Effects of Copper and/or Cholesterol Overload on Mitochondrial Function in a Rat Model of Incipient...

Hindawi Publishing CorporationInternational Journal of Alzheimerrsquos DiseaseVolume 2013 Article ID 645379 14 pageshttpdxdoiorg1011552013645379

Research ArticleEffects of Copper andor CholesterolOverload on Mitochondrial Function in a Rat Model ofIncipient Neurodegeneration

Nathalie Arnal1 Omar Castillo2 Mariacutea J T de Alaniz1 and Carlos A Marra13

1 Instituto de Investigaciones Bioquımicas de La Plata (INIBIOLP) CCT La Plata CONICET-UNLP Catedra de Bioquımica y BiologıaMolecular Facultad de Ciencias Medicas Universidad Nacional de La Plata 60 y 120 1900 La Plata Argentina

2 Centro de Investigaciones Cardiovasculares (CIC) CCT-CONICET 1900 La Plata Argentina3 INIBIOLP Catedra de Bioquımica Facultad de Ciencias Medicas Universidad Nacional de La PlataCalles 60 y 120 1900 La Plata Argentina

Correspondence should be addressed to Carlos A Marra contactocarloshotmailcom

Received 4 August 2013 Accepted 13 September 2013

Academic Editor Renato Polimanti

Copyright copy 2013 Nathalie Arnal et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Copper (Cu) and cholesterol (Cho) are both associatedwith neurodegenerative illnesses in humans and animalsmodelsWe studiedthe effect in Wistar rats of oral supplementation with trace amounts of Cu (3 ppm) andor Cho (2) in drinking water for 2months Increased amounts of nonceruloplasmin-bound Cu were observed in plasma and brain hippocampus together with ahigher concentration of ceruloplasmin in plasma cortex and hippocampus Cu Cho and the combined treatment Cu + Cho wereable to induce a higher Chophospholipid ratio in mitochondrial membranes with a simultaneous decrease in glutathione contentThe concentration of cardiolipin decreased and that of peroxidation products conjugated dienes and lipoperoxides increasedTreatments including Cho produced rigidization in both the outer and inner mitochondrial membranes with a simultaneousincrease in permeability No significant increase in Cyt C leakage to the cytosol was observed except in the case of cortex from ratstreated with Cu and Cho nor were there any significant changes in caspase-3 activity and the BaxBcl2 ratio However the A120573(1ndash42)(1ndash40) ratio was higher in cortex and hippocampus These findings suggest an incipient neurodegenerative process induced byCu or Cho that might be potentiated by the association of the two supplements

1 Introduction

It is well known that copper (Cu) is an essential transitionmetal for all living organisms functioning as cofactor formany enzymes [1ndash3] However we and other laboratorieshave demonstrated in vivo and in vitro that excess inorganicCu produces increased levels of reactive oxygen species(ROS) and damage to biomolecules ultimately promotingcell death [4ndash7] Humans are continuously at risk fromexcess Cu due to involuntary exposure to pollution (contami-nated water food) professional activities [8ndash10] ingestion ofdietary supplements [10ndash12] and prolonged use of intrauter-ine devices [13 14] Elevated Cu plasma levels especiallyof free-Cu or the so-called nonceruloplasmin-bound Cu(NCBC) have been associated with neurodegenerative dam-age [10 15 16] In recent years there has been a considerable

increase in the number of published papers relating Cuto the neurodegenerative process [15ndash18] In line with thisBrewer [10] hypothesized that ingestion of inorganic Cu fromdifferent sources is at least a partial cause ofAlzheimer disease(AD) in developed countries Squitti et al [17ndash19] reportedthat NCBC which is loosely bound to molecules such asserum albumin and other proteins is one of the main riskfactors involved in AD development They suggest that itis the ratio of CP to Cu that is the crucial biomarker forinterpreting Cu-associated features in live AD patients

AD has multiple conditioning and causative factorsHowever it was reported that inorganic Cu in conjunctionwith a high fat diet sets the stage for the development of ADparticularly if other risk factors are present [19] Pappolla etal [20] proposed that hypercholesterolemia accelerates thebiochemical neuropathologic damage observed in transgenic

2 International Journal of Alzheimerrsquos Disease

mice In addition Notkola et al [21] found that plasmacholesterol (Cho) levels were predictors of AD prevalence ina population-based sample of 444 men (aged 70ndash89 years)More recently in vivo models of AD-like neurodegenerativediseases demonstrated that the association of Cu and Choincreases the risk of the development of neurodegenerativedamage [22ndash24]

Cu overload has been extensively associated with ROSoverproduction in vitro and in vivo [6 7 25] Oxidative stressis considered a primary event in the development of AD [26]Pope et al [27] reported that ROS overproduction due tothe dysfunction of the electron transport chain is causativeof neurodegeneration associated with AD and ParkinsonrsquosdiseaseMoreover some experimental evidence indicates thatalterations in mitochondrial energetics occur along with theaccumulation of oxidative damage before the cardinal signsof AD pathogenesis in the brains of transgenic animalmodelsand human patients [28 29]

As reviewed by Paradies et al [30] the mitochondrionis considered the most important cellular organelle con-tributing to the neurodegenerative process mainly throughrespiratory chain dysfunction and the formation of reactiveoxygen species Factors that increase the rate of mitochon-drial ROS production lead to mitochondrial dysfunctionbecause of the oxidative-induced damage caused to vari-ous crucial biomolecules such as mtDNA lipids and pro-teins thus increasing mitochondrial deterioration in a self-perpetuating process [31] Together with ROS accumulationmitochondrial dysfunction is one of the earliest and mostprominent features of AD [27 28] and this condition isstrongly associated with multiple negative consequencesDysfunctional mitochondria generate high levels of ROS thatare ultimately toxic to cells particularly those with a longlifespan and low antioxidant defense system such as neurons[28 29] At the same timemitochondria are also targets of theROS produced by pro-oxidative factors such as Cu overloador increased levels of NCBC

Among the mitochondrial lipids directly affected byROS overproduction cardiolipin (CL) is one of the mostdeeply involved in both synapses and neuronal loss [31]CL is linked to the endogenous proapoptotic cascade thateventually leads to the integration of the apoptosome and thesubsequent activation of the effector caspase system [28ndash31]Moreover CL is involved in the regulation of the bioenergeticprocess and in the integrity of mitochondrial membranesPeroxidation of CL directly affects the biochemical functionsthat depend on the inner and outermitochondrialmembranecomposition and structure Experimental evidence clearlyindicates that this lipid represents the most important targetof ROS attack [30]

In summary there is conclusive evidence linking ROShyperproduction with mitochondrial dysfunction Oxidativestress is also directly involved in neurodegeneration andCu overload in association with cholesterol apparently actsas a synergistic risk factor in the etiology of neurologicaldisorders All the foregoing evidence suggests the need formore in-depth study of this phenomenon In view of theabsence of conclusive studies on the biochemical effects

of the Cu + Cho (CuCho) association on mitochondrial-associated dysfunction especially in relation to those aspectsinvolved in programmed cell death we aimed to investigatethe effects of CuCho on mitochondria isolated from cortexand hippocampus of Wistar rats and explore (i) the ChoPiratio and steady-state fluorescence anisotropy of the outerand inner mitochondrial membranes in order to assess theirfluidities (ii) mitochondrial membrane integrity by meansof a probe that measures trans-membrane potential (iii)the concentration of the main mitochondrial antioxidantglutathione (iv) the concentration of the key mitochondrialphospholipid cardiolipin and the possible pro-oxidativedamage it causes (v) the activity of the apoptotic pathwaydepending on mitochondrial membrane integrity (caspase-3 and the BaxBcl-2 ratio) and (vi) the concentration of 120573-amyloid (A120573) in the two brain zones and in peripheral plasmaas a biomarker of a proneurodegenerative effect

2 Materials and Methods

21 Chemicals All chemicals used were of analytical gradeand obtained from Sigma Chem Co (Buenos AiresArgentina or USA)Merck (Darmstadt Germany) and CarloErba (Milan Italy)

22 Animals and Treatments Certified pathogen-free maleWistar rats were used The rats were maintained at a con-trolled temperature (25∘C) and relative humidity of 60with forced ventilation under a normal photoperiod of12 h darkness and 12 h light The health of the animalswas monitored in accordance with the internationally rec-ommended practices of the Institute of Laboratory AnimalResources Commission of Life Sciences National ResearchCouncil (ILAR) Solid food and drinkingwater were providedad libitum The diets for the experiments were prepared inour laboratory according to the recommendations for Wistarrats [32] All procedures for handling the animals followedthe NIH regulations [33] The experimental protocol wasreviewed and approved by the Bioethics Committee of theFaculty of Medical Sciences UNLP (number 0038211)

23 Experimental Protocols Rats (21 days old)were randomlyassigned (ten animals per group) to the protocols detailed asfollows and treated for eight weeks (2months) Selecting veryyoung rats lets us discard any neurodegenerative (unknown)event(s) associated to the age of the animal By the way thisapproach may be extrapolated to humans that are exposedto copper from the very beginning of their life In additionin previous experiments (not shown) we noted thatmdashat lestduring the first year of lifemdashthat we have no differencesin the response of the model as a function of the startingage of treatment The control group (C) was maintainedon lab-prepared pellets as recommended for normal growthcontaining 7 ppm of Cu [34 35] The Cu-supplementedexperimental group (Cu) was fed on control pellets and tapwater supplemented with 3mgL (or ppm) of Cu in the formof ultrapure CuSO

4(Merck Darmstadt Germany)TheCho-

supplemented group (Cho) was fed on pellets containing

International Journal of Alzheimerrsquos Disease 3

2 (WW) of Cho (87 pure) (obtained from Saporiti SRLBuenos Aires AR) and the Cu + Cho-supplemented group(CuCho) was simultaneously treated with Cu in water +Cho in food Rats were monitored during the experimentalperiod to observe their behavior quantify water and foodconsumption and determine their body weight gain TotalCu concentration in tap water supplemented with CuSO wasdetermined by means of atomic absorption methodologyand was 342 plusmn 021 ppm (means of all daily measurementstaken along the experimental period) Considering that eachanimal imbibed between 49plusmn04 and 150plusmn11mLwaterday(at the beginning and the end of the protocol resp) a max-imum of 001 to 005mgCuday was acquired from water (adose equivalent to 006 and 018mgCuKg live animal resp)Linear regression curves and ANOVA test for Cu content infood demonstrated that there were no significant variationsbetween the 6 preparations used for the experiments (722 plusmn031 ppm or mgCuKg diet) Fe and Zn content (determinedby atomic absorption spectrometry) was the same in allpreparations (459 plusmn 08 and 666 plusmn 20 ppm resp) Ingestionof solid food along the experiments varied from 116 plusmn 08to 297 plusmn 28 grat implying that the oral ingestion of Cuwas in the range from 008 to 021mgCudayrat (00032 to00008mgCuKg live animal)

24 Sample Collection At the end of the treatmentsanimals were deeply anesthetized with ketamine (70mgKg)and xylazine (5mgKg) applied intramuscularly and thensacrificed by decapitation Brainswere removed and dissectedinto two zones cortex and hippocampus using the atlas ofPaxinos andWatson [36] as a guide for tissue dissection Bothbrain regions were washed weighed and homogenized usinga TrisHCl buffer (10mMpH 74) with sucrose (70mM)mannitol (230mM) ethylenediaminetetraacetic acid(EDTA) (1Mm) and dithiothreitol (DTT) (1mM) (Buffer I)Homogenates were centrifuged at 2∘C 700timesg for 10min andthe supernatants recentrifuged at 8000timesg (10min2∘C) toobtain the cytoplasm fraction Pure mitochondrial fractionswere isolated from the pellets of the previous centrifugationby resuspending them in 3mL of buffer I and centrifugingduring 5min at 1000timesg (2∘C)Thepellets were discarded andthe supernatants re-centrifuged for 10min at 11000timesg (2∘C)Finally the final pellet (mitochondrial fraction) was resus-pended in buffer II (buffer I with 0015 mg pure albumin(Sigma Chem Co Buenos Aires AR)) Cytosol fractionswere prepared by ultracentrifugation of the supernatants at110000timesg for 1 h at 2∘C During the sacrifice of the animalsblood was also collected using heparin as anticoagulant(1 IU5mL) in ice-cold polypropylene tubes The plasmasamples were immediately prepared by centrifugation in thecold (4000timesg 10min) and stored at minus70∘C until analyzed

25 Atomic Absorption Measurements Aliquots of samplewere digested with a mixture of 4mL of HNO

3(c) and

1mL HClO4(Aldrich or Sigma Chem Co Buenos Aires

Argentina) by heating at 120∘C for 60min in a mineralizationblock [37] The digests were cooled diluted with ultrapurewater (18mΩ cm Carlo Erba Milan Italy) and ultrafiltered

with a 022120583m Millipore membrane (Milli-Q PurificationSystem fromMillipore CA USA) Ultrafiltered dissolutionswere directly aspirated into the flame of a PerkinElmer 1100B Spectrophotometer equipped with a Perkin-Elmer cathodelamp (Perkin-Elmer Corp Norwalk CT USA) at a spectralwidth of 1 nm Standard solutions of 100 ppm Zn and Fefrom HCR Inc (QuimiNet Buenos Aires AR) were usedCu determinations were calibrated with a standard solu-tion (200 ppm) of Cu(NO

3)2in HNO

305N (Titrisol from

Merck Co Darmstadt Germany) All measurements werecarried out in peak height mode (3247 nm line) The intra-[(SDx) sdot 100] and inter-[(ΔSDΔx) sdot 100] assay coefficientsof variation were 155 and 60 respectively We routinelyobtained a similar equation for the calibration curve (IR =55 sdot 10minus5 + 0048 [Cu mgL]) and the statistical analysesdemonstrated a correlation coefficient always between 095and 099 In addition we explored the so-called matrixeffects that might have modified the slopes of the standardregressions In spiked samples the obtained values varyingfrom 48 to 60 sdot 10minus5 were very similar to those of Cu standardsolutions indicating that the matrix effect was considerednonsignificant or was negligible The mean for recovery andRSD for spiked samples was 997 and 33 respectivelyand the detection limit was 009mgL In order to verify theaccuracy of the method we explored the influence of timeafter dilution temperature of acid digestion and concentra-tion of HNO

3HClO

4following the suggestions of Terres-

Martos et al [38] We also checked our results with biologicalsamples (plasma and homogenates) against a SeronormTraceElements Serum (from Sero Labs Billingstad Norway) andfound no significant differences between the obtained and thedeclared (certified) concentrations

26 Ceruloplasmin (CP) Levels and Nonceruloplasmin-BoundCopper (NCBC) Samples were analyzed by the enzyme con-version of p-phenylenediamine into a blue-colored product[39]whichwas thenmeasured at 550 nmReaction proceededat 37∘C in buffer glacial aceticsodium acetate (50mM pH55) directly into flat-bottomed plates using a MicroplateReader SpectraMax M2m2e model from Molecular DevicesAnalytical Technologies (Sunnyvale CA USA) for 3minIntra- and interassay coefficients of variation were 83 and44 respectively CP concentrations were calculated bycomparison with the reaction rate of pure CP standard(Sigma Chem Co Buenos Aires Argentina) Using the Cuand CP data we calculated the non-CP-bound Cu (NCBC orso-called free Cu) as described by Brewer [19] by subtractingthe amount of Cu bound to each mg of CP from data of totalCu This parameter can be easily expressed in percentagesusing the formula (([Cu] minus 472 times [CP]) times 100[Cu]) whereCu is in 120583molL and CP in gL [40]

27 Lipid Analysis Total lipids were extracted by themethod of Folch et al [41] An aliquot of each Folchextract was evaporated and the residue dissolved in 50mMsodium phosphate buffer (pH 74) containing digitonin1 Aliquots of this solution were taken to enzymaticallymeasure cholesterol (Cho) and phospholipids (PL) usingcommercial kits from Wienner Lab (Rosario Argentina)


To determine the mitochondrial cardiolipin (CL) contentsamples were separated by high-performance thin-layerchromatography (HPTLC) on precoated silica gel plates (10 times20 cm) with concentration zone from Whatman Schleicherand Schuell (Maidstone England) The mobile phase waschloroform methanol ammonium hydroxide (65 25 4 byvolume) Spots were localized using iodine vapor on alateral lane spotted with authentic standards of CL mono-and dilyso-CL and other reference phospholipids (AvantiPolar Lipids Ontario Canada) The rest of the plate wascovered with a glass to avoid possible spontaneous oxidationThe CL and lyso-CL zones were scraped off the platesand eluted from the silica with ldquoinverserdquo Folch extraction(chloroform methanol 1 2) After evaporation under nitro-gen at room temperature the selected zones were dissolvedin 200120583L of Folch solvent mixture The total amount ofCL and lyso-CL was quantified by means of phosphorousmeasurement using the method of Chen et al [42]

28 Mitochondrial Membrane Integrity The mitochondrialmembrane potential (Δ120595) was measured by estimating theintegrity of the mitochondrial membrane using the iso-lated mitochondrial staining kit from Sigma-Aldrich Inc(Buenos Aires AR) This kit is based on the uptake ofthe lipophilic cationic probe 551015840661015840-tetrachloro-111015840331015840-tetraethyl-benzimidazolcarbocyanine iodide dye (JC-1) intothe mitochondrial matrix which directly depends on the Δ120595In healthy cells the dye concentrates in the matrix whereit forms bright red fluorescent agglomerates Uptake by themitochondria of JC-1 can be utilized as an effective distinctionbetween apoptotic and healthy cells Any event that dissipatesthe Δ120595 prevents the accumulation of the JC-1 dye in themitochondria and the dye is thus dispersed in the cytoplasmleading to a shift from red (JC-1 agglomerated) to greenfluorescence (JC-1 monomers) To measure the samples weused a PerkinElmer LS 55 Fluorescence Spectrometer set at525 nm excitation wavelength and 590 nm emission

29 Steady-State Fluorescence Anisotropy of DPH and TMA-DPH Submitochondrial fractions (inner and outer mem-branes) were obtained following the method described byFraser and Zammit [43] with the modifications of Pellon-Maison et al [44] Prior to membrane fluidity assays totalprotein concentration was determined in each suspensionand adjusted to approximately 300 120583g proteinmL using5mM TRISHCL buffer pH 740 Membrane fluidity wasdetermined by the fluorescence anisotropy (or polarization)technique using two fluorescent probes 16-diphenyl-135-hexatriene (DPH Sigma-Aldrich St Louis MO) and 1-[4-(trimethylamino)phenyl]-6-phenyl-135-hexatriene (TMA-DPH Molecular Probes Eugene OR) TMA-DPH was usedto monitor fluidity near the surface of the cell membraneThe polar region of this probe is anchored at the lipid-water interface and the hydrocarbon moiety enters the lipidpart of the membrane The length of the hydrophobic partof the TMA-DPH molecule is approximately equivalent tothat of a plasma membrane [45] DPH is incorporated intothe hydrophobic regions of the lipid bilayer [46] DPH or

TMA-DPH anisotropy is inversely correlatedwithmembranefluidity [47] Anisotropy measurements were performed aspreviously described [48] with slight modifications Brieflymembranes were vigorously homogenized using a vortexvibrator for 1min and subjected to mild sonication in aBranson sonifier 450 (Branson Ultrasonic SA CA USA) (setat 40 output) following a 3min incubation period on iceThe sonication cycle was controlled by the turbidity of thesuspension as evaluated at 600 nm and was interrupted whenan absorbance value of 02 or less was reachedThis proceduredoes not interfere with the transition of lipid bilayers butdisperses aggregates facilitating fluorescence readings anddecreasing light scattering [49] The samples for the DPHand TMA-DPH assays (3mL) contained 5mM TrisndashHClbuffer pH 74 60 120583g of protein from diluted suspensionsand 50 120583M MDPH in tetrahydrofuran or 25 120583M TMA-DPHin dimethylformamide and were incubated for 45min at37∘CThe steady-state anisotropywasmeasured in aHe12058210S120573spectrofluorophotometer (Thermoelectron Corp SydneyAustralia) equipped with a thermostated cell holder Thesamplersquos temperature was checked to an accuracy of plusmn01∘Cusing a thermistor thermometer Excitation and emissionwere set at 357435 nm and 358428 nm for DPH and TMA-DPH (resp) using 5 nm excitation and emission slits Sampleswere illuminated with linear (vertically V or horizontally ℎ)polarized monochromatic light and the fluorescence intensi-ties emitted (119868 in arbitrary units) parallel or perpendicularto the direction of the excitation beam were recorded Blanksamples without the addition of the probe(s) were alsomeasured to estimate unspecific light that might reach thedetector The fluorescence anisotropy of the probes (119883) wascalculated as 119903(119883) = [119868VVminus119868Vℎtimes119866] = [119868VV+2119868Vℎtimes119866] where119868VV and 119868Vℎ are the intensities of the fluorescence emittedrespectively parallel and perpendicular to the direction of thevertically polarized excitation light G is the correction factor(119866 = 119868ℎV119868ℎℎ) for the optical system given by the ratio of thevertically to the horizontally polarized emission componentswhen the excitation light is polarized in the horizontaldirection and119883 represents TMA-DPHorDPHAccording toShinitzky and Barenholz [50] fluorescence anisotropy valuesare inversely proportional to cell membrane fluidity Thusa high degree of fluorescence anisotropy represents a highstructural order or low cell membrane fluidity

210Mitochondrial Reduced (GSH) andOxidized (GSSG)Glu-tathione Content Total glutathione was determined by theglutathione reductasedithionitrobenzoic (DTNB) methodthat can measure both GSH and GSSG [51] MitochondrialGSH (mGSH) were calculated by subtracting GSSG fromthe total glutathione content (GSH + GSSG) For this reasonsamples were run in the presence and absence of 2mMdivinylpyridine

211 Programmed Cell Death Biomarkers

2111 Caspase-3Activity Caspase-3 activitywasmeasured bya colorimetric assay kit (CASP-3-C) based on the hydrolysisof the synthetic peptide substrate acetyl-Asp-Glu-Val-Asp-p-nitroaniline (Ac-DEVD-pNA) by caspase-3 (Sigma Chem


Co Buenos Aires Argentina) The resulting p-nitroaniline(p-NA) released was monitored at 405 nm Each assay wasrun in parallel with inhibitor-treated cell lysate (to measurethe nonspecific hydrolysis of the substrate) and caspase-3 positive control (using commercial caspase-3 5mgmLprovided by the kit manufacturer) A calibration curve usinga standard solution of p-nitroaniline (p-NA) was also run foreach assay to calculate the activity of the protease expressedas 120583mol p-NA releasedmin sdotmL of sample

2112 BaxBcl-2 Ratio Western blot assays of Bax and Bcl-2were performed to calculate the BaxBcl-2 ratio Equal quan-tities (30 120583g protein per lane) of total protein were separatedby SDS-PAGE (15 gels) under reducing conditions and aMiniprotean VI unit (Bio-Rad CA USA) The proteins werethen electrophoretically transferred to PVDF membranes(IPVH00010 Immobilon Millipore USA) The membraneswere blocked with 5 skimmed milk and incubated withanti-Bcl-2 and anti-Bax antibodies respectively (1 1000 sc-492 rabbit polyclonal and sc-493 rabbit polyclonal from SantaCruz CA USA resp) at 4∘C overnight This was followedby incubation with goat anti-rabbit secondary antibody con-jugated with horseradish peroxidase (1 20000 Santa CruzCA USA) Photographs were taken using the MolecularImager Gel Doc XR and ChemiDoc XRS systems (Bio-Rad)and the optical densities of the bands were quantified

212 Beta-Amyloid Peptides (1ndash40) and (1ndash42) Beta-amyloidpeptides (A120573) (1ndash40) and (1ndash42) were measured using theHumanRat120573Amyloid-40 ELISAkitWako II and theHumanAmyloid-42 ELISA kit Wako High-sensitive respectivelyBefore the assay the samples were centrifuged (2∘C) at5000timesg for 15min and the supernatants diluted 1 1 withthe buffer provided by the manufacturer Results for plasmawere expressed in pmolesL and for brain tissues in pmolmgprotein The A120573(1ndash42)(1ndash40) ratios were calculated fromeach individual pair of data

213 Biomarkers of Cardiolipin Peroxidation

2131 Authentic Lipoperoxides Lipoperoxides (LPOO) incardiolipin extracts (CL) were determined according to themethod of Nourooz-Zadeh et al [52] Results were expressedas 120583moles LPOO120583moles CL (for calculation we used anextinction coefficient of 460 (10minus4) sdotmolminus1 sdot cmminus1 at 560 nm)

2132 Conjugated Dienes The spectrophotometric detec-tion of conjugated dienes was performed according to themethodology described by Recknagel and Glende [53] Thespectral register between 300 and 220 nm was obtained andsubtracted from a blank spectrum of pure cyclohexaneConjugated dienes show a typical absorption band in therange of 230ndash240 nm The results were expressed as relativeunits of optical density (UDO)mg mitochondrial CL

214 Statistical Analysis All values represent the mean of10 rats assayed in triplicate expressed as mean plusmn standarddeviation (SD) Data were analyzed by ANOVA plus the

Tukey test with the aid of SPSS 1101 software (SPSS IncChicago IL) To analyze the bands obtained by western blotwe used the Image J software for image processing (NationalInstitute of Health USA) Results were also plotted andanalyzed using Sigma Scientific Graphing Software (version110) from Sigma Chem Co (St Louis MO) The statisticalsignificance (119875 le 001) of differences is indicated bysuperscript letters (values with distinct letters are statisticallydifferent between them)

3 Results

Growth parameters (final weight gain rate of body weightfood efficiency ratio etc) showed no significant differencesbetween the experimental groups (data not shown) Alsowater intake among the experimental groups was essentiallyidentical which is a reason to assume that the rats did notnote any difference in the palatability of the supplementedwater compared with the nonsupplemented one The samehappened with solid food

31 Copper Content in Mitochondrial Suspensions Atomicabsorption measurements demonstrated that the total Cucontent in digested mitochondrial suspensions did not differsignificantly among the experimental groups Howevermeanvalues for cortex were higher than those for hippocampus(59plusmn04 versus 40plusmn02 gmgmitochondrial proteins resp)

32 Nonceruloplasmin-Bound Copper (NCBC) and Cerulo-plasmin (CP) Concentrations Figure 1(a) shows the concen-tration of free Cu (expressed as NCBC) in plasma andin both brain zones analyzed Plasma and hippocampusshowed significant increases after Cu and CuCho treatmentscompared to the control group In cortex we observed nosignificant differences

We also analyzedCP concentration in plasma cortex andhippocampus after the experimental diets (Figure 1(b)) CPincreased significantly after Cu and CuCho supplementationin plasma and in both brain zones Cortex and hippocampusfrom rats fed on the CuCho diet also showed significantincreases with respect to the Cu treatment alone

33 Effect of Diets on Mitochondrial Membrane Physicochem-ical Properties Figure 2 shows the ratio between Cho andtotal phospholipids (measured as total inorganic phosphateor Pi) in cortex and hippocampus In both brain zonesCu treatment produced a significant increase in this ratioSupplementation with Cho and with Cu + Cho (CuCho) alsoproduced significant increases in the ChoPi ratio comparedto the control or Cu treatment alone

The mitochondrial membrane potential was estimatedby fluorimetric determination of the degree of aggregationof the JC-1 dye in mitochondrial suspensions from braincortex and hippocampus (Figure 3) Supplementation withCu alone produced no significant changes in hippocampusand a discrete although significant increase in the membranepermeability in cortex In both brain zones cholesterol sup-plementation also produced a clear loss of integritywhichwas


aa

a a a a aa

b b

b b

NCB

C (n

gm

g pr

otei

n)

25

20

15

10

05

00Plasma Cortex Hippocampus

ControlCu

ChoCuCho

(a)

a a

a aa a

b b

bbc c

Plasma Cortex Hippocampus

CP (n

gm

g pr

otei

n)

60

50

40

30

20

10

0

ControlCu

ChoCuCho

(b)

Figure 1 Free Cu (NCBC) (a) and ceruloplasmin (CP) (b) levels inbrain cortex and hippocampus homogenates (ngmg total protein)Treatments are indicated with different colors control (white bars)Cu (black bars) Cho (gray bars) and CuCho (dark gray bars)Samples were analyzed as indicated in Sections 25 and 26 Resultsare expressed as mean of 10 rats assayed in triplicate plusmn standarddeviation (SD) Significant differences among the experimentalgroups are indicated with superscript letters (values with distinctletters are significantly different between them 119875 lt 001)

more pronounced when the lipid was associated with Cu indrinking water (especially in hippocampus)

34 Glutathione Content and Biomarkers of Lipid Peroxi-dation Cho and CuCho treatments produced a significantdecrease in total mitochondrial glutathione (GSH + GSSG)in both brain zones compared to the control data and tosupplementation with Cu alone (Figure 4) Reductions weremore evident in hippocampus than in cortex Addition ofinorganic Cu to drinking water did not produce significantchanges with respect to the control group

ControlCu

ChoCuCho

aa

b b

cc

cc

20

15

10

05

00Cortex Hippocampus

[Cho

Pi]middot103

Figure 2 Cholesterol (Cho) and total phospholipids (measuredas total inorganic phosphate or Pi) ratio (ChoPi) in brain cortexand hippocampus homogenates (ngmg total protein) Sampleswere analyzed as indicated in Section 27 Treatments are indicatedwith different colors control (white bars) Cu (black bars) Cho(gray bars) and CuCho (dark gray bars) Results are expressed asmean of 10 rats assayed in triplicate plusmn standard deviation (SD)Significant differences among the experimental groups are indicatedwith superscript letters (values with distinct letters are significantlydifferent between them 119875 lt 001)

Figure 5 shows the concentration of two products of CLfatty acids oxidation lipoperoxides (LPOO) and conjugateddienes (Figures 5(a) and 5(b) resp) Significant increasesin LPOO were observed after Cu addition in hippocampusand in cortex with respect to control data Cho treatmentsproduced a significant decrease in this biomarker in hip-pocampus A very significant increase was observed for theCuCho treatment with respect to supplementation with Cuor Cho alone for both brain zones After Cu and CuChotreatments the amount of conjugated dienes was also highercompared to control data in hippocampus and in cortex

35 Cardiolipin (CL) and Lyso-Cardiolipin (LCL) Contentin Mitochondrial Suspensions Figure 6(a) shows the totalconcentration of mitochondrial CL in 120583mol of inorganicphosphate (Pi)mg protein After Cu treatment CL con-tent decreased in both brain zones analyzed The Cho-supplemented diet increased CL in both cortex and hip-pocampus whereas cosupplementation with Cu and Choproduced no significant changes with respect to the controldiet Figure 6(b) shows the levels of lyso-CL (LCL) afterdietary treatments All diets induced significant increases inLCL concentrations in hippocampus and cortex compared tocontrol data however the higher increases were observed forthe groups treated with Cu (alone or in combination withCho)

36 Mitochondrial Membrane Apparent Microviscosities Iso-lated outer and inner mitochondrial membranes were exam-ined for their apparentmicroviscocities using two fluorescent

International Journal of Alzheimerrsquos Disease 7Fl

uore

scen

ce ra

tiom

g pr

otei

n

0

25

50

75

100

ab c

c

a a a

b

ControlCu

ChoCuCho

Cortex Hippocampus

Figure 3 Mitochondrial membrane potential in brain cortex andhippocampus Treatments are indicated with different colors con-trol (white bars) Cu (black bars) Cho (gray bars) and CuCho (darkgray bars) Sampleswere analyzed as indicated in Section 28 Resultsare expressed as mean of 10 rats assayed in triplicate plusmn standarddeviation (SD) Significant differences among the experimentalgroups are indicated with superscript letters (values with distinctletters are significantly different between them 119875 lt 001)

Table 1 Caspase-3 activity in brain cortex and hippocampus fromthe experimental diets

Diets Cortex HippocampusC 21 plusmn 01 14 plusmn 01Cu 23 plusmn 02 15 plusmn 01Cho 20 plusmn 01 14 plusmn 03CuCho 24 plusmn 01 17 plusmn 01Results were obtained as described in Section 2111 They were expressed asthe mean of 10 independent determinations plusmn SD There are no statisticaldifferences ascribed to the experimental diet

probes (DPH or TMA-DPH) that explore different zones ofthe lipid bilayer Figures 7 (cortex) and 8 (hippocampus)show the values of the anisotropies which are inverselycorrelated with (outer and inner) membrane fluidities Inboth brain regions and with both probes tested we observeddecreased fluidities and increased fluorescence anisotropiesproduced by cholesterol supplementation either alone or incombination with Cu

37 Programmed Cell Death Biomarkers Despite the damageobserved in the integrity of the inner mitochondrial mem-brane (JC-1 measurements) we did not detect any significantincrease in Cyt C in cytosol fractions with the sole exceptionof a slightmdashalthough significantmdashincrease in brain cortexfrom rats simultaneously treated with Cu and Cho (data notshown) In agreement with this finding caspase-3 activitywas not significantly modified in any experimental group(Table 1) nor was there any significant alteration in theBaxBcl2 ratio (Figure 9)

Cortex0

25

50

75

100

ControlCu

ChoCuCho

aa

b ba

a

bc

Hippocampus

mG

SH (120583

gm

gmiddotpr

ot)middot10minus3

Figure 4 Mitochondrial glutathione (mGSH) content in braincortex and hippocampus (ngmg total protein) Treatments areindicated with different colors control (white bars) Cu (blackbars) Cho (gray bars) and CuCho (dark gray bars) Sampleswere analyzed as indicated in Section 210 Results are expressedas mean of 10 rats assayed in triplicate plusmn standard deviation (SD)Significant differences among the experimental groups are indicatedwith superscript letters (values with distinct letters are significantlydifferent between them 119875 lt 001)

Table 2 Ratio A120573(1ndash42)(1ndash40) in plasma and brain cortex andhippocampus after the experimental diets

Diets Plasma BrainCortex Hippocampus

C 65 plusmn 10a 73 plusmn 09a 63 plusmn 07a

Cu 68 plusmn 08a 92 plusmn 05b 66 plusmn 04a

Cho 71 plusmn 11a 92 plusmn 06b 96 plusmn 05b

CuCho 85 plusmn 08a 109 plusmn 05c 93 plusmn 06b

Data were obtained as described in Section 212 They correspond to themean of 10 individual determinations assayed in triplicate plusmn SD Differencesstatistically significant among the different diets are indicated with distinctsuperscript letters

38 Biomarker of Neurodegeneration Table 2 shows theA120573(1ndash42)(1ndash40) ratio in plasma and in cortex and hippocam-pus homogenates as a biomarker of neurodegenerationCu or Cho treatments produced a significant increase inthis biomarker in brain cortex and the CuCho associationproduced an even more significant increase Cho supple-mentation increased the ratio in hippocampus with a valueindistinguishable from that observed for the simultaneoustreatment with Cu and ChoThese changes were not reflectedin peripheral plasma

4 Discussion

Inappropriate dietary copper and cholesterol intakes areimplicated in the development of Alzheimerrsquos disease Thisstudy follows previous experimental evidence reporting

8 International Journal of Alzheimerrsquos DiseaseLP

OO

(120583m

oles120583

mol

es C

L)

0

2

4

6

a

a

ab

b

c

c

d

Cortex

ControlCu

ChoCuCho

Hippocampus

(a)

0

5

10

15

20

25

30

a

d

c

b

a

b

c

a

Cortex

ControlCu

ChoCuCho

Hippocampus

UD

Omiddot103

(mg

CL)

(b)

Figure 5 Levels of lipoperoxides (LPOO) (a) and conjugated dienes(b) in mitochondrial fractions from brain cortex and hippocampusResults are expressed as 120583moles of LPOO120583Moles of Pi in CL orUDOmg protein respectively Treatments are indicated with dif-ferent colors control (white bars) Cu (black bars) Cho (gray bars)and CuCho (dark gray bars) Samples were analyzed as indicated inSections 2131 and 2132 Results are the mean of 10 rats assayed intriplicate plusmn standard deviation (SD) Significant differences amongdata are indicatedwith superscript letters (valueswith distinct lettersare significantly different between them 119875 lt 001)

the impact of dietary Cu and Cho manipulations as pro-neurodegenerative cause in animal models

The impact of elevated inorganic Cu Cho or a combina-tion of both in the diet (for 2 months) significantly modifiesthemitochondrial function Results suggest that these dietarysupplements are able to increase oxidative damage in braincortex and hippocampus (with a substantial increment ofthe lipoperoxides and conjugated dienes in the cardiolipinsubfraction) increase the proportion Chophospholipidsand consequently decrease the concentration of mGSH

0

40

80

120

a

b

ba

a

b

ab

ControlCu

ChoCuCho

Cortex Hippocampus

CL (120583

mol

Pim

g pr

otei

n)

(a)

0

4

8

12

a

bc

d

b

a

b

c

ControlCu

ChoCuCho

Cortex Hippocampus

L-CL

(120583m

oles

Pim

g m

itoch

ondr

ial p

rot)

(b)

Figure 6 Cardiolipin (CL) (a) and lyso-CL (LCL) (b) levels inmito-chondrial fractions from brain cortex and hippocampus (120583moles ofPimg total protein) Treatments are indicated with different colorscontrol (white bars) Cu (black bars) Cho (gray bars) and CuCho(dark gray bars) Samples were analyzed as indicated in Section 27Results are the mean of 10 rats assayed in triplicate plusmn standarddeviation (SD) Significant differences among the experimentalgroups are indicated with superscript letters (values with distinctletters are significantly different between them 119875 lt 001)

decrease the membrane potential modify the proportion ofcardiolipin and lyso-derivatives alter inner and outer mito-chondrial membrane fluidities and increase A120573(1ndash42)(1ndash40)ratio

It is well known that the metabolism of Cu in humansis under strictly regulated control The usual concentrationof total Cu in human plasma (as determined by us andother groups) is in the range of 03 to 21mgL for intakesof 14 to 20mgCuday [16] However the amount of Cu notbound to ceruloplasmin (NCBC) is very low and has beenassociated with some of the features of neurodegenerative


uore

scen

ce an

isotro

py

00

02

04

06

08

TMA-DPHDPH

aa

b

a

b

a

b b

C Cu Cho CuCho

(a) Cortex (OMM)

Fluo

resc

ence

aniso

tropy

00

02

04

06

08

a a

b

a

a b

b

b

TMA-DPHDPH

C Cu Cho CuCho

(b) Cortex (IMM)

Figure 7Mitochondrial outer (a) and inner (b)membrane fluidities(fluorescence anisotropy) in suspension isolated from brain cortexTreatments are indicated with different colors control (white bars)Cu (black bars) Cho (gray bars) and CuCho (dark gray bars)Samples were analyzed as indicated in Section 29 Results areexpressed as the mean plusmn standard deviation (SD) of 10 ratsSignificant differences among the experimental groups are indicatedwith superscript letters (values with distinct letters are significantlydifferent between them 119875 lt 001)

disorders such as AD [16 17] Cholesterol was also associatedwith the etiology or increased risk of developing AD [54ndash57]Thus our experimental system was designed to investigatethe possible concurrence of these two factors in promotingneurodegeneration with a focus on their role in mito-chondrial function Mitochondrial dysfunction due to ROSoverproduction andor the alteration of the physicochemicalproperties of their membranes (mainly lipid composition)was reported to be the main factor in the advance of ADfeaturing early on in the progression of the disease [25ndash27]

Fluo

resc

ence

aniso

tropy

00

02

04

06

08

aa

a a

bb

bb

TMA-DPHDPH

C Cu Cho CuCho

(a) Hippocampus (OMM)

Fluo

resc

ence

aniso

tropy

00

02

04

06

08

aa

aa

b

b

bb

TMA-DPHDPH

C Cu Cho CuCho

(b) Hippocampus (IMM)

Figure 8Mitochondrial outer (a) and inner (b)membrane fluidities(fluorescence anisotropy) in suspension isolated from brain hip-pocampus Treatments are indicated with different colors control(white bars) Cu (black bars) Cho (gray bars) andCuCho (dark graybars) Samples were analyzed as indicated in Section 29 Resultsare expressed as the mean plusmn standard deviation (SD) of 10 ratsSignificant differences among the experimental groups are indicatedwith superscript letters (values with distinct letters are significantlydifferent between them 119875 lt 001)

In discussing the validity andor limitations of our exper-imental system it is necessary to consider the level of thesupplementation with Cu and Cho and the characteristicsof the metabolism of these two dietary supplements Withrespect to Cu overload using oral administration our exper-imental conditions were based on previous work [23 24] andresemble the Cu levels commonly found as a consequence ofinvoluntary exposure through air food and water pollution[8 58ndash60] ingestion of dietary mineral supplements andin professionals engaged in agrochemical activities [9ndash12] orfemale users of Cu-based intrauterine devices [13 14] Studies


0

1

2

3

ControlCu

ChoCuCho

Cortex Hippocampus

Bax

Bcl-2

Figure 9 BaxBcL-2 ratio in brain cortex and hippocampusTreatments are indicated with different colors control (white bars)Cu (black bars) Cho (gray bars) and CuCho (dark gray bars)Samples were analyzed as indicated in Section 2111 Results areexpressed as the mean plusmn standard deviation (SD) of 10 rats Therewere no significant differences between data

performed in rats demonstrated that Cu metabolism andhomeostasis are essentially identical to those in humans [61]Similarly oral Cho supplementation was selected on the basisof previous experiments by other researchers [22ndash24] andthe plasma Cho concentration represents an approximately30 increase over themediumvalues obtained for the controlgroup This increase is frequently observed in humans withdyslipemia [62] We chose a treatment duration equivalent toapproximately 6 years in human terms and therefore assumethat the experimental exposure conditions can reasonably beextrapolated to human populations The known differencesbetween the metabolism of lipoproteins in rats as opposed tohumans could be considered a limitation to this assumptionbut this can be taken into account as in the case of otherexperimental models (rabbits or hamsters eg) It is impor-tant to recall that rats are quite unique in terms of their sterolmetabolism since in other species studied previously (rabbitguinea pig hamster and squirrel monkey) the liver displaysa secondary function whereas extrahepatic tissues play aquantitatively major role in whole animal sterol biosynthesisand metabolism [63]

Cu supplementation in the trace amounts used here didnot substantiallymodify the concentration of themetal insidethe mitochondria isolated from cortex or hippocampusHowever after two months of treatment we observed anincrease in CP concentration in both brain zones in thoseexperimental groups that received Cu We attribute thisincrease mainly to a response to the proinflammatory condi-tion induced by the Cu-induced oxidative stress as describedin our previous work [64] We were unable to measurethe NCBC inside the purified mitochondrial fraction mainlydue to the lack of CP or to the presence of this protein inamounts beyond detection by our method of measurementHowever in plasma and in whole homogenates we found that

NCBC was increased by Cu administration even in the smallamounts used here Concomitantly theCP concentrationwasalso higher in all samples except cortex which probably has astrong homeostatic system to control Cu levels In agreementwith previous findings Das et al [65] reported that increasesof free Cu (NCBC) stimulate the expression of CP throughthe activation of AP-1

Experimental diets also produced significant changesin the lipid composition of mitochondrial fractions Cusupplementation increased the Chophospholipid ratio in thecortex and hippocampus of Wistar rats This increase waseven more pronounced after Cho or CuCho addition Thesechanges must obviously be associated with the modificationof themetabolism (uptake biosynthesis andor degradation)of both subtypes of lipids The elucidation of these effectsremains to be investigated in detail opening a new avenue ofresearch of potential importance in the search for therapeutictargets in neurodegenerative processes As a consequence ofthe aforementioned changes we observed significant modifi-cations in the physicochemical properties of mitochondrialmembranes The transmembrane potential was altered inCu- and Cu + Cho-treated rats Furthermore the apparentmicroviscocity of both mitochondrial membranes (outerand inner) was profoundly altered by Cho and CuChotreatments Generalized and extensive damage in the formof increased permeability (JC-1) and rigidization was mainlydemonstrated by the use of two probes with substantiallydifferent capacities of lipid bilayer exploration (DPH andTMA-DPH)These findings are in agreement with previouslyreported data associating the loss of membrane fluidity withan increase in the ChoPi ratio [66] These changes are ofrelevance from the physiological point of view It is knownthat many membrane protein functions including carriersenzymes and receptors among others can be modulatedby the microenvironment of the membrane where they areembedded [66] Since ROS overproduction has a key role inAD and other neurodegenerative illnesses [67 68] we willfocus on the possible consequences of changes in membraneproperties on mitochondrial redox homeostasis and thepossible activation of programmed cell death signals

Mitochondrial glutathione (mGSH) is considered thekey antioxidant for mitochondrial functioning and survivalIt is the main line of defense for the maintenance of theappropriate mitochondrial redox environment by prevent-ing or repairing oxidative modifications that could lead tomitochondrial dysfunction and cell deathThe importance ofmGSH lies not only in its abundance but also in its extremeversatility to counteract hydrogen peroxide lipid hydroper-oxides or xenobiotics mainly as a cofactor of enzymessuch as glutathione peroxidase or glutathione-S-transferaseMany cell-inducing stimuli such as excess NCBC or othertransition metals that induce oxidative stress reduce thelevels of mGSH and sensitize the mitochondria to additionalinsults (eg excess cholesterol in the composition of theirbiomembranes) [66] Interestingly the transport of GSH intothe mitochondria is critically dependent on the maintenanceof physiologically regulatedmembrane parameters especiallythe ChoPi ratio and the apparent microviscocity of the innermitochondrial membrane [66ndash70] The dicarboxylate carrier


05 06 07 08 14 16 18200

400

600

800

1000m

GSH

[120583m

olesm

g pr

ot]

Cortex

+Cho (r2 = 096)

minusCho (r2 = 092)

Cho middot 103Pi

(a)

02 04 06 08 10 12 14 160

200

400

600

800

mG

SH[120583

mol

esm

g pr

ot]

Hippocampus

+Cho (r2 = 098)

minusCho (r2 = 091)

Cho middot 103Pi

(b)Figure 10 Lineal correlation coefficients for the relationship between mGSH and ChoPi ratio in cortex (a) and hippocampus (b) groupedaccording to two groups of data independently of Cu supplementation one for rats that did not receive cholesterol (minusCho) and the other foranimals fed on diets supplemented with Cho (+Cho) The values of the 1199032 for each curve adjustment appear in parenthesis

and the 2-oxoglutarate carrier have been shown to functionas GSH transporters highly sensitive to the physiochemicalstate of mitochondrial membranes [69]The activities of bothGSH carriers were inhibited for a decrease in the membranefluidity In agreement with these facts we not only observeda decrease in mGSH in the Cho-supplemented rats but also astrong negative correlation between mGSH and the ChoPiratio in mitochondrial membranes (Figure 10) Cu supple-mentation also produced an increase in the ChoPi ratiothough not sufficiently to significantly modify the membranefluidity leaving it unable to modify the mGSH levels

Depletion of the main antioxidant defense system inmitochondria is directly linked to increased lipid peroxida-tion A pro-oxidative environment clearly promotes the oxi-dation of CL and also stimulates its regeneration or remodel-ing activity by increasing the intermediary metabolites lyso-CL (mono- and dilyso-CL in cortex and hippocampus) Inline with this we found higher LPOO and conjugated dieneproduction in isolated CL subfractions after Cu additionthat was even more pronounced after cosupplementationwith Cho (CuCho) Peroxidized CL decreases the pool ofCL available in mitochondria thus preventing the formationof the transition pore and the subsequent leakage of Cyt Cto the cytosol Moreover oxidized CL was able to producemitochondrial dysfunction preventing electron transportthrough the protein complexes [28 68] Our finding of lowermitochondrial CL content after Cu treatment in both brainszones is in agreement with previous observations of Yurkovaet al [71] They reported that Cu ions possess the abilityto fragment CL with the subsequent formation of phospha-tidic acid and phosphatidyl hydroxyacetone in mitochondriafrom a mouse model of Wilsonrsquos disease Interestingly Chosupplementation produced an increase in CL levels prob-ably through an ROS-dependent overstimulation of acyl-transferase-1 activity [72]The fact that no significant changeswith respect to control rats were observed under CuChotreatment could be explained in terms of a compensatory

mechanism to counteract the opposite effects produced byCuand Cho supplementations

Cho enrichment of mitochondrial membranes has otherimportant consequences Alterations in the physical proper-ties of mitochondrial membranes such as those revealed bythe response to DPH and TMA-DPH probes could modifymitochondrial dynamics which are critical for the main-tenance of mitochondrial integrity and functions includingenergy production ROS generation and apoptosis regulation[26ndash31] Modifications in mitochondrial dynamics lead tostructural changes in cristae formation and the assembly ofelectron transport complexes compromising bioenergeticsand causing calcium dyshomeostasis increased oxidativestress (consumption ofmGSH)mitochondrial DNAdamageand synaptic dysfunction [28ndash30]

We have mentioned that the pro-oxidative conditioninduces CL peroxidation and in consequence diminishes CLrsquosability join to proteins like Cyt C Montero et al [73] andEckmann et al [74] emphasized that even though only 15of the Cyt C is strongly bound to CL the oxidation of minoramounts of this phospholipid could trigger the mobilizationof the Cyt C from the mitochondrial inner membrane tothe intermembrane space and then to the cytosol Howeverwe found no significant changes in Cyt C release to thecytoplasm after any treatments except in the case of CuChoaddition to the cortexThere were also no significant changesin the BaxBcL-2 ratio and in caspase-3 activity indicatingthat the intrinsic apoptotic pathway was still not significantlyactivatedThe pro-oxidative conditions likely induced by Cutogether with the enrichment of cholesterol that reducesmGSH levels produce CL peroxidation however it appearsthat the level of damage is insufficient to produce a signif-icant stimulation of apoptosome formation and subsequentactivation of the caspase-3 pathway [73] Our lab is nowstudying the effect of a more prolonged time of exposure inorder to investigate the chronic effects of these experimentaltreatments


Lu et al [24] reported that Cu is associated with A120573in senile plaques and that this complex can recruit Chomolecules oxidizing them and producing even more ROSDespite the clear activation of the proapoptotic cascade wefound an increase in A120573 formation in the brain a conditionwhich is a recognized biomarker of neurodegenerationThesechanges in cortex and hippocampus were not reflected inperipheral plasma most likely because the latter is lesssensitive or the duration of the treatment was not sufficient toaffect the proportion between A120573(1ndash42)(1ndash40) in peripheralplasma or both Oxidative stress induces A120573 accumulationin mitochondrial membranes causing structural and func-tional damage Amyloid-120573 interacts with the mitochondrialprotein A120573-binding alcohol dehydrogenase (ABAD) whichis upregulated in the temporal lobe of AD patients as wellas in A120573PP transgenic mice [28] This complex preventsthe binding of nicotinamide adenine dinucleotide to ABADthereby changing mitochondrial membrane permeability (aswe observed in our model) and reducing the activities ofrespiratory enzymes triggering elevated ROS production[28ndash30] Increased A120573 production and A120573-ROS-dependentformation are linked to other important issues such asthe inactivation of the presequence protease (PreP) one ofthe most important proteins involved in A120573 degradationalteration of mitochondrial dynamics (previously discussed)increased nitrosative stress activation of cyclophilin D (anintegral part of the permeability transition pore or mPTP)potentiation of synaptic failure and cytoskeletal aberrationsamong others [26ndash31] All these facts indicate the importanceof monitoring the level of A120573 production not only at the local(SNC) but also at the systemic (peripheral plasma) level

5 Conclusions

Our work demonstrates that dietary supplementation withtrace amounts of inorganic Cu in drinking water + Chosupplementation in solid food causes oxidative stress in braincortex and hippocampus by depletion of mGSH in an inverseChoPi-dependent fashion CL peroxidation and stimulationof the remodeling route major alterations in mitochondrialmembrane integrity and fluidity and a higher A120573(1ndash42)(1ndash40) ratio as a biomarker of neurodegenerationThese changesmay be additive and could leadmdashunder more prolongedexposuremdashto activation of the proapoptotic cascade On-going research will shed further light on the real significanceof the present results and establish the intimate mechanismsunderlying the damage induced by these two very commonfactors (Cu and Cho overload) with a view to definingpotential preventive strategies in human populations at risk

Abbreviations

Cu CopperCL CardiolipinCP CeruloplasminCho CholesterolLPOO LipoperoxidesmGSH Mitochondrial glutathioneROS Reactive oxygen species

Conflict of Interests

The authors declare that there is no conflict of interests

Disclosure

All authors disclose any financial and personal relationshipswith other people or organisations that could inappropriatelyinfluence this work

Acknowledgments

This study was supported by a Grant from Consejo Nacionalde Investigaciones Cientıficas y Tecnicas (CCT-CONICET)PIP no 0697 and UNLPM-145 The authors would like tothank Ms Norma Cristalli Ms Cristina Pallanza and MsEva Illara de Bozzolo for their excellent technical assistance

References

[1] H Tapiero D M Townsend and K D Tew ldquoTrace elementsin human physiology and pathology Copperrdquo Biomedicine andPharmacotherapy vol 57 no 9 pp 386ndash398 2003

[2] C G Fraga ldquoRelevance essentiality and toxicity of traceelements in human healthrdquo Molecular Aspects of Medicine vol26 no 4-5 pp 235ndash244 2005

[3] V Vassiliev Z L Harris and P Zatta ldquoCeruloplasmin inneurodegenerative diseasesrdquoBrainResearchReviews vol 49 no3 pp 633ndash640 2005

[4] C Gocmen B Giesselman and W C de Groat ldquoEffect ofneocuproine a copper (I) chelator on rat bladder functionrdquoJournal of Pharmacology and Experimental Therapeutics vol312 no 3 pp 1138ndash1143 2005

[5] M E Letelier A M Lepe M Faundez et al ldquoPossiblemechanisms underlying copper-induced damage in biologicalmembranes leading to cellular toxicityrdquo Chemico-BiologicalInteractions vol 151 no 2 pp 71ndash82 2005

[6] N Arnal M J de Alaniz and C A Marra ldquoCytotoxic effectsof copper overload on human-derived lung and liver cells inculturerdquo Biochimica et Biophysica Acta vol 1820 pp 931ndash9392012

[7] N Arnal M J de Alaniz and C A Marra ldquoEffect of copperoverload on the survival of HepG2 and A-549 human-derivedcellsrdquo Human amp Experimental Toxicology vol 32 pp 299ndash3152013

[8] R Newhook H Hirtle K Byrne and M E Meek ldquoReleasesfrom copper smelters and refineries and zinc plants in Canadahuman health exposure and risk characterizationrdquo Science of theTotal Environment vol 301 no 1ndash3 pp 23ndash41 2003

[9] N Arnal M Astiz M J T de Alaniz and C AMarra ldquoClinicalparameters and biomarkers of oxidative stress in agriculturalworkers who applied copper-based pesticidesrdquo Ecotoxicologyand Environmental Safety vol 74 no 6 pp 1779ndash1786 2011

[10] G J Brewer ldquoCopper toxicity in Alzheimerrsquos disease cognitiveloss from ingestion of inorganiccopperrdquo Journal of Trace Ele-ments in Medicine and Biology vol 26 pp 89ndash92 2012

[11] F Y Leung ldquoTrace elements in parenteral micronutritionrdquoClinical Biochemistry vol 28 no 6 pp 561ndash566 1995

[12] G Hardy AMMenendez andWManzanares ldquoTrace elementsupplementation in parenteral nutrition pharmacy posology


andmonitoring guidancerdquoNutrition vol 25 no 11-12 pp 1073ndash1084 2009

[13] N Arnal M J T de Alaniz and C A Marra ldquoAlterations incopper homeostasis and oxidative stress biomarkers in womenusing the intrauterine device TCu380Ardquo Toxicology Letters vol192 no 3 pp 373ndash378 2010

[14] D de la Cruz A Cruz M Arteaga L Castillo and H TovalinldquoBlood copper levels in Mexican users of the T380A IUDrdquoContraception vol 72 no 2 pp 122ndash125 2005

[15] R Squitti G Barbati L Rossi et al ldquoExcess of noncerulo-plasmin serum copper in AD correlates with MMSE CSF 120573-amyloid and h-taurdquo Neurology vol 67 no 1 pp 76ndash82 2006

[16] N Arnal D O Cristalli M J T de Alaniz and C A MarraldquoClinical utility of copper ceruloplasmin and metallothioneinplasma determinations in human neurodegenerative patientsand their first-degree relativesrdquo Brain Research vol 1319 pp118ndash130 2010

[17] R Squitti P Pasqualetti G Dal Forno et al ldquoExcess of serumcopper not related to ceruloplasmin in Alzheimer diseaserdquoNeurology vol 64 no 6 pp 1040ndash1046 2005

[18] R Squitti F Bressi P Pasqualetti et al ldquoLongitudinal prog-nostic value of serum ldquofreerdquo copper in patients with Alzheimerdiseaserdquo Neurology vol 72 no 1 pp 50ndash55 2009

[19] G J Brewer ldquoIssues raised involving the copper hypotheses inthe causation of Alzheimerrsquos diseaserdquo International Journal ofAlzheimerrsquos Disease vol 2011 Article ID 537528 11 pages 2011

[20] M A Pappolla M A Smith T Bryant-Thomas et al ldquoCholes-terol oxidative stress and Alzheimerrsquos disease expanding thehorizons of pathogenesisrdquo Free Radical Biology and Medicinevol 33 no 2 pp 173ndash181 2002

[21] I-L Notkola R Sulkava J Pekkanen et al ldquoSerum totalcholesterol apolipoprotein E 1205764 allele and Alzheimerrsquos diseaserdquoNeuroepidemiology vol 17 no 1 pp 14ndash20 1998

[22] D L Sparks and B G Schreurs ldquoTrace amounts of copperin water induce 120573-amyloid plaques and learning deficits in arabbitmodel of Alzheimerrsquos diseaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 100 no19 pp 11065ndash11069 2003

[23] J Lu Y-L Zheng D-M Wu D-X Sun Q Shan and S-HFan ldquoTrace amounts of copper induce neurotoxicity in thecholesterol-fed mice through apoptosisrdquo FEBS Letters vol 580no 28-29 pp 6730ndash6740 2006

[24] J Lu D-M Wu Y-L Zheng et al ldquoTrace amounts ofcopper exacerbate beta amyloid-induced neurotoxicity in thecholesterol-fed mice through TNF-mediated inflammatorypathwayrdquo Brain Behavior and Immunity vol 23 no 2 pp 193ndash203 2009

[25] N Arnal L Dominici M J de Alaniz and C A MarraldquoCopper-induced alterations in rat brain depends on the routeof overload and basal copper levelsrdquo Nutrition In press

[26] D J Bonda X Wang G Perry et al ldquoOxidative stress inAlzheimer disease a possibility for preventionrdquo Neuropharma-cology vol 59 no 4-5 pp 290ndash294 2010

[27] S Pope J M Land and S J R Heales ldquoOxidative stress andmitochondrial dysfunction in neurodegeneration cardiolipin acritical targetrdquo Biochimica et Biophysica Acta vol 1777 no 7-8pp 794ndash799 2008

[28] J Yao R W Irwin L Zhao J Nilsen R T Hamilton andR D Brinton ldquoMitochondrial bioenergetic deficit precedesAlzheimerrsquos pathology in female mouse model of Alzheimerrsquosdiseaserdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 106 no 34 pp 14670ndash14675 2009

[29] V Garcıa-Escudero P Martın-Maestro G Perry and J AvilaldquoDeconstructing mitochondrial dysfunction in Alzheimer dis-easerdquo Oxidative Medicine and Cellular Longevity vol 2013Article ID 162152 13 pages 2013

[30] G Paradies G Petrosillo M Pistolese N di Venosa A Fed-erici and F M Ruggiero ldquoDecrease in mitochondrial complexI activity in ischemicreperfused rat heart involvement ofreactive oxygen species and cardiolipinrdquo Circulation Researchvol 94 no 1 pp 53ndash59 2004

[31] S L Iverson and S Orrenius ldquoThe cardiolipin-cytochromec interaction and the mitochondrial regulation of apoptosisrdquoArchives of Biochemistry and Biophysics vol 423 no 1 pp 37ndash46 2004

[32] P G Reeves F H Nielsen and G C Fahey Jr ldquoAIN-93 purifieddiets for laboratory rodents final report of the American insti-tute of nutrition ad hoc writing committee on the reformulationof the AIN-76A rodent dietrdquo Journal of Nutrition vol 123 no11 pp 1939ndash1951 1993

[33] National Institute of Health Guide for the Care and Use ofLaboratoryAnimals no 85-23 (rev)National ResearchCouncilBethesda Md USA 1985

[34] K A Cockell A T L Wotherspoon B Belonje et al ldquoLimitedeffects of combined dietary copper deficiencyiron overload onoxidative stress parameters in rat liver and plasmardquo Journal ofNutritional Biochemistry vol 16 no 12 pp 750ndash756 2005

[35] C D Davis and S Newman ldquoInadequate dietary copperincreases tumorigenesis in the Min mouserdquo Cancer Letters vol159 no 1 pp 57ndash62 2000

[36] G Paxinos and C WatsonThe Rat Brain in Stereotaxic Coordi-nates Academic Press 4th edition 1998

[37] M Berkovitch E Heyman R Afriat et al ldquoCopper and zincblood levels among children with nonorganic failure to thriverdquoClinical Nutrition vol 22 no 2 pp 183ndash186 2003

[38] C Terres-Martos M Navarro-Alarcon F Martın-Lagos et alldquoDetermination of copper levels in serum of healthy subjects byatomic absorption spectrometryrdquo Science of the Total Environ-ment vol 198 pp 97ndash103 1997

[39] S Martınez-Subiela F Tecles and J J Ceron ldquoComparison oftwo automated spectrophotometric methods for ceruloplasminmeasurement in pigsrdquoResearch inVeterinary Science vol 83 no1 pp 12ndash19 2007

[40] P J Twomey A S Wierzbicki I M House A Viljoen and TM Reynolds ldquoPercentage non-caeruloplasmin bound copperrdquoClinical Biochemistry vol 40 no 9-10 pp 749ndash750 2007

[41] J Folch M Lees and G H Sloane Stanley ldquoA simple methodfor the isolation and purification of total lipides from animaltissuesrdquo The Journal of Biological Chemistry vol 226 no 1 pp497ndash509 1957

[42] P S Chen Jr T Y Toribara and H Warner ldquoMicrodetermi-nation of phosphorusrdquo Analytical Chemistry vol 28 no 11 pp1756ndash1758 1956

[43] F Fraser andVA Zammit ldquoEnrichment of carnitine palmitoyl-transferases I and II in the contact sites of rat liver mitochon-driardquo Biochemical Journal vol 329 no 2 pp 225ndash229 1998

[44] M Pellon-Maison M A Montanaro R A Coleman and MR Gonzalez-Baro ldquoMitochondrial glycerol-3-P acyltransferase1 is most active in outer mitochondrial membrane but notin mitochondrial associated vesicles (MAV)rdquo Biochimica etBiophysica Acta vol 1771 no 7 pp 830ndash838 2007


[45] F G Prendergast R P Haugland and P J Callahan ldquo1-[4-(Tri-methylamino)phenyl]-6-phenylhexa-135-triene synthe-sis fluorescence properties and use as a fluorescence probe oflipid bilayersrdquo Biochemistry vol 20 no 26 pp 7333ndash7338 1981

[46] N Masumoto K Tasaka J Mizuki M Tahara A Miyakeand O Tanizawa ldquoDynamics of exocytosis endocytosis andrecycling in single pituitary gonadotropesrdquo Biochemical andBiophysical Research Communications vol 197 no 1 pp 207ndash213 1993

[47] B R Lentz ldquoUse of fluorescent probes to monitor molecularorder and motions within liposome bilayersrdquo Chemistry andPhysics of Lipids vol 64 no 1ndash3 pp 99ndash116 1993

[48] G P Eckert J-H Keller C Jourdan et al ldquoHyperforinmodifiesneuronal membrane properties in vivordquo Neuroscience Lettersvol 367 no 2 pp 139ndash143 2004

[49] A R Andrade Pires G Rodrıgues Noleto A Echevarrıa et alldquoInteraction of 134-thiadiazolium mesoionic derivatives withmitochondrial membrane and scavenging activity involvementof their effects on mitochondrial energy-linked functionsrdquoChemico-Biological Interactions vol 189 pp 17ndash25 2011

[50] M Shinitzky and Y Barenholz ldquoFluidity parameters of lipidregions determined by fluorescence polarizationrdquo Biochimica etBiophysica Acta vol 515 no 4 pp 367ndash394 1978

[51] M E Anderson and A Meister ldquoEnzymic assay of GSSG plusGSHrdquoMethods in Enzymology vol 105 pp 448ndash450 1984

[52] J Nourooz-Zadeh J Tajaddini-Sarmadi S McCarthy D JBetteridge and S P Wolff ldquoElevated levels of authentic plasmahydroperoxides in NIDDMrdquo Diabetes vol 44 no 9 pp 1054ndash1058 1995

[53] R O Recknagel and E A Glende Jr ldquoSpectrophotometricdetection of lipid conjugated dienesrdquo Methods in Enzymologyvol 105 pp 331ndash337 1984

[54] G di Paolo and T-W Kim ldquoLinking lipids to Alzheimerrsquosdisease cholesterol and beyondrdquo Nature Reviews Neurosciencevol 12 no 5 pp 284ndash296 2011

[55] M Stefani and G Liguri ldquoColesterol in Alzheimerrsquos diseaseunresolved questionsrdquo Current Alzheimer Research vol 6 pp1ndash17 2009

[56] B Schereurs ldquoThe effects of cholesterol on learning and mem-oryrdquo Neuroscience amp Biobehavioral Reviews vol 34 pp 1366ndash1379 2010

[57] M C Morris D A Evans C C Tangney et al ldquoDietarycopper and high saturated and trans fat intakes associated withcognitive declinerdquoArchives of Neurology vol 63 no 8 pp 1085ndash1088 2006

[58] K A Cockell J Bertinato and M R LrsquoAbbe ldquoRegulatoryframeworks for copper considering chronic exposures of thepopulationrdquoThe American Journal of Clinical Nutrition vol 88pp 863Sndash866S 2008

[59] M Costa O Cantoni M de Mars and D E SwartzendruberldquoToxic metals produce an S-phase-specific cell cycle blockrdquoResearch Communications in Chemical Pathology and Pharma-cology vol 38 no 3 pp 405ndash419 1982

[60] N Arnal M J de Alaniz and C A Marra ldquoInvolvementof copper overload in human diseasesrdquo in Metals in BiologySystems M S Gimenez Ed Research Signpost Kerala India2010

[61] J A Cuthbert ldquoWilsonrsquos disease a new gene and an animalmodel for an old diseaserdquo Journal of Investigative Medicine vol43 no 4 pp 323ndash336 1995

[62] T Planella M Cortes C Martınez-Bru et al ldquoCalculation ofLDL-cholesterol by using apolipoprotein B for classification ofnonchylomicronemicdyslipemiardquo Clinical Chemistry vol 43pp 808ndash815 1997

[63] H R Koelz B C Sherrill S D Turley and J M DietschyldquoCorrelation of low and high density lipoprotein binding in vivowith rates of lipoprotein degradation in the rat A comparison oflipoproteins of rat and human originrdquoThe Journal of BiologicalChemistry vol 257 no 14 pp 8061ndash8072 1982

[64] N Arnal G Morel M J de Alaniz L Dominici and C AMarra ldquoRole of copper and cholesterol association in neu-rodegenerative processrdquoThe International Journal of AlzheimerrsquosDisease vol 2013 Article ID 414817 15 pages 2013

[65] D Das N Tapryal S K Goswami P L Fox and C KMukhopadhyay ldquoRegulation of ceruloplasmin in human hep-atic cells by redox active copper identification of a novel AP-1site in the ceruloplasmin generdquo Biochemical Journal vol 402no 1 pp 135ndash141 2007

[66] M Marı A Morales A Colell C Garcıa-Ruiz and J CFernandez-Checa ldquoMitochondrial glutathione a key survivalantioxidantrdquo Antioxidants and Redox Signaling vol 11 no 11pp 2685ndash2700 2009

[67] M C Vazquez E Balboa A R Alvarez and S ZanlungoldquoOxidative stress a pathogenic mechanism for Niemann-Picktype C diseaserdquo Oxidative Medicine and Cellular Longevity vol2012 Article ID 205713 11 pages 2012

[68] M Jimenez-Del-Rio and C Velez-Pardo ldquoThe bad the goodand the ugly about oxidative stressrdquo Oxidative Medicine andCellular Longevity vol 2012 Article ID 163913 13 pages 2012

[69] O Coll A Colell C Garcıa-Ruiz N Kaplowitz and J CFernandez-Checa ldquoSensitivity of the 2-oxoglutarate carrierto alcohol intake contributes to mitochondrial glutathionedepletionrdquo Hepatology vol 38 no 3 pp 692ndash702 2003

[70] K D Hauff and G M Hatch ldquoCardiolipin metabolism andBarth Syndromerdquo Progress in Lipid Research vol 45 no 2 pp91ndash101 2006

[71] I L Yurkova J Arnhold G Fitzl and D Huster ldquoFragmenta-tion of mitochondrial cardiolipin by copper ions in the Atp7b-- mouse model of Wilsonrsquos diseaserdquo Chemistry and Physics ofLipids vol 164 no 5 pp 393ndash400 2011

[72] Y Y Tyurina M A Tungekar M-Y Jung et al ldquoMitochondriatargeting of non-peroxidizable triphenylphosphonium conju-gated oleic acid protects mouse embryonic cells against apop-tosis role of cardiolipin remodelingrdquo FEBS Letters vol 586 no3 pp 235ndash241 2012

[73] J Montero M Mari A Colell et al ldquoCholesterol and per-oxidized cardiolipin in mitochondrial membrane propertiespermeabilization and cell deathrdquo Biochimica et Biophysica Actavol 1797 no 6-7 pp 1217ndash1224 2010

[74] J Eckmann S H Eckert K Leuner et al ldquoMitochondria mito-chondrial membranes in brain ageing and neurodegenerationrdquoThe International Journal of Biochemistry amp Cell Biology vol 45pp 76ndash80 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


mice In addition Notkola et al [21] found that plasmacholesterol (Cho) levels were predictors of AD prevalence ina population-based sample of 444 men (aged 70ndash89 years)More recently in vivo models of AD-like neurodegenerativediseases demonstrated that the association of Cu and Choincreases the risk of the development of neurodegenerativedamage [22ndash24]

Cu overload has been extensively associated with ROSoverproduction in vitro and in vivo [6 7 25] Oxidative stressis considered a primary event in the development of AD [26]Pope et al [27] reported that ROS overproduction due tothe dysfunction of the electron transport chain is causativeof neurodegeneration associated with AD and ParkinsonrsquosdiseaseMoreover some experimental evidence indicates thatalterations in mitochondrial energetics occur along with theaccumulation of oxidative damage before the cardinal signsof AD pathogenesis in the brains of transgenic animalmodelsand human patients [28 29]

As reviewed by Paradies et al [30] the mitochondrionis considered the most important cellular organelle con-tributing to the neurodegenerative process mainly throughrespiratory chain dysfunction and the formation of reactiveoxygen species Factors that increase the rate of mitochon-drial ROS production lead to mitochondrial dysfunctionbecause of the oxidative-induced damage caused to vari-ous crucial biomolecules such as mtDNA lipids and pro-teins thus increasing mitochondrial deterioration in a self-perpetuating process [31] Together with ROS accumulationmitochondrial dysfunction is one of the earliest and mostprominent features of AD [27 28] and this condition isstrongly associated with multiple negative consequencesDysfunctional mitochondria generate high levels of ROS thatare ultimately toxic to cells particularly those with a longlifespan and low antioxidant defense system such as neurons[28 29] At the same timemitochondria are also targets of theROS produced by pro-oxidative factors such as Cu overloador increased levels of NCBC

Among the mitochondrial lipids directly affected byROS overproduction cardiolipin (CL) is one of the mostdeeply involved in both synapses and neuronal loss [31]CL is linked to the endogenous proapoptotic cascade thateventually leads to the integration of the apoptosome and thesubsequent activation of the effector caspase system [28ndash31]Moreover CL is involved in the regulation of the bioenergeticprocess and in the integrity of mitochondrial membranesPeroxidation of CL directly affects the biochemical functionsthat depend on the inner and outermitochondrialmembranecomposition and structure Experimental evidence clearlyindicates that this lipid represents the most important targetof ROS attack [30]

In summary there is conclusive evidence linking ROShyperproduction with mitochondrial dysfunction Oxidativestress is also directly involved in neurodegeneration andCu overload in association with cholesterol apparently actsas a synergistic risk factor in the etiology of neurologicaldisorders All the foregoing evidence suggests the need formore in-depth study of this phenomenon In view of theabsence of conclusive studies on the biochemical effects

of the Cu + Cho (CuCho) association on mitochondrial-associated dysfunction especially in relation to those aspectsinvolved in programmed cell death we aimed to investigatethe effects of CuCho on mitochondria isolated from cortexand hippocampus of Wistar rats and explore (i) the ChoPiratio and steady-state fluorescence anisotropy of the outerand inner mitochondrial membranes in order to assess theirfluidities (ii) mitochondrial membrane integrity by meansof a probe that measures trans-membrane potential (iii)the concentration of the main mitochondrial antioxidantglutathione (iv) the concentration of the key mitochondrialphospholipid cardiolipin and the possible pro-oxidativedamage it causes (v) the activity of the apoptotic pathwaydepending on mitochondrial membrane integrity (caspase-3 and the BaxBcl-2 ratio) and (vi) the concentration of 120573-amyloid (A120573) in the two brain zones and in peripheral plasmaas a biomarker of a proneurodegenerative effect

2 Materials and Methods

21 Chemicals All chemicals used were of analytical gradeand obtained from Sigma Chem Co (Buenos AiresArgentina or USA)Merck (Darmstadt Germany) and CarloErba (Milan Italy)

22 Animals and Treatments Certified pathogen-free maleWistar rats were used The rats were maintained at a con-trolled temperature (25∘C) and relative humidity of 60with forced ventilation under a normal photoperiod of12 h darkness and 12 h light The health of the animalswas monitored in accordance with the internationally rec-ommended practices of the Institute of Laboratory AnimalResources Commission of Life Sciences National ResearchCouncil (ILAR) Solid food and drinkingwater were providedad libitum The diets for the experiments were prepared inour laboratory according to the recommendations for Wistarrats [32] All procedures for handling the animals followedthe NIH regulations [33] The experimental protocol wasreviewed and approved by the Bioethics Committee of theFaculty of Medical Sciences UNLP (number 0038211)

23 Experimental Protocols Rats (21 days old)were randomlyassigned (ten animals per group) to the protocols detailed asfollows and treated for eight weeks (2months) Selecting veryyoung rats lets us discard any neurodegenerative (unknown)event(s) associated to the age of the animal By the way thisapproach may be extrapolated to humans that are exposedto copper from the very beginning of their life In additionin previous experiments (not shown) we noted thatmdashat lestduring the first year of lifemdashthat we have no differencesin the response of the model as a function of the startingage of treatment The control group (C) was maintainedon lab-prepared pellets as recommended for normal growthcontaining 7 ppm of Cu [34 35] The Cu-supplementedexperimental group (Cu) was fed on control pellets and tapwater supplemented with 3mgL (or ppm) of Cu in the formof ultrapure CuSO

4(Merck Darmstadt Germany)TheCho-

supplemented group (Cho) was fed on pellets containing





3(c) and




3)2in HNO

305N (Titrisol from


3HClO






















3 Results








aa

a a a a aa

b b

b b

NCB

C (n

gm

g pr

otei

n)

25

20

15

10

05


ControlCu

ChoCuCho

(a)

a a

a aa a

b b

bbc c


CP (n

gm

g pr

otei

n)

60

50

40

30

20

10

0

ControlCu

ChoCuCho

(b)




ControlCu

ChoCuCho

aa

b b

cc

cc

20

15

10

05


[Cho

Pi]middot103






uore

scen

ce ra

tiom

g pr

otei

n

0

25

50

75

100

ab c

c

a a a

b

ControlCu

ChoCuCho

Cortex Hippocampus






Cortex0

25

50

75

100

ControlCu

ChoCuCho

aa

b ba

a

bc

Hippocampus

mG

SH (120583

gm

gmiddotpr

ot)middot10minus3










4 Discussion



OO

(120583m

oles120583

mol

es C

L)

0

2

4

6

a

a

ab

b

c

c

d

Cortex

ControlCu

ChoCuCho

Hippocampus

(a)

0

5

10

15

20

25

30

a

d

c

b

a

b

c

a

Cortex

ControlCu

ChoCuCho

Hippocampus

UD

Omiddot103

(mg

CL)

(b)




0

40

80

120

a

b

ba

a

b

ab

ControlCu

ChoCuCho

Cortex Hippocampus

CL (120583

mol

Pim

g pr

otei

n)

(a)

0

4

8

12

a

bc

d

b

a

b

c

ControlCu

ChoCuCho

Cortex Hippocampus

L-CL

(120583m

oles

Pim

g m

itoch

ondr

ial p

rot)

(b)





uore

scen

ce an

isotro

py

00

02

04

06

08

TMA-DPHDPH

aa

b

a

b

a

b b

C Cu Cho CuCho

(a) Cortex (OMM)

Fluo

resc

ence

aniso

tropy

00

02

04

06

08

a a

b

a

a b

b

b

TMA-DPHDPH

C Cu Cho CuCho

(b) Cortex (IMM)



Fluo

resc

ence

aniso

tropy

00

02

04

06

08

aa

a a

bb

bb

TMA-DPHDPH

C Cu Cho CuCho


Fluo

resc

ence

aniso

tropy

00

02

04

06

08

aa

aa

b

b

bb

TMA-DPHDPH

C Cu Cho CuCho





0

1

2

3

ControlCu

ChoCuCho

Cortex Hippocampus

Bax

Bcl-2








05 06 07 08 14 16 18200

400

600

800

1000m

GSH

[120583m

olesm

g pr

ot]

Cortex

+Cho (r2 = 096)

minusCho (r2 = 092)

Cho middot 103Pi

(a)

02 04 06 08 10 12 14 160

200

400

600

800

mG

SH[120583

mol

esm

g pr

ot]

Hippocampus

+Cho (r2 = 098)

minusCho (r2 = 091)

Cho middot 103Pi









5 Conclusions


Abbreviations




Disclosure


Acknowledgments


References



















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3(c) and




3)2in HNO

305N (Titrisol from


3HClO






















3 Results








aa

a a a a aa

b b

b b

NCB

C (n

gm

g pr

otei

n)

25

20

15

10

05


ControlCu

ChoCuCho

(a)

a a

a aa a

b b

bbc c


CP (n

gm

g pr

otei

n)

60

50

40

30

20

10

0

ControlCu

ChoCuCho

(b)




ControlCu

ChoCuCho

aa

b b

cc

cc

20

15

10

05


[Cho

Pi]middot103






uore

scen

ce ra

tiom

g pr

otei

n

0

25

50

75

100

ab c

c

a a a

b

ControlCu

ChoCuCho

Cortex Hippocampus






Cortex0

25

50

75

100

ControlCu

ChoCuCho

aa

b ba

a

bc

Hippocampus

mG

SH (120583

gm

gmiddotpr

ot)middot10minus3










4 Discussion



OO

(120583m

oles120583

mol

es C

L)

0

2

4

6

a

a

ab

b

c

c

d

Cortex

ControlCu

ChoCuCho

Hippocampus

(a)

0

5

10

15

20

25

30

a

d

c

b

a

b

c

a

Cortex

ControlCu

ChoCuCho

Hippocampus

UD

Omiddot103

(mg

CL)

(b)




0

40

80

120

a

b

ba

a

b

ab

ControlCu

ChoCuCho

Cortex Hippocampus

CL (120583

mol

Pim

g pr

otei

n)

(a)

0

4

8

12

a

bc

d

b

a

b

c

ControlCu

ChoCuCho

Cortex Hippocampus

L-CL

(120583m

oles

Pim

g m

itoch

ondr

ial p

rot)

(b)





uore

scen

ce an

isotro

py

00

02

04

06

08

TMA-DPHDPH

aa

b

a

b

a

b b

C Cu Cho CuCho

(a) Cortex (OMM)

Fluo

resc

ence

aniso

tropy

00

02

04

06

08

a a

b

a

a b

b

b

TMA-DPHDPH

C Cu Cho CuCho

(b) Cortex (IMM)



Fluo

resc

ence

aniso

tropy

00

02

04

06

08

aa

a a

bb

bb

TMA-DPHDPH

C Cu Cho CuCho


Fluo

resc

ence

aniso

tropy

00

02

04

06

08

aa

aa

b

b

bb

TMA-DPHDPH

C Cu Cho CuCho





0

1

2

3

ControlCu

ChoCuCho

Cortex Hippocampus

Bax

Bcl-2








05 06 07 08 14 16 18200

400

600

800

1000m

GSH

[120583m

olesm

g pr

ot]

Cortex

+Cho (r2 = 096)

minusCho (r2 = 092)

Cho middot 103Pi

(a)

02 04 06 08 10 12 14 160

200

400

600

800

mG

SH[120583

mol

esm

g pr

ot]

Hippocampus

+Cho (r2 = 098)

minusCho (r2 = 091)

Cho middot 103Pi









5 Conclusions


Abbreviations




Disclosure


Acknowledgments


References



















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































3 Results








aa

a a a a aa

b b

b b

NCB

C (n

gm

g pr

otei

n)

25

20

15

10

05


ControlCu

ChoCuCho

(a)

a a

a aa a

b b

bbc c


CP (n

gm

g pr

otei

n)

60

50

40

30

20

10

0

ControlCu

ChoCuCho

(b)




ControlCu

ChoCuCho

aa

b b

cc

cc

20

15

10

05


[Cho

Pi]middot103






uore

scen

ce ra

tiom

g pr

otei

n

0

25

50

75

100

ab c

c

a a a

b

ControlCu

ChoCuCho

Cortex Hippocampus






Cortex0

25

50

75

100

ControlCu

ChoCuCho

aa

b ba

a

bc

Hippocampus

mG

SH (120583

gm

gmiddotpr

ot)middot10minus3










4 Discussion



OO

(120583m

oles120583

mol

es C

L)

0

2

4

6

a

a

ab

b

c

c

d

Cortex

ControlCu

ChoCuCho

Hippocampus

(a)

0

5

10

15

20

25

30

a

d

c

b

a

b

c

a

Cortex

ControlCu

ChoCuCho

Hippocampus

UD

Omiddot103

(mg

CL)

(b)




0

40

80

120

a

b

ba

a

b

ab

ControlCu

ChoCuCho

Cortex Hippocampus

CL (120583

mol

Pim

g pr

otei

n)

(a)

0

4

8

12

a

bc

d

b

a

b

c

ControlCu

ChoCuCho

Cortex Hippocampus

L-CL

(120583m

oles

Pim

g m

itoch

ondr

ial p

rot)

(b)





uore

scen

ce an

isotro

py

00

02

04

06

08

TMA-DPHDPH

aa

b

a

b

a

b b

C Cu Cho CuCho

(a) Cortex (OMM)

Fluo

resc

ence

aniso

tropy

00

02

04

06

08

a a

b

a

a b

b

b

TMA-DPHDPH

C Cu Cho CuCho

(b) Cortex (IMM)



Fluo

resc

ence

aniso

tropy

00

02

04

06

08

aa

a a

bb

bb

TMA-DPHDPH

C Cu Cho CuCho


Fluo

resc

ence

aniso

tropy

00

02

04

06

08

aa

aa

b

b

bb

TMA-DPHDPH

C Cu Cho CuCho





0

1

2

3

ControlCu

ChoCuCho

Cortex Hippocampus

Bax

Bcl-2








05 06 07 08 14 16 18200

400

600

800

1000m

GSH

[120583m

olesm

g pr

ot]

Cortex

+Cho (r2 = 096)

minusCho (r2 = 092)

Cho middot 103Pi

(a)

02 04 06 08 10 12 14 160

200

400

600

800

mG

SH[120583

mol

esm

g pr

ot]

Hippocampus

+Cho (r2 = 098)

minusCho (r2 = 091)

Cho middot 103Pi









5 Conclusions


Abbreviations




Disclosure


Acknowledgments


References



















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















aa

a a a a aa

b b

b b

NCB

C (n

gm

g pr

otei

n)

25

20

15

10

05


ControlCu

ChoCuCho

(a)

a a

a aa a

b b

bbc c


CP (n

gm

g pr

otei

n)

60

50

40

30

20

10

0

ControlCu

ChoCuCho

(b)




ControlCu

ChoCuCho

aa

b b

cc

cc

20

15

10

05


[Cho

Pi]middot103






uore

scen

ce ra

tiom

g pr

otei

n

0

25

50

75

100

ab c

c

a a a

b

ControlCu

ChoCuCho

Cortex Hippocampus






Cortex0

25

50

75

100

ControlCu

ChoCuCho

aa

b ba

a

bc

Hippocampus

mG

SH (120583

gm

gmiddotpr

ot)middot10minus3










4 Discussion



OO

(120583m

oles120583

mol

es C

L)

0

2

4

6

a

a

ab

b

c

c

d

Cortex

ControlCu

ChoCuCho

Hippocampus

(a)

0

5

10

15

20

25

30

a

d

c

b

a

b

c

a

Cortex

ControlCu

ChoCuCho

Hippocampus

UD

Omiddot103

(mg

CL)

(b)




0

40

80

120

a

b

ba

a

b

ab

ControlCu

ChoCuCho

Cortex Hippocampus

CL (120583

mol

Pim

g pr

otei

n)

(a)

0

4

8

12

a

bc

d

b

a

b

c

ControlCu

ChoCuCho

Cortex Hippocampus

L-CL

(120583m

oles

Pim

g m

itoch

ondr

ial p

rot)

(b)





uore

scen

ce an

isotro

py

00

02

04

06

08

TMA-DPHDPH

aa

b

a

b

a

b b

C Cu Cho CuCho

(a) Cortex (OMM)

Fluo

resc

ence

aniso

tropy

00

02

04

06

08

a a

b

a

a b

b

b

TMA-DPHDPH

C Cu Cho CuCho

(b) Cortex (IMM)



Fluo

resc

ence

aniso

tropy

00

02

04

06

08

aa

a a

bb

bb

TMA-DPHDPH

C Cu Cho CuCho


Fluo

resc

ence

aniso

tropy

00

02

04

06

08

aa

aa

b

b

bb

TMA-DPHDPH

C Cu Cho CuCho





0

1

2

3

ControlCu

ChoCuCho

Cortex Hippocampus

Bax

Bcl-2








05 06 07 08 14 16 18200

400

600

800

1000m

GSH

[120583m

olesm

g pr

ot]

Cortex

+Cho (r2 = 096)

minusCho (r2 = 092)

Cho middot 103Pi

(a)

02 04 06 08 10 12 14 160

200

400

600

800

mG

SH[120583

mol

esm

g pr

ot]

Hippocampus

+Cho (r2 = 098)

minusCho (r2 = 091)

Cho middot 103Pi









5 Conclusions


Abbreviations




Disclosure


Acknowledgments


References



















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















uore

scen

ce ra

tiom

g pr

otei

n

0

25

50

75

100

ab c

c

a a a

b

ControlCu

ChoCuCho

Cortex Hippocampus






Cortex0

25

50

75

100

ControlCu

ChoCuCho

aa

b ba

a

bc

Hippocampus

mG

SH (120583

gm

gmiddotpr

ot)middot10minus3










4 Discussion



OO

(120583m

oles120583

mol

es C

L)

0

2

4

6

a

a

ab

b

c

c

d

Cortex

ControlCu

ChoCuCho

Hippocampus

(a)

0

5

10

15

20

25

30

a

d

c

b

a

b

c

a

Cortex

ControlCu

ChoCuCho

Hippocampus

UD

Omiddot103

(mg

CL)

(b)




0

40

80

120

a

b

ba

a

b

ab

ControlCu

ChoCuCho

Cortex Hippocampus

CL (120583

mol

Pim

g pr

otei

n)

(a)

0

4

8

12

a

bc

d

b

a

b

c

ControlCu

ChoCuCho

Cortex Hippocampus

L-CL

(120583m

oles

Pim

g m

itoch

ondr

ial p

rot)

(b)





uore

scen

ce an

isotro

py

00

02

04

06

08

TMA-DPHDPH

aa

b

a

b

a

b b

C Cu Cho CuCho

(a) Cortex (OMM)

Fluo

resc

ence

aniso

tropy

00

02

04

06

08

a a

b

a

a b

b

b

TMA-DPHDPH

C Cu Cho CuCho

(b) Cortex (IMM)



Fluo

resc

ence

aniso

tropy

00

02

04

06

08

aa

a a

bb

bb

TMA-DPHDPH

C Cu Cho CuCho


Fluo

resc

ence

aniso

tropy

00

02

04

06

08

aa

aa

b

b

bb

TMA-DPHDPH

C Cu Cho CuCho





0

1

2

3

ControlCu

ChoCuCho

Cortex Hippocampus

Bax

Bcl-2








05 06 07 08 14 16 18200

400

600

800

1000m

GSH

[120583m

olesm

g pr

ot]

Cortex

+Cho (r2 = 096)

minusCho (r2 = 092)

Cho middot 103Pi

(a)

02 04 06 08 10 12 14 160

200

400

600

800

mG

SH[120583

mol

esm

g pr

ot]

Hippocampus

+Cho (r2 = 098)

minusCho (r2 = 091)

Cho middot 103Pi









5 Conclusions


Abbreviations




Disclosure


Acknowledgments


References



















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















OO

(120583m

oles120583

mol

es C

L)

0

2

4

6

a

a

ab

b

c

c

d

Cortex

ControlCu

ChoCuCho

Hippocampus

(a)

0

5

10

15

20

25

30

a

d

c

b

a

b

c

a

Cortex

ControlCu

ChoCuCho

Hippocampus

UD

Omiddot103

(mg

CL)

(b)




0

40

80

120

a

b

ba

a

b

ab

ControlCu

ChoCuCho

Cortex Hippocampus

CL (120583

mol

Pim

g pr

otei

n)

(a)

0

4

8

12

a

bc

d

b

a

b

c

ControlCu

ChoCuCho

Cortex Hippocampus

L-CL

(120583m

oles

Pim

g m

itoch

ondr

ial p

rot)

(b)





uore

scen

ce an

isotro

py

00

02

04

06

08

TMA-DPHDPH

aa

b

a

b

a

b b

C Cu Cho CuCho

(a) Cortex (OMM)

Fluo

resc

ence

aniso

tropy

00

02

04

06

08

a a

b

a

a b

b

b

TMA-DPHDPH

C Cu Cho CuCho

(b) Cortex (IMM)



Fluo

resc

ence

aniso

tropy

00

02

04

06

08

aa

a a

bb

bb

TMA-DPHDPH

C Cu Cho CuCho


Fluo

resc

ence

aniso

tropy

00

02

04

06

08

aa

aa

b

b

bb

TMA-DPHDPH

C Cu Cho CuCho





0

1

2

3

ControlCu

ChoCuCho

Cortex Hippocampus

Bax

Bcl-2








05 06 07 08 14 16 18200

400

600

800

1000m

GSH

[120583m

olesm

g pr

ot]

Cortex

+Cho (r2 = 096)

minusCho (r2 = 092)

Cho middot 103Pi

(a)

02 04 06 08 10 12 14 160

200

400

600

800

mG

SH[120583

mol

esm

g pr

ot]

Hippocampus

+Cho (r2 = 098)

minusCho (r2 = 091)

Cho middot 103Pi









5 Conclusions


Abbreviations




Disclosure


Acknowledgments


References



















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















uore

scen

ce an

isotro

py

00

02

04

06

08

TMA-DPHDPH

aa

b

a

b

a

b b

C Cu Cho CuCho

(a) Cortex (OMM)

Fluo

resc

ence

aniso

tropy

00

02

04

06

08

a a

b

a

a b

b

b

TMA-DPHDPH

C Cu Cho CuCho

(b) Cortex (IMM)



Fluo

resc

ence

aniso

tropy

00

02

04

06

08

aa

a a

bb

bb

TMA-DPHDPH

C Cu Cho CuCho


Fluo

resc

ence

aniso

tropy

00

02

04

06

08

aa

aa

b

b

bb

TMA-DPHDPH

C Cu Cho CuCho





0

1

2

3

ControlCu

ChoCuCho

Cortex Hippocampus

Bax

Bcl-2








05 06 07 08 14 16 18200

400

600

800

1000m

GSH

[120583m

olesm

g pr

ot]

Cortex

+Cho (r2 = 096)

minusCho (r2 = 092)

Cho middot 103Pi

(a)

02 04 06 08 10 12 14 160

200

400

600

800

mG

SH[120583

mol

esm

g pr

ot]

Hippocampus

+Cho (r2 = 098)

minusCho (r2 = 091)

Cho middot 103Pi









5 Conclusions


Abbreviations




Disclosure


Acknowledgments


References



















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

ControlCu

ChoCuCho

Cortex Hippocampus

Bax

Bcl-2








05 06 07 08 14 16 18200

400

600

800

1000m

GSH

[120583m

olesm

g pr

ot]

Cortex

+Cho (r2 = 096)

minusCho (r2 = 092)

Cho middot 103Pi

(a)

02 04 06 08 10 12 14 160

200

400

600

800

mG

SH[120583

mol

esm

g pr

ot]

Hippocampus

+Cho (r2 = 098)

minusCho (r2 = 091)

Cho middot 103Pi









5 Conclusions


Abbreviations




Disclosure


Acknowledgments


References



















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















05 06 07 08 14 16 18200

400

600

800

1000m

GSH

[120583m

olesm

g pr

ot]

Cortex

+Cho (r2 = 096)

minusCho (r2 = 092)

Cho middot 103Pi

(a)

02 04 06 08 10 12 14 160

200

400

600

800

mG

SH[120583

mol

esm

g pr

ot]

Hippocampus

+Cho (r2 = 098)

minusCho (r2 = 091)

Cho middot 103Pi









5 Conclusions


Abbreviations




Disclosure


Acknowledgments


References



















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusions


Abbreviations




Disclosure


Acknowledgments


References



















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Effects of Copper and/or Cholesterol Overload on Mitochondrial Function in a Rat Model of Incipient...

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