Neurotransmitter Receptors in Brain Regions of Acrylamide-Treated Rats . I : Effects of a Single Exposure to Acrylamide 1

AGRAWAL, A. K , P. K SETH, R E. SQUIBB, H. A TILSON, L. L. UPHOUSE ANDS. C BONDY Neurotransmitter receptors m brain regwns of acrylam1de-treated rats. I Effects of a single exposure to acrylamide PHARMAC. BIOCHEM. BEHAV 14(4) 527-531, 1981.-A smgle oral dose of acrylam1de (25 or 50 mg/kg) increased the level of striatal H-spiroperidol bmding in six week old male rats. This enhanced dopamme receptor act1v1ty was specific smce treatment caused no significant changes in glycine, serotonm, and muscarinic cholmerg1c bmdmg. At the highest acrylamide dose tested (100 mg/kg), elevations of the medullary glycme and frontal cortical serotonm receptors were also found. Pretreatment of animals with a blocker of hepatic mixed funct10n ox1dase (SKF 525a) or a th1ol blocker (methylmercunc chlonde) prevented the acrylam1de-induced elevat10n of stnatal sp1ropendol bmding, ind1catmg that the causative agent was a secondary metabohte of acrylam1de Apomorphme-mduced motJhty was significantly attenuated by 24 hr predosmg with acrylam1de, suggesting a change m the sensitivity of the dopamme receptor The behav10ral relevance of observed b1ochem1cal changes was thus shown by the altered response of treated animals to apomorphine.

ACRYLAMIDE has been shown to cause damage to the peripheral nervous system, in humans and experimental animals [11,16,18,21]. Other signs of acrylam1de intoxication such as tremor, pupillary dilatation, and excess salivation suggest hyperactivity of the catecholammergic system [4]. More recently central nervous tissue has also been shown to be injured by systemically administered acrylam1de [3, 18,19]. In this laboratory, the striatal dopamine receptor of animals given a single dose of acrylamide has been shown to be increased in density and to have a heightened affinity for a labeled neuroleptic agent. These effects are greater following a two week dosing schedule containing 10 separate exposures to acrylamide [13].
The purpose of this work was to determine the specificity of the effect of a single oral dose acrylamide treatment upon the dopamine receptor. In addition, further information was sought concerning the nature of the active agent causing changes within the bram after exposure to acrylamide. These data indicate the dopamine receptors to be unusually susceptible to acrylamide exposure. The toxic effect appeared to be due to a metabolically formed derivative of acrylamide rather than directly to the unaltered molecule. Moreover, acrylam1de-treated rats were also found to be less sensitive to the behavioral effects of apomorphine, indicating that acrylamide might have altered the functional sensitivity of the dopamine receptor.

Binding Assay
Six-week old male Fischer rats were used m this study. After decapitation, brain regions were dissected by the method of Iversen and Glowinski [IO]. A crude membrane fraction was prepared from frozen bram regions by homogenization of tissue in 19 volumes of0.32 M sucrose followed by centrifugation (50,000 g, IO min). The precipitate from this step was then homogenized m 40 mM tris pH 7.4 and recentrifuged. This procedure combined with the pnor freezing step causes major lysis of structural cell components such as mitochondna or nerve endings. The final pellet was suspended in the tris-HCl pH 7.4 buffer at a concentration representing 50 mg original tissue/ml.
Bmdmg incubations were carried out in tnplicate in a final volume of l ml containing 40 mM tris-HCl (pH 7.4) together '  with appropriate labeled and unlabeled pharmacological agents. The incubation mixture used in the assay of serotonin also included 10-5 M pargyline, 4x 10-3 M CaC1 2 and 5.7x 10-3 M ascorbic acid. The amount of tissue used per tube corresponded to 5-10 mg original wet weight and contained 300-400 µ,g protein as determined by the method of Lowry et al. [14]. At the end of a 15 min incubation at 37° samples were filtered on glass fiber discs (25 mm diameter, 0.3 µ, pore size, Gelman Inc., Ann Arbor, Ml) and washed twice rapidly with 5 ml tris buffer. In the case of strychnine bindmg assay, only one wash was used. Filter discs were then dried and counted in 5 ml of a scintillation mixture (Aquasol, New England Nuclear. Boston, MA) using a scintillation counter (Packard Tri Carb 2660) at an efficiency of 38-43%. Table I presents the final concentrations of radioactive ligands used, the receptor class that they were mtended to assay for. and the final concentrations of competing compounds that were present in control mcubations in order to determine the extent of non-specific bindmg. Control incubations were carried out simultaneously with the experimental senes contaming unlabeled competing ligand The level of non-specific binding to tissue was between 5% and 29% of total binding. The method used was essentially similar to other filtrat10n bmding methods [22]. However, we felt 1t necessary to establish basic binding characteristics pnor to studies on ammals treated with acrylam1de. These included delineation of saturability, specificity, reversibility, and regional distribution [1, 5,6]. Differences between groups were assessed using Fisher's Least Sigmficant Difference Test after a one-way analysis of variance [15]. The accepted level of sigmficance in all cases was p<0.05 using a two-tailed test. There was always experimental variance between var-10us groups of control animals tested in different weeks. For this reason all treated and control rats that were to be compared were maintained simultaneously and membrane preparations and binding studies were always conducted in parallel. Each data point represents values derived from 6-8 individual animals

Drug and Auylamide Admuustration
Methylmercuric chloride (CH 3 HgCI) was injected intraperitoneally (14 mg/kg) daily for three days preceding 2-diethylammoethyl-2, 2-diphenylvalerate HCl (SKF 525A), was injected twice intrapentoneally (50 mg/kg) at 10 a.m. and 4 p.m. on the day before acrylamide dosmg of 100 mg/kg body weight. Acrylamide dissolved in water was administered orally by gavage in a volume of 5 ml/kg body weight. Control rats received an equivalent volume of distilled water. Animals were killed 24 hr after administration of ac-rylam1de in all cases.

Apomorphine Challenge
Thirty male Fischer rats were randomly assigned to receive either 0, 25, or 100 mg/kg ofacrylam1de by gavage in a volume of 5 ml/kg body weight. Twenty-four hr later, the rats were put individually mto plastic domiciliary cages havmg 0.5 mches of com cob bedding and a wire screen hd; they were then placed inside light and sound attenuating chambers containing activity monitors (Automex, Columbus Instruments, Columbus, Oh10). The animals were allowed to acclimate for 10 min and then mjected IP with 1 mg/kg of apomorphine hydrochloride dissolved in isotonic saline. Apomorphine-induced motility was measured for a period of 1 hr In a second experiment, 20 rats received either 0 or 100 mg/kg of acrylamide by gavage and were challenged with 1 mg/kg of apomorphine as described in the previous experiment. In the present study, the animals were also assessed for apomorphine-mduced stereotypy using a rating scale similar to that of Reinstein et al [17]; activity was also measured by the automated activity monitors. Behavioral observations were recorded at 1 min intervals by an observer unaware of pretreatment of the animals (i.e., acrylamide or vehicle pretreatment). Behavioral ratings of forwardcrawling, paddling, wall climbmg, forward-sniffing, and rearing were combined for a quantitative measure of'' Activity''. "Inactivity" was defined as sitting, lying, displaying odd posture, and sitting-sniffing. A third category, "sniffing". consisted of ratings of forward and sitting-sniffing.
The total number of counts accumulated by the automex momtor were square root transformed [12] and were tested for statistical significance using a one-way analysis of vanance. post hoc comparisons between groups were made using Fisher's Least S1gmficant Difference Test [15]. In the case where there were only two treatment groups, Student's t-test was used to test for differences between means [15]. All tests were two-tailed and p <0.05 was set as the accepted level of significance.
The number of movements rated as "activity", "inactivity", or "sniffing" were accumulated for the 60 min observation period. Differences between treatment groups were tested for statistical significance using two-tailed Mann-Whitney U-tests [20]. Correlation between activity counts measured by the automex with behavioral categones of "activity". "inactivity", and "sniffing" were determined usmg Spearman's Rank correlation coefficient [20].

RESULTS AND DISCUSSION
An increase in 3 H-spiroperidol bindmg within the corpus striatum of treated animals was found 24 hr after administration of acrylamide at all dose levels studied. However, at the two lower doses, the intensity of binding of several other labeled ligands was not significantly changed ( Table 2). The lack of an effect upon the striatal muscannic receptor showed that it was the dopamine binding site rather than the entire striatum that was mitially the most sensitive to the treatment. This selectivity was lost at the highest dose used (100 mg/kg). In this latter case, increased glycinergic and serotonergic binding were also apparent. These data suggest that the dopaminergic circuitry may be unusually susceptible to disruption by acrylamide treatment.
Twenty-four hr after the administration of acrylamide, the number of activity counts generated durmg the 10 mm acclimation was not affected significantly (Fig. 1). However. pretreatment with acrylamide significantly affected the number of apomorphme-induced activity counts (Fig. 1). Animals receiving 25 or 100 mg/kg acrylamide had significantly fewer counts than those receiving distilled water. The ammals receiving the higher dose of acrylamide also differed significantly from the animals receiving 25 mg/kg.
In the second experiment in which a rating scale was used, acrylamide pretreatment was also found to antagonize the effects of apomorphine (Fig. 2). The number of counts FIG. J. The effects of acrylamide pretreatment on motor activity J 0 mm before InJect10n (acchmation) and for J hr after tnJect1on of J mg/kg apomorphme. Data are average counts (square root transformed) ±SE for JO rats per group Overall s1gmficance was assessed by a one-way analysis of vanance A stensks md1cate s1gmficant difference form control (0 mg/kg) (Fisher's Least Significant Difference Test, p<0.05) rated as "activity" or "sniffing" were significantly decreased by acrylamide pretreatment. while the number of ratmgs in the "inactivity" category were significantly increased by acrylam1de. The number of activity counts measured by the automex was positively correlated with ratings of "activity" (r=.572, p<0.05) and "sniffing" (r=.482, p<0.05), but was inversely correlated with "inactivity" (r= -.594, p<0.05).
The elevation of dopamine receptors m conjunction with a reduced behavioral response to a dopammergic agonist can best be explained in terms of destruction of or damage to the dopamine neurons of the nigrostnatal pathways. This would reduce the magnitude of stereotypic mduct1on by apomorphine and also explain the receptor increase m terms of a denervat1on supersens1tivity of the postsynaptic cell. The correlation of transmitter receptor binding data with behavioral changes should be viewed with caution, since some active neuroleptics such as disulfuride bmd poorly to the dopamine receptor while certain psychogenically mact1ve butyrophenones compete strongly with 3 H-spiroperidol bindmg [13]. Another study was d!fected toward ascertaining whether 1t was acrylam1de, one of its metabolites or a conjugate involving interact10n with sulfhydryl groups that was the causative agent m effecting the observed changes. This was earned out by pretreatment of rats with an inhibitor of hepatic induction of mixed function ox1dases (SKF 525A) or an agent reacting with th10l groups (methylmercuric chloride). The acrylamide-induced changes of striatal dopaminergic receptors were completely prevented by either SKF 525A or methylmercunc chlonde. However, neither agent had. by itself, any significant effect upon the dopamine receptor (Table 3). The results with SKF 525A suggested that the effect of acrylam1de on the stnatum was not direct but involved the production of an active agent from acrylamide by catabolic enzymes. The results with the methylmercunc AGRAWAL ET AL. chlonde might be due to blockage of mteraction of acrylamide with certain sulfhydryl residues [7]. It may be that the effect of acrylamide upon the dopamine receptor induces reaction of key sulfhydryl residues with acrylamide or an acrylamide-metabolite This is in contrast with reports that SKF 525A pretreatment worsens the peripheral effects of and the lethality of acrylam1de [9]. However, phenobarbital mduct10n of hepatic enzymes does not alter the rate of onset of acrylamide induced peripheral neuropathy in hens [8]. Thus, acrylamide may act directly as a toxic substance but some of its effects may also be attnbuted to metabolic breakdown products The lack of effect of I 0-5 M acrylam1de m vitro upon the striatal binding of3H-spiroperidol [Agrawal, A.K., unpublished result] substantiates this idea. The relation between the neurotoxicity of acrylamide and its capacity to link to -SH residues remains unclear smce N(hydroxymethyl) acrylam1de which apparently does not injure the peripheral nervous system, is as avid as acrylamide in attaching itself to -SH groups [9]. However, the effects of this denvative upon the central nervous system have not been determined. The nature of the chemicals produced by acrylamide catabolism is presently being studied