nose and society - toxicologic pathology anatomy of the respiratory tract alveolus ... in...

7/20/2012

1

Jack R. HarkemaJuly 26, 2012

Respiratory Response to Toxic Injury: Upper Respiratory Tract

(Nasal Toxicology)

Respiratory Response to Toxic Injury: Upper Respiratory Tract

(Nasal Toxicology)

Industrial Toxicology and Pathology Short Course 2012

Nose and Society• 40 million cases of chronic sinusitis

• Cases of allergic rhinitis also increasing

• Increase in the identification of nasal toxicants

• New understanding of the molecular makeup of olfaction: 2004 Nobel Prize (Drs. Axel and Buck)

Outline

• Nasal Airway Anatomy and Histology

• Nasal Toxicity of Inhaled Ozone

• Nasal Toxicity of Inhaled Nanoparticles

• Olfactory Toxicity of Inhaled Satratoxin‐G (Mycotoxin in Stachybotris chatarum)

• Future Studies, Summary and Conclusions

7/20/2012

2

Comparative Anatomy of the Respiratory Tract

Alveolus

Alveolar Duct

Respiratory Bronchiole

Terminal Bronchiole

Bronchus

Larynx

Rat Human

Pulmonary Artery

Pulmonary Vein

Alveolar Capillary Bed

Nasopharynx

Nasal Airway

Trachea

Comparative Nasal Airway Structure and Function

Human Monkey Rat

Volume

(cm3) 16‐19 8 0.4

Turbinate

Anatomy Simple Simple Complex

Olfactory

Epithelial

Surface Area

Small

<10%

Moderate

20‐30%

Large

50%

Breathing Oronasal Oronasal Nasal

Computer Assisted Reconstruction of Rat Nasal MRI

Proximal T2 Nasal Section

Distal T4 Nasal Section

7/20/2012

3

Comparative Anatomy of Nasal Airways

Harkema JR et al. Chapter 6 Nose, Sinus, Pharynx and Larynx. In Comparative Anatomy and Histology: A Mouse and Human Atlas, Eds. Treuting PM and Dintz SM, Academic Press, 2012

Nasal Airway of the Monkey Nose


Nasal Mucosa of the Monkey Nose

7/20/2012

4

Proximal Nasal Airway of the Mouse


Proximal Nasal Airway of the Mouse


Distal Nasal Airways of the Mouse

Harkema JR et al. Chapter 6 Nose, Sinus, Pharynx and Larynx. In Comparative Anatomy and

7/20/2012

5

Olfactory System

1) Main Olfactory Organ (ordors in general)

2) Vomeronasal Organ (chemicals in social and sexual activities)

3) Septal Olfactory Organ (same odors as main olfactory system and an alerting role)

• Olfactory Epithelium– Olfactory sensory neurons

(OSN)• Axons are unmyelinated(0.1‐ 0.7 mm in diameter)

– Sustentacular cells– Basal cells – Microvillous cells

• Lamina Propria– Olfactory nerve fascicles

(bundles of OSN axons)– Schwann cells surrounding

bundles of OSN axons– Bowman’s Glands– Blood vessels– Connective tissue

Olfactory Mucosa

Farbman AI. Cell Biology of Olfaction, Cambridge University Press,1992

Unique Characteristics of the Olfactory Chemosensory Neurons

• Dendrites exposed to the external environment

• Axons project directly to the forebrain without synapsing in the thalamus (conduit to the CNS)

• Capacity for continued postnatal neurogenesis

7/20/2012

6

ON

Dendritic Cilia

OSN

SC

BC

Olfactory Marker Protein

Farbman AI. Cell Biology of Olfaction, Cambridge University Press,1992

Olfactory Sensory Neuron (Receptor Cells)Dendrite: Knob and Cilia

1-2 m

0.1 m

50 mm

0.3 m

9(2)+2

No dynein arms∴ not motile

2004 Nobel AwardDrs. Axel and Buck

Odorant Receptors and Organization of the Olfactory System

7/20/2012

7

Olfactory Genes & Receptors

• 3% of human genome devoted to identifying odors (1000 odorant receptor genes ORG)

• Humans and mice have 5 million olfactory sensory neurons (OSN)

• Bloodhound has 220 million OSN

• 1 ORG per OSN

• 1,800 olfactory axon convergence centers in the Olfactory Bulb = Glomeruli

• Each ORG corresponds to 2 glomeruli

Ozone (O3)• One of the most reactive

chemicals

• Oxidant gas in photochemical smog

• 131 million people (50% of the U.S. population) live in communities where average ambient concentrations exceed the NAAQS

• Respiratory toxicant causing airway inflammation and remodeling

• Long-term exposure causes an increase in mortality

Comparative Nasal Toxicity of Ozone

Harkema et al. Am. J. Pathol. 127:90‐96, 1987

Harkema et al. Am. J. Pathol. 128:29‐44, 1987

Calderon‐Garciduenaset al. Am. J. Pathol. 140: 225‐32, 1992

Harkema et al. Toxicol. Pathol. 17: 525‐535, 1989

7/20/2012

8

O3‐Induced Remodeling of Maxilloturbinate in Rat

Kinetics of Nasal Tissue Responses to Inhaled Ozone

Kinetics of Ozone‐Induced Mucous Cell Metaplasia in Rat Transitional Epithelium

Cho HY et al., Toxicol Sci 51:135‐145, 1999

7/20/2012

9

Schematic of Ozone Exposure Protocol for Infant Monkeys

Carey SA et al. Am J Physiol Lung Cell Mol Physiol 2011;300:L242‐L254

Imaging, 3D modeling, & Histopathologic Methods

Carey SA et al. Toxicol Pathol 2007;35:27‐40

Site‐ and Exposure‐Specific Nasal Mucosal Injury

Carey SA et al. Am J Physiol Lung Cell Mol Physiol 2011;300:L242-L254

7/20/2012

10

Nasal Morphometry of Ozone‐Induced Nasal Epithelial Injury in Infant Rhesus Monkeys


Effect of 1‐ and 11‐cycle O3 exposure on the intracellular concentrations of GSH (A), GSSG (B), ascorbate (AH2; C), and uric acid (UA; D) in the

nasal mucosa from the anterior MT, posterior MT, and anterior ET of infant monkeys.


Correlations Among Sites of Ozone‐Induced Nasal Airway Injury, Predicted Ozone Flux,

and Epithelial Type

Carey SA et al. Toxicol Pathol 2007;35:27‐40

7/20/2012

11

Fractional Deposition of Inhaled Particles in the Human Respiratory Tract (ICRP Model, 1994; Nose‐breathing)

Inhaled Particle Deposition in Airways

Is the nose a potential target for toxic injury caused by inhaled

nanoparticles?

Carbon Black Nanoparticles

• Combustion derived nanoparticles

• Industrially produced for colouring enamels, acrylics, and plastics, as well as inks and paints

• Low toxicity, low solubility without organics or metals

• Pulmonary carcinogen in rats with long‐term exposures and under overload conditions

Donaldson et al. Particle and Fibre Toxicology 2:1‐14, 2005

7/20/2012

12

• Female F344 rats, B6C3F1 mice, and Syrian Golden Hamsters

• Rodents exposed by inhalation to 0, 1, 7, or 50 mg/m3 of high surface area carbon black (HSCB; 17nm primary particle diameter) for 6 h/day, 5 days/wk for 13 wk

• Some rats exposed to 50 mg/m3 of low surface area CB (LSCB; 70nm primary diameter) for 6 h/day, 5 days/wk for 13 wk

• Rodents were sacrificed 1 day, 13 wk, or 11 mo post‐exposure

• Nasal and pulmonary airways were processed for microscopic and morphometric examination

Effects of Inhaled Carbon Black in Three Rodent Species: Study Design and Methods

Elder et al., Toxicol Sci. 2005; 88(2), 614‐629

Pulmonary Effects of Inhaled Carbon Black in Laboratory Rats

• Initial lung burdens were similar in high‐dose HSCB and LSCB

• Surface area burdens were equivalent for mid‐dose HSCB and high‐dose LSCB

• LSCB cleared faster from the lung (less retention) than HSCB

• More pulmonary pathology in HSCB than in LSCB

• Particle surface area is an important factor in target tissue dose and, therefore, toxic effects

• Toxicity in Rats>Mice>Hamsters

Elder et al., Toxicol Sci. 2005; 88(2), 614‐629

Nasal Histopathology in Rodents Exposed to Inhaled Carbon Black

• Particles in nasal mucosa

• Epithelial hyperplasia

• Mucous cell metaplasia

• Inflammation (rhinitis)

• Atrophy of turbinate bone

• Hyalinosis (chitinase)

• Dose‐dependent severity

• No LSCB related lesions

7/20/2012

13

HSCB‐Induced Histopathology in Maxilloturbinates of Rats

H&E

AB/PAS

Air Control HSCB 50mg/m3

HSCB in Nasal Mucosa

In Mucosal Macrophage

In Nasal Transitional and Respiratory Mucosa

In Olfactory Mucosa

OE

tb

bg

100 m

HSCB in Olfactory Epithelium, Nerves, and Bulb

DorsalMeatus

Olfactory Epithelium

Olfactory Nerves

CP

CP

CB

Olfactory Bulb

50 m 100 m

7/20/2012

14

11 Months After HSCB Exposure: Maxilloturbinate

tb

e

lp

Airborne Carbon Black Concentration (mg/m3)

0 1 7 50 50

*

Vo

lum

e D

ensi

ty (

nl/m

m2 )

0

1

2

3

4

5HSCB

LSCB

Air (0 mg/m3) HSCB (50 mg/m3) LSCB (50 mg/m3)

HSCB‐Induced Mucous Cell Metaplasia in Transitional Epithelium Lining the Rat Maxilloturbinates

0

Vo

lum

e D

ensi

ty (

nL

/mm

2 )

0

1

2

3

4

5

5071


RatsMiceHamsters

a

ab

*

*

Mucous Cell Metaplasia in Rats and Mice, but not Hamsters, Exposed to HSCB

H

R

M

0 mg/m3

tb

e tb

bv

e

bv

tbe

tb

e

0 mg/m3 Air 50 mg/m3 HSCB

ee

tb

tbbv

bv

7/20/2012

15

Species T1 T2 T3 T4

Rat ↑↑ ↑↑↑↑ ↑↑↑↑ ↑↑

Mouse ↑ - ↑ -

Hamster - - - ↑

Interspecies Differences in the Severity of HSCB‐Induced Mucous Cell Metaplasia at One Day after Exposure

Nasal Tissue Sections

Proximal Distal

Air control (0 mg/m3) HSCB (50 mg/m3)

HSCB‐Induced Rhinitis in Rats, but not in Mice or Hamsters

0

20

40

60

80

100

120RatsMice

Neutrophils in Nasal Mucosa of the Midseptum (T2) of Rodents Exposed to Carbon Black Particles

Nu

mer

ic C

ell D

ensi

ty (

neu

tro

ph

ils

/mm

BL

)

Time Post Exposure: 1 day

0 50 HSCB

*Hamsters

ND ND


0

20

40

60

80

100

120

1 day PE13 weeks PE

Neutrophils in Nasal Mucosa Lining the Midseptum (T2) of Rats Exposed to Carbon Black


Nu

me

ric

Ce

ll D

en

sity

(N

eu

tro

ph

ils/m

m B

asa

l Lam

ina

)

0 Air 50 HSCB 50 LSCB

*

Summary of Results

• Rats exposed to 7 or 50 mg/m3 HSCb had chronic rhinitis, epithelial hyperplasia, and mucous cell metaplasia at one day post‐exposure

• Magnitude of HSCb‐induced nasal lesions was site‐and dose‐dependent, and attenuated with time post exposure

• Aggregates of HSCB found in nasal mucosa, olfactory nerves and olfactory bulb

• Rats exposed to 1 mg/m3 HSCb had no nasal lesions

• Rats exposed to 50 mg/m3 LSCb had minimal nasal lesions

• HSCB‐induced nasal toxicity was species dependent: Rat > Mice > Hamsters

7/20/2012

16

Conclusions

• The nose is a potential target tissue for nanoparticle‐induced toxicity and a possible route of exposure to the brain

• As in the lung, particle surface area is an important determinant in the nasal toxicity caused by inhaled carbon black

• Future studies are needed to determine the underlying mechanisms responsible for nasal toxicity caused by nanoparticles

• The effects of nanoparticles on the human nasal airways is yet unknown.

Black Mold, Trichothecenes and Satratoxins

• ‘Black Mold’ Stachybotryschatarum – saprophytic fungus that grows on cellulosic materials

• Trichothecenes – low molecular weight (200‐500D) mycotoxins produced by Stachybotrys, Fusarium, and other molds

• Bind to ribosomes, inhibit protein synthesis, and induce ribotoxic stress

• Satratoxins – macrocyclictrichothecenes produced by Stachybotrys

Experimental Design and Methods

• 7‐8 wk old, C57Bl/6 Mice

• Single intranasal instillation of Satratoxin G (SG; 500 µg/kg BW) in saline or saline alone (controls)

• Necropsy at 6h, 1 day, 3 days, 7days or 28 days after instillation

• Nasal and olfactory bulb tissues were processed for microscopic, morphometric, and molecular analysis

Islam Z, Harkema JR, and Pestka JJ. Environ Health Perspect. 2006; 114: 1099‐1107

7/20/2012

17

Summary of SG‐Induced Histopathology in Nose and Brain


Kinetics of SG‐Induced Atrophy of OE

Days Post Instillation

Epi

thel

ial T

hick

ness

(m

)

0

10

20

30

40

50

60

70

1 3 7 6

AA A A

B

E

D

F

Single InstillationMultiple

Instillations

B

C

28

Satratoxin-G Control


C

Pro-/Anti-Apoptotic Genes

FasFas

Lp7

5p5

3Bax

Bcl-2

Apaf-1

Cas-3

Cad

Re

lativ

e m

RN

A E

xpre

ssio

n (F

old

Inc

reas

e)

0

5

10

15

20 Control Satratoxin G

* *

*

* **

*

SG‐Induced Apoptosis of OE at 24h Post Instillation


7/20/2012

18

Days Post Instillation

OM

P P

ositi

ve C

ells

/mm

0

100

200

300

400

500

600

700

1 3 7

A

AA

B

D

C

Single Instillation

A

C

6

MultipleInstillations

A

B

28

SG‐induced Loss of OMP‐Positive Olfactory Sensory Neurons


A

B

SG‐Induced Atrophy of Olfactory Marker Protein (OMP)‐Positive Olfactory Sensory Neurons

C

A) Normal OMP expression in olfactory nerve and glomerular layers of olfactory bulb (OB)

B) Expression of OMP in OB 7 days after SG instillation

C) Site of SG‐induced atrophy of OB (*)


Deoxynivalenol

Satratoxin G

Isosatratoxin F

Verrucarin A

T‐2 toxin

AA B

O

O

O

HO

H2CHO

8

1 2

3

45

6

79 10

11

1213

14

15

16

OH

H

H

H

H

O

O

O

O

O

O

O

CH3

O

OH

O

H

4

15

O

OO

4

15O

O

OH

O

O

O

5

O

O

O

O

O

O

O

CH3

OH

OH

O

H

4

15

O

O

CH2

8

1 2

3

45

6

79 10

11

1213

15

16

OC

O

CH2

CHH3C

CH3

O

C

CH3

O

O

OH

H

H

C

CH3

O

Effects of Trichothecenes on OE

7/20/2012

19

A C D

E

G

F

H

Rel

ativ

e m

RN

A E

xpre

ssio

n

(Fo

ld In

crea

se)

1

10

100

1000 ControlSatratoxin-G

*

*

**

*

IL-1

IL-1

IL-6

TNF-M

IP-2

Inflammatory Cytokines

Rel

ativ

e m

RN

A E

xpre

ssio

n

(Fol

d In

crea

se)

0

1

2

3

4

5 ControlSatratoxin-G

*

**

IL-1

IL-1

IL-6

TNF-M

IP-2

B

Olfactory Epithelium

Olfactory Bulb

STG‐Induced Inflammatory Responses in Nose and Brain


Carey SA et al. Toxicol Pathol 2012 online, in press

Satratoxin G‐Induced Olfactory Injury in Rhesus Monkeys

Carey SA et al. Toxicol Pathol. 2012; online, in press

7/20/2012

20

Carey SA et al. Toxicol Pathol. 2012; online, in press.

Satratoxin G‐Induced Loss of OSN in Rhesus Monkeys


Satratoxin G‐Induced Apoptosis of OSN in Rhesus Monkeys


Satratoxin G‐Induced Rhinitis in Rhesus Monkeys

7/20/2012

21

• Nasal airways and brain are potential target sites for toxicity caused by inhalation exposure to macrocylic trichothecenemycotoxins that may be found at high concentrations in the indoor air of mold‐contaminated, water‐damaged buildings.

• Quantitative assessments of human exposures and epidemiological studies are needed to determine the potential risks to human health.

Conclusions

Future Studies: Multi‐Scale Modeling for Airway Dosimetry

Rat vs. Human Nasal Breathing

0.6 ppm Acrolein13.8 L/min flow

Slide courtesy of Dr. Rick Corley

0.6 ppm Acrolein434 ml/min flow

Summary and Conclusions Nasal Airway Anatomy and Histology

Nasal Toxicity of Inhaled Ozone

Nasal Toxicity of Inhaled Nanoparticles

Olfactory Toxicity of Inhaled Satratoxin‐G (Mycotoxin in Stachybotris chatarum)

• The nose is a target organ for many inhaled toxicants.

• Nasal responses to toxicants are dependant on many factors (e.g., physicochemical characteristics, dosimetry, tissue sensitivity, host, species).

• Results from future laboratory animal studies will be able to better predict human risk to toxic injury.

nose and society - toxicologic pathology anatomy of the respiratory tract alveolus ... in...

Documents