1. Department of Environmental Health
The Role of Glutathione S-Transferase Variants in Global and Gene-Specific Methylation in Children Exposed to Chronic Diesel Exhaust
Kelly Brunst BA
PhD Candidate
EPA Star Fellow
Department of Environmental Health

2. Presentation Outline Background Introduction to the CCAAPS cohort
Diesel exhaust particles
Role of DNA methylation in DEP-induced health effects
Introduction to the CCAAPS cohort
Eligibility and recruitment
Clinical evaluations
DEP exposure Assessment
Study sample
Selection process and inclusion criteria
Global methylation assessment
Genotyping analysis
Statistical methodology
Results
Conclusions

3. Risk Factors: Childhood Asthma
Demographic, Social, and Genetic Factors
Gender
Stress, violence
Genetics / epigenetics
Clinical Predictors
Reduced lung function in infancy
Airway hyperreactivity
Atopy or elevated total IgE
Allergic rhinitis, atopic dermatitis
Early respiratory infections
Environmental Factors
Indoor allergen exposure
Environmental tobacco smoke
Endotoxin and microbial exposure
Indoor NO2 and particulate matter
Traffic Related Air Pollution and Diesel Exhaust

4. Diesel Exhaust Particles (DEP)
Inert carbon core with large surface area per unit of mass
Ideal for absorbing heavy metals and organic compounds
Polycyclic aromatic hydrocarbons (PAHs)
Major source of ambient PM2.5
~25% of all PM from fuel combustion is derived from diesel
Size
92% ultrafine particles (UFPs)
Enhances allergen specific IgE production in the upper airway after intranasal allergen exposure
Increased TH2 cytokine production after in vivo nasal challenge with DEP and ragweed pollen
DEP can bind with allergens and be co-deposited into the airways
Generate reactive oxygen species (ROS)
Inflammation and lung damage

5. PM Size Definitions Coarse particles (PM10) Fine particles (PM2.5)
Diameter > 2.5 microns and < 10 microns
Produced primarily by mechanical processes (tire wear, grinding, re-suspension of ground particles)
Fine particles (PM2.5)
Diameter < 2.5 microns
Produced primarily from combustion sources
Ultrafine particles (PM0.1)
Diameter < 0.1 micron (100 nm)
PM10
PM2.5
PM0.1

6. Increased surfaced area Increased surface molecules
Impact of PM Size
Increased surfaced area
Increased surface molecules
Respiratory deposition
Oberdorster et al. Environ Health Perspect. 2005;113:

7. Role of DNA Methylation
DNA Methylation: Addition of a methyl group to the 5 position of cytosine pyrimidine ring
CpG dinucleotide or site: cytosine next to a guanine
CpG Island: Genomic region that contains high frequency of CpG sites
Hypomethylation ~ Result in successful transcription of a gene
Hypermethylation ~ Inhibits transcription of a gene
Global hypomethylation is common in many complex diseases such as cancer and Alzheimer's
Global Hypomethylation =Genomic Instability

8. Why study global methylation patterns among DEP exposed children?
Generation of Reactive Oxygen Species (ROS)
DEP
Reactive with DNA
DNA Damage
ROS
Obstruct DNA methyltransferases from binding
Global hypomethylation
ROS-mediated DNA Damage
In vitro

9. Human Studies (Adults)
STUDY
PM Pollutant
Findings
Normative Aging Study (2009)
PM2.5, Black carbon, and sulfates
Hypomethylation of Line1 (PM2.5 and BC)
Electric Furnace Steel Plant (2009)
PM10
Hypomethylation of Line1 and Alu
Normative Aging Study (2011)
Hypomethylation of Line1 and Alu (prolonged BC and sulfate)
Coke-oven workers and controls (2009)
PAH
Hypermethylation of Line1 and Alu

10. Human Studies (Children)
STUDY
PM Pollutant
Findings
Children’s Health Study (2009)
Maternal Smoking
Hypomethylation of Alu
(buccal cells)
New York Women’s Birth Cohort (2011)
Prenatal Tobacco Smoke
Hypomethylation of Sat2 but not Line1 or Alu
(peripheral blood granulocytes)
Columbia Children’s Center (2011)
PAH
Global hypomethylation of cytosines
(cord blood)
Surrogate tissues??

11. Role of Glutathione S-Transferase (GST) Variants
Both variants diminish Glutathione S-Transferase enzyme activity
Increase susceptibility to oxidative stress
STUDY
PM Pollutant/SNP
Findings
Cincinnati Childhood Allergy and Air Pollution Study –CCAAPS (2009)
DEP
GSTP1 Ile105Val polymorphism
Increased risk for wheezing in children exposed to DEP among children with Val105
Children’s Health Study (2009)
Maternal Smoking
GSTM1 deletion
Hypomethylation of Line1 in GSTM1-null children exposed to maternal smoking
Normative Aging Study (2011)
PM2.5, Black carbon, and sulfates
GSTM1-null genotype strengthened the association between BC and Alu methylation in adults
DEP exposure in children??

12. Why study IFN-g methylation patterns among DEP exposed children?
IFN-g expression regulated via DNA methylation
IFN-gamma
Induce hypermethylation of IFN-g promoter in vitro and mouse models
DEP
(in vitro/in vivo)
Induce hypermethylation of IFN-g promoter
Co-exposures (allergens)
PAH/DEP (human studies)
First human study of children exposed to PAH

13. Study Questions
Does chronic exposure to DEP among children result in global DNA hypomethylation or IFN-g hypermethylation?
Do GSTP1 and GSTM1 variants modify the relationship between DEP and DNA methylation in children?
Is there a gene-dose effect?
Common variants in GST genes on various clinical outcomes are likely have a gene-dose effect and are best described in a dosage specific manner

14. Cincinnati Childhood Allergy and Air Pollution Study (CCAAPS)
Are children who are exposed to diesel exhaust particles (DEP) at increased risk for developing allergic disease and asthma?
Examine additional environmental exposures that may impact the development of disease or modify the effect of DEP
Determine if this effect is modified in a genetically at-risk population

15. CCAAPS Eligibility and Recruitment
Full-term infants born in the Ohio/Kentucky River Valley region were identified from birth records ( )
Parents recruited at infants’ age 2-7 months
Study Eligibility
Birth records were geocoded and initially grouped:
Exposed: Infants residing < 400 meters (m) from major road (> 1000 trucks/day)
Unexposed: Infants residing > 1500 m from major road
Parental report of allergy symptoms and SPT+
Study Recruitment
2265 / 7352 completed eligibility survey
1879 Eligible = ‘Yes’ response to allergy symptoms
1152 parents underwent allergy skin prick testing to 15 common aeroallergens
881 parents were SPT+
762 infants enrolled (SPT to 15 aeroallergens and milk and egg)

16. CCAAPS Clinical Evaluations
Ages 1, 2, 3, 4 and 7
Questionnaire
Skin prick testing
Physical examination
Hair sample
Nicotine / Cotinine
Saliva sample
DNA Isolation
Blood Collection
Exhaled Nitric Oxide (eNO)
Spirometry
Bronchodilator
MCCT
BASC-2

17. CCAAPS Ambient Air Sampling (2001 – 2006)
24 monitoring stations to measure daily ECAT…Notice the 5-fold distribution in ECAT (with stations having high exposures and low exposures)

18. Estimating Individual Exposure to DEP
Sampling Sites ?
Homes

19. Land-Use Regression (LUR)
Predict pollution concentrations at a given location based on surrounding land and traffic data
Average daily ECAT at each sampling site serve as the dependent variable and nearby land-use and traffic variables serve as independent variables in a multiple linear regression model
Predict ECAT at each sampling site

20. Visualizing LUR Results

21. Time-Weighted Daily Exposure
Geocoding all addresses where a child was reported spending more than 8 h/wk in the previous 12 months
Daycare, relatives, babysitter, school, etc.

22. How Has Exposure to DEP Changed from Birth to Age 7?
ECAT (Birth) μg/m3
ECAT (Birth) μg/m3
To assess chronic DEP exposure we had to make sure we chose kids with similar exposures through out their childhood
ECAT (7) μg/m3
ECAT (1) μg/m3

23. Study Sample Workflow Methylation CCAAPS Random Sample (n=92)
CCAAPS (n=604)
No second hand smoke exposure (n=522)
Constant DEP exposure (n=376)
Random selection
(n=100)
Hair Cotinine <0.2 ng/mg (n=92)
Random Sample (n=92)
Inclusion Criteria Definitions
SHS: Parental report of cigarettes smoked daily at home=0
DEP: ECAT varied by less than 0.05 standard deviations from birth through age 7
8 Children excluded due to hair cotinine levels higher than 0.2 ng/mg
** Parental report of cigarettes smoked daily by member of household > 0

24. Global Methylation: MethylFlash Global DNA Quantification Kit--Fluorometric) (Epigentek Group Inc, NY)
DNA source: Saliva (age 7)
Quantity of DNA
Qubit Quantification
Quality of DNA
Agarose Gel Electrophoresis
DNA Input
50 ng (in duplicate)--Manufacture ng
Two 96-well plates, including negative and positive controls
Output
Fluorescence spectrophotometer reads the relative fluorescence units
Amount of methylated DNA is proportional to the fluorescence intensity
Total methylated cytosine (mC) / 50ng of total DNA
global methylation is detected using capture and detection antibody and then quantified fluorometrically by reading the relative fluorescence units with a fluorescence spectrophotometer. The amount of methylated DNA is proportional to the fluorescence intensity measured. Readings were normalized to the positive control

25. IFN-g Methylation: Methylation Specific qPCR
Saliva genomic DNA (500ng) from each sample was bisulfite-converted using the EZ Methylation Kit (Zymo Research, Irvine, CA).
MS-qPCRs were carried out in a total volume of 10 ul
5 ul 2× power syber green master mix (Invitrogen)
0.5 ul of 10 uM “methylation-specific” or “unmethylated-specific” primers (Table 3)
0.2 ul ROX, 1.3 ul H2O, and 3 ul of bisulfite-converted DNA template
ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA).
50 °C for 2 min followed by 45 cycles of 95 °C for 15 sec and 59 °C for 1 min.
Data was analyzed using Sequence Detector v2.3 Analysis Software (Applied Biosystems, Foster City, CA).
Each sample was run in duplicate for analysis.
Percentage of methylation in each sample is calculated by the equation:
% meth = 100/[1+2ΔCt(meth-unmeth)]%

26. Genotyping Analysis: GSTP1/GSTM1
Buccal cells Genotyping for GSTP1
LightTyper platform (Roche Diagnostics, GmbH, Mannheim, Germany)
The PCR primers and hybridization probes were designed with the LightCycler Probe Design Software 2.0 (Roche Diagnostics, GmbH, Mannheim, Germany) (Table 3).
Genotyping of GSTM1
LightCycler 480 in a multiplex reaction using a hydrolysis probe assay
The primers used are listed in Table 3.
20% of the sampled genotypes were verified by PCR and gel electrophoresis with an error rate of 7%.
Genotypes were dichotomized to carriers and noncarriers of the Val105 allele and as either GSTM1-present or GSTM1-null.

27. Statistical Methods Descriptive Statistics
Chi-square tests and T-tests: investigate possible differences in categorical and continuous predictors,
Univariate associations were examined for the primary outcomes with DEP exposure and all potential covariates using generalized linear regression (link=log)
Global and IFNg methylation were modeled separately and covariates were removed by backwards elimination, beginning with the highest p-value.
If a change in the parameter estimate of DEP exceeded 10% after removal, the variable remained in the model. The final models included variables significant at the 20% level.
The statistical interaction between each variant (GSTM1 and GSTP1) and DEP exposure was evaluated by including the corresponding product term to each regression model. If a product term was significant or borderline significant (0.05 < p <0.20), the modifying effects of GSTM1 and GSTP1 were then assessed by model stratification.
All tests assumed a two-sided alternative hypothesis, a 0.05 significance level, and were conducted using SAS 9.2 (SAS Institute Inc., Cary, NC, USA).

29. Global Methylation: Results
Chronic exposure to DEP was associated with global hypomethylation
(B,-2.13; p<0.01).
GSTM1*DEP (p=0.15) and GSTP1*DEP (p=0.18) were borderline significant
Model Stratification

30. IFN-g Methylation: Results
Chronic DEP exposure was associated with hypermethylation of IFN-g promoter
(B, 1.62; 95%CI: 0.47, 2.78).
Carriers of the Val105 allele and GSTM1 null children were not at increased risk for IFN-g promoter hypermethylation as a result of chronic DEP exposure

31. Gene-Dose Effect?

32. Conclusions
Chronic DEP exposure alters global and IFNg methylation in children and these changes can be detected in DNA from saliva
The effects of DEP on global methylation varied among children with common GST variants and suggest a gene-dose effect of GSTP1 and GSTM1
Two different mechanisms may be responsible for regulating DNA methylation at the global and gene-specific level with glutathione deficiency potentially playing a major role in regulating global but not IFNg methylation

33. Study Limitations/Advantages
Sample Size
Inclusion criteria key advantage
Established a constant exposure (Chronic)
Changes in global methylation are more likely to be from DEP rather than residual confounding by other PM exposures (such as SHS)
“Clean” population
Source of DNA
Oral cavity provides an important first line of defense against DEP exposure
A potential approach for measuring the effects of air pollution (via cytokines levels) in children
Positive association between DNA methylation levels found in peripheral blood mononuclear cells and saliva

34. Acknowledgements University of Cincinnati
Grace LeMasters PhD (mentor)
Shuk-mei Ho PhD (advisor)
Ricky Leung PhD (committee member)
Linda Levin PhD (committee member)
David Bernstein MD
Jeff Burkle
Sergey Grinshpun PhD
James Lockey MD, MS
Tiina Reponen PhD
Sang Young Son PhD
Cincinnati Children’s Hospital Medical Center
Patrick H. Ryan PhD (advisor)
G. Khurana Hershey MD PhD (committee member)
Jocelyn Biagini-Myers PhD
Mark Ericksen
Abraham Research
Manuel Villareal MD
Missy Randolph, CRC

35. Extra Slides Allergic Sensitization age 7
TRAP (or DEP) and longitudinal wheeze
Future Directions (Foxp3)

36. SPT Results (Age 7)

37. Frequency of Positive SPT at Age 7

38. Are children exposed to TRAP at increased risk for wheezing throughout childhood?
Longitudinal analysis of wheezing at ages 1, 2, 3, 4, and 7
Childhood wheeze phenotypes defined at age 7
Transient
Parental report of wheeze at ages 1/2/3/4 and not 7
Late-onset
Parental report of wheeze at age 7 and no wheeze prior
Persistent
Parental report of wheeze at age 7 and at least 1/2/3/4
Does early childhood represent the most susceptible time period for exposure to TRAP?

39. Childhood Exposure to TRAP and Wheezing Phenotypes at Age 7: Preliminary Results
Birth
Age of ECAT Exposure
Unadjusted Associations Between ECAT Exposure* and Wheezing Phenotypes at Age Seven
*ECAT dichotomized at the 75% at each age
of exposure

40. Childhood Exposure to TRAP and Asthma at Age 7: Preliminary Results
Unadjusted Associations Between ECAT Exposure and Asthma at Age Seven
Birth
Age of ECAT Exposure

41. Future Directions (FOXP3)
Why Foxp3?
Important in Treg-cell development and function (Treg important in allergy and asthma)
Expression of Foxp3 is regulated by DNA methylation
Asthmatic children exposed to high levels of PAH have increased Foxp3 methylation
Study Questions:
Is chronic DEP exposure associated with Foxp3 DNA methylation?
Are the methylation patterns in Foxp3 associated with persistent and/or early transient wheezing during childhood or a diagnosis of asthma at age 7?