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Markers of Pulmonary Toxicity. Gary E. Hatch, Ph.D. Pulmonary Toxicology Branch Experimental Toxicology Division Email: [email protected] Phone: 919-541-2658 October 3, 2006. IMPORTANT TERMS. Biological marker or Biomarker : 'Indicator signaling events in a biological system'

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Markers of

Pulmonary Toxicity

Gary E. Hatch, Ph.D.

Pulmonary Toxicology Branch

Experimental Toxicology Division

Email: [email protected]

Phone: 919-541-2658

October 3, 2006


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IMPORTANT TERMS

Biological marker or Biomarker:

'Indicator signaling events in a biological system'

Exposure markers

Effect markers

Susceptibility 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


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DESIRABLE QUALITIES

in a Marker of Toxicity

- Rapid, simple, easy

- Predictive of human pathology

- Non-invasive

- Sensitive

- Validated scientifically

- Accepted by scientists and public


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POSSIBLE TEST QUESTIONS

- Definitions

- What are the desirable qualities of biomarkers of

toxicity?

- Choose one marker and evaluate its strengths

and weaknesses.

- Outline a protocol for an experiment that could

either 1) remove a weakness or reduce an

uncertainty in an existing marker or

2) find a new marker.


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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


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EXAMPLES: MARKERS OF PULMONARY TOXICITY

Gross appearance of the lung

Clinical signs: whole body

Microscopic changes

Lung physiology changes

Lung and lung lavage fluid

Cellular

Biochemical

Genomic

Markers of Dose

Markers of Susceptibility

Bacterial infectivity marker

Non-invasive markers

Blood and urine


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GROSS

APPEARANCE

of the LUNG

HUMAN

LUNG

Normal Lung

Emphysema

Fibrosis, cut away

Fibrosis


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GROSS

APPEARANCE

of the LUNG

ASBESTOSIS

in the

HUMAN LUNG


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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


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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


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NITROGEN DIOXIDE

OZONE

PHOSGENE

CARBON

CARBON

TETRACHLORIDE

TETRACHLORIDE

CHLORINE DIOXIDE

CHLORINE

Prototype Oxidant Pollutant Molecules

NITROGEN DIOXIDE

NITROGEN DIOXIDE

OZONE

OZONE

PHOSGENE

PHOSGENE

CHLORINE DIOXIDE

CHLORINE DIOXIDE

CHLORINE

CHLORINE


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CLINICAL SIGNS: Whole Body

INFARED THERMAL CAMERA PHOTOS OF

A MOUSE EXPOSED TO PHOSGENE

Control air exposed

Same mouse ~3 minutes in

subclinical level of phosgene


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CLINICAL SIGNS: Whole Body

INVASIVE CORE TEMPERATURE

USING PERITONEAL RADIO-TELIMETER


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LUNG PHYSIOLOGY CHANGES: Human

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.


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LUNG PHYSIOLOGY CHANGES: HUMAN FEV1, AGING, SMOKING


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LUNG PHYSIOLOGY CHANGES: Animal: "PenH"

Assessment of Airway Obstruction in

Unanesthetized Unrestrained Rodents (Buxco)

analysis software

aerosol chamber

control unit

nebulizer

plethysmograph

preamplifier

flow regulator

Courtesy: Steve Gavett, EPA, 541-2555


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Wheel Running Activity of Mice Exposed to Ozone

Night-time wheel running

Decreased as ozone

concentrations increased

Black = ozone exposure

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


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CLINICAL SIGNS: Whole Body

PERITONEAL IRRITANCY ASSAY

Control

CdSO4 150 ug /kg i.p.

Mice injected I.P. with and irritating test material develop visible indentations

above the hind legs.

Sensory irritation induced by metal containing fly ash

(Hatch et al, 1982 Fundam. Appl. Toxicol. 2:77)


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MICROSCOPIC CHANGES: Light Microscopy of rat lung

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


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MICROSCOPIC CHANGES: Light Microscopy

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

Trichrome Stain


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LUNG BIOCHEMICAL MARKERS: Chronic effect: FIBROSIS

Biochemical Determination of Hydroxyproline

Phosgene

Dose

12 weeks

12 weeks

+ recovery

4 weeks

Kodavanti et al, 1992


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MICROSCOPIC CHANGES: Scanning Electron Microscopy

Rat terminal airway epithelium

1 day post exposure to

1 ppm ozone for 8 hr.

Normal rat terminal broncheolar epithelium

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


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MICROSCOPIC CHANGES

"Comet Assay" for detection

of single cell DNA strand breaks

Normal cell

Severe injury

Mild injury

Cells are suspended in agar and electrophoresed in the presence of

NaOH then stained for DNA. Broken DNA strands migrate into the gel.

[[Ref.


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MICROSCOPIC CHANGES

HISTOCHEMICAL STAINING FOR APOPTOSIS

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.


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MICROSCOPIC CHANGES: Dividing cells -- Repair

VISUALIZING PROLIFERATING CELLS: BRDU labeling

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)


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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 > 2x

76 decreased

Many oncogenes activated

OF NOTE:

- Insulin like growth factor

- VEGF receptor

- c-fos, c-met, c-jun, ras

Nadadur, Hatch., 2005


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1.0 ppm

0.5 ppm

0.25 ppm

Air

Exposure: 4 hr

TOXIC EFFECT MARKER: Lung lavage fluid protein

concentration

PHOSGENE DOSE RESPONSE in Rats

2000

*

1800

*

1600

*

*

1400

*

1200

BAL Protein, mg/ml

*

1000

*

*

*

800

600

400

200

72

0

24

48

Hours Post Exposure

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


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LUNG LAVAGE MARKERS:

Inflammatory cell changes

Guinea Pig Alveolar Macrophages

eosinophils

macrophages


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100

Time Course Dose Response: Rat lung lavage neutrophils

following phosgene inhalation

80

60

1.0 ppm

% NEUTROPHILS

40

0.5 ppm

20

0.25 ppm

0

72

24

48

Hours post Exposure

Air

Exposure: 4 hr

LUNG LAVAGE FLUID TOXICITY MARKER:

Inflammatory cell influx

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


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MARKERS of INTERNAL DOSE: Labeled phosgene

AUTORADIOGRAPHY OF PHOSGENE REACTION PRODUCT AT THE

NASAL EPITHELIAL SURFACE

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 (Cl214C=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.).


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Ozone Generator

BIOMARKERS of INTERNAL DOSE: Oxygen-18 labeled ozone

GENERATION OF 18O3 FOR INHALATION EXPOSURES

Air

5% 18O2 in argon

Inhalation

Chamber

18O3


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Resting

Exercising

Resting

F344 Rat

Human

F344 Rat

(0.4 ppm,

(0.4 ppm,

(2.0 ppm,

2 hours)

2 hours)

2 hours)

BIOMARKERS OF INTERNAL DOSE: Linking animal to human

Rats underestimate human ozone dose:

18O incorporation into lung following 18o3

50

Bronchoalveolar lavage cells

BAL High Speed Pellet

BAL High Speed Supernatant

Lavaged Lung

Excess 18O, ug / g dry

25

0

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


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MARKERS OF SUSCEPTIBILITY

ANTIOXIDANT SUBSTANCES in LUNG and LINING FLUID

Ascorbate Urate Glutathione

Alpha-tocopherol



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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

Clearance

Effect spectrum

Lung

Bacteria

Morbidity

viruses

pollutants

“stress”

diet,

age,

genetics

Mortality



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NO

2

COMPARISON OF TWO MARKERS: Sensitivity of dose response

Streptococcus Infectivity vs. Lung Permeability

100

2.5

Phosgene

90

Phosgene

80

2.0

70

NO2

60

1.5

BAL Fluid Protein, mg/ml

Ozone

% Excess Mortality Due to Infection

50

40

1.0

Ozone

30

20

0.5

10

0

0.0

0.01

0.10

1.00

10.00

100.00

Approximate Amount in Lungs,

m

g / mouse


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NON-INVASIVE MARKER OF OXIDATIVE STRESS: ISOPROSTANE

Fatty acid: Arachidonate radical

OXIDATION

Type III Isoprostane, measured by GCMS or ELISA


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

25

20

Urine from18O3exposed

15

EXCESS 18O ug / g dry

10

5

Urine from untreated rats

0

Post-exposure time, hr

-5

10

20

30

40

50

60

70

80

90

100

NON-INVASIVE MARKER: Urinary products of lung repair

from labeled ozone


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SUMMARY: MARKERS OF PULMONARY TOXICITY

Gross appearance of the lung

Clinical signs: whole body

Microscopic changes

Lung physiology changes

Lung and lung lavage fluid

Cellular

Biochemical

Genomic

Markers of Dose

Markers of Susceptibility

Bacterial infectivity marker

Non-invasive markers

Blood and urine


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SUMMARY

DESIRABLE QUALITIES

in a Marker of Toxicity

- Rapid, simple, easy

- Predictive of human pathology

- Non-invasive

- Sensitive

- Validated scientifically

- Accepted by scientists and public


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