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1193Current Trends in Biotechnology and PharmacyVol. 5 (2) 1193-1205 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)

Calcium protection against lead and manganese toxicity

AbstractChronic exposure to lead (Pb) or

manganese (Mn) is known to alter variety ofneurological and behavioral functions. In thisstudy, we have examined the neuro-behavioralperturbations in rats exposed to both Pb and Mn,and the protective effect of calcium supplement.Rats (3 months old) were exposed to Pb (0.2%through drinking water) and Mn (intraperitoniallydaily at a concentration of 2.5 mg/kg body wt)for a period of 3 weeks.

A separate batch of animals receivedcalcium (0.02%) in drinking water together withPb and exposed to Mn. Following exposures,the neurochemical alterations were assessed bydetermining the activity changes in synaptosomalacetyl cholinesterase (AChE) and mitochondrialATPases in different brain regions (cortex,hippocampus and cerebellum). Behavioral studiesincluded both cognitive (water maze tasks) andnon-cognitive (open field, exploratory andlocomotory tasks). Combined exposure to Pb andMn resulted in behavioral dysfunctions (decreasein latency, swim distance, swim speed in watermaze task and decrease in open field behaviorand locomotor activity). Similar to the changesobserved in motor and cognitive behavior,decrease in the activity of both AChE andATPases were also observed in the brain regions

Calcium Addition Potentially Reverses Lead and ManganeseInduced Enzymatic and Behavioral Alterations in Rats

M. Ram Kumar, K. Praveen Kumar, V. Kavitha and G. Rajarami Reddy*Department of Zoology, Sri Venkateswara University, Tirupati – 517502, India

*For Correspondence - [email protected]

of Pb+Mn exposed rats. However, the animalswhich received calcium together with Pb+Mnshowed reversal effect in behavioral as well asthe activity of the enzymes suggesting protectiverole of calcium supplementation against the Pband Mn induced neurotoxicity. The results of thisstudy support our earlier findings on the protectiverole of calcium and zinc against Pb and Mninduced neurotoxicity.

Keywords: Behavioral perturbations, Watermaze, Lead, Manganese, Locomotor activity,cholinergic system, Calcium protection.

IntroductionThe nervous system is the primary target

for the low levels of Pb-exposure and thedeveloping brain appears to be especiallyvulnerable to Pb/Mn-neurotoxicity (1-6). Pb-exposure at very low doses produces seriousadverse effects on the central nervous system ofchildren and infants, and these effects last forseveral years (7). Perinatal exposure to low levelsof Pb has been shown to exert behavioral andneurochemical alterations in both suckling andadult rats (8). Pb is known to exert its neurotoxiceffects by competing with calcium for calciumreceptors coupled with second messengerfunctions (9) and in some cases, to inhibit theactions of Ca2+ as a regulator of cell function (9).


Ram Kumar et al

Manganese (Mn) is an essential tracemetal that is present in all tissues of mammals.This element can act both as a nutrient and atoxicant to the brain (10). The higher intake ofMn, either through the air or the diet, may resultin severe pathologies, particularly in the centralnervous system (CNS) leading to manganism, aParkinson-like disorder (11). The toxic effects ofMn have been widely studied using animalmodels, notably concerning the accumulation ofMn in tissues and interactions with various otherelements (e.g., iron [Fe], zinc [Zn] copper [Cu])after subchronic or chronic administration (12).

The adverse effects of Mn on thenervous system probably result from the failureof protective enzymes capable to detoxify criticalamounts of Mn or to alter its oxidation potential.A multifactor hypothesis is more likely, and couldinvolve iron-induced oxidative stress and thedirect interaction of Mn with dopaminergicterminal mitochondria, leading to selectivemitochondrial dysfunction and subsequentexcitotoxicity (13,14). In this study, we haveexamined the alterations in brain enzymes andbehavioral perturbations in rats exposed to bothlead and manganese since both metals coexist inenvironment originating from different exposuresources. We have also studied the protectiveeffects of calcium against the alterations causedby Pb+Mn.

Materials and MethodsProcurement and maintenance ofexperimental animals: Young albino rats(Wistar) of 3 months age were purchased fromIISc, Bangalore and maintained in the animalhouse of Dept. of Zoology, S.V. University. Theanimals were housed in clear plastic cages withhardwood bedding in a room maintained at 28o ±2o C and relative humidity 60 ± 10% with a 12hour light/day cycle. The animals were fed withstandard pellet diet supplied by Sri VenkateswaraTraders, Bangalore and water ad libitum.

Animal exposure to Pb and Mn: Animals wereexposed to 0.2% lead acetate (Sigma) by addingPb- acetate to de-ionized drinking water and Mn(2.5 mg/kg) (Sigma) intraperitonially daily for aperiod of 21 days. Control rats received onlydeionized water without Pb, intraperitonially saline(0.9%) injections daily up to 21 days.

Calcium (Ca2+) was supplemented as0.02% in 0.2% Pb- water. The behavioral tasksin control, Pb+Mn-exposed and calciumsupplemented 3 months old rats were observedin the open-field, water maze, exploratorybehavior chamber and locomotory activityrecorder. The brain regions (cerebral cortex,cerebellum and hippocampus) of control, Pb+Mn-exposed, and Ca2+ supplemented rats werecollected and used for biochemical assays.

Behavioural StudiesOpen-Field Behavior: The open field test hasbeen widely used to assess emotional reactivity/anxiety. It provides measures of locomotoractivity. The horizontally directed activity (orlocomotion) is measured by the number of linecrossings, and vertically directed activity (orexploration) is measured by the frequency ofrearings (15). The open-field behaviour of threemonths age rats was assessed in a wooden boxmeasuring 90 x 90 x 30 cm high. The floor of thearena was divided into 36 equal squares by blacklines. Immediately after a rat was placed in thecentre of the open field, the movement of theanimal was scored. The number of squarescrossed with all paws (crossings), the standingson the hind legs (rearings), placing the nose againstwall or floor (sniffing), wiping, licking, combingor scratching of any part of the body (grooming)were counted in all sessions. All the activitiesmeasured were combined together to assess themean total behaviour in each session. Testing wascarried out on five consecutive days in five minutesessions in control, Pb-exposed and supplementedanimals.


Locomotory Activit: Locomotor activity of therat was studied with Opto-Varimex mini(Columbus Instruments, USA). The data wastaken as total duration 30minutes to each animalduring the 5 minutes session interval.

Exploratory Behaviour: Exploratory behaviourwas evaluated in the hole board. The apparatuswas an open-field arena with four equally spacedholes (3 cm in diameter) in the floor. Each ratwas placed individually in the centre of the arenafor 5 min, during which we recorded head-dipcount and head-dipping duration, in seconds. Ahead dip was scored if both eyes disappearedinto the hole. Head-dipping duration data areexpressed as total duration during the 5-minsession. The results for head dip are expressedas number of counts, and for head-dippingduration in seconds.

Morris water maze: Spatial discriminationlearning not only involves place learning, whichis learning a position in space which in this caseis the position of the hidden platform in the Morriswater escape task, but also involves non-spatialcomponents like procedural learning (such aslearning to search for an escape platform) andvisual or other sensorimotor processes togetherwith possible motivational/emotional processesnecessary for executing the task. After acquisitionof the spatial water escape task, a probe trial canreveal whether the rats have actually learned theposition of the platform. Furthermore, a spatialdiscrimination reversal task after the acquisitionof the spatial task, measures mainly placelearning because the rats are already familiarwith the procedural component.

The water maze is a circular water tankmeasuring 1.85 m in diameter and 0.7 m deepconstructed according to a basic design similar tothat of Morris (16). Four points along thecircumference of the water tank were designatedarbitrarily North (N), South (S), East (E), and West

(W), thus dividing the maze into four quadrants.The pool was filled to a depth of 30 cm with watermade opaque with white, non toxic water-basedpaint. A circular submerged platform (diameter12.5 cm) remained below the surface of water.All parameters involving time and distance aremeasured in seconds. Testing was carried outon five consecutive days. Control, Pb+Mn treatedand Ca2+ supplemented rats were subjected towater maze learning tasks.

Biochemical StudiesPreparation of mitochondrial fraction:Mitochondrial fractions were prepared byfollowing the method of Lai and Clark (17) byhomogenizing in 5 volumes (w/v) of Sucrose-EDTA-Tris buffer (SET) buffer (0.25 M sucrose,10 mM Tris-HCl, and 1 mM EDTA, pH 7.4). Thehomogenate was first centrifuged at 800 g for 10min at 4°C, and then the supernatant wascentrifuged at 10,000g for 20 min. Then the pelletof mitochondrial fraction was suspended in SETbuffer.

Estimation of AChE: The specific activity ofAChE was determined as described by Ellmanet al., (18). The reaction mixture contained 3.0ml of 0.1M phosphate buffer (pH 8.0), 20µl of0.075 M acetylthiocholine iodide and 100 µl of0.01 M 5, 5-dithiobis 2-nitrobenzoic acid. Thereaction was initiated with the addition of 100 µlof crude homogenate. The contents wereincubated for 30min at room temperature and thecolor absorbance was measured at 412nm inspectrophotometer (Hitachi, Model U-2000). Theenzyme activity was expressed as µ moles of AChhydrolyzed /mg protein/h.

Estimation of Adenosine Triphosphatase(ATPase) activity: Na+K+ and Mg2+ATPaseactivities in the tissues were estimated followingthe method of Tirri et al., (19). Briefly, 1%homogenates of the tissues were prepared in 0.25M ice cold sucrose solution. Homogenates were



divided into two parts. One part was centrifugedat 1400g and the supernatant thus obtained wasused as an enzyme source for Mg2+ATPase, whilethe other part of the homogenate was used forthe estimation of the total ATPase.

Mg2+ATPase: The reaction mixture forMg2+ATPase assay contained 0.5 ml of tris buffer(0.13 M; pH 7.4), 0.4 ml of substrate ATP, 0.5 mlof Magnesium chloride (0.05 M MgCl

2) and 0.2

ml of mitochondrial fraction (enzyme source). Thecontents were incubated at 37° C for 15 minutesand the reaction was stopped by the addition of10% TCA. Zero time controls were maintainedby adding TCA prior to the addition ofhomogenate/mitochondrial fraction. The contentswere centrifuged at 1000g for 15 minutes andthe inorganic phosphate was estimated in thesupernatant fraction following the method of Fiskeand Subbarow (20).

Na+K+ATPase: 1% (W/V) homogenate alreadyset apart was used for the total ATPase assay.The reaction mixture in a final volume of 2.6 mlcontained, 0.5 ml of Tris buffer (0.13 M; pH 7.4),0.4 ml of substrate ATP, 0.5 ml MgCl

2 (0.05 M),

0.5 ml potassium chloride (KCl, 0.05 M), 0.5 mlof sodium chloride (NaCl, 0.05 M) and 0.2 ml ofcrude homogenate/ mitochondrial fraction(enzyme source). The contents were incubatedat 37° C for 15 minutes and the reaction wasarrested by the addition of 1.5 ml of 10% TCAprior to the addition of homogenate. The contentswere centrifuged and the inorganic phosphate wasestimated in the supernatant fraction (Na+

K+ATPase = Total ATPase - Mg2+ATPase).

Estimation of protein content: Protein contentof the tissues was estimated by the method ofLowry et al. (21).

Analysis of Data: The data was subjected tostatistical analysis (ANOVA and t-test asapplicable) using standard statistical procedures.

ResultsOpen field behavior:The results presented inFig.1 show decreased locomotor behavior in allresponses (crossings, rearings, sniffings,groomings) of the open-field in Pb+Mn exposedrats compared to controls. The control rats showedhigher locomotor activity (111.6 crossings, 14.2rearings, 19.2 sniffings, 19.2 groomings) comparedto Pb+Mn treated rats (31.7 crossings, 3.1rearings, 3.7 sniffings, 4.3 groomings). However,the addition of calcium to Pb+Mn, showed amarginal reversal in the alterations observed inthe locomotor behavior (46.88 crossings, 4.33rearings, 6.77 sniffings, 7.33 groomings) ofPb+Mn treated rats.

Locomotor activity: The results of locomotoractivity (Fig.2) showed a decrease in the totalnumber of movements in the Pb+Mn treated ratsthan the control rats. The administration ofcalcium along with the Pb+Mn, showed increasein the locomotory activity than Pb+Mn exposedrats.

Fig.1. Open-field behavior of control, Pb+Mn exposedand Ca2+ supplemented rats. Rats were exposed to0.2% lead acetate through drinking water and Mn (2.5mg/kg) intraperitonially daily for a period of 21 days.Calcium (0.02%) was added in 0.2% Pb water. Each barrepresents mean ± SD (n = 6). The values marked withasterisk (*) are significantly different from controls atp<0.05 – p<0.001.

Ram Kumar et al


than the control rats (head dip duration: 41.625sec/5min and the number of head dippings: 5.875times/5min). However, the administration ofcalcium showed a marginal increase in both headdip duration (5.66 sec/ 5 min) and number of headdippings (3.166 times/5 min) than the Pb+Mn alonetreated rats.

Water maze learning: Behavioral assessmentson water maze confirmed the impairment inperformance of the water maze acquisition,reversal and working memory tasks in Pb+Mnexposed rats (Figs 4 to 6). All the rats easilylearned to find the submerged platform exceptfor the first day of training when all subjectsperformed at chance level. The Pb+Mn exposedrats took significantly longer time than controlanimals to find the hidden platform.

Acquisition phase : From the Fig. 4, it evidentthat Pb+Mn exposure significantly impaired thewater maze acquisition performance. In the firstday of training session, no specific alterations were

Fig.2. Total locomotor activity of control, Pb+Mnexposed and Ca2+ supplemented rats. Rats wereexposed to 0.2% lead acetate through drinking waterand Mn (2.5 mg/kg) intraperitonially daily for a periodof 21 days. Calcium (0.02%) was added in 0.2% Pbwater. Each bar represents mean ± SD (n = 6). Thevalues marked with asterisk (*) are significant atp<0.001

Exploratory behavior: From the figure 3, it isevident that Pb+Mn treated rats showed adecrease in head dip duration (3.52 sec/5 min)and number of head dippings (1.91 times/ 5 min)

Fig.3. Exploratory behavior of control, Pb+Mnexposed and Ca2+ supplemented rats. Rats wereexposed to 0.2% lead acetate through drinking waterand Mn (2.5 mg/kg) intraperitonially daily for a periodof 21 days. Calcium (0.02%) was added in 0.2% Pbwater. Each bar represents mean ± SD (n = 6). Thevalues marked with asterisk (*) are significant atp<0.001

Fig.4. Average escape latency (time in seconds) of control,Pb+Mn exposed and Ca2+ supplemented rats to find thehidden plat form during the acquisition phase (the platformremained in the same position). Rats were exposed to 0.2%lead acetate through drinking water and Mn (2.5 mg/kg)intraperitonially daily for a period of 21 days. Calcium(0.02%) was added in 0.2% Pb water. Each bar representsmean ± SD (n = 6). The changes are significant at P<0.05-0.001.

Control animals, Lead and manganese exposedanimals, Calcium Supplemented Animals


Days

Seco

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found between control and treated rats. Later,control rats quickly proceeded to the northquadrant where the platform is located andidentified the hidden platform, where as Pb+Mntreated rats took longer time to reach the hiddenplatform. Pb+Mn-exposed rats exhibited thetendency of peripheral swimming, thereby tooklonger time to reach the platform. The meanescape latency of controls was 12.17±3 on 1st

day, 11.62±2.9 on 2nd day, 9.42±2.4 on 3rd dayand 8.67±2.2 on 4th day. The Pb+Mn treatedyoung rats exhibited increase in the escapelatencies (24.56±1.2 on 1st day, 24.2±1.8 on 2nd

day, 21.02±2.2 on 3rd day and 21.3±1.9 on 4th

day). The escape latency however greatlyreduced in Pb+Mn-exposed rats supplementedwith Ca2+ (16.54±2.3 on 1st day, 17.86±1.7 on 2nd

day, 16.32±2.3 on 3rd day and 16.2±3.1 on 4th

day).

Reversal phase: During the spatial discriminationof reversal task, the submerged platform wasshifted to south quadrant. All the groups initiallyspent more time in north quadrant and thenreached to the south quadrant. In the first session,there was not much difference in all treated andcontrol groups. From the Fig. 5, it is evident thatthe control rats showed mean latencies of10.29±1.2 on 1st day, 10.01±1.5 on 2nd day,8.27±0.9 on 3rd day and 8.72±0.9 on 4th day. ThePb+Mn-treated young rats exhibited increase inthe escape latencies (19.7±1.3 on 1st day,18.02±1.9 on 2nd day, 18.01±2.1 on 3rd day and16.09±2.2 on 4th day). The escape latencyhowever greatly reduced in Pb+Mn-exposed ratssupplemented with Ca2+ (14.408±1.5 on 1st day,14.02±1.7 on 2nd day, 12.02±1.9 on 3rd day and11.62±2.2 on 4th day).

Working memory: As the platform is changedto different quadrants in working memory, theescape latencies were greater than acquisitionand reversal phases. However, all the control aswell as Pb+Mn-exposed rats took lesser time to

Fig.5. Average escape latency (time in seconds) of control,Pb+Mn exposed and Ca supplemented rats to find the hiddenplat form during the reversal phase (the platform waschanged to opposite quadrant). Rats were exposed to 0.2%lead acetate through drinking water and Mn (2.5 mg/kg)intraperitonially daily for a period of 21 days. Calcium(0.02%) was added in 0.2% Pb water. Each bar representsmean ± SD (n = 6). The changes are significant at P<0.05-0.001. : Control Animals, : Lead and Manganese ExposedAnimals, : Calcium supplemented animals

Fig.6. Average escape latency (time in seconds) of control,Pb+Mn exposed and Ca2+ supplemented rats to find thehidden plat form during the working memory phase (theplatform was kept in four different quadrant). Rats wereexposed to 0.2% lead acetate through drinking water andMn (2.5 mg/kg) intraperitonially daily for a period of 21days. Calcium (0.02%) was added in 0.2% Pb water. Eachbar represents mean ± SD (n = 6). The changes are significantat P<0.005-0.01.(a): Control animals, (b): Pb+Mn exposed animals and (c):Ca2+ supplemented animals : Escape stand hidden in southside and rats were allowed from north, east and west : Escapestand hidden in east side and rats were allowed from west,south and north : Escape stand hidden in west side and ratswere allowed from east, south and north

Ram Kumar et al

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Days

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(a) (b) (c)


locate the hidden platform kept in north quadrantfrom all the three directions suggesting theretention of earlier acquisition (Fig. 6). As in thecase of acquisition and reversal phases, Pb+Mn-exposure exhibited greater impairments in workingmemory also.

Acetylcholinesterase: Among the different brainregions studied, the specific activity ofsynaptosomal AChE in control rats was recordedhighest in hippocampus, followed by cerebralcortex and cerebellum. Incase of Pb+Mn treatedanimals, a decrease in the activity of AChE wasobserved in all the three brain regions i.e.,hippocampus followed by cerebral cortex andcerebellum.The animals which received calciumtogether with Pb+Mn indicated significantrecovery in AChE activity in all the three brainregions (Fig. 7).

Adenosine triphosphatases: The specificactivity of Na+K+ ATPase was recorded highestin cerebral cortex (0.282), followed byhippocampus (0.217) and cerebellum (0.146) inthe control rats. Incase of Pb+Mn treated rats, adecrease in the Na+K+ ATPase activity wasrecorded in all the brain regions, i.e., cerebralcortex (0.211) followed by hippocampus (0.172)and cerebellum (0s.101). However, calciumsupplementation to Pb+Mn, reversed the inhibitoryeffect of Pb+Mn in all the brain regions; i.e.,cerebral cortex (0.256) followed by hippocampus(0.198) and cerebellum (0.124) (Fig.8).

In the control rats, the activity of Mg2+

ATPase was recorded highest in hippocampus(0.894) followed by cerebral cortex (0.783) andcerebellum (0.624). Pb+Mn treated rats exhibitessignificant decrease in Mg2+ ATPase activity inall the three brain regions. i.e., hippocampus(0.717) followed by cerebral cortex (0.712) andthen cerebellum (0.526). Calcium supplementationto Pb+Mn, however, reversed this effectproducing a marginal increase in the Mg2+ ATPase

activity, i.e., hippocampus (0.768) followed bycerebral cortex (0.754) and cerebellum (0.601)(Fig.9).

Fig.7. Effect of Pb+Mn exposure on AChE activity incerebral cortex, hippocampus and cerebellum. Ratswere exposed to 0.2% lead acetate through drinkingwater and Mn (2.5 mg/kg) intraperitonially daily for aperiod of 21 days. Calcium (0.02%) was added in 0.2%Pb water. Each bar represents mean ± SD (n = 6). Thechanges (values marked with asterisk *) are significantat p<0.001.

Fig.8. Effect of Pb+Mn exposure on Na+K+ ATPaseactivity in cerebral cortex, hippocampus andcerebellum. Rats were exposed to 0.2% lead acetatethrough drinking water and Mn (2.5 mg/kg)intraperitonially daily for a period of 21 days. Calcium(0.02%) was added in 0.2% Pb water. Each barrepresents mean ± SD (n = 6). The changes (valuesmarked with asterisk *) are significant at p<0.001.



DiscussionOur recent work (22, 23) has shown

inhibitory effect of Pb on AChE activity in thedeveloping nervous system and reversal effectwith calcium supplementation. Keeping in viewof these findings, the present study was aimed atexamining the combined effect of Pb and Mn-exposure on the activity of AChE and ATPasesas well as locomotor and cognitive behaviour, andthe protective effect of calcium supplementation.

Control animals showed higherfrequency of all the open field responses(crossings, rearings, sniffings and grooming)where as significant decrease was observed inopen field responses of Pb+Mn-exposed ratscompared to controls. The open field test has beenwidely used to assess emotional reactivity/anxiety.It provides measures of locomotor activity (15).The memory of habituation in the open field isprocessed by the hippocampus, which is believedto be a target for metal neurotoxicity.

In the present study, the cognitive deficitsin Pb+Mn-exposed rats were examined by watermaze swim tasks. As the platform is changed todifferent quadrants in working memory, theescape latencies in working memory were greaterthan acquisition and reversal phases. However,all the control as well as Pb+Mn-exposed ratstook lesser time to locate the hidden platform keptin north quadrant from all the three directionssuggesting the retention of earlier acquisition.

The impaired performance in water mazeswim tasks of Pb+Mn exposed rats can beattributed to motor impairments because therewere significant differences between swimlatencies in control and Pb+Mn exposed groups.These results demonstrate that chronic exposureto Pb+Mn, causes learning impairment by wayof deficits in both working memory and referencememory. Thus, the results of our behavioralstudies indicate that the ability needed to solve acomplex task is more affected by the combinedexposure of Pb and Mn. However Ca2+

supplementation significantly reversed the Pb+Mninduced behavioral impairments.

The nervous system is the primary targetfor the Pb-exposure and the developing brainappears to be especially vulnerable to Pbneurotoxicity (3-5). Pb has high affinity for thefree sulfhydril groups in enzymes and proteinsand its binding can alter their correct function innumerous processes (24, 25). This may be thereason for the observed inhibition of AChEactivity. The alteration in the motor activity isone of the most studied effect of the intoxication,but learning impairments, in particular, caused byPb has been described at very low doses andincluded a decrease in the intelligence quotientas well as memory (26). Therefore, a significantdecrease in open field behavior and cognitivebehavior in Morris water maze were observedwith combined exposure to Pb+Mn.

Fig.9. Effect of Pb+Mn exposure on Mg2+ ATPaseactivity in cerebral cortex, hippocampus andcerebellum. Rats were exposed to 0.2% lead acetatethrough drinking water and Mn (2.5 mg/kg)intraperitonially daily for a period of 21 days. Calcium(0.02%) was added in 0.2% Pb water. Each barrepresents mean ± SD (n = 6). All values marked withasterisk (*) are significantly (p<0.01) different fromcontrol.

Ram Kumar et al


A generalised reduction in brain cholinergicfunction has been reported in Pb-treated rats.Exposure to Pb resulted in a decrease in the AChEactivity in cerebellum and hippocampus at variouspost natal time points (22). Neonatal exposure toPb altered the muscarinic receptor density (27)which account for the deficits in central cholinergicfunctions (28). The results of our present studyare in agreement with these findings.

The alterations in AChE activity duringearly postnatal development could be related withthe fact that Pb crosses the blood brain barrierquite readily (29). The inhibitory effect of Pb onAChE activity also reflected in alterations in themotor activity (30, 5). The results of the presentstudy showed maximum alterations in AChEcontent in hippocampus of Pb-treated rats. Thecholinergic synapses are more in hippocampusas compared to cerebral cortex and cerebellum(31). AChE inhibition in this area leads tocognitive and non cognitive alterations as this isthe principle area for memory and cognition. Thefact that the hippocampus develops late (32) andsequester, Pb, primarily in the mossy fibrepathway (33) would put this structure at particularrisk for damage following early Pb-exposure.

Since the cholinergic system isresponsible for the behavioral manifestations, anyalteration in the cholinergic system would bereflected in the behavior. In the present study,the observed alterations in cholinergic systemhave greatly influenced the open field behaviorof rats exposed to Mn and Pb. The memory andspatial discrimination tasks in Morris water mazealso decreased suggesting the hippocampaldamage due to Pb+Mn exposure.

Supplementation with Ca2+ reversed the Pband Mn induced inhibition in AChE activity. Thereversal of inhibition in the activity of AChE bysupplementation with Ca2+ may be due tocompetition of these metals for similar binding

sites and reducing the availability of binding sitesfor Pb or Mn.

Ca2+ is a divalent cation just like Pb.Because the same transport mechanism isoperative for absorption of Pb and Ca2+ from thegastrointestinal tract there is resulting competitiveinteraction between Pb and Ca2+ (34). Studieshave shown that Pb has an inhibitory effect onthe peripheral nervous system through stimuluscoupled or Ca2+ dependent release ofacetylcholine (35) and this inhibitory effect of Pbat the neuromuscular junction and the ganglionwas similar to the effect of reducing theconcentration of Ca2+ in bathing media of neuralpreparations; so it is not surprising that thisinhibitory effect of Pb can be overcome by theaddition of Ca2+. Absorption of Pb bygastrointestinal tract is inversely related to theamount of Ca2+ present (36, 37). FurthermoreCa2+ supplements had a protective effect bysignificantly reducing blood Pb levels in pregnantwomen whose diets were deficient in Ca2+ (38,39). Ziegler et al., (40) observed an inverserelationship between dietary Ca2+ and Pbretention and absorption in young infants.

Pb+Mn-exposure exerted inhibitoryaction on the activity of enzymes Mg2+, andNa+K+ATPases in the developing brain. Heavymetals such as Pb can bind to a number of siteson proteins including imidazole, histodyl, carbonyland especially sulfhydryl side chains (41). Heavymetals have great affinity for ATPase system andthey interact with enzyme molecule resulting inthe inhibition (42). Pb has been reported to inhibitNa+K+ATPase of mammalian tissues (43, 44) andalso interferes with mitochondrial function andblocks the O

2 uptake. Bhaumik and Raychudhari

(45) reported that the inhibition of Na+K+ATPasemay be due to the flow of the Na+ K+ ions fromthe tissues to the blood. The decrease in theMg2+ATPase activity might be due to lowoperation of oxidative pathway, resulting in



decreased formation of free energy and alteredcellular energy metabolism (46).

The combination of Pb2+ and Mn2+

produced a pronounced decrease in the activityof Na+ K+-ATPase, but the magnitude of thechange was the sum of the individual metaleffects. (47). It is known that brain derivedNa+K+ATPase is among the enzymes particularlyaffected by Pb (48-50). The decrease ofNa+K+ATPase activity can change the gradientsof Na+ and K+ across the cell membrane and canbe the cause of the disturbances inneurotransmitters levels (50).

The observed high activity of ATPase inthe cortex, cerebellum and hippocampus regionsof the brain suggests the involvement of theseregions in different behavioral functions. It isknown that the level of ATPase parallels themetabolic demands of different regions of rat brainand the differential sensitivity to Pb+Mnneurotoxicity in these brain regions is not due to apreferential metal accumulation, but is possiblydue to alteration of biochemical or cellularprocesses that are uniquely associated with, orgreatly enhanced in a particular region (51, 8).Regional variations in AChE and ATPases activitylevels observed in different brain regions couldbe due to structural and functional differences inbrain regions. Addition of Ca2+ reduced theeffects of Pb+Mn on the activities of AChE andATPases in brain regions. The supplemented Ca2+

may compete for similar binding sites as that ofPb and Mn. Supplementation with Ca2+ decreasesPb gastrointestinal absorption and decreasestissue accumulation (52). Thus Ca2+ replaces Pband Mn in the body reducing the Pb+Mn-burdenin the body.

Thus, from the present study, it is evidentthat exposure to a combination of Pb and Mninhibited the activities of both Mg2+ATPase andNa+K+ATPase enzymes as well as AChE leading

to decrease in motor and cognitive functions.Calcium supplementation significantly reversedthese alterations in enzyme activities as well asbehavioral functions suggesting therapeutic roleof calcium for Pb+Mn induced neurotoxicity.

AcknowledgementsThe authors acknowledge the financial

support from CSIR Grant No. 37(1349)/08/EMR-II and DST Grant No.SR/SO/AS-72/2008.

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