Hepatic and Hippocampus Iron Status is not Alteredin Response to Increased Serum Ceruloplasmin and Serum“Free” Copper in Wistar Rat Model for Non-Wilsonian BrainCopper Toxicosis

Amit Pal & Rakesh kumar Vasishta & Rajendra Prasad

Received: 7 May 2013 /Accepted: 3 July 2013 /Published online: 20 July 2013# Springer Science+Business Media New York 2013

Abstract Copper and iron dyshomeostasis has been impli-cated directly or indirectly in the pathogenesis of neurodegen-erative diseases. Previously, we have shown the first in vivoevidence of significant increase in the hippocampus copperand zinc content with spatial memory impairments, astrocytesswelling (Alzheimer type-II cells) coupled with increase in thenumber of astrocytes, copper deposition in the choroid plexus,and degenerated neurons in copper-intoxicated Wistar rats. Incontinuation with our previous study, the aim of this study wasto further investigate the effects of intraperitoneally injectedcopper lactate (0.15 mg Cu/100 g body weight) daily for90 days on serum “free” copper levels, iron levels in the liver,and hippocampus by atomic absorption spectrophotometryand histopathological study of the liver and brain tissues ofWistar rats using Perls' Prussian blue (PPB) stain. A massivesignificant increase in serum “free” copper (79.48 % increase)along with strong correlation (r=0.978) was found betweenserum copper and serum “free” copper in copper-intoxicatedrats. No significant difference was detected in hepatic andhippocampus iron levels between control and copper-intoxicated rats. PPB stain demonstrated very few scatteredgrade 1 haemosiderin deposits within sinusoidal cells predom-inantly Kupffer cells; however, brain sections were negativelystained with PPB stain. In conclusion, the current study dem-onstrates that chronic copper toxicity causes increase in serum“free” copper, which may serve as predisposing factor for thedevelopment of neurodegeneration and memory deficits, and

grade 1 haemosiderin deposition in Kupffer cells withoutaltering hepatic and hippocampus iron levels in male Wistarrats.

Keywords Copper intoxication . Serum “free” copper .

Ceruloplasmin . Neurodegeneration

Introduction

Copper (Cu) and iron (Fe) are essential trace elements respon-sible for the normal functioning of brain, many cellular en-zymes, and proteins; however, Cu and Fe become toxic, when-ever there is an excessive accumulation of these metals insidethe cell [1]. Neonates are particularly vulnerable to Cu toxicosisbecause of the immaturity of the biliary system, which is themain route for excretion of Cu along with the humans whichare either exposed to Cu contaminated water/milk causingIndian childhood cirrhosis or having ATP7B gene mutationresulting in Wilson's disease (WD) [2]. Cu and Fe are redoxactive and very effective catalyst for Haber–Weiss or Fentontype reactions producing damaging hydroxyl radicals, whichculminate with serious oxidative damage. Cells have conse-quently established a sophisticated machinery to ensure tightcontrol over Cu and Fe homeostasis within the cell [1, 3]. Avast amount of experimental evidence suggests that homeosta-sis of Cu and Fe in the body is physiologically intertwined[4, 5] with ceruloplasmin and hephaestin, representing the bestrecognized links between Cu and Femetabolism. Ceruloplasminis a liver-derived circulating ferroxidase protein that is vital forFe release from certain tissues. Notwithstanding, ceruloplasmincontains the predominance of serum Cu, the absence of cerulo-plasmin does not lead to perturbations in Cu homeostasis; rather,it exerts preferential effects on Fe homeostasis [6].

A. Pal : R. Prasad (*)Department of Biochemistry, PGIMER, Chandigarh 160012, Indiae-mail: fateh1977@yahoo.com

R. kumar VasishtaDepartment of Histopathology, PGIMER, Chandigarh, India

Biol Trace Elem Res (2013) 154:403–411DOI 10.1007/s12011-013-9753-1

Cu is often conjugated with proteins in the serum andcellular compartments, such as ceruloplasmin, albumin, andmetallothionein. Approximately 90 % of blood Cu in humansis covalently bound to ceruloplasmin, a ferroxidase, necessaryfor the oxidation of Fe2+ to Fe3+ and subsequent binding ofFe3+ to transferrin. The remaining 10 % of Cu is loosely boundto albumin and micronutrients (peptide and amino acids)known as non-ceruloplasmin bound Cu or serum “free” Cu,which participates in generating reactive oxygen species (ROS)throughout life, and it is only with aging or under neurodegen-erative settings that these ROS results in compromised protec-tion by antioxidant defense mechanisms causing oxidativestress [7–10].

Deficiency or excess of either Cu or Fe is known to be anetiological factor in the initiation and/or progression of severalneurodegenerative disorders, including Alzheimer's disease(AD), Parkinson's disease (PD), amyotrophic lateral sclerosis,Huntington's disease, and genetic disorders likeWD. All theseneurodegenerative diseases share a conspicuous common fea-ture of selective neuronal loss [1, 3, 11]. Serum “free” Cu hasbeen shown to be correlated with cognitive waning and withCu, h-tau, and Aβ in the cerebrospinal fluid (CSF) in ADpatients [7]. Furthermore, Salustri et al. (2010) established thatcognitive function is inversely correlated with serum “free”Cu levels in normal subjects [12].

Although, there are reports in scientific literature whereinCu intoxication has been shown to induce cognitive declineand/or neurotoxicity with an increase in brain Cu content inexperimental animals; however, the animals used in thesestudies were either genetically compromised [13] or were fedwith high cholesterol diet [14]. Moreover, recently, Mao et al.[15] and An et al. [16] have used bilateral common carotid arteryocclusion (2VO) and CuO nanoparticles, respectively, to induceCu toxicity-incited neurotoxicity and/or neurobehavioral impair-ments in animals. Nonetheless, as far as authors are aware,relations of serum “free” Cu with memory deficit/neurotoxicityand/or augmented brain Cu levels have not been documented inpreviously mentioned animal models. Earlier, we haveestablished a Wistar rat model for non-Wilsonian brain Cutoxicosis, documenting the first in vivo evidence of impairedspatial memory and neuromuscular coordination, swelling ofastrocytes coupled with increase in the number of astrocytes incerebral cortex, Cu deposition in the choroid plexus, decreasedserum acetylcholinesterase activity, degenerated neurons show-ing pyknotic nuclei with dense eosinophilic cytoplasm, andaugmented levels of Cu and zinc (Zn) in the hippocampus ofchronically Cu-intoxicated male Wistar rats [17].

While Cu and Fe interactions are well documented, thequestions as to whether escalation in serum “free” Cu can leadto neurodegeneration, and Fe dyshomeostasis in the brain uponchronic Cu intoxication are virtually unexplored in animalmodels of neurodegenerative diseases and Cu toxicosis; so, itwill be intriguing to examine the role of serum “free” Cu in our

previously reported animal model for non-Wilsonian brain Cutoxicosis. Therefore, the present study sought to further ascer-tain the effects of chronic Cu intoxication on hepatic andhippocampus Fe content along with serum “free” Cu levels inWistar rats. Here, we have reported the effect of chronic Cuintoxication on Fe levels and relation of serum “free” Cu levelswith the development of neurodegeneration and memory im-pairments in Wistar rat model for non-Wilsonian brain Cutoxicosis.

Materials and Methods

Chemicals

Copper chloride, histological grade formalin, Fe standard solu-tions assigned for atomic absorption spectrophotometry (AAS)(Sigma–Aldrich Co., Germany), and lactic acid (Qualigens finechemicals) were purchased. All the other reagents andchemicals used in this study were of analytical grade.

Animals and Experimental Design

Male Wistar rats in the weight range of 60–80 g were procuredfrom the institute animal house, PGIMER, Chandigarh, India.All the rats were housed in the polypropylene cages (oneanimal per cage), kept in well-ventilated rooms maintained at20 to 24 °C. The rats were fed with standard rat chow andwaterad libitum. Institutional animal ethical committee (IAEC-161)consent was taken, and IAEC guidelines were strictly followedfor all the animal experimentation.

The animals were divided into two groups, each consistingof eight animals:

Control group animals were given intraperitoneal (i.p.)injection of isotonic sodium chloride solution daily forthe period of 90 days.

Cu intoxication group animals were given i.p. injection ofCu lactate solution (0.15 mg Cu/100 g. BW) daily for theperiod of 90 days. For ethical reasons, the group receivingsodium lactate solution was not kept, as it has been reportedpreviously that it does not alter any vital biochemical param-eters and Cu levels in various tissues in Wistar rats [17, 18].

Collection and Preservation of Blood and Tissue AutopsySamples

The serum was separated from blood obtained from animalsby retrobulbar blood vessel stab with a glass capillary on the90th day of the study in sterile containers. All the animalswere sacrificed at the end of the 99th day of the study underether anesthesia after completion of neurobehavioral stud-ies [17]. Liver and brain tissue autopsies samples for Fe

404 Pal et al.

measurement were collected in normal saline solution andstored at −80 °C till further processing. Liver and brain tissueswere fixed in formalin for histopathological studies. All theglassware used in Fe estimation were soaked in concentratedHCl for 24 h and rinsed several times in deionized waterbefore use. Tissues were cut with clean stainless steel scalpelblades.

Measurement of Biochemical Parameters

Lactate dehydrogenase (LDH) and alkaline phosphatase(ALP) were determined spectroscopically using autoanalyzer(Modular P800, Roche, Germany).

Measurement of Serum “Free” Cu (Non-CeruloplasminBound Cu)

The amount of Cu associated with ceruloplasmin is approxi-mately 3.15 μg of Cu per milligram of ceruloplasmin. Serum“free Cu” (non-ceruloplasmin bound Cu) was calculated bysubtracting ceruloplasmin-bound Cu (3.15 multiplied by ce-ruloplasmin in mg/L equals the amount of ceruloplasmin-bound Cu in μg/L) from the total serum Cu concentration(in micrograms per liter; serum Cu in micromoles per litermultiplied by 63.5 equals serumCu inmicrograms per liter) asreported in book chapter [19, 20] by Danks. In the previousstudy, we have reported serum ceruloplasmin and serum Culevels in Cu-intoxicated Wistar rats [17].

Measurement of Fe Concentration

The concentrations of Fe was measured by a flame (an air–acetylene burner) AAS (AAnalyst 400, PerkinElmer) [17, 21].For tissue samples, ~50 mg of tissue was digested with 1:1perchloric acid and nitric acid in hot air oven and diluted fivetimes with 10 mM HNO3 before analysis by AAS [17]. Theinstrument was calibrated using the Sigma standards of Fe.Bovine liver standard reference material SRM1577 obtainedfrom the National Bureau of Standard (Washington, DC) wasused as standard reference material for quality control. Eachsample was analyzed in triplicate and reported as mean, andthe concentration of Fe was derived by comparing absorptionwith the calibration curve. The Fe estimations were carried outin a blinded experiment.

Histopathological Studies

Tissues from the liver and brain were obtained as 5-mm thicksections, fixed with 10 % neutral formalin for more than 12 h,dehydrated, embedded in paraffin, sectioned at 5 μm andmounted on glass slides, and stained with hematoxylin andeosin stain (H&E) and Perls' Prussian blue (PPB) stain for Fe[22]. Liver section of a hepatic Fe overload patient enrolled in

the histopathology department of PGIMER, Chandigarh wastaken as positive control for PPB stain evaluation. For all thestaining studies, negative control tissues (tissues from controlanimals) were also stained and evaluated. All H&E and PPBstained slides were evaluated by a FRCP qualified pathologist,which was not aware of tissue Fe quantification studiesresults.

All H&E stained slides were semiquantitatively scoredon a 0 to 4 scale, where 0=no staining/background staining,1=minimal staining, 2=mild staining, 3=moderate staining,and 4=severe staining. Minimal staining was defined as theleast increase in brown pigment staining that could be clearlydistinguished from background staining, whereas severe stain-ing was defined as a significant increase in the amount andintensity of staining that covered a large area of the liverlobule. Mild and moderate staining were defined as levels ofstaining that could be clearly distinguished from each otherand intermediate between minimal and severe [23].

Histological sections, which were stained by PPB stain,and the stainable Fe content of the parenchymal cells was gradedaccording to the criteria of Scheuer, Williams, and Muir [24].By this method, grade 0 represents absence of stainable cellFe and grades 1–4 represents increasing degrees of paren-chymal siderosis, ranging from a weakly positive Perls' reac-tion through to the massive Fe deposition found in fullydeveloped haemochromatosis. When the intensity of thesiderosis was regarded as intermediate between two grades,the higher value was accepted; trace amounts of stainable Felocalized to a few parenchymal cells were designated as grade1 siderosis [25].

Statistical Analysis

All values were expressed as mean±standard error of the mean(SEM) of eight animals in each group. Unpaired Student's t testandMann–Whitney rank sum test were used for analysis of thedata, and values with p<0.05 were considered statisticallysignificant. For correlation analysis, Pearson's correlation andregression was used, and correlation was considered significantat <0.05 level (two-tailed). All the calculations were carried outby SPSS version 16 software.

Results

Chronic Cu Toxicity Increases the Serum LDH, ALP,and Serum “Free” Cu (Non-Ceruloplasmin bound Cu)Concentrations

Abnormally increased levels of serum LDH andALP have beenestablished as indicators of widespread tissue damage, and inorder to access the extent of Cu-induced tissue damage, levels ofserumLDH andALPweremeasured using autoanalyzer. Serum

Hepatic and Hippocampus Iron Status 405

LDH and ALP levels were significantly increased in Cu intox-ication group compared to control group on the 90th day ofthe study (80.74 and 46 % increase, respectively), whichindicates widespread tissue damage due to chronic Cu toxic-ity. Previously, we have reported that serum ceruloplasminand serum Cu levels were considerably increased in Cu-intoxicated animals compared to control animals (serumceruloplasmin (in milligrams per deciliter), 29.3±0.5 in thecontrol group compared to 47.9±1.1 in the Cu-intoxicatedgroup; p<0.01 with respect to the controls and serum Cu (inmicrograms per deciliter), 140.9±1.1 in the control groupcompared to 386.3±17.9 in the Cu-intoxicated group;p<0.01 with respect to the controls) [17]. A significant79.48 % increase in serum “free” Cu (non-ceruloplasminbound Cu) levels was documented (Fig. 1) in conjunctionwith the increase of serum Cu levels in Cu-intoxicated groupcompared to the control group on 90th day of the study. Cuassociated with ceruloplasmin showed a significant 38.78 %increase in parallel with increase in serum ceruloplasminlevels in Cu-intoxicated group compared to control group onthe 90th day of the study (Table 1).

Serum “Free” Cu (Non-Ceruloplasmin Bound Cu)is Significantly Correlated with Serum Cu

Serum “free” Cu (non-ceruloplasmin bound Cu) was directlyproportional to serum Cu levels as shown in Fig. 2. Notably,a strong degree of correlation (r=0.978) was observed be-tween serum “free” Cu (non-ceruloplasmin bound Cu) andserum Cu in Cu-intoxicated group, which suggests that withincrease in serum Cu levels, the amount of serum “free” Cu(non-ceruloplasmin bound Cu) also increased proportionately(Fig. 2). However, no significant correlation was found be-tween serum Cu and serum ceruloplasmin and between serumceruloplasmin and serum “free” Cu (non-ceruloplasminbound Cu) in Cu-intoxicated group (data not shown).

Hepatic and Hippocampus Fe Content

Fe levels in liver and hippocampus were measured using AASwhich demonstrated nonsignificant changes in the hepatic Fecontent between Cu-intoxicated and control group (p>0.05).Similarly, Fe content in the hippocampus of Cu-intoxicatedgroup was comparable with that of control group (p>0.05;Table 1).

Cu-Intoxicated Rats Demonstrated Grade 1 HaemosiderinDeposition in Kupffer Cells

H&E and PPB stain have been routinely used for studyingtissue histology and Fe deposition, respectively. H&E stainshowed minimal staining (score 1) of brown pigments (Fig. 3a)which was further confirmed as scattered traces of grade 1haemosiderin within sinusoidal cells, predominantly Kupffercells, by PPB stain (Fig. 4a) in very few areas of liver sectionsof Cu-intoxicated rats. However, H&E (Fig. 3b) and PPB(Fig. 4b) staining of liver sections from control rats did notreveal any brown pigments or haemosiderin, respectively. Brainsections were negatively stained with PPB stain suggesting noFe deposition (Fig. 5).

Discussion

Results described herein provide evidence of grossly aug-mented levels of serum “free” Cu (Fig. 1) along with elevatedserum Cu levels and serum ceruloplasmin concentration inCu-intoxicated maleWistar rats [17], which concurs well withvery recent scientific finding by Ranganathan et al. that ceru-loplasmin activity and expression are increased in response tohepatic Cu loading in rats [6].Moreover, high serum “free”Cuhas been detected in AD and PD patients [7, 10, 12, 26–28],which strongly supports the possible role of Cu toxicity,especially, serum “free" Cu of being partially responsible for

Fig. 1 Effect of 90 days of chronic copper intoxication on the serum“free” copper levels in rats. Values are expressed as the mean±SEM ofeight animals. ***p<0.001, significantly different from the controlgroup

Table 1 Biochemical parameters with liver and hippocampus Fe con-tent of rats exposed to chronic Cu toxicity (90 days)

Parameters Control group Cu-intoxicated group

ALP (U/L) 150.16±3.42 278.10±6.24***

LDH (U/L) 170.55±4.19 885.89±35.54***

Cu associated withceruloplasmin (μg/dl)

92.47±0.53 151.07±1.06***

Liver Fe 289.54±7.98 296.80±5.71#

Hippocampus Fe 27.38±0.23 26.75±0.47#

Fe content of tissue expressed as (μg Fe/g of wet tissue weight)

Values are expressed as mean±SEM (n=8/group)

Statistical significance ***p<0.001 with respect to controls; # notsignificant

406 Pal et al.

neurodegeneration in AD [9, 29]. In addition, a strong corre-lation was documented between serum Cu and serum “free”Cu in Cu-intoxicated animals (Fig. 2). The present study alsodemonstrated that there is no significant effect of chronic Cuintoxication on hepatic and hippocampus Fe levels corrobo-rated by nonsignificant changes in Fe levels quantified byAAS (Table 1). However, grade 1 haemosiderin was observedin the Kupffer cells of Cu-intoxicated rats (Fig. 4).

Ceruloplasmin is synthesized and secreted by astrocytes,choroid plexus, hepatocytes, and Sertoli cells in addition toexisting as a glycosylphosphatidylinositol (GPI)-linked cerulo-plasmin on both Sertoli cells and astrocytes [30]. The holo formof ceruloplasmin enzyme, which is the functional form ofceruloplasmin, has a half-life of 5 days [31], whereas that ofthe apo form of ceruloplasmin enzyme is estimated to be lessthan 6 h [32]. Thus, majority of ceruloplasmin in the circulationis holo-ceruloplasmin [6]. Serum ceruloplasmin is also an acute

phase reactant; accordingly, serum ceruloplasmin concentrationincreases during several conditions associated with oxidativestress. In these situations, the acute elevation of ceruloplasminconcentration is generally attributed to increased transcriptionof the ceruloplasmin gene by cytokines, especially interleukin-6 [8, 33]. The causal molecular mechanism for increasedceruloplasmin expression and activity in the observations re-ported by Ranganathan et al. may be correlated to augmentedmetallation of the ceruloplasmin leading to higher circulatinglevels of the holoenzyme form of the ceruloplasmin [6].Increased ceruloplasmin activity has also been reported in ahuman patient suffering from acute Cu toxicosis [34].

In the previous study, we have reported that chronic Cutoxicity resulted in the increase of hippocampus Cu contentin Cu-intoxicated Wistar rats [17]. The observed grosslyelevated levels of serum “free” Cu (Fig. 1) along with a strongcorrelation between serum Cu and serum “free” Cu (Fig. 2)

Fig. 2 Correlation betweenserum copper and serum “free”copper in copper intoxicated ratstreated with copper lactate for90 days. The straight line isplaced through the data by linearregression analysis. Correlationis significant at the 0.01 level(two-tailed)

Fig. 3 Photomicrograph ofhistopathological study of livertissue of animals. a Liver sectionshowing brown pigmentation incopper-intoxicated rat treatedwith copper lactate for 90 days(arrow). H&E stain ×20. b Liversection of a control group ratshowing no brown pigmentation.H&E stain ×20

Hepatic and Hippocampus Iron Status 407

indicates that with chronic Cu intoxication, levels of serumCu increases, which in turn causes rise in serum “free” Cu,which, according to the findings of Monnot et al. [35] andChoi et al. [36], is the primary Cu species crossing theblood–brain barrier and blood–cerebrospinal fluid barrierand getting access to brain parenchyma. Various studieshave linked the increase in serum “free” Cu levels withmemory deficits in human neurodegenerative diseases.Most direct evidence came from the study of Salustriet al. [12] who have reported that cognitive function isinversely correlated with serum “free” Cu levels in normalsubjects. In addition, extensive studies by Squitti et al. [26, 27]have demonstrated that in AD, patient's serum “free” Cuis slightly increased. More importantly, serum “free” Cu isassociated with cognitive decline and with Cu, h-tau, andAβ in the CSF in AD patients [7]. Of interest was theobservation that in PD patients, serum ceruloplasmin concen-tration and serum “free” Cu has been found to be increasedwith the deterioration of disease [28]. The serum “free” Cuconcentration has been proposed as a diagnostic test forWD [37], which manifests as a chronic liver disease and/or

neurological impairment/renal dysfunction. In most untreatedWD patients, serum “free” Cu was found to be elevated(>200 μg/L) [20]. Considering the paramount importanceof serum “free” Cu in human neurodegenerative diseases,more studies are warranted to study serum “free” Cu in Cudyshomeostasis associated neurodegenerative diseases inanimals.

H&E stain showed least of brown pigments (Fig. 3a)[23], and PPB stain revealed scattered traces of grade 1haemosiderin deposits in Kupffer cells in very few hepaticareas (Fig. 4a); whereas, most of the other liver section areasdemonstrated grades 3 and 4 Cu depositions by rhodaninestain coupled with grade 1 Cu-associated protein in the liversections of Cu-intoxicated rats [17]. The liver is one of themain organs for storage of excess Fe. Fe may be stored in cellsas a soluble compound called ferritin or an insoluble formcalled haemosiderin; however, unlike ferritin, haemosiderin isstainable with PPB stain [25]. Haemosiderin is defined as anintracellular and extracellular granule composed of complexof ferritin, denatured ferritin, and other material, particu-larly present in old areas of hemorrhage or extravasation ofblood. Certain cells normally contain some water-insolublehaemosiderin, which is particularly evident in tissues whereFe is being recycled. Grade 1 siderosis corresponds to liver Felevels in the upper half of the control range and to marginallyelevated values. Earlier, stainable Fe was regarded as anunusual finding in a normal liver, and its presence was gener-ally thought to represent Fe excess [25]. Subsequently, thestudies of MacDonald [38, 39] have established that small ormodest siderosis is relatively common, and stainable liver Fecan occur in essentially healthy subjects with normal hepaticFe concentrations [25, 40]. The occasional presence of smallquantities of haemosiderin in normal hepatocytes is not sur-prising, in view of the turnover of Fe containing enzymes andof the production of ferritin in these cells. In normal individ-uals, some Kupffer cells regularly contain haemosiderin.Nonetheless, normal levels for tissue haemosiderin in animalsor humans have not been established. It is worth noting thatabsence of PPB stainable Fe corresponded to Fe concentra-tions within 10 to 181 μg/100 mg dry weight [25]. Fe may

Fig. 4 Photomicrograph of histopathological study of liver tissue ofanimals. aLiver section showing grade 1 haemosiderin deposits in Kupffercells in copper-intoxicated rat treated with copper lactate for 90 days. NoteFe is dark blue in color (arrow). Perls' Prussian blue stain ×20. b Liver

section of a control group rat showing no haemosiderin deposition. Perls'Prussian blue stain ×20. c Positive control from the liver section of apatient suffering from hepatic iron overload. Note massive grade 4 irondeposition in hepatocytes. Perls' Prussian blue stain ×20

Fig. 5 Photomicrograph of cerebral cortex section of a rat treated withcopper lactate for 90 days. No Fe deposition was observed. Perls'Prussian blue stain ×20

408 Pal et al.

also accumulate in Kupffer cells in secondary hemosiderosis,in which there is an underlying cause for Fe accumulationsuch as hemolysis which might be the case in Cu-intoxicatedanimals (Fig. 4a). However, hepatic Fe levels were compara-ble between control and Cu-intoxicated group, which is inaccordance to the findings of MacDonald, as mentioned pre-viously [38, 39]. It should be noted that the association be-tween liver Fe concentration and stainable liver Fe does nothave a normal linear form [25].

Fe is involved in the pathophysiology of AD as suggestedby positive correlation between iron levels in the hippocam-pus and the mini-mental state examination of AD patients[41] in addition to its presence in senile plaques and neurofi-brillary tangles in postmortem brains from AD patients [3, 42].Redox active Fe has also been associated with free radicalproduction through the Fenton reaction, leading to the pro-duction of the extremely reactive hydroxyl radicals not onlyaffecting membrane lipids, proteins, and nucleic acids but alsocontribute in Aβ aggregation by promoting covalent bindingbetween peptide monomers [3, 43]. The resulting associationbetween Fe and Cu and Aβ may also lead to the synthesis ofhydrogen peroxide, thereby aggravating the oxidative damage[3, 44, 45]. Nevertheless, neither Fe deposition in the brain(Fig. 5) nor increase in hippocampus Fe content (Table 1) wasobserved in young Cu-intoxicated rats compared to control rats.Negative PPB staining of brain sections in Cu-intoxicated ani-mals can be justified by the sensitivity of the PPB [25]. Riedereret al. [46] have shown nonsignificant difference of Fe content inPD brain demonstrating moderate neurodegeneration; thus, itcould be considered that Fe buildup in PDmight be the result ofthe underlying mechanisms of neuronal cell death, since, other-wise, Fe levels should be expected to be increased since theinitial periods of the disease; thus, certain mechanisms mayinitiate neuronal cell death at the early stages of the disorderand with aging leads to Fe amassing that, in turn, may furtherexacerbate oxidative damage [3]. In the context of non-Wilsonian brain Cu toxicosis model [17], it can be interpretedthat the observed swelling as well as increase in number ofastrocytes was due to the build-up of Cu in the brain, whichmight, in turn, have resulted in astrocytic dysfunction, leadingand/or contributing to neurodegenerative processes andmemoryimpairments [47, 48] without any involvement of Fe, as sug-gested by nonsignificant changes in hippocampus Fe levels inCu-intoxicated animals compared to control animals (Table 1).In addition, astrocytes have a significant role in learning andmemory consolidation [49]. Recent exciting findings havehighlighted that astrocytes possess GPI-linked ceruloplasmin[8], amyloid precursor protein (APP)which is a Cu bindingprotein [50, 51], APP-695 which exhibits ferroxidase activity[52], and ferritin-reactive microglia which surrounds thedegenerating neurons [53]; thus, the role of astrocytes, APP-695 and microglia, deserves further investigation in Cu toxicityelicited neurodegeneration. LDH and ALP levels were

significantly increased in the serum of Cu-intoxicated rats(Table 1), which in addition to elevated SGOT and SGPT [17]further corroborates the widespread tissue damage due to chron-ic Cu intoxication (Table 1).

In conclusion, the findings herein demonstrates that maleWistar rats chronically injected with Cu lactate exhibited gross-ly elevated levels of serum “free” Cu along with increasedserum ceruloplasmin which might precede the clinical onsetof neurotoxicity and may help in predicting early risk of Cutoxicity-induced neurodegeneration. In addition, data presentedhere also support the possibility that ceruloplasmin and serum“free” Cu are determined by hepatic Cu overload due to Cuintoxication. However, more studies are needed to utterly re-veal the conundrum of Cu toxicity-induced neurodegenerationand memory discrepancies with particular emphasis on the roleof type of astrocytes involved [54], ceruloplasmin linked toastrocytes, APP, APP-695 ferroxidase in Cu, and Fe homeo-stasis in the brain.

Acknowledgments The authors acknowledge the financial assistanceprovided by the Indian Council of medical Research (ICMR, New Delhi)as JRF to Mr. Amit Pal [3/1/3/JRF-2009/MPD-13 (11279)]. Specialthanks to Dr. J.K. Pradhan of Jaypee University, Solan for providingfacilities and guidance to carry out tissue Fe estimations. Authors alsoacknowledge the support of Mr. Charan Singh (for staining studies) andMr. Rakesh Mohindra (for statistics).

Conflict of interest Authors declare no conflict of interest.

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Hepatic and Hippocampus Iron Status 411