Graphene oxide nanoparticles induce hepatic dysfunction through the regulation of innate immune signaling in zebrafish (Danio rerio)

Purpose: The goals of this study are to investigate the toxic effects and molecular mechanisms of GO exposure in adult zebrafish liver by transcriptome profiling (RNA-seq) Methods: Liver mRNA profiles of three-month-old control (CK) and GO-exposed (GO) zebrafish were generated by deep sequencing, in triplicate, using Illumina Hiseq X ten. The sequence reads that passed quality filters were analyzed at the gene level with two methods: RSEM and HISAT followed by Ballgown. qRT-PCR validation was performed using SYBR Green assays Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the zebrafish genome (GRCz11) and identified 43,106 genes in the livers of CK and GO zebrafish with RSEM and HISAT2 workflow. RNA-seq data confirmed stable expression of 10 known housekeeping genes, and 6 of these were validated with qRT-PCR. Approximately 0.7% of the genes showed differential expression between the CK and GO liver, with a fold change =1.5 and p value <0.05. Hierarchical clustering of differentially expressed genes uncovered several genes that may contribute to function in liver inflammation and lipid disorder. Conclusions: Our study represents the detailed analysis of zebrafish liver transcriptomes after GO exposure, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that steroid hormone biosynthesis, lipoprotein metabolic process and PPAR signaling pathway were signifificantly enriched. Most of the lipid metabolism genes were down-regulated while majority of the immune genes were up-regulated after GO treatment. Overall design: Liver mRNA profiles of three-month old control (CK) and GO-exposed (GO) adult zebrafish were generated by deep sequencing, in triplicate, using Illumina Hiseq X Ten.