Reactive Oxygen Species (ROS) Reactive Oxygen Species (ROS) ––a potential new health relevant metrica potential new health relevant metric

Frank KellyMRC-HPA Centre for Environment & Health

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London London –– summer 2007summer 2007Exposure to particulate air pollution is associated with an average loss of life expectancy of six‐eight months in Europe due to adverse cardio‐respiratory health effects

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Ranking of estimates for allRanking of estimates for all‐‐cause mortality by cause mortality by annual average levels of PM10annual average levels of PM10

Anderson et al, 2004

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Ambient particles carry ‘oxidants’ or components which generate ‘oxidants’

Fe

Ca

CaCa

Cl

Cl

Cl

Cl

Cl

VV

V

VFe

Cu

Cu

Cu

Cu

S

S

SO4.Zn

Zn

Zn

NO3

Fe

Fe

Fe

Fe

CaTransition metals

Quinones, PAHs

Biological material

Hydroxyl radicals

Organic radicals

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PM carries oxidants into the lungPM carries oxidants into the lung

H2O2OH•

O2-•

InterstitiumBloodEndotheliumEpithelium

Respiratory Tract Lining Fluid

AirwayH2O2

OH•OH•

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InterstitiumBloodEndotheliumEpithelium

Respiratory Tract Lining Fluid

Airway

H2O2OH•

O2-•

Lung Defence MechanismLung Defence Mechanism

H2O2

OH•OH•

Low Molecular Antioxidants Ascorbate Urate Glutathione

Antioxidants PM Oxidants

Response magnitude

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37 Co

0h 4hControl

0h 4h 0h 4h 0h 4h

200uM AA/UA/GSH(pH 7.4)

Carbon black

ROFA PM2.5 &10

Artificial lung fluid

Particle Exposure Model

All incubations at 50μg/ml

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PM oxidative potential at various sites across PM oxidative potential at various sites across LondonLondon

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Regional and Temporal Variation in PM2.5 oxidative activity across Europe

Environ Health Perspect. 2006;114:684-90ECHRS

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High Activity Sites

Environ Health Perspect. 2006;114:684-90

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Low Activity Sites

Environ Health Perspect. 2006;114:684-90

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Popular Acellular Models for Measuring OPPopular Acellular Models for Measuring OP

1. Lung lining fluid model – measures both GSH and ascorbate oxidation

2. Thiol Oxidation

3. Ascorbate Oxidation

4. Electron Paramagnetic Resonance

5. Fluorescent Probes – DCFH Oxidation /Profluorescent  nitroxides 

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II. Thiol OxidationII. Thiol Oxidation

Kumagai et al., (2002) Chem. Res. Toxicol. 15: 483.

PM metal and quinone induced ROS

DTT (dithiothreitol) Thiyl

PMn PMn-1

O2O2•-

Unreacted DTT

+ DTNB 5,5’-dethiobis-(2-nitrobenzoic acid) Fluorescent NTB2-

λ= 412 nm

PM incubations at 37 °C 60 minutes

Oxidative Potential = DTT oxidation

[nmol (µg PM)-1]

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III. Ascorbate OxidationIII. Ascorbate OxidationPM induced ROS: metals ONLY

Ascorbate (AA) Dehydroascorbate (DHA)

DiStefano et al., (2009) Inhal. Toxicol. 21: 731.

PMn PMn-1

O2O2•-

H2O2OH•, OH-

+salincylate

+2,5-DHBA

(dihydroxybenzoate)2,3-DHBA

Measure conc loss = non-metal specific AA oxidation

Metal only

Metal andQuinone

Oxidative Potential = DHBA formation[nmol (µg PM)-1]

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IV. Electron Paramagnetic Resonance (EPR)IV. Electron Paramagnetic Resonance (EPR)

Shi et al., (2003) J. Environ. Monit. 5: 550; Valavanidis et al (2006) Environ. Chem. 3:118.

PM bound and metal/quinone induced ROS

EPR

Sig

nal (

Arb

itrar

y U

nits

)

Diesel Exhaust PM

GasolineExhaust PM

Cigarette Tar Extract

PM2.5

PM10

TSP

Diesel Central Heating

Cu2+ Fe2+ Fe3+ Ni2+ Zn2+ V2+ V5+

OH• generation by metal coated PM

• PM bound ROSDMPO + free radical (OH•, O2

•-) → DMPO-OH•

OH• generation by PM spin trapped using DMPO (5,5-dimethyl-1-pyrroline-N-oxide);

Oxidative Potential = EPR spectra measured of DMPO-OH adducts [Arbitrary Units]

EPR

Sig

nal (

A.U

.)• PM metal induced ROS productionH2O2 + Fe2+ → Fe3+ + OH• + OH-

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Va. Fluorescent ProbesVa. Fluorescent Probes

PMn-1

DCFH (2’-7’-dichlorofluorescin)

Fluorescent DCFλ= 525 nm

Peroxid

ase

H2O2

Non-specific ROS mediated oxidation of DCFH

1. PM bound ROS Organic free radicals

2. PM metal induced ROS(+ peroxidase)

OP by DCFH oxidation can be used to indicate:

LeBel et al., (1992) Chem. Res. Toxicol. 5: 227.

•OH

PM bound and metal induced ROS

PMn

PMn

1. 2.

PMn-1

Oxidative Potential = H2O2 concentration

equivalent [nM (µg PM)-1]

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Vb. Fluorescent ProbesVb. Fluorescent Probes

BPEAnit(9,10-bis(phenylethynl)anthracene +

nitroxide ring

λ= 485-513 nm

PM bound ROS Organic free radicals

OP by BPEAnit oxidation can be used to indicate:

Miljevic et al., (2010) Atmos Environ. 44: 2224-30.

PM bound and metal induced ROS

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Measurement of PM Oxidative Potential Measurement of PM Oxidative Potential (OP) with Acellular Models (OP) with Acellular Models 

• Rapid screening technique which serves as a biologically pertinent index of PM toxicity integrating heterogeneities in PM size, surface area and composition into a single metric.

• Acellular  OP models quantify the ability of PM to drive damaging biological oxidation mediated by inherently redox active PM chemical constituents.   

• PM bound reactive oxygen species• Organic free radicals

• PM induced production of reactive oxygen species• Transition Metals• Quinones + other organics

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AcknowledgementsAcknowledgements

• Chrissi Dunster, Sean Duggan, Krystal Godri, Ian Mudway, David Green & Ben Barrett, King’s College London

• MRC• Defra• Department of Health• European Commission• Health Effects Institute (US)

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THANK YOU FOR YOUR ATTENTION!