Paradigms for Assessing NM Toxicity

Air pollution and mineral dust particles have been implicated in a number of adverse biological effects and disease outcomes. Major disease outcomes include the exacerbation of airway inflammation, asthma, interstitial pulmonary fibrosis, atherosclerosis, ischemic cardiovascular events, and cardiac arrhythmias. Fortunately, no clinically recognizable disease outcomes have so far been reported for manufactured NM to date. Although a household-cleaning product Magic Nano was recently implicated in respiratory symptoms, closer investigation failed to reveal a link to NM. However, we are just entering the nano-revolution and it is quite possible that clinically relevant NM toxicity could emerge. While such outcomes will no doubt launch intensive investigations into NM toxicity, a retroactive approach could be disastrous in terms of public perception and possibly harm the nanotechnology industry. It makes far more sense to instigate preventative measures to avert such a disaster.

Is it possible to formulate a preemptive approach to the potential danger(s) of NM? In our opinion the answer is yes, since the potential mech-anism(s) of injury can be studied by a science-based approach. One of the key mechanisms by which ambient particulate matter (PM) causes tissue injury and cardiopulmonary disease is through the generation of the reactive oxygen species (ROS) and oxidative stress. Since the oxidative stress paradigm has evolved into a comprehensive disease model, it illustrates the type of approach that could be used to develop a predictive paradigm for the NM toxicity testing.

Biological systems are generally able to integrate multiple pathways of injury into a limited number of pathological outcomes, including

TABLE 6.1 NM Effects as the Basis for Pathophysiology and Toxicity

Experimental NM Effects

Possible Pathophysiological Outcomes

ROS generation* Oxidative stress*

Mitochondrial perturbation*

Inflammation*

Uptake by reticulo-endothelial system*

Protein denaturation, degradation* Nuclear uptake* Uptake in neuronal tissue* Perturbation of phagocytic function,* "particle overload," mediator release* Endothelial dysfunction, effects on blood clotting* Generation of neo-antigens, breakdown in immune tolerance Altered cell cycle regulation DNA damage

Protein, DNA & membrane injury,* oxidative stress^ Phase II enzyme induction, inflammation^, mitochondrial perturbation* Inner membrane damage,* PTP opening,* energy failure,* apoptosis,* apo-necrosis, cytotoxicity Tissue infiltration with inflammatory cells^, fibrosis^, granulomas^, atherogenesis^, acute phase protein expression (e.g., C-reactive protein) Asymptomatic sequestration and storage in liver,* spleen, lymph nodes^, possible organ enlargement and dysfunction Loss of enzyme activity,* auto-antigenicity

DNA damage, nucleoprotein clumping,* autoantigens Brain and peripheral nervous system injury Chronic inflammation^, fibrosis^, granulomas^, interference in clearance of infectious agents^

Atherogenesis,* thrombosis,* stroke, myocardial infarction Autoimmunity, adjuvant effects

Proliferation, cell cycle arrest, senescence Mutagenesis, metaplasia, carcinogenesis

Adapted from [11].

*Limited experimental evidence

^Limited clinical evidence inflammation, apoptosis, necrosis, fibrosis, hypertrophy, metaplasia, and carcinogenesis (Table 6.1). Although oxidative stress feeds into most of these outcomes, it is important to mention that it is by no means the only injury mechanism that will cause such pathological outcomes. Other forms of injury include the disruption of biological membranes, protein denaturation, DNA damage, immune reactivity, and the formation of foreign body granulomas. In fact, one of the first biological interactions that take place when a nanoparticle penetrates or enters a tissue is contact with the surface membrane of the target cell. This interaction can lead to membrane damage based on particle properties such as hydrophobicity, cationic charge, or detergent activity that allow the particle contact, penetration, or disruption of membrane integrity. The cell may respond by leakage of intracellular content, intracellular Ca2+ release, and the induction of apoptosis. To mention but one example, some cationic dendrimers are capable of disrupting cell membranes by being able to pull off lipid molecules, leading to the formation of

Figure 6.1 Particle-Related Characteristics Module. Nanoparticle physico-chemical characterization can be divided into modules to clarify how primary, secondary, and tertiary particle characteristics may lead to adverse biological effects. Adapted from [42].

membrane defects. This leads to the release of cellular enzymes that can be assayed for [13]. Protein denaturation or degradation at the organic/inorganic interphase can lead to functional or structural changes in proteins at the primary, secondary, or tertiary level; this could manifest as interference in enzyme function or exposure to antigenic sites [14]. This damage may result from splitting of covalent bonds that are responsible for protein structure, such as disulfide bonds (Figure 6.1). There is also some evidence that certain nanoparticle characteristics facilitate cellular uptake and access to the nucleus, where DNA damage can result [15, 16]. In addition to this list of potential NM injuries, it is conceivable that new material properties may emerge that could lead to a new mechanism of toxicity.

Oxidative stress as a predictive paradigm for ambient ultrafine particle toxicity

Although the manufacture of NM is a relatively new scientific development, particle toxicity in response to ambient PM or mineral dust particles is a mature science. Animal exposure to these particles has shown linkage of pulmonary inflammation to the ability of the particles to induce ROS production and biologically relevant levels of oxidative stress [17, 18]. More recently, PM exposure has also been linked to systemic inflammatory effects in the cardiovascular system; this could be due to either the ability of ambient PM to induce pulmonary inflammation or the ability of the ultrafine particles (aerodynamic diameter <100 nm) to gain access to the systemic circulation. Cellular studies have generally supported the role of oxidative stress, pro-inflammatory cytokines, and programmed cell death as relevant mechanisms of PM injury [19, 20]. Ambient ultrafine particles have a higher pro-oxidative potential than ambient particles of larger size [19].

In contrast to the heterogeneous characteristics of ambient PM, manufactured NM are more homogeneous in shape, size, and form. In spite of these differences, research into ambient PM has helped to establish a number of principles that can be used to study NM toxicity. These include recognition that small particle size, large surface area, chemical composition, and ability to catalyze ROS production are important properties that determine PM-induced oxidant injury and inflammation [6,7]. Additional NM properties may contribute to ROS generation and oxidant injury and will be discussed throughout this chapter.

Oxidative stress refers to a state in which cellular GSH is depleted while oxidized glutathione (GSSG) accumulates. Under normal coupling conditions, ROS are generated at low frequency, mostly in mitochondria, and are easily neutralized by antioxidant defense mechanisms such as the glutathione (GSH)/glutathione disulfide (GSSG) redox couple. Ambient nanoparticles can elicit further ROS production in mitochondria, in addition to ROS generation by catalytic conversion pathways and NADPH oxidase activation [19, 21]. PM-induced ROS production is dependent on the particles themselves, as well as the redox cycling organic chemicals and transition metals that coat the particle surface [19, 21]. In addition to intrinsic redox cycling capabilities, the metabolic transformation of these chemicals and their ability to elicit intracellular calcium flux, disrupt electron flow in the mitochon-drial inner membrane, perturb the permeability transition pore, and deplete cellular GSH content could contribute to cellular ROS generation. While small amounts of ROS could be buffered by the antioxidant defense pathways in the cell, excess amounts of ROS could lead to a drop in the GSH/GSSG ratio. This elicits additional cellular responses.

Only a limited number of manufactured NM have so far been shown to exert toxicity in tissue culture and animal experiments, and usually at high doses. A recent study shows that the biological response of BV2 microglia to noncytotoxic concentrations of TiO2 induced a rapid (<5 minutes) and sustained (120 minutes) release of reactive oxygen species [22]. The kinetics of ROS production suggests that TiO2 stimulates immediate oxidative burst activity in microglia and can also interfere in mitochondrial energy production. Carbon nanostructures represent another material type that has undergone some toxicity testing [11]. Water-soluble, monodisperse or colloidal fullerene aggregates induce O2-anions, lipid peroxidation in cells and tissue as well as the ability to affect GSH depletion and cytotoxicity [23]. In vitro incubation of keratinocytes and bronchial epithelial cells with relatively high doses of single-wall carbon nanotubes (SWNT) results in ROS generation, lipid peroxidation, oxidative stress, mitochondrial dysfunction, and changes in cell morphology [24,25]. MWNT also elicits pro-inflammatory effects in keratinocytes [26].

Oxidative stress elicits quantifiable cellular responses that can be used to study NM toxicity

Cells respond to oxidative stress by mounting a number of protective and injurious responses that, depending on the stress level, can lead to a protected or an injurious outcome [7, 11]. The protective responses are elicited by even minor changes in the cellular redox equilibrium. This sensitivity is rooted in the behavior of the cap 'n' collar transcription factor, Nrf-2, that operates on the antioxidant response element (ARE) in the promoter of > 200 phase II enzymes [27]. These phase II enzymes exert antioxidant, detoxification and anti-inflammatory effects that are responsible for preventing or slowing the effects of oxidant injury [27]. Examples of phase II enzymes include HO-1, glutathione-S-transferase, NADPH quinone oxidoreductase, catalase, superoxide dismutase, glutathione peroxidase, and UDP-glucoronosyltransferase [27, 28]. A reduced or compromised phase II response promotes susceptibility to oxi-dant injury. In studies conducted in epithelial cells and macrophages, phase II enzyme expression is the most sensitive oxidative response parameter and has also been referred to as Tier 1 of the hierarchical oxidative stress response [29—31]. A higher level of oxidative stress can lead to a Tier 2 response, which is characterized by pro-inflammatory effects that follow the activation of intracellular signaling cascades, including the MAP kinase and NF-kB signaling cascades [30-32]. These signaling cascades are responsible for the activation of a number of cytokines, chemokines, and adhesion molecules that play a role in local and systemic inflammatory responses [32-34]. The dynamic equilibrium between the protective (Tier 1) and pro-inflammatory responses (Tier 2) determines the outcome of the oxidative stress response and the likelihood that this will lead to injury.

Tier 3 responses involve mitochondrial perturbation, Ca2+ flux, and activation of apoptosis pathways [21, 30, 32, 35]. Although the in vivo significance of the mitochondrial pathway is still under investigation, it has been demonstrated in tissue culture cells that ambient PM interferes in mitochondrial electron transfer, thereby leading to dissipation of the mitochondrial membrane potential, increased ROS production, and the induction of programmed cell death [30, 32, 35]. The basis for the mitochondrial response may be direct oxidant injury by free radicals, as well as the effect of free ionized calcium, which acts as an intra-cellular pathway by which oxidative stress can indirectly impact mitochondrial function. Research on ambient UFP also reveals the interesting possibility that the mitochondrion could be directly targeted by nanoparticles [19, 21, 36]. Ambient UFP lodge in the damaged mitochondria of exposed macrophages and epithelial cells [19, 21, 37]. Other nano-sized materials have been demonstrated to target mitochondria. As early as 1970, de Lorenzo found that colloidal gold particles (50 nm) delivered intra-nasally in the squirrel monkey cross the olfactory nerve/mitral cell synapse and have the capability to lodge in mitochondria of the mitral cells [38]. Afullerene derivative, C61(CO2H)2, has been shown capable of crossing the surface membrane with preferential localization in mitochondria [39]. The same observation was made with block copolymers, which are water-soluble biocompatible nano-containers that can be used for drug delivery. Fluorescent-labeled block copolymer micelles localize in a variety of cytoplasmic organelles, including mitochondria [40]. In summary, mitochondrial targeting and damage could constitute an important mechanism of NM toxicity. Changes in mitochondrial membrane potential, calcium uptake, O2 production, car-diolipin integrity, and induction of cellular apoptosis represent testable responses.

Need for standardized materials

Well-characterized NM that have undergone rigorous biological testing to show reproducible biological effects are required as standard reference materials to compare the effects of newly introduced NM. These benchmark materials will help to prevent the discrepancy and paradoxical findings that arise from the study of NM types in different hands. The choice of such standards should be based on the physico-chemical properties of the material, frequency of use, volume of production, and likelihood of release as a singlet substance to which humans and the environment may be exposed. Carbon black is a bulk-manufactured material that is in widespread use, including as a powder that can be inhaled. In a highly purified form, carbon black is incapable of ROS generation and devoid of cytotoxicity [41]. In their unadulterated form these could serve as a benchmark material that does not engage in ROS production and oxidant injury. TiO2 nanoparticles, also widely used and bulk-produced, can be considered as a representative material capable of ROS production under abiotic conditions [41]. Carbon nan-otubes could be used as a NM that, due to their large aspect ratios, could act as biopersistent fibers. A fullerene could also be considered for the development as a carbon-based reference standard NM. No doubt as we undertake a more deliberate exploration of NM properties that lead to adverse biological events, we may be able to develop reference materials with predictive toxicity. The establishment of benchmark standards will rapidly expand our knowledge of NM toxicity.

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