Proteomic Analyses of Lung Lysates from Short-Term Exposure of Fischer 344 Rats to Cigarette Smoke

A short-term 5 day mainstream cigarette smoke exposure study was conducted in Fischer 344 rats to identify changes in lung proteins. Groups of 10 male and female rats at 5 weeks of age were assigned to one of four exposure groups. Animals received either nose-only filtered air (Air Control) or 75, 200, or 400 mg total particulate matter (TPM)/m(3) of diluted cigarette smoke. Exposures were conducted for 3 h per day, for 5 consecutive days. One lung per animal was frozen in liquid nitrogen and processed for proteomic analyses. Lung lysates from control verses treated animals were screened with 650 antibodies for changes in signaling protein levels and phosphorylation using antibody microarray technology, and then over 100 of the top protein hits were assessed by immunoblotting. The top smoke-altered proteins were further evaluated using reverse lysate microarrays. Major protein changes showed medium to strong bands on Western blots, depended on dose and gender, and included protein-serine kinases (Cot/Tpl2, ERK1/2, GSK3α/β, MEK6, PKCα/γ, RSK1), protein phosphatases (PP4/A'2, PP1Cβ), and other proteins (caspase 5, CRMP2, Hsc70, Hsp60, Rac1 and STAT2). The most pronounced changes occurred with 75 mg TPM/m(3) exposed females and 200 mg TPM/m(3) exposed males. Smoke-altered proteins regulate apoptosis, stress response, cell structure, and inflammation. Changes in identified proteins may serve as early indicators of lung damage.