Open Access

Review

Physicochemical characteristics as determinants of biologic activity.

The small size and corresponding large specific surface area of solid NSPs (Table 2) confer specific properties to them, for example, making them desirable as catalysts for chemical reactions. The importance of surface area becomes evident when considering that surface atoms or molecules play a dominant role in determining bulk properties (Amato 1989); the ratio of surface to total atoms or molecules increases exponentially with decreasing particle size (Figure 2). Increased surface reactivity predicts that NSPs exhibit greater biologic activity per given mass compared with larger particles, should they be taken up into living organisms and provided they are solid rather than solute particles. This increased biologic activity can be either positive and desirable (e.g., antioxidant activity, carrier capacity for therapeutics, penetration of cellular barriers for drug delivery) or negative and undesirable (e.g., toxicity, induction of oxidative stress or of cellular dysfunction), or a mix of both. Not only may adverse effects be induced, but interactions of NSPs with cells and subcellular structures and their biokinetics are likely to be very different from those of larger-sized particles. For example, more than 60 years ago virologists described the translocation of 30- to 50-nm-sized virus particles along axons and dendrites of neurons and across epithelia (Howe and Bodian 1940), whereas first reports about increased inflammatory activity and epithelial translocation of man-made 20- and 30-nm solid particles appeared only more recently (Ferin et al. 1990; Oberdörster et al. 1990).

The characteristic biokinetic behaviors of NPs are attractive qualities for promising applications in medicine as diagnostic and therapeutic devices and as tools to investigate and understand molecular processes and structures in living cells (Akerman et al. 2002; Foley et al. 2002; Kreuter 2001; Li et al. 2003). For example, targeted drug delivery to tissues that are difficult to reach [e.g., central nervous system (CNS)], NPs for the fight against cancer, intra-vascular nanosensor and nanorobotic devices, and diagnostic and imaging procedures are presently under development. The discipline of nanomedicine—defined as medical application of nanotechnology and related research—has arisen to design, test, and optimize these applications so that they can eventually be used routinely by physicians (Freitas 1999).

However, in apparent stark contrast to the many efforts aimed at exploiting desirable properties of NPs for improving human health are the limited attempts to evaluate potential undesirable effects of NPs when administered intentionally for medicinal purposes, or after unintentional exposure during manufacture or processing for industrial applications. The same properties that make NPs so attractive for development in nanomedicine and for specific industrial processes could also prove deleterious when NPs interact with cells. Thus, evaluating the safety of NPs should be of highest priority given their expected worldwide distribution for industrial applications and the likelihood of human exposure, directly or through release into the environment (air, water, soil). Nanotoxicology—an emerging discipline that can be defined as “science of engineered nanodevices and nanostructures that deals with their effects in living organisms”—is gaining increased attention. Nanotoxicology research not only will provide data for safety evaluation of engineered nanostructures and devices but also will help to advance the field of nanomedicine by providing information about their undesirable properties and means to avoid them.

Human exposure to nanosized materials.

In addition to natural and anthropogenic sources of UFPs in the ambient air, certain workplace conditions also generate NSPs that can reach much higher exposure concentrations, up to several hundred micrograms per cubic meter, than is typically found at ambient levels. In contrast to airborne UFP exposures occurring via inhalation at the workplace and from ambient air, not much is known about levels of exposure via different routes for NPs, whether it is by direct human exposure or indirectly through contamination of the environment. For example, are there or will there be significant exposures to airborne singlet engineered carbon nanotubes or C₆₀ fullerenes? First measurements at a model workplace gave only very low concentrations, < 50 μg/m³, and these were most likely in the form of aggregates (Maynard et al. 2004). However, even very low concentrations of nanosized materials in the air represent very high particle number concentrations, as is well known from measurements of ambient UFPs (Hughes et al. 1998). For example, a low concentration of 10 μg/m³ of unit density 20-nm particles translates into > 1 × 10⁶ particles/cm³ (Table 2). Inhalation may be the major route of exposure for NPs, yet ingestion and dermal exposures also need to be considered during manufacture, use, and disposal of engineered nanomaterials, and specific biomedical applications for diagnostic and therapeutic purposes will require intravenous, subcutaneous, or intramuscular administration (Table l). It can be assumed, however, that the toxicology of NPs can build on the experience and data already present in the toxicology literature of ambient UFPs. [Additional details provided in Supplemental Material available online (http://ehp.niehs.nih.gov/members/2005/73​39/supplemental.pdf ).]

Manufactured nanomaterials in the environment.

Manufactured nanomaterials are likely to enter the environment for several reasons. Some are and others will be produced by the ton, and some of any material produced in such mass quantities is likely to reach the environment from manufacturing effluent or from spillage during shipping and handling. They are being used in personal-care products such as cosmetics and sunscreens and can therefore enter the environment on a continual basis from washing off of consumer products (Daughton and Ternes 1999). They are being used in electronics, tires, fuel cells, and many other products, and it is unknown whether some of these materials may leak out or be worn off over the period of use. They are also being used in disposable materials such as filters and electronics and may therefore reach the environment through landfills and other methods of disposal.

Scientists have also found ways of using nanomaterials in remediation. Although many of these are still in testing stages (Chitose et al. 2003; Jaques and Kim 2000; Joo et al. 2004; Nagaveni et al. 2004; Nghiem et al. 2004; Tungittiplakorn et al. 2004), dozens of sites have already been injected with various nanomaterials, including nano-iron (Mach 2004). Testing to determine the safety of these NPs used for remediation to environmentally relevant species has not yet been done. Although most people are concerned with effects on large wildlife, the basis of many food chains depends on the benthic and soil flora and fauna, which could be dramatically affected by such NP injections. In addition, as noted by Lecoanet et al. (2004), nanosized materials may not migrate through soils at rapid enough rates to be valuable in remediation. Future laboratory and field trials will help clarify the line between remediation and contamination [Supplemental Material available online (http://ehp.niehs.nih.gov/members/2005/73​39/supplemental.pdf )].

Toxicology of Airborne UFPs Top

In recent years, interest in potential effects of exposure to airborne UFPs has increased considerably, and studies have shown that they can contribute to adverse health effects in the respiratory tract as well as in extrapulmonary organs. Results on direct effects of ambient and model UFPs have been reported from epidemiologic studies and controlled clinical studies in humans, inhalation/instillation studies in rodents, or in vitro cell culture systems. For example, several epidemiologic studies have found associations of ambient UFPs with adverse respiratory and cardiovascular effects resulting in morbidity and mortality in susceptible parts of the population (Pekkanen et al. 1997; Penttinen et al. 2001; Peters et al. 1997a, 1997b; von Klot et al. 2002; Wichmann et al. 2002), whereas other epidemiologic studies have not seen such associations (Pekkanen et al. 1997; Tiittanen et al. 1999). Controlled clinical studies evaluated deposition and effects of laboratory-generated UFPs. High deposition efficiencies in the total respiratory tract of healthy subjects were found, and deposition was even greater in subjects with asthma or chronic obstructive pulmonary disease. In addition, effects on the cardiovascular system, including blood markers of coagulation and systemic inflammation and pulmonary diffusion capacity, were observed after controlled exposures to carbonaceous UFPs (Anderson et al. 1990; Brown et al. 2002; Chalupa et al. 2004; Henneberger et al., 2005; Jaques and Kim 2000; Pekkanen et al. 2002; Pietropaoli et al. 2004; Wichmann et al. 2000).

Studies in animals using laboratory-generated model UFPs or ambient UFPs showed that UFPs consistently induced mild yet significant pulmonary inflammatory responses as well as effects in extrapulmonary organs. Animal inhalation studies included the use of different susceptibility models in rodents, with analysis of lung lavage parameters and lung histopathology, effects on the blood coagulation cascade, and translocation studies to extra-pulmonary tissues (Elder et al. 2000, ²⁰⁰² , 2004; Ferin et al. 1991; Ferin and Oberdörster 1992; Kreyling et al. 2002; Li et al. 1999; Nemmar et al. 1999, 2002a, 2002b, 2003; Oberdörster et al. 1992a, 1995, 2000, 2002, 2004; Semmler et al. 2004; Zhou et al. 2003).

In vitro studies using different cell systems showed varying degrees of proinflammatory-and oxidative-stress–related cellular responses after dosing with laboratory-generated or filter-collected ambient UFPs (Brown et al. 2000, 2001; Li et al. 2003). Collectively, the in vitro results have identified oxidative-stress–related changes of gene expression and cell signaling pathways as underlying mechanisms of UFP effects, as well as a role of transition metals and certain organic compounds on combustion-generated UFPs (Figure 3). These can alter cell signaling pathways, including Ca²⁺ signaling and cytokine signaling (e.g., interleukin-8) (Donaldson et al. 2002; Donaldson and Stone 2003). Effects were on a mass basis greater for model UFPs than for fine particles, whereas effects for ambient UFP cellular responses were sometimes greater and sometimes less than those of fine and coarse particles. The interpretation of the in vitro studies is oftentimes difficult because particles of different chemical compositions were used, target cells were different, and duration, end points, and generally high dose levels also differed. Results from high doses in particular should be viewed with caution if they are orders of magnitude higher than predicted from relevant ambient exposures (see “Exposure dose–response considerations,” below). [Supplemental Material available online (http://ehp.niehs.nih.gov/members/2005/73​39/supplemental.pdf ).]

Concepts of Nanotoxicology Top

Laboratory rodent studies.

With respect to potential health effects of NSPs, two examples should serve to illustrate a) that NSPs have a higher inflammatory potential per given mass than do larger particles, provided they are chemically the same, and b) that NSPs generated under certain occupational conditions can elicit severe acute lung injury.

The first example involves studies with ultrafine and fine titanium dioxide (TiO₂) particles, which showed that ultrafine anatase TiO₂ (20 nm), when instilled intratracheally into rats and mice, induced a much greater pulmonary-inflammatory neutrophil response (determined by lung lavage 24 hr after dosing) than did fine anatase TiO₂ (250 nm) when both types of particles were instilled at the same mass dose (Figure 4A,C). However, when the instilled dose was expressed as particle surface area, it became obvious that the neutrophil response in the lung for both ultrafine and fine TiO₂ fitted the same dose–response curve (Figure 4B,D), suggesting that particle surface area for particles of different sizes but of the same chemistry, such as TiO₂, is a better dosemetric than is particle mass or particle number (Oberdörster G 2000). Moreover, normalizing the particle surface dose to lung weight shows excellent agreement of the inflammatory response in both rats and mice [Figure S-2 in Supplemental Material available online (http://ehp.niehs.nih.gov/members/2005/73​39/supplemental.pdf )]. The better fit of dose–response relationships by expressing the dose as surface area rather than mass when describing toxicologic effects of inhaled solid particles of different sizes has been pointed out repeatedly, especially when particles of different size ranges—nano to fine—were studied (Brown et al. 2001; Donaldson et al. 1998, 2002; Driscoll 1996; Oberdörster and Yu 1990; Oberdörster et al. 1992a; Tran et al. 1998, 2000) [Supplemental Material available online (http://ehp.niehs.nih.gov/members/2005/73​39/supplemental.pdf )].

Particle chemistry, and specifically surface chemistry, plays a decisive role in addition to particle size, as shown in the second example: exposure of rats to polytetrafluoroethylene (PTFE) fume. PTFE fume (generated by heating PTFE) has long been known to be of high acute toxicity to birds and mammals, including humans (Cavagna et al. 1961; Coleman et al. 1968; Griffith et al. 1973; Nuttall et al. 1964; Waritz and Kwon 1968). Analysis of these fumes revealed the nanosized nature of the particles generated by heating PTFE to about 480°C, with a count median diameter (CMD) of 18 nm. They were highly toxic to rats, causing severe acute lung injury with high mortality within 4 hr after a 15-min inhalation exposure to 50 μg/m³ (Oberdörster et al. 1995). This short exposure resulted in an estimated deposited dose in the alveolar regions of only approximately 60 ng. In humans, acute lung injury, known as polymer fume fever, can result from exposures to PTFE fumes (Auclair et al. 1983; Goldstein et al. 1987; Lee et al. 1997; Williams et al. 1974; Woo et al. 2001). Additional rat studies showed that the gas phase alone was not acutely toxic and that aging of the PTFE fume particles for 3 min increased their particle size to > 100 nm because of accumulation, which resulted in a loss of toxicity (Johnston et al. 2000). However, it is most likely that changes in particle surface chemistry during the aging period contributed to this loss of toxicity, and that this is not just an effect of the accumulated larger particle size [Supplemental Material available online (http://ehp.niehs.nih.gov/members/2005/73​39/supplemental.pdf )].

These examples seem to represent the extremes of NSPs in terms of pulmonary toxicity, one (TiO₂) being rather benign yet still inducing significantly greater inflammatory effects on a mass basis than fine particles of the same chemical makeup, the other (PTFE fumes) inducing very high acute toxicity, possibly related to reactive groups on the large surface per unit mass.

Engineered nanomaterials can have very different shapes, for example, spheres, fibers, tubes, rings, and planes. Toxicologic studies of spherical and fibrous particles have well established that natural (e.g., asbestos) and man-made (e.g., biopersistent vitreous) fibers are associated with increased risks of pulmonary fibrosis and cancer after prolonged exposures [Greim et al. 2001; International Agency for Research on Cancer (IARC) 2002]. Critical parameters are the three Ds: dose, dimension, and durability of the fibers. Fibers are defined as elongated structures with a diameter-to-length ratio (aspect ratio) of 1:3 or greater and with a length of > 5 μm and diameter ≤3 μm [World Health Organization (WHO) 1985]. Carbon nanotubes have aspect ratios of up to ≥100, and length can exceed 5 μm with diameters ranging from 0.7 to 1.5 nm for single-walled nanotubes, and 2 to 50 nm for multiwalled nanotubes. Results from three studies using intratracheal dosing of carbon nanotubes in rodents indicate significant acute inflammatory pulmonary effects that either subsided in rats (Warheit et al. 2004) or were more persistent in mice (Lam et al. 2004; Shvedova et al. 2004b). Administered doses were very high, ranging from 1 to 5 mg/kg in rats; in mice doses ranged from 3.3 to 16.6 mg/kg (Lam et al. 2004) or somewhat lower, from 0.3 to 1.3 mg/kg (Shvedova et al. 2004a). Granuloma formation as a normal foreign body response of the lung to high doses of a persistent particulate material was a consistent finding in these studies. Metal impurities (e.g., iron) from the nanotube generation process may also have contributed to the observed effects. Although these in vivo first studies revealed high acute effects, including mortality, this was explained by the large doses of the instilled highly aggregated nanotubes that caused death by obstructing the airways and should not be considered a nanotube effect per se (Warheit et al. 2004). In vitro studies with carbon nanotubes also reported significant effects. Dosing keratinocytes and bronchial epithelial cells in vitro with single-walled carbon nanotubes (SWNTs) resulted in oxidative stress, as evidenced by the formation of free radicals, accumulation of peroxidative products, and depletion of cell antioxidants (Shvedova et al. 2004a, 2004b). Multiwalled carbon nanotubes (MWNTs) showed pro-inflammatory effects and were internalized in keratinocytes (Monteiro-Riviere et al. 2005). Again, the relatively high doses applied in these studies need to be considered when discussing the relevancy of these findings for in vivo exposures. A most recent study in macrophages comparing SWNTs and MWNTs with C₆₀ fullerenes found a cytotoxicity ranking on a mass basis in the order SWNT > MWNT > C₆₀. Profound cytotoxicity (mitochondrial function, cell morphology, phagocytic function) was seen for SWNTs, even at a low concentration of 0.38 μg/cm². The possible contribution of metal impurities of the nanotubes still needs to be assessed. Therefore, whether the generally recognized principles of fiber toxicology apply to these nanofiber structures needs still to be determined (Huczko et al. 2001).

Future studies should be designed to investigate both effects and also the fate of nanotubes after deposition in the respiratory tract, preferentially by inhalation using well-dispersed (singlet) airborne nanotubes. In order to design the studies using appropriate dosing, it is necessary to assess the likelihood and degree of human exposure. It is of utmost importance to characterize human exposures in terms of the physicochemical nature, the aggregation state, and concentration (number, mass, surface area) of engineered nanomaterials and perform animal and in vitro studies accordingly. If using direct instillation into the lower respiratory tract, a large range of doses, which include expected realistic exposures of appropriately prepared samples, needs to be considered [Supplemental Material available online (http://ehp.niehs.nih.gov/members/2005/73​39/supplemental.pdf )].

Ecotoxicologic studies.

Studies have been carried out to date on only a few species that have been accepted by regulatory agencies as models for defining ecotoxicologic effects. Tests with uncoated, water-soluble, colloidal fullerenes (nC₆₀) show that the 48-hr LC₅₀ (median lethal concentration) in Daphnia magna is 800 ppb (Oberdörster E 2004b), using standard U.S. EPA protocols (U.S. EPA 1994). In largemouth bass (Micropterus salmoides), although no mortality was seen, lipid peroxidation in the brain and glutathione depletion in the gill were observed after exposure to 0.5 ppm nC₆₀ for 48 hr (Oberdörster E 2004a). There are several hypotheses as to how lipid damage may have occurred in the brain, including direct redox activity by fullerenes reaching the brain via circulation or axonal translocation (see also “Disposition of NSPs in the respiratory tract,” below) and dissolving into the lipid-rich brain tissue; oxyradical production by microglia; or reactive fullerene metabolites may be produced by cytochrome P450 metabolism. Initial follow-up studies using suppressive subtractive hybridization of pooled control fish versus pooled 0.5-ppm–exposed fish liver mRNA were also performed. Proteins related to immune responses and tissue repair were up-regulated, and several proteins related to homeostatic control and immune control were down-regulated. A cytochrome P450 (CYP2K4) involved in lipid metabolism was up-regulated [Supplemental Material available online (http://ehp.niehs.nih.gov/members/2005/73​39/supplemental.pdf )]. In addition to these biochemical and molecular-level changes in fish, bactericidal properties of fullerenes have also been reported and are being explored as potential new antimicrobial agents (Yamakoshi et al. 2003). Engineered nanomaterials used as antimicrobials may shift microbial communities if they are released into the environment via effluents. As we know from anthropogenic endocrine-disrupting compounds, interference of signaling between nitrogen-fixing bacteria and their plant hosts could be extremely harmful both ecologically and economically in terms of crop production (Fox et al. 2001).

Aqueous fullerenes and coated SWNTs are stable in salt solutions (Cheng et al. 2004; Warheit et al. 2004), cell culture media (Lu et al. 2004; Sayes et al. 2004), reconstituted hard water, and MilliQ water (Dieckmann et al. 2003; Oberdörster E 2004a, 2004b). NSPs will tend to sorb onto sediment and soil particles and be immobilized because of their high surface area:mass ratio (Lecoanet and Wiesner 2004). Biologic transport would occur from ingested sediments, and one would expect movement of nanomaterials through the food chain (Figure 5).

To make engineered nanomaterials more biocompatible, both surface coatings and covalent surface modifications have been incorporated. Some studies have shown that both the surface coating and the covalent modifications can be weathered either by exposure to the oxygen in air or by ultraviolet (UV) irradiation for 1–4 hr (Derfus et al. 2004; Rancan et al. 2002). Therefore, although coatings and surface modifications may be critically important in drug-delivery devices, the likelihood of weathering under environmental conditions makes it important to study toxicity under UV and air exposure conditions. Even coatings used in drug delivery of NPs may not be bio-persistent or could be metabolized to expose the core NP material [Supplemental Material available online (http://ehp.niehs.nih.gov/members/2005/73​39/supplemental.pdf )].

Reactive oxygen species mechanisms of NSP toxicity.

Both in vivo and in vitro, NSPs of various chemistries have been shown to create reactive oxygen species (ROS). ROS production has been found in NPs as diverse as C₆₀ fullerenes, SWNTs, quantum dots, and UFPs, especially under concomitant exposure to light, UV, or transition metals (Brown et al. 2000, 2001; Derfus et al. 2004; Joo et al. 2004; Li et al. 2003; Nagaveni et al. 2004; Oberdörster E 2004a; Rancan et al. 2002; Sayes et al. 2004; Shvedova et al. 2004a, 2004b; Wilson et al. 2002; Yamakoshi et al. 2003). It has been demonstrated that NSPs of various sizes and various chemical compositions preferentially mobilize to mitochondria (de Lorenzo 1970; Foley et al. 2002; Gopinath et al. 1978; Li et al. 2003; Rodoslav et al. 2003). Because mitochondria are redox active organelles, there is a likelihood of altering ROS production and thereby overloading or interfering with antioxidant defenses (Figure 3).

Figure 6 diagrams some of the antioxidant defense systems that occur in animals, and possible areas where NSPs may create oxyradicals. The C₆₀ fullerene is shown as a model NP producing superoxide, as has been shown by Yamakoshi et al. (2003). The exact mechanism by which each of these diverse NPs cause ROS is not yet fully understood, but suggested mechanisms include a) photo excitation of fullerenes and SWNTs, causing intersystem crossing to create free electrons; b) metabolism of NPs to create redox active intermediates, especially if metabolism is via cytochrome P450s; and c) inflammation responses in vivo that may cause oxyradical release by macrophages. Other mechanisms will likely emerge as studies on NP toxicity continue.

The small size and respective large specific surface area of NPs, like those of ambient airborne UFPs, give them unique properties with respect to a potential to cause adverse effects. Certainly, as shown in studies with UFPs, chemical composition and other particle parameters are additional important effect modifiers. Results from these studies will therefore serve as a basis for future studies in the field of nanotoxicology, for example, the propensity of NSPs to translocate across cell layers and along neuronal pathways (see “Disposition of NSPs in the respiratory tract” below).

Exposure dose–response considerations.

A careful evaluation of exposure–dose–response relationships is critical to the toxicologic assessment of NSPs. This includes not only questions about the dosemetric—mass, number, or surface of the particles, as discussed above—but most important, also the relevance of dose levels. For example, it is tempting, and a continual practice, to dose primary cells or cell lines in vitro with very high doses without any consideration or discussion of realistic in vivo exposures; for instance, 100 μg NSPs/mL culture medium—labeled as a low dose—is extremely high and is unlikely to be encountered in vivo. Likewise, intratracheal instillations of several hundred micrograms into a rat does not resemble a relevant in vivo inhalation exposure; both dose and dose rate cause high bolus dose artifacts. Although such studies may be used in a first proof-of-principle approach, it is mandatory to follow up and validate results using orders of magnitude lower concentrations resembling realistic in vivo exposures, including worst-case scenarios. The 500-year-old phrase “the dose makes the poison” can also be paraphrased as “the dose makes the mechanism.” The mechanistic pathways that operate at low realistic doses are likely to be different from those operating at very high doses when the cell’s or organism’s defenses are overwhelmed.

Therefore, in vivo and in vitro studies will provide useful data on the toxicity and mode of action of NSPs only if justifiable concentrations/doses are considered when designing such studies. This approach is particularly important for the proper identification of the dose–response curve. When data are generated only at high concentrations/doses, it is difficult to determine whether the dose–response curve in question is best described by a linear (no threshold), supralinear, threshold, or hormetic model (Figure 7). Study designs should include doses that most closely reflect the expected exposure levels. A critical gap that urgently needs to be filled in this context is the complete lack of data for human or environmental exposure levels of NSPs. Furthermore, some knowledge about the biokinetics of NSPs is required in order to estimate appropriate doses. Do specific NPs reach certain target sites? If so, what are the doses, dose rates, and their persistence? Further, although it may be tempting to extrapolate from in vitro results to an in vivo risk assessment, it is important to keep in mind that in vitro tests are most useful in providing information on mechanistic processes and in elucidating mechanisms/mode of actions suggested by studies in whole animals. A combination of in vitro and in vivo studies with relevant dose levels will be most useful in identifying the potential hazards of NPs, and a thorough discussion and justification of selected dose levels should be mandatory.

Portals of Entry and Target Tissues Top

Most of the toxicity research on NSPs in vivo has been carried out in mammalian systems, with a focus on respiratory system exposures for testing the hypothesis that airborne UFPs cause significant health effects. With respect to NPs, other exposure routes, such as skin and GI tract, also need to be considered as potential portals of entry. Portal-of-entry–specific defense mechanisms protect the mammalian organism from harmful materials. However, these defenses may not always be as effective for NSPs, as is discussed below.

Respiratory Tract

In order to appreciate what dose the organism receives when airborne particles are inhaled, information about their deposition as well as their subsequent fate is needed. Here we focus on the fate of inhaled nanosized materials both within the respiratory tract itself and translocated out of the respiratory tract. There are significant differences between NSPs and larger particles regarding their behavior during deposition and clearance in the respiratory tract [Supplemental Material available online (http://ehp.niehs.nih.gov/members/2005/73​39/supplemental.pdf )].

Efficient deposition of inhaled NSPs.

The main mechanism for deposition of inhaled NSPs in the respiratory tract is diffusion due to displacement when they collide with air molecules. Other deposition mechanisms of importance for larger particles, such as inertial impaction, gravitational settling, and interception, do not contribute to NSP deposition, and electrostatic precipitation occurs only in cases where NSPs carry significant electric charges. Figure 8 shows the fractional deposition of inhaled particles in the nasopharyngeal, tracheobronchial, and alveolar regions of the human respiratory tract under conditions of nose breathing during rest, based on a predictive mathematical model (International Commission on Radiological Protection 1994). These predictions apply to particles that are inhaled as singlet particles of a given size and not as aggregates; the latter obviously will have larger particle size and different deposition site. In each of the three regions of the respiratory tract, significant amounts of a certain size of NSPs (1–100 nm) are deposited. For example, 90% of inhaled 1-nm particles are deposited in the nasopharyngeal compartment, only approximately 10% in the tracheobronchial region, and essentially none in the alveolar region. On the other hand, 5-nm particles show about equal deposition of approximately 30% of the inhaled particles in all three regions; 20-nm particles have the highest deposition efficiency in the alveolar region (~ 50%), whereas in tracheobronchial and nasopharyngeal regions this particle size deposits with approximately 15% efficiency. These different deposition efficiencies should have consequences for potential effects induced by inhaled NSPs of different sizes as well as for their disposition to extrapulmonary organs, as discussed further below.

Disposition of NSPs in the respiratory tract.

In the preceding section we summarized data demonstrating that inhaled NSPs of different sizes can target all three regions of the respiratory tract. Several defense mechanisms exist throughout the respiratory tract aimed at keeping the mucosal surfaces free from cell debris and particles deposited by inhalation. Several reviews describe the well-known classic clearance mechanisms and pathways for deposited particles (Kreyling and Scheuch 2000; Schlesinger et al. 1997; U.S. EPA 2004), so here we only briefly mention those mechanisms and point out specific differences that exist with respect to inhaled NSPs [Supplemental Material available online (http://ehp.niehs.nih.gov/members/2005/73​39/supplemental.pdf )].

Once deposited, NSPs—in contrast to larger-sized particles—appear to translocate readily to extrapulmonary sites and reach other target organs by different transfer routes and mechanisms. One involves transcytosis across epithelia of the respiratory tract into the interstitium and access to the blood circulation directly or via lymphatics, resulting in distribution throughout the body. The other is a not generally recognized mechanism that appears to be distinct for NSPs and that involves their uptake by sensory nerve endings embedded in airway epithelia, followed by axonal translocation to ganglionic and CNS structures.

Classical clearance pathways.

The clearance of deposited particles in the respiratory tract is basically due to two processes (Table 3): a) physical translocation of particles by different mechanisms and b) chemical clearance processes. Leaching refers to loss of elements from a particle matrix (e.g., loss of sodium from asbestos fibers due to dissolution in intra-or extracellular milieu). Chemical dissolution is directed at biosoluble particles or components of particles that are either lipid soluble or soluble in intracellular and extracellular fluids. Solutes and soluble components can then undergo absorption and diffusion or binding to proteins and other subcellular structures and may be eventually cleared into blood and lymphatic circulation. Chemical clearance for biosoluble materials can happen at any location within the three regions of the respiratory tract, although to different degrees, depending on local extracellular and intracellular conditions (pH). In contrast, a number of diverse processes involving physical translocation of inhaled particles exist in the respiratory tract and are different in the three regions. Figure 9 summarizes these clearance processes for solid particles. As discussed further below, some of them show significant particle-size–dependent differences, making them uniquely effective for a certain particle size but very inefficient for other sizes.

The most prevalent mechanism for solid particle clearance in the alveolar region is mediated by alveolar macrophages, through phagocytosis of deposited particles. The success of macrophage–particle encounter appears to be facilitated by chemotactic attraction of alveolar macrophages to the site of particle deposition (Warheit et al. 1988). The chemotactic signal is most likely complement protein 5a (C5a), derived from activation of the complement cascade from serum proteins present on the alveolar surface (Warheit et al. 1986; Warheit and Hartsky 1993). This is followed by gradual movement of the macrophages with internalized particles toward the mucociliary escalator. The retention half-time of solid particles in the alveolar region based on this clearance mechanism is about 70 days in rats and up to 700 days in humans. The efficacy of this clearance mechanism depends highly on the efficiency of alveolar macrophages to “sense” deposited particles, move to the site of their deposition, and then phagocytize them. This process of phagocytosis of deposited particles takes place within a few hours, so by 6–12 hr after deposition essentially all of the particles are phagocytized by alveolar macrophages, to be cleared subsequently by the slow alveolar clearance mentioned above. However, it appears that there are significant particle-size–dependent differences in the cascade of events leading to effective alveolar macrophage-mediated clearance.

Figure 10 displays results of several studies in which rats were exposed to different-sized particles (for the 3- and 10-μm particles, 10-μg and 40-μg polystyrene beads, respectively, were instilled intratracheally) (Kreyling et al. 2002; Oberdörster et al. 1992b, 2000; Semmler et al. 2004). Twenty-four hours later, the lungs of the animals were lavaged repeatedly, retrieving about 80% of the total macrophages as determined in earlier lavage experiments (Ferin et al. 1991). As shown in Figure 10, approximately 80% of 0.5-, 3-, and 10-μm particles could be retrieved with the macrophages, whereas only approximately 20% of nanosized 15–20-nm and 80-nm particles could be lavaged with the macrophages. In effect, approximately 80% of the UFPs were retained in the lavaged lung after exhaustive lavage, whereas approximately 20% of the larger particles > 0.5 μm remained in the lavaged lung. This indicates that NSPs either were in epithelial cells or had further translocated to the interstitium [Supplemental Material available online (http://ehp.niehs.nih.gov/members/2005/73​39/supplemental.pdf )].

Epithelial translocation.

Because of the apparent inefficiency of alveolar macrophage phagocytosis of NSPs, one might expect that these particles interact instead with epithelial cells. Indeed, results from several studies show that NSPs deposited in the respiratory tract readily gain access to epithelial and interstitial sites. This was also shown in studies with ultrafine PTFE fumes: shortly after a 15-min exposure, the fluorine-containing particles could be found in interstitial and submucosal sites of the conducting airways as well as in the interstitium of the lung periphery close to the pleura (Oberdörster G 2000). Such interstitial translocation represents a shift in target site away from the alveolar space to the interstitium, potentially causing direct particle-induced effects there.

In a study evaluating the pulmonary inflammatory response of TiO₂ particles, ranging from NP TiO₂ to pigment-grade TiO₂ (12–250 nm), a surprising finding was that, 24 hr after intratracheal instillation of different doses, higher doses induced a lower effect (Oberdörster et al. 1992a). This was explained by the additional finding that at the higher doses (expressed as particle surface area) of the nanosized TiO₂, ≥50% had reached the pulmonary interstitium, causing a shift of the inflammatory cell response from the alveolar space to the interstitium [Supplemental Material available online (http://ehp.niehs.nih.gov/members/2005/73​39/supplemental.pdf )]. The smaller particle size of 12 and 20 nm versus 220 and 250 nm also means that the administered particle number was more than three orders of magnitude higher for the NSPs, a factor that seems to be an important determinant for particle translocation across the alveolar epithelium, as are the delivered total dose and the dose rate (Ferin et al. 1992). Because interstitial translocation of fine particles across the alveolar epithelium is more prominent in larger species (dogs, nonhuman primates) than in rodents (Kreyling and Scheuch 2000; Nikula et al. 1997), it is reasonable to assume that the high translocation of NSPs observed in rats occurs in humans as well [Supplemental Material available online (http://ehp.niehs.nih.gov/members/2005/73​39/supplemental.pdf )].

Translocation to the circulatory system.

Once the particles have reached pulmonary interstitial sites, uptake into the blood circulation, in addition to lymphatic pathways, can occur; again, this pathway is dependent on particle size, favoring NSPs. Berry et al. (1977) were the first to describe translocation of NSPs across the alveolar epithelium using intratracheal instillations of 30-nm gold particles in rats. Within 30 min postexposure, they found large amounts of these particles in platelets of pulmonary capillaries; the researchers suggested that this is an elimination pathway for inhaled particles that is significant for transporting the smallest air pollutant particles—in particular, particles of tobacco smoke—to distant organs. They also hypothesized that this “might predispose to platelet aggregation with formation of microthrombi atheromatous plaques” (Berry et al. 1977).

Since then, a number of studies with different particle types have confirmed the existence of this translocation pathway, as summarized in Table 4. Collectively, these studies indicate that particle size and surface chemistry (coating), and possibly charge, govern translocation across epithelial and endothelial cell layers. In particular, the studies summarized by Mehta et al. (2004) and those performed by Heckel et al. (2004) using intravenous administration of albumin-coated gold nanoparticles in rodents demonstrated receptor-mediated transcytosis (albumin-binding proteins) via caveolae (Figure 11). These 50–100 nm vesicles, first described by Simionescu et al. (1975), form from indentations of the plasmalemma and are coated with the caveolin-1 protein. Albumin, as the most abundant protein in plasma and interstitium, appears to facilitate NP endocytosis, as does lecithin, a phospholipid: even 240-nm polystyrene particles translocated across the alveolo-capillary barrier when coated with lecithin, whereas uncoated particles did not (Kato et al. 2003). The presence of both albumin and phospholipids in alveolar epithelial lining fluid may, therefore, be important constituents for facilitated epithelial cell uptake of NSPs after deposition in the alveolar space.

Rejman et al. (2004) reviewed a number of different endocytic pathways for internalization of a variety of substances, including phagocytosis, macropinocytosis, clathrin-mediated endocytosis, and caveolae-mediated endocytosis. They found in nonphagocytic cells in vitro that internalization via clathrin-coated pits prevailed for latex microspheres < 200 nm, whereas with increasing size up to 500 nm, caveolae became the predominant pathway. However, as shown in Table 4, surface coating of NSPs with albumin clearly causes even the smallest particles to be internalized via caveolae. The presence of caveolae on cells differs: they are abundant in lung capillaries and alveolar type l cells but not in brain capillaries (Gumbleton 2001). In the lung, during inspiratory expansion and expiratory contraction of the alveolar walls, caveolae with openings around 40 nm disappear and reappear, forming vesicles that are thought to function as transport pathways across the cells for macromolecules (Patton 1996). Knowledge from virology about cell entry of biologic NSPs (viruses) via clathrin-coated pits and caveolae mechanisms should also be considered (Smith and Helenius 2004) and can shed light on the mechanism by which engineered NPs may enter cells and interact with subcellular structures.

Evidence in humans for the translocation of inhaled NSPs into the blood circulation is ambiguous, with one study showing rapid appearance in the blood and significant accumulation of label in the liver of humans inhaling ⁹⁹Tc-labeled 20-nm carbon particles (Nemmar et al. 2002a), whereas another study using the same labeled particles reported no such accumulation (Brown et al. 2002). Taking into consideration all of the evidence from animal and human studies for alveolar translocation of NSPs, it is likely that this pathway also exists in humans; however, the extent of extrapulmonary translocation is highly dependent on particle surface characteristics/chemistry, in addition to particle size. Translocation to the blood circulation could provide a mechanism for a direct particle effect on the cardiovascular system as an explanation for epidemiologic findings of cardiovascular effects associated with inhaled ambient UFPs (Pekkanen et al. 2002; Wichmann et al. 2000) and for results of clinical studies showing vascular responses to inhaled elemental carbon UFPs (Pietropaoli et al. 2004). In addition to direct alveolar translocation of NSPs, cardiovascular effects may also be the corollary of a sequence of events starting with particle-induced alveolar inflammation initiating a systemic acute phase response with changes in blood coagulability and resulting in cardiovascular effects (Seaton et al. 1995).

Once NSPs have translocated to the blood circulation, they can be distributed throughout the body. The liver is the major distribution site via uptake by Kupffer cells, followed by the spleen as another organ of the reticulo-endothelial system, although coating with polyethylene glycol (PEG) almost completely prevents hepatic and splenic localization so that other organs can be targeted (Akerman et al. 2002). Distribution to heart, kidney, and immune-modulating organs (spleen, bone marrow) has been reported. For example, several types of NPs, ranging from 10 to 240 nm, localized to a significant degree in bone marrow after intravenous injection into mice (Table 5). Such target specificity may be extremely valuable for drug delivery; for example, drug delivery to the CNS via blood-borne NPs requires NP surface modifications in order to facilitate translocation across the tight blood–brain barrier via specific receptors (e.g., apolipoprotein coating for LDL-receptor–mediated endocytosis in brain capillaries) (Kreuter 2001, 2004; Kreuter et al. 2002). Such highly desirable properties of NPs must be carefully weighed against potential adverse cellular responses of targeted NP drug delivery, and a rigorous toxicologic assessment is mandatory [Supplemental Material available online (http://ehp.niehs.nih.gov/members/2005/73​39/supplemental.pdf )].

Neuronal uptake and translocation.

A translocation pathway for solid particles in the respiratory tract involving neuronal axons is apparently specific for NSPs. Respective studies are summarized in Table 6. This pathway was described > 60 years ago, yet it has received little or no attention from toxicologists. This pathway, shown in Figure 9 for the nasal and tracheobronchial regions, comprises sensory nerve endings of the olfactory and the trigeminus nerves and an intricate network of sensory nerve endings in the tracheobronchial region. These early studies concerned a large series of studies with 30-nm polio virus intranasally instilled into chimpanzees and rhesus monkeys (Bodian and Howe 1941a, 1941b; Howe and Bodian 1940). Their studies revealed that the olfactory nerve and olfactory bulbs are, indeed, portals of entry to the CNS for intranasally instilled nanosized polio virus particles, which could subsequently be recovered from the olfactory bulbs. The close proximity of nasal olfactory mucosa and olfactory bulb requires only a short distance to be covered by neuronal transport (Figure 12). Bodian and Howe (1941b) determined the transport velocity of the virus in the axoplasm of axons to be 2.4 mm/hr, which is very well in agreement with neuronal transport velocities measured later by Adams and Bray (1983) for solid particles (up to 500 nm) directly microinjected into giant axons of crabs, and by de Lorenzo (1970) for silver-coated colloidal gold (50 nm) in squirrel monkeys.

The de Lorenzo (1970) study demonstrated in squirrel monkeys that intranasally instilled silver-coated colloidal gold particles (50 nm) translocated anterogradely in the axons of the olfactory nerves to the olfactory bulbs. The 50-nm gold particles even crossed synapses in the olfactory glomerulus to reach mitral cell dendrites within 1 hr after intranasal instillation. An interesting finding in this study—and important for potential adverse effects—was that the NSPs in the olfactory bulb were no longer freely distributed in the cytoplasm but were preferentially located in mitochondria (see also “Reactive oxygen species mechanisms of NSP toxicity,” above).

Newer studies indicated that this translocation pathway is also operational for inhaled NSPs. Inhalation of elemental ¹³C UFPs (CMD = 35 nm) resulted in a significant increase of ¹³C in the olfactory bulb on day 1, which increased further throughout day 7 post-exposure (Oberdörster et al. 2004). Results of another inhalation study with solid nanosized (CMD = 30 nm) manganese oxide (MnO₂) particles in rats showed after a 12-day exposure a more than 3.5-fold significant increase of Mn in the olfactory bulb, compared with only a doubling of Mn in the lung. When one nostril was occluded during a 6-hr exposure, Mn accumulation in the olfactory bulb was restricted to the side of the open nostril only (Figure 13) (Feikert et al. 2004). This result contrasts with 15-day inhalation of larger-sized MnO₂ particles in rats (1.3 and 18 μm mass median aerodynamic diameter) where no significant increases in olfactory Mn was found (Fechter et al. 2002). This was to be expected given that the individual axons of the fila olfactoria (forming the olfactory nerve) are only 100–200 nm in diameter (de Lorenzo 1957; Plattig 1989).

Collectively, these studies point out that the olfactory nerve pathway should also be considered a portal of entry to the CNS for humans under conditions of environmental and occupational exposures to airborne NSPs. However, there are important differences between rodents and humans. The olfactory mucosa of the human nose comprises only 5% of the total nasal mucosal surface as opposed to 50% in rats—which in addition are obligatory nose breathers (Table 7). One can argue that the olfactory route may therefore be an important transfer route to the CNS for inhaled NSPs in animals with a well-developed olfaction system, yet at the same time its importance for humans with a more rudimentary olfactory system can be questioned. However, estimates using a predictive particle deposition model and data from Table 7 show that concentrations of 20-nm translocated particles in the human olfactory bulb can, indeed, be 1.6–10 times greater than in rats [Supplemental Material available online (http://ehp.niehs.nih.gov/members/2005/73​39/supplemental.pdf )].

Translocation into deeper brain structures may possibly occur as well, as shown in rats for soluble Mn (Gianutsos et al. 1997), but requires further confirmatory studies with respect to solid NSPs. Further evidence for movement of NSPs along axons and dendrites in humans is provided by knowledge accumulated by virologists who have long understood the movement of human meningitis virus through olfactory and trigeminal neurons and, similarly, herpes virus movement up and down the trigeminal neuron to trigger outbreaks of herpes cold sores in humans (Kennedy and Chaudhuri 2002; Terasaki et al. 1997).

There are additional neuronal translocation pathways for solid NSPs via the trigeminus nerve and tracheobronchial sensory nerves (Table 6). A study by Hunter and Dey (1998) in rats demonstrated the translocation of intranasally instilled rhodamine-labeled microspheres (20–200 nm) to the trigeminal ganglion inside the cranium via uptake into the ophthalmic and maxillary branches of the trigeminus nerve that supplies sensory nerve endings throughout the nasal mucosa. In another study, Hunter and Undem (1999) instilled the same microparticles intratracheally into guinea pigs; they found neuronal translocation of these solid microparticles to the ganglion nodosum in the neck area that is networked into the vagal system. This finding may be relevant for ambient UFPs because it can be hypothesized that cardiovascular effects associated with ambient particles in epidemiologic studies (Utell et al. 2002) are in part due to direct effects of translocated UFPs on the autonomic nervous system via sensory nerves in the respiratory tract.

In the context of potential CNS effects of air pollution, including ambient UFPs, two recent studies with exposures of mice to concentrated ambient fine particles and UFPs should be mentioned. Campbell et al. (2005) and Veronesi et al. (in press) found significant increases of tumor necrosis factor-α or decreases in dopaminergic neurons, supporting the hypothesis of ambient PM causing neuro-degenerative disease. A study by Calderon-Garcidueñas et al. (2002) may also point to an interesting link between air pollution and CNS effects: these authors described significant inflammatory or neurodegenerative changes in the olfactory mucosa, olfactory bulb, and cortical and subcortical brain structures in dogs from a heavily polluted area in Mexico City, whereas these changes were not seen in dogs from a less-polluted rural control city. However, whether direct effects of airborne UFPs are the cause of these effects remains to be determined.

Although the existence of neuronal translocation of NSPs has been well established, size alone is only one particle parameter governing this process. Surface characteristics of NSPs (chemistry, charge, shape, aggregation) are essential determinants as well, and it should not be assumed that all NSPs, when inhaled, will be distributed by the mechanism described here. It should be kept in mind, however, that the unique biokinetic behavior of NSPs—cellular endocytosis, transcytosis, neuronal and circulatory translocation and distribution—which makes them desirable for medical therapeutic or diagnostic applications—may be associated with potential toxicity. For example, NP-facilitated drug delivery to the CNS raises the question of the fate of NPs after their translocation to specific cell types or to subcellular structures in the brain. For example, does mitochondrial localization induce oxidative stress? How persistent is the coating or the core of the NPs? A respective safety evaluation is key [Supplemental Material available online (http://ehp.niehs.nih.gov/members/2005/73​39/supplemental.pdf )].

Exposure via GI Tract and Skin

NSPs cleared from the respiratory tract via the mucociliary escalator can subsequently be ingested into the GI tract. Alternatively, nanomaterials can be ingested directly, for example, if contained in food or water or if used in cosmetics or as drugs or drug delivery devices. Only a few studies have investigated the uptake and disposition of nanomaterials by the GI tract, and most have shown that NSPs pass through the GI tract and are eliminated rapidly. In rats dosed orally with radiolabeled functionalized C₆₀ fullerenes, water solubilized using PEG and albumin (18 kBq in 100 μL), 98% were cleared in the feces within 48 hr, whereas the rest was eliminated via urine, indicating some uptake into the blood circulation (Yamago et al. 1995). In contrast, in this same study, 90% of the same radiolabeled fullerenes administered intravenously (9.6 kBq in ~ 50 μL or 14–18 kBq in 215 μL) were retained after 1 week, with most (73–80%, depending on time course) found in the liver. Studies by Kreyling and colleagues (Kreyling et al. 2002; Semmler et al. 2004) using ultrafine ¹⁹²Ir did not show significant uptake in the GI tract, whereas earlier studies with larger TiO₂ particles (150–500 nm) found uptake into the blood and movement to the liver (Böckmann et al. 2000; Jani et al. 1994). Likely there are differences in GI tract uptake dependent on both particle surface chemistry and particle size. Indeed, after oral dosing in rats, Jani et al. (1990) found a particle-size–dependent uptake of polystyrene particles (ranging from 50 to 3,000 nm) by the GI mucosa. This uptake (6.6% of the administered 50 nm, 5.8% of the 100 nm NSP, 0.8% of 1 μm particles, and 0% for 3 μm particles) was mainly via the Peyer's patches with translocation into the mesenteric lymph and then to systemic organs (i.e., liver, spleen, blood, bone marrow, and kidney).

A potentially important uptake route is through dermal exposure. The epidermis, consisting of the outer horny layer (stratum corneum), the prickle cell layer (stratum spinosum), and basal cell layer (stratum basale), forms a very tight protective layer for the underlying dermis (Figure 14). The dermis has a rich supply of blood and tissue macrophages, lymph vessels, dendritic cells (Langerhans, also in stratum spinosum of epidermis), and five different types of sensory nerve endings. Broken skin represents a readily available portal of entry even for larger (0.5–7 μm) particles, as evidenced by reports about accumulation of large amounts of soil particles in inguinal lymph nodes of people who often run or walk barefoot; this can be associated with elephantiatic lymphedema (podoconiosis; Corachan et al. 1988; Blundell et al. 1989). Tinkle et al. (2003) hypothesized that unbroken skin when flexed—as in wrist movements—would make the epidermis permeable for NSPs. They demonstrated in a proof-of-concept experiment that, indeed, flexing the skin, but not flat skin, resulted in penetration of even 1 μm fluorescent beads to the dermis. The follow-up question about access of particles in the dermis to the circulation is answered by the aforementioned reports of podoconiosis, that is, uptake into the lymphatic system and regional lymph nodes. Subsequent translocation of NSPs beyond lymph nodes to the blood circulation is likely to occur as well, as shown in studies with small asbestos fibers (Oberdörster et al. 1988).

Newer studies by Kim et al. (2004) in mice and pigs with intradermally injected near-infrared quantum dots confirmed that NPs, once in the dermis, will localize to regional lymph nodes, which makes these particles very useful for in vivo imaging. Likely transport mechanisms to the lymph nodes are skin macrophages and dendritic (Langerhans) cells (Ohl et al. 2004; Sato et al. 1998); this raises a question about potential modulation of immune responses, after interaction of these NP-containing macrophages and dendritic cells with T lymphocytes. For example, Chen et al. (1998) were able to raise antibodies in mice specific for C₆₀ after intraperitoneal injections of C₆₀ conjugated to thyroglobulin and serum albumin. Clearly, research is needed to determine whether and under what conditions NPs can be recognized by the immune system, following any route of uptake into the organism.

Another question relates to the potential of sensory skin nerves to take up and translocate NPs. Given that this mechanism has been demonstrated for the nasal and tracheobronchial regions of the respiratory tract, how likely is this to occur in the dermis layer of the skin with its dense supply of different types of sensory nerves? It may be conceivable, considering data on neuronal uptake and translocation of NSPs after intramuscular injection. For example, nanosized ferritin and iron-dextran, after injection into the tongue of mice, labeled the neurons of the hypoglossal nuclei, and injection of both of these NSPs into facial muscles of mice also resulted in synaptic uptake; cationized ferritin was also detected in cell bodies of facial neurons, indicating that electrical charge is of importance for incorporation into axons and axonal transport (Arvidson 1994; Malmgren et al. 1978; Olsson and Kristensson 1981). Other studies using intramuscular injection of ferritin (~ 112 nm), iron-dextran (11 or 21 nm), and gold protein (20–25 nm) NSPs also showed rapid penetration through the basal lamina into the synaptic clef of the neuromuscular junction, but this was restricted to only the smaller nanoparticles, implying that there may be a size-dependent penetration of the basal lamina with a threshold somewhere between 10 and 20 nm (Oldfors and Fardeau 1983).

Neuronal transport of NSPs along sensory skin nerves is well established for herpes virus. After passing through the skin—especially broken skin—the viruses are transported retrogradely along dendrites of sensory neurons to the dorsal root ganglion, where they remain dormant until a stress situation triggers antero-grade translocation along the dendrites back to the skin (Kennedy and Chaudhuri 2002; Terasaki et al. 1997). Future studies need to determine whether and to what degree such translocation along sensory skin neurons also occurs with NPs penetrating the epidermis.

Risk Assessment Top

The lack of toxicology data on engineered NPs does not allow for adequate risk assessment. Because of this, some may even believe that engineered NPs are so risky that they call for a precautionary halt in NP-related research. However, the precautionary principle should not be used to stop research related to nanotechnology and NPs. Instead, we should strive for a sound balance between further development of nanotechnology and the necessary research to identify potential hazards in order to develop a scientifically defensible database for the purpose of risk assessment. To be able to do this, a basic knowledge about mammalian and ecotoxicologic profiles of NPs is necessary, rather than attempting to assess NP risks based on some popular science fiction literature. Most important, sufficient resources should be allocated by governmental agencies and industries to be able to perform a scientifically based risk assessment and then establish justifiable procedures for risk management. The data needed for this risk assessment should be determined a priori so that limited resources can be used efficiently to develop useful and well-planned studies.

At this point, governmental regulation is not possible, given the lack of needed information on which to base such regulations. However, academia, industry, and regulatory governmental agencies should seriously consider the view that NPs have new and unique biologic properties and that the potential risks of NPs are not the same as those of the bulk material of the same chemistry. Assigning a unique identifier to nanosized materials would indicate that the toxicology profile of the material in question may not be the same as the bulk material. Toxicologic tests and the resulting database would provide information for material safety data sheets for NPs as well as a basis for potential NP risk assessments and risk management. Obviously, this approach may not be appropriate for all NPs, for example, when embedded in a matrix, and the feasibility of this proposed strategy needs to be thoroughly discussed and considered. For discussing this, and for developing and deciding upon a reasonable battery of tests for toxicologic profiling, it would be very useful to convene international multidisciplinary workshops of experts from industry, academia, and regulatory agencies (including material scientists, chemists, chemical engineers, toxicologists, physicians, regulators, statisticians, and others) to establish an NP classification scheme and testing guidelines. A multidisciplinary and multinational collaborative team approach is critical. Respective efforts have been initiated nationally by the American National Standards Institute (ANSI 2004) and internationally by the International Council on Nanotechnology (ICON 2004) as well as the International Organization for Standardization (Geneva, Switzerland).

Because many regulatory agencies do not consider a nanotechnologically manufactured substance different from the conventional substance, the manufacture and use of nanotechnology products are currently not specifically regulated. Typically, nanosized substances are treated as variations of the technical material or existing formulation and thus do not require a separate registration. A main reason for producing a nanosize form of a registered substance, however, is that conversion of a substance to a nanoparticle imparts new properties to the substance (e.g., enhanced mechanical, electrical, optical, catalytic, biologic activity). Thus, as stated above, although the toxicology of the base material may be well defined, the toxicity of the nanosize form of the substance may be dramatically different from its parent form. As a result, new toxicology data on the nanosize form of a substance is likely to result in a different hazard assessment for the NPs. Figure 15 shows a risk assessment/risk management paradigm that points out different steps and data required for this process.

As described in the preceding sections, the difference in toxicologic profile of NPs compared with its parent form is due to not only its intrinsic chemical properties but also to a large degree to its differing kinetics in vivo. Although larger particles may not enter the CNS, the potential exists for inhaled NSPs to be translocated to the CNS via the axons of sensory neurons in the upper respiratory tract. Furthermore, although the toxicity per unit mass of a particular substance may vary depending on the nano versus larger form, it will be important to take into account not only new biologic activities but also potential new target organs and routes of exposure. To what degree does the nanoform of a substance have enhanced dermal penetration, or increased systemic uptake via the lung or GI tract? What determines how many nanoparticles that enter the systemic circulation will distribute throughout the body, reach the bone marrow, cross the blood–brain barrier, cross the placenta to affect the developing offspring, or sequester effectively in the liver? Do nanoparticles released into the environment affect species that are important in food chain dynamics? What are the long-term consequences of exposure to nanoparticles? Changes in toxicity profile and new target organs can be expected, and it will then be necessary to establish new risk assessments for nanoparticles in addition to the bulk material. Currently there exists a paucity of data to effectively address these questions, but it will be important to determine whether there exist common modes of action/behavior of NPs to establish baseline assumptions for use in risk assessments.

The use of nanotechnology products will likely increase dramatically over the next decade. In fact, nanomaterials are already being used in applications ranging from burn and wound dressings to dental-bonding agents to sunscreens and cosmetics to fuel cells, tires, optics, clothing, and electronics. Although currently there exists little public awareness of nanotechnology in everyday life (e.g., stain-free clothing), it would be prudent to examine and address environmental and human health concerns before the widespread adoption of nanotechnology. Both the societal benefits and potential risks of nanotechnology should be evaluated and clearly communicated to the general public and regulators. This type of open communication and risk/benefit evaluation will avoid the pitfalls encountered with genetically modified organisms recently experienced in the field of biotechnology. In that instance, the benefits of the emerging field of biotechnology were not communicated effectively before the introduction of the technology. As the public’s awareness of this new technology grew, regulators and producers of biotechnology failed to effectively acknowledge public concerns that genetically modified organisms could adversely affect ecosystem balance. As a result, the public support of genetically modified organisms, particularly in the European Union, is low. For nanomaterial producers, it will be important to demonstrate that what they may perceive as a new and potentially harmless form of a familiar material has, indeed, an acceptable risk profile. If such proactive steps are not taken, nanomaterials may be regarded as dangerous by the public and regulators, which could lead to inappropriate categorization and unnecessarily burdensome regulations. Such action (or inaction on the side of producers), in turn, could result in significant barriers to commercialization and the widespread acceptance of otherwise useful nanotechnology materials.

Summary and Outlook Top

Research on ambient UFPs has laid the foundation for the emerging field of nanotoxicology, with the goal of studying the biokinetics and the potential of engineered nanomaterials (particles, tubes, shells, quantum dots, etc.) to cause adverse effects. Major differences between ambient UFPs and NPs are the polydisperse nature of the former versus the monodisperse size of the latter, and particle morphology, oftentimes a branched structure from combustion particles versus spherical form of NPs, although other shapes (tubes, wires, rings, planes) are also manufactured. In addition, combustion-derived volatile organic compounds and inorganic constituents (e.g., metals, nitrates, sulfates) of different solubilities on UFPs predict differences in the toxicologic profile between UFPs and NPs. However, as far as the insoluble particle is concerned, concepts of NSPs kinetics, including cell interactions, will most likely be the same for UFPs and NPs (Figure 16).

The introduction of nanostructured materials for biomedical and electronics applications opens tremendous opportunities for biomedical applications as therapeutic and diagnostic tools as well as in the fields of engineering, electronics, optics, consumer products, alternative energy, soil/water remediation, and others. However, very little is yet known about their potential to cause adverse effects or humoral immune responses once they are introduced into the organism—unintentionally or intentionally. Nanomedicine products will be well tested before introduction into the marketplace. However, for the manufacturers of most current nanotechnology products, regulations requiring nanomaterial-specific data on toxicity before introduction into the marketplace are an evolving area and presently under discussion (Bergeson and Auerbach 2004; Foresight and Governance Project 2003). During a product’s life cycle (manufacture, use, disposal), it is probable that nanomaterials will enter the environment, and currently there is no unified plan to examine ecotoxicologic effects of NPs. In addition, the stability of coatings and covalent surface modifications need to be determined both in ecologic settings and in vivo. [Supplemental Material available online (http://ehp.niehs.nih.gov/members/2005/73​39/supplemental.pdf ).]

Results of older biokinetic studies and some new toxicology studies with NSPs (mostly ambient UFPs) can be viewed as the basis for the expanding field of nanotoxicology. These studies showed that the greater surface area per mass renders NSPs more active biologically than larger-sized particles of the same chemistry, and that particle surface area and number appear to be better predictors for NSPs-induced inflammatory and oxidative stress responses. The following emerging concepts of nanotoxicology can be identified from these studies:

The biokinetics of NSPs are different from larger particles. When inhaled, they are efficiently deposited in all regions of the respiratory tract; they evade specific defense mechanisms; and they can translocate out of the respiratory tract via different pathways and mechanisms (endocytosis and transcytosis). When in contact with skin, there is evidence of penetration to the dermis followed by translocation via lymph to regional lymph nodes. A possible uptake into sensory nerves needs to be investigated. When ingested, systemic uptake via lymph into the organism can occur, but most are excreted via feces. When in blood circulation, they can distribute throughout the organism, and they are taken up into liver, spleen, bone marrow, heart, and other organs. In general, translocation rates are largely unknown; they are probably very low but are likely to change in a compromised/diseased state.

The biologic activity and biokinetics are dependent on many parameters: size, shape, chemistry, crystallinity, surface properties (area, porosity, charge, surface modifications, weathering of coating), agglomeration state, biopersistence, and dose. These parameters are likely to modify responses and cell interactions, such as a greater inflammatory potential than larger particles per given mass, translocation across epithelia from portal of entry to other organs, translocation along axons and dendrites of neurons, induction of oxidative stress, pro-oxidant and antioxidant activity of NSPs in environmentally relevant species, binding to proteins and receptors, and localization in mitochondria.

The principles of cellular and organismal interactions discussed in this article should be applicable for both ambient UFPs and NPs, even if the latter are coated with a bio-compatible material. Knowledge about the bio-persistence of this coating is as essential as is knowledge about the bioavailability of the core material that could have intrinsic toxic properties, for example, semiconductor metal compounds in sub-10-nm quantum dots consisting of cadmium and lead compounds. The very small size of these materials makes them available to the same translocation processes described here for polydisperse NSPs, possibly even in a more efficient way because of their uniform size. When studying biologic/toxicologic effects, new processes of interactions with subcellular structures (e.g., microtubuli, mitochondria) will likely be discovered. The diversity of engineered nanomaterials and of the potential effects represents major challenges and research needs for nanotoxicology, including also the need for assessing human exposure during manufacture and use. The goal to exploit positive aspects of engineered nanomaterials and avoid potential toxic effects can best be achieved through a multidisciplinary team effort involving researchers in toxicology, materials science, medicine, molecular biology, bioinformatics, and their subspecialties.

Correction Top

The authors found additional information on GI tract uptake of NSPs that was not in the original manuscript published online. This information has been included here in “Exposure via GI Tract and Skin.”

Figures and Tables Top

Figure 1.

Idealized size distribution of traffic-related particulate matter (U.S. EPA 2004). D _p , particle diameter. The four polydisperse modes of traffic-related ambient particulate matter span approximately four orders of magnitude from < 1 nm to > 10 μm. Nucleation- and Aitken-mode particles are defined as UFPs (< approximately 100 nm). Source-dependent chemical composition is not well controlled and varies considerably. In contrast, NPs (1–100 nm) have well-controlled chemistry and are generally monodispersed.

Figure 2.

Surface molecules as a function of particle size. Surface molecules increase exponentially when particle size decreases < 100 nm, reflecting the importance of surface area for increased chemical and biologic activity of NSPs. The increased biologic activity can be positive and desirable (e.g., antioxidant activity, carrier capacity for therapeutics, penetration of cellular barriers), negative and undesirable (e.g., toxicity, induction of oxidative stress or of cellular dysfunction), or a mix of both. Figure courtesy of H. Fissan (personal communication).

Figure 3.

Hypothetical cellular interaction of NSPs (adapted from Donaldson and Tran 2002). EGFR, epidermal growth factor receptor. Inflammation and oxidative stress can be mediated by several primary pathways: a) the particle surface causes oxidative stress resulting in increased intracellular calcium and gene activation; b) transition metals released from particles result in oxidative stress, increased intracellular calcium, and gene activation; c) cell surface receptors are activated by transition metals released from particles, resulting in subsequent gene activation; or d) intracellular distribution of NSPs to mitochondria generates oxidative stress.

Figure 4.

Percentage of neutrophils in lung lavage of rats (A,B) and mice (C,D) as indicators of inflammation 24 hr after intratracheal instillation of different mass doses of 20-nm and 250-nm TiO₂ particles in rats and mice. (A,C) The steeper dose response of nanosized TiO₂ is obvious when the dose is expressed as mass. (B,D) The same dose response relationship as in (A,C) but with dose expressed as particle surface area; this indicates that particle surface area seems to be a more appropriate dosemetric for comparing effects of different-sized particles, provided they are of the same chemical structure (anatase TiO₂ in this case). Data show mean ± SD.

Figure 5.

Routes of exposure, uptake, distribution, and degradation of NSPs in the environment. Solid lines indicate routes that have been demonstrated in the laboratory or field or that are currently in use (remediation). Magenta lettering indicates possible degradation routes, and blue lettering indicates possible sinks and sources of NSPs.

Figure 6.

NPs have been shown to release oxyradicals [pictured here is the mechanism of C₆₀ as determined by Yamakoshi et al. (2003)], which can interact with the antioxidant defense system. Abbreviations: GPx, glutathione peroxidase; GSH, reduced glutathione; GSSG, oxidized glutathione; ISC, intersystem crossing; R, any organic molecule; SOD, superoxide dismutase. In addition to fullerenes, metals such as cadmium, iron, or nickel quantum dots, or iron from SWNT manufacturing, could also act in Fenton-type reactions. Phase II biotransformation, ascorbic acid, vitamin E, beta carotene, and other interactions are not shown.

Figure 7.

Some basic shapes of exposure–response or dose–response relationships. Abbreviations: H, hormetic (biphasic); L, linear (no threshold); S, supralinear; T, threshold. Prerequisites for establishing these relationships for NSPs from in vitro or in vivo studies include a sufficient number of data points, that is, over a wide range of exposure concentrations or doses; knowledge about exposure levels; and information about correlation of exposure with doses at the organismal or cellular level (an exposure is not a dose). Dose–response curves of different shapes can be extrapolated when only response data at high dose levels (indicated by dashed oval) are available. Lack of data in the low—oftentimes the most relevant—dose range can result in severe misinterpretation if a threshold or even a hormetic response is present. Consideration also needs to be given to the likelihood that the shape or slope of exposure–dose–response relationships change for susceptible parts of the population.

Figure 8.

Predicted fractional deposition of inhaled particles in the nasopharyngeal, tracheobronchial, and alveolar region of the human respiratory tract during nose breathing. Based on data from the International Commission on Radiological Protection (1994). Drawing courtesy of J. Harkema.

Figure 9.

Pathways of particle clearance (disposition) in and out of the respiratory tract. There are significant differences between NSPs and larger particles for some of these pathways (see “Disposition of NSPs in the respiratory tract”). Drawing courtesy of J. Harkema.

Figure 10.

In vivo retention of inhaled nanosized and larger particles in alveolar macrophages (A) and in exhaustively lavaged lungs (epithelial and interstitial retention; B) 24 hr postexposure. The alveolar macrophage is the most important defense mechanism in the alveolar region for fine and coarse particles, yet inhaled singlet NSPs are not efficiently phagocytized by alveolar macrophages.

Figure 11.

Different forms of caveolae and cellular tight junctions function as translocation mechanisms across cell layers. Depending on particle surface chemistry, NSPs have been shown to transcytose across alveolar type I epithelial cells and capillary endothelial cells (Table 4), but not via cellular tight junctions in the healthy state (A). However, in a compromised or disease state (e.g., endotoxin exposure; B) translocation across widened tight junction occurs as well (Heckel et al. 2004). This indicates that assessing potential effects of NSPs in the compromised state is an important component of nanotoxicology. Adapted from Cohen et al. (2004).

Figure 12.

Close proximity of olfactory mucosa to olfactory bulb of the CNS. Inhaled NSP[s], especially below 10 nm, deposit efficiently on the olfactory mucosa by diffusion, similar to airborne “smell” molecules which deposit in this area of olfactory dendritic cilia. Subsequent uptake and translocation of solid NSP[s] along axons of the olfactory nerve has been demonstrated in non-human primates and rodents. Surface chemistry of the particles may influence their neuronal translocation. Copyright © the McGraw-Hill Companies, Inc. Reproduced from Widmaier et al. (2004) with permission from McGraw-Hill.

Figure 13.

Occlusion of the right nostril of rats during 6-hr inhalation of nanosized MnO₂ particles (~ 30 nm CMD, ~ 450 μg/m³) resulted in accumulation of Mn only in the left olfactory bulb only at 24 hr after dosing. Data are mean ± SD. Data from Feikert et al. (2004).

Figure 14.

The epidermis represents a tight barrier against NSP penetration. Quantitatively, dermal translocation will therefore be minimal or nonexistent under normal conditions but increases in areas of skin flexing (Tinkle et al. 2003) and broken skin. Once in the dermis, lymphatic uptake is a major translocation route, likely facilitated by uptake in dendritic cells (epidermis) and macrophages; other potential pathways may include the dense networks of blood circulation and sensory nerves in the dermis. Adapted from Essential Day Spa (2005) with permission from www.essentialdayspa.com.

Figure 15.

Risk assessment (NRC 1983) and risk management paradigm for NPs. Risk assessment requires answers to the following questions: Do NPs have adverse effects? What are the dose–response relationships? What are occupational/environmental levels in different media? What is the calculated risk? Once a risk is determined, a risk management decision can be established, including exposure standards and regulations and efforts for effective risk communication. Modified from Oberdörster (1994).

Figure 16.

Biokinetics of NSPs. PNS, peripheral nervous system. Although many uptake and translocation routes have been demonstrated, others still are hypothetical and need to be investigated. Translocation rates are largely unknown, as are accumulation and retention in critical target sites and their underlying mechanisms. These, as well as potential adverse effects, largely depend on physicochemical characteristics of the surface and core of NSPs. Both qualitative and quantitative changes in NSP biokinetics in a diseased or compromised organism also need to be considered.

Table 1.

UFPs/NPs (< 100 nm), natural and anthropogenic sources.

Table 2.

Particle number and particle surface area per 10 μg/m³ airborne particles.

Table 3.

Clearance mechanisms for inhaled solid particles in the respiratory tract.

Table 4.

Particle size and surface chemistry-related alveolar–capillary translocation.

Table 5.

Translocation of NSPs in the blood circulation to bone marrow in mice.

Table 6.

Studies of neuronal translocation of UFPs from respiratory tract.

Table 7.

Rat versus human nasal and olfactory parameters.

Supplemental Material Top

(616 KB) PDF.

References Top

1. Adams RJ, Bray D 1983. Rapid transport of foreign particles microinjected into crab axons Nature 303:718–720.6190095 Find this article online
2. Akerman MA, Chan WCW, Laakkonen P, Bhatia SN, Ruoslahti E 2002. Nanocrystal targeting in vivo Proc Natl Acad Sci USA 99:12617–12621.12235356 Find this article online
3. Amato I 1989. Making the right stuff Sci News 136:108–110. Find this article online
4. Anderson PJ, Wilson JD, Hiller FC 1990. Respiratory tract deposition of ultrafine particles in subjects with obstructive or restrictive lung disease Chest 97:1115–1120.2331906 Find this article online
5. ANSI 2004. Home page. American National Standards Institute. Available: http://www.ansi.org. [accessed 16 March 2005]
6. Arvidson B 1994. A review of axonal transport of metals Toxicology 88:1–14.8160191 Find this article online
7. Auclair F, Baudot P, Beiler D, Limasset J 1983. Accidents benines et mortetels dus aux “traitement” du polytetrafluoroethylene en millieu industriel: observations cliniques et measures physiochemiques des atmosphere pollues Toxicol Eur Res 1:43–48. Find this article online
8. Ballou B, Lagerholm BC, Ernst LA, Bruchez MP, Waggoner AS 2004. Non-invasive imaging of quantum dots in mice Bioconjug Chem 15:79–86.14733586 Find this article online
9. Bazile DV, Ropert C, Huve P, Verrecchia T, Marlard M, Frydman A, et al. 1992. Body distribution of fully biodegradable [14C]-poly(lactic acid) nanoparticles coated with albumin after parenteral administration to rats Biomaterials 13(15):1093–1102.1493193 Find this article online
10. Bergeson LL, Auerbach B 2004. The Environmental Regulatory Implications of Nanotechnology. Daily Environment Report No. 71. Washington, DC:Bureau of National Affairs, Inc. Available: http://www.lawbc.com/other_pdfs/The%20En​vironmental%20Regulatory%20Implications%​20of%20Nanotechnology.pdf [accessed 26 April 2005]
11. Berry JP, Arnoux B, Stanislas G, Galle P, Chretien J 1977. A microanalytic study of particles transport across the alveoli: role of blood platelets Biomedicine 27:354–357.606312 Find this article online
12. Blakemore R 1975. Magnetotactic bacteria Science 190:377–379.170679 Find this article online
13. Blundell G, Henderson WJ, Price EW 1989. Soil particles in the tissues of the foot in endemic elephantiasis of the lower legs Ann Trop Med Parasitol 83 (4):381–385.2604475 Find this article online
14. Böckmann J, Lahl H, Eckert T, Unterhalt B 2000. Titan-Blutspiegel vor und nach Belastungsversuchen mit Titandioxid [in German] Pharmazie 55:140–143.10723775 Find this article online
15. Bodian D, Howe HA 1941a. Experimental studies on intraneural spread of poliomyelitis virus Bull Johns Hopkins Hosp 69:248–267. Find this article online
16. Bodian D, Howe HA 1941b. The rate of progression of poliomyelitis virus in nerves Bull Johns Hopkins Hosp 69:79–85. Find this article online
17. Brand P, Gebhart J, Below M, Georgi B, Heyder J 1991. Characterization of environmental aerosols on Helgoland Island Atmos Environ 25A(3/4):581–585. Find this article online
18. Brown DM, Stone V, Findlay P, MacNee W, Donaldson K 2000. Increased inflammation and intracellular calcium caused by ultrafine carbon black is independent of transition metals or other soluble components Occup Environ Med 57:685–691.10984341 Find this article online
19. Brown DM, Wilson MR, MacNee W, Stone V, Donaldson K 2001. Size-dependent proinflammatory effects of ultrafine polystyrene particles: a role for surface area and oxidative stress in the enhanced activity of ultrafines Toxicol Appl Pharmacol 175:191–199.11559017 Find this article online
20. Brown JS, Zeman KL, Bennett WD 2002. Ultrafine particle deposition and clearance in the healthy and obstructed lung Am J Respir Crit Care Med 166:1240–1247.12403694 Find this article online
21. Cagle DW, Kenmnel SJ, Mirzadeh S, Alford JM, Wilson LJ 1999. In vivo studies of fullerene-based materials using endohedral metallofullerene radiotracers Proc Natl Acad Sci USA 96:5182–5187.10220440 Find this article online
22. Calderon-Garcidueñas L, Azzarelli B, Acuna H, Garcia R, Gambling TM, Osnaya N, et al. 2002. Air pollution and brain damage Toxicol Pathol 30(3):373–389.12051555 Find this article online
23. Campbell A, Oldham M, Becaria A, Bondy SC, Meacher D, Sioutas C, et al. 2005. Particulate matter in polluted air may increase biomarkers of inflammation in mouse brain Neurotoxicology 26:133–140.15527881 Find this article online
24. Cavagna G, Finulli M, Vigliani EC 1961. Studio sperimentale sulla patogenesi della febre da inalazione di fumi di Teflon (polite-trafluoroetilene) [in Italian] Med Lavoro 52:251–261.13691688 Find this article online
25. Chalupa DC, Morrow PE, Oberdörster G, Utell MJ, Frampton MW 2004. Ultrafine particle deposition in subjects with asthma Environ Health Perspect 112:879–882.15175176 Find this article online
26. Chen B, Wilson S, Das M, Coughlin D, Erlanger B 1998. Antigenicity of fullerenes: antibodies specific for fullerenes and their characteristics Proc Natl Acad Sci USA 95:10809–10813.9724786 Find this article online
27. Cheng X, Kan A, Tomson M 2004. Napthalene adsorption and desorption from aqueous C60 fullerene J Chem Engineer Data 49:675–678. Find this article online
28. Chitose N, Ueta S, Seino S, Yamamoto T 2003. Radiolysis of aqueous phenol solutions with nanoparticles. 1. Phenol degradation and TOC removal in solutions containing TiO₂ induced by UV, gamma-ray and electron beams Chemosphere 50:1007–1013.12531706 Find this article online
29. Cohen A, Hnasko R, Schubert W, Lisanti M 2004. Role of caveolae and caveolins in health and disease Physiol Rev 84:1341–1379.15383654 Find this article online
30. Coleman WE, Scheel LD, Gorski CH 1968. The particle resulting from polytetrafluorethylene (PTFE) pyrolysis in air AIHA J 29:54–60. Find this article online
31. Corachan M, Tura JM, Campo E, Soley M, Traveria A 1988. Poedoconiosis in Aequatorial Guinea. Report of two cases from different geological environments Trop Geogr Med 40:359–364.3227560 Find this article online
32. Cyrys J, Stolzel M, Heinrich J, Kreyling WG, Menzel N, Wittmaack K, et al. 2003. Elemental composition and sources of fine and ultrafine ambient particles in Erfurt, Germany Sci Tot Environ 305:143–156. Find this article online
33. Daughton C, Ternes T 1999. Pharmaceuticals and personal care products in the environment: agents of subtle change? Environ Health Perspect 107(suppl 6):907–938.10592150 Find this article online
34. de Lorenzo AJ 1957. Electron microscopic observations of the olfactory mucosa and olfactory nerve J Biophys Biochem Cytol 3:839–850.13481018 Find this article online
35. de Lorenzo AJ 1970. The olfactory neuron and the blood-brain barrier. In: Taste and Smell in Vertebrates (Wolstenholme G, Knight J, eds). London:Churchill, 151–176
36. Derfus AM, Chan WCW, Bhatia SN 2004. Probing the cytotoxicity of semiconductor quantum dots Nano Lett 4(1):11–18. Find this article online
37. Dieckmann G, Dalton A, Johnson P, Razal J, Chen J, Giordano GM, et al. 2003. Controlled assembly of carbon nanotubes by designed amphiphilic peptide helices J Am Chem Soc 125:1770–1777.12580602 Find this article online
38. Dobson J 2001. Nanoscale biogenic iron oxides and neuro-degenerative disease FEBS Lett 496:1–5.11343696 Find this article online
39. Donaldson K, Brown D, Clouter A, Duffin R, MacNee W, Renwick L, et al. 2002. The pulmonary toxicology of ultrafine particles J Aerosol Med 15:213–220.12184871 Find this article online
40. Donaldson K, Li XY, MacNee W 1998. Ultrafine (nanometre) particle mediated lung injury J Aerosol Sci 29:553–560. Find this article online
41. Donaldson K, Stone V 2003. Current hypotheses on the mechanisms of toxicity of ultrafine particles Ann Ist Super Sanita 39:405–410.15098562 Find this article online
42. Donaldson K, Tran C-L 2002. Inflammation caused by particles and fibers Inhal Toxicol 14:5–27.12122558 Find this article online
43. Donlin M, Frey R, Putnam C, Proctor J, Bashkin J 1998. Analysis of iron in ferritin, the iron-storage protein J Chem Educ 75:437–441. Find this article online
44. Driscoll KE 1996. Role of inflammation in the development of rat lung tumors in response to chronic particle exposure Inhal Toxicol 8(suppl):139–153. Find this article online
45. Dunn JR, Fuller M, Zoeger J, Dobson J, Heller F, Hamnmann J, et al. 1995. Magnetic material in the human hippocampus Brain Res Bull 36:149–153.7895092 Find this article online
46. Elder ACP, Gelein R, Azadniv M, Frampton M, Finkelstein J, Oberdorster G 2002. Systemic interactions between inhaled ultrafine particles and endotoxin Ann Occup Hyg 46(suppl 1):231–234. Find this article online
47. Elder ACP, Gelein R, Azadniv M, Frampton M, Finkelstein J, Oberdorster G 2004. Systemic effects of inhaled ultrafine particles in two compromised, aged rat strains Inhal Toxicol 16(6/7):461–471.15204762 Find this article online
48. Elder ACP, Gelein R, Finkelstein JN, Cox C, Oberdörster G 2000. Pulmonary inflammatory response to inhaled ultrafine particles is modified by age, ozone exposure, and bacterial toxin Inhal Toxicol 12(suppl 4):227–246.12881894 Find this article online
49. Essential Day Spa 2005. Skin Anatomy and Physiology. Available: http://www.essentialdayspa.com/Skin_Anat​homy_and_Physiology.htm [accessed 26 April 2005]
50. Evelyn A, Mannick S, Sermon PA 2003. Unusual carbon-based nanofibers and chains among diesel-emitted particles Nano Lett 3:63–64. Find this article online
51. Fechter LD, Johnson DL, Lynch RA 2002. The relationship of particle size to olfactory nerve uptake of a non-soluble form of manganese into brain Neurotoxicology 23:177–183.12224759 Find this article online
52. Feikert T, Mercer P, Corson N, Gelein R, Opanashuk L, Elder A, et al. 2004. Inhaled solid ultrafine particles (UFP) are efficiently translocated via neuronal naso-olfactory pathways [Abstract] Toxicologist 78(suppl 1):435–436. Find this article online
53. Ferin J, Oberdörster G 1992. Translocation of particles from pulmonary alveoli into the interstitium J Aerosol Med 5:179–187. Find this article online
54. Ferin J, Oberdörster G, Penney DP 1992. Pulmonary retention of ultrafine and fine particles in rats Am J Resp Cell Mol Biol 6:535–542. Find this article online
55. Ferin J, Oberdörster G, Penney DP, Soderholm SC, Gelein R, Piper HC 1990. Increased pulmonary toxicity of ultrafine particles? I. Particle clearance, translocation, morphology J Aerosol Sci 21:381–384. Find this article online
56. Ferin J, Oberdörster G, Soderholm SC, Gelein R 1991. Pulmonary tissue access of ultrafine particles J Aerosol Med 4(1):57–68. Find this article online
57. Foley S, Crowley C, Smaihi M, Bonfils C, Erlanger BF, Seta P, et al. 2002. Cellular localisation of a water-soluble fullerene derivative Biochem Biophys Res Commun 294:116–119.12054749 Find this article online
58. Foresight and Governance Project 2003. Nanotechnology & Regulation: A Case Study using the Toxic Substance Control Act (TSCA): A Discussion Paper. Publication 2003-6. Washington, DC:Woodrow Wilson International Center for Scholars. Available: http://www.nanotechcongress.com/Nanotech​-Regulation.pdf [accessed 26 April 2005]
59. Fox JE, Starcevic M, Kow KY, Burow ME, McLachlan JA 2001. Nitrogen fixation: endocrine disrupters and flavonoid signalling Nature 413:128–129.11557969 Find this article online
60. Freitas RA Jr 1999. Nanomedicine, Volume I: Basic Capabilities. Georgetown, TX:Landes Bioscience
61. Gianutsos G, Morrow GR, Morris JB 1997. Accumulation of manganese in rat brain following intranasal administration Fundam Appl Toxicol 37:102–105.9242582 Find this article online
62. Gibaud S, Andreux JP, Weingarten C, Renard M, Couvreur P 1994. Increased bone marrow toxicity of doxorubicin bound to nanoparticles Eur J Cancer 30A:820–826.7917543 Find this article online
63. Gibaud S, Demoy M, Andreux JP, Weingarten C, Gouritin B, Couvreur P 1996. Cells involved in the capture of nanoparticles in hematopoietic organs J Pharm Sci 85:944–950.8877884 Find this article online
64. Gibaud S, Rousseau C, Weingarten C, Favier R, Douay L, Andreux JP, et al. 1998. Polyalkylcyanoacrylate nanoparticles as carriers for granulocyte-colony stimulating factor (G-CSF) J Control Release 52:131–139.9685943 Find this article online
65. Goldstein M, Weiss H, Wade K, Penek J, Andrews L, Brandt-Rauf P 1987. An outbreak of fume fever in an electronics instrument testing laboratory J Occup Med 29:746–749.3681507 Find this article online
66. Gopinath PG, Gopinath G, Kumar A 1978. Target site of intranasally sprayed substances and their transport across the nasal mucosa: a new insight into the intranasal route of drug delivery Curr Ther Res 23 (5):596–607. Find this article online
67. Greim H, Borm P, Schins R, Donaldson K, Driscoll K, Hartwig A, et al. 2001. Toxicity of fibers and particles—report of the workshop held in Munich, Germany, 26–27 October 2000 Inhal Toxicol 13:737–754.11498804 Find this article online
68. Griffith FD, Stephens SS, Tayfun FO 1973. Exposure of Japanese quail and parakeets to the pyrolysis products of fry pans coated with teflon and common cooking oils Am Ind Hyg Assoc J 34:176–178.4723395 Find this article online
69. Gumbleton M 2001. Caveolae as potential macromolecule trafficking compartments within alveolar epithelium Adv Drug Deliv Rev 49:281–300.11551400 Find this article online
70. Hautot D, Pankhurst QA, Khan N, Dobson J 2003. Preliminary evaluation of nanoscale biogenic magnetite in Alzheimer’s disease brain tissue Proc R Soc Lon B 270(suppl 1):S62–S64. Find this article online
71. Heckel K, Kiefmann R, Dorger M, Stoeckelhuber M, Goetz AE 2004. Colloidal gold particles as a new in vivo marker of early acute lung injury Am J Physiol Lung Cell Mol Physiol 287:L867–L878.15194564 Find this article online
72. Henneberger A, Zareba W, Ibald-Mulli A, Ruckerl R, Cyrys J, Couderc JP, et al. 2005. Repolarization changes induced by air pollution in ischemic heart disease patients Environ Health Perspect 113:440–446.15811835 Find this article online
73. Howe HA, Bodian D 1940. Portals of entry of poliomyelitis virus in the chimpanzee Proc Soc Exp Biol Med 43:718–721. Find this article online
74. Huczko A, Lange H, Calko E, Grubek-Jaworska H, Droszcz P 2001. Physiological testing of carbon nanotubes: are they asbestos-like? Fullerene Sci Technol 9:251–254. Find this article online
75. Hughes LS, Cass GR, Gone J, Ames M, Olmez I 1998. Physical and chemical characterization of atmospheric ultrafine particles in the Los Angeles area Environ Sci Technol 32:1153–1161. Find this article online
76. Hunter DD, Dey RD 1998. Identification and neuropeptide content of trigeminal neurons innervating the rat nasal epithelium Neuroscience 83:591–599.9460765 Find this article online
77. Hunter DD, Undem BJ 1999. Identification and substance P content of vagal afferent neurons innervating the epithelium of the guinea pig trachea Am J Respir Crit Care Med 159:1943–1948.10351943 Find this article online
78. IARC (International Agency for Research on Cancer). 2002. Manmade Vitreous Fibres IARC Monogr Eval Carcinog Risks Hum 81:1–418.12458547 Find this article online
79. ICON 2004. International Council on Nanotechnology Home page. Available: http://icon.rice.edu [accessed 16 March 2005]
80. International Commission on Radiological Protection. 1994. Human respiratory model for radiological protection Ann ICRP 24:1–300. Find this article online
81. Jani P, Halbert GW, Langridge J, Florence AT 1990. Nanoparticle uptake by the rat gastrointestinal mucosa: quantitation and particle size dependency J Pharm Pharmacol 42:821–826.1983142 Find this article online
82. Jani PU, McCarthy DE, Florence AT 1994. Titanium dioxide (rutile) particle uptake from the rat GI tract and translocation to systemic organs after oral administration Int J Pharm 105:157–168. Find this article online
83. Jaques PA, Kim CS 2000. Measurement of total lung deposition of inhaled ultrafine particles in healthy men and women Inhal Toxicol 12:715–731.10880153 Find this article online
84. Johnston CJ, Finkelstein JN, Mercer P, Corson N, Gelein R, Oberdorster G 2000. Pulmonary effects induced by ultrafine PTFE particles Toxicol Appl Pharmacol 168:208–215.11042093 Find this article online
85. Joo SH, Feitz AJ, Waite TD 2004. Oxidative degradation of the carbothioate herbicide, molinate, using nanoscale zerovalent iron Environ Sci Technol 38:2242–2247.15112830 Find this article online
86. Kato T, Yashiro T, Murata Y, Herbert DC, Oshikawa K, Bando M, et al. 2003. Evidence that exogenous substances can be phagocytized by alveolar epithelial cells and transported into blood capillaries Cell Tiss Res 311:47–51. Find this article online
87. Kennedy P, Chaudhuri A 2002. Herpes simplex encephalitis J Neurol Neurosurg Psychiatry 73:237–238.12185148 Find this article online
88. Keyhani K, Scherer PW, Mozell MM 1997. A numerical model of nasal odorant transport for the analysis of human olfaction J Theor Biol 186:279–301.9219668 Find this article online
89. Kim S, Lim YS, Soltesz EG, De Grand AM, Lee J, Nakayama A, et al. 2004. Near infrared fluorescent type II quantum dots for sentinel lymph node mapping Nat Biotechnol 22(1):93–97.14661026 Find this article online
90. Kimbell JS, Godo MN, Gross EA, Joyner DR, Richardson RB, Morgan KT 1997. Computer simulation of inspiratory airflow in all regions of the F344 rat nasal passages Toxicol Appl Pharmacol 145:388–398.9266813 Find this article online
91. Kirschvink JL, Kobayashi-Kirschvink A, Woodford BJ 1992. Magnetite biomineralization in the human brain Proc Natl Acad Sci USA 89:7683–7687.1502184 Find this article online
92. Kirschvink J, Walker M, Diebel C 2001. Magnetite-based magneto-reception Curr Opin Neurobiol 11:462–468.11502393 Find this article online
93. König MF, Lucocq JM, Webel ER 1993. Demonstration of pulmonary vascular perfusion by electron and light microscopy J Appl Physiol 75(4):1877–1883.8282645 Find this article online
94. Kreuter J 2001. Nanoparticulate systems for brain delivery of drugs Adv Drug Deliv Rev 47:65–81.11251246 Find this article online
95. Kreuter J 2004. Influence of the surface properties on nanoparticle-mediated transport of drugs to the brain J Nanosci Nanotech 4:484–488. Find this article online
96. Kreuter J, Shamenkov D, Petrov V, Ramge P, Cychutek K, Koch-Brandt C, et al. 2002. Apolipoprotein-mediated transport of nanoparticle-bound drugs across the blood-brain barrier J Drug Target 10:317–325.12164380 Find this article online
97. Kreyling WG, Scheuch G 2000. Clearance of particles deposited in the lungs. In: Particle-Lung Interactions (Gehr P, Heyder J, eds). New York:Marcel Dekker Inc., 323–376
98. Kreyling W, Semmler M, Erbe F, Mayer P, Takenaka S, Schulz H, et al. 2002. Translocation of ultrafine insoluble iridium particles from lung epithelium to extrapulmonary organs is size dependent but very low J Toxicol Environ Health 65A:1513–1530. Find this article online
99. Kulmala M 2004. Formation and growth rates of ultrafine atmospheric particles: a review of observations J Aerosol Sci 35:143–176. Find this article online
100. Lam CW, James JT, McCluskey R, Hunter RL 2004. Pulmonary toxicity of single-wall carbon nanotubes in mice 7 and 90 days after intratracheal instillation Toxicol Sci 77:126–134.14514958 Find this article online
101. Lecoanet H, Bottero J, Wiesner M 2004. Laboratory assessment of the mobility of nanomaterials in porous media Environ Sci Technol 38:5164–5169.15506213 Find this article online
102. Lecoanet H, Wiesner M 2004. Velocity effects on fullerene and oxide nanoparticle deposition in porous media Environ Sci Technol 38(16):4377–4382.15382867 Find this article online
103. Lee CH, Guo YL, Tsai PJ, Chang HY, Chen CR, Chen CW, et al. 1997. Fatal acute pulmonary oedema after inhalation of fumes from polytetrafluoroethylene (PTFE) Eur Res J 10:1408–1411. Find this article online
104. Li N, Sioutas C, Cho A, Schmitz D, Misra C, Sempf J, et al. 2003. Ultrafine particulate pollutants induce oxidative stress and mitochondrial damage Environ Health Perspect 111:455–460.12676598 Find this article online
105. Li X, Brown D, Smith S, MacNee W, Donaldson K 1999. Short-term inflammatory responses following intratracheal instillation of fine and ultrafine carbon black in rats Inhal Toxicol 11:709–731.10477444 Find this article online
106. Lu Q, Moore JM, Huang G, Mount AS, Rao AM, Larcom LL, et al. 2004. RNA polymer translocation with single-walled carbon nanotubes Nano Lett 4:2473–2477. Find this article online
107. Mach R 2004. Nanoscale Particle Treatment of Groundwater. Federal Remedial Technology Roundtable: Naval Facilities Engineering Command. Available http://www.frtr.gov/pdf/meetings/l--mach​_09jun04.pdf [accessed 16 March 2005]
108. Malmgren L, Olsson Y, Olsson T, Kristensson K 1978. Uptake and retrograde axonal transport of various exogenous macromolecules in normal and crushed hypoglossal nerves Brain Res 153:477–493.81088 Find this article online
109. Maynard AD, Baron PA, Foley M, Shvedova AA, Kisin ER, Castranova V 2004. Exposure to carbon nanotube material: aerosol release during the handling of unrefined single-walled carbon nanotube material J Toxicol Environ Health A 67:87–107.14668113 Find this article online
110. McMurry PH, Woo KS 2002. Size distributions of 3 to 100 nm urban Atlanta aerosols: measurement and observations J Aerosol Med 15(2):169–178.12184867 Find this article online
111. Mehta D, Bhattacharya J, Matthay MA, Malik AB 2004. Integrated control of lung fluid balance Am J Physiol Lung Cell Mol Physiol 287:L1081–L1090.15531757 Find this article online
112. Monteiro-Riviere N, Nemanich R, Inman A, Wang Y, Riviere J 2005. Multi-walled carbon nanotube interactions with human epidermal keratinocytes Toxicol Lett 155:377–384.15649621 Find this article online
113. Nagaveni K, Sivalingam G, Hegde M, Madras G 2004. Photocatalytic degradation of organic compounds over combustion-sized nano-TiO₂ Environ Sci Technol 38:1600–1604.15046366 Find this article online
114. Nemmar A, Delaunois A, Nemery B, Dessy-Doize C, Beckers JF, Sulon J, et al. 1999. Inflammatory effect of intratracheal instillation of ultrafine particles in the rabbit: role of C-fiber and mast cells Toxicol Appl Pharmacol 160:250–261.10544059 Find this article online
115. Nemmar A, Hoet PHM, Vanquickenborne B, Dinsdale D, Thomeer M, Hoylaerts MF, et al. 2002a. Passage of inhaled particles into the blood circulation in humans Circulation 105:411–414. Find this article online
116. Nemmar A, Hoylaerts MF, Hoet PHM, Dinsdale D, Smith T, Xu H, et al. 2002b. Ultrafine particles affect experimental thrombosis in an in vivo hamster model Am J Respir Crit Care Med 166:998–1004. Find this article online
117. Nemmar A, Hoylaerts MF, Hoet PHM, Vermylen J, Nemery B 2003. Size effect of intratracheally instilled particles on pulmonary inflammation and vascular thrombosis Toxicol Appl Pharmacol 186:38–45.12583991 Find this article online
118. Nghiem LD, Schäfer AI, Elimelech M 2004. Removal of natural hormones by nanofiltration membranes: measurement, modeling, and mechanisms Environ Sci Technol 38:1888–1896.15074703 Find this article online
119. Nikula KJ, Avila KJ, Griffith WC, Mauderly JL 1997. Lung tissue responses and sites of particle retention differ between rats and cynomolgus monkeys exposed chronically to diesel exhaust and coal dust Fundam Appl Toxicol 37:37–53.9193921 Find this article online
120. NNI (National Nanotechnology Initiative) 2004. What Is Nanotechnology? Available: http://www.nano.gov/html/facts/whatIsNan​o.html [accessed 16 March 2005]
121. NRC (National Research Council) 1983. Risk Assessment in the Federal Government: Managing the Process. Washington, DC:National Academy Press
122. Nuttall JB, Kelly RJ, Smith BS, Whiteside CK Jr 1964. Inflight toxic reactions resulting from fluorocarbon resin pyrolysis Aerospace Med 35:676–683. Find this article online
123. Oberdörster E 2004a. Manufactured nanomaterials (fullerenes, C60) induce oxidative stress in brain of juvenile largemouth bass Environ Health Perspect 112:1058–1062. Find this article online
124. Oberdörster E 2004b. Toxicity of nC60 fullerenes to two aquatic species: Daphnia and largemouth bass [Abstract]. In: 227th American Chemical Society National Meeting, 27 March–1 April 2004, Anaheim, CA. Washington, DC:American Chemical Society, IEC 21
125. Oberdörster G 2000. Toxicology of ultrafine particles: in vivo studies Philos Trans R Soc Lond A 358:2719–2740. Find this article online
126. Oberdörster G, Ferin J, Finkelstein J, Wade P, Corson N 1990. Increased pulmonary toxicity of ultrafine particles? II. Lung lavage studies J Aerosol Sci 21:384–387. Find this article online
127. Oberdörster G, Ferin J, Gelein R, Soderholm SC, Finkelstein J 1992a. Role of the alveolar macrophage in lung injury: studies with ultrafine particles Environ Health Perspect 97:193–197. Find this article online
128. Oberdörster G, Ferin J, Morrow PE 1992b. Volumetric loading of alveolar macrophages (AM): a possible basis for diminished AM-mediated particle clearance Exp Lung Res 18:87–104. Find this article online
129. Oberdörster G, Finkelstein JN, Johnston C, Gelein R, Cox C, Baggs R, et al. 2000. Acute pulmonary effects of ultrafine particles in rats and mice Res Rep Health Eff Inst 96:5–74.11205815 Find this article online
130. Oberdörster G, Gelein RM, Ferin J, Weiss B 1995. Association of particulate air pollution and acute mortality: involvement of ultrafine particles? Inhal Toxicol 7:111–124.11541043 Find this article online
131. Oberdörster G, Morrow PE, Spurny K 1988. Size dependent lymphatic short term clearance of amosite fibers in the lung Ann Occup Hyg 32(suppl 4):149–156. Find this article online
132. Oberdörster G, Sharp Z, Atudorei V, Elder A, Gelein R, Kreyling W, et al. 2004. Translocation of inhaled ultrafine particles to the brain Inhal Toxicol 16(6/7):437–445.15204759 Find this article online
133. Oberdörster G, Sharp Z, Atudorei V, Elder A, Gelein R, Lunts A, et al. 2002. Extrapulmonary transloction of ultrafine carbon particles following whole-body inhalation exposure of rats J Toxicol Environ Health 65A:1531–1543. Find this article online
134. Oberdörster G, Yu CP 1990. The carcinogenic potential of inhaled diesel exhaust: a particle effect? J Aerosol Sci 21(suppl 1):S397–S401. Find this article online
135. Ohl L, Mohaupt M, Czeloth N, Hintzen G, Kiafard Z, Zwirner J, et al. 2004. CCR7 governs skin dendritic cell migration under inflammatory and steady-state conditions Immunity 21:279–288.15308107 Find this article online
136. Oldfors A, Fardeau M 1983. The permeability of the basal lamina at the neuromuscular junction. An ultrastructural study of rat skeletal muscle using particulate tracers Neuropathol Appl Neurobiol 9:419–432.6656996 Find this article online
137. Olsson T, Kristensson K 1981. Neuronal uptake of iron: somatopetal axonal transport and fate of cationized and native ferretin, and iron-dextran after intramuscular injections Neuropath Appl Neurobiol 7:87–95. Find this article online
138. Patton JS 1996. Review—mechanisms of macromolecule absorption by the lungs Adv Drug Deliv Rev 19:3–36. Find this article online
139. Pekkanen J, Peters A, Hoek G, Tiittanen P, Brunekreef B, de Hartog J, et al. 2002. Particulate air pollution and risk of ST-segment depression during repeated submaximal exercise tests among subjects with coronary heart disease. The Exposure and Risk Assessment for Fine and Ultrafine Particles in Ambient Air [ULTRA] study Circulation 106:933–938.12186796 Find this article online
140. Pekkanen J, Timonen KL, Ruuskanen J, Reponen A, Mirme A 1997. Effects of ultrafine and fine particles in urban air on peak expiratory flow among children with asthmatic symptoms Environ Res 74:24–33.9339211 Find this article online
141. Penttinen P, Timonen KL, Tiittanen P, Mirme A, Ruuskanen J, Pekkanen J 2001. Ultrafine particles in urban air and respiratory health among adult asthmatics Eur Resp J 17:428–435. Find this article online
142. Peters A, Doring A, Wichmann H-E, Koenig W 1997a. Increased plasma viscosity during an air pollution episode: a link to mortality? Lancet 349:1582–1587. Find this article online
143. Peters A, Wichmann HE, Tuch T, Heinrich J, Heyder J 1997b. Respiratory effects are associated with the number of ultrafine particles Am Respir Crit Care Med 155:1376–1383. Find this article online
144. Pietropaoli AP, Frampton MW, Oberdörster G, Cox C, Huang L-S, Marder V, et al. Blood markers of coagulation and inflammation in healthy human subjects exposed to carbon ultrafine particles. In: Effects of Air Contaminants on the Respiratory Tract - Interpretations from Molecular to Meta Ananlysis (Heinrich U, ed). INIS Monographs. Stuttgart, Germany:Fraunhofer IRB Verlag, 181–194
145. Plattig K-H 1989. Electrophysiology of taste and smell Clin Phys Physiol Meas 10:91–126.2663324 Find this article online
146. Rancan F, Rosan S, Boehm F, Cantrell A, Brellreich M, Schoenberger H, et al. 2002. Cytotoxicity and photocytotoxicity of a dendritic C(60) mono-adduct and a malonic acid C(60) tris-adduct on Jurkat cells J Photochem Photobiol B 67(3):157–162.12167314 Find this article online
147. Rejman J, Oberle V, Zuhorn IS, Hoekstra D 2004. Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis Biochem J 377:159–169.14505488 Find this article online
148. Rodoslav S, Laibin L, Eisenberg A, Dusica M 2003. Micellar nanocontainers distribute to defined cytoplasmic organelles Science 300:615–618.12714738 Find this article online
149. Sato K, Imai Y, Irimura RT 1998. Contribution of dermal macrophage trafficking in the sensitization phase of contact hypersensitivity J Immunol 161:6835–6844.9862715 Find this article online
150. Sayes C, Fortner J, Guo W, Lyon D, Boyd AM, Ausman KD, et al. 2004. The differential cytotoxicity of water-soluble fullerenes Nano Lett 4:1881–1887. Find this article online
151. Schlesinger RB, Ben-Jebria A, Dahl AR, Snipes MB, Ultman J 1997. Disposition of inhaled toxicants. In: Handbook of Human Toxicology (Massaro EJ, ed). New York:CRC Press, 493–550
152. Schultheiss-Grassi PP, Wessiken R, Dobson J 1999. TEM investigations of biogenic magnetite extracted from the human hippocampus Biochim Biophys Acta 1426:212–216.9878742 Find this article online
153. Seaton A, MacNee W, Donaldson K, Godden D 1995. Particulate air pollution and acute health effects Lancet 345:176–178.7741860 Find this article online
154. Semmler M, Seitz J, Erbe F, Mayer P, Heyder J, Oberdörster G, et al. 2004. Long-term clearance kinetics of inhaled ultrafine insoluble iridium particles from the rat lung, including transient translocation into secondary organs Inhal Toxicol 16:453–459.15204761 Find this article online
155. Shi JP, Evans DE, Khan AA, Harrison RM 2001. Sources and concentration of nanoparticles (< 10 nm diameer) in the urban atmosphere Atmos Environ 35:1193–1202. Find this article online
156. Shvedova AA, Kisin E, Keshava N, Murray AR, Gorelik O, Arepalli S, et al. 2004a. Cytotoxic and genotoxic effects of single wall carbon nanotube exposure on human keratinocytes and bronchial epithelial cells [Abstract]. In: 227th American Chemical Society National Meeting, 27 March–1 April 2004, Anaheim, CA. Washington, DC:American Chemical Society, IEC 20
157. Shvedova AA, Kisin E, Murray A, Schwegler-Berry D, Gandelsman V, Baron P, et al. 2004b. Exposure of human bronchial cells to carbon nanotubes caused oxidative stress and cytotoxicity. In: Proceedings of the Meeting of the SFRR Europe 2004, Ioannina, Greece. Philadelphia:Taylor & Francis Group, 91–103
158. Silva VM, Corson N, Elder A, Oberdörster G In press. The rat ear vein model for investigating in vivo thrombogenicity of ultrafine particles (UFP). Toxicol Sci
159. Simionescu N, Simionescu M, Palade GE 1975. Permeability of muscle capillaries to small heme-peptides J Cell Biol 64:586–607.239003 Find this article online
160. Smith AE, Helenius A 2004. How viruses enter animal cells Science 304:237–242.15073366 Find this article online
161. Terasaki S, Kameyama T, Yamamoto S 1997. A case of zoster in the 2nd and 3rd branches of the trigeminal nerve associated with simultaneous herpes labialis infection—a case report Kurume Med J 44(1):61–66.9154763 Find this article online
162. Tiittanen P, Timonen KL, Ruuskanen J, Mirme A, Pekkanen J 1999. Fine particulate air pollution, resuspended road dust and respiratory health among symptomatic children Eur Resp J 13(2):266–273. Find this article online
163. Tinkle SS, Antonini JM, Rich BA, Roberts JR, Salmen R, DePree K, et al. 2003. Skin as a route of exposure and sensitization in chronic beryllium disease Environ Health Perspect 111:1202–1208.12842774 Find this article online
164. Tran CL, Buchanan D, Cullen RT, Searl A, Jones AD, Donaldson K 2000. Inhalation of poorly soluble particles. II. Influence of particle surface area on inflammation and clearance Inhal Toxicol 12:1113–1126.11114784 Find this article online
165. Tran CL, Jones AD, Cullen RT, Donaldson K 1998. Influence of particle characteristics on the clearance of low toxicity dusts from lungs J Aerosol Sci 29(suppl 1):S1269–S1270. Find this article online
166. Tungittiplakorn W, Lion LW, Cohen C, Kim J-Y 2004. Engineered polymeric nanoparticles for soil remediation Environ Sci Technol 38(5):1605–1610.15046367 Find this article online
167. Turetsky BI, Moberg PJ, Arnold SE, Doty RL, Gur RE 2003. Low olfactory bulb volume in first-degree relatives of patients with schizophrenia Am J Psychiatry 160(4):703–708.12668359 Find this article online
168. U.S. EPA 1994. 10-Day Chronic Daphnia magna or Daphnia pulex. SOP 2028. Washington DC:U.S. Environmental Protection Agency. Available: http://www.ert.org/products/2028.pdf [accessed 16 March 2005]
169. U.S. EPA 2004. Air Quality Criteria for Particulate Matter. Vol 3. 600/P-95-001cF. Washington DC:U.S. Environmental Protection Agency, Office of Research and Development
170. Utell M, Frampton M, Zareba W, Devlin R, Cascio W 2002. Cardiovascular effects associated with air pollution: potential mechanisms and methods of testing Inhal Toxicol 14:1231–1247.12454788 Find this article online
171. Veronesi B, Makwana O, Pooler M, Chen LC In press. Effects of subchronic exposure to CAPs in ApoE^−/− mice: VII. Degeneration of dopaminergic neurons. Inhal Toxicol
172. von Klot S, Wolke G, Tuch T, Heinrich J, Dockery DW, Schwartz J, et al. 2002. Increased asthma medication use in association with ambient fine and ultrafine particles Eur Respir J 20:691–702.12358349 Find this article online
173. Warheit DB, Hartsky MA 1993. Role of alveolar macrophage chemotaxis and phagocytosis in pulmonary clearance responses to inhaled particles: comparisons among rodent species Microsc Res Tech 26:412–422.8286787 Find this article online
174. Warheit DB, Hill LH, George G, Brody AR 1986. Time course of chemotactic factor generation and the corresponding macrophage response to asbestos inhalation Am Rev Respir Dis 134:128–133.3729150 Find this article online
175. Warheit DB, Laurence BR, Reed KL, Roach DH, Reynolds GAM, Webb TR 2004. Comparative pulmonary toxicity assessment of single-wall carbon nanotubes in rats Toxicol Sci 77:117–125.14514968 Find this article online
176. Warheit DB, Overby LH, George G, Brody AR 1988. Pulmonary macrophages are attracted to inhaled particles on alveolar surfaces Exp Lung Res 14:51–66.2830106 Find this article online
177. Waritz RS, Kwon BK 1968. The inhalation toxicity of pyrolysis products of polytetrafluoroethylene heated below 500 degrees centigrade Am Ind Hyg Assoc J 29:19–26.5667186 Find this article online
178. WHO 1985. Reference Methods for Measuring Airborne Man-Made Mineral Fibers. Environmental Health Series 4. Copenhagen:World Health Organization
179. Wichmann H-E, Cyrys J, Stölzel M, Spix C, Wittmaack K, Tuch T, et al. 2002. Sources and elemental composition of ambient particles in Erfurt, Germany. In: Fortschritte in der Umweltmedizin (Wichmann HE, Schlipköter HW, Fülgraff G, eds). Erfurt, Germany:Ecomed Publishers
180. Wichmann H-E, Spix C, Tuch T, Wolke G, Peters A, Heinrich J, et al. 2000. Daily Mortality and Fine and Ultrafine Particles in Erfurt, Germany. Part I: Role of Particle Number and Particle Mass Res Rep Health Eff Inst 98:5–86.11918089 Find this article online
181. Widmaier EP, Raff H, Strang KT 2004. Vander, Sherman & Luciano's Human Physiology: The Mechanisms of Body Functions. 9th ed. New York:McGraw Hill
182. Williams N, Atkinson G, Patchefsky A 1974. Polymer fume fever: not so benign J Occup Med 16:519–522.4843400 Find this article online
183. Wilson M, Lightbody J, Donaldson K, Sales J, Stone V 2002. Interactions between ultrafine particles and transition metals in vivo and in vitro Toxicol Appl Pharmacol 184(3):172–179.12460745 Find this article online
184. Woo KS, Chen D-R, Pui D, McMurry P 2001. Measurement of Atlanta aerosol size distributions: observations of ultrafine particle events Aerosol Sci Technol 34:75–87. Find this article online
185. Yamago S, Tokuyama H, Nakamura E, Kikuchi K, Kananishi S, Sueki K, et al. 1995. In vivo biological behavior of a water-miscible fullerene: ¹⁴C labeling, absorption, distribution, excretion and acute toxicity Chem Biol 2:385–389.9383440 Find this article online
186. Yamakoshi Y, Umezawa N, Ryu A, Arakane K, Miyata N, Goda Y, et al. 2003. Active oxygen species generated from photo-excited fullerene (C₆₀) as potential medicines: O₂⁻* versus ¹O₂ J Am Chem Soc 125:12803–12809.14558828 Find this article online
187. Zhou Y-M, Zhong C-Y, Kennedy IM, Leppert VJ, Pinkerton KE 2003. Oxidative stress and NFκB activation in the lungs of rats: a synergistic interaction between soot and iron particles Toxicol Appl Pharmacol 190:157–169.12878045 Find this article online
188. Zhu Y, Hinds WC, Kim S, Shen SK, Sioutas C 2002. Study of ultrafine particles near a major highway with heavy-duty diesel traffic Atmos Environ 36:4323–4335. Find this article online