Ethoxyresorufin-O-deethylase enzyme activities and accumulation of secondary/tertiary lysosomes in rabbitfish Siganus oramin as biomarkers for xenobiotic exposures.

The sensitivities of using hepatic and intestinal ethoxyresorufin-O-deethylase (EROD) activities and hepatic accumulation of secondary/tertiary (2 degrees/3 degrees) lysosomes to detect xenobiotic exposures were assessed in the rabbitfish Siganus oramin in a metropolitan harbour, subtropical Hong Kong, over a complete seasonal cycle of one year. Additional information on the body-burden pollutants and physiological indices in S. oramin, and seasonal variables in seawater quality, were extracted from published data and re-analyzed. Under the influences of pollutant cocktail and seasonal factors, neither the hepatic nor intestinal EROD activity was indicative of total polycyclic aromatic hydrocarbons (Sigma PAH), total polychlorinated biphenyls, condition factor and hepatosomatic index (HSI) in S. oramin. However, the relative ratio of hepatic to intestinal EROD activities provided an indication to differentiate the xenobiotic intake route in the fish through diffusion via gills/skin or consumption of contaminated food. In addition, the elevated hepatic accumulation of 2 degrees/3 degrees lysosomes was closely associated with the dominant temporal trends of zinc and Sigma PAH, as well as reduced HSI, in S. oramin. Being minimally influenced by any investigated seasonal factors, the hepatic 2 degrees/3 degrees lysosomes in S. oramin was recommended as an effective biomarker of xenobiotic exposures and toxic effects for use in coastal pollution monitoring programmes.

Japanese medaka: a new vertebrate model for studying telomere and telomerase biology.

A good understanding of telomeres and telomerase biology is crucial for unraveling mechanisms related to aging and cancer. However, in vivo vertebrate studies of telomere biogenesis and telomerase function have been limited by the development of appropriate animal model systems. The present study aims to demonstrate evolutionary conservation of telomerase in vertebrate species, supporting the potential application of fish as vertebrate model for studying telomeres and telomerase function. Comparison of genomic and protein information among vertebrate TERTs (TElomerase Reverse Transcriptase), the Japanese medaka Oryzias latipes shares the highest similarity to that of the human than the other small size fish species studied (including pufferfish and zebrafish). The ubiquitous expression of TERT mRNA, the high constitutive level of telomerase activity, and the humanized telomere lengths further substantiate that Japanese medaka is an ideal vertebrate model for the study of telomere and telomerase-related mechanisms in vivo. Moreover, medaka exhibits fast, invariable growth and is able to provide a variety of useful developmental and reproductive endpoints for lifelong and multi-generational experiments. Our earlier and present findings support the use of medaka for studying organismal aging, tissue regeneration and carcinogenesis.

Hypoxia induces telomerase reverse transcriptase (TERT) gene expression in non-tumor fish tissues in vivo: the marine medaka (Oryzias melastigma) model.

BACKGROUND: Current understanding on the relationships between hypoxia, hypoxia-inducible factor-1 (HIF-1) and telomerase reverse transcriptase (TERT) gene expression are largely based on in vitro studies in human cancer cells. Although several reports demonstrated HIF-1- mediated upregulation of the human TERT gene under hypoxia, conflicting findings have also been reported. Thus far, it remains uncertain whether these findings can be directly extrapolated to non-tumor tissues in other whole animal systems in vivo. While fish often encounter environmental hypoxia, the in vivo regulation of TERT by hypoxia in non-neoplastic tissues of fish remains virtually unknown. RESULTS: The adult marine medaka (Oryzias melastigma) was employed as a model fish in this study. We have cloned and characterized a 3261-bp full-length TERT cDNA, omTERT, which encodes a protein of 1086 amino acids. It contains all of the functional motifs that are conserved in other vertebrate TERTs. Motif E is the most highly conserved showing 90.9-100% overall identity among the fish TERTs and 63.6% overall identity among vertebrates. Analysis of the 5'-flanking sequence of the omTERT gene identified two HRE (hypoxia-responsive element; nt. - 283 and - 892) cores. Overexpression of the HIF-1alpha induced omTERT promoter activity as demonstrated using transient transfection assays. The omTERT gene is ubiquitously expressed in fish under normoxia, albeit at varying levels, where highest expression was observed in gonads and the lowest in liver. In vivo expression of omTERT was significantly upregulated in testis and liver in response to hypoxia (at 96 h and 48 h, respectively), where concomitant induction of the omHIF-1alpha and erythropoietin (omEpo) genes was also observed. In situ hybridization analysis showed that hypoxic induction of omTERT mRNA was clearly evident in hepatocytes in the caudal region of liver and in spermatogonia-containing cysts in testis. CONCLUSION: This study demonstrates for the first time, hypoxic regulation of TERT expression in vivo in a whole fish system. Our findings support the notion that hypoxia upregulates omTERT expression via omHIF-1 in non-neoplastic fish liver and testis in vivo. Overall, the structure and regulation of the TERT gene is highly conserved in vertebrates from fish to human.

Effects of moderate and substantial hypoxia on erythropoietin levels in rainbow trout kidney and spleen.

Erythropoietin (EPO) is a glycoprotein hormone that regulates the proliferation and differentiation of erythroid progenitor cells in mammals. Although EPO has been identified in fish, the specific function and effects of hypoxia have not been investigated previously. In this study, we have demonstrated a relationship between increases in renal EPO levels and decreases in spleen EPO levels and spleen-somatic index (SSI), with increases in haemoglobin (Hb) concentration in the blood during hypoxia exposure in rainbow trout. Splenic contraction and the subsequent red blood cell release accounts for the initial increase in Hb concentration in the blood, whereas EPO action probably accounts for the later increases in hemoglobin concentration in the blood. Our data indicate that fish and mammalian erythropoietic systems are similar in response to hypoxia, in that erythropoiesis in fish is influenced by EPO.

Isolation, characterization and expression analysis of a hypoxia-responsive glucose transporter gene from the grass carp, Ctenopharyngodon idellus.

Glucose transporters (GLUTs) have been implicated in adaptive and survival responses to hypoxic stress in mammals. In fish, the expression and regulation of GLUT in relation to hypoxia remains unexplored. Here we describe the identification of a hypoxia-responsive glucose transporter gene (gcGLUT) and the corresponding full-length cDNA from the grass carp. The gene spans approximately 11 kb of genomic sequence and consists of 12 exons and 11 introns, and an open reading frame (ORF) of 1599 bp encoding a polypeptide of 533 amino acids, with a predicted molecular mass of approximately 57 kDa and a pI of 8.34. blastx analysis showed that the ORF shared high sequence identity with the GLUT1 (57-59%), GLUT3 (59-60%) and GLUT4 (55-59%) proteins from different vertebrates. Comparative analysis of GLUT genomic structures showed that the arrangement of exons and position of split codons are highly conserved amongst members of the class I GLUTs suggesting that these genes share a common ancestor. Phylogenetic analysis indicated that gcGLUT is most closely related to the GLUT3 proteins. Northern blot analysis showed that the 3.1-kb gcGLUT transcript was most abundantly expressed and responsive to hypoxia in kidney. Up-regulated expression by hypoxia was also evident in eye and gill, but differential patterns of expression were observed. Low expression levels detected in brain, heart, liver and muscle were not responsive to hypoxic stress.