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Unravelling the mechanisms of PFOS toxicity by combining morphological and transcriptomic analyses in zebrafish embryos

ABSTRACT: Purpose: the goal of this project is to study the effects of the PFOS (perfluorooctanesulfonate) in the transcriptome profiling (RNA-seq) of exposed zebrafish larvae. Methods: Total RNA was isolated from the samples using AllPrep DNA/RNA Mini Kit (Qiagen, CA, USA) as described by the manufacturer. Three high quality sample per condition were chosen to the mRNA enrichment using KAPA Stranded mRNA-Seq Kit Illumina® Platforms (Kapa Biosystems). Transcriptomic profiles were generated by deep sequencing using Illumina TruSeq SBS Kit v3-HS (pair-ended; 2x76bp) on a HiSeq2000 sequencing system. Image analysis, base calling and quality scoring of the run were processed using the manufacturer’s software Real Time Analysis (RTA 1.13.48) and followed by generation of FASTQ sequence files by CASAVA. Statistical analysis: RNA-seq reads were aligned to the D. rerio reference genome (GRCz10) using STAR version 2.5.1b . Genes annotated in GRCz10.84 were quantified using RSEM version 1.2.28 with default parameters. Differential expression analysis between all PFOS conditions was performed with the DESeq2 (v.1.10.1) R package with the Likelihood ratio test option. ANOVA-PLS was performed on the normalized data using the lmdme package in R (v. 1.0.136, R Core Team). Results: We generated on average 39 million paired-end reads for each sample and identified aproximatelly 24500 transcripts. 1434 differentially expressed genes (DEGs) were detected which could be divided in 2 clusters including 767 and 667 genes, respectively. Affected metabolic pathways were analyzed from the DEGs: lipid transport and metabolism, protein ubiquination, antigen processing, immune system, apoptosis, trans-membrane, cell matrix, Zn-ion binding, cytokines and JAK-STAT signaling pathways', among others, were down or upregulated. Conclusions: Our results suggest a complex, multiple endocrine disruption-like toxic effects at a concentrations well bellow the 1 mg/L, considered as the LOAEC/NOAEC for many of the macroscopic effects traditionally linked to PFOS toxicity in zebrafish embryos. While our results confirm the known effect of PFOS in lipid metabolism, we found a clear decrease on expression of many genes related to natural immunity and defense against infections. We propose that this transcriptional pattern may be a marker for the immunotoxic effects of PFOS and other related substances in fish and other vertebrates, including humans. We concluded that our analysis allowed us the identification of underlying molecular mechanisms occurring simultaneously at the exposed animals. While this approach is very useful to analyze the effects of compounds, like PFOS, able to interact with different cellular targets, we believe that it can be also applied to the characterization of the different toxic components present in complex natural mixtures.

ORGANISM(S): Danio rerio

PROVIDER: GSE125072 | GEO | 2019/04/28

REPOSITORIES: GEO

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Project description:Purpose: the goal of this project is to study the effects of the bisphenol A (BPA) in the transcriptome profiling (RNA-seq) of exposed zebrafish larvae. Methods: Total RNA was isolated from the samples using AllPrep DNA/RNA Mini Kit (Qiagen, CA, USA) as described by the manufacturer. Three high quality sample per condition were chosen to the mRNA enrichment using KAPA Stranded mRNA-Seq Kit Illumina® Platforms (Kapa Biosystems). Transcriptomic profiles were generated by deep sequencing using Illumina TruSeq SBS Kit v3-HS (pair-ended; 2x76bp) on a HiSeq2000 sequencing system. Images analysis, base calling and quality scoring of the run were processed using the manufacturer’s software Real Time Analysis (RTA 1.13.48) and followed by generation of FASTQ sequence files by CASAVA. Statistical analysis: RNA-seq reads were aligned to the D. rerio reference genome (GRCz10) using STAR version 2.5.1b . Genes annotated in GRCz10.84 were quantified using RSEM version 1.2.28 with default parameters. Differential expression analysis between all BPA conditions was performed with the DESeq2 (v.1.10.1) R package with the Likelihood ratio test option. ANOVA-PLS was performed on the normalized data using the lmdme package in R (v. 1.0.136, R Core Team). Results: We generated on average 40 million paired-end reads for each sample and identified aproximatelly 25000 transcripts. 2539 differentially expressed genes (DEGs) were detected which could be divided in 3 clusters including 960, 1132 and 447 genes, respectively. Affected metabolic pathways were analyzed from the DEGs: liver development, lipid transport, lysosome and protein glycosylation, metabolic pathways for lipids, glutathione, retinol, and steroid hormones, and cytochrome P450-mediated metabolism in adittion to protein ubiquitination and glycolysis/gluconeogenesis pathways' were down or upregulated. Conclusions: The results suggest the interaction of BPA with different signaling pathways, being the estrogenic and retinoid receptors two likely BPA targets. We concluded that our analysis allowed us the identification of underlying molecular mechanisms occurring simultaneously at the exposed animals. While this approach is very useful to analyze the effects of compounds, like BPA, able to interact with different cellular targets, we believe that it can be also applied to the characterization of the different toxic components present in complex natural mixtures.

Project description:Purpose: the goal of this project is to study the effects of the TBT (tributyltin) in the transcriptome profiling (RNA-seq) of exposed zebrafish larvae. Methods: Total RNA was isolated from the samples using AllPrep DNA/RNA Mini Kit (Qiagen, CA, USA) as described by the manufacturer. Three high quality sample per condition were chosen to the mRNA enrichment using KAPA Stranded mRNA-Seq Kit Illumina® Platforms (Kapa Biosystems). Transcriptomic profiles were generated by deep sequencing using Illumina TruSeq SBS Kit v3-HS (pair-ended; 2x76bp) on a HiSeq2000 sequencing system. Image analysis, base calling and quality scoring of the run were processed using the manufacturer’s software Real Time Analysis (RTA 1.13.48) and followed by generation of FASTQ sequence files by CASAVA. Statistical analysis: RNA-seq reads were aligned to the D. rerio reference genome (GRCz10) using STAR version 2.5.1b . Genes annotated in GRCz10.84 were quantified using RSEM version 1.2.28 with default parameters. Differential expression analysis between all TBT conditions was performed with the DESeq2 (v.1.10.1) R package with the Likelihood ratio test option. ANOVA-PLS was performed on the normalized data using the lmdme package in R (v. 1.0.136, R Core Team). Results: We generated on average 42 million paired-end reads for each sample and identified aproximatelly 24420 transcripts. 3238 differentially expressed genes (DEGs) were detected which could be divided in 2 clusters including 1703 and 1535 genes, respectively. Affected metabolic pathways were analyzed from the DEGs: DNA-binding, domain transcription factors, transcription, nucleus, regulation of cell proliferation, cell cycle and differentiation, NAD(P)-binding domain, carbon metabolism, transferases, nucleotide binding, catalytic activity, and specially steroid metabolism and biosynthesis, among others, were down or upregulated. Conclusions: The dose-response setup allowed us to determine the most sensitive pathways affected by TBT, mostly related to the steroid biosynthesis, and general cell viability and development. Its covalent binding to RXR receptors seems to be the responsible for their specific disruption but also could be involved in its general toxicity along with an unspecific heavy metal poisoning. Transcriptomic general toxicity was related to a developmental delay which was in agreement with previous morphometric results. Other properties traditionally attributed to TBT as obesogenity only appeared at higher concentrations which were relative close to lethal levels. Other biological levels were also affected at concentrations relative closer to lethality than other endocrine disruptors, suggesting than its mode of action and/or lethality could differ from other EDCs. The present work contributes in the understanding of the environmental hazards associated to TBT.

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Project description:Perfluorooctanesulfonate (PFOS) has been widely used in a variety of industrial and commercial applications as a surfactant and stain repellent. PFOS causes liver damage (including liver tumors) in experimental animals, primarily via interaction with PPARa and CAR/PXR. We investigated the involvement of microRNAs (miRNAs) in PFOS-induced hepatotoxicity, and mechanisms involved in abnormal TH homeostasis, in the livers of adult male rats exposed in feed to 50 mg PFOS/kg diet for 28 days. PFOS-treated rats exhibited expected histopathological and clinical chemistry changes. Global gene expression changes were consistent with the involvement of PPARα and CAR/PXR in PFOS-induced effects. Thirty-eight miRNAs were significantly altered. Three members of the miR-200 family were the most increased, while miR-122 and miR-21 were the most decreased, in PFOS-treated relative to control rats. Expression of the miR-23b/27b/24 cluster also decreased in PFOS-treated animals. Pathway analysis of miRNAs and associated gene expression changes demonstrated enrichment of transcripts involved in epithelial to mesenchymal transition (EMT), which is a primary process involved in tumor cell motility and cancer metastasis. Liver expression analysis revealed transcripts that may mediate PFOS effects on thyroid hormone (TH) homeostasis including: activation of the CAR/PXR pathway, phase II/III enzymes, and deiodinase. These changes are consistent with low serum TH due to enhanced metabolic clearance of TH. However, most TH hepatic target genes were not altered in a manner consistent with reduced TH signalling; suggesting that PFOS exposure did not induce functional hypothyroidism. Collectively, PFOS-induced miRNA perturbations were strongly associated with EMT suggesting an important role for miRNAs in PFOS-induced hepatotoxicity. The work also provides novel insights into the effects of PFOS on TH homeostasis. Total RNAs from liver samples (4 animals from each group) were labelled using the miRNA Complete Labelling and Hybridization kit (Agilent Technologies). Labelled RNA was hybridized on 8 X 15K Agilent rat miRNA microarray slides. Arrays were scanned using an Agilent G2505B scanner (5 μm resolution). Agilent Feature Extraction (version 10.7.3.1) was used to acquire the fluorescence intensity of each probe. The quality of the microarray data was evaluated using Agilent Feature Extraction quality control metrics. Data were normalized in R using cyclic-lowess (Bolstad et al., 2003). Ratio-intensity plots, boxplots and cluster analyses were used to identify potential outliers. One control sample failed the quality control tests and was eliminated for subsequent analyses. Statistically significantly altered miRNAs were identified using the methods described above for gene expression.

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Unravelling the mechanisms of PFOS toxicity by combining morphological and transcriptomic analyses in zebrafish embryos

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