Copper (Cu) and cholesterol (Cho) are both associated with neurodegenerative illnesses in humans and animals models. We studied the effect in Wistar rats of oral supplementation with trace amounts of Cu (3？ppm) and/or Cho (2%) in drinking water for 2 months. Increased amounts of nonceruloplasmin-bound Cu were observed in plasma and brain hippocampus together with a higher concentration of ceruloplasmin in plasma, cortex, and hippocampus. Cu, Cho, and the combined treatment Cu + Cho were able to induce a higher Cho/phospholipid ratio in mitochondrial membranes with a simultaneous decrease in glutathione content. The concentration of cardiolipin decreased and that of peroxidation products, conjugated dienes and lipoperoxides, increased. Treatments including Cho produced rigidization in both the outer and inner mitochondrial membranes with a simultaneous increase in permeability. No significant increase in Cyt C leakage to the cytosol was observed except in the case of cortex from rats treated with Cu and Cho nor were there any significant changes in caspase-3 activity and the Bax/Bcl2 ratio. However, the Aβ(1–42)/(1–40) ratio was higher in cortex and hippocampus. These findings suggest an incipient neurodegenerative process induced by Cu 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 transition metal for all living organisms, functioning as cofactor for many enzymes [1–3]. However, we and other laboratories have demonstrated in vivo and in vitro that excess inorganic Cu produces increased levels of reactive oxygen species (ROS) and damage to biomolecules, ultimately promoting cell death [4–7]. Humans are continuously at risk from excess Cu due to involuntary exposure to pollution (contaminated water, food), professional activities [8–10], ingestion of dietary supplements [10–12], and prolonged use of intrauterine devices [13, 14]. Elevated Cu plasma levels, especially of free-Cu or the so-called nonceruloplasmin-bound Cu (NCBC), have been associated with neurodegenerative damage [10, 15, 16]. In recent years, there has been a considerable increase in the number of published papers relating Cu to the neurodegenerative process [15–18]. In line with this, Brewer [10] hypothesized that ingestion of inorganic Cu from different sources is at least a partial cause of Alzheimer disease (AD) in developed countries. Squitti et al. [17–19] reported that NCBC, which is loosely bound to molecules such as serum albumin and other proteins, is one of the main risk factors
References
[1]
H. Tapiero, D. M. Townsend, and K. D. Tew, “Trace elements in human physiology and pathology. Copper,” Biomedicine and Pharmacotherapy, vol. 57, no. 9, pp. 386–398, 2003.
[2]
C. G. Fraga, “Relevance, essentiality and toxicity of trace elements in human health,” Molecular Aspects of Medicine, vol. 26, no. 4-5, pp. 235–244, 2005.
[3]
V. Vassiliev, Z. L. Harris, and P. Zatta, “Ceruloplasmin in neurodegenerative diseases,” Brain Research Reviews, vol. 49, no. 3, pp. 633–640, 2005.
[4]
C. G？？men, B. Giesselman, and W. C. de Groat, “Effect of neocuproine, a copper (I) chelator, on rat bladder function,” Journal of Pharmacology and Experimental Therapeutics, vol. 312, no. 3, pp. 1138–1143, 2005.
[5]
M. E. Letelier, A. M. Lepe, M. Faúndez et al., “Possible mechanisms underlying copper-induced damage in biological membranes leading to cellular toxicity,” Chemico-Biological Interactions, vol. 151, no. 2, pp. 71–82, 2005.
[6]
N. Arnal, M. J. de Alaniz, and C. A. Marra, “Cytotoxic effects of copper overload on human-derived lung and liver cells in culture,” Biochimica et Biophysica Acta, vol. 1820, pp. 931–939, 2012.
[7]
N. Arnal, M. J. de Alaniz, and C. A. Marra, “Effect of copper overload on the survival of HepG2 and A-549 human-derived cells,” Human & Experimental Toxicology, vol. 32, pp. 299–315, 2013.
[8]
R. Newhook, H. Hirtle, K. Byrne, and M. E. Meek, “Releases from copper smelters and refineries and zinc plants in Canada: human health exposure and risk characterization,” Science of the Total Environment, vol. 301, no. 1–3, pp. 23–41, 2003.
[9]
N. Arnal, M. Astiz, M. J. T. de Alaniz, and C. A. Marra, “Clinical parameters and biomarkers of oxidative stress in agricultural workers who applied copper-based pesticides,” Ecotoxicology and Environmental Safety, vol. 74, no. 6, pp. 1779–1786, 2011.
[10]
G. J. Brewer, “Copper toxicity in Alzheimer's disease: cognitive loss from ingestion of inorganiccopper,” Journal of Trace Elements in Medicine and Biology, vol. 26, pp. 89–92, 2012.
[11]
F. Y. Leung, “Trace elements in parenteral micronutrition,” Clinical Biochemistry, vol. 28, no. 6, pp. 561–566, 1995.
[12]
G. Hardy, A. M. Menendez, and W. Manzanares, “Trace element supplementation in parenteral nutrition: pharmacy, posology, and monitoring guidance,” Nutrition, vol. 25, no. 11-12, pp. 1073–1084, 2009.
[13]
N. Arnal, M. J. T. de Alaniz, and C. A. Marra, “Alterations in copper homeostasis and oxidative stress biomarkers in women using the intrauterine device TCu380A,” Toxicology Letters, vol. 192, no. 3, pp. 373–378, 2010.
[14]
D. de la Cruz, A. Cruz, M. Arteaga, L. Castillo, and H. Tovalin, “Blood copper levels in Mexican users of the T380A IUD,” Contraception, vol. 72, no. 2, pp. 122–125, 2005.
[15]
R. Squitti, G. Barbati, L. Rossi et al., “Excess of nonceruloplasmin serum copper in AD correlates with MMSE, CSF β-amyloid, and h-tau,” Neurology, vol. 67, no. 1, pp. 76–82, 2006.
[16]
N. Arnal, D. O. Cristalli, M. J. T. de Alaniz, and C. A. Marra, “Clinical utility of copper, ceruloplasmin, and metallothionein plasma determinations in human neurodegenerative patients and their first-degree relatives,” Brain Research, vol. 1319, pp. 118–130, 2010.
[17]
R. Squitti, P. Pasqualetti, G. Dal Forno et al., “Excess of serum copper not related to ceruloplasmin in Alzheimer disease,” Neurology, vol. 64, no. 6, pp. 1040–1046, 2005.
[18]
R. Squitti, F. Bressi, P. Pasqualetti et al., “Longitudinal prognostic value of serum “free” copper in patients with Alzheimer disease,” Neurology, vol. 72, no. 1, pp. 50–55, 2009.
[19]
G. J. Brewer, “Issues raised involving the copper hypotheses in the causation of Alzheimer's disease,” International Journal of Alzheimer's Disease, vol. 2011, Article ID 537528, 11 pages, 2011.
[20]
M. A. Pappolla, M. A. Smith, T. Bryant-Thomas et al., “Cholesterol, oxidative stress, and Alzheimer's disease: expanding the horizons of pathogenesis,” Free Radical Biology and Medicine, vol. 33, no. 2, pp. 173–181, 2002.
[21]
I.-L. Notkola, R. Sulkava, J. Pekkanen et al., “Serum total cholesterol, apolipoprotein E ε4 allele, and Alzheimer's disease,” Neuroepidemiology, vol. 17, no. 1, pp. 14–20, 1998.
[22]
D. L. Sparks and B. G. Schreurs, “Trace amounts of copper in water induce β-amyloid plaques and learning deficits in a rabbit model of Alzheimer's disease,” Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 19, pp. 11065–11069, 2003.
[23]
J. Lu, Y.-L. Zheng, D.-M. Wu, D.-X. Sun, Q. Shan, and S.-H. Fan, “Trace amounts of copper induce neurotoxicity in the cholesterol-fed mice through apoptosis,” FEBS Letters, vol. 580, no. 28-29, pp. 6730–6740, 2006.
[24]
J. Lu, D.-M. Wu, Y.-L. Zheng et al., “Trace amounts of copper exacerbate beta amyloid-induced neurotoxicity in the cholesterol-fed mice through TNF-mediated inflammatory pathway,” Brain, Behavior, and Immunity, vol. 23, no. 2, pp. 193–203, 2009.
[25]
N. Arnal, L. Dominici, M. J. de Alaniz, and C. A. Marra, “Copper-induced alterations in rat brain depends on the route of overload and basal copper levels,” Nutrition. In press.
[26]
D. J. Bonda, X. Wang, G. Perry et al., “Oxidative stress in Alzheimer disease: a possibility for prevention,” Neuropharmacology, vol. 59, no. 4-5, pp. 290–294, 2010.
[27]
S. Pope, J. M. Land, and S. J. R. Heales, “Oxidative stress and mitochondrial dysfunction in neurodegeneration; cardiolipin a critical target?” Biochimica et Biophysica Acta, vol. 1777, no. 7-8, pp. 794–799, 2008.
[28]
J. Yao, R. W. Irwin, L. Zhao, J. Nilsen, R. T. Hamilton, and R. D. Brinton, “Mitochondrial bioenergetic deficit precedes Alzheimer's pathology in female mouse model of Alzheimer's disease,” Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 34, pp. 14670–14675, 2009.
[29]
V. García-Escudero, P. Martín-Maestro, G. Perry, and J. Avila, “Deconstructing mitochondrial dysfunction in Alzheimer disease,” Oxidative Medicine and Cellular Longevity, vol. 2013, Article ID 162152, 13 pages, 2013.
[30]
G. Paradies, G. Petrosillo, M. Pistolese, N. di Venosa, A. Federici, and F. M. Ruggiero, “Decrease in mitochondrial complex I activity in ischemic/reperfused rat heart: involvement of reactive oxygen species and cardiolipin,” Circulation Research, vol. 94, no. 1, pp. 53–59, 2004.
[31]
S. L. Iverson and S. Orrenius, “The cardiolipin-cytochrome c interaction and the mitochondrial regulation of apoptosis,” Archives of Biochemistry and Biophysics, vol. 423, no. 1, pp. 37–46, 2004.
[32]
P. G. Reeves, F. H. Nielsen, and G. C. Fahey Jr., “AIN-93 purified diets for laboratory rodents: final report of the American institute of nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet,” Journal of Nutrition, vol. 123, no. 11, pp. 1939–1951, 1993.
[33]
National Institute of Health, Guide for the Care and Use of Laboratory Animals, no. 85-23 (rev), National Research Council, Bethesda, Md, USA, 1985.
[34]
K. A. Cockell, A. T. L. Wotherspoon, B. Belonje et al., “Limited effects of combined dietary copper deficiency/iron overload on oxidative stress parameters in rat liver and plasma,” Journal of Nutritional Biochemistry, vol. 16, no. 12, pp. 750–756, 2005.
[35]
C. D. Davis and S. Newman, “Inadequate dietary copper increases tumorigenesis in the Min mouse,” Cancer Letters, vol. 159, no. 1, pp. 57–62, 2000.
[36]
G. Paxinos and C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, 4th edition, 1998.
[37]
M. Berkovitch, E. Heyman, R. Afriat et al., “Copper and zinc blood levels among children with nonorganic failure to thrive,” Clinical Nutrition, vol. 22, no. 2, pp. 183–186, 2003.
[38]
C. Terrés-Martos, M. Navarro-Alarcón, F. Martín-Lagos, et al., “Determination of copper levels in serum of healthy subjects by atomic absorption spectrometry,” Science of the Total Environment, vol. 198, pp. 97–103, 1997.
[39]
S. Martínez-Subiela, F. Tecles, and J. J. Ceron, “Comparison of two automated spectrophotometric methods for ceruloplasmin measurement in pigs,” Research in Veterinary Science, vol. 83, no. 1, pp. 12–19, 2007.
[40]
P. J. Twomey, A. S. Wierzbicki, I. M. House, A. Viljoen, and T. M. Reynolds, “Percentage non-caeruloplasmin bound copper,” Clinical Biochemistry, vol. 40, no. 9-10, pp. 749–750, 2007.
[41]
J. Folch, M. Lees, and G. H. Sloane Stanley, “A simple method for the isolation and purification of total lipides from animal tissues,” The Journal of Biological Chemistry, vol. 226, no. 1, pp. 497–509, 1957.
[42]
P. S. Chen Jr., T. Y. Toribara, and H. Warner, “Microdetermination of phosphorus,” Analytical Chemistry, vol. 28, no. 11, pp. 1756–1758, 1956.
[43]
F. Fraser and V. A. Zammit, “Enrichment of carnitine palmitoyltransferases I and II in the contact sites of rat liver mitochondria,” Biochemical Journal, vol. 329, no. 2, pp. 225–229, 1998.
[44]
M. Pellon-Maison, M. A. Montanaro, R. A. Coleman, and M. R. Gonzalez-Baró, “Mitochondrial glycerol-3-P acyltransferase 1 is most active in outer mitochondrial membrane but not in mitochondrial associated vesicles (MAV),” Biochimica et Biophysica Acta, vol. 1771, no. 7, pp. 830–838, 2007.
[45]
F. G. Prendergast, R. P. Haugland, and P. J. Callahan, “1-[4-(Trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene: synthesis, fluorescence properties, and use as a fluorescence probe of lipid bilayers,” Biochemistry, vol. 20, no. 26, pp. 7333–7338, 1981.
[46]
N. Masumoto, K. Tasaka, J. Mizuki, M. Tahara, A. Miyake, and O. Tanizawa, “Dynamics of exocytosis, endocytosis and recycling in single pituitary gonadotropes,” Biochemical and Biophysical Research Communications, vol. 197, no. 1, pp. 207–213, 1993.
[47]
B. R. Lentz, “Use of fluorescent probes to monitor molecular order and motions within liposome bilayers,” Chemistry and Physics of Lipids, vol. 64, no. 1–3, pp. 99–116, 1993.
[48]
G. P. Eckert, J.-H. Keller, C. Jourdan et al., “Hyperforin modifies neuronal membrane properties in vivo,” Neuroscience Letters, vol. 367, no. 2, pp. 139–143, 2004.
[49]
A. R. Andrade Pires, G. Rodrígues Noleto, A. Echevarría, et al., “Interaction of 1,3,4-thiadiazolium mesoionic derivatives with mitochondrial membrane and scavenging activity: involvement of their effects on mitochondrial energy-linked functions,” Chemico-Biological Interactions, vol. 189, pp. 17–25, 2011.
[50]
M. Shinitzky and Y. Barenholz, “Fluidity parameters of lipid regions determined by fluorescence polarization,” Biochimica et Biophysica Acta, vol. 515, no. 4, pp. 367–394, 1978.
[51]
M. E. Anderson and A. Meister, “Enzymic assay of GSSG plus GSH,” Methods in Enzymology, vol. 105, pp. 448–450, 1984.
[52]
J. Nourooz-Zadeh, J. Tajaddini-Sarmadi, S. McCarthy, D. J. Betteridge, and S. P. Wolff, “Elevated levels of authentic plasma hydroperoxides in NIDDM,” Diabetes, vol. 44, no. 9, pp. 1054–1058, 1995.
[53]
R. O. Recknagel and E. A. Glende Jr., “Spectrophotometric detection of lipid conjugated dienes,” Methods in Enzymology, vol. 105, pp. 331–337, 1984.
[54]
G. di Paolo and T.-W. Kim, “Linking lipids to Alzheimer's disease: cholesterol and beyond,” Nature Reviews Neuroscience, vol. 12, no. 5, pp. 284–296, 2011.
[55]
M. Stefani and G. Liguri, “Colesterol in Alzheimer's disease: unresolved questions,” Current Alzheimer Research, vol. 6, pp. 1–17, 2009.
[56]
B. Schereurs, “The effects of cholesterol on learning and memory,” Neuroscience & Biobehavioral Reviews, vol. 34, pp. 1366–1379, 2010.
[57]
M. C. Morris, D. A. Evans, C. C. Tangney et al., “Dietary copper and high saturated and trans fat intakes associated with cognitive decline,” Archives of Neurology, vol. 63, no. 8, pp. 1085–1088, 2006.
[58]
K. A. Cockell, J. Bertinato, and M. R. L'Abbé, “Regulatory frameworks for copper considering chronic exposures of the population,” The American Journal of Clinical Nutrition, vol. 88, pp. 863S–866S, 2008.
[59]
M. Costa, O. Cantoni, M. de Mars, and D. E. Swartzendruber, “Toxic metals produce an S-phase-specific cell cycle block,” Research Communications in Chemical Pathology and Pharmacology, vol. 38, no. 3, pp. 405–419, 1982.
[60]
N. Arnal, M. J. de Alaniz, and C. A. Marra, “Involvement of copper overload in human diseases,” in Metals in Biology Systems, M. S. Giménez, Ed., Research Signpost, Kerala, India, 2010.
[61]
J. A. Cuthbert, “Wilson's disease: a new gene and an animal model for an old disease,” Journal of Investigative Medicine, vol. 43, no. 4, pp. 323–336, 1995.
[62]
T. Planella, M. Cortés, C. Martínez-Brú, et al., “Calculation of LDL-cholesterol by using apolipoprotein B for classification of nonchylomicronemicdyslipemia,” Clinical Chemistry, vol. 43, pp. 808–815, 1997.
[63]
H. R. Koelz, B. C. Sherrill, S. D. Turley, and J. M. Dietschy, “Correlation of low and high density lipoprotein binding in vivo with rates of lipoprotein degradation in the rat. A comparison of lipoproteins of rat and human origin,” The Journal of Biological Chemistry, vol. 257, no. 14, pp. 8061–8072, 1982.
[64]
N. Arnal, G. Morel, M. J. de Alaniz, L. Dominici, and C. A. Marra, “Role of copper and cholesterol association in neurodegenerative process,” The International Journal of Alzheimer's Disease, vol. 2013, Article ID 414817, 15 pages, 2013.
[65]
D. Das, N. Tapryal, S. K. Goswami, P. L. Fox, and C. K. Mukhopadhyay, “Regulation of ceruloplasmin in human hepatic cells by redox active copper: identification of a novel AP-1 site in the ceruloplasmin gene,” Biochemical Journal, vol. 402, no. 1, pp. 135–141, 2007.
[66]
M. Marí, A. Morales, A. Colell, C. García-Ruiz, and J. C. Fernández-Checa, “Mitochondrial glutathione, a key survival antioxidant,” Antioxidants and Redox Signaling, vol. 11, no. 11, pp. 2685–2700, 2009.
[67]
M. C. Vázquez, E. Balboa, A. R. Alvarez, and S. Zanlungo, “Oxidative stress: a pathogenic mechanism for Niemann-Pick type C disease,” Oxidative Medicine and Cellular Longevity, vol. 2012, Article ID 205713, 11 pages, 2012.
[68]
M. Jimenez-Del-Rio and C. Velez-Pardo, “The bad, the good, and the ugly about oxidative stress,” Oxidative Medicine and Cellular Longevity, vol. 2012, Article ID 163913, 13 pages, 2012.
[69]
O. Coll, A. Colell, C. García-Ruiz, N. Kaplowitz, and J. C. Fernández-Checa, “Sensitivity of the 2-oxoglutarate carrier to alcohol intake contributes to mitochondrial glutathione depletion,” Hepatology, vol. 38, no. 3, pp. 692–702, 2003.
[70]
K. D. Hauff and G. M. Hatch, “Cardiolipin metabolism and Barth Syndrome,” Progress in Lipid Research, vol. 45, no. 2, pp. 91–101, 2006.
[71]
I. L. Yurkova, J. Arnhold, G. Fitzl, and D. Huster, “Fragmentation of mitochondrial cardiolipin by copper ions in the Atp7b-/- mouse model of Wilson's disease,” Chemistry and Physics of Lipids, vol. 164, no. 5, pp. 393–400, 2011.
[72]
Y. Y. Tyurina, M. A. Tungekar, M.-Y. Jung et al., “Mitochondria targeting of non-peroxidizable triphenylphosphonium conjugated oleic acid protects mouse embryonic cells against apoptosis: role of cardiolipin remodeling,” FEBS Letters, vol. 586, no. 3, pp. 235–241, 2012.
[73]
J. Montero, M. Mari, A. Colell et al., “Cholesterol and peroxidized cardiolipin in mitochondrial membrane properties, permeabilization and cell death,” Biochimica et Biophysica Acta, vol. 1797, no. 6-7, pp. 1217–1224, 2010.
[74]
J. Eckmann, S. H. Eckert, K. Leuner, et al., “Mitochondria: mitochondrial membranes in brain ageing and neurodegeneration,” The International Journal of Biochemistry & Cell Biology, vol. 45, pp. 76–80, 2013.
