19/04/2016

Investigation of air pollution

biomarkers for use in the SCAMP study

Kings College London

Rosamund Dove

Project Overview:

Theme 4, Project 2: Neurocognitive and behavioural impacts of traffic derived pollutants in

children.

Ian Mudway (King’s); Mireille Toledano (Imperial); Karen Exley, PHE

• Investigate the potential of urinary metal concentrations as biomarkers of

traffic exposure

• Investigate the impact of air pollution on cognitive development during

adolescence

Dispersion models are now available for 2014 and 2015; these models can be used to estimate annual exposures

(PM2.5, PM10, NOx and NO2) for the 12 months prior to cognitive assessment (SCAMP - Theme III/Project 2 –

Electromagnetic fields; Mireille Toledano (Imperial), Simon Bouffler (PHE))

Background – Cognitive effect?

LIFE

Long term

BIRTH

Early LifePrenatal

• IQ (Perera 2009)• “Cognitive

dysfunction” (Jedrychowski 2015)

• Autism (Wong 2015)

• “cognitive performance” (Wang 2009, Freire 2010, Suglia 2008, Harris 2015, Calderon-Garciduenas 2011)

• ADHD (Newman 2013, Siddique 2011)• Autism (Becerra 2013, Roberts 2013, Volk

2011, 2013)

• “cognitive function” (Schikowski2015, Weuve 2012, Power 2011, Ranft 2009, Porta 2015, Sunyer 2015)

• Stroke (Kettunen 2007)• Depression (Banarjee 2012, Lim 2012)• Dementia (Chang 2014, Oudin 2015)• Alzheimer’s (Oudin 2015)• Parkinson’s – (Ritz 2015)

Sensory

Language

Higher

cognitive

functions

Cognitive Impact?• Verbal/non-

verbal

intelligence

• Processing

speed

• Working

memory

• Short-term

memory

• Sustained

attention

• Cognitive

flexibility

• Visual-motor

integration

• Fluid reasoning

• sample size

• co-variates

(noise, diet etc),

• Longitudinal

study design

• PM10

• PM2.5

• NO2

• NOx

• Black carbon

• Ozone

• CO

• Distance to

major road

• Home/school;

indoor/outdoor

Background – Cognitive effect?

SCAMP – Study Design

Baseline (Year 1/HPRU Year 2) and Follow-up (Year 3/HPRU Year 4)

Exposure

modelling

(NO2, NOx,

PM10, PM2.5)

SCAMP team (school visits)

• Dr. Gemma Knowles (Imperial)

• Charlotte Fleming (Imperial)

• Irene Chang (Imperial)

• Dr. Rosamund Dove (KCL)

• Mark Ellis (Imperial)

• Rosi Hirst (Imperial)

Parent mailing

SCAMP Cohort = All Year 7 pupils from participating schools (n = ~5500)

School assessment

Child online surveyParental consent Cognitive ability, mobile phone

use, and lifestyle

Medical history, lifestyle,

demographics Routine records:

1. Educational

achievement

2. Traffic data

3. Health data

Parent online

survey

Subset of the SCAMP

cohort (~200)In-depth personal

radiofrequency exposure

monitoring (opt-in)

Traffic data

Smartphone ‘apps’

Personal & environmental measurements

QuestionnairesActivi

ty diary GPS

Biosamples

SCAMP – Personal Monitoring

To take place during

SCAMP year 2/HPRU

Year 3

Theme 3,

Project 2:

• Stress

biomarkers

• ETS

• hormones

Theme 4,

Project 2:

• Oxidative

stress

• Urinary

biomarkers

Biomarkers

Response biomarkers

(generic)• Oxidative stress: 8-Oxo-2'-

deoxyguanosine, 8-isoprostane

• Systematic inflammation: IL-6, CRP

• Neuroinflammation/injury: neuron specific

enolase, S100B

Exposure

biomarkers• PAHs and hydroxylated PAHs

• Metals

• Cotinine

• Chemical entity that demonstrates a gradient with respect to

traffic

• Stable/readily measurable in accessible biofluids

• Should align with established exposure metrics

• High throughput/low cost

• Sensitive/specific methodology

• Reproducible

Biomarkers

PM10, PM2.5, PNC, black

carbon

PM10, PM2.5,

NO2, NOx

EXHALE

Proxy personal monitoring data

Urinary metals - (2008-2011)

Modelled exposure(using home

address)

Urinary metals - (2012)

Metal Analysis – (ICPMS)

• Brake-wear – Ba, Cu, Sb

• Mechanical wear – Fe, Mn,

Sn, Zn

• Exhaust wear – Cr, Mo, Ni,

Pb, V

Exposure Biomarkers

Association of urinary Fe with

modelled pollutant exposures

Roadside PM10 Fe (2008/9) • Modelled annual exposure

(20x20m resolution) based

on residential address.

• Shorter duration exposure

estimated based on

NOWCAST (24 h and 7

days pre-visit) adjustment of

the annual model.

• Generalized linear latent and

mixed model assuming a

gamma distribution with log

link

• Adjusted for air pollutant,

sex, ethnicity, ETS and

study year with a random

effect for school

Exposure Biomarkers

Association of urinary Cu

with modelled pollutant

exposures

Roadside PM10 Cu (2008/9)

Exposure Biomarkers

Roadside PM10 Mn (2008/9)

Association of urinary Mn

with modelled pollutant

exposures

Biomarkers – Personal Exposure

Children’s routes

were selected based

on GIS modelling of

shortest route

between the school

and the home

address

Frequency of routes

walked

PM2.5

Biomarkers – Personal Exposure

Children’s routes

were selected based

on GIS modelling of

shortest route

between the school

and the home

address

Black Carbon Ultra fine particle No

Future Work

• Analysis of the EXHALE Year 4 samples will be completed in April 2016.

• 3rd data set – Airway Cells and Air Pollution (ACAP) study examined the effect

of PM on airway macrophages and dendritic cells in children aged 8-14 years.

Sputum samples were analysed for the presence of black carbon in

macrophages. Urinary metal analysis will completed April/May 2016

• Personal monitoring and sample collection for Year 2 of the SCAMP study is

ongoing. Analysis of these samples is expected to begin October/November

2016

• Hydroxylated PAHs – method development for determination in low volume

samples based on solid phase extraction, dansyl chloride derivatization and

liquid chromatography-tandem mass spectrometry detection. (Yao L et al.

Anal. Methods, 2014, 6, 6488)

Acknowledgements

KCL:

• Frank kelly

• Ian Mudway

• Heather Walton

• Chrissi Dunster

• Ana Oliete

Dominguez

• Andrew

Cakebread

• Asra Alwandi

ACAP:

• Prof Jonathan

Grigg

• Dr Abigail

Whitehouse

SCAMP Team

Imperial:

• Mireille Toledano

• Paul Elliott

• Charlotte Fleming

• Irene Chang

• Will Mueller

• Iroise Dumontheil (Birkbeck)

• Michael Thomas (Birkbeck)

• Martin Röösli (Swiss TPH)

19/04/2016

Response of human airway epithelial cells to

diesel exhaust particles and cerium dioxide

and silver nanoparticles

Chang Guo (PHE), Kirsty Meldrum (IC/PHE), Lareb Dean (IC),

Martin Leonard (PHE), Rachel Smith (PHE), Terry Tetley (IC)

Theme IV – Project 4

Health Impacts - Nanoparticles

Introduction

• Health impact of nanomaterials – to understand the potential health effects of

nanoparticle exposure and to address the knowledge gaps

• Animal models used but have disadvantages as well as advantages

• In vitro modelling is useful to identify pathways and therapeutic targets

• Use of novel “human” organotypic experimental models becoming more relevant

Theme IV – Project 4

Health Impacts - Nanoparticles

Progress FY15/16

• Milestone 1 – The ALI-AE system/protocols have been modified/developed to

facilitate robust operation. A549 cells, human primary small airway epithelial

(HPSAE) cells and organotypic reconstituted 3D HPSAE cultures have been

exposed to AgNPs and characterised for adverse effects (general cytotoxicity

and expression of select mRNAs/miRNAs).

• Milestone 2 – In vitro system to explore the effect of NPs on allergic effects in

development - initially maintaining ALI control cultures of organotypic

reconstituted 3D human bronchial epithelium.

• Milestone 3 - In vitro 3D co-culture model of the human alveolar unit (comprising air

and blood compartments) established, characterised and development completed.

• Milestone 4 - Consolidated studies of the effects of carbon nanotubes on cell

viability, oxidative stress, inflammation, and pro-thrombotic mediators.

Project 4 – Milestone 1

• The increasing use of silver nanoparticles (AgNPs) in consumer products

has raised concerns about potential toxic effects through inhalation.

• Our previous in vivo study indicates the small airways are important for

AgNP exposure.

• Develop exposure system combined with optimised human cultures to

provide important toxicity relevant information.

ALI-AE System(Air Liquid Interface - Aerosol Exposure System)

AgNP aerosol

deposition device

in vitro cell models

Cell culture media

Precipitator module

Charger module

ALI-AE System Schematic

Exposure conditions:Electrostatic charger voltage: -150 V

Deposition efficiency: ~40%

Cell temperature: 37 ºC

Delivered gas CO2 content: 5%

Delivered gas O2 content: 20%

Delivered gas relative humidity: >80%

0.005lpm 0.005lpm

0.005lpm

1lpm

Gas monitors:

RH=>80%

O2=20%

CO2=5%

24mm Transwell inserts

AgNP aerosol deposition checked by

Laser Ablation ICP-MS

12mm Transwell inserts

Ag

10

7A

g 1

07

Diameter (mm)

Diameter (mm)

+AgNPs + Air

6.5mm Transwell inserts

AgNP Aerosol Characterisation and Dose

Group Exposure Length

(mins)

Mass conc.

(µg/m3)

Number conc.

(#/cm3)

CMD

(nm)

GSD Dose

(particles/cell*)

Ag dose 1

(LD)7 180 1.5 x 106 15.5 1.36 ~220

Ag dose 2

(HD)20 180 1.5 x 106 15.5 1.36 ~660

Air 7 - 50 - - 0

Air 20 - 50 - - 0

*Assuming 2 x 106 cells/well

Average number-weighted aerosol size distribution for all exposures measured with an SMPS.

Representative

TEM image of

particles

deposited onto

grids placed at

the bottom of the

wells (with no

cells). Larger

objects are part

of grid structure.

In vitro models used in the ALI-AE system

A549 cells

Small Airway Epithelia

H/E Alcian Blue staining

CC-10 Staining

(marker of Club Cells)

MUC-5AC staining

(marker of Goblet cells)

Small airways

Internal diameter <2mm

Generation >8

Non-cartilagineous

Relevant diseases :

brochiolitis, asthma, COPD

General toxicity of epithelial cells exposed

to AgNP aerosols in ALI-AE system

0

1

2

3

Air AgNPs

24 hrs post exposure

No

rmal

ized

cel

l via

bili

tyN

orm

aliz

ed c

ell v

iab

ility

0

0.5

1

1.5

2

unexposed Air AgNPs unexposed Air AgNPs

6 hrs post exposure 24 hrs post exposure

LD

HD

A549 cells

Small Airway Epithelia (one donor)

*

Selected mRNA and miRNA expression in

epithelial cells exposed to AgNP aerosols

A549 cells

6 hrs

LD

HD

24 hrs

LD

HD

5.08

18

509

58A

lox1

5

1.9

54

76

301

De

fb4

-1.03

39

45

86

4B

atf

-1.6

27

608

045C

hi3

l1

2.3

92

46

84

69C

3

-1.1

71

101

58

9C

elc

10a

-1.6

48

77

95

57C

cl17

-1.8

202

68

44

2C

lec4

a

5.1

61

45

096

2C

cl2

-2.2

97

71

98

24C

elc

5a

-1.3

53

74

28

98C

cl24

1.1

54

27

19

62C

xcl1

-1.4

81

19

076

4C

cl7

-1.7

57

87

55

65C

xcl2

1.1

91

92

77

24C

xcl5

-2.02

46

41

59

1It

gam

-1.4

96

28

507

9D

uo

x1

-1.7

78

48

75

21H

mo

x1

-5.04

55

83

45

9Fa

bp

5

-1.8

11

25

52

58Li

lib

4

-1.5

72

91

25

87Fc

gr2b

-1.4

606

96

87

6M

mp

12

-2.8

36

95

64

66Fo

xp3

-1.7

802

63

24

1Lc

n2

1.2

48

29

89

33M

uc1

1.5

32

47

46

92N

oxo

1

-4.2

605

43

058Ll

ib

-1.1

63

103

16

8M

t1a

1.6

46

91

12

04M

t2a

-2.9

27

49

12

03Sl

c39a

1.7

108

41

67

2Sf

tpd

-1.1

88

42

602

1Sl

c26a

-1.8

31

36

37

84R

ab7b

-1.7

18

905

57

2Ta

c1

-1.4

096

96

77

9R

nas

e9

3.1

85

78

33

73Ta

c4

-9.1

18

39

45

1S1

00a9

1.1

63

19

27

75Tl

r10

-3.04

64

45

039Se

pin

b10

-1.1

58

28

43

21Tn

faf8

-1.8

22

94

77

91O

rm1

-1.4

38

27

79

49Tr

em

1

-1.2

59

62

21

47C

cl22

-3.1

44

86

94

32Tr

em

2

-1.06

93

013

61Sp

p1

-1.5

53

17

301

4C

hia

-4.1

22

29

25

77M

reg

-1.7

47

98

21

59Il

r2

-4.4

58

16

47

2C

ep

55

-2.01

66

53

19m

ir12

98

2.5

49

52

44

91m

ir14

6b

-1.7

38

600

18

9m

ir22

4

-1.1

41

67

309

8m

ir21

24 hrs

HD

Small Airway Epithelia

Alo

x15

De

fb4

Bat

f

Ch

i3l1

C3

Ce

lc10

a

Ccl

17

Cle

c4a

Ccl

2

Ce

lc5a

Ccl

24

Cxc

l1

Ccl

7

Cxc

l2

Cxc

l5

Itga

m

Du

ox1

Hm

ox1

Fab

p5

Lili

b4

Fcgr

2b

Mm

p12

Foxp

3

Lcn

2

Mu

c1

No

xo1

Llib

Mt1

a

Mt2

a

Slc3

9a

Sftp

d

Slc2

6a

Rab

7b

Tac1

Rn

ase

9

Tac4

S100

a9

Tlr1

0

Sep

inb

10

Tnfa

f8

Orm

1

Tre

m1

Ccl

22

Tre

m2

Spp

1

Ch

ia

Mre

g

Ilr2

Ce

p55

mir

1298

mir

146b

mir

224

mir

21

-2.485145696

-1.372825537

2.252008228

1.364345806

-1.166046226

-1.045613486

1.710371124

-1.046524504

-1.428463259

-1.039023095

1.869185915

1.202048617

-1.102204453

1.128706376

1.723235074

-1.121214365

-1.214062688

-1.142229781

2.673690562

1.035631405

1.057389708

1.09017957

1.958289113

1.048984713

1.102792228

1.176931416

2.445641281

1.673126441

-1.005609924

1.327102666

2.426105853

1.284554068

1.414018939

-1.456239172

1.482868051

1.28415847

-1.032868429

-1.005109018

2.140320474

-1.029464188

1.168252888

1.297743773

2.046380908

1.174367029

1.289545631

-1.130463769

1.619601385

1.071380629

1.001724208

1.004343886

1.471610368

1.289044114

-1.05837093

1.151278222

2.039954991

1.354821872

1.052327031

-1.148620578

1.657912887

1.165026675

1.347474235

1.625521308

4.183313531

-1.001605856

1.037868982

1.199571983

2.045289825

1.026582479

1.880766243

1.338837462

1.584156204

-1.230141873

-1.021895275

1.803776389

2.273164385

1.421749839

-2.430285306

1.185384829

2.569043965

-3.061887022

1.034764071

1.159345065

2.093764798

1.132277921

7.786700218

-1.998510833

1.327587854

1.493454502

-1.386660887

1.165865336

1.714695996

1.325271222

1.697022934

1.280948922

-1.153736757

-1.153736757

-1.151307696

1.349065807

2.375417472

1.038059017

-1.461160478

-3.206707836

7.854610461

3.356945276

1.021072457

1.037773552

1.111885699

1.132698684

1.350957666

1.233304076

1.245042353

-1.123805525

-2.120976922

-1.644621317

2.973292506

1.239298363

-1.028013697

-1.172574418

3.106925136

1.214804997

-2.255142814

-2.537732765

2.393173391

1.226625976

-2.729103026

-2.460612146

2.555738153

2.028560514

-1.679977838

-1.824107693

2.507135954

1.088554946

-1.342985189

-1.240666984

5.238212433

1.290698485

-1.768810275

-1.57982454

2.154128153

1.476421077

-1.17811014

-1.28276879

5.050926526

1.461712363

-1.419214198

-1.345806777

3.00689699

1.12021115

-1.441180516

-1.392554688

1.614099319

-1.021454449

-2.911577529

-2.587593681

5.463250401

1.793842874

-1.302768624

-1.002232997

5.061631573

1.355528308

-1.79182945

-1.644878596

2.876206681

1.292727071

-1.296440388

-1.243858539

2.670416766

-1.571115623

-1.016410737

-1.028129865

2.547385959

1.291115261

-1.078969138

1.108417865

1.661554512

1.004345052

1.056937376

1.034587806

1.364670868

-1.061624485

1.024595706

1.412203885

1.597749036

1.188259368

-1.073809951

1.033303915

1.983499067

-1.033690741

-1.229538935

-1.005406092

2.050223806

1.084492081

-2.757774051

1.100221251

1.485606566

-1.019928958

-1.979862603

-1.92977435

-2.383655035

-2.940424196

-1.440349575

-2.667523141

-1.62046781

-1.372818362

-1.120358123

-2.464025542

-1.388162553

-1.529254489

-1.235223873

-2.84264225

-1.321816086

1.063853226

Summary and Future Work

• Finalize optimisation of ALI-AE system.

• Complete exposures of the Small Airway Epithelia.

• Identify key pathways reflective of specific toxicity relevant for human

hazard identification.

• To give a more complete picture of the hazard posed by these NPs.

• Investigate other nanomaterials using the system

Project 4 – Milestone 2

• The increasing use of cerium dioxide nanoparticles (CeO2NPs) as

catalysts within diesel engines

– Have been used as a catalyst since 1999

– Changes what is expelled from the exhaust

– Bus fleets in Newcastle and London

• Using optimised human cultures to investigate potential allergic

effects addition of cerium dioxide NPs to diesel engines may cause

• Aim to determine whether cerium dioxide impacts on DEP-induced

toxicity in a model of primary human bronchial airway epithelium

PE membrane

Transwell insert

Apical chamber

HPBE

Basal chamber

Diesel (NIST 2975)

CeO2NPs (Sigma <25nm)

DEP+CeO2NPs

24 hours

Basal

compartment

mediumApical

compartment

medium

CCL20

IL-6

CCL20

IL-6Human Primary

Bronchial

Epithelial Cells

(HPBECs)

Experimental model DEP, CeO2NPs and

DEP+CeO2NPs exposure, at concentrations

relevant to normal Envirox use.

IL-6 - pleiotropic cytokine; CCL20 – dendritic cell chemoattractant

Basal and apical release of CCL20 (green) and IL-6

(pink) by HPBECs following exposure to DEP,

CeO2NPs and DEP/CeO2NP

DEP

CeO2NP

DEP/CeO2NP

Summary and Future Work

• IL-6 release decreased with exposure of DEP/CeO2NPs when compared to

CeO2NPs alone

• Basal, but not apical, release of CCL20 was blocked on exposure to

DEP/CeO2NPs but not on exposure to DEP or CeO2NPs alone.

• Significant effect of addition of CeO2NPs to diesel fuel on large airway

inflammation and allergic responses?

• Fully characterize HPBEC model

• Study other markers of inflammation and allergy – IL-8, TSLP etc

• Continue to investigate the potential hazards the addition of these NPs may

pose

• Preparation of a review article in progress (nanoparticles and asthma)

Thanks for Listening

Chang Guo (PHE), Kirsty Meldrum (IC/PHE),

Lareb Dean (IC), Martin Leonard (PHE),

Rachel Smith (PHE), Terry Tetley (IC)