cell type: primary human hepatocytes main source: liver study type: Wash out effect assay type: ex vivo strain: Hu4227, Hu8150, Hu4197 agent: DMSO dose: 0 dose unit: % dose duration: 5 duration unit: days dose frequency: daily vehicle: DMSO route: medium
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) 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, 0.3 µM of AFB1, was selected for the main experiment.
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.
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 the treatment was removed for 3 days (washout period), then cells lysates were harvested for DNA and total RNA isolation. At the end of washout, 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.
High-density oligonucleotide GeneChips from Affymetrix were used to measure gene expression levels (Human Genome U133 Plus 2.0 array (604,258 probes)). Targets for these arrays were prepared, hybridized and scanned according to the Affymetrix protocol (Affymetrix).Normalization quality controls appeared to be within acceptable limits for almost all chips.
Amplified, biotinylated and fragmented targets were then hybridized on to the arrays.
After hybridization, arrays were washed and stained using an Affymetrix fluidics station and scanned by use of an Affymetrix GeneArray scanner. A total of 6 RNA samples was prepared and analyzed on GeneChip arrays (treated and control samples from each time point were at least in triplicate). Normalization quality controls, including scaling factors, average intensities, present calls, background intensities, noise, and raw Q values, appeared to be within acceptable limits for all chips. Hybridization controls BioB, BioC, BioD, and CreX were called present on all chips and yielded the expected increases in intensities.
BrainArray custom CDF v15.1.0
The Arrayanalysis.org web service was used for quality control (27) and all microarrays except one were of high quality which was omitted from the analyses. CEL files were imported into R v2.15.3 (http://www.r-project.org) using the “affy” library (28) within BioConductor (v2.9) (29). Probe re-annotation, normalization and data filtering was performed as previously described (30).