A pulmonary rat gene array for screening altered expression profiles in air pollutant-induced lung injury.
Pulmonary tissue injury and repair processes involve complex and coordinated cellular events such as necrosis, inflammation, cell growth/differentiation, apoptosis, and remodeling of extracellular matrix. These processes are regulated by expression of multiple mediator genes. Commercially available microarray blots and slides allow screening of hundreds to thousands of genes in a given tissue or cell preparation. However, often these blots do not contain cDNAs of one's interest and are difficult to interpret. In order to analyze the tissue expression profile of a large number of genes involved in pulmonary injury and pathology, we developed a rat gene array filter using array technology. This array consisted of 27 genes representing inflammatory and anti-inflammatory cytokines, growth factors, adhesion molecules, stress proteins, transcription factors and antioxidant enzymes; 3 negative controls, and 2 blank spots. Using rat gene-specific polymerase chain reaction (PCR) primer pairs, cDNAs for these genes were amplified and cloned into a TA vector. Plasmids with recombinant cDNA inserts were purified and blotted onto a nylon membrane. Lung total RNA was isolated at 3 or 24 h following intratracheal (IT) exposure of male Sprague Dawley rats to either saline (control), residual oil fly ash (ROFA; 3.3 mg/kg) or metals found in one instillate of ROFA: nickel (NiSO(4); 1. 3 micromol/kg) or vanadium (VSO(4); 2.2 micromol/kg). (32)P-Labeled cDNA was generated from RNA samples in a reverse transcriptase reaction and subsequently hybridized to array blots. Densitometric scans of array blots revealed a twofold induction of interleukin (IL)-6 and TIMP-1 at 24 h post ROFA or Ni exposure. The pulmonary expressions of cellular fibronectin (cFn-EIIIA), ICAM-1, IL-1beta, and iNOS genes were also increased 24 h post ROFA-, V-, or Ni-exposure. Consistent hybridization of beta-actin in all array blots and absence of hybridization signals in negative controls indicated gene specific hybridization. ROFA or metal-induced increase in the expression of IL-6 observed in array blot was validated by Northern blot hybridization. Developing a pulmonary rat gene array may provide a tool for screening the expression profile of tissue specific markers following exposure to toxic air contaminants.
Nadadur
S S
SS
Pulmonary Toxicology Branch, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, MD-82, Research Triangle Park, NC 27711, USA. nadadur.srikanth@epa.gov
Schladweiler
M C
MC
Kodavanti
U P
UP
D000393:Air Pollutants / Q000633:toxicity*; D000818:Animals; D015152:Blotting, Northern; D005786:Gene Expression Regulation / Q000187:drug effects*; D008168:Lung / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008297:Male; D009532:Nickel / Q000633:toxicity; D051381:Rats; D017207:Rats, Sprague-Dawley; D014639:Vanadium / Q000633:toxicity
D000393:Air Pollutants; D014639:Vanadium; D009532:Nickel