In this study, we demonstrated that gene promoter methylation occurred frequently in DNA extracted from sputum of individuals exposed to smoky coal emissions in XWC, with a frequency varying from 51.4% (55/107) for p16 gene, to 29.9% (32/107) for RASSF1A gene, 17.8% (19/107) for DAPK gene, and 15.9% (17/107) for MGMT gene. Furthermore, 3 individuals (2.8%) showed the co-occurrence of promoter methylation of all four genes, while 8 (7.5%), 21 (19.6%), and 41 (38.3%) other individuals showed this alteration in 3 genes, 2 genes, and 1 gene, in their sputum, respectively. The clinical implication of the co-occurrence of promoter methylation on lung cancer risk remains unclear since these subjects were anonymous and were not followed up further. However, results from previous studies of smokers from Europe and the United States suggested that gene promoter methylation in sputum may provide a useful predictive biomarker for lung cancer. For example, Palmisano et al reported that aberrant methylation of the p16 and MGMT was detected in sputum of all patients with squamous cell lung carcinoma up to 3 years before clinical diagnosis. Moreover, the prevalence of these markers in sputum from cancer-free, high-risk subjects approximates lifetime risk for lung cancer [7
]. A study by Kersting et al of p16 gene promoter methylation, and p53 and K-ras mutations in exfoliated cells from 51 NSCLC patients and 25 chronic smokers showed that 8 of the chronic smokers harbored a genetic and/or epigenetic alteration, and 3 of whom were subsequently diagnosed with lung cancer [8
]. Two recent studies, both from the Belinsky’s group, showed that the co-occurrence of promoter methylation of three genes and of more than 3 genes in sputum was associated with a 3.6- and 6.5-fold increased risk of lung cancer, respectively [10
]. Therefore, individuals with sputum positive for aberrant promoter methylation, especially those showing such an alteration in multiples genes, may be at high risk for lung cancer.
In this study, the gene promoter methylation frequencies found in sputum of the XWC population are higher than those reported previously in sputum from smokers from Europe and the United States [7
]. For instance, Destro et al reported that only 4 cases (4%) among 100 heavy smokers (age>60, >20 cigarettes/day last at least 20 years) showed p16 promoter methylation [26
]. This discrepancy was unlikely attributable to technical problems because similar detection methods were used in both our present study and these studies. Ethnic differences might play a factor to account for this difference. It has been reported that there is a relationship between gene methylation and geography [28
] and the Chinese lung cancer patients harboured higher frequency of gene hypermethylation in their tumor tissues, plasma and sputum [29
] and bronchoaleolar lavage than Western cases [30
]. Furthermore, in this study, there were no differences in promoter methylation frequencies for any of the genes in sputum between the nonsmokers and the smokers, by using either univariate () or logistic () analysis. This result is in disagreement with the significantly higher promoter methylation frequencies observed for p16 and RASSF1A genes in sputum samples from smokers, compared with nonsmokers [31
]. Taken together, the higher promoter methylation frequencies found in sputum of the XWC individuals, compared with those found in sputum of the European and American smokers, and the similar promoter methylation frequencies between smokers, mostly men, and nonsmokers, mostly women, in the XWC population, may be due to the exposure of the XWC subjects to smoky coal combustion. These emissions contained 81% of organic matter, of which 43% were PAHs [32
] and were previously associated with the detection of a higher level of benzo(a)pyrene-adducted guanine in urine from nonsmokers exposed smoky coal emissions, compared with urine from smokers not exposed to these emissions [33
]. Furthermore, exposure to these emissions has been associated with high frequencies of p53 and K-ras mutations in lung tumors from lung cancer patients [17
] and in sputum from individuals who showed no evidence of lung cancer [34
] in XWC.
Finally, there were no differences in the frequencies of promoter methylation in any of the genes between the groups of individuals diagnosed with chronic bronchitis and the group of those without such a symptom, by using either univariate () or logistic () analysis. This result is in line with our previous study of these same sputum samples showing no differences in p53 mutation frequencies between these two groups of individuals [34
]. These results suggests that promoter methylation of these genes, like p53 mutations, was associated primarily with exposure to smoky coal emissions.
Taken together, these results suggest that chemicals in the smoky coal emissions, particularly the high concentration of PAHs and other chemicals [35
], may play a primary role in the formation of genetic and epigenetic alterations found in sputum from individuals without evidence of lung cancer in XWC.