In the present study, we showed that the mean methylation levels of promoter CpG sites were significantly associated with the exposure to PM and certain metal components. In particular, the mean DNA methylation levels of the CpG sites measured in APC or p16 promoter increased significantly in post-exposure DNA samples. By contrast, the mean of p53 or RASSF1A promoter CpG sites methylation decreased in post-exposure DNA samples. CpG site-specific analyses did not show consistent patterns of DNA methylation changes in any of the four genes.
Evidence in human subjects is rapidly mounting to establish associations of DNA methylation alterations with environmental exposures. Such methylation changes can persist over time even in the absence of the conditions that established them and even accumulate in response to continuous exposure [24–26]. Exposure to air pollution, particularly to ambient PM, has been associated with increased lung cancer risk [1, 2]. Aberrant DNA promoter methylation of tumor suppresser genes, including hypermethylation of p16 [27, 28], APC  and RASSF1A [28, 30], and hypomethylation of p53 , has been observed in blood leukocyte DNA from lung cancer patients, suggesting that PBL DNA methylation may serve as a cancer-related biomarker.
The four genes of interest in our study are involved in cell-cycle control (p16), invasion and metastasis (APC), apoptosis (p53) and Ras signaling (RASSF1A) and have been shown to be altered in lung cancer patients' blood DNA [27–31]. Some of our findings are in line with previous reports. Zhang et al and Chanda et al found hypermethylation of p16 tumor suppressor gene in blood DNA from individuals exposed to emissions from indoor unventilated-stove coal usage  and individuals exposed to high-level arsenic . Expression of p16, one of the most promising early epigenetic markers for the detection of lung cancer , was demonstrated to be regulated by promoter methylation . Hypermethylation of p16 has been detected in blood leukocyte DNA from cancer-free smokers , lung cancer smokers [27, 28] and in lung tissue from diesel exhaust-exposed rats . APC promoter hypermethylation, another early event of tumorigenesis, has been extensively studied in lung tumors . APC promoter hypermethylation has also been seen in lung tissue in healthy subjects exposed to cigarette smoke  and blood in lung cancer patients . Our finding, for the first time, shows that air pollution may induce blood leukocyte APC promoter hypermethylation. p53 is a key factor in DNA damage-signaling pathway, and p53 hypomethylation is associated with DNA double strand breaks and chromosomal instability . In a previous human study, we observed p53 hypomethylation in blood DNA in Polish male non-smoking coke-oven workers exposed to polycyclic aromatic hydrocarbons (PAHs) [38, 39], which is similar to findings in smoker lung cancer patients . Our finding further suggests that p53 blood leukocyte hypomethylation may also occur in healthy subjects who are exposed to PM. Although hypermethylated RASSF1A promoter is frequently observed in the blood DNA of lung cancer patients [28, 30], we found an inverse association between mean promoter RASS1FA methylation and PM exposure. It is possible that our results might represent tissue-specific effects of PM exposures on blood leukocytes, as well as a false positive finding due to the limited sample size. Different tumor suppressor genes may behave differently with respect to carcinogens. Air pollution may cause hypo- or hypermethylation in each individual gene depending on the role of the gene in cancer development. In our study, we observed positive association of p16 methylation with zinc, and negative association of RASSF1A methylation with zinc in both unadjusted and adjusted regressions. Zinc is a major component in PM in steel-production plants, and was present at high levels in PM measured in the present study (Table 1). Zinc inhalation has been shown to induce inflammation and oxidative stress in animal studies [40, 41]. Kodavanti et al have also demonstrated that leachable zinc from PM induced both pulmonary and systemic changes in multiple in vivo toxicology experiments . Furthermore, a study has shown that inhalation of soluble zinc sulfate, even at low levels (10 μg/m3), caused gene expression change in heart tissue in healthy rats .
PM and its metal components may affect DNA methylation through several cellular processes, including oxidative stress/reactive oxygen species (ROS) generation and systemic inflammation/immune deficiency [44, 45], the two major components in the etiology of cancer . These cellular processes have been shown to be induced by exposure to PM [47, 48] and associated with altered DNA methylation patterns .
Our study was based on occupationally PM-exposed volunteers who worked in several work areas of the same factory and did not include a different population of subjects without a specific condition of exposure to PM. Limiting our investigation to individuals who have all been working in the same work facility avoided potential concerns related to the selection of external referents who might have differed from the exposed population in terms of socioeconomic factors and other characteristics determining hiring into the plant . The differences in the individual levels of exposure in our study group were large, which provided sufficient contrast for identifying exposure-related changes in DNA methylation. For example, the lowest level of PM10 observed in our study population (73.72 μg/m3) was only marginally higher than ambient PM10 levels measured in the geographic area where the plant is located [average annual ambient PM10 levels between 41 and 57 μg/m3 were recorded in the year of the study by different ambient monitoring stations in Brescia area] , whereas the highest level was 1220.17 μg/m3. It is worth noting that, although our study was based on a group of foundry workers with higher average exposures than the general population, the levels of exposure to metals in our study were all lower than the commonly accepted threshold limits for industrial settings . As foundry workers may have additional exposures [53–57], we cannot exclude that exposures other than PM might have contributed to the observed effects, although study subjects in our study were in a modern facility with state-of-the-art systems for exposure reduction. A limitation of the study is that it did not include an unexposed comparison group. We cannot exclude that effects of potential changes in life-style from the start to the end of the week might have played a role in generating the observed associations. However, this group of foundry workers showed wide differences in exposures between individuals. The dose-response associations that we found between PM and APC methylation, and between zinc and p16 and RASSF1A methylation all hint to specific exposure-related effects.
In summary, we observed hypermethylation of p16 and APC, and hypomethylation of RASSF1A and p53 in a group of healthy foundry workers who had higher levels of exposure to PM compared to the general populations. Altered DNA methylation of tumor suppressor genes in easily obtainable cells such as PBLs may have potentials for developing biomarkers to detect biological alterations in PM-exposed subjects. These results suggest that such methylation alterations may reflect processes related to PM-induced lung carcinogenesis. However, because our study did not include lung cancer or other carcinogenesis-related endpoints, we cannot make inference on the biological significance of our findings. Further studies in larger populations are required before any firm conclusion could be reached on whether PM exposed individuals with such DNA methylation alterations are at higher risk for lung cancer.