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Pulmonary Toxicology: Animal Models and Their Utility in the Assessment of Toxic
Inhalants – ENVR430
Nov. 6, 8, &10, 2006
Dan Costa, Sc.D.Pulmonary Toxicology BranchExperimental Toxicology Division NHEERL/[email protected]
541-2532
Lecture OutlineNovember 6, 2006• Basic lung biology – cross-species perspective• Principles of particle and gas entry into the lungNovember 8, 2006• Basic features of lung toxicity• Acute vs chronic outcomesNovember 10, 2006• Example: PM health issue today• Example: Air Toxics – risk assessment
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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
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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 (TOXICOKINETIC STUDIES)• EXPOSURE (DOSE)-RESPONSE RELATIONSHIPS
– USE OF MULTIPLE SPECIES INCREASES CONFIDENCE IN EXTRAPOLATIONS TO MAN
IN VITRO STUDIES:ROLE IN INHALATION TOXICITY ASSESSMENTS
• Provides basic mechanistic information needed for extrapolation and fundamental understanding – e.g. ALVEOLAR MACROPHAGES
• PHAGOCYTIC CAPACITY• CYTOTOXICITY
– OTHER ISOLATED CELLS AND TISSUES (e.g. epithelium)• CYTOTOXICITY & METABOLISM
– ISOLATED PERFUSED LUNG OR HEART• METABOLISM • EVALUATION OF FUNCTION
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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
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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)
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Particulate Matter
• Size• Shape • Composition• Solid/liquid• Primary sources • Secondary products
Mineral FiberMineral Fiber Natural FiberNatural Fiber
PollenPollenAnthropogenic PMAnthropogenic PM
10/30/03 10:27:18 AM
http://www.firedetect.ssd.nesdis.noaa.gov/viewer.htm
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CIIT Model PM Deposition PredictionsCIIT Model PM Deposition Predictions
AsgharianAsgharian, et al., 1999, et al., 1999
HumanHuman RatRat
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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
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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
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Chronic Diseases
• Chronic exposure – tobacco smoke• Episodic – coal; silica (miners); air pollution• Single exposure w/ chronic outcome – silica; MIC• Idiopathic disease or other risk elements - "1-AT
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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 ProblemThe Problem: : A Historical A Historical PerspectivePerspective
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Classical Air PollutionClassical Air Pollution
Reducing SmogReducing Smog -- Acrid, Acrid, smokeysmokey emissions of coal & fossil fuel emissions of coal & fossil fuel combustion: Industry and domestic heatingcombustion: Industry and domestic heating
Sulfur dioxide (SOSulfur dioxide (SO22 ))Particulate matter (PM) Particulate matter (PM) -- complex soot; acidic & metal sulfates (SOcomplex soot; acidic & metal sulfates (SO44
==))Carbon monoxide (CO)Carbon monoxide (CO)
Oxidant SmogOxidant Smog -- EyeEye--irritating, haze of sunny suburbanized cities: irritating, haze of sunny suburbanized cities: AutomobileAutomobile
Nitrogen oxides (Nitrogen oxides (NONOxx))Partially combusted organics (PICS)Partially combusted organics (PICS)Ozone (OOzone (O33) ) -- photochemical reaction product of photochemical reaction product of NONOxx and and PICsPICsCarbon monoxide (CO)Carbon monoxide (CO)
Modern Dimensions of Air PollutionModern Dimensions of Air Pollution
Air ToxicsAir ToxicsFugitive & Fugitive & accidentialaccidential industrial releasesindustrial releases
Volatile Organic Compounds (Volatile Organic Compounds (VOCsVOCs))Polycyclic Aromatic Hydrocarbons (Polycyclic Aromatic Hydrocarbons (PAHsPAHs))Metal compoundsMetal compoundsMineral dustsMineral dusts
Indoor AirIndoor AirStealth, underestimated source of personal exposureStealth, underestimated source of personal exposure
Penetration of outdoor air pollutantsPenetration of outdoor air pollutantsEnvironmental Tobacco Smoke (ETS)Environmental Tobacco Smoke (ETS)NOxNOx -- cooking and heating sourcescooking and heating sourcesVOCsVOCs -- household productshousehold productsBiologicalsBiologicals -- insect dusts, molds, fungiinsect dusts, molds, fungi
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Interaction of Host and EnvironmentInteraction of Host and EnvironmentRegional exposure Regional exposure -- site monitorsite monitor
vs. Personal exposure vs. Personal exposure -- integrated outdoor / indoorintegrated outdoor / indoorvs. Dose vs. Dose -- targettarget
Exposure ScenarioExposure ScenarioAcute responsesAcute responsesAdaptationAdaptationChronic effects / diseaseChronic effects / disease
SusceptibilitiesSusceptibilitiesGenetics / gender / race / ageGenetics / gender / race / ageDiet / behaviorsDiet / behaviors
An International ProblemAn International Problem
Industrialized nationsIndustrialized nations -- obvious improvementsobvious improvementsTechnologyTechnologyPublic educationPublic educationWealthWealth
Developing nationsDeveloping nations -- urbanization and deterioration of urbanization and deterioration of air qualityair quality
Population growthPopulation growthEnergy consumptionEnergy consumption
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From the Plane out of Dehli
Smaller PM sizes relate better to the observed effect.
Dockery, et al., 1993
Cardiopulmonary diseases predispose to PM effects.
Schwartz et al., 1992
Regarding Mortality…
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Association Between Long Term Exposure to PM and Mortality
Dockery et al., 1993
Regarding Morbidity…
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• Classical toxicology has largely focused on relatively young, homogeneous, healthy lab animals.
• The human population is heterogeneous, hosting a spectrum of susceptibility factors.
• Studies in animal models offer tools for controlled investigation of specific “susceptibility” factors and has both acute and chronic applicability.
EpidemiologyAnimal
Toxicology
Mechanisms
Identifying Those Affected: A Needle In A Haystack?
Given:• 50 natural deaths / day for Philadelphia County: 1.6 million people
Assume: • A 3-day smog episode envelops the city • The PM10 concentration increases from 30 to 130 ug/m3
• A relative risk of 1.05 per 100 ug/m3 change in [PM]
Result:• 2.5 extra deaths per day or about an excess of 8 deaths among 158
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HealthyAnimal
DiseasedAnimal
HealthyHuman
DiseasedHuman
Toxicological Paradigm for Use of Susceptible Disease Models
f’(x)f(x)
g(x)
g’(x)
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%
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PM10
(μg/
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
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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
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GeneticsMonogenic/polygenic-Species/strain-Gender
Environmental -Exposures-Infections-Nutrition
AgeDisease
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?
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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
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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
BALF
Pro
tein
(µg/
ml)
#~600ug/m3 x 2 days
CAPs
Kodavanti et al., 2000
0
500
Air CAPs Air
BALF
Pro
tein
(µg/
ml)
#~500ug/m3 x 3 days
CAPs
1500
2500
3500
4500
Boston RTP
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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)
EELECTROCARDIOGRAPHIC LECTROCARDIOGRAPHIC AANALYSISNALYSIS
SINGLE ELECTROCARDIOGRAPHIC WAVEFORMSINGLE ELECTROCARDIOGRAPHIC WAVEFORM
ADULT RATADULT RATHUMANHUMAN
R-aT
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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 Electrical 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
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772 MI patients
OR = 1.69 (1.13-2.34) for a 20 μg/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
Designer Mice
EC-SOD overexpressing mouse exposed to ROFA PM shows a strong role of oxidant toxicity
Ghio et al., 2002
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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 theresponse 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?
NAS Risk AssessmentNAS Risk Assessment ParadigmParadigm
DoseDose--ResponseResponseAssessmentAssessment
Hazard Hazard IdentificationIdentification
RiskRiskCharacterizationCharacterization
ExposureExposureAssessmentAssessment
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EPA Risk Assessment Guidelines EPA Risk Assessment Guidelines for:for:CancerCancer
Developmental ToxicityDevelopmental ToxicityReproductive ToxicityReproductive Toxicity
Inhalation ToxicityInhalation ToxicityNeurotoxicityNeurotoxicity
Chemical MixturesChemical MixturesExposureExposure
EcosystemsEcosystems
Inhalation Reference Inhalation Reference Concentration (Concentration (RfCRfC))
ororOral Reference Dose (Oral Reference Dose (RfDRfD))
RfCRfC/D = /D = NOAELNOAELUFUF
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Use of Uncertainty Factors in Deriving Inhalation Reference Concentration (RfC)
H= Human to sensitive human
Use a 10-fold factor when extrapolating from valid experimental results using prolonged exposure to average healthy humans. Used to account for sensitivity/susceptibility
Uncertainty factor Process considered
PharmacokineticsSensitivityDifferences in mass (e.g. children)Concomitant exposuresActivity patterns
Use of Uncertainty Factors in Deriving Inhalation Reference Concentration (RfC)
A= Animal to human
Use a 3-fold factor when extrapolating from animals experiments to humans. Can be extended to 10 with judgement.
Uncertainty factor Process considered
PharmacokineticsRelevance of animal modelsSpecies sensitivity
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Use of Uncertainty Factors in Deriving Inhalation Reference Concentration (RfC)
S= Subchronic to chronic
Use a 10-fold factor when extrapolating from less than chronic animal experiments to humans. Can be extended to 10 with judgment.
Uncertainty factor Process considered
Accumulation/cumulative damageSeverity of effectRecoveryDuration of StudyConsistency of effect with duration
Use of Uncertainty Factors in Deriving Inhalation Reference Concentration (RfC)
L= LOAEL[HEC] to NOAELHEC]
Use a 10-fold factor when deriving an RfC from a LOAEL instead of a NOAEL.
Uncertainty factor Process considered
SeveritySlope of dose responseTrend, consistency of effectRelationship of endpointsFunctional vs histopath
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Use of Uncertainty Factors in Deriving Inhalation Reference Concentration (RfC)
D= Incomplete data base
Use a 10-fold factor when data are incomplete. e.g. lack of replicate studies, sparse endpoints
Uncertainty factor Process considered
Quality of critical studyData gapsPower of critical studyGender
Use of Uncertainty Factors in Deriving Inhalation Reference Concentration (RfC)
MF= Modifying factor
Use professional judgment to determine whether another uncertainty factor (<10) is needed. Magnitude depends on other scientific uncertainties not treated in other decisions. e.g. number of animals, quality of exposure characterization
Uncertainty factor
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RfCRfC = = NOAELNOAELadjustedadjusted
UFUFTotalTotal x MFx MFwhere:where:
NOAEL = No Observed Adverse Effect LevelNOAEL = No Observed Adverse Effect Level(adjusted for duration, ventilation rate, (adjusted for duration, ventilation rate, dosimetrydosimetry))
UFUFTotalTotal = Total Uncertainty Factor:= Total Uncertainty Factor:
Susceptible subpopulationsSusceptible subpopulations
Extrapolation from animals to humansExtrapolation from animals to humans
SubchronicSubchronic to chronicto chronic
Data base adequacyData base adequacy
MF = Modifying Factor (special considerations)MF = Modifying Factor (special considerations)
RfCRfC for MTBEfor MTBE
259 mg/m259 mg/m33
1010Subpop.Subpop. x 10x 10Interspp.Interspp.
= 3 mg/m= 3 mg/m33 (0.8 (0.8 ppmppm))
RfCRfC ==