c e n t r a t i o n s of toluene, only at the 1000-ppm concentration was the increase significant ( P < 0.05). N o s i g n i f i c a n t e f f e c t s n o r t r e n d s w e r e o b s e r v e d w i t h N E or 5 - H T a t a n y c o n c e n t r a t i o n of t o l u e n e t e s t e d . T h e e f f e c t of e x p o s u r e to t o l u e n e (1000 p p m ) on b i o g e n i c a m i n e c o n c e n t r a t i o n s in d i s s e c t e d b r a i n p a r t s is p r e s e n t e d in T a b l e II. T h e s p e c i f i c b r a i n regions studied were cerebellum, medulla, midbrain, striatum, hypot h a l a m u s , c o r t e x , a n d h i p p o c a m p u s . A s i g n i f i c a n t i n c r e a s e in D A c o n c e n t r a t i o n s w a s o b s e r v e d in t h e s t r i a t u m . A l t h o u g h w h o l e b r a i n c o n c e n t r a t i o n s of N E o r 5 . H T w e r e n o t s i g n i f i c a n t l y a l t e r e d , a s i g n i f i c a n t i n c r e a s e of N E c o n c e n t r a t i o n s in t h e m e d u l l a a n d m i d b r a i n w a s d e t e c t e d . S i g n i f i c a n t i n c r e a s e s in 5 - H T c o n c e n t r a t i o n s w e r e o b s e r v e d in t h e c e r e b e l l u m , m e d u l l a , and striatum. No gross behavioral changes were observed.
DISCUSSION The present study has indicated that toluene inhalation can cause significant increases in DA, NE, and 5-HT concentrations in discrete regions of the rat brain. While whole brain concentrations of D A were elevated, such changes in N E or 5-HT may have been masked by the whole brain study. A report has indicated an increase in D A and N E in the median eminence after inhalation of 500 pprn and 1000 p p m of toluene, respectively, by male rats [10]. In the above mentioned study catecholamine concentrations were reported to be measured, and D A and N E concentrations were inferred by discrete brain location [10]. In contrast, the present study differentiated D A and N E by assay, and not by measurement of catecholamine (e.g. both D A and NE) concentration. In the present study increased D A concentrations were observed in the striatum; however, no changes were observed in N E or D A in the hypothalamic region. Accordingly, results of the reported effects of toluene on D A and N E are consistent only in effect and not in discrete brain location. A review of the literature has indicated an absence of published reports to 146
198 "- 34" 285 _+50 (5) 490 _+71 (5) 1899-+371 1555 *_ 197 (5) 399 *- 86 356 z 112
Cerebellum Medulla Midbrain Striatum Hypothalamus C or te x Hippocampus
260 " 122 (5) 324 -* 75 (5) 501 -+ 114 2615-+514 b 1283 ± 352 367 _+41 287 _~55
I000 131 444 401 299 1606 200 305
0 + 14 _* 70 (5) +_91 (5) _-x62 ± 324 +_ 25 _*59
NE(ng/g)
*Mean -+S.D., n = 6, except where indicated. bSig~ificantly different from controls (0 ppm) (P < 0.05).
0
Brain part
D A (ng/ g)
T o l u e n e c o n c e n t r a t i o n (ppm)
123 : 4 l 541 _+66 b 545 +- 91 b 333±42 1267 ± 298 198 ± 35 284 _+57
I000
90 ± 8 498 ~_48 687 -- 160 436-+44 802 "_ 232 305 ~ 49 375 z 96
0
5-HT(ng/g}
116 = 20 b 607 :~ 47 b 832 -~ 233 536-+40 b 643 _* 283 341 ~ 32 574 ~ 207
1000
E F F E C T S O F I N H A L A T I O N O F T O L U E N E F O R 8 H O N L E V E L S O F D O P A M I N E (DA), N O R E P I N E P H R I N E (NE), A N D 5 - H Y D R O X Y T R Y I : C r A M I N E (5-HT) IN B R A I N P A R T S O F T H E RA T
T A B L E II
contrast the effects of increased N E concentrations in the midbrain and medulla, and increased 5-HT concentrations in the cerebellum, medulla, and striatum. Reported actions of toluene m a y be correlated with its effects on biogenic amine functions in the CNS. For example, toluene has been reported to cause "forced turning" in rats [28]. That D A m a y be involved in such actions is supported by the observation that D A agonists such as apomorphine cause rotational behavior [29]. Amphetamine, which also causes similar stereotypic behavior [29],exerts its actions by releasing biogenic amine, e.g. DA, from its storage sites [30].The releasing ability of amphetamine m a y be related to its ability to effectivelyblock D A re-uptake [31].However, no stereotypicbehavior was indicated by gross observation with the concentrations utilized in the present investigation. A n available clinical report also supports indirectly the possible involvement of D A in the actions of toluene. In this instance [18], toluene poisoning was reported to be misdiagnosed as schizophrenia. This is consistent with recent hypotheses which state that schizophrenia may be related to DAmediated activity [32]. Since this was a deliberate inhalation exposure by a solvent abuser, the reported effect of a schizophrenic symptomology attributed to toluene must by interpreted with caution. Amphetamine has also been reported to induce schizophrenic symptomology, presumably through its effect on the dopaminergic system centrally [33]. Thus, experimental evidence and possibly a clinical report of toluene induced schizophrenic symptomology support the involvement of the dopaminergic system in the actions of toluene. A review of the literature indicated an absence of reports which could be related to the observation of increased NE and 5-HT concentrations. Toluene has been reported to distribute rapidly to the CNS with uptake being highest in the cerebrum and lowest in the spinal cord of the rat [34]. Similarly, toluene has also been reported to be rapidly eliminated from brain tissue in the rat [34]. Thus, the eiTects of toluene, if dependent solely upon dose delivered to the site of action, may be expected to be reversible. However, the possible role of oxidative metabolism and tissue binding in the CNS has been suggested as a factor for toluene in particular [34], and solvents in general [35]. A review of the literature has indicated an absence of published reports concerning whether in fact toluene action on the biogenic amines is reversible. If toluene affects the biogenic amines by action on cellular membranes dependent upon thermodynamic activity, then other aromatic hydrocarbons would be expected to have similar action and effect. However, kinetic constants of activation reactions have been reported to be important in the amount of metabolite formation in vivo following inhalation exposure [36]. Thus, different substituted groups resulting in unique metabolic characteristics may have the effect of determining the amount of parent compound or metabolite at the site of action. Furthermore, aromatic hydrocarbons may have a similar mechanism at the site of action as suggested [5], but different
148
e f f e c t s m a y s t i l l o c c u r in v i v o d u e to " e x t r a n e u r o l o g i c " m e t a b o l i s m o r pharm a c o k i n e t i c s a f f e c t i n g t i s s u e c o n c e n t r a t i o n s a t t h e s i t e of a c t i o n . In summary, uncertainty exists concerning the mechanism by which t o l u e n e a f f e c t s t h e c o n c e n t r a t i o n s of t h e b i o g e n i c a m i n e s in r a t b r a i n t i s s u e . Such action may possibly be membrane mediated, metabolic, or both. Further i n t e g r a t i o n of d a t a f r o m in v i v o n e u r o b e h a v i o r a l a s s a y , p h a r m a c o k i n e t i c s , a n d n e r v o u s s y s t e m m o l e c u l a r e v e n t s m a y p r o v i d e r e l e v a n t i n f o r m a t i o n to realistically evaluate the potential for any neurochemical or behavioral risk of t o l u e n e a t low c o n c e n t r a t i o n s s u c h a s t h o s e e n c o u n t e r e d in c o m m o n - u s e c o n d i t i o n s . A s i m i l a r i n t e g r a t i o n of d a t a h a s b e e n s u g g e s t e d a s a n a p p r o a c h to d e t e r m i n e h u m a n c a r c i n o g e n i c r i s k [37]. ACKNOWLEDGEMENTS
T h i s s t u d y w a s s u p p o r t e d b y t r a i n i n g G r a n t 5 - T 3 2 - E S 0 7 0 3 9 f r o m t h e U.S. P u b l i c H e a l t h S e r v i c e , N a t i o n a l I n s t i t u t e of E n v i r o n m e n t a l H e a l t h S c i e n c e s , U.S. P u b l i c H e a l t h S e r v i c e . REFERENCES
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