Pesticide-induced decrease in rat testicular steroidogenesis is differentially prevented by lipoate and tocopherol.

We have previously demonstrated that the sub-chronic administration of low doses of Toc or α-Toc, glyphosate and zineb to rats (i.p. 1/250 LD50, three times a week for 5 weeks) provoked severe oxidative stress (OS) in testicles. These effects were also reflected in plasma. Lipoic acid (LA) and α-tocopherol are considered as antioxidants due to their ability to neutralize reactive oxygenated species (ROS) and reset endogenous antioxidant levels. To investigate the possible protective effect on reproductive function, LA and Toc (i.p. 25, 50 and 100mg/kg) were administered simultaneously with the pesticide mixture (PM) for 5 weeks. Both drugs prevented OS and the damage to proteins and lipids caused by PM in a dose-dependent manner. The PM-induced increase levels of prostaglandins E2 and F2α was completely restored by LA but not by Toc. Similarly, only LA was able to restore the inhibition of testosterone production, the decrease of 3ß- and 17ß-hydroxysteroid dehydrogenases activities, and the elevation of gonatropins (FSH and LH) levels produced by PM. Furthermore, LA was more efficient than Toc in normalizing the histological alterations produced by PM administration, suggesting that pesticides act though other mechanisms that generate oxidative stress. In our experimental model LA displayed a higher protective role against pesticide-induced damage than that observed by Toc administration. Our results suggest that LA administration is a promising therapeutic strategy for coping with disorders suspected to be caused by OS generators - such as pesticides - in male reproductive system.

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.

Microtubule depolymerization modifies the incorporation of fatty acids into glycerolipids.

BACKGROUND: The influence of cytoskeletal integrity on fatty-acid (FA) metabolism is an almost unexplored field of biochemical research. This study therefore investigated the influence of cytoskeletal integrity on the incorporation of palmitate and eicosa-8,11,14-trienoate into glycerolipids of Hep G2 human hepatoma cells. MATERIAL/METHODS: Attached cultures and suspended cells were exposed to colchicine (COL, 10 microM) or dihydrocytochalasin B (DHCB, 20 microM) and supplemented with [14C]FAs bound to delipidated BSA or [14C]glycerol during 0-300 min of incubation. Various key enzymes of lipid metabolism were also determined after COL or DHCB treatment. RESULTS: Incorporation of both FAs into phospholipids (PLs) was strongly reduced by COL treatment especially in the PE and PC subfractions at short incubation times and in PS and SM for 300 min. COL also produced increased incorporation of both FAs into neutral lipids (NL), especially in TG and its precursors (MG and DG). DHCB increased the labeling into lyso-PL and reduced incorporation into PE and SM. However, this drug did not modify the [14C]NL to [14C]PL ratio. DG-acyltransferase and phosphatidate phosphohydrolase were stimulated by COL treatment. Phospholipase A2 activity was reduced significantly by COL and stimulated by DHCB treatment. CONCLUSIONS: It was demonstrated that the microtubule and microfilament network is involved in the incorporation of FAs and in its channeling to neutral lipids and phospholipids. These effects had differential characteristics depending on the type of FA involved and may have potential significance in the understanding of physiological and/or pathological processes.

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 Parkinson's 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.

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.

Dietary lipids modify redox homeostasis and steroidogenic status in rat testis.

OBJECTIVE: The present study explored the effect of dietary oils on lipid composition, antioxidant status, and the activity of the main steroidogenic enzymes in the testis. METHODS: Forty Wistar rats were randomly assigned to one of four groups (n = 10) fed for 60 d on the same basal diet plus different lipid sources as commercial oils: soybean, olive, coconut, or grapeseed. After sacrifice, testicular lipids and fatty acid composition, free radical biomarkers, antioxidant levels, hormones, and steroidogenic enzymes were determined. RESULTS: The lipid composition of diets produced significant changes in neutral/phospholipids, free/esterified cholesterol, and plasmalogen proportion. Fatty acid patterns of these lipids were also strongly modified, influencing the double bond index. We also found a close correlation between the type of diet and the generation of free radicals. The oxidative stress in testes was higher with the grapeseed oil-supplemented diet and decreased with the other diets in this order: soybean oil > olive oil > coconut oil. Animals fed with the olive oil and coconut oil diets showed the highest testicular levels of antioxidants in addition to significantly high levels of testosterone and 3beta- or 17beta-hydroxysteroid dehydrogenase enzymes. CONCLUSION: Different oils in the diets strongly modified the homeostasis of the testicular antioxidant defense system and, in consequence, affected steroidogenic function, showing a clear correlation with the damage induced. According to our results, an appropriate mixture of olive and soybean oils could be a healthy recommendation.

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.

Lipid metabolism in rats is modified by nitric oxide availability through a Ca++-dependent mechanism.

We studied lipid metabolism and the antioxidant defense system in plasma and liver of rats fed diets supplemented with L(omega)-nitro-L-arginine methyl ester (L-NAME), isosorbide dinitrate (DIS), L-arginine (Arg), or the associations of these drugs. Liver hydroperoxide and thiobarbituric-acid-reactive substance (TBARS) levels were decreased by Arg and increased by L-NAME or DIS treatments. Oxidized glutathione and conjugated dienes were increased by DIS. Nitrate + nitrite levels and serum calcium ([Ca(++)]) were incremented by Arg or DIS and reduced by L-NAME. Superoxide dismutase and catalase activities decreased under Arg treatment, while L-NAME or DIS caused stimulation. Liver high-density lipoprotein (HDL) cholesterol was increased by DIS or NAME (alone or associated with Arg). Free fatty acids and neutral and polar lipids were increased by Arg, L: -NAME, and DIS. However, predominating phospholipid synthesis increased the neutral/polar ratio. Decreased levels of nitric oxide (NO) (low [Ca(++)]) was directly associated with increased fatty acid synthetase, decreased phospholipase A(2), carnitine-palmitoyl transferase, and fatty acid desaturase activities. Raised NO (high [Ca(++)]) inversely correlated with increased phospholipase-A(2) and acyl-coenzyme A (CoA) synthetase and decreased fatty acid synthetase and beta-oxidation rate. Arg or DIS produced changes that were partially reverted by association with L-NAME. Based on these observations, prolonged therapeutical approaches using drugs that modify NO availability should be carefully considered.

Neutral and polar lipid metabolism in liver microsomes of growing rats fed a calcium-deficient diet.

We studied the incorporation of (14)C-labeled fatty acids and glycerol into different classes of glycerolipids in an in vitro system containing liver microsomes from growing Wistar rats fed a calcium-deficient (CaD; 0.5 g/kg) diet for a 60-day period. Desaturase activities and incorporation of the elongation-desaturation metabolites into specific neutral and polar glycerolipids were also studied and correlated with the activities of various enzymes involved in complex lipid metabolism (acyl-CoA synthase, acyl-CoA hydrolase, DAG-acyltransferase, DAG-kinase, lysophospatidate-acyl-CoA transferase, phosphatidate-phosphohydrolase and phospholipase A(2)). Low calcium condition led to a significant increase in the incorporation (relative amounts and specific activities) of both labeled fatty acids and glycerol with a preferential increase of labeling in neutral lipids rather than in phospholipids. Acyl-CoA synthetase, diacylglycerol acyltransferase and diacylglycerol-3-P acyltransferase activities were increased in low calcium microsomes while diacylglycerol kinase, phospholipase A(2) and palmitoyl-, stearoyl-, linoleyl-, alpha-linolenyl, and eicosatrienoyl-desaturases were decreased. The modifications observed in the interlipid and lipid/protein relationships, enzyme activities, and pattern of incorporation of labeled precursors into each glycerolipid class, suggest that decreased intake of calcium should be considered as a harmful risk factor for the development of cardiovascular diseases.

Clues on the role of Beauveria bassiana catalases in alkane degradation events.

Entomopathogenic fungi adapt to growth in a culture medium containing an insect-like hydrocarbon as the sole carbon source inducing the beta-oxidation pathway during the alkane degradation. The effect of two carbon sources on the catalase activity was studied in the entomopathogenic fungus Beauveria bassiana. Catalase activity was detected both in the peroxisomal and cytosolic fraction. A significant increment in the specific activity of the peroxisomal fraction (12.6-fold) was observed when glucose was replaced by an insect-like hydrocarbon, whereas the specific activity in the cytosol diminished more than 1.2-fold in the same culture condition. After purification to homogeneity by gel filtration and strong anion exchange chromatography, an apparent molecular mass of 54.7 and 84.0 kDa per subunit were determined respectively for the peroxisomal and cytosolic catalase. The enzymes showed different biochemical and kinetic characteristics, but both were inhibited by 3-amino-1,2,4 triazole. Peroxisomal catalase was sensitive to pH, heat and high concentration of the hydrogen peroxide substrate. Inversely the cytosolic isoform exhibited a broad range of optimal pH (6.0-10.0), high thermostability (<55 C) and remained fully active at least up to 70 mM hydrogen peroxide. Measurement of catalase activity is a new approach for evaluating fungal ability to degrade hydrocarbons.

Microtubular integrity differentially modifies the saturated and unsaturated fatty acid metabolism in cultured Hep G2 human hepatoma cells.

The influence of cytoskeleton integrity on the metabolism of saturated and unsaturated FA was studied in surface cultures and cell suspensions of human Hep G2 hepatoma cells. We found that colchicine (COL), nocodazol, and vinblastin produced a significant inhibition in the incorporation of labeled saturated FA, whereas incorporation of the unsaturated FA remained unaltered. These microtubule-disrupting drugs also diminished Delta9-, Delta5-, and Delta6-desaturase capacities. The effects produced by COL were dose (0-50 microM) and time (0-300 min) dependent, and were antagonized by stabilizing agents (phalloidin and DMSO). Dihydrocytochalasin B (20 microM) was tested as a microfilament-disrupting drug and produced no changes in either the incorporation of [14C] FA or the desaturase conversion of the substrates. We hypothesized that the interactions between cytoskeleton and membrane proteins such as FA desaturases may explain the functional organization, facilitating both substrate channeling and regulation of unsaturated FA biosynthesis.

Liver desaturase activities and FA composition in monkeys. Effect of a low-protein diet.

The aim of the present study was to measure delta9-, delta6-, and delta5-desaturase activities in liver microsomes, as well as phospholipid FA composition of liver and erythrocytes in monkeys fed a control or low-protein diet during the postweaning period. Ten Saimiri sciureus boliviensis (Cebidae) of both sexes were employed; at 12 mon of age they were separated into two groups fed ad libitum on a control or a low-protein diet for 24 mon. Saimiri sciureus had active delta9, delta6, and delta5 liver desaturase enzymes, and these activities were influenced by the diet. A low-protein diet produced a significant reduction in delta5-desaturation capacity, an increase in delta9-desaturase activity, and no change in delta6-desaturase activity (P < 0.05). These changes, evoked by protein deprivation, were reflected in the liver phospholipid FA composition. Increases in the proportion of saturated FA and in monounsaturated oleic acid (18:1n-9) and a decrease in the proportion of PUFA of the n-6 and n-3 series were produced in the animals fed a low-protein diet (P < 0.0001).

Correlation between fatty acyl composition in neutral and polar lipids and enzyme activities from various tissues of calcium-deficient rats.

In this study we investigated the changes induced by feeding rats a calcium-deficient diet (0.5 g Ca/kg diet) during 65 d after weaning. Phospholipase A2, acyl-Co synthetase and FA delta9-, delta6-, and delta5-desaturase activities were also determined. Calcium deficiency evoked a general alteration in the quality and proportion of the FA chains acylated to neutral and polar lipids from liver, lungs, spleen, brain, kidneys, fat, articular cartilage, erythrocyte ghosts, and plasmas, characterized by an increment of saturated FA and a significant depletion of polyunsaturated acids derived from linoleate and alpha-linolenate. Several interlipid and lipid/protein relationships were also modified in microsomes from calcium-deprived rats, with a concomitant reduction in the rotational mobility of the probe diphenylhexatriene. Phospholipase A2 and acyl-CoA synthetase activities were also decreased and increased, respectively, in some tissues from calcium-deficient rats, whereas delta9-, delta6- and delta5-desaturases were significantly depressed. We conclude that changes in tissue fatty acyl composition evoked by calcium deprivation are due to alterations in the acylation/deacylation cycles via inhibition of the phospholipase A2. These changes were reflected in the physicochemical properties of the membranes, which in turn inhibits desaturase activities. A possible failure in the transcriptional rate for desaturase-mRNA was also discussed.

Dietary lipid-induced changes in enzymes of hepatic lipid metabolism.

OBJECTIVE: To investigate the effect of different dietary oils on the main hepatic enzymes involved in metabolism and their impact on oxidative stress status. METHODS: Twenty-four male Wistar rats were fed for 60 d on the same basal diet plus different lipid sources from commercial oils: soybean (S), olive (O), coconut (C), and grape seed (G). After sacrifice, the liver lipid fatty acid composition, enzymatic and non-enzymatic components of the antioxidant defense system, and the activity of enzymes involved in lipid metabolism were determined. The concentration of Ca(2+) in plasma and liver homogenates was also measured. RESULTS: The diets produced significant changes in the total and polar lipid fatty acid compositions and alterations in key enzyme activities involved in lipid metabolism. The S and G groups showed significantly increased oxidative stress biomarkers. The enzymatic and non-enzymatic components of the antioxidant defense system were increased in the O and C groups. The highest levels of nitrite plus nitrate were observed in the S and G groups compared with the O and C groups in plasma and in liver homogenates. These were directly correlated with the Ca(2+) concentration. The most beneficial effects were obtained with olive oil. However, it is necessary to study in more detail appropriate mixtures of olive and soybean oils to provide an adequate balance between ω-3 and ω-6 fatty acids. CONCLUSION: Different dietary oils modify the lipid composition of the plasma and liver, local and systemic antioxidant statuses, and the activity of the key enzymes of lipid metabolism. The interrelation between Ca(2+) and nitrite plus nitrate could be the causal factor underlying the observed changes.

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 A ß 1-42/A ß 1-40 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.

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 A ß (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.

The oxidative damage and inflammation caused by pesticides are reverted by lipoic acid in rat brain.

We have previously demonstrated that the administration of low doses of dimethoate, glyphosate and zineb to rats (i.p. 1/250 LD50, three times a week for 5weeks) provokes severe oxidative stress (OS) in specific brain regions: substantia nigra, cortex and hippocampus. These effects were also observed in plasma. Lipoic acid (LA) is considered an "ideal antioxidant" due to its ability to scavenge reactive species, reset antioxidant levels and cross the blood-brain barrier. To investigate its protective effect we administered LA (i.p. 25, 50 and 100mg/kg) simultaneously with the pesticide mixture (PM) for 5weeks. After suppression of PM administration, we evaluated the restorative effect of LA for a further 5weeks. LA prevented OS and the production of nitrites+nitrates [NOx] caused by PM in a dose-dependent manner. The PM-induced decrease in reduced glutathione and α-tocopherol levels in all brain regions was completely restored by LA at both high doses. PM administration also caused an increase in prostaglandins E(2) and F(2α) in brain that was reduced by LA in a dose-dependent fashion. Taking into account the relationship between OS, inflammation and apoptosis, we measured caspase and calpain activity. Only milli- and micro-calpain isoforms were increased in the PM-treated group and LA reduced the activities to basal levels. We also demonstrated that interrupting PM administration is not enough to restore the levels of all the parameters measured and that LA is necessary to achieve basal status. In our experimental model LA displayed a protective role against pesticide-induced damage, suggesting that LA administration is a promising therapeutic strategy to cope with disorders suspected to be caused by OS generators, especially in brain.

Exogenous arachidonate restores the dimethoate-induced inhibition of steroidogenesis in rat interstitial cells.

The present work studies the potential restorative effect of polyunsaturated fatty acids (PUFA, 5 µM/24 h) on the dimethoate (DMT)-induced inhibition of testosterone biosynthesis in Leydig cells isolated from rat testes. Various fatty acids (FA) from the n-6 (18:2, 20:3, 20:4, 22:4 and 22:5) and n-3 (18.3, 20:5, 22:5, 22:6) series were assayed in Leydig cells, alone (as delipidated BSA complexes) and in combination with DMT (1 ppm). The n-6 FA stimulated lipid peroxidation (LPO) and inhibited the activities of steroidogenic enzymes (3ß- and 17ß-hydroxysteroid dehydrogenases). The n-3 FA exerted an anti-oxidant effect, decreasing the production of thiobarbituric-acid reactive substances (TBARS) and inhibiting phospholipase A(2) activity. The biosynthesis of testosterone in DMT-treated cultures was completely normalized by ARA (20:4n-6) and partially restored by the addition of 20:3n-6, increasing ARA content inside the mitochondria. The other FA assayed failed to restore androgenesis. COX-2 protein and prostaglandin F2α and E2 production were stimulated by 20:3n-6, ARA, 18:3n-3 and 20:5 n-3. COX-2 protein decreased upon addition of 22:5n-3 and 22:6n-3. StAR protein was increased by ARA and partially increased by 20:3n-6, likely due to its metabolic conversion into ARA. Both FA increased the mitochondrial cholesterol pool available for testosterone biosynthesis. The rate of androgenesis is likely the result of various regulatory factors acting concomitantly on the physiology of Leydig cells.

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.

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.