Statistical Results
In this observational study, serum PAH levels were assessed in patients diagnosed with COPD at various stages of the disease. A number of 15 PAHs were investigated for 102 patients diagnosed with COPD and other respiratory diseases, hospitalized at the Clinical Hospital of Pulmonary Diseases, Iasi, Romania. The study mainly focuses on the quantification of PAHs in the blood of patients diagnosed with COPD and the assessment of the admission of these contaminants according to smoking status and exposure to air pollution. The quantification of oxidative stress in smokers/non-smokers/former smokers was evaluated based on PAHs concentrations, malondialdehyde and serum uric acid levels for COPD patients. The clinical and demographic characteristics of the investigated patients are presented in
Table 1. The COPD group patients had an average age of 59.48 ± 10.96, and 92% of the patients were male and 8% female. From the total patients investigated, 49% were from rural areas and 51% were from urban areas, and following the interview, some stated an occupational exposure (16%) or a passive exposure to tobacco smoke (10%) (
Table 1).
The profiles of the levels of PAHs determined in the blood samples for the control group and the group of patients with COPD are presented in
Table 2 and
Table 3. Bellow can be found the names and abbreviations of the 16 PAHs determined in the present study.
The International Agency for Research on Cancer, 1986, classified PAHs into carcinogenic and non-carcinogenic compounds. Six PAHs have been classified as possible carcinogenic compounds to humans: BaA, B(b), Flu, B(k)Flu, BaPy, Db(a, h)A and IPy. The results obtained in this study show an admission of pollutants according to smoking status (former smokers/smoker/non-smokers) quantified in average total concentrations for the group of patients with COPDs of 4.12 ng/mL, 6.76 ng/mL, and 6.04 ng/mL (
Table 2). In the case of the control group, the admission of carcinogenic PAHs was determined at mean concentrations of 1.20 ng/mL, 1.67 ng/mL and 1.99 ng/mL for former smokers/smokers/non-smokers, and these values were much smaller than in the case of the COPD group patients. Similar results for levels of PAHs in the blood have been reported by Neal et al. [
18].
The evaluation of PAHs in the investigated samples revealed high concentrations for patients with COPD stage II and III compared to the values of the concentrations in patients with stage IV (
Figure 1 and
Figure 2). These findings can be explained by the fact that patients with stage IV COPD, due to the extremely decreased lung function and the need for supplemental oxygen, have a very low physical activity capacity, which is reflected in an inability to leave the house, thus being much less exposed to outdoor pollution [
19,
20]. Lung function can also be affected/diminished by dominant involvement in forms of passive leisure, an aspect that generates a sedentary lifestyle [
21]. Physical sports and active leisure activities have a beneficial role in optimizing or improving respiratory function, as a compensation mechanism for problems generated by various vices (smoking, alcohol, drugs, etc.) [
22]. Globally, it has been found that young people are doing less and less movement in an organized or freeway [
23]. The human being is a whole, and the human body is an organism, not a mechanism, so the synergic concept is required in our mode of understanding [
24].
The statistical data show a high Spearman correlation between PAHs in the blood serum of patients with COPD. Significant positive correlations were observed between compounds with four (BaA, r = 0.83,
p < 0.005), five (BaPy, r = 0.83,
p < 0.005) and six rings (BghiPy, r = 0.98,
p < 0.005) in smoker patients (
Table 4). In the case of non-smoker patients with COPD, important correlations were observed between compounds with four and five rings (r > 0.98,
p < 0.005) (
Table 5).
For the group of patients diagnosed with COPD, the most common non-cancerous PAHs were naphthalene (mean concentration = 7.27 ng/mL) and phenanthrene (mean concentration = 2.79 ng/mL) and these were determined in almost all samples analyzed (frequency = 90–100%). Some two-ringed PAHs, such as naphthalene, detected at relatively high concentrations in the present study have also been found at high levels in environmental samples collected in the same area [
25].
At the same time, it can be easily observed that the levels of non-cancerous PAHs are higher in non-smokers and former smokers than in smokers, and the order of admission is PAHs non-smoker > PAHs former smoker > PAHs smokers. These results imply a double exposure of patients to PAHs—exposure to tobacco smoke but also to air pollution [
26].
In the case of carcinogenic PAHs, the most abundant compound was DahA, with a mean concentration of 1.79 ng/mL for the non-smoking group. DahA has been detected in compost, wood preservative sludge, gasoline (high octane number), exhaust condensate of gasoline engines, airborne coal tar emissions, and in airborne coke oven emissions [
27,
28]. The potency values for DahA are known to vary with the route of exposure [
29], and DahA has been reported to be up to 10 times more potent than BaPy [
30].
BaPy was significantly higher in the group of smokers diagnosed with COPD (mean concentration = 1.21 ng/mL) and was higher than in the group of former smokers (mean concentration of 0.56 ng/mL), but also compared to the group of non-smoker patients (mean concentration = 0.87 ng/mL). BaPy is one of the most investigated PAHs in both biological and environmental samples [
31].
Thus, the profile of carcinogenic PAHs concentrations shows high concentrations in smokers (ΣPAHs = 6.76 ng/mL) compared to non-smokers and ex-smokers, and the order of accumulation is ΣPAHs carcinogenic smokers > ΣPAHs carcinogenic non-smokers > ΣPAHs carcinogenic former smokers [
32]. Most of the time, identifying the sources of exposure to pollutants remains the most important step in research studies. Thus, the evaluation of some statistical correlations between compounds (
Table 4 and
Table 5—Spearman correlation), applications of factorial analysis to identify possible exposure factors, or even the calculation of molecular ratios are attempted.
Regarding serum PAHs intake, six different ratios have been proposed that could suggest the source of exposure. In the present study, molecular ratios were investigated for the control group and COPD group (A/A + P; Flu/Flu + Py; BaA/BaA + Py; BaPy/BghiPy and IPy/IPy + BghiP, Fl/Fl + Py proposed by KG Koukoulakis et al. (2020)) to identify the main sources of exposure to PAHs.
Figure 3a shows levels of these ratios for the control group and the COPD group. It is thus observed that in the case of the COPD group, the content of PAHs in the blood could be due to PAHs emissions because the ratios Fl/Fl + Py suggest diesel emissions, and the ratios BaA/BaA + Ch and IPy/IPy + BghiPy are derived from vehicle emissions and fuel combustion [
33]. The BaPy/BghiPy ratio indicates non-traffic emissions, and it is observed that in the present study the values are much higher in the group of patients with COPD versus the control group, and this could suggest smoking as the main source of exposure. In
Figure 3 it can be observed that smokers in the control group had a high ratio of IPY/IPY + BghiPy (
Figure 3b), and in the COPD group, smokers had a high ratio of BaPy/BghiPy (
Figure 3c).
There are 16 US EPA PAHs classified as priority pollutants with toxicity and high levels of exposure to humans, 8 of which are considered possible human carcinogens, which include BaA, BaPy, B(b)Flu, B(k)Flu, Ch, DahA and IPy [
34,
35]. Additionally, PAHs are classified in group 2A (probably carcinogenic) or 2B (possibly carcinogenic). In the present study, PAHs from group 2A (BaA, BaPy, DahA) were quantified, and slightly increased concentrations were observed in smoking patients. For the compounds found in group 2B (B(b)Flu, B(k)Flu, IPy) higher concentrations were observed in the group of patients with COPD. The International Agency for Research on Cancer (IARC) considers exposure to PAHs to be the major risk factor for the development of lung cancer, as well as other pulmonary diseases [
36].
The factorial analysis applied to the dataset of patients with COPD highlighted three factors that significantly contribute to the separation of the dataset. Thus, with a variance of 52%, the first factor was mainly represented by PAHs (Py, BaA, Ch, DahA, B(b,k)Flu, Ipy) originating from fuel combustion and traffic emissions, but also non-traffic BghiPy and BaPy. The second factor accounts for 13% of the total variation in the dataset and is represented by FL and A, and the third factor is represented by phenanthrene and accounts for 9% of the variation in the dataset. Identifying the factors that lead to the admission of PAHs in the body is difficult to achieve, even if in the present research the content of non-carcinogenic PAHs predominates, and they could be the source of traffic emissions. At the same time, there is a big difference between the concentrations of carcinogenic PAHs between the COPD group and the control group (
Table 2), a difference that could be due to exposure to tobacco smoke.PAHs and nicotine induce the production of oxidative stress markers and reduce the number of antioxidants, contributing greatly to the production of oxidative stress due to cigarette smoke [
37,
38,
39].
In the present study, the quantification of oxidative stress was analyzed by assessing PAHs concentrations, MDA and serum uric acid levels in smokers and non-smokers diagnosed with COPD.
Table 6 shows mean, median, stdev and range characteristics of serum MDA, uric acid values and lipid profile parameters of smokers/non-smokers/former smokers diagnosed with COPD.
The results in
Table 6 show serum uric acid levels in former smokers/smokers/non-smokers patients diagnosed with COPD at different stages of the disease. The descriptive statistics show, in the case of smoking patients, values of the average uric acid concentration of 5.21 mg/dL, results that are much lower compared to the group of non-smoking patients (6.50 mg/dL) but also compared to the group of former smokers (6.09 mg/dL). Similar results have been reported in other studies [
13,
40], where a reduction in serum uric acid in smokers indicates that oxidative stress is intensified with each cigarette smoked.
Uric acid is the end product of purine metabolism, and is a non-enzymatic antioxidant with an important role in assessing oxidative stress in the body [
12]. Lifestyle (diet, age, smoking, alcohol consumption, exercise) and genetic factors may influence serum uric acid levels, as these factors are directly associated with pathophysiology and oxidative stress. Increased oxidative stress can lead to the depletion of antioxidants, including uric acid, and increased oxidants levels.
In the present study, high concentrations of malondialdehyde were quantified for smoking patients diagnosed with COPD (2.72 µmol/L) compared to former smokers (2.43 µmol/L) and non-smoking patients (2.32 µmol/L) (
Table 6). Increased lipid peroxidation in the peripheral blood may indicate a systemic increase in oxidative stress largely characterized by the deficiency of the antioxidant system assessed in this study by determining uric acid concentrations. In fact, cigarette smoke, due to the large number of chemicals resulting from combustion (4700 substances, including polycyclic aromatic hydrocarbons, heavy metals, NO) leads to the formation of strong chemical oxidative stress involving virtually all compounds in tobacco, leading to the formation of free radicals [
41]. Several previous studies have demonstrated that PAH exposure may induce oxidative stress [
32].
In the case of the evaluation of the lipid profile, significant differences were observed for the levels of serum triglycerides in smokers (226 mg/dL) compared to non-smokers (111.91 mg/dL) and former smokers (103.9 mg/dL). Similar values for the level of serum triglycerides in the case of smoking subjects were also obtained in other studies [
42], which tried to explain the physiological consequences of exposure to tobacco smoke.
The statistical difference between the investigated groups was tested with the Mann–Whitney U test. Thus, the statistical results show a statistically significant difference (
p < 0.05) between the groups of patients with stage II COPD and stage IV COPD for the following investigated parameters: P (
p = 0.02), BaA (
p = 0.04), BaPy (
p = 0.03) and uric acid (
p = 0.0003) (
Table 7).
In the present study, the quantification of oxidative stress was studied by analyzing the content of PAHs, serum uric acid values and PAHs in patients diagnosed with COPD. The linear regression model applied to the dataset shows a positive correlation of serum uric acid with BaPy levels (
p < 0.05, r = 0.30) in smokers and non-smokers diagnosed with COPD, and also with the smoking status of the patients by association with PY (packs-years) (
p < 0.05, r = 0.35) in smokers diagnosed with COPD (
Figure 4 and
Figure 5).
The pathogenetic mechanism of oxidative stress caused by exposure to tobacco smoke has not been fully elucidated. A basic hypothesis concludes that free radicals produced by smoking induce oxidative disorders at the macromolecular level (lipids, proteins and DNA). Thus, the results presented in
Table 6 quantify high serum levels for triglyceride and low serum levels for HDL-C in smokers (mean triglyceride concentration = 226.99 mg/dL, HDL = 44.9) compared to the group of non-smoker patients (triglycerides = 111, 91 mg/dL, HDL-C = 48.08 mg/dL). Smoking is associated with an atherogenic lipid profile, which can also contribute to the production of oxidative stress. Smoking, in its various forms, leads to an increased risk of high total cholesterol serum levels, as well as high triglycerides levels [
40].