We found that personal exposure to BC, as a marker for TRAP, was positively associated with FeNO at lag periods of less than 12 h in children with asthma. To better understand the mode of action by which exposure to TRAP may increase FeNO, we examined associations between exposure to BC and DNA methylation levels in promoter regions of NOS and ARG genes. We found that exposure to BC was negatively associated only with methylation of the NOS3 gene, suggesting that modulation of this “constitutive” isoform of NOS may be involved in short-term changes in FeNO associated with exposure to TRAP.
Consistent with previous studies, we observed a significant increase in FeNO in response to BC exposure in children with asthma. As a non-invasive biomarker of inflammation, FeNO has been reported to be positively associated with TRAP in epidemiological studies, especially in patients with preexisting asthma and COPD [3, 8] . Nitric oxide normally functions as a key physiological mediator in human immune responses and smooth muscle relaxation. However, overproduction of NO may mediate cellular toxicity and cause inflammation [19, 20]. Airway inflammation is a key mechanism in the pathogenesis of asthma exacerbation . However, the biological mechanisms linking the TRAP exposure to airway inflammation and increased FeNO remain unclear.
Results from prior human studies have been inconsistent, but most studies revealed that exposure to particulate air pollutants decreased NOS2A promoter methylation and increased ARG methylation. In a cohort of 163 urban children, Jung et al. found that higher level of 24-h BC measured by personal monitors were associated with reduced methylation of NOS2A 5 days later . The magnitude of association was stronger among the seroatopic and cockroach-sensitized children compared to non-sensitized children. In the Southern California Children’s Health Study cohort, Salam et al. reported that elevated 7-day average exposure to PM2.5 was associated with a statistically significant decrease in NOS2A methylation in 940 children . Using the same cohort, Breton et al. found significantly lower NOS2A methylation and higher NOS3 methylation in response to acute and chronic exposure to particulate matter . Similar results were observed in panel studies of exposure to PM in China among 43 healthy adults and 30 adults with chronic obstructive pulmonary disease [8, 11] . However, one panel study revealed that occupational exposure to fine PM was positively associated with increased NOS2A methylation in 38 male boilermaker welders .
Based on these previous studies, we hypothesized, that exposure to TRAP would be negatively associated with DNA methylation in NOS genes, especially NOS2A, and positively associated with methylation in ARG genes in children with asthma exposed to BC. We found statistically significant negative associations between exposure to BC and NOS3 promoter methylation only. In contrast, in Breton et al., PM2.5 was associated with increased methylation levels in the NOS3 gene among children. Unlike previous studies, we did not find significant changes in the methylation level in other NOS and ARG isoforms in response to TRAP . These inconsistent results could be due to several differences between our study and the previous studies, including differences in the study populations (children vs. adults, and asthma vs. other health conditions), exposure assessment approaches (personal vs. fixed-site monitoring) and the selection of CpG sites tested [8, 10, 11, 22, 23].
To further explore the role of DNA methylation in the associations of TRAP and FeNO in children with asthma, we examined the changes of NOS methylation and FeNO levels in response to TRAP at different time-lag periods. We found that the negative effect of BC on NOS3 methylation at position 1 was stronger in the first 24 h. The largest effect of BC on FeNO was found within 12 h, and then both strengths of association decreased and became statistically insignificant. Unlike NOS2A, which is inducible, NOS3 is expressed constitutively. In our previous controlled experimental studies, we found an immediate but transient increase in nitrite, a stable metabolite of NO, in the exhaled breath condensate (EBC) of healthy young adults after exposure to TRAP particles and adults with asthma after exposure to diesel engine exhaust [24, 25]. Such transient increases in EBC nitrite could be explained by rapid increases in either NO production or oxidation of NO to nitrite. However, in the current study, we did not find the expected association between methylation at the NOS3 position 1 locus and FeNO at lag day 1.
Our study has several strengths. We followed our subjects for up to 30 consecutive days. This study design addresses the temporality between exposure and outcomes, and the repeated sampling of the same subject increased the study power. Furthermore, personal real-time BC measurement likely reduced exposure measurement error compared with data from fixed-site monitors that were used in most of the previous studies. The non-differential exposure measurement error could severely attenuate exposure-disease associations in epidemiological studies. For example, Niu et al. compared the associations of DNA methylation in NOS2A and ARG2 with personal and central-site monitored ozone exposure and found much stronger associations of ozone using personal measurements . To avoid multiple pairwise comparisons, we applied the MANOVA test at multiple CpG loci in NOS and ARG genes. To reduce the unknown impacts of time-varying patterns, we collected buccal cells and FeNO on various day-of-week. Unlike most studies that only focused on CpG sites in NOS2A, our study tested multiple CpG loci at all isoforms of NOS and ARG, both of which are involved in the regulation of NO production. Finally, we focused on children with asthma living in environmental justice communities. This specific subpopulation may be more sensitive to short-term changes in exposure to TRAP.
Nonetheless, this study has certain limitations. Our study had very limited sample size, which was justified using power calculation that assumed one outcome and exposure at one lag period. In the analysis, we controlled for multiple testing across responses by using a Multivariate Analysis of Variance, such that the overall test of all responses for an exposure was conducted at the 0.05 level before looking at individual responses. Therefore, our study may have been underpowered for looking at the responses relative to an exposure. Because the BC collected at each lag period were highly correlated we could not include multiple lags in the regression models at once and we were not able to conclusively say that one lag was more influential than another. Due to the limited sample size, we were unable to check if the time lag pattern between exposure to TRAP and DNA methylation level varies by the degree of asthma.
Besides DNA methylation, there are several types of epigenetic modifications, including histone modification and miRNA expression, which could be impacted by air pollution exposures and could affect the development and exacerbation of asthma [26,27,28]. However, this is beyond the scope of this paper and future studies should be done in assessing the impacts of personal TRAP exposure on histone mondification and miRNA levels in children with asthma. We used buccal cells as surrogates for cells from bronchial tissue due to the feasibility of collection in a community based study of children with asthma. Studies have found similar gene expression, as well as methylation level in buccal cells and cells from the respiratory tract in response to tobacco, supporting the potential of the oral epithelium as a surrogate tissue for respiratory epithelium [29, 30]. Other studies, noted above, have found associations between exposure to air pollution and changes in methylation in NOS genes from buccal cells [9,10,11,12, 22].