Markers of Pulmonary Toxicity

Gary E. Hatch, Ph.D.Pulmonary Toxicology BranchExperimental Toxicology Division

Email: Hatch.Gary@epa.govPhone: 919-541-2658October 3, 2006

IMPORTANT TERMSBiological marker or Biomarker:

'Indicator signaling events in a biological system'Exposure markersEffect markersSusceptibility markers

Non-invasive: Measurable in blood or urine

Homologous: Applicable across species

Harmonized: Markers predictive of both cancer and non-cancer effects

Mechanism of action: The complete sequence of biological events --> toxic effect

Mode of action: Less-detailed than 'mechanism'

Source: Human Health Research Strategy, U.S. E.P.A., ORD, 2003

DESIRABLE QUALITIES in a Marker of Toxicity

- Rapid, simple, easy- Predictive of human pathology- Non-invasive- Sensitive- Validated scientifically- Accepted by scientists and public

POSSIBLE TEST QUESTIONS

- Definitions

- What are the desirable qualities of biomarkers oftoxicity?

- Choose one marker and evaluate its strengthsand weaknesses.

- Outline a protocol for an experiment that couldeither 1) remove a weakness or reduce an uncertainty in an existing marker or 2) find a new marker.

READING MATERIAL

Examples of experiments on markers:

Finding a new marker: intratracheal instillation with bacterial infectivity

Linking rat to human: ozone dose

Linking acute to chronic: phosgene toxicity

EXAMPLES: MARKERS OF PULMONARY TOXICITY

Gross appearance of the lungClinical signs: whole bodyMicroscopic changesLung physiology changesLung and lung lavage fluid

CellularBiochemicalGenomic

Markers of DoseMarkers of SusceptibilityBacterial infectivity markerNon-invasive markers

Blood and urine

Normal Lung

Emphysema

Fibrosis

GROSS APPEARANCEof the LUNG

HUMANLUNG

Fibrosis, cut away

GROSSAPPEARANCEof the LUNG

ASBESTOSISin theHUMAN LUNG

THE NEED FOR MARKERS To predict and prevent bad experiences, such as:

Crystalline silica: miners silicosis

Tobacco smoke: cancer, emphysema, COPD

Asbestos: cancer

Beryllium metal: fibrosis

Paraquat (herbicide) : edema, fibrosis

How were these agents first established as respiratory toxicants?

Human Epidemiology - Polycyclic aromatic hydrocarbons (roofing tar) - Airborne particulate matter - Tobacco smoke - Asbestos

Animal studies - Ozone - Aldehydes - Carbon tetrachloride - Nitrogen oxides

OZONE NITROGEN DIOXIDE PHOSGENE

CHLORINE CHLORINE DIOXIDE

OZONEOZONE NITROGEN DIOXIDENITROGEN DIOXIDE PHOSGENEPHOSGENE

CHLORINECHLORINE CHLORINE DIOXIDECHLORINE DIOXIDECARBON

TETRACHLORIDECARBON

TETRACHLORIDE

Prototype Oxidant Pollutant Molecules

INFARED THERMAL CAMERA PHOTOS OFA MOUSE EXPOSED TO PHOSGENE

Control air exposed Same mouse ~3 minutes in subclinical level of phosgene

CLINICAL SIGNS: Whole Body

Mouse Strains Differ in Ability to Lower Core BodyTemperature During O3 Exposure (2.0 ppm)

0 10 20 30 40 50 60 70 80 90 100 110 120 130 14026

27

28

29

30

31

32

33

34

35

36

37

38

O3 Exposure

B6

C3

- 253 58 min C

- 432 65 min C

Area

O3

Tem

pe

ratu

re, C

temp1.pzmSource: R. Slade, W.P. Watkinson, G.E. Hatch, 1997, Am. J. Physiol., 272:L73.

Time, min.

CLINICAL SIGNS: Whole Body

INVASIVE CORE TEMPERATUREUSING PERITONEAL RADIO-TELIMETER

FEV1 - "FORCED EXPIRATORY VOLUME IN ONE SECOND"

Measures "the inability to take a deep breath."

Affected by asthma, aging, irritant exposure, COPD

Might sometimes be interpreted as a protective response.

LUNG PHYSIOLOGY CHANGES: Human

LUNG PHYSIOLOGY CHANGES: HUMAN FEV1, AGING, SMOKING

Assessment of Airway Obstruction in Unanesthetized Unrestrained Rodents (Buxco)

analysis software

plethysmographpreamplifier

aerosol chamber control unit

flow regulator

nebulizer

Courtesy: Steve Gavett, EPA, 541-2555

LUNG PHYSIOLOGY CHANGES: Animal: "PenH"

Enhanced Pause (PenH)

Te - RTRT

PEFPIF

PenH =

-1

-0.5

0

0.5

1

exp

iratio

nin

spir

atio

nBOX PRESSURE (equiv. to FLOW (dV/dt))

Te

RT(35% VT)

PEF

PIF

Ti

Enhanced Pause (PenH)

Te - RTRT

PEFPIF

PenH = Te - RT

RTPEFPIF

PenH =

-1

-0.5

0

0.5

1

exp

iratio

nin

spir

atio

nBOX PRESSURE (equiv. to FLOW (dV/dt))

-1

-0.5

0

0.5

1

exp

iratio

nin

spir

atio

nBOX PRESSURE (equiv. to FLOW (dV/dt))

Te

RT(35% VT)

PEF

PIF

Ti

Methacholine Challenge

0.0

2.0

4.0

6.0

8.0

10.0

0 5 10 15 20 25 30 35 40 45

Time (min)

Pen

H

Base Saline 4 mg/mL

8 mg/mL

16 mg/mL32 mg/mL

Saline, Mch aerosol: 1 min neulizer.

Methacholine Challenge

0.0

2.0

4.0

6.0

8.0

10.0

0 5 10 15 20 25 30 35 40 45

Time (min)

Pen

H

Base Saline 4 mg/mL

8 mg/mL

16 mg/mL32 mg/mL

Saline, Mch aerosol: 1 min neulizer.

0.0

2.0

4.0

6.0

8.0

10.0

0 5 10 15 20 25 30 35 40 45

Time (min)

Pen

H

Base Saline 4 mg/mL

8 mg/mL

16 mg/mL32 mg/mL

0.0

2.0

4.0

6.0

8.0

10.0

0 5 10 15 20 25 30 35 40 45

0.0

2.0

4.0

6.0

8.0

10.0

0 5 10 15 20 25 30 35 40 45

Time (min)

Pen

H

Base Saline 4 mg/mL

8 mg/mL

16 mg/mL32 mg/mL

Saline, Mch aerosol: 1 min neulizer.

Wheel Running Activity of Mice Exposed to Ozone

Tepper et al, T.A.P. 64: 317, 1982

Night-time wheel runningDecreased as ozone concentrations increased

Black = ozone exposure

PERITONEAL IRRITANCY ASSAY

Sensory irritation induced by metal containing fly ash(Hatch et al, 1982 Fundam. Appl. Toxicol. 2:77)

CdSO4 150 ug /kg i.p.

Mice injected I.P. with and irritating test material develop visible indentationsabove the hind legs.

CLINICAL SIGNS: Whole Body

Control

CONTROL LUNG TISSUE SECTION, 10 X, trichrome stain for collagen

MICROSCOPIC CHANGES: Light Microscopy of rat lung

12 WEEKS EXPOSURE of RATS to 0.2 ppm phosgene (20x) green = collagen

MICROSCOPIC CHANGES: Light Microscopy

Trichrome Stain

Biochemical Determination of Hydroxyproline

LUNG BIOCHEMICAL MARKERS: Chronic effect: FIBROSIS

PhosgeneDose

Kodavanti et al, 1992 4 weeks 12 weeks 12 weeks + recovery

Normal rat terminal broncheolar epithelium

MICROSCOPIC CHANGES: Scanning Electron Microscopy

Rat terminal airway epithelium 1 day post exposure to1 ppm ozone for 8 hr.

Paige and Plopper, Air Pollution and Health, 1999 p.539

Normal cell Mild injury Severe injury

"Comet Assay" for detection of single cell DNA strand breaks

Cells are suspended in agar and electrophoresed in the presence of NaOH then stained for DNA. Broken DNA strands migrate into the gel.

[[Ref.

MICROSCOPIC CHANGES

17 day pregnant mouse mammary glands of the p53Arg-Leu transgenic (left) and a control (right) mouse stained using the TUNEL assay indicate apoptotic cells. Only one apoptotic cell is present in the upper right hand corner of the control, but approximately 20% of the cells stained positively in the line expressing the p53Arg-Leu transgene.

From: B Li, FS Kittrell, D Medina, JM Rosen (1995). Delay of dimethylbenz(a)anthracene-induced mammary tumorigenesis in transgenic mice by apoptosis induced by an unusual mutant p53 protein. Mol. Carcinogenesis 14:75-83.

HISTOCHEMICAL STAINING FOR APOPTOSIS

MICROSCOPIC CHANGES

Light micrographs of the centriacinar regions of the lung with immunohistochemical identification of proliferating cells. (A) filtered air-exposed mouse, (D) ozone-exposed mouse. Proliferating cells are recognized as densely BrdU labeling-positive (seen as dots centering around the bronchiolar-alveolar duct junctions). Scale bar = 100 µm.

From: M Yu, X Zheng, H Witschi, K E Pinkerton. The Role of Interleukin-6 in Pulmonary Inflammation and Injury Induced by Exposure to Environmental Air Pollutants. Toxicological Sciences 68, 488-497 (2002)

VISUALIZING PROLIFERATING CELLS: BRDU labeling

MICROSCOPIC CHANGES: Dividing cells -- Repair

GENOMIC MARKERS: Whole lung homogenate

Results from a 2 hr ozone exposure (5 ppm, 2 hr) Clontech array of 588 genes. Lung homogenate (~40 cell types).

67 increased > 2x76 decreased Many oncogenes activated

OF NOTE:- Insulin like growth factor- VEGF receptor- c-fos, c-met, c-jun, ras

Nadadur, Hatch., 2005

0 24 48 72

200

400

600

800

1000

1200

1400

1600

1800

2000

Hours Post Exposure

BA

L P

rote

in,

g/m

l

Exposure: 4 hr

1.0 ppm

0.5 ppm

0.25 ppm

Air

*

**

*

*

*

**

*

PHOSGENE DOSE RESPONSE in Rats

TOXIC EFFECT MARKER: Lung lavage fluid protein concentration

Hatch et al, 2001. Toxicol Indust. Health 17: 285-293

LUNG LAVAGE MARKERS:Inflammatory cell changes

macrophages

eosinophils

Guinea Pig Alveolar Macrophages

Time Course Dose Response: Rat lung lavage neutrophils following phosgene inhalation

0 24 48 72

20

40

60

80

100

Hours post ExposureExposure: 4 hr

1.0 ppm

0.5 ppm

0.25 ppm

Air

% N

EU

TR

OP

HIL

SLUNG LAVAGE FLUID TOXICITY MARKER:

Inflammatory cell influx

Hatch et al, 2001. Toxicol Indust. Health 17: 285-293

Visualization of the reaction product of inhaled labeled phosgene on the surface of the rat nasal cavity. A transverse section of the nasal turbinate is shown. The dark line on most of the airway surface (except in the pocket) is carbon-14 containing reaction product of carbon-14 labeled phosgene (Cl2

14C=O). Rats were exposed for 3 minutes to 1 ppm of the labeled phosgene and killed immediately after exposure. (Hatch, G.E. and Morgan, K.).

AUTORADIOGRAPHY OF PHOSGENE REACTION PRODUCT AT THE NASAL EPITHELIAL SURFACE

MARKERS of INTERNAL DOSE: Labeled phosgene

GENERATION OF 18O3 FOR INHALATION EXPOSURES

5% 18O2 in argon Inhalation Chamber

18O3

Ozone Generator Air

BIOMARKERS of INTERNAL DOSE: Oxygen-18 labeled ozone

0

25

50

BAL High Speed Pellet

BAL High Speed Supernatant

Bronchoalveolar lavage cells

Lavaged Lung

ExercisingHuman

(0.4 ppm,2 hours)

RestingF344 Rat(0.4 ppm,2 hours)

RestingF344 Rat(2.0 ppm,2 hours)

Rats underestimate human ozone dose: 18O incorporation into lung following 18o3

Ex

ces

s 1

8O

, ug

/ g

dry

Hatch et al, 1994 Am J. Respir. Crit Care Med 150: 676

BIOMARKERS OF INTERNAL DOSE: Linking animal to human

ANTIOXIDANT SUBSTANCES in LUNG and LINING FLUID

Ascorbate Urate Glutathione

Alpha-tocopherol

MARKERS OF SUSCEPTIBILITY

MARKERS OF SUSCEPTIBILITY: Species comparisons

Bacteria

diet,age,genetics

Lung

“stress”

virusespollutants

Clearance

Morbidity

Mortality

Effect spectrum

FEATURES Defense Occurs at Air-Liquid Interface Phagocyte mediators are important Extracellular defenses are important

BACTERIAL INFECTIVITY ANIMAL MODEL

Pollutants increase susceptibility to bacterial infection

MOUSE BACTERIAL INFECTIVITY MODEL

CD-1 Mouse

3 Hr Exposureto Air orPollutant

20 Min.Exposure

to StreptococcusAerosol

14 DayExamination

Period

All anim als get sick0 - 10% m orta lity in a ir-exposed

IMMUNE BIOMARKERS OF PULMONARY TOXICITY

Gardner, et al 1982

Streptococcus Infectivity vs. Lung Permeability

0.01 0.10 1.00 10.00 100.000

10

20

30

40

50

60

70

80

90

100

0.0

0.5

1.0

1.5

2.0

2.5

Phosgene

NO2

NO2% E

xc

es

s M

ort

ali

ty D

ue

to

In

fec

tio

nB

AL

Flu

id P

rote

in, m

g/m

l

Approximate Amount in Lungs, g / mouse

Ozone

Phosgene

Ozone

COMPARISON OF TWO MARKERS: Sensitivity of dose response

Fatty acid: Arachidonate radical

Type III Isoprostane, measured by GCMS or ELISA

NON-INVASIVE MARKER OF OXIDATIVE STRESS: ISOPROSTANE

OXIDATION

Post-exposure time, hr

10 20 30 40 50 60 70 80 90 100-5

0

5

10

15

20

25

Urine from untreated rats

18O in urine of rats exposed to 5.0 ppm 18O3 (2 hr)

EX

CE

SS

18O

u

g /

g d

ry

Urine from 18O3 exposed

NON-INVASIVE MARKER: Urinary products of lung repair from labeled ozone

SUMMARY: MARKERS OF PULMONARY TOXICITY

Gross appearance of the lungClinical signs: whole bodyMicroscopic changesLung physiology changesLung and lung lavage fluid

CellularBiochemicalGenomic

Markers of DoseMarkers of SusceptibilityBacterial infectivity markerNon-invasive markers

Blood and urine

DESIRABLE QUALITIES in a Marker of Toxicity

- Rapid, simple, easy- Predictive of human pathology- Non-invasive- Sensitive- Validated scientifically- Accepted by scientists and public

SUMMARY