Nanotoxicology: a definition

Another illustration of the scope of current research activity (and hence of future published material) can be obtained from the recently released National Nanotechnology Initiative report from the Nanotechnology Environmental & Health Implications (NEHI) working group (NNI, 2008). Although this report covers only US based research, the general trends presented in the review are relevant also to current European research activity.

Nanotoxicology can be defined as the study of the interactions of nanoparticles with biological systems with an emphasis on establishing the relationship, if any, between the physical and chemical properties of nanoparticles and the induction of toxicological responses. The term ‘nanoparticle’ is used here to refer to free nanoparticles that are present in air or suspended in liquid (Hansen et al.,2007’s suggested Category IIIb-d), but does not include those bound to surfaces, suspended in solids, or components of structured surfaces, films, etc, for which toxicology data is mostly lacking. This may be an important gap in deriving risks related to the lifetime usage of a product, e.g. the adherence to and leaching of MNPs from material surfaces.

There is a correspondingly large and rapidly expanding published literature on the toxicology of manufactured nanoparticles (MNPs) that deserves a few words of overview.

Depending on the particular combinations(s) of keywords used, the relevant current literature relating to MNP toxicology consists of between 400 and 800 papers, the vast majority of which concern the cytotoxic effects of MNPs in cell culture systems, mostly mammalian.

A commendable percentage of the multi million dollar US budget is devoted to improved instrumentation and analytical technologies for characterising particles and for studying their interactions with biological materials and the consequences for human health. An extremely small percentage of funding effort is devoted to environmentally realistic exposure assessment and risk management protocols. A similar conclusion has been reached by Grieger et al., (2007), who note the majority of ecotoxicology-relevant publications to be in the areas of regulation, characterisation of nanoparticles and testing (not always together), with a complete absence of exposure assessment. This emphasises that when reviewing the literature relating to proposed mechanisms of cellular uptake and pathways of toxicity, it is also necessary to consider what types of exposure would lead to these effects in real world situations.

An accompanying review (S. Holgate, 2008) provides an excellent overview of the literature relevant to mechanisms of toxicity of MNPs in mammalian systems and in particular in relation to lung and inhalation toxicology.

http://www.rcep.org.uk/novel%20materials/Literature%20Review_Ecotoxicology%20of%20Nanomaterials.pdf




The behaviour of manufactured NPs in environmental matrices such as natural waters, sediments or soils

There is now a wider debate about the risks and benefits of the many manufactured NMs and consumer products (Royal Society 2004; US EPA 2005; Owen and Depledge 2005; Handy and Shaw 2007; Owen and Handy 2007)

Clearly, the scientific debate on the environmental safety of NMs needs to adopt a multi-disciplinary approach involving physicists, chemists, material scientists, biologists, toxicologists, risk assessors, regulators and policy makers. There was a seminar, which took place in Museum of Science in London where for the first time gathered diverse people from chemistry, biology, and risk assessment issues together in one volume.

Nanoparticles are no new phenomena, being a natural product of sea waves, volcanic eruption, etc. If we consider atmospheric dust alone, estimates indicate about one billion metric tons per year are produced globally (Kellogg andGriffin 2006). There is also incidental production of NPs from human activity (e.g.,wear of car tyres, urban air pollution) that may also present a toxicological risk (reviewed in Handy and Shaw 2007). Their effects on human, however, depend on their shape, size, surface energy, and chemistry. Their behaviour in agglomeration, or other properties such as the attractive-repulsive properties of the particles in collision and its frequencies, and also their colloidal environment is important as medium to define further interactions, and agglomeration. After an initial collision, particles may remain in aqueous phase as single particles, or form particle–particle, particle–cluster and cluster–cluster aggregates. The forces involved in the collisions include Borne repulsion, diffuse double layer potential, and van der Waals attraction. These are the cause for particles attachment to the walls of equipments, and aggregation in natural waters, or more often on the organisms which may have toxicological implications regarding fate and behaviour of the materials, and the types of ecosystems and organisms exposed. Nanoparticles themselves may be coated by natural organic matter that leaves them dispersed for longer period of time. For example, additions of negatively charged
humic and fulvic acids to positively charged mineral NPs in natural freshwater. Other components of water such as Ca2+ hugely affect surface charge. For changing the shape or surface chemistry detergents or surfactants were added to the water, for instance to wash away SWCNTs, SDS were experimented. Ecotoxicology have been considering surface behaviour for a long time (Handy and Eddy 1991). The surface of the organism may present a complex unstirred layer (USL), which could result in shear forces that either cause particle aggregation (peri-kinetic aggregation, Handy et al. 2008). Such processes have already been implicated in TiO2 NP toxicity to trout (Federici et al. 2007). Solid–liquid and air–water interfaces in the environment may show similar properties that attract NPs to agglomerate on species, and act as transporter. Baun et al. (2008) recently showed that the uptake of phenanthrene by Daphnia magna was much faster in the presence of C60 NPs and was probably due to the NPs enabling delivery of the phenanthrene to the test organism.
Respiratory toxicology and inflammation reactions to NP exposure are important routes in the uptake of NPs and in relation to NPs impacts in ecotoxicology. Biochemical change and genotoxicity also require investigation in wildlife. There has been evidence of impacts on Ti particles hampering algae growth, but concentration of NPs on soil showed to have no impact. Moore (2006) also raised concerns about NPs acting as delivery vehicles for other chemicals via endocytosis pathways. Manufactured nanoparticles have not shown dispersion in water so far. For example, carbon nanotubes are almost impossible to disperse in water by physical methods such as sonication or stirring alone, and may require the use of a dispersing agent (e.g., Smith et al. 2007).
The main requirement is categorisation of NPs for the purposes of ecotoxicological risk assessments in analysis of products life cycle that would release NPs into the environment. We should recognise that the behaviour of NPs in the marine environment is likely to be very different from some freshwaters, and that a fundamental understanding of natural NPs and colloids may be a prerequisite to elucidating the fate and behaviour of manufactured NPs in complex environmental matrices. Ecotoxicologists therefore need to learn some physico-chemistry, and work more closely with physicists, chemists, and material scientists to achieve the correct interpretation of data from ecotoxicity experiments.

Handy R D et al, The ecotoxicology of nanoparticles and nanomaterials: current status, knowledge gaps, challenges, and future needs, Ecotoxicology Journal, 17:315-325, Springer, April 2008


Toxic potential of materials at nano-level

Although the extraordinary characteristics of NP may necessitate novel investigation approach to assess their hazard potential, particle toxicology is a mature science that addresses the mechanisms of lung injury by inhaled particles (4–6). Inhaled or instilled ambient ultrafine particles (particulate matter with an aerodynamic diameter G 100 nm) can induce pulmonary inflammation, oxidative stress, and distal organ involvement. In a similar fashion, occupational exposure to quartz, mineral dust particles (e.g., coal and silicates), and asbestos fibers induce oxidative injury, inflammation, fibrosis, cytotoxicity, and mediator release from lung target cells (4–8). The same holds true for experimental instillation of titanium dioxide (TiO2) and carbon black nanoparticles in animal lungs. Tissue and cell culture analysis support the physiological response seen in animal models, pointing to the role of oxidative stress in the production of inflammatory cytokines and cytotoxic cellular responses.

In addition to the paradigm of oxidative stress and inflammation, it is important to consider that some of the NM interactions depicted may also results in other forms of injury, such as protein denaturation, membrane damage, DNA damage, immune reactivity, and the formation of foreign body granulomas. It is also possible that new NM properties may emerge that can lead to novel mechanisms of toxicity.

Carbon nanotubes are long carbon-based tubes that can be either single- or multiwalled and have the potential to act as biopersistent fibers. Nanotubes have aspect ratios Q 100, with lengths of several mm and diameters of 0.7 to 1.5 nm for single-walled nanotubes (SWNT) and 2 to 50 nm for multiwalled nanotubes (MWNT). In vitro incubation of keratinocytes and bronchial epithelial cells with high doses of SWNT results in ROS generation, lipid peroxidation, oxidative stress, mitochondrial dysfunction, and changes in cell morphology (19).

The biological impacts of NM and the biokinetics of nanoparticles are dependent on size, chemical composition, surface structure, solubility, shape, and aggregation. These parameters can modify cellular uptake, protein binding, translocation from portal of entry to the target site, and the possibility of causing tissue injury (4).

Although inhalation is a less likely route for engineered NM exposure compared with ambient or mineral dust particles, this can happen during bulk manufacture and handling of freely dispersable nanoparticles. Inhaled nanoparticles are efficiently deposited by diffusional mechanisms in all regions of the lung (4).

It has been proposed that Radio-labeled ultrafine carbon black may translocate through the respiratory epithelial layer to reach the lung interstitium or the blood and lymph circulations, but this finding has been refuted by others (25, 26).

....the state of particle aggregation or dispersion is important in cellular interactions as exemplified by the finding that, if nanoparticles are coated with lung surfactant before cellular incubation, the cellular fate differs from that of uncoated particles. The assessment of nanomaterial inorganic and organic coatings and state of aggregation are therefore important considerations in evaluating NM toxicity.

.......However, given the unique characteristics of NM, this will necessitate new test strategies to delineate the novel mechanisms of injury that may arise from these materials. More refined approaches for NM characterization and toxicological evaluations will emerge with time, for example, use of nanosensors to detect ROS generation by nanoparticles. This could make these evaluations cost effective, facilitating new product development.

What type of NM testing should be performed? The National Toxicology Program (NTP) in the United States has been established as an interagency program to evaluate chemical agents that are of public health concern by implementing modern toxicology tools. [Other governmental agencies, such as the Environmental Protection Agency (EPA) and the National Institute of Occupational Safety and Health (NIOSH) also have important roles in assessing nanomaterial safety in the United States, which will not be discussed here]. Although it is still questionable whether NM should be treated as commercial or industrial chemicals, the preferred NTP approach to chemical toxicity is a predictive scientific model that focuses on target-specific, mechanism-based biological observations, rather than a descriptive approach.

...among the 80,000 chemicals that are currently registered for commercial use in the United States, only 530 have undergone long-term and 70 short-term testing by the
NTP. Moreover, the resource-intensive nature of these studies puts the cost of each bioassay at $2 to $4 million and takes over 3 years to complete.

Much can be learned from research into the adverse health effects of ambient PM, where progress was slow until major mechanistic hypotheses were introduced. Armed with the knowledge that particle size, surface area, and chemical composition are important for ROS generation as a key toxicity principal, it has become
easier to design in vivo studies in at-risk populations (8). The extent to which this or other paradigms of injury (Table 2) apply to a wide range of NM needs to be determined.

Although it is not possible to provide detailedprotocols for nanotoxicity testing here, it will suffice to mention that the three key elements of a toxicity screening strategy should include physicochemical characterization of NM, in vitro assays (cellular and noncellular), and in vivo studies (40). There is a strong likelihood that biological activity will depend on physicochemical characteristics that are not usually considered in toxicity screening studies. Thus, any test paradigm must attempt to characterize the test material with respect to size (surface area, size
distribution), chemical composition (purity, crystallinity, electronic properties, etc.), surface structure (surface reactivity, surface groups, inorganic/organic coatings, etc.), solubility, shape and aggregation. This should be done at the time of NM administration as well as at the conclusion, if possible. It is beyond the scope of this paper to discuss the scientific methods for NM characterization except to comment that standard reference materials (e.g., TiO2, carbon black, quartz) are essential to compare material behavior. Cellular assays should reflect portal-of-entry toxicity in lungs, skin, and mucus membranes as well as noxious effects on target tissue such as endothelium, blood cell elements, spleen, liver, nervous system, heart, and kidney. Noncellular assays could include protein interactions and
pro-oxidant activity. The in vivo studies can make use of disease-specific animal models that assess portal of entry and target organ injury, as well as animal models in which live imaging can be used to show the activation of oxidative stress and redox signaling pathways that are involved in particle-induced tissue injury. When
in vivo toxicity is observed, it may also be appropriate to proceed with studies that formerly assess the absorption, distribution, metabolism, and elimination of NM. Because NM have the potential to spread beyond the portal of entry, it is important to assess systemic responses. Examples include assays for oxidative stress (e.g., lipid peroxidation), C-reactive protein, immune and inflammatory responses, and cytotoxicity (e.g., release of liver enzymes and glial fibrillary acidic protein). The biological studies can be strengthened by the use of discovery tools such as proteomics and genomics to develop biomarkers for toxicity screening (12).

As testing proceeds, it will be important to incorporate these data into a knowledge base that allows investigators to classify NM as safe or possibly hazardous. Negative data should be reported to show which materials are devoid of
toxic effects. This could represent the majority of NM. Potential difficulties may be encountered in conducting in vitro and in vivo studies with engineered NM. These include problems with dosimetry, state of agglomeration (singlets versus aggregates), impact of material coating, and lack of knowledge of real-world exposures to NM. Detection methods need to be developed for exposure assessment and dosimetry calculation. Current state-of-the-art methods to detect airborne nanoparticles should enable personal monitoring devices to be developed to assess these exposures.

Andre Nel et al, Science 311, 622 (2006)


Cell Physiology

Treating cells with a cytotoxic compound can result in a variety of cell fates. The cells may undergo necrosis, in which they lose membrane integrity and die rapidly as a result of cell lysis. The cells can stop actively growing and dividing (a decrease in cell viability), or the cells can activate a genetic program of controlled cell death (apoptosis).

Cells undergoing necrosis typically exhibit rapid swelling, lose membrane integrity, shut down metabolism and release their contents into the environment. Cells that undergo rapid necrosis in vitro do not have sufficient time or energy to activate apoptotic machinery and will not express apoptotic markers.[1] Apoptosis is characterized by well defined cytological and molecular events including a change in the refractive index of the cell, cytoplasmic shrinkage, nuclear condensation and cleavage of DNA into regularly sized fragments.[2] Cells in culture that are undergoing apoptosis eventually undergo secondary necrosis. They will shut down metabolism, lose membrane integrity and lyse.[2][3]

[edit] Measuring Cytotoxicity

Cytotoxicity assays are widely used by the pharmaceutical industry to screen for cytotoxicity in compound libraries. Researchers can either look for cytotoxic compounds, if they are interested in developing a therapeutic that targets rapidly dividing cancer cells, for instance; or they can screen "hits" from initial high-throughput drug screens for unwanted cytotoxic effects before investing in their development as a pharmaceutical.

Assessing cell membrane integrity is one of the most common ways to measure cell viability and cytotoxic effects. Compounds that have cytotoxic effects often compromise cell membrane integrity. Vital dyes, such as trypan blue or propidium iodide are normally excluded from the inside of healthy cells; however, if the cell membrane has been compromised, they freely cross the membrane and stain intracellular components.[3] Alternatively, membrane integrity can be assessed by monitoring the passage of substances that are normally sequestered inside cells to the outside. One commonly measured molecule is lactate dehydrogenase (LDH).[4] Protease biomarkers have been identified that allow researchers to measure relative numbers of live and dead cells within the same cell population. The live-cell protease is only active in cells that have a healthy cell membrane, and loses activity once the cell is compromised and the protease is exposed to the external environment. The dead-cell protease cannot cross the cell membrane, and can only be measured in culture media after cells have lost their membrane integrity.[5]

Cytotoxicity can also be monitored using the MTT or MTS assay. This assay measures the reducing potential of the cell using a colorimetric reaction. Viable cells will reduce the MTS reagent to a colored formazan product. A similar redox-based assay has also been developed using the fluorescent dye, resazurin. In addition to using dyes to indicate the redox potential of cells in order to monitor their viability, researchers have developed assays that use ATP content as a marker of viability.[3] Such ATP-based assays include bioluminescent assays in which ATP is the limiting reagent for the luciferase reaction.[6]

Cytotoxicity can also be measured by the Sulforhodamine B (SRB) assay, WST assay and clonogenic assay.

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