In-Vitro BioaccessibilityIn Vitro Bioaccessibility Studies of POPs
Renli Ma Jane Entwistle and John R DeanRenli Ma, Jane Entwistle and John R. Dean
Biomolecular and Biomedical Research CentreSchool of Applied SciencesSchool of Applied Sciences,Northumbria University, UK
P i t t O i P ll t t St diPersistent Organic Pollutants Studies- Risk Assessment
Bi ibilitBioaccessibility
• Approaches to simulate the consequences to humans of consuming contaminated gmaterials.– Estimated using simulated in-vitroEstimated using simulated in vitro
gastro-intestinal extraction (also known as the physiologically-based extractionas the physiologically-based extraction test or PBET).POP i l t d il– POPs in plants and soils
Physiology of human contaminant uptake
Mouth 10 s to 2 mins
Stomach 8 mins to 3 hours
Duodenum30 mins to 45 mins Duodenum
Jejunum Small intestines
30 mins to 45 mins
1.5 to 2 hours
Ileum4 to 7 hours
Physiology of the human digestive tract
• Mouth: intake of material followed by mastication (often excluded from PBETmastication (often excluded from PBET process).
• Gastric: mechanical and chemical breakdown of material in the stomach.
• Intestines: Absorption of components through membranethrough membrane.
PBET: Important variablesp• Temperature (37 °C)• Solid to solution ratio (1:2 through to 1:150 w/v)• Solid to solution ratio (1:2 through to 1:150 w/v)• Mixing - how? orbital or horizontal; stirred; inert
atmosphereatmosphere• Incubation time (1 to 5 hours)
Additi f f d t i l ilk d• Addition of food materials e.g. milk powder• Enzymes used (gastric juice – pepsin 1.25 to 10
/ l d h d hl i id t H f 1 1 t 4mg/ml and hydrochloric acid at pH from 1.1 to 4; intestinal juice – pancreatin and bile salt, pH 5-8)S ti t h i ( t if ti /filt ti )• Separation technique (centrifugation/filtration)
• Extraction: SPE or LLE• Analytical Technique: GC-MS
A current applicationA current application
• Pesticides, phenols and BNAs in soil CRMs
Methodology:1 E tract soil CRMs b PFE and So hlet e traction to1. Extract soil CRMs by PFE and Soxhlet extraction to
obtain baseline data on total POP content.
2 E bli h LLE f i /i i l fl id2. Establish LLE recovery from gastric/intestinal fluids
3. Subject soil samples to PBET.
4. Extract gastric and intestinal fluid phases with LLE; Extract residual fraction with PFE. Produce results.
What is PFE?Load sample into cell + drying agent+ copper?
Fill cell with solvent~0.5 min
Heat (40-200°C) and pressurize (1000-3000psi)
Cell ~5 minCell 5 min
Hold sample at pressureand temperature
Static extraction (5-10 min)
Pump clean solvent into cell (0 min) ll l0.6 x cell volume
Purge with N2 (1-2 min)
Analysis
1. Extract soil CRMs by PFE and Soxhlet extraction to obtain baseline data on total POP content.
25
30
/kg)
Pesticides in CRM 805-050
5
10
15
20
once
ntra
tion
(mg/
PFESoxhletcertificate
0
linda
ne
endo
sulph
an I
endri
n
p,p'-D
DE
p,p'-D
DD
endo
sulph
an II
co
60708090
mg/
kg)
BNAs in CRM 107-100
Phenols in CRM 401-225 0102030405060
conc
entra
tion
(m PFESoxhletcertificate
3000
3500
4000
/kg)
Phenols in CRM 401-225 0
hexa
chlor
oethan
e
acen
apthen
e
dibenz
ofuran
fluore
nehe
xach
loroeth
ane
1000
1500
2000
2500
conc
entr
atio
n (m
g/
PFESoxhletcertificate
0
500
cresol 2,4,6-trichlorophenol PCP
c
2 Establish LLE recovery from gastric/intestinal fluids2. Establish LLE recovery from gastric/intestinal fluids
PBET requires some form of liquid – liquid extraction prior to chromatographic analysis.extraction prior to chromatographic analysis.
Average LLE recoveries (using 3 x 10 mL DCM) f t i d i t ti l fl idDCM) from gastric and intestinal fluids was 88.9 ± 3.3 % (n = 27).
3. Subject soil samples to PBET.
S lSample(5 g soil)
G t i J iGastric Juice[1% w/v pepsin in saline at pH 1.8 (with HCl), shake at
37 °C for 3 hours]37 C for 3 hours]
Intestinal Juice[3% w/v pancreatin and 1% w/v amylase in saline and 0.15% w/v bile
l i li H 7 h k 37 C f 3 h ]salts in saline at pH 7, shake at 37°C for 3 hours]
Pesticides % Recoveries from a soil CRM 805-050 and analysis by LLE/PFE-GC-MS
pesticide Gastric Intestinal ResidualLindane 0 5.3 94.70 5.3 94.7
Endosulphan I 0 6.6 93.4
Endrin 0 6.4 93.6
DDE 0 8.0 92.0
DDD 0 7 9 92 1DDD 0 7.9 92.1
Endosulphan II 0 12.8 87.2
Phenols % Recoveries from a soil CRMPhenols % Recoveries from a soil CRM 401-225 and analysis by LLE/PFE-GC-MS
Phenol Gastric Intestinal Residual
Cresol 2.0 4.1 93.9
TCP 1.6 4.6 93.8
PCP 1.9 5.4 92.7
BNAs % Recoveries from a soil CRM 107-BNAs % Recoveries from a soil CRM 107100 and analysis by LLE/PFE-GC-MS
BNA Gastric Intestinal ResidualHexachlorethane 4.0 9.2 86.8
Acenaphthene 2 5 7 8 89 7Acenaphthene 2.5 7.8 89.7
Dibenzofuran 1 4 6 6 92 01.4 6.6 92.0
Fluorene 3.6 7.4 89.0
Hexachlorobenzene 2.0 12.7 85.3
ConclusionsConclusions• Simulated in-vitro gastro-intestinalSimulated in vitro gastro intestinal
extraction (PBET) is a useful approach to estimate the bioaccessibility of POPsto estimate the bioaccessibility of POPs.
• Majority of POPs present are not bioaccessible (<20%).
• Differences noted between pesticidesDifferences noted between pesticides and phenols/BNAs in gastric fluid.I i l fl id h i• Intestinal fluid the most important.
Future issues (1)( )• Does PBET adequately model the risk to q y
human health?– Are POPs much more complicated than metalsAre POPs much more complicated than metals
to apply to a PBET approach?– Is the current PBET methodology for metalsIs the current PBET methodology for metals
sophisticated enough for POPs?– What needs to be done to improve theWhat needs to be done to improve the
approach?• What about potential POPs – gastric /• What about potential POPs – gastric /
intestinal fluid interactions? (GC-MS is often used in SIM mode)used in SIM mode).
Future issues (2)• No consideration given to the potential for
utu e ssues ( )g p
microbial degradation of POPs in the intestinal phase.p
• Further work is needed to ‘standardise’ on the approaches possible to perform in-vitrothe approaches possible to perform in vitro gastro-intestinal extraction (PBET).How can we obtain reliable data to be able• How can we obtain reliable data to be able to transfer methodology from lab to lab
ith t d fi d t l?without a defined protocol?• Development of PBET CRMs for POPs /
PAHs.
Future issues (3)• Influence of soil parameters (e.g. soil
utu e ssues (3)( g
organic matter, clay content etc.) on bioaccessibility not yet investigated.y y g
• Evaluation of most effective extraction methodology required (LLE versus SPE)methodology required (LLE versus SPE).
• Could the process be modelled using physical constants e g Kow etcphysical constants e.g. Kow etc.
Sponsors of PBET workSponsors of PBET work
• EPSRC Industrial case award in collaboration with LGC limited, London, with support from the Department of Trade and Industry under the National MeasurementTrade and Industry under the National Measurement System Valid Analytical Measurement (VAM) Programme. g
• The Royal Thai Government • Northumbria Universityo u b a U e s y• British Geological Survey
Selected Publications• "Methods for environmental trace analysis", J.R. Dean, John Wiley and Sons, (2003)
• “Recent developments to assess the bioavailability of persistent organic pollutants in the environment” J R Dean• Recent developments to assess the bioavailability of persistent organic pollutants in the environment , J.R. Dean and W.C. Scott, Trends in Analytical Chemistry, 23, 609-618 (2004).
• “An assessment of the bioavailability of persistent organic pollutants from contaminated soil”, W.C. Scott and J.R. Dean, Journal of Environmental Monitoring, 7, 710-715 (2005).Dean, Journal of Environmental Monitoring, 7, 710 715 (2005).
• “Uptake and bioavailability of endosulfan by lettuce grown in contaminated soil”, F.A. Esteve-Turrillas, W.C. Scott, A. Pastor and J.R. Dean, Journal of Environmental Monitoring, 7, 1093- 1098 (2005).
• “Uptake of heavy metals by vegetable plants grown on contaminated soil and their bioavailability in the human gastro-intestinal tract”, M. Intawongse and J.R. Dean, Food Additives and Contaminants, 23(1), 36-48 (2006).
• “In-vitro testing for assessing oral bioaccessibility of trace metals in soil and food samples”, M. Intawongse and g g y p , gJR Dean, Trends in Analytical Chemistry, 25(9), 876-886 (2006)
• “Bioavailability, bioaccessibility and mobility of environmental contaminants”, JR Dean, John Wiley and Sons Ltd., Chichester.
• “Approaches to assess the oral bioaccessibility of persistent organic pollutants: A critical review “, JR Dean and R. Ma, Chemosphere, 68, 1399-1407 (2007)
• “Use of the physiologically-based extraction test to assess the oral bioaccessibility of metals in vegetable plants grown in contaminated soil”, M. Intawongse and JR Dean, Environmental Pollution, in press.
Funding Opportunity - Leverhulme TrustFunding Opportunity Leverhulme Trust
• Calling for expressions of interest in theCalling for expressions of interest in the development and support of international networks for academic collaborationnetworks for academic collaboration. – between one or more UK HEIs and two or
more overseas institutions. • The award will be up to £125k, and last forThe award will be up to £125k, and last for
up to three years. O tli li ti d b tl• Outline applications now and subsequently invited full applications by 21 March, 1 September and 1 December 2008.
