Potassium bromide (KBr) is a chemical compound isolated from the Mediterranean Sea in 1826(1). KBr and other inorganic bromide salts produce sedative effects(2). Therefore, it was prescribed as an anxiolytic, sedative-hypnotic or antiepileptic agent(2). It is still indicated for the treatment of refractory seizures in children(3). These effects are primarily mediated through the activation of GABAA receptor. These receptors are ionotropic channels which, upon activation, increase chloride permeation and cause hyperpolarization of neurons(4). This is the main cause of KBr-induced inhibitory effects in the CNS. In this regard, KBr is reported to cause depression, weakness, fatigue, lethargy, coma and other symptoms related to CNS suppression(5). KBr was withdrawn from the pharmaceutical market because of low efficacy, adverse effects, and the discovery of more effective hypnotic and antiepileptic drugs(2). However, it is illegally used for material processing in food industry. For example, oxygenated KBr is added to enhance the volume and mature the flour in bakery. Indeed, it helps oxidize the sulfhydryl groups of the gluten into disulphide bridges, and traps carbon dioxide in the dough. This improves the appearance and elasticity of the dough(67). Likewise, potassium bromate is used as a preservative to keep the concentrated fish paste(7). As such, brominated vegetable oil is frequently used as a clouding agent in soft drinks(8). Therefore, people may intake different amount of bromide via food products. Following the ingestion of bromide, it enhances GABA effects in the CNS(4), which can influence neurobehavioral processes like learning and memory(910).
Memory formation and retrieval are complex processes that involve neuronal networks and multiple pre- and post- synaptic events. Several literatures have investigated the molecular mechanisms underlying activity-dependent synaptic changes during memory formation(1112). GABA is a key mediator in several brain regions involved in memory(13). In addition, drugs stimulating the GABA receptor activity including antiepileptic agents are reported to negatively influence learning and memory(9).
This study aimed to investigate the potential effects of KBr on memory function. In particular, acute and long-term effects of KBr on spatial memory were studied in object recognition task (ORT). ORT is based on the spontaneous behavior of rats to explore a novel object more intensely than a familiar one. It is a non-rewarded, ethologically relevant, relatively simple test. In addition, a large body of evidence have demonstrated that spontaneous exploratory activity in this task can be used as a valid measure of memory function(1415).
MATERIALS AND METHODS
The experiments were carried out on male Wistar rats weighing 200 ± 20 g obtained from the animal house of School of Pharmacy and Pharmaceutical Sciences at Isfahan University of Medical Sciences (Isfahan, Iran). The animals had free access to food and water during the experiment and were kept at a constant room temperature (22 ± 1 ºC) under a 12-12 h light/dark cycle. All experimental procedures were conducted during the light phase of the cycle. The study protocol was approved by the Bioethics Committee of Isfahan University of Medical Sciences (Registration No. 394070), and performed in accordance with National Institute of Health Guide for the Care and Use of Laboratory Animals.
Apparatus and objects
The apparatus consisted of a circular arena of 83 cm in diameter and walls of 40 cm high which was made of white polyvinyl chloride(1617). Two different sets of objects consisted of a massive aluminum cube (10 × 5 × 7.5 cm) and a massive aluminum cube with a tapering top (13 × 8 × 8 cm) were used in the task. The objects could not be displaced by rat and each object was available in triplicate(1615).
Animals were randomly divided into 6 groups of 7 each. In the acute study, rats received a single dose of 50, 100 or 150 mg/kg of KBr per oral. In the long term study, animals were treated with 50 mg/kg of KBr for 28 consecutive days. KBr was dissolved freshly in saline, and the control groups received saline as the vehicle.
The animals underwent the ORT 90 min post-treatment in either acute or chronic experiments. ORT consisted of three defined phases, (T1) a training session or first trial; and a training-test interval, and (T2) a test session or second trial.
These phases last for 5, 60 and 5 min, respectively(18). During the T1 phase, animals encountered with two identical objects (A1 and A2) which were placed in a symmetrical position about 10 cm away from the wall. In the T2 trial, they faced with one identical and one novel object (A and B) to explore. Exploration was defined as directing the nose to the object at a distance of no more than 2 cm and/or touching the object with the nose, and sitting on the object was not considered an exploratory behavior.
The exploration time (s) for each object was recorded and the following memory indicating factors were calculated. e1, is the total exploration time for both objects in the first trial (eA1+ eA2); e2, is the total exploration time for both objects in the second trial (eA + eB); and DI is (eB – eA)/(eB + eA). In this set, DI (discrimination index) indicates the discrimination between new and familiar objects. Its’ values vary between +1 and -1, where a positive score indicates more time spent with the novel object, a negative score indicates more time spent with the familiar object, and a zero score shows a null preference.
F, is the frequency of the object exploration. Another measure of the ORT is the recognition index (RI) which is the time spent to explore the novel object relative to the both object exploration time and calculated as,
RI = eB/ (eB + eA)
In every section, animals who explored less than 10 s in the second trial (e2 < 10) were excluded from the study(15161719).
The data are presented as mean ± SEM. The values were analyzed using t-test or one-way analysis of variance (ANOVA) according to the number of comparable groups. Multiple comparisons were accomplished using Tukey post-hoc test and P < 0.05 was considered as statistically significant. Statistical analysis was conducted using Graph pad prism V.5.
Acute effects of KBr on exploratory behavior in T2 trial
A single dose of 150 mg/kg of KBr administered 90 min prior to the T1 trial significantly decreased the DI compared to the control group (P < 0.01) (Fig. 1A). However, lower doses of KBr (50 or 100 mg/kg) did not alter this index (Fig. 1A).
In addition, a single dose of 100 (P < 0.05) or 150 (P < 0.01) mg/kg of KBr remarkably decreased RI in the T2 trial in comparison to the control group (Fig. 1B), whereas, KBr at 50 mg/kg failed to produce a significant effect on RI (Fig. 1B).
Acute treatment with KBr at doses as high as 150 mg/kg did not change the frequency of novel object exploration in the T2 trial compared to the control group (Fig. 2A).
Long-term effects of KBr on exploratory behavior in T2 trial
Daily administration of 50 mg/kg KBr for 28 consecutive days before ORT considerably (P < 0.05) reduced the DI and RI in T2 trial compared to the control (Fig. 3A, 3B). As such, it significantly decreased the frequency of exploration of new object in comparison with the control group (P < 0.05) (Fig. 2B).
In the present study, the effect of KBr, as an illegal additive of bakery products, in a single or repeated dose/s was evaluated on the spatial memory using ORT. The ORT is a non-rewarded, stress free task which could study the spatial memory of rodent correctly(1415).
The results showed that single doses of 50 or 100 mg/kg of KBr did not disrupt the memory in rats. However, single dose of 150 mg/kg KBr significantly hampered memory formation compared to the control group. Furthermore, long term administration of KBr at the dose of 50 mg/kg/day for 28 days led to the cognitive impairment in rats. In addition, we observed that the memory impairment induced by KBr is a dose related effect. Specifically, doses of 50 or 100 mg/kg of KBr in the acute treatment failed to influence cognitive function. Our findings are in agreement with studies demonstrating the disruptive effects of different antiepileptic drugs on memory. For instance, Tsutsumi, et al. reported cognitive impairment in rat offspring exposed prenatally to phenytoin during 7th to 18th day of gestation(20). Likewise, exposure to phenobarbital, carbamazepine, valproate, and topiramate are associated with impairment in learning and memory (21222324).
KBr could easily reach CNS, and influence the physiological function of neurotransmitters such as GABA. So, it can potentially alter neurological processes as well as behavioral activities. KBr is proven to stimulate the GABAA receptor which is a chloride channel. Upon activation, it leads to the hyperpolarization of neurons and induces an inhibitory effect in the brain(4). Memory impairment in this study can be related to KBr-induced activation of GABA receptor(25). Based on a great number of literatures, increase of GABA concentration or receptor activity results in memory impairment(9).
It has been fully demonstrated that the cholinergic system plays a key role in learning and memory processes(26). In particular, disruption of cholinergic activity impairs cognitive function both in animal and human subjects. On the other hand, enhancement of cholinergic function is meant to improve cognitive function in dementia related disorders(27). In this regard, bromide decreases acetylcholine release from presynaptic cells(28). This inhibitory effect can explain, to some extent, the memory impairment observed in rats treated with KBr.
Alteration of serotonergic signaling may also play a role in bromide-induced cognitive dysfunction. In this regard, in vitro studies have demonstrated that bromide inhibits serotonin release(28). In addition, serotonin plays an important role in a great variety of behaviors such as learning and memory, particularly by interacting with cholinergic, glutamatergic, dopaminergic or GABAergic systems(293031). Therefore, bromide inhibition of serotonergic function can translate to modification of other neurotransmitters, and finally result in memory dysfunction. This may also explain KBr-induced memory impairment in this research, and requires further investigation.
Taken together, we demonstrated that single high dose as well as multiple low dose administration of KBr can cause memory impairment in ORT. The stimulation of GABAergic transmission as well as inhibition of cholinergic or serotonergic activity could possibly explain KBr-induced cognitive dysfunction.
The content of this paper is extracted from the MSc thesis NO. 394070 submitted by F. Safdari which was financially supported by the Research Department of Isfahan University of Medical Sciences, Isfahan, I.R. Iran. We greatly thank Dr. Mohammad Seyedabadi due to his valuable helps.
1. Ryan M, Baumann RJ. 1999. Use and monitoring of bromides in epilepsy treatment Pediatr Neurol. 1999;21(2):523–528
2. Ban TA. The role of serendipity in drug discovery Dialogues Clin Neurosci. 2006;8(3):335–344
3. Baird-Heinz HE, Van Schoick AL, Pelsor FR, Ranivand L, Hungerford LL. 2012. A systematic review of the safety of potassium bromide in dogs J Am Vet Med Assoc. 2012;240(6):705–715
4. Tan JS, Lin F, Tanouye MA. Potassium bromide, an anticonvulsant, is effective at alleviating seizures in the Drosophila bang-sensitive mutant bang senseless Brain Res. 2004;1020(1-2):45–52
5. Mangurten HH, Ban R. Neonatal hypotonia secondary to transplacentalbromism J Pediatr. 1974;85(3):426–428
6. Oloyede OB, Sunmonu TO. Potassium bromate content of selected bread samples in Ilorin, Central Nigeria and its effect on some enzymes of rat liver and kidney Food Chem Toxicol. 2009;47(8):2067–2070
7. Alli AL, Nwegbu MM, Inyang BI, Nwachukwu KC, Ogedengbe JO, Onaadepo O, et al Assessment of bread safety: determination of potassium bromate in selected bread samples in Gwagwalada, Abuja Int J Health Nutr. 2013;4:15–20
8. Bendig P, Maier L, Vetter W. Brominated vegetable oil in soft drinks–an underrated source of human organobromine intake Food Chem. 2012;133:678–682
9. Mathew J, Kumar TP, Khan RS, Paulose CS. Behavioral deficit and decreased GABA receptor functional regulation in the cerebellum of epileptic rats: Effect of Bacopa monnieri and bacoside A Epilepsy Behav. 2010;17(4):441–447
10. Sałat K, Podkowa A, Mogilski S, Zaręba P, Kulig K, Sałat R, et al The effect of GABA transporter 1 (GAT1) inhibitor, tiagabine, on scopolamine-induced memory impairments in mice Pharmacol Rep. 2015;67(6):1155–1162
11. Abel T, Lattal KM. Molecular mechanisms of memory acquisition, consolidation and retrieval Curr Opin Neurobiol. 2001;11:180–187
12. Hosseini-Sharifabad A, Ghahremani MH, Sabzevari O, Naghdi N, Abdollahi M, Beyer C, et al Effects of protein kinase A and G inhibitors on hippocampal cholinergic markers expressions in rolipram-and sildenafil-induced spatial memory improvement Pharmacol Biochem Behav. 2012;101(3):311–319
13. Jafari-Sabet M, Jannat-Dastjerdi I. Muscimol state-dependent memory: Involvement of dorsal hippocampal μ-opioid receptors Behav Brain Res. 2009;202(1):5–10
14. Grayson B, Idris NF, Neill JC. Atypical antipsychotics attenuate a sub-chronic PCP-induced cognitive deficit in the novel object recognition task in the rat Behav Brain Res. 2007;184(1):31–38
15. Sik A, Nieuwehuyzen PV, Prickaerts J, Blokland A. Performance of different mouse strains in an object recognition task Behav Brain Res. 2003;147:49–54
16. Rutten K, Prickaerts J, Hendrix M, van der Staay FJ, Şik A, Blokland A. Time-dependent involvement of cAMP and cGMP in consolidation of object memory: studies using selective phosphodiesterase type 2, 4 and 5 inhibitors Eur J Pharmacol. 2007;558(1-3):107–112
17. Hosseini-Sharifabad A, Rabbani M, Sharifzadeh M, Bagheri N. Acute and chronic tramadol administration impair spatial memory in rat Res Pharm Sci. 2016;11(1):49–57
18. Bertaina-Anglade V, Enjuanes E, Morillon D, Drieu la Rochelle C. The object recognition task in rats and mice: a simple and rapid model in safety pharmacology to detect amnesic properties of a new chemical entity J Pharmacol Toxicol Methods. 2006;54(2):99–105
19. Antunes M, Biala G. The novel object recognition memory: neurobiology, test procedure, and its modifications Cogn Process. 2012;13(2):93–110
20. Tsutsumi S, Akaike M, Ohno H, Kato N. Learning/memory impairments in rat offspring prenatally exposed to phenytoin Neurotoxicol Teratol. 1998;20(2):123–132
21. Frankel S, Medvedeva N, Gutherz S, Kulick C, Kondratyev A, Forcelli PA. Comparison of the long-term behavioral effects of neonatal exposure to retigabine or phenobarbital in rats Epilepsy Behav. 2016;57:34–40
22. Reeta KH, Mehla J, Gupta YK. Curcumin ameliorates cognitive dysfunction and oxidative damage in phenobarbitone and carbamazepine administered rats Eur J Pharmacol. 2010;644(1-3):106–112
23. Stepien KM, Tomaszewski M, Luszczki JJ, Czuczwar SJ. The interactions of atorvastatin and fluvastatin with carbamazepine, phenytoin and valproate in the mouse maximal electroshock seizure model Eur J Pharmacol. 2012;674(1):20–26
24. Faught E. Topiramate in the treatment of partial and generalized epilepsy Neuropsychiatr Dis Treat. 2007;3(6):811–821
25. Balcar VJ, Erdö SL, Joó F, Kása P, Wolff JR. Neurochemistry of GABAergic system in cerebral cortex chronically exposed to bromide in vivo
J Neurochem. 1987;48(1):167–169
26. Batool Z, Sadir S, Liaquat L, Tabassum S, Madiha S, Rafiq S, et al Repeated administration of almonds increases brain acetylcholine levels and enhances memory function in healthy rats while attenuates memory deficits in animal model of amnesia Brain Res Bull. 2016;120:63–74
27. Francisa PT, Palmerb AM, Snapeb M, Wilcockc GK. The cholinergic hypothesis of Alzheimer’s disease: a review of progress J Neurol Neurosurg Psychiatry. 1999;66:137–147
28. Kasa P, Toldi J, Farkas Z, Joó F, Wolff JR. Inhibition by sodium bromide of acetylcholine release and synaptic transmission in the superior cervical ganglion of the rat Neurochem Int. 1987;11:443–449
29. Buhot MC, Martin S, Segu L. Role of serotonin in memory impairment Ann Med. 2000;32(3):210–221
30. Seyedabadi M, Fakhfouri G, Ramezani V, Mehr SE, Rahimian R. The role of serotonin in memory: interactions with neurotransmitters and downstream signaling Exp Brain Res. 2014;232(3):723–38
31. Whitaker-Azmitia PM. Serotonin and brain development: role in human developmental diseases Brain Res Bull. 2011;56:479–485