Post on 13-Mar-2018
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