The exposure to the selected chemicals was done for a period of 72h at IC10 concentrations.
Growth protocol
Hepatocytes were isolated by using a two-step collagenase perfusion technique . The viability was assessed by trypan blue exclusion and the cells (≥85% viability) were cultured as a monolayer at a density of 0.57x105 cells/cm2 in William’s E medium supplemented with 10%v/v fetal bovine serum, 292mg/ml L-glutamine, 7ng/ml glucagon and antibiotics (7.33IU/ml benzylpenicillin sodium, 50µg/ml kanamycin monosulphate, 50µg/ml streptomycin sulphate, and 10µg/ml ampicillin sodium) in an incubator at 37°C (5% CO2 and 95% air, 100% humidity). After 4 hours, the medium was renewed with serum-free culture medium, supplemented with 25µg/ml hydrocortisone hemisuccinate and 1mg/ml bovine insulin. The hES-Hep cells were derived, cultured and characterized from the human embryonic stem cell (hESC) line SA002 (Cellartis AB, Sweden), as previously described, with an additional step of enzymatic passaging for further expansion before the onset of hepatic differentiation (Heins et al. 2006). The differentiation of SA002 cells into hepatocyte-like cells was achieved by the sequential exposure to growth factors, such as human growth factor and differentiation-promoting factors, including hydrocortisone and insulin (Brolen et al. 2010). The hepatocellular carcinoma-derived HepG2 cell line (Rockville, USA) was cultured and handled as previously described (Tolosa et al. 2011). Human hepatoma HepaRG cells (Biopredic International, France) were cultivated as previously described (Gripon et al. 2002; Guillouzo et al. 2007). At day 13 of cultivation, low DMSO-containing medium was added for a period of 7 days.
Extracted molecule
total RNA
Extraction protocol
Samples for RNA isolation were collected after 72 hours of exposure. Minimum three independent biological experiments were conducted for each compound. The total RNA extraction (RNA extraction kit, Qiagen), including a DNase digestion step, was done according to the manufacturer’s instructions.
Label
biotin
Label protocol
We used standard Affymetrix labeling protocols
Hybridization protocol
We used standard Affymetrix hybridisation rotocols
Scan protocol
We used standard Affymetrix scanning parameters
Description
Treatment with Tetraphorbolacetate
Data processing
The Affymetrix IDs were alternatively remapped to the alternative IDs from Brainarray (http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/CDF_download.asp), thus the ensemb version 61 was used (http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/14.1.0/ensg.download/HGU133Plus2_Hs_ENSG_14.1.0.zip). All samples were further normalized by GC-RMA algorithm. In the hES-Heps 2NF, BaP, MAN, MPH, NIF, PIPB, SDF and SPB shared one control whereas AFB, CND, TPA, TOL, WYE and CYCLO shared another control. In the HepaRG system several compounds shared also one control. The groups were divided as follows: 1) 2NF, BaP, MPH, NIF, PIPB and SPB; 2) WYE, AFB, CND, CYCLO, TPA and TOL; 3) MAN and SDF; 4) NNK. In the HepG2 system there were shared control among several compounds also. 2NF, BaP, NIF, PIPB and SPB shared one control, AFB, CND, CYCLO, TPA, TOL and WYE shared another control. The third group was MAN, MPH and SDF which had their own control whereas NNK had its own control. In the HepsC and the HepsT systems there were also several groups of compounds having the same control.The compounds were separated as follows: 1)2NF, NIF and PIPB sharing one control; 2)NNK, MAN, SDF and SPB sharing one control; 3)AFB, CND, CYCLO, TPA, TOL and WYE sharing one control; 4) BaP and MPH sharing one control.
