Roel Schins

IUF - Leibniz Research Institute for

Environmental Medicine, Dusseldorf

Bijeenkomst CGC & NVT-AT; Den Bosch, 14-3-2013

Stofdeeltjes:

Toxicokinetiek & -dynamiek

Dust (matter),

particles,

fine particulate matter (PM)

Poussière (matière),

particules,

particules fines

Stof (materie),

deeltjes, fijnstof

Staub (Materie/Stoff),

Teilchen/Partikel,

Feinstaub

http://geodata.rivm.nl/gcn/

(2011)

Dose?

AMBIENT EXPOSURE

(PM10/PM2.5)

Adverse effects in susceptible groups:

Asthma attacks, exacerbations of COPD

and cardiovascular disease

Immunological effects

Cancer

(Diabetes, Alzheimer‘s disease…?)

Typical exposures: g/m3 Typical exposures: mg/m3

OCCUPATIONAL EXPOSURE

Pneumonitis

Chronic obstructive pulmonary

disease (COPD):

emphysema

chronic bronchitis

Pneumoconioses

(silicosis, asbestosis,..)

Lung cancer

Oberdörster et al. EHP 2005

Courtesy: Gunter Oberdorster

Clearance via the muco-ciliary escalator

Deposition on the

conducting airways

Bronchial epithelium

Courtesy: Ken Donaldson/Peter Gehr

alveolar space

alveolar septum

capillary

red blood cell

alveolar space

alveolar septum

capillary

red blood cell

Clearance by alveolar

macrophage phagocytosis

Deposition on the

terminal bronchioles/

proximal alveoli

Interstitial

pathway via

lymph to the

lymph nodes

Clearance mechanisms of particles

Group Treatment Tumour (%)

Control Room air 3

Quartz 1 mg/m3 DQ12 6 h/day 5 days/week 24 months 19

Muhle et al. 1989 Am J Ind Hyg

Silica and cancer - toxicological evidence

(inhalation studies in rats)

0

2

4

6

8

10

12

14

16

18

20

0 20 40 60 80

Days exposure

Bronchoalveolar

Lavage neutrophils

(x106)

Donaldson et al. 1988 Thorax

Silica and inflammation: toxicological evidence Inflammatory effects of inhaling 10 mg/m3 quartz in rats

Exposure 7 h/day

5 days/week for

up to 75 days

Macrophage

Alveolus

Neutrophils

Lung epithelial

cell

Toxicology of inhaled particles

Exposure chronic inflammation fibrosis, cancer

alveolar space

alveolar septum

capillary

red blood cell

alveolar space

alveolar septum

capillary

red blood cell

epithelium

O2.-/H2O2

Recruitment of

inflammatory cells: Macrophages,

Polymorphonuclear

Neutrophils(PMN)

NFB

O2.-/H2O2

Oxidative damage

(proteins, lipids, DNA)

Proliferation

H2O2 .OH

O2.-/H2O2

Modified from: Stone, Johnson and Schins (2009) Crit Rev Toxicol

Reactive oxygen species generation (ROS) and

induction of “oxidative stress”

Phospho-IB

(Ser32/36)

μφ

Quartz (2mg i.t.)

NF-κB RelA

Epithelial type II cells

Epithelial type II cells

μφ

Control

Oxidative stress and NFB pathway activation

Cytosol

iNOS, COX-2, TNFα,

IL-1β, IL-6, IL-8 / MIP-2,

GM-CSF, ICAM-1, etc.

NucleusRelA

p50

RelA

p50

IκBαRelA

p50

RelA

p50

IKKγ

IKKα

IKKβ

26S

β-TrCP

IκBα

IκBα

P

U

IKKγ

Cytosol

iNOS, COX-2, TNFα,

IL-1β, IL-6, IL-8 / MIP-2,

GM-CSF, ICAM-1, etc.

NucleusRelA

p50

RelA

p50

RelA

p50

IκBαIκBαRelA

p50

RelA

p50

RelA

p50

RelA

p50

IKKγIKKγ

IKKα

IKKβ

26S

β-TrCP

IκBαIκBα

IκBαIκBα

P

UU

IKKγIKKγ

Van Berlo et al. (2010) Part Fibre Toxicol

Oxidative stress

NFB

activation

Cytokines/

chemokines

Phagocyte activation

(inflammation)

ROS production

Antioxidant

depletion

Oxidative stress and inflammation

Coutesy: Aalt Bast

Borm et al. (2004) Toxicol Appl Pharmacol

Carcinogenicity studies with particles in the rat

Tumour formation in rat inhalation bioassays is associated

with durability of the material (biopersistence)

Genotoxicity tests with epithelial cells

(HPRT, Micronuclei, strand breaks)

Percoll gradient

epithelial cells

surfactant

Purification

Harvesting

& Analysis

Trypsin

Digestion

Genotoxicity of poorly soluble particles in vivo (inhalation or intratracheal instillation of particles in rat models)

Lavage, isolation

Bronchoalveolar lavage fluid analysis

(inflammatory cell counts, toxicity)

Driscoll et al. 1997; Knaapen et al. 2002; De Boeck et al. 2004; Muller et al., 2008; Albrecht et al., 2009

0

50

100

150

200

0 20 40 60 80

% neutrophils in BAL

HP

RT

/10

e6 c

ells

TiO2

Carbon Black (NP)

Quartz

Intratracheal instillation of particles

Isolation of type II lung epithelial cells

(15 months after i.t. application)

Cell culturing for selection of HPRT-

mutants (i.e. growth in 6-thioguanine

containing medium)

Driscoll et al. (1997) Carcinogenesis

Mutagenesis in rat lungs is associated with severity and

persistence of pulmonary inflammation

Chronic inflammation and release of reactive

oxygen and nitrogen species (ROS/RNS)

Genotoxicity proliferation

Tumour formation

Particle exposure

apoptosis

cell cycle arrests

DNA repair

Schins and Knaapen (2007) Inhal Toxicol

secondary

primary

Primary vs. secondary genotoxicity of particles

Dose metric?

0 200 400 600 800 0

0.5

1

1.5

2

2.5

3

Particle surface area (cm 2 ) N

um

ber

of

PM

Ns

(x

10

6 )

Burden expressed as surface area

TiO2 and

BaSO4

75mg/m3 BaSO4

50mg/m3 BaSO4

0 2 4 6 8 10 12 0

0.5

1

1.5

2

2.5

3

Lung burden (mg)

Nu

mb

er o

f P

MN

s (

x1

0 6

)

Burden expressed as mass

TiO2

BaSO4

50mg/m3 TiO2

25mg/m3 TiO2

Tran et al, 2000 Inhal Toxicol

Poorly soluble low toxicity particles

(PSP) Threshold of overload = 200-300

cm2 of particle surface in the rat lung

Chronic inflammation and lung burden of TiO2 and BaSO4

Düsseldorf

Ambient particulate air pollution

Aeroallergen Metal Salt

Non-toxic

particles

Soot

“Combustion derived

nanoparticles“

The PM mixture

Van Berlo et al EXS 2012

Extraction of PM from inert filters

(e.g. Teflon)

Human studies Rodent toxicology Cell culture Cell free assays

Sampling

of PM

Using PM samples in toxicology studies

Van Berlo et al EXS 2012

3.7 ± 2.7 4.6 ± 2.4 5.6 ± 3.0 * 4.6 ± 3.0 TNF

8.8 ± 2.4 14.7 ± 14.4 14.2 ± 5.2 * 8.6 ± 2.2 IL-6

Saline Zerbst Hettstedt Baseline

% Neutrophils 3.2 ± 3.6 3.7 ± 4.6 6.2 ± 7.1 1.2 ± 1.2

Control

(Saline)

PM2.5

Hettstedt

(Metal-rich)

PM2.5

Zerbst

Baseline

lavage

Lavage at t=24 h

Segmental instillation of “Hettstedt”- and

“Zerbst”- PM2.5 in human volunteers

Schaumann et al (2004) Am J Respir Crit Care Med

% Monocytes (FACS)

Baseline Saline H Z 0.0

2.5

5.0

7.5

10.0 Baseline Saline H Z %

p=0.02 p=0.01

% Monocytes (BAL)

**

*

********

********

0

1

2

3

4

3 - 7 µm 1.5 - 3 µm 0.95 - 1.5 µm 0.5 - 0.95 µm < 0.5 µm

Lo

g E

SR

(a.u

.)

RB

UB

BR

QR

Queensway underpass (A38),

Birmingham, UK

Mace Head Research station,

Connemara, Ireland (west coast)

OXIDANT GENERATION AND TOXICITY OF SIZE-FRACTIONATED

AMBIENT PARTICLES IN HUMAN LUNG EPITHELIAL CELLS.

RB = Remote background

UB = Urban background

BR = Bristol Road

QR = Tunnel

PM (size fraction)

DMPO

Wessels et al (2010) Environ Sci Technol

r = 0.70

p < 0.001

n = 44

0

10

20

30

40

50

60

0 1 2 3 4

Log ESR (a.u.)

Ox

ida

tiv

e D

NA

da

ma

ge

(a

.u.)

*

*

0

100

200

300

400

3 - 7 µm 1.5 - 3 µm 0.95 - 1.5 µm 0.5 - 0.95 µm < 0.5 µm

IL-8

(%

co

ntr

ol)

RB

UB

BR

QR

*

***

# #

***

0

10

20

30

40

50

60

3 - 7 µm 1.5 - 3 µm 0.95 - 1.5 µm 0.5 - 0.95 µm < 0.5 µm

Oxid

ati

ve D

NA

dam

ag

e (

a.u

.)

RB

UB

BR

QR

Oxidative DNA damage (A549 cells)

Oxidant generation / DNA damage

Interleukin-8 release (A549 cells)

Wessels et al (2010) Environ Sci Technol

Air

Respiratory

tract

Mucociliar clearance

Gastrointestinal

tract

Faeces

Particle effects beyond the respiratory tract

lymph

Liver

blood

brain

blood blood

Gehirn

Blut blood

brain

blood blood Brain

Blood

Kidney Urine

Inhaled ultrafine particles / nanoparticles

Food, water

ingestion

Dermal exposure Drug delivery

skin

Modified from Oberdorster et al EHP 2005

Summary

Particles can trigger oxidative stress and activation of NFB

signalling activation of the innate immune system

(macrophages, neutrophils)

Particle properties (e.g. surface reactivity, solubility) drive

inflammation (level and persistence)

Tumourigenesis in rat lungs by poorly soluble particles of low

toxicity (PSP) is driven by the “overload” phenomenon

(saturation due to macrophage clearance impairment)

Dose metrics other than mass should be considered:

surface area dose, number dose, surface reactivity

http://conference.iuf-duesseldorf.com/

Email: conference@iuf-duesseldorf.de

SCIENTIFIC COMMITTEE

Vicki Stone (Edinburgh) & Roel Schins (Düsseldorf)

SCIENTIFIC COMMITTEE

Paul Borm (Netherlands); Ken Donaldson (UK); Flemming Cassee (Netherlands); Vincent Castranova (USA); Bice Fubini (Italy); Mary Gulumian (South Africa); Marie-Claude Jaurand (France); Wolfgang Kreyling (Germany); Jean Krutmann (Germany); Dominique Lison (Belgium); Brooke Mossman (USA); Gunter Oberdörster (USA); Lang Tran (UK); Håkan Wallin (Denmark); David Warheit (USA)

MAIN TOPICS

• Toxicological testing and material/exposure characterization

• Target specific effects including respiratory tract, cardiovascular system, gastrointestinal tract, liver, central nervous system and reproductive organs

• Safe design of nanomaterials through hazard testing, modelling and QSAR type approaches

• Relating hazard study design and interpretation to regulatory requirements

Materials to be covered include crystalline silica, asbestos, environmental particulate matter, engineered nanomaterials as well as pharmaceutical particles.