This study aimed to evaluate the relationship between systemic oxidative stress and intensity of smoking exposure. The main finding was that the smokers exhibited significantly higher secretion of H2O2 than the healthy controls and that there was a significantly positive association of oxidative stress with pack-years of smoking. These results confirm that smoking itself is an important determinant of oxidative stress and that a higher consumption of cigarettes is associated with higher levels of oxidative stress
[12, 14, 20]. We also reinforce previous findings that smokers exhibit evidence of systemic inflammation compared with healthy controls
The relationship between intensity of smoking exposure, through the evaluation of smoking pack-years, and oxidative stress has previously been investigated in the literature. However, our study is the first to show a significant association between pack-years of smoking and the secretion of H2O2 by PBMs. Thomassen et al.
 did not find a correlation between the number of pack-years of smoking and the secretion of H2O2 by alveolar macrophages when assessing smokers and non-smokers. Similarly, Puri et al.
 found no correlation between exhaled ethane air levels and cumulative smoking status. It is difficult to compare our results with those of these studies. The study by Thomassen et al.
 used different cultures of cells to evaluate the secretion of H2O2. Those authors assessed alveolar macrophages, whereas we evaluated PBM cultures. Similarly, Puri et al. used a different outcome to quantify oxidative stress. However, the sample size of these studies was smaller than that in our study.
In this study, we demonstrated the higher secretion of H2O2 in smokers by PBM cultures, either spontaneously or after stimulation. Nowak et al.
 demonstrated increased content of H2O2 in the expired breath condensate of cigarette smokers compared with controls. Our results are also in agreement with those of Ishida et al.
, who identified higher H2O2 production in alveolar macrophage cultures of rats exposed to cigarette smoke. In contrast to our results, van Beurden et al.
 did not detect differences in the release of superoxide dismutase by PBM cultures, either spontaneously or after stimulation with PMA, between COPD current/ex-smoker patients and healthy controls, and the smoking history did not influence the results when compared by covariance analysis between groups. The authors concluded that superoxide dismutase is only one of the oxidants that are produced by PBM culture; these cells also produce H2O2 and hydroxyl radicals. Therefore, the fact that the secretion of H2O2 by PBM culture is higher in smokers may indicate an imbalance in which the production of reactive oxygen species exceeds the capacity of the antioxidant defense systems in the systemic microenvironment. The oxidative stress may already be occurring to produce oxygen free radicals
Our results showed higher secretion in H2O2 after stimulation with PMA in both groups, and the smokers exhibited higher production than the controls. This result indicates that the smokers included in our study still have monocytes with efficient immune capacity
. We can speculate that monocyte phagocytosis in smokers is still functional, although we did not evaluate the immune capacity in our study. In contrast, Correa et al. showed lower fungicidal activity and secretion of H2O2 by the monocytes of patients with peripheral atherosclerosis obliterans compared with those of control subjects.
Cigarette smoke induces the enhanced recruitment of mononuclear phagocytes and polymorphonuclear cells into the lower airways
[2, 12], and these cells modify oxygen metabolism and release additional H2O2 and other reactive oxygen species
. In our results, we identified a statistically higher number of neutrophil cells in the peripheral blood of smokers in comparison with that of healthy controls and a positive association between the numbers of leukocytes cells and pack-years of smoking. These observations are in accordance with a previous study that demonstrated an accelerated release of polymorphonuclear leukocytes from the bone marrow after smoking exposure
The systemic inflammation induced by smoking includes neutrophilia and cytokines
[6, 27, 28]. In our study, we found higher levels of TNF-α in smokers compared with healthy controls. Similarly, Tanni et al.
 and Pretescu et al.
 showed higher TNF-α levels in smokers compared with healthy non-smokers. We did not find differences in the cytokine secretion by the PBM culture in both groups, and the literature reveals controversial results that are dependent on the cell culture methods
[7, 21]. In agreement with our results, Ryder et al.
 utilized cell cultures without stimulus and did not find increased values of TNF-α production by PBM cultures in smokers compared with controls. In contrast, Zeidel et al.
 identified increased production of the pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) in smokers compared with non-smoking subjects, however, these authors analyzed cell cultures stimulated with lipopolysaccharide.
The limitations of our study need to be addressed. First, this study is cross sectional, and we are not explaining the cause-and-effect relationships among events. Second, we did not evaluate whether different smoking pack-years or cigarettes-day can induce greater secretion of H2O2 to confirm our results. Third, we did not assess the other biomarkers of oxidative stress and antioxidants to better understand the biological response to smoking pack-years.
In conclusion, we identified an association between higher secretions of H2O2 and smokers compared with healthy controls. The influence of pack-years smoking may be a key modifiable factor in oxidative stress associated to smoking.