For the chemicals used in this study, appropriate dosages were derived from previously performed dose range finding (DRF) studies (2-AAF, Phe, CsA, DEHP, DES, E2, PBB, Res, WY, D-man (van Kreijl et al. 2001; de Vries et al. 1997)) or if not known yet, identified by performing additional 28-day DRF studies prior to the short-term exposure studies (see supplemental information 1 for DRF studies using AFB1, CPPD, BPA, DIDP, SD and TBTO). In short, eight to ten weeks old male C57BL/6J mice (n = 10 per group) were exposed to chemicals, using multiple dosages based on literature or expert advice. Body weight dynamics during the first week and over the full 28-day exposure were monitored to extract suitable (non- or slightly cytotoxic) doses (Supplemental information 1). If body weight dynamics were not conclusive, the liver was studied macroscopically (data not shown). Exposure through feed was continuously during the experiment, application using i.p. injection occurred at day 0, 3 and 6 (autopsy on day 7) and exposure using gavage at day 0, 2, 4 and 6 (autopsy on day 7). Body weights were recorded during this 7-day exposure period (Supplemental information 2). Comparison of different control groups (gavage, i.p. injection or feed) showed no significant differential effect on transcriptional levels (Luijten et al. in preparation), hence only food administrated control samples were implemented in this study
Growth protocol
Six-week-old male WT mice (C57BL/6J, n=4 per group) were acclimated for 2 weeks and subsequently treated for 7 days with a GTXC, NGTXC or NC through feed, gavage or i.p. injection. From the day of weaning, the health status of the mice was monitored daily and mice were weighed weekly starting at acclimation. Animals were kept in the same stringently controlled (specific pathogen-free, spf) environment, fed ad libitum and kept under a normal day/night rhythm. After 7 days of exposure, mice were sacrificed at a fixed time of the day. During autopsy, several organs (including the liver), were isolated and stored according to protocol using RNAlater (Qiagen, Valencia, CA, USA).
Extracted molecule
total RNA
Extraction protocol
Hepatic total RNA was isolated using the miRNeasy kit (Qiagen, Valencia, CA, USA) and the QIAcube (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. All samples passed RNA quality control using capillary gel electrophoresis (RIN > 7.6) (Bioanalyzer 2100; Agilent Technologies, Amstelveen, the Netherlands). The same total RNA isolates as used for mRNA were used for isolation of microRNAs. MicroRNA profiling was performed as previously described (Pothof et al. 2009).
Label
biotin
Label protocol
MRNA was amplified and labeled with the GeneChip Expression 3'-Amplification One-Cycle cDNA Synthesis Kit and GeneChip Expression 3'-Amplification Reagents For IVT Labeling according to the manufacturer’s instructions (Eukaryotic Sample and Array Processing 701025 Rev.5; Affymetrix Inc., Santa Clara, CA, USA).
Hybridization protocol
Amplified materials were hybridized to Mouse Genome 430 2.0 Array for 16 hours at 45°C, subsequently washed and stained with the EukGE-WS2v5_450 protocol
Scan protocol
Samples were finally scanned using the GeneChip Scanner 3000-7G (Affymetrix Inc., Santa Clara, CA, USA). Image generation and feature extraction were performed using Affymetrix GCOS Software v1.4.0.036.
Description
Quality Control: Approved
Data processing
Quality control and correction (correcting significant hybridization and experimental blocking effects), annotation, RMA normalization and subsequent data analysis was performed as previously described (Schaap et al. 2012). Only the annotated Entrez genes were used for further analysis. Functional genomics analyses, using the top 1000 FDR-ranked genes, were performed using Metacore (version 6.11 build 41105, GeneGo Inc. St. Joseph, MI, USA) to assess the biological response upon each chemical exposure
