TK6 cells were exposed to 3 concentrations of the test chemical as follows: 15, 20 and 30 µg/ml, in addition to vehicle and positive controls (water (+S9), and BaP(+S9), respectively). Cells were co-exposed to each chemical (+S9) for four hours, then the media was replaced and cells were collected 3 to 4 hours later. All exposures were done in the presence of 2% aroclor-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. 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 cells were then re-suspended in 3 ml of media and re-incubated for an additional 3 to 4 hours before being harvested for RNA extraction. Cells dedicated for the purposes of RNA extraction were collected from each well (4 ± 0.5 x 106 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 ± 1oC and 6 ± 1% CO2 in air. Immediately prior to chemical exposure, cells were seeded at a density of 4 (± 0.5) x 105 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 compound requires metabolic activation to form reactive metabolites, all cellular exposures were conducted in the presence of 2% Aroclor 1254-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 chemical exposures, total RNA was isolated from treated and control TK6 cells between 7 and 8 hours post-exposure. 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 8.0 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 the 7-8 hour time points from the definitive studies (n=3 per concentration and time point), along with the corresponding 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).
TK6 cells were exposed to 3 concentrations of the test chemical as follows: 15, 20 and 30 µg/ml, in addition to vehicle and positive controls (water (+S9), and BaP(+S9), respectively). Cells were co-exposed to each chemical (+S9) for four hours, then the media was replaced and cells were collected 3 to 4 hours later. All exposures were done in the presence of 2% aroclor-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. 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 cells were then re-suspended in 3 ml of media and re-incubated for an additional 3 to 4 hours before being harvested for RNA extraction. Cells dedicated for the purposes of RNA extraction were collected from each well (4 ± 0.5 x 106 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 ± 1oC and 6 ± 1% CO2 in air. Immediately prior to chemical exposure, cells were seeded at a density of 4 (± 0.5) x 105 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 compound requires metabolic activation to form reactive metabolites, all cellular exposures were conducted in the presence of 2% Aroclor 1254-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 chemical exposures, total RNA was isolated from treated and control TK6 cells between 7 and 8 hours post-exposure. 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 8.0 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 the 7-8 hour time points from the definitive studies (n=3 per concentration and time point), along with the corresponding 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 or B, Agilent Microarray Design ID: 028004 or 039494; 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
Co-hybridized with negative vehicle control pool (DMSO +S9)
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.
