Neurotransmitter Receptors in Brain Regions of Acrylamide-Treated Rats. II: Effects of Extended Exposure to Acrylamide

BONDY, S. C., H A. TILSON AND A K. AGRAWAL. Neurotransmllter receptor~ in hrmn re{?tons ofacry/amtde treated rats. II EffecH of extended exposure to acrylamtde PHARMAC. BIOCHEM BEHAV. 14(4) 533-537. 1981. Acrylam1de was admmistered orally to 6 week old male rats m ten doses, spread over a two week penod. At the two lower doses (5 and IO mg/kg, total dose 50 and 100 mg/kg) effects on neurotransmitter receptor sites appeared confined to the stnatum where both the dopamme and muscarimc acetylcholine receptors exhibited enhanced bmdmg twenty four hours after the last acrylam1de dose. Other receptor sites w1thm the frontal cortex, cerebellum, and medulla were not significantly altered. At the highest dose (20 mg/kg, given ten times), increases were also found for frontal cortical serotonm, medullary glycme, and cerebellar GABA receptor sites. The only unaffected receptor found was the cortical site for benzodiazepene One week after the final acrylamide dose, the mtensity of bmding of all ligands studied was not s1gmficantly different m treated and control groups. Thus, effects appeared reversible Since stnatal membrane protem concentrat10n was reduced by treatment of rats with acrylam1de, the observed increase m act1v1ty ofmuscarinic receptors could be best accounted for in terms of loss of stnatal non-receptor protem rather than mcreased bmdmg However, the magmtude of mcreased stnatal 'H-sp1ropendol bmding m treated ammals suggested an mcrease m overall binding capacity An effect on dopamme neurons was also suggested by a decreased responsiveness to apomorphme m rats treated with acrylam1de at IO mg/kg for IO successive days; however, the effect had d1ss1pated by 8 days after the final mJect1on of acrylamide Acrylam1de

IN VIEW of its known neurotox1city in humans, the effects of acrylamide treatment upon high affinity bmdmg sites within the brain has been the subject of several studies from this laboratory [21]. Initial studies indicated an unusual sensitivity of the striatal dopamine receptor to systemic treatment of rats with acrylamide [4]. A single oral exposure to acrylam1de caused a specific increase in the capacity of striatal membranes to bmd 3 H-spiroperidol. This increase could be correlated with behavioral changes and was attributed to the action of a metabolic breakdown product of acrylamide rather than to the intact chemical [2]. Gestational exposure to acrylamide also caused changes in the dopamine receptors of neonates [3].
The purpose of the present work was to determme whether repeated exposure to acrylamide would affect a wider range of transmitter bmding sites than that observed after a smgle dose. The reversibllity of any changes was also studied. Data were analyzed so as to reveal whether the overall binding capacity of the striatum was altered or whether acrylamide-mduced changes reflected variation of the level of non-receptor protein within tissues. 'To whom reprmt requests should be addressed. 533 METHOD Six-week old male Fischer rats were used in this study. Acrylamide dissolved in water was administered orally by gavage daily in a volume of 5 ml/kg body weight. Control rats received an equivalent volume of distilled water. Animals were killed 24 hours or eight days after administration of acrylamide. After decapitation, brain regions were dissected by the method of Iversen and Glowinski [10]. A crude membrane fraction was prepared from frozen brain regions by homogenization of tissue in 19 volumes of 0.32 M sucrose followed by centrifugation (50,000 g, IO min). The precipitate from this step was then homogenized in 40 mM tns pH 7.4 and recentrifuged. The final pellet was suspended in the tris-HCI buffer at a concentration representing 50 mg original tissue/ml.
Binding incubations were carried out m triplicate ma final volume of 1 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-s M pargyline, 4x 10-3 M CaCl 2 and Bmdmg expressed as pmol/g protem±S.E *Differs from zero-dose (p<0.05, Fisher's Least Sigmficant Difference Test) Assays were conducted on membranes prepared from rats 24 hours after the last acrylam1de dose Expenmental details are descnbed m the text 5-7 x 10-3 M ascorbic acid. The amount of tissue used per tube corresponded to 5-10 mg origmal wet weight and contained 3~00 µg protein as determined by the method of Lowry et al [12]. 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 rapidly two times with 5 ml tris buffer. In the case of the strychnine bmding assay only one wash was used. Filter discs were then dried and counted in 5 ml of a scintillat10n mixture using a scintillation counter at an efficiency of 38--43%. Control incubations were carried out in order to determine the extent of non-specific binding simultaneously with the experimental series. The final concentration of unlabeled competmg compounds in control incubations was 10-s M. The assay of the dopamine receptor was performed using 10-9 M [1-phenyl-4 3 H]-spiroperidol (23 Ci/mmol) as the binding ligand and haloperidol as the competmg compound in control tubes. In a parallel manner 10-9 MDL [benzilic-4, 4' -3 H] quinuchdinyl benzilate (29 Ci/mmol) was used to measure muscarinic sites with atropine as a competitor. Benzodiazepene sites were estimated with 0.7x 10-9 M (methyl-3H)-diazepam (73 Ci/mmol) and the unlabeled compound as competitor. 8.0x 10-9 M [methylene-3H(N)]muscimol (7.3 Ci/mmol) and unlabeled GABA were used in GABA binding site assays. For the glycine site, nonradioactive strychnine was used to complete with 6.0x 10-9 M [G-3 H]-strychnme sulphate (13 Ci/mmol) 3.1x10-9 M [ l ,2 3 H(N) ]-serotonin (30 C1/mmol) was used together with unlabeled serotonm in the assay of this receptor. The method used was essentially similar to other filtration binding methods [23]. However, we felt it necessary to establish basic bindmg characteristics prior to studies on animals treated with acrylamide. These included delineation of saturability, specificity, reversibility, and reg10nal distribution [1, 5,6]. Differences between groups were assessed using Fisher's Least Significant Difference Test after a one-way analysis of variance. The accepted level of significance in all cases was p<0.05 usmg a one-tailed test. Each data point represents values drived from 6-8 individual animals processed separately.
In a separate expenment, 20 male Fischer rats were given either 0 or 10 mg/kg of acrylamide orally for a penod of 10 consecutive days. Twenty-four hours and 8 days later, they were tested for responsiveness to the dopamine agomst apomorphine using a method described m detail elsewhere [2]. Motility was assessed usmg an automated monitor (Automex, Columbus Instruments, Columbus, Ohm). Rats were allowed to acclimate for a period of 10 minutes m plastic cages positioned atop of the monitors and then Injected with 1 mg/kg of apomorphine, IP; motility was recorded for a period of 60 mm.

RESULTS
Ten doses of acrylam1de as low as 5 mg/kg resulted in significant elevations of 3 H-spiroperidol and 3 H-QNB bmdmg to striatal membranes. Twenty-four hours after the last dose at 5 and 10 mg/kg levels, no other significant changes in receptor binding were observed, but at 20 acrylamide mg/kg, mcreased binding of several tritiated ligands were seen in cortex, cerebellum, and medulla (Table 1). The only receptor class examined that was not increased was the receptor for the non-neurotransmitter, benzodiazepene. All receptor sites thought to be directly associated with neurotransmission were elevated at the 20 mg/kg dose while no decreases in bmdmg were observed.
An analysis was made of the total membrane protein recoverable from each brain area 24 hours after the last acrylamide dose. The concentration of membrane protein was reduced in the corpus stnatum of acrylam1de-treated rats but no major change was seen in protein content of other brain regions at any acrylamide dose used (Table 2). This difference between the striatum and other cerebral areas is additional evidence for the unusual sensitivity of the basal ganglia to the treatment with acrylamide. Taking into account the reduced striatal membrane content of treated animals, the observed changes in muscarinic binding are largely attributable to a loss of non-receptor protem rather than mcreased overall receptor capacity. However, the elevated dopaminergic binding in treated animals 1s too large to be totally accounted for in this manner. Together with previous work [4] the data mdicate that a real mcrease in the dopamme receptor capacity occurs in treated ammals.   The effects of repeated exposure to acrylam1de on responsiveness to apomorphine Rats were dosed with either 0 or 10 mg/kg of acrylanude, orally for 10 consecutive days Twenty-four and 192 hours later, they were challenged with 1 mg/kg of apomorphme, IP and motthty recorded as descnbed elsewhere [2]. Data are mean ± SE motthty counts, square root transformed for 10 rats per group. The astensk md1cates s1gmficant difference from control (Student's t-test, p<0.05, two-tailed) Ammals treated with the intermediate dose of acrylamide (10 mg/kg, ten times) were tested for their responsiveness to the dopamine receptor agonist apomorphine (1 mg/kg). Experimental animals were significantly less susceptible to such drug-induced behavioral changes and the effect was reversed by 8 days after the last dose of acrylamide (Fig. 1). Thus, the observed alterations of striatal receptors in treated animals appeared to lead to modified behavior in response to a pharmacological challenge. Taken together, the data suggest an impaired functioning of dopamine neurons in Bmdmg expressed as pmol/g protein±S.E *Differs from zero-dose (p<O 05, Fisher's Least S1gmficant Difference Test). Bmdmg assays were conducted on membranes prepared from rats 8 days after the last acrylamide dose. Expenmental detatls are descnbed in the text. treated rats. These observed differences in the apomorphine induced responses of treated rats were not detectable eight days after the last acrylamide dose. In a study involving greater expsoure to acrylamide, behav10ral indices of peripheral nerve impairment were found no longer observed at 5 weeks, but were still present at 1 week after cessation of dosing [21].
All observed changes m striatal protein content were reversed within one week after cessation of the dosing schedule (data not presented). The binding of ligands to nonstriatal regions was also not significantly different in dosed and control animals eight days after the last acrylamide administration. However, at the two higher doses of acrylamide used, dopaminergic binding was still significantly elevated, and muscarinic binding tended to remain higher after this eight day interval (Table 3). This increase in acrylamide binding although significant was never greater than 18% and thus markedly less than the 32-57% increases found one day post-dosing Thus, much of the imtial effect of acrylamide treatment appeared to be reversed, although a residual abnormality was still detectable. DISCUSSION Acrylamide treatment is known to rapidly depress the rate of protein synthesis in several tissues including bram [8,18]. However, this general effect may not totally account for the more localized reduction of striatal protem content that we have found Taken together with the binding data, our results imply an unusual susceptibility of the dopaminergic innervation of the striatum to acrylamide. The persistence of effects induced by the higher acrylamide doses could perhaps be related to the presence of residual acrylam1de or metabolites, smce this chemical 1s known to be present m nerve tissue for some weeks after exposure [9,17]. A similar reversibility has been found in young rats after gestation exposure to acrylam1de [3] and after neonatal exposure (Gerhart and Tilson, unpublished observation). The central nervous system appears to possess sufficient regenerative capacity to at least partially restore normal metabolism after limited exposure to this toxicant. However, it 1s not known whether more prolonged acrylamide treatment could result in permanent damage to central nervous tissue. The repeated treatment reported here resulted in more widespread changes (both chemically and anatomically) than the single dose reported in the precedmg chapter [2].
The increased striatal 3 H-spiroperidol bmding level and concurrent decrease in responsiveness to apomorphine may appear to be paradoxical. However, our results are analogous to those reported by Owen et al. [14]. These authors found that chronic haloperidol treatment of rats lead to an increase of3H-spiroperidol bmdmg sites m the striatum but a decrease in apomorphine induced stereotypy. Both their results and those reported in this paper might be best explamed by postulatmg damage to, rather than hypoactivity of, the dopammergic pathways. In this case the postsynaptic nerve cells might develop a supersensitivity but apomorphine would not be able to elicit a major response even in the BONDY, TILSON AND AGRAWAL presence of an excess number of dopamine receptors. The specificity of the stnatum may be due to some vulnerable biochemical aspect of the dopamine system. Another possibility 1s that the mean length of dopamme neurons is greater than that of neurons contammg the other neurotransmitter species studied here Dopamine neurons run m relatively long tracts and shorter dopamme intemeurons do not appear to exist. This excess length might cause such nerve cells to be more easily damaged by acrylam1de. Thi~ could most readily be detected by receptor binding studies in areas where the target cells of tracts occupy a relatively compact area such as the stnatum. This idea 1s supported by the known deleterious effect of acrylamide upon axonal transport processes in peripheral nerves [ 15]. Such impairment might be expected to be especially mJunous to relatively long axons Defective axonal transport mduced by acrylamide may be a secondary reflection of general damage to anabolic processes [22].
From this emerges the concept of a relat10n between the size of neurons and their vulnerability to a vanety of toxicants. This relationship could m part explam the unusual sensitivity of dopamine neurons to toxicants such as manganese [7] and to anoxia [ 13]. The sens1t1V1ty of the optic nerve and long spmal tracts to vanous tox1cants m humans and experimental ammals, also supports this idea [11,16,20] The lack of a clear effect of acrylamide m i·itro upon protein synthes1'> in the sciatic dorsal root ganglion [8], upon the striatal dopamine receptor (A. K. Agrawal, unpubhshed data) suggests that acrylamide itself 1s not neurotox1c directly This 1s supported by the observation that the effect of acrylamide treatment upon the dopamme receptor, is abohshed by pre-treatment with an inhibitor of mixed function oxidases [2]. However, acrylamide has been found to be cytotoxic m chick ganglion cultures at relatively high concentrations above 10-r. M [18].