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Paride Mantecca University of Milano Bicocca,
Department of Earth and Environmental Sciences
Research Centre POLARIS,
Milan, Italy
Biological Effects of Nanoparticles
“Nanoparticles in the environment: fate and effects”,
Tallin, 29 May 2014
The story of the University of Milano Bicocca
Pirelli industrial site
Campus Bicocca
http://www.polaris.unimib.it/index.php/en/who-we-are/nanotoxicology
www.polaris.unimib.it
RESEARCHES
The story of the POLARIS Research Centre (Dept. Earth and Environmental Sciences, University of Milano Bicocca)
From tire particles analysis
…and toxicity
To Nanoparticle biological
effects and MOA
Through Particulate Matter
health effects
Milan
The inhalable fraction of Tire Particles induce (lung) toxicity
… and the effects are related to size
The fine fraction resulted enriched with sub-micrometric
(nanometric) particles
In London, in 1952, 3-4.000 people died as a
consequence of 3 days of great smog of PM and
sulphur dioxide.
London’s Great Smog
Statistical average loss of life expectancy in months in Europe
due to anthropogenic PM 2.5 (air pollution)
The spectre
PM is a relevant environmental and health concern
Stimulus also for the scientific community:
PM effects may be related not only to mass…
Press and
public opinion
asked for
evidences…
TOSCA project: Toxicity of Particulate Matter and molecular Markers of Risk
Urban PM10 and PM2.5 sampled in
Milan in summer and winter
PM10
PM2.5
Teflon filter
BEAS-2B
Exposure of in vitro
systems representative
of the lung epithelia
A549
THP-1
In vivo
toxicology
Clinical and epidemiological
studies
Bacteria Virus
Natural and anthropogenic particles
Cigarette smoke
Allergens
PM is a complex mixture of particles (μm and nm scale)
from different sources
Oberdorster et al., 2005 (EHP)
Penetration of particles in the respiratory system
Per cent distribution of emissions in Lombardy for 2010 by fuel
Fuel / Pollutnat PM2.5 PM10 TSP
gasol ine 0,52% 0,45% 0,39%
coal 0,72% 0,73% 0,99%
diesel 19,65% 17,02% 14,51%
refinery gas 0,50% 0,43% 0,37%
gasoi l 0,58% 0,50% 0,42%
LPG 0,04% 0,03% 0,03%
kerosene 0,09% 0,09% 0,08%
wood 56,14% 49,51% 44,66%
natura l gas 1,01% 0,94% 0,87%
fuel oi l 1,26% 1,13% 1,01%
others 1,75% 1,74% 1,85%
no fuel 17,73% 27,42% 34,83%
Total 100,00% 100,00% 100,00%
Combustion processes – fine and ultrafine (nano) particles
Buzea et al., 2007
31.3
73.4
16.8
52.2
10.4
32.3
4.3 6.50
0
10
20
30
40
50
60
70
80
90
100
ESTATE INVERNO
mg
m-3
Concentrazioni
PM10
PM2.5
PM1
PM0.4
18% 15%
20% 34%
16%
28%
39%23%
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
ESTATE INVERNO
Distribuzione dimensionale
PM(10-2.5) PM(2.5-1) PM1 PM0.4
31.3
73.4
16.8
52.2
10.4
32.3
4.3 6.50
0
10
20
30
40
50
60
70
80
90
100
ESTATE INVERNO
mg
m-3
Concentrazioni
PM10
PM2.5
PM1
PM0.4
18% 15%
20% 34%
16%
28%
39%23%
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
ESTATE INVERNO
Distribuzione dimensionale
PM(10-2.5) PM(2.5-1) PM1 PM0.4
Fine and Ultrafine Particles abundant in urban PM
Prog TOSCA, 2011
… also in Milan, especially in Winter
Background
Summer PM10
SO4=
15,4
NO3-
5,4
NH4+
6,4
EC
10,1
Metals
4,8
Inorganic
ions
4,8
OC
33,0
unaccounted
20,0
Winter PM10
unaccounted
28,2
OC
33,0
Inorganic
ions
5,4
Metals
2,5
EC
8,2
NH4+
5,4
NO3-
13,5
SO4=
4,3
Bacterial components (endotoxin content)
Chemical characterization (mass %)
Summer PM10 effect
Pro-inflammatory effect
release of the pro-inflammatory
mediator IL-1β
Release of IL-1β triggered by Milan summer PM10: molecular pathways involved in the cytokine release. R
Bengalli, E Molteni, E Longhin, M Refsnes, M Camatini, M Gualtieri. http://dx.doi.org/10.1155/2013/158093
Low doses
(5 and 2.5 μg/cm2)
Summer PM10 characterization
Background
Summer PM2.5
unaccounted
14,8
OC
28,8
Inorganic
ions
2,0Metals
2,3
EC
14,6
NH4+
11,6
NO3-
3,6
SO4=
22,4
Winter PM2.5
SO4=
6,4
NO3-
20,6
NH4+
9,1
EC
11,8
Metals
0,9
Inorganic
ions
3,2
OC
35,9
unaccounted
12,1
Chemical characterization (mass %) PAHs (OC %)
OC
%
Winter PM2.5 characterization
Winter PM2.5 effect
0
10
20
30
40
50
60
70
SubG1 G0/G1 S G2/M
Cel
ls %
Ctrl Summer PM2.5 Winter PM2.5
0
2
4
6
Control summer winter
PM2.5
Flu
ore
scen
ce a
.u.
0
2
4
6
Control summer winter
PM2.5
flu
ore
scen
ce a
.u.
ROS formation DNA damage γH2AX expression
Cell cycle alteration
PM dose: 10 μg/cm2 *p<0.05 vs Ctrl
Increased mitotic cells
* * *
*
0
10
20
30
40
50
60
70
80
90
subG1 G0/G1 S G2/M
Ce
ll n
um
be
r (%
)
*
*
0
10
20
30
40
50
60
70
80
90
subG1 G0/G1 S G2/M
Cell
num
ber
(%)
Control whole washed organic
0
10
20
30
Control whole washed organic
PM2.5
Flu
ore
sce
nce
(a
.u.)
*
*
ROS formation Cell cycle alteration
* p<0.05 vs Ctrl
Whole FP Acetonitrile,
centrifuge (15min, 13000rpm)
Organic fraction
resuspended in
DMSO
Washed FP
resuspended in
H2O
How we obtained
the organic
fraction Carbon cores
Adsorbed hydrocarbons
The organic soluble fraction of PM2.5 is responsible for the
genotoxic effects. Washed particles (BC?) are ineffective
Toxicity of UFP from Diesel with different additives
Carbon
cores
Adsorbed
hydrocarbons
Diesel emissions:
NOC vapour
phase
hydrocarbo
ns
Cell viability
0,0
0,2
0,4
0,6
0,8
1,0
1,2
Diesel Diesel + alkyl benzene Diesel + naphteneF
old
In
cre
ase
1ppm 3ppm 7ppm
0,0
0,2
0,4
0,6
0,8
1,0
1,2
Diesel Diesel + Alkyl
benzene
Diesel +
naphtene
Biodiesel
Fo
ld In
cre
ase
ROS formation
0
1
2
3
4
5
6
7
Ctrl Diesel Diesel + alkyl
benzene
Diesel + naphtene
RO
S (
a.u.)
*
*
*
0,0
0,2
0,4
0,6
0,8
1,0
1,2
Diesel Diesel + alkyl benzene Diesel + naphtene
Fo
ld In
cre
ase
1ppm 3ppm 7ppm
Winter FP organic
compounds /
PAHs
DNA damage CELL
NUCLEUS
Cell cycle
alteration
ROS
3. Mitotic spindle involvement
in cell cycle alteration
2. ROS/CYP
induced DNA
damage
1. AhR/CYP
activation
Cell death and
carcinogenesis
AhR
AhR
Possible mechanism of action of fine PM (and combustion derived UFPs)
The PM toxicity depends not only from the concentration
Biological effects vary according to the particle size and season of collection
PM chemical composition (and thus the contribution of different emission
sources) stands at the base of the cell response variability
Coarse PM-induced responses are much more related to acute inflammatory
events
Fine and ultrafine fractions mainly originate from combustion sources and
their biological activity is mainly related to pro-carcinogenic events
Data on health effects of UFPs from different combustion sources are
incosistent. There’s the need to develop new toxicity paradigm and
technology
Summary of the PM and UFP biological effects
Exposures to airborne nanosized particles (NSPs; < 100
nm) have been experienced by humans throughout their
evolutionary stages, but it is only with the advent of the
industrial revolution that such exposures have increased
dramatically because of anthropogenic sources such as
internal combustion engines, power plants, and many other
sources of thermodegradation.
The rapidly developing field of nanotechnology is likely to
become yet another source for human exposures to
NSPs—engineered nanoparticles (NPs)—by different
routes: inhalation (respiratory tract), ingestion
[gastrointestinal (GI) tract], dermal (skin), and injection
(blood circulation).
Nanotoxicology was born (term coined in 2004-2005)
When peculiar phisical chemical properties of NPs
suggested unpredictable interactions with living systems
Evolution of “traditional” particle and fibre toxicology
Donaldson et al., 2004 – Occup. Environ. Med. 61:727-28
Oberdorster et al., 2005 – Environ. Health Perspect. 113:823-39
Seaton and Donaldson, 2005 – Lancet 365:923-24
From coal minings to nanotechnology
… and the advent of nanotoxicology
Biological interactions and effects of Engineered Nanoparticles (ENPs)
NP properties and biological interactions
Nanomaterials properties driving
adverse effects
Correlations between phisico-chemical properties
of NMs and biological and toxicological outcomes
(Schvedova et al., 2010)
•Dose-dependent toxicity
not always NP effects are correlated with particle
mass dose (high conc, high agglomeration?!)
•Size-dependent toxicity
•Surface-area-dependent toxicity
•Crystalline-structure-dependent toxicity
•Surface-coating dependent toxicity
Figure 4. Percentage of neutrophils in lung lavage of rats (A,B) and mice (C,D) as
indicators of inflammation 24 hr after intratracheal instillation of different mass doses
of 20-nm and 250-nm TiO2 particles in rats and mice. (A,C) The steeper dose
response of nanosized TiO2 is obvious when the dose is expressed as mass. (B,D)
The same dose response relationship as in (A,C) but with dose expressed as particle
surface area; this indicates that particle surface area seems to be a more appropriate
dosemetric for comparing effects of different-sized particles, provided they are of the
same chemical structure (anatase TiO2 in this case). Data show mean ± SD.
Oberdorster et al., 2005 (EHP)
NP properties relevant for toxicological studies
Griffitt et al., 2007. Environ. Sci. Technol. 41:8178-8186
Looking for sensible markers able to discriminate the effects of NM
Toward a biology of nanosystems
… where nanotoxicology is defined
as the study of the interference of
man-made nanomaterials with
endogenous (cellular)
nanostructures.
(Shvedova et al., 2010)
“Nanovision”
Living cells as a miniature factory that
contain a large collection of dedicated
protein machines of nano-scale
dimensions, optimized by billions of years
of evolution.
Alberts B., 1998. The cells as a collection of
protein machines: preparing the next generation
of molecular biologists. Cell 92:291-94
The evolution of nanotoxicolgy into a predictive science as
apposed to being merely descriptive science
Nanostructural Biology: a new frontier for nanotoxicology?
Nanotoxicology Unit Ref. Paride Mantecca (Dept. Earth and Environmental Sciences, University of Milano Bicocca)
In vitro nanotoxicology M. Gualtieri, C. Urani, E. Longhin, R. Bengalli,
L. Capasso, M. Camatini
Cell models available:
- Human lung cells (A549, NCI-H441;
BEAS-2B) ;
-human monocytes (THP-1)
-Human lung microvascular endothelial
cells (HPMEC-ST1.6R)
- mouse fibroblasts (C3H) – to study
general cytotoxicity and cytoskeletal
remodelling
Applications:
- NP-induced cytotoxic effects
- Production of inflammatory mediators
- NP-induced genotoxicity
- NP properties driving cytotoxicity
(size, shape, ion dissolution, surface
reactivity)
Aquatic nanotoxicology A. Colombo, P. Bonfanti, E. Moschini
Collaborators: R. Bacchetta, P. Tremolada, L.
Del Giacco (University of Milan)
Aquatic models available:
- Frog Embryo Teratogenesis Assay –
Xenopus, FETAX – (amphibian
developmental toxicity test, ASTM
E1439-98)
- Daphnia magna acute and chronic
toxicity test (ISO 6341, 10706)
Applications:
- Acute, chronic and developmental
toxicity of nude and functionalized NPs;
NPs and soluble contaminants leached
from nanostructured materials; effluents
and contaminated surface waters.
- Biological mechanisms inducing NP
toxicity in aquatic organisms
Microscopy and Spectroscopy of
cell-NP interactions G. Chirico, M. Collini, L. D’Alfonso, S. Freddi
Collaborators: U. Fascio, N. Santo (CIMA,
University of Milan)
Techniques
- Conventional and analytical light and
electron microscopy
- Confocal laser microscopy
- Two Photon Correlation Spectroscopy
- Stimulated emission depletion (STED)
microscopy (nanoscopy)
-Dynamic Light Scattering
Applications:
-Modality of cell-particle interaction
-- NP tracking and translocation in fixed
and living systems
- Structural characterization of NP
suspensions.
1- Toxicity and mode of action of metal oxide NPs in human lung cells
MeO NPs (CuO in particular) are highly toxic to lung cells
Cytotoxicity is driven by abundant NP internalization and a Trojan horse-mediated
autophagic cell death
2- Toxicity and mode of action of metal oxide NPs in embryos
Comparative embryotoxicity: the teratogenic potential of ZnO NPs and the dissolved ions-
mediated effects of nCuO
Permeation and disruption of the intestinal barrier: a specific MOA for nZnO?
3- Toward the reduction of NP toxicity: surface coating and crystallinity
modulation
Does nZnO capping by polymers help in reducing toxicity?
Lower the crystallinity, lower the toxicity of CuO NPs
4- Metal oxide nanocomposite showing enhanced reactivity… and
toxicity?
… more effective than CuO and ZnO, less toxic than Zn
Biological effects of ENPs: the case study of nano metal oxide
**
**
*
* *
0
20
40
60
80
100
120
0 1 5 10 50 100
nCuO (ug/ml)
24h
-Via
bil
ity%
(M
TT
)
nCuO Cu++ Dissolved Cu++
- CuO NPs are highly cytotoxic
- Toxicity depends on NP
internalization and reactivity
The modality of cell-NP interaction drives nCuO toxicity in A549 cells
Moschini et al. 2013. Toxicology Letters 222: 102– 116
nCuO
nTiO2
1- Toxicity and MOA of metal oxide NPs in human lung cells
Lysosome
Late
En
do
so
me
Lyso
so
mes
Moschini et al. 2013.
Toxicology Letters 222: 102– 116
Trojan horse mechanism and autophagic cell death in nCuO exposed cells
- CuO NPs endocytosis and
lysosomal destabilization
- NP intracellular dissolution
and autophagy induction
1- Toxicity and MOA of metal oxide NPs in human lung cells
- No mortality
- Significant malformation rates
- Significant growth inhibition
Co CuO
Co ZnO
2- Toxicity and MOA of metal oxide NPs in embryos
Comparative embryotoxicity of Metal oxide NPs (nCuO, nZnO, nTiO2)
ZnO NPs are potential teratogen.
Malformations specifically affect
intestine
Bacchetta et al., 2012. Nanotoxicology 6 (4): 381-398
0
20
40
60
80
100
Co
CuO
100
CuO
500
Cu+
+
ZnO
100
ZnO
500
Zn+
+
malf
orm
ed
%
* #
*
* *
C
o CuO Cu++
ZnO
Zn++
CuO NPs effects are mediated by
dissolved ions
F-actin
ZO-1
2- Toxicity and MOA of metal oxide NPs in embryos
ZnO<50 ZnO<100
nZnO MOA: permeation and disruption of the intestinal barrier
bZnO Co sZnO
TBARS assay
RT-PCR
4-HNE immunofluorescence
Bacchetta et al., 2013. Nanotoxicology, Aug, early on line
1- NPs penetrate apical membrane 2- … walk along the lateral membrane
and paracellular space…
3- … reach basal membrane and
underneath connective tissue
4- … induce swelling and finally
cell death
- Nano-sized ZnO is absorbed by epithelial cells and
damages intestinal mucosa reaching underlying tissues
- The mechanism involves oxidative stress, disruption of
cytoskeleton and cell junctions, independently from
dissolved Zn++.
- The loss of cell junction integrity facilitates the NP
translocation and leads to cell death.
Bacchetta et al., 2013. Nanotoxicology
2- Toxicity and MOA of metal oxide NPs in embryos
0
20
40
60
80
100
Control SZnO 50 BZnO 50 SZnO 50 BZnO 50 SZnO 50 BZnO 50
Nude PVP PEG
Experimental groups
ma
lfo
rme
d e
mb
ryo
s (
%)
?
Small/round Big/rod
*
*
*
**
0
20
40
60
80
100
Control SZnO
[1]
SZnO
[10]
SZnO
[50]
SZnO
[100]
BZnO
[1]
BZnO
[10]
BZnO
[50]
BZnO
[100]
Experimental groups
%
Mortality (%) Malformed larvae (%)
? ?
Differently shaped nZnOs displayed comparable effects (?)
Modification of ZnO NPs size and surface coating
Polymer-capped ZnO NPs produced effects comparable to
those of nude ones (?)
Lower the crystallinity, lower the toxicity of CuO NPs
CuO and ZnO NPs sonochemically synthesized (Bar-Ilan Univ)
crystallites with more defects and less organized structure are
more toxic to the bacteria…
… and sono-CuO NPs are less toxic to Xenopus embryos and
human lung cells…
Toward the reduction of NP toxicity
-1
0
1
2
3
4
5
6
7
8
9
10
CuO - com CuO - sono
An
tib
acte
rial
act
ivit
y va
lue
(A
)
0
1
2
3
4
5
6
7
ZnO - com ZnO - sono
Ant
ibac
teri
al a
ctiv
ity
valu
e (A
)
CuO
ZnO
Commercial
Sono
DSC Antimicrobial activity
Sono
Sono
(Perelshtein et al. Nano Research, in press)
Striped bars= Sono Solid bars=commercial
A metal oxide nanocomposite with enhanced antibacterial properties
… and toxicity?
CuO
ZnO
Cu0.89Zn0.11O
b
Zn-doped CuO nanocomposite (Zn-CuO)
Eradication of Multi-Drug
Resistant Bacteria by a Zn-
doped CuO Nanocomposite
Malka et al. 2013. Small (in
press)
S. aureus E.coli
MRSA MDR E.coli
*
*
*
0
20
40
60
80
100
Contr
ol
0,1 1
10
100
0,1 1
10
100
0,1 1
10
100
CuO ZnO Zn-CuO
Malf
orm
ed
larv
ae (
%)
Experimental group
0
20
40
60
80
100
120
con
trol 1
10
100 1
10
100 1
10
100
CuO MAE ZnO MAE Zn-CuO
Via
bil
ity%
NP (ug/ml)
3h 6h 24hOn amphibian development On cell viability
Need for advanced in vitro models for assessing NPs toxicity
NPs-induced cardio-vascular effects likely involve ABB alterations. NPs may exert its effects through the ABB translocation or by inflammatory mediators released by the epithelium.
NPs
1. Local effects:
- inflammation
- oxidative stress
- DNA damage
2. Systemic effect:
cardio-
vascular
diseases
Possible mechanisms of PM
cardio-vascular effects:
• ABB translocation
• Release of inflammatory mediators by
epithelium and endothelium
A model of air-blood barrier
In vitro reconstructed Air-Blood Barrier (ABB): 3D co-cultures
NCI-H441
HPMEC-ST1.6R
THP-1
Lung epithelium
Lung endothelium
Monocytes
What is still almost unknown in UFP and NP toxicology
Carcinogenic and neurodegenerative effects
Paracelsus Philippus Aureolus Theophrastus
Bombastus von Hohenheim (1493 – 1541)
Funder of the discipline of toxicology.
« Omnia venenum sunt: nec sine veneno
quicquam existit. Dosis sola facit, ut venenum
non fit »
«"All substances are poisons; there is none
which is not a poison. The right dose
differentiates a poison…."
The dose makes the poison…
… for toxicologists since 1500… what’s now for NPs?
Concluding remarks
- Different incidental and engineered NPs show different mode of action in different biological
models
- Toxicity levels vary according with NP properties, like size, extra- or intra-cellular solubility,
crystallinity and chemicals associated (or adsorbed onto) primary particles
- The knowledge of the modality of cell-NP interactions and the molecular pathways driving
the biological effects are crucial in assessing NP toxicity
-The development and use of more complex in vitro systems are necessary to predict the
complex biological responses to NPs in vivo
- Only addressing the complexity of the biological responses to NPs it is possible to generate
toxicological data useful in risk assessment. At present many gaps have to be filled in NP
exposure monitoring and toxicity during the NP entire life cycle (especially for ENPs)
- Special attention should be devoted to the effects after prolonged exposure periods to low
NP doses (in mass), but high in number.
- A strong multidisciplinary approach is the only possible way toward succeeding in limiting
environmental and human health consequences to NP exposure.
Current projects in which we are trying to address these issues - Biological effects and human health impacts of ultrafine particles sources (new generation diesel and biomasses)
- Do new generations of nano-antibacterials OVERcome the epithelial barriers posing human health at risk? A predictive
nanoTOXicology study (OVER NanoTOX)
- The transfer of engineered nanomaterials from wastewater treatment & stormwater to rivers (ENTER) EU-COST Action ES1205
- Mode of action of engineered nanoparticles (MODENA). EU-COST Action
(DISAT)
Marina Camatini
Maurizio Gualtieri
Eleonora Longhin
Elisa Moschini
Laura Capasso
Rossella Bengalli
(DSS)
Paola Palestini
Giulio Sancini
Alberto Pesci
Francesca
Farina
Particle Toxicity Group
Paride Mantecca, Università degli Studi di Milano Bicocca, Dipartimento di Scienze dell’Ambiente e del Territorio e di Scienze
della Terra, 1, piazza della Scienza, 20126 – Milano. Tel. 02 6448 2916, e.mail: paride.mantecca@unimib.it
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