Nanotoxicology: Science at the interphases, 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 of Molecular Genetics, Tallinn, Estonia

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

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

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 for study of the mechanisms of toxic action of chemicals and/or nanoparticles Manifestation of toxic effects on physiological endpoints of micro-organisms. Intracellular homeostasis. Bioavailability and its mechanisms

Environmental risk assessment

3R, QSAR, REACH

Since 2006 -

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

ISI Web of Science (1995-2008)
Peer-reviewed scientific papers

Hazard to the environment? Toxicity mechanisms?

???

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 luminescent bacteria - for mechanistic toxicological profiling of eNPs.

Are the NPs more toxic than the same bulk formulation?
Do the NPs have different toxicity mechanism? If yes, is it nanospecific?

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

PROKARYOTIC

Daphnia magna

Thamnocephalus Tetrahymena platyurus thermophila

Pseudokirchneriella subcapitata

Saccharomyces cerevisiae

Vibrio fischeri

Escherichia coli

CRUSTACEANS

PROTOZOA

ALGAE

YEAST

BACTERIA naturally and recombinant luminescent bacteria

Consumers

Primary produces

Destructors

Toxicity mechanism based profiling of nanoparticles: set of gene modified microbial cells
Recombinant luminescent Escherichia coli strains
Wild type Superoxide dismutase (sod)
mutants

Specific sensor strains - Metal specific (Cu, Zn, Ag, Hg)

Catalase (cat)

- ROS specific

Responding to toxic compounds by decreasing their luminescence

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 and solubilised metals. Anal Bioanal Chem 398:701716.

Uptake of the NPs: Two different types of cellular models

Particle ingesting organisms
Particle non-ingesting organisms (particle resistant cells)

10 m

Bakter Vetikas 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.

Possible toxic effect of metal/metal oxide NPs

Aggregation
NPs

Test medium (modulating effects?)

Ions (Cu2+, Ag+)

ROS

Adsorption

Metal oxide nanoparticles: ZnO and CuO

Scanning electron microscopy (SEM) of TiO2, ZnO and CuO suspensions

The same nominal concentration

Nano-size metal oxides

Micro-size metal oxides

Ionic forms of respective metals: ZnSO4*7H2O, CuSO4

Size effect control Ionic effect control

New physicochemical properties Increased toxicity?? Increased bioavailability??

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

Solubility??

Solubility of ZnO and CuO

Yeast growth medium
Bioavailable zinc (mg Zn/l)

100 75 50
Nano ZnO

Bioavailable copper (mg Cu/l)

50 40 30 20 10 0 1 10 100 1000 CuO (mg Cu/l) 10000

Nano CuO Bulk CuO

25 0 10 100 ZnO (mg Zn/l)

Bulk ZnO

1000

Kasemets, et al (2009). Toxicity of nanoparticles of ZnO, CuO and TiO2 to yeast Saccharomyces cerevisiae. Toxicology in vitro 23, 1116 1122.

Toxicity of ZnO NPs, EC50

Test organism
Bacteria (V. fischeri) Algae (P. subcapitata) Yeast (S. cerevisiae) Protozoa (T. thermophila)

Nano ZnO ppm

1.8 0.037 121 4.3

Bulk ZnO ppm

1.9 0.042 134 3.9

Crustacean (D. magna)

3.2

8.8

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

Toxicity of CuO NPs, EC50

Test organism
Bacteria (V. fischeri) Algae (P. subcapitata) Yeast (S. cerevisiae) Protozoa (T. thermophila)

Nano CuO ppm

79 0.710 21 127

Bulk CuO ppm

3811 11.5 2031 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)

Characterization of nano CuO

24 h
MQ NaCl 0.9% NaCl 2%

MQ

Osterhouts medium
0 hour (100 ppm nCuO)

MQ water (100 ppm) Hydrodynamic diameter, nm

0 hour 16711 27 1 0 hour 1613357 n.d

24 hours 21821 19 2 24 hours 4136 675 n.d

MQ NaCl

0.9% NaCl 2%

Zeta potential, mV 0.9% NaCl (100 ppm) Hydrodynamic diameter, nm Zeta potential, mV

24 hours

Case study 1

Daphnia magna
Do CuO NPs enter Daphnis via gut epithelial cells?

Equitoxic concentrations (EC50)

Nano CuO

Bulk CuO

4 mg/L

175 mg/L

Exposure up to 48 h

Alive daphnids were fixed and analysed by the TEM

Margit Heinlaan, Tours University, France

Nano CuO dispersed, bulk CuO clumped in the midgut lumen

No uptake of nano CuO by the intestinal cells

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

PhD Thesis of Margit HEINLAAN (Dec, 2010)

Case study 2

Toxic effect of nano CuO on membranes of T. thermophila (protozoa)

NanoCuO

+
10 m

24 h

The effects of nCuO and bulk on the fatty acid composition in T. thermophila were measured after 2 h and 24 h exposure.

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

Mortimer et al. (2011). Exposure to CuO Nanoparticles Changes the Fatty Acid Composition of Protozoa Tetrahymena thermophila. Environ. Sci. Technol. 2011, 45, 66176624. PhD Thesis of Monika MORTIMER (august, 2011)

Case study 2

Saccharomyces cerevisiae: Phenotype analysis - comparison of sensitivity of the mutated and non-mutated strains pre-screening with 10 delete strains

Wild type
Oxidative stress response deficient strains Elevated copper ions stress response deficient strains

EC50

The complete collection of open reading frame deletion mutants (~6000 singlegene mutants) have been generated by the Saccharomyces Gene Genome Deletion Project (EUROSCARF collection).

Sensitivity correlations
Nano CuO
30,0 25,0 y = 24,854x + 2,7931 R2 = 0,775 SOD

Bulk CuO
1000 y = 906,83x + 86,214 R = 0,8064 GSH1 500 CCS1
2

SOD 2

Nano CuO EC50, ppm

Bulk CuO EC50, ppm

20,0 GSH 15,0 CCS1 10,0 5,0 CUP2 0,0 0,00 0,20 0,40 Cu2+ EC50, ppm 0,60 0,80

750

250 CUP2 0 0,00 0,20 0,40 Cu2+ EC50, ppm 0,60 0,80

Copper ions versus Nano CuO

Copper ions versus Bulk CuO

Kasemets et al. (2011). In preparation

Saccharomyces cerevisiae BY4741: dyed by the Trypan Blue (cell viability dye)

Cells have been exposed to the CuO nanoparticles for 24 hours

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 genemodified microbial cells provides new possibilities for rapid toxicological profiling of NPs.

Nano publications
1.
2. 3.

4. 5. 6.

7.
8. 9. 10. 11.

Mortimer et al. (2011). Exposure to CuO Nanoparticles Changes the Fatty Acid Composition of Protozoa Tetrahymena thermophila. Environ. Sci. Technol. 2011, 45, 66176624. Heinlaan M, et al (2011). Changes in the Daphnia magna midgut upon ingestion of copper oxide nanoparticles: a transmission electron microscopy study. Water Research, 45: 179-190. 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. Mortimer M, et al (2010). Toxicity of ZnO and CuO nanoparticles to ciliated protozoa Tetrahymena thermophila. Toxicology 269, 182-189. Blinova I, et al (2009).. Ecotoxicity of nanoparticles of CuO and ZnO in natural water. Environ. Pollut. Environmental Pollution 15, 41-47. 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 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. Aruoja V, et al (2009). Toxicity of nanoparticles of CuO, ZnO and TiO2 to microalgae Pseudokirchneriella subcapitata. Sci. Total. Environ. 407, 1461-1468. Kahru A, et al (2008). Biotests and biosensors for ecotoxicology of metal oxide nanoparticles: a minireview. Sensors 8, 5153 - 5170. Mortimer M, et al (2008). High-throughput kinetic Vibrio fischeri bioluminescence inhibition assay for study of toxic effects of nanoparticles. Toxicology in Vitro 22, 1412-1417. Heinlaan M. et al (2008). Toxicity of nanosized and bulk ZnO, CuO and TiO2 to bacteria Vibrio fischeri and crustaceans Daphnia magna and Thamnocephalus platyurus. Chemosphere 71, 1308 1316.

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 (Caphosphates, PbS), nanocellulosic materials, polystyrene, dendrimers, ceramics, nanoclays.

Acknowledgements

THANK YOU!

Research group of In vitro and Ecotoxicology

Financial support: Estonian target funding project 0690063s08 and Estonian Science Foundation (Grant 7686)

Tetrahymena thermophila exposed to carbon nanotubes (2800 mg/L)

Photo: Monika Mortimer

Movie made by Monika Mortimer and HC Dubourguier