* CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709;
Syngenta, Macclesfield, Cheshire, England SK10 4TJ;
DuPont Haskell Laboratories, Newark, Delaware 19714; and
GlaxoSmithKline, Research Triangle Park, North Carolina 27709
Received August 12, 2003; accepted October 12, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: ultrafine titanium dioxide; inhalation; lung response; rats; mice; hamsters.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chronic ultrafine TiO2 (uf-TiO2) inhalation exposures have been conducted and the pulmonary toxicological effects assessed. Wistar rats exposed to uf-TiO2 for 24 months at an average concentration of 10 mg/m3 developed pulmonary tumors (Heinrich et al., 1995). Similarly, chronic exposure to other PSP such as carbon black, diesel exhaust, pigmentary TiO2, and talc led to the development of lung tumors in rats but not hamsters or mice (Heinrich et al., 1986
; Hext, 1994
; Lee et al., 1985
; Mauderly et al., 1987
, 1994
; Muhle et al., 1998
). The pulmonary response of rats to high, chronic doses of low solubility particles include findings of bronchoalveolar hyperplasia and metaplasia, fibrosis, and pulmonary tumorigenesis. These responses to the inhalation of PSP appear to be unique to the rat, relative to other rodent species, and limited to scenarios where there are substantial particle lung burdens and a concomitant impairment of alveolar macrophage-mediated lung clearance (pulmonary overload).
Impairment of pulmonary particle clearance and an attendant pulmonary inflammatory response have been shown to occur in rats exposed subchronically by inhalation to concentrations of uf-TiO2 and other PSP known to induce pulmonary overload under a chronic exposure regimen (Cullen et al., 2000; Ferin et al., 1992
; Warheit et al., 1997
). Persistent inflammation has been hypothesized to occupy a central role in the pathogenesis of PSP-induced epithelial changes leading to lung tumorigenesis in rats (Donaldson, 2000
). Notably, rats appear to mount greater inflammatory and epithelial responses than other laboratory rodent species following the inhalation of pigmentary TiO2 and other PSP (Bermudez et al., 2002
; Donaldson and Tran, 2002
).
Ultrafine particles, defined as having a diameter of less than 0.1 µm, have been shown to have a greater capacity to induce inflammation of the lung than fine particles. Ferin and coworkers have shown that uf-TiO2 has a greater capacity than fine mode TiO2 to induce an inflammatory response of the lung in rats exposed to equivalent aerosol concentrations (Ferin et al., 1992). Although the properties of uf-TiO2 underlying the heightened biological responses of rats relative to fine TiO2 are unknown, it is hypothesized that increased surface area of this material and oxidative stress are important players (Donaldson et al., 2002
).
Data regarding the pulmonary responses of rodents to ultrafine materials have been in large part collected using the rat. Studies examining the differences in species responses to ultrafine particulates, uf-TiO2 in particular, are few. The present study was one of two, identical in design (Bermudez et al., 2002), conducted to carry out a systematic comparison of the pulmonary responses of laboratory rodent species to inhaled TiO2 particles and to test the hypothesis that the pulmonary responses of rats to these particles differ from those of mice and hamsters. Subchronic inhalation exposures of mice, rats, and hamsters to equivalent aerosol concentrations of uf-TiO2 were conducted and the pulmonary pathobiological responses during recovery compared.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aerosol generation and monitoring.
Uf-TiO2 (P25, average primary particle size of 21 nm) was obtained from DeGussa-Hüls AG (Frankfurt, Germany). Aerosol generation was accomplished using a brush generator (CR-9100 Aerosol Generator, CR Equipment SA, Tannay, Switzerland) with dispersion of the particles by jet streams of air and passage through a mixing chamber prior to delivery to the exposure chambers. Inhalation exposures were conducted in 1 m3 H-1000 stainless steel chambers (Lab Products, Maywood, NJ). During the exposures, particle concentrations were continuously monitored using light scatter (Model RAM-S, Monitoring Instruments for the Environment, Inc., Bedford, MA), and the time-averaged concentration was recorded at least six times over the 6 h exposure period. The particle size distribution of the aerosol was measured at least twice per exposure level per chamber (excluding the control chambers) during the course of the study. Measurements were made using a MOUDI (Micro-Orifice Uniform Deposit Impactor, model 100, MSP Corporation, Minneapolis, MN). Mean mass-median aerodynamic diameter was 1.37 µm (see Results).
Experimental design.
Animals were exposed to 0.5, 2.0, or 10 mg/m3 uf-TiO2 for 6 h/day, 5 days/week, for 13 weeks. Controls were exposed to filtered air only. Hamsters were exposed separately from the mice and rats due to health considerations. Groups of 25 animals for each species and time point were used. Animals were sacrificed immediately following completion of the 13-week exposure periods and additional recovery groups were held for postexposure periods of 4, 13, 26, or 52 (49 for hamster) weeks in clean air. Following exposure and at each recovery time, the uf-TiO2 burdens in the combined right lung lobes and in the lung-associated lymph nodes were determined. The left lungs of these animals were used to assess lung cell proliferation and histopathological end points. The inflammatory status of the lung was assessed at each time point using a separate group of animals subjected to bronchoalveolar lavage. The bronchoalveolar lavage fluid (BALF) was assayed for lactate dehydrogenase (LDH) and total protein levels and the differential cytology of the recovered cells was determined.
Titanium burden analysis.
Burden analysis was performed according to the method of Levine et al.(2003). At necropsy, right lung and lymph node tissue samples were collected and stored frozen until ready for titanium burden analysis. Prior to burden analysis, the tissues were thawed, weighed, dried overnight in a muffle furnace at 37°C, desiccated, and weighed again. Dry tissue samples were digested overnight in a mixture of nitric acid and hydrofluoric acid and then further digested in a microwave oven. The sample digests were diluted with deionized water to 25 ml, and samples were analyzed using an inductively coupled plasma optical emission spectrometer (ICP-OES; Thermo Jarrell Ash AtomScan 16, Thermo Elemental, Franklin, MA). Data for lung tissue are expressed as milligrams of uf-TiO2 per gram dry lung. For lymph node tissues, the data are expressed as the total micrograms of uf-TiO2 present because the harvest of lymph node tissues was subject to collection of extraneous tissue to ensure collection of the nodes. The method quantitation limit (MQL) was 0.05 µg Ti/ml sample digest. The minimum detectable concentrations (MDC) of uf-TiO2 in pulmonary tissues were calculated using the MQL in combination with the average control lung weight; for lung, these were 0.14, 0.03, and 0.03 mg uf-TiO2/g dry weight for mouse, rat, and hamster, respectively, and, for lymph nodes, it was 2.09 µg uf-TiO2/sample. Values above the MDC were observed in control mouse lung tissues at the end of the exposures and reflect an increase in the baseline for that run of the assay rather than the presence of uf-TiO2 in control tissues. Retention half-times for uf-TiO2 in lung were calculated using the best-fit equations to the lung burden data from all time points.
Bronchoalveolar lavage.
Lungs were lavaged five times with equal volumes of phosphate-buffered saline. Fluid from the first two lavages was recovered, pooled, and placed on ice. The subsequent three lavages were pooled and also placed on ice. Recovered cells from all lavages were collected by centrifugation, resuspended in cell culture medium, and counted using an automated cell counter (Model ZM, Coulter, Marietta, GA). Cell differential counts were performed on Wright-Giemsastained cytocentrifuge slide preparations. LDH and total protein levels in cell-free fluid from the first two pooled lavages were quantitated spectrophotometrically using a COBAS FARA II automated analyzer (Roche Diagnostic Systems Inc., Montclair, NJ).
Lung cell proliferation.
Five days prior to euthanasia, animals were subcutaneously implanted with osmotic pumps (Alza, Palo Alto, CA) containing bromodeoxyuridine (BrdU, Sigma Chemical Co., St. Louis, MO). Rats and hamsters were implanted with model 2ML1 (10 µl/h) pumps containing 5 mg/ml BrdU. Mice were implanted with model 2001 (1 µl/h) pumps containing 16 mg/ml BrdU. At necropsy, left lungs were pressure-infused intratracheally (20 cm H2O for mice and 30 cm H2O for rats and hamsters) with 10% neutral-buffered formalin. Lungs were fixed for approximately 48 h and then changed to 70% ethanol. Subsequently, the lungs were embedded in paraffin, sectioned at 5 µm, and stained for BrdU by established methods (Rutten et al., 1994). Terminal bronchiolar and alveolar cell labeling indices were determined for each animal, and the mean labeling index was calculated for each group of five animals.
Histopathology.
Paraffin-embedded left lung tissues were sectioned at 5 µm and stained with Massons trichrome. The trichrome-stained lung sections were evaluated for particle-induced histopathological changes.
Statistical methods.
All data were tested for normality and homogeneity of variance. If the hypotheses for these assumptions were rejected (p < 0.01), common transformations (e.g., log, square root, arc sine) were applied and the data were retested. Comparisons to controls were made using Dunnetts test. The software package JMP (SAS Institute, Cary, NC) was used for the statistical analysis.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Body Weights
Body weights were collected weekly for the exposure phase of the study and biweekly from week 17 through the end of the postexposure period. A depression in body weight gain was noted in all groups of mice and rats following the end of the exposure period with recovery occurring over the next 3 to 4 weeks (data not shown). Frank body weight loss was observed in hamsters at the end of the exposure (915%) with a slow recovery over the remainder of the study (data not shown). Body weight fluctuations were more frequent and abrupt in hamsters than in mice or rats (data not shown). A likely reason for the body weight effects in the three species was the need for a period of acclimation to changes in environmental conditions experienced by the animals during the postexposure recovery period, when they were no longer housed in inhalation exposure chambers and were moved to new housing.
Uf-TiO2 Burdens
There were dose-related changes in uf-TiO2 lung burdens for all three species. Following 13 weeks of exposure, rats and mice exhibited equivalent uf-TiO2 lung burdens at all exposure concentrations (Fig. 1). Lung burdens of uf-TiO2 in hamsters were approximately two- to five-fold lower than those of rats and mice. Particle burdens decreased in the lung with time postexposure in mice, rats, and hamsters (Fig. 1
). Decreases in lung burdens with time after exposure were sharply different among exposure concentration groups in both rats and mice. In mice, the high-dose burdens decreased slowly to approximately 46% of the initial burden, whereas low- and mid-dose burdens were at undetectable levels by the end of the recovery period. Similarly, rats of the high-dose group retained approximately 57% of the initial lung burden, whereas the lung burdens in animals of the low- and mid-dose groups decreased to 10 and 25%, respectively, by the end of the recovery period. There were very significant differences among species in the pulmonary clearance kinetics of uf-TiO2. Whereas the lung burdens of uf-TiO2 in mice and rats of the high-dose group decreased in a linear fashion during the recovery period to approximately 50% of the lung burden at the end of the exposure, retained lung burdens in hamsters declined in a biphasic manner to just 3% of the initial burden. For animals of the mid- and low-dose groups, the change in lung burdens was also biphasic; however, in rats and not mice or hamsters, there were detectable concentrations of uf-TiO2 at the end of the recovery period.
|
|
Significant increases in the total number of cells recovered (number ± one standard deviation) were observed at the end of exposures in rats (5.3 x 106 ± 1.2) and mice (4.7 x 105 ± 1.1) of the high-dose group. These elevations in the number of cells recovered from uf-TiO2exposed rats and mice declined with time after exposure to control levels in rats by 26 weeks postexposure but remained significantly (p < 0.05) different (3.8 x 105 ± 0.11) from concurrent controls in mice at 52 weeks postexposure.
Statistically significant changes in the cytological profile of the cells recovered by BAL were limited to the mice, rats, and hamsters of the high-dose group. Mice had significantly elevated numbers of macrophages, neutrophils, and lymphocytes at the end of the exposure period (Fig. 3). Over the recovery period of 52 weeks, the numbers of these cell types and the percentages of the populations that they comprised remained relatively constant and were significantly different from concurrent controls at the 52 week time point (Figs. 3
and 4
). Rats also had significantly elevated numbers of macrophages, neutrophils, and lymphocytes at the end of the exposure period. Unlike mice, in rats the numbers of these cell types had returned to control levels by 26 weeks of recovery (Fig. 3
), although the proportion of the population that was made up of macrophages and neutrophils remained significantly different from concurrent controls at 52 weeks of recovery (Fig. 4
). Significant changes in recovered cell populations from the hamsters were limited to an increase in the number of neutrophils (0.3 x 106 ± 0.1) at the end of the exposures. At this same time point, the percentage of neutrophils was increased but not statistically significant, mostly due to the large variation among animals (10.2% ± 14.7).
|
|
|
|
|
|
|
Other than alveolar and interstitial macrophages containing particles and the occasional aggregation of particle-laden macrophages in the high concentrationexposed group, there was no pathology associated with uf-TiO2 exposure in the hamsters (Fig. 6C).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Particle concentrations in the aerosols for the exposures of each species were within 11% of the target concentrations of 0.5, 2, and 10 mg/m3. The aerodynamic diameter of the particles was found to be in the range of 1.29 to 1.44 µm and did not significantly differ between exposures. Considering the primary particle size of approximately 0.02 µm, the aerosol generated was made up of particle aggregates, probably due to agglomeration. This is a common occurrence in the generation of aerosols of these types of particles, and it is assumed that once the particles are deposited there is disaggregation of the primary particles.
There were species differences in retained lung burdens. Lung burdens of uf-TiO2 were increased in a concentration-dependent manner in rats, mice, and hamsters after 13 weeks of exposure. Initial lung burdens per gram of lung were similar in rats and mice; however, hamsters had approximately 23% of the rat and mouse burdens. The fact that hamsters had lower initial lung burdens is indicative of the ability of hamsters to efficiently clear particles from the lung during exposure (Bermudez et al., 2002; Creutzenberg et al., 1998
). The retained lung burden in rats exposed to 10 mg/m3 was 2.1 mg/lung and was comparable, adjusting for dose and daily length of exposure, to lung burdens found in other studies using this material (Ferin et al., 1992
; Heinrich et al., 1995
). Similarly, uf-TiO2 lung burdens of 0.42 mg/lung in mice of the high-dose group were comparable to the mice in the study by Heinrich et al. (1995)
, when adjusted for dose and daily length of exposure.
Determinations of lung burdens subsequent to exposure showed a decrease with time. Uf-TiO2 was detectable in the lungs of rats of all dose groups at the end of the postexposure period, whereas this was true only for mice and hamsters of the high-dose group. A comparison of the percentages of initial lung burden remaining in the lungs of the high-dose animals at the end of the postexposure period reveals that rats and mice were approximately equivalent with respect to particle retention (57 and 45%, respectively). In contrast, particle retention in hamsters was much lower (3%). The percentage of the initial burden retained in rats is in good agreement with that found in the study by Ferin et al. (1992). Calculation of retention half-times from the lung burden data of the low-, mid-, and high-dose groups showed the following, respectively: rats had half-times of 63, 132, and 395 days; mice had half-times of 48, 40, and 319 days; and hamsters had half-times of 33, 37, and 39 days. The evident prolongation of particle clearance from the lungs of the high-dose rats and mice, such that clearance of the particle burden would require more than the animals lifetime, is indicative of pulmonary overload.
The overload of the lung noted in mice and rats was reflected in the translocation of particles to lung-associated lymph nodes. The lack of particle translocation to the lymph nodes in hamsters reflects the short clearance half-times/low lung burdens in these animals. Rats and mice of the high-dose groups had significant lymph node burdens that continued to increase with time postexposure, consistent with the declining lung burdens and indicating an ongoing translocation of particles to these tissues. These results are consistent with other studies, using various particulates, showing that at lung burdens of 1 mg/g lung or greater there is a prolonged retention of particles (Morrow, 1992).
Pulmonary inflammation is a common response to the inhalation of various types of particles and has been closely associated with other chronic pathological outcomes, such as fibrosis and cancer, following extended exposure to these materials (Bajpai et al., 1992; Driscoll et al., 1990
; Muhle et al., 1998
; Oberdorster et al., 1992
; Warheit et al., 1997
). Characterization of the inflammatory response is usually accomplished by the assessment of changes in lung cell populations and biomarkers of inflammation in parenchymal lung tissue and BAL fluid. In the present study, histopathology of lung sections and cytological and biochemical markers of toxicity in BAL fluid (LDH and protein) were examined. Significant alterations in inflammatory parameters were observed only in those animals exposed to the high concentration (10 mg/m3) of uf-TiO2. In general, the magnitude of the responses was greatest in rats and least in hamsters.
Mice of the high-dose group had mild, persistent inflammation of the lung following 13 weeks of exposure. This inflammation was characterized by elevated concentrations of BALF protein and increased numbers of macrophages and neutrophils. With time postexposure, LDH transiently increased then returned to control levels, whereas BALF protein concentrations and inflammatory cell numbers remained relatively constant.
Inflammation in rats of the high-dose group following 13 weeks exposure was more severe than in mice in that all the measured parameters were significantly increased, particularly the number of neutrophils (65% of the recovered cells). Upon cessation of the exposure, there was a decline in all measured parameters with BALF LDH and protein and macrophage numbers returned to control levels by 26 weeks postexposure. The number of neutrophils remained elevated at 52 weeks postexposure and at the same level as mice (12%), indicating the presence of mild inflammation. These results are similar to those reported by Ferin et al. (1992), who observed a persistent increase in neutrophils in rats 52 weeks after a 12-week exposure to uf-TiO2.
Pulmonary inflammation was absent in hamsters, as evidenced by control levels of biochemical and cytological parameters. This is not surprising in light of the relative low lung burdens noted at the end of the exposure and the short retention half-times of the particles for this species.
Bronchiolar cells of rats, mice, and hamsters of the high-dose group exhibited increased cell replication following the cessation of exposure. This response was transient and is consistent with the clearance of particles or inflammation of the airway and is believed to represent a lesion separate from cell proliferation of alveolar epithelial cells.
Rat alveolar cells had significantly elevated (two- to three-fold) cell replication in animals of the mid- and high-dose groups at the end of exposure. This increased cell replication persisted through 13 weeks postexposure and correlated well with proliferative lesions observed in these animals. These results are similar to the two- to three-fold elevations of alveolar cell replication noted in rats exposed to fine grade TiO2 (Bermudez et al., 2002; Warheit et al., 1997
). Quantitative differences in levels of cell replication noted in control populations between the present study and that of Warheit et al. (1997)
are probably due to the differences in the methods of label administration (pulse vs. continuous administration of BrdU). Persistent alveolar cell replication in rats 1 year after exposure to 23.5 mg/m3 uf-TiO2, using the same exposure regimen as in the present study, has been reported (Baggs et al., 1997
). The genesis of the observed alveolar cell replication is unclear, as there was a good correlation of this parameter with lung burdens and neutrophil numbers; however, persistent replication of parenchymal cells with attendant macromolecular damage and impairment of clearance may lead to the induction of pulmonary tumors.
Mice also had elevated cell replication of the alveolar cells, however the pattern was somewhat different than in rats. Significantly elevated alveolar cell replication was first observed 13 weeks after cessation of the exposure and had returned to control values by 52 weeks postexposure. Although cell replication correlated fairly well with neutrophil numbers (r2 = 0.717), it did not correlate with lung burdens. This suggests that in mice the augmentation of cell replication is related to the particle-induced inflammatory response rather than being a direct effect of particles.
Unlike mice and rats, hamsters demonstrated no change in alveolar cell replication indices. This was consistent with the lack of an inflammatory response and low lung burdens in these animals.
Histopathological findings for mice, rats, and hamsters exposed to uf-TiO2 differed. Rats exposed to 10 mg/m3 exhibited septal thickening and slight proliferation of type II cells but little metaplasia at the end of the exposure period. With time postexposure, these lesions progressed such that by 13 weeks there was increased thickening of the interstitium, epithelial cell proliferation, and metaplasia of alveolar epithelium. At subsequent time points, there was a diminution of the epithelial response and focal clustering of particle-laden macrophages, mostly alveolar intraluminal but with increased aggregation in interstitial regions. At the final time point, tight clusters of particle-laden macrophages were seen in some alveoli and substantial interstitialization of such macrophages was apparent. Occasional small foci of epithelial hyperplasia and hypertrophy and minimal metaplasia were present. A minimal fibrotic response was noted as thickening of alveolar septae in centriacinar regions associated with particle aggregation. There was, in general, little epithelial or fibroproliferative reaction in regions of intraluminal or interstitial aggregation of particle-laden macrophages. In mice exposed to 10 mg/m3 uf-TiO2, particle-laden macrophages and aggregates of free particles were present in centriacinar regions with no epithelial response. Interstitialization of these macrophages was apparent at the 13-week time point but again no epithelial response was observed. At the final time point, intraluminal accumulations of particle-laden macrophages were still present but, again, in the absence of an epithelial response. The rapid clearance of particles from the lungs of hamsters resulted in relatively few particle-associated lesions. Occasional particle-laden macrophages were present in alveoli adjacent to and along the alveolar ducts, and occasional particles were noted either free or cell-associated in alveolar and bronchiolar interstitium. Clearance of particles in hamsters continued into the postexposure period such that there were virtually no particles or particle-laden macrophages visible by 26 weeks.
Experimental exposure to fine and ultrafine modes of some particulates leads to differential pulmonary effects (Donaldson et al., 1998; Li et al., 1999
). Titanium dioxide in the ultrafine mode has been shown to result in more lung injury and pathology than equivalent deposited mass concentrations of pigmentary TiO2 (Ferin et al., 1992
; Janssen et al., 1994
). Comparisons between these two particle sizes of TiO2 on a mass basis do not correlate well with the observed tissue responses; however, comparisons using the surface area per unit mass have yielded an improved correlation of the rat data for some end points (Oberdorster, 1996
). Previous work in this laboratory examined the pulmonary effects of three concentrations of inhaled pigment grade TiO2 in mice, rats, and hamsters using the same end points as in the present study (Bermudez et al., 2002
). Of the three airborne concentrations used in that experiment, two (50 and 250 mg/m3) resulted in pulmonary overload in rats and mice. A comparison of the lung burdens between the two studies, using surface area as the dose-metric, reveals that the lung burdens in those animals exposed for 13 weeks to 10 mg/m3 uf-TiO2 or to 50 mg/m3 pigmentary TiO2 were approximately the same for all three species. When the pulmonary responses of each species to 50 mg/m3 pigmentary and 10 mg/m3 uf-TiO2 were compared, there was concordance in the BALF indicators of toxicity (i.e., LDH and total protein). This conclusion is based on an examination of the magnitude of the pulmonary responses in dosed animals relative to the concurrent controls.
In comparing the two 90-day interspecies inhalation studies, one with 0, 10, 50, and 250 mg/m3 pigment-grade TiO2 particles and the other with 0, 0.5, 2, and 10 mg/m3 ultrafine TiO2 particles, the results demonstrate many similarities. In the pigment-grade study, exposures to 50 and 250 mg/m3 produced particle overload in rats and mice but not in hamsters. Lung inflammation was measured in all three species at 50 and 250 mg/m3, but the ranking of sensitivity comparisons were (rats > mice > hamsters). Pulmonary lesions were most severe in rats, manifested by progressive epithelial metaplastic and fibroproliferative changes concomitant with enhanced alveolar epithelial cell proliferation measured in the group exposed to 250 mg/m3. In the studies with ultrafine TiO2 particles reported herein, particle overload was measured in rats and mice but not hamsters exposed to 10 mg/m3. The ranking of severity of the lung inflammatory responses at 10 mg/m3 was as follows: rats > mice > hamsters. Progressive epithelial and fibroproliferative changes were observed in rats but not in mice exposed to 10 mg/m3. These lesions were characterized by foci of alveolar epithelial proliferation of metaplastic epithelial cells circumscribing the aggregated foci of heavily particle-laden macrophages. Thus, to summarize the findings of the two studies, clear species differences were observed and measured in pulmonary responses to both pigment-grade (50 and 250 mg/m3) and ultrafine (10 mg/m3) titanium dioxide particles. Moreover, there was consistency among the species in responses to both particle types. Clearly, the rat is the most sensitive species in the pulmonary responses to both pigment-grade and ultrafine TiO2 particles.
In summary, inhalation of 10 mg/m3 uf-TiO2 for 13 weeks resulted in pulmonary overload in rats and mice but not in hamsters where the lung burdens were approximately 23% of the other species. While there were various responses in mice and rats, hamsters had very limited responses probably due to the low lung burdens and rapid clearance of particles in these animals. Responses in mice were limited to animals exposed to 10 mg/m3, whereas in rats responses were also observed in animals exposed to 2 mg/m3. The magnitude and spectrum of responses were, in general, equivalent in rats and mice. The extent and character of the inflammatory responses in rats differed from mice; in rats the responses had a greater neutrophilic component that diminished with time, whereas in mice significantly increased neutrophil and macrophage numbers remained relatively constant. Histopathological examination of rats and mice uncovered progressive fibroproliferative lesions in rats but not in mice. Taken together, the species differences observed in this study reflect the outcome of previously reported chronic exposures to poorly soluble particulates for each species and suggest that susceptibility of the rat, under pulmonary overload conditions, to the induction of lung tumors by these materials has underlying components of dosimetry and biological response.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bajpai, R., Waseem, M., Gupta, G. S., and Kaw, J. L. (1992). Ranking toxicity of industrial dusts by bronchoalveolar lavage fluid analysis. Toxicology 73, 161167.[CrossRef][ISI][Medline]
Bermudez, E., Mangum, J. B., Asgharian, B., Wong, B. A., Reverdy, E. E., Janszen, D. B., Hext, P. M., Warheit, D. B., and Everitt, J. I. (2002). Long-term pulmonary responses of three laboratory rodent species to subchronic inhalation of pigmentary titanium dioxide particles. Toxicol. Sci. 70, 8697.
Bingham, E., Corhssen, B., and Powell, C. H., Eds. (2002). Pattys Toxicology, Vol. 2. Wiley, New York.
Creutzenberg, O., Bellmann, B., Heinrich, U., Fuhst, R., Koch, W., and Muhle, H. (1990). Clearance and retention of inhaled diesel exhaust particles, carbon black, and titanium dioxide in rats at lung overload conditions. J. Aerosol Sci. 21, S455S458.[CrossRef][ISI]
Creutzenberg, O., Bellmann, B., Muhle, H., Dasenbrock, C., Morrow, P., and Mermelstein, R. (1998). Lung clearance and retention of toner, TiO2, and crystalline silica, utilizing a tracer technique during chronic inhalation exposure in Syrian golden hamsters. Inhal. Toxicol. 10, 731751.[CrossRef][ISI]
Cullen, R. T., Tran, C. L., Buchanan, D., Davis, J. M., Searl, A., Jones, A. D., and Donaldson, K. (2000). Inhalation of poorly soluble particles. I. Differences in inflammatory response and clearance during exposure. Inhal. Toxicol. 12, 10891111.[CrossRef][ISI][Medline]
Donaldson, K., Li, X. Y., and MacNee, W. (1998). Ultrafine (nanometre) particle mediated lung injury. J. Aerosol Sci. 29, 553560.[CrossRef][ISI]
Donaldson, K. (2000). Nonneoplastic lung responses induced in experimental animals by exposure to poorly soluble nonfibrous particles. Inhal. Toxicol. 12, 121139.[Medline]
Donaldson, K., and Tran, C. L. (2002). Inflammation caused by particles and fibers. Inhal. Toxicol. 14, 527.[CrossRef][ISI][Medline]
Donaldson, K., Brown, D., Clouter, A., Duffin, R., MacNee, W., Renwick, L., Tran, L., and Stone, V. (2002). The pulmonary toxicology of ultrafine particles. J. Aerosol Med. 15, 213220.[CrossRef][ISI][Medline]
Driscoll, K. E., Lindenschmidt, R. C., Maurer, J. K., Higgins, J. M., and Ridder, G. (1990). Pulmonary response to silica or titanium dioxide: Inflammatory cells, alveolar macrophagederived cytokines, and histopathology. Am. J. Respir. Cell Mol. Biol. 2, 381390.[ISI][Medline]
Ferin, J., Oberdorster, G., and Penney, D. P. (1992). Pulmonary retention of ultrafine and fine particles in rats. Am. J. Respir. Cell Mol. Biol. 6, 535542.[ISI][Medline]
Heinrich, U., Muhle, H., Takenaka, S., Ernst, H., Fuhst, R., Mohr, U., Pott, F., and Stober, W. (1986). Chronic effects on the respiratory tract of hamsters, mice, and rats after long-term inhalation of high concentrations of filtered and unfiltered diesel engine emissions. J. Appl. Toxicol. 6, 383395.[ISI][Medline]
Heinrich, U., Fuhst, R., Rittinghausen, S., Creutzenberg, O., Bellmann, B., Koch, W., and Levsen, K. (1995). Chronic inhalation exposure of Wistar rats and two different strains of mice to diesel engine exhaust, carbon black, and titanium dioxide. Inhal. Toxicol. 7, 533556.[ISI]
Hext, P. M. (1994). Current perspectives on particulate induced pulmonary tumours. Hum. Exp. Toxicol. 13, 700715.[ISI][Medline]
Janssen, Y. M., Marsh, J. P., Driscoll, K. E., Borm, P. J., Oberdorster, G., and Mossman, B. T. (1994). Increased expression of manganese-containing superoxide dismutase in rat lungs after inhalation of inflammatory and fibrogenic minerals. Free Radic. Biol. Med. 16, 315322.[CrossRef][ISI][Medline]
Lee, K. P., Trochimowicz, H. J., and Reinhardt, C. F. (1985). Pulmonary response of rats exposed to titanium dioxide (TiO2) by inhalation for two years. Toxicol. Appl. Pharmacol. 79, 179192.[ISI][Medline]
Levine, K. E., Fernando, R. A., Lang, M., Essader, A., and Wong, B. A. (2003). Development and validation of a high-throughput method for the determination of titanium dioxide in rodent lung and lung-associated lymph node tissues. Anal. Lett. 36, 563576.[CrossRef][ISI]
Li, X. Y., Brown, D., Smith, S., MacNee, W., and Donaldson, K. (1999). Short-term inflammatory responses following intratracheal instillation of fine and ultrafine carbon black in rats. Inhal. Toxicol. 11, 709731.[CrossRef][ISI][Medline]
Mauderly, J. L., Jones, R. K., Griffith, W. C., Henderson, R. F., and McClellan, R. O. (1987). Diesel exhaust is a pulmonary carcinogen in rats exposed chronically by inhalation. Fundam. Appl. Toxicol. 9, 208221.[ISI][Medline]
Mauderly, J. L., Snipes, M. B., Barr, E. B., Belinsky, S. A., Bond, J. A., Brooks, A. L., Chang, I. Y., Cheng, Y. S., Gillett, N. A., Griffith, W. C., et al. (1994). Pulmonary toxicity of inhaled diesel exhaust and carbon black in chronically exposed rats. Part I. Neoplastic and nonneoplastic lung lesions. Res. Rep. Health Eff. Inst. 68, 175; discussion 7797.[Medline]
Morrow, P. E. (1992). Dust overloading of the lungs: Update and appraisal. Toxicol. Appl. Pharmacol. 113, 112.[ISI][Medline]
Muhle, H., Bellmann, B., Creutzenberg, O., Koch, W., Dasenbrock, C., Ernst, H., Mohr, U., Morrow, P., and Mermelstein, R. (1998). Pulmonary response to toner, TiO2, and crystalline silica upon chronic inhalation exposure in Syrian golden hamsters. Inhal. Toxicol. 10, 699729.[CrossRef][ISI]
Muhle, H., Creutzenberg, O., Bellmann, B., Heinrich, U., and Mermelstein, R. (1990). Dust overloading of lungs: Investigations of various materials, species differences, and irreversibility of effects. J. Aerosol Med. 3, S111S128.[ISI]
Oberdorster, G., Ferin, J., Gelein, R., Soderholm, S. C., and Finkelstein, J. (1992). Role of the alveolar macrophage in lung injury: Studies with ultrafine particles. Environ. Health Perspect. 97, 193199.[ISI][Medline]
Oberdorster, G. (1996). Significance of particle parameters in the evaluation of exposure-dose-response relationships of inhaled particles. Inhal. Toxicol. 8, 7389.[Medline]
Rutten, A. A., Bermudez, E., Mangum, J. B., Wong, B. A., Moss, O. R., and Everitt, J. I. (1994). Mesothelial cell proliferation induced by intrapleural instillation of man-made fibers in rats and hamsters. Fundam. Appl. Toxicol. 23, 107116.[CrossRef][ISI][Medline]
Warheit, D. B., Hansen, J. F., Yuen, I. S., Kelly, D. P., Snajdr, S. I., and Hartsky, M. A. (1997). Inhalation of high concentrations of low toxicity dusts in rats results in impaired pulmonary clearance mechanisms and persistent inflammation. Toxicol. Appl. Pharmacol. 145, 1022.[CrossRef][ISI][Medline]