Unknown,Transcriptomics,Genomics,Proteomics

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Effect of waterborne exposure to carcinogenic PAHs on adult zebrafish hepatic transcriptome

ABSTRACT: Several environmental pollutants, especially organic compounds such as polycyclic aromatic hydrocarbons (PAHs) are potent carcinogenic chemicals. Exposure of different fish species to PAHs causes a high prevalence and variety of tumor lesions. To identify and compare the mode of action (MOA) of different model carcinogenic PAHs, a transcriptomic study was carried out in adult zebrafish (Danio rerio) after exposure to 0.3 mg/l of benzo(a)pyrene (B(a)P) or to 7,12-dimethylbenz(a)anthracene (DMBA), both of them dissolved in dimethyl sulfoxide (DMSO; final concentration of 0.01%). Samples for microarray analysis were taken after 1 and 2 weeks of exposure. The changes in the mRNA transcription levels over the corresponding controls were compared among treatments. Correspondence analysis was able to discriminate among treatments; factor 1 separated both sampling times while factor 2 separated the compounds. A total number of 3043 transcripts was shown to be differentially regulated in at least one of the treatments. B(a)P treatment produced regulation of sequences related to GO categories associated to cell cycle and cell division whereas DMBA regulated GO terms related to oxidation/reduction responses, responses to chemicals and response to bacterium. Both compounds were able to alter many xenobiotic metabolism related genes, especially upregulating the transcription of phase I metabolism-related genes such as cytochrome P450 members (cyp1a or cyp1b), and many cancer-related genes such as the Jun B proto-oncogene (junb). Overall, these results indicated that the exposure to both PAHs was able to differentially alter the transcription levels of genes involved in both the detoxification metabolism and the cell-cycle control, including different tumor-supressor genes and oncogens. These alterations have been often related to the appearance of tumor lesions. Zebrafish were waterborne exposed to 0.3ppm BaP or DMBA for 1 and 2 weeks. After 1 and 2 weeks hepatic samples were collected from each treatment group as well as from the control group. 3 biological replicates have been collected per exposure group and time point. Each biological replicate is a pool of 5 livers. Samples from the different treatments were hybridized in an n+2 (n = 9) A-optimal loop design. Additionally, these two designs were linked by two extra hybridizations to enable comparison between time points

ORGANISM(S): Danio rerio

SUBMITTER: Unai Vicario

PROVIDER: E-GEOD-43675 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:This study aims to evaluate the transcriptome alterations, through cDNA libraries, associated with the effects of the combination of two PAHs, benzo[a]pyrene (0.5M-NM-<g/L) and phenanthrene (50M-NM-<g/L), present in crude oil, on specimens of Symphysodon aequifasciatus (discus fish) after 48h of exposure. The cDNA libraries were constructed according to the SOLiDTM SAGETM protocol for sequencing in the SOLiD v.3 Plus sequencer. The results were analyzed by bioinformatics and differentially expressed genes were categorized using the gene ontology program (AmiGO v.1.8). The functional categories (terms) found in the gene ontology and the gene network generated using STRING software were used to predict the adverse effects in the liver. In the present study, 27,127 genes (compared to Danio rerio database) were identified. Considering only those genes with a p-value less than or equal to 0.05 and a greater than or equal to two-fold change in expression across libraries, we found 804 genes, 438 down-regulated (54%) and 366 up-regulated genes (46%), in the experimental group compared to the control group. Out of this total, 327 genes were successfully categorized, 174 down-regulated and 153 up-regulated genes, using gene ontology tool. The final confidence gene network, analyzed in STRING, was composed by 199 nodes of proteins, 124 of them resulted in 274 interactions. The results showed that even an acute exposure of 48 h caused metabolic change in response to environmental contaminants, resulting in changes of cell integrity, changes in oxidation-reduction processes, disturbances of intracellular signaling and changes in the immune response of discus fish. Also the gene network interactions have showed no central interplay cluster, exhibiting interconnected clusters interactions and sparsely connected sub-networks. These findings highlight that even an acute and sublethal exposure of PAHs can cause metabolism changes that may affect this fish species survival rates. Our findings using the SAGE-method and the SOLiD sequencer showed that this is a powerful tool for gene expression analysis in discus fish, a non-model organism. Transcriptome analysis of differentially expressed genes in liver in benzo[a]pyrene and phenanthrene-exposed and non-exposed fish (N=6 per treatment)

Project description:Despite recent knowledge of the potential environmental impact that compounds present in municipal wastewater effluents, including contaminants of emerging concern (CECs), may have, the implications of fish exposure to this contaminant mixtures are not completely understood. The effects caused by effluent CECs may be subtle and diverse, thus the need for sensitive and comprehensive tools such as gene expression to detect such responses. In this study, we conducted laboratory exposures that examined plasma concentrations of vitellogenin (VTG), changes in secondary sexual characteristics and gene expression in sexually mature male fathead minnows (Pimephales promelas) exposed to environmentally realistic (0.5%) and higher (5%) concentrations of municipal wastewater effluents. Secondary and primary treated effluents were used. Several of the 32 CECs investigated were detected, including pharmaceuticals, personal care products, hormones, current use pesticides and industrial compounds. The percent of males with detectable levels of VTG was higher in fish exposed to effluent treatments. An increased number of males with changes in secondary sexual characteristics (e.g. development of ovipositors), was observed in fish exposed to 5% effluent treatments. Gene expression data indicated that overall expression patterns were characteristic to each effluent. Higher numbers of differentially expressed genes were observed in fish exposed to primary treated effluent when compared to controls. Differentially expressed genes belonged to several functional categories, including xenobiotic metabolism, estogenicity and energy/metabolism processes. Gene expression data provided information to understand some of the mechanisms behind the effects observed at higher biological levels. To investigate gene expression responses resulting from exposure to POTW effluents, two laboratory experiments were conducted using effluent from San Diego (Point Loma; SD) and Los Angeles (Hyperion; LA). The LA effluent received secondary treatment and the SD effluent received advanced primary treatment. Treatments used during exposures consisted of negative controls (moderately hard water), positive controls (E2), and 0.5% and 5% effluent concentrations. The 0.5% concentration of effluent represented an environmentally realistic exposure level. The 5% effluent concentration represented a higher level at which we expected biological responses. The exposures lasted 14 days. Treatments: EFFHa = 5% primary treated effluent EFFHb = 5% secondary treated effluent EFFLa = 0.5% primary treated effluent E2a = Estradiol, positive control for primary effluent E2b = Estradiol, positive control for secondary effluent CTRLa = Moderately hard water, negative control for primary effluent CTRLb = Moderately hard water, negative control for secondary effluent

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Effect of waterborne exposure to carcinogenic PAHs on adult zebrafish hepatic transcriptome

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