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