cell type: primary human hepatocytes study type: Single Dose Toxicity assay type: ex vivo strain: Hu4227, Hu8150, Hu4197 compound: DMSO dose: 0.5 dose unit: % dose duration: 5 duration unit: days dose frequency: daily vehicle: DMSO route: medium
Treatment protocol
After quick thawing in a water bath at 37°C, viability of the cells was checked by a Trypan blue (CAS no. 72-57-1, Sigma–Aldrich) exclusion test as instructed in the supplier’s protocol (Invitrogen, 2012). All viability scores after thawing were in agreement with those listed by the supplier. Before AFB1 treatment, cells were allowed to acclimatize for 48 hours. This is needed for the hepatocytes to restore an in vivo like cellular configuration and enzyme expression levels as optimally as possible.AFB1 doses causing minimal (IC20) and moderate (IC40) cytotoxicity after 5 days of repetitive daily exposure were established by means of the MTT assay (23). Furthermore, crucial liver function enzymes such as lactate dehydrogenase (LDH) and alanine transaminase (ALT) were measured. LDH and ALT were spectrophotometrically determined on a Cobas 8000 Modular Analyser (Roche Diagnostics, Basel, Switzerland). Based on this data, two doses, 0.3 µM and 1 µM of AFB1, were selected for the main experiment.
Growth protocol
Cryopreserved primary human hepatocytes (PHH) were purchased from Life Technologies. Cells were cultured in pre-coated 24-well and 6-well plates (700,000 cells/ml) in a 2-layer collagen sandwich (A11428-02, Gibco), according to the supplier’s protocol (Invitrogen, 2012). The following culture media were used: Hepatocyte Thawing Medium (HTM) for thawing (CM7500, Gibco), Williams’ Medium E (1x, no phenol red) (A1217601, Gibco) + Cell Maintenance supplement B kit (CM4000, Gibco) for plating and incubation.
Extracted molecule
total RNA
Extraction protocol
Cells were cultured in triplicate in 24 wells (RNA) or 6 wells (DNA) in a collagen sandwich layer. Following 5 days of repetitive daily exposure, cells lysates were harvested for DNA and total RNA isolation. At the end of treatment, medium was removed and PHH were harvested in Qiazol (Qiagen). Total RNA was isolated using a miRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol and followed by DNase I (Qiagen) treatment. Upon purification, RNA concentrations were measured by means of a NanoDrop® ND-1000 spectrophotometer (Thermo Scientific) at 260 and 280 nm. RNA quality and integrity were assessed by using automated gel electrophoresis on an Agilent 2100 Bioanalyzer system (Agilent Technologies). 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
Cy3
Label protocol
standard Agilent labeling kit
Hybridization protocol
MicroRNA expression profiling was performed using Agilent Sureprint G3 Unrestricted Human miRNA V19 8 × 60 K microarrays. The hybridization was performed following standard protocols, after which the microarray slides were washed and scanned using a DNA microarray scanner (Agilent Technologies).
The scanned images were converted into TXT files using the Feature Extraction Software v10.7.3.1 from Agilent Technologies, which were imported in R 2.15.3 (http://www.r-project.org) for quality control with an in-house developed pipeline. Filtering and normalization was performed using AgiMicroRna (32). Total gene signals were log2-transformed and quantile-normalized. The selection criteria for determining differentially expressed microRNAs (DE-miRs) were identical to those for DEGs.
