Françoise Croute

Implication of free radicals and glutathione in the mechanism of cadmium-induced expression of stress proteins in the A549 human lung cell-line

Cellular mechanisms underlying the expression of stress proteins (HSP) were studied in the human cell-line A549 submitted to a pollutant, cadmium, in the presence of several agents which modulate the glutathione level and, supposedly, the effects of this metal in the cell. It was observed that HSP 90, HSP 72 and HSP 27 are significantly over-expressed after exposure to cadmium chloride for 24 h. Low cadmium concentrations (i.e. from 1 to 10 microM) also triggered a slight accumulation of glutathione, whereas this compound was depleted after exposure to higher cadmium concentrations (25-100 microM). When 50 microM diethyl-maleate, which traps glutathione, was added together with cadmium, the over-expression of HSP 72 and HSP 90 was much stronger. Treatment of cells with 20 or 40 mM N-acetyl-L-cysteine, which traps free radicals, was found to increase by 30% the glutathione level and to suppress the HSP over-expression. From our results, it is suggested that HSP induction by cadmium in A549 cells is due, at least in part, to the oxidative stress consisting in formation of reactive oxygen species and inhibition of peroxides detoxification. Due to this oxidative status within the cell, more proteins would be damaged inducing the HSP over-expression.

Volatile organic compounds cytotoxicity and expression of HSP72, HSP90 and GRP78 stress proteins in cultured human cells.

The aim of this study was to determine whether overexpression of stress proteins (SPs) could be a sensitive biomarker for cell injury due to exposure to low doses of volatile organic compounds (VOCs) such as benzene, ethylbenzene, toluene, xylene, and chlorinated derivatives (ClB). Sublethal and cytotoxic threshold concentrations of the VOCs were determined by studying the growth rate of normal (fibroblasts) or tumor-derived human cell lines (A549, HepG2) exposed for 4 days to VOCs. Changes in SP expression as a function of concentrations were investigated by Western blotting.VOC toxicity was found to be correlated with their degree of chlorination and their hydrophobicity. Cytotoxic threshold concentrations (no-observed effect concentration, NOEC) were found to be similar for the three cell lines. It was observed that using a mixture of VOCs, each of them at concentration below the NOEC, resulted in an actual toxicity to the cells. This finding reveals a synergistic effect and should be taken into account when assessing threshold risk and exposure limit values in the workers environment when several pollutants may be present. HSP72 and HSP90 expression levels were not affected whereas GRP78 expression was increased by all the VOCs. Taking into account the specific molecular function of GRP78, it suggests that VOC exposure results in misfolded or underglycosylated protein accumulation in the endoplasmic reticulum. GRP78 overexpression was closely related to the magnitude of growth inhibition due to increasing concentrations of each VOC. The overexpression was found to be significant for concentrations 5 to 30 times higher than NOEC, indicating that, under our experimental conditions, GRP78 expression cannot be considered as a sensitive biomarker of exposure to environmental VOCs.

Long-term Exposure to Low Lithium Concentrations Stimulates Proliferation, Modifies Stress Protein Expression Pattern and Enhances Resistance to Oxidative Stress in SH-SY5Y Cells

SH-SY5Y cells, derived from a human neuroblastoma, were submitted to short- or long-term exposures to lithium carbonate concentrations ranging from 0.5 to 8xa0mM. Short-term exposures (4xa0days) to concentrations higher than 6xa0mM were found to reduce cell growth rate while exposure to 8xa0mM resulted in significant cell mortality. These ranges of concentrations induced an overexpression of (1) the HSP27 stress protein, (2) a 108xa0kDa protein (P108) recognized by an anti-phospho-HSP27(Ser78) antibody, and probably corresponding to a phosphorylated HSP27 tetramer, (3) a 105xa0kDa protein (P105), possible glycosylated or phosphorylated form of the GRP94 stress protein and (4) a phosphorylated (inactivated) form of glycogen synthase kinase (GSK3α/β) SH-SY5Y cells, when cultured in the presence of 0.5xa0mM lithium for 25xa0weeks, displayed interesting features as compared to controls: (1) higher cell growth rate, (2) increased resistance toward the inhibitory effects of high lithium concentrations on cell proliferation, (3) lower basal level of lipid peroxidation (TBARS) and improved tolerance to oxidative stress induced by high lithium concentrations, (5) reduced expression of monomeric HSP27 versus an increase of corresponding tetrameric protein (P108) and (6) overexpression of a 105xa0kDa protein (P105). In conclusion, our study suggests that chronic treatment (over several months) by therapeutic relevant lithium concentrations could favour neurogenesis, decrease the vulnerability of neuronal cells to oxidative stress and induce posttranslational changes of molecular chaperones.

Protective effect of cactus (Opuntia ficus indica) cladode extract upon nickel-induced toxicity in rats

The purpose of this study carried out on male Wistar rats, was to evaluate the protective effects of regular ingestion of juice from the prickly pear cactus (Opuntia ficus indica) cladodes against nickel chloride toxicity. Rats were given either normal tap water or water containing 25% of cactus juice for one month. Then, rats of each group were injected daily, for 10 days, with either NiCl(2) solution (4mg (30micromol)/kg body weight) or with the same volume of saline solution (300mM NaCl). Significant increases of lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase activities and of cholesterol, triglycerides and glucose levels were observed in blood of nickel-treated rats. In the liver, nickel chloride was found to induce an oxidative stress evidenced by an increase in lipid peroxidation and changes in antioxidant enzymes activities. Superoxide-dismutase (SOD) activity was found to be increased whereas glutathione peroxidase and catalase activities were decreased. These changes did not occur in animals previously given cactus juice, demonstrating a protective effect of this vegetal extract.

Neuroprotective effects of chronic exposure of SH-SY5Y to low lithium concentration involve glycolysis stimulation, extracellular pyruvate accumulation and resistance to oxidative stress

Recent studies suggest that lithium protects neurons from death induced by a wide array of neurotoxic insults, stimulates neurogenesis and could be used to prevent age-related neurodegenerative diseases. In this study, SH-SY5Y human neuronal cells were cultured in the absence (Con) or in the presence (Li+) of a low lithium concentration (0.5 mm Li2CO3, i.e. 1 mm lithium ion) for 25-50 wk. In the course of treatment, growth rate of Con and Li+ cells was regularly analysed using Alamar Blue dye. Resistance to oxidative stress was investigated by evaluating: (1) the adverse effects of high concentrations of lithium (4-8 mm) or glutamate (20-90 mm) on cell growth rate; (2) the levels of lipid peroxidation (TBARS) and total glutathione; (3) the expression levels of the anti-apoptotic Bcl-2 protein. In addition, glucose metabolism was investigated by analysing selected metabolites in culture media and cell extracts by 1H-NMR spectroscopy. As compared to Con, Li+ cells multiplied faster and were more resistant to stress, as evidenced by a lower dose-dependent decrease of Alamar Blue reduction and dose-dependent increase of TBARS levels induced by toxic doses of lithium and glutamate. Total glutathione content and Bcl-2 level were increased in Li+ cells. Glucose consumption and glycolytic activity were enhanced in Li+ cells and an important release of pyruvate was observed. We conclude that chronic exposure to lithium induces adaptive changes in metabolism of SH-SY5Y cells involving a higher cell growth rate and a better resistance to oxidative stress.

The effects of subchronic lithium administration in male Wistar mice on some biochemical parameters

Lithium salts are efficiently used for treatment of psychiatric disorders. However, prolonged treatment frequently involves adverse side effects. In this study, effects of lithium carbonate administration on some biochemical parameters were studied in male mice. Lithium carbonate (20, 40, or 80 mg/kg body weight corresponding to 3.77, 7.54, or 15.08 mg Li element/kg body weight, respectively) was injected daily for 14 or 28 days. The following parameters were recorded: drinking water consumption, body weight, lithium and testosterone serum concentrations, activities of catalase (CAT), superoxide-dismutase (SOD), and glutathione-peroxidase (GPX), and level of lipid peroxidation (expressed as TBARS) in liver was performed. Lithium treatment, especially at the highest dose for 28 days, was found to induce weight gain and polydipsia and a significant decrease of plasma testosterone level. Lipid peroxidation level and activities of SOD and GPX were increased in liver, which suggests a perturbation of the antioxidative status. Our results indicate that subchronic exposure to lithium, which induces weight gain and polydipsia under our experimental conditions, also damages the male reproductive system and triggers an oxidative stress in the liver.

Chronic lithium administration triggers an over-expression of GRP94 stress protein isoforms in mouse liver

Moderate doses of lithium were chronically administered to mice in order to verify whether the cytoprotective effects of lithium could be in part attributed to a molecular protection conferred by stress proteins/chaperones accumulation. In order to reach serum lithium levels within the common therapeutic values, mice were fed for 6 months on food pellets contained 1 g (L1 group) or 2 g (L2 group) lithium carbonate/kg, resulting in serum concentrations of 0.5 and 0.9 mM Li, respectively. Under these experimental conditions, no clinical side-effects were observed. Urea and creatinine concentrations in serum, lipids peroxidation level and activities of catalase, superoxide-dismutase and glutathione-peroxidase in liver and kidney were not significantly different from control values. Although the expression level of the constitutive HSP73 was not significantly modified, HSP72 was found to be down-regulated in kidney after 1 month. In liver, three protein bands were immunodetected by the anti-GRP94 antibody: 98 kDa and 96 kDa proteins corresponding to more or less glycosylated forms and/or phosphorylated forms of GRP94 and a 80 kDa protein probably being a cleavage product of GRP94. The 96 kDa and 80 kDa proteins were significantly up-regulated in liver of lithium-treated mice as compared to controls.

Cytoskeleton involvement in lithium-induced SH-SY5Y neuritogenesis and the role of glycogen synthase kinase 3β.

Lithium modulates signals impacting on the cytoskeleton, a dynamic system contributing to neural plasticity at multiple levels. In this study, SH-SY5Y human neuronal cells were cultured in the absence (C) or in presence (Li) of a 0.5xa0mM Li2CO3 (i.e. 1xa0mM lithium ion) for 25–50xa0weeks. We investigated the effect of this treatment on (1) morphological changes of cells observed using Hemalun eosin staining assay, (2) cytoskeletal changes by indirect immunofluorescence (IIF) staining of microtubules (α-tubulin) and heavy neurofilaments subunits (NF-H) and by measuring the expression rate changes of genes coding for receptor for activated C kinase (RACK1), casein kinase2 (CK2) and thymosine beta-10 using cDNA arrays technology, (3) cell adhesion properties by IIF staining of β-catenin protein. Besides, we have tried to understand the molecular mechanism of lithium action that triggers changes in cytoskeleton and neurites outgrowth. Thus, we examined the effect of this treatment on glycogen synthase kinase 3 (GSK3) expression and activity using western blotting of GSK3 and phosphorylated β-catenin, a downstream GSK3 target protein. Our results showed that lithium treatment reduces axon length, increases axonal spreading, enhances neurites growth and neurites branching with an increase of growth cone size. Moreover, genes coding for CK2 and thymosine beta-10 were significantly up-regulated, however, that coding for RACK1 was down-regulated. The most interesting result in this work is that mechanism underlying lithium action was not related to the inhibition of GSK3 activity. In fact, neither expression rate nor activity of this protein was changed.

Chronic neuroprotective effects of low concentration lithium on SH-SY5Y cells: possible involvement of stress proteins and gene expression

To investigate the molecular mechanism underlying the neuroprotective effect of lithium on cells, in this study, we exposed SH-SY5Y cells to 0.5 mmol/L lithium carbonate (Li2CO2) for 25–50 weeks and then detected the expression levels of some neurobiology related genes and post-translational modifications of stress proteins in SH-SY5Y cells. cDNA arrays showed that pyruvate kinase 2 (PKM2) and calmodulin 3 (CaM 3) expression levels were significantly down-regulated, phosphatase protein PP2A expression was lightly down-regulated, and casein kinase II (CK2), threonine/tyrosine phosphatase 7 (PYST2), and dopamine beta-hydroxylase (DBH) expression levels were significantly up-regulated. Besides, western blot analysis of stress proteins (HSP27, HSP70, GRP78 and GRP94) showed an over-expression of two proteins: a 105 kDa protein which is a hyper-phosphorylated isoform of GRP94, and a 108 kDa protein which is a phosphorylated tetramer of HSP27. These results suggest that the neuroprotective effects of lithium are likely related to gene expressions and post-translational modifications of proteins cited above.

Incidences de la restriction calorique et du Nickel sur l'aromatase testiculaire du rat

ResumeDes études épidémiologiques chez l’homme ont montré qu’il existait, depuis 1960, des signes d’augmentation de certaines pathologies génitales masculines. La contamination croissante de l’environnement par des composés chimiques semble être un facteur de causalité. Par ailleurs, divers auteurs avancent l’hypothèse que la restriction calorique a un effet bénéfique sur la reproduction masculine.Ce travail a pour objectif de comparer les effets du nickel sur les processus de reproduction, entre autres l’étude de l’aromatase, chez des rats mâles nourris soit tous les jours, soit un jour sur deux, afin d’évaluer les possibles effets bénéfiques, ou non, d’une restriction calorique sur la fertilité des rats mâles.Dans ce but, nous avons utilisé des rats mâles de souche « Wistar » nourris, soit tous les jours (groupe N), soit un jour sur deux (jeûne intermittent) (groupe J). Après un mois de ce traitement, les rats des deux lots (N) et (J) sont répartis chacun en deux groupes: l’un injecté intra-péritonéalement avec du NaCI 9‰ (groupes NO et JO), l’autre injecté intra-péritonéalement avec du NiCl2 à raison de 4mg/kg (groupes NNi et JNi). Le jeûne intermittent se poursuit parallèlement au traitement par le nickel, et ceci durant 1, 3, 5 et 10 jours.Nous avons choisi pour notre étude comme biomarqueurs sexuels: dosage sérique des oestrogènes, dosage des ARN totaux et l’exploration moléculaire de l’ARNm de l’aromatase testiculaire.L’étude de l’aromatase montre que le nickel seul stimule l’activité de l’aromatase testiculaire et provoque la dégradation des ARN totaux durant le 3èmeet le 10ème jours de traitement.Le jeûne intermittent seul n’induit pas de variations significatives. Dans le cas où le nickel est associé au jeûne intermittent, il semble que pour le taux des ARN totaux testiculaires, l’effet du jeûne l’emporte, pas de dégradation des ARN totaux, d’où ses effets bénéfiques en protégeant les ARN totaux des effets délétères du nickel. On note aussi une surexpression de l’ARNm de l’aromatase vers le 10ème jours de traitement.L’effet hypocalorique du jeûne intermittent pourrait être à l’origine de l’inhibition des effets cytotoxiques du nickel, métal classé parmi les stress oxydants.AbstractThis study was designed to determine whether intermittent fasting induces malnutrition that, according to many authors, accentuates the cytotoxic effects of environmental pollutants, or caloric restriction that reduces these effects.Ninety six male Wistar rats (180g) were divided into two groups: one group was fed daily (N) and the other group was fed every second day (J) for one month. At the end of one month, each group was then divided into two subgroups, one subgroup received an injection of 0.9% NaCI (groups NO and JO), the other subgroup received an injection of 4 mg/kg NiCIb2 (groups NNi and JNi).Intermittent fasting was continued in parallel to treatment for 1, 3, 5 and 10 days.Under these experimental conditions, nickel increased testicular aromatase activity and altered total RNA, while no alteration of these biomarkers was observed with intermittent fasting.The combination of these two factors, nickel and intermittent fasting, did not amplify these effects. In contrast, protection of RNA by intermittent fasting was observed, especially overexpression of aromatase mRNA.