A New Approach Methodology (NAM) Based Assessment of Butylated hydroxytoluene (BHT) for Endocrine Disruption (ED) Potential

We tested the hypothesis that BHT is an endocrine disruptor by using a Next Generation Risk Assessment (NGRA) method. Four different cell lines: A549, HCC1428, HepG2 and MCF7 were treated with BHT and a series of BHT analogs at 5 different concentrations, RNA was isolated from cell extracts and run on the L1000 gene array platform. A toxicogenomics-based assessment was performed by comparing BHT’s unique genomic signature to a large external database containing signatures of other compounds (including many known endocrine disruptors) to identify if any endocrine disruption-related modes of action (MoAs) are prevalent among BHT and other compounds with similar genomic signatures. In addition, we performed a toxicogenomics-based structure activity relationship (SAR) assessment of BHT and a series of structurally similar analogues to understand if endocrine disruption is a relevant MoA for chemicals that are considered suitable analogs to BHT using the P&G read across framework (Wu et al., 2010). The platform is GPL20573: Broad Institute Human L1000 epsilon http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL20573 Four different cell lines: A549, HCC1428, HepG2 and MCF7 were treated with BHT and 15 BHT analogs (16 chemicals/perturbagens) at 5 different concentrations: 50, 100, 250, 500 and 1000 µM for 6 hours in quadruplicate wells per dose. Detailed cell culture, plating, treatment and lysis protocols are described in https://assets.clue.io/resources/sop-cell.pdf. RNA was isolated from cell extracts and run on the L1000 gene array platform. The data were processed through a computational system, that converts raw fluorescence intensities into differential gene expression signatures. The data at each stage of the pre-processing are available: Level 1 (LXB) - raw, unprocessed flow cytometry data from Luminex scanners. One LXB file is generated for each well of a 384-well plate, and each file contains a fluorescence intensity value for every observed analyte in the well. Level 2 (GEX) - gene expression values per 1,000 genes after deconvolution from Luminex beads. Level 3 (Q2NORM) - gene expression profiles of both directly measured landmark transcripts plus inferred genes. Normalized using invariant set scaling followed by quantile normalization. Level 4 (Z-SCORES) - signatures with differentially expressed genes computed by robust z-scores for each profile relative to control (PC relative to plate population as control; VC relative to vehicle control). Level 5 (SIG) consists of the replicates, usually 3 per treatment, aggregated into a single differential expression vector derived from the weighted averages of the individual replicates. Raw data files can be analyzed at: https://github.com/cmap/cmapJ where the Broad Institute of MIT and Harvard provided the Connectivity Map software for Java to read and store lxb and GCTX files.