Biological response to nano-scale titanium dioxide (TiO2): role of particle dose, shape, and retention

Abstract

Titanium dioxide (TiO2) is one of the most widely used nanomaterials, valued for its highly refractive, photocatalytic, and pigmenting properties. TiO2 is also classified by the International Agency for Research on Cancer (IARC) as a possible human carcinogen. The objectives of this study were to (1) establish a lowest-observed-effect level (LOEL) for nano-scale TiO2, (2) determine TiO2 uptake in the lungs, and (3) estimate toxicity based on physicochemical properties and retention in the lungs. In vivo lung toxicity of nano-scale TiO2 using varying forms of well-characterized, highly dispersed TiO2 was assessed. Anatase/rutile P25 spheres (TiO2-P25), pure anatase spheres (TiO2-A), and anatase nanobelts (TiO2-NB) were tested. To determine the effects of dose and particle characteristics, male Sprague-Dawley rats were administered TiO2 (0, 20, 70, or 200 μg) via intratracheal instillation. Bronchoalveolar lavage fluid (BALF) and lung tissue were obtained for analysis 1 and 7 d post exposure. Despite abundant TiO2 inclusions in all exposed animals, only TiO2-NB displayed any significant degree of inflammation seen in BALF at the 1-d time point. This inflammation resolved by 7 d, although TiO2 particles had not cleared from alveolar macrophages recovered from the lung. Histological examination showed TiO2-NB produced cellular changes at d 1 that were still evident at d 7. Data indicate TiO2-NB is the most inflammatory with a LOEL of 200 μg at 1 d post instillation.

Figure 1. SEM images of all studied nanomaterials in pristine form

From left to right: TiO₂-P25, TiO₂-A, and TiO₂-NB. Spherical particle types, TiO₂-P25 and TiO₂-A, averaged approximately 26 nm in diameter. TiO₂-NB averaged 7000 nm in length and 200 nm in width, and individual nanobelts were approximately 10 nm thick. Scale indicated by the white bars. Reproduced with permission from Environmental Health Perspectives.

Figure 2. Stimulation of neutrophilic inflammation in the lungs of rats by TiO₂ engineered nanomaterials

Percentages of neutrophils (PMNs) in BALF 1 day (left panels) and 7 days (right panels) after IT exposure to P25 nanospheres (top), anatase nanospheres (middle), or anatase nanobelts (bottom). *P<0.05 compared to DM-exposed controls.

Figure 3. Instilled TiO₂-A causes moderate but sustained particle loading in alveolar macrophages

Cells recovered from BALF at 7 days post exposure to TiO₂-P25 (left), TiO₂-A (middle) and TiO₂-NB (right) by intratracheal instillation. Panels are Phase Contrast microscopy images of representative cells from rats given a single dose of TiO₂. BAL cells were stained with Diff Qwik®. Arrows indicate TiO₂ nanoparticles. Scale indicated by black bar.

Figure 4. TiO₂ engineered nanomaterials uptake by macrophages in the lungs

Percentages of macrophages in BALF with visible particle inclusions 1 day (left panels) and 7 days (right panels) after intratracheal instillation exposure to TiO₂-P25 nanospheres (top) or TiO₂-A nanospheres (bottom). *P<0.05 compared to DM-exposed controls.

Figure 5. TEM images of TiO₂ nanobelts (TiO₂-NB) in alveolar macrophages

Cells recovered from BALF at 1 (left) and 7 (right) days post instillation. Panels are images of representative cells from rats given 200 µg TiO₂-NB. White arrows indicate TiO₂-NB aggregates. Scale indicated by the white bars.

Figure 6. TiO₂ causes inflammatory infiltrates in the left lung at 1 day post exposure

Histopathological findings from exposure to 200 µg/250 µL of TiO₂-P25 (left), TiO₂-A (middle) and TiO₂-NB (right) by intratracheal instillation. Panels are Brightfield microscopy images of representative lung tissues from rats given a single dose of TiO₂. Tissues were stained with hematoxylin and eosin. Closed arrow = inflammatory cell infiltrate; Open arrow = particle laden cells; TB = terminal bronchiole. Scale indicated by black bar.

Figure 7. Inflammatory cells can be seen at 1 and 7 days post exposure to TiO₂-NB

Histopathological findings from exposure to 200 µg/250 µL of TiO₂-NB by intratracheal instillation. Panels are Brightfield microscopy images of representative lung tissues from rats given a single dose of TiO₂. Tissues were stained with hematoxylin and eosin. Closed arrow = mixed inflammatory cell infiltrate; BV = blood vessel. Scale indicated by black bar.

Figure 8. TiO₂ lung burden in the right cranial and caudal lobes

Average mass of TiO₂ observed in the cranial (top panel) and caudal (bottom panel) lung lobes 1 day and 7 days after intratracheal instillation exposure to TiO₂-P25 nanospheres, TiO₂-A nanospheres, or TiO₂-NB. *,**P<0.05 compared to method blank (control) from 1 day and 7 days, respectively.

Figure 9. TiO₂ lung burden in the right cranial and caudal lobes normalized by lung mass

Normalized mass of TiO₂ observed in the cranial (top) and caudal (bottom) lung lobes 1 and 7 days after a single IT exposure.

Figure 10. Deep lung penetration of TiO₂ nanobelts at 1 day post exposure

Histopathological findings from exposure to 200 µg/250 µL of TiO₂-NB by intratracheal instillation. Panels are Brightfield microscopy images of representative lung tissues from rats given a single dose of TiO₂. Tissues were stained with hematoxylin and eosin. Open arrow = TiO₂-NB aggregates. Scale indicated by black bar.