Heart rate variability and DNA methylation levels are altered after short-term metal fume exposure among occupational welders: a repeated-measures panel study

Numerous air pollution studies have shown that both acute and cumulative exposures to fine particulate matter (PM_2.5) are associated with increased risk of adverse cardiovascular events, such as the onset of atrial fibrillation (AF), incidence and recurrences of myocardial infarction, heart failure and stroke, and mortality from cardiovascular disease [1‐4]. Heart rate variability (HRV) has been used as an early disease marker of adverse cardiovascular outcomes and as an indicator of cardiac autonomic function. It is believed that reductions in HRV alter the heart’s ability to properly respond to external signals, leading to myocardial infarction [5].

In occupational settings, welders are exposed to high levels of PM_2.5 generated during the welding process. The metal components of PM_2.5 play an important role in its toxicity [6]; metal-rich particle exposure related to welding has been associated with increased systemic inflammation, inflammation-related endothelial dysfunction, and elevated oxidative stress [7, 8]. For example, C-reactive protein (CRP), an inflammatory risk factor of endothelial dysfunction, is associated with fatal and non-fatal coronary artery disease (CAD) events in a healthy population [9, 10]. Studies have shown an association between increased circulating levels of CRP and decreased HRV in middle-aged men free of CAD, which suggests a relationship between ANS dysfunction induced and systemic inflammation [11]. Previous studies from the same cohort have reported an association between HRV and welding PM_2.5 levels [12] and the concentration of various metal components of welding fumes [13].

Not only is inflammation associated with PM_2.5, epigenetic changes have been implicated in PM-related conditions [14]. Epigenetic patterns are known to be sensitive to environmental exposures throughout the lifespan [15]. In a previous study, acute and chronic exposure to welding PM_2.5 was associated with altered gene methylation [16]. DNA methylation in transposable elements, such as Alu and long interspersed nuclear elements-1 (LINE-1), which has been shown to be altered by PM_2.5 exposure [17, 18].

Alu and LINE-1 elements are widely represented across the human genome and DNA methylation in which maintains transcriptional inactivation and integrity of the genome [19]. Studies show that decreased methylation in LINE-1 elements is associated with ischemic heart disease and stroke, as well as cardiovascular disease risk factors, such as high levels of low-density lipoprotein (LDL) and low levels of high-density lipoprotein (HDL) [20‐23]. Decreased methylation has also been shown to be associated with increased vascular cell adhesion molecule-1 (VCAM-1), a biomarker of vascular inflammation [20‐23], which suggests a potential functionality of DNA methylation in cardiovascular disease. A study has further reported that genetic variations in the methionine cycle affect heart rate variability which suggests the role of methylation in cardiac autonomic dysfunction and lower intake of nutritional methyl supplement is associated with the negative effect of PM_2.5 on HRV [24]. However, whether DNA methylation levels in systemic circulation mediate the PM-induced cardiovascular effect measured in HRV is not known.

To investigate the cardiac and epigenetic changes in response to metallic welding fume exposure, we investigated the short-term effect of welding PM_2.5 on resting HRV and blood DNA methylation in Alu and LINE-1 elements in a cohort of 66 male occupational welders. In addition, we tested the association between PM_2.5-induced DNA methylation changes and HRV to characterize the mediation effect of DNA methylation on the relationship between welding PM_2.5 and heart rate variability.

We demonstrated a statistically significant effect of PM_2.5 on a reduction in HRV measured by HF, pNN10, and pNN20, and in SDNN and rMSSD that showed a marginally significant effect. Our results confirm an inverse exposure-response relationship between PM_2.5 exposure and HRV and further support evidence of an observed short-term HRV change following an average of five hours of welding exposure.

These findings also confirm a previous study showing a steep short-term decline in hourly SDNN index (SDNNi) in the first few hours post-exposure to welding PM_2.5, followed by a plateau and a second period of decline in the 9–10 hours post-exposure [28]. The variability of results seen in studies where PM_2.5 induces HRV alterations may be due to differences in sources and components of PM_2.5. A study of highway patrol troopers with exposure to PM_2.5 originating from traffic combustion showed a post-shift increase in SDNN and pNN50 [32]. The assessment of PM_2.5 metal exposure from another study confirmed the inverse relationship between manganese (Mn) and night-time rMSSD [33]. However, with increases in lead and vanadium (Pb and V) concentration, statistically significant mean increases in day-time SDNNi were also reported from the same cohort [13].

A subset of the participants (47 out of 66 participants) were also monitored on non-welding days prior to welding days. We tested the differences between pre and post-shift HRV measurements and the results did not show significant changes between pre and post-shift HRV (Table 4.). However, the higher prevalence of current smokers in this subgroup may be a confounding factor in this non-significant differences between pre and post-shift HRV on non-welding days. Due to incomplete data, we were not able to control the baseline HRV measurements for all participants in the final model. Based on the results and the assumption of missing at random, we assumed the variability of observed HRV measurements on welding days were unlikely due to circadian variation which is not confounding the observed negative effect of PM_2.5 in this study.

Transition metal components of PM_2.5 are inhaled and delivered into the airways and can catalyze the Fenton reaction to generate reactive oxygen species (ROS), leading to oxidative stress, although the exact mechanism remains unclear [34]. Oxidative stress can cause endothelial injury and inflammation followed by cardiac autonomic dysfunction, which can then be visualized in an altered heart rate pattern [35]. Recent studies have suggested that oxidative stress as a consequence of ROS accumulation induces epigenetic profile alterations in peripheral blood leukocytes to further interfere with DNA, leading to changes in gene expression and, eventually, adverse cardiovascular outcomes [19, 21]. In addition, welding particles has been associated with increased systemic inflammation and study has reported that DNA methylation is associated with ROS and inflammatory exposure [7, 36]. In this study we investigated the epigenetic effect of welding PM_2.5, and found a significant association between welding PM_2.5 and increased blood methylation level of LINE-1. Transposable element LINE-1 has more complete retrotransposon structures thus it has different biological functions from Alu, and studies have suggested that Alu and LINE-1 methylation responded differently to environmental factors [17, 37]. Our results suggest that LINE-1 might be more sensitive to short-term exposure whereas Alu is more susceptible to cumulative exposure over time [7]. We tested the association between blood cell types and blood methylation levels in Alu and LINE-1 and found that the percentage of either neutrophils or lymphocytes, which are the major differentials in peripheral blood, was not significantly associated with methylation levels. Hence, although DNA methylation is cell-type specific, our analysis shows that blood cell differentials were not confounding the observed epigenetic effect of PM_2.5 in this study.

It is generally understood that PM-induced oxidative DNA damage can interfere with the ability of DNA methyltransferase to interact with DNA to reduce methylation [38]. However, one study showed an increase in global methylation in sperm from mice exposed to particulate air pollution in an urban/industrial location, supporting the positive association of global hypermethylation and particulate exposures [39]. Along with our results, the positive association between LINE-1 methylation and welding PM_2.5 suggests other factors that may play a role in PM-induced ROS. Another possible explanation may be the complex toxicity of welding exposure compositions. For example, a coke-oven worker study has reported a significant association between increased methylation of LINE-1 and exposure to PAHs [40], which are also generated from welding processes and whose genotoxic risk is well established [41]. In addition to organic chemicals, nickel-induced higher global methylation was also found in Chinese hamster G12 cells [42]. Unfortunately, there is a limitation in this study that concomitant pollutants including manganese, nickel and chromium from the practicing on a mix of standard and stainless steel welding are potential confounders that we were unable to control the unmeasured pollutants or perform a compositional exposure assessment of to further extricate the observed epigenetic responses to welding fume. In addition, smoking was only controlled as a dichotomous variable because the smoking behavior information was collected through a lifestyle questionnaire so that the lack of accuracy might not provide us to evaluate the effect of smoking measured in quantity. Also, the possible influences of electromagnetic fields generated from welding activities (in the absence of welding fume exposures) on the HRV measurements has not been assessed.

Many studies have shown a link between altered repetitive element methylation and cardiovascular diseases [23], yet the underlying epigenetic regulatory pathways have not been identified. To understand the role of DNA methylation between PM exposure and reduced HRV, we performed mediation analysis [43] to investigate whether LINE-1 methylation is a mediator of the association between PM_2.5 exposure and HRV outcomes. This method allowed us to decompose a total effect of exposure on an outcome into a direct effect of the exposure and an indirect effect of the exposure through a mediator’s pathway [44]. Mediation analysis usually requires a significant association between the exposure and the mediator, and a significant association between the mediator and the outcome [43]. However, the data presented here show a non-significant positive association between LINE-1 methylation level and HRV after adjustment of PM_2.5, therefore we did not see any significant mediation effect. The association between LINE-1 methylation and HRV was tested after a few hours of welding exposure, which might not capture the best timing of a dose–response relationship but still suggests a link between decreased LINE-1 methylation and adverse cardiac outcomes. A limitation of this study is the impossibility of collecting tissue-specific heart cells from human subjects and lack of data on gene-specific methylation such as genes involved in regulating oxidative stress and inflammatory, since LINE-1 is a transposable element and lacks the specificity necessary to serve as a mediator in a biological pathway. In addition, the relatively small sample size of this study, though adequate for repeated-measures panel results, may be a limitation in estimating the mediation effect.

In summary, our results show the acute decline of HRV following the exposure of metal-rich welding PM_2.5 and support evidence of a short-term cardiac response to welding exposure. We also show a short-term increase in DNA methylation in LINE-1 elements following welding exposure. These results support a systemic epigenetic response to short-term exposure to high-level metal-rich particulates, however no mediation effect of LINE-1 methylation was supported from the study. Further studies involving specific tissues and gene methylation are required to establish a more detailed mechanism for the observed cardiac responses.