Environ. Sci. Technol. 2009, 43, 7939–7945
Nanoscale and Fine Zinc Oxide Particles: Can in Vitro Assays Accurately Forecast Lung Hazards following Inhalation Exposures? D. B. WARHEIT,* C. M. SAYES,† AND K. L. REED DuPont Haskell Global Centers for Health and Environmental Sciences, Newark, DE
Received May 23, 2009. Revised manuscript received August 15, 2009. Accepted August 25, 2009.
The development of accurate in vitro screening assays to assess lung hazard potential of nanomaterials is a highly desirable goal. However, some studies have noted little correlation between in vitro and in vivo results. Moreover, a recent National Academy of Sciences report predicts that future hazard testing will be conducted primarily using cell culture assays. The three major objectives of this study were to compare lung toxicity impacts of nanoscale (NZnO) vs fine zinc oxide (FZnO) particulates, assess predictability of in vitro cell culture systems, and compare effects of instillation vs inhalation exposures in rats. Physicochemical aspects of ZnO particle types were rigorously characterized and did not agree with specifications provided by the supplier; i.e., the ZnO particle types were closer in size than advertised. Rats were exposed in vivo either by intratracheal instillation to 1 or 5 mg/kg of nanoscale or fine size zinc oxide particle types or by inhalation to aerosols of 25 or 50 mg/m3 for 1 or 3 h. Lung inflammation, cytotoxicity, and histopathological endpoints were assessed at several time points postexposure. Three different in vitro culture conditions were utilized. Cultures of (1) rat lung epithelial cells, (2) primary alveolar macrophages, and (3) alveolar macrophages-L2 lung epithelial cell cocultures were incubated with fine or nano ZnO particles and evaluated for cytotoxicity biomarkers (LDH) and proinflammatory cytokines (MIP-2 and TNF-R). In vivo exposures to instilled or inhaled fine or nanoscale ZnO produced “metal fume fever” responses, characterized by transient, short-term lung inflammatory or cytotoxic responses. Alternatively, in vitro exposures to fine or nanoscale ZnO particles produced minor cytotoxic responses at 4 and 24 h, only in cocultures and at the highest (particle overload) dose with little detectable proinflammatory cytokine generation (MIP-2, and TNF-R). To summarize, the comparisons of in vivo and in vitro toxicity measurements following nano or fine ZnO particle exposures demonstrated little convergence and few differences in potency.
Introduction Exposures to welding fumes or zinc oxide particles in workers are known to produce metal fume fever-type responses, characterized by a transient fever which resolves after a few * Corresponding author phone: (302) 366-5322; fax: (302) 3665211; e-mail:
[email protected]. † Current address: Texas A&M University, College Station, TX. 10.1021/es901453p CCC: $40.75
Published on Web 09/08/2009
2009 American Chemical Society
days (1, 2). In many rodent species, exposures to zinc oxide aerosols or welding fumes produce transient pulmonary inflammatory responses which likewise are not persistent and generally recover to control levels within a few days (3, 4). Unlike crystalline silica particle exposures, the shortterm effects following zinc oxide exposures are not sustained and resolution may be due to the high solubility of ZnO particle types and/or to the binding of zinc particles by metallothionein. Nonetheless, the dual and reversible nature of the pulmonary response to ZnO particulates can be problematic for modeling when in vitro screening effects are used, particularly in the absence of time course studies. The development of predictive in vitro toxicity assays for screening nanoscale materials could obviate the need for animal testing, particularly during the incipient phases of hazard evaluations. Accordingly, from a cost, timing, material, and animal use perspective, it would be beneficial if the early stages of hazard screening assessments could be carried out using relatively inexpensive in vitro assays. Moreover, accurate data generated from these tests could serve as a bridge to the subsequent implementation of longer-term particle inhalation studies, thereby obviating the need for acute hazard studies. In addition, the U.S. National Academy of Sciences has recently published a report entitled “Toxicity Testing in the 21st Century: A Vision and a Strategy”, which predicts that most hazard evaluations will be implemented using in vitro methodologies in the near term (5, 6). A major drawback thus far, however, has been the lack of efficacy and reliability of pulmonary toxicity findings from in vitro screening studies with both particles and fibers (7). The aims of this study were to compare and assess the predictability of in vitro lung cellular responses to nanoscale and/or fine-sized zinc oxide (ZnO) particles when compared to effects measured following two in vivo methods of exposures, namely intratracheal instillation and inhalation. In addition, we tested the hypothesis that nanoscale ZnO particles produce greater pulmonary inflammation and cytotoxicity when compared to fine-sized zinc oxide particle types at equivalent mass dose concentrations. Ultrafine and nanoparticles are thought to have greater potency for hazard effects compared to fine-sized particulates of similar or identical chemical composition. Unfortunately, rigorous characterization of the NZnO and FZnO particle types revealed that the differences in particle sizes of the two purchased samples were not as substantially dissimilar as had been advertized by the supplier. Nonetheless, the measured surface area and particle sizes for the NZnO in the dry state were calculated to be 12.1 m2/g and 90 nm (still in the nanoscale range), respectively, while the FZnO data measured 9.6 m2/g and 111 nm. The lesson to be learned: data provided by the supplier should be reconfirmed by investigators.
Materials and Methods Experimental Design. One of the goals of this study was to evaluate pulmonary hazard effects to ZnO particulates following three different methods of exposure, namely, lung cell cultures, intratracheal instillation exposures, and physiologically relevant inhalation exposures. For both in vitro and in vivo exposures to fine-sized or nanoscale zinc oxide particle types, pulmonary toxicity measurements were conducted using well established inflammatory and cytotoxicity biomarkers. ZnO particle types were carefully characterized, both in the wet and dry states for in vitro and instillation exposures (particle suspensions in water, phosphate-buffered saline, and culture media), as well as in the VOL. 43, NO. 20, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
7939
TABLE 1. Physicochemical Characteristics of Fine and Nano ZnO Particles Particle Characterization in the Wet State
nano ZnO fine ZnO
solvent
avg particle size in soln (nm) DLS
surface charge (mV)
water PBS F-12K media water PBS F-12K media
168 ( 16% 314 ( 31% 283 ( 45% 243 ( 19% 319 ( 35% 372 ( 45%
-54.51 -28.5 -5.7 -55.76 -14.9 -9.4
Particle Characterization in the Dry State reported primary calculated particle area size in size (nm) surface density dry state by supplier (m2/g) (g/mL) (nm) crystallinity nano ZnO fine ZnO
50-70 99.0%, purity). The particle samples supplied by Sigma were produced by grinding or milling a bulk material into an ultrafine dust. Stock suspensions (10 mg/mL) of each of the particle types was prepared in ultrapure Milli-Q water, phosphate buffered saline (PBS) solution (sterile filtered), and F-12K cell culture media, and each particle preparation was probe sonicated (Ultrasonic Processor, 50 W/60 Hz) for 5 min prior to exposure and characterization. For the in vivo intratracheal instillation studies, stock solutions were diluted serially to achieve doses of 1 and 5 mg/kg animal body weight. For the in vitro studies, each of the stock solutions was diluted serially to yield concentrations ranging from 0.1 mg/mL () 0.052 µg/cm2 microgram of particle sample to area of culture dish (µg/ cm2)) to 100 mg/mL () 52 µg/cm2). Particle overload concentrations were determined to be 100 mg/mL or 52 µg/ cm2 (9). Calculations for this conclusion were based on conversion of µg/cm2 dose units to cm2/cm2 units (i.e., µg/ cm2 × surface area of the particle). Surface areas are reported in Table 1. Particle characterization in the dry state included, surface area (BET (10)), density, calculated size, crystallinity (X-ray diffraction), and chemical composition of each particle sample. Particle characterization in the wet phase included 7940
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 20, 2009
FIGURE 1. Transmission electron micrographs of fine (FZnO) (a) and nanoscale (b) zinc oxide (NZnO) particles. DLS (dynamic light scattering (11)) and measurements in water, PBS, and cell culture media (Table 1). Transmission electron micrographs of nano and fine-sized zinc oxide particles are presented in Figure 1, along with particle size distributions in various suspensions (Table 1). Animals. Groups of male Crl:CD(SD)IGS BR rats (Charles River Laboratories, Inc., Raleigh, NC) were used in this study. The rats were approximately 8 weeks old at the start of the study (mean weights in the range of 240-255 g). All procedures using animals were reviewed and approved by the Institutional Animal Care and Use Committee and the animal program is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). In Vivo: Inhalation exposure system. Atmosphere Generation. Atmospheres of either nanoscale or fine zinc oxide particles or magnesium oxide particulates were generated by suspending the test substance in air. The test materials were metered into a Fluid Energy model 00 Jet-O-Mizer jetmill with a K-Tron model T-20 twin screw volumetric feeder. Highpressure house air was fed into the Jet-O-Mizer and then metered into the jetmill which swept the resulting atmosphere into a 1000 mL glass cyclone and then into the exposure chamber. Chamber atmospheres of aerosolized particle types were controlled by varying the test substance feed rate to the atmosphere generator.
All exposure chambers were constructed of stainless steel and glass (NYU style) with a nominal internal volume of approximately 150 L. Directly inside the chamber inlet, a stainless steel baffle was positioned to provide uniform distribution of the test substance within the chamber. During exposure periods, three rats/exposure concentration/postexposure time point were individually restrained in perforated stainless steel cylinders with conical nose pieces (i.e., nose-only exposures). The restrainers were inserted into the faceplate of the exposure chamber so that only the nose of each rat extended into the chamber. The faceplates used to support the animal restrainers were constructed of polymethylmethacrylate. The restrainers containing the rats were rotated each exposure day to ensure homogeneity of exposures. Groups of sham exposed rats exposed to room air served as controls. Bronchoalveolar lavage fluids recovered from the lungs of air or particle-exposed rats were analyzed at 24 h, 72, and 192 h (i.e., 8 days) postinhalation exposure. In Vivo: Intratracheal Instillation Exposures. Methodologies for intratracheal instillation/in vivo pulmonary toxicity measurements have previously been described (8). Groups of rats (5 rats/group/dose/time point) were intratracheally instilled with nanoscale or fine ZnO particle doses of 1 or 5 mg/kg in PBS. Groups of PBS instilled rats served as vehicle controls and Min-U-Sil quartz particles were utilized as benchmark positive controls. Following exposures, bronchoalveolar lavage fluids from the lungs of PBS and particleexposed rats were analyzed at 24 h, 1 week, 1 month, and 3 month time points postinstillation exposure. The lungs of control and particle-exposed rats were lavaged with a phosphate-buffered saline (PBS) solution as described previously (12). Methodologies for cell counts, cell differentials, and pulmonary cytotoxicity biomarkers in lavaged fluids were conducted as previously described (12). Briefly, the first 12 mL of lavaged fluids recovered from the lungs of PBS or particulate-exposed rats was centrifuged at 700 g, and 2 mL of the supernatant was removed for biochemical studies. All biochemical assays were performed on BAL fluids using a Roche Diagnostics (BMC)/Hitachi 717 clinical chemistry analyzer using Roche Diagnostics (BMC)/Hitachi reagents. Lactate dehydrogenase (LDH) and lavage fluid protein activities were measured using Roche Diagnostics (BMC)/ Hitachi reagents. Lactate dehydrogenase is a cytoplasmic enzyme and is used as an indicator of cell injury/cytotoxicity. Increases in BAL fluid protein concentrations generally are consistent with enhanced permeability of vascular proteins into the alveolar regions, indicating a breakdown in the integrity of the alveolar-capillary barrier. When the F test from ANOVA was significant, the Dunnett’s or Dunn’s test was used to compare means from the control group and each of the groups exposed to particles. Statistical significance vs PBS controls was established as p < 0.05. Statistical tests were performed with SAS 9.1 software (SAS Institute Inc., Cary, NC). Morphological/Histopathology Studies. The lungs of rats exposed to particulates or PBS controls were weighed and prepared for microscopy by airway infusion of formaldehyde under pressure (21 cm H2O) at 24 h, 1 week, 1 month, and 3 months postexposure. Sagittal sections of the left and right lungs were made with a razor blade. Tissue blocks were dissected from left, right upper, and right lower regions of the lung and were subsequently prepared for light microscopy (paraffin embedded, sectioned, and hematoxylin-eosin stained) (12, 13). In Vitro Exposures: Cell Culture System. Three different immersion-type cell culture systems were used in this study: (1) rat L2 lung epithelial cell lines (American Type Culture Collection, Manassas, VA), (2) primary rat lung alveolar macrophages lavaged from the lungs of rats, and (3) L2 lung epithelial-alveolar macrophage cocultures (14). All cell
culture systems were suspended in F-12K medium (Kaighn’s modification of Ham’s F-12 medium) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. All experiments were conducted at a cellular density of 200 000 cells/cm2. The epithelial cells were confluent, and the macrophages were adherent 45 min prior to particle exposures. Tests for LDH release and production of inflammatory mediators MIP-2 and TNF-R were performed on all three in vitro cell culture systems and done in triplicate. Cells were seeded in 24-well plates, exposed to increasing concentrations of particle suspensions (0.01 mg/mL (0.0052 µg/ cm2) to 100 mg/mL (i.e., 52 µg/cm2)), where 100 mg/mL was calculated to be a particle overload dose, and incubated for two different time points at 4 and 24 h. This conclusion was based on calculations to convert mg/mL to µg/cm2 dose units to cm2/cm2 units (i.e., µg/cm2 × surface area of the particle). Accordingly, using calculations, we converted the dose at 100 mg/mL to 6.29 (NZnO) and 5.0 (FZnO) cm2/cm2, whereas 1-3 cm2/cm2 is considered to be an overload dose for low toxicity dusts (9). After incubation, the plate was centrifuged at 1900 rpm for 4 min. The media was transferred into a fresh 24-well plate and analyzed for LDH release as described earlier. The Rat Macrophage Inflammatory Protein-2 (Rt MIP2) Enzyme Immunometric Assay kit (Alpco Diagnostics, Salem, New Hampshire) was used to evaluate the production of MIP-2). Cells grown in 24-well plates were exposed to varying particle concentrations at the 24 h time point (15-17). Statistical analyses were performed as described earlier, i.e., statistical significance vs a control of µg/cm2 dose was established as p < 0.05. In vitro experiments were conducted with replicates, and the studies were repeated at least two times. The Tumor Necrosis Factor-R (TNF-R) Enzyme Immunometric Assay Kit (Assay Designs) was used to determine the level of TNF-R in the biological samples. Cells grown in 24-well plates were exposed to varying particle concentrations at the 24 h time point (18, 19).
Results Particle Characterization. It is important to note that the mean particle size data provided by the manufacturer were significantly different than actual measured sizes using BET and XRD analyses in the native dry state and dynamic light scattering (DLS) methods in the wet state, under three different suspensions. For example, with regard to the nanoscale zinc oxide sample, the supplier reported a particle size of 50-70 nm. However, using two different methods, it was determined that the nanoscale ZnO particulate samples ranged from 90 nm (dry state, calculated using density and surface area) to 168 nm (wet state, measurements DLS in water), which were not substantially different from the measured particle sizes of the fine-zinc oxide particle samples (111 and 243 nm, respectively). The supplier reported that the mean size of fine ZnO particle types was 25% neutrophils through 3 months postexposure (Figure 3a). Similarly, BAL fluid LDH (Figure 3b) and microprotein (Figure 3c) values in nano and fine ZnO-exposed rats followed transient patterns similar to the pulmonary inflammatory responses; with dose response increases at 24 h pe and a corresponding full recovery at the remaining time points postexposure. Intratracheal exposures to Min-U-Sil silica particles produced sustained cytotoxic responses in the lungs of exposed rats (Figures 3b and 3c). In Vitro Cytotoxicity Studies: LDH studies (4 and 24 h). LDH values from various doses of nanoscale and fine zinc oxide particle types at two different time points (4 and 24 h) were compared with corresponding culture media (control) values. Significant increases in LDH activity were measured only in epithelial-macrophage cocultures and only at the highest particulate dose, 100 mg/mL (Figure 4a). This dose was considered to be a 3× particle-overload dose (see Methods section). No increases vs controls in LDH activity were measured in L2 rat epithelial cells (Figure 4a) or in alveolar macrophages (data not shown) at any of the tested doses at the 4 and 24 h incubation time periods. In Vitro: Inflammatory Mediator Studies. MIP-2 production by cells exposed in vitro for 24 h to NZnO or FZnO particles is shown in Figure 4b. Little MIP-2 production was measured in the culture fluids of particle-exposed lung epithelial cells at any concentration. Macrophages and epithelial-macrophage cocultures exposed to NZnO or FZnO particle types were more active constitutively but demon7942
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 20, 2009
FIGURE 2. (a) Pulmonary inflammation in rats exposed by inhalation for 1 or 3 h to NZnO, FZnO, or magnesium oxide particles. Air-exposed rats served as controls (all separate experiments). Values given are mean % neutrophils ( SD. Exposures to either nanoscale or fine ZnO particles produced short-term, inflammatory responses, with recovery by 8 days postexposure. The exposures to MgO particles produced no discernible effects. *p < 0.05 vs air controls. (b, c) BAL fluid lactate dehydrogenase or microprotein values in rats exposed by inhalation for 1 or 3 h to NZnO, FZnO, or MgO particles (all separate experiments). Values given are means ( SD. Exposures to either NZnO or FZnO particles produced transient pulmonary inflammatory and cytotoxic responses, with recovery by 8 days postexposure. *p < 0.05 vs air controls. strated no dose-response or statistically significant relationships (Figure 4b). TNF-R production by cells exposed in vitro for 24 h to nanoscale ZnO, or fine ZnO particles is shown in Figure 4c. Exposures of lung epithelial cells, macrophages, or cocultures to NZnO or FZnO did not produce any significant secretions of TNF-R in the cell culture fluid, even at particle overload doses. To summarize the results of in vitro cytokine genera-
FIGURE 3. (a) Pulmonary inflammation in rats exposed by intratracheal instillation to NZnO or FZnO particles or Min-U-Sil crystalline silica particles (positive control). High dose intratracheal instillation exposures of NZnO and FZnO particle types produced transient lung inflammation, measured at 24 h and 1 week postexposure, with recovery at 1 and 3 months postexposure. In contrast, exposures to quartz particles (1 and 5 mg/kg) produced sustained pulmonary inflammatory responses, as measured through 3 months postexposure. (b, c) BAL fluid lactate dehydrogenase or microprotein values in rats exposed by intratracheal instillation to NZnO or FZnO particles or Min-U-Sil crystalline silica particles (positive control). High dose intratracheal instillation exposures of NZnO and FZnO particle types produced transient lung injury and cytotoxic responses, measured at 24 h and 1 week postexposure, with recovery at 1 and 3 months postexposure. In contrast, exposures to quartz particles (1 and 5 mg/kg) produced sustained pulmonary cytotoxic responses, as measured through 3 months postexposure. For all panels, PBS served as vehicle controls. Values given are means ( SD at 24 h, 1 week, 1 month, and 3 months postexposure (pe). *p < 0.05 vs PBS controls.
FIGURE 4. (a) Lacate dehydrogenase values in cells exposed in culture to FZnO or NZnO particles. Values given are after 4 and 24 h of treatment. NZO and FZO particles produced increased LDH levels in cocultured cells at 4 or 24 h only at 100 mg/mL (52 µg/cm2), which was calculated to be a particle overload concentration. (b, c) Proinflammatory cytokine generation in nanoscale or fine zinc oxide treated cells in vitro. MIP-2 production by cells exposed in vitro for 24 h to NZnO or FZnO particles is shown in (b). Little or no MIP-2 production was measured in the culture fluids of lung epithelial cells exposed to any of the particle types. Similarly, rat alveolar macrophages or epithelial-macrophage cocultures exposed to NZnO or FZnO particles did not generate MIP-2 in the culture fluid. TNF-r production by cells exposed in vitro for 24 h to nanoscale or fine zinc oxide particle types is shown in (c). Treatment of lung epithelial cells, macrophages, or cocultures with zinc oxide particles did not produce any significant secretion of TNF-r in the cell culture fluid. tion studies as potential surrogates for in vivo inflammation (measured in the in vivo studies), exposures to NZnO or FZnO particle types did not produce enhanced secretions by VOL. 43, NO. 20, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
7943
cultured cells of two representative proinflammatory chemokines, namely MIP-2 or TNF-R generation in culture fluids. Histopathological Evaluation. Histopathological analyses of lung tissues at several times postexposure revealed that pulmonary exposures to nanoscale or fine zinc oxide particles via either intratracheal or inhalation exposure routes produced no significant adverse effects when compared to corresponding controls, as evidenced by the normal lung architecture observed in the exposed animals at postinstillation exposure time periods ranging from 24 h to 3 months or at 8 days postinhalation exposures (data not shown). Histopathological analyses of lung tissues revealed that pulmonary exposures to quartz particles in rats produced dose-dependent lung inflammatory responses characterized by neutrophils and foamy (lipid-containing) alveolar macrophage accumulation. In addition, lung tissue thickening as a prelude to the development of fibrosis was evident and progressive (data not shown).
Discussion The objectives of this study were to compare, using in vitro vs in vivo methodologies, the pulmonary hazard potentials following exposures to nanoscale or fine zinc oxide (ZnO) particulates. A secondary aim was to compare the lung responses to nanoscale vs fine zinc oxide particle exposures. In addition, the patterns and relevance of intratracheal exposure effects vs inhalation exposures in rats was assessed. The physicochemical characteristics of both zinc oxide particle samples were extensively characterized. Our first conclusion (and minor disappointment) was that the material characterization of both nanoscale and fine ZnO samples provided by the same supplier were not compatible with the measurements made in our laboratory. Indeed, the nano zinc oxide was advertised to be in the 50-70 nm particle size range (and accordingly the purchase price was substantially more expensive when compared to the fine ZnO samples). However, our measurements conducted in both wet and dry phases indicated that the particle sizes ranged from ∼90-300 nm, although it is certain that the wet phase (DLS) measurements included particle agglomeration in solvents. Alternatively, the measured fine zinc oxide particle sizes clearly were substantially less than the
