Richard Man Kit Yu

Effects of 20 PBDE metabolites on steroidogenesis in the H295R cell line

Polybrominated diphenyl ethers (PBDEs) are additive flame retardants that have been found in the environment as well as human tissues. Environmental concentrations of these compounds have been increasing in many parts of the world in recent years. Due to their structural similarity, PBDEs are believed to have similar toxicity to PCBs, but their toxicological properties are still being determined. In this study, the steroidogenic effects of hydroxylated, methoxylated and/or chlorinated derivatives of PBDEs were assessed at both the gene and enzyme/hormone levels in the H295R human adrenocortical carcinoma cell line. The expression levels of 10 steroidogenic genes were measured using quantitative real-time PCR (Q-RT-PCR). Aromatase activity in the cells and sex steroid (testosterone (T) and 17beta-estradiol (E2)) concentrations in the culture medium were also measured. CYP11B2, which regulates the synthesis of aldosterone, was the most sensitive gene and was induced by most of the compounds tested in this study. CYP19 gene expression, aromatase activity, and E2 production were also affected by several metabolites, but no consistent relationship was observed between these endpoints. Several PBDE metabolites showed some potential ability to interfere with steroidogenesis, including 5-Cl-6-OH-BDE-47, a biologically relevant BDE-47 metabolite, which significantly decreased aromatase activity and E2 production at a concentration of 10 microM. The results of this study indicate that PBDE metabolites affect steroidogenesis in vitro and that they may have the potential to affect steroidogenesis and reproduction in whole organisms.

Enhancement of hypoxia-induced gene expression in fish liver by the aryl hydrocarbon receptor (AhR) ligand, benzo[a]pyrene (BaP).

Fish in polluted coastal habitats commonly suffer simultaneous exposure to both hypoxia and xenobiotics. Although the adaptive molecular responses to each stress have been described, little is known about the interaction between the signaling pathways mediating these responses. Previous studies in mammalian hepatoma cell lines have shown that hypoxia-inducible factor (HIF)- and/or aryl hydrocarbon receptor (AhR)-activated gene expression is suppressed following co-exposure to hypoxia and the hallmark AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, whether similar crosstalk exists in the non-tumor liver tissues of fish and whether other non-TCDD ligands also play the same inhibitory role in this crosstalk remain unknown. Here, the in vivo hepatic mRNA expression profiles of multiple hypoxia- and AhR-responsive genes (later gene expression=mRNA expression of the gene) were examined in the orange-spotted grouper (Epinephelus coioides) upon single and combined exposures to hypoxia and benzo[a]pyrene (BaP). Combined exposure enhanced hypoxia-induced gene expression but did not significantly alter BaP-induced gene expression. Protein carbonyl content was markedly elevated in fish subjected to combined exposure, indicating accumulation of reactive oxygen species (ROS). Application of diethyldithiocarbamate (DDC) to hypoxia-treated grouper liver explants similarly exaggerated hypoxia-induced gene expression as in the combined stress tissues in vivo. These observations suggest that ROS derived from the combined hypoxia and BaP stress have a role in enhancing hypoxia-induced gene expression.

Leptin: clue to poor appetite in oxygen-starved fish

Hypoxia is the most widespread deleterious consequence of eutrophication and has become a major cause of fishery decline. One feature of chronic exposure to hypoxia in fish is inhibition of feeding. In this study, we investigated if the gene that encodes the appetite-suppressing hormone leptin is regulated by hypoxia in zebrafish (Danio rerio). Exposure of adult zebrafish to hypoxic conditions (1+/-0.2 mg O(2) L(-1)) for 4 and 10 days significantly increased leptin-a (zlep-a) mRNA levels in the liver. To evaluate the role of hypoxia-inducible factor 1 (HIF-1) in regulating zlep-a expression, zebrafish embryos were exposed to cobalt chloride (CoCl(2), a HIF-1 inducer) and overexpressed with HIF-1alpha mRNA. Both CoCl(2) treatment and HIF-1alpha overexpression markedly increased zlep-a expression in developing embryos, indicating the possible involvement of HIF-1 in zlep-a regulation. In vivo promoter analysis indicated that zlep-a promoter activity is found in the muscle fibers of zebrafish embryos and enhanced by CoCl(2). This is the first report to show that leptin gene expression in fish is regulated by hypoxia possibly via the involvement of HIF-1.

Leptin-Mediated Modulation of Steroidogenic Gene Expression in Hypoxic Zebrafish Embryos: Implications for the Disruption of Sex Steroids

Hypoxia can impair reproduction of fishes through the disruption of sex steroids. Here, using zebrafish (Danio rerio) embryos, we investigated (i) whether hypoxia can directly affect steroidogenesis independent of pituitary regulation via modulation of steroidogenic gene expression, and (ii) the role of leptin in hypoxia-induced disruption of steroidogenesis. Exposure of fertilized zebrafish embryos to hypoxia (1.0 mg O(2) L(-1)) from 0-72 h postfertilization (hpf), a developmental window when steroidogenesis is unregulated by pituitary influence, resulted in the up-regulation of cyp11a, cyp17, and 3β-hsd and the down-regulation of cyp19a. Similar gene expression patterns were observed for embryos exposed to 10 mM cobalt chloride (CoCl(2), a chemical inducer of hypoxia-inducible factor 1, HIF-1), suggesting a regulatory role of HIF-1 in steroidogenesis. Testosterone (T) and estradiol (E2) concentrations in hypoxic embryos were greater and lesser, respectively, relative to the normoxic control, thus leading to an increased T/E2 ratio. Expression of the leptin-a gene (zlep-a) was up-regulated upon both hypoxia and CoCl(2) treatments. Functional assays suggested that under hypoxia, elevated zlep-a expression might activate cyp11a and 3β-hsd and inhibit cyp19a. Overall, this study indicates that hypoxia, possibly via HIF-1-induced leptin expression, modulates sex steroid synthesis by acting directly on steroidogenic gene expression.

Two genes encoding protein phosphatase 2A catalytic subunits are differentially expressed in rice

Type 2A serine/threonine protein phosphatase (PP2A) plays a variety of regulatory roles in metabolism and signal transduction. Two closely related PP2A catalytic subunit (PP2Ac) genes, OsPP2A-1 and OsPP2A-3, have been isolated from the monocot Oryza sativa. Both genes contain six exons and five introns which intervene at identical locations, suggesting they have descended from a recent duplication event. Their encoded proteins share 97% sequence identity and are highly similar (94–96%) to a PP2Ac subfamily (AtPP2A-1, -2 and -5) identified in Arabidopsis thaliana. Both OsPP2A-1 and OsPP2A-3 are ubiquitously expressed, with the expression levels high in stems and flowers and low in leaves. OsPP2A-1, but not OsPP2A-3, is also highly expressed in roots. Transcript levels of OsPP2A-1 in roots and OsPP2A-3 in stems are elevated at the maturation and young stages, respectively. Drought and high salinity upregulate both genes in leaves, whereas heat stress represses OsPP2A-1 in stems and induces OsPP2A-3 in all organs. These findings indicate that the two PP2Ac genes are subjected to developmental and stress-related regulation. In situ hybridization results show that both transcripts exhibit nearly identical cellular distribution, except in leaves, and are abundant in meristematic tissues including the young leaf blade of stems and the root tip.

Hypoxia Impairs Primordial Germ Cell Migration in Zebrafish (Danio rerio) Embryos

Background As a global environmental concern, hypoxia is known to be associated with many biological and physiological impairments in aquatic ecosystems. Previous studies have mainly focused on the effect of hypoxia in adult animals. However, the effect of hypoxia and the underlying mechanism of how hypoxia affects embryonic development of aquatic animals remain unclear. Methodology/Principal Findings In the current study, the effect of hypoxia on primordial germ cell (PGC) migration in zebrafish embryos was investigated. Hypoxic embryos showed PGC migration defect as indicated by the presence of mis-migrated ectopic PGCs. Insulin-like growth factor (IGF) signaling is required for embryonic germ line development. Using real-time PCR, we found that the mRNA expression levels of insulin-like growth factor binding protein (IGFBP-1), an inhibitor of IGF bioactivity, were significantly increased in hypoxic embryos. Morpholino knockdown of IGFBP-1 rescued the PGC migration defect phenotype in hypoxic embryos, suggesting the role of IGFBP-1 in inducing PGC mis-migration. Conclusions/Significance This study provides novel evidence that hypoxia disrupts PGC migration during embryonic development in fish. IGF signaling is shown to be one of the possible mechanisms for the causal link between hypoxia and PGC migration. We propose that hypoxia causes PGC migration defect by inhibiting IGF signaling through the induction of IGFBP-1.

Structure, evolution and expression of a second subfamily of protein phosphatase 2A catalytic subunit genes in the rice plant (Oryza sativa L.).

Protein phosphatase 2A (PP2A) is one of the major serine/threonine protein phosphatases in the cell and plays a variety of regulatory roles in metabolism and signal transduction. Previously, we described the structure and expression of two genes encoding PP2A catalytic subunits (PP2Ac)—OsPP2A-1 and OsPP2A-3—in the rice plant (Yu et al. 2003). Here, we report the isolation and characterisation of a second structurally distinguishable PP2Ac subfamily comprised of three additional isogenes, OsPP2A-2, OsPP2A-4 (each containing ten introns) and OsPP2A-5 (which contains nine introns). Northern blot analysis demonstrated that the three isogenes are ubiquitously expressed in all rice tissues during plant development, and differentially expressed in response to high salinity and the combined stresses of drought and heat. Phylogenetic analyses indicated that the two PP2Ac subfamilies are descended from two ancient lineages, which derived from gene duplications that occurred after the monocotyledon–dicotyledon split. In the second subfamily, it is proposed that two duplication events were involved; in which, the initial duplication of a ten-intron primordial gene yielded OsPP2A-2 and the progenitor of OsPP2A-4 and OsPP2A-5. The OsPP2A-4/OsPP2A-5 progenitor, in turn, underwent a second duplication event, resulting in the present day OsPP2A-4 and OsPP2A-5. It is proposed that loss of the 5′-most intron from OsPP2A-5 occurred after these two duplication events.

Hypoxia disrupts gene modulation along the brain-pituitary-gonad (BPG)-liver axis.

Hypoxia alters sex hormone concentrations leading to reproductive impairment in fish; however the mechanisms underlying these effects remain largely unknown. Using zebrafish (Danio rerio), this study is the first to demonstrate that hypoxia causes endocrine disruption by simultaneously acting on multiple targets along the brain-pituitary-gonadal (BPG)-liver axis in fish. Alterations in the expression of key genes associated with reproductive endocrine pathways in the brain (sGnRH), pituitary (FSHβ and LHβ), gonads (FSH-R, LH-R, HMGR, StAR, CYP19A, CYP11A, CYP11β and 20β-HSD), and liver were correlated with significant reductions of estradiol in females and testosterone in males. Hypoxia also induced sex-specific and tissue-specific changes in the expression of estrogen, androgen, and membrane progestin receptors along the BPG axis, suggesting disruption of the feedback and synchronization of hormone signals. Furthermore, the hypoxia-induced upregulation of hepatic sex hormone-binding globulin suggests an increase in hormone transport and reduced bioavailability in blood, while upregulation of hepatic CYP3A65 and CYP1A in females suggests an increase in estrogen biotransformation and clearance. Given that the regulation of reproductive hormones and the BPG-liver axis are highly conserved, this study provides new insights into the hypoxia-induced endocrine disrupting mechanisms and reproductive impairment in other vertebrates.

In situ hybridization to detect spatial gene expression in medaka

A whole-animal tissue section in situ hybridization (ISH) system with radio-labeled probes was developed to detect differential gene expression among tissues of the small, oviparous teleost fish, Japanese medaka (Oryzias latipes). Because of its tissue- and gender-specific expression, gonadal aromatase (CYP19a) was selected as a model gene to demonstrate the potential of the system. The ISH system was validated with a 7d exposure to the model aromatase inhibitor, fadrozole. Fadrozole did not affect the magnitude of gene expression in testes, but significantly up-regulated CYP19a gene expression in ovaries. These results were confirmed with quantitative real-time-polymerase chain reaction (RT-PCR). Histological evaluation revealed that females exposed to 100microg/L fadrozole lacked mature oocytes. Male gonadal morphology was normal in all treatments. The ISH method developed in this study allowed tissue-specific resolution of gene expression in a whole animal model, as well as the ability to analyze cellular morphological detail in the same organism.

Transcriptional regulation and functional implication of the grass carp CITED1 (gcCITED1) in the negative regulation of HIF-1.

Hypoxia triggers a broad range of gene responses that are primarily mediated by the transcription factor, hypoxia-inducible factor-1 (HIF-1) that complexes with the transcriptional coactivator CREB-binding protein/p300 (CBP/p300). In mammals, members of the CBP/p300-interacting transactivators with ED-rich tail (CITED) family, such as CITED2 and CITED4, bind CBP/p300 with high affinity and thereby negatively regulate HIF-1 transactivation. In fish, we have previously shown that two CITED3 homologues from the hypoxia-tolerant grass carp (Ctenopharyngodon idellus) are induced by hypoxia/HIF-1 and able to inhibit HIF-1 transactivation. Here we report the identification and functional characterization of the grass carp CITED1 (gcCITED1) protein as a new repressor of HIF-1-mediated transcriptional activity. Expression of gcCITED1 mRNA was increased in heart, kidney and liver in vivo after exposure to hypoxia. Luciferase reporter and ChIP assays, respectively, indicated the inducibility of the gcCITED1 promoter by gcHIF-1 and the in vivo binding of gcHIF-1 to the gcCITED1 promoter. Ectopic overexpression of gcCITED1 significantly attenuated HIF-1-dependent transactivation of a HRE-luciferase reporter gene. Furthermore, GST pull-down confirmed that gcCITED1 specifically binds via its CR2 domain to the CH1 region of the grass carp p300 coactivator. Overall, our findings suggest that the hypoxia/gcHIF-1-inducible gcCITED1 may function in a negative feedback loop to regulate gcHIF-1 activity in response to hypoxia stress.