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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/EPAcosta.dan@epa.gov

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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The Respiratory System

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Weibel et al., 1981

Design Parameters of the Mammalian Lung

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Airway surface ~5% total SA

Air-blood barrier ~1:m

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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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Dynamics of Atmospheric PM

Wilson et al., 1977

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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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Emphysema (COPD)Emphysema (COPD)

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Emphysema: Loss of Elastic Recoil

Costa, 1985

Compliance Changes with Disease

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Volume and Diffusion Changes with Disease

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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

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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

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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 ==