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Current food?Nanotechnology in the Food ChainNanotechnology in the Food Chain Brussels, Nov. 24/2010Brussels, Nov. 24/2010
MycotoxinsMycotoxins
P ti idP ti id
Heavy metalsHeavy metals AlcaloidsAlcaloids
PesticidesPesticides
PAHPAHDrugsDrugs
PCB & dioxinsPCB & dioxinsCarbohydrates, Carbohydrates, lipids,lipids,proteinsproteins
NanoparticlesNanoparticles?? Vitamins,Vitamins,ii
proteinsproteins
Drugs and Drugs and veterinary veterinary hh
ions, …ions, …
Bioactive Bioactive All All maymayi t ti t t!!hormoneshormones compoundscompoundsinteractinteract!!
Toxicodynamic aspects of nanoparticles in food:
interactions with the intestinal barrierinteractions with the intestinal barrier
Y J S h id A BY.-J. Schneider, A. BazesInstitut des Sciences de la Vie & UCLouvain,L i l N B l iLouvain-la-Neuve, Belgium
A B J G J M t & L P iA. Brasseur, J. Geys, J. Mast & L. PussemierCODA-CERVA, Veterinary and Agrochemical R h C t B l B l iResearch Centre, Brussels, Belgium
<<yjs@uclouvain beyjs@uclouvain be>><<yjs@uclouvain.beyjs@uclouvain.be>>Nanotechnology in the Food ChainNanotechnology in the Food Chain Brussels, Nov. 24/2010Brussels, Nov. 24/2010
Nanoparticles (NPs) in the Food Chain
Nanotechnology in the Food ChainNanotechnology in the Food Chain Brussels, Nov. 24/2010Brussels, Nov. 24/2010
Nanoparticles (NPs) in the Food Chain
"Classical" toxicology: active substances in solution"Classical" toxicology: active substances in solution • Toxicodynamy is based on interactions with receptors,
enzymes, genes, …enzymes, genes, …• Toxicokinetics is based on mass concentrations during
absorption, distribution, metabolism, elimination (ADME)
Nanoparticle toxicology: insoluble substances • Toxicodynamy and toxicokinetics are further based on• Toxicodynamy and toxicokinetics are further based on
surface area, surface chemistry, tissue distribution, …
NPs toxicology
Nanotechnology in the Food ChainNanotechnology in the Food Chain Brussels, Nov. 24/2010Brussels, Nov. 24/2010
NPs toxicology
From Gornati R. et al., in Nanotoxicity , Sahu S.C. & Casciano D.A., Wiley & Sons, 2009
Evaluation of NPs in vivo and in vitro
Nanotechnology in the Food ChainNanotechnology in the Food Chain Brussels, Nov. 24/2010Brussels, Nov. 24/2010
Evaluation of NPs in vivo and in vitro
Main techniquesMain techniques• Physico-chemical properties
Surface area and propertiesSize, aggregates, …Size, aggregates, …Chemical compositionand concentration Lung toxicity of
carbon nanotubes• Biological aspects
Optical microscopy
Muller et al., Toxicol. Appl. Pharmacol.207, 221-231, 2005
Evaluation of NPs in vivo and in vitro
Nanotechnology in the Food ChainNanotechnology in the Food Chain Brussels, Nov. 24/2010Brussels, Nov. 24/2010
Evaluation of NPs in vivo and in vitro
Main techniquesMain techniques• Physico-chemical properties
Surface area and propertiesSize, aggregates, …Size, aggregates, …Chemical compositionand concentration Lung toxicity of
carbon nanotubes• Biological aspects
Optical microscopyConfocal microscopy
Uptake of silicananotubes by cultured breastpy cultured breastcancer cells
Gormley & Ghandehariin Nanotoxicity , Sahu S.C. & Casciano D A
Muller et al., Toxicol. Appl. Pharmacol.207, 221-231, 2005
& Casciano D.A., Wiley & Sons, 2009
Evaluation of NPs in vivo and in vitro
Nanotechnology in the Food ChainNanotechnology in the Food Chain Brussels, Nov. 24/2010Brussels, Nov. 24/2010
Evaluation of NPs in vivo and in vitro
Main techniquesMain techniques• Physico-chemical properties
Surface area and propertiesSize, aggregates, …Size, aggregates, …Chemical compositionand concentration Uptake of Aluminium
NPs by cultured cells• Biological aspects
Optical microscopyConfocal microscopy
NPs by cultured cells
pyTransmission electronmicroscopy
Powers KW et al. in Nanotoxicity , Sahu S.C. & Casciano D.A., Wiley & Sons, 2009
Evaluation of NPs in vivo and in vitro
Nanotechnology in the Food ChainNanotechnology in the Food Chain Brussels, Nov. 24/2010Brussels, Nov. 24/2010
Evaluation of NPs in vivo and in vitro
Main techniquesMain techniques• Physico-chemical properties
Surface area and propertiesSize, aggregates, …Size, aggregates, …Chemical compositionand concentration Uptake of Cobalt
Oxide NPs by• Biological aspects
Optical microscopyConfocal microscopy
Oxide NPs by cultured cells
pyTransmission electronmicroscopyScanning electronmicroscopy Gornati R. et al.
in Nanotoxicity , SahuS.C. & Casciano D.A., Wiley & Sons, 2009
Evaluation of NPs in vivo and in vitro
Nanotechnology in the Food ChainNanotechnology in the Food Chain Brussels, Nov. 24/2010Brussels, Nov. 24/2010
Evaluation of NPs in vivo and in vitro
Main techniquesMain techniques• Physico-chemical properties
Surface area and propertiesSize, aggregates, …Size, aggregates, …Chemical compositionand concentration
AFM of Cobalt OxideNP• Biological aspects
Optical microscopyConfocal microscopy
NPs
pyTransmission electronmicroscopyScanning electronmicroscopyAtomic force microscopy…
Gornati R. et al.in Nanotoxicity , SahuS.C. & Casciano D A& Casciano D.A., Wiley & Sons, 2009
NPs in vivo
Nanotechnology in the Food ChainNanotechnology in the Food Chain Brussels, Nov. 24/2010Brussels, Nov. 24/2010
NPs in vivo
Advantages Advantages • Integrative models• Toxicokinetic parameters• Tissue distribution and histological analysis• Tissue distribution and histological analysis• Acute and chronic toxicity
DrawbacksDrawbacks • Increase in the use of animals• Interspecies variations
Model of in vitro digestion
Nanotechnology in the Food ChainNanotechnology in the Food Chain Brussels, Nov. 24/2010Brussels, Nov. 24/2010
• Food matrix: pure material, enriched feed, spiked solutions
Model of in vitro digestion
1 , p• Salivary digestion: amylase
1
•• GastricGastric digestion: digestion: pepsinpepsin2
•• PancreaticPancreatic digestion: digestion: pancreatinpancreatin and bileand bileI t ti l di tiI t ti l di ti
3
4 •• Intestinal digestion: Intestinal digestion: enterocytesenterocytes brushbrush borderborder
•• MicrobialMicrobial fermentationfermentation
45
•• MicrobialMicrobial fermentationfermentation
PhysicoPhysico chemicalchemical analysisanalysis ofof•• PhysicoPhysico--chemicalchemical analysisanalysis of of nanoparticlesnanoparticles
NPs and intestinal barrier
Nanotechnology in the Food ChainNanotechnology in the Food Chain Brussels, Nov. 24/2010Brussels, Nov. 24/2010
Nanoparticles
NPs and intestinal barrier
Intestinal Flow
MΦ + B & T cells
Adapted from The genetics and immunopathogenesisof inflammatory bowel disease Judy H. Cho Nature Reviews Immunology 8, 458-466 (June 2008)
B & T cells
Tools to investigate the intestinal function
Nanotechnology in the Food ChainNanotechnology in the Food Chain Brussels, Nov. 24/2010Brussels, Nov. 24/2010
Tools to investigate the intestinal function• In vitro models of the
h i t ti lhuman intestinal barrier
UpperUpper compartmentcompartment::apical poleapical pole lumenlumen
•• CacoCaco--2 cell line2 cell linedifferentiation into differentiation into
apical pole apical pole lumenlumen
LowerLowercompartmentcompartment::
MonolayerMonolayer
Ti htTi ht j tij ti
enterocytesenterocytes
EE
compartmentcompartment::basolateralbasolateral pole pole
bloodbloodMicroporousMicroporousfilterfilter •• Bicameral insertBicameral insert
EnterocyteEnterocyteMicrovillositiesMicrovillosities
TightTight junctionsjunctions Enzymes Enzymes •• Bicameral insertBicameral insertabsorption absorption
studiesstudiesTransportersTransporters
In vitro models of human M cells
Nanotechnology in the Food ChainNanotechnology in the Food Chain Brussels, Nov. 24/2010Brussels, Nov. 24/2010
In vitro models of human M cells
• Based on Gullberg et al. work, (BBRC, 2000), a cell Based on Gullberg et al. work, (BBRC, 2000), a cell culture system was optimized
Additi fC 2
3 days21 days14 days
Addition ofRaji cells
Caco-2 cells
Microporous Membrane
21 days14 days
Mixture of proteins(Matrigel®)
d Ri A Fi Vd Ri A Fi V t lt l EE JJ PhPh S iS i 20072007( g )
des Rieux A., Fievez V.des Rieux A., Fievez V. et alet al., ., EurEur J J PharmPharm SciSci. 2007. 2007
• Caco-2 cell conversion assessed by:TEER decreaseTEER decreasefunctionality test: transport of model nanoparticles
In vitro models of goblet cells
Nanotechnology in the Food ChainNanotechnology in the Food Chain Brussels, Nov. 24/2010Brussels, Nov. 24/2010
In vitro models of goblet cells
HT29-5M21 cellsCaco-2 HT29 5M21 cells(mucus secreting)
Caco 2 cells
Mucin detectionCell
Caco-2 cells Caco-2/HT29-5M21monoculture cells coculture
Cellmonolayer
Serum-free mediummedium
Nollevaux et al., BMC Cell Biol., 7, 20-31, 2006
In vitro model of absorption
Nanotechnology in the Food ChainNanotechnology in the Food Chain Brussels, Nov. 24/2010Brussels, Nov. 24/2010
In vitro model of absorption
1•• CacoCaco--2 cells based 2 cells based
culture systemsculture systemsUpperUpper compartmentcompartment::
1
1.1. Addition of digestion Addition of digestion d t t i ld t t i l
UpperUpper compartmentcompartment::apical pole apical pole lumenlumen
LowerLowerMonolayerMonolayer
2
products to apical products to apical polepole
22 Incubation at 37Incubation at 37°°
LowerLowercompartmentcompartment::basolateralbasolateral pole pole
bloodbloodMicroporousMicroporous filterfilter 3 2.2. Incubation at 37Incubation at 373.3. Collect of cell Collect of cell
contentcontent
4
Analysis5
contentcontent4.4. Collect of Collect of basolateralbasolateral
polepole
Analysis
polepole5.5. AnalysisAnalysis
Evaluation of NPs effects in vitro
Nanotechnology in the Food ChainNanotechnology in the Food Chain Brussels, Nov. 24/2010Brussels, Nov. 24/2010
Evaluation of NPs effects in vitro
Acute toxicityAcute toxicity• Cell necrosis• Apoptosis
Chronic toxicity• Inflammation• Oxidative stressOxidative stress• Biotransformation activities• Transport
Gene expression• Transcriptomic• ProteomicProteomic • Apoptosis
Others • …
Evaluation of NPs in vitro
Nanotechnology in the Food ChainNanotechnology in the Food Chain Brussels, Nov. 24/2010Brussels, Nov. 24/2010
Evaluation of NPs in vitro
Advantages Advantages • 3Rs technology ("reduce, refine and replace" animal toxicology)• Mechanistic studies
Drawbacks • Reductionist approach
Further developments • Improved cell culture systemsp o ed ce cu tu e syste s• Validated detection and characterization of NPs
Conclusions: urgent need to evaluate Nanotechnology in the Food ChainNanotechnology in the Food Chain Brussels, Nov. 24/2010Brussels, Nov. 24/2010
possible toxicity of NPs• Use of matrix as close as possible of real food or feedUse of matrix as close as possible of real food or feed• Physical and chemical characterization of NPs
before and after administration: need of appropriate tools
• In vivo approaches ADMETissue distribution and histopathological examination
• In vitro approaches ? f ?Absorption ? Possible modifications ?
Molecular effects: acute vs. chronic effects
O t h• Our current approach Nanorisk: methods for risk assessment of NPS in foodOpen to further collaborations
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