Many studies have shown that ambient pollution is consistently associated with adverse health outcomes [1-6], but mechanisms accountable for these associations have not been fully elucidated. Suggested biological mechanisms linking air pollution and cardiovascular diseases include direct effect on the myocardium, disturbance of the cardiac autonomic nervous system, pulmonary and systematic oxidative stress and inflammatory response that triggers endothelial dysfunction, atherosclerosis and coagulation/thrombosis [7]. Understanding relative roles of such potential is a priority of recent air pollution epidemiology.

Some studies have demonstrated that exposures to particulate matter (aerodynamic diameter ≤2.5 μm, PM_2.5) and ozone are associated with global oxidative stress [7-11]. Others reported that the exposures were associated with heart rate variability (HRV), plasma homocysteine and C-reactive protein and such effects were modified by genetic polymorphisms related to oxidative defenses [12-16]. In living cells, reactive oxygen species (ROS) are continuously generated as a consequence of metabolic reactions, which may cause oxidative damage to nucleic acids. DNA damage may be repaired by the base excision repair pathway. The resulting repair product, 8-Hydroxy-2'-deoxyguanosine (8-OHdG), is the most common DNA lesion [17] and is not affected directly by either diet or cell turnover [18]. Therefore, 8-OHdG is a good biomarker for ROS or oxidative stress.

A limited number of epidemiological studies reported that 8-OHdG was associated with exposures to indoor and ambient pollution or smoking, but they were conducted among a small number of children or occupationally exposed employees [9,10,19]. Oxidative stress caused by air pollution may be implicated in the development of respiratory disease, cardiovascular disease, lung cancer and other diseases [20-22]. Our recent study found that the elevated urinary 8-OHdG was associated with pollutants often thought of as secondary or formed through photochemical reactions after emission (PM_2.5, nitrogen dioxide, NO₂, maximal one-hour ozone, O₃, sulfate, SO₄^2-or organic carbon, OC), but not with directly emitted primary pollutants (black carbon, BC, carbon monoxide, CO or elemental carbon, EC), suggesting that secondary pollution plays a stronger role in oxidative stress [23].

Several studies have demonstrated that certain genetic polymorphisms related to oxidative stress modified effects of PM on cardiovascular responses [6,13,14], but a set of examined single nucleotide polymorphisms (SNPs) was very limited. Further, these studies only indirectly implicated oxidative stress as none of these outcomes was a direct measure of oxidative stress. For example, some studies reported that associations between exposure to PM_2.5 and heart rate variability (HRV) were modified by polymorphisms of the glutathione-S-transferase M1 (GSTM1) gene [14] or heme oxygenase-1 (HMOX) [15], enzymes that reduce impacts of ROS. Our previous studies examined a set of genotypes related to oxidative stress and found that polymorphisms of hemochromatosis (HFE) and glutathione S-transferase T1 (GSTT1) significantly modified associations of PM_2.5 with plasma homocysteine [12]. Anh et al. [24] reported that vitamin D-related genes (group-specific component, GC) were significantly associated with the serum D-vitamin concentrations that related to prostate cancer.

However, the selection of certain genes is somewhat arbitrary and the use of an array of genes is vulnerable to false positives from multiple comparisons, a major issue in genetic association studies. In this study, we aimed to examine whether daily ambient OC, SO₄^2- and maximal one-hour O₃ were associated with urinary 8-OHdG based on our previous findings [23] and such associations were modified by genotypes related to oxidative stress in the Normative Aging Study population (NAS). Because of multiple comparisons, we used the Multiple Testing Procedures (MTP) modified by our team, multtest in the R project http://www.r-project.org to identify significant SNPs from a set of candidate genes [25-28].

Table 1 shows the descriptive statistics of the demographic characteristics, health and environmental variables among the NAS population during 2006-2008 at visit (n = 320). There were no repeated measurements in this study. Table 2 shows distributions of polymorphisms of candidate genes. Among 320 participants, wild types were dominant for CATs, HFEs, GSTP1 (rs1799811), HMOX (rs2071749) and GCLC, but the situation varied for other candidate genes. There were no obvious differences for the distributions of wild and heterozygous types in GCLM, GC and GSTP1 (rs1695). Heterozygous types for HMOX (rs2071746 and rs2071749) consisted of large components. 80.9% and 48.8% of subjects were classified as non-deletions for GSTT1 and GSTM1, respectively. Mean of the HMOX-1 GC repeated number was 25.8 (SD: 3.3) with median 24.

We first fit the linear regression model to estimate associations of OC, SO₄^2- and maximal one-hour O₃ with 8-OHdG using moving averages of pollutants up to four weeks. Results show that main effects varied across different day moving averages and 24-, 20- and 18-day moving averages were strongest associated with SO₄^2-, OC and maximal one-hour O₃, respectively, which were used to assess effect modifications. The detailed information has been reported elsewhere [23]. For an IQR increases in 24-, 20- and 18-day moving averages of daily SO₄^2-, OC and maximal one-hour O₃, urinary 8-OHdG increased by 29.0% (95% CI: 5.9%, 52.1%), 27.6% (95% CI: 3.6%, 51.6%) and 54.3% (95% CI: 7.6%, 100.9%), respectively.

Before examining effect modification, we categorized each candidate gene into a dummy variable so that the gene and the pollutant of interest only have one interaction term. We combined the homozygous and heterozygous types for appropriate genes known as the non-wild type (dominant model) due to small number of the homozygous type. We also combined the homozygous and heterozygous short repeat for HMOX-1, referred to as any short (Table 2). Then, we identified candidate genes that executed significant effect modification as aforementioned. Adjusted p-values in MTP model show that GSTP1 A114V (rs1799811) marginally significantly modified the effect of SO₄^2- on 8-OHdG (adjusted p = 0.091). CAT (rs2286367) (adjusted p = 0.037), GSTM1 (adjusted p = 0.037), GC (rs2282679) (adjusted p = 0.025) and GC (rs1155563) (adjusted p = 0.027) significantly modified effects of OC on 8-OHdG. There was no significant effect modification for O₃ (Table 3). As sensitive analyses, we used different options in MTP for typeone (type I error) (tail probabilities for error rate, TPPER; false discovery rate, FDR) and methods (single-step maximum T, ss.maxT; single-step minimum P ss.minP; step-down minimum P, ss.minP). Similar trends were found in spite of some variations. We also categorized pack-years of cigarettes smoked using tertiles as cut-off and re-ran MTP model. Results were similar to those using continuous variable for pack-years of cigarettes smoked. Figure 1 shows the estimated effects of OC or SO₄^2- on 8-OHdG across subpopulations carrying different genotypes, for those SNPs where an interaction with p < 0.10 was found.

We found that associations of the secondary pollutants, specifically OC and SO₄^2-, with 8-OHdG, a direct oxidative stress-related biomarker, were modified by polymorphisms in genes related to oxidative defenses. This is significant for several reasons. First, the finding that genetic polymorphisms in the oxidative defense pathway modified the association suggests that it is not due to chance or confounding, since neither should be associated with the genotypes of the individuals. Second, while considerable focus has been placed recently on freshly generated traffic particles, such as BC or ultrafine particle number, this study confirms that particles, including particles from coal burning power plants, play a role in increasing systemic oxidative stress.

The specific polymorphisms that modified the associations were GSTP1 (rs1799811), GSTM1, CAT (rs1799811) and GC (rs22826799, rs1155563). We found 8-OHdG was more strongly associated with SO₄^2- among those carrying the wild type of the GSPT1, and more strongly associated with OC among those carrying the wild type of CAT (rs2284367), the non-deletion of GSTM1 and the non-wild type of the GCs (rs2282679 and rs1155563) comparing with other types of the corresponding genes (Figure 1). Based on our knowledge, it is the first time that MTP has been used to identify significant gene-environment interactions. MTP has advantages over some other approaches to controlling for false discovery rates in which a group of fixed covariates are adjusted for while a set of variables were compared.

Several studies have examined effect modification and found that people carrying variants of oxidative stress-related genes are differentially susceptible to air [12-14,16,48]. Human GSTs are subdivided into several classes, among which GSTT1, GSTM1 and GSTP1 have been extensively investigated [12,14,49,50]. GSTM1 or GSTT1 catalyzes the conjugation of glutathione to numerous potentially genotoxic compounds [50]. Individuals with the deletion of GSTM1 or GSTT1 have been shown to reduce GST activity and thus may be unable to eliminate toxins as efficiently when they expose to oxidative pollutants [50]. Schwartz et al. [14] found that PM_2.5 was significantly associated with high frequency of HRV among those without the GSTM1 allele, but not for those with the allele. Gilliland et al. [48] reported that exposure to in utero maternal smoking was associated with increased prevalence of early onset asthma among those without GSTM1 allele, but not for those with GTSM1 allele. Similarly, Romieu et al. [51] found that GSTM1 null children were more sensitive to ozone exposure. However, all the aforementioned studies did not report whether there were significant effect modifications. Differential results from these stratification analyses might also be attributed to statistical powers across subpopulations or differential distributions of other controlled or uncontrolled covariates across subpopulations. This study observed that GSTM1 significantly modified associations of OC with 8-OHdG, but paradoxically that the GSTM1 null allele provided protection against exposure. Our recent study examined whether variations of a set of genes altered effects of black carbon and PM_2.5 on plasma homocysteine in this population and found that GSTT1 (but not GSTM1) significantly modified associations between pollutants and homocysteine. PM_2.5 and black carbon were more strongly associated with homocysteine among those carrying GSTM1 allele comparing those without the allele although no significant interactive effects were found [12]. Different findings of effect modification by GSTM1 variation across studies may reflect differences of exposure, outcome and population, measurement errors in exposure or phenotype, and by chance. Similar situations also appeared in other studies [52,53]. Therefore, statistical effect modification may be inconsistent with biological interaction. Further research or meta-analysis is needed for GSTM1.

In contrast, few studies have examined the function of GSTP1 A114V (rs1799811) on diseases with inconsistent results [54-57]. None of these studies found the GSTP1 is significantly associated with the outcomes of interest although some studies found positive trends. Therefore, the functions of the polymorphisms have not been determined. Several studies examined effect modifications of GSTT1 on various endpoints but no significant effect modification was found [58-60]. For example, Melén et al. [59] examined whether GST modified traffic-related pollution effect on childhood allergic disease and found that carriers with variants of GSTP1 (rs1799811) were higher susceptible to NO_x. Our study found the variation of GSTP1 showed a protective effect of SO₄^2- on 8-OHdG. However, other two studies did not find any evidence that the GSTP1 modified effects of black carbon or smoking on blood pressure or Parkinson's disease occurrence [58,60]. Inconsistent observed findings may be attributable to various sources as aforementioned. In this study, it may also related to the small number of variants in this population, which probably lead to unstable estimates. Therefore, its functions remain to be clarified by others (Table 2).

GC, vitamin D-related genes, is related to the vitamin D metabolism [61]. Vitamin D is activated to form 1, 25-dihydroxyvitamin D in the liver and kidney and then transported in serum to different tissues by the vitamin D-binding protein, which is encoded by GC [61]. Studies show that polymorphisms of vitamin D-related genes are associated with various cancers, cardiovascular diseases and respiratory diseases [62-64]. Ahn et al. [61] examined variations of 212 SNPs related to vitamin D metabolism and found that all four SNPs of GC (rs1212631, rs2282679, rs7041, rs1155563) are significantly associated with the concentration of serum vitamin D. When these four SNPs were simultaneously included in the multivariate model, only two SNPs (rs22679, rs1155563) were significantly associated with vitamin D. In this study, we found that the two SNPs of GC (rs22679, rs1155563) were associated with 8-OHdG in this study. The mechanisms remain to be clarified yet.

Catalase is a protein of 526 amino acids, encoded by the catalase gene with 34 kb pairs of nuclear acids [65]. Catalase is the main regulator of hydrogen peroxide metabolism [66]. Catalase enzyme mutations may reduce its activity and probably results in the increase of the hydrogen peroxide concentrations in the tissues [62]. Inherited catalase deficiency results in acatalasemia (homozygous state) and hypocatalasemia (heterozygous) and is related to increased plasma homocysteine concentrations [42,67,68]. Our previous study reported that the variation of CAT modified associations between particle matter and plasma homocysteine concentrations [12].

Experimental toxicology studies have shown that air pollutants act via the oxidative stress pathway [8,36,69]. Ghio et al. [36] found that homozygous Belgrade rats functionally deficient in divalent metal transporter-1 display decreased metal transport from the lower respiratory tract and have stronger lung injury than control littermates, when exposed to oil fly ash containing iron. Belgrade rats cannot transport iron and other divalent metals across membranes via HFE gene regulated processes. They also reported that healthy volunteers exposed to concentrated ambient air particles had increased concentrations of blood fibrinogen and induced mild pulmonary inflammation [8]. Tamagawa et al. [69] reported that five-day and four-week exposures to PM₁₀ caused acute and chronic lung and systematic inflammation of New Zealand rabbits.

There are several strengths in this study. First, we used MTP model to identify the significance of a group of candidate genes while we examined effect modification by genes on air pollution effects. This method overcame some problems in this kind of studies, such as arbitrary selection of a few significant genes or high false discovery rate when individually examining a set of genes. Secondly, this study was conducted in a relatively large population. Information of participants was well measured and collected. However, several limitations also exist with this study. First, we used air pollution data collected from a single monitoring site for personal pollution exposure and therefore, some extent misclassification might happen. A recent study compared ambient concentrations with personal exposures with monitoring measurement and results show that ambient measures were good surrogates for PM_2.5 and SO₄^2- in both winter and summer, but O₃ was only good in summer, not well in winter [70]. Nevertheless, with non-differential misclassification, any potential bias would be expected toward the null. Second, MTP has several options to select type I error and several methods to calculate adjusted p-values. Using bootstrap re-sampling methods will result in different estimates when a MTP model is rerun. These will introduce the uncertainties in model selections [25-28]. In addition, the NAS consists of an aged population and non-Hispanic white men were dominant. Thus, the findings are not well generalizable to other populations.

This study found that variations of oxidative stress-related genes modified effects of OC or SO₄^2- on 8-OHdG. This suggests that effects of OC or SO₄^2- on 8-OHdG and other endpoints may be through the oxidative stress pathway.

BC: black carbon; OC: organic carbon; EC: element of carbon; SNP: single nucleotide polymorphism; NO2: nitrogen dioxide; CO: carbon monoxide; O3: ozone; 8-OHdG: 8'-hydroxy-2'-deoxyguanosine; PM_2.5: particulate matter ≤2.5 μm in aerodynamic diameter; GST: glutathione S-tranferase; CAT: catalase; GC: group-specific component; HFE: hemochromatosis; HOMX: heme oxygenase-1; GCLC: glutamate cysteine ligase catalytic subunit; GCLM: glutamate cysteine ligase modifier;

The authors declare that they have no competing interests.

CR was responsible for study design, data analyses, result interpretation and manuscript writing. JS was responsible for study design, data collection and result interpretation. Other coauthors participated in the study design, data collection and result interpretation. All authors read and approved the final manuscripts.