Pulmonary Inflammation and Rat Lung Cancer

Implications for Dust Exposure Limits

Kevin E. Driscoll, Ph.D.

Titanium Dioxide

Talc

Diesel Soot

Carbon Black

Coal Dust

Magnitite

Hematite

Exposure to Poorly Soluble Particles and Rat Lung Cancer

1000100101.10

10

20

30

40

Lu

ng

tu

mo

r in

cid

en

ce (

%)

lung burden (mg/lung)

background tumor incidence

TiO2

carbon black

TiO2

talc

diesel

carbon black

diesel

carbon black

diesel

coal

Lung Tumor Response and Lung Dose – mass (mg/lung)

Adapted from Driscoll, Inhalation Tox, 1996

1000001000010001001010

10

20

30

40

Particle Surface Area (m^2)/Lung

Lu

ng

tu

mo

r in

cid

en

ce

(%

)

background tumor incidence

TiO2

talc

carbon black

diesel TiO2

carbon black

diesel

diesel

carbon black

Lung Tumor Response and Lung Dose – Surface Area

Adapted from Driscoll, Inhalation Tox, 1996

Rat Lung after Chronic Talc Inhalation

Particle Exposure

Recruitment & Activation

of Inflammatory Cells

Clearance by Macrophages

No Adverse Effect

Oxidants Proteases Growth Factors Bioactive Lipids

& Cytokines

Cell Injury

Cell ProliferationExtracellular

Matrix Synthesis

DNA Damage

Carcinogenesis Fibrosis

Low

Dose

High

Dose

Adapted from: Driscoll KE. Mechanisms of rat lung tumors after chronic particle

exposure:role of persistent inflammation. Inhalation Toxicol. 1996.

Comparison of Rat Lung Particle Responses

- Inflammation & Mutation -

Silica

Carbon Black

Titanium Dioxide

0.9 mg/lung

39 mg/lung

665 mg/lung

19 % tumors

15 % tumors

26 % tumors

Study Design

Instill rats with 10 or 100 mg/kg particles

Sacrifice rats 17 months after exposure

Perform BAL and characterize cells

Isolate and culture alveolar epithelial cells

Select for mutation in the hprt gene

Isolate epithelial cells

In RLE-6TN cells exposed to BAL cells

Saline

Silica

10 mg.kg

100 mg/kg

Carbon Black

10 mg/kg

100 mg/kg

Titanium Dioxide

10 mg/kg

100 mg/kg

Total Cells (x106) Neutrophils (%)

4.5 + 0.7

19.0 + 3.7

52.8 + 15.9

5.5 + 1.4

12.0 + 2.4

3.0 + 0.7

6.0 + 0.8

1.6 + 0.7

57.0 + 1.9

64.5 + 2.8

19.0 + 0.9

60.0 + 3.0

3.0 + 1.5

41.0 + 5.8

*

*

*

*

**

*

*

Inflammatory Response to Instilled Particles (BAL Cells)

* different from saline, p<0.05Adapted from: Driscoll et al. Mutagenic effects of mineral particles in vivo and of

particle elicited inflammatory cells ex vivo. Carcinogenesis 1997.

Characterization of Mutation in Rat Lung Epithelial Cells

Driscoll et al. Characterizing mutagenesis in the hprt gene of rat alveolar

epithelial cells. Exp. Lung. Res. 21:941-956, 1995.

Mutant Frequency in Lung Epithelial Cells

After Particle Instillation

0

60

120

180

240

Mu

tan

ts/1

0^

6 c

ells

saline

Silica (mg/kg)

10 100

CB (mg/kg)

10 100

TiO2 (mg/kg)

10 100

*

**

*

* denotes different from saline, p<0.05

Adapted from: Driscoll et al. Mutagenic effects of mineral particles in vivo and of particle elicited

inflammatory cells ex vivo. Carcinogenesis 1997.

Co-Culture of Particle-Elicited Inflammatory Cells

with RLE-6TN Cells – Effect on Mutant Frequency

0

10

20

30

40

Mu

tan

ts/1

0^

6 c

ells

saline

Silica (mg/kg)

10 100

CB (mg/kg)

10 100

TiO2 (mg/kg)

10 100

*

**

*

* denotes different from saline, p<0.05

Adapted from: Driscoll et al. Mutagenic effects of mineral particles in vivo and of particle elicited

inflammatory cells ex vivo. Carcinogenesis 1997.

saline BALCells BALCells+ CAT

BALCells +inactivatedCAT

0

10

20

30

40

50 **

Mu

tan

ts/1

0^

6 c

ells

Mutant Frequency in RLE-6TN Cell Exposed to

Quartz-Elicited BAL Cells In Vitro

60504030201000

100

200

300

400

(mutants/106 cells)

In Vitro Response to Inflammatory Cells

In V

ivo

Resp

on

se t

o P

art

icle

s

(muta

nts

/106

ce

lls

)

R2 = 0.70

p<0.001

In Vivo Mutation Frequency and Mutagenic

Activity of Inflammatory Cells

0

80

160

240

0 20 40 60 80

Neutrophils (%)

Mu

tan

ts/1

0^

6 c

ells *

**

*

Silica

Carbon Black

Titanium Dioxide

Saline

Relationship Between Epithelial Cell Mutant Frequency

And Pulmonary Neutrophilic Inflammation

Chronic Inhalation Exposure to PSPs – Inflammation and Tumors

- Carbon black, Diesel exhaust, Toner, Talc -

From - Watson and Valberg, 2002

Weeks of Exposure/Recovery

-----------------------------------------

Treatment Group 13 32

-------------------------------------------------------------------

1.1 mg/m3 354 ± 42 126 ± 12*

7.1 mg/m3 1826 ±260 1184 ± 249*

52.8 mg/m3 7861 ±504 5916 ±1162

--------------------------------------------------------------------

* different from week 13 lung burdens; p<0.05.

Expose rats to CB

by Inhalation

(6h/d, 5/wk)

Sacrifice rats

after 13 wks

Characterize

inflammation

Isolate RLE &

select for hprt

Lung Burden (mg/Lung)

Adapted from: Elder et al. Effects of subchronically inhaled carbon black in three species. I. Retention kinetics,

lung inflammation, and histopathology. Toxicol Sci. 2006.

0

10

20

30

40

Ne

utr

op

hils

(%

)

air 1 7 50

Carbon Black (mg/m3)

*

*

0

25

50

Mu

tan

ts/1

0^

6 e

pit

helial cells

air 1 7 50

Carbon Black (mg/m3)

*

*

BAL Fluid Neutrophils HPRT Mutant Frequency

Expose rats to CB

by Inhalation

(6h/d, 5/wk)

Sacrifice rats

after 13 wks

Characterize

inflammation

Isolate RLE &

select for hprt

Adapted from: Driscoll et.al. Pulmonary inflammatory and mutagenic responses to subchronic inhalation

of carbon black by rats. Toxicol. Appld. Pharm. 1996.;

Effect of Glutathione on Particle-Induced

Lung Inflammation

Pretreatment - i.p. saline, 0.4mmol/kg BSO, 0.1mol/kg NAC

Treatment - i.t. saline or 2 mg carbon black

Timepoint - 28 day pretreatment, 7 day treatment

BALF AnalysisLDH, Total Protein

Total cell number

% Neutrophils

NPSHs

Ex vivo BAL cellsSuperoxide

Hydrogen Peroxide

Mutagenicity

TissueType II cell mutant

frequency

NPSHs

NPSHs after Pretreatment with BSO or NAC

0

2

4

6

8

10

12

14

16

18

20

saline carbon

black

nm

ol/m

l

Tissue

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2.0

saline carbon

black

nm

ol/

ml

saline

BSO

NAC

BALF

*

Y

*

*

*

Y

0

10

20

30

40

50

60

saline carbon

black

LD

H(U

/L)

saline

BSO

NAC

BAL LDH

*

Y

0

2

4

6

8

10

12

14

16

18

saline carbon

black

% n

eu

tro

ph

ils

BAL Neutrophils

*

Y

BAL Fluid Analysis

BAL Cell Oxidants

0

2

4

6

8

10

12

14

16

18

20

saline carbon

black

nm

ol/

mg

pro

tein

saline

BSO

NAC

Superoxide anion

0

2

4

6

8

10

12

14

16

18

saline carbon

black

nm

ol/m

g p

rote

in

Hydrogen peroxide

*

Y

*

Y

In Vivo Mutant Frequency- Lung Epithelial Cells -

0

6

9

12

15

saline carbon black

mu

tan

ts (

x 1

0^

6)

saline

BSO

NAC

3

*

Y

Talc

Diesel

Exhaust

Diesel

Exhaust

Titanium

Dioxide

Quartz

Inhalation

Inhalation

Inhalation

Inhalation

Instillation

Yes

Yes

Yes

Yes

Yes

No

No

No

No

No

?

No

?

?

No

NTP, 1992

Heinrich et. al, 1996

Mauderly et. al, 1997

Heinrich et. al, 1993

Saffiotti et. al, 1992

Test

Material Exposure

Lung Tumor Response

Rat Mouse Hamster Reference

Lung Tumor Response to Particle Exposure

Species Differences

Balance Between Pro- and Anti-

Inflammatory Processes

H2O2

O2-

peroxynitrite

MIP-2

CINC

TNF

metallothionein

glutathione

catalase

SOD

IL-10

200

0

100

*

*

U/m

g p

rote

in

Catalase

0.2

0

0.1

*Glutathione Px

Species Differences in Lung Anti-OxidantsLung Tissue Anti-Oxidant Enzymes

From: Bryon and Jenkinson, 1987

U/m

g p

rote

in

Comparison of Rat and Hamster Responses

to Particles

Test Particles - quartz, carbon black

Treatment - 0.2, 2.0 and 20.0 mg i.t.

Evaluate response 7 days after exposure

Endpoints

BAL fluid analysis

LDH

Cell # and type

BAL cells (ex vivo)

Hydrogen peroxide

Nitric Oxide

Mutagenicity

Adapted from: Carter and Driscoll. The role of inflammation, oxidative stress and proliferation in silica-induced lung disease: a species

comparison. J Environ Pathol Toxicol Oncol 2001; Driscoll, Carter and Borm. Antioxidant defense mechanisms and the toxicity of fibrous

and nonfibrous particles. Inhal. Toxicol. 2002.

BAL LDH BAL Neutrophils

0

100

200

300

400

*

*

*

Silica (mg)

0 0. 2 2. 0 20 .0 0

40

80

0 0. 2 2. 0 20 .0

Silica (mg)

hamster

rat *l

l

*

l

l

BAL LDH BAL Neutrophils

0

1 0 0

2 0 0

3 0 0

4 0 0

Carbon black (mg)

0 0. 2 2. 0 20 .0 0

4 0

8 0

0 0. 2 2. 0 20 .0

Carbon black (mg)

hamster

rat

l

l

**

**

l

BAL Cell Hydrogen Peroxide

saline 0.2 2.0 20 0.2 2.0 20

a-quartz (mg) carbon black (mg)

mM

H2O

2

0

10

20

30

40

50 rat

hamster

*

*

l

*l

*

l

nm

ol N

O2

0

1

2

3

4

5

6rat

hamster

saline 0.2 2.0 20 0.2 2.0 20

a-quartz (mg) carbon black (mg)

BAL Cell Nitric Oxide

+

+

+

+

+ *

*

*

ll

l

l

BAL Cell ex vivo Mutant Frequency

saline 0.2 2.0 20 0.2 2.0 20

carbon black (mg)

mu

tan

t fr

eq

ue

nc

y

0

5

10

15

20 rat

hamster

* *

**

a quartz (mg)

Summary

A common mechanism for the rat lung tumor response to non-genotoxic PSPs

is suggested by the correlation between surface area dose rat lung tumors

Studies on PSP exposure, inflammation and mutation indicate the mechanism

for rat lung tumors is dependent on inflammation and cell proliferation

Inherent in an inflammation-dependent mechanism is a threshold for the rat

lung tumor response

The high sensitivity of the rat to PSP-induced lung tumors may be due, in part,

to differences in the balance between pro- and anti-oxidant processes

The basis for differences between the rat and human requireds further study

and analysis

Screening of New PSPs - Proposal

For preliminary safety assessments on new PSPs:

1. Assess genotoxicity screen or use existing data on composite materials

2. Benchmark inherent toxicity again PSPs for which extensive data exist using:

• In vitro screening for toxicity – macrophages or epithelial cells

• In vivo short-term (1-2 week) intratrachael instillation or inhalation study

using bronchoalveolar lavage fluid analysis and histopathology to compare

the new PSP to a positive ad negative control (e.g., crystalline silica,

titanium dioxide).

If results for the above screening studies demonstrate the

new material behaves similar to the control PSP (nature,

magnitude and temporal), set interim exposure limits at

levels similar to the control PSP.

Janet Carter

James Maurer

Tim Bertram

Diana Hassenbein

Brian Howard

Brooke Mossman

Gunter Oberdorster

Paul Borm

Alison Elder

Jack Finkelstein

Jack Harkema

George Leikauf

Debra Laskin

Acknowledgments

Selected References• Driscoll KE. Mechanisms of rat lung tumors after chronic particle exposure:role of persistent inflammation. Inhalation

Toxicol. 8:139-153, 1996.

• Driscoll, K.E., Deyo, L.C., Howard, B.W., Poynter and J., Carter, J.M. Characterizing mutagenesis in the hprt gene of

rat alveolar epithelial cells. Exp. Lung. Res. 21:941-956, 1995.

• Driscoll, KE. Carter, JM, Howard, BW, Hassenbein, D, Pepelko, W, Baggs, R and Oberdorster, G. Pulmonary

inflammatory and mutagenic responses to subchronic inhalation of carbon black by rats. Toxicol. Appld. Pharm. 136:

372-380, 1996.

• Borm, P.A. and Driscoll, K.E. Particles, inflammation and respiratory tract carcinogenesis. Toxicology 110:1-5, 1996

• Driscoll, KE, Carter, JM, Howard, BW, Hassenbein, DG and Bertram TA. Mutagenic effects of mineral particles in vivo

and of particle elicited inflammatory cells ex vivo. Carcinogenesis 2:423-430, 1997.

• Carter, J.M. and Driscoll K.E. The role of inflammation, oxidative stress and proliferation in silica-induced lung

disease: a species comparison. J Environ Pathol Toxicol Oncol 20 (Suppl. 1): 33-43, 2001

• Driscoll, K.E. Carter, J.M. and Borm, P.J.A. Antioxidant defense mechanisms and the toxicity of fibrous and nonfibrous

particles. Inhal. Toxicol. 14:101-118, 2002.

• Elder A., Gelein R., Finkestein, JN., Harkema J., and Oberdorster, G. Effects of subchronically inhaled carbon black in

three species. I. Retention kinetics, lung inflammation, and histopathology. Toxicol Sci. 88(2):614-19, 2006.

• Carter, J.M., Corson, M., Driscoll, K.E., Elder, A., Finkelstein, J.N., Harkema, J.N., Gelein, R., Wade-Mercer, P.,

Nguyen, K., and Oberdorster, G. A Comparative Dose-Related Response of Several Key Pro- and Antiinflammatory

Mediators in the Lungs of Rats, Mice, and Hamsters After Subchronic Inhalation of Carbon Black J Occup and

Environ Med 48(12):1265-78, 2007