PBDE-47 exposure causes gender specific effects on apoptosis and heat shock protein expression in marine medaka, Oryzias melastigma.

Marine medaka (Oryzias melastigma) was fed with a low and high dose of dietary 2,2',4,4'-tetra-bromodiphenyl ether (PBDE-47), over 21 days. Gender specific changes in caspases 3 and 8 in medaka were found as activities in male medaka were significantly increased in both liver and muscle at both low and high exposure levels whereas caspase activity in female medaka tissue remained unchanged. Results of HSP90 and HSP70 immunoassays also showed gender specific related changes as both HSP families were unchanged in liver and muscle of male medaka but significantly increased in liver and muscle of female medaka, following PBDE-47 exposure. The gender specific effects of PBDE-47 on HSP expression profiles could not be explained by inherent differences in the heat shock response of male and female marine medaka, as the HSP profiles in liver and muscle, induced by acute heat shock, were similar in both sexes. The findings from this study provide evidence that PBDE-47 can cause gender specific modulatory effects on mechanisms critical to the apoptotic cascade as well as HSP regulation and expression.

Aquaporin 1a expression in gill, intestine, and kidney of the euryhaline silver sea bream.

This study aimed to investigate the effects of chronic salinity acclimation, abrupt salinity transfer, and cortisol administration on aquaporin 1 (AQP1) expression in gill, intestine, and kidney of silver sea bream (Sparus sarba). An AQP1a cDNA was cloned and found to share 83-96% amino acid sequence identity with AQP1 genes from several fish species. Tissue distribution studies of AQP1a mRNA demonstrated that it was expressed in gill, liver, intestine, rectum, kidney, heart, urinary bladder, and whole blood. Semi-quantitative RT-PCR analysis was used to measure AQP1a transcript abundance in sea bream that were acclimated to salinity conditions of 0, 6, 12, 33, 50, and 70 ppt for 1 month. The abundance of gill AQP1a transcript was highest in sea bream acclimated to 0 ppt whereas no differences were found among 0-50 ppt groups. For intestine, the highest AQP1a transcript amounts were found in sea bream acclimated to 12 and 70 ppt whereas the transcript abundance of kidney AQP1a was found to be unchanged amongst the different salinity groups. To investigate the effects of acute salinity alterations on AQP1a expression, sea bream were abruptly transferred from 33 to 6 ppt. For intestine AQP1a levels were altered at different times, post transfer, but remained unchanged in gill and kidney. To study the effects of cortisol on AQP1a expression, sea bream were administered a single dose of cortisol followed by a 3-day acclimation to either 33 or 6 ppt. The findings from this experiment demonstrated that cortisol administration resulted in alterations of AQP1a transcript in gill and intestine but not in kidney.

Growth hormone attenuates branchial HSP70 expression in silver sea bream.

In this study the effects of growth hormone (GH) on silver sea bream branchial heat-shock protein 70 (HSP70) expression was investigated using in-vivo and in-vitro experiments. For in-vivo experiments, sea bream were administered recombinant bream GH or the GH secretagogue hexarelin. Pituitary levels of GH were unchanged in fish administered exogenous GH but decreased on hexarelin administration, in comparison with saline controls. Levels of HSP70 were measured using immunoanalysis and it was found that both GH and hexarelin administration caused a significant decrease in branchial HSP70 abundance. For in-vitro analysis, branchial filaments were exposed to a range of GH concentrations (1, 10, and 100 ng/ml) and it was found that HSP70 levels were significantly lowered in all cases. This study adds to the growing body of evidence surrounding the importance of hormones in regulating heat-shock protein expression in fish.

Impact of nitrite exposure on endocrine, osmoregulatory and cytoprotective functions in the marine teleost Sparus sarba.

The effects of nitrite, at varying concentrations (0, 25 and 50mg/l), on silver sea bream (Sparus sarba), was assessed after 7 days exposure. Nitrite exposure resulted in an elevated renosomatic index in parallel with increased kidney water content. Measurements of serum thyroid hormones demonstrated that levels of thyroxine (T(4)) were decreased upon nitrite exposure whereas triiodothyronine (T(3)) concentrations remained unchanged. Nitrite did not affect serum K and Na levels but did cause an increase in gill sodium pump (Na(+)-K(+)-ATPase) activity. Using immunoassays, it was found that the abundance of the water channel protein, aquaporin 3 (AQP3) was unchanged in gills but decreased in kidneys of sea bream upon nitrite exposure. Immunoassay analysis also demonstrated that the amount of the heat shock protein 70 (HSP70) family were increased in gills, kidney and liver during nitrite exposure whereas amounts of the heat shock protein 90 (HSP90) family increased in kidneys and liver. Taken together, the findings from this study provide new insights into how nitrite affects osmoregulatory, endocrine processes and heat shock protein expression in a marine fish.

Effects of mitogenic hormones on HSP70 expression in a silver sea bream fibroblast cell line and a primary macrophage preparation.

This study was performed in order to elucidate the role and importance of three mitogenic hormones [growth hormone (GH), insulin-like growth factor-I (IGF-1) and prolactin (PRL)] on heat shock protein 70 (HSP70) expression in a silver sea bream fibroblast cell line and a primary macrophage preparation. Fibroblasts and macrophages that were exposed to GH at concentrations of 1-1000ng/ml did not exhibit modulated HSP70 expression, whereas GH at an exposure concentration of 10ng/ml lowered HSP70 levels in macrophages. Upon exposure to IGF-1 it was found that HSP70 expression remained unchanged in fibroblasts but was significantly decreased in macrophages at exposure concentrations of 1-10ng/ml. Finally, and using a preparation of ovine PRL it was found that HSP70 expression decreased in fibroblasts at exposure concentrations of 1-1000ng/ml and also decreased in macrophages at exposure concentrations of 1-100ng/ml.

Cortisol can be pro- or anti-apoptotic in sea bream cells: potential role of HSP70 induction for cytoprotection.

Cortisol, and heat shock protein 70 (HSP70) are known to perform key roles as part of the fish stress response. In the present study, two in vitro systems were used to investigate a possible cortisol-HSP70-apoptosis regulatory relationship. Using a developed silver sea bream fibroblast cell line (SSF), cortisol was found to induce HSP70 synthesis with a concomitant protection against camptothecin induced apoptosis. The induction of HSP70 synthesis using azetidine was also found to protect SSF against apoptosis. A primary culture of silver sea bream macrophages (SSM) displayed reduced HSP70, underwent apoptosis and displayed reduced phagocytic activity upon exposure to cortisol. The effect of cortisol on HSP70 expression in both SSF and SSM were blocked by the glucocorticoid antagonist, RU486. Treatment of SSM with azetidine protected against apoptosis and also enhanced phagocytic activity. The data from this study demonstrates for the first time that cortisol can be either anti- apoptotic or pro-apoptotic in different fish cells and such actions can be mediated via HSP70 induction or suppression respectively.

Tissue distribution, effects of salinity acclimation, and ontogeny of aquaporin 3 in the marine teleost, silver sea bream (Sparus sarba).

The purpose of the present study was to ascertain the tissue-specific expression of the water channel protein, aquaporin 3 (AQP3), during salinity acclimation and larval development of silver sea bream (Sparus sarba). A cDNA fragment encoding aquaporin 3 (aqp3) from silver sea bream gill was cloned and from the deduced amino acid sequence a polyclonal antibody was prepared. AQP3 was found to be present in gill, kidney, liver, brain, heart, and spleen but not in whole blood. The abundance of AQP3 was significantly highest in gills of hypoosmotic (6 ppt) and isoosmotic (12 ppt) acclimated sea bream when compared to seawater (33 ppt) and hypersaline (50 ppt)- acclimated sea bream. Spleen tissue also displayed significantly high levels of AQP3 protein in hypoosmotic and isoosmotic salinities whereas the AQP3 abundance in brain, liver, heart, and kidney remained unchanged across the range of salinities tested. The ontogenetic profile of AQP3 was also investigated from developing sea bream larvae and AQP3 was first detected at 14 days posthatch (dph) and increased steadily up to 28-46 dph. In conclusion, this study has demonstrated that AQP3 expression is modulated in gill and spleen tissue of salinity acclimated sea bream and that it can be detected relatively early during larval development.

Impact of heavy metals and organochlorines on hsp70 and hsc70 gene expression in black sea bream fibroblasts.

The aim of the present study was to investigate the effects of environmentally important heavy metals and organochlorines on the transcriptional profiles of genes coding for heat shock cognate 70 (hsc70) and inducible heat shock protein 70 (hsp70) in a black sea bream fibroblast cell line. Using the nucleotide sequence information, from the cloned genes, specific reverse transcriptase-polymerase chain reaction (RT-PCR) methods were devised to test the effects of heavy metals (Cd2+, Cu2+ and Ni2+) and organochlorines (aroclor 1254, hexachlorobenzene and 2-4-dichloroaniline) on the cell stress response. Hsp70 was induced in fibroblasts upon heavy metal exposure concentrations as low as 0.01 microM whereas hsc70 expression was induced upon organochlorine exposure concentrations as low as 0.001 microM. Overall, our findings demonstrate that gene members of the HSP70 family are responsive to environmentally important chemicals.

Molecular cloning of growth hormone from silver sea bream: effects of abiotic and biotic stress on transcriptional and translational expression.

The pituitary growth hormone (GH) gene of silver sea bream (Sparus sarba) was cloned and characterized and found to be 615 base pairs encoding a protein of 204 amino acids. Using a bacterial expression system, recombinant protein was prepared and rabbit polyclonal antibody was raised. Transcript and protein amounts of GH were measured in fish that were adapted to a range of salinities, acclimated to different temperatures, or undergoing a natural time course of Vibrio alginolyticus infection. Isoosmotic salinity (12 ppt) adaptation resulted in increased GH transcript and protein in comparison to freshwater (0 ppt) and seawater (33 ppt) adapted fish. It was also found that cold temperature (12 degrees C) acclimated sea bream had higher amounts of pituitary GH transcript and protein when compared to warm temperature (25 degrees C) acclimated fish. Finally, the amounts of GH transcript and protein were found to be rapidly downregulated from an early stage of disease. The results from the present study demonstrate how GH can be modulated during both abiotic and biotic stress in fish.

Expression studies on glucose-6-phosphate dehydrogenase in sea bream: effects of growth hormone, somatostatin, salinity and temperature.

In the present study, the gene encoding hepatic glucose-6-phosphate dehydrogenase (G6PDH) was cloned and characterized from silver sea bream (Sparus sarba). The deduced amino acid sequence from sea bream g6pdh shared 78-84% homology with deduced amino acid sequences from previously cloned teleost g6pdh genes. A reverse transcriptase polymerase chain reaction (RT-PCR) coupled with radioisotope hybridization method was used for assessment of g6pdh expression and it was found that administration of growth hormone to sea bream increased g6pdh transcript and G6PDH activity whereas injections of somatostatin decreased both of these parameters. It was also found that sea bream maintained at an isoosmotic salinity (12 ppt) and cold temperature (12 degrees C) displayed upregulated g6pdh expression and enhanced G6PDH activtity. The results from this study demonstrate that g6pdh expression can be mediated by both hormonal and environmental factors in teleosts.

Growth hormone increases hsc70/hsp70 expression and protects against apoptosis in whole blood preparations from silver sea bream.

Preparations of whole blood, from silver sea bream (Sparus sarba), were used for heat shock protein 70 (hsp70) and apoptosis studies. It was found that the expression of both gene members of the hsp70 family (hsc70 and hsp70) were upregulated during acute heat shock. The transcript abundance of both of these genes was increased when cells were exposed to growth hormone (GH) at concentrations of 10 and 100 ng/mL. It was also found that GH at concentrations of 10 and 100 ng/mL could protect whole blood from camptothecin-induced apoptosis as determined by DNA fragmentation analysis.

Upregulation of the somatotropic axis is correlated with increased G6PDH expression in Black Sea bream adapted to iso-osmotic salinity.

Black sea bream (Mylio macrocephalus) were adapted to salinities of 0 ppt (freshwater), 6 ppt (hypo-osmotic), 12 ppt (iso-osmotic), 33 ppt (seawater), and 50 ppt (hypersaline) for 1 month. Using RT-PCR assays, the expression of pituitary growth hormone (GH) and hepatic insulin-like growth factor 1 (IGF-1) genes were studied. It was found that the transcripts for both of these genes were highest in fish maintained at iso-osmotic salinity. To correlate the expression of GH and IGF-1 with an index of growth, we also measured the transcript levels of glucose-6-phosphate dehydrogenase (G6PDH) in liver. It was found that this transcript was also elevated in iso-osmotically adapted black sea bream.

Cloning and characterization of sea bream Na+-K+-ATPase alpha and beta subunit genes: in vitro effects of hormones on transcriptional and translational expression.

The full length genes encoding the catalytic alpha and glycosylated beta subunits of the sodium pump (Na+-K+-ATPase) were cloned and characterized from silver sea bream gill. Using in vitro preparations of gill tissue it was found that growth hormone (10 and 100 ng/ml) caused an increase in subunit transcription, translation, and Na+-K+-ATPase enzyme activity. Similarly, insulin-like growth factor 1 (10 and 100 ng/ml) also caused an increase in Na+-K+-ATPase subunit amounts and enzyme activity. Cortisol (10 and 100 ng/ml) increased alpha subunit transcript and protein but did not modulate beta subunit expression or enzyme activity. Ovine prolactin did not cause any changes in Na+-K+-ATPase subunit transcription, translation or enzyme activity. This study is the first to describe how both Na+-K+-ATPase alpha and beta subunits are modulated at transcriptional and translational levels in fish osmoregulatory tissue upon exposure to hormones.

Cloning and characterization of the hsp70 multigene family from silver sea bream: Modulated gene expression between warm and cold temperature acclimation.

The genes encoding heat shock cognate 70 (hsc70) and inducible heat shock protein 70 (hsp70) were cloned and characterized from silver sea bream liver. Upon acute heat shock (+7 degrees C), the transcript abundance of hsc70 was increased 1.7-fold whereas the transcript abundance of hsp70 increased 6.7-fold. The chronic acclimation of sea bream to cold temperature (12 degrees C) resulted in a downregulation of hsc70 and an upregulation of hsp70 in comparison to levels in sea bream kept at a warmer temperature (25 degrees C). The expression of heat shock transcription factor I was also increased during cold temperature acclimation. Increased amounts of hepatic insulin-like growth factor 1 transcript, serum thyroxine (T4), and triiodothyronine (T3) were also found during cold temperature acclimation whereas serum cortisol remained unchanged. The results from this study demonstrate how temperature acclimation, in fish, can affect the regulation of the hsp70 multigene family and hormonal factors that are associated with anabolism.

Differential gene expression associated with euryhalinity in sea bream (Sparus sarba).

Certain fish have the remarkable capability of euryhalinity, being able to withstand large variations in salinity for indefinite periods. Using the highly euryhaline species, silver sea bream (Sparus sarba), as an experimental model, some of the molecular processes involved during ion regulation (Na+-K+-ATPase), cytoprotection [heat shock protein (hsp) 70], and growth (somatotropic axis) were studied. To perform these studies, seven key genes involved in these processes were cloned, and the tissue-specific expression profiles in fish adapted to salinities of 6 parts per thousand (ppt; hypoosmotic), 12 ppt (isoosmotic), 33 ppt (seawater), and 50 ppt (hypersaline) were studied. In gills, the transcriptional and translational expression profiles of Na+-K+-ATPase alpha- and beta-subunit genes were lowest in isoosmotic-adapted fish, whereas in kidneys the expression of the beta-subunit increased in seawater- and hypersaline-adapted groups. The hsp70 multigene family, comprising genes coding for heat shock cognate (hsc70), inducible heat shock protein (hsp70), and a heat shock transcription factor (hsf1), was found to be highly upregulated in gills of seawater- and hypersaline-adapted fish. In liver, hsc70 expression was lowest in isoosmotic groups, and in kidneys the hsp70 multigene family remained unchanged over the salinity range tested. The regulation of the somatotropic axis was studied by measuring pituitary growth hormone expression and liver IGF-I expression in salinity-adapted fish. The expression amounts of both genes involved in the somatotropic axis were highest in fish maintained at an isoosmotic salinity. The results of this study provide new information on key molecular processes involved in euryhalinity of fish.

Modulated heat shock protein expression during pathogenic Vibrio alginolyticus stress of sea bream.

The effects of infective Vibrio alginolyticus and its extracellular product (ECP) on host fish function are not well understood. In this study a partial biochemical characterization of the ECP from an infective strain of V. alginolyticus isolated from diseased silver sea bream Sparus sarba was achieved and the effects of live V. alginolyticus and ECP on hepatic heat shock protein (hsp) expression was compared. The ECP fraction was found to contain several hydrolytic enzymes including both haemolytic and proteolytic activities. Intramuscular administration of ECP to sea bream resulted in vibriosis with similar pathological signs as those observed with live V. alginolyticus administration. Using quantitative immunoassays we assessed the levels of the major hsp families, hsp90, hsp70 and hsp60, in hepatic tissue of diseased sea bream between 12 and 48 h post-infection. Throughout the infective period, live V. alginolyticus did not alter hsp90 whereas ECP significantly reduced hepatic hsp90 during the late stages of acute infection. The levels of hsp70 were found to be rapidly and drastically increased with both live V. alginolyticus and ECP. The transcript levels of both gene members of the hsp70 family (hsc70 and hsp70) were significantly increased with both live V. alginolyticus and ECP. The levels of hsp60 remained unchanged with both live V. alginolyticus and ECP. The data presented in this study is the first report describing an effect of both live V. alginolytus and ECP on hsp expression in diseased fish.

Ontogeny of thyroid hormones, cortisol, hsp70 and hsp90 during silver sea bream larval development.

We studied the profiles of silver sea bream (Sparus sarba) thyroxine (T(4)), triiodothyronine (T(3)), cortisol and the heat shock protein (hsp) families hsp70 and hsp90 during larval development. Eggs from sexually mature female sea bream were fertilized and larvae were collected at incremental time intervals between 1-46 days post hatch (dph). Both T(4) and T(3) were detected in 1 dph larvae and it was found that both increased as development progressed with a distinct surge in amounts between 21-35 dph, a time associated with direct development of larvae to juveniles. Cortisol increased from 1 dph reaching a maximum and constant level from 35 dph onwards. Using RT-PCR coupled with radioisotope hybridization of immobilized cDNA we assessed the transcript levels of hsp70 and it was found that transcript remained unaltered between 1-14 dph before progressively increasing. Immunoblotting was used to study the larval concentrations of hsp70 and hsp90 and it was found that hsp70 was not significantly changed between 1-14 dph whereas hsp90 increased from 1 dph onwards. These findings suggest an important role for hsp90 in the corticosteroid receptor complex during silver sea bream larval development.

Chronic salinity adaptation modulates hepatic heat shock protein and insulin-like growth factor I expression in black sea bream.

Black sea bream ( Mylio macrocephalus) hepatic heat shock proteins hsp90, hsp70, and hsp60 were found to be thermally and reversibly inducible as they were elevated 2.0, 3.2, and 2.1 fold, respectively, on acute heat shock and returned to pre-heat-shock levels after a 40-hour recovery period. To establish whether salinity plays a role in regulating heat shock protein (hsp) and insulin-like growth factor-I (IGF-I) expression in a euryhaline marine fish, we adapted groups of juvenile black sea bream to salinities of 50 ppt (hypersaline), 33 ppt (seawater), 12 ppt (isoosmotic), and 6 ppt (hypoosmotic) for 8 months. The lowest levels of hsps were found in fish reared in an isoosmotic salinity and the highest in those adapted to hypersaline and hypoosmotic salinities. Hepatic beta-actin messenger RNA abundance remained unchanged in all groups during salinity adaptation, whereas IGF-I mRNA abundance was highest in isoosmotic adapted black sea bream. This study is the first report of an effect of salinity ranging from hypersaline to hypoosmotic on the expression of different hsp forms and IGF-I in fish, and the possible relationship between environmental salinity, hepatic IGF-I expression, and hsp regulation is discussed.