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
Cy3
Label protocol
Total RNA was labeled using the Createch Cy3 label kit (Createch), 1ug of total RNA was used.
Hybridization protocol
Labeled materials were hybridized to Custom Exiqon LNA probes for 16 hours at 60°C, subsequently washed and scanned
Scan protocol
Samples were finally scanned using the ScanArray Express HT (Tecan). Image generation and feature extraction were performed using Imagene 6.0.
Data processing
The raw microRNA data, median signal minus median background, were normalized using quantile normalization. For the CsA, WY and CDDP exposed groups quality control discarded one outlier per group. Duplicate spots on the array were averaged and the normalized values were analyzed for differentially expressed microRNAs using a linear model (bioconductor package Limma; Smyth 2005) and corrected for multiple testing (Benjamini and Hochberg 1995) was applied. Only the spots corresponding to mouse microRNAs were used. Note: the annotation is updated for a couple of spots after signal extraction by Imagene. Therefore the annotation in the EL**.txt raw files are not completely correct. The new annotation is available in a separate text file (see 'annotation.txt' linked as supplementary file on Series record).
