Comparison of the uptake of fine and ultrafine TiO2 in a tracheal explant system

A. Churg, B. Stevens, and J. L. Wright

Department of Pathology, University of British Columbia, Vancouver, British Columbia, Canada V6T 2B5

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

To examine the relationship between particle uptake by pulmonary epithelial cells and particle size, we exposed rat tracheal explants to fine particles (FPs; 0.12 µm) or ultrafine particles (UFPs; 0.021 µm) of titanium dioxide for 3 or 7 days. By electron microscopy, particles were found in the epithelium at both time points, but in the subepithelial tissues, they were found only at 7 days. The volume proportion of both FPs and UFPs in the epithelium increased from 3 to 7 days; it was greater for UFPs at 3 days but was greater for FPs at 7 days. The volume proportion of particles in the subepithelium at 7 days was equal for both dusts, but the ratio of epithelial to subepithelial volume proportion was ~2:1 for FPs and 1:1 for UFPs. Mean volume of individual particle aggregates was similar for both dusts at 3 days but was markedly smaller for FPs at 7 days. These observations suggest that the behavior of particles of different size is complex: UFPs persist in the tissues as relatively large aggregates, whereas the size of FP aggregates becomes smaller over time. UFPs appear to enter the epithelium faster, and once in the epithelium, a greater proportion of them is translocated to the subepithelial space compared with FPs. However, if it is assumed that the volume proportion is representative of particle number, the number of particles reaching the interstitial space is directly proportional to the number applied; i.e., overall, there is no preferential transport from lumen to interstitium by size.

mineral particle; epithelial uptake; titanium dioxide; ultrafine particle; ambient atmospheric particle

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE UPTAKE OF MINERAL PARTICLES by pulmonary epithelial cells is a widely recognized phenomenon, but little is known about the mechanisms that control this process (2). One parameter that has been proposed as an important determinant of uptake and transport is particle size; in particular, it has been claimed that smaller particles enter epithelial cells and are transported through the cells to the interstitium to a much greater extent than are larger particles of the same mineral type (1-3, 9). The entry of particles into epithelia and subepithelial tissues is known to be associated with a variety of deleterious effects including release of inflammatory and fibrogenic cytokines, oxidative cell injury, cell death, genotoxicity, and eventual interstitial fibrosis (briefly reviewed in Ref. 2). The notion that ultrafine particles (UFPs; i.e., those smaller than 0.1 µm) have particularly high tissue access and cause particularly high levels of mediator release has been proposed as one of the mechanisms behind the increases in morbidity and mortality associated with increases in ambient atmospheric particle levels (10, 11). As well, Ferin and colleagues (3-5) and Oberdorster et al. (10) have suggested that high access of UFPs to the interstitium results in increased levels of interstitial fibrogenic reactions.

Although the idea that there is a relationship between particle size and uptake is often repeated, the actual published data are difficult to interpret. In some experiments, not only are the sizes of dust administered differently, but the types of dusts are also different (13), thus confounding issues of size with issues of particle type-specific uptake (see DISCUSSION). Even where the same mineral species is used for large and small particles, dusts are usually administered at equal weight concentrations so that the number of small particles in many experiments exceeds the number of large particles by three to four orders of magnitude; because particle uptake appears to be directly related to the number of particles persisting in air spaces (1-3), the observed differences require detailed quantitative analysis to determine whether an apparently greater uptake of small particles is simply a reflection of administration of greater numbers of small particles rather than an effect of particle size.

Interpretation of the published data is also complicated by the general use of intact whole animal systems: quantitative determination of particle uptake and particle location in the lung in such systems presents problems of both technical approach and inter- pretation (see DISCUSSION). One important confounder is the inflammatory reaction evoked by dust particles. As a general rule, this reaction parallels particle number and appears to be more intense with UFPs compared with fine particles (FPs) (3-5, 10), although it is again unclear whether this difference really is a specific effect of UFPs or merely reflects administration of very large numbers of particles. Mediators released from inflammatory cells may affect particle uptake; exogenous oxidants, for example, have been shown to enhance particle uptake in vitro (2), and the same phenomenon presumably occurs with inflammatory cell-derived oxidants as well. Thus, in vivo, the effects of inflammatory cells on particle uptake are difficult to sort out from the effects intrinsic to the epithelium.

In this study, we have approached this problem by applying two sizes of titanium dioxide (TiO2; anatase) particles to rat tracheal explants, a system in which there are no air space inflammatory cells and one in which relative uptake between dusts is easy to quantify by morphometric techniques.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Sources of materials. TiO2 with a geometric mean particle diameter of 0.12 µm (geometric SD = 1.4) was obtained from Aldrich (Milwaukee, WI). This dust is referred to as "fine" (FP) TiO2 in this paper. TiO2 with a geometric mean particle diameter of 0.021 µm (geometric SD = 1.7) was a kind gift of Dr. G. Oberdorster. This dust is referred to as "ultrafine" (UFP) TiO2 in this paper.

Preparation of explants. Rat tracheal explants measuring ~2 × 2 mm were prepared by a modification of the method of Mossman et al. (9) as previously described. Multiple 250-g Sprague-Dawley rats were killed, and the segments from the different tracheae were mixed and then randomly assigned to treatment groups. Each treatment group contained four or five segments. The freshly prepared explants were submerged, epithelial side up, in a 5 mg/ml suspension of either fine or ultrafine TiO2 in Dulbecco's minimal Eagle's medium without serum for 1 h and then were lifted from the medium; the serosal surfaces were blotted and then placed on agarose-Dulbecco's modified Eagle's medium plates supplemented with 1% glutamine, 1% penicillin-streptomycin-Fungizone, 1 µg/ml of insulin, 0.1 µg/ml of hydrocortisone, 1.5× amino acids, and 10% chicken serum. The explants were maintained in a 95% air-5% CO2 atmosphere for 3 or 7 days to allow time for particle uptake to occur.

Preparation of explants for electron microscopy. After 3 or 7 days of culture, explants were fixed in buffered 4% paraformaldehyde-0.5% glutaraldehyde followed by 1% osmium tetroxide, dehydrated, embedded in epoxy resin, and sectioned at right angles to the tracheal length for electron microscopy. Each explant was embedded and cut as one piece. To avoid areas that might have been damaged when the trachea was cut into segments, the blocks were cut in at least 100 µm from the edge of the tissue before sections were taken for analysis. The cartilage was trimmed away, and a section that encompassed the entire epithelium plus most of the subepithelial tissues was cut and mounted on a single (slot)-hole grid. Sections were examined unstained or were lightly counterstained with lead citrate and uranyl acetate.

Determination of volume proportion of TiO2. We used the epithelial basement membrane as a dividing line: all tissues above the basement membrane were counted as epithelium, and all tissues between the basement membrane and the cartilage were counted as subepithelium. Almost all TiO2 in the tissues was found as aggregates of particles and only rarely as single particles. After initial examination to determine optimum conditions, the entire epithelium and attached subepithelium were photographed as contiguous photographs, and each negative was printed to a final magnification of ×5,000. A 420-point grid was then placed over each photograph, and points on TiO2 particles and points on the tissue were counted separately for the epithelium and subepithelium to obtain a volume proportion of TiO2; counts were summed over all the photographs to obtain values for each segment. To ensure that this fairly low magnification did not miss particles, two additional measurements were made. First, for each segment, the immediate subapical cytoplasm of each epithelial cell was searched on screen at a total magnification of ×250,000. Other than the occasional particle aggregate (which was visible in the ×5,000 photograph), no individual particles, and particularly no individual UFPs, were seen at this magnification. Second, random fields of epithelium and subepithelium were photographed and printed to a final magnification of ×25,000 and carefully searched for particles. All aggregates and individual FPs identified at a magnification of ×25,000 were also readily visible in the ×5,000 photographs. Individual UFPs were not seen at a magnification of ×5,000, but it was apparent from the higher power images that individual particles were sufficiently rare that missing them did not affect any of the calculations.

Determination of mean aggregate volume. With the use of a ×10 magnifier with an engraved scale, the maximum diameter of every particle aggregate or individual particle was measured from the photographs. The assumption was made that all aggregates were really spherical, and the volume was calculated from the measured radius.

Statistics. Data were log transformed for normalization, and differences among groups were determined by analysis of variance with SYSTAT (14). Data are presented graphically as arithmetic means ± SD because these provide a reasonable visual sense of the data.

    RESULTS
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Introduction
Materials & Methods
Results
Discussion
References

To determine how much aggregation of particles occurs in the dust suspension, fine and ultrafine dust suspensions were allowed to sediment onto electron microscope grids for 1 h to mimic the exposure conditions for the tracheal explants. Figure 1 shows the resulting appearance: both dusts tend to aggregate, but the aggregates of UFPs are larger and encompass greater numbers of particles.


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Fig. 1.   Electron micrographs of fine particle (A) and ultrafine particle (B) dusts after being allowed to settle from suspension onto coated electron microscope grids for 1 h, thus mimicking settling of dust onto surface of explants. It is apparent that both dusts tend to aggregate, although this phenomenon is more striking for ultrafine particles. Magnification ×12,000.

Figure 2 shows the volume proportion of TiO2 in the tissues at 3 and 7 days. At 3 days, particles were found only in the epithelium; at 7 days, particles were present in both the epithelium and subepithelium. The volume proportion of particles in the epithelium (VVE) increased from 3 to 7 days for both FPs and UFPs. The VVE was greater for UFPs at 3 days but was greater for FPs at 7 days. The volume proportion of particles in the subepithelium (VVI) at 7 days was the same for both dusts.


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Fig. 2.   Volume proportion of titanium dioxide (TiO2) in tissues at 3 (A) and 7 (B) days. Values are means ± SD. Error bars for subepithelium at 3 days indicate a 0 value. Volume proportion increased over time for both dusts. At 3 days, particles were found only in epithelium; at 7 days, particles were present in both epithelium and subepithelium. Volume proportion of particles in epithelium was greater for ultrafine particles at 3 days but for fine particles at 7 days. Volume proportion of particles in subepithelium was the same for both dusts. * P < 0.05 for fine compared with ultrafine dust.

Dust was almost always seen in epithelial cells as aggregates and very rarely as individual particles; there was no evidence of pericellular transport of particles to the interstitium. Figure 3 shows the mean volume of individual aggregates of particles at 3 and 7 days. At 3 days, there was no statistical difference in mean aggregate volume between the dusts. The mean aggregate volume in the epithelium did not change between 3 and 7 days for ultrafine dust but decreased markedly for fine dust. No statistical differences between the dusts were seen for subepithelial particles, but the mean volume of individual aggregates of ultrafine dust in the subepithelium was significantly smaller than the volume in the epithelium.


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Fig. 3.   Mean volume of individual aggregates of particles at 3 (A) and 7 (B) days. Values are means ± SD. Error bars for subepithelium at 3 days indicate a 0 value. At 3 days, there was no statistical difference in mean aggregate volume between dusts, but at 7 days, mean volume of epithelial ultrafine aggregates was much greater than that of fine aggregates. No statistical differences were seen for subepithelial particles. * P < 0.05 for fine compared with ultrafine dust. Ultrafine aggregates in subepithelium are also significantly smaller than those in epithelium.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

As noted in the introduction, determination of the effects of particle size on uptake, although seemingly straightforward, has proved to be technically difficult. Adamson and Bowden (1) administered smaller and larger (0.1 vs. 1.0 µm) latex spheres to mice by intratracheal instillation, and Mossman et al. (9) exposed hamster tracheal explants to smaller and larger (0.5-1.0 vs. >15 µm) carbon black particles. Both sets of authors reported that relatively few of the larger compared with the smaller particles entered the pulmonary or tracheal epithelial cells. However, both dusts were administered on an equal weight basis, and if it is assumed for convenience that the dusts were close-packed spheres, the smaller particle carbon black dose was 3,400-27,000 times the number concentration of the larger particle dose; similarly, the smaller latex particles were administered at 2,000 times the number concentration of the larger particles. Thus, although the authors' conclusions about the uptake of large and small particles in these experiments may be correct, absent actual quantitative data, apparently greater uptake of smaller particles might simply be a function of a vastly (2,000-30,000 times) greater particle number. To further complicate matters, in some reports, the small and large dusts have not been the same type of mineral. For example, Takenaka et al. (13) used ultrafine TiO2 and 1- to 2-µm fly ash particles; thus their observations that most of the fly ash ended up in alveolar macrophages and most of the TiO2 ended up in interstitial macrophages could as well be an effect of particle type as of particle size.

Ferin and colleagues (3-5) attempted to get around this problem by using carefully sized and well-characterized particles of TiO2 (0.012-0.030 vs. 0.23-0.25 µm, with control of mineral type) or aluminum oxide (0.020 vs. 0.50 µm) at equal weight concentration administered to rats by either inhalation or instillation. Multiple lavages were used to remove dust and dust-laden macrophages from the alveoli, and residual dust retained within the lavaged lung and in the lymph nodes was determined by atomic absorption spectroscopy. With this approach, intratracheal instillation of ultrafine dusts resulted in an ~50% greater retention (i.e., nonlavageable fraction) at 1 day but not thereafter. With inhalation exposure, the retained nonlavageable fraction was the same until inhalation ceased; thereafter, the clearance half-time and lymph node burden were significantly greater for UFPs over the course of ~1 yr.

Again, these experiments are not easy to interpret. An important question is where the various dust fractions actually are located. Dust that is unlavageable may or may not be located within epithelial cells and interstitium; it may simply be bound (in macrophages?) to the alveolar surface. Moreover, Ferin et al. (3) pointed out that the lymph node burdens accounted for many of the differences between fine and ultrafine dusts. Unfortunately, the fact that dust accumulates in lymph nodes to a greater or lesser extent provides no information about the route the dust took to the lymph nodes (i.e., by passage through the epithelium to the interstitium vs. transport within macrophages or some other route), and, in fact, Ferin et al. speculated that particles entering the interstitium were also phagocytosed by interstitial macrophages and then transported back to the airways at the site of peribronchial lymphoid tissue. These theories may be correct, but because the measurements are all indirect, they are very difficult to prove.

In this paper, we have attempted to approach this problem using a rat tracheal explant model. This model offers the advantage that particle uptake, although tedious to measure, is accurately quantifiable by morphometric techniques. Because the system is free of alveolar or air space inflammatory cells, it is also possible to determine whether differences in particle uptake by size are intrinsic features of tracheal epithelial cells.

We have made the assumption that because the rats are very close in weight at the time of death and because the tracheal segments for each treatment group are randomly selected from an aggregated collection of segments from all the rats, the average volume of the epithelial and subepithelial tissue compartments is the same in each treatment group. Thus the VVE and VVI provide a measure of the relative volume of dust of each type in each compartment; because the dusts are the same mineral species and have the same density and if it is assumed that the dust aggregates consist of close-packed spheres, the volume proportions also provide a measure of the relative number of FPs and UFPs in each location. This approach allows us to determine whether the relative number of particles of different size entering the epithelium or interstitium is different from the relative number applied to the explants. Because we used equal weight concentrations of FPs and UFPs, the number of particles applied to the explants in these experiments is ~200 times greater for UFPs compared with FPs.

It is important to consider possible technical artifacts in this system. One is the entry of particles into the epithelium or subepithelial tissues from the cut sides of the explant. Although theoretically possible, we believe this is unlikely. For one thing, when the explants are lifted from the dust suspension, visible dust remains on the apical surface, but the suspension drains off the sides of the tissue and no dust is visible on the sides. Second, to avoid areas of tissue damaged by the process of cutting the tracheae into segments, we routinely cut the tissue blocks in at least 100 µm from the tissue edge before taking sections for analysis. It appears unlikely that any particles could penetrate that great a distance in a horizontal fashion from the edges of the explants. Last, uptake of particles by tracheobronchial epithelium and subsequent transport of particles through the epithelium to the interstitium have been well documented in vivo (6, 12), and the fact that, in our system, particles first appear in the epithelium and then in the subepithelial tissues supports the idea that the explants mimic the in vivo situation.

Another consideration is the possible overloading of the explants with dust. Although overload (i.e., failure of macrophage-mediated clearance) is a well-defined condition in the alveolar space (8), there is no equivalent for tracheobronchial epithelium. One of the peculiarities of the tracheal explant system is the requirement for relatively high particle concentrations to produce conditions in which there is any significant uptake. We have worked out the present dust concentrations empirically to avoid tissue necrosis (which will occur if the FP concentration is increased by a factor of three or more) but to produce sufficient uptake for accurate counting. Because not every cell takes up particles, it is not likely that the system is overloaded, but it is clear that, in vivo, similarly high dust concentrations simply cannot occur.

Last, we were administering dusts in liquid suspension, and this is clearly not the same situation as inhalation. Both dusts tend to aggregate in solution, and the UFP dust also tends to aggregate in air (3-5). The effect of this phenomenon on uptake is unknown, although it should be pointed out that small mineral particles of any type that enter epithelial cells always tend to end up aggregated in lysosomes whether they are taken up as individual particles or aggregates.

Our actual results are complex. First, it is clear that VVE and VVI increase sequentially over time for both fine and ultrafine dusts, but they do so in a different pattern. At 3 days, VVE is greater for UFPs, suggesting that they enter the epithelium faster than FPs. However, by 7 days, VVE is greater for FPs. A partial explanation of this observation may be present in the ratio of VVE to VVI. For FPs at 7 days, this ratio is ~2:1, whereas for UFPs, the ratio is ~1:1. This may indicate that once UFPs enter the epithelium, they are more rapidly translocated to the interstitium, whereas FPs, even though they are taken up more slowly, tend to remain in the epithelium and thus become relatively concentrated.

An additional peculiarity is the marked change in the mean volume of individual dust aggregates over time. At 3 days, this value is similar for FPs and UFPs, but by 7 days, aggregate size has markedly decreased for FPs. The mechanism behind this effect is not obvious. It is possible that aggregate size may be important in the transport of particles from the epithelium to the interstitium.

Are there differences in the relative number of FPs and UFPs entering the tissues? From the logic described above and a ratio of UFPs to FPs of 200:1 applied to the tissues, our findings suggest that, over time, there is a slight preferential accumulation of FPs in the epithelium so that the ratio of UFPs to FPs drops to 100:1. However, the VVI is the same for both dusts, and thus the ratio of UFP to FP in the interstitium is 200:1, the same value as was present in the dusts applied to the explants. These findings do not support the idea that FPs show preferential transport to the interstitial tissues but rather indicate that transport is simply proportional to applied particle number. Given the rather small (twofold) difference between the VVE values for FPs and UFPs at 7 days, the same conclusion may apply to epithelial uptake. Although these results would suggest that there is no intrinsic preferential uptake of different-size particles in vitro, they do not rule out the possibility that, in vitro, inflammatory cell-derived mediators may change the relative uptake.

    ACKNOWLEDGEMENTS

This work was supported by Grants 8051 and 7820 from the Medical Research Council of Canada.

    FOOTNOTES

Address for reprint requests: A. Churg, Dept. of Pathology, Univ. of British Columbia, 2211 Wesbrook Mall, Vancouver, BC, Canada V6T 2B5.

Received 29 May 1997; accepted in final form 28 September 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Adamson, I. Y. R., and D. H. Bowden. Dose response of the pulmonary macrophagic system to various particulates and its relationship to transepithelial passage of free particles. Exp. Lung Res. 2: 165-175, 1981[Medline].

2.   Churg, A. The uptake of mineral particles by pulmonary epithelial cells. Am. J. Respir. Crit. Care Med. 154: 1124-1140, 1996[Medline].

3.   Ferin, J., G. Oberdorster, and D. P. Penney. Pulmonary retention of ultrafine and fine particles in rats. Am. J. Respir. Cell Mol. Biol. 6: 535-542, 1992[Medline].

4.   Ferin, J., G. Oberdorster, D. P. Penney, S. C. Soderholm, R. Gelein, and H. C. Piper. Increased pulmonary toxicity of ultrafine particles. 1. Particle clearance, translocation, morphology. J. Aerosol Sci. 21: 381-384, 1990.

5.   Ferin, J., G. Oberdorster, S. C. Soderholm, and R. Gelein. Pulmonary tissue access of ultrafine particles. Aerosp. Med. 4: 57-68, 1991.

6.   Gore, D. J., and G. Patrick. A quantitative study of the penetration of insoluble particles into the tissue of the conducting airways. Ann. Occup. Hyg. 26: 149-161, 1982[Medline].

7.   Keeling, B., J. Hobson, and A. Churg. Effects of cigarette smoke on tracheal epithelial uptake of non-asbestos mineral particles in organ culture. Am. J. Respir. Cell Mol. Biol. 9: 335-340, 1993[Medline].

8.   Morrow, P. E. Dust overloading of the lungs: update and appraisal. Toxicol. Appl. Pharmacol. 113: 1-12, 1992[Medline].

9.   Mossman, B. T., K. B. Adler, and J. E. Craighead. Interaction of carbon particles with tracheal epithelium in organ culture. Environ. Res. 8: 110-122, 1978.

10.   Oberdorster, G., R. M. Gelein, J. Ferin, and B. Weiss. Association of particulate air pollution and acute mortality: involvement of ultrafine particles? Inhalation Toxicol. 7: 111-124, 1995.[Medline]

11.   Seaton, A., W. MacNee, K. Donaldson, and D. Godden. Particulate air pollution and acute health effects. Lancet 345: 176-178, 1995[Medline].

12.   Stirling, C., and G. Patrick. The localisation of particles retained in the trachea of the rat. J. Pathol. 131: 309-320, 1980[Medline].

13.   Takenaka, S., H. Dornhoffer-Takenaka, and H. Muhle. Alveolar distribution of fly and of titanium dioxide after long-term inhalation by Wistar rats. J. Aerosol Sci. 17: 361-364, 1986.

14.   Wilkinson, L. SYSTAT: The System for Statistics. Evanston, IL: SYSTAT, Inc., 1991.


AJP Lung Cell Mol Physiol 274(1):L81-L86
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