Effects of cigarette smoking on the human urinary proteome Luisa Airoldi * , Cinzia Magagnotti, Angela Rita Iannuzzi, Cristina Marelli, Renzo Bagnati, Roberta Pastorelli, Alessandro Colombi, Stefano Santaguida, Chiara Chiabrando, Silvia Schiarea, Roberto Fanelli Department of Environmental Health Sciences, Istituto di Ricerche Farmacologiche Mario Negri, via Giuseppe La Masa, 19, 20156 Milan, Italy article info Article history: Received 10 February 2009 Available online 15 February 2009 Keywords: 2-Dimensional electrophoresis Cigarette smoking Mass spectrometry Pathway analysis Proteomics Urine Western blotting abstract In this pilot study we used a proteomic approach to compare the urinary protein patterns of healthy smokers and non-smokers. Proteins were resolved by two-dimensional gel electrophoresis and identified by mass spectrometry. The relative abundance of three inflammatory proteins (S100A8, inter-alpha-tryp- sin inhibitor heavy chain 4, CD59) and that of two isoforms of pancreatic alpha amylase was significantly higher in smokers. Zinc-alpha-2-glycoprotein was the only protein down-regulated in smokers. Its abun- dance was significantly correlated with urinary glucocorticoids. Most of the proteins identified may be non-specific biomarkers of tobacco effects, since they are involved in inflammatory responses associated with several diseases. Of greater interest are the changes in abundance of pancreatic alpha amylase and zinc-alpha-2-glycoprotein, which after proper validation, might be candidate biomarkers of diseases resulting from exposure to tobacco smoke. The data also show for the first time that smoking can affect the expression profile of urinary proteins. Ó 2009 Elsevier Inc. All rights reserved. Introduction Cigarette smoking attributable deaths are projected to rise by from 5.4 million in 2005 to 6.4 million in 2015 under the present scenario [1]. Because of the wide spread of the smoking habit and its consequent impact on human health, interventions to re- duce tobacco-related diseases have high priority. Obviously, quitting smoking is usually the best preventive ac- tion, but most smokers are unable or unwilling to do this, so a number of biomarkers have been used to detect exposure and/or individual susceptibility to tobacco smoke carcinogens, with the ultimate goal of avoiding health problems. It is assumed that exogenous compounds may become toxic or carcinogenic by interacting with receptors, proteins, gene regula- tory elements, DNA, and various biological processes. The result may be dysregulation of the expression of proteins on which cell physiological functions depend. Establishing a relationship be- tween exposure to toxic compounds and protein markers might provide information to lead to new or refined hypotheses on the mechanisms of toxic or carcinogenic effects of xenobiotics and help prevent adverse health effects. Therefore studying the proteome of readily obtainable human biological samples, such as body fluids, is challenging. Urine, the most accessible body fluid, has been pre- viously studied to search for protein markers of a pathological sta- tus or exposure to toxic compounds [2, and references herein]. Lifestyle, however, although recognized as having important ef- fects on protein expression, has been only marginally considered [3]. This pilot study assessed whether the human urinary proteome is affected by cigarette smoking and whether the proteins whose expression was changed could be candidate biomarkers with po- tential clinical value in relation to tobacco-related diseases. Pro- teins were separated by two-dimensional gel electrophoresis (2DE) and identified by mass spectrometry (MS) coupled to a data- base search. Materials and methods (see Supplementary materials and methods for details) Recruitment of subjects Urine specimens were collected from twenty healthy male do- nors, ten smokers and ten non-smokers, with no history of kidney or extra-renal disease. Each subject signed an informed consent and answered a questionnaire about personal data, smoking, alco- holic drinks and coffee consumption, and use of prescription or over-the-counter medications. Sample preparation The second morning urine (about 150–200 mL) was collected from consenting individuals on one tablet of Complete Protease Inhibitor Cocktail (Roche Diagnostics Italia, Milan, Italy). Urine 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.02.055 * Corresponding author. Fax: +39 02 39001916. E-mail address: airoldi@marionegri.it (L. Airoldi). Biochemical and Biophysical Research Communications 381 (2009) 397–402 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc