Olivier Toussaint

IMPORTANCE OF SE-GLUTATHIONE PEROXIDASE, CATALASE, AND CU/ZN-SOD FOR CELL SURVIVAL AGAINST OXIDATIVE STRESS

Eukaryotic cells have to constantly cope with highly reactive oxygen-derived free radicals. Their defense against these free radicals is achieved by natural antioxidant molecules but also by antioxidant enzymes. In this paper, we review some of the data comparing the efficiency of three different antioxidant enzymes: Cu/Zn-superoxide dismutase (Cu/Zn-SOD), catalase, and selenium-glutathione peroxidase. We perform our comparison on one experimental model (human fibroblasts) where the activities of these three antioxidant enzymes have been modulated inside the cells, and the repercussion of these changes was investigated in different conditions. We also focus our attention on the protecting role of selenium-glutathione peroxidase, because this enzyme is very rarely studied due to the difficulties linked to its biochemical properties. These studies evidenced that all three antioxidant enzymes give protection for the cells. They show a high efficiency for selenium-glutathione peroxidase and emphasize the fact that each enzyme has a specific as well as an irreplaceable function. They are all necessary for the survival of the cell even in normal conditions. In addition, these three enzymes act in a cooperative or synergistic way to ensure a global cell protection. However, optimal protection is achieved only when an appropriate balance between the activities of these enzymes is maintained. Interpretation of the deleterious effects of free radicals has to be analyzed not only as a function of the amount of free radicals produced but also relative to the efficiency and to the activities of these enzymatic and chemical antioxidant systems. The threshold of protection can indeed vary dramatically as a function of the level of activity of these enzymes.

Protocols to detect senescence-associated beta-galactosidase (SA-βgal) activity, a biomarker of senescent cells in culture and in vivo

Normal cells can permanently lose the ability to proliferate when challenged by potentially oncogenic stress, a process termed cellular senescence. Senescence-associated beta-galactosidase (SA-βgal) activity, detectable at pH 6.0, permits the identification of senescent cells in culture and mammalian tissues. Here we describe first a cytochemical protocol suitable for the histochemical detection of individual senescent cells both in culture and tissue biopsies. The second method is based on the alkalinization of lysosomes, followed by the use of 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG), a fluorogenic substrate for βgal activity. The cytochemical method takes about 30 min to execute, and several hours to a day to develop and score. The fluorescence methods take between 4 and 8 h to execute and can be scored in a single day. The cytochemical method is applicable to tissue sections and requires simple reagents and equipment. The fluorescence-based methods have the advantages of being more quantitative and sensitive.

Cellular and molecular mechanisms of stress-induced premature senescence (SIPS) of human diploid fibroblasts and melanocytes

Replicative senescence of human diploid fibroblasts (HDFs) or melanocytes is caused by the exhaustion of their proliferative potential. Stress-induced premature senescence (SIPS) occurs after many different sublethal stresses including H(2)O(2), hyperoxia, or tert-butylhydroperoxide. Cells in replicative senescence share common features with cells in SIPS: morphology, senescence-associated beta-galactosidase activity, cell cycle regulation, gene expression and telomere shortening. Telomere shortening is attributed to the accumulation of DNA single-strand breaks induced by oxidative damage. SIPS could be a mechanism of accumulation of senescent-like cells in vivo. Melanocytes exposed to sublethal doses of UVB undergo SIPS. Melanocytes from dark- and light- skinned populations display differences in their cell cycle regulation. Delayed SIPS occurs in melanocytes from light-skinned populations since a reduced association of p16(Ink-4a) with CDK4 and reduced phosphorylation of the retinoblastoma protein are observed. The role of reactive oxygen species in melanocyte SIPS is unclear. Both replicative senescence and SIPS are dependent on two major pathways. One is triggered by DNA damage, telomere damage and/or shortening and involves the activation of the p53 and p21(waf-1) proteins. The second pathway results in the accumulation of p16(Ink-4a) with the MAP kinase signalling pathway as possible intermediate. These data corroborate the thermodynamical theory of ageing, according to which the exposure of cells to sublethal stresses of various natures can trigger SIPS, with possible modulations of this process by bioenergetics.

Low levels of reactive oxygen species as modulators of cell function.

In this paper, we present various arguments supporting the hypothesis that reactive oxygen species (ROS) could be responsible for the modulation of various cellular functions, besides their well known toxic effects. We first review the recent evidence indicating that ROS are able to modulate genome expression through specific and precise mechanisms during cell activation. The role of the nitrogen reactive radicals such as nitric oxide is separately analyzed because of its specific role in the nervous and vascular systems. The action of the other ROS on gene activation will then be reviewed by first looking at their possible involvement in the activation of transcription factors like NF-kappa B. Arguments will then be developed in favor of the implication of the ROS in the cellular effects of PMA, TNF-alpha and other cytokines on the modulation of the genetic expression. Possible mechanisms will be presented for linking the production of the ROS with cell activation. In a general way we postulate that ROS can play a role of secondary messengers in several cell responses to external stimuli. In the second part of the paper, we will examine the long term influence of ROS and their possible roles in cellular aging. Different links exist between ROS and aging and the relationship between them is probably indirect. We propose to consider the effect of ROS as one of the multiple challenges that cells have to face, the cell being considered as a global system which must optimize its energy expenditure for carrying out its basic functions such as turnover, differentiated phenotype functions, multiplication, defense and repair processes. This thermodynamic point of view will help to understand the effect of low ROS stresses, among others, on accelerated aging.

Repeated exposure of human skin fibroblasts to UVB at subcytotoxic level triggers premature senescence through the TGF-β1 signaling pathway

Premature senescence of human diploid fibroblasts (HDFs) can be induced by exposures to a variety of oxidative stress and DNA damaging agents. In this study we developed a robust model of UVB-induced premature senescence of skin HDFs. After a series of 10 subcytotoxic (non-proapoptotic) exposures to UVB at 250 mJ/cm2, the so-called biomarkers of senescence were markedly expressed: growth arrest, senescence-associated β-galactosidase activity, senescence-associated gene overexpression, deletion in mitochondrial DNA. A set of 44 stress- and senescence-associated genes were found to be differentially expressed in this model, among which clusterin/apolipoprotein J (apo J) and transforming growth factor-β1 (TGF-β1). Transfection of apo J cDNA provided protection against premature senescence-inducing doses of UVB and other stressful agents. Neutralizing antibodies against TGF-β1 or its receptor II (TβRII) sharply attenuated the senescence-associated features, suggesting a role for TGF-β1 in UVB-induced premature senescence. Both the latent and active forms of TGF-β1 were increased with time after the last UVB stress. Proteasome inhibition was ruled out as a potential mechanism of UVB-induced stress-induced premature senescence (SIPS). This model represents an alternative in vitro model in photoaging research for screening potential anti-photoaging compounds.

Wheat-derived arabinoxylan oligosaccharides with prebiotic effect increase satietogenic gut peptides and reduce metabolic endotoxemia in diet-induced obese mice

Background:Alterations in the composition of gut microbiota —known as dysbiosis— have been proposed to contribute to the development of obesity, thereby supporting the potential interest of nutrients acting on the gut microbes to produce beneficial effect on host energetic metabolism. Non-digestible fermentable carbohydrates present in cereals may be interesting nutrients able to influence the gut microbiota composition.Objective and design:The aim of the present study was to test the prebiotic potency of arabinoxylan oligosaccharides (AXOS) prepared from wheat bran in a nutritional model of obesity, associated with a low-grade chronic systemic inflammation. Mice were fed either a control diet or a high fat (HF) diet, or a HF diet supplemented with AXOS during 8 weeks.Results:AXOS supplementation induced caecal and colon enlargement associated with an important bifidogenic effect. It increased the level of circulating satietogenic peptides produced by the colon (peptide YY and glucagon-like peptide-1), and coherently counteracted HF-induced body weight gain and fat mass development. HF-induced hyperinsulinemia and the Homeostasis Model Assessment of insulin resistance were decreased upon AXOS feeding. In addition, AXOS reduced HF-induced metabolic endotoxemia, macrophage infiltration (mRNA of F4/80) in the adipose tissue and interleukin 6 (IL6) in the plasma. The tight junction proteins (zonula occludens 1 and claudin 3) altered upon HF feeding were upregulated by AXOS treatment suggesting that the lower inflammatory tone was associated with the improvement of gut barrier function.Conclusion:Together, these findings suggest that specific non-digestible carbohydrates produced from cereals such as AXOS constitute a promising prebiotic nutrient in the control of obesity and related metabolic disorders.

HAGR: the Human Ageing Genomic Resources

The Human Ageing Genomic Resources (HAGR) is a collection of online resources for studying the biology of human ageing. HAGR features two main databases: GenAge and AnAge. GenAge is a curated database of genes related to human ageing. Entries were primarily selected based on genetic perturbations in animal models and human diseases as well as an extensive literature review. Each entry includes a variety of automated and manually curated information, including, where available, protein–protein interactions, the relevant literature, and a description of the gene and how it relates to human ageing. The goal of GenAge is to provide the most complete and comprehensive database of genes related to human ageing on the Internet as well as render an overview of the genetics of human ageing. AnAge is an integrative database describing the ageing process in several organisms and featuring, if available, maximum life span, taxonomy, developmental schedules and metabolic rate, making AnAge a unique resource for the comparative biology of ageing. Associated with the databases are data-mining tools and software designed to investigate the role of genes and proteins in the human ageing process as well as analyse ageing across different taxa. HAGR is freely available to the academic community at http://genomics.senescence.info.

Anti-inflammatory effects of dietary phenolic compounds in an in vitro model of inflamed human intestinal epithelium.

Phenolic compounds (PCs) are considered to possess anti-inflammatory properties and therefore were proposed as an alternative natural approach to prevent or treat chronic inflammatory diseases. However their effects are not fully understood, particularly at the intestinal level. To further understand their mode of action at the molecular level during intestinal inflammation, an in vitro model of inflamed human intestinal epithelium was established. Different representative dietary PCs, i.e. resveratrol, ellagic and ferulic acids, curcumin, quercetin, chrysin, (-)-epigallocatechin-3-gallate (EGCG) and genistein, were selected. To mimic intestinal inflammation, differentiated Caco-2 cells cultivated in bicameral inserts, in a serum-free medium, were treated with a cocktail of pro-inflammatory substances: interleukin (IL)-1β, tumor necrosis factor-α, interferon-γ and lipopolysaccharides. The inflammatory state was characterized by a leaky epithelial barrier (attenuation of the transepithelial electrical resistance) and by an over-expression at the mRNA and protein levels for pro-inflammatory markers, i.e. IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1), quantified by ELISA and by gene expression analysis using a low-density array allowing the evaluation of expression level for 46 genes relevant of the intestinal inflammation and functional metabolism. Treatment with PCs, used at a realistic intestinal concentration, did not affect cell permeability. In inflamed cells, the incubation with genistein reduced the IL-6 and MCP-1 overproduction, to ca. 50% of the control, whereas EGCG provoked a decrease in the IL-6 and IL-8 over-secretion, by 50 and 60%, respectively. This occurred for both flavonoids without any concomitant inhibition of the corresponding mRNA expression. All the PCs generated a specific gene expression profile, with genistein the most efficient in the downregulation of the expression, or over-expression, of inflammatory genes notably those linked to the arachidonic metabolism pathway. In conclusion, this study provides evidence that genistein and EGCG downregulate the inflammatory response in inflamed intestinal epithelial cells by a pathway implicating largely a post-transcriptional regulatory mechanism.

Deoxynivalenol affects in vitro intestinal epithelial cell barrier integrity through inhibition of protein synthesis

Deoxynivalenol (DON), one of the most common mycotoxin contaminants of raw and processed cereal food, adversely affects the gastrointestinal tract. Since DON acts as a protein synthesis inhibitor, the constantly renewing intestinal epithelium could be particularly sensitive to DON. We analyzed the toxicological effects of DON on intestinal epithelial protein synthesis and barrier integrity. Differentiated Caco-2 cells, as a widely used model of the human intestinal barrier, were exposed to realistic intestinal concentrations of DON (50, 500 and 5000 ng/ml) during 24h. DON caused a concentration-dependent decrease in total protein content associated with a reduction in the incorporation of [(3)H]-leucine, demonstrating its inhibitory effect on protein synthesis. DON simultaneously increased the paracellular permeability of the monolayer as reflected through a decreased transepithelial electrical resistance associated with an increased paracellular flux of the tracer [(3)H]-mannitol. A concentration-dependent reduction in the expression level of the tight junction constituent claudin-4 was demonstrated by Western blot, which was not due to diminished transcription, increased degradation, or NF-kappaB, ERK or JNK activation, and was also observed for a tight junction independent protein, i.e. intestinal alkaline phosphatase. These results demonstrate a dual toxicological effect of DON on differentiated Caco-2 cells consisting in an inhibition of protein synthesis as well as an increase in monolayer permeability, and moreover suggest a possible link between them through diminished synthesis of the tight junction constituent claudin-4.

Replicative aging down-regulates the myogenic regulatory factors in human myoblasts.

Background information. Aging of human skeletal muscle results in a decline in muscle mass and force, and excessive turnover of muscle fibres, such as in muscular dystrophies, further increases this decline. Although it has been shown in rodents, by cross‐age transplantation of whole muscles, that the environment plays an important role in this process, the implication of proliferating aging of the muscle progenitors has been poorly investigated, particularly in humans, since the regulation of cell proliferation differs between rodents and humans. The myogenic differentiation of human myoblasts is regulated by the muscle‐specific regulatory factors. Cross‐talk between the muscle‐specific regulatory factors and the cell cycle regulators is essential for differentiation. The aim of the present study was to determine the effects of replicative senescence on the myogenic programme of human myoblasts.