The present study showed lower MtDNAcn in association with increased levels of personal EC measured during work hours and of ambient PM10 averaged over 5 and 8 days before the MtDNAcn examination days. We found no significant associations of MtDNAcn with personal measures of PM2.5 taken during work hours on the day of the examination, nor with PM10 on the examination day and the 2-day mean. MtDNAcn was similar in truck drivers and office workers. Thus our results do not support effects of work-related long-term differences in exposure to air particles.
Research on MtDNAcn in relation to air pollution is still limited with inconsistent results. Two studies have demonstrated that individuals exposed to higher ambient benzene exhibited higher MtDNAcn than participants with lower exposure [21, 22]. In our previous study of healthy steel workers in Northern Italy, personal PM10 and PM1 were associated with increased MtDNAcn . In this group of Italian steel foundry workers with high exposure to metal-rich PM, MtDNAcn was determined from blood DNA obtained on the 1st and 4th day of the same work week after two off-work days . Exposures to PM10, PM1, and coarse particles showed a dose–response relationship with increased MtDNAcn on both the 1st and 4th days of the study week, indicating that the correlations between PM exposure and MtDNAcn were the result of a more protracted exposure to PM, rather than of the acute exposure between the two days. In the present study, we observed significant negative association of 5- and 8-day means of ambient PM10 with MtDNAcn in office workers, but not in truck drivers. It should be noted that the dose–response slope between particles and cardiovascular mortality has been shown to be nonlinear, with lower slopes at higher particle concentrations . Therefore, PM effects might be substantial at low- to middle-range doses and taper off at higher concentrations. Also, these results suggest that MtDNA may react differently under the influence of different environmental factors, leading to differences in copy numbers. Compared with the present study, higher PM10 level (median=179.44 μg/m3), metal components (i.e., chromium, lead, arsenic, nickel, manganese), older age (mean=44 years), and different race in steel foundry workers might have all contributed to the discrepancy in the results between the two studies. In the present study, levels of EC during the work hours, taken as a tracer of exposure to traffic particles, were associated with decreased MtDNAcn. This finding indicated effects on MtDNAcn from traffic particles that appeared on the same day of the study. The finding of stronger effects of EC in office workers than that in truck drivers suggests that the decrease in MtDNAcn did not result from the effects of work-related exposure to traffic air particles. Compared to truck drivers, the office workers may represent a more homogenous group in relation to different variables, such as socioeconomic status, smoking, and other factors that may have the potential to influence MtDNAcn. Also, unmeasured exposures or differences in particle content may have influenced MtDNAcn more in office workers than in truck drivers. However, we conducted a test for interaction to determine whether the effects of air particle exposures on MtDNAcn in office workers were different from those in truck drivers. We did not observe any significant interaction (p>0.05) (data not shown), indicating no significant difference in the slope of the relation between air particle exposures and MtDNAcn between office workers and truck drivers. Therefore, the observed stronger association and larger effect in office workers than truck drivers may mostly reflect imprecision in the regression estimates due to limited sample size. Larger studies are warranted in larger groups with different sources of exposure.
MtDNAcn is dependent on oxidative stress level, cell antioxidant capacity, and quality of mitochondria and MtDNA . Mild oxidative stress may stimulate MtDNAcn synthesis, while high exposure may result in decreased or no synthesis, owing to severe oxidative damage of cells. PM exposure, particularly from traffic sources, induces systemic inflammation , which may lead to decreased MtDNAcn. Haden et al. found that MtDNAcn was reduced in the early phase of sepsis as a consequence of the sepsis-induced oxidative damage and inflammation in mice . Pyle et al. observed decreased MtDNAcn in the blood of sepsis patients compared with controls .
MtDNA is dynamic and can be influenced by multiple factors. We observed inverse associations of MtDNAcn with factors that can cause oxidative stress, including cigarette smoking and BMI (see Additional file 1: Table S3). The observed negative association of MtDNAcn with pack-years of smoking is consistent with previous findings that have associated pack-years of smoking with lower blood MtDNAcn in men . We also found a negative association of BMI with MtDNAcn. A strong inverse association was reported between BMI and MtDNAcn in adipocytes in healthy subjects . In this study population, we have previously reported an association of increased BP with ambient PM10 over 5 or 8 days before the examination days . In the present analysis, we observed decreased MtDNAcn with increasing systolic and diastolic BP (see Additional file 1: Table S3). This finding is in line with previous studies showing a negative correlation between MtDNAcn in blood and BP [12, 20]. Also, previous investigations have associated decreased blood MtDNAcn with PM-related diseases, such as cognitive function . Decreased MtDNAcn in placental tissues has been associated with prenatal PM10 exposure (mean=22.7 ± 3.7 in whole pregnancy)  and maternal smoking .
Our additional analysis on male and female have found higher mtDNAcn in women than that in men, which is consistent with two previous studies [40, 41]. Thyagarajan et al. found that individuals with high dietary intake of α- and β-carotenes had slightly higher MtDNAcn, and suggested that lifestyle factors may contribute to the gender-related difference . However, the exact mechanisms underlying the sex-related differences are not well understood. Further, in our stratified analysis on the effect of exposures by sex, we found that the effect of exposure, including personal EC and PM10 (2-day, 5-day, and 8-day means), on MtDNAcn appears to be stronger in men than in women. Similary, Purdue et al. has reported a stronger association of MtDNAcn with renal cell carcinoma in men than women . However, due to the limited sample size, particularly, in the female group, our results need to be interpreted with caution. Larger studies are needed to examine sex-differences in susceptibility toward air pollution in the future.
Some previous investigations have suggested that MtDNA has a long-half life and low turnover rates. For instance, Collins et al. showed a half-life of approximately 350 days for MtDNA in rats . However, other studies have shown more rapid half-lives, varying between 7 and 31 days . It has been postulated that the MtDNA turnover rates are variable depending on the tissue investigated as well as the effects of environmental factors . Our data indicate that relatively rapid changes in MtDNAcn may occur in a cell type with high turnover rates, such as neutrophils with an average half-life of 5–6 days , at high levels of exposure to air particles.
Our study had the advantage of having both personal and ambient measures of air pollution. Our technical validation of personal PM2.5 measures showed high reproducibility (r=0.944). We also recognize that our study is subject to a number of limitations. In our investigation, we did not examine inflammatory markers, which may play important roles in both mtDNAcn changes and PM-related diseases, thus limiting our ability of understanding how inflammation may affect PM-related MtDNAcn changes. Further studies are needed to examine cytokines, in particular, IL2, IL4, IL6, IL8, and TNFα that have been previously associated with PM exposure [46–48], and high-sensitivity C-reactive protein (hs-CRP)  to clarify the role of inflammation in the relation of MtDNAcn with PM exposure. Because of the relatively small sample size, we cannot exclude false negative or chance findings. In addition to using personal PM2.5 measures, we used stationary measures of ambient PM10 to represent exposures, which are just a proxy of personal exposure. However, simulation studies have shown that the error introduced by using data from stationary monitors is highly unlikely to bias away from the null, and indicated that this exposure misclassification may lead to an underestimation of the health effects of air pollution . In addition, serial measures of ambient particulate concentrations have been shown to be representative of variations in personal exposures , particularly in the presence of high ambient PM levels . The observed decreased MtDNAcn in our study suggests that high doses of exposures, such as those found in Beijing, might lead to clearance of cells with highly damaged mitochondria. However, the clinical significance of such small changes in MtDNA content remains largely unknown. More studies are warrented to examine the risk of future disease in large cohorts with clinical disease outcomes.
Overall, our investigation provided evidence that short-term exposure to air pollution is associated with decreased MtDNAcn. These findings suggest that damaged mitochondrial DNA, as reflected in peripheral blood MtDNAcn, may reflect recent toxic exposures and potentially contribute to the etiology of PM-related diseases. Further studies are required to validate the present findings, as well as to better elucidate the time relationships between PM exposure and MtDNAcn.