María J.T. de Alaniz

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.

Effect of pesticides on cell survival in liver and brain rat tissues

Pesticides are the main environmental factor associated with the etiology of human neurodegenerative disorders such as Parkinsons disease. Our laboratory has previously demonstrated that the treatment of rats with low doses of dimethoate, zineb or glyphosate alone or in combination induces oxidative stress (OS) in liver and brain. The aim of the present work was to investigate if the pesticide-induced OS was able to affect brain and liver cell survival. The treatment of Wistar rats with the pesticides (i.p. 1/250 LD50, three times a week for 5 weeks) caused loss of mitochondrial transmembrane potential and cardiolipin content, especially in substantia nigra (SN), with a concomitant increase of fatty acid peroxidation. The activation of calpain apoptotic cascade (instead of the caspase-dependent pathway) would be responsible for the DNA fragmentation pattern observed. Thus, these results may contribute to understand the effect(s) of chronic and simultaneous exposure to pesticides on cell survival.

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.

Antioxidant defense system in rats simultaneously intoxicated with agrochemicals

The effect of dimethoate, zineb and glyphosate administered alone or in combination on liver, kidney, brain and plasma antioxidant defense system was investigated. Lipid peroxidation, and RNS production were increased in all tissues studied, especially in those groups that received a combination of drugs. Intoxicated rats exhibited lower antioxidant ability, higher oxidized protein and glutathione levels in plasma with a decreased concentration of α-tocopherol in brain and liver, between 30% and 60% of control. Superoxide dismutase was decreased in liver and brain. Glutathione reductase was inhibited in liver while glutathione peroxidase and transferase were unaffected. Plasma lactate dehydrogenase and γ-glutamyl transpeptidase activities were both increased. The associations of drugs produce more damage than individual administration being the effects observed strongly dependent on the kind of tissue analyzed. In conclusion, the present paper evidenced both the role of the oxidative stress as a mechanism of action of some pesticides and the potential additive effects of a simultaneous exposure to more than one compound. In addition, results suggest a potential contribution of pesticide mixtures to the aetiology of some neurodegenerative diseases.

The action of insulin and dibutyryl cyclic AMP on the biosynthesis of polyunsaturated acids ofα-linolenic acid family in HTC cells

SummaryIncubation of HTC cells (7288 C) with 114C-α-linolenic acid in Swims 77 medium during 24 hours converted the fatty acid to octadeca-6,9,12,15-tetraenoic acid, eicosa-11,14,17-trienoic acid, eicosa-8,11,14,17-tetraenoic acid, eicosa-5,11,14,17-tetraenoic acid, eicosa-5,8,11,14,17-pentaenoic acid and unsaturated acids of 22 carbons. The existence of two pathways was recognized: one initiated by a Δ6-desaturation and the other by an elongation ofα-linolenic acid. Incubation of the cells with insulin and dibutyryl cyclic AMP modified both pathways in different ways. HTC cells were sensitive to insulin which enhanced the de-saturating route increasing eicosapentaenoic acid synthesis and depressed the elongating route decreasing eicosatrienoic acid. In an opposite way, dibutyryl cyclic AMP decreased eicosapentaenoic acid synthesis and increased eicosatrienoic acid.

Effect of catecholamines and β-blockers on linoleic acid desaturation activity.

The effect of catecholamines and adrenergic blocking agents on the oxidative desaturation of linoleic acid in rat liver microsomes was studied. Epinephrine (1 mg/kg/body weight) produced a significant decrease on the conversion of [1-14C]linoleic acid to γ-linolenic acid. The effect of epinephrine was blocked by single injections of the β blockers propranolol (10 mg/kg body weight) or dichloroisoproterenol 30 min before the hormone treatment. Isoproterenol (100 μg/kg body weight) produced a significant decrease on the activity of the linoleyl-CoA desaturase. The effect of the catecholamines was postulated to be mediated through β receptors by an enhancement of the intracellular levels of cyclic AMP.

A Reliable Biomarker Derived from Plasmalogens to Evaluate Malignancy and Metastatic Capacity of Human Cancers

Antigen tumor markers employed in monitoring therapeutical approaches are limited by their specificity (Sp) and sensitivity (Se). The aim of this study was to investigate the suitability of a lipid tumor marker derived from ether-linked phospholipids and to compare it with others usually assayed in clinical practice. Complex lipids from normal and pathological breast, lung, and prostate tissue were isolated and analyzed by TLC and c-GLC methods. Results were compared as pooled samples, or by means of the averaged percent changes with respect to the composition observed in the normal tissue of the same patient. Sp, Se, negative-predictive (NPV) and positive- predictive values (PPV) were established for conventional markers and for the proposed lipid-derived marker. Results demonstrated that the content of monoenoic fatty acyl chains was significantly increased in total lipids, phosphatidylethanolamine, and especially in ethanolamine-containing ether lipids of neoplastic tissues with respect to their corresponding normal ones. Major changes were observed in the plasmalogen sub-fraction where the ratio monoenoic/saturated fatty acids can distinguish with high Se normal tissues from either benign or neoplastic tissues from breast, lung, or prostate lesions. Analyses of fatty acyl chains from ethanolamine-containing plasmalogens provided a reliable tumor marker that correlated with high Se and linearity with metastases spreading. This fact may be useful in prognosis of the most frequently observed human cancers.

Hepatic recovery after damage produced by sub-chronic intoxication with the cyanotoxin microcystin LR

The effect of sub-chronic exposure of intraperitoneal (i.p.) injections of microcystin-LR (MC-LR) on microscopic tissue architecture, hepatic function and lipid peroxidation has been studied in liver and kidney of mice. Mice were treated i.p. with 25 microg of pure MC-LR/kg body weight or saline solution for 1 month (every 2 days) with the aim of producing an inflictive stage with evident damage. Histopathological analysis of dissected livers of mice showed a disrupted lobar architecture and the development of cytoplasmatic vacuoles. According to this, a significant increase in hepatic lipid content and in lipid peroxidation levels in liver and kidney was found in MC-LR-treated animals when compared with controls. Moreover, serum alkaline phosphatase and aspartate aminotransferase activities showed a significant alteration in MC-LR-treated animals. After damage, progression or recovery was studied for 1 and 2 months of wash-out. The recovery from liver damage was evident at the cytological and physiological level, only the recovery of lobar architecture was incomplete along the period investigated. In conclusion, the present study demonstrates the ability of hepatic tissue to recover from damage produced by sub-chronic MC-LR administration. The dynamic interplay between damage and tissue-repairing response in determining the ultimate outcome of toxicity should be considered in risk-assessment studies.

Comparative effect of glucagon, dibutyryl cyclic AMP, and epinephrine on the desaturation and elongation of linoleic acid by rat liver microsomes.

The effect of glucagon, dibutyryl cyclic adenosine 3′,5′-monophosphate, and epinephrine on the biosynthesis of polyunsaturated fatty acids of the linoleic acid family was studied. The incubations were performed with rat liver microsomes and labeled linoleic acid under desaturating and elongating conditions. Under desaturating conditions, linoleic acid was converted to γ-linolenic acid, whereas under elongating conditions it was converted to 20∶2ω6. Glucagon, dibutyryl cyclic AMP, and epinephrine decreased the oxidative desaturation of linoleic acid to γ-linolenic acid while the elongating reaction was not modified in the experimental conditions tested. Consequently, the results support the hypothesis that the oxidative desaturation of linoleic acid to γ-linolenic acid is the main controllable step in the biosynthesis of polyunsaturated fatty acids of the linoleic acid family in the microsomes.

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.