Non-Invasive Methods for Biomonitoring Trace Element Exposure
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11
RSC TOXICOLOGY GROUP and
SOCIETY FOR BROWNFIELD RISK ASSESSMENTCurrent Issues in Contaminated Land Risk Assessment - 2013
Dr Chris Harrington
Deputy Director
SAS Trace Element Laboratory,
Royal Surrey County Hospital
Non-Invasive Methods for
Biomonitoring Human Exposure to
Trace Elements
2
Human Health Effects
3
Biomonitoring Model
3
Two classes of biomarker can be measured:
Biomarkers of exposure and biomarkers of effect.
55
Biomarkers of Exposure
• Random urine: guidance values for occupational
exposure and defined species eg organic Pb.
Requires creatinine correction.
• Venous blood: guidance values for specific elements
(Ag, Cd, Hg, Mn and Pb).
• Hair: reference ranges available for some
populations. Provides a timeline of exposure.
Problems with surface contamination and cosmetic
treatments.
• Nails: similar to hair. Toe-nails better than finger-nails
as less affected by contamination.
• Zinc protoporphyrin in blood: blood drop from finger-
tip. Screening method for Pb and iron-deficiency
anaemia.
66
Study One: ArsenicDietary Effects and Ethnicity
77
Objectives
Small scale preliminary study looking at:
Control groups from Leicester.
As in hair, urine and nails.
Development of life-style questionnaire.
Methods for cleaning hair and nails
prior to analysis.
Total As and As speciation by ICP-MS
and HPLC-ICP-MS.
8
Results 1.
Total As concentration in
urine, hair and fingernail of
three ethnic groups:
Asian (n = 21), Somali (n =
22) and White (n = 20).
(A) Total As concentration
(μg/g creatinine) in urine.
(B) Total As concentration
(μg/kg) in hair and
fingernail.
E. I. Brima, P. I. Haris, R.O. Jenkins, A. G. Gault, D. A. Polya, C.
F. Harrington. Understanding arsenic metabolism through a
comparative study of arsenic levels in the urine, hair and
fingernails of healthy volunteers from three unexposed ethnic
groups in the United Kingdom. Toxicology and Applied
Pharmacology, Vol. 216, 122-130, (2006).
9
Results 2.
Proportions of As species in the
urine of three ethnic groups.
Differences are predominantly
related to dietary exposure: all
participants refrained from
seafood for 3 days prior to
sampling.
Higher proportion of DMA in
Somali urine could relate to
higher protein intake which
promotes methylation.
1010
Study Two: ArsenicDevon Great Consols, Cornwall
1111
Objectives
Small scale preliminary study looking at:
Exposure to As from abandoned mine site.
As in human hair, urine and nails.
Use of worms as sentinel organisms.
Use of bioaccessible fraction.
Used preparation methods and
questionnaire from previous study.
Total As and As speciation by ICP-MS and
HPLC-ICP-MS.
12
Total As (mg/kg)
255 - 289
331- 439
913 - 1005
1564 - 2980
5141 - 12466 0 0.50.25 Kilometers'
Devon Great Consols, Cornwall
Devon Great Consuls
Bath
Exeter
Taunton
Swansea
Bristol
Weymouth
Plymouth
Penzance
CheltenhamCarmarthen
´0 25 50 75 10012.5Kilometers
Nottingham garden
used as control site.
•Total As in soil
determined via ICP-MS
following acid digestion.
•Bioaccessible fraction
by PBET.
•SGV 32 – 640 mg/kg
*
13
Devon Great Consols: Sampling
14
Pathways of Exposure to Contaminated Soil
1. Inhalation
2. Dermal uptake
3. Ingestion- Soil particles adhere to
vegetables
- Geophagia
- Hand-to-mouth activities
(‘Pica’ Children)
15
Unified Bioaccessibility Method
Stomach
Phase
Stomach + Intestine
Phase
Bioaccessible = Maximum concentration of arsenic available
for gastro-intestinal absorption.
(Bioavailable – Contaminant fraction that reaches the systemic
circulation).
16
Results: Stomach vs. Intestine
Sample site Total As (mg/kg-1)
Stomach Only
Stomach + Intestine
Bioaccessible As (%)
1 2 3 5 6 7 9
10 11 12 13 15
Control
3058 1564 996 238
11939 313 272 452 282 5232 2870 876 15.4
578 298 308 44.2 340 94.8 60.9 43.8 36.5 1595 848 158 4.6
588 303 329 36.8 1603 73.0 72.2 40.5 35.3 1116 690 113 6.7
19.2 19.4 33.1 18.5 13.4 30.3 26.6 9.68 12.9 30.5 29.6 18.1 43.4
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Toe-Nail Levels: DGC
Exposed Group Control Group
Mean±SD min max Mean±SD min max
Age 46 ± 26 11 67 41 ± 13 25 55
Male/Female (n) 5/3 6/3
Time outdoors (hr/wk) 11 ± 7 3 21 5 ± 2 2 10
Toenail As (ug/kg) 5406 858 25981 122 73 273
Exogen. TN As (ug/kg) 506 102 3784 4.0 2.1 13
Total conc. As in toe-nail as a biomarker of exposure.
Levels much higher in participants living near to DGC.
Small cohort sizes.
18
Publications From this Project
M.J. Watts, M. Button, T.S Brewer, G.R.T. Jenkin and C. F. Harrington. The speciation of arsenic in two species of
earthworms from a former mine site. Journal of Environmental Monitoring, Vol 10, 753-759, (2008).
M.J. Watts, M. Button, T.S Brewer, C. F. Harrington and G.R.T. Jenkin. Toenails as a biomarker of exposure to
elevated environmental arsenic levels in residents of an abandoned mine site, Devon, UK. Journal of Environmental
Monitoring. Vol. 11, 610-617. (2009).
Mark Button, Mark Cave, Chris F. Harrington, Michael J. Watts. Earthworms and in vitro physiologically based
extraction tests: complimentary tools in a holistic approach towards understanding risk at arsenic contaminated sites.
Environmental Geochemistry and Health, 31, 273-282, (2009).
Button M, Jenkin GRT, Bowman, KJ, Brewer TS, Harrington CF, Jones GDD and Watts MJ (2008). Assessment of
resistance to arsenic in earthworms from genotoxic contaminated soils using the Comet assay. Mutation Research:
Genetic Toxicology and Environmental Mutagenesis, 696, 95-100, (2010).
1919
Study Three: MultielementalPanasqueira Mine, Portugal
Panasquei
ra Mine
Castelo
dam
20
Objectives
Investigate the effects on population health caused by mine
waste contamination
using a multistage approach
integrate different biomarkers
a better characterization of the risk
Three groups: control (n = 35); occup. exp. (n = 34); environ. exp (n = 33).
21
São Francisco de
Assis village
Geochemical sampling
campaign undertaken
in the vicinity of São
Francisco de Assis
village
Soil samples (when compared to local
background)
As - 36x
Cd - 4x
Stream sediments (when compared to
local background)
As – 117x
Cd – 23x
Mining
site
Village
22
- Genotoxicity:
- T-cell receptor (TCR) mutation assay
- Micronucleus (MN)
- Chromosomal Aberrations (CA)
- Comet Assay
- Immunotoxicity-> Lymphocyte subset frequency
- Susceptibility-> genetic polymorphisms (enzymes
involved in the metabolism of metal(loid)s and
DNA repair)
Biomarkers of Effect
23
COLLECTED SAMPLES / METHODS
Blood
Urine
Nails
Hair
Digestion in
microwave
vessels with
HNO3 (conc.)
GFAAS and ICP-MS analysis
ICP-MS analysis
ICP-MS and
ICP-OES
analysis
24
RESULTS – ICP-MS
Confounding factors
Gender
- Cd in WB (↑ in males - E.E.)
- Pb in WB (↑ in males - C. and E.E.)
Age
- As in U
- Hg in U
- Se in U
- Se in FN - ↑ younger individuals
Smoking Habits
- Cd in WB (↑ in males - E.E.)
↑ older individuals
RESULTS – ICP-MS
C. vs. E.E. E.E. vs. O.E. C. vs. O.E.
As↑ WB*, FN* &
TN* - ↑ FN*
Cd ↑ FN* ↓ FN*,↑ H* ↑ H*
Cr ↑ WB** ↓ WB** -
Hg ↓ TN** & H* ↑ TN* & H* ↑ H*
Mn - ↓ U*, ↑ H* ↑ H*
Ni - - -
Pb - ↑ WB* & H** ↑ WB*
Se - ↓ FN* -
* p<0,05 * *p<0,001
C. – Contol Group
E.E. – Environmental Exposed Group
O.E. – Occupational Exposed Group
WB – Whole Blood
FN – Fingernails
TN – Toenails
H – Hair
U – Urine
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RESULTS – ICP-MS
Correlations between elements & matrices
Significant correlations between:
• different elements / same matrix
• same element / different matrices
• different elements / different matrices were found for
the majority of the elements showing a good synergy
between these biomarkers.
Elements concentrations vs. Reference/Published ranges
Several elements exceed the reference ranges for
WB and U samples, and for FN, TN and H the
published ranges for non-exposed populations.
27
Initial Conclusions
Preliminary results for the biomarkers of effect:
• Increased MN frequency -> genotoxicity
• Elevated TCR mutation frequency -> mutagenicity
• Alterations in the percentages of lymphocytes subsets ->
immunotoxicity
Related to the metal(loid) contamination
from Panasqueira mine activities
FURTHER DATA ANALYSIS IS
UNDERWAY
Increased risk of effects on health
28
Publications From this Project
Patrícia Clara dos Santos Coelho, Solange Costa, Susana Silva, Alan Walter, James Ranville, Ana Sousa, Carla da
Costa, Marta Isabel Correia Coelho, Julia García-Lestón, M. Ramiro Pastorinho, Blanca Laffon, Eduardo Pásaro
Mendez, Chris Harrington, Andrew Taylor and João Paulo Teixeira. Metal(loid) levels in biological matrices from
human populations exposed to mining contamination - Panasqueira Mine (Portugal). Journal of Toxicology and
Environmental Health, Part A. 2012, 75(13-15), 893-908.
P. Coelho, S. Costa, C. Costa, S. Silva, A. Walter, J. Ranville, M.R. Pastorinho, C. Harrington, A. Taylor, V. Dall'Armi,
R. Zoffoli, C. Candeias, E. Ferreira da Silva, S. Bonassi, B. Laffon J.P. Teixeira. Impact of Panasqueira mine
activities on populations environmentally and occupationally exposed – quantification of several metal(loid)s in
different biological matrices. Environmental Geochemistry and Health, published on-line.
All publications relating to this work are available to download from Research Gate :
www.researchgate.net/profile/Chris_Harrington/?ev=hdr_xprf
2929
Future Work: Vulnerable populations
Ethically unacceptable to perform wide
scale biomonitoring on children using
invasive methods.
3030
Acknowledgements
All colleagues and collaborators who helped with the
different studies:
Study One: De Montfort University Studentship
(Dr Eid Brima).
Study Two: British Geological Survey, NERC
Award (Dr Mark Button).
Study Three: FCT Portugal (Dr Patricia Coelho).
Current work: NHS for on-going instrument time.
All publications relating to this work are available to
download from Research Gate : www.researchgate.net/profile/Chris_Harrington/?ev=hdr_xprf