Reactive Oxygen Species (ROS) - AirMonTech...Reactive Oxygen Species (ROS) – a potential new...
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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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7
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!