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Exploring the genotoxic potential of nanoparticles using amorphous silica nanoparticles

vrijdag, 27 mei, 2011 - 16:00
Campus: Brussels Humanities, Sciences & Engineering campus
Faculteit: Science and Bio-engineering Sciences
E
0.04
Laetitia Gonzalez
doctoraatsverdediging

Nanomaterials, generally defined as materials with at least one dimension smaller than 100 nm, have
become of great interest for industrial and biomedical applications in the last years. With this
increased interest, concerns have been raised about their potential adverse health effects. Several
reports have indicated that these gnewh materials induce effects different from their micrometersize
counterparts. Therefore the need for hazard assessment and risk characterisation has become
evident.
The main objective of this study was to assess the genotoxic potential of nanoparticles and explore
their modes of action. With this aim, insoluble amorphous silica nanoparticles (SNPs) were chosen, as
a model material because amorphous silica is regarded as an inert material. Furthermore SNPs are
relatively easily tunable which allowed us to test particles with different physico]chemical properties
(e.g. size). In addition the SNPs used in this thesis were synthesized as monodisperse suspensions of
non]aggregated particles, allowing the assessment of the effects of nano]size particles, rather than
aggregates of particles.
Because nanomaterials are new materials, with physico]chemical properties and behaviour different
from bulk material or chemicals, the experimental design and used methodologies need careful
consideration. Therefore in the first part of this study the applied in vitro methodologies, i.e.
cytotoxicity assays, in vitro micronucleus (MN) assay and the alkaline comet assay, were evaluated at
different levels. The suitability of the assays to detect the induced effects, also taking into account
the potential modes of action, the experimental conditions that are able to influence the outcome of
the test and potential interferences between the test method and the SNPs were evaluated. We
demonstrated that for in vitro assays the nominal dose is the most appropriate metric for SNPs (Lison
et al. 2008). This holds true for MN induction since both nominal or cellular dose in presence of
serum correlated in the same way with the genotoxic effects (Gonzalez et al. 2010b). The
experimental conditions modulating experimental outcome were defined for cytotoxicity assays and
the in vitro MN assay. They were both influenced by the serum conditions, with a greater extent of
the effect in serum]free conditions. Considering the in vitro MN assay, the use of cytochalasin]B was
shown to influence the uptake of SNPs. Also, the exposure protocol and appropriate choice of cell
lines were emphasised. No interferences were observed between the SNPs and the cytotoxicity
assays, as comparable results were obtained using different assays, or between SNPs and
cytochalasin]B. A low interference was detected between the SNPs and FPG, an enzyme used for the
detection of oxidised purines when combined with the alkaline comet assay. Overall, the assays were
esteemed suitable for assessing the cellular responses of SNPs.
Applying the in vitro MN assay (cytokinesis]block MN and flow cytometry based MN assay) the
genotoxic potential was assessed of SNPs ranging from 12 to 174 nm. For most SNPs an induction of
MN was observed, except for S]28 and S]139 in the absence of serum. No biologically significant
induction of MN was observed for any of the SNPs tested, either in presence or absence of serum.
MN induction by SNPs did not show a clear dose]dependent or size]dependent relationship when
considering mass dose. Several dose]effect curves for nanoparticles show a plateau for a given range
of doses, which might reflect dynamic steady state cellular dose as a consequence of
endocytosis/exocytosis or saturation of uptake. However, we showed that, when expressing MN data
for the 16, 60 and 104 nm SNPs together, a statistically significant correlation was observed between
the fold MN induction and total surface area dose and the dose expressed as particle number,
indicating that these metrics determine the in vitro induction of MN in presence of serum (Gonzalez
et al. 2010b). Therefore the use of appropriate dose metrics is of great importance for the
interpretation of results.
Potential modes of action, i.e. the generation of reactive oxygen species and mechanical interference
of cellular components were explored, after treatment of A549 cells with 16, 60 and 104 nm SNPs in
presence of serum. Results showed weak and not statistically significant induction of DNA strand
breaks and oxidized purines. Furthermore, chromosome loss, mitotic arrest and mitotic slippage
were induced by SNPs, although not statistically significantly, indicative of spindle interference
(Gonzalez et al. 2010b).
The hypothesis of spindle interference was further investigated in situ and in absence of serum. No
clear interference of the mitotic spindle or preferential distribution of the fluorescently labeled SNPs
was observed. Furthermore no induction of multi]polar spindles was recorded. The main finding was
that treatment of A549 cells with S]28, S]59 and S]174 induced a promotion of microtubule
repolymerisation after cold treatment (Gonzalez et al. 2011 d) and this at doses where no cell toxicity
was observed. This in situ promotion of microtubule repolymerisation was not associated with
increased acetylation.
In conclusion, the cytotoxic and genotoxic potential of SNPs was assessed after evaluation of the
applied tests for their suitability, the experimental conditions influencing their outcome and possible
interferences with SNPs. Both cytotoxic and genotoxic effects were induced by SNPs. The mode of
action of SNPs responsible for the observed weak genotoxic effects were investigated and revealed
non]statistically significant increases of oxidised purines, chromosome loss, mitotic arrest and mitotic
slippage. The latter three point to spindle interference. Examination of the effects of SNPs on
microtubules showed an in situ promotion of microtubule repolymerisation.