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Biochemical alterations in rat brainfollowing aluminum exposure:Protection with centrophenoxineBimla Nehru a & Punita Bhalla aa Department of Biophysics , Panjab University , Chandigarh, 160014, IndiaPublished online: 23 Sep 2010.

To cite this article: Bimla Nehru & Punita Bhalla (2007) Biochemical alterations in rat brainfollowing aluminum exposure: Protection with centrophenoxine, Toxicological & EnvironmentalChemistry, 89:3, 577-585, DOI: 10.1080/02772240701228763

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Toxicological & Environmental Chemistry, July–Sep. 2007; 89(3): 577–585

Biochemical alterations in rat brain following

aluminum exposure: Protection with centrophenoxine

BIMLA NEHRU & PUNITA BHALLA

Department of Biophysics, Panjab University, Chandigarh 160 014, India

(Received 10 January 2007; revised 24 May 2007; in final form 24 May 2007)

AbstractAluminum (Al) is a neurotoxicant potentially affecting ionic, cholinergic, and dopaminergicneurotransmission in the central nervous system. These alterations are known to be associated withlearning ability, adaptive responses, and other aspects of behavior. The present experiment wasdesigned to study the neurotoxic consequences of Al exposure on neurotransmitters like dopamine(DA), serotonin (5-HT), norepinephrine (NE) along with the activity of acetylcholinesterase (AChE).Furthermore, Centrophenoxine (CpH) was administered as a post treatment to evaluate its potentialin Al-induced neurotoxicity. The cognitive functions and memory loss were also studied after bothAl and CpH administration. Al was administered orally at a dose of 40 mg kg�1 day�1 for a period of8 weeks, whereas CpH was administered intraperitoneally at a dose of 100�1 mg kg�1 day�1 for aperiod of 6 weeks. The study was carried out in four regions of the brain, namely cerebrum,cerebellum, medulla oblongata, and hypothalamus. A significant reduction in AChE activity anddifferent neurotransmitters was observed after Al exposure in the regions. CpH as a post treatmentproved beneficial in restoring these alterations. Al exposure also affected the cognitive functions andshort-term memory, which were significantly improved following CpH post treatment.

Keywords: Aluminum, behavior studies, centrophenoxine, dopamine, serotonin, norepinephrine

Introduction

There is unequivocal evidence that Aluminum (Al) is a potent neurotoxic agent [1,2].Elevated levels of Al were reported in the autopsied brain samples of patients with certainneurological disorders such as amyotrophic lateral sclerosis [3], Alzheimer’s disease [4,5],and amyotrophic lateral sclerosis-Parkinson’s disease complex of Guam [6]. One of theimportant pathological hallmarks of Al-associated neurotoxicity is the formation ofneurofibrillary tangles affecting the anterograde transport of neurotransmitters and otherprecursor/metabolites derived neurotransmitters.

Correspondence: Bimla Nehru, Reader, Department of Biophysics, Panjab University, Chandigarh 160 014, India. Tel.: 91-172-534119. E-mail: bnehru@pu.ac.in

ISSN 0277-2248 print/ISSN 1029-0486 online � 2007 Taylor & FrancisDOI: 10.1080/02772240701228763

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Al is a potent inhibitor of the uptake of choline [7] and dopamine (DA) [8]. Both are vitalendogenous substances released during nerve impulse transmission, as well as nerveimpulse conduction to muscles and various glands. As a result, the presence of Al ions in thebrain may exert an adverse impact on thinking and reasoning processes as well as short-termmemory. Additionally, studies have shown that Al interfered with the calcium–calmodulinpool [9]. Al induced conformational changes in calmodulin and blocked calmodulindependent calcium–magnesium triphosphatase, which is important for extrusion of calciumions from cells [10]. Impairment in cognitive functions and encephalopathy are otherreported manifestations attributed to Al toxicity.

Centrophenoxine (CpH) is a known nootropic drug, used in psychogeriatric treatment ofvarious disorders in elderly patients. CpH is an ester of p-chlorophenoxyacetic acid (PCPA)and dimethylaminoethanol (DMAE). CpH is well absorbed orally and after absorption ismetabolized in the liver to yield DMAE and PCPA. The latter compound is quicklyexcreted while the former remains in the body for a relatively longer time. Beneficialtherapeutic effects of CpH were observed in various human disorders such as cerebralatrophy, brain injury, post-apolectic status, chronic alcoholism, and barbiturates intoxica-tion [11]. The unusual affinity of CpH to the brain is exhibited by rapid binding to neuronaltissue after administration and by a regulatory action in neuronal metabolism [12]. CpHstimulating effect on motor activity in other species was also demonstrated [13], and is oftenused in human therapy.

Therefore, the present study was designed to investigate the effect of CpH administrationon various neurotransmitters (serotonin (5-HT), norepinephrine (NE), and dopamine(DA)) as well as on the activity of acetylcholinesterase (AChE) following Al administration.Active and passive avoidance tests were also conducted to assess the short-term memoryand cognitive behavior of animals after Al exposure.

Material and methods

Female Sprague–Dawley rats weighing between 160 and 200 g were obtained from thecentral animal house of Panjab University. The animals were housed in polypropylene cagesunder hygienic conditions, and acclimatized in the departmental animal quarters for 1 weekbefore being subjected to various treatment schedules. All procedures were done inconsideration of ethical guidelines for the care and use of laboratory animals, and approvedby the local care of experimental animals committee.

For the present experiment, a total of 24 animals were used which were divided intofour different groups with six animals each. (a) Group I – served as normal control andwere fed on standard animal chow and water ad-libitum; (b) Group II – animals wereadministered Al at a dose of 40 mg kg�1 body weight for a period of 8 weeks daily throughoral gavage; (c) Group III – animals first received Al for a period of 8 weeks as was given togroup II animals followed by a post treatment with CpH for another 6 weeks as given togroup IV animals; (d) Group IV – animals were administered CpH at a dose of 100 mg kg�1

body weight daily, intraperitoneally (ip) for a period of 6 weeks.Al was administered in the form of AlCl3 and was dissolved in normal drinking water

whereas CpH was dissolved in normal saline and was injected ip. CpH (trade names,Lucidril, and Meclofenoxate) is 2-dimethylaminoethanol-4-chloro phenoxyacetal, andhydrochloride purchased from Sigma Chemical Company; Al chloride and other reagentswere purchased from Merck Company.

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At the end of various treatments, active and passive avoidance tests were carried out toassess alterations in cognitive behavior and short-term memory in all groups includingnormal controls.

Active avoidance test

Cognitive behavior was assessed by the number of times the animal escapes, in 10 test trials.The apparatus for this test consists of two chambers separated by a partition. A lamp lightsone chamber. Animals are placed in the lit compartment. After 10 s, the buzzer is set on,and after 10 s an electric shock at 100 V is given. If the animal jumps to the othercompartment as soon as the buzzer is set on, it means the animal has avoided the test.However, if the animal jumps to the other compartment after shock or tries to hide bysome other means, this is termed escapism. A total of 10 trials are given to every animal.To qualify, the animal jumps to avoid shock at least 8 times out of 10.

Passive avoidance test

This test was used to assess short-term memory; the animals were exposed to light stimulus;avoiding this, the animals entered the maze fitted with the electric grid, centrally located in ashock-free zone. The apparatus for this test consist of two open chambers and one closedchamber. The three chambers are interconnected. The animal is placed in the openchamber. Light from the table lamp is made to fall on the rat. The time taken by the rat toenter the closed chamber from the open lit chamber is noted, which is termed as acquisitiontrial time. The shutter is closed, and the shock of 100 V was given for 5 s. Then the animal istaken out through the second open chamber. After 24 h, the animal is again put in the litchamber and the time is noted. The time taken by the animal to enter the closed chamber istaken as retention trial time. For retention trial time the animal is allowed to stay in the openlit chamber for a maximum 300 s.

Twenty-four hours after completion of behavior studies, animals were sacrificed bydecapitation, brains were dissected out and cerebrum cerebellum, medulla oblongata, andhypothalamus regions were excised immediately and weighed. A portion of brain was keptfor estimation of AChE activity and other half is kept for neurotransmitters measurements.For AChE estimation, all tissues (10% w/v) were homogenized in 10 mM phosphate buffersaline (pH 7.4). Homogenate was centrifuged first at 1000 g for 20 min at 4�C. Thesupernatant so obtained was again homogenized at 10,000 g for 20 min to obtain post-mitochondrial supernatant (PMS).

AChE activity

The activity of AChE was determined by the modified method of Ellman et al. [14]. Theresults were expressed as nanomoles of acetylcholine hydrolyzed mg�1 protein min�1. Thesubstrate used in the assay system is acetylthiocholine. The enzyme activity was measuredby the increase in absorbance at 412 nm for 3 min at an interval of 30 s.

Neurotransmitters (NE, DA, and 5-HT)

The concentrations of neurotransmitters NE, DA, and 5-HT were estimated by the methodof Cox and Perhach [15]. These neurotransmitters were extracted in butanol and laterlayered in a mixture of heptane and water in the ratio of 1 : 2. The aqueous phase obtainedfrom this was transferred to 200 mg of alumina, which was shaken gently for 10 min.A portion of the aqueous alumina was later used for the estimation of 5-HT, which after

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treatment with ninhydrin, showed fluorescence read at the activation and emissionwavelengths set at 360 nm and 460 nm. For the estimation of DA and NE, the aluminawas treated with 0.1 N acetic acid and the aqueous phase obtained was mixed with EDTA,iodine, and alkaline sulfite. The mixture was later heated with the addition of 0.2 mL of 0.5 Nacetic acid for 2 min at 100�C and fluorescence was read with the activation and emissionwavelengths set at 385 nm and 485 nm, respectively. For DA, the mixture was reheated andthen cooled, and fluorescence was read at 320 nm and 370 nm as activation and emissionwavelengths. The results were expressed as mg g�1 of wet tissue weight. The overall sensitivityof the method was in the range of 30–100 ng and reproducibility was 65–70%.

Proteins

Proteins were estimated in the samples using the method of Lowry et al. [16].

Statistical analysis

The data above were expressed as mean�SD. The statistical significance of the resultsobtained for various comparisons was estimated by applying Student‘s t-test. The criterionfor significance was set at p� 0.05.

Results

A significant decrease (p� 0.01) in AChE activity was observed after Al treatment in all fourregions of the brain. CpH post treatment significantly restored the enzymic activity with themore profound effect seen in the cerebrum region with an increase up to 93% (Figure 1).CpH treatment alone has no effect on the enzyme activity. Furthermore, treatment with Alfor 8 weeks produced a significant decrease in the concentration of DA, NE and 5-HT(Figures 2–4). However, CpH post treatment proved beneficial in restoring the altered

Figure 1. Activity of acetylcholinesterase in different regions of rat brain in treated and control group.Values are mean�SD of six observations. *p< 0.05: Treatment compared to control group. p< 0.05:AlþCpH treated compared to Al alone.

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concentrations of DA, NE, and 5-HT in all brain regions studied. NE concentration wasalso observed to be increased in the CpH only group (Figure 2). Futhermore, NEconcentration was found to be maximum in the hypothalamus region compared to otherregions in normal control animals. 5-HT concentration was reported to be the same in allthe four regions under study, whereas DA concentration was reported to be higher in thecerebrum as compared to other brain regions.

Cognitive behavior was assessed by the number of times the animal escaped in the 10 testtrials (Figure 5). The present study indicated that during the long term exposure of 8 weeks,

Figure 2. Concentration of norepinephrine in different regions of rat brain in the control and treatedgroup. Values are mean�SD of six observations. *p< 0.05: Treatment compared with Control.p< 0.05: AlþCentrophenoixne treated group compared with Al treated alone.

Figure 3. Concentration of Serotonin in different regions of rat brain in the control and treated group.Values are mean�SD of six observations. *p< 0.05: Treatment compared with Control. p< 0.05:AlþCentrophenoxine treated compared with Al treated alone.

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the animal escaped the maze an average of six times as compared to the normal control,which escaped twice in the given set of 10 trials. CpH post treatment was able to improvethe functions in animals and they escaped three times, which was comparable to control.CpH treatment alone has no effect on cognitive behavior. A passive avoidance test was usedto assess short-term memory. The animals were exposed to light stimulus; to avoid this, theanimal entered the maze fitted with an electric grid, centrally located in a shock-freezone. The animal was kept for 300 s and the procedure was repeated 24 h after the firstexposure to electric shock (Figure 6). The animals exposed to Al entered the cage within226 s. CpH post treatment significantly improved this function and so the animal avoidedthe test after 24 h.

Figure 4. Concentration of Dopamine in different regions of rat brain in the control and treatedgroup. Values are mean�SD of six observations. *p< 0.05: Treatment compared with Control.p< 0.05: AlþCentrophenoxine treated compared with Al treated alone.

Figure 5. The response of control and treated animals to active avoidance test.

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Discussion

Administration of Al for 8 weeks resulted in a significant reduction in AChE activity in thebrain tissue and the results are in accordance with the observations of Ravi et al. [17]. AChEactivity is dependent on the availability of the acetylcholine (ACh), which is synthesized bythe process of acetyl-Co-A and choline. Acetyl-Co-A is synthesized from pyruvate, which isan energy dependent process [18]. Studies showed that Al exposure significantly reduceshexokinase activity, which in turn may decrease pyruvate formation, thereby reducingacetylcholine formation. In addition, it may also be postulated that since Al is a potentinhibitor of the uptake of choline, this metal may limit the synthesis ACh [7] and thus mayaccount for the decreasing AchE activity. However, post treatment with CpH significantlyrestored enzyme activity. CpH is considered as a cholinergic drug that gets hydrolyzedin vivo into two components DMAE and PCPA. DMAE serves as an important precursorof choline and hence may contribute to increased ACh levels, which in turn increasesAChE activity [19].

Several authors linked the reduction of ACh with short-term memory disturbance, whichmay result in incomplete understanding and consequently abnormal communication skillsas seen in our studies. Long-term exposure of Al for 8 weeks resulted in a significantimpairment in memory and learning ability. Connor et al. [19] demonstrated impairedperformance of rats on a passive avoidance task after administration of Al in drinking water.Similarly, Schwartz et al. [20] suggested that cognitive function may progressively declinebecause of post-occupational exposure to a neurotoxicant. However, post treatment withCpH resulted in a significant improvement in the cognitive behavior of animals as theyescaped only 3.2 trials as compared to Al exposed animals. Zs-Nagy et al. [21] also reportedthat CpH supplementation produced marked improvement in learning, memory, andmental alertness. Similarly, Nandy [22] reported a significant improvement of symptomssuch as fatigue, irritability, confusional states, and loss of memory in the geriatric patienttreated with CpH. According to his studies CpH-treated mice did better on learning tasksas compared to control mice.

A significant decrease in the concentration of other neurotransmitters such as DA, NE,and 5-HT was reported after long-term Al exposure. Similarly, Ravi et al. [17] also found a60% reduction in NE in the striatum and a 20% decrease in 5-HT levels in cortex, septum,striatum, and hippocampus. 5-HT is an excitatory neurotransmitter synthesized from

Figure 6. The response of control and treated animals to the passive avoidance test.

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tryptophan via the intermediate 5-hydroxytrytophane. Al exerted an inhibitory effect on5-HT system due to the withdrawal of cholinergic input and thus decreased levels of 5-HTin cerebral cortex, hippocampus, and in cerebellum regions after 60 days exposure [23].All these biogenic amines are involved in a number of pathways of the brain. The center forNE functions is the locus ceruleus in the brain stem that controls overall activity, mood,and wakefulness. On the other hand, DA, which is secreted by neurons that originate inthe substantia nigra, acts as an inhibitory transmitter in a number of pathways and isresponsible for coordination on muscular movements. In the present study, after Alexposure a decrease in DA concentration was found. CpH supplementation was able torestore the altered balance of neurotransmitters in all four regions studied. However, themost consistent findings were observed in the hypothalamus region, which was alsoobserved in the studies of Petkov et al. [26]. Similarly, Stancheva et al. [24] reported thatafter the oral administration of CpH 5-HT levels in the striatum and cortex increased.

Deloncle et al. [25] postulated that Al in vivo (1) yields stable complexes with asparticand glutamic acid, (2) crosses the blood brain barrier, and (3) is deposited in the brain.Within the brain, it forms a complex with L-glutamic acid, which then is unable to detoxifyammonia from neurons to produce L-glutamine. An accumulation of ammonia issubsequently responsible for the neuronal death affecting the whole neurotransmittersystem. This in turn affects the cognitive functions and short-term memory.

From the present study one may conclude that Al affects the activation/deactivation ofneurotransmitters such as ACh, NE, DA, and 5-HT, which are manifested in alteredcognitive, memory, and neuromuscular functions. Furthermore, CpH proved beneficial inrestoring the altered concentrations of various neurotransmitters as well as restoring thememory defects seen in Al exposed rats.

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