PB (Sigma [St Louis, MO] #04710, 0.05% [wt/vol] in drinking water) was administered to one group through ad libitum access to drinking water. Mice were checked daily for activity and behavior and sacrificed on the indicated dates.
Growth protocol
29- to 32-day-old male B6C3F1/Crl (C57BL/6 female × C3H/He male) mice were obtained from Charles River Laboratories (Germany). Animals were allowed to acclimatize for 5 days prior to being randomly divided into treatment groups.
Extracted molecule
total RNA
Extraction protocol
Blood was withdrawn for PK analysis, and liver was removed, split into several sections, either frozen in liquid nitrogen and stored at −80°C for subsequent analyses or fixed in neutral phosphate-buffered formalin, and embedded in paraffin wax. To ensure sample homogeneity for different molecular profiling methods, frozen liver samples were reduced to powder with Covaris Cryoprep (Covaris Inc., Woburn, MA) system and aliquoted on dry ice. Frozen liver samples were homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA) and subsequently purified on a silica-gel-based-membrane (RNeasy, Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. RNA quality was assessed by measuring the RIN (RNA Integrity Number) using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).
Label
biotin
Label protocol
Processing of GeneChip® experiments was conducted as recommended by the manufacturer of the GeneChip® system (Affymetrix, Santa Clara, CA). For tissue samples, double stranded cDNA was synthesized with a starting amount of 0.1 µg total RNA. For RNA reverse transcription, the GeneChip® 3' IVT Express Labelling Assay (lot ID 0904012, Affymetrix) was used in the presence of a T7-(dT)24 DNA oligonucleotide primer (Affymetrix). The cDNA was then transcribed in vitro in the presence of biotinylated ribonucleotides to form biotin-labelled amplified RNA (aRNA). The labelled aRNA was then purified and quantified by UV spectrophotometry at 260 nm and fragmented.
Hybridization protocol
10 µg of fragmented, biotinylated aRNA were hybridized for approximately 16 hrs at 45ºC to the GeneChip® Mouse430_2 arrays. The arrays were then washed and stained with the GeneChip® Hybridization Wash and Stain kit (Affymetrix). The washing and staining steps were performed with GeneChip® Fluidics Workstation 450 (Affymetrix).
Scan protocol
Arrays were then scanned using a solid-state laser scanner (GeneArray® Scanner 3000 combined with the GeneChip® autoloader, Affymetrix). The Affymetrix GeneChip® Operating Software (GCOS) was used to generate the primary and secondary raw data files.
Data processing
The raw microarray data was normalized using the RMA method implemented in R/Bioconductor.
