RNA-Seq Provides New Insights in the Transcriptome Responses Induced by the Carcinogen Benzo[a]pyrene

Abstract

Whole-genome transcriptome measurements are pivotal for characterizing molecular mechanisms of chemicals and predicting toxic classes, such as genotoxicity and carcinogenicity, from in vitro and in vivo assays. In recent years, deep sequencing technologies have been developed that hold the promise of measuring the transcriptome in a more complete and unbiased manner than DNA microarrays. Here, we applied this RNA-seq technology for the characterization of the transcriptomic responses in HepG2 cells upon exposure to benzo[a]pyrene (BaP), a well-known DNA damaging human carcinogen. Based on EnsEMBL genes, we demonstrate that RNA-seq detects ca 20% more genes than microarray-based technology but almost threefold more significantly differentially expressed genes. Functional enrichment analyses show that RNA-seq yields more insight into the biology and mechanisms related to the toxic effects caused by BaP, i.e., two- to fivefold more affected pathways and biological processes. Additionally, we demonstrate that RNA-seq allows detecting alternative isoform expression in many genes, including regulators of cell death and DNA repair such as TP53, BCL2 and XPA, which are relevant for genotoxic responses. Moreover, potentially novel isoforms were found, such as fragments of known transcripts, transcripts with additional exons, intron retention or exon-skipping events. The biological function(s) of these isoforms remain for the time being unknown. Finally, we demonstrate that RNA-seq enables the investigation of allele-specific gene expression, although no changes could be observed. Our results provide evidence that RNA-seq is a powerful tool for toxicology, which, compared with microarrays, is capable of generating novel and valuable information at the transcriptome level for characterizing deleterious effects caused by chemicals.

High-throughput data integration of RNA–miRNA–circRNA reveals novel insights into mechanisms of benzo[a]pyrene-induced carcinogenicity

“…All samples considered, 1.89 billion reads were produced, in pair-end, with an average of 79.1 million reads per sample. Because of the sequencing of non-coding repetitive elements, 35.5% of the reads (on average, corresponding to 27.9 million reads per sample) could be mapped to the Ensembl human genome (v73, with masqued repetitive element), to be compared with the 60% of poly-A enriched RNA-SEQ which appeared mappable in our previous study (6). …”

“…Previously, we applied this technology to assess the mRNA expression profile of HepG2 cells exposed to the prototypical human carcinogen benzo[a]pyrene (BaP) over a period of 24 h (6). BaP is a polycyclic aromatic hydrocarbon (PAH) widespread in the environment and in food, and is formed mainly during incomplete combustion of organic material (such as fossil fuels) or by smoking.…”

RNA Sequencing Identifies Dysregulation of the Human Pancreatic Islet Transcriptome by the Saturated Fatty Acid Palmitate

“…Different from microarrays, it allows detection of high-and low-abundance genes, alternative splice variants, and novel transcripts, identifying 25-75% more genes than conventional arrays and more differentially expressed transcripts (25,(66)(67)(68). We used it here to map the global response of human islets facing metabolic stress induced by palmitate.…”

Comparison of toxicogenomics and traditional approaches to inform mode of action and points of departure in human health risk assessment of benzo[a]pyrene in drinking water

“…Activation of the AHR by BaP leads to its own metabolism (Section 2.3.3) creating reactive metabolites that produce its subsequent genotoxic MOA. In addition, BaP–AHR activation causes transcriptional changes in genes related to carcinogenesis (Labib et al, 2012; Van Delft et al, 2012) and largely accounts for the nongenotoxic MOAs of BaP. Mice devoid of the AHR do not develop BaP-induced tumors, highlighting the receptor's role in BaP-induced carcinogenicity (Shimizu et al, 2000).…”