The purpose of this protocol is to assess whether epigenetic factors in healthy individuals
make a person more or less responsive to lung inflammation following ozone exposures.
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.
1. Normal baseline 12-lead resting EKG.
2. Normal lung function, defined by NHANES III as:
- FVC of > 80 %.
- FEV1 of > 80 %.
- FEV1/FVC ratio of > 80 %.
3. Oxygen saturation of > 96 %.
4. Ability to complete the exposure exercise regimen without reaching 80% of predicted
maximal heart rate.
1. 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.
2. Asthma or a history of asthma.
3. A Framingham risk score ≥10.
4. Women who are pregnant, attempting to become pregnant, or breastfeeding.
5. An allergy to any medications which may be used or prescribed in the course of this
6. Cannot refrain from taking vitamins C or E (or multivitamins which contain Vitamins C
or E) for 7 days prior to all visits.
7. 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.
8. Untreated hypertension (≥ 150 systolic or ≥ 90 diastolic blood pressure).
10. Unspecified illnesses, which in the judgment of the investigator or medical staff
might increase the risk associated ozone inhalation challenge or exercise.
11. A history of skin allergies to adhesives used in securing EKG electrodes.
12. Do not understand or speak English.
13. Chronic and continuous allergic rhinitis.
14. Unable to perform the moderate exercise required for the study.
15. 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
16. Those currently taking or have taken anti-coagulant medication in the week prior to
17. 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.
18. A history of fainting in response to blood being drawn or other medical procedures.
19. 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.
20. 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.
1. Viral upper respiratory tract infection or any acute infection within 6 weeks of
2. Current exacerbation of allergic rhinitis and or use of antihistamines during one week
prior to exposure.
3. Recent or recurring exposure to pollutants or irritants.
Exclusion criteria for bronchoscopy:
1. Any food or fluids after midnight prior to bronchoscopy.
2. FEV1 decrement of >10% from baseline on AM of bronchoscopy.
3. Use of aspirin ≥ 81 mg daily, or other nonsteroidal anti-inflammatory drugs within one
week of bronchoscopy.
4. You are unwilling or unable to take nothing by mouth after midnight the night before
5. 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.