RICERCA ITALIANA     

Interaction of novel nanoparticulate materials with biological systems: testing models for human health risk assessment

Università degli Studi di Parma

Abstract

Nanostructured materials (NM) play a key role in nanotechnology based innovations and their production rates are expected to increase exponentially in the next few years. NM exhibit entirely new physicochemical properties as compared to bulk materials. Carbon nanotubes (CNT), nanowires, quantum dots and metal oxides have received enormous attention since their use may lead to the creation of new analytical tools for biotechnology and life sciences. However, the size of nanoparticles (NP, particles smaller than 100 nanometers in at least one dimension) and their surface characteristics may profoundly affect cell behaviour. Thus, concerns have been raised about NP adverse effects on biological systems and, possibly, consequent health risks. There is evidence that NP entry the body through a number of routes, including inhalation and skin permeation. Once in the body, NP seem to penetrate into the cells more rapidly than larger particles and, therefore, to move more easily to distant sites within the body. This fact may explain why NP, e.g of metallic elements or CNT, exert greater cytotoxic and genotoxic effects than larger particles of the same substance at the same mass concentration. Although the mechanism of this toxicity is not fully established, an accepted hypothesis is that NP induce reactive oxygen and nitrogen species production, thus leading to oxidative stress both in lipis and nucleic acids and influencing calcium and sulphydryl homeostasis.
Relying on the collaboration of researchers with different backgrounds (Occupational Medicine and Industrial Toxicology, Cell Biology, Genetics, Physics), the present project is aimed at characterizing the mechanisms of toxicity of newly synthesized NP, like single-wall and multiple-wall CNT and metal oxides (mainly titanium, cobalt and platinum), with both conventional and new testing methods and models. To produce data comparable among different laboratories and to ensure reproducible results in biological assays, the same metal NP and CNT will be synthesized, characterized, purified, and distributed to different Units. Such NMs should allow to characterize a battery of in vitro tests enabling routine measurements of representative effects, including genotoxicity, following exposure to NP of occupational and environmental concern. To this purpose, different kinds of cell types will be used: human peripheral lymphocytes, U937, A549, CaLu3, HUVECs, THP-1, HACAT, and human stem cells (CD34+) will be employed for being at the interface between exposure and the human body (“ports of entry” models) or involved in immunity. NP will be localized and their distribution in the cellular compartment will be analyzed. Cell proliferation (through viability assays like MTT, XTT, or Trypan blue exclusion), the induction of survival/apoptotis pathways (through microarray techniques applied to the MAPK pathway), and the redox modulation (through the assessment of Ca2+ and pH homeostasis, as well as ROS production) will be investigated. The induction of oxidative damage to sensitive targets will be also evaluated with well validated approaches (Comet assay). Trans-epithelial migration of NP will be assessed using respiratory epithelial cells, grown on permeable filters, co-cultured with endothelial and monocytic cells in a double-chamber culture system. NP will be added in the upper compartment, while their passage across the epithelium will be documented by their presence/activity in the lower compartment. In vivo experiments carried out in a rat model will deal with events following systemic translocation of inhaled NM and will focus on perturbation of autonomic nervous system, alterations in blood coagulation and inflammation, and endothelial dysfunction. The results of mechanistic studies, combined with the preliminary assessment of exposure at workplace, will contribute to the characterization of NP-dependent risk for human health and provide novel methods to assess NP toxicity. <<<

Principal Investigator

Enrico Bergamaschi Università degli Studi di PARMA

Research Objectives

Relying on the collaboration of researchers with different scientific backgrounds (Occupational Medicine, Industrial Toxicology, Biology, General Pathology, Genetics, Physics), the present research project is aimed at:
a)clarifying the mechanisms underlying the toxicity of different nanoparticles (NP), such as carbon nanotubes or metal oxide NP, so as to yield a solid toxicologic rationale based on structure-function relationships and on relevant biological responses;
b)developing experimentally validated, reliable in vitro methods to assess NP toxicity, so as to characterize a battery of tests suitable for human health risk assessment of newly synthesized nanomaterials.
These two strictly related aims will be pursued by the Units through the achievements of intermediate endpoints:
i)the synthesis and chemical functionalization of new NPs (OUs 2 and 5);
ii)the identification of the structural determinants that influence the trans-epithelial permeability of NP across the airway epithelium (OUs1, 2, and 5);
iii)the characterization of the effects of distinct types of NP on biologically relevant cell types: airway epithelial cells (OUs1 and 5), inflammatory cells of monocyte-macrophage lineage (OUs 1 and 4), cord blood-derived stem cells differentiating into T or NK lymphocytes (OU3), human endothelial cells (OU1), human cheratinocytes (OU5), human peripheral lymphocytes (OU5);
iv)the analysis of the mechanisms of the alterations in cell survival/apoptosis, cell cycle/proliferation, expression of genes involved in the inflammatory response and oxidative stress (UO 4);
v)the evaluation of oxidative DNA damage by single cell assays (Comet and micronucleus) and the determination of the total antioxidant capacity of cells against different classes of generated oxyradicals (UO 4 and 5);
vi)the identification of toxicological parameters, obtained from in vitro models, consistent with toxic effects observed with in vivo (OU1) or in ex vivo cell models (OUs3 and 5)
vii)the identification of the subcellular and molecular targets of NP toxicity, also by using advanced microscopy techniques (AFM, TEM, SEM, CLSM) (OUs 1, 4, and 5);
viii)the quantitative assessment of the actual exposure levels to airborne nanomaterials in different work environments (OU2). <<<

Timescale

24 months

National and international background

Nanostructured materials (NM) play a key role in most of the innovations based on nanotechnology and their production rates is expected to increase exponentially in the next few years. By tailoring the structure at the nanoscale, it is indeed possible to engineer novel materials that have entirely new physicochemical properties as compared to bulk materials. The unusual physicochemical properties of NM are attributable to the higher surface to volume ratio associated with nanoparticles (NP, i.e. particles smaller than 100 nanometres in size in at least one dimension) and the quantum effects that occur in the nanometre scale, but chemical composition, surface structure (reactivity, surface groups, inorganic or organic coatings), solubility, shape and aggregation should also be considered. Although impressive from a physicochemical point of view, the novel properties of NM raise concerns about adverse effects on biological systems, since they may favour an opportunity for enhanced uptake and interaction with several cell types and tissues [1, 2].

Most of the data available on NP concern the respiratory system where NP exert greater toxic effects than larger particles of the same substance at the same mass concentration [3]. Toxicological studies have shown that airborne ambient ultrafine particles (UFP, i.e. particulate matter smaller than 100 nm in all dimensions) produced by combustion processes [3], can induce pulmonary inflammation, oxidative stress and distal organ involvement [4-6]. Indeed, UFP can cross the airway barrier, reach the circulation [6], and exert several toxic effects on the endothelium, as well as on other tissues [3, 5-8].

Cardiovascular dysfunction and coagulation disorders have been also observed upon UFP exposure and explained with the persistent inflammatory reactions in the lungs leading to the release of mediators, but the hypothesis of a direct influence of the particles translocated from the lungs into the systemic circulation on haemostasis or cardiovascular integrity has been also put forward [5]. Recent data indicate that also the autonomic nervous system is adversely affected by inhaled particulates [9, 10]. The activation of neural reflexes may alter the autonomic tone, and may contribute to plaque instability or to the development of cardiac arrhythmias during episodes of air pollution [11].

These studies mostly concern NP of environmental origin, heterogeneous for composition and size, and not industrial NP of defined composition and surface chemistry. Although they may have similar size limits, ambient and engineered NP may significantly differ for biological activity and toxicological properties [3]. Indeed, in spite of their collective definition, each material made at nanoscale should be treated as an individual entity when possible health risks are to be ascertained.

The interpretation of toxicological data on engineered NM is made difficult by three main reasons: (i) different types of NM may exert different effects; (ii) most preparation of NM used in bioassays are not pure; for instance, CNT are frequently contaminated with iron, which may be responsible for the observed effects; (iii) many different modalities of preparation of NM are used, each resulting in different types of contaminants. Metallic impurity comes from catalysers and can be very toxic. However, methods are now available to eliminate these metallic impurities. In particular by the joint use of surfactants, ultrasonication and ultracentrifugation fluorescent impurities can be separated and subsequently characterized by spectroscopy, transmission electron microscopy (TEM), high resolution transmission electron microscope (HRTEM) and microanalysis for both hydrophobic and oxidized nanotubes [12]. This perspective should lead to use standardised materials, and to pay attention to the type of material used in the toxicological tests when making comparisons.

Thus, while conventional toxicological tests have already been proven their usefulness in evaluating the hazards of NP, some methods may require modification and some new testing methods and approaches may also be needed, especially to enable routine measurement, in various media, of representative effects following NP exposure in occupational and environmental fields. In this perspective, it will be mandatory to distinguish not only among different types of NM, but also to consider whether they are dispersed in gaseous, liquid or solid phase, whether they occur as single-particles or as agglomerates, whether they are untreated of surface modified [13].
Carbon nanotubes are a man-made form of carbon that did not previously exist in the environment. Due to their unique chemical, physical, optical and magnetic properties, CNT have found many uses in industrial products and in the field of nanotechnologies, including nanomedicine and pharmacology, since they can easily cross cell membranes.
Since CNT are essentially novel molecules and humans have never been exposed to them so far, it is mandatory to examine the effects that such an exposure may exert on health and environment. It is known that CNT react with important classes of biological molecules, such as DNA [14] and several amino acid residues in proteins [15, 16]. These properties of CNT have suggested a possible use in biotechnology, e.g., for targeting compounds in specific areas of the body, or for immobilizing cells for further treatments [17], or as biosensors after proper mixing with reacting materials [18]. A further, very attractive development in biotechnology comes from the possibility of "functionalysing" CNT, by preparing hybrid or composite materials, where nanotubes are linked, (either covalently or non-covalently) to a different material of biological interest [19]. The possibility that CNT, as such or after functionalisation, react with specific target molecules and selectively interact with one or the other cell types/structures, implies multiple possible uses in biotechnology and drug delivery. Moreover, small sized CNT may enter cells more easily, due to the hydrophobic moiety of the molecules. The wide diffusion that CNT are likely to have in the next few years prompts a thorough analysis of different cellular parameters following passive entry, or active delivery, of CNT into the cell. Few studies have been published on CNT in these last years, mostly focused on the toxicological analysis on epithelial cells, as they are the first barrier of the organism to external insults. These studies have reported a slight toxic effect on renal cells, human epidermal keratinocytes and lymphocytes [20-22], which exhibit an enhanced induction of apoptosis. Toxic properties of MWCNT can be modulated by modifying surface characteristics. Indeed, the toxicity of pristine CNT on human T cell leukaemia Jurkat line was enhanced by the presence of carboxylic groups [22].
Single-walled carbon nanotubes, instilled in airways at a concentration of 1-5 mg/kg, led to the development of lung granulomas, suggesting peculiar interactions with inflammatory cells of monocyte-macrophage lineage [23, 24]. Nanotubes may exert more subtle effects on cells, i.e., by eliciting cell-protective stress responses, such as the induction of NFkB [25] or the heat shock response, or metabolic/genetic alterations that, without compromising cell viability, may nonetheless compromise cells functionality, alter redox equilibrium or the cell cycle [26].

Another type of NP of particular interest is represented by metal containing MN. At present, metal oxides, in particular titanium (TiO2), alumina (Al2O3), iron oxides (Fe3O4, Fe2O3) occupy the first position in terms of economic importance within the range of inorganic NP. Whereas iron oxides have a commercial history spanning half of century, other nanocristalline metal oxides have entered the market more recently. The main application fields of metal oxide NP are electronics, pharmacy, cosmetics and catalysis. Titanium and zinc oxides are increasingly used as UV light absorbing components in sunscreens. However, upon absorption of UV radiation, they release free radicals, which can damage DNA; they can also penetrate much deeper than microparticles and can interfere with the immune system [27]. Although metal toxicology is a mature science, and occupational and environmental exposures to metals have been associated with various diseases, the molecular mechanisms underlying the carcinogenesis caused by metals are still unclear. Metal-mediated formation of free radicals, reactive oxygen species (ROS) and reactive nitrogen species (RNS), may cause various modifications to DNA bases, enhance lipid peroxidation, and induce changes in calcium and sulphydryl homeostasis [28]. Increasing evidence indicates that ROS generated by metals may play an important role in the aetiology of neurodegenerative diseases and cancer. Many studies have focused on metal-induced toxicity and carcinogenicity, emphasising their role in the generation of ROS and RNS in biological systems, and the significance of this effect [29-31]. Metals/metal oxides of NP have been investigated in the BRL 3A cell line by analyzing the cell morphology and viability, the reduction of glutathione (GSH) and ROS formation after a 24-hour treatment [32]. The results clearly indicated a significant reduction of the mitochondrial functionality following the treatment with AgNP (5-50 micrograms/ml), whereas Al, Fe3O4, MoO3 and TiO2 (10-50 µg/ml) had no effect. Morphological studies showed that the treatment with high doses of NP both cell size and shape became very irregular; moreover, Ag NP induced a significant depletion of GSH and an increase in ROS formation, confirming that the oxidative stress is the mechanism for the cytotoxicity triggered by this NP.

From the above findings, it is clear that a number of cells and systems may be potentially affected by the interaction with NM of different composition. For this reason, in addition to oxidative stress and inflammation, it is important to consider that some of the NM exposure, e.g. to metal oxides and CNT, may also results in other forms of injury, such as protein denaturation, membrane and DNA damage [14-16, 28-32] or enhanced immune reactivity [33].

The effect of the interaction with immune system appear of particular interest and can be assessed with an innovative in vitro model based on human stem cells that differentiate into immune cells [34, 35]. This model represents a reliable and innovative tool for studying carcinogenesis and immunotoxicity. This model also offers the opportunity to evaluate in vitro the toxicity of NP during the cell differentiation process, a quite similar condition to that encountered “in vivo”. Finally, thanks to their multipotent characteristics, stem cells allow the study of the toxicity induced by distinct metals and chemical substances on different outcomes. Indeed, immune cells derived from stem cells in this experimental model exert important functions not only in the defence against viral infections (T CD8+ lymphocytes) but also against neoplastic diseases (NK cells).

No definite information is available on the mechanisms underlying the translocation of engineered NP of different physico-chemical properties across the airway epithelium, the first and fundamental barrier that nanoparticulate materials have to cross to exert toxic effects on other cell types and systems. Although it is generally believed that NP are endowed with very high permeability through biological membranes, this property should not be assumed per se as a bona fide index of toxicity in the absence of experimental evidence [1, 2]. However, the capacity to cross cell membranes and overcome cell junctions may increase the burden of reactive, non inert and biopersistent xenobiotics in human body fluids. Thus, rapid access to the intracellular compartment and absence of a clear-cut sequestration in phagocytic vacuoles [36] may represent important factors for enhanced toxic effects of nanostructures.

In the assessment of the health effects of inhaled NP the evaluation of translocation from airways to the systemic circulation is an important issue. Nevertheless, the relevant literature is limited and often conflicting [6, 36, 37]. Some studies [37-39] have reported extra-pulmonary translocation of ultrafine particles after intra-tracheal instillation or inhalation. However, the reported amount of ultrafine particles that translocate into blood and extrapulmonary organs differed widely among these studies. A morphologic study has shown that inhaled polystyrene particles are transported into the pulmonary capillary space, presumably by trans-cytosis [40].

It would be also very important to know how and to what extent lung inflammation modulates the extra-pulmonary translocation and effects of particles. Particle-induced pulmonary and systemic inflammation, accelerated atherosclerosis, and altered cardiac autonomic function may be part of the patho-physiological pathways, linking particulate air pollution with cardiovascular mortality. It has been shown that particles deposited in the alveoli lead to activation of cytokine production by alveolar macrophages and epithelial cells and to recruitment of inflammatory cells. Nemmar et al [41, 42] showed that polystyrene particles of 60 nm diameter (neutral, negative or positive charged) have a direct effect on haemostasis by the intravenous injection but also by the intratracheal administration; positively charged amine-particles led to a marked increase in prothrombotic tendency, resulting from platelet activation. The lack of effect of the larger particles on thrombosis, despite their marked effect on pulmonary inflammation, suggests that pulmonary inflammation by itself was insufficient to influence peripheral thrombosis.
To clarify the mechanisms of nanoparticle permeability, both their transepithelial transport and the factors governing their translocation - such as dose, size, chemical composition and surface chemistry - as well as time course should be investigated. <<<