Lung toxicity and biodistribution of Cd/Se-ZnS quantum dots with different surface functional groups after pulmonary exposure in rats
© Roberts et al.; licensee BioMed Central Ltd. 2013
Received: 3 July 2012
Accepted: 23 February 2013
Published: 4 March 2013
The potential use of quantum dots (QD) in biomedical applications, as well as in other systems that take advantage of their unique physiochemical properties, has led to concern regarding their toxicity, potential systemic distribution, and biopersistence. In addition, little is known about workplace exposure to QD in research, manufacturing, or medical settings. The goal of the present study was to assess pulmonary toxicity, clearance, and biodistribution of QD with different functional groups in rats after pulmonary exposure.
QD were composed of a cadmium-selenide (CdSe) core (~5nm) with a zinc sulfide (ZnS) shell functionalized with carboxyl (QD-COOH) or amine (QD-NH2) terminal groups. Male Sprague–Dawley rats were intratracheally-instilled (IT) with saline, QD-COOH, or QD-NH2 (12.5, 5.0, or 1.25 μg/rat). On days 0, 1, 3, 5, 7, 14, and 28 post-IT, the left lung, lung-associated lymph nodes (LALN), heart, kidneys, spleen, liver, brain, and blood were collected for metal analysis of Cd content by neutron activation to evaluate clearance and biodistribution. One right lobe was ligated and fixed for microscopy and histopathological analysis. The remaining right lobes from rats in each group were subjected to bronchoalveolar lavage (BAL) to retrieve BAL fluid and cells for analysis of injury and inflammation.
Lung injury and inflammation was found to be dose-dependent and peaked at days 7 and 14 post-exposure for both forms of QD, with slight variations in degree of toxicity at early and later time points. Both QD appeared to lose their fluorescent properties and destabilize after 1 week in the lung. Cd persisted up to 28 days for both forms of QD; however, clearance rate was slightly greater for QD-COOH over time. No Cd was detected in the liver, spleen, heart, brain, or blood at any time point. Cd appeared in the LALN and kidneys beginning at 1–2 weeks post-exposure.
QD-COOH and QD-NH2 differed in clearance rate and differed slightly in degree of toxicity at different time points; however, the overall pattern of toxicity and biodistribution was similar between the two particles. Toxicity may be dependent on the dissolution rate and bioavailability of free Cd.
Quantum dots (QD), also referred to as nanocrystals, are fluorescent semiconductor nanomaterials (~2-100 nm in diameter) commonly used in the biomedical and electronic industries [1, 2]. In terms of structure, QD are nanocrystals composed of a semiconductor core [e.g., cadmium-selenide (CdSe)] encased within a shell comprised of a second semiconductor material [e.g., zinc sulfide (ZnS)] . Due to their small size, QD have unique optical and electronic properties that impart the nanoparticle with a bright, high intensity fluorescence , which persists as long the QD remain stable. The emission wavelength of the QD is specific to the size and composition of the nXanocrystal, where increasing size results in increasing emission wavelength. Because of their large surface area, QD are readily functionalized with targeting ligands for site-directed activity. Biomedical applications exploit the fluorescent properties of QD for diagnostic tools (e.g., imaging), therapeutic purposes (e.g., drug delivery and cancer treatment), live cell labeling, and tracking over long periods of time . QD are also being incorporated into solar cell and electronic devices, such as LEDs, where their electronic and optical properties provide advantages.
Due to the presence of Cd, a highly reactive and potentially toxic metal, there is concern about the health risks associated with exposure to QD. In vitro studies indicate that some QD are cytotoxic in short-term, cell culture-based assays (as reviewed by [1, 3]). QD composition, size, charge, exposure concentration, outer coating bioactivity (e.g., treatment with functional groups) and oxidative properties, and mechanical stability have been implicated as possible causative factors in the cytotoxicity of QD . Potential conditions of exposure to QD include therapeutic, diagnostic, environmental, and occupational . Workplace QD exposures (e.g., engineers, researchers, clinicians) may result from inhalation, ingestion, and dermal contact.
Concerning inhalation, numerous animal studies have examined the toxicology of other nanoparticles after pulmonary exposure –; however, little is known about the lung toxicity of QD. Ma-Hock et al.  performed a short-term (6 hr/day for 5 days) inhalation study in which Wistar rats were exposed to 4.1 mg/m3 (0.52 mg Cd/m3) of a liquid aerosol of Cd-based QD (cadmium sulfide core with a cadmium hydroxyl shell). A local inflammatory response was observed at 8 days after exposure that regressed after a 3-week recovery period with limited translocation of Cd from the lungs to systemic organs. In addition, QD were observed within phagolysosomes of alveolar macrophages (AM) after inhalation exposure. Clift et al. [11, 12] demonstrated that QD, functionalized with an organic coating or COOH or NH2 (PEG) (polyethylene glycol) moieties, can cause oxidative stress, leading to modulated Ca2+ signaling in a macrophage cell line after prolonged in vitro exposure to sub-lethal concentrations, and that toxicity is largely dependent on surface coating.
The goal of the current study was to expand upon the limited information that is known about the in vivo effects after pulmonary exposure to QD. Pulmonary toxicity and lung distribution, clearance, and translocation of QD functionalized with different surface moieties, resulting in different surface charges, were examined. Commercially-available QD, composed of a CdSe (~5 nm diameter) core with a ZnS shell, were used. The QD were functionalized with either amine (QD-NH2) or carboxyl (QD-COOH) groups to form positively- or negatively-charged QD, respectively. Rats were intratracheally instilled with various doses of QD-NH2, QD-COOH, or phosphate-buffered saline (PBS; vehicle control). Lung injury and inflammation were measured at 2 hr and at 1, 3, 5, 7, 14, and 28 days post-exposure. Sites of particle deposition and distribution in the lungs and particle uptake by macrophages were assessed using autometallographic silver enhancement of QD in tissue, laser scanning confocal microscopy (LSCM), and transmission electron microscopy (TEM). Particle translocation from the lung was evaluated using neutron activation analysis of Cd in lungs, blood, heart, lung-associated lymph nodes (LALN), brain, kidneys, liver, and spleen.
Relative to the initial amount deposited, rats exposed to QD-COOH were able to clear more QD from the lungs over time when compared to rats exposed to QD-NH2; however, the majority of particles in both groups persisted out to day 14 in the lungs. No Cd was measured above the limit of detection in the blood, brain, heart, liver, or spleen in either treatment group (data not shown), either due to the lack of translocation or the relatively small amount of Cd that may have translocated relative to the overall organ weight due to the limit of detection in rats exposed to QD-COOH, CD appeared in the LALN and kidneys as early as day 7, and ~ 3-5% of Cd instilled could be detected in the LALN while ~10-20% was located in the kidney by day 14 (Figure 6A). By day 28, increased clearance from the lungs and increased deposition in LALN and kidneys was evident. The pattern of clearance for rats exposed to QD-NH2 was similar; however, the time frame for clearance to the kidney and LALN was slower. In rats treated with QD-NH2, ~20% of the instilled Cd was located in the kidneys by day 14, and no Cd was detected in the LALN until day 28 (Figure 6B).
QD are being developed and used for a variety of applications (e.g., drug delivery, diagnostic procedures) that may lead to human exposure in medical settings. In addition, the number of workers producing or using nanomaterials, such as QD, is growing substantially. Unfortunately, little information exists about the potential health hazards associated with medical, environmental, or occupational lung exposures to QD. One important occupational health issue that is being addressed by the National Institute for Occupational Safety and Health (NIOSH) Nanotechnology Research Center is determining the toxic potential and fate of nanomaterials in the body after exposure . Therefore, there is interest in assessing the potential adverse health outcomes that may occur following pulmonary QD exposure, and the mechanisms underlying these responses. NIOSH is actively performing field studies to evaluate the potential exposure of workers in the QD industry (personal communication with Kenneth Martinez, 2012).
In vivo animal studies assessing the pulmonary responses after exposure to QD are limited. The QD samples examined in the current study were composed of a CdSe core. Cd is a widespread environmental and occupational pollutant and has been classified as a type I carcinogen by the International Agency for Cancer Research (IARC ). Inhalation of Cd-containing pollutants can result in metal fume fever, chemical pneumonitis, pulmonary edema, and pleural effusion . In addition to composition and the presence of potentially toxic metals, surface coating, charge, and size may influence QD toxicity. In vivo lung toxicology studies have shown that nanoparticles of identical composition and mass may be more toxic (Brown et al. [16, 17]) when compared to their larger counterparts. In vitro studies indicate that Cd-containing QD with a smaller diameter had greater toxicity and distributed to the nucleus of the cell compared to a larger QD of the same composition that were less cytotoxic and remained in the cell cytoplasm . Functionality and surface charge of QD are also of important in regard to conjugation to other particles or molecules for targeted delivery of substances in the body or tracking of substances. The QD in the current study were functionalized with different surface moieties by the manufacturer to examine the effect of surface charge on toxicity and were determined to be in the ultrafine size range with core size diameters of 5–10 nm.
A significant increase in lung injury was observed at different time points after a single intratracheal instillation of the QD-COOH and QD-NH2 samples in the current study. Lung injury parameters, LDH and albumin, were significantly increased immediately after treatment with the 12.5 μg dose, peaked at day 7 post-exposure, and remained elevated throughout the 28 day period, even though the response at 28 days was subsiding. Evidence for lung injury was not apparent until 5 days after treatment with the 5 μg dose of QD-NH2 sample where it was found to be more potent than the QD-COOH treatment. At 7 days post-exposure, the 5 μg dose of the QD-COOH sample began to exhibit toxicity and was more potent than the QD-NH2 sample. The difference in temporal response between the two doses may be related to the lungs responding earlier to a substantially greater number of particles at the higher 12.5 μg dose (e.g., dose effect), whereas the later injury response after treatment with the 5.0 μg dose may better reflect a potential mechanism of toxicity, the in situ dissolution of the QD by AM, leading to the direct exposure of the lung cells to free Cd (e.g., chemical effect). Moreover, the QD-NH2 sample induced significantly more lung injury at earlier time points after treatment compared to the QD-COOH, which may be due to a difference in particle distribution related to charge or a slightly faster disruption of the QD-NH2 structure, allowing for cellular exposure to free Cd sooner, or a combination of the two. Multiple studies have indicated that coating affects destabilization of QD and that uncoated Cd-containing QD released free Cd from the QD core intracellularly and were cytotoxic in vitro [19, 20]. In addition, Derfus et al.  demonstrated that the encapsulation of Cd/Se QD by a ZnS shell greatly reduced both the amount of free Cd released and the production of free radicals by QD.
In terms of lung inflammation as determined by the number of PMN recovered from the lungs, a significant inflammatory response was observed at 1 day after treatment with the 12.5 μg dose, likely due to a dose/bolus effect because this initial response subsided by 3 days after treatment, before increasing again and peaking at day 7. In addition, the lower dose QD treatment groups had no effect on inflammation at 1, 3, and 5 days after treatment. As was seen with the lung injury response, the number of recovered lung PMN was significantly greater at earlier time points (days 3 and 5) after treatment 12.5 μg dose of QD-NH2 sample compared to the QD-COOH sample, whereas PMN influx was greater in the QD-COOH-treated rats at days 14 and 28 when compared to those treated with QD-NH2, again implicating a likely difference in the dissolution rate between the two QD samples and the release of free Cd. Despite these differences, the pattern of inflammation is similar between QD groups, with peak responses between 7–14 days after treatment for the 5 and 12.5 μg dose groups for both QD samples. In addition to PMN influx, AM infiltration peaked at these two time points as well. In agreement with the inflammatory response seen in the current study, Ma-Hock et al.  also observed a transient local PMN inflammation in the lungs of rats that was associated with increases in inflammatory chemokines, MCP-1 and MIP-2, after a short-term inhalation of Cd-containing QD (cadmium sulfide core with a cadmium hydroxyl shell – CdS/Cd(OH)2)). This delay in inflammatory cell influx into the lungs after treatment is additional evidence that free Cd released from the dissolution of the QD structure is likely a causative factor responsible for the pulmonary responses observed. The lung inflammation pattern was similar to that of lung injury and appeared to be transient with the response subsiding (even at the highest dose) by 28 days after treatment with both QD samples.
Regarding lung deposition and clearance, QD-COOH and QD-NH2 were both rapidly phagocytized in vivo by AM in a dose-dependent manner as early as 2 hr after intratracheal instillation. Cd burden in LALN beginning 1 week after exposure suggests that at least a portion of the QD in the alveolar region are cleared via scavenging phagocytes which then migrate out of the lung through the lymphatics. Ma-Hock et al.  also observed AM clearance to the LALN as well. Translocation from the lung and clearance via the blood is also a possible route of clearance for particles in the lung. Confocal microscopy revealed some QD to have deposited on the epithelium outside AM and move to the interstitium, giving this portion of QD the potential of reaching the systemic circulation and translocating to other organs. However, measureable levels of Cd as determined by neutron activation did not appear in a systemic organ until 7 and 14 days after treatment with the QD-COOH and QD-NH2 samples, respectively. Using an in vitro model, Geys et al.  examined the effect of surface charge on the translocation of non-functionalized QD, QD-COOH, and QD-NH2 through a tight monolayer of primary rat alveolar epithelial cells. They observed that QD did not translocate through the tight junctions of alveolar epithelial cells, regardless of surface charge, without disruption of the cell-to-cell barrier induced by oxidative stress. In the current study, Cd depositing in extrapulmonary organ systems corresponds to the time points post-exposure where the greatest lung injury is observed, rendering it likely that the translocation of QD is occurring, and that this may be a passive process via “leaky” cell junctions, rather than an active process.
Neutron activation showed that at day 1 there was more Cd measured in the lungs of rats treated with QD-COOH when compared to those treated with QD-NH2. Uneven distribution due to route of administration (intratracheal instillation resulting in more QD going to one bronchus or the other), differences in deposition pattern (greater deposition in upper airways versus parenchyma), or differences in destabilization and translocation of the particles in the first 24 hours may contribute to this discrepancy. Differences in the rate of clearance were evident with greater percentage of QD cleared from the lungs by day 28 in the QD-COOH group compared to the QD-NH2 group, relative to early deposition amounts. However, the pattern of clearance via the lymphatics, and translocation to the kidneys via the blood stream, were similar between the two QD, and neutron activation analysis showed that Cd persisted in the lungs at least 28 days after exposure for both QD. This data is in agreement with other studies. Using inductively coupled plasma optical emission spectroscopy (ICP-OES) as opposed to neutron activation, Ma-Hock et al.  also observed that Cd levels in the lungs did not decline during a 3-week recovery period after inhalation exposure to water soluble CdS/Cd(OH)2.
Silver enhancement staining of lung tissue recovered from the QD-treated animals in the current study provided additional evidence that Cd persisted in the lungs at 28 days after treatment. The silver staining, used to image deposited metal particles, taken into consideration with the diminished fluorescent signal observed 1 week after exposure to QD using confocal microscopy, is another indication that free Cd had dissociated from the QD beginning 5–7 days after treatment. Although both forms of QD persisted in the lung up to 28 days, the rate of clearance appears to differ between the positively and negatively charged particles, with a slower clearance rate observed for QD-NH2 particles. This difference in the clearance patterns may be due to different dissolution rates of Cd from the QD after AM phagocytosis and enzymatic digestion because of the different surface charges and properties of the two QD samples as discussed above. In addition, a greater influx of phagocytes was observed at the later time points post-exposure to QD-COOH when compared to QD-NH2, resulting in a greater capacity for clearance. Also, it should be noted that initial Cd deposition in the lungs was greater with the QD-COOH sample compared to the QD-NH2 sample and this may contribute to a greater initial clearance of QD-COOH. As mentioned above, for both QD, a portion of the clearance appeared to occur via mechanical clearance by AM, as well as by extrapulmonary translocation.
The kidney, a primary target organ of Cd toxicity, was the only extrapulmonary organ in which Cd was measured in the current study. Relatively small concentrations of Cd appeared in the kidneys at 7 days after QD-COOH treatment and at 14 days after QD-NH2 treatment with levels increasing at 28 days for both groups. Ma-Hock et al.  also observed small amounts of Cd in kidney, which increased very slightly over the post-exposure recovery period. Importantly, histological analysis of kidney in their study did not indicate any morphological abnormalities after short-term QD inhalation. The Cd levels measured in the kidney of the current study were substantially lower than the levels of 104–252 μg/g wet kidney that were observed to cause renal dysfunction in rats after oral Cd treatment . The presence of Cd in the kidney is in line with the findings of Chen et al.  and Lin et al.  who observed the accumulation of QD in the kidney of rats and mice, respectively, after intravenous injection. However, Ma-Hock et al.  measured Cd in the liver after inhalation, and both Chen et al.  and Lin et al.  observed QD in the liver after intravenous exposure. Measureable levels of Cd were not detected in the liver after intratracheal treatment of the different QD samples in the current study, likely due to dose and route of administration combined. In agreement with the Ma-Hock et al.  study, lung treatment did not lead to the accumulation of Cd in the brain or spleen in the current study.
Based on the findings of the current study and others, several conclusions can be made concerning the toxicity and fate of QD after pulmonary treatment. Lung exposure to high enough concentrations of CdSe QD can cause transient lung inflammation and injury that appears to be related to the dissolution of the QD structure and removal of the ZnS shell coating, allowing for the release of free Cd. Different surface charges due to the addition of different functional groups attached to the surface of the QD may influence both the toxic pulmonary response and the persistence and clearance of the Cd from the lungs. QD-NH2 appeared to induce more injury and inflammation at earlier time points post-exposure where epithelial cell necrosis and type II cell hyperplasia were minimal but present; whereas QD-COOH cause greater injury and cellular influx at the later time points post-exposure. Although differences exist between the two particles, the pattern of toxicity was primarily the same for both QD with toxicity peaking at days 7 and 14 and beginning to subside over time. This is in agreement with Clift et al. , who found that QD with organic coatings were more toxic than QD-COOH and QD-NH2(PEG) with only slightly more toxicity present with the carboxyl groups versus the amine groups. In addition, toxicity appears to be dependent on the availability of free Cd for both QD, as well, although dissolution rates may vary. Overall, despite a relatively high pulmonary load, the systemic availability of the QD appears to be low. No free Cd was measured in blood, brain, liver, and heart in the current study. Measureable, but relatively small, amounts of free Cd did accumulate in the kidney, the main storage organ of Cd in the body.
Overall, both forms of QD induced lung damaged which was more severe one week after exposure, with QD-NH2 having induced more toxicity at the early time points post-exposure and QD-COOH having been more toxic at the later time points. However, the peak damage for both QD is around day 7 and sustained until day 14. This damage coincided with the lack of fluorescence in lung tissue under confocal microscopy, which indicates destabilization of the QD, and with evidence that Cd still persisted in the lungs (silver staining of tissues) at the times of peak lung damage. The data suggests that Cd became bioavailable over time due to destabilization of the QD in the lung, and induced injury as it became available and while it persisted there. Althought the rate of clearance of the QD-NH2 and QD-COOH differed, indicating a potentially different destabilization rate, the pattern of clearance over time was similar, primarily via the lymphatics and translocation systemically to the kidneys.
Quantum dots and characterization
Water-soluble quantum dots (Evident Technologies, Inc., Troy, NY) (QD), with a hydrodynamic diameter in the range of 30–50 nm, were composed of a CdSe core with a ZnS shell and had an emission peak of 620nm. The surface of the QD particles was functionalized with either carboxylic terminal groups (QD-COOH; stock = 2.1 nmol/ml water equivalent to 250 μg/ml) or amine terminal groups (QD-NH2; stock = 3.4 nmol/ml water equivalent to 250 μg/ml) by the manufacturer and were supplied in water (no toluene present). Transmission electron microscopy (TEM) was performed to detect individual and agglomerated QD in suspension. QD were diluted in deionized water to a concentration of 2.5 μg/ml and evaluated using a JEOL-1220 transmission electron microscope (JEOL, Inc., Tokyo, Japan). In addition QD suspended in PBS at a final instillate concentration were also imaged. Field emission scanning electron microscopy (FESEM) was used to obtain an energy dispersion X-ray (EDX) to illustrate elemental composition. For FESEM, QD were suspended in deionized water at a concentration of 2.5 μg/ml, dried onto carbon planchets, and imaged using a Hitachi Model S-4800 microscope (Hitachi High Technologies America, Inc., Gaithersburg, MD) operated between 5 and 20 kV. In addition, QD suspended in phosphate-buffered saline (PBS) at the instillate concentration for groups exposed to the high dose of particles were also analyzed using a dynamic light scatterer (DLS; Microtrac Inc., Largo, FL) to assess aggregation in the delivery vehicle. TEM of the instillate was also performed to further analyze aggregation of particles.
Male Sprague–Dawley [Hla:(SD)CVF] rats (Hilltop Laboratories, Scottdale, PA) weighing 250–300 g, approximately 10 weeks old, were used for all experiments in accordance with a protocol approved by the institute’s Animal Care and Use Committee (ACUC). Animals were given a Teklad 2918 diet and tap water ad libitum, housed in a clean air and viral- and antigen-free room with restricted access in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-approved animal facility. All animals were allowed to acclimate for one week before use. The rats were monitored and found to be free of endogenous viral pathogens, parasites, mycoplasms, Helicobacter, and CAR Bacillus.
In vivo exposure and study design
On day 0, male Sprague–Dawley rats were lightly anesthetized by an intraperitoneal (i.p.) injection of 0.6 ml of a 1% solution of sodium methohexital (Brevital, Eli Lilly, Indianapolis, IN), and intratracheal instillation (IT) was performed with a volume of 300 μl of QD-NH2 or QD-COOH diluted in PBS at doses of 12.5 μg, 5.0 μg, or 1.25 μg /rat. Vehicle control rats received an equivalent volume (300 μl) of sterile PBS by IT. Treated animals then were sacrificed with an overdose of sodium pentobarbital (>100 mg/kg bwt; Sleepaway, Fort Dodge Animal Health, Wyeth, Madison, NJ) followed by exanguination at 1, 3, 5, 7, 14, or 28 days after QD exposure. An additional 2 hour time point was conducted with the 12.5 μg dose to evaluate lung tissue for QD deposition and biodistribution. Bronchoalveolar lavage (BAL) was performed on right lungs with one lobe ligated for later microscopic preparation and analysis. To assess lung damage, lactate dehydrogenase (LDH) and albumin levels were measured in the first fraction of acellular BAL fluid (BALF). Recovered BAL cells were differentiated to assess lung inflammation, as well as seeded onto glass coverslips and fixed for laser scanning confocal microscopy (LSCM) analysis. Additional aliquots of BAL cells were suspended in Karnovsky’s fixative and imaged using TEM. The ligated right lobe of rats treated with 12.5 μg of QD was either: (1) inflated with a Tissue-Tek O.C.T. Compound (Sakura Finetek Inc., Torrance, CA) + 20% sucrose solution, cryo-preserved, and sectioned for LSCM or (2) pressure inflated with 10% formalin, embedded, sectioned, and stained for histological evaluation or autometallographic silver enhancement. To assess biodistribution and clearance of QD, left lungs, lung-associated lymph nodes (LALN), blood, brains, hearts, kidneys, livers, and spleens were analyzed using neutron activation.
BAL cell and fluid collection
BAL was performed by washing the lungs of treated rats with aliquots of PBS in order to obtain pulmonary cells for morphological and functional analysis. The acellular BALF was retained for analysis of indicators of tissue damage and cellular activity. Rats were euthanized with an i.p. overdose of sodium pentobarbital (>100 mg/kg body weight; Sleepaway, Fort Dodge Animal Health, Wyeth, Madison, NJ), the trachea was cannulated, the chest cavity was opened, the left lung was clamped off, one right lobe was ligated, and BAL was performed on the right lung via the tracheal cannula at different time points after IT. The acellular fraction of the first BAL was obtained by filling the right lung with 1 ml/100 g body weight of PBS, massaging for 30 seconds, withdrawing, and repeating the process one more time. This concentrated aliquot was withdrawn, retained, kept separately, and was designated as the first fraction of BALF. The following lavage aliquots were 6 ml in volume, instilled once with light massaging, withdrawn, and combined until a 30 ml volume was obtained. For each animal, both fractions of BAL were centrifuged, the cell pellets were combined and resuspended in 1ml of PBS, and the acellular fluid from the first fraction was retained for further analysis.
Evaluation of lung injury and inflammation
LDH and albumin
The presence of albumin and LDH in the acellular BALF of all treatment groups was measured to evaluate the loss of integrity of the alveolar-capillary barrier and general cytotoxicity, respectively. Measurements of both albumin and LDH in the acellular fluid were obtained using a Cobas Mira analyzer (Roche Diagnostic Systems, Montclair, IN). Albumin was determined by spectrophotometric measurement of the reaction product of albumin with bromcresol green (628 nm) according to a method by Sigma Diagnostics (St. Louis, MO). LDH activity was quantified by detection of the oxidation of lactate coupled to the reduction of NAD+ at a spectrophotometric setting of 340 nm.
Monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-2 were measured in the acellular BALF of rats pre-treated with QD or PBS using commercially available enzyme-linked immunosorbent assay (ELISA) kits (BioSource International Inc., Camarillo, CA).
Total BAL cells collected from rats treated with QD or PBS were counted using a Coulter Multisizer II (Coulter Electronics, Hialeah, FL). Cell differentials were performed to determine the total number of AM, polymorphonuclear leukocytes (PMN), and lymphocytes. Briefly, 1 × 105 cells from each rat were spun down onto slides with a Cytospin 3 centrifuge (Shandon Life Sciences International, Cheshire, England) and labeled with Leukostat stain (Fisher Scientific, Pittsburgh, PA) to differentiate cell types. Two hundred cells per slide were counted, and the percentage of alveolar macrophages (AM), polymorphonuclear cells (neutrophils, PMN), and lymphocytes were multiplied by the total number of cells to calculate the total number of each cell type.
The formalin fixed right lobe from animals in each group was embedded in paraffin, sectioned onto slides, and stained with hematoxylin and eosin (H&E). H&E-stained slides were qualitatively analyzed for indications of inflammation and injury by a certified veterinary pathologist at Charles River Laboratories (Wilmington, MA) who was blinded to the treatment groups. Indices of inflammation and injury were scored on scale of 0–5, where 0 = no observed effect, 1 = minimal response, 2 = mild response, 4 = moderate response, and 5 = severe response.
QD lung deposition, clearance, and biodistribution
Left lung, LALN, blood, brain, heart, kidney, liver, and spleen tissue were collected from a set of animals from each treatment group (n = 3–4). Tissues were freeze-dried by lyophilization, weighed, and analyzed for Cd content by North Carolina State University Nuclear Reactor Program, Department of Nuclear Engineering (Raleigh, NC) using neutron activation. Briefly, tissue samples and standards were irradiated in a PULSTAR reactor rotating exposure port for 12 MW-hr. Samples decayed for approximately 1 wk and were counted for 10 min each on a gamma spectroscopy system analyzing for Cd.
Cryo-preserved lung tissue was sectioned, and lung tissue sections and BAL cells were imaged with a LSCM (Carl Zeiss, Inc., Thornwood, NY). Tissue sections were scanned at 63x with a 543nm laser and an LP585 filter to detect the QD (red), and tissue autofluorescence was detected with a 488nm laser (green). Cells were counter-stained with CellTracker green (Invitrogen Corp., Carlsbad, CA) and imaged with a 63x or 100x objective at similar laser settings.
BAL cells were preserved with Karnovsky’s fixative, post-fixed in osmium tetroxide, and embedded in epoxy resin. QD were imaged in AM using a JEOL 1220 transmission electron microscope (JEOL, Inc., Tokyo, Japan) at 80 KV.
Tissue autometallographic silver enhancement
For light microscope visualization of QD, paraffin-embedded lung tissue sections were deparaffinized and dehydrated. The tissue sections were then washed with multiple changes of deionized water. Sections were developed with a silver enhancement kit according to the manufacturer’s instructions (Polysciences, Inc., Warrington, PA). The reaction was terminated after 30 min of development by washing in deionized water, and then the lung tissue sections were lightly counter-stained with Sirius Red .
Results are expressed as means ± standard error of measurement (SE). Statistical analyses were carried out with the SigmaStat 3.1 statistical program (Chicago, IL). The significance of the interaction among different treatment groups for the different parameters at each time point was assessed using analysis of variance (ANOVA). The significance of difference between individual groups was analyzed using the Holm-Sidak method with the criterion of significance set at p<0.05.
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.
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