Translating proteomics into environmental health : biomarkers discovery for tobacco smoke-induced biological damage

Tese de doutoramento, Biologia (Biologia Molecular), Universidade de Lisboa, Faculdade de Ciências, 2015

Tobacco smoke is the number one preventive cause of death and causes massive losses in health and productivity worldwide, each year. Since 1964, near two million non-smokers died from involuntary exposure to tobacco smoke. When people burn tobacco products, secondhand smoke (SHS) spreads, fills and accumulates in enclosed spaces. Nearly 50% of children regularly breathe air polluted by tobacco smoke in public places and over 40% have at least one smoking parent. As a result, in a single year more than 160 thousand children have died from SHS related illness. Tobacco smoke contains more than seven thousand chemicals, including 70 carcinogens. Polyaromatic hydrocarbons, a group of environmental pollutants produced during incomplete combustion of organic materials, are among those. Exposure to these chemicals significantly increase the risk for several diseases, namely ischemic heart disease, stroke, lower respiratory infections, lung cancer and chronic obstructive pulmonary disease. All are top major killers of the last decades. According with the World Health Organization, there is no safe level for SHS exposure and some groups, such as the personnel working in leisure venues where smoking indoors is allowed, are particularly at risk. In 2008, smoking in public places was partially banned, under the implementation of a new legislation in Portugal (Law 37/2007). Accordingly, leisure venues owners could choose if it would be entirely allowed or prohibited to smoke indoors. Other venues became a mixture including both designated areas for smokers and for non-smokers inside their premises. A year after, the will to understand the health impact of this new legislation, in these venues workers, driven this project. For that purpose the indoor air of restaurants in Lisbon was evaluated for tobacco smoke contaminants, followed by the search for biomarkers of SHS exposure, in these venues workers. In total 25 restaurants were included in the study, where 13 were mixed with both smoking and non-smoking designated areas, SDA and NSDA, and 12 were entirely smoke-free. Four canteens were also included as a reference of smoke-free places, considering that they have been free from SHS since 1984. Furthermore, the fact that outdoor contaminants easily penetrate indoors, 16 of the 25 restaurants were monitored outdoors as well. A total of 97 employees were recruited. In summary 87 provided a urine sample, 81 provided blood and 27 provided cells from nasal epithelia. Additionally workers filled a detailed lifestyle survey and went through clinical evaluation. All subjects with respiratory disease were excluded from the study. To evaluate possible SHS-indoor air contamination, fine particles, carbon dioxide and carbon monoxide concentrations were monitored and polyaromatic hydrocarbon levels were estimated. Information regarding typology and ventilation was also recorded. The results from indoor air monitoring demonstrated high contamination with fine particles in all restaurants with SDA, in comparison with NSDA or canteens. Moreover, carbon monoxide median level was higher in SDA. Importantly, the urinary concentration of cotinine, a metabolite of nicotine, was increased in workers from SDA, compared with NSDA and entirely smoke-free restaurants, regardless of their smoking status. All together, these results demonstrated that exposure to hazardous SHS levels remain high in restaurants where smoking is still allowed, regardless of the protective measures adopted. Moreover, it was proved that workers in SDA are still effectively exposed to SHS, inhaling tobacco combustion sub-products. The fact that these workers are clinically healthy, motivated the search for molecular changes that could be induced by occupational SHS exposure, mostly in subjects that never smoked. After monitoring two oxidative markers, namely the plasma total antioxidant status and serum 8-hydroxy-2-deoxy-guanosine, we found no significant differences between SHS-exposed and unexposed workers, conversely to the analysis of DNA and proteins. After challenging leucocytes with a mutagenic agent, a comet assay revealed that SHS exposed workers had a higher acute DNA repairing response. The plasma samples were enriched with the medium and lower abundant proteins, by the immunodepletion of the higher abundant proteins. Then the samples were pooled, and run by two dimensional difference in gel electrophoresis (2D-DIGE). The pattern with the separated protein spots, in the gel image, was analyzed with SameSpots software and those indicated to be significantly differentiated run in a tandem mass spectrometry approach in, a MALDI - TOF/TOF. This approach identified nine proteins that significantly differentiated the workers that were exposed to SHS. Out of these, two acute-phase inflammation proteins, ceruloplasmin (CP) and inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), became prominent. For the first time, to our knowledge, both were represented with a high number of isoforms. In addition, they exhibited a common expression pattern, where all high-molecular-weight isoforms suffered a decrease in their concentration, while low-molecular-weight isoforms suffered an increase, in SHS exposed workers. In order to complement and corroborate these results, a second proteomics approach was performed in individual samples of plasma and nasal epithelia, by liquid chromatography coupled with a tandem mass spectrometry instrument, a ESI-LTQ-Orbitrap. These experiments generated huge amounts of data that was submitted to Progenesis QI software, for a proper examination. In total 521 proteins were identified in depleted plasma samples and other 3384, in nasal epithelia. All in all, 38 proteins were significantly different in SHS-exposed workers, compared with the unexposed. Functional analysis revealed that these proteins are involved in various processes already proved to be induced by exposure to tobacco smoke. Therefore a subset of six proteins, was submitted to an additional validation step. Aided by a targeted specific reaction monitoring technique we were able to validate five, as candidate biomarkers namely gelsolin (GSN), alpha-1-acid glycoprotein (ORM2), elongation factor 2 (EF2) and ribosomal 40S S8 (RPS8) and 60S L13 (RPL13) proteins. With this work and dissertation, it is expected to widen the knowledge of the SHS-induced molecular mechanisms. The fact that these molecular mechanisms precede the first clinical symptoms of disease, makes their routine use in health checkups critical, towards the protection of occupational health. Furthermore, to fully protect workers, or public health in general, from SHS exposure, a law revision is urgently advised in Portugal.

Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/74857/ 2010)