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Pulmonary Toxicology: Animal Models and Their Utility in the Assessment of Toxic
Inhalants – ENVR430
Nov 16, 18, & 20, 2009
Dan Costa, Sc.D.Director of Air Research USEPAcosta.dan@epa.gov
919-541-2532
Lecture OutlineNovember 16, 2009• Basic lung biology – cross-species perspective• Principles of particle and gas entry into the lungNovember 18, 2009• Basic features of lung toxicity• Acute vs chronic outcomesNovember 20, 2009• Example: PM health issue today
• Witschi H. Chap 15 Toxic Responses of the Respiratory Tract, Casarett & Doull's Toxicology: The Basic Science of Poisons - 6th Ed. (2006).• Costa, DL. Chapter 28 Air Pollution Casarett & Doull's Toxicology: The Basic Science of Poisons - 7th Ed. (2007).• Pope CA, III, Dockery D.: 2006 Critical Review - Health Effects of Fine Particulate Air Pollution: Lines that Connect. JAWMA 56: 709-748, 2006.•Ghio AJ, Devlin RB. Inflammatory lung injury after bronchial instillation of air pollution particles. Am J Respir CritCare Med. Aug 15;164(4):704-8, 2001.• Dye J. et al. Acute pulmonary toxicity of particulate matter filter extracts in rats: coherence with epidemiologic studies in Utah Valley residents. Environ Health Perspect. 109 Suppl 3:395-403, 2001.
Leading Causes of Death in the
U.S. 2003
Accidents: Leading Cause of death <34 yrs of age
Distribution of Asthma in the U.S. Population
The Challenge of Air Pollution Health Sciences
• Exposures to air pollution are inherently “mixtures”• Exposures have temporal and spatial variability• Health-relevant airborne concentrations of most individual
pollutants are quite low - exception is ozone.• In most cases, the gradient of exposure concentrations from
those most exposed to least is small• Impact of confounding factors (e.g., tobacco smoke) is
substantial• The disease outcomes are not uniquely related to air
pollution• Host-pollution interaction may be critical (i.e., susceptibility)
HUMAN STUDIES: EPIDEMIOLOGY & CLINICAL STUDIES
• Should be used whenever possible– Right species– Exposures most relevant – real world
• Limitations of epidemiology – Exposure data are often weak – dosimetry even weaker / assumed– Difficult to establish cause-effect relationships:
• Confounding factors – cigarette smoke; diet; occupational• Effects of various pollutants may be similar• Sometimes the interval between exposure and effect is long
• Controlled Human Studies– Right species but ethical limitations – Study groups may not reflect the demographics– Controlled exposure may need extrapolation
LABORATORY ANIMAL STUDIES: ROLE IN INHALATION TOXICITY ASSESSMENTS
– Defined Subjects: Species; Strain; Age; Sex; Health Status– Control Exposure Conditions
• Specific Compound or Mixture• Well Characterized Atmosphere
– Concentration– Particle Size Distribution– Temperature & Humidity
– Allow Assessment of…• Exposure-dose Relationships (Toxicokinetics)• Exposure (Dose)-Response Relationships (Pharmacokinetics)
– Use of Multiple Species Increases Confidence in Extrapolation
IN VITRO STUDIES:ROLE IN INHALATION TOXICITY ASSESSMENTS
• Provides Basic Mechanistic Information Needed For Extrapolation and Fundamental Understanding – Isolated Perfused Lung or Heart
• Metabolism • Evaluation of Function
– Isolated Cells or Tissues (examples)• Epithelium – function, cytotoxicity, intracellular processes• Alveolar Macrophages - Phagocytic Capacity, Cytotoxicity
– Screening potential – “Toxicology for the 21st Century” (NAS)
The Lung is a Multifunctional Organ• Well-designed for its primary function (O2 CO2)
• All cardiac output passes through the lungs• Primary target for anything in inhaled air
• Maintains blood pH• Exocrine functions –
angiotension, biogenic amines
• Metabolic functions – P450’s• Excretory function –
CO, NH3, organic vapors
The Respiratory System
Weibel et al., 1981
Design Parameters of the Mammalian Lung
Airway surface ~5% total SA
Air-blood barrier ~1m
Comparative Nasal Airway Structure and Function
0.4816-19Volume(cm3)
NasalOronasalOronasalBreathing
Large50%
Moderate20-30%
Small<10%
OlfactoryEpithelialSurface Area
ComplexSimpleSimpleTurbinateAnatomy
RatMonkeyHuman
Harkema et al., The Nose Revisited: A Brief Review of the Comparative Structure,Function, and Toxicologic Pathology of the Nasal Epithelium Tox. Pathology, 34:252–269, 2006
Ventilation• Divisions of lung volume• Tidal Volume (Vt)• Frequency of breathing (f)• Vt x f = Minute Ventilation (VE)
• Transpulmonary pressure (PL)• Computation: Lung Resistance (RL) and Lung
Compliance (Cdyn)
28U.S. Environmental Protection AgencyOffice of Research and Development
PM is a complex mixture of solid, semi-volatile and aqueous materials of various sizes found in the air.
Mineral Fiber Natural Fiber
PollenAnthropogenic
Ultrafine PM (nano)
2.4 million = 1
Particulate Matter
29U.S. Environmental Protection AgencyOffice of Research and Development
NucleationMode
Ultrafine
AccumulationMode
CoarseMode
Fine (PM 2.5) 2.5
PM 10
Los AngelesEastern USMineralsSulfateAmmoniumNitrate
Organic CarbonElemental Carbon
Unknown
Airborne PM is itself a
complex mix of size and chemistry
Gases
Numbers and Surface Area of Different Sizes of Particles of Unit Density at a Mass Concentration of 10 µg/m3
241.22.5
60191.0
1201530.5
60019,1000.1
30162,400,0000.02
Particles Surface Area (µm2/cm3 )
Particle Number (per cm3)
Particle Diameter (µm)
CIIT Model PM Deposition Predictions
Asgharian, et al., 1999Asgharian, et al., 1999
Human Rat
Gases & Vapors
• Gases exist in gas phase at room temperature
• Vapors can coexist as gas and/or liquid at room temperature (have lower vapor pressure)
• Diffusion is a major factor in dispersion in the air
The higher the blood/air partition coefficient, the less desorbed on the ensuing breath –hence accumulation of the vapor by the body (blood and fat stores)
ASTM – E981-84 Mouse Irritancy Bioassay
Assesses the irritant potency of sensory irritants and segregates these responses from pulmonary irritants by examining changes in breathing frequency
Chronic Diseases• Chronic exposures – tobacco smoke (MS & ETS);
outdoor and indoor pollutants• Episodic (often chronic) – occupational (coal, silica
miners); air pollution• Single exposure w/ chronic outcome – accidental
releases (MIC – Bhopal India 1980); occupational; avocation (hobbies)
• Idiopathic disease (unknown origins)• Gene – Environment Interations - (1-AT; GSTM-1)
Emphysema (COPD)Emphysema (COPD)
Emphysema: Loss of Elastic Recoil
Costa, 1985
Functional Outcomes
Compliance Changes with Disease
Volume and Diffusion Changes with Disease
London's "killer smog" of 1952 was so thick that busses had to be escorted by men walking alongside with lanterns.This photo was taken around 10:30 AM.
The Problem: 20th Century Perspective
4-8000 deaths
Donora, PA – 1948
20-50 deaths
London Smog 1873
Houses of Parliament, Claude Monet
Ambient PM Derive from Diverse Sources
AgricultureFine particles can be emitted directly
or formed in the air from gases.
Power Plants
Africa
10/30/03 10:27:18 AM
http://www.firedetect.ssd.nesdis.noaa.gov/viewer.htm
General PM Sources and Composition
PM Composition Varies by Size Mode
““ClassicalClassical”” Air PollutionAir Pollution
Reducing SmogReducing Smog -- Acrid, smoky emissions of coal & fossil Acrid, smoky emissions of coal & fossil fuel combustion: Industry and domestic heatingfuel combustion: Industry and domestic heating•• Particulate matter (PM) Particulate matter (PM) -- complex soot; acidic & metal sulfates (SOcomplex soot; acidic & metal sulfates (SO44
==))•• Sulfur dioxide (SOSulfur dioxide (SO22 ))•• Carbon monoxide (CO)Carbon monoxide (CO)
Birmingham – 1972New York City – 1966
““ClassicalClassical”” Air PollutionAir Pollution
Oxidant SmogOxidant Smog -- EyeEye--irritating, haze of sunny suburbanized irritating, haze of sunny suburbanized cities: Automobile emissionscities: Automobile emissions•• Nitrogen oxides (NONitrogen oxides (NOxx))•• Volatile organic chemicals (Volatile organic chemicals (VOCsVOCs))•• Ozone (OOzone (O33) ) -- photochemical reaction product of NOphotochemical reaction product of NOxx and and VOCsVOCs•• Partially combusted organics (Partially combusted organics (PICsPICs) ) –– often mixed with often mixed with VOCsVOCs and Rx productsand Rx products•• Carbon monoxide (CO)Carbon monoxide (CO)
The Air of our Parents:The US Air Quality Management Program has come a long way
The Modern Dimensions of Air Pollution
• Most urban and suburban environments have oxidizing atmospheres comprised of ozone, primary and secondary particles, and a varying mix of copollutants– Regional pollutants (long range transport) – power industry– Transportation – auto, diesel – Fugitive and accidental releases – VOCs, PAHs, metal cpds. etc.
• Indoor Air exposure is ‘stealth’ and often underappreciated– Penetration of outdoor pollutants– Environmental tobacco smoke– Indoor sources – NOx, VOCs, – Biologicals – molds, dust and cockroach mites etc.
An International Problem
WHO (2002) estimates 2M premature deaths / year
Industrialized nations – show obvious improvementsAdvanced TechnologiesPublic educationGrowing Wealth
Developing nations - urbanization ►deterioration of AQ Population growthUncontrolled IndustrializationEnergy consumptionQuest for rapid wealth
Delhi, 2000
Beijing, 2001
PM Problems Persist Even in the U.S
Atlanta, 2003
75U.S. Environmental Protection AgencyOffice of Research and Development
Size mattersDockery, et al., 1993
Susceptibility
Schwartz et al., 1992
Life-Shortening
In 1997, the PM Epidemiology Was
Compelling…(but with many uncertainties)
Regarding Morbidity…
Rolling Stone Magazine
Science March 25, 2005 (Vol 307) pp.1858-1861
What is it about PM that Makes it a Significant Health Hazard?
• Is it the size and/or composition of PM?• How does PM lead to mortality?• Are there chronic effects?• Who is at risk?• Are there host factors? • Is there a source hierarchy?• Do co-pollutants have a role?• Are there other environmental factors?
HealthyAnimal
DiseasedAnimal
HealthyHuman
DiseasedHuman
Toxicological Paradigm for Use of Susceptible Disease Models
f’(x)f(x)
g(x)
g’(x)
• The human population is heterogeneous, hosting a spectrum of susceptibility factors: gene-environment (including epigenetic); disease; age; life-style…
• New tools open many possibilities
• Classical toxicology has largely focused on relatively young, homogeneous, healthy lab animals. Expanded use of genetic and other models…
• Studies in animal models offer tools for controlled investigation of specific “susceptibility” factors and has both acute and chronic applicability.
EpidemiologyAnimal
Toxicology
Mechanisms
Acidic particles
Transition metals
Organics
Hydrogen peroxide
Ultrafines
Bioaerosols
Dose distribution
Proposed Attribute “Hypotheses”
Sources of PM10 Pollution in the Utah Valley (1985-87)Geneva Steel 82% of industrial emissions when operating
47- 80% of total emissionsWood Burning 16%Road Dust 11%Diesel Fuel 7%Oil Combustion 7%
PM10
(g/
m3 )
0
25
50
75
100
125
150
1985 1986 1987 1988
PM10 Concentrations, Lindon Site
Steel MillClosed
PM10 Levels Correlate with Hospital Admissions for Pneumonia & Pleurisy; Bronchiolitis & Asthma
0
10
20
30
40
50
60
7080
1985 1986 1987 1988
Monthly Bronchiolitis & Asthma Hospital Admissions: All Ages
Steel MillClosed
Pope, Am J Public Health 79:623, 1989
Utah Valley filter extracts
40 mL
Pooled and Lyophilized
Aqueous extracts
Utah Valley filter extract metal analysis
1986 1987 19880.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
CopperZincIronLeadStrontiumArsenicNickelManganeseVanadium
Met
al (m
g) p
rese
nt in
filte
r ext
ract
s Utah Valley extract (12 TSP filters/year)
Dye et al, EHP 2001
Effects on Lung Permeability
1986 1987 19880
50
100
150
200
250
300 Saline(contralateral)Extract
Saline 1986 1987 19880
100
200
300
400
500
Prot
ein
(µg/
mL
BA
L fl
uid)
Rats Humans
Dye et al, EHP 2001 Ghio et al, 2001
(BAL Total Protein)
Prot
ein
(µg/
mL
BA
L fl
uid)Saline
Extract
0
100
200
300
400
500 g/ml250 g/ml100 g/ml0 g/ml
IL-6
(pg/
wel
l)Frampton et al., 1999
198619871988
Proinflammatory Marker
GeneticsMonogenic/polygenic-Species/strain-Gender
Environmental -Exposures-Infections-Nutrition
AgeDisease
Susceptibility – “No longer data outliers”
• Young versus old – excesses at both extremes• Asthma & other preexisting cardiopulmonary disease• Preexisting lung inflammation – infection• Diabetics – systemic inflammation / oxidant load• Genetics - gene-environment interactions (e.g.GST-M1)
Is there a common factor(s)?• Red-Ox imbalances• Loss of compensation / recoverability• Interactions with dosimetry• Multiple factors
Exposure Dose of an Air Pollutant
Homeostasis
Effect
Injury
Clinical Effect
Leve
l of B
iom
arke
r
Severe Effect
How Do We Describe Susceptibility?
Rodent Models of Cardiopulmonary Diseases
• Bronchitis / Emphysema / Fibrosis• Systemic hypertension• Aging / Cardiomyopathy• Allergic asthma• Pulmonary vasculitis / hypertension• Bacterial / viral infections• Genetic and transgenic disabilities
Bronchitis Models
• 200 ppm SO2 6 h/d, 5 d/wk, 6 wks LPS (Gordon & Harkema,’94)
Bronchitis
SO2
Sprague Dawley RatAir
Smith et al., 2002
Airway Cell Metaplasia in SH Rats Following 8-week ETS Exposure
(70-80 mg/m3 6 hrs/day 3 d/wk, 8 wks)
PM Deposition in the Bronchitis
Sweeney et al., 1995
NORMAL
RAT
SO2 BRONCHITIS
HUMAN
Bennett et al., personal communication
BRONCHITIS
Clarke et al.,1999
Impact of CAPS (PM2.5) in Bronchitic Rats
0100
300
500
700
900
Air CAPs Air
BA
LF P
rote
in (µ
g/m
l)
#~600ug/m 3 x 2 days
CAPs
Kodavanti et al., 2000
0
500
Air CAPs Air
BA
LF P
rote
in (µ
g/m
l)
#~500ug/m 3 x 3 days
CAPs
1500
2500
3500
4500
Boston RTP
PM Pulmonary-Cardiac Interactions
PM Exposure
DepositionClearance & ∆PFTs
VentilationCNS
InflammationEicosanoidsCyto/ChemokinesGrowth Factors Reactive O2 & N2
Proteases
PM Dissolution
Bio/Chemical Interactions (endotoxin, metals, PAH’s, reactive O2 & N2)
Chronic Impact Lung Remodeling Cancer
Allergenic / Immune Rxs
A “New” Dimension to Air Pollution
(Pope, 2000)
ELECTROCARDIOGRAPHIC ANALYSIS
SINGLE ELECTROCARDIOGRAPHIC WAVEFORMSINGLE ELECTROCARDIOGRAPHIC WAVEFORM
ADULT RATADULT RATHUMANHUMAN
R-aT
Lo Mid High
SO2 0.05 0.25 1.25 ppm
NO 0.05 0.25 1.25 ppm
NO2 0.04 0.20 1.00 ppm
CO 7.2 36.0 180.0 ppm
CH4 2.7 14.0 67.5 ppm
(NH4) 2SO4 ~25 ~125 ~625 ug/m3
Mid High
Normal rats (30wk) 2/20 1/20
Thrombogenic rats (16wk) 6/10 6/10
Hypertensive rats (32wk) 9/12 11/11
Anemic rats (16wk) 1/8 0/8
Hartroft et al., The Institute of Elect and Electronics Engineers, USA Publication #75CH1004-134-5:1, 1976
Susceptible Rats and Complex Atmospheres
Inhaled ROFA (15 mg/m3 6 hr/d, 3d)-induced hemolysis and BAL RBC’s
SH Rats Exhibit Greater Lung Injury Following PM Exposure
WKY-AIR
WKY-ROFA
SH-AIR
SH-ROFA
Kodavanti et al., 2000
772 MI patients
OR = 1.69 (1.13-2.34) for a 20 g/m3 increment in 24-hour PM2.5
Peters et al., 2001
-0.6-0.4-0.20.00.20.40.6
Healthy Subjects
Compromised Subjects
HF LF HF LF
Heart Rate Variability
Liao et al., 2000
Evidence that PM affects the Cardiovascular System
Watkinson et al., 1998
ECG Abnormalities and death in fly ash exposed hypertensive rats
Important Generic Questions to Ask of Animal Models
Given: The pathophysiology of human disease is variable….
At what pathophysiologic stage does susceptibility become evident? Is the response coherent with that of the human - esp. mechanistically? What are the implications of a genetically homogeneous host on the
response being studied? Do host attributes interact? (lung & heart; age; systemic factors)
Does the underlying responsiveness reflect altered dosimetry, a unique mechanism for the condition, or a loss of functional reserve?
Fine-Particulate Air Pollution and Life Expectancy in the United StatesPope, Ezzati, & Dockery, N Engl. J.Med. 2009;360:376-86
How do we know PM reductions actually have public health benefit?
Accountability
0 5 1 0 1 5 2 0 2 5 3 0 3 50 .7
0 .8
0 .9
1
1 .1
1 .2
1 .3
P
T W
HL
S
T
W
H
L
S
W
L
HS
TW
LH
S
T
Annual Average PM Concentration, µg/m3
Mor
talit
y R
isk
Rel
ativ
e to
Por
tage
, WI
Follow-up to the Harvard Six Cities Study Indicates Reduced Air Pollution Results in
Lowered Health Risks.
Laden et al, 2006
0
1
2
3
4
5
0 2 4 6 8 10 12 14 16Reduction inPM2.5 (g/m3)
L
ife E
xpec
tanc
yLife Expectancy vs PM2.5, 1980-2000
Slope +.61 yr/10 g/m3
71
72
73
74
75
76
77
78
79
80
81
5 10 15 20 25 30 35PM2.5 (g/m3)
Life
Exp
ecta
ncy
Slope -2.1 yr/10 g/m3
EPA NAAQS
Life Expectancy vs PM2.5, 1997-2001
106U.S. Environmental Protection AgencyOffice of Research and Development
• What does this finding mean relative to other life-expectancy factors?– E.g., relative to the impact of obesity?
PM Slope +.61 yr/10 g/m3
Obesity
Olshansky et al., NEJM 352:11 (2005)
Pope et al., NEJM 360:376 (2009)
What do we understand about PM risk and biologic plausibility?
• Attributes of PM composition/size (perhaps in concert with copollutants?) drives the health responses.
• “Dose” is fundamental to response and often goes un-appreciated in the context of susceptibility.
• Host attributes may be the primary determinant of risk.
• Impacts may not necessarily be greater than in healthy individuals, but may be additive with pre-existing factors or simply may overwhelm reserve and compensatory capacities.
• Accountability – science is showing that regulatory approaches are making a difference; will there be a “climate penalty”?
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