The p53 binding protein PDCD5 is not rate-limiting in DNA damage induced cell death

The tumour suppressor p53 is an important mediator of cell cycle arrest and apoptosis in response to DNA damage, acting mainly by transcriptional regulation of specific target genes. The exact details how p53 modulates this decision on a molecular basis is still incompletely understood. One mechanism of regulation is acetylation of p53 on lysine K120 by the histone-acetyltransferase Tip60, resulting in preferential transcription of proapoptotic target genes. PDCD5, a protein with reported pro-apoptotic function, has recently been identified as regulator of Tip60-dependent p53-acetylation. In an effort to clarify the role of PDCD5 upon DNA damage, we generated cell lines in which PDCD5 expression was conditionally ablated by shRNAs and investigated their response to genotoxic stress. Surprisingly, we failed to note a rate-limiting role of PDCD5 in the DNA damage response. PDCD5 was dispensable for DNA damage induced apoptosis and cell cycle arrest and we observed no significant changes in p53 target gene transcription. While we were able to confirm interaction of PDCD5 with p53, we failed to do so for Tip60. Altogether, our results suggest a role of PDCD5 in the regulation of p53 function but unrelated to cell cycle arrest or apoptosis, at least in the cell types investigated.FP06 RTN ‘ApopTrain’Tyrolean Science FundKrebshilfe-Tyro

Additive effect of calcium depletion and low resource quality on Gammarus fossarum (Crustacea, Amphipoda) life history traits

International audienceGammarus fossarum is an often-abundant crustacean detritivore that contributes importantly to leaf litter breakdown in oligotrophic, mainly heterotrophic, headwater streams. This species requires large amounts of Ca to moult, thus allowing growth and reproduction. Because resource quality is tightly coupled to the organism's growth and physiological status, we hypothesised that low Ca concentration [Ca] and low food resource quality (low phosphorus [P] and/ or reduced highly unsaturated fatty acid [HUFA] contents) would interactively impair molecular responses (gene expression) and reproduction of G. fossarum. To investigate the effects of food resources quality, we experimentally manipulated the P content of sycamore leaves and also used diatoms because they contain high amounts of HUFAs. Three resource quality treatments were tested: low quality (LQ, unmanipulated leaves: low P content), high quality 1 (HQ1; P-manipulated leaves: high P content), and high quality 2 (unmanipulated leaves supplemented with a pellet containing diatoms: high P and HUFA content). Naturally, demineralised stream water was supplemented with CaSO4 to obtain three Ca concentrations (2, 3.5, and 10.5 mg Ca

Impact of intracellular metallothionein on metal biouptake and partitioning dynamics at bacterial interfaces

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A sub-individual multilevel approach for an integrative assessment of CuO nanoparticle effects on Corbicula fluminea

International audienceBecause they are widely used, copper oxide nanoparticles (CuO NPs) are likely to enter the aquatic environment and then reach the sediment. We have examined the effect of CuO NPs in the freshwater endobenthic bivalve Corbicula fluminea. Some previous studies have investigated effects at biochemical and physiological levels, but molecular endpoints are still poorly studied despite they are sensitive in early detection of NPs effect. In the present study, we have investigated short-term effects (96 h) of CuO NP (12, 30 nm; 0, 20 and 100 mg/L) using molecular endpoints as well as more conventional biochemical and physiological markers. The expression of antioxidant (CuZnSOD, MnSOD, Cat, Se-GPx, Trxr) and antitoxic (GST-Pi, HSP70, MT, Pgp, MRP1) related genes was measured at the mRNA level while anti-oxidant (SOD, TAC) and antitoxic (GST, ACP) defenses, energetic reserves and metabolism (ETS, Tri, LDH), and cellular damages (LPO) were assessed using a biochemical approach. The filtration rate measured at 96 h provided information at the physiological scale. Gene expression and filtration rate were responsive to CuO NPs but the effects differed according to the NP size. The results suggest that defense mechanisms may have been set up following 30 nm-NP exposure. The response to 12 nm-NP was lower but still showed that exposure to 12 nm-NP led to activation of cellular elimination mechanisms. The lowering of the filtration rate may have protected the organisms from the contamination. However, this raised the question of further repercussions on organism biology. Together, the results (i) indicate that CuO NP may exert effects at different levels even after a short-term exposure and (ii) point out the precocity of molecular response

Genotoxicity and physiological effects of CeO2 NPs on a freshwater bivalve (Corbicula fluminea)

International audienceThe rapid development of nanotechnology and the increased use of nanomaterials in products used in everyday life have raised the question of the potential release of nanoparticles into the aquatic environment. Their fate and effects in natural ecosystems are not currently well understood but harmful effects of nanoparticles have been demonstrated at low concentrations on some freshwater and marine species. Cerium dioxide nanoparticles (CeO2 NPs) are produced in large quantities and used in products in many different fields, such as automotives or optics. Because of their widespread use in daily products, CeO2 NPs are included in the OECD priority list of manufactured nanomaterials for human and environmental assessment. Indeed some studies have been conducted to assay various enzymatic biomarkers, which showed the CeO2 NPs potential to modify anti-oxidative defenses and cellular membrane stability. Nevertheless, only a few studies were performed on their genotoxic potential. The aim of this work was to evaluate the genotoxic and physiological effects of CeO2 NPs on a widespread freshwater bivalve Corbicula fluminea by using comet assay and a multi-enzymatic biomarker approach. Exposure to two CeO2 NP concentrations during a short term experiment (6 days) was set up. The first one (10 μg/L) was chosen in order to work with low but measurable concentrations whereas the second one was ten times higher (100 μg CeO2 NPs/L). DNA damage was significantly more pronounced compared with control for both concentrations tested as early as two days of exposure and seemed to increase with time. Some enzymatic biomarkers of anti-oxidative defenses (total antioxidant capacity, catalase activity), anti-toxic mechanisms (glutathione-S-transferase activity, caspase-3 activity) or metabolism (lactate dehydrogenase activity) tended to increase after 6 days of exposure but only the induction of caspase pathway and DNA damages appeared significant for exposed organisms. In this study, time and concentration effects of CeO2 NPs were highlighted by coupling genotoxic and cellular biomarker assessments

Integrated assessment of ceria nanoparticle impacts on the freshwater bivalve Dreissena polymorpha

International audienceExposures in realistic environmental conditions are essential to properly assess the effects of emerging pollutants on ecosystems. While ceria nanoparticles (nCeO 2) production and use are expanding quickly, ecotoxicity studies remain very scarce. In this work, we set up experimental systems reproducing a simplified ecosystem to assess the effects of a chronic exposure to citrate-coated nCeO 2 (ci-CeO 2) and bare nCeO 2 (ba-CeO 2) on the freshwater mussel Dreissena polymorpha using an inte‐ grated multibiomarker approach. The fate of nanoparticles was tightly monitored to properly characterize the exposure. Organisms were exposed for three weeks and sampled weekly for biomarker analysis. Mussel filter-feeding activity resulted in significant removal of nCeO 2 from the water column. At the same time, bioaccumu‐ lation was low, reaching its maximum in the first week. Mussels bioaccumulated ci-CeO 2 three times more than ba-CeO 2 , probably due to coating-related differences in their behavior in the water column and in organisms. Meanwhile, biomarker results were integrated and synthesized using linear discriminant analysis, highlighting that pi-glutathione-S-transferase (piGST) mRNA, catalase (CAT) activity, and lysosomal system were the most impacted of the seven biomarkers singled out by the discriminant analysis. These biomarker responses indicated that mussels exposed to both forms of nCeO 2 were stressed and differentiate from the controls. Moreover,theyresponded differently to ba-CeO 2 and ci-CeO 2 exposure. How‐ ever, biomarkers used in the experimental conditions of the present study did not indicate severe nCeO 2 toxicity on mussels, as cellular damage biomarkers and mussel filtering activity were left unimpaired. However, further studies are needed to investigate if the slight perturbations observed could lead to populational impacts in the long-term

Caspase-2 activation in the absence of PIDDosome formation

International audiencePIDD (p53-induced protein with a death domain [DD]), together with the bipartite adapter protein RAIDD (receptor-interacting protein-associated ICH-1/CED-3 homologous protein with a DD), is implicated in the activation of pro–caspase-2 in a high molecular weight complex called the PIDDosome during apoptosis induction after DNA damage. To investigate the role of PIDD in cell death initiation, we generated PIDD-deficient mice. Processing of caspase-2 is readily detected in the absence of PIDDosome formation in primary lymphocytes. Although caspase-2 processing is delayed in simian virus 40–immortalized pidd−/− mouse embryonic fibroblasts, it still depends on loss of mitochondrial integrity and effector caspase activation. Consistently, apoptosis occurs normally in all cell types analyzed, suggesting alternative biological roles for caspase-2 after DNA damage. Because loss of either PIDD or its adapter molecule RAIDD did not affect subcellular localization, nuclear translocation, or caspase-2 activation in high molecular weight complexes, we suggest that at least one alternative PIDDosome-independent mechanism of caspase-2 activation exists in mammals in response to DNA damage