in quest of new fingerprints of exposure to vocs from...

In Quest of New Fingerprints

of Exposure to VOCs from

Consumer Products

Dr Juana Maria Delgado-Saborit

Division of Environmental Health and Risk Management

University of Birmingham, UK

CEFIC-LRI 2010 Innovative Science Award

1. INTRODUCTION

2. HYPOTHESIS AND OBJECTIVES

3. METHODOLOGY

4. PROGRESS UPDATE

5. FUTURE STEPS

6. RESEARCH TEAM

INDEX

INTRODUCTION

1. INTRODUCTION

Are VOC concentrations indoors

relevant to human exposure?

1. INTRODUCTION

CONTRIBUTION OF INDOOR AIR TO PERSONAL EXPOSURES (PE)

TIME SPENT IN MICROENVIRONMENTS

Indoors 80–93%

In Transit –vehicles 1–7%

Outdoors 2–7%

(Harrison et al. 2009, Brunekreef et al. 2005)

MICROENVIRONMENT

CONTRIBUTION TO PE

Indoors 68% - 77%

In Transit 30% - 6%

Outdoors 2% - 3% (Bruinen de Bruin et al. 2008, Harrison et al. 2009)

CAR

FRIENDS' HOUSE

HOME

PUB

RESTAURANT

STREET

OFFICE

OTHERS

INDOOR SOURCE OF VOCs

Consumer Products

1. INTRODUCTION

Can we trace exposures to VOCs

in the human body?

1. INTRODUCTION

PATHWAY OF VOCs IN THE BODY

RESPIRATORY SYSTEM

Inhalation

CIRCULATORY SYSTEM

Distribution

DIGESTIVE SYSTEM – LIVER

Metabolism

RENAL SYSTEM

Excretion

IN

OUT

• Personal

Exposures

• Lung Doses

• Biomarkers of

Exposure

• Biomarkers of

Effect

TRACERS ?

– Searching for the tracers

1. INTRODUCTION

PERSONAL EXPOSURES VS LUNG DOSE

PERSONAL EXPOSURES

Measured at the breathing area

2 – 5 µg/m3 in Europe

LUNG DOSE

Mass of pollutant inhaled in the lungs

100 (70 – 500) µg/day in Europe

Depends on:

• Concentration in the breathing area (PE)

• Duration of exposure

• Minute ventilation

• Age

• Gender

• Fitness

• Level of exertion

• Presence of disease

1. INTRODUCTION

(Nerbert et al 2002; ATSDR 2007)

Biomarkers of

Exposure

?

BIOMARKERS OF EXPOSURE Exogenous substance within the system, the interactive product between xenobiotic

compounds and endogenous components ... related to the exposure.

1. INTRODUCTION

BIOMARKERS OF EXPOSURE

(Rappaport et al. 2010. Chem-Biolog Interact: 189)

(Harrison et al. 2009. HEI)

(Farmer et al. 2005. Chem-Biolog Interact : 97)

HIGH EXPOSURES

LOW EXPOSURES

1. INTRODUCTION

BIOMARKERS OF EXPOSURE

(Nerbert et al 2002; ATSDR 2007)

Biomarkers of

Exposure at low

concentrations

?

www.genome.jp/kegg/pathway/map/map01100.html Image may be subject to copyright.

1. INTRODUCTION

BIOMARKERS OF EFFECT

• Recognise changes in pattern of endogenous metabolites (natural

occurring substances) after xenobiotic exposure

• Connects molecular events to those at the macro level

Xenobiotic (e.g. pollutants) metabolism

might affect other metabolic processes...

... Metabolomics might help identify new

biomarkers...

METABOLOMICS

Indicator of an endogenous component of the

biological system, a measure of the functional

capacity of the system, or an altered state of

the system that is recognized as impairment or

disease.

http://www.genome.jp/kegg/pathway/map/map01100.html

1. INTRODUCTION

Source

Emissions

Micro-

Environment

concentrations

PE exposure Lung Dose

Biomarkers of

Exposure

Biomarkers of

Effect

Human

Exposure Health Effects

MARKERS

Source Emissions

Transport & Transformation

Human

Exposure

Bio-

availability

METABOLISM

Accumulation

Transformation

Elimination

Early

Expression

of Disease

Altered

Function

Clinical

Disease

1. INTRODUCTION

EXPOSURE-EFFECTS CONTINUUM

Micro-

Environment

concentrations

VOC indoors

PE

exposure

VOC PE

Lung

Dose

VOC

inhaled

Biomarkers of

Exposure

U-BZ

1,4-BQ

EXOGENOUS

Biomarkers of

Effect

Metabolic

Profiles

ENDOGENOUS

HYPOTHESIS AND

OBJECTIVES

Source Emissions

Transport & Transformation

Human

Exposure

Bio-

availability

METABOLISM

Accumulation

Transformation

Elimination

Early

Expression

of Disease

Altered

Function

Clinical

Disease


HYPOTHESIS: EXPOSURE-EFFECTS CONTINUUM

The general population is exposed to different VOC emitted from

consumer products and building materials.

Doses of inhaled VOCs are metabolised producing biomarkers of

exposure (exogenous) and effect (endogenous), that can be

detected from urine samples


Characterise VOC concentrations in indoor microenvironments

relevant to personal exposures (home and workplace)

OBJECTIVES

Characterise personal exposures to a common range of chemicals

used in consumer products and building materials

Model inhalation doses and personal exposures using

microenvironment concentrations and subject information

Characterise biomarkers of exposure to low-level VOC

concentrations

Discover biomarkers of effect using metabolomics

MARKERS

Micro-

Environment

concentrations

VOC indoors

PE

exposure

VOC PE

Lung

Dose

VOC

inhaled

Biomarkers of

Exposure

U-BZ

1,4-BQ

EXOGENOUS

Biomarkers of

Effect

Metabolic

Profiles

ENDOGENOUS

METHODOLOGY

WP2:

MicroEnvironment

Sampling

WP 5: Urine

Sampling

WP3: PE & ME

VOC analysis

PE

concentration

3. METHODOLOGY

WP1:Subject

Recruitment

WP 2: Personal

Exposure Sampling

WP2: PE & ME data

collection

WP5:Metabol-

omic analysis

WP5:Quinone

urine analysis

WP5: VOC

urine analysis

ME

concentration

WP 4:

Lung

Dose

Biomarker of

Exposure

Biomarker of

Effect

WP 7: Data

Analysis

QA/QC

WP 8:

Dissemination

PROGRESS UPDATE

WP2:

MicroEnvironment

Sampling

WP 5:Urine

Sampling

WP3: PE & ME

VOC analysis

PE

concentration

WP1:Subject

Recruitment

WP 2: Personal

Exposure Sampling

WP2: PE & ME data

collection

WP5:Metabol-

omic analysis

WP5:Quinone

urine analysis

WP5: VOC

urine analysis

ME

concentration

WP 4:

Lung

Dose

Biomarker of

Exposure

Biomarker of

Effect

WP 7: Data

Analysis

QA/QC

WP 8:

Dissemination

4. PROGRESS UPDATE

SUBJECT RECRUITMENT

GROUP 1 (N=15)

Occupationally

Exposed

GROUP 2 (N=15)

Medium – VOC

exposed

GROUP 3 (N=15)

Control

• Workers from steel foundries, oil storage

depots, petrol station attendants,

motor mechanics, bus drivers.

• “Birmingham is the city of all trades”

• Subjects living / working in new or

redecorated / refurbished buildings.

• New Hospital, Muirhead tower

• No occupationally exposed subjects, nor

living/working in new or redecorated buildings.

• Big database of subjects from previous studies

4. PROGRESS UPDATE

http://www.abcteach.com/free/n/newspaper_bw.jpg

• 24- hour personal sample

• Samples collected

•VOC in sorbent tubes

• PM2.5 in Teflon filters

•PAH, quinones

• BC real time - microAethalometer

• Urine collected the day after

SAMPLING EQUIPMENT

• Group 1 & 3. Sampled once

• Group 2. Sampled twice:

•1st : when building new/redecorated

• 2nd : One year later

SAMPLING STRATEGY • Concurrent Measurement

• PERSONAL EXPOSURE

• HOME

• WORK

• URINE COLLECTION

4. PROGRESS UPDATE

4. PROGRESS UPDATE RECRUITMENT AND SAMPLING

GROUP 1 (N=15)

Occupationally

Exposed

GROUP 2 (N=15)

Medium – VOC

exposed

GROUP 3 (N=15)

Control

0

10

20

30

40

50

60

70

80

90

100

G1 Occupational Exposed

G2 New Buildings G3 Control Group

Perc

en

tag

e o

f Targ

et

(%)

Recruited Sampled

RECRUITMENT AND SAMPLING GROUP 1 (N=15)

Occupationally

Exposed Recruitment Steps

• PETROCHEMICAL INDUSTRY

• Discussions with a UK petrochemical company

• Failed

• Next step – Contact UK Petroleum Industry

Association (UKPIA)

• OTHER INDUSTRIES

• Arizona State University – UoB pilot campaign

• Test Suitability of Gardeners, Mechanics &

Decorators/Painters

4. PROGRESS UPDATE

http://www.asu.edu/

• Goal:

– To test various occupational

exposures

• Equipment:

– Hybrid Device – ASU design

– Shoulder bag

• Procedure:

– Device is given to user to

sample their occupational

exposure for approximately 15

minutes

– Device is taken back by the

researchers and set down for

approximately 45 minutes as

the device runs

The device was fixed in a

bag so the user could

easily carry the device as

they worked.

Inlet

RECRUITMENT AND SAMPLING

Arizona State University / UoB pilot campaign – Occupational Testing

4. PROGRESS UPDATE

• Three different occupational exposures were considered:

– Gardening

– Garage/mechanics

– Painting



4. PROGRESS UPDATE



0

20

40

60

80

100

120

140

160

180

200

Be

nze

ne

Co

nc

en

tra

tio

ns

(p

pb

) 4. PROGRESS UPDATE

G1 Target - Mechanics and Gardeners

Capleton and Levy (2005) Chem-Biolog Interactions 153-54: 43-53

WP2:

MicroEnvironment

Sampling

WP 5: Urine

Sampling

WP3: PE & ME

VOC analysis

PE

concentration

WP1:Subject

Recruitment

WP 2: Personal

Exposure Sampling

WP2: PE & ME data

collection

WP5:Metabol-

omic analysis

WP5:Quinone

urine analysis

WP5: VOC

urine analysis

ME

concentration

WP 4:

Lung

Dose

Biomarker of

Exposure

Biomarker of

Effect

WP 7: Data

Analysis

QA/QC

WP 8:

Dissemination

4. PROGRESS UPDATE

QUINONE URINE ANALYSIS

Expected Range of Concentrations

4. PROGRESS UPDATE

• Non Exposed 0.15 – 4.3 µg/mL (HQ)

• Exposed 0.26 – 47 µg/mL (HQ) Kim et al (2006) Carcinogenesis 27 (4): 772-781

Standard preparation

• BQ & BQ-d4 in ethanol

• 5 calibration standards ranging 0.1 – 200 µg/mL (BQ) & 200 µg/mL (BQ-d4)

• Matrix – artificial urine (Putnal (1971) NASA report)

Proposed methods

• Reversed-phase Solid Phase Extraction

• Liquid – Liquid Extraction


Reversed Solid Phase Extraction

4. PROGRESS UPDATE

Column selected C-18 Paci et al (2007) Biomarkers 14 (7): 502-512

SPE extraction Test 1

• Condition the column with acidified methanol (1% acetic)

• Rinse the column with acidified water (1% acetic)

• Load the column with the sample

• Rinse with acidified water

• Elute with methanol

No results – possible explanations:

• The phenol from the artificial urine is an interfering peak with the BQs

• The phenol interacts with the column, displacing the less hydrophobic

compounds (e.g. BQ)

• The ethanol in which the standards are dissolved affects the BQ – HQ

equilibrium


BQ – HQ equilibrium in different solvents

4. PROGRESS UPDATE

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Pentane DCM Ethanol Nonane Ethyl Acetate

BQ – HQ equilibrium Percentage of response

% of HQ % of BQ

-

20,000,000

40,000,000

60,000,000

80,000,000

100,000,000

120,000,000

140,000,000

160,000,000

Pentane DCM Ethanol Nonane Ethyl Acetate

BQ – HQ equilibrium Response

HQ Response/Weight BQ Response/Weight


Solid Phase Extraction

4. PROGRESS UPDATE

SPE extraction Test 2

• Standards are prepared with BQ or HQ in water and spiked in artificial urine

• Condition the column with ethyl acetate (pure, not acidified)

• Rinse the column with acidified water (0.1% HCl)

• Load the column with the sample (with or without acidification)

• Rinse with acidified water (0.1% HCl)

• Elute with ethyl acetate

0

2,000

4,000

6,000

8,000

p-benzoquinone Hydroquinone

Resp

on

se

SPE Artificial Urine

BQ Standard HQ Standard

0

5,000

10,000

15,000

20,000

25,000


Resp

on

se

SPE Acidified Artificial Urine



Liquid- Liquid Extraction

4. PROGRESS UPDATE

Standards are prepared with BQ or HQ in water and spiked in artificial urine

Extraction of free metabolites

• Add ethyl acetate

• Shake 5 min

• Recover the ethyl acetate

• Dry with Na2SO4

0

1,000,000

2,000,000

3,000,000


Resp

on

se

L-L Artificial Urine


0

400,000

800,000

1,200,000

1,600,000


Resp

on

se

L-L Acidified Artificial Urine



Extractions from Real Urine

4. PROGRESS UPDATE

0

8,000

16,000


Re

sp

on

se

L-L Real Urine

0

2,500

5,000


Res

po

nse

L-L Acidified Real Urine

0

1,000

2,000


Res

po

ns

e

SPE Real Urine

0

400

800


Res

po

nse

SPE Acidified Real Urine

FUTURE STEPS

5. FUTURE STEPS SAMPLING

GROUP 1 (JAN-APR ’12)

• RECRUITMENT

• Contact UKPIA

• Contact Grounds Maintenance Service at UoB (target gardeners)

• Contact local Car Garages (target mechanics)

• SAMPLING

• Sampling scheduled for February – April ‘12

GROUP 2 / 3 (ongoing)

• RECRUITMENT

• Include new channels to advertise the recruitment (local newspaper)

• SAMPLING

• Continue sampling of Group 2 (new volunteers) and 3

• Repeat sampling of Group 2 (former volunteers) – August’12- onwards

ANALYSIS - ENVIRONMENTAL SAMPLES • Environmental Samples Analysis : May – Jun’12 & Feb-Mar’13

BQ-HQ LIQUID- LIQUID EXTRACTION (DEC-MAR ’12)

• FIRST STEP

• Redo synthetic urine removing phenol (old NASA recipe)

• Redo standards and internal standards dissolved in water (not ethanol)

• Ascertain the HQ-BQ partitioning at the extraction conditions

• Prepare calibration curves with Std spiked in phenol-free artificial urine

• Re-Test the method in real urine

• Extract real urine

• SECOND STEP

• Recover the conjugated HQ – heat 100oC for 1 h to hydrolise the conjugates

5. FUTURE STEPS URINE ANALYSIS AND METHOD DEVELOPMENT

U-BZ HS-GCMS ANALYSIS (APR-JLY ‘ 12)

• Apply the method of Fustinoni et al (2010) Chem-Biol Interactions

METABOLOMICS DEVELOPMENT ( SEP-DEC’12)

• NMR analysis Viant et al (2007) Metabolomics

RESEARCH TEAM

RESEARCH TEAM

Dr. Peyton Jacob III (UCSF)

Researcher specialist in

urinary biomarker analysis

Prof Roy Harrison (UoB)

Head of Division Env Health & Risk Manag.

UK NERC Env. Poll & Human Health Theme Leader

Dr. Mark Viant (UoB)

Director of UK NERC Biomolecular

Analysis Facilities Metabolomics

ADVISORY BOARD

Dr. Juana Mari Delgado-Saborit

Research Fellow

Research Coordinator

Mr. Massimiliano Mascelloni

PhD student – 2010 LRI Award funded

Urine Analysis and Method Development

Mrs. Barbara Macias Hernandez

PhD student – Mexican Government funded

Sampling and Environmental Analysis

Thanks for your attention

Thanks to CEFIC for the 2010 LRI Innovative

Science Award http://www.gees.bham.ac.uk/research/projects/airtoxics/index.shtml

in quest of new fingerprints of exposure to vocs from...

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