The Cellular and Molecular Toxicity of Low Solubility ... · NAPIER UNIVERSITY EDINBURGH The Cellular and Molecular Toxicity of Low Solubility Nanoparticles Prof Vicki Stone Centre

NAPIER UNIVERSITYEDINBURGH

The Cellular and Molecular Toxicity of Low Solubility

Nanoparticles

Prof Vicki StoneCentre for Health and Environment

Napier University, Edinburghhttp://www.lifesciences.napier.ac.uk/Research/CHE1.htm

Safety of nanomaterials Interdisciplinary Research Centrehttp://www.snirc.org/


PM10

Carbon based particles•Traffic and industrial•Many ultrafine•Transition metals

Sulphates/Nitrates•Traffic/photochemical•Mainly ultrafine

Wind blown dust•Mainly coarse

Biological components•Spores•Pollen•Mainly fine and course

*** ***

500nm


Lung Defence Mechanisms

Blood vesselGas exchange

Particles trapped and cleared on

mucociliaryescalator

Airway

AlveolusParticles

phagocytosedand cleared

mucus cilia

Macrophage••••

••••

Particles

Antioxidants


The Ultrafine Particle Hypothesis

Seaton al. 1995 The Lancet 345: 176-178•

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Particles

Release of mediators by

macrophages and epithelial cells

Impaired movement

Blood vessel

INFLAMMATION

Particles cross the epithelial barrier

••••


Ultrafine particle toxicology

0

20

40

60

80

100

0 10 20 30 40 50 60 70Time from start of exposure (weeks)

Neutrophils in bronchoalveolar

lavage fluidX 105

21 nm TiO2

250 nmTiO2

Ferin et al 1992Am.J.Respir.Cell.Mol.Biol. 6: 535-542

‘Accidental’ versus ‘engineered’nanoparticles

Nanoparticles Source Exposure Toxicology

Accidental Fossil fuel combustionE.g. Traffic, cooking.

Low exposure to everyone

Lots e.g. diesel, CB, welding fume etc

Purposeful Bulk use of nanoparticles in industry e.g. carbon black

Nanotechnology

High exposure toworkers followed by low exposure to everyone

Virtually none


Adapted from Ken Donaldson


CB

100nm

100nm

14 nm254 m2/g

260 nm8 m2/g

Carbon black

535nm

202nm

64nm

500nm

Polystyrene beads

Model nanoparticles

Low solubility, low toxicity materials.

Factors involved in NP toxicity

..

10μm 1 μm 0.1μm

Bronchial epithelium

0.1μm1.0 μm

Cilia 0.25μm diameter


1. SIZE

2. PARTICLE NUMBERThere are thousands more ultrafine particles / mg of dust than larger, respirable particles.


Factors involved in NP toxicity

3. SURFACE AREAParticle surface area / mg is much greater for ultrafineparticles than for larger, respirable particles.

Fine carbon black 7.9 m2/gUltrafine carbon black 253.9 m2/g


Particle surface area and surface reactivity

Low toxicity low solubility particles •straight line relationship between SA and neutrophils.

Highly pathogenic particles •highly reactive surface (eg quartz). •do not sit on same line as low toxicity particles.

0

5

10

0 200 400 600 800 1000Surface Area instilled (cm2)

Neutrophils in BAL (x106)

Relatively low toxicity particles

Quartz

Duffin et al. (2002) Ann.Occ.Hyg.46;242-245


Nanoparticles, ROS and oxidative stress.


Reactive oxygen species production by carbon particles

Stone et al. 1998 Toxicol In Vitro 12: 649-659.

0

2

4

6

8

10

12

1 5 10Particle dose / 290 μl plasmid

Supercoiledband intensity x 103

260 nm CB14 nm CB

* *


Reactive oxygen species produced by nanoparticles

Wilson et al. 2002 TAP 184: 172-179.

50

100

150

0 0.5 1.0 1.5 2.0 2.5

Particle dose (mg/ml)

DC

F Fl

uore

scen

ce In

tens

ity

260 nm CB

14 nm CB***

***

**

-5

5

15

25

35

45

55

64 nm 202 nm 535 nmPolystyrene particle diameter

DC

F Fl

uore

scen

ce In

tens

ity

***

Brown et al. 2001 TAP 175: 191-199.


Effect of carbon particles on intracellular antioxidant content of A549 cells

Stone et al. 1998 TIV 12: 649-659.0.78 μg/mm2

14 nm CB260 nm CBControl

40

50

60

70

80

90

100

110

120

0 2 4 6 8 Time (hours)

GSH (% of control)

*


Effects of Nanoparticles and PM10 on Intracellular Signalling Pathways


Effect of particles on Ca2+ signalling in human MonoMac 6 cells

Stone et al. 2000 Eur Resp J

15: 297-303.

0

20

40

60

80

100

120

140

160

Control 14 nmCB

260 nmCB

[Ca2+]c

2000 sec

**


Ca 2+

THAPSIGARGIN

Ca 2+

Ca 2+VERAPAMIL

0 200 400 600 800 1000 1200

Control

14 nmCB

260 nm CB

[Ca2+]c nM after treatment with thapsigargin

***

Particles Particles + verapamil

The response to thapsigargin in human MM6 cells in the presence of CB and verapamil

Stone et al. 2000 Eur Resp J 15: 297-303.

2000 sec


1500 sec Stone et al. 2000 Inhal Tox 12 (suppl 3): 345-351.

[Ca2+]c nM after treatment with thapsigargin0 100 200 300 400 500 600 700 800 900

Control

64 nm

202 nm

535 nm

***

Pol

ysty

rene

bea

d di

amet

er

The effect of polystyrene nanoparticles on calcium signalling in macrophages


[mM]

Ca 2+

Calciumchannel

[nM]

Ca 2+

Ca 2+BAPTACa chellator

Calmodulin

SignallingW-7

Endoplasmicreticulum

VerapamilCa2+

channel blocker

Calcium Signalling – Inhibitors


TNFα pg/ml

0

50

100

150

200

250

Control 14nmCB

260nmCB

14nmCB

Verap

Verap 14nmCB

BAPTA

BAPTA

Rat alveolar macrophages4 hour treatments

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Brown et al. 2004 AJP 286; L344-L353

The role of Ca2+ in the induction of TNFαexpression by carbon nanoparticles


The role of Ca2+ in the induction of TNFαmRNA expression by CB nanoparticles

GAPDH

TNFαControl 14nm

CB260nm

CB14 CBVerap

Verap 14 CBBAPTA

BAPTA LPS

TNFα mRNA(% GAPDH)

05

1015202530

Control 14nmCB

260nmCB

14nm CBVerap

Verap 14nm CBBAPTA

BAPTA

***

**

Brown et al. 2004 AJP 286; L344-L353


Brown et al. 2004 AJP 286; L344-L353

The effect of antioxidants on CB induced TNFα expression

0

50

100

150

200

250

Control 14nmCB

14nmCB

Nacystelin

Nacystelin

TNFα pg/ml

**

0

5

10

15

20

25

30

Control14nmCB

14nmCB

Nacystelin

Nacystelin

TNFα mRNA(% GAPDH)

******


200

100

150

50

0Control 14 nm CB CB CB CB

CB + verap + BAPTA + W-7 + Trolox

TNFα pg/ml

Particle induced TNFα expression – role of calcium and oxidants

**Human monocyte derived macrophages

4 hour treatments

Brown et al. 2004 AJP 286; L344-L353


Activation of NFκB and promoter binding

Phosphorylation

DNA

Promoter

Nuclear membrane

IκB

NFκB IκB

NFκB

PIκB

NFκB

P

NFκB

mRNA

Degraded

TRANSCRIPTION

P

U


Control

14 nm CB

Immunofluorescent staining of the p65 subunit of NFκB in human monocyte/macrophages

Brown et al. 2004 AJP 286; L344-L353


40

80

120

160

Control CB CB CB CB CB CB + verap + W-7 + BAPTA + Nac + Trolox

Nuclear p65 intensity(% control)

*

Brown et al. 2004 AJP 286; L344-L353

Nuclear localisation of the p65 subunit of NFκB in human monocytes


Nanoparticles, calcium and oxidants

Ultrafine, nanoparticles

Macrophages

Oxidative Calcium stress signalling

Activation of NF-κB

Production of pro-inflammatory proteins


J774 macrophages exposed to 14nm CB

CONTROL

14 nm CB

Wilson et al., manuscript in prep


Twisting field BTW

Fluxgate sensor

Θ

Step twist

6 HzMagnetic particleswithin phagosomes Macrophage

•Magnetic twisting device•Aligned ferromagnetic microparticles ingested by macrophages,•Detection by an array of magnetic fluxgate sensors (Förster device).

Measurement of cytoskeletal stiffness using ferromagnetic particles


0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

14nmCB

EC90 DEP UD

Relaxation b5 (drug/control)

*

*

320 μg/million cells EC90

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6Relaxation b5 (drug/control)

EC90 Verap BAPTA W7

$ $ $*

Impact of particles on the macrophage phagosome transport

Moeller et al., 2003 Manuscript submitted


Effects of nanoparticles and PM10 on migration and phagocytosis


Chemotaxis Chamber

Actual Filter

Filter (5 µm pore)

Chemoattractant

Macrophages (J774.2)

Direction of migration


Chemotaxis of J774 macrophages in response to epithelial cell conditioned medium

50

100

150

200

ZAS 250 nmTiO2

20 nmTiO2

260 nmCB

14 nmCB

Chemotaxis% of Control Particles &

CellsParticles Only

*

Barlow et al. 2005 Particle Fibre Toxicol 2:11.


Chemotactic response of J774 macrophages to epithelial cell conditioned medium

100

120

140

160

180

200

ZAS >30 10-30 5-10 <5

% of Control

Molecular Weight Fraction of Conditioned Medium kDa

Barlow et al. 2005 Particle Fibre Toxicol 2:11.


Chemotactic response of J774 macrophages to serum treated with particles

260nm CB

90

110

130

150

170

190

210

ZAS 1.51 51.51 51.51 5 mg/ml1.51

% o

f Neg

ativ

e C

ontro

l

14nm CB 21nm TiO2 250nm TiO2

5

***

***

Barlow et al. 2005 Toxicol Lett 155:397-401.


Nanoparticle uptake by macrophages


Phagocytosis

Barlow et al. 2006 Submitted.


Effect of TiO2 and CB on phagocytosis

Renwick et al. 2001 Toxicol Applied Pharmacol. 172: 119-127

% of cells that phagocytose >2 beads8h exposure


Non-phagocytic cells

Renwick et al. 2001 Toxicol Applied Pharmacol. 172: 119-127


Cambridge multiwalled carbon nanotube


Nottingham Multiwalled carbon nanotubes


Control

Nottingham

Cambridge

Commercial

Carbon nanotubes


Effects of nanotubes on cell viability

0

5

10

15

20

25

30

35

40

Control E21 3-01 PR19 1-03 9-01 UfCB LFA Triton TNF

Nanotube Treatment

% T

otal

Cel

ls

NecroticApoptotic

15.625ug/ml Nanotubes

0

5

10

15

20

25

30

35

40

Control E21 3-01 PR19 1-03 9-01 UfCB LFA Triton TNF

Nanotube treatment

% T

otal

Cel

ls

NecroticApoptotic

31.25ug/ml nanotubes

16μg/ml

31μg/ml

ApoptosisNecrosis

Nanotubes/fibres CB LFA Triton TNFαControl

% total cells40

30

20

10

0

40

30

20

10

0Nanotubes/fibres CB LFA Triton TNFαControl

% total cells


Effect of nanotubes and nanofibres on phagocytosis

0102030405060708090

Medium E21

PR19Sam

ple 9-0

1Sam

ple 3-0

1Sam

ple 1-0

3

Uf CB

LFA

Treatment

Phag

ocyt

osis

(% T

otal

)

15.625ug/ml31.25ug/ml62.5ug/ml

All treatments induced a significant (p<0.05) inhibition


Summary

Low toxicity, low solubility NP:

•Inflammation is related to surface area dose.

•Generate ROS leading to oxidative stress in epithelial and macrophage cells.

•Induce Ca2+ influx in macrophages in vitro resulting in; •activation of NFκB and AP1•up-regulation of pro-inflammatory cytokine expression•Inhibition of phagosome transport


Summary

Low toxicity, low solubility NP:

•Stimulate production of chemotaxins by epithelial cells.

•Rapidly taken up into macrophages.

•Inhibit subsequent phagocytosis of larger particles

MWCNT:

•Morphology determines uptake by macrophages.

•Inhibit subsequent phagocytosis of E.Coli.


Napier UniversityDr David BrownDr Martin WilsonMarieke TuinmanDr Suzanne PattersonJanis VamvakopolousDr Peter BarlowMartin Clift

University of EdinburghProf Ken Donaldson

University of CambridgeProf Alan WindleDr Ian Kinloch

UIF DusseldorfDr Rodger DuffinDr Roel SchinsDr Paul BormDr Markus Denhart

GSF MunichDr Winfried Moeller

Nottingham UniversityDan WaltersTHE COLT FOUNDATION

http://www.royalsoc.ac.uk/

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The Cellular and Molecular Toxicity of Low Solubility ... · NAPIER UNIVERSITY EDINBURGH The Cellular and Molecular Toxicity of Low Solubility Nanoparticles Prof Vicki Stone Centre