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.

Antioxidant treatment prevents the development of fructose-induced abdominal adipose tissue dysfunction.

In the present study, we tested the effect of OS (oxidative stress) inhibition in rats fed on an FRD [fructose-rich diet; 10% (w/v) in drinking water] for 3 weeks. Normal adult male rats received a standard CD (commercial diet) or an FRD without or with an inhibitor of NADPH oxidase, APO (apocynin; 5 mM in drinking water; CD-APO and FRD-APO). We thereafter measured plasma OS and metabolic-endocrine markers, AAT (abdominal adipose tissue) mass and cell size, FA (fatty acid) composition (content and release), OS status, LEP (leptin) and IRS (insulin receptor substrate)-1/IRS-2 mRNAs, ROS (reactive oxygen species) production, NADPH oxidase activity and LEP release by isolated AAT adipocytes. FRD-fed rats had larger AAT mass without changes in body weight, and higher plasma levels of TAG (triacylglycerol), FAs, TBARS (thiobarbituric acid-reactive substance) and LEP. Although no significant changes in glucose and insulin plasma levels were observed in these animals, their HOMA-IR (homoeostasis model assessment of insulin resistance) values were significantly higher than those of CD. The AAT from FRD-fed rats had larger adipocytes, higher saturated FA content, higher NADPH oxidase activity, greater ROS production, a distorted FA content/release pattern, lower insulin sensitivity together with higher and lower mRNA content of LEP and IRS-1-/2 respectively, and released a larger amount of LEP. The development of all the clinical, OS, metabolic, endocrine and molecular changes induced by the FRD were significantly prevented by APO co-administration. The fact that APO treatment prevented both changes in NADPH oxidase activity and the development of all the FRD-induced AAT dysfunctions in normal rats strongly suggests that OS plays an important role in the FRD-induced MS (metabolic syndrome) phenotype.

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.

Hepatic and intestine alterations in mice after prolonged exposure to low oral doses of Microcystin-LR.

Oral intake of Microcystin-LR (MC-LR) is the principal route of exposure to this toxin, with prolonged exposure leading to liver damage of unspecific symptomatology. The aim of the present paper was therefore to investigate the liver and intestine damage generated by prolonged oral exposure to low MC-LR doses (50 and 100 µg MC-LR/kg body weight, administrated every 48 h during a month) in a murine model. We found alterations in TBARS, SOD activity and glutathione content in liver and intestine of mice exposed to both doses of MC-LR. Furthermore, the presence of MC-LR was detected in both organs. We also found hepatic steatosis (3.6 ± 0.6% and 15.3 ± 1.6%) and a decrease in intraepithelial lymphocytes (28.7 ± 5.0% and 44.2 ± 8.7%) in intestine of 50- and 100-µg MC-LR/kg treated animals, respectively. This result could have important implications for mucosal immunity, since intraepithelial lymphocytes are the principal effectors of this system. Our results indicate that prolonged oral exposure at 50 µg MC-LR/kg every 48 h generates significant damage not only in liver but also in intestine. This finding calls for a re-appraisal of the currently accepted NOAEL (No Observed Adverse Effect Level), 40 µg MC-LR/kg body weight, used to derive the guideline value for MC-LR in drinking water.

Dietary fats significantly influence the survival of penumbral neurons in a rat model of chronic ischemic by modifying lipid mediators, inflammatory biomarkers, NOS production, and redox-dependent apoptotic signals.

OBJECTIVE: Brain stroke is the third most important cause of death in developed countries. We studied the effect of different dietary lipids on the outcome of a permanent ischemic stroke rat model. METHODS: Wistar rats were fed diets containing 7% commercial oils (S, soybean; O, olive; C, coconut; G, grape seed) for 35 d. Stroke was induced by permanent middle cerebral artery occlusion. Coronal slices from ischemic brains and sham-operated animals were supravitally stained. Penumbra and core volumes were calculated by image digitalization after 24, 48, and 72 h poststroke. Homogenates and mitochondrial fractions were prepared from different zones and analyzed by redox status, inflammatory markers, ceramide, and arachidonate content, phospholipase A2, NOS, and proteases. RESULTS: Soybean (S) and G diets were mainly prooxidative and proinflammatory by increasing the liberation of arachidonate and its transformation into prostaglandins. O was protective in terms of redox homeostatic balance, minor increases in lipid and protein damage, conservation of reduced glutathione, protective activation of NOS in penumbra, and net ratio of anti-to proinflammatory cytokines. Apoptosis (caspase-3, milli- and microcalpains) was less activated by O than by any other diet. CONCLUSION: Dietary lipids modulate NOS and PLA2 activities, ceramide production, and glutathione import into the mitochondrial matrix, finally determining the activation of the two main protease systems involved in programmed cell death. Olive oil appears to be a biological source for the isolation of protective agents that block the expansion of brain core at the expense of penumbral neurons.

Biomarkers of prolonged exposure to microcystin-LR in mice.

The effects of prolonged exposure to microcystins (MCs) on health are not yet sufficiently understood and this type of poisoning is often undiagnosed. Even though chronic exposure has been linked with liver cancer and alterations have been described in liver damage marker enzymes in exposed populations, there are not profile parameters that indicate prolonged exposure to microcystins. The aim of this work is to determine, based on an animal model of prolonged exposure to successive i.p. doses of 25 µg MC-LR/kg body weight, several plasma parameters which could be useful as exposure biomarkers. Hemoglobin (Hb) and methemoglobin (MetHb) levels were determined on blood samples. We also studied plasma levels of hydroperoxides (ROOHs), α-tocopherol, glutathione and lipid profile as well as superoxide dismutase (SOD) and catalase (CAT) erythrocyte activities. In addition, the determination of MC-LR levels in liver, kidney, plasma, urine and feces of treated mice was carried out. We found that alteration in MetHb, ROOHs, glutathione, α-tocopherol levels, SOD activity and plasma lipid profile, correlates with those expected if the alteration derived from hepatic damage. The alterated plasma paramenters together with MC-LR determination could be used as biomarkers, helpful tools in screening and epidemiological studies.

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.

Natural polyphenols may ameliorate damage induced by copper overload.

The effect of the simultaneous exposure to transition metals and natural antioxidants frequently present in food is a question that needs further investigation. We aimed to explore the possible use of the natural polyphenols caffeic acid (CA), resveratrol (RES) and curcumin (CUR) to prevent damages induced by copper-overload on cellular molecules in HepG2 and A-549 human cells in culture. Exposure to 100µM/24h copper (Cu) caused extensive pro-oxidative damage evidenced by increased TBARS, protein carbonyls and nitrite productions in both cell types. Damage was aggravated by simultaneous incubation with 100µM of CA or RES, and it was also reflected in a decrease on cellular viability explored by trypan blue dye exclusion test and LDH leakage. Co-incubation with CUR produced opposite effects demonstrating a protective action which restored the level of biomarkers and cellular viability almost to control values. Thus, while CA and RES might aggravate the oxidative/nitrative damage of Cu, CUR should be considered as a putative protective agent. These results could stimulate further research on the possible use of natural polyphenols as neutralizing substances against the transition metal over-exposure in specific populations such as professional agrochemical sprayers and women using Cu-intrauterine devices.

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.

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 Alzheimer's disease (AD), Parkinson's 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.

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.

Influence of commercial dietary oils on lipid composition and testosterone production in interstitial cells isolated from rat testis.

The aim of this study was to examine the influence of dietary fat on lipid composition, as well as on the steroidogenic function of interstitial cells isolated from Wistar rats that had been fed semi-synthetic diets containing four different commercial oils (S soybean, O olive, C coconut, and G grape seed). Steroidogenic enzyme activities, lipid composition, and androgen production were measured in testicular interstitial cells. Lipid analysis included measurement of the contents of major lipid subclasses (neutral lipids, polar lipids, free and esterified cholesterol), as well as principal polar and neutral lipid fatty acyl compositions. Significant differences in lipid composition were observed among the groups, most of them reflecting the specific fatty acyl composition of the diet tested. Testosterone concentration was higher in O and C groups compared with S or G. In agreement with this observation, the activity of both key enzymes involved in testosterone biosynthesis (3-beta-HSD and 17-beta-HSD) was higher in O and C groups with significant differences between them (O > C). A significant negative correlation was found between cellular testosterone production and cellular cholesterol ester content. Additionally, testosterone concentration directly correlated with cholesterol levels. We conclude that dietary oils qualitatively and quantitatively modified the lipid composition of interstitial cells, producing either a direct or indirect regulatory effect on testicular steroidogenic function.

Involvement of lipids in dimethoate-induced inhibition of testosterone biosynthesis in rat interstitial cells.

The mechanism involved in the inhibition of testosterone (Te) biosynthesis after a sub-chronic exposure to low doses of dimethoate (D) was studied in rat interstitial cells (IC). Expression of COX-2 in IC isolated from D-treated rats increased by 44% over C data, while transcription of StAR decreased by approx. 50% and the expression of this protein was diminished by approximately 40%. PGE(2) and PGF(2alpha) were increased by 61 and 78%, respectively. Te concentration decreased by 49% in IC homogenates. Concomitantly, plasma concentration of LH and FSH both increased. Araquidonate (ARA) and C(22) fatty acyl chains in phospholipids from IC mitochondrial fraction decreased by approx. 30% after D treatment. Protein carbonyls, lipoperoxides and nitrite content increased while alpha-tocopherol and the antioxidant capacity of the soluble cellular fraction decreased significantly. Stimulation with h-CG 10 nM overnight failed to overcome the inhibition caused by D on both Te biosynthesis and 3beta- and 17beta-hydroxysteroid dehydrogenases. Decreased Te biosynthesis may be attributed to (1) inhibition of StAR protein activity due to the stimulation of COX-2 and the overproduction of PGF(2alpha), (2) decreased stimulatory effect of ARA on StAR with a subsequent reduction in the availability of CHO for the androgenic pathway, and/or (3) indirect inhibition of steroidogenic enzymes by a lower transcriptional rate caused by elevated PGF(2alpha). Rofecoxib administration prevents the deleterious effect(s) exerted by D.

Biomarcadores emergentes para diferentes patologías humanas / Emerging biomarkers for different human pathologies / Biomarcadores emergentes para diferentes doenças humanas

El objetivo de esta actualización fue revisar los hallazgos concernientes a la posible utilidad clínica de nuevos biomarcadores de estrés oxidativo-nitrosativo, especialmente aquellos derivados del metabolismo lipídico, como herramientas a implementar en el diagnóstico diferencial, la prognosis, el riesgo o la evaluación de estrategias terapéuticas, en enfermedades crónicas de tipo degenerativo. Se analizó la información disponible sobre el uso de marcadores emergentes en casos de infertilidad masculina por varicocele, pacientes con neurodegeneración debida a mal de Alzheimer, de Parkinson, o demencias de origen vascular, enfermos con cáncer de pulmón, próstata o mama, diabéticos tipo 2 y personas expuestas a agroquímicos en forma profesional. Como conclusión surge la posibilidad de implementar muchos de estos biomarcadores en el laboratorio bioquímico-clínico con promisoria y confiable utilidad en varias de estas situaciones patológicas, considerando que la mayoría de ellos se pueden determinar con relativa baja dificultad y requieren muestras obtenibles por métodos no invasivos o mínimamente invasivos.

This review aimed to analyze the possible clinical utility of various oxidative-nitrosative stress biomarkers, mostly derived from lipid metabolism, as a tool to be implemented in the differential diagnosis, prognosis, risk assessment, or the evaluation of the therapeutical strategies for chronic illnesses of the degenerative type. The available information concerning the utility of the biomarkers on male infertility due to varicocele, neurodegenerative disease such as Alzheimer, Parkinson or vascular dementia, prostate, lung and breast cancer, professional sprayers exposed to agrochemicals or type 2 diabetes patients was discussed. The main conclusion is that many of these emerging biomarkers could be implemented in clinica /biochemical analyses of human samples due to the fact that they can be obtained from non-invasive or minimal-invasive procedures and are easily determined.

O objetivo desta atualização é rever os achados sobre a possível utilidade clínica de novos biomarcadores de estresse oxidativo-nitrosativo, especialmente aqueles derivados de metabolismo lipídico, como ferramentas para implementar no diagnóstico diferencial, prognose, risco, ou avaliação de estratégias terapêuticas, em doenças crônicas de tipo degenerativo. Foi analisada a informação disponível sobre o uso de marcadores emergentes em casos de infertilidade masculina por varicocele, pacientes com neurodegeneração devida à doença de Alzheimer, de Parkinson ou demência de origem vascular, pacientes com câncer do pulmão, próstata ou mama, diabéticos tipo 2 e pessoas expostas a produtos agroquímicos devido a sua profissão. Como conclusão surge a possibilidade de implementar muitos destes biomarcadores no laboratório bioquímico- clínico com promissora e confiável utilidade em várias dessas situações patológicas, considerando-se que a maior parte deles pode ser determinada com relativa baixa dificuldade e requerem amostras obtidas através de métodos não invasivos ou minimamente invasivos.

Sobre la regulación de la biosíntesis de ácidos grasos por hormonas esteroides / Effect of steroid hormones on the biosynthesis of fatty acids

Se sumarizan los conocimientos reunidos en los últimos años sobre la regulación del metabolismo de ácidos grasos por hormonas esteroides