cell line: TK6 cell type: human lymphoblastoid cells exposed to: 24 µg/ml Cisplatin harvest time: after 20h recovery in fresh media
Treatment protocol
TK6 cells were exposed to 3 concentrations of AFB1 (n = 3 for AFB1 at 4, 8 & 24h) as follows: 0.025 μM (low), 0.075 μM (medium) and 0.1 μM (high). Separate plates were used for exposures to negative and positive controls (NC (±S9), VC (±S9), PC-1 (-S9) and PC-24(-S9)). Time points were named as follows: 4h = four hours of exposure and immediate sample collection; 8h = four hours of exposure, media replaced and sampled 4 hours later; 24h = four hours of exposure, media replaced, and sampling 20 hours later. All exposures were done in the presence of 1% BF/PB-induced rat liver S9 with NADPH generating system cofactors to act as a metabolic activation system (MAS), as TK6 cells do not have inherent metabolic capacity (except for the direct-acting positive control and its associated negative and vehicle controls). Exponentially growing cells were exposed to each chemical for 4 ± 0.5 hours. At the end of each 4h exposure, the exposed and control TK6 cells for each time point were removed by centrifugation and the cells were rinsed in PBS. The 4h samples were immediately harvested and flash frozen for RNA collection and a portion was used for measurement of cytotoxicity. The 8 and 24h samples were re-suspended in 3 ml of media and re-incubated for an additional 4 ± 0.5 and 20 ± 0.5 hours, respectively, before being harvested for RNA and cytotoxicity/MN assays. Cells dedicated for the purposes of RNA extraction were collected from each well (4 ± 0.5 x 10^6 cells), pelleted by centrifugation and flash frozen in liquid nitrogen. Cells were stored at -80oC until RNA extraction was performed.
Growth protocol
TK6 cells, a human lymphoblastoid cell line, were obtained from American Type Culture Collection (ATCC# CRL-8015; ATCC, Manassas, VA, USA). TK6 cells were maintained as described elsewhere [Ellinger-Ziegelbauer et al., 2009]. Briefly, cells were cultured and maintained in RPMI 1640 medium containing 10% heat inactivated horse serum, in addition to 0.1% pluronics, sodium pyruvate and antibiotics (penicillin at 20 units/ml and streptomycin at 20 µg/ml) at 37 ± 1C and 6 ± 1% CO2 in air. Immediately prior to chemical exposure, cells were seeded at a density of 4 (± 0.5) x 10^5 cells/ml in twelve-well plates with a final volume of 3 ml per well. Given that TK6 cells are not metabolically competent cells and that BaP requires metabolic activation to form reactive metabolites, all cellular exposures were conducted in the presence of 1% 5,6 benzoflavone-/phenobarbital-induced rat liver S9 (Moltox, Boone, NC, USA) with NADPH generating system cofactors. The in vitro cellular exposures were performed by Integrated Laboratory Systems, Inc. (ILS; Research Triangle Park, NC, USA), in addition to the associated cytotoxicity and genotoxicity testing.
Extracted molecule
total RNA
Extraction protocol
Following AFB1 cellular exposures, total RNA was isolated from treated and control TK6 cells at the 4, 8 and 24h time points. An RNeasy Mini Kit (Qiagen, Toronto, ON, Canada) was used to isolate and purify total RNA using the spin technology protocol with an on-column DNase I digestion, following the manufacturer’s instructions. Total RNA was quantified and assessed for purity and integrity using a NanoDrop® ND-1000 spectrophotometer and an Agilent 2100 Bioanalyzer. Only high quality total RNA samples, with RNA Integrity Numbers (RINs) ranging from 8.6 to 10 and A260/280 absorbance ratios >2.0 were used for gene expression analysis.
Label
Cy3,Cy5
Label protocol
Gene expression profiles were generated using a two-colour dye swap design [Kerr and Churchill, 2001]. Microarray analysis was conducted on all time points from the definitive studies (n=3 per concentration and time point), along with the corresponding pooled vehicle and positive controls. Agilent Low-Input Quick Amp Labelling kits were used to ultimately generate cyanine-3 and cyanine-5 labelled cRNA from 100 ng of total RNA, according to the manufacturer’s instructions (Agilent Technologies, Mississauga, ON, Canada).
cell line: TK6 cell type: human lymphoblastoid cells sample type: negative vehicle control pool (DMSO -S9)
Treatment protocol
TK6 cells were exposed to 3 concentrations of AFB1 (n = 3 for AFB1 at 4, 8 & 24h) as follows: 0.025 μM (low), 0.075 μM (medium) and 0.1 μM (high). Separate plates were used for exposures to negative and positive controls (NC (±S9), VC (±S9), PC-1 (-S9) and PC-24(-S9)). Time points were named as follows: 4h = four hours of exposure and immediate sample collection; 8h = four hours of exposure, media replaced and sampled 4 hours later; 24h = four hours of exposure, media replaced, and sampling 20 hours later. All exposures were done in the presence of 1% BF/PB-induced rat liver S9 with NADPH generating system cofactors to act as a metabolic activation system (MAS), as TK6 cells do not have inherent metabolic capacity (except for the direct-acting positive control and its associated negative and vehicle controls). Exponentially growing cells were exposed to each chemical for 4 ± 0.5 hours. At the end of each 4h exposure, the exposed and control TK6 cells for each time point were removed by centrifugation and the cells were rinsed in PBS. The 4h samples were immediately harvested and flash frozen for RNA collection and a portion was used for measurement of cytotoxicity. The 8 and 24h samples were re-suspended in 3 ml of media and re-incubated for an additional 4 ± 0.5 and 20 ± 0.5 hours, respectively, before being harvested for RNA and cytotoxicity/MN assays. Cells dedicated for the purposes of RNA extraction were collected from each well (4 ± 0.5 x 10^6 cells), pelleted by centrifugation and flash frozen in liquid nitrogen. Cells were stored at -80oC until RNA extraction was performed.
Growth protocol
TK6 cells, a human lymphoblastoid cell line, were obtained from American Type Culture Collection (ATCC# CRL-8015; ATCC, Manassas, VA, USA). TK6 cells were maintained as described elsewhere [Ellinger-Ziegelbauer et al., 2009]. Briefly, cells were cultured and maintained in RPMI 1640 medium containing 10% heat inactivated horse serum, in addition to 0.1% pluronics, sodium pyruvate and antibiotics (penicillin at 20 units/ml and streptomycin at 20 µg/ml) at 37 ± 1C and 6 ± 1% CO2 in air. Immediately prior to chemical exposure, cells were seeded at a density of 4 (± 0.5) x 10^5 cells/ml in twelve-well plates with a final volume of 3 ml per well. Given that TK6 cells are not metabolically competent cells and that BaP requires metabolic activation to form reactive metabolites, all cellular exposures were conducted in the presence of 1% 5,6 benzoflavone-/phenobarbital-induced rat liver S9 (Moltox, Boone, NC, USA) with NADPH generating system cofactors. The in vitro cellular exposures were performed by Integrated Laboratory Systems, Inc. (ILS; Research Triangle Park, NC, USA), in addition to the associated cytotoxicity and genotoxicity testing.
Extracted molecule
total RNA
Extraction protocol
Following AFB1 cellular exposures, total RNA was isolated from treated and control TK6 cells at the 4, 8 and 24h time points. An RNeasy Mini Kit (Qiagen, Toronto, ON, Canada) was used to isolate and purify total RNA using the spin technology protocol with an on-column DNase I digestion, following the manufacturer’s instructions. Total RNA was quantified and assessed for purity and integrity using a NanoDrop® ND-1000 spectrophotometer and an Agilent 2100 Bioanalyzer. Only high quality total RNA samples, with RNA Integrity Numbers (RINs) ranging from 8.6 to 10 and A260/280 absorbance ratios >2.0 were used for gene expression analysis.
Label
Cy5,Cy3
Label protocol
Gene expression profiles were generated using a two-colour dye swap design [Kerr and Churchill, 2001]. Microarray analysis was conducted on all time points from the definitive studies (n=3 per concentration and time point), along with the corresponding pooled vehicle and positive controls. Agilent Low-Input Quick Amp Labelling kits were used to ultimately generate cyanine-3 and cyanine-5 labelled cRNA from 100 ng of total RNA, according to the manufacturer’s instructions (Agilent Technologies, Mississauga, ON, Canada).
Hybridization protocol
Labelled cRNA samples (325 ng of cyanine-3 labelled cRNA and 325 ng of cyanine-5 labelled cRNA) were hybridized to Agilent SurePrint G3 Human GE 8x60K oligonucleotide microarrays (Agilent Catalogue No: G4851A, Agilent Microarray Design ID: 028004; Agilent Technologies, Mississauga, ON, Canada) at 65oC for 17 hours.
Scan protocol
Slides were washed according to the manufacturer’s specifications and scanned using an Agilent G2505C DNA Microarray Scanner at 3 μm resolution. Agilent Feature Extraction (version 11.0.1.1) was used for data extraction purposes from the generated image files and to generate QC reports for each array.
Description
Sample 23
Data processing
The log2 of the ratio using the median signal intensities were normalized using lowess using the transform.madata function in the maanova library in R. After normalization the log2 ratios for the dye swap were averaged yielding a ratio representing the treatment relative to the vehicle control [the sample-dye assignment information for each of the raw data files is included in the 'readme.txt' (available as Series supplementary file)].
Integration of metabolic activation with a predictive toxicogenomics signature to classify genotoxic versus nongenotoxic chemicals in human TK6 cells [AFB1]
