Nathalie Arnal

Clinical utility of copper, ceruloplasmin, and metallothionein plasma determinations in human neurodegenerative patients and their first-degree relatives

The concentration of plasma copper, ceruloplasmin (CRP), non-ceruloplasmin-bound Cu (NCBC), and metallothioneins (MTs) were studied as putative biomarkers for neurodegenerative diseases in patients and in their first-degree relatives. We found increased levels of Cu in the plasma of Alzheimers disease (AD), Parkinsons disease (PD), and vascular dementia (VD) patients, and the increase observed in VD group was linked to the evolution of the disease. CRP was also elevated in response to the inflammatory component of the diseases, however, a correlation with illness progression was only observed in VD patients. The level of MTs is proportional to the evolution of VD. The Cu/CRP and Cu/MTs ratios are both indicative of disease progression for AD patients but not for those with PD or VD. Moreover, there is a correlation between the NCBC levels and the cognitive impairment estimated through the Mini-mental State Examination (MMSE) scale. This dependence is linear for AD and PD patients and non-linear for the VD ones. The relative values of NCBC showed dependence on the disease duration, especially for AD. Copper measurement and the Cu/CRP ratio may be predictive markers of risk for the first-degree relatives of AD patients. We believe that these results are valuable as a reliable clinical tool.

Peripheral markers in neurodegenerative patients and their first-degree relatives

We have determined various biomarkers in the peripheral blood of Alzheimer, Parkinson and vascular dementia patients by comparing the samples with those of first-degree relatives and control subjects. Our results, together with correlation studies using data from the Mini-Mental State Examination (MMSE), suggest that the clinical evaluation of the nitrite (NOx) concentration in Alzheimer patients should be complemented by assays of protein carbonyls (PCs) levels, the ratio of reduced to oxidized glutathione (GSH/GSSG) in plasma, PCs in erythrocytes and PCs and calcium content in leukocytes. For Parkinson patients it would be useful to determine NOx, thiobarbituric-acid reactive substances (TBARS) and PCs in erythrocytes, and NOx and TBARS en leukocytes. For vascular-demented (VD) patients, determination of NOx, Cu, and GSH/GSSG in plasma and TBARS, and PCs in erythrocytes together with PCs in leukocytes should be assayed. Relatives of Alzheimer patients showed alterations in plasma Se and Zn concentrations, catalase (CAT) activity in erythrocytes and calcium content in leukocytes as possible predictive markers of the disease. Relatives of Parkinson patients showed elevated levels of NOx in leukocytes. In the case of vascular-demented patients we suggest NOx, GSH/GSSG and α-tocopherol in plasma, the CAT/superoxide dismutase ratio in erythrocytes and TBARS, GSSG and glutathione reductase in leukocytes as predictive markers. Large-scale longitudinal population-based studies using these suggested biomarkers are necessary in order to assess their level of reliability and specificity in clinical practice.

Cytotoxic effects of copper overload on human-derived lung and liver cells in culture.

BACKGROUND Copper (Cu) is an essential trace metal used as a catalytic cofactor for many enzymes. However, it can have nocive effects when it participates in the Fenton reaction, producing reactive oxygen species (ROS). Excess Cu is present in the plasma of patients with diseases in which cell survival is crucial. In order to investigate the effect of Cu overload on the induction of cellular damage we chose two human cell lines derived from liver (HepG2) and lung (A-549) as representative cells exposed to exogenous (polluted air) and/or endogenous (systemic) Cu overload. METHODS We studied ROS production using thiobarbituric acid reactive substances (TBARS) and fluorimetric measurements with dichlorofluorescein, cell viability by the trypan dye exclusion test, the methyltetrazolium (MTT) and lactate dehydrogenase leakage (LDH) assays, various cytotoxic indexes, and caspasa-3 and calpain-dependent activation as the main signals involved in the apoptosis pathway. RESULTS Cu overload induces cell death by a differential activation of calpains (m- and μ-) and caspase-3, and modifies various proliferative indexes in a cell-type and concentration-dependent manner. The involvement of these two protease systems and the response of the two main Cu homoestatic proteins ceruloplasmin and metallothioneins are specific to each cell type. We demonstrated that Cu can trigger cell death by activation of specific protease systems and modify various proliferative indexes in a cell-type and concentration-dependent manner. GENERAL SIGNIFICANCE These findings contribute to understanding the diverse effects of Cu overload on the pathogenesis of human diseases like cancer, cirrhosis and degenerative disorders.

Clinical parameters and biomarkers of oxidative stress in agricultural workers who applied copper-based pesticides.

Copper based-pesticides are widely used in agricultural practice throughout the world. We studied the (i) concentration of Cu and proteins involved in Cu homeostasis, (ii) plasma redox status, and (iii) biomarkers of exposure in Cu-based pesticide applicators in order to compare them with clinical biochemical tests. Thirty-one professional applicators and 32 control subjects were recruited. Oxidative stress biomarkers, ceruloplasmin (CRP), metallothioneins (MTs), copper, hematological parameters, and biochemical markers for pancreatic, hepatic and renal function were measured in plasma. Copper was increased in the exposed group compared to the control group concomitantly with TBARS, protein carbonyls, and nitrate+nitrite levels. In the exposed group, α-tocopherol and the FRAP assay were lower and LDH, transaminases, GGT, ALP, urea, creatinine, CRP and MTs were higher than in the control group. The relative leukocyte subclasses were also different between the two groups. Clinical chemistry tests did not surpass the upper reference limit. Our results suggest that the incorporation of oxidative stress biomarkers to biochemical/clinical tests should be considered for validation and included in the human health surveillance protocols.

Alterations in copper homeostasis and oxidative stress biomarkers in women using the intrauterine device TCu380A.

Copper ions participate in the Häber-Weiss reaction to produce ROS, which can be toxic when in excess. The purpose of this study was to measure the copper concentration (Cu) in the plasma of women using Cu-IUDs and determine (i) the effect of Cu on oxidative stress biomarkers, (ii) the levels of copper transport proteins in the plasma and (iii) the status of some liver damage markers in relation to the length of the intrauterine device use. Thirty-nine controls and 35 T380-IUD users were recruited. Various oxidative stress biomarkers, ceruloplasmin (CRP), metallothioneins (MTs), Cu and enzyme activities involved in liver function were measured in the plasma. The Cu concentration was higher in women with IUDs, concomitantly with time-dependent increases in the main oxidative stress biomarkers (TBARS, protein carbonyls, glutathione and nitrates+nitrites), hepatic enzymes (LDH and transaminases), MTs and CRP. We concluded that the use of Cu-IUDs for more than 2 consecutive years should be avoided in order to prevent oxidative damage.

Occupational exposure characterization in professional sprayers: clinical utility of oxidative stress biomarkers.

The impact of involuntary exposure to pesticides was studied in a group of professional sprayers (S) (25±5 years old) exposed to various agrochemicals for about 10 years. The results were compared with a group of non exposed people (C). S group showed hematological, renal, pancreatic and hepatic biomarkers within the reference values established for the general population, including cholinesterase activity. In spite of that, all the biochemical tests were statistically different compared to C. On the other hand, oxidative stress biomarkers (OSB) such as plasma tocopherol and the total reducing ability of plasma were significantly decreased, while protein carbonyls, thiobarbituric acid-reactive substances, total glutathione and the sum of nitrites and nitrates were increased in the exposed group. Results demonstrated that screening laboratory tests could not be fully sensitive in detecting sub-clinical exposure to pesticides, and also suggest that OSB could be validated and included in health surveillance protocols.

Copper-induced alterations in rat brain depends on route of overload and basal copper levels

OBJECTIVES Copper (Cu) is widely used in industry for the manufacture of a vast range of goods including Cu-intrauterine devices (IUDs), electronic products, agrochemicals, and many others. It is also one of the trace elements essential to human health in the right measure and is used as a parenteral supplement in patients unable to ingest food. Elevated Cu levels have been found in the plasma of women using Cu-IUDs and in farmers working with Cu-based pesticides. However, possible alterations due to Cu overload in the brain have been poorly studied. Therefore, the aim of this study was to investigate the effects of Cu administration on rat brain in Cu-sufficient and Cu-deficient animals fed on semi-synthetic diets with different doses of Cu (7 or 35 ppm). METHODS We aimed to investigate the effects of Cu administration using two routes of administration: oral and intraperitoneal (IP). Male Wistar rats were feeding (one month) a complete (7 ppm) or a deficient (traces) Cu diets subdivided into three categories oral-, intraperitoneal- (or both) supplemented with copper carbonate (7 to 35 ppm). Cu content in plasma, brain zones (cortex and hippocampus), antioxidant enzyme activities, and protease systems involved in programmed cell death were determined. RESULTS The results show that Cu levels and the concentration of Cu in plasma and brain were dose-dependent and administration route-dependent and demonstrated a prooxidative effect in plasma and brain homogenates. Oxidative stress biomarkers and antioxidative enzyme activity both increased under Cu overload, these effects being more noticeable when Cu was administered IP. Concomitantly, brain lipids from cortex and hippocampus were strongly modified, reflecting Cu-induced prooxidative damage. A significant increase in the activities of calpain (milli- and micro-) and caspase-3 activity also was observed as a function of dose and administration route. CONCLUSION The findings of this study could be important in evaluating the role of Cu in brain metabolism and neuronal survival.

Role of Copper and Cholesterol Association in the Neurodegenerative Process

Age is one of the main factors involved in the development of neurological illnesses, in particular, Alzheimer, and it is widely held that the rapid aging of the world population is accompanied by a rise in the prevalence and incidence of Alzheimer disease. However, evidence from recent decades indicates that Cu and Cho overload are emerging causative factors in neurodegeneration, a hypothesis that has been partially investigated in experimental models. The link between these two variables and the onset of Alzheimer disease has opened up interesting new possibilities requiring more in-depth analysis. The aim of the present study was therefore to investigate the effect of the association of Cu + Cho (CuCho) as a possible synergistic factor in the development of an Alzheimer-like pathology in Wistar rats. We measured total- and nonceruloplasmin-bound Cu and Cho (free and sterified) contents in plasma and brain zones (cortex and hippocampus), markers of oxidative stress damage, inflammation, and programmed cell death (caspase-3 and calpain isoforms). The ratio beta-amyloid (1-42)/(1-40) was determined in plasma and brain as neurodegenerative biomarker. An evaluation of visuospatial memory (Barnes maze test) was also performed. The results demonstrate the establishment of a prooxidative and proinflammatory environment after CuCho treatment, hallmarked by increased TBARS, protein carbonyls, and nitrite plus nitrate levels in plasma and brain zones (cortex and hippocampus) with a consequent increase in the activity of calpains and no significant changes in caspase-3. A simultaneous increase in the plasma Aβ1-42/Aβ1-40 ratio was found. Furthermore, a slight but noticeable change in visuospatial memory was observed in rats treated with CuCho. We conclude that our model could reflect an initial stage of neurodegeneration in which Cu and Cho interact with one another to exacerbate neurological damage.

Carnosine and neocuproine as neutralizing agents for copper overload-induced damages in cultured human cells

Copper is dangerous when it is present in excess, mainly because it can participate in the Fenton reaction, which produces radical species. As a consequence of copper pollution, people are involuntarily exposed to a copper overload under sub-clinical and sub-symptomatological conditions, which may be very difficult to detect. Thus, we investigated (i) the possible use of the chelator molecules carnosine and neocuproine to prevent the Cu overload-induced damage on cellular lipids and proteins, as tested in human cell culture systems, and (ii) the differential response of these two chelating agents in relation to their protective action, and the type of copper ion involved in the process, by using two types of human cultured cells (HepG2 and A-549). Cu treatment clearly enhanced (p<0.01) the formation of protein carbonyls, thiobarbituric acid-reactive substances (TBARS) and the concentration of nitrate plus nitrites, with a concomitant decrease in cell survival, as estimated by the trypan dye exclusion test and lactate dehydrogenase leakage. Simultaneous treatment with Cu and carnosine or neocuproine indicated that carnosine is more efficient than neocuproine in protecting both types of cells from the effect of cupric ions on both the cell-associated damages and the decrease in the cellular viability. This observation was supported by the fact that carnosine is not only a complexing agent for Cu(II), but also an effective antioxidant that can dismutate superoxide radicals, scavenge hydroxyl radicals and neutralize TBARS formation. Carnosine should be investigated in more detail in order to establish its putative utility as an agent to prevent copper-associated damages in biological systems.

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

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.