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Dose-dependent transcriptomic responses of zebrafish embryos to Bisphenol A

ABSTRACT: 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.

ORGANISM(S): Danio rerio

PROVIDER: GSE113676 | GEO | 2018/09/19

REPOSITORIES: GEO

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Project description: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.

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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Dose-dependent transcriptomic responses of zebrafish embryos to Bisphenol A

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