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

Abstract

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.

Tracheal Morphologic and Protein Alterations FollowingShort-Term Cigarette Mainstream Smoke Exposure to Rats

“…Cigarette smoke activated ERK 1/2 in human bronchial epithelial cells 18 and significantly increased phosphorylation of ERK 1/2 in human aortic smooth muscle cells 19 . In lung lysates from Fischer rats from the same exposure regimen as in the present study, ERK2 increased 79% in 75 mg TPM/m 3 females and 41% in 200 mg TPM/m 3 males 6 . Taken together, cigarette smoke has a prominent modulatory effect on ERK protein levels in respiratory system tissues.…”

“…Caspase 5 mediates apoptosis and is an inflammatory caspase regulated by lipopolysaccharide and interferon γ 24 . Caspase 5 p20 is a fully cleaved or activated form of capase 5 and increased in tracheal lysates (200 and 400 mg TPM/m 3 in both genders vs controls) while in lung lysates from Fischer rats from the same study, caspase 5 decreased 55% in 75 mg TPM/m 3 females and 63% in 200 mg TPM/m 3 males 6 . Caspase 5 intermediate domain (30 kDa) does not fully lead to a similar expression of p20 caspase 5 in tracheal lysates, especially not in males.…”

“…We are evaluating a newer mechanistic approach that focuses on induction of toxicological changes relevant to disease such as cell proliferation, chronic inflammation, and inhibition of apoptosis 5 , 6 . Toxicoproteomics is being employed to evaluate the protein changes associated with these functional and phenotypic changes.…”

Metabolomics Approach for Hazard Identification in Human Health Assessment of Environmental Chemicals

“…Additionally, metabolomics has identified biochemical alterations in pathways associated with oxidative stress, inflammation, cell proliferation, and cytotoxicity in human lung epithelial cells exposed to cigarette smoke (Vulimiri et al, 2009). These observations are supported by data from genomic (Harvey et al, 2007;Meng et al, www.intechopen.com Spira et al, 2004) and proteomic (Carter et al, 2011;Kelsen et al, 2008;Zhang et al, 2008) investigations that also detected markers associated with the pathways affected by exposure to cigarette smoke.…”

Farklı Yaşlardaki Saanen Keçilerinin Farkli Mevsimlerdeki HSP 60 ve HSP70 için mRNA Gen Ekspresyon Profilleri

“…These proteins, called as Heat Shock Proteins (HSPs), have been determined to have increased not only due to high environmental or body temperatures but due to the stress factors, such as viral infections, heavy metals, chemical matterials, pesticides, oxygen and glucose deficiency as well [11,12] . Plenty of studies have recently been conducted on various diseases, particularly in humans, appearing with the aging due to dropping down HSP60 levels [13,14] .…”