Pulmonary response to intratracheal instillation of ultrafine versus fine titanium dioxide: role of particle surface area
© Sager et al; licensee BioMed Central Ltd. 2008
Received: 17 September 2008
Accepted: 01 December 2008
Published: 01 December 2008
The production and use of nanoparticles is growing rapidly due to the unique physical and chemical properties associated with their nano size and large surface area. Since nanoparticles have unique physicochemical properties, their bioactivity upon exposure to workers or consumers is of interest. In this study, the issue of what dose metric (mass dose versus surface area dose) is appropriate for toxicological studies has been addressed. Rats were exposed by intratracheal instillation to various doses of ultrafine or fine TiO2. At 1, 7, or 42 days post-exposure, inflammatory and cytotoxic potential of each particle type was compared on both a mass dosage (mg/rat) as well as an equal surface area dosage (cm2 of particles per cm2 of alveolar epithelium) basis.
The findings of the study show that on a mass basis the ultrafine particles caused significantly more inflammation and were significantly more cytotoxic than the fine sized particles. However, when doses were equalized based on surface area of particles delivered, the ultrafine particles were only slightly more inflammogenic and cytotoxic when compared to the fine sized particles. Lung burden data indicate that ultrafine TiO2 appears to migrate to the interstitium to a much greater extent than fine TiO2.
This study suggests that surface area of particles may be a more appropriate dose metric for pulmonary toxicity studies than mass of particles.
Nanoscience and nanotechnology offer new opportunities for making superior materials for use in industrial and health applications [1, 2]. As the field of nanotechnology vastly expands, many questions involving the effects of nanomaterials on the environment as well as human health have been raised which warrant investigation. The primary problem plaguing the field of nanotechnology is the possibility that nanoparticles can become suspended in air during production, incorporation into consumer products, and use or disposal of such products. Therefore, manufactured nanoparticles can become a component of the indoor and outdoor environments and, thus, the air we breathe .
A number of toxicology studies have suggested that for some classes of materials, biological response following deposition in the lungs is dependent on particle surface area [3, 4]. Materials of interest typically possess characteristics such as low solubility, which leads to extended persistence in the lungs. These materials can be produced as ultrafine particles, which have high specific surface areas . Titanium dioxide (TiO2) is an example of a fine, low solubility particle which was considered to exhibit relatively low toxicity . However, in a key study, Ferin et al.  demonstrated that ultrafine TiO2 (UFTiO2) caused more inflammation in rat lungs than exposure to the same airborne mass concentration of fine TiO2 (FTiO2). Up until this point in time, TiO2 often had been used as a low solubility, low toxicity, control dust in many studies. Therefore, this report was highly influential in highlighting that a material that was low in solubility and toxicity in the form of fine particles could be bioactive in the form of ultrafine particles .
In the last decade, it has become clear from rat studies that chronic exposure to high airborne mass concentrations of low solubility, low toxicity particles, such as carbon black and titanium dioxide, can lead to development of the features characteristic of "rat lung overload" . A breakdown in normal alveolar macrophage- mediated clearance is seen in overload. This particle-induced depression of clearance was proposed to be a consequence of volumetric overload of the alveolar macrophages and the resulting loss of alveolar macrophage mobility . This overload phenomenon was thought to be a problem related to particles occupying a large volume inside each macrophage and, thus, preventing them from phagocytosing additional particles and moving to the mucociliary escalator to be cleared. This was known as the volumetric overload hypothesis [8, 10]. However, more recent studies suggest that volumetric overload is not a complete explanation for the toxicological response to ultrafine particles. Recent evidence has suggested that surface area may drive inflammation . Exposure of rats to nanoparticles, with a large surface area per mass, showed features of poor clearance and inflammation at a lower lung burden, in terms of mass and volume of particles, compared with larger size particles with the same mineral and chemical properties .
A study conducted by Tran et al.  compared the inflammatory response to inhalation of two sizes of two very different types of low solubility, low toxicity particles. At an equal lung mass burden, the smaller particles were more inflammatory than the larger particles. However, when the lung burden was expressed as total particle surface area delivered, the inflammatory potential of the large and small particles was similar . This supports the hypothesis that surface area of the particle may be an important component of toxic potential. In contrast, in vitro studies by Monteiller et al.  utilized insoluble quartz, which has a very high surface reactivity. Due to this very high surface reactivity, quartz stimulated release of inflammatory mediators at a much lower mass and surface area dose than did low solubility, low toxicity particles. This is consistent with the high inflammatory potential of quartz in animal models. The outcome of this study indicates that, while surface area is important, surface activity is also a critical factor to assess pulmonary toxicity of particles. Studies supporting an important role of particle surface area in the bioactivity of low solubility, low toxicity particles, such as ultrafine carbon black and ultrafine titanium dioxide, impact assessment of workplace and environmental exposure to these nanoparticles, since regulations are currently based on mass of airborne particles .
In summary, several studies have concluded that on an equal mass dose basis ultrafine or nano-sized particles are uniquely more toxic than fine-sized particles of the same composition due to a high particle surface area for the nanoparticles [4, 7–9, 11]. Recently, this conclusion has been questioned [12, 13]. It is possible that these conflicting results may be due to the use of highly agglomerated UFTiO2 samples in the later two studies [12, 13]. Therefore, the aim of the present study was to readdress the issue of the relative toxicity of UFTiO2 vs FTiO2 on a mass dose or equivalent particle surface area delivered dose basis using alveolar lining fluid, which our laboratory has shown previously greatly improves nanoparticles dispersion , as the particle suspension medium. In the present study, in vivo intratracheal exposures of rats to ultrafine and fine titanium dioxide were conducted. Animals were given a dose of particles based on mass, and dose was normalized to surface area of particles administered. Particle-induced changes in several pulmonary toxicity parameters were compared on a mass dose and surface area of particles administered basis to determine which dose metric is more appropriate in nanoparticle toxicology studies.
Comparison of pulmonary toxicity of UFTiO2 and FTiO2
Potency difference between UFTiO2 and FTiO2 when analyzed on a mass vs. surface area basis.
PMN (10 6 )
Zym. Stim. Chemi.
NO Dep. Chemi.
Effect of Exposure to UFTiO2 vs. FTiO2 (High Dose) on Pulmonary Responses.
PMN (10 6 )
1.4 ± 0.1
0.78 ± 0.07
0.88 ± 0.10
23.8 ± 1*
25.5 ± 3*
18.3 ± 1*
FTiO 2 high
15.6 ± 0.9*
19.8 ± 0.7*
10.8 ± 0.4*
46.3 ± 2
39.5 ± 1.35
37.3 ± 2
337.4 ± 3*
323.5 ± 3*
239.9 ± 5*
FTiO 2 high
307.6 ± 5*
288.1 ± 5*
175.6 ± 4*
0.07 ± 0.03
0.08 ± 0.003
0.09 ± 0.007
0.38 ± 0.04*
0.51 ± 0.02*
0.40 ± 0.01*
FTiO 2 high
0.21 ± 0.055*
0.40 ± 0.01*
0.30 ± 0.01*
25.7 ± 0.6
23.1 ± 0.7
25.86 ± 0.8
67.4 ± 2.2*
73.5 ± 1.5*
88.5 ± 4.4*
FTiO 2 high
60.7 ± 1.7*
58.5 ± 2.8*
55.4 ± 1.3*
517.9 ± 12
601.8 ± 26
643.6 ± 7
802.9 ± 28*
886.9 ± 21*
893.5 ± 26*
FTiO 2 high
695.6 ± 15*
847.1 ± 43*
852.1 ± 22*
75.9 ± 6
82.2 ± 6
91.9 ± 8
167.5 ± 13*
230.4 ± 14*
273.6 ± 14*
FTiO 2 high
119.9 ± 13
131.5 ± 11*
180.3 ± 14*
Zym. Stim. Chemi.
0.53 ± .08
0.50 ± .05
0.51 ± .05
4.4 ± .51*
4.4 ± .47*
4.2 ± .48*
FTiO 2 high
2.6 ± .26*
2.8 ± .35*
1.4 ± .27
NO Dep. Chemi.
4.3 ± .06
0.45 ± .11
0.39 ± .08
3.3 ± .11*
2.2 ± .27*
1.0 ± .12*
FTiO 2 high
2.6 ± .20*
0.28 ± .04
0.230 ± .04
Alveolar macrophage zymosan-stimulated and NO-dependent chemiluminescence were measured to assess reactive oxygen species production after UFTiO2 and FTiO2 exposures. On a mass dose basis, UFTiO2 and FTiO2 both caused dose-dependent increases in zymosan-stimulated and NO dependent chemiluminescence (data analogous to Figures 2, 4 and 6 not shown). The responses to the high dose are shown in Table 2. A significantly greater mass dose of FTiO2 was required to produce comparable chemiluminescence levels as elicited by UFTiO2 exposure. The highest doses of UFTiO2 caused a significant increase in zymosan and NO-dependent chemiluminescence levels over control at all post-exposure time points (Table 2). The high dose of FTiO2 caused a significant increase over control in zymosan-stimulated chemiluminescence at 1 and 7 days post-exposure and in NO-dependent chemiluminecence only at 1 day post-exposure (Table 2).
On a mass dose basis, in regards to zymosan-stimulated chemiluminescence levels, UFTiO2 was shown to be 82 times more potent than FTiO2 at all post-exposure time points (Table 1). However, when dose was normalized to surface area of particles administered, UFTiO2 zymosan-stimulated chemiluminecence levels were at most only 3 fold higher than FTiO2 zymosan-stimulated chemiluminecence levels (Table 1). When the surface area zymosan-stimulated chemiluminescence dose response curves were analyzed using a linear regression curve with a 95% confidence interval (data not shown), there was no significant difference between the UFTiO2 and FTiO2 dose response curves at 1 day or 7 days post-exposure.
On a mass dose basis, in regards to NO-dependent chemiluminescence levels, UFTiO2 was 41 times more potent than FTiO2 at 1 day post-exposure and was 82 times more potent at 7 and 42 days post-exposure. However, when dose was normalized to surface area of particles administered, UFTiO2 NO-dependent chemiluminescence levels were only 1.5-fold greater than FTiO2 at 1 day post-exposure but were 8-fold greater at 42 days post-exposure (Table 1). When the surface area NO-dependent chemiluminescence dose response curves were analyzed using a linear regression curve with a 95% confidence interval (data not shown), there was no significant difference between the UFTiO2 and FTiO2 dose response curves at 1 day post-exposure.
At 42 days post-exposure, lungs were evaluated for pulmonary fibrosis by histological examination of lung tissue stained with Masson's trichrome. No excess fibrotic tissue was noted in lungs exposed to either UFTiO2 or FTiO2 (micrographs not shown).
Comparison of lung burden data from TiO2 metal analysis
Decline in lung burden from 7 to 42 days post – exposure
51 ± 4%
12 ± 1%
Comparison of lymph node burden data from TiO2 metal analysis
Increase in lymph node burden from 7 to 42 days post-exposure
246 ± 118%
134 ± 2%
Comparison of lavaged lung burden data from TiO2 metal analysis
Percent lavagable vs non-lavagable TiO2 in the lung
7 Days post
42 Days post
16% non- lavagable
Fine TiO2 particles as well as other low-solubility, low-toxicity particles have long been of interest in particle toxicology. Historically, fine TiO2 particles were utilized as negative controls in numerous pulmonary toxicity studies. However, the current study shows that FTiO2 at relatively high mass lung burdens can induce pulmonary inflammation and damage, which are sustained through 42 days post-exposure. Using a mass dose metric, UFTiO2 was significantly more potent (at least 41 fold) than FTiO2 in causing sustained lung damage and inflammation. When exposure dose was normalized to equivalent surface area of particles delivered, no significant difference in potency between UFTiO2 and FTiO2 was observed. These findings help support the hypothesis that surface area of particles administered may be the more appropriate dose metric for nanoparticle pulmonary toxicity studies. This conclusion, although not novel, has been recently disputed [12, 13].
Two similar studies were conducted by Warheit et al. [12, 13] to assess the lung toxicity of intratracheally instilled nano-sized and fine-sized TiO2. In both studies, they instilled fine and ultrafine TiO2 suspended in PBS into rats (1 or 5 mg/kg) and compared measured pulmonary parameters to animals instilled with crystalline silica (used as a positive control). They monitored inflammation as PMN influx and cytotoxicity as LDH activity at 1 day, 1 week, 1 month and 3 months post-exposure in both studies. Warheit et al. [12, 13] concluded that exposures to nanosized TiO2 had no significant long term adverse pulmonary effects. They also reported similar acute levels of inflammation and cytotoxicity for both nano-sized and fine-sized TiO2, with only the highest dose (5 mg/kg) at 1 day post-exposure, causing a significant increase in inflammation for both nano- and fine-sized TiO2 exposures. Warheit and colleagues argued that the transient, reversible inflammation seen at 1 day post-exposure with both nano-sized and fine-sized TiO2 was due primarily to the method of exposure (intratracheal instillation) rather than the effects of the nano-sized particles in the lung. The inflammation shown at 1 day post-exposure in the Warheit et al. [12, 13] studies quickly resolved to control levels by 1 week post-exposure.
In the first study, Warheit et al.  concluded that their results "run counter to the postulation that surface area is a major factor associated with the pulmonary toxicity of nanoscale particle-types". They argued that their study was instrumental in demonstrating that the fact that nanoparticles have larger surface areas does not necessarily indicate that they produce increased pulmonary inflammation and cytotoxicity when compared to fine-sized particles of similar composition. Warheit et al. , using very similar data to the Warheit et al.  study, expanded on the conclusions from the initial study. They again concluded that the fact that nanoparticles have larger surface areas does not necessarily indicate that they produce increased pulmonary inflammation and cytotoxicity when compared to fine-sized particles of similar composition.
The findings of the current study disagree with the findings of Warheit et al. [12, 13]. The results of the current study indicate that surface area of the nanoparticles does in fact play a role in pulmonary toxicity, and that low mass burdens (as low as 0.26 mg/rat) of UFTiO2 can cause persistent lung damage and inflammation over a 42 day post-exposure study period. The results of the current study support the fact that on a mass dose basis nano-sized particles are more toxic than fine-sized particle of the same composition. However, when dose is normalized to surface area of particles administered, the difference in toxicity responses between UFTiO2 and FTiO2 for all pulmonary parameters measured becomes substantially less. This, therefore, indicates that surface area of particles is a critical driver of toxicity and may be an appropriate dose metric for pulmonary toxicity studies. The discrepancies between the two studies are believed to be due to the fact that Warheit et al. [12, 13] reports extreme agglomeration of the PBS-suspended nano-sized TiO2 utilized in their studies. In addition, significant agglomeration was also reported for the suspension of fine TiO2. This agglomeration issue leads to an over-estimation of delivered surface area of the particles. Indeed, their dynamic light scattering data indicate that once suspended in PBS both ultrafine and fine TiO2 had agglomerated particle sizes of > 2 μm. One would argue that since delivered particle size was equivalent, toxicity would not be expected to differ. In the present study, efforts were made to substantially improve the dispersion of UFTiO2 and FTIO2 by using rat bronchoalveolar lavage fluid (BALF) as the suspension medium. Results from our laboratory indicate that dispersion was vastly improved for both UFTiO2 and FTiO2 in BALF compared to PBS . Our laboratory has reported that the diameter determined by dynamic light scattering of UFTiO2 suspended in a BALF model medium was 204 nm . Therefore, in the present study, a potency difference between UFTiO2 and FTiO2 (on a mass basis) was observed because the physical surface areas of the UF vs F structures were different.
A recent study by Duffin et al.  further supports the conclusions of the current study. They investigated the relationship between inflammation, as indicated by neutrophil content of the bronchoalveolar lavage of rats 18–24 hours after particle instillation, and particle size. Rats were instilled with ultrafine and fine-sized carbon black or titanium dioxide and then assessed for PMN influx into the lung. Duffin et al.  reported that the extent of inflammation was a function not of mass dose instilled but of the surface area of particles instilled. Likewise, the findings of the current study support the conclusion that inflammatory potential of TiO2 is a function of surface area of the particles administered.
Secondly, studies by Warheit et al. [12, 13] report that UFTiO2 at lung burdens of 1–5 mg/kg did not cause persistent lung damage and inflammation. In contrast, data from the present study indicate that lung burdens as low as 1 mg/kg UFTiO2 caused significant lung damage and inflammation which persisted through 42 days post-exposure. In the present study, efforts were made to improve dispersion of UFTiO2 by using BALF as the suspension medium, as reported previously . Shvedova et al.  reported that improved dispersion of ultrafine carbon black (UFCB) particles in BALF increased the inflammatory and damage potency compared to UFCB suspended in PBS; i.e., intratracheal instillation of a 30 fold greater mass of poorly dispersed UFCB suspended in PBS was required to attain the same level of pulmonary damage (LDH activity) and inflammation (PMN level) as well dispersed UFCB suspended in BALF in a rat model. Therefore, poor dispersion of UFTiO2 in PBS (diameters of over 2 μm determined by dynamic light scattering) may account for the low biological activity previously reported by Warheit et al. [12, 13].
Warheit et al.  suggested that particle characteristics, such as surface reactivity, crystal structure, surface pH, surface coating, shape, etc., may play important roles in the pulmonary toxicity of nanoparticles. They have demonstrated that UFTiO2 in the uncoated, 80/20 anatase/rutile form exhibited greater surface activity than alumina-coated rutile as judged by ability to deplete vitamin C in an acellular assay. In addition, Sayes et al.  demonstrated that 80/20 anatase/rutile generated 6 fold more reactive species (measured as acellular luminol-enhanced chemiluminescence) than rutile after UV irradiation. They argued that this increased surface reactivity resulted in greater in vitro cytotoxicity for the anatase form of UFTiO2. In the present study, the UFTiO2 used was 80/20 anatase/rutile, while the FTiO2 was the rutile crystal form. However, the particle samples were not irradiated with UV light in this study. In the absence of UV irradiation, Sayes et al.  reported the UFTiO2 in the 80/20 anatase/rutile form generated only twice the reactive species as ultrafine rutile. Such as small difference in surface reactivity can not account for the 40 fold greater potency of ultrafine vs. fine TiO2 on an equal mass basis reported in the present study. However, a 2 fold difference in surface activity may explain why normalizing dose to equivalent particle surface area delivered did not result in identical potency for the ultrafine and fine particle samples. Recent results indicate that the shape of nanoparticles can affect toxic potential. Porter et al.  demonstrated that TiO2 nanowires exhibited greater toxicity in vitro and greater inflammatory potential in vivo than TiO2 nanospheres of the same crystal structure and diameter. Therefore, although the present study concentrated on the role of particle surface area in nanoparticle toxicity, results do not discount the importance of other particle characteristics in nanoparticle toxicity.
Results of lung burden studies suggest that although a large portion of FTiO2 appears to have been engulfed by lavagable alveolar macrophages (Table 5) clearance of FTiO2 was relatively low, 12% from 7 to 42 days post-exposure (Table 3). One, thus, could conclude that the lungs were in overload at this FTiO2 burden (10.7 mg/rat). As proposed by Morrow , when alveolar macrophages have engulfed a large volume of particles, the phagocytes become immobile and clearance fails. In contrast, macrophage clearance apparently is relatively robust after exposure to 0.52 mg/rat UFTiO2; i.e., 51% decline in lung burden is observed from 7 to 42 days post-exposure (Table 3). This clearance is only partially due to migration of UFTiO2 to the lymph nodes (Table 4). In addition, migration of UFTiO2 into the alveolar walls is far more substantial than that for FTiO2 (Table 5). A high rate of interstitilization of UFTiO2 vs FTiO2 has been reported previously .
In summary, exposure of rats by intracheal instillation to a well-dispersed suspension of UFTiO2 caused dose dependent pulmonary damage and inflammation, which persisted 42 days post-exposure. On a mass dose basis, UFTiO2 was at least 41 fold more potent than FTiO2. When exposure dose was normalized to surface area of particles delivered, this potency difference was no longer significant. Burden data indicate that UFTiO2 migrates to the interstitium to a far greater extent than FTiO2. Results support the use of particle surface area as a dose metric for evaluation of pulmonary response to particles.
Recently, the hypothesis that nanoparticles exhibit unique bioactivity due to their large surface area per mass has been questioned [12, 13]. The aim of the present study was to readdress the issue of the relative toxicity of UFTiO2 vs FTiO2 on a mass dose or equivalent particle surface area delivered dose basis. In this study, particle dispersion was substantially improved by suspension of particles in diluted alveolar lining fluid obtained by bronchoalveolar lavage of naïve rats as described previously . Rats were exposed to various mass concentrations of UFTiO2 vs FTiO2 by intratracheal instillation. Mass doses of UFTiO2 (0.26, 0.52 and 1.04 mg/rat) and FTiO2 (5.35, 10.7 and 21.41 mg/rat) were chosen to result in equivalent total particle surface area delivered doses (0.0313, 0.0625 and 0.125 cm2 of particles administered per cm2 of alveolar epithelial surface) for UFTiO2 and FTiO2. Rats were sacrificed at 1 day, 7 days, and 42 days post-exposure, and markers of inflammation (PMN number or inflammatory mediator levels in BAL samples), lung injury (LDH activity or albumin levels in BAL samples), and macrophage activity (zymosan-stimulated or NO-dependent chemiluminescence) were monitored. The magnitude of pulmonary responses to exposure to UFTiO2 vs FTiO2 was then compared on a mass dose and an equivalent surface area of particles administered dose basis to evaluate the role of particle surface area in the pulmonary response.
Animals for in vivo exposures
The rats used for the in vivo experiments were male Fischer CDF (F344/DuCrl) rats weighing 200–300 g (~10 weeks old at arrival) obtained from Charles River (Raleigh, NC). The animals were housed in an AAALAC-accredited; specific pathogen-free, environmentally controlled facility. The animals were monitored to be free of endogenous viral pathogens, parasites, mycoplasms, Helicobacter and CAR Bacillus. Animals were housed in ventilated cages which were provided HEPA-filtered air, with Alpha-Dri virgin cellulose chips and hardwood Beta-chips used as bedding. The rats were maintained on a Teklad 2918 diet and tap water, both of which were provided ad libitum.
Bronchoalveolar fluid collection for particle suspension media
Rats were euthanized with an i.p. injection of sodium pentobarbital (>100 mg/kg body weight) and exsanguinated by cutting the descending aorta. A tracheal cannula was inserted and bronchoalveolar lavage was conducted . A 6 ml aliquot of cold Ca+2 and Mg+2-free phosphate-buffered saline (PBS) was used for the lavage wash. The cold PBS was flushed into and out of the lungs two times before the lavage fluid was collected. The bronchoalveolar lavage (BAL) from five rats was combined and centrifuged at 600 × g for 10 minutes using a Sorvall RC 3B Plus centrifuge (Sorvall Thermo Electron Corporation, Asheville NC). The supernatant was decanted into a new tube, while the pellet was discarded. This acellular bronchoalveolar lavage fluid (BALF) was then used as the vehicle for particle suspensions. The BALF was collected fresh the same day that the particulate suspensions were made.
UFTiO2 (Aeroxide TiO2 P-25) was obtained as a gift from Degussa Corporation (Parsippany, NJ). UFTiO2 had a primary particle size of 21 nm and was a 80/20 mixture of anatase/rutile. FTiO2 (titanium (IV) oxide, #224227) was purchased from Sigma-Aldrich (St. Louis, MO). FTiO2had a primary particle size of 1 μm and was 100% rutile.
Suspension of UFTiO2 and FTiO2 in rat BALF
Ultrafine titanium dioxide (UFTiO2) and fine titanium dioxide (FTiO2) particles were sieved using a Retsch AS 200 Sieve (Retsch GmbH, Haann, Germany) through 1.18 mm, 250 μm, and 45 μm mesh screens. Particles were weighed and suspended in rat BALF to obtain the desired concentration. Once the particles were added to the BALF, the suspensions were pulse sonicated with 5 individual pulses at a duty cycle setting of 10% and an output setting of 1 with a Branson 450 Sonifier probe sonicator (Branson Ultrasonics Corporation, Danbury CT.). This method has been reported previously by our laboratory to result in well dispersed suspensions as determined by light and electron microscopy . Dynamic light scattering of UFTiO2 suspended in a BALF model medium indicated that ultrafine particles had a diameter of 204 ± 18 nm .
In vivo exposures
To receive their respective dose of particles, each rat was anesthetized with an intraperitoneal (i.p.) injection of methohexital sodium (30–40 mg/kg body weight; Monarch Pharmaceuticals, Bristol, TN). Each animal was then instilled via intratracheal instillation (IT) using a 20-gauge 4-inch ball tipped animal feeding needle . Each animal was instilled with 0.3 ml of UFTiO2 or FTiO2 suspended in BALF. BALF (0.3 ml) was instilled as the control. Results from our laboratory indicate that instillation of BALF does not cause lung inflammation . In addition, suspension of quartz in BALF did not alter its inflammatory potency .
Bronchoalveolar lavage and cell differentials
At 24-hours, 7 days, and 42 days post-IT, the animals were euthanized with an i.p. injection of sodium pentobarbital (>100 mg/kg body weight) and exsanguinated by cutting the descending aorta. A tracheal cannula was inserted and bronchoalveolar lavage (BAL) was conducted . A 6 ml aliquot of cold Ca+2 and Mg+2 free PBS was used for the first lavage wash. The cold PBS was flushed into and out of the lungs two times before the lavage fluid was collected. After the first lavage wash was collected and stored separately, the BAL continued with 8 ml aliquots of cold Ca+2 and Mg+2-free PBS until an additional 80 ml of bronchoalveolar lavage was collected. The BAL was then centrifuged at 600 × g for 10 minutes using a Sorvall RC 3B Plus centrifuge (Sorvall Thermo Electron Corporation, Asheville NC). After centrifugation, the supernatant from the first lavage wash was decanted into a clean conical vial and was stored on ice to be used for lung damage/cytotoxicity analysis. The remaining supernate from the lavages was discarded, and the cells remaining were washed with cold Ca+2 and Mg+2-free PBS and spun again at 600 × g for 10 minutes. After this, the supernatant was discarded and the cells were resuspended in 1 ml of HEPES-buffered medium. Using these lavage samples, polymorphonuclear neutrophil (PMN) and alveolar macrophage (AM) counts were determined to assess inflammation. The number of AM and PMN was determined according to their unique cell diameters, using an electronic cell counter equipped with a cell sizing attachment (Beckman Coulter Multisizer 3 Counter, Hialeah, FL).
BAL fluid lactate dehydrogenase activity and albumin concentration
The degree of cytotoxicity induced by the instilled particles was determined by lactate dehydrogenase (LDH) activity in the BALF. LDH activity was measured using a Roche COBAS MIRA Plus chemical analyzer (Roche Diagnostic Systems Inc., Branchburg, NJ) as described previously by our laboratory . Albumin concentrations in BALF were assessed to examine if instilled particle exposures had compromised the integrity of the alveolar air/blood barrier. Albumin concentrations were also measured using a Cobas Fara II Analyzer (Roche Diagonostic Systems, Montclair, NJ) as previously described by our laboratory .
Mediator measurements in bronchoalveolar lavage fluid
The presence of inflammatory mediators in the BALF was analyzed by enzyme-linked immunosorbent assay (ELISA). The levels of mediators present were measured using commercially available ELISA kits (BioSource International Inc., Camarillo, CA). Three mediators were quantified: tumor necrosis factor-α (TNF-α), interleukin (IL)-β, and macrophage-inflammatory protein-2 (MIP-2).
Zymosan-stimulated and NO-dependent alveolar macrophage chemiluminescence
Reactive oxygen species production was determined by measuring AM chemiluminescence. According to Van Dyke et al. , only AM will generate reactive oxygen species in response to unopsonized zymosan in the chemiluminecence assay procedure. The AM chemiluminescence assay was conducted in the same manner as previously described by our laboratory . Briefly, resting AM chemiluminescence was determined by incubating 1.0 × 106 AM/ml at 37°C for 20 minutes, followed by the addition of 5-amino-2,3-dihydro-1,4, phthalazinedione (luminol) to a final concentration of 0.08 μg/ml. This was then followed by the measurement of chemiluminescence generated over 15 minutes.
Zymosan-stimulated chemiluminescence was determined by adding unopsonized zymosan (2 mg/ml) to the AM samples immediately prior to measurement of chemiluminescence. NO-dependent chemiluminescence was determined by adding the unopsonized zymosan as well as N-nitro-L-arginine methyl ester HCL (L-NAME) to the AM samples immediately prior to measurement of chemiluminescence. Zymosan-stimulated (zymosan – rest) and NO-dependent (zymosan – zymosan with L-NAME) chemiluminescence were both measured using a Berthold automated luminometer (Berthold Autolumat LB 953, EG&G, Gaithersburg, MD) at 390–620 nm for 15 minutes.
Tissue collection and analysis for titanium dioxide burden analysis
Immediately after the 7 and 42- day exposure time periods, rats were euthanized with an i.p. injection of sodium pentobarbital (>100 mg/kg body weight). The left and right lungs as well as the tracheobronchial (TBL) and thymic (TLN) lymph nodes were removed for titanium dioxide burden analysis. After removal, the lungs and lymph nodes were weighed and then frozen at -80°C. The lungs and lymph nodes were then lyophilized. The lyophilized samples were sent to Bureau Veritas North America, Inc. (Novi, MI) for TiO2 metal analysis.
Immediately after the 7 and 42 days exposure, rats were euthanized with i.p. injection of sodium pentobarbital (> 100 mg/kg body weight). The lungs were removed and infused to 25 cm H2O with 10% buffered neutral formalin. Paraffin-embedded histologic sections were stained with hematoxylin and eosin (H&E) or Masson's trichrome for light microscopic examination by a board-certified veterinary pathologist.
Statistical differences between control groups and treatment groups for the in vivo experiments examining the toxicity of titanium dioxide were determined using an analysis of variance (ANOVA). Individual means were compared using the Student-Newman-Keuls Method multiple comparison procedure with an overall significance level of p= 0.05. A linear regression curve analysis with a 95% confidence interval was also conducted on the surface area data for each pulmonary parameter measured.
The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the National Institute for Occupational Safety and Health.
Association for Assessment and Accreditation of Laboratory Animal Care
Analysis of variance
Bronchoalveolar lavage fluid
Certified virus free
Fine carbon black
Fine titanium dioxide
Hematoxylin and eosin
Macrophage inflammatory protein
Tracheo-bronchial lymph node
Thymic lymph node
Tumor necrosis factor
Ultrafine carbon black
Ultrafine titanium dioxide.
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