Establishment of killer whale (Orcinus orca) primary fibroblast cell cultures and their transcriptomic responses to pollutant exposure.

Populations of killer whale (Orcinus orca) contain some of the most polluted animals on Earth. Yet, the knowledge on effects of chemical pollutants is limited in this species. Cell cultures and in vitro exposure experiments are pertinent tools to study effects of pollutants in free-ranging marine mammals. To investigate transcriptional responses to pollutants in killer whale cells, we collected skin biopsies of killer whales from the Northern Norwegian fjords and successfully established primary fibroblast cell cultures from the dermis of 4 out of 5 of them. Cells from the individual with the highest cell yield were exposed to three different concentrations of a mixture of persistent organic pollutants (POPs) that reflects the composition of the 10 most abundant POPs found in Norwegian killer whales (p,p'-DDE, trans-nonachlor, PCB52, 99, 101, 118, 138, 153, 180, 187). Transcriptional responses of 13 selected target genes were studied using digital droplet PCR, and whole transcriptome responses were investigated utilizing RNA sequencing. Among the target genes analysed, CYP1A1 was significantly downregulated in the cells exposed to medium (11.6 µM) and high (116 µM) concentrations of the pollutant mixture, while seven genes involved in endocrine functions showed a non-significant tendency to be upregulated at the highest exposure concentration. Bioinformatic analyses of RNA-seq data indicated that 13 and 43 genes were differentially expressed in the cells exposed to low and high concentrations of the mixture, respectively, in comparison to solvent control. Subsequent pathway and functional analyses of the differentially expressed genes indicated that the enriched pathways were mainly related to lipid metabolism, myogenesis and glucocorticoid receptor regulation. The current study results support previous correlative studies and provide cause-effect relationships, which is highly relevant for chemical and environmental management.

Gene expression analysis and clinical diagnosis.

BACKGROUND: A new basis for diagnostic tests is being provided by the vast amount of data on gene expression that are now becoming available through large-scale measurement of mRNA abundance. The insights gained from these resources are most likely going to provide both a better basic understanding of disease mechanisms, and to identify molecular markers for more precise diagnoses and for prediction of prognosis and treatment response. METHODS: Some quantitative RT-PCR assays are utilized today for diagnosis of both malignant and non-malignant disease, but the use of gene expression measurements in clinical medicine can be expected to increase dramatically. CONCLUSIONS: There are important technical issues that must be adequately solved in order to obtain robust assays, such as standardized protocols with appropriate quality controls that ensure reliable data for the specific samples being analysed and good inter-laboratory reproducibility.

Identification of novel growth factor-responsive genes in neuroendocrine gastrointestinal tumour cells.

Targeting growth-regulatory pathways is a promising approach in cancer treatment. A prerequisite to the development of such therapies is characterisation of tumour growth regulation in the particular tumour cell type of interest. In order to gain insight into molecular mechanisms underlying proliferative responses in neuroendocrine (NE) gastrointestinal (GI) tumours, we investigated gene expression in human carcinoid BON cells after exposure to gastrin, hepatocyte growth factor (HGF), pituitary adenylate cyclase-activating polypeptide or epidermal growth factor. We particularly focused on gastrin- and HGF-induced gene expression, and identified 95 gastrin- and 101 HGF-responsive genes. The majority of these genes are known mediators of processes central in tumour biology, and a number of them have been associated with poor prognosis and metastasis in cancer patients. Furthermore, we identified 12 genes that were regulated by all four factors, indicating that they may be universally regulated during NE GI tumour cell proliferation. Our findings provide useful hypotheses for further studies aimed to search for new therapeutic targets as well as tumour markers in NE GI tumours.

Effects of 4-nonylphenol on gene expression of pituitary hormones in juvenile Atlantic salmon (Salmo salar).

Alkylphenols such as 4-nonylphenol (NP) are one of the wide variety of environmental chemicals reported to have estrogenic effects in both in vitro and in vivo studies. Induction of eggshell zona radiata proteins (Zrp) and vitellogenin (Vtg) mRNA and protein synthesis in the liver are widely used biomarkers for xenoestrogen exposure in fish. However, little work has been done to characterize the molecular effects of xenoestrogens on other potential target organs such as the pituitary. To evaluate pituitary effects and develop new potential biomarkers for xenoestrogens, the influences of NP and 17beta-estradiol (E2) on the mRNA levels of pituitary gonadotropic hormone (GTH) beta subunits [leutinizing hormone beta (LH beta or GTH II beta) and follicle stimulating hormone beta (FSH beta or GTH I beta)], prolactin (PRL), growth hormone (GH) and the pituitary specific transcription factor (Pit-1) were investigated in individual male and female juvenile Atlantic salmon (Salmo salar), 3 days after a single intraperitoneal (i.p.) injection. In one experiment, fish were injected with NP (125 mg/kg body weight (BW)) or E2 (5 mg/kg BW) and a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method was used to analyze LH beta and FSH beta mRNA levels. In the second experiment, fish were injected with three doses of NP (10, 50, 125 mg/kg BW) or a single dose of E2 (5 mg/kg BW) and Northern blot analysis was used to quantify LH beta, FSH beta, PRL, GH and Pit-1 mRNAs. Both NP (50 and 125 mg/kg BW) and E2 significantly induced LH beta mRNA levels (P<0.01), but only in females. The highest dose of NP (125 mg/kg BW) significantly induced Pit-1 mRNA in males (P<0.01). NP did not have significant effects on any of the other pituitary transcripts. NP induced LH beta mRNA synthesis in females by up to 6-fold and the changes appeared to correlate with the increases in hepatic Vtg and Zrp mRNA levels. The results show that LH beta mRNA assay in female juvenile salmonids may be used as a marker for pituitary effects of xenoestrogens. The data also suggest that NP may have the potential to perturb the regulation of LH beta gene expression by mimicking E2.

In vivo modulation of nonylphenol-induced zonagenesis and vitellogenesis by the antiestrogen, 3,3'4,4'-tetrachlorobiphenyl (PCB-77) in juvenile fish.

Zonagenesis and vitellogenesis (eggshell zona radiata protein (Zrp) and vitellogenin (Vtg) production, respectively), are two estrogen-regulated processes in oviparous vertebrates that are crucial for oocyte maturation. Treatment of juvenile Atlantic salmon (Salmo salar) with nonylphenol (NP; 25 mg kg(-1)) alone or in combination with 3,3',4,4'-tetrachlorobiphenyl (TCB; 0.1 mg kg(-1)) resulted in pronounced elevations of plasma eggshell Zrp and Vtg and their respective liver mRNA levels in two separate experiments. TCB treatment alone caused the elevation of CYP1A mRNA, protein and enzyme levels (7-ethoxyresorufin O-deethylase (EROD)). In experiment 3, which also included the time factor, exposure of juvenile salmon to 10 and 25 mg NP per kg in combination with TCB generally resulted in reduced plasma Zrp and Vtg levels, compared with NP treatments alone. In a fourth experiment, juvenile salmon were exposed to different doses of TCB either 2 days before or 2 days after a single dose (25 mg kg(-1)) of NP. Samples were always collected 5 days after the NP exposure and analyzed for mRNA and protein levels. Generally, TCB doses given 2 days after NP exposure resulted in the elevation of Vtg and Zrp protein and mRNA levels. Vtg and Zrp mRNA levels were also elevated in the groups treated with 0.1 mg TCB 2 days before NP exposure. In all experiments, TCB injection resulted in the induction of liver CYP1A mRNA, CYP1A protein and EROD activity, but no Zrp or Vtg protein/mRNA inductions were observed when given alone. The present study documents for the first time the apparent stimulation of xenoestrogen-induced responses by an antiestrogenic CYP1A-inducer, in fish or any other lower vertebrate. However, the stimulatory or inhibitory effect of TCB on NP-induced responses appear to be dependent on the ratio of NP and TCB doses, and temporal sequence of exposure. Fish hepatic zonagenesis and vitellogenesis continue to provide interesting models for further studies on the mechanisms and possible interactions between endocrine disruptors and CYP1A-inducers, their antiestrogenic and/or estrogen potentiating effects.

Induction of hepatic estrogen receptor in juvenile Atlantic salmon in vivo by the environmental estrogen, 4-nonylphenol.

Alkylphenol ethoxylate degradation products such as nonylphenol and octylphenol are shown to have estrogenic effects. Nonylphenol induces synthesis of vitellogenin (a precursor of egg yolk proteins) and zona radiata proteins (eggshell proteins) in juvenile and/or male fish. Little is known about the molecular mechanisms of estrogenicity of environmental chemicals such as nonylphenol. To study the mechanisms of estrogenic effects of 4-nonylphenol (NP), we examined its in vivo effects on the expression of the estrogen receptor (ER), vitellogenin (Vtg) and zona radiata protein (Zrp) genes in juvenile Atlantic salmon liver. We show that the ER mRNA synthesis is induced by NP in a dose-dependent manner in juvenile Atlantic salmon liver. The induction of the ER mRNA synthesis is followed by the induction of Zrp and Vtg mRNA synthesis. The ER transcripts reach peak levels earlier than the Zrp and Vtg mRNA and proteins, which is in agreement with the physiological effects of estradiol during zonagenesis and vitellogenesis. Various studies have also shown that NP competitively inhibits the binding of 17 beta-estradiol (E2) to ER. Our results further suggest that NP directly mimics E2 in inducing the ER, Zrp and Vtg genes in salmon liver.

Salmon Eggshell Protein Expression: A Marker for Environmental Estrogens.

: A liver complementary DNA expression library from Atlantic salmon (Salmo salar) pretreated with estradiol-17beta (E2) was constructed and screened with antibodies raised against salmon eggshell (zona radiata) proteins. Two clones, SalZr2_19 and SalZr2_23 were sequenced and shown to encode proteins of approximately 50 kDa. SalZr2_23 contains 12 octamer sequence lpqr/kpa/vq repeats also found in SalZr2_19, but only twice. Alignment reveals that the two salmon sequences are similar to piscine zona radiata proteins and mammalian zona pellucida proteins. Several transcripts ranging from 2.3 to 12 kb appeared in liver extracts of E2-treated fish. Juvenile fish treated with E2 or 4-nonylphenol showed strong induction of zona radiata protein. The use of egg envelope transcriptional and translational induction in male or juvenile fish as a biological marker of environmental estrogens is discussed.