Rasoul Nourizadeh-Lillabadi

Differential gene expression and biomarkers in zebrafish (Danio rerio) following exposure to produced water components

The main effluent from oil and gas production is produced water (PW), a waste that contains low to moderate concentrations of oil-derived substances such as polycyclic aromatic hydrocarbons (PAHs) and alkylphenols (APs). PW components may be present in seawater at low concentrations over large areas in the vicinity of oil and gas production facilities. In this study, zebrafish (Danio rerio) were exposed to control and three treatments (high-, pulsed-, low-dose) of a synthetic PW mixture for 1, 7 and 13 weeks. The aim was to investigate the development of transcriptome and biomarker responses as well as relationships between early responses and population-relevant effects. The synthetic PW contained a mixture of low-molecular-weight PAHs (<5 ring) and short-chain APs (C1-C4). The water-borne exposure levels (sum PAH) ranged from 0.54 ppb (low dose) to 5.4 ppb (high dose). Bile pyrene metabolites ranged from 17-133 ng g(-1) bile in the control group to 23-1081 ng g(-1) bile in the high exposure group. Similar levels have been observed in wild fish, confirming an environmentally relevant exposure. The expression of mRNAs of hepatic genes was investigated in the high exposure group using the Zebrafish OligoLibrary from Compugen. Functional clustering analysis revealed effects in the reproductive system, the nervous system, the respiratory system, the immune system, lipid metabolism, connective tissue and in a range of functional categories related to cell cycle and cancer. The majority of differentially expressed mRNAs of genes were down-regulated, suggesting reduction in gene transcription to be as relevant as up-regulation or induction when assessing biological responses to PW exposure. Biomarkers for effects of PAHs (cytochrome P450 1A) and environmental estrogens (vitellogenin) did not appear to be affected by the chronic exposure to low concentration of PW components. Effects at the population level included a reduction in condition factor in male fish from all exposed groups and spinal column deformations in the F1 generation of exposed groups. The different exposure regimes did not produce any significant differences in reproduction or recruitment. The results from this study demonstrate that environmentally relevant concentrations of PW affect gene expression and population-relevant endpoints in zebrafish, although links between the two were not obvious.

Natural mixtures of POPs affected body weight gain and induced transcription of genes involved in weight regulation and insulin signaling

Obesity is reaching epidemic proportions worldwide, and is associated with chronic illnesses such as diabetes, cardiovascular disease, hypertension and dyslipidemias (metabolic syndrome). Commonly held causes of obesity are overeating coupled with a sedentary lifestyle. However, it has also been postulated that exposure to endocrine disrupting chemicals (EDCs) may be related to the significant increase in the prevalence of obesity and associated diseases. In the present study, developmental and reproductive effects of lifelong exposure to environmentally relevant concentrations of two natural mixtures of persistent organic pollutants (POPs) were investigated using classical and molecular methods in a controlled zebrafish model. The mixtures used were extracted from burbot (Lota lota) liver originating from freshwater systems in Norway (Lake Mjøsa and Lake Losna). The concentration of POPs in the zebrafish ranged from levels detected in wild fish (Lake Mjøsa and Lake Losna), to concentrations reported in human and wildlife populations. Phenotypic effects observed in both exposure groups included (1) earlier onset of puberty, (2) elevated male/female sex ratio, and (3) increased body weight at 5 months of age. Interestingly, genome-wide transcription profiling identified functional networks of genes, in which key regulators of weight homeostasis (PPARs, glucocoricoids, CEBPs, estradiol), steroid hormone functions (glucocoricoids, estradiol, NCOA3) and insulin signaling (HNF4A, CEBPs, PPARG) occupied central positions. The increased weight and the regulation of genes associated with weight homeostasis and insulin signaling observed in the present study suggest that environmental pollution may affect the endocrine regulation of the metabolism, possibly leading to increased weight gain and obesity.

Natural Mixtures of Persistent Organic Pollutants (POP) Increase Weight Gain, Advance Puberty, and Induce Changes in Gene Expression Associated with Steroid Hormones and Obesity in Female Zebrafish

In the present study, developmental and reproductive effects of lifelong exposure to environmental relevant concentrations of two natural mixtures of persistent organic pollutants (POP) were investigated using classical and molecular methods in a controlled zebrafish model. The mixtures used were extracted from burbot ( Lota lota ) liver originating from freshwater systems in Norway: one mixture with high levels and one mixture with background levels of polybrominated diphenyl ethers (PBDE), polychlorinated biphenyls (PCB), and dichlorodiphenyltrichloroethane metabolites (DDT). The concentration of POP measured in the zebrafish ranged from levels detected in wild fish from Lake Mjøsa to concentrations reported in human and wildlife populations, indicating that the experimental fish were exposed to concentrations comparable with wild fish. Phenotypic effects observed in both exposure groups included earlier onset of puberty, increased male/female sex ratio, and differences in body weight at 5 mo of age. Interestingly, genome-wide transcription profiling showed changes in regulation of genes involved in endocrine signaling and growth. The transcriptomics changes include key regulator genes for steroid hormone functions ( ncoa3 ), and growth (c/ebp, ncoa3 ). The effects observed in the experimental zebrafish model raise the question whether chemical pollution represents a risk to reproductive health of wild fish inhabitating the freshwater system.

In silico and in situ characterization of the zebrafish (Danio rerio) gnrh3 (sGnRH) gene

BackgroundGonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).ResultsWe have characterized a zebrafish [Trp7, Leu8] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.ConclusionsThe characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

Transcriptional Regulation in Liver and Testis Associated with Developmental and Reproductive Effects in Male Zebrafish Exposed to Natural Mixtures of Persistent Organic Pollutants (POP)

Persistent organic pollutants (POP) occur as mixtures in nature and it is difficult to predict the toxicity of such mixtures based on knowledge about toxicity and mechanisms of action for single compounds. The present knowledge on the combined toxic effects and modes of actions of exposure to mixtures is limited. Thus, the scientifically based hazard and risk assessment of POP requires analytical and toxicological data from studies with environmental mixtures of POP. The application of genome wide transcription profiling in toxicology, in combination with classical endpoints, will improve the current understanding of the mechanisms of toxic processes. Furthermore, gene expression data may be useful in establishing new hypothesis and discovering new biomarkers for known toxicity as well as not yet recognized toxicity endpoints. In the present study, developmental and reproductive effects of lifelong exposure to environmental relevant concentrations of two natural mixtures of POP were investigated using classical and molecular methods in a controlled zebrafish model. The mixtures used were extracted from burbot (Lota lota) liver originating from freshwater systems in Norway: one mixture with high levels and one mixture with background levels of polybrominated diphenyl ethers (PBD), polychlorinated biphenyls (PCB), and DDT. The concentration of POP in the zebrafish ranged from levels detected in wild fish from Lake Mjøsa, to concentrations reported in human and wildlife populations. Phenotypic effects observed in both exposure groups included (1) reduced survival, (2) earlier onset of puberty, (3) increased male/female sex ratio, and (4) differences in body weight at 5 mo of age. Interestingly, genome-wide transcription profiling showed changes in regulation of genes involved in endocrine signaling and growth. The transcriptomics changes included (1) key regulator genes for steroid and thyroid hormone functions (cga, ncoa3), (2) insulin signaling and metabolic homeostasis (pik3r1, pfkfb3, ptb1), and (3) p53 activation (mdm4). The effects observed in the experimental zebrafish model raise the question of whether chemical pollution represents a risk to the reproductive health of wild fish inhabiting the freshwater system.

Parental exposure to natural mixtures of POPs reduced embryo production and altered gene transcription in zebrafish embryos.

Determination of toxicity of complex mixtures has been proposed to be one of the most important challenges for modern toxicology. In this study we performed genome wide transcriptome profiling to assess potential toxicant induced changes in gene regulation in zebrafish embryos following parental exposure to two natural mixtures of persistent organic pollutants (POPs). The mixtures used were extracted from burbot (Lota lota) liver originating from two lakes (Lake Mjøsa and Lake Losna) belonging to the same freshwater system in Norway. The dominating groups of contaminants were polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane metabolites (DDTs). Because both mixtures used in the present study induced similar effects, it is likely that the same toxicants are involved. The Mjøsa mixture contains high levels of PBDEs while this group of pollutants is low in the Losna mixture. However, both mixtures contain substantial concentrations of PCB and DDT suggesting these contaminants as the predominant contributors to the toxicity observed. The observed effects included phenotypic traits, like embryo production and survival, and gene transcription changes corresponding with disease and biological functions such as cancer, reproductive system disease, cardiovascular disease, lipid and protein metabolism, small molecule biochemistry and cell cycle. The changes in gene transcription included genes regulated by HNF4A, insulin, LH, FSH and NF-κB which are known to be central regulators of endocrine signaling, metabolism, metabolic homeostasis, immune functions, cancer development and reproduction. The results suggest that relative low concentrations of the natural mixtures of POPs used in the present study might pose a threat to wild freshwater fish living in the lakes from which the POPs mixtures originated.

Molecular characterization of three GnRH receptor paralogs in the European eel, Anguilla anguilla: tissue-distribution and changes in transcript abundance during artificially induced sexual development.

Gonadotropin-releasing hormone receptor (GnRH-R) activation stimulates synthesis and release of gonadotropins in the vertebrate pituitary and also mediates other processes both in the brain and in peripheral tissues. To better understand the differential function of multiple GnRH-R paralogs, three GnRH-R genes (gnrhr1a, 1b, and 2) were isolated and characterized in the European eel. All three gnrhr genes were expressed in the brain and pituitary of pre-pubertal eels, and also in several peripheral tissues, notably gills and kidneys. During hormonally induced sexual maturation, pituitary expression of gnrhr1a (female) and gnrhr2 (male and female) was up-regulated in parallel with gonad development. In the brain, a clear regulation during maturation was seen only for gnrhr2 in the midbrain, with highest levels recorded during early vitellogenesis. These data suggest that GnRH-R2 is the likely hypophysiotropic GnRH-R in male eel, while both GnRH-R1a and GnRH-R2 seems to play this role in female eels.

Early Embryonic Gene Expression Profiling of Zebrafish Prion Protein (Prp2) Morphants

Background The Prion protein (PRNP/Prp) plays a crucial role in transmissible spongiform encephalopathies (TSEs) like Creutzfeldt-Jakob disease (CJD), scrapie and mad cow disease. Notwithstanding the importance in human and animal disease, fundamental aspects of PRNP/Prp function and transmission remains unaccounted for. Methodology/Principal Findings The zebrafish (Danio rerio) genome contains three Prp encoding genes assigned prp1, prp2 and prp3. Currently, the second paralogue is believed to be the most similar to the mammalian PRNP gene in structure and function. Functional studies of the PRNP gene ortholog was addressed by prp2 morpholino (MO) knockdown experiments. Investigation of Prp2 depleted embryos revealed high mortality and apoptosis at 24 hours post fertilization (hpf) as well as impaired brain and neuronal development. In order to elucidate the underlying mechanisms, a genome-wide transcriptome analysis was carried out in viable 24 hpf morphants. The resulting changes in gene expression profiles revealed 249 differently expressed genes linked to biological processes like cell death, neurogenesis and embryonic development. Conclusions/Significance The current study contributes to the understanding of basic Prp functions and demonstrates that the zebrafish is an excellent model to address the role of Prp in vertebrates. The gene knockdown of prp2 indicates an essential biological function for the zebrafish ortholog with a morphant phenotype that suggests a neurodegenerative action and gene expression effects which are apoptosis related and effects gene networks controlling neurogenesis and embryo development.

Parental exposure to natural mixtures of persistent organic pollutants (POP) induced changes in transcription of apoptosis-related genes in offspring zebrafish embryos

ABSTRACT Apoptosis is an integral element of development that may also be initiated by environmental contaminants. The aim of the present study was to assess potential changes in the regulation of apoptotic genes in zebrafish embryos following parental exposure to two natural mixtures of persistent organic pollutants (POP). The mixture from Lake Mjøsa contained exceptionally high concentrations of polybrominated diphenyl ethers (PBDE), as well as relatively high levels of polychlorinated biphenyls (PCB) and dichlorodiphenyltrichloroethane (DDT). The mixture from Lake Losna contained background concentrations of POP. Genes involved in the apoptotic machinery were screened for their expression profile at four time points during embryonic development. Thirteen and 15 genes involved in apoptosis were found to be significantly upregulated in the high-exposure and background exposure groups, respectively, compared with controls. Modulation of apoptotic genes was restricted only to the first time point, which corresponds with the blastula stage. Although there were substantial differences in POP concentrations between mixtures, genes underlying the apoptosis process showed almost similar responses to the two mixtures. In both exposure groups the main executors of apoptosis p53, casp 2, casp 6, cassp 8, and BAX displayed upregulation compared to controls, suggesting that these POP induce apoptosis via a p53-dependent mechanism. Upregulation of genes that play a critical role in apoptosis suggests that disturbance of normal apoptotic signaling during gametogenesis and embryogenesis may be one of the central mechanisms involved in adverse reproductive effects produced by POP in zebrafish.

Endocrine effects of real-life mixtures of persistent organic pollutants (POP) in experimental models and wild fish.

ABSTRACT A series of studies have assessed the occurrence, levels, and potential adverse effects of persistent organic pollutants (POP) in fish from Lake Mjøsa. In this lake, high levels of various POP were detected in biota. Fish from the nearby Lake Losna contain background levels of POP and served as reference (controls) in these studies. Significantly higher prevalence of mycobacteriosis and pathological changes were documented in burbot (Lota lota) from Mjøsa compared to burbot from Losna. Further, transcriptional profiling identified changes in gene expression in burbot from Mjøsa compared to burbot from Losna associated with drug metabolism enzymes and oxidative stress. POP extracted from burbot liver oil from the two lakes was used to expose zebrafish (Danio rerio) during two consecutive generations. During both generations, POP mixtures from both lakes increased the rate of mortality, induced earlier onset of puberty, and skewed sex ratio toward males. However, opposite effects on weight gain were found in exposure groups compared to controls during the two generations. Exposure to POP from both lakes was associated with suppression of ovarian follicle development. Analyses of genome-wide transcription profiling identified functional networks of genes associated with weight homeostasis, steroid hormone functions, and insulin signaling. In human cell studies using adrenocortical H295R and primary porcine theca and granulosa cells, exposure to lake extracts from both populations modulated steroid hormone production with significant difference from controls. The results suggest that POP from both lakes may possess the potential to induce endocrine disruption and may adversely affect health in wild fish.