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Interactions between handling and acrylamide on the striatal dopamine receptor
Brain Research, 221 (1981) 421-424 Elsevier/North-Holland Biomedical Press
421
Interactions between handling and acrylamide on the striatal dopamine receptor
LYNDA L. UPHOUSE Laboratory of Behavioral and Neurological Toxicology, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709 (U.S.A.)
(Accepted June 4th, 1981) Key words: acrylamide - - dopamine - - handling receptors - - striatum
Rats were either handled for one week or were left undisturbed in their home cages. Twentyfour hours after exposure to 100 mg/kg acrylamide, rats were sacrificed and spiroperidol binding investigated in the striatum. In handling-gentled rats, spiroperidol binding was reduced by acrylamide. In non-handled animals, no overall drug effects were seen, but the effect of acrylamide varied with the time of sacrifice. These results demonstrate the importance of environmental factors in the response to neurotoxicants.
Several studies have suggested a relatively specific effect of acrylamide on striatal dopamine receptors1,2,11. Other neurotoxicants (e.g. Kepone, manganese, lead compounds) also influence dopamine receptors 1-8. However, dopaminergic systems can be modified by variations in the social environment. Guisado et al. 6 reported increased spiroperidol binding in long-term isolated rats and Corda et al. 4 presented evidence for an effect of handling on the response to dopaminergic drugs. These studies suggested that past experience modified the organism's response to dopaminergic manipulation. In the following study, the effect of acrylamide on striatal receptors of rats gentled by handling and of non-handled animals was examined 24 h after exposure to a nonlethal dose of the compound. Forty-eight male, Fischer inbred rats (Harlan Laboratories) were used in the experiments. Rats arrived in the laboratory at 6 weeks of age, were housed four to a cage, and were allowed one week to adapt to colony conditions (12-12 light-dark cycle with lights on at 7 a.m.; 50 -4- 10 % humidity and 21 q- 2 °C). One half of the animals were then individually handled for 3 min a day for 7 days. Twenty-four hours after the last handling experience, rats were dosed orally by gavage either with 100 mg/kg acrylamide (1 g/50 ml in H20) or an equivalent volume of water or were left undisturbed in their home cages. Twenty-four d: 2 h later, all rats were sacrificed by decapitation and striata were removed and frozen in liquid nitrogen. The order of sacrifice was counterbalanced so that within any one hour, representatives of all six conditions were sacrificed.
422 Membranes were prepared and spiroperidol binding was performed as described by Agrawal et al. 1. Specific binding was determined by subtracting the binding of [3H]spiroperidol (1 nM; 25 Ci/mmol; New England Nuclear) in the presence of 10-6 M haloperidol (McNeil Laboratories) from binding in the absence of the competitor. Specific binding was corrected for membrane protein as determined by the method of Lowry et al. a. Data were analyzed by two-way ANOVAs. Differences between individual means were assessed by Least Squares Difference Test 7. An alpha level of 0.05 was required for rejection of the null hypothesis. As seen in Table I, there were no significant differences between gentled and naive animals in spiroperidol binding. There was, however, an effect of acrylamide but the effect of the compound on spiroperidol binding depended on the pre-injection condition. In gentled animals, acrylamide led to a significant decrease in spiroperidol binding compared to either non-gavaged or water-treated controls (respectively t -2.41 and 2.76, df z 13, P < 0.05, LSD test). No significant effect of acrylamide on spiroperidol binding was seen in non-handled animals (P > 0.05). A similar dependence on past experience was seen in the effect of acrylamide on membrane protein (see Table II). Gentled animals given acrylamide had less protein in the membrane fraction than non-gavaged or water controls (respectively t = 2.89 and 2.44, df -- 13, P < 0.05, LSD test) but non-handled animals were not significantly different from each other.
TABLE I Effects of handling and acrylamide on spiroperidol binding Drug Treatment Handling Treatment
X 4- S.E.M.* (pmbound/gprotein) No Garage
Water Garage /lcrylamide
Handling (gentled) No handling
432 ± 24 431 i 13
442 ± 23 410 ± 19
354 4- 22** 383 t 2l
* average of 7-8 animals. ** indicates significant differencefrom water control.
TABLE II Effects of handling and acrylamide on striatal membraneprotein Drug Treatment Handling Treatment
~ ! S.E.M.* (t~gprotein/5 mg striatum) No Garage Water Garage Acrylamide
Handling (gentled) No handling
195 i 6 189 ± 7
191 ± 6 183 ± 6
* average of 7-8 animals. ** indicates significantdifferencefrom water control.
168 ± 5** 178 t 8
423 These data demonstrate that past experience of the organism influenced the CNS response to a neurotoxicant. Such a finding is in agreement with Corda's et al. 4 suggestion that handling-habituated animals may be better models than naive animals for the study of perturbations of the nervous system. In particular, they argued that handlinginduced stress of naive animals could confound biochemical changes related to the experimental procedures. The present results, however, are not totally explained by the differential effects of the drug administration on handled and non-handled animals. Even though there was a tendency for the water-gavaged animals to have less binding than non-handled controls, water-gavaged and non-gavaged animals did not differ in spiroperidol binding. Furthermore, previous studies have demonstrated that the gavaging experience does not influence spiroperidol binding over the 24-h period p o s t - g a v a g O t The accentuation of the effects of acrylamide in handling-gentled animals is not, therefore, due simply to different effects of the gavaging experience in handled and non-handled animals. Very little is known about the mechanism(s) by which acrylamide interacts with the CNS. However, recent findings from this laboratory suggest that acrylamide may influence pituitary secretions (in particular, prolactin levels) and these may in turn influence the CNS (Uphouse, personal observation). As more is discovered about the various organismic effects of acrylamide, the interactions between environmental factors and acrylamide-toxicity can be clarified. In contrast to previous studiesl, T M an acrylamide-induced elevation of spiroperidol binding was not observed in non-handled animals. However, recent observations in this laboratory suggest that the elevated spiroperidol binding seen 24 h after a single acrylamide exposure may be attenuated by a variety of factors. For example, in the present study, an unplanned observation was the finding that animals sacrificed from 10 to 12 a.m. were different from those sacrificed from 1 to 3 p.m. (Table III). A three-way A N O V A showed time of sacrifice to be a significant factor (F = 7.54, df = 1,35, P < 0.01). Furthermore, there was a significant interaction between the time of sacrifice and the drug treatment (F = 5.30, df = 2,35, P < 0.01). Specifically, in the non-handled condition, acrylamide-treated animals sacrificed from 1 to 3 p.m. had TABLE III Effects of time of sacrifice on spiroperidol binding in the striatum Drug Treatment Handling Treatment
Handling (gentled) a.m. p.m. No handling a.m. p.m.
}( ~- S.E.M.* (pmbound/gprotein) No Garage
Water Garage .4crylamide
439 ~ 19 448 ± 5
447 dz 43 439 d: 32
324 d- 20 384 ~ 34
411 ± 39 404 ~ 35
377 -4- 13 443 ~ 29
316 ± 15 450 _k 29**
* average of 3 ~ animals. * * indicates significant difference from a.m. group.
424 significantly m o r e spiroperidol binding t h a n d i d those sacrificed f r o m 10 to 12 a.m. (t = 3.38, d f = 6, P < 0.05, L S D test). T h e control g r o u p was not significantly affected b y the time o f sacrifice (P > 0.05). Previous studies have shown a d i u r n a l v a r i a t i o n in spiropCridol binding in the s t r i a t u m 9. Interestingly these investigators r e p o r t e d a n a d i r in binding at 10 a.m. a n d a p e a k at 2 p.m. in animals housed in conditions similar to those used in the present study. I t is possible, therefore t h a t the time o f sacrifice differences are related to this d i u r n a l rhythmicity, a n d p r i o r experience influences the effectiveness o f the drug in m o d i f y i n g the d i u r n a l rhythm. It is also possible t h a t the time course o f a c r y l a m i d e ' s effect on striatal d o p a m i n e depends on e x t r a n e o u s events. In a previous study, spiroperidol binding was increased 30 min after exposure to a c r y l a m i d e a n d was still elevated at 24 h 11. However, since spiroperidol binding was n o t m o n i t o r e d later t h a n 24 h following exposure to acrylamide, the time at which the binding returns to control levels is u n k n o w n . E n v i r o n mental factors m a y affect the rate at which the o r g a n i s m recovers from this acute exposure to the chemical. In s u m m a r y , the effects o f p r i o r h a n d l i n g on a c r y l a m i d e - i n d u c e d alterations in the striatal d o p a m i n e receptor were investigated. In handling-gentled a n i m a l s acryla m i d e reduced spiroperidol binding to striatal m e m b r a n e s b u t in n o n - h a n d l e d animals, no significant effect o f a c r y l a m i d e was seen. These findings suggest t h a t h a b i t u a t i o n to handling accentuates the effects o f a c r y l a m i d e a n d m a y even influence t h e directional a d a p t a t i o n o f the d o p a m i n e receptor. A u t h o r expresses a p p r e c i a t i o n to Michael Russell for technical assistance a n d to Mrs. N a n c y Mitchell for t y p i n g the manuscript. 1 Agrawal, A. K., Seth, P. K., Squibb, R. E., Tilson, H. A., Uphouse, L. L. and Bondy, S. C., Neurotransmitter receptors in brain regions of acrylamide-treated rats. I. Effects of a single exposure to acrylamide, Pharmaeol. Biochem. Behav., submitted. 2 Agrawal, A. K., Squibb, R. E. and Bondy, S. C., The effects of acrylamide treatment upon the dopamine receptor, Pharmacol. Biochem. Behav., submitted. 3 Bondy, S. C., Harrington, M. E., Anderson, C. L. and Prasad, K. N., The effect of low concentrations of an organic lead compound on the transport and release of putative neurotransmitters, Toxicol. Lett., 3 (1979) 35-41. 4 Corda, M. G., Biggio, G. and Gessa, G. L., Brain nucleotides in naive and handling-habituated rats: differences in levels and drug-sensitivity, Brain Research, 188 (1980) 287-290. 5 Goldman, M., Levo-dihydroxyphenylalanine, parkinson's disease and manganese poisoning, lndust. Med., 41 (1972) 12-15. 6 Guisado, E., Fernandez-Tome, P., Garzon, J. and Del Rio, J., Increased dopamine receptor binding in the striatum of rats after long-term isolation, Europ. J. PharmacoL, 65 (1980) 463-464. 7 Keppel, G., Design and Analysis: A Researcher's Handbook, Prentice-Hall, Englewood Cliffs, N.J., 1973. 8 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 9 Naber, D., Wirz-Justice, A., Kafka, M. S. and Wehr, T. A., Dopamine receptor binding in rat striatum; ultradian rhythm and its modification by chronic imipramine, Psychopharmacology, 68 (1980) I-5. 10 Seth, P. K., Agrawal, A. K. and Bondy, S. C., Biochemical changes in the brain consequent to dietary exposure of developing and mature rats to chlordecone (Kepone), submitted. 11 Uphouse, L. L. and Russell, M., Rapid effects of acrylamide on spiroperidol and serotonin binding in neural tissue, Pharmaeol. Bioehem. Behav., submitted.
421
Interactions between handling and acrylamide on the striatal dopamine receptor
LYNDA L. UPHOUSE Laboratory of Behavioral and Neurological Toxicology, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709 (U.S.A.)
(Accepted June 4th, 1981) Key words: acrylamide - - dopamine - - handling receptors - - striatum
Rats were either handled for one week or were left undisturbed in their home cages. Twentyfour hours after exposure to 100 mg/kg acrylamide, rats were sacrificed and spiroperidol binding investigated in the striatum. In handling-gentled rats, spiroperidol binding was reduced by acrylamide. In non-handled animals, no overall drug effects were seen, but the effect of acrylamide varied with the time of sacrifice. These results demonstrate the importance of environmental factors in the response to neurotoxicants.
Several studies have suggested a relatively specific effect of acrylamide on striatal dopamine receptors1,2,11. Other neurotoxicants (e.g. Kepone, manganese, lead compounds) also influence dopamine receptors 1-8. However, dopaminergic systems can be modified by variations in the social environment. Guisado et al. 6 reported increased spiroperidol binding in long-term isolated rats and Corda et al. 4 presented evidence for an effect of handling on the response to dopaminergic drugs. These studies suggested that past experience modified the organism's response to dopaminergic manipulation. In the following study, the effect of acrylamide on striatal receptors of rats gentled by handling and of non-handled animals was examined 24 h after exposure to a nonlethal dose of the compound. Forty-eight male, Fischer inbred rats (Harlan Laboratories) were used in the experiments. Rats arrived in the laboratory at 6 weeks of age, were housed four to a cage, and were allowed one week to adapt to colony conditions (12-12 light-dark cycle with lights on at 7 a.m.; 50 -4- 10 % humidity and 21 q- 2 °C). One half of the animals were then individually handled for 3 min a day for 7 days. Twenty-four hours after the last handling experience, rats were dosed orally by gavage either with 100 mg/kg acrylamide (1 g/50 ml in H20) or an equivalent volume of water or were left undisturbed in their home cages. Twenty-four d: 2 h later, all rats were sacrificed by decapitation and striata were removed and frozen in liquid nitrogen. The order of sacrifice was counterbalanced so that within any one hour, representatives of all six conditions were sacrificed.
422 Membranes were prepared and spiroperidol binding was performed as described by Agrawal et al. 1. Specific binding was determined by subtracting the binding of [3H]spiroperidol (1 nM; 25 Ci/mmol; New England Nuclear) in the presence of 10-6 M haloperidol (McNeil Laboratories) from binding in the absence of the competitor. Specific binding was corrected for membrane protein as determined by the method of Lowry et al. a. Data were analyzed by two-way ANOVAs. Differences between individual means were assessed by Least Squares Difference Test 7. An alpha level of 0.05 was required for rejection of the null hypothesis. As seen in Table I, there were no significant differences between gentled and naive animals in spiroperidol binding. There was, however, an effect of acrylamide but the effect of the compound on spiroperidol binding depended on the pre-injection condition. In gentled animals, acrylamide led to a significant decrease in spiroperidol binding compared to either non-gavaged or water-treated controls (respectively t -2.41 and 2.76, df z 13, P < 0.05, LSD test). No significant effect of acrylamide on spiroperidol binding was seen in non-handled animals (P > 0.05). A similar dependence on past experience was seen in the effect of acrylamide on membrane protein (see Table II). Gentled animals given acrylamide had less protein in the membrane fraction than non-gavaged or water controls (respectively t = 2.89 and 2.44, df -- 13, P < 0.05, LSD test) but non-handled animals were not significantly different from each other.
TABLE I Effects of handling and acrylamide on spiroperidol binding Drug Treatment Handling Treatment
X 4- S.E.M.* (pmbound/gprotein) No Garage
Water Garage /lcrylamide
Handling (gentled) No handling
432 ± 24 431 i 13
442 ± 23 410 ± 19
354 4- 22** 383 t 2l
* average of 7-8 animals. ** indicates significant differencefrom water control.
TABLE II Effects of handling and acrylamide on striatal membraneprotein Drug Treatment Handling Treatment
~ ! S.E.M.* (t~gprotein/5 mg striatum) No Garage Water Garage Acrylamide
Handling (gentled) No handling
195 i 6 189 ± 7
191 ± 6 183 ± 6
* average of 7-8 animals. ** indicates significantdifferencefrom water control.
168 ± 5** 178 t 8
423 These data demonstrate that past experience of the organism influenced the CNS response to a neurotoxicant. Such a finding is in agreement with Corda's et al. 4 suggestion that handling-habituated animals may be better models than naive animals for the study of perturbations of the nervous system. In particular, they argued that handlinginduced stress of naive animals could confound biochemical changes related to the experimental procedures. The present results, however, are not totally explained by the differential effects of the drug administration on handled and non-handled animals. Even though there was a tendency for the water-gavaged animals to have less binding than non-handled controls, water-gavaged and non-gavaged animals did not differ in spiroperidol binding. Furthermore, previous studies have demonstrated that the gavaging experience does not influence spiroperidol binding over the 24-h period p o s t - g a v a g O t The accentuation of the effects of acrylamide in handling-gentled animals is not, therefore, due simply to different effects of the gavaging experience in handled and non-handled animals. Very little is known about the mechanism(s) by which acrylamide interacts with the CNS. However, recent findings from this laboratory suggest that acrylamide may influence pituitary secretions (in particular, prolactin levels) and these may in turn influence the CNS (Uphouse, personal observation). As more is discovered about the various organismic effects of acrylamide, the interactions between environmental factors and acrylamide-toxicity can be clarified. In contrast to previous studiesl, T M an acrylamide-induced elevation of spiroperidol binding was not observed in non-handled animals. However, recent observations in this laboratory suggest that the elevated spiroperidol binding seen 24 h after a single acrylamide exposure may be attenuated by a variety of factors. For example, in the present study, an unplanned observation was the finding that animals sacrificed from 10 to 12 a.m. were different from those sacrificed from 1 to 3 p.m. (Table III). A three-way A N O V A showed time of sacrifice to be a significant factor (F = 7.54, df = 1,35, P < 0.01). Furthermore, there was a significant interaction between the time of sacrifice and the drug treatment (F = 5.30, df = 2,35, P < 0.01). Specifically, in the non-handled condition, acrylamide-treated animals sacrificed from 1 to 3 p.m. had TABLE III Effects of time of sacrifice on spiroperidol binding in the striatum Drug Treatment Handling Treatment
Handling (gentled) a.m. p.m. No handling a.m. p.m.
}( ~- S.E.M.* (pmbound/gprotein) No Garage
Water Garage .4crylamide
439 ~ 19 448 ± 5
447 dz 43 439 d: 32
324 d- 20 384 ~ 34
411 ± 39 404 ~ 35
377 -4- 13 443 ~ 29
316 ± 15 450 _k 29**
* average of 3 ~ animals. * * indicates significant difference from a.m. group.
424 significantly m o r e spiroperidol binding t h a n d i d those sacrificed f r o m 10 to 12 a.m. (t = 3.38, d f = 6, P < 0.05, L S D test). T h e control g r o u p was not significantly affected b y the time o f sacrifice (P > 0.05). Previous studies have shown a d i u r n a l v a r i a t i o n in spiropCridol binding in the s t r i a t u m 9. Interestingly these investigators r e p o r t e d a n a d i r in binding at 10 a.m. a n d a p e a k at 2 p.m. in animals housed in conditions similar to those used in the present study. I t is possible, therefore t h a t the time o f sacrifice differences are related to this d i u r n a l rhythmicity, a n d p r i o r experience influences the effectiveness o f the drug in m o d i f y i n g the d i u r n a l rhythm. It is also possible t h a t the time course o f a c r y l a m i d e ' s effect on striatal d o p a m i n e depends on e x t r a n e o u s events. In a previous study, spiroperidol binding was increased 30 min after exposure to a c r y l a m i d e a n d was still elevated at 24 h 11. However, since spiroperidol binding was n o t m o n i t o r e d later t h a n 24 h following exposure to acrylamide, the time at which the binding returns to control levels is u n k n o w n . E n v i r o n mental factors m a y affect the rate at which the o r g a n i s m recovers from this acute exposure to the chemical. In s u m m a r y , the effects o f p r i o r h a n d l i n g on a c r y l a m i d e - i n d u c e d alterations in the striatal d o p a m i n e receptor were investigated. In handling-gentled a n i m a l s acryla m i d e reduced spiroperidol binding to striatal m e m b r a n e s b u t in n o n - h a n d l e d animals, no significant effect o f a c r y l a m i d e was seen. These findings suggest t h a t h a b i t u a t i o n to handling accentuates the effects o f a c r y l a m i d e a n d m a y even influence t h e directional a d a p t a t i o n o f the d o p a m i n e receptor. A u t h o r expresses a p p r e c i a t i o n to Michael Russell for technical assistance a n d to Mrs. N a n c y Mitchell for t y p i n g the manuscript. 1 Agrawal, A. K., Seth, P. K., Squibb, R. E., Tilson, H. A., Uphouse, L. L. and Bondy, S. C., Neurotransmitter receptors in brain regions of acrylamide-treated rats. I. Effects of a single exposure to acrylamide, Pharmaeol. Biochem. Behav., submitted. 2 Agrawal, A. K., Squibb, R. E. and Bondy, S. C., The effects of acrylamide treatment upon the dopamine receptor, Pharmacol. Biochem. Behav., submitted. 3 Bondy, S. C., Harrington, M. E., Anderson, C. L. and Prasad, K. N., The effect of low concentrations of an organic lead compound on the transport and release of putative neurotransmitters, Toxicol. Lett., 3 (1979) 35-41. 4 Corda, M. G., Biggio, G. and Gessa, G. L., Brain nucleotides in naive and handling-habituated rats: differences in levels and drug-sensitivity, Brain Research, 188 (1980) 287-290. 5 Goldman, M., Levo-dihydroxyphenylalanine, parkinson's disease and manganese poisoning, lndust. Med., 41 (1972) 12-15. 6 Guisado, E., Fernandez-Tome, P., Garzon, J. and Del Rio, J., Increased dopamine receptor binding in the striatum of rats after long-term isolation, Europ. J. PharmacoL, 65 (1980) 463-464. 7 Keppel, G., Design and Analysis: A Researcher's Handbook, Prentice-Hall, Englewood Cliffs, N.J., 1973. 8 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 9 Naber, D., Wirz-Justice, A., Kafka, M. S. and Wehr, T. A., Dopamine receptor binding in rat striatum; ultradian rhythm and its modification by chronic imipramine, Psychopharmacology, 68 (1980) I-5. 10 Seth, P. K., Agrawal, A. K. and Bondy, S. C., Biochemical changes in the brain consequent to dietary exposure of developing and mature rats to chlordecone (Kepone), submitted. 11 Uphouse, L. L. and Russell, M., Rapid effects of acrylamide on spiroperidol and serotonin binding in neural tissue, Pharmaeol. Bioehem. Behav., submitted.