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Low-density lipoprotein (LDL) oxidation is a key step in atherogenesis, promoting the formation of lipid-laden macrophages. Here, we compared the effects of copper-oxidized LDLs (OxLDLs) and of the more physiologically relevant myeloperoxidase-oxidized LDLs (MoxLDLs) in murine RAW264.7 macrophages and in human peripheral blood monocyte-derived macrophages. Both oxidized LDLs, contrary to native LDLs, induced foam cell formation and an intracellular accumulation of reactive oxygen species (ROS). This oxidative stress was responsible for the activation of the NF-E2-related factor 2 (Nrf2) transcription factor, and the subsequent Nrf2-dependent overexpression of the antioxidant genes, Gclm and HO-1, as evidenced by the invalidation of Nrf2 by RNAi. MoxLDLs always induced a stronger response than OxLDLs. These differences could be partly explained by specific ROSproducing mechanisms differing between OxLDLs and MoxLDLs. Whereas both types of oxidized LDLs caused ROS production partly by NADPH oxidase, only MoxLDLs-induced ROS production was dependent on cytosolic PLA2. This study highlights that OxLDLs and MoxLDLs induce an oxidative stress, through distinct ROSproducing mechanisms, which is responsible for the differential activation of the Nrf2 pathway. These data clearly suggest that results obtained until now with copper oxidized-LDLs should be carefully reevaluated, taking into consideration physiologically more relevant oxidized LDLs. Antioxid. Redox Signal. 13, 1491-1502.

Apoptosis has become recognized as a crucial mechanism involved in a wide range of physiological and pathological processes. Following an initial pro-apoptotic signal, controlling phases allow the cell to reinforce or downgrade signals leading to the irrevocable entry into apoptosis.

The potential genotoxic and carcinogenic properties reported for malachite green (MG) and the frequent detection of MG residues in fish and fish products, despite the ban of MG, have recently generated great concern. Additional toxicological data are required for a better understanding of the mechanism of action and a more comprehensive risk assessment for the exposure of fish to this fungicide. To date, the use of fish peripheral blood mononuclear cells (PBMCs) has not been exploited as a tool in the assessment of the toxicity of chemicals. However, PBMCs are exposed to toxicants and can be easily collected by blood sampling. The present study aims at better understanding the effects of MG by a proteomic analysis of primary cultured PBMC from the Asian catfish, Pangasianodon hypophthalmus, exposed to MG. The two lowest concentrations of 1 and 10 ppb were selected based on the MTS (water soluble tetrazolium salts) cytotoxicity test. Using a proteomic analysis (2D-DIGE), we showed that 109 proteins displayed significant changes in abundance in PBMC exposed during 48 h to MG. Most of these proteins were successfully identified by nano LC-MS/MS and validated through the Peptide and Protein Prophet of Scaffold TM software, but only 19 different proteins were considered corresponding to a single identification per spot. Our data suggest that low concentrations of MG could affect the mitochondrial metabolic functions, impair some signal transduction cascades and normal cell division, stimulate DNA repair and disorganize the cytoskeleton. Altogether, these results confirm that the mitochondrion is a target of MG toxicity. Further studies on the identified proteins are needed to better understand the mechanisms of MG toxicity in fish produced for human consumption.

Using a proteomic approach, we characterized different protein expression profiles in anterior gills of the Chinese mitten crab, Eriocheir sinensis, after cadmium (Cd) exposure. Two experimental conditions were tested: (i) an acute exposure (i.e. 500 g Cd l −1 for 3 days) for which physiological, biochemical and ultrastructural damage have been observed previously; (ii) a chronic exposure (i.e. 50 g Cd l −1 for 30 days) resulting in physiological acclimation, i.e. increased resistance to a subsequent acute exposure. Two-dimensional gel electrophoresis (2-DE) revealed six protein spots differentially expressed after acute, and 31 after chronic Cd exposure. From these spots, 15 protein species were identified using MS/MS micro-sequencing and MS BLAST database searches. Alpha tubulin, glutathione S-transferase and crustacean calcium-binding protein 23 were downregulated after an acute exposure, whereas another glutathione S-transferase isoform was up-regulated. Furthermore, analyses revealed the over-expression of protein disulfide isomerase, thioredoxin peroxidase, glutathione S-transferase, a proteasome subunit and cathepsin D after chronic exposure. Under the same condition, ATP synthase beta, alpha tubulin, arginine kinase, glyceraldehyde-3-phosphate dehydrogenase and malate dehydrogenase were down-regulated. These results demonstrate that acute and chronic exposure to waterborne Cd induced different responses at the protein expression level. Protein identification supports the idea that Cd mainly exerts its toxicity through oxidative stress induction and sulfhydryl-group binding. As a result, analyses showed the up-regulation of several antioxidant enzymes and chaperonins during acclimation process. The gill F. Silvestre et al. / Aquatic Toxicology 76 (2006) 46-58 47 proteolytic capacity seems also to be increased. On the other hand, the clearly decreased abundance of several enzymes involved in energy transfer suggests that chronic metal exposure induced an important metabolic reshuffling.

The environmental persistence, bioaccumulative tendency and potential toxicity of perfluorooctane sulfonate (PFOS) have generated great concern. This study aimed at evaluating the toxicity of short-term PFOS exposure in gills of the European bullhead Cottus gobio, a candidate sentinel species, by monitoring the response of some enzymes (citrate synthase CS, cytochrome c oxidase CCO, and lactate dehydrogenase LDH), and by undertaking a proteomic analysis using 2D-DIGE. First, a 96-h exposure to 1 mg PFOS/L significantly altered the activity of mitochondrial CS and CCO. Second, 2D-DIGE gels were used to compare gills from the control fish group with tissues from fish exposed for 96 h to either 0.1 or 1 mg PFOS/L. From the 27 protein spots displaying significant changes in abundance following PFOS exposure, a total of 20 different proteins were identified using nano LC-MS/MS and the Peptide and Protein Prophet of Scaffold software. The differentially expressed proteins that were identified are involved in the general stress response, ubiquitin-proteasome system, energy metabolism, and actin cytoskeleton, which provide clues on the cellular pathways and components mainly affected by PFOS. Moreover, our results showed that most proteins were differentially expressed at the low but not at the high PFOS concentration. This work provides insights into the biochemical and molecular events in PFOS-induced toxicity in gill tissue, and suggests that further studies on the identified proteins could provide crucial information to better understand the mechanisms of PFOS toxicity in fish.

The master regulator of the adaptive response to hypoxia is HIF-1. However, while some data show that HIF-1 can control more than 80% of the genes induced under hypoxia, other experiments clearly demonstrate that a part of the hypoxic response is independent of HIF-1. The goal of this study was to identify some of these HIF-1 independent factors and to investigate their functional role in the adaptation of tumor cells to hypoxia. We show that the cytoplasmic dynein intermediate chain 2 (DH IC-2), a component of an intracellular ATPase minus-end directed tubulin-based motile complex, was stabilized and posttranslationally modified under hypoxia in a HIF-1 independent way. We identified this modification as a phosphorylation by protein kinase C, which is inhibited under hypoxia. In parallel, the migration of HepG2 cells was enhanced under hypoxia. Cell migration was also increased, to the same extent, by the invalidation of DH IC-2 using siRNA. Taken together, these results suggest that under hypoxia, a specific modification of DH IC-2 may modulate its activity, and in turn promote cell migration. These results are important to better understand cancer development since they highlight a HIF-1 independent mechanism, which may be involved in metastasis.

Hypoxia-inducible factor-1 (HIF-1) is the major transcription factor speci®cally activated by hypoxia. It induces the expression of different genes whose products play an adaptive role for hypoxic cells and tissues. Besides these protective responses, HIF-1 and/or hypoxia have also been shown to be either anti-apoptotic or pro-apoptotic, according to the cell type and experimental conditions. More severe or prolonged hypoxia rather induces apoptosis that is, at least in part, initiated by the direct association of HIF-1a and p53 and p53induced gene expression. On the other hand, HIF-1a dimerized with ARNT, as an active transcription factor, can protect cells from apoptosis induced by several conditions. This review is aimed to describe the different mechanisms that account for these opposite effects of HIF-1a. #

Nuclear factor kappa B (NF-KB) is a potent and pleiotropic transcription factor that can be activated by a wide variety of inducers, including interleukin-1 (IL-l). Although the detailed activation mechanism of NF-KB is still under investigation, it requires both phosphorylation and degradation of its inhibitory subunit IKB and the presence of an oxidative environment.

Changes in endothelium functions during ischemia are thought to be of importance in numerous pathological conditions, with, for instance, an increase in the release of in¯ammatory mediators like prostaglandins. Here, we showed that hypoxia increases phospholipase A 2 (PLA 2 ) activity in human umbilical vein endothelial cells. Both basal PLA 2 activity and PG synthesis are sensitive to BEL and AACOCF3, respectively, inhibitors of calcium-independent PLA 2 (iPLA 2 ) and cytosolic PLA 2 (cPLA 2 ), while OPC, an inhibitor of soluble PLA 2 (sPLA 2 ) only inhibited the hypoxia-induced AA release and PGF 2a synthesis. Hypoxia does not alter expression of iPLA 2 , sPLA 2 and cPLA 2 and cycloheximide did not inhibit PLA 2 activation, indicating that hypoxia-induced increase in PLA 2 activity is due to activation rather than induction. However, mRNA levels for sPLA 2 displayed a 2-fold increase after 2 hr incubation under hypoxia. BAPTA, an intracellular calcium chelator, partially inhibited the AA release in normoxia and in hypoxia. Direct assays of speci®c PLA 2 activity showed an increase in sPLA 2 activity but not in cPLA 2 activity after 2 hr hypoxia. Taken together, these results indicate that the hypoxia-induced increase in PLA 2 activity is mostly due to the activation of sPLA 2 . #