Inhibition of glucocorticoid synthesis alleviates cognitive impairment in high-fat diet-induced obese mice.

In addition to cardiovascular diseases, metabolic syndrome and type 2 diabetes mellitus, obesity is associated with cognitive deficits. In rodents, it has been shown that long-term high-fat diet (HFD) consumption leads to the alteration of hypothalamic-pituitary-adrenal (HPA) axis resulting in increased corticosterone release. However, mechanisms underpinning cognitive impairments induced by long-term HFD intake are unclear. Herein we evaluated the effects of systemic administration of glucocorticoid synthesis inhibitor metyrapone on cognitive performance assessed by novel object recognition test and plasma corticosterone levels evaluated by enzyme-linked immunosorbent assay in HFD-induced obese mice. We found that metyrapone treatment alleviated recognition memory impairments in HFD-induced obese mice. Furthermore, glucocorticoid synthesis inhibitor also lowered plasma corticosterone levels in HFD-induced obese mice. Our findings indicate that hyperactivation of HPA axis resulting in elevated circulating glucocorticoid levels leads to memory impairments in HFD-induced obese mice. We identify glucocorticoid system as a potential therapeutic target for treating cognitive deficits associated with obesity condition.

Toxicity of DDT to the hooded oyster Saccostrea cucullata: Mortality, histopathology and molecular mechanisms as revealed by a proteomic approach.

Dichlorodiphenyltrichloroethane (DDT), a persistent organochlorine pesticide, has been linked to adverse biological effects in organisms. However, there is limited knowledge about its toxic effects on marine organisms and the underlying molecular mechanisms. This study investigated the toxic effects of DDT in the hooded oyster Saccostrea cucullata. The oysters were exposed to DDT at concentrations of 0, 10, 50, 100, 500, 1000 and 2000 µg/L for 96 h and the LC50 (96 h) was 891.25 µg/L. Two sublethal concentrations (10 and 100 µg/L) were used to investigate the histopathological effects and the proteome response. Histopathological results showed that DDT caused the alteration of mantle tissue. This included the induction of mucocytes in the epithelium and the inflammatory effect in the connective tissue indicated by the enlargement of blood sinus and hemocyte aggregation within the sinus. Proteomic results showed that, amongst approximately 500 protein spots that were detected across 2DE gels, 51 protein spots were differentially expressed (P < 0.01; fold change > 1.2). Of these, 29 protein spots were identified by LC-MS/MS. These included 23 up-regulated, 5 down-regulated and 1 fluctuating spots. Thus, we observed that stress response and cytoskeletal proteins are the central targets of DDT action. Furthermore, DDT alters the expression of proteins involved in energy metabolism, calcium homeostasis and other proteins of unknown function. Additionally, proteomic results clearly elucidated the molecular response of the histopathological changes which were driven by the alteration of cytoskeletal proteins. Our results improve the current knowledge of toxicity of the DDT to histology and molecular response of oyster proteome to DDT exposure. In addition, histopathological changes will be beneficial for the development of an appropriate guideline for health assessment of this species in ecotoxicological context.

A monoclonal antibody against Lates calcarifer vitellogenin and a competitive ELISA to evaluate vitellogenin induction after exposure to xenoestrogen.

A monoclonal antibody specific to sea bass (Lates calcarifer) vitellogenin (VTG) was developed, for use as a tool for monitoring endocrine disrupting chemicals (EDCs). VTG was induced in sea bass by intramuscular injection of 17ß-estradiol (E2: 2 mg/kg) every three days. Blood was collected three days after the last injection. Plasma VTG was then purified by chromatography in hydroxyapatite and a sephacryl-S300 column. Characterizations of purified VTG were done by phospholipoglycoprotein staining on a native-PAGE with confirmation by mass spectrometry (LC-MS/MS). Antibody was raised in mice by injection of purified VTG. After monoclonal antibody production, the hybridoma clone No. 41 (MAb-sea bass VTG 41) was selected and developed for quantification of VTG by competitive enzyme-linked immunosorbent assay (ELISA). The ELISA method was sensitive with a detection limit of VTG 40 ng/mL. MAb-sea bass VTG 41 was specific to VTG from E2-treated sea bass and others EDCs (Nonylphenol, Benzo[a]pyrene and CdCl2). Moreover, cross-reactivity was also found in E2-treated coral grouper (Epinephelus corallicola). The ELISA method obtained from this work can be further applied for the assessment of EDCs in Thailand and Southeast Asia's aquatic environment.

Histopathological effect and stress response of mantle proteome following TBT exposure in the Hooded oyster Saccostrea cucullata.

Tributyltin (TBT), an environmental pollutant in marine ecosystems, is toxic to organisms. Although contamination by and bioaccumulation and toxicity of this compound have been widely reported, its underlying molecular mechanisms remain unclear. In the present study, we exposed the Hooded oyster Saccostrea cucullata to TBT to investigate histopathological effects and proteome stress response. Animals were exposed to three TBT sub-lethal concentrations, 10, 50 and 150 µg/l for 48 h. TBT produced stress leading to histopathological changes in oyster tissues including mantle, gill, stomach and digestive diverticula. TBT induced mucocyte production in epithelia and hemocyte aggregation in connective tissue. Cell necrosis occurred when exposure dosages were high. Comparative proteome analyses of mantle protein of oysters exposed to 10 µg/l and control animals were analyzed by a 2-DE based proteomic approach. In total, 32 protein spots were found to differ (p < 0.05). Of these, 17 proteins were identified which included 14 up-regulated and 3 down-regulated proteins. TBT induced the expression of proteins involved in defensive mechanisms (HSP-78, HSP-70, aldehyde dehydrogenase and catalase), calcium homeostasis (VDAC-3), cytoskeleton and cytoskeleton-associated proteins, energy metabolism and amino acid metabolism. Our study revealed that TBT disturbs calcium homeostasis via VDAC-3 protein in mantle and this probably is the key molecular mechanism of TBT acting to distort shell calcification. Moreover, proteins involved in cell structure (tubulin-alpha and tubulin-beta) and protein synthesis were reduced after TBT exposure. Additionally, differential proteins obtained from this work will be useful as potential TBT biomarkers.

Proteomic identification of protein associated to mature spermatozoa in the Pacific oyster Crassostrea gigas.

Knowledge of sperm maturation process is limited in the Pacific oyster Crassostrea gigas and major factors of fertilization success of this free spawning animal are unknown. We investigated proteins associated to spermatozoa by analyzing two cellular fractions obtained from a 40-80% Percoll gradient fractioning of germ cell of mature male gonads. Mature spermatozoa were enriched in the lower Percoll fraction while the upper fraction contained less mature or earlier germ cells. A 2-DE proteomic approach was used to identify differentially expressed proteins in both fractions. We screened out 31 differential proteins (P<0.05) which included 14 up-regulated and 17 down-regulated proteins. Using MALDI-TOF/TOF MS and bioinformatics search against a C. gigas database, 13 and 8 proteins were identified for the up-regulated and down-regulated groups, respectively. In the spermatozoa enriched fraction, proteins regarding flagellum formation and control, energy production and Proteosome subunit beta were increased. In less mature germ cell fraction, proteins regarding developmental processes and chaperon molecules were mainly increased. Our results improve current knowledge of proteins associated with spermatozoa maturation related to zootechnical practices used in mollusk hatcheries. BIOLOGICAL SIGNIFICANCE: This is the revised version of the manuscript "Proteomic identification of protein associated to mature spermatozoa in the Pacific oyster Crassostrea gigas" by Kingtong et al. to the Journal of Proteomics. The corrections have been done by the team carefully. This work highlight the enrichment method of spermatozoa of Pacific oyster from stripped complex sample using Percoll gradient. The results reflexed developmental stages of germ cells in gonadal tubules of this species. We have used proteomic approach to identify differentially expressed proteins in mature spermatozoa fraction compared to less mature spermatozoa fraction which provided candidates of protein associated to mature spermatozoa in the Pacific oyster. This work improves the current knowledge in marine bivalve hatchery.

ATP-binding cassette multidrug transporters in Indian-rock oyster Saccostrea forskali and their role in the export of an environmental organic pollutant tributyltin.

ATP-binding cassette (ABC) multidrug transporters confer resistance in human cancer cells as well as in pathogenic microorganisms by mediating the extrusion of various chemotherapeutic drugs out of the cell. In aquatic invertebrates, the presence of ABC transporters which are involved in the multi-xenobiotic resistance has been demonstrated. However, most studies have been confined to the MDR1 subfamily. In the present study, we characterized the expression and localization of the ABC multidrug transporters including MDR1, MRP1 and BCRP subfamily in the Indian-rock oyster Saccostrea forskali. To our knowledge, these data represent one of the first reports on the orthologues of MRP1 and BCRP in marine invertebrates. Furthermore, the observations of (i) the expression of the ABC multidrug proteins in detoxifying tissues; (ii) the induction of these transporters upon exposure to an environmental organic pollutant tributyltin (TBT); and (iii) the concentration-dependent inhibition of rhodamine efflux by TBT imply a possible role of these proteins in the export of TBT. Our findings along with previous studies suggest that the ABC multidrug transporters act as a detoxifying mechanism of various toxic agents including TBT in aquatic organisms.