SRA

Transcriptomic signatures, or biomarkers, of toxicity can facilitate rapid mechanistic analysis of chemicals using high-content transcriptomic data and identification of potential hazards to human health. We developed an 81-gene transcriptomic biomarker, named TGx-HDACi, to detect histone deacetylase inhibitors (HDACi) in TK6 human lymphoblastoid cells after 4 hours of chemical exposure. This work leveraged an established transcriptomic biomarker of DNA damage, TGx-DDI, which was developed by Heng Hong Li et al. (2015); TGx-DDI contains 63 genes and can distinguish between DNA damage-inducing (DDI) and non-DDI agents after 4 hours of exposure in TK6 cells. TGx-HDACi was derived from Templated Oligo-Sequencing (TempO-Seq) whole transcriptome gene expression profiles of 20 reference compounds, consisting of 10 HDACi and 10 non-HDACi compounds. Fourteen of the TGx-HDACi reference compounds are also part of the 28-compound reference set for TGx-DDI. The nearest shrunken centroid (NSC) method was applied to the gene expression profiles and 81 genes were identified that accurately identified HDACi compounds in the NSC probability analysis. The classification performance of TGx-HDACi was validated using an additional set of 11 test compounds (4 HDACi and 7 non-HDACi). Thus far, TGx-HDACi has demonstrated 100% accuracy. The availability of TGx-HDACi increases the diversity of tools that can facilitate mode of action analysis of toxicants using gene expression profiling. Overall design: TK6 human lymphoblastoid cells were exposed to the 20 TGx-HDACi reference compounds (10 HDACi and 10 non-HDACi) and 11 validation compounds (4 HDACi and 7 non-HDACi) for 4h and whole transcriptomes were profiled using a TempO-Seq whole transcriptome kit (BioSpyder, Carlsbad, CA). Prior to whole transcriptome profiling, range finding experiments were performed to select one concentration for each compound. TK6 cells were exposed to increasing concentrations of each chemical for 4h. Cell viability was measured at 24h (4h exposure followed by 20h recovery in media) by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and gene expression of three indicator genes was measured at 4h by TaqMan qPCR assays. For HDACi compounds, RGL1, NEU1, and GRP183 were used as indicators of transcriptional response to HDACi exposure at 4h. The three genes were selected based on DNA microarray profiles of TK6 cells exposed to four HDACi compounds (trichostatin A, oxamflatin, apicidin, and HC toxin) for 4h, generated in a previous study [Li et al. 2015]; the three genes had the largest fold change in expression in the four microarray profiles and were not altered by non-HDACi exposures in the study. For non-HDACi compounds, cellular stress response genes ATF3, CDKN1A, and GADD45A were used as indicators genes [Li et al. 2015]. The selected concentrations for TempO-Seq analysis induced the highest fold changes in the three indicator genes after 4h exposure, without overt cytotoxicity in the MTT assay after a 20h-recovery (less than 50% reduction in viability at 24 h). Cells were exposed to the selected concentration of each compound on three separate occasions to generate three replicate RNA samples for each treatment.