Presentation_Kasemets Et Al

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Nanotoxicology: Science at theinterphases, Estonian perspective

Anne Kahru, Kaja Kasemets

, Angela Ivask, Irina Blinova, Olesja Bondarenko,Monika Mortimer, Margit Heinlaan1, Aleksandr Kkinen, Villem Aruoja

National Institute of Chemical Physics and Biophysics, Laboratory ofMolecular Genetics, Tallinn, Estonia

Nordic NanoNet Workshop and EDC discussion, Espoo, Finland, October 11-13, 2011


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Main research areas and key-words: Elucidation of mechanisms of toxic effects of chemicals (e.g.,

nanoparticles) using in vitro tests (luminescent bacteria,algae, protozoa, animal/human cell cultures etc)

Construction of new recombinant luminescent bacteria forstudy of the mechanisms of toxic action of chemicals and/ornanoparticles

Manifestation of toxic effects on physiological endpoints ofmicro-organisms. Intracellular homeostasis.

Bioavailability and its mechanisms

Environmental risk assessment

3R, QSAR, REACH

Research Group of In vitro toxicology andecotoxicology (head Dr. A. Kahru)


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Nano(eco)toxicology studies: ZnO, CuO, TiO2

Since 2006 -

P

eer-reviewedscientificpapers

ISI Web of Science (1995-2008)

Hazard to the environment?

Toxicity mechanisms?

???


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Main directions of the nano-research:

Evaluation of the hazard of eNPs: EC50, EC20, NOEC,LOEC. How toxic?

Evaluation of the mechanisms of toxic effect (solubilisation,ROS production). Why toxic?

Construction of new tools - recombinant luminescentbacteria - formechanistic toxicological profiling ofeNPs.

Are the NPs more toxic than the same bulkformulation?

Do the NPs have different toxicity mechanism? If yes,is it nanospecific?


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(In vitro) toxicity testing:test organisms at the various levels of the food-web

Escherichia coli

CRUSTACEANS PROTOZOA ALGAE YEAST

Daphniamagna

Thamnocephalusplatyurus

Tetrahymenathermophila

Pseudo-kirchneriellasubcapitata

Saccharomycescerevisiae

Vibrio fischeri

EUKARYOTIC ORGANISMS PROKARYOTIC

BACTERIA

naturally and recombinant

luminescent bacteria

ConsumersPrimary

producesDestructors


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Toxicity mechanism based profiling ofnanoparticles: set of gene modified microbial cells

Recombinant luminescentEscherichia colistrains

Wild type

Superoxide dismutase (sod)

Catalase (cat)

mutants

Responding to toxic compounds by

decreasing their luminescence

Specific sensor strains

- Metal specific (Cu, Zn, Ag, Hg)

- ROS specific

Bioluminescence is induced by

specific compounds (Cu, H2O2)

Inask et al. (2010). Profiling of the reactive oxygen species-related ecotoxicity of CuO, ZnO, TiO2, silver and fullerene

nanoparticles using a set of recombinant luminescent Escherichia coli strains: differentiating the impact of particles andsolubilised metals. Anal Bioanal Chem 398:701716.


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Uptake of the NPs:Two different types of cellular models

Bakter

Vetikas

10 m10 m

Good models for the

studying the toxic effects

of ingested NPs.

Good models for studying the

toxic effects of NPs caused by

the solubilised fraction,

external ROS effects,

adsorption onto the cell

surface etc.

Particle ingesting organisms Particle non-ingesting

organisms (particle resistant

cells)
http://www.pmf.unsa.ba/biologija/talofiti/Saccharomyces-cerevisiae.jpg


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Possible toxic effect of metal/metal oxide NPs

NPs

ROS

Aggregation

Ions (Cu2+, Ag+)

Adsorption

Test medium

(modulating effects?)


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Metal oxide nanoparticles: ZnO and CuO

Scanning electron microscopy (SEM) of

TiO2, ZnO and CuO suspensions

Nano-size metal oxides

Micro-size metal oxides

Size effect

control

Ionic forms of

respective metals:

ZnSO4*7H2O, CuSO4

Ionic effect

control

SEM photo: Kahru et al. (2008) Sensors, 8, 5153 - 5170

New physicochemical

propertiesIncreased toxicity??

Increased bioavailability??

The same nominal

concentration


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Solubility??


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Solubility of ZnO and CuO

0

25

50

75

100

10 100 1000

ZnO (mg Zn/l)

Bioavailablezinc(mgZn/l)

Nano ZnO

Bulk ZnO

Yeast growth medium

0

10

20

30

40

50

1 10 100 1000 10000

CuO (mg Cu/l)

Bioa

vailablecopper(mgCu/l)

Nano CuO

Bulk CuO

Kasemets, et al (2009). Toxicity of nanoparticles of ZnO, CuO and TiO2 to yeast Saccharomyces cerevisiae.

Toxicology in vitro 23, 1116 1122.


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Toxicity of ZnO NPs, EC50

Test organism Nano ZnOppm

Bulk ZnOppm

Bacteria (V. fischeri) 1.8 1.9

Algae (P. subcapitata) 0.037 0.042

Yeast (S. cerevisiae) 121 134

Protozoa (T. thermophila) 4.3 3.9

Crustacean (D. magna) 3.2 8.8

Toxicity of nano ZnO was comparable to bulk formulation andmainly due to the dissolved zinc ions (Zn-sensor data).


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Toxicity of CuO NPs, EC50

Test organism Nano CuOppm

Bulk CuOppm

Bacteria (V. fischeri) 79 3811

Algae (P. subcapitata) 0.710 11.5

Yeast (S. cerevisiae) 21 2031

Protozoa (T. thermophila) 127 1580

Crustacean (D. magna) 4.0 175

Nano CuO was more toxic than bulk CuO.

Toxicity of bulk CuO due to the dissolved copper ions (Cu-sensor data)


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Characterization of nano CuO

0 hour (100 ppm nCuO)

MQ 0.9% NaCl 2%

NaCl

24 hours

MQ 0.9% NaCl 2%

NaCl

MQ

Osterhouts medium

MQ water (100 ppm) 0 hour 24 hours

Hydrodynamic diameter, nm 16711 21821

Zeta potential, mV 27 1 19 2

0.9% NaCl (100 ppm) 0 hour 24 hours

Hydrodynamic diameter, nm 1613357 4136 675

Zeta potential, mV n.d n.d

24 h


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Daphnia magna

Do CuO NPs enter Daphnis via gut epithelial

cells?

Equitoxic

concentrations(EC50)

Nano CuO

4 mg/L

Bulk CuO

175 mg/L

Exposure up to 48 h

Alive daphnids were fixed and

analysed by the TEM

Margit Heinlaan, Tours

University, France

Case study 1


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Nano CuO dispersed, bulk CuO clumped in the

midgut lumen


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No uptake of nano CuO by

the intestinal cells


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Lot of bacteria in the lumen of intestine: only in thecase of exposure to nano CuO

PhD Thesis of Margit HEINLAAN (Dec, 2010)


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Toxic effect of nano CuO on membranes of T.

thermophila (protozoa)

The effects of nCuO and bulk on the fatty acid composition in T. thermophila were

measured after 2 h and 24 h exposure.

NanoCuO

24 h

10 m

+

Case study 2


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CuO NPs make the membranes

of protozoa more rigid

Control Nano CuO Bulk CuO

Unsaturated fatty acids

Saturated fatty acids

Increase in the

membrane rigidity

PhD Thesis of Monika MORTIMER (august, 2011)

Mortimer et al. (2011). Exposure to CuO Nanoparticles Changes the Fatty Acid Composition of

Protozoa Tetrahymena thermophila. Environ. Sci. Technol. 2011, 45, 66176624.


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Saccharomyces cerevisiae:Phenotype analysis - comparison of sensitivity of the

mutated and non-mutated strains

pre-screening with 10 delete strains

The complete collection of open reading frame deletion mutants (~6000 single-

gene mutants) have been generated by the Saccharomyces Gene Genome Deletion

Project (EUROSCARF collection).

Wild type

Oxidative stress response deficient strains

Elevated copper ions stress response deficient strains

EC50

Case study 2


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y = 24,854x + 2,7931

R2

= 0,775

0,0

5,0

10,0

15,0

20,0

25,0

30,0

0,00 0,20 0,40 0,60 0,80

Cu2+ EC50, ppm

NanoCu

O

EC50,pp

CUP2

GSH

CCS1

SOD

Sensitivity correlations

y = 906,83x + 86,214

R2

= 0,8064

0

250

500

750

1000

0,00 0,20 0,40 0,60 0,80

Cu2+ EC50, ppm

BulkCuO

EC50,ppm

CCS1

GSH1

CUP2

SOD

Copper ions versus Nano CuO Copper ions versus Bulk CuO

Nano CuO Bulk CuO

Kasemets et al. (2011). In preparation


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Saccharomyces cerevisiae BY4741:

dyed by the Trypan Blue (cell viability dye)

Cells have been exposed to the CuO nanoparticles for 24 hours


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Conclusions

With few exceptions, the solubility seems to be the

key determinant of the toxicity of metal-containing

NPs. Thus, for the toxic outcome the NPs do not

necessarily have to enter the cell/organism, as the

metal-ions will do the job.

Tailored construction, modification and use of gene-

modified microbial cells provides new possibilities

for rapid toxicological profiling of NPs.


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Nano publications

1. Mortimer et al. (2011). Exposure to CuO Nanoparticles Changes the Fatty Acid Composition of Protozoa

Tetrahymena thermophila. Environ. Sci. Technol. 2011, 45, 66176624.

2. Heinlaan M, et al (2011). Changes in the Daphnia magna midgut upon ingestion of copper oxidenanoparticles: a transmission electron microscopy study. Water Research, 45: 179-190.

3. Ivask A, et al (2010). Profiling of the reactive oxygen species-related ecotoxicity of CuO, ZnO, TiO2, silver

and fullerene nanoparticles using a set of recombinant luminescent Escherichia coli strains: differentiating

the impact of particles and solubilised metals. Anal Bioanal Chem, Anal Bioanal Chem 398:701-16.

Kahru A, Dubourguier H-C (2010). From ecotoxicology to nanoecotoxicology. Toxicology 269:105-119.

4. Mortimer M, et al (2010). Toxicity of ZnO and CuO nanoparticles to ciliated protozoa Tetrahymena

thermophila. Toxicology 269, 182-189.

5. Blinova I, et al (2009).. Ecotoxicity of nanoparticles of CuO and ZnO in natural water. Environ. Pollut.Environmental Pollution 15, 41-47.

6. Kasemets K, et al (2009).Toxicity of nanoparticles of ZnO, CuO and TiO2 to yeast Saccharomyces

cerevisiae, Toxicology in Vitro, Volume 23, Issue 6, p. 1116-1122

7. Ivask A, et al (2009). A suite of recombinant luminescent bacterial strains for the quantification of

bioavailable heavy metals and toxicity testing. BMC Biotechnol. 9: 41.

8. Aruoja V, et al (2009). Toxicity of nanoparticles of CuO, ZnO and TiO2 to microalgae Pseudokirchneriella

subcapitata. Sci. Total. Environ. 407, 1461-1468.9. Kahru A, et al (2008). Biotests and biosensors for ecotoxicology of metal oxide nanoparticles: a minireview.

Sensors 8, 5153 - 5170.

10. Mortimer M, et al (2008). High-throughput kinetic Vibrio fischeribioluminescence inhibition assay for study of

toxic effects of nanoparticles. Toxicology in Vitro 22, 1412-1417.

11. Heinlaan M. et al (2008). Toxicity of nanosized and bulk ZnO, CuO and TiO2 to bacteria Vibrio fischeriand

crustaceans Daphnia magna and Thamnocephalus platyurus. Chemosphere 71, 1308 1316.


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EU FP7 NMP NanoValid 2011-2014

November 2011

35 partners. Coordinator Dr. Rudolf Reuther,

NordMilj, Sweden.

Priotity 1 test materials: metal oxides (SiO2, TiO2, ZnO, CuO4), metals (Ag, Au

and Pd), CNTs, (SWCNTs and MWCNTs) and fullerenes.

Priority 2 test materials: quantum dots (CdSe, CdS, CeO2), salts (Ca-

phosphates, PbS), nanocellulosic materials, polystyrene, dendrimers, ceramics,

nanoclays.


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Acknowledgements

Financial support:

Estonian target funding project 0690063s08 and Estonian Science Foundation

(Grant 7686)

Research group ofIn vitro and Ecotoxicology

THANK YOU!


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Tetrahymena thermophila exposed tocarbon nanotubes (2800 mg/L)

Photo: Monika Mortimer

Movie made by Monika Mortimer and HC Dubourguier

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