Cobalt metal dust was produced by OMG Kokkola Chemicals Oy (Kokkola, Finland) and was provided by the Cobalt Development Institute via PEL Technologies in one lot (P32 3040-1) that was used in the 2-week, 3-month, and 2-year studies. Groups of 50 male and 50 female mice were exposed to cobalt metal particulate aerosol by inhalation at concentrations of 0, 1.25, 2.5, or 5 mg/m3, 6 hours plus T90 (12 minutes) per day, 5 days per week for up to 105 weeks.
At the start of the study, the animals were 5–6 weeks of age. The animals were housed 2 per cage. Tap water and NTP-2000 diet (Zeigler Brothers, Inc. Gardners, PA) were made available ad libitum. The care of animals on this study was according to National Institutes of Health (NIH) procedures as described in the ‘‘The U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals,’’ available from the Office of Laboratory Animal Welfare, NIH, Department of Health and Human Services, RKLI, Bethesda, MD or online at http://grants.nih.gov/grants/olaw/olaw.htm#pol.
The lung was harvested and weighed, and then, a representative sections are collected and fixed in 10 % neutral buffered formalin for histology. Hematoxylin and eosin (H&E)-stained sections (5 μm) were examined by a board certified pathologist. Selected lung tumors are dissected from the remaining lung parenchyma and flash-frozen in liquid nitrogen and stored at −80 °C until RNA extraction. RNA was extracted from frozen samples using the Invitrogen PureLink Mini kit (Invitrogen cat# 12183-018A, Carlsbad, CA) according to the manufacturer’s protocol. RNA concentration and quality were measured on a Bioanalyzer (Agilent Technologies, Santa Clara, CA). Samples were aliquoted and stored at −80 °C until they were analyzed for gene expression studies.
One hundred nanograms of total RNA was amplified as directed in the Affymetrix 3′ IVT Plus kit protocol. Fifteen micrograms of amplified biotin-aRNAs was fragmented, and 12.5 μg was hybridized to each array for 16 h at 45 °C in a rotating hybridization oven using the Affymetrix Eukaryotic Target Hybridization Controls and protocol.
Labeled cRNA was fragmented and hybridized to the Affymetrix Rat Genome 230 2.0 GeneChip arrays (Affymetrix, Santa Clara, CA). Array slides were stained with streptavidin/phycoerythrin utilizing a double-antibody staining procedure and then washed for antibody amplification according to the GeneChip Hybridization, Wash and Stain Kit and user manual.
Affymetrix Scanner 3000
cobalt spontaneous tumor M. musculus sample
Data were processed using R/affy software. In brief, the RMA method adjusts the background of perfect match (PM) probes, applies a quantile normalization of the corrected PM values, and calculates final expression measures with the Tukey median polish algorithm. Data were normalized across all three groups.