Jean-Pierre Soleilhavoup

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.

Expression of Stress-related Genes in a Cadmium-resistant A549 Human Cell Line

This study was designed to explain the basis for Cd-acquired tolerance of A549 cells cultured in the presence of Cd. Thirty-day exposure of cultured human pneumocytes (A549 cell line) to 10 μ M Cd was previously found to induce an acquired resistance persisting over several weeks of culture. Moreover, these Cd-resistant cells (R-cells) were found to proliferate faster than controls. No difference was found between R-cells and control cells (S-cells) concerning the basal and Cd-induced level of metallothioneins expression. However, after exposure to Cd, cell glutathione levels were unchanged in R-cells while they were either increased (at 10 μM Cd) or decreased (at 25 μM Cd) in S-cells. cDNA array analysis showed that genes encoding for (GPx1) glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase were similarly expressed in R- and S-cells, whereas the gene of (GPx2) glutathione peroxidase was overexpressed in R-cells. Most genes encoding stress proteins were similarly expressed, except for HSP27 and GRP94 genes, which were respectively under- (ratio 0.5 ± 0.1) and over- (1.8 ± 0.5) expressed in R-cells. Acute exposure to Cd was found to trigger the upregulation of genes encoding the chaperone proteins HSP90A, HSP27, HSP40, GRP78, HSP72, and HO-1 in S-cells. In R-cells, only HO-1 and HSP72 were overexpressed but at a lower level. This suggests that the Cd-related adverse conditions, leading to protein misfolding, are lowered in R-cells. It is likely that the upregulation of GPx2 in R-cells leads to a higher antioxidant defense in these cells. This research was supported by ASUPS from the University Paul Sabatier Toulouse III.

Modulation by hypergravity of extracellular matrix macromolecules in in vitro human dermal fibroblasts

In vitro human dermal fibroblasts were submitted to normal gravity (1 g) or to chronic hypergravity (20 g) over a period of 8 days. Changes in organization of extracellular matrix molecules were seen by indirect immunofluorescence. In the fibronectin layer, bundles of fibrils were gathered together leading to a disorganisation of the normal parallel pattern of fibers seen in control cultures. Type I collagen fibrils appeared with wooly outlines in controls whereas thick fibers were closely packed in 20-g cultures. A moderate increase of type III collagen fibril density was observed. No elastic fibers were seen in control or in 20-g cultures. In the culture medium, the release of soluble elastin (ELISA) and type I and III collagens (RIA) was undisturbed. Assays of enzymes involved in the remodeling of extracellular matrix showed an increase of cellular elastase activity (10%) and a decrease of the spontaneously active collagenase. Nevertheless, the total collagenase activity, (activated by trypsin), was increased by up to 30%. These data show a significant rise of the latent collagenase activity and suggest that release of the tissue inhibitor of metalloproteinase (TIMP1) was enhanced by hypergravity.

Impact du thé vert “Camellia sinensis” sur les effets du vanadium sur la croissance et l’appareil génital du ratWistar mâle

ResumeCertains métaux lourds sont susceptibles de générer un stress oxydant et d’altérer les fonctions sexuelles et reproductrices mâles. L’apport d’antioxydants dans la nourriture semble être un moyen de limiter les effets cytotoxiques du stress oxydant.Dans ce cadre nous nous sommes proposés d’explorer l’impact de la consommation du thé vert «Camellia sinensis» riche en antioxydants (poly phénols …) sur les effets du vanadium sur la croissance corporelle et l’appareil génital du rat mâle.Pour cela, nous avons soumis pendant 90 jours des rats mâles de soucheWistar à un traitement par le méta vanadate d’ammonium (0,46g/l) en présence ou en absence de thé vert.Chez les rats exposés au toxique seul, l⇃sorption chronique, par voie orale, de vanadium a induit un ralentissement de la croissance corporelle et des organes génitaux (testicule, épididyme, prostate et vésicules séminales). Parallèlement, une diminution de la motilité et du nombres des spermatozoïdes a été observée. L’étude histologique des testicules a montré que les tubes séminifères étaient atrophiés, les différents stades de la spermatogenèse étaient perturbés, ce qui conduisait à une absence de spermatozoïdes dans plus de la moitié des tubes séminifères. Par ailleurs, nos résultats montrent que les taux de testostérone sérique, évalués par un dosage radio-immunologique, étaient diminués du 2ème au 20ème jour de traitement. Ces taux sont, chez les témoins, de 0,717±0,107 ng/ml de sérum; 4,366±0,666 et 1,979±0,42 respectivement à 2j, 10j et 20j. Après traitement par le vanadium, ces taux passent à 0.043±0.012; 2,494±0,17 et 1,086±0,53 respectivement pour les mêmes dates de traitement.Il faut cependant noter que la plupart de ces perturbations morphologiques (poids), histologiques et fonctionnelles (spermatozoïdes immatures, baisse du taux de testostérone) ne sont significatives qu’au début du traitement (2ème au 10ème jour). Du 30ème au 90ème jour, ces perturbations tendent à disparaître, ce qui est en faveur d’un phénomène d’adaptation.L’absorption orale de vanadium chez des rats buvant du thé n’a pas entraîné de modification de la croissance corporelle. Au niveau du tractus génital, seule l’étude histologique a mis en évidence une augmentation modérée de la fréquence de tubes séminifères atrophiés avec absence de spermatozoïdes.Nos résultats sont en faveur d’un effet protecteur du thé vis à vis du vanadium lorsqu’ils sont pris par voie orale. Le thé vert est riche en poly phénols qui peuvent chélater le fer et former des complexes insolubles. Nous suggérons que ces poly phénols peuvent également former avec le vanadium des complexes insolubles qui sont éliminés au niveau des fèces, ce qui pourrait expliquer la diminution et/ou la disparition des effets nocifs du vanadium en présence du thé.AbstractSome heavy metals are known to exert harmful effects by generating an oxidative stress which, in turn, can affect the sexual and reproductive functions of male animals. The addition of antioxidants to the diet could decrease the cytotoxic effect related to oxidative stress (in the presence of heavy metals as food or water contaminants).As a contribution to this problem, the protective effect ofCamellia sinensis green tea, which is know to be rich in antioxidant compounds (polyphenols, etc.), was studied in vanadium-treated adult male rats, with particular attention to growth and genital tract function.White male Wistar rats were given ammonium metavanadate in drinking water (0.46 g/L) for 90 days One group of animals received green tea supplement in drinking water and the control group did not.Chronic vanadium intoxication (without green tea supplement) induced a low growth rate and relative atrophy of the testes, epididymis, prostate and seminal vesicles. Motility and number of spermatozoa were also decreased. Histological examination of the testes revealed atrophy of the seminiferous tubules and defects of spermatogenesis leading to the absence of spermatozoa in 50% of seminiferous tubules. Blood testosterone levels, evaluated by radioimmunoassay, were also decreased from day 2 to day 20. In control animals, these levels were 0.717±0.107 ng/ml; 4.366±0.666 ng/ml and 1.979±0.42 ng/ml on day 2, day 10 and day 20, respectively. After vanadium treatment, they were reduced to 0.043±0.012 ng/ml, 2.494±0.17 ng/ml and 1.086±0.53 ng/ml, respectively, at the same periods.These morphological, histological and functional disorders mostly occured during the first phase of the intoxication period (day 2 to day 10) and were subsequently attenuated, indicating adaptation to the poisoning.In rats receiving green tea, vanadium ingestion did not modify growth rate compared to control animals. Very minor changes were observed in the genital tract. Testicular atrophy and absence of spermatozoa were observed in only some seminiferous tubules.Our results underscore the protective effect of green tea on vanadium poisoning. Polyphenols, which are abundant in green tea, are known to chelate iron. It is proposed that polyphenols may also form insoluble complexes with vanadium, allowing it to be eliminated in the feces. This could explain the decreased effects of vanadium poisoning under our experimental conditions.