Nanotechnology in the Food Chain Brussels, Nov. 24/2010 ... · Nanotechnology in the Food Chain Brussels, Nov. 24/2010 Evaluation of NPs in vivo in vitro MMainain tectechniqueshniques

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>><<[email protected]@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


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



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





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





Surface area and propertiesSize, aggregates, …Size, aggregates, …Chemical compositionand concentration Uptake of Aluminium

NPs by cultured cells• Biological aspects


NPs by cultured cells

pyTransmission electronmicroscopy

Powers KW et al. in Nanotoxicity , Sahu S.C. & Casciano D.A., Wiley & Sons, 2009





Surface area and propertiesSize, aggregates, …Size, aggregates, …Chemical compositionand concentration Uptake of Cobalt

Oxide NPs by• Biological aspects


Oxide NPs by cultured cells

pyTransmission electronmicroscopyScanning electronmicroscopy Gornati R. et al.

in Nanotoxicity , SahuS.C. & Casciano D.A., Wiley & Sons, 2009





Surface area and propertiesSize, aggregates, …Size, aggregates, …Chemical compositionand concentration

AFM of Cobalt OxideNP• Biological aspects


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


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


• 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


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


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


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


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


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


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


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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Nanotechnology in the Food Chain Brussels, Nov. 24/2010 ... · Nanotechnology in the Food Chain Brussels, Nov. 24/2010 Evaluation of NPs in vivo in vitro MMainain tectechniqueshniques