Project description:Thicklip grey mullet hepatic transcriptome comparission from PASAIA harbour, Basque Country Active biomonitoring using sentinel species caged in differentially polluted aquatic environments is being implemented worldwide. To determine the biological effects of pollutants in a heavily polluted Basque harbour (Pasaia, 431700N 015500W), two cages specially designed to deploy thicklip grey mullets (Chelon labrosus) were placed in the inner- and outer-harbour. Cages were deployed for 5 and 21 days to determine possible differences between sites, linked to specific chemical burdens. Hepatic gene expression profiles were studied using Q-PCR and a toxicology tailored low density mullet microarray (160 genes). Chemical analysis showed high concentrations of metals, PAHs, PCBs, phthalates, and organometallic compounds at both sites, most chemicals showing slightly higher concentrations in the inner-harbour. Q-PCR expression studies confirmed chemical data as metallothionein and CYP1A showed significant expression upregulation at day 21 at both sites, but showed no differences between sites. The microarray showed gene expression profiles that were able to distinguish mullets according to the site they were deployed in and the day they were captured. Regarding sites, 39 genes showed significant differences at p<0.05 on the last sampling day. Two families of genes are to be highlighted. Phase I and II biotransformation genes CYP2, CYP3 and UGT were upregulated in the inner-harbour together with AhR2 and AhRR. Similarly, TBT binding protein and genes involved in lipid metabolism such us PPARa and CYP7 were upregulated. The latter two genes have also been reported to be regulated by TBT. In summary, the low density microarray designed for mullets proved to be useful to unveil even slight differences in pollutant burdens in field conditions. 24 samples:6 hepatic samples from Inner harbour after 3 days of exposure, 6 hepatic samples from outer harbour after 3 days of exposure, 6 hepatic samples from Inner harbour after 15 days of exposure and 6 hepatic samples from outer harbour after 15 days of exposure

Project description:Ecotoxicogenomics in field experiments have yielded valuable mechanistic information for organisms present in polluted environments. The Queen conch (Strombus gigas) is a threatened species and populations are declining due to anthropogenic impact that includes pollution from boating activities. In the British Virgin Islands (BVI), local Queen conch populations have exhibited imposex, a condition in which both male and female gonadal characteristics are present and studies in the BVI suggest that tributyl tin (TBT), a chemical used in boat paint, is correlated to increased incidence imposex. This present study utilized a previously validated 8 x 15K Queen conch microarray to characterize the response of the ovarian transcriptome in conch found in polluted environments with high TBT in the BVIs. There polluted sites, Road Harbour (RH) and Trellis Bay (TB), are harbours with high boating activity while the reference sites, Guana Island (GI) and Anegada (AN), are areas with low boating activity. Microarray analysis revealed that there were 17 transcripts with high homology to known genes that were differentially expressed in the environments with high TBT and these included 6 induced and 11 down-regulated transcripts (p<0.01). These differentially expressed transcripts included phosphoenolpyruvate carboxylase, transposase, and high-affinity phosphate transporter PT1. When considering both RH and TB together in comparison to GI, functional enrichment showed that the biological processes and molecular functions of calcium ion binding, immune response, and negative regulation of cell proliferation were over represented in the polluted sites. Gene set enrichment analysis revealed that transcripts involved in the biological processes of general metabolism, immune, lipid metabolism, and stress were affected in polluted environments. Although difficult to directly link changes at the transcriptomics level to TBT in the harbour, this analysis provides novel insight into pathways impacted in regions that experience heavy boating activity in the BVIs. Adult female queen conchs were collected from field sites in the British Virgin Islands with varying levels of TBT contamination and imposex incidence. Contaminated sites included Rhode Harbour (RH, n=3) and Trellis Bay (TB, n=3), and the low-pollution site was Guana Island (GA, n=5). Ovaries were preserved in RNAlater, transported to the laboratory, and RNA was prepared using a phenol-chloroform extraction and a CsCl gradient. RNA was labeled with Cy3 for a single-dye design, per the Agilent protocol.

Project description:Ecotoxicogenomics in field experiments have yielded valuable mechanistic information for organisms present in polluted environments. The Queen conch (Strombus gigas) is a threatened species and populations are declining due to anthropogenic impact that includes pollution from boating activities. In the British Virgin Islands (BVI), local Queen conch populations have exhibited imposex, a condition in which both male and female gonadal characteristics are present and studies in the BVI suggest that tributyl tin (TBT), a chemical used in boat paint, is correlated to increased incidence imposex. This present study utilized a previously validated 8 x 15K Queen conch microarray to characterize the response of the ovarian transcriptome in conch found in polluted environments with high TBT in the BVIs. There polluted sites, Road Harbour (RH) and Trellis Bay (TB), are harbours with high boating activity while the reference sites, Guana Island (GI) and Anegada (AN), are areas with low boating activity. Microarray analysis revealed that there were 17 transcripts with high homology to known genes that were differentially expressed in the environments with high TBT and these included 6 induced and 11 down-regulated transcripts (p<0.01). These differentially expressed transcripts included phosphoenolpyruvate carboxylase, transposase, and high-affinity phosphate transporter PT1. When considering both RH and TB together in comparison to GI, functional enrichment showed that the biological processes and molecular functions of calcium ion binding, immune response, and negative regulation of cell proliferation were over represented in the polluted sites. Gene set enrichment analysis revealed that transcripts involved in the biological processes of general metabolism, immune, lipid metabolism, and stress were affected in polluted environments. Although difficult to directly link changes at the transcriptomics level to TBT in the harbour, this analysis provides novel insight into pathways impacted in regions that experience heavy boating activity in the BVIs.

Project description:The widely distributed thicklip grey mullet (Chelon labrosus) has been proposed as a suitable sentinel of pollution since it is able to survive heavily polluted marine/estuarine waters. Previous studies applying molecular to histological level biomarkers have indicated that mullets respond to exposure to chemical compounds. Microarrays are used to identify gene pathways responsive to specific chemical exposures. In this context, fragments of 129 genes relevant in peroxisome proliferation, detoxification, lipid metabolism, inflammatory/immune response, metal sequestration, oxidative and general stress, cell cycle regulation and proliferation, apoptosis, protein synthesis/degradation, and endocrine disruption were cloned through homological cloning using degenerate primers for microchip creation. Additional 31 sequences available in databases belonging to mugilid fishes were included in the final Agilent custom-microchip design. Female multitissue transcritome analysis was performed in mullets from Ondarrua. 108 genes showed differential expression when comparing female brain, gonad, gill and liver. Typical brain transcripts such as aromatase or dopamine receptor were expressed preferentially in the brain, whereas liver specific genes were detected in the liver; choriogenin-L, vitellogenins, fibrinogens or hepatocyte growth-factor. Genes related to peroxisome proliferation were systematically overrepresented in gonads. Female thicklip grey mullet tissue transcriptome comparission from Ondarrua, Basque Country GSM861357-GSM861379: 23 samples: 5 female samples from Gonad: female-Ondarru (F-L); 6 female samples from gonad: female-Ondarru (F-GO); 6 female samples from gill: female-Ondarru (F-GI); 6 female samples from brain: female-Ondarru (F-B); GSM886139-GSM886153: 15 samples: 2 intersex samples from Ondarru (I-L); 2 intersex gonad samples: intersex-Ondarru (I-GO); 6 female hepatic samples: female-Arriluze (F-A); 5 male hepatic samples: male-Arriluze (M-A) Immature Thicklip grey mullets were exposed to accetone, cadmium, benzo(a)pyrene and to nonylphenol and hepatic transcriptome was compared GSM886154-GSM886183: 30 samples: immature thicklip grey mullets hepatic samples 6 control seawater exposed samples, 6 acetone exposed samples; 6 cadmium exposed samples, 6 benzo(a)pyrene exposed samples (BAP) and 6 nonylphenol (NP) exposed samples.

Project description:Organotin compounds are highly persistent organic pollutants that bioaccumulate in marine biota, behaving as potent endocrine disruptors. Tributyltin (TBT) has been described as an environmental obesogen, as it contributes in mammals to lipid accumulation in a mechanism mediated by PPARÎ³ and RXR. With the aim of studying the molecular mechanisms that elicit TBT-induced pathogenesis in fish, thicklip grey mullets Chelon labrosus were exposed to low (10 ng/L) and high (500 ng/L) TBT concentrations for 1, 7 and 21 days, and gene transcription and metabolome profiles were studied. With this purpose, the multitissue transcriptome of mullet was sequenced by 454 pyrosequencing obtaining 126 Mb of sequence information. Assembly and annotation allowed spotting 8330 gene signatures on an oligonucleotide microarray that was used to identify hepatic gene transcription profiles specific to each TBT exposure set-up. Functional pathway analysis revealed that early profiles were characterized by genes related to response to organic substances and to oxidative stress and glucose metabolic processes. After 21 days of exposure, enriched GO terms in both concentrations were related to lipid homeostasis with a significant regulation of steroid metabolic and cholesterol biosynthetic processes. Also, the androgen receptor signalling pathway was regulated while PPAR signalling appeared as the most significantly regulated KEGG pathway. Neutral lipid accumulation measured by Oil-Red-O histochemistry did not show any treatment-related effect but hepatic and plasma NMR and GC-MS metabolomic analysis revealed distinctive metabolites. Aqueous profiles were characterised by lactate, glucose and alanine while GC-MS revealed a whole set of discriminating polyunsaturated fatty acids. Thus, TBT pathogenesis involves alterations of lipid metabolism through a mechanism that could involve PPARÎ³-regulated processes confirming the obesogen hypothesis for TBT in fish. Thicklip grey mullets were exposed to tributyltin (TBT). 3 treatments: control, TBT low dose (10ng/L), TBT high dose (500ng/L); 3 sampling times: 1 day, 1 week, 3 weeks. 6 mullets were dissected in each sampling point and group.

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Active biomonitoring of a polluted harbour using gene expression profiles in caged sentinel thicklip grey mullets (Chelon labrosus).

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