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Toxicity and biological impact of metal and metal oxide nanoparticles: Focus on the vascular toxicity of ultra-small titanium dioxide nanoparticles
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Susana Cristobal)
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The application of nanoparticles (NPs) in different technologies has led to tremendous advancement in those fields.  Moreover, there is growing interest in application of ultra-small NPs (USNPs) at 1-3 nm due to their distinct molecule like features. Parallel to these promises, there is a growing concern regarding their safety. The main goal of this thesis was to investigate the toxicity and underlying mechanisms following exposure to different metal and metal oxide NPs as well as USNPs. Their effects were studied on Saccharomyces cerevisiae, on hepatocytes and endothelial cells and finally in vivo on zebrafish embryos (Danio rerio). By selecting the rutile form of titanium dioxide (TiO2-USNPs) without intrinsic or intracellular reactive oxygen species (ROS) production, we could study biological impacts solely due to size and direct interaction with the cells. We showed that TiO2-USNPs were not cytotoxic but induced DNA damage. They had anti-angiogenic effects both in vitro and in vivo. Also, at high concentrations they caused complete mortality in zebrafish embryos exposed in water, while at lower concentrations induced delay in hatching. When injected they caused malformations. They specifically induced the differential overexpression of transcripts involved in lipid and cholesterol metabolism in endothelial cells. In hepatocytes they induced the overexpression of proteins in the electron transport chain and decreased lipids in lipid rafts. At 30 nm, TiO2-NPs, were also not cytotoxic but were genotoxic. They had no effects in vivo or on angiogenesis. However, they induced differential expression of transcripts involved in endoplasmic reticulum (ER) stress and heat shock response as well as cholesterol metabolism. This suggests a more toxic response in the cells compared to TiO2-USNPs.  Single walled carbon nanotubes (SWCNTs) despite having the highest oxidative activity among the NPs studied, were not severely cyto- or genotoxic but induced expression of transcripts involved in early ER stress response. Copper oxide (CuO-NPs) was the most toxic NPs studied due to both ion release and ROS production, affecting lipid metabolism of the cells. Silver (Ag-NPs) were also cytotoxic and caused the disruption of cellular components and lipids. ZnO-NPs were not cytotoxic, did not affect cellular lipids but they increased the size of vacuoles in yeast cells. Finally by using superparamagnetic iron oxide NPs (SPIONs) with different coatings, and using a mathematical model, a nano impact index (INI) was developed as a tool to enable the comparison of nanotoxicology data.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2015. , 78 p.
Keyword [en]
Nanoparticles, Nanotoxicology, Ultra-small nanoparticles
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-116708ISBN: 978-91-7649-193-5 (print)OAI: oai:DiVA.org:su-116708DiVA: diva2:807532
Public defence
2015-06-02, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (Swedish)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Accepted. Paper 3: Manuscript. Paper 4: Manuscript. Paper 5: Manuscript.

Available from: 2015-05-11 Created: 2015-04-23 Last updated: 2015-05-12Bibliographically approved
List of papers
1. The effects of engineered nanoparticles on the cellular structure and growth of Saccharomyces cerevisiae
Open this publication in new window or tab >>The effects of engineered nanoparticles on the cellular structure and growth of Saccharomyces cerevisiae
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2014 (English)In: Nanotoxicology, ISSN 1743-5390, E-ISSN 1743-5404, Vol. 8, no 4, 363-373 p.Article in journal (Refereed) Published
Abstract [en]

In order to study the effects of nanoparticles (NPs) with different physicochemical properties on cellular viability and structure, Saccharomyces cerevisiae were exposed to different concentrations of TiO2-NPs (1-3 nm), ZnO-NPs (<100 nm), CuO-NPs (<50 nm), their bulk forms, Ag-NPs (10 nm) and single-walled carbon nanotubes (SWCNTs). The GreenScreen assay was used to measure cyto- and genotoxicity, and transmission electron microscopy (TEM) used to assess ultrastructure. Cu-ONPs were highly cytotoxic, reducing the cell density by 80% at 9 cm(2)/ml, and inducing lipid droplet formation. Cells exposed to Ag-NPs (19 cm(2)/ml) and TiO2-NPs (147 cm(2)/ml) contained dark deposits in intracellular vacuoles, the cell wall and vesicles, and reduced cell density (40 and 30%, respectively). ZnO-NPs (8 cm(2)/ml) caused an increase in the size of intracellular vacuoles, despite not being cytotoxic. SWCNTs did not cause cytotoxicity or significant alterations in ultrastructure, despite high oxidative potential. Two genotoxicity assays, GreenScreen and the comet assay, produced different results and the authors discuss the reasons for this discrepancy. Classical assays of toxicity may not be the most suitable for studying the effects of NPs in cellular systems, and the simultaneous assessment of other measures of the state of cells, such as TEM are highly recommended.

Keyword
nanoparticles, genotoxicity, cytotoxicity, cellular structure, transmission electron microscopy, lipid droplets, vacuoles
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-99859 (URN)10.3109/17435390.2013.788748 (DOI)000328126700002 ()
Funder
Swedish Research CouncilCarl Tryggers foundation VINNOVA
Note

AuthorCount:5;

Available from: 2014-01-24 Created: 2014-01-20 Last updated: 2017-12-06Bibliographically approved
2. Proteomic and lipidomic analysis of primary mouse hepatocytes exposed tometal and metal oxide nanoparticles
Open this publication in new window or tab >>Proteomic and lipidomic analysis of primary mouse hepatocytes exposed tometal and metal oxide nanoparticles
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2015 (English)In: Journal of Integrated Omics, ISSN 2182-0287, Vol. 5, no 1Article in journal (Refereed) Published
Abstract [en]

The global analysis of the cellular lipid and protein content upon exposure to metal and metal oxide nanoparticles (NPs) can provide an overview of the possible impact of exposure. Proteomic analysis has been applied to understand the nanoimpact however the relevance of the alteration on the lipidic profile has been underestimated. In our study, primary mouse hepatocytes were treated with ultra-small (US) TiO2-USNPs as well as ZnO-NPs, CuO-NPs and Ag-NPs. The protein extracts were analysed by 2D-DIGE and quantified by imaging software and the selected differentially expressed proteins were identified by nLC-ESI-MS/MS. In parallel, lipidomic analysis of the samples was performed using thin layer chromatography (TLC) and analyzed by imaging software. Our findings show an overall ranking of the nanoimpact at the cellular and molecular level: TiO2-USNPs<ZnO-NPs<Ag-NPs<CuO-NPs. CuO-NPs and Ag-NPs were cytotoxic while ZnO-NPs and CuO-NPs had oxidative capacity. TiO2-USNPs did not have oxidative capacity and were not cytotoxic.  The most common cellular impact of the exposure was the down-regulation of proteins. The proteins identified were involved in urea cycle, lipid metabolism, electron transport chain, metabolism signaling, cellular structure and we could also identify nuclear proteins. CuO-NPs exposure decreased phosphatidylethanolamine and phosphatidylinositol and caused down-regulation of electron transferring protein subunit beta. Ag-NPs exposure caused increased of total lipids and triacylglycerol and decrease of sphingomyelin. TiO2-USNPs also caused decrease of sphingomyelin as well as up-regulation of ATP synthase and electron transferring protein alfa. ZnO-NPs affected the proteome in a concentration-independent manner with down-regulation of RNA helicase.  ZnO-NPs exposure did not affect the cellular lipids. To our knowledge this work represents the first integrated proteomic and lipidomic approach to study the effect of NPs exposure to primary mouse hepatocytes in vitro.

Keyword
nanoparticles, hepatocytes, proteomics, lipidomics, mass spectrometry, toxicity
National Category
Biochemistry and Molecular Biology Cell Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-117027 (URN)10.5584/jiomics.v5i1.184 (DOI)
Available from: 2015-05-05 Created: 2015-05-05 Last updated: 2015-05-12Bibliographically approved
3. Vascular toxicity of ultra-small TiO2 nanoparticles and single walled carbon nanotubes in vitro and in vivo
Open this publication in new window or tab >>Vascular toxicity of ultra-small TiO2 nanoparticles and single walled carbon nanotubes in vitro and in vivo
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Ultra-small nanoparticles (USNPs) at 1-3 nm are a subset of nanoparticles (NPs) that exhibit intermediate physicochemical properties between molecular dispersions and larger NPs. Despite interest in their utilization in applications such as theranostics, limited data about their toxicity exist. Here the effect of TiO2-USNPs on endothelial cells in vitro, and zebrafish embryos in vivo, was studied. The findings were compared to TiO2-NPs (30 nm) and single walled carbon nanotubes (SWCNTs). TiO2-USNPs were not cytotoxic, had no oxidative ability yet were genotoxic in vitro. They caused mortality at high concentrations in water possibly by acidifying the water and caused malformations in the form of pericardial edema when injected in early developing zebrafish embryos. Myo1C involved in glomerular development of zebrafish embryos was upregulated in embryos exposed to TiO2-USNPs. They also exhibited anti-angiogenic effects both in vitro and in vivo plus decreased nitric oxide concentration. TiO2-NPs were genotoxic but not cytotoxic. SWCNTs were cytotoxic in vitro and had the highest oxidative ability. Neither of these NPs had significant effects in vivo. To our knowledge this is the first study evaluating the effects of TiO2-USNPs on vascular toxicity in vitro and in vivo, demonstrating their potency and necessity for more focus in nanotoxicology.

Keyword
Ultra small nanoparticles, Zebrafish. Endothelium, Angiogenesis, Genotoxicity
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-117024 (URN)
Available from: 2015-05-05 Created: 2015-05-05 Last updated: 2016-01-29Bibliographically approved
4. The effects of ultra-small TiO2 nanoparticle and single walled carbon nanotubes on endothelial cells: next generation sequencing and transcriptome sequencing (RNA-seq) analysis
Open this publication in new window or tab >>The effects of ultra-small TiO2 nanoparticle and single walled carbon nanotubes on endothelial cells: next generation sequencing and transcriptome sequencing (RNA-seq) analysis
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The cardiovascular system is a key route of exposure to nanoparticles (NPs). The exposure could costendothelial cell dysfunction and impairment in blood circulation that could lead to cardiovascular diseasessuch as atherosclerosis. Currently, ultra-small nanoparticles (USNPs) at 1-3 nm, are receiving growingattention due to their unique properties. Emerging application for rutile TiO2-USNPs in medicine areexploring due to their insoluble nature, lack of oxidative activity and strong luminescence not observed inlarger NPs. On organic nanoparticle side, single walled carbon nanotubes (SWCNTs) are candidatemolecules for drug delivery from the chemical perspective. However their potential applications arehindered by their high oxidative activity and potential toxicity. Here we used transcriptome sequencing(RNA-seq) to evaluate the effects of exposure to sub-lethal concentration of TiO2-USNPs, TiO2-NPs andSWCNTs on human dermal microvascular endothelial cells. Specific toxicological effects were inferredfrom the functions of genes whose transcripts either increased or decreased. Our results show that TiO2-USNPs mostly induced the up-regulation of transcripts involved in lipid and cholesterol metabolism.TiO2-NPs induced the highest number of differentially expressed transcripts involved in cellularsenescence, endoplasmic reticulum (ER) stress, and heat shock responses as well lipid metabolism.Finally, SWCNTs affected to those genes involved in early stress and inflammatory responses.

Keyword
Nanoparticles, ultra-small nanoparticles, RNA, endothelial cells, toxicity, lipid metabolism
National Category
Biochemistry and Molecular Biology Cell Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-117025 (URN)
Available from: 2015-05-05 Created: 2015-05-05 Last updated: 2016-01-29Bibliographically approved
5. Assessment of the safety of functionalized iron oxide nanoparticles in vitro: introduction to integrated nanoimpact index
Open this publication in new window or tab >>Assessment of the safety of functionalized iron oxide nanoparticles in vitro: introduction to integrated nanoimpact index
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Functionalization of super paramagnetic iron oxide NPs (SPIONs) with different coatings renders them with unique physicochemicalproperties that allow them to be used in a broad range of applications such as drug targeting and water purification. However, it is required tofill the gap between the promises of any new functionalized SPIONs and the effects of these coatings on the NPs safety. Nanotoxicology isoffering diverse strategies to assess the effect of exposure to SPIONs in a case-by-case manner but an integrated nanoimpact scale has notbeen developed yet. We have implemented the classical integrated biological response (IBR) into an integrated nanoimpact index (INI) as anearly warning scale of nano-impact based on a combination of toxicological end points such as cell proliferation, oxidative stress, apoptosisand genotoxicity. Here, the effect of SPIONs functionalized with tri-sodium citrate (TSC), polyethylenimine (PEI), aminopropyltriethoxysilane(APTES) and Chitosan (chitosan) were assessed on human keratinocytes and endothelial cells. Our results show thatendothelial cells were more sensitive to exposure than keratinocytes and the initial cell culture density modulated the toxicity. PEI-SPIONshad the strongest effects in both cell types while TSC-SPIONS were the most biocompatible. This study emphasizes not only the importanceof surface coatings but also the cell type and the initial cell density on the selection of toxicity assays. The INI developed here could offer aninitial rationale to choose either modifying SPIONs properties to reduce its nanoimpact or performing a complete risk assessment to definethe risk boundaries.

National Category
Biochemistry and Molecular Biology Cell Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-117026 (URN)
Available from: 2015-05-05 Created: 2015-05-05 Last updated: 2016-01-29Bibliographically approved

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