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Behavioral effects of chronic toluene exposure in the developing rat
Neurotoxicologyand Teratology, Vol. 12, pp. 353-357. ©Pergamon Press plc, 1990. Printed in the U.S.A.
0892-0362/90 $3.00 + .00
Behavioral Effects of Chronic Toluene Exposure in the Developing Rat MARTE LORENZANA-JIMENEZ
a AND MANUEL
SALAS
Departamento de Farmacologfa, Facultad de Medicina and Departamento de Fisiologfa, Instituto de Investigaciones Biom(dicas, Universidad Nacional Autdnorna de M(xico, M~xico 04510, D. F. Mdxico R e c e i v e d 28 S e p t e m b e r 1987
LORENZANA-JIMENEZ, M. AND M. SALAS. Behavioral effects of chronic toluene exposure in the developing rat. NEUROTOXICOL TERATOL 12(4) 353-357, 1990.--The effects of acute and chronic toluene exposure on the hypnotic effect, the righting reflex latencies and the blood and tissue toluene contents were studied in rats during development. The data showed a progressive significant prolongation of the hypnotic effect latencies until the third and fourth postnatal weeks, followed by a significant continuous declining trend until the eighth week postpartum. The measure of the righting reflex latencies followed an opposite temporal course compared to that of hypnotic effect measurements. The acute and chronic toluene exposure did not reveal significant differences in toluene concentrations of blood, brain and liver tissues. The data suggest that chronic toluene treatment may probably be inducing behavioral manifestations of a tolerance phenomenon combined with maturational influences in the developing rat. Behavioral tolerance
Rats
Neonatal toluene exposure
METHOD
A number of studies have suggested that repetitive glue sniffing or the chronic exposure to industrial solvents such as the thinner mixture, toluene~and benzene, results in a consistent behavioral tolerance to these products (4, 6, 10, 18, 19, 26). Thus, the abusers usually initiate the addiction by inhaling these compounds once or twice a week, but shortly this practice becomes very frequent and compulsive (3,18). By contrast, different studies employing animal models (9,14) have suggested that chronic exposure to these industrial solvents in the infant, juvenile or adult rats do not cause clear behavioral signs of tolerance as evidenced by the lack of effects on locomotor activity (15, 22, 27). The low cost, availability in numerous domestic products, broken home environment and the poor legal regulation in the distribution and selling of cements and other volatile solvents have increased the population of young boys (below I0 years old) involved in the glue-sniffing practice (6). To our knowledge, there is a lack of evidence concerning the mechanisms through which the industrial solvents interfere with behavioral and other brain functions in adults and, moreover, how these compounds may be affecting the appearance and maturation of different motor adaptive mechanisms at critical stages in early life. The aim of this study was to obtain additional information about the tolerance phenomenon produced by chronic exposure to toluene by using the hypnotic effect and the righting reflex latencies, as well as their possible correlation with the blood, brain and liver toluene concentrations in the rat.
Animals Behavioral measurements were undertaken in rats derived from a total of twenty-five Wistar rats between 100-120 days of age. Rats were maintained in a 14/10 hr light-dark cycle with lights on scheduled at 0700 hr, with Purina rat chow and water ad lib in a room thermostatically maintained at 2 4 - 2 ° C throughout the experiment. The females were mated with normal breeder males of similar age. Pregnant rats were removed from the breeding cages and each placed in Plexiglas maternity cages (45 x 30 × 20 cm) at least 3-5 days before the scheduled parturition. At birth (Day 0) litters were adjusted to 8 male pups per mother; when necessary, the rats were completed with animals taken from other litters born on the same day. A total of five litters were assigned to each of the three different toluene dose groups. The groups were weaned at 25 days of age, and subsequently allowed free access to water and food (Purina chow). After weaning, the animals were kept in groups of 4-45 rats. The breeding cages were cleaned at birth and every two weeks throughout the experimental period. All subjects were kept in an air-conditioned colony room maintained at constant temperature and humidity, and on a 14/10 hr light-dark cycle (lights on at 0700 hr). Body weights were recorded on 2, 7, 14, 21, 28, 35, 42, 49 and 56 days of age.
~Some of the data were presented at the Symposium Avances en el mecanismo de acci6n de fgtrmacos, Mrxico, D. F., November, 1983. 2Requests for reprints should be addressed to Marte Lorenzana-Jimrnez, Departamento de Farmacologia, Facultad de Medicina, Universidad Nacional Aut6noma de Mrxico, A. Postal 70297, Mrxico 04510, D. F. Mrxico.
353
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LORENZANA-JIMENEZ AND SALAS
TOLUENE (pprn) c
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EXPOSURES
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FIG. 1. Hypnotic effect latencies in rats exposed to different toluene concentrations during the first 8 weeks postnatally (A), and at 8 months of age (B) for a 3-day period. Each point in the left graph represents the average of 40 animals, and 7 subjects to the right panel. Vertical lines on the curves indicate the mean standard errors. The horizontal lines limited by arrows below the curves represent the period of toluene exposure.
Solvent Exposure From the third day of age until 8 weeks postnatally, each experimental rat was subjected once a day to a 15-min exposure period to toluene (Merck Co.) 5 days a week, in a glass chamber (2774 ml air capacity) containing 10,000, 20,000 and 40,000 ppm liquid toluene (31.7, 62.5 and 125 mg/liter of air, respectively). In all cases each rat was exposed to only one of the different toluene concentrations each day. The toluene concentrations were initially achieved in the chamber, but were progressively diluted during the 15-min exposure period. Toluene vaporized in a 2-min period, as a gas chromatography report has indicated (8). The cages were removed singly from the colony room to an adjacent room during the experimental procedure. The liquid toluene concentrations (0.1, 0.2 and 0.4 ml) were introduced directly into the chamber with a syringe underneath a false wire lattice serving as a floor, and left to diffuse spontaneously. In all cases toluene was introduced 5 sec after the rat was placed into the chamber, so the rat was indirectly in contact with the toluene. Concentrations in the glass chamber were monitored by gas chromatography as reported elsewhere (8,22).
Behavioral Procedure Behavioral measurements were performed in 120 male Wistar rats during the first eight weeks postpartum. The hypnotic effect latency was defined as the time elapsed between the liquid toluene introduction into the chamber and the time to complete prostration of subjects on the floor. The righting reflex latency was evaluated immediately following toluene exposure, by resting the rat on its back in a warm (___24°C) plastic cage (40 x 40 x 20 cm) and measuring the time of appearance of righting response. In all cases the differences in latencies between the three toluene groups ( n = 4 0 rats per group) were evaluated by using a two-way ANOVA. Because the behavioral measurements may not reflect a tolerance phenomenon, but alterations of the responses to toluene as a function of age, only two additional groups of rats (n = 7 per group) of 8 months of age were exposed twice a day for a 3-day period at 0.1 and 0.2 ml of toluene, respectively, and the behavioral responses evaluated.
Toluene Concentrations Blood and tissue toluene concentrations were evaluated in
twenty-seven male Wistar rats. Thus, three different groups of 9 rats each were exposed daily from 2-30 days postpartum to 0.1, 0.2 and 0.4 ml of toluene during a 15-min period. Moreover, three additional groups of 3 rats each were exposed to a single 15-min solvent exposure at 30 days postnatally. In all cases following chronic or acute toluene exposures, the animals were sacrificed on day 30 postpartum by cervical dislocation, and 0.5 ml of blood was obtained with a heparinized syringe from an external jugular vein. The blood was then deposited in 15-ml hermetically sealed glass vials. Moreover, the brain and liver were dissected immediately and a piece of tissue was taken (1-2 g). The tissues were washed with tap water and introduced into 26-ml sealed glass vials. The vials contained 4 steel spheres to aid in the homogenization of the tissue; this was achieved by careful shaking of each vial for 3 min. To equilibrate the concentration of toluene in the samples with that of the overlaying gaseous phase (27), the vials were placed in a water bath at 40°C for 30 min. A 0.5-ml sample of the gaseous phase was withdrawn and injected into a Hewlett Packard model 5840A gas chromatograph. The apparatus was equipped with a flame ionization detector and a 6' × 1/8" stainless steel column packed with 10% Carbowax 20 M in Chromosorb WHP 80/100 and operated at 70°C. The carder gas was nitrogen flowing at a rate of 22 ml/min. The temperature of both the injector and the detector was 150°C. Calibration curves were obtained with blood and tissue samples containing known amounts of toluene and handled as described. In all cases I% isopropanol was used as an internal standard. The analysis of score differences between acute and chronic experimental groups was compared by using the Student's t-test. RE SU L T S
During the first week of toluene exposure to 10,000, 20,000 and 40,000 ppm of the solvent concentrations, latencies of 9.2, 8.6 and 5.4 min in the hypnotic effect, respectively, resulted (Fig. 1A). These scores then progressively increased and achieved their highest values around the fourth week of toluene exposure (15, 12 and 10 min, respectively). By contrast, during the following weeks of exposure, there was a progressive declining in their values, that in the last week were very similar to those observed at the beginning of the experiment (20,000 and 40,000 ppm), or were still increased around 50% of its initial value (10,000 ppm).
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FIG. 2. Rightingreflex latenciesin animals exposed to differenttoluene concentrationsduring the first 8 weeks postpartum (A), and at 8 monthsof age (B) for a 3-day period. Numberof subjectsand symbols in graphs as in Fig. 1. The analysis of the mean hypnotic effect scores between the different toluene concentrations throughout the study yielded the main effect of treatment, F(2,189)= 752.98, p<0.05. Additionally, a significant treatment × weeks interaction, F(14,189)= 8.44, p<0.05, was found. The analysis of variance in each week of the study revealed significant differences between the three experimental groups throughout the period analyzed (p<0.05). Figure 1B shows that toluene exposures (10,000 and 20,000 ppm) throughout a 3-day period in rats of 8 months of age did not provoke significant differences in the hypnotic effect latencies. During the first week of the experiment, the 10,000 or 20,000 ppm of toluene exposure provoked in the animals a righting reflex latency of 7 and l0 min, respectively (Fig. 2A), followed by a progressive decline in their values until week 4 of the experiment. Although in the case of the 10,000 ppm toluene dose, the righting reflex latency had a tendency to recover, however, their values were still shorter (2 min) in week 8 of the study. The use of a dose of 20,000 ppm toluene caused a clear recovery in the fighting reflex latency from week 4 onward, although their values were not in the range of the first 2 weeks. Concerning the exposure to 40,000 ppm of toluene, there was a clear prolongation in the fighting reflex latency during the first week (19 min), compared with that of the other 2 toluene doses. However, these scores were progressively decreasing in value until week 4 of the study (6.5 min), and thereafter they increased until week 8 of the experiment, where the fighting reflex latency attains a 12-min value (Fig. 2A). As in the case of the other two toluene doses, the fighting reflex latency increments seen from week 4 of the study did not achieve the values observed during the first 2 weeks of the experiment. The analysis of the mean righting reflex latency scores between the three different toluene groups revealed a significant main effect of treatment, F(2,147) = 359.65, p<0.05, and a significant treatment × weeks interaction, F(14,147) = 5.50, p<0.05. The analysis of variance on each week of the experirnent showed significant differences between the three experimental groups (p<0.05) throughout the period studied. Figure 2B indicates that toluene
exposure (10,000 and 20,000 ppm) throughout a 3-day period in rats of 8 months of age did not provoke significant differences in the righting response latencies. Blood, brain and liver toluene concentrations to a single or repetitive solvent exposures are presented in Table 1. There was a progressive increment to toluene concentrations in the different tissues, correlated with the increased solvent concentrations during a 15-min exposure. The comparisons between the acute vs. chronic toluene concentrations in each type of tissue, and the three different toluene doses here employed did not show significant score differences (p>0.05). The analysis of variance performed on body weight measurements did not reveal significant differences between the three different toluene groups, F(1,126)= 2.96, p=0.09. Body weight comparisons between toluene-exposed and nonexposed subjects have not shown significant differences (16). DISCUSSION
The present data suggest that chronic exposure to different toluene concentrations during the pre- and postweaning periods in the rat results in behavioral signs of a tolerance phenomenon, as evidenced by a significant progressive prolongation of the hynotic effect and a reduction of the fighting reflex latencies. This tolerance was apparently more consistent for the fighting reflex measurements in response to 10,000 ppm of toluene, particularly around the 8th week of the study, where the fighting reflex latency was 21% of its initial value. By contrast, the fighting response for the 20,000 and 40,000 ppm of toluene concentrations was approximately 60% and 63% of their initial scores, respectively. The fact of a progressive appearance of the behavioral tolerance phenomenon achieving its maximum effects around the third and fourth postnatal weeks, and then followed by a decrement in its values around the eighth week of the study is still a matter of speculation. The remarkable resistance of altricial newborns to extreme temperatures, pain, anoxia, starvation, and the imma-
356
LORENZANA-JIMENEZ AND SALAS
TABLE 1 BLOOD, BRAIN AND LIVER TOLUENE CONCENTRATIONS ( ~- SDM) OF 3* AND 9t RATS PER GROUP, RESPECTIVELY, AFTER 15 MIN OF SOLVENT EXPOSURE Blood p.cg/ml
Brain p.g/ml
Liver Ixcg/ml
Toluene Group mg/1 Air (ppm)
Acute*
Chronic1-
p:~
Acute*
Chronic~
p$
Acute*
31.7 (10,000)
94.19 _ 12.19
95.98 _+10.92
NS
132.62 -+ 12.98
131.22 -- 11.45
NS
62.5 (20,000)
125.62 --- 16.70
108.04 -+23.17
NS
152.40 -+26.10
127.87 -+33.50
125.0 (40,000)
198.60 -+36.86
160.49 +4.35
NS
250.03 ---25.40
246.18 ---42.30
Chronic#
p:~
477.28 _+33.75
402.36 ---72.76
NS
NS
656.77 -+ 189.11
584.00 - 134.90
NS
NS
745.41 -+ 189.71
719.44 -+ 139.71
NS
*Single exposure at 30 days old. tDaily exposures, 2-30 days postpartum SNS, not significant differences (Student's t-test).
ture response to stress (1, 11, 12, 25) perhaps may play a fundamental role in the temporal course of the presumably chronic toluene tolerance phenomenon. The possibility of a progressive maturational effect upon the specific enzymatic systems subserving the hepatic elimination of toxic compounds could perhaps be discarded because of the lack of significant differences in liver toluene contents, following the acute and/or chronic solvent intoxication. However, the lack of a significant reduction in both the hypnotic effect and the righting response latencies in 8-monthold toluene-exposed rats suggests that the behavioral tolerance phenomenon before two months of age is concurrent with maturational changes. Nevertheless, it may be necessary to analyze behavioral responses throughout longer periods of chronic toluene exposure and the temporal course of acute and chronic toluene detoxification processes in these tissues before eliminating the influence of a maturational effect and a pharmacodynamic or a pharmacokinetics tolerance phenomena (2, 13, 21). Although the increased use of a substance does not necessarily indicate the development of tolerance, but also a positive reinforcement to the same, at present we cannot discriminate among them because, through reflex measurements, it is difficult to recognize a compulsive toluene abuse which requires complex limbic and corticosubcortical circuitry for its neural integration. Several studies have suggested that during brain maturation the excitatory circuits develop earlier than the inhibitory ones (5, 7, 20). The centers maturating later probably exert modulatory influences upon neural structures previously developed. Thus,
adaptive behaviors such as locomotion, swimrmng, righting responses, etc., are largely modulated through different brain stem and telencephalic mechanisms, becoming efficient around the first month of life in the rat (17, 22-24). The finding of an altered hypnotic effect and righting reflex latencies as behavioral manifestations of a possible tolerance phenomenon to the chronic toluene exposure disagrees with similar studies showing no significant effects on the spontaneous locomotor activity measurements in adult rats (15,28). It is possible, then, that a differential threshold of activation in the brain substrate underlying the spontaneous locomotor activity, hypnotic effect and fighting reflex latencies, and/or a disturbance of the normal balance between the excitatory and inhibitory circuits subserving the integration of these locomotor patterns, may account for the behavioral effects provoked by the postnatal toluene exposure. Finally, while this study has focused on the effects of postnatal toluene exposure in rats, the behavioral responses described here can easily be employed in the assessment of behavioral tolerance following chronic exposure to different chemical compounds and industrial solvents during development. ACKNOWLEDGEMENTS The authors wish to thank Dr. V. Colotla for critical reading of the manuscript; A. Jimtnez for statistical advice; M. Rivero Murphy for technical assistance and M. del Pozo and B. Huerta for the preparation of the manuscript.
REFERENCES 1. Adolph, E. F. The ontogeny of physiological regulations in the rat. Q. Rev. Biol. 32:89-137; 1957. 2. Auerbach, V. H.; Waisman, H. A. Tryptophan peroxidase-oxidase, histidase and transaminase activity in liver of developing rat. J. Biol. Chem. 234:304-306; 1959. 3. Aviado, D. M. Pharmacology of abused inhalants. In: Sharp, C. M.; Carrol, L. T., eds. Voluntary inhalation of industrial solvents. Rockville, MD: National Institute on Drug Abuse; 1978:303-311. 4. Bass, M. Sudden sniffing death. JAMA 212:2075-2079; 1970. 5. Blozovski, D.; Bachevalier, J. Effects of atropine on behavioral arousal in the developing rat. Dev. Psychobiol. 8:97-102; 1975. 6. Brozovsky, M.; Winkler, E. G. Glue sniffing in children and adolescents. NY State J. Med. 1:1984-1989; 1965. 7. Campbell, B. M.; Lytle, L. D.; Fibiger, H. C. Ontogeny of adrenergic arousal and cholinergic inhibitory mechanisms in the rat. Science
166:637-638; 1969. 8. Colotla, V. A.; Bautista, S.; Lorenzana-Jimtnez, M.; Rodriguez, R. Effects of solvents on schedule-controlled behavior. Neurobehav. Toxicol. l(Suppl. 1):113-118; 1979. 9. Contreras, C. M.; Gonz~lez-Estrada, T.; Zarabozo, D.; Fem~indezGuardiola, A. Petit mal and grand mal seizures produced by toluene or benzene intoxication in the cat. Electroencephalogr. Neurophysiol. 46:290-301; 1979. 10. Corlis, L. M. A review of the evidence on glue sniffing a persistent problem. J. Sch. Health 35:442-449; 1965. 11. Dawes, G. S.; Handier, J. J.; Mott, J. C. Some cardiovascular responses in fetal and adult rabbits. J. Physiol. 139:123-136; 1957. 12. Fazekas, J. F.; Alexander, F. A. D.; Himwich, H. E. Tolerance of the newborn to anoxia. Am. J. Physiol. 134:281-287; 1941. 13. Geller, E.; Yuwiler, A.; Schapiro, S. Comparative effects of a stress
TOLUENE AND BEHAVIORAL DEVELOPMENT
14. 15. 16. 17. 18.
19. 20. 21.
and cortisol upon some enzymatic activities. Biochem. Biophys. Acta 93:311-315; 1964. Guzm~in-Flores, C. Neurobiologia del tlner: alteraciones conductuales producidas a largo plazo. Cuadernos Cientfficos, CEMEF (M6x) 2:49-58; 1975. Lorenzana-Jim~nez, M.; Salas, M. Effects of neonatal exposure to paint thinner on the development of swimming in rats. Neurobehav. Toxicol. 2:1-5; 1980. Lorenzana-Jim6nez, M.; Salas, M. Neonatal effects of toluene on the locomotor behavioral development of the rat. Neurobehav. Toxicol. Teratol. 5:295-299; 1983. McCance, R. A. The maintenance of stability in the newly born. I. Chemical exchange. Arch. Dis. Child. 34:361-370; 1959. Messengale, O. N.; Glaser, H. H.; Lelievre, R. E.; Dodds, J. B.; Klock, M. E. Physical and psychologic factors in glue sniffing. N. Engl. J. Med. 269:1340-1344; 1963. Merry, J.; Zachariadis, N. Addiction to glue-sniffing. Br. Med. J. 11:1448; 1962. Moorcrofi, W. H.; Lytle, L. D.; Campbell, B. A. Ontogeny of starvation-induced behavioral arousal in the rat. J. Comp. Physiol. Psychol. 75:59-67; 1971. Poczopko, P. A contribution to the studies on changes of energy metabolism during postnatal development. J. Cell. Comp. Physiol.
357
57:175-184; 1961. 22. Rodriguez, R.; Lorenzana-Jim6nez, M.; Manjarrez, A.; G6mez-Rufz, H. Behavioral effects from the acute and chronic inhalation of thinner in rats of various ages. In: Sharp, C. W.; Carrol, L. T., eds. Voluntary inhalation of industrial solvents. Rockville, MD: National Institute on Drug Abuse; 1978:213-225. 23. Salas, M. Effects of early malnutrition on the development of swimming ability in the rat. Physiol. Behav. 8:119-122; 1972. 24. Schapiro, S.; Salas, M.; Vukovich, K. Hormonal effects on ontogeny of swimming ability in the rat: assessment of central nervous system development. Science 168:147-151; 1970. 25. Schapiro, S.; Yuwiler, A.; Geller, E. Maturation of a stress-activated mechanism inhibiting induction of tyrosine transaminase. Science 152:1642-1643; 1966. 26. Torres-Rulz, A. Sintomas clinicos de usadores y/o abusadores de inhalantes vol~itiles. Cuadernos Cientfficos CEMEF (M6x.) 2:73-84; 1975. 27. Yamamura, H.; Wakasugi, B.; Sato, S.; Takebe, Y. Gas chromatographic analysis of inhalation anesthetics in whole blood by an equilibration method. Anaesthesiology 27:311-317; 1966. 28. Yamawaki, S.; Segawa, T.; Sarai, K. Effects of acute and chronic toluene inhalations on behavior and (3H) serotonin binding in rat. Life Sci. 30:1997-2002; 1982.
0892-0362/90 $3.00 + .00
Behavioral Effects of Chronic Toluene Exposure in the Developing Rat MARTE LORENZANA-JIMENEZ
a AND MANUEL
SALAS
Departamento de Farmacologfa, Facultad de Medicina and Departamento de Fisiologfa, Instituto de Investigaciones Biom(dicas, Universidad Nacional Autdnorna de M(xico, M~xico 04510, D. F. Mdxico R e c e i v e d 28 S e p t e m b e r 1987
LORENZANA-JIMENEZ, M. AND M. SALAS. Behavioral effects of chronic toluene exposure in the developing rat. NEUROTOXICOL TERATOL 12(4) 353-357, 1990.--The effects of acute and chronic toluene exposure on the hypnotic effect, the righting reflex latencies and the blood and tissue toluene contents were studied in rats during development. The data showed a progressive significant prolongation of the hypnotic effect latencies until the third and fourth postnatal weeks, followed by a significant continuous declining trend until the eighth week postpartum. The measure of the righting reflex latencies followed an opposite temporal course compared to that of hypnotic effect measurements. The acute and chronic toluene exposure did not reveal significant differences in toluene concentrations of blood, brain and liver tissues. The data suggest that chronic toluene treatment may probably be inducing behavioral manifestations of a tolerance phenomenon combined with maturational influences in the developing rat. Behavioral tolerance
Rats
Neonatal toluene exposure
METHOD
A number of studies have suggested that repetitive glue sniffing or the chronic exposure to industrial solvents such as the thinner mixture, toluene~and benzene, results in a consistent behavioral tolerance to these products (4, 6, 10, 18, 19, 26). Thus, the abusers usually initiate the addiction by inhaling these compounds once or twice a week, but shortly this practice becomes very frequent and compulsive (3,18). By contrast, different studies employing animal models (9,14) have suggested that chronic exposure to these industrial solvents in the infant, juvenile or adult rats do not cause clear behavioral signs of tolerance as evidenced by the lack of effects on locomotor activity (15, 22, 27). The low cost, availability in numerous domestic products, broken home environment and the poor legal regulation in the distribution and selling of cements and other volatile solvents have increased the population of young boys (below I0 years old) involved in the glue-sniffing practice (6). To our knowledge, there is a lack of evidence concerning the mechanisms through which the industrial solvents interfere with behavioral and other brain functions in adults and, moreover, how these compounds may be affecting the appearance and maturation of different motor adaptive mechanisms at critical stages in early life. The aim of this study was to obtain additional information about the tolerance phenomenon produced by chronic exposure to toluene by using the hypnotic effect and the righting reflex latencies, as well as their possible correlation with the blood, brain and liver toluene concentrations in the rat.
Animals Behavioral measurements were undertaken in rats derived from a total of twenty-five Wistar rats between 100-120 days of age. Rats were maintained in a 14/10 hr light-dark cycle with lights on scheduled at 0700 hr, with Purina rat chow and water ad lib in a room thermostatically maintained at 2 4 - 2 ° C throughout the experiment. The females were mated with normal breeder males of similar age. Pregnant rats were removed from the breeding cages and each placed in Plexiglas maternity cages (45 x 30 × 20 cm) at least 3-5 days before the scheduled parturition. At birth (Day 0) litters were adjusted to 8 male pups per mother; when necessary, the rats were completed with animals taken from other litters born on the same day. A total of five litters were assigned to each of the three different toluene dose groups. The groups were weaned at 25 days of age, and subsequently allowed free access to water and food (Purina chow). After weaning, the animals were kept in groups of 4-45 rats. The breeding cages were cleaned at birth and every two weeks throughout the experimental period. All subjects were kept in an air-conditioned colony room maintained at constant temperature and humidity, and on a 14/10 hr light-dark cycle (lights on at 0700 hr). Body weights were recorded on 2, 7, 14, 21, 28, 35, 42, 49 and 56 days of age.
~Some of the data were presented at the Symposium Avances en el mecanismo de acci6n de fgtrmacos, Mrxico, D. F., November, 1983. 2Requests for reprints should be addressed to Marte Lorenzana-Jimrnez, Departamento de Farmacologia, Facultad de Medicina, Universidad Nacional Aut6noma de Mrxico, A. Postal 70297, Mrxico 04510, D. F. Mrxico.
353
354
LORENZANA-JIMENEZ AND SALAS
TOLUENE (pprn) c
A
15
•
I0 0 0 0
B
)-
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13
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2
3 AGE
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4 5 (weeks)
t
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]
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6
7
8
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EXPOSURES
(doysl
FIG. 1. Hypnotic effect latencies in rats exposed to different toluene concentrations during the first 8 weeks postnatally (A), and at 8 months of age (B) for a 3-day period. Each point in the left graph represents the average of 40 animals, and 7 subjects to the right panel. Vertical lines on the curves indicate the mean standard errors. The horizontal lines limited by arrows below the curves represent the period of toluene exposure.
Solvent Exposure From the third day of age until 8 weeks postnatally, each experimental rat was subjected once a day to a 15-min exposure period to toluene (Merck Co.) 5 days a week, in a glass chamber (2774 ml air capacity) containing 10,000, 20,000 and 40,000 ppm liquid toluene (31.7, 62.5 and 125 mg/liter of air, respectively). In all cases each rat was exposed to only one of the different toluene concentrations each day. The toluene concentrations were initially achieved in the chamber, but were progressively diluted during the 15-min exposure period. Toluene vaporized in a 2-min period, as a gas chromatography report has indicated (8). The cages were removed singly from the colony room to an adjacent room during the experimental procedure. The liquid toluene concentrations (0.1, 0.2 and 0.4 ml) were introduced directly into the chamber with a syringe underneath a false wire lattice serving as a floor, and left to diffuse spontaneously. In all cases toluene was introduced 5 sec after the rat was placed into the chamber, so the rat was indirectly in contact with the toluene. Concentrations in the glass chamber were monitored by gas chromatography as reported elsewhere (8,22).
Behavioral Procedure Behavioral measurements were performed in 120 male Wistar rats during the first eight weeks postpartum. The hypnotic effect latency was defined as the time elapsed between the liquid toluene introduction into the chamber and the time to complete prostration of subjects on the floor. The righting reflex latency was evaluated immediately following toluene exposure, by resting the rat on its back in a warm (___24°C) plastic cage (40 x 40 x 20 cm) and measuring the time of appearance of righting response. In all cases the differences in latencies between the three toluene groups ( n = 4 0 rats per group) were evaluated by using a two-way ANOVA. Because the behavioral measurements may not reflect a tolerance phenomenon, but alterations of the responses to toluene as a function of age, only two additional groups of rats (n = 7 per group) of 8 months of age were exposed twice a day for a 3-day period at 0.1 and 0.2 ml of toluene, respectively, and the behavioral responses evaluated.
Toluene Concentrations Blood and tissue toluene concentrations were evaluated in
twenty-seven male Wistar rats. Thus, three different groups of 9 rats each were exposed daily from 2-30 days postpartum to 0.1, 0.2 and 0.4 ml of toluene during a 15-min period. Moreover, three additional groups of 3 rats each were exposed to a single 15-min solvent exposure at 30 days postnatally. In all cases following chronic or acute toluene exposures, the animals were sacrificed on day 30 postpartum by cervical dislocation, and 0.5 ml of blood was obtained with a heparinized syringe from an external jugular vein. The blood was then deposited in 15-ml hermetically sealed glass vials. Moreover, the brain and liver were dissected immediately and a piece of tissue was taken (1-2 g). The tissues were washed with tap water and introduced into 26-ml sealed glass vials. The vials contained 4 steel spheres to aid in the homogenization of the tissue; this was achieved by careful shaking of each vial for 3 min. To equilibrate the concentration of toluene in the samples with that of the overlaying gaseous phase (27), the vials were placed in a water bath at 40°C for 30 min. A 0.5-ml sample of the gaseous phase was withdrawn and injected into a Hewlett Packard model 5840A gas chromatograph. The apparatus was equipped with a flame ionization detector and a 6' × 1/8" stainless steel column packed with 10% Carbowax 20 M in Chromosorb WHP 80/100 and operated at 70°C. The carder gas was nitrogen flowing at a rate of 22 ml/min. The temperature of both the injector and the detector was 150°C. Calibration curves were obtained with blood and tissue samples containing known amounts of toluene and handled as described. In all cases I% isopropanol was used as an internal standard. The analysis of score differences between acute and chronic experimental groups was compared by using the Student's t-test. RE SU L T S
During the first week of toluene exposure to 10,000, 20,000 and 40,000 ppm of the solvent concentrations, latencies of 9.2, 8.6 and 5.4 min in the hypnotic effect, respectively, resulted (Fig. 1A). These scores then progressively increased and achieved their highest values around the fourth week of toluene exposure (15, 12 and 10 min, respectively). By contrast, during the following weeks of exposure, there was a progressive declining in their values, that in the last week were very similar to those observed at the beginning of the experiment (20,000 and 40,000 ppm), or were still increased around 50% of its initial value (10,000 ppm).
TOLUENE AND BEHAVIORAL DEVELOPMENT
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FIG. 2. Rightingreflex latenciesin animals exposed to differenttoluene concentrationsduring the first 8 weeks postpartum (A), and at 8 monthsof age (B) for a 3-day period. Numberof subjectsand symbols in graphs as in Fig. 1. The analysis of the mean hypnotic effect scores between the different toluene concentrations throughout the study yielded the main effect of treatment, F(2,189)= 752.98, p<0.05. Additionally, a significant treatment × weeks interaction, F(14,189)= 8.44, p<0.05, was found. The analysis of variance in each week of the study revealed significant differences between the three experimental groups throughout the period analyzed (p<0.05). Figure 1B shows that toluene exposures (10,000 and 20,000 ppm) throughout a 3-day period in rats of 8 months of age did not provoke significant differences in the hypnotic effect latencies. During the first week of the experiment, the 10,000 or 20,000 ppm of toluene exposure provoked in the animals a righting reflex latency of 7 and l0 min, respectively (Fig. 2A), followed by a progressive decline in their values until week 4 of the experiment. Although in the case of the 10,000 ppm toluene dose, the righting reflex latency had a tendency to recover, however, their values were still shorter (2 min) in week 8 of the study. The use of a dose of 20,000 ppm toluene caused a clear recovery in the fighting reflex latency from week 4 onward, although their values were not in the range of the first 2 weeks. Concerning the exposure to 40,000 ppm of toluene, there was a clear prolongation in the fighting reflex latency during the first week (19 min), compared with that of the other 2 toluene doses. However, these scores were progressively decreasing in value until week 4 of the study (6.5 min), and thereafter they increased until week 8 of the experiment, where the fighting reflex latency attains a 12-min value (Fig. 2A). As in the case of the other two toluene doses, the fighting reflex latency increments seen from week 4 of the study did not achieve the values observed during the first 2 weeks of the experiment. The analysis of the mean righting reflex latency scores between the three different toluene groups revealed a significant main effect of treatment, F(2,147) = 359.65, p<0.05, and a significant treatment × weeks interaction, F(14,147) = 5.50, p<0.05. The analysis of variance on each week of the experirnent showed significant differences between the three experimental groups (p<0.05) throughout the period studied. Figure 2B indicates that toluene
exposure (10,000 and 20,000 ppm) throughout a 3-day period in rats of 8 months of age did not provoke significant differences in the righting response latencies. Blood, brain and liver toluene concentrations to a single or repetitive solvent exposures are presented in Table 1. There was a progressive increment to toluene concentrations in the different tissues, correlated with the increased solvent concentrations during a 15-min exposure. The comparisons between the acute vs. chronic toluene concentrations in each type of tissue, and the three different toluene doses here employed did not show significant score differences (p>0.05). The analysis of variance performed on body weight measurements did not reveal significant differences between the three different toluene groups, F(1,126)= 2.96, p=0.09. Body weight comparisons between toluene-exposed and nonexposed subjects have not shown significant differences (16). DISCUSSION
The present data suggest that chronic exposure to different toluene concentrations during the pre- and postweaning periods in the rat results in behavioral signs of a tolerance phenomenon, as evidenced by a significant progressive prolongation of the hynotic effect and a reduction of the fighting reflex latencies. This tolerance was apparently more consistent for the fighting reflex measurements in response to 10,000 ppm of toluene, particularly around the 8th week of the study, where the fighting reflex latency was 21% of its initial value. By contrast, the fighting response for the 20,000 and 40,000 ppm of toluene concentrations was approximately 60% and 63% of their initial scores, respectively. The fact of a progressive appearance of the behavioral tolerance phenomenon achieving its maximum effects around the third and fourth postnatal weeks, and then followed by a decrement in its values around the eighth week of the study is still a matter of speculation. The remarkable resistance of altricial newborns to extreme temperatures, pain, anoxia, starvation, and the imma-
356
LORENZANA-JIMENEZ AND SALAS
TABLE 1 BLOOD, BRAIN AND LIVER TOLUENE CONCENTRATIONS ( ~- SDM) OF 3* AND 9t RATS PER GROUP, RESPECTIVELY, AFTER 15 MIN OF SOLVENT EXPOSURE Blood p.cg/ml
Brain p.g/ml
Liver Ixcg/ml
Toluene Group mg/1 Air (ppm)
Acute*
Chronic1-
p:~
Acute*
Chronic~
p$
Acute*
31.7 (10,000)
94.19 _ 12.19
95.98 _+10.92
NS
132.62 -+ 12.98
131.22 -- 11.45
NS
62.5 (20,000)
125.62 --- 16.70
108.04 -+23.17
NS
152.40 -+26.10
127.87 -+33.50
125.0 (40,000)
198.60 -+36.86
160.49 +4.35
NS
250.03 ---25.40
246.18 ---42.30
Chronic#
p:~
477.28 _+33.75
402.36 ---72.76
NS
NS
656.77 -+ 189.11
584.00 - 134.90
NS
NS
745.41 -+ 189.71
719.44 -+ 139.71
NS
*Single exposure at 30 days old. tDaily exposures, 2-30 days postpartum SNS, not significant differences (Student's t-test).
ture response to stress (1, 11, 12, 25) perhaps may play a fundamental role in the temporal course of the presumably chronic toluene tolerance phenomenon. The possibility of a progressive maturational effect upon the specific enzymatic systems subserving the hepatic elimination of toxic compounds could perhaps be discarded because of the lack of significant differences in liver toluene contents, following the acute and/or chronic solvent intoxication. However, the lack of a significant reduction in both the hypnotic effect and the righting response latencies in 8-monthold toluene-exposed rats suggests that the behavioral tolerance phenomenon before two months of age is concurrent with maturational changes. Nevertheless, it may be necessary to analyze behavioral responses throughout longer periods of chronic toluene exposure and the temporal course of acute and chronic toluene detoxification processes in these tissues before eliminating the influence of a maturational effect and a pharmacodynamic or a pharmacokinetics tolerance phenomena (2, 13, 21). Although the increased use of a substance does not necessarily indicate the development of tolerance, but also a positive reinforcement to the same, at present we cannot discriminate among them because, through reflex measurements, it is difficult to recognize a compulsive toluene abuse which requires complex limbic and corticosubcortical circuitry for its neural integration. Several studies have suggested that during brain maturation the excitatory circuits develop earlier than the inhibitory ones (5, 7, 20). The centers maturating later probably exert modulatory influences upon neural structures previously developed. Thus,
adaptive behaviors such as locomotion, swimrmng, righting responses, etc., are largely modulated through different brain stem and telencephalic mechanisms, becoming efficient around the first month of life in the rat (17, 22-24). The finding of an altered hypnotic effect and righting reflex latencies as behavioral manifestations of a possible tolerance phenomenon to the chronic toluene exposure disagrees with similar studies showing no significant effects on the spontaneous locomotor activity measurements in adult rats (15,28). It is possible, then, that a differential threshold of activation in the brain substrate underlying the spontaneous locomotor activity, hypnotic effect and fighting reflex latencies, and/or a disturbance of the normal balance between the excitatory and inhibitory circuits subserving the integration of these locomotor patterns, may account for the behavioral effects provoked by the postnatal toluene exposure. Finally, while this study has focused on the effects of postnatal toluene exposure in rats, the behavioral responses described here can easily be employed in the assessment of behavioral tolerance following chronic exposure to different chemical compounds and industrial solvents during development. ACKNOWLEDGEMENTS The authors wish to thank Dr. V. Colotla for critical reading of the manuscript; A. Jimtnez for statistical advice; M. Rivero Murphy for technical assistance and M. del Pozo and B. Huerta for the preparation of the manuscript.
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