Metal Particles Are Inappropriate for Testing a Postulate of Extrapulmonary Transport

Referencing: Translocation of Inhaled Ultrafine Manganese Oxide Particles to the Central Nervous System

Exposure to ambient air pollution particles has been associated with increased human morbidity and mortality, much of which is nonpulmonary. One proposed explanation of this extrapulmonary tissue injury is a transport of the particles outside of the respiratory tract. In the August 2006 issue of EHP, Elder et al. (2006) tested a postulate of extrapulmonary transport of particulate matter (PM). Specifically, the authors focused on the potential translocation of particles by olfactory neuronal pathways to the central nervous system. Comparable to previous studies on systemic transport of PM, they used a metal particle (i.e., a manganese oxide). Elder et al. measured tissue Mn concentrations in an effort to establish transport of the particle.

Past research has repeatedly demonstrated that components of PM can be solubilized, mobilized, and transported to tissues outside the respiratory tract independently of the original particle (e.g., nicotine transport from the cigarette-smoke particle to the blood and central nervous system of the smoker). Metals are among those components that can be solubilized and mobilized from particles and distributed systemically without the translocation of the particle from the original site of deposition in the respiratory tract. To meet the demands of growth and homeostasis, living systems frequently acquire metals from particles using some combination of chemical reduction and direct chelation (Currie and Briat 2003). Elder et al. (2006) reported that the transport of Mn, with elevated concentrations of the metal in extrapulmonary tissues, supports a translocation of the original PM into the central nervous system. However, their use of Mn oxide to test any postulate of extrapulmonary transport of a particle is inappropriate as a result of the in vivo availability of both reductants (e.g., superoxide, ascorbate, glutathione) and chelators (e.g., transferrin, lactoferrin, citrate, urate) in the mammalian respiratory tract. These reductants were not available in their in vitro solubility tests (performed in normal saline), the results of which they used to support their conclusion of direct in vivo translocation of particles. Elevated concentrations of the metal in extrapulmonary tissues do not prove direct translocation of the original PM, but rather reflect solubilization and mobilization of the Mn from the oxide particle, with subsequent distribution. Additionally, the rapidity of change in metal concentration at an extrapulmonary site should not support a direct translocation because the time required for solubilization, mobilization, and systemic transport of a metal from any particle has never been defined; the required time for such transport is predicted to be short for ultrafine particles, which have an increased surface area available for rapid interactions of the PM with endogenous reductants and chelators. What is required to prove the existence of an extrapulmonary transport of PM is employment of a particle with components that cannot be independently solubilized, mobilized, and transported from the site of its original deposition. A carbon-based PM would be appropriate.

When evaluating previous investigations for evidence of extrapulmonary transport of carbon-based particles, there is little to support translocation of such PM outside the respiratory tract. A recent study by Mills et al. (2006) showed no translocation of insoluble, radiolabeled ultrafine carbon particles from the lung into the bloodstream of humans. The small amount of radioactivity they found in the bloodstream and other organs within 6 hr of inhalation could be attributed entirely to the small amount of leached, soluble radiolabel from the particles. Among miners who were exposed to coal dust at 1,000 times the concentrations of ambient air pollution PM, LeFevre et al. (1982) observed the particle in only the lung and the reticuloendothelial system; the presence of the particle in the reticuloendothelial system reflects the transport of PM after sequestration within phagocytes, because the particles are always detected within these cells at these sites.

Finally, decades of research have provided no evidence of an extrapulmonary transport (including via olfactory neuronal pathways) of particles associated with cigarette smoking; smoking exposes the individual to literally kilograms of a particulate combustion product that includes ultrafine particles. While again demonstrating that a metal component of a particle can be solubilized, mobilized, and transported from the site of original deposition, Elder et al. (2006) provided no evidence to support a disbursal of the actual PM to tissues outside the respiratory tract.

The authors declare they have no competing financial interests.

Metal Particles and Extrapulmonary Transport: Oberdörster and Elder Respond

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We appreciate the opportunity to address the points raised by Ghio and Bennett in their letter. The pH of our cell-free dissolution studies (Elder et al. 2006) was appropriate because the nasal cavity surface is neutral and does not have airway macrophages and the phagolysosomal pH of nasal epithelial cells is neutral (Johnson 1994). Acidic pH, as in the phagolysosome of alveolar macrophages, dissolves manganese oxide, resulting in increased levels of blood-borne nonparticulate Mn. Our neutral pH buffer dissolved only 1.5% of Mn oxide nanoparticles in 24 hr. The dissolution buffer was not "normal saline," as stated by Ghio and Bennett; it was physiologic saline, a "simulated lung fluid" used for decades in studies of man-made fiber dissolution (Potter and Mattson 1991). It was an oversight on our part to omit the exact composition from our article (Elder et al. 2006), which includes citrate (model organic acid) and glycine (model protein component). Citrate is a stronger metal chelator than glycine.

If only soluble Mn translocates, the time required for solubilization would significantly retard the increase of Mn in the olfactory bulb of administered Mn oxide nanoparticles. Instead, we found no difference between the two forms of Mn when we directly compared the translocation rate of the Mn oxide nanoparticles to soluble Mn chloride (Elder et al. 2006), indicating a direct uptake of the Mn oxide nanoparticles by olfactory neuronal structures and subsequent translocation. It is conceivable that subsequent dissolution in the olfactory system occurs. The rapidity of solid nanoparticle transport along neuronal axons is, indeed, remarkable (~ 2.5 mm/hr), as demonstrated earlier by the arrival in the olfactory bulb of 50 nm gold particles within 30 min after intranasal instillation. This and other studies with gold nanoparticles using transmission electron microscopy detection (reviewed by Oberdörster et al. 2005) demonstrate unequivocally that some metal particles are indeed appropriate for demonstrating solid particle transport across epithelial barriers, refuting the absolute statement in the title of Ghio and Bennett's letter.

Ghio and Bennett suggest that a carbon-based particle would be appropriate for studying ultrafine particle transport and translocation. This is true for elemental and organic carbon only if insoluble in vivo. Indeed, study with inhaled ultrafine elemental carbon particles (¹³C) confirmed their translocation to the olfactory bulb of rats (Oberdörster et al. 2004). In contrast, labeled elemental carbon is problematic, as pointed out by Ghio and Bennett regarding Technegas (^99mTc labeled-ultrafine carbon). Recent studies using inhaled Technegas (Mills et al. 2006; Wiebert et al. 2006) showed no translocation in humans, contradicting earlier work (Nemmar et al. 2002). Both leaching of the soluble radiolabel and the inability of the -camera to detect small amounts (≤ 1%) of the deposited dose in extrapulmonary organs are significant limitations with this noninvasive technique, resulting in misinterpretions suggesting either significant particle translocation or lack thereof.

Ghio and Bennett cite LeFevre et al. (1982), apparently as evidence that inhaled carbon-based particles do not undergo extrapulmonary transport. However, LeFevre et al. interpreted their findings of high black pigment scores in liver and spleen of coal miners differently—namely, as migration of coal dust in the pneumoconiotic lung into pulmonary lymphatics and then to the systemic circulation, and also as migration of coal mine dust–laden macrophages through the walls of pulmonary blood vessels. Whatever the mechanism, their findings clearly indicate extrapulmonary transport. Heavy silica inhalation exposure has also been found to result in particle accumulation in liver and other extrapulmonary tissues in humans and nonhuman primates (Carmichael et al. 1980; Rosenbruch 1990; Slavin et al. 1985). Ghio and Bennett further state that

Decades of research have provided no evidence of an extrapulmonary transport (including via olfactory neuronal pathways) of particles associated with cigarette smoking.

How many investigators have tried to find cigarette smoke particles in extrapulmonary tissues? We are aware of only one study in rats in which a 25-min inhalation exposure to ¹⁴C cigarette smoke resulted in 0.24–0.83% of retained ¹⁴C in the liver within 15 min after the short-term exposure (Chen et al. 1989). Does this indicate extrapulmonary particle transport? Possibly yes.

We conclude that the physicochemical characteristics of a nanoparticle—whether metal or not—and the physiologic milieu at the site of deposition in the respiratory tract determine whether extrapulmonary translocation occurs as particle or as solute. A prerequisite for noninvasively measuring this is that the detection method has sufficient sensitivity for identifying the analyte at expected low translocation rates.

Regarding our nasal translocation study in rats (Elder et al. 2006), we conclude that inhaled Mn oxide nanoparticles are taken up by the nasal olfactory neuronal pathway as solid particles rather than being slowly dissolved first at neutral pH. For the alveolar region where dissolution is expected to be more rapid, this does not apply.