treatment: Nonylphenol (9-nonylphenol) Tergitol NP-9_2 dose: 1 ppm time: 24h cell type: primary rat hepatocytes, rtNHeps gender: Male
Treatment protocol
The chemicals for the entire OMICS study were purchased from various sources: Energetics from Chem Service (West Chester, PA) and SRI International (Menlo Park, CA); remaining chemicals from Sigma-Aldrich (St. Louis, MO) and Fisher Scientific (Fair Lawn, NJ). The purity of all these compounds was equal or greater than 98%. Prior to testing, the compounds were made up and serially diluted in dimethyl sulfoxide, DMSO (Fisher Scientific, Fair Lawn, NJ). Metal compounds were made up and serially diluted in 0.1Fm-filtered, ultra-pure water before testing. The 24-h toxicity of each OMICS compound was first determined in the human hepatocyte cell line HepG2 using the Neutral Red cytotoxicity kit (In Vitro Toxicology Kit Tox-4) obtained from Sigma-Aldrich.Primary rtNHeps cells collected at the end of the 24h chemical exposure were immediately stored in RNA Later (7021) purchased from Ambion (Austin, TX). The primary rtN Heps cells were reconstituted in HCM supplemented with ascorbic acid, fatty acid-free bovine serum albumin, transferrin, insulin, recombinant human epidermal growth factor, hydrocortisone 21 hemisuccinate, Gentamicin sulfate, and Amphotericin B immediately upon receipt. An aliquot of the cell suspension was stained in a 0.05% Trypan Blue solution and counted under an inverted microscope. The cells were then seeded at 3 x 106 cells per (Type 1 collagen-coated) T-75 flask. The flasks were left in a 37C, 5% CO2 incubator overnight to allow for cell attachment. The cells were replenished with fresh HCM and dosed in triplicate flasks with the non-toxic concentration of each compound (pre-determined in the HepG2 cells) at 1% DMSO (v/v) or with solution at 1% water (v/v). Another set of triplicate flasks were dosed with the appropriate solvent control. Hence, for every 3 chemicals and a solvent control, a total of 12 flasks were used.After dosing, the flasks were returned to the incubator and incubated for 24 hours. The cells were washed with 1x PBS and detached with 1x trypsin-EDTA. The cells were left in tryspin-EDTA no longer than 5 minutes. Freshly warmed HCM was then added to neutralize the trypsin. The cells were then transferred into a tube and centrifuged. RNA Later was added the cell pellet and stored at –20C until genomic analysis
Growth protocol
The primary rat hepatocytes, rtNHeps (AC-2630), isolated from male Sprague Dawley and its hepatocyte culture medium (HCM), and supplements and growth factors (CC-3198) were purchased from Cambrex BioScience (Walkersville, MD). Freshly isolated hepatocytes were shipped overnight on ice and processed immediately upon receipt. These cells were seeded on BioCoat Type 1 Collagen-coated T-25 (BD356484) or T-75 (BD356485) flasks, or clear, flat-bottom, 96-well plates (BD356407) that were purchased from BD Biosciences (Palo Alto, CA). Phosphate buffered saline (PBS, 21600-010), Trypsin-EDTA (15400-054), Trypan Blue stain (15250-061), Leibovitz medium (12448-015) were obtained from GIBCO-InVitrogen (Grand Island, NY).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from primary rat hepatocytes cell pellet. Cell pellet were homogenized in the lysis buffer with FAST Prep-24 from MP at speed 6.0/s twice, each last 30s before using RNeasy kits (Qiagen). Total RNA concentrations were measured using NanoDrop® ND-1000 Spectrophotometer (NanoDrop technologies, Wilmington, DE, USA). The integrity and quality of total RNA was checked on an Agilent 2100 Bioanalyzer (Palo Alto, CA). The gel-like images generated by the Bioanalyzer show that total RNAs have two bands, represent 18S and 26S RNA of mammalian RNA . Nuclease-free water (Ambion) was used to elute total RNA.
Label
CY3
Label protocol
Rat whole genome oligo arrays in the format of 4X44K were purchased from Agilent. Sample cRNA synthesis, labeling, hybridization and microarray processing were performed according to manufacturer’s protocol One-Color Microarray-Based Gene Expression Analysis (version 1.0). 1ug of total RNA was used. The Agilent One-Color Spike-Mix (part number 5188-5282) was diluted 5000-fold and 5 μL of the diluted spike-in mix was added to 1000 ng of each of the total RNA samples prior to labeling reactions. The labeling reactions were performed using the Agilent Low RNA Input Linear Amplification Kit in the presence of cyanine 3-CTP.
Hybridization protocol
1.65 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
Scan protocol
After washing, the arrays were scanned at PMT levels 350 using GenePix 4200AL scanner (Molecular Device Inc.),
Description
Gene Expression after 24h exposed to 14_Nonylphenol_90-2 in rat liver cell
Data processing
The Feature extraction software (V. 9.5.1) from Agilent was used to automatically find and place microarray grids, reject outlier pixels, accurately determine feature intensities and ratios, flag outlier pixels, and calculate statistical confidences. First normalize be median perchip, then by median per gene, which means normalization by median value across all samples, then transformed to log2 based value.
Identification of biomarkers that distinguish chemical contaminants using a gradient feature selection method
Data table header descriptions
ID_REF
VALUE
If the scanned intensity was less than 5.0 for a probe, it was transformed to 5. A perchip (within) array normalization was performed using 50 percentile values of all the probe values in the array. Data were subsequently log (base 2) transformed for statistical analyses.
