After a recovery period of 40-42h, the Dulbecco's Modified Eagle's (Gibco BRL, Breda, The Netherlands) culture medium was replaced by culture medium containing either one of the 21 compounds or a vehicle control (0.5% of DMSO, PBS, EtOH). Seventeen chemicals were obtained through Sigma-Aldrich, TCDD through Cerilliant, DMBA through ICN, E2 through Steraloids Inc. and PhB through Brocacef Intramuraal bv. A dose causing minimal cytotoxicity after 24h of exposure thus resulting in ±80% viability, was established by the MTT assay (Mosmann, 1983). Next, cells were treated with an IC20-24h dose of di(2-ethylhexyl)phthalate(DEHP), 4-Acetylaminofluorene(4AAF), Curcumin(Cur), Phenobarbital(PhB), Reserpine(Res), 7,12-Dimethylbenzanthracene (DMBA), Resorcinol(Resorcinol), Para-cresidine(pCres), Phenacetin(Phen), Diclofenac(diclo), Wy 14643(Wy), Tributyltinoxide(TBTO), Benzo[a]pyrene(BaP), 8-Hydroxyquinoline [AKA 8-quinolinol](8HQ), 17-b-estradiol(E2), ampicillin(AmpC), cisplatin(CisPl), Aflatoxin B1(AFB1), Cyclosporin A(CsA), 2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD), Quercetin(Que) for 24 or 48 h before being harvested. Independent biological experiments (at least in triplicate) with hepatocytes from different mice were obtained for every time point.
Growth protocol
PMH were isolated from adult male C57BL/6 mice using a perfusion method and cultured between a collagen-collagen sandwich formation in two six wells plates as described before.
Extracted molecule
total RNA
Extraction protocol
At the end of treatment the medium was removed and PMH were harvested in Qiazol (QIAGEN Benelux B.V., Venlo, The Netherlands). Total RNA was isolated using a miRNeasy Mini Kit (QIAGEN Benelux B.V., Venlo, The Netherlands) according to the manufacturer’s protocol and followed by DNase I (Qiagen, Inc) treatment. Following purification, RNA concentrations were measured by means of a NanoDrop® ND-1000 spectrophotometer (Thermo Scientific, Wilmington, USA) at 260 and 280 nm. RNA quality and integrity were assessed by using automated gel electrophoresis on an Agilent 2100 Bioanalyzer system (Agilent Technologies Netherlands B.V., Amstelveen, The Netherlands). Only RNA samples which showed clear 18S and 28S peaks and with an RNA integrity number (RIN) higher than 8 were used. Samples were stored at -80ºC until RNA hybridization.
Label
Hy3
Label protocol
The miRCURYTM locked-nuclei acid array (LNA), 5th and 7th generation (Exiqon, Denmark) contains probes that detect mature forms of all microRNAs present in miRBase 20.0 (http://www.mirbase.org/) (Griffiths-Jones et al, 2008). The Exiqon platform has been validated and is shown to reliably detect microRNA expression. The 5th and 7th generation microarray contains 9,360 and 14,708 reporters, which represent several control probes and sequences of mature human/mouse/rat specific microRNA (4 technical replicates). In our analysis, 661 unique mouse reporters (measurable on both generation arrays) were used. cDNA was generated using 1 μg of total RNA per sample. RNA was labeled only with Hy3 containing dye (single color) using the mercury LNA™ microRNA Hy3 Power labeling kit (Exiqon, Denmark) according to the manufacturer’s protocol in a total volume of 12.5 μl.
Hybridization protocol
To adjust the volume to 200 μl, nuclease free water was added to the labeled samples. Thereafter 200 μl hybridization buffer provided in miRCURY LNA™ microRNA Array, 5th generation kit (Exiqon, Denmark) was added. In a total volume of 400 μl the sample was denatured for 2 minutes at 95ºC, incubated on ice for at least 15 minutes and then spun down. Manual hybridization was performed using an Agilent hybridization SureHyb chamber kit and gasket slide kit at 56 °C for 16h in a hybridization oven with rotation. Following manual hybridization the arrays were automatically washed in a Tecan HS4800 Pro Hybridization station according to Exiqon instructions. For each biological experiment, one hybridization per time point was conducted and one sample per array.
Scan protocol
After washing, the arrays were scanned using the GenePix 4000A scanner (Axon Instruments, Foster City, CA).
Data processing
To quantify the signals, the images were processed in to GPR (5th) and TXT (7th) files through the GenePix Pro Sofware Suite version 3.0 by using the GAL file for probe annotation, as obtained from Exiqon. The GAL file is based on miRBase version 20.0. These GPR or TXT files were then imported in to R 2.11.0, for quality control and statistical analysis. First, the quality of all arrays was inspected using arrayQC, an in-house quality control pipeline that generates virtual images, boxplots, correlation plots, clustering images, MvA and PCA plots. Spike-ins were used for all arrays as an extra quality check. Within this dataset, no arrays were deviated technically. During data analysis the spot intensities were background-corrected and then filtered for low signals (i.e. intensity < 10). Only the mouse microRNA reporter intensities (661 unique reporters x 4 technical replicates) were used for further analyses. If for a reporter less than 3 of the 4 technical replicates were left, then the reporter intensity on that specific reporter was set to NA. The data were log2 transformed, followed by a quantile normalization and a summarization step. In the summarization step, the median intensity from the remaining technical replicates per condition was calculated. Finally, for each reporter a cut-off filtering was applied using the median of the three biological replicates. Only reporters that had a median intensity ≥50 for at least one condition of the 9 compounds tested, or their respective controls, passed this filtering per time point. After filtering, log2 ratios were calculated between average intensities of the treatment of its respective control.
Exploiting microRNA and mRNA profiles generated in vitro from carcinogen-exposed primary mouse hepatocytes for predicting in vivo genotoxicity and carcinogenicity (miRNA)
Exploiting microRNA and mRNA profiles generated in vitro from carcinogen-exposed primary mouse hepatocytes for predicting in vivo genotoxicity and carcinogenicity
