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MRNA-Seq of Lemna minor exposed to different concentrations of the pharmaceutical atorvastatin against untreated control groups

ABSTRACT: In this study, Lemna minor was used to investigate ecotoxic modes-of-action (MoA) at the gene expression level. For this purpose, mRNA sequencing was applied to plant RNA extracted from L. minor exposed to the pharmaceutical atorvastatin in a shortened version of the OECD guideline test No. 221 (OECD TG 221). L. minor is commonly used as a non-target model organism to determine ecotoxicological effects of xenobiotics. Since atorvastatin (and statins in general) are frequently used pharmaceuticals, they can be found in the aquatic environment. In humans, they are used as cholesterol-lowering agents due to their hydroxymethylglutaryl-CoA reductase (HMGR) inhibitory effect. Analogously, in L. minor, atorvastatin can also inhibit the plant’s HMGR, which is involved in phytosterol synthesis. The aim of our study was to determine a molecular fingerprint of the substance in order to identify potential biomarkers for its MoA. Therefore, we applied bioinformatics approaches to functionally annotate the previously unannotated reference genome of L. minor. Our functional annotation pipeline is in principle applicable to any organism with an available reference genome and thus greatly facilitates the identification of gene functions for poorly annotated organisms. The observed effects at the molecular level showed promising results for the development of OMICs as screening methods as well as for the identification of biomarkers for the toxicity of atorvastatin in L. minor.

INSTRUMENT(S): Illumina HiSeq 4000

ORGANISM(S): Lemna minor

SUBMITTER: Alexandra Loll

PROVIDER: E-MTAB-11459 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Within the regulatory framework on the approval of new substances, the assessment of immunotoxic modes of action (MoA) is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound-specific response of zebrafish embryos to two immunomodulative reference substances, clobetasol propionate (CP, CAS 25122-46-7) and imiquimod (IMQ, CAS 99011-02-6), on the global level of gene expression. The obtained results were used to (i) identify potential biomarker candidates for the assessment of immunotoxic MoAs, (ii) identify compound-specific molecular signatures, (iii) evaluate the reliability of data acquisition using OMICs-coupled approaches and (iv) to assess the suitability of the zebrafish embryo as an alternative model for the human-representative investigation of psoriatic effects. For this, the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP-induced immunosuppression in absence or presence of an IMQ-induced immune challenge, i.e. the simulation of a resting and an activated immune system. In a modified version of the zebrafish embryo toxicity test (OECD 236), 15 fertilized eggs were exposed to either CP (250 nM) or to IMQ (4000 nM) or to a combination of both under semi static conditions. Exposure to CP started at 2 hours post fertilization (hpf) until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without CP nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf, all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit (Macherey-Nagel) following the manufacturer’s protocol. RNA quality was assessed with a 2100 Bioanalyzer system (Agilent) before coding RNA was purified (PolyA selection with TruSeq RNA Library Prep Kit v2) and sequenced on an Illumina NovaSeq 6000 System (Illumina) in 150 bp paired-end mode, producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.

Dataset Information

MRNA-Seq of Lemna minor exposed to different concentrations of the pharmaceutical atorvastatin against untreated control groups

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