Robert Devlin
Human Studies DivisionNational Health and Environmental
Effects Research LaboratoryUS Environmental Protection Agency
Comparison of Health Effects of Coarse Comparison of Health Effects of Coarse Particles with Effects of Fine and Particles with Effects of Fine and
Ultrafine Particles: Que es Mas Macho?Ultrafine Particles: Que es Mas Macho?
Three ApproachesThree Approaches
• Expose humans to size fractionated ambient PM (CAPS)
• Use Panel studies to associate health effects with exposure to PM of different sizes
Populations which cannot be intentionally exposedExposure scenarios that cannot be easily replicated in chambers
• Collect size fractionated particles for toxicology studiesUnderstand mechanisms by which each size fraction causes adverse health
effectsCompare the relative potency of particles collected from different
geographical locations and across seasons
Exposure of Healthy Volunteers to Exposure of Healthy Volunteers to Concentrated Coarse Ambient Air ParticlesConcentrated Coarse Ambient Air Particles
Coarse CAPS Study DesignCoarse CAPS Study Design
2 Hr ExposureClean Air and CAPS
Pre 0 Hr Post 24 hr Post
Lung functionBlood Draw
HRVDLCO
Lung functionBlood Draw
HRVDLCO
BAL
Lung functionBlood Draw
HRVDLCO
VE 20 L/min-m2,
Subject # 15 Age 18-35PM2.5-10 (g/m3) 89.0 ± 13.2PM10 105.1 ± 15.6PM Size (MMAD) 2.59 ± 0.58
Particle CharacterizationParticle Characterization
MassFilters, TEOM, Data RAM
SizeMOUDI APS
Chemical CompositionElements - XRF, ICP-MS (water soluble metals)Anions - IC (sulfate, nitrate), pHCarbon - organic and elementalSEM/EXD (coarse)
BiologicalsLPS
GasesOzone, NO2
Exposure of Healthy Volunteers to Coarse, Exposure of Healthy Volunteers to Coarse, Fine, and Ultrafine CAPS: Summary of EffectsFine, and Ultrafine CAPS: Summary of Effects
Pulmonary Function
BAL Fluid Cells
BAL FluidMarkers
Cardiac Endpoints
Plasma Markers
Fine No Effect PMNsMonocytes IL-8
FibrinogenNo Effect Fibrinogen
WBCsLDH
Coarse No Effect PMNsMonocytes
Protein SDNN tPAD-dimer
Ultrafine No Effect No Effect IL-8 HFLF
Mean QTNorm. QT var
D-DimertPA
Tryglyceridescholesterol
RTP Asthmatic Panel StudyRTP Asthmatic Panel Study
• Biological materials are found predominately in coarse PM
• Biological materials can exacerbate asthma
• Therefore, we hypothesized that asthmatics should be more responsive to coarse, rather than fine, PM.
Days 1-4 Week1 Wk2 Wk3 Wk4 Wk5
Exposure
Outcomes
PM Outside residence
PM Personal monitoring
PM Central site
Respiratory and Cardiopulmonary OutcomesRespiratory and Cardiopulmonary Outcomes
• Respiratory symptoms and asthma medication use
• Pulmonary function
• Markers of airway inflammation Induced sputum, exhaled nitric oxideInduced sputum, exhaled nitric oxide
• Vascular markersClotting/coagulation factorsClotting/coagulation factorsLipids Lipids (triglycerides, VLDL)(triglycerides, VLDL)Cells Cells (circulating eosinophils) (circulating eosinophils)
• Heart rate variability (SDNN, PNN50, HF)
Associations were only observed with coarse, not fine, PM.
Ongoing StudiesOngoing Studies
• Response of mild asthmatics to fine and coarse CAPS
• Panel study comparing the response of healthy individual, and those with mild or severe asthma to fine and coarse PMIn vitro challenge of cultured cells from these indivuals with In vitro challenge of cultured cells from these indivuals with
PMPM
Health Effects Associated with Health Effects Associated with Different Size PM From Chapel HillDifferent Size PM From Chapel Hill
CoarseCoarse FineFine
UltrafineUltrafine• Particles collected in weekly
batches for a year, using a ChemVol sampler (pooled to obtain 12 monthly samples)
• UF, F, C fractions extracted and used for in vitro and animal instillation studies
Coarse PM Elicits Larger ILCoarse PM Elicits Larger IL--8 Production 8 Production Than Fine or Ultrafine PM at 24 HrsThan Fine or Ultrafine PM at 24 Hrs
October PM
0 100 200 3000.0
2.5
5.0
7.5
10.0CoarseFineUltrafine
g/ml PM
Incr
ease
ove
r C
ontr
ol
PROTEIN
October PM
0 100 200 3000
10
20
30
40
50CoarseFineUltrafine
g/ml PM
Incr
ease
ove
r C
ontr
ol
mRNA
Human primary airway epithelial cells exposed to UF, F, C particles
Coarse PM Elicits Larger GMCoarse PM Elicits Larger GM--CSF and CSF and Cox2 mRNA Production at 24 HrsCox2 mRNA Production at 24 Hrs
GM-CSF
24hr treatment
0 100 200 300 400 5000
10
20
30
CoarseFineUltrafine
g/ml PM
Incr
ease
ove
r Con
trol
Cox2 mRNA
24hr exposure
0 100 200 3000
5
10
CoarseFineUltrafine
g/ml PM
Incr
ease
Ove
r Con
trol
Coarse PM Causes More Inflammation in MiceCoarse PM Causes More Inflammation in Mice
0
10
20
30
40
50
60
70
Control Coarse Fine Ultrafine
Neu
trop
hils
(%)
0
10
20
30
Control Coarse Fine Ultrafine
TNF
alph
a (p
g/m
l)
Saline and 100 g coarse, fine, or ultrafine PM were instilled into C3H/HeJ mice.
Lavage was performed 24 hrs later and % neutrophils assessed, as well as BAL TNF levels.
Is LPS Responsible for Coarse Effects?Is LPS Responsible for Coarse Effects?
Neither Polymixin B nor E5531 Inhibit IL-8 Production by PM in NHBE cells
Ctrl Coarse Fine Ultrafine0
1000
2000
3000
4000 MediumPolymixin BE5531
IL-8
(pg/
ml)
PMN Response to Coarse PM in LPS PMN Response to Coarse PM in LPS Sensitive and Insensitive MiceSensitive and Insensitive Mice
No strain-specific differences in pulmonary neutrophils were evident across the various size fractions suggesting that LPS was not required for the this effect.
Control Coarse LPS0.0
0.1
0.2
0.3
C3H/HeJC3H/ouJ
Tota
l Cel
l Num
ber (
103 )
TNF Response to Coarse PM in LPS TNF Response to Coarse PM in LPS Sensitive and Insensitive MiceSensitive and Insensitive Mice
Control Coarse Fine Ultrafine LPS0
100
200
750
1000
1250
1500TN
F-
(pg/
ml B
AL)
C3H/HeJ
C3H/ouJ
• TNF is likely produced by alveolar macrophages (known to respond to LPS) whereas the PMN response is likely mediated by IL-8 produced by epithelial cells (which do not respond to LPS).
Coarse PM Stimulates More Cytokine Coarse PM Stimulates More Cytokine Production in Alveolar MacrophagesProduction in Alveolar Macrophages
0 50 100 150 200 2500
10000
20000
30000
40000CoarseFine
PM (g/ml)
IL-6
(pg/
ml)
Ultrafine
LPS Inhibitors Block Production of ILLPS Inhibitors Block Production of IL--6 by AM6 by AM
Ctr C F UF0
10000
20000
30000
40000
50000
IL-6
(pg/
ml)
Ctr C F UF
Poly B
Ctr C F UF
E5531
Heat Inactivation of Coarse PM Does Not Heat Inactivation of Coarse PM Does Not Diminish PMN Response in Human VolunteersDiminish PMN Response in Human Volunteers
• Coarse PM was collected in Chapel Hill and UV treated to inactivate viable bacteria.
• A portion was baked at 120oC for 20hrs to inactivate LPS.
• PM was administered to volunteers via nebulization
• Each subject was exposed 3 times: once to unbaked PM, once to baked PM, and once to saline.
• Induced sputum was collected 3hrs after exposure.
Neutrophils
0
10
20
30
40
50
SalinePMcf+PMcf-
**
%
Heat Inactivation Does Diminish TNF Heat Inactivation Does Diminish TNF and HLAand HLA--DR ResponseDR Response
TNF mRNA
0
1
2
3
4
5
6
7
8
9
10salinePMcf+PMcf-
*
Rel
ativ
e Le
vels
to G
APD
H
HLA-DR
0
50
100
150
200
250
300
350
400
* SalinePMcf+PMcf-
Macrophages
MFI
*p<0.05 vs saline; # vs PMcf-
#
• LPS present on coarse PM does not appear to affect processes driven by epithelial cells
• It does, however, stimulate macrophages.
Is Coarse PM Always More Potent than Is Coarse PM Always More Potent than Fine or Ultrafine PM?Fine or Ultrafine PM?
2 hr exposure
0 100 200 3000
25
50
75Coarse
Fine
Ultrafine
g/ml PM
Incr
ease
ove
r Con
trol
Heme Oxygenase mRNA
C F UF
Langendorff Perfusion SystemLangendorff Perfusion System
Ultrafine PM Reduced Baseline Coronary Flow Rate
Ultrafine PM Increased Cardiac Necrosis
Saline NRC FRC NRF FRF NRU FRU0
1
2
3
4
5
6
* *
Cor
onar
y Fl
ow R
ate
(mL/
min
)
Saline NRC FRC NRF FRF NRU FRU0
10
20
30
40
50
60
*
LVD
P R
ecov
ery
(% o
f Ini
tial)
Ultrafine PM Decreased Recovery of Post-Ischemic Cardiac Function
Saline NRC FRC NRF FRF NRU FRU0
10
20
30
40
50
60 *
Infa
rct S
ize
(% o
f Who
le H
eart
)
Seasonal Variation in ILSeasonal Variation in IL--8 Response8 Response
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
Oct Dec Feb Apr June Aug
Coarse Fine Ultrafine
SummarySummary
• Size fractionated particles collected in Chapel Hill, NC cause different effects, depending on the cell type.
• Coarse particles seem to cause more inflammation, which does not seem to be driven by LPS.
• Fine and ultrafine particles cause more oxidative stress
• Ultrafine particles cause cardiac effects.
Beyond PM Mass Beyond PM Mass
• Particles were collected in 7 US cities• Cities were chosen because they have different source profiles
Seattle
Provo Sterling ForestSouth Bronx
Phoenix
Hunter College
Chapel Hill
ILIL--8 Levels in C, F, UF PM Collected 8 Levels in C, F, UF PM Collected from MAPS Citiesfrom MAPS Cities
0
2
4
6
8
10
12
14
16
18
Ut a h S e a t t l e P hoe ni x S t e r l i ngFor e st
S out hBr onx
Hunt e rCol l e ge
Cha pe lH i l l
Fold
Incr
ease
Coarse
Fine
Ult raf ine
CoarseCoarseSeattle Utah
South Bronx Sterling Forest
Organic Carbon
Elemental Carbon
Ammonium Ion
Nitrate
Sulfate
Sum Method 200.7Elements, no Na
Unknown
Chloride
Sodium
Source Profile of PM in MAPS CitiesSource Profile of PM in MAPS Cities
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Utah Seattle Phoenix SterlingForest
SouthBronx
HunterCollege
Unexplained MassMarineRoad SaltTire WearSoil DustSteel FurnanceMotor VehiclePaved Road Dust
Association of Biological Changes to Association of Biological Changes to Sources in MAPS CitiesSources in MAPS Cities
IL-8 Protein vs. Coal Combustion
y = 1.5072x + 1.073R2 = 0.7239
0.001.002.003.004.005.006.007.008.009.00
0.00 1.00 2.00 3.00 4.00 5.00
Coal Combustion (ug/m3)
IL-8
Pro
tein Salt Lake City
SeattlePhoenix
Sterling Forest
Hunter College
South Bronx
ZerbstHettstedt
HOX1 vs. Oil Refinery
y = 115.69x + 3.2957R2 = 0.9879
0.00
4.00
8.00
12.00
16.00
20.00
0.00 0.02 0.04 0.06 0.08 0.10 0.12
(ug/m3)
HO
X1
Seattle
Phoenix
Salt Lake City
HOX1 is correlated with Oil Refinery (r2=0.98)
IL-8 protein is correlated with coal combustion (r2=0.72) and residual oil combustion (r2=0.84)
IL-8 Protein vs. Residual Oil Combustion
y = 14.816x + 4.2156R2 = 0.8399
0.00
2.00
4.00
6.00
8.00
10.00
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35
Residual Oil Combustion (ug/m3)
IL-8
Pro
tein
Sterling Forest
Hunter CollegeSouth Bronx
AcknowledgementsAcknowledgements
CAPs StudiesDonald GrafJim SametTony HuangAndrew GhioAna Rappold
Asthmatic Panel StudiesKaren YeattsLucas NeasAna RappoldDave PedenNeil AlexisPeg Herbst
In Vitro StudiesLisa DaileyMike SchmittJoleen SoukupMary DanielsLiz Boykin
Animal and Human Instillation StudiesIan GilmourHaiYan TongSusanne BeckerNeil Alexis
Source Apportionment StudiesRachelle DuvallGary Norris
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