Epigenetic Effect Modifications With Ozone Exposure on Healthy Volunteers

Controlled human exposure studies to ozone have reported decreases in lung function (Devlin et al. 2012; Kim et al. 2011) and increased inflammation (Kim et al. 2011; Koren et al. 1991; Liu et al. 2009; Romieu et al. 2008). However, the range of response to ozone in healthy young volunteers is an order of magnitude, and if individuals are exposed to ozone some months later they retain their hierarchy on the response curve, suggesting that long-lived factors are responsible. Several studies have demonstrated that polymorphisms in oxidative stress genes such as GSTM1 or NQO1 may be associated with responsiveness to air pollutants (Bergamaschi et al. 2001; Corradi et al. 2002). However, within the past decade, many researchers have started exploring the epigenome as a possible link between exposures to environmental toxicants and disease. Epigenetics refers to non-genetic mechanisms influencing gene expression and phenotype (Cortessis et al. 2012). Commonly studied epigenetic changes include DNA methylation, histone modification, and non-coding RNA expression (i.e. micro-RNA). Recently, work conducted at the Harvard School of Public Health looked at DNA methylation as an effect modifier to air pollution-induced adverse health effects (Bind et al. 2012). This group, using a cohort representing previous war veterans from the VA Normative Aging Study, observed stronger effects in cardiovascular disease-related blood biomarkers with DNA methylation status, both globally and within candidate genes. Additionally, Salam et al. found that fractional exhaled nitric oxide, a marker of lung inflammation, was interrelated with short-term PM 2.5 concentration as well as NOS2 epigenetic and genetic variations in children (2012). Thus, these studies suggest epigenetic changes could impact susceptibility to pollutants. Additionally, acute epigenetic changes, which are potential pathways of air pollution-induced health effects, have been associated with the inhalation of particulate matter and ambient gaseous pollutants (Baccarelli et al. 2009; Bellavia et al. 2013; Bollati et al. 2010; De Prins et al. 2013; Madrigano et al. 2011; Tarantini et al. 2009). Therefore, it is possible that an individual's epigenetic profile could make them more or less responsive to ozone, and that ozone exposure itself could cause acute changes in the epigenome which could in turn affect ozone-responsiveness.

Previous studies that have looked at epigenetic changes associated with air pollutants have difficultly disentangling the role of genetic and epigenetic factors. One way to do this is to study identical (MZ) twins. MZ twins arise when two or more daughter cells split from a single zygote during embryonic development, forming two individuals with identical genetic sequences (Fraga et al. 2005) but dissimilar epigenomes (Li et al. 2013; Szyf 2007). A number of diseases in which MZ twins are discordant, such as bipolar and schizophrenia disorders (Bonsch et al. 2012; Dempster et al. 2011), asthma (Runyon et al. 2012), autism spectrum disorder (Wong et al. 2013), and breast cancer (Heyn et al. 2013), implicate epigenetic variability as the cause. Therefore, as discordance for disease status has already been linked with epigenetic changes, this adds further plausibility to the notion that epigenetics could be responsible for the susceptibility of some subjects to ozone exposures while others seem non-responsive. By using MZ twins as one target population for this study, variability due only to epigenetics, without the influence of genetics, can be fully explored.

For this study, the investigators will measure changes in pulmonary inflammation after a controlled exposure in healthy subjects and healthy twin pairs to clean air and ozone. This endpoint was chosen because previous work has shown that the epithelial cells lining the airways are the first target of ozone and respond by making pro-inflammatory cytokines such as IL-6 and IL-8. Epigenetic changes are dependent on tissue type, and airway epithelial cells can be obtained by brush biopsies during bronchoscopy and assayed for epigenetic changes. The investigators will determine whether differences in baseline epigenetic profiles between subjects are associated with responsiveness to ozone and whether ozone exposure itself causes acute changes in a subject's epigenome.

Inclusion criteria

• Normal baseline 12-lead resting EKG.
• Normal lung function, defined by NHANES III as:

• FVC of > 80 %.
• FEV1 of > 80 %.
• FEV1/FVC ratio of > 80 %.
• Oxygen saturation of > 96 %.
• Ability to complete the exposure exercise regimen without reaching 80% of predicted maximal heart rate.

Exclusion criteria

• A history of acute and/or chronic illnesses such as diabetes, rheumatological diseases, immunodeficiency state, neurological disease, renal disease, liver disease, endocrinological disease, malignancy, cardiovascular disease, chronic respiratory diseases, and lung cancer.
• Asthma or a history of asthma.
• A Framingham risk score ≥10.
• Women who are pregnant, attempting to become pregnant, or breastfeeding.
• An allergy to any medications which may be used or prescribed in the course of this study.
• Cannot refrain from taking vitamins C or E (or multivitamins which contain Vitamins C or E) for 7 days prior to all visits.
• Cannot refrain from taking supplements for 7 days prior to all visits that contain homeopathic/naturopathic medicines or medications which may impact the results of the ozone challenge or interfere with any other medications potentially used in the study. Medications not specifically mentioned here may be reviewed by the investigators and medical staff prior to inclusion in the study.
• Untreated hypertension (≥ 150 systolic or ≥ 90 diastolic blood pressure).
• Dementia.
• Unspecified illnesses, which in the judgment of the investigator or medical staff might increase the risk associated ozone inhalation challenge or exercise.
• A history of skin allergies to adhesives used in securing EKG electrodes.
• Do not understand or speak English.
• Chronic and continuous allergic rhinitis.
• Unable to perform the moderate exercise required for the study.
• Those that are unwilling or unable to refrain from the following medications for the week prior to each exposure: anti-inflammatory agents such as ibuprofen, naproxen, or aspirin.
• Those currently taking or have taken anti-coagulant medication in the week prior to each exposure.
• Currently smoker or has a smoked within the last 2 years, or if you have a smoking history > 1 pack-years or are living with a smoker that smokes inside the house.
• A history of fainting in response to blood being drawn or other medical procedures.
• Unwilling or unable to stay for a suitable observation period after the procedure at the discretion of the physician involved, and not ride a bicycle or motorcycle home.
• You are unwilling or unable to refrain from strenuous exercise for 24 hours prior to and after all visits, consuming caffeine for 12 hours prior to all study visits, using of antihistamines for one week prior to exposures, and drinking alcohol 24 hours before all visits.

Temporary exclusions:

• Viral upper respiratory tract infection or any acute infection within 6 weeks of bronchoscopy.
• Current exacerbation of allergic rhinitis and or use of antihistamines during one week prior to exposure.
• Recent or recurring exposure to pollutants or irritants.

Exclusion criteria

for bronchoscopy:

• Any food or fluids after midnight prior to bronchoscopy.
• FEV1 decrement of >10% from baseline on AM of bronchoscopy.
• Use of aspirin ≥ 81 mg daily, or other nonsteroidal anti-inflammatory drugs within one week of bronchoscopy.
• You are unwilling or unable to take nothing by mouth after midnight the night before bronchoscopy.
• You are unwilling or unable to stay in the local Raleigh/Durham/Chapel Hill area for 24 hours after the procedure.

Use of other medications will be evaluated on a case-by-case basis. There is the potential that an individual's current medication use will preclude them from participating in the study at the current time, but they may be reassessed and potentially rescheduled for participation at a later time.