Brian A. Wong

Inhaled carbon nanotubes reach the subpleural tissue in mice

Summary Carbon nanotubes have fibre-like shape1 and stimulate inflammation at the surface of the peritoneum when injected into the abdominal cavity of mice2, raising concerns that inhaled nanotubes3 may cause pleural fibrosis and/or mesothelioma4. Here we show that multi-walled carbon nanotubes reach the sub-pleura in mice after a single inhalation exposure of 30 mg/m3 for 6 hours. Nanotubes were embedded in the sub-pleural wall and within sub-pleural macrophages. Mononuclear cell aggregates on the pleural surface increased in number and size after 1 day and nanotube-containing macrophages were observed within these foci. Sub-pleural fibrosis increased after 2 and 6 weeks following inhalation. None of these effects were seen in mice that inhaled carbon black nanoparticles or a lower dose of nanotubes (1 mg/m3). This work advances a growing literature on pulmonary toxicology of nanotubes5 and suggests that minimizing inhalation of nanotubes during handling is prudent until further long term assessments are conducted.

Inhaled Multiwalled Carbon Nanotubes Potentiate Airway Fibrosis in Murine Allergic Asthma

Carbon nanotubes are gaining increasing attention due to possible health risks from occupational or environmental exposures. This study tested the hypothesis that inhaled multiwalled carbon nanotubes (MWCNT) would increase airway fibrosis in mice with allergic asthma. Normal and ovalbumin-sensitized mice were exposed to a MWCNT aerosol (100 mg/m(3)) or saline aerosol for 6 hours. Lung injury, inflammation, and fibrosis were examined by histopathology, clinical chemistry, ELISA, or RT-PCR for cytokines/chemokines, growth factors, and collagen at 1 and 14 days after inhalation. Inhaled MWCNT were distributed throughout the lung and found in macrophages by light microscopy, but were also evident in epithelial cells by electron microscopy. Quantitative morphometry showed significant airway fibrosis at 14 days in mice that received a combination of ovalbumin and MWCNT, but not in mice that received ovalbumin or MWCNT only. Ovalbumin-sensitized mice that did not inhale MWCNT had elevated levels IL-13 and transforming growth factor (TGF)-beta1 in lung lavage fluid, but not platelet-derived growth factor (PDGF)-AA. In contrast, unsensitized mice that inhaled MWCNT had elevated PDGF-AA, but not increased levels of TGF-beta1 and IL-13. This suggested that airway fibrosis resulting from combined ovalbumin sensitization and MWCNT inhalation requires PDGF, a potent fibroblast mitogen, and TGF-beta1, which stimulates collagen production. Combined ovalbumin sensitization and MWCNT inhalation also synergistically increased IL-5 mRNA levels, which could further contribute to airway fibrosis. These data indicate that inhaled MWCNT require pre-existing inflammation to cause airway fibrosis. Our findings suggest that individuals with pre-existing allergic inflammation may be susceptible to airway fibrosis from inhaled MWCNT.

Particle Deposition in Human Nasal Airway Replicas Manufactured by Different Methods. Part II: Ultrafine Particles

Information on the deposition efficiency of aerosol particles in the nasal airways is used for optimizing the delivery of therapeutic aerosols into the nose and for risk assessment of toxic airborne pollutants inhaled through the nose into the respiratory system. Nasal particle deposition is often studied using plastic replicas of nasal airways. Deposition efficiency in a nasal replica manufactured by stereolithography has not been reported to date. We determined the inertial particle deposition efficiency of two replicas of the same nasal airways manufactured by different stereolithography machines and compared results with deposition efficiencies reported for models manufactured by other techniques from the same magnetic resonance imaging scans. Deposition in the replicas was measured for particles of aerodynamic diameter between 1 and 10 μm and constant inspiratory flow rates ranging from 20–40 Ipm. Deposition efficiency of the replicas increased from nearly 0–100% with increasing particle inertia. For a range of particle inertias, particle deposition in the replica made with higher resolution stereolithography machine was slightly less than in the replica made with a lower resolution stereolithography process. These data showed lower deposition efficiency when compared with other deposition studies in nasal replicas based on the same magnetic resonance imaging data. The differences in deposition efficiency can be attributed in part to differences in methods used to manufacture the replicas. There was little or no difference in deposition due to cutting tool size, some difference due to the use of assembly plates, and some difference due to surface roughness. These associations suggest that inertial nasal particle deposition is significantly influenced by small differences in nasal airways.

OLFACTORY TRANSPORT: A DIRECT ROUTE OF DELIVERY OF INHALED MANGANESE PHOSPHATE TO THE RAT BRAIN

Experiments examining the dosimetry of inhaled manganese generally focus on pulmonary deposition and subsequent delivery of manganese in arterial blood to the brain. Growing evidence suggests that nasal deposition and transport along olfactory neurons represents another route by which inhaled manganese is delivered to certain regions of the rat brain. The purpose of this study was to evaluate the olfactory uptake and direct brain delivery of inhaled manganese phosphate ( 54 MnHPO 4 ). Male, 8-wk-old, CD rats with either both nostrils patent or the right nostril occluded underwent a single, 90-min, nose-only exposure to a 54 MnHPO 4 aerosol (0.39 mg 54 Mn/m 3 ; MMAD 1.68 w m, σ g 1.42). The left and right sides of the nose, olfactory pathway, striatum, cerebellum, and rest of the brain were evaluated immediately after the end of the 54 MnHPO 4 exposure and at 1, 2, 4, 8, and 21 d postexposure with gamma spectrometry and autoradiography. Rats with two patent nostrils had equivalent 54 Mn concentrations on both sides of the nose, olfactory bulb, and striatum, while asymmetrical 54 Mn delivery occurred in rats with one occluded nostril. High levels of 54 Mn activity were observed in the olfactory bulb and tubercle on the same side (i.e., ipsilateral) to the open nostril within 1-2 d following 54 MnHPO 4 exposure, while brain and nose samples on the side ipsilateral to the nostril occlusion had negligible levels of 54 Mn activity. Our results demonstrate that the olfactory route contributes to 54 Mn delivery to the rat olfactory bulb and tubercle. However, this pathway does not significantly contribute to striatal 54 Mn concentrations following a single, short-term inhalation exposure to 54 MnHPO 4 .

Inhalation Exposure Systems: Design, Methods and Operation

The respiratory system, the major route for entry of oxygen into the body, provides entry for external compounds, including pharmaceutic and toxic materials. These compounds (that might be inhaled under environmental, occupational, medical, or other situations) can be administered under controlled conditions during laboratory inhalation studies. Inhalation study results may be controlled or adversely affected by variability in four key factors: animal environment; exposure atmosphere; inhaled dose; and individual animal biological response. Three of these four factors can be managed through engineering processes. Variability in the animal environment is reduced by engineering control of temperature, humidity, oxygen content, waste gas content, and noise in the exposure facility. Exposure atmospheres are monitored and adjusted to assure a consistent and known exposure for each animal dose group. The inhaled dose, affected by changes in respiration physiology, may be controlled by exposure-specific monitoring of respiration. Selection of techniques and methods for the three factors affected by engineering allows the toxicologic pathologist to study the reproducibility of the fourth factor, the biological response of the animal.

Benzene-Induced Hematotoxicity and Bone Marrow Compensation in B6C3F1 Mice

Long-term inhalation exposure of benzene has been shown to cause hematotoxicity and an increased incidence of acute myelogenous leukemia in humans. The progression of benzene-induced hematotoxicity and the features of the toxicity that may play a major role in the leukemogenesis are not known. We report the hematological consequences of benzene inhalation in B6C3F1 mice exposed to 1, 5, 10, 100, and 200 ppm benzene for 6 hr/day, 5 days/week for 1, 2, 4, or 8 weeks and a recovery group. There were no significant effects on hematopoietic parameters from exposure to 10 ppm benzene or less. Exposure of mice to 100 and 200 ppm benzene reduced the number of total bone marrow cells, progenitor cells, differentiating hematopoietic cells, and most blood parameters. Replication of primitive progenitor cells in the bone marrow was increased during the exposure period as a compensation for the cytotoxicity induced by 100 and 200 ppm benzene. In mice exposed to 200 ppm benzene, the primitive progenitor cells maintained an increased percentage of cells in S-phase through 25 days of recovery compared with controls. The increased replication of primitive progenitor cells in concert with the reported genotoxicity induced by benzene provides the components necessary for producing an increased incidence of lymphoma in mice. Furthermore, we propose this mode of action as a biologically plausible mechanism for benzene-induced leukemia in humans exposed to high concentrations of benzene.

A 90-Day Chloroform Inhalation Study in F-344 Rats: Profile of Toxicity and Relevance to Cancer Studies

Chloroform acts via a nongenotoxic-cytotoxic mode of action to produce cancer if given in doses and at dose rates sufficiently high to produce organ-specific toxicity. In a recent study, chloroform failed to induce cancer in male or female F-344 rats when administered by inhalation for 2 years at 90 ppm, 5 days/week. The present study was undertaken to define the concentration-response curves for chloroform-induced lesions and regenerative cell proliferation in the F-344 rat when exposed by inhalation and to correlate those patterns of toxicity with the results from the inhalation cancer bioassay. Male and female F-344 rats were exposed to airborne concentrations of 0, 2, 10, 30, 90, or 300 ppm chloroform 6 hr/day, 7 days/week for 4 days or 3, 6, 13 weeks. Additional treatment groups were exposed 5 days/week for 13 weeks or were exposed for 6 weeks and held until Week 13. Bromodeoxyuridine was administered via osmotic pumps implanted 3.5 days prior to necropsy and the labeling index (LI, percentage of nuclei in S-phase) was evaluated immunohistochemically. A full-screen necropsy identified the kidney, liver, and nasal passages as the only target organs. This study confirmed that 300 ppm is extremely toxic and would be inappropriate for longer-term cancer studies. The primary target in the kidney was the epithelial cells of the proximal tubules of the cortex, with significantly elevated increases in the LI at concentrations of 30 ppm and above. However, only a marginal increase in the renal LI in the males was seen after exposures of 90 ppm, 5 days/week. Chloroform induced hepatic lesions in the midzonal and centrilobular regions with increases in the LI throughout the liver, but only at 300 ppm exposures. An additional liver lesion seen only at the highly hepatotoxic concentration of 300 ppm was numerous intestinal crypt-like ducts surrounded by dense connective tissue. Enhanced bone growth and hypercellularity in the lamina propria of the ethmoid turbinates of the nose occurred at the early time points at concentrations of 10 ppm and above. At 90 days there was a generalized atrophy of the ethmoid turbinates at concentrations of 2 ppm and above. Cytolethality and regenerative cell proliferation are necessary but not always sufficient to induce cancer because of tissue, sex, and species differences in susceptibility. A combination of a lack of direct genotoxic activity by chloroform, only a marginal induction of cell proliferation in the male rat kidney, and lower tissue-specific susceptibility in the female rat is apparently responsible for the reported lack of chloroform-induced cancer in a long-term inhalation bioassay with F-344 rats.

In vivo mutagenicity of ethylene oxide at the hprt locus in T-lymphocytes of B6C3F1 lacI transgenic mice following inhalation exposure

Ethylene oxide (EO) is a direct-acting alkylating agent with the potential to induce cytogenetic alterations, mutations, and cancer. In the present study, the in vivo mutagenicity of EO at the hypoxanthine guanine phosphoribosyltransferase (hprt) locus of T-lymphocytes was evaluated following inhalation exposure of male B6C3F1 lacI transgenic mice. For this purpose, groups of male Big Blue mice at 6-8 (n = 4/group) and 8-10 (n = 5/group) weeks of age were exposed to 0, 50, 100, or 200 ppm EO for 4 weeks (6 h/day, 5 days/week). At necropsy, T-cells were isolated from thymus and/or spleen and cultured in the presence of concanavalin A, IL-2, and 6-thioguanine [Skopek, T.R., V.E. Walker, J.E. Cochrane et al. (1992) Proc. Natl. Acad. Sci. USA, 89, 7866-7870]. The time course for expression of hprt-negative lymphocytes in thymus was determined in mice necropsied 2 h, 2 weeks, and 8 weeks after exposure to 200 ppm EO. The dose-response for hprt mutant T-cells in thymus and spleen was defined in mice necropsied 2 and 8 weeks post-exposure, respectively. The hprt mutant frequency (Mf) in thymus of exposed mice was increased 2 h after exposure and reached a maximum of 7.5 +/- 0.9 x 10(-6) (average Mf +/- SE) at 2 weeks post-exposure, compared with 2.3 +/- 0.8 x 10(-6) in thymus of control mice. Dose-related increases in hprt Mfs were found in thymus from mice exposed to 100 and 200 ppm EO. In addition, a nonlinear dose-dependent increase in hprt Mfs was observed in splenic T-cells, with greater mutagenic efficiency (mutations per unit dose) found at higher concentrations than at lower concentrations of EO. Average induced Mfs (i.e. induced Mf = treatment Mf - background Mf) in splenic T-cells were 1.6, 4.6, and 11.9 x 10(-6) following exposures to 50, 100, or 200 ppm EO, respectively, while the average control Mf value was 2.2 +/- 0.3 x 10(-6). In aliquots of lymphocytes (both B- and T-cells) isolated from spleen for analysis of lacI mutations in the same animals, only two of three EO-exposed mice at the 200 ppm exposure level demonstrated an elevated lacI Mf and these elevations were apparently due to the in vivo replication of preexisting mutants and not due to the induction of new mutations associated with EO exposure [Sisk, S., L.J. Pluta, K.G. Meyer and L. Recio (1996) Mutation Res., submitted]. These data demonstrate that repeated inhalation exposures to high concentrations of EO produce dose-related increases in mutations at the hprt locus of T-lymphocytes in male lacI transgenic mice of B6C3F1 origin.

Respiratory Tract Responses in Male Rats Following Subchronic Acrolein Inhalation

The goal of this study was to characterize the respiratory tract toxicity of acrolein, including nasal and pulmonary effects, in adult male F344 rats. Animals underwent whole-body exposure to 0, 0.02, 0.06, 0.2, 0.6, or 1.8 ppm acrolein for 6 hr/day, five days/week for up to 65 exposure days (13 exposure weeks). Respiratory tract histopathology was evaluated after 4, 14, 30, and 65 exposure days, as well as 60 days after the end of the 13 week exposure. Acrolein exposure was associated with reduced body weight gain. Rats exposed to ≥ 0.06 ppm acrolein had depressed terminal body weights when compared with air-exposed controls. Histologic evaluation of the nasal cavity showed olfactory epithelial inflammation and olfactory neuronal loss (ONL) following exposure to 1.8 ppm acrolein. Moderately severe ONL in the dorsal meatus and ethmoid turbinates occurred within four days while septal involvement developed with ongoing exposure. A rostral-caudal gradient in lesion severity was noted, with the anterior portion of the nasal cavity being more severely affected. Acrolein exposure was associated with inflammation, hyperplasia, and squamous metaplasia of the respiratory epithelium. The lateral wall was amongst the most sensitive locations for these responses and increased respiratory epithelial cell proliferation occurred at this site following 4 to 30 days of exposure to ≥ 0.6 ppm acrolein. The NOAEL for nasal pathology seen in this study was 0.2 ppm acrolein.

Inhaled iron, unlike manganese, is not transported to the rat brain via the olfactory pathway.

Iron and manganese share structural, biochemical, and physiological similarities. The objective of this study was to determine whether iron, like manganese, is transported to the rat brain via the olfactory tract following inhalation exposure. Eight-week-old male CD rats were exposed to approximately 0.31 mg Fe per m(3) (mass median aerodynamic diameter = 2.99 microm; geometric standard deviation = 1.15) via inhalation for a target duration of 90 min. Following exposure, rats were euthanized immediately (0) or at 1, 2, 4, 8, or 21 days postexposure. In addition to nasal and regional brain tissues, blood, and viscera were also collected. 59Fe concentrations were determined by gamma spectrometry. Further, heads were collected and frozen, and autoradiograms were prepared to visualize the location of 59Fe from the nose to the brain. Finally, olfactory mucosa samples collected at 0, 2, 4, and 21 days postexposure were further analyzed using high-performance liquid chromatography (HPLC) plus gamma spectroscopy to determine the association between 59Fe and transferrin. Data obtained from gamma spectrometry revealed that most of the iron remained in the nasal regions of the olfactory system and that less than 4% of iron deposited on the olfactory mucosa was observed in the olfactory bulb. Autoradiograms confirmed the data obtained from gamma spectrometry. 59Fe activity was absent in the olfactory regions of the brain even 4 days postexposure. Further, HPLC-gamma spectroscopy analyses indicated that 59Fe in the olfactory mucosa was coeluted with transferrin. Hence iron, unlike manganese, is not readily transported to the brain via the olfactory tract.