Proteomic and phosphoproteomic analysis of cellular responses in medaka fish (Oryzias latipes) following oral gavage with microcystin-LR.

Chronic and subchronic toxicity resulting from exposure to microcystins (MCs) receives increasing attention due to the risk of bioaccumulation of these toxins by aquatic animals, including fish. The mechanisms of action of MCs that target the liver, involve modifications of protein phosphorylation resulting from phosphatases 1 and 2A inhibition. Therefore, studying phosphoprotein modifications by using a specific phosphoprotein stain Pro-Q Diamond in fish liver contaminated with MC-leucine-arginine (MC-LR), the most toxic MC, should help dissecting disturbed signaling and metabolic networks. We have recently used this technology to identify several proteins that are modulated either in expression or phosphorylation in the liver of medaka following short-term exposure to MC-LR by balneation. In the present study, we have decided to use an alternative way of introducing the toxin into fish; that is by gavage (force-feeding). This was first achieved using tritiated MC-LR and allowed us to quantify the quantity of toxin incorporated into fish and to demonstrate that the toxin is mainly accumulated in liver. Afterwards a proteomics study limited to liver cytosolic proteins of contaminated animals showed that several proteins were up or down regulated either in quantity or in phosphorylation or both. Some of them had been previously detected as modified in balneation experiments but new molecules were identified as involved in signal transduction pathways activated by the toxin. In addition, in the conditions used (5 microg toxin/g body weight) anatomopathological studies supported a process of apoptonecrosis established after 24h, which was suggested to proceed by the evolution of some of the proteins after 2h contamination.

The COX-2 inhibitor nimesulide suppresses superoxide and 8-hydroxy-deoxyguanosine formation, and stimulates apoptosis in mucosa during early colonic inflammation in rats.

As we have shown previously [Tardieu,D., Jaeg,J.P., Cadet,J., Embvani,E., Corpet,D.E. and Petit,C. (1998) Cancer Lett, 134, 1-5], a 48-h treatment of 6% dextran sodium sulphate (DSS) in drinking water led to a reproducible 2-fold increase of the mutagenic oxidative lesion 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in colonic mucosa DNA of rats in vivo. The aim of this study was to test the effect of nimesulide, a preferential COX-2 inhibitor, on the DSS-induced 8-oxodGuo increase. We show that nimesulide when administered orally, simultaneously with DSS at 5 mg/kg/day, not only totally prevents 8-oxodGuo formation but also suppresses the 5-fold increase of superoxide induced by DSS in the colonic mucosa. This was measured by in vivo formazan blue precipitation (P < 0.01 in the Wilcoxon test). Moreover, nimesulide enhances apoptosis by approximately 30% as compared with the already high level induced by DSS treatment (P < 0.01). It is suggested that the significant increase in mutagenic oxidative DNA damage, produced by mild acute colonic inflammation, could be important in the initiation of colon cancer in both animals and man. These effects may explain at least partly the well-documented protective action towards colon cancer by preferential COX-2 inhibitors, either xenobiotics such as nimesulide or natural nutrients.

Dextran sulfate enhances the level of an oxidative DNA damage biomarker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, in rat colonic mucosa.

Dextran sodium sulfate (DSS) given in drinking water can induce colonic inflammation and produce colorectal tumors in rodents, although it is not directly genotoxic. The hypothesis that DSS can produce free radicals and induce oxidative DNA damage in colonic mucosa has been tested. In rats fed for 2 days with water containing 3% and 6% DSS, colonic inflammation manifestations were recorded and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), a major biomarker of oxidative DNA damage, was assayed in colonic mucosa. As compared with control rats given pure water, inflammatory manifestations were seen in rats given DSS. At the same time, 8-oxodGuo levels in colonic mucosa were doubled (P < 0.001). These results suggest that formation of oxidative DNA damage in colonic mucosa depends on inflammation and maybe on the production of reactive oxygen species. This study shows that DSS can induce oxidative DNA damage within only 2 days, which could explain in part its carcinogenic properties.

In vitro eukaryotic DNA excision repair assays: an overview.

Great progress is being made in understanding the process of nucleotide excision repair (NER) in eukaryotes. Different lines of research have been developed, among them an in vitro assay with cell-free extracts has played a major role. This in vitro repair assay takes advantage of a cell-free system that can mediate DNA excision-repair by transcriptionally active protein extracts from mammalian cells incubated in the presence of two plasmids of different sizes, one damaged and the other undamaged as internal control. The extent of repair activity is determined by following the level of radiolabeled incorporation during the repair synthesis step consecutive to the excision of DNA lesions. We discuss the interest and drawbacks of this biochemical assay in light of the main results obtained. We report the modifications that we have undertaken in order to determine repair synthesis activity in a chemiluminescent-directed reaction as well as to assess incision activity in protein extracts.

UV induction of excision repair enzymes detected in protein extracts from Schizosaccharomyces pombe.

Induction of genes and proteins after DNA damaging treatment is well documented in various biological systems. In order to monitor repair activity in Schizosacchromyces pombe, we adapted the biochemical assay that allowed specific quantification of excision repair in mammalian cells (Wood et al. 1988, Cell, 53, 97-106) to yeast-free extracts. Repair synthesis determined on UV-damaged plasmid DNA with S. pombe total protein extract relied on base excision repair and not nucleotide excision repair. Under conditions that allowed optimal repair activity, an enhanced repair synthesis was found with extract from yeast previously irradiated with UV light (254 nm). A 4-fold induction factor was obtained with 70 J/m2 irradiation dose after 40 min incubation post-irradiation. This base excision repair activity on UV photoproducts was transiently induced since it returned to the level of untreated yeast after about 2 hours post-irradiation.

Variations of plasma creatine kinase in rabbits following repetitive blood sampling effects of pretreatment with acepromazine, carazolol and dantrolene.

Plasma creatine kinase activity increased significantly (P less than 0.001) in rabbits sampled every two hours for 12 hours (mean from 509 to 2242 U/l), but did not change when rabbits had been accustomed to laboratory handling procedures for two weeks. This increase was not alleviated or only moderately by pretreatment with acepromazine (per os, 2.5 mg.kg-1), carazolol (intravenously, 0.05 mg.kg-1) or dantrolene (intravenously, 1.0 mg.kg-1). Thus, when using plasma creatine kinase in rabbits, e.g. to test muscle damage or local tolerance of drugs, animals should be made familiar with laboratory procedures before any experiment.