Single-walled carbon nanotube-induced mitotic disruption.

Carbon nanotubes were among the earliest products of nanotechnology and have many potential applications in medicine, electronics, and manufacturing. The low density, small size, and biological persistence of carbon nanotubes create challenges for exposure control and monitoring and make respiratory exposures to workers likely. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to 24, 48 and 96 µg/cm(2) single-walled carbon nanotubes (SWCNT). To investigate mitotic spindle aberrations at concentrations anticipated in exposed workers, primary and immortalized human airway epithelial cells were exposed to SWCNT for 24-72 h at doses equivalent to 20 weeks of exposure at the Permissible Exposure Limit for particulates not otherwise regulated. We have now demonstrated fragmented centrosomes, disrupted mitotic spindles and aneuploid chromosome number at those doses. The data further demonstrated multipolar mitotic spindles comprised 95% of the disrupted mitoses. The increased multipolar mitotic spindles were associated with an increased number of cells in the G2 phase of mitosis, indicating a mitotic checkpoint response. Nanotubes were observed in association with mitotic spindle microtubules, the centrosomes and condensed chromatin in cells exposed to 0.024, 0.24, 2.4 and 24 µg/cm(2) SWCNT. Three-dimensional reconstructions showed carbon nanotubes within the centrosome structure. The lower doses did not cause cytotoxicity or reduction in colony formation after 24h; however, after three days, significant cytotoxicity was observed in the SWCNT-exposed cells. Colony formation assays showed an increased proliferation seven days after exposure. Our results show significant disruption of the mitotic spindle by SWCNT at occupationally relevant doses. The increased proliferation that was observed in carbon nanotube-exposed cells indicates a greater potential to pass the genetic damage to daughter cells. Disruption of the centrosome is common in many solid tumors including lung cancer. The resulting aneuploidy is an early event in the progression of many cancers, suggesting that it may play a role in both tumorigenesis and tumor progression. These results suggest caution should be used in the handling and processing of carbon nanotubes.

Induction of aneuploidy by single-walled carbon nanotubes.

Engineered carbon nanotubes are newly emerging manufactured particles with potential applications in electronics, computers, aerospace, and medicine. The low density and small size of these biologically persistent particles makes respiratory exposures to workers likely during the production or use of commercial products. The narrow diameter and great length of single-walled carbon nanotubes (SWCNT) suggest the potential to interact with critical biological structures. To examine the potential of nanotubes to induce genetic damage in normal lung cells, cultured primary and immortalized human airway epithelial cells were exposed to SWCNT or a positive control, vanadium pentoxide. After 24 hr of exposure to either SWCNT or vanadium pentoxide, fragmented centrosomes, multiple mitotic spindle poles, anaphase bridges, and aneuploid chromosome number were observed. Confocal microscopy demonstrated nanotubes within the nucleus that were in association with cellular and mitotic tubulin as well as the chromatin. Our results are the first to report disruption of the mitotic spindle by SWCNT. The nanotube bundles are similar to the size of microtubules that form the mitotic spindle and may be incorporated into the mitotic spindle apparatus.

Changes in asthma-like responses after extended removal from exposure to trimellitic anhydride in the Brown Norway rat model.

BACKGROUND: Organic acid anhydride-induced occupational asthma is considered to be IgE-mediated. Airway and skin exposure are the two main routes of sensitization in the work place. Recently we developed an allergic asthmatic Brown Norway rat model sensitized by dermal exposure to trimellitic anhydride (TMA) using an occlusion patch application. OBJECTIVES: The objectives of this study were (1) to develop a model of non-occluded dermal exposure leading to allergic sensitization and (2) to examine the effect of extended removal from exposure on persistence of both specific IgE and TMA aerosol-induced airway responses in this model. METHODS: TMA powder (4 or 40 mg) was applied, unoccluded, to the skin of rats for 4 h, once/week for 4 weeks. Rats were given a 10-min aerosol challenge to 40 mg/m(3) TMA 2 weeks after the last dermal exposure (day 35). Another group was challenged on day 35 and again 18-24 months later. Respiratory enhanced pause (Penh), pulmonary histopathology and inflammation and specific IgE titres were measured. RESULTS: Rats produced dose-dependent specific IgE titres after exposure and developed early-phase (EAR) and late-phase airway responses (LAR) after airway challenge to TMA aerosol as well as airway eosinophilic inflammation. Specific airway responses were still manifested after a second TMA airway challenge given 18-24 months following the initial airway challenge. While persistent, airway inflammation, specific IgE and EAR were significantly attenuated following the second TMA challenge. LAR remained robust at 18-24 months and was not significantly different from the response on day 35. CONCLUSIONS: These results demonstrate the persistence of chemical sensitization and further suggest that IgE is not essential for LAR.

Inhalation vs. aspiration of single-walled carbon nanotubes in C57BL/6 mice: inflammation, fibrosis, oxidative stress, and mutagenesis.

Nanomaterials are frontier technological products used in different manufactured goods. Because of their unique physicochemical, electrical, mechanical, and thermal properties, single-walled carbon nanotubes (SWCNT) are finding numerous applications in electronics, aerospace devices, computers, and chemical, polymer, and pharmaceutical industries. SWCNT are relatively recently discovered members of the carbon allotropes that are similar in structure to fullerenes and graphite. Previously, we (47) have reported that pharyngeal aspiration of purified SWCNT by C57BL/6 mice caused dose-dependent granulomatous pneumonia, oxidative stress, acute inflammatory/cytokine responses, fibrosis, and decrease in pulmonary function. To avoid potential artifactual effects due to instillation/agglomeration associated with SWCNT, we conducted inhalation exposures using stable and uniform SWCNT dispersions obtained by a newly developed aerosolization technique (2). The inhalation of nonpurified SWCNT (iron content of 17.7% by weight) at 5 mg/m(3), 5 h/day for 4 days was compared with pharyngeal aspiration of varying doses (5-20 microg per mouse) of the same SWCNT. The chain of pathological events in both exposure routes was realized through synergized interactions of early inflammatory response and oxidative stress culminating in the development of multifocal granulomatous pneumonia and interstitial fibrosis. SWCNT inhalation was more effective than aspiration in causing inflammatory response, oxidative stress, collagen deposition, and fibrosis as well as mutations of K-ras gene locus in the lung of C57BL/6 mice.

Popcorn worker's lung: in vitro exposure to diacetyl, an ingredient in microwave popcorn butter flavoring, increases reactivity to methacholine.

Workers who inhale microwave popcorn butter flavorings experience decrements in lung function and can develop clinical bronchiolitis obliterans, i.e., "popcorn worker's lung" (Kreiss, K., Gomaa, A., Kullman, G., Fedan, K., Simoes, E.J., Enright, P.L., 2002. Clinical bronchiolitis obliterans in workers at a microwave-popcorn plant. N. Engl. J. Med. 347, 330-338.). In a rat inhalation model, vapors of an artificial butter flavoring damaged the epithelium of the upper and lower airways (Hubbs, A.F., Battelli, L.A., Goldsmith, W.T., Porter, D.W., Frazer, D., Friend, S., Schwegler-Berry, D., Mercer, R.R., Reynolds, J.S., Grote, A., Castranova, V., Kullman, G., Fedan, J.S., Dowdy, J., Jones, W.G., 2002. Necrosis of nasal and airway epithelium in rats inhaling vapors of artificial butter flavoring. Toxicol. Appl. Pharmacol. 185, 128-135.). Diacetyl, a butter flavoring component, is a major volatile ketone in the popcorn-processing workplace. We investigated the effects of diacetyl on epithelium of guinea pig isolated airway preparations and the effects of diacetyl in vitro on reactivity to bronchoactive agents. In the isolated, perfused trachea preparation, diacetyl added to the intraluminal (mucosal) bath elicited responses that began with contraction (threshold ca. 3 mM) and ended with relaxation. After a 4-h incubation with intraluminal diacetyl (3 mM), contractions to extraluminal (serosal) methacholine (MCh) were slightly increased; however, sensitivity to intraluminally (mucosally) applied MCh was increased by 10-fold. Relaxation responses of MCh (3 x 10(-7) M)-contracted tracheas to extraluminally applied terbutaline and intraluminally applied 120 mM KCl, to evoke epithelium-derived relaxing factor release, were unaffected by diacetyl. Exposure of the tracheal epithelium in Ussing chambers to diacetyl decreased transepithelial potential difference and resistance. These findings suggest that diacetyl exposure compromised epithelial barrier function, leading to hyperreactivity to mucosally applied MCh. The respiratory epithelium appears to serve as an initial target for the toxic effects of diacetyl in the airways.

Necrosis of nasal and airway epithelium in rats inhaling vapors of artificial butter flavoring.

As the result of a high prevalence of fixed airways obstruction in workers at a microwave popcorn manufacturing plant, we examined the hypothesis that vapors of butter flavoring used in the manufacture of microwave popcorn and other foods can produce airway injury in rats. Rats were exposed to vapors liberated from heated butter flavoring. Rats were exposed for 6 h by inhalation and were necropsied 1 day after exposure. The exposure was found by GC-MS analysis to be a complex mixture of various organic gases with the major peaks consisting of diacetyl (2,3-butanedione), acetic acid, acetoin (3-hydroxy-2-butanone), butyric acid, acetoin dimers, 2-nonanone, and delta-alkyl lactones. Diacetyl was used as a marker of exposure concentration. In the lung, butter flavoring vapors containing 285-371 ppm diacetyl caused multifocal, necrotizing bronchitis, which was most consistently present in the mainstem bronchus. Alveoli were unaffected. Butter flavoring vapors containing 203-371 ppm diacetyl caused necrosuppurative rhinitis, which affected all four levels of the nose. Within the posterior two nasal levels (T3 and T4), necrosis and inflammation was principally localized to the nasopharyngeal duct. Control rats were unaffected. Therefore, concentrations of butter flavoring vapors that can occur during the manufacture of foods are associated with epithelial injury in the nasal passages and pulmonary airways of rats.

Time course of pulmonary response of rats to inhalation of crystalline silica: histological results and biochemical indices of damage, lipidosis, and fibrosis.

Previous studies have determined that alpha-quartz (crystalline silica) can cause pulmonary inflammation, damage, and fibrosis. However, the temporal relationship between silica inhalation and pulmonary inflammation, damage, and fibrosis has not been fully examined. To address this gap in our knowledge of silica-induced pulmonary fibrosis, a chronic inhalation study using rats was designed. Specifically, rats were exposed to a silica aerosol (15 mg/m3 silica, 6 h/d, 5 d/wk, 116 d), and measurements of pulmonary inflammation, damage, and fibrosis were monitored throughout the study. We report (1) data demonstrating that the silica aerosol generation and exposure system produced a consistent silica aerosol of respirable size particles; (2) the time course of silica deposition in the lung; (3) calculations that demonstrate that the rats were not in pulmonary overload; (4) histopathological data demonstrating time-dependent enhancement of silica-induced alveolitis, epithelial hypertrophy and hyperplasia, alveolar lipoproteinosis, and pulmonary fibrosis in the absence of overload; and (5) biochemical data documenting the development of lipidosis, lung damage, and fibrosis.

Biological and statistical approaches to predicting human lung cancer risk from silica.

Chronic inflammation is a key step in the pathogenesis of particle-elicited fibrosis and lung cancer in rats, and possibly in humans. In this study, we compute the excess risk estimates for lung cancer in humans with occupational exposure to crystalline silica, using both rat and human data, and using both a threshold approach and linear models. From a toxicokinetic/dynamic model fit to lung burden and pulmonary response data from a subchronic inhalation study in rats, we estimated the minimum critical quartz lung burden (Mcrit) associated with reduced pulmonary clearance and increased neutrophilic inflammation. A chronic study in rats was also used to predict the human excess risk of lung cancer at various quartz burdens, including mean Mcrit (0.39 mg/g lung). We used a human kinetic lung model to link the equivalent lung burdens to external exposures in humans. We then computed the excess risk of lung cancer at these external exposures, using data of workers exposed to respirable crystalline silica and using Poisson regression and lifetable analyses. Finally, we compared the lung cancer excess risks estimated from male rat and human data. We found that the rat-based linear model estimates were approximately three times higher than those based on human data (e.g., 2.8% in rats vs. 0.9-1% in humans, at mean Mcrit lung burden or associated mean working lifetime exposure of 0.036 mg/m3). Accounting for variability and uncertainty resulted in 100-1000 times lower estimates of human critical lung burden and airborne exposure. This study illustrates that assumptions about the relevant biological mechanism, animal model, and statistical approach can all influence the magnitude of lung cancer risk estimates in humans exposed to crystalline silica.

IgG subclass responses in experimental silicosis.

Silicosis is a crippling fibrotic lung disease induced by inhaling crystalline silica. In addition to fibrosis, silica inhalation by humans is associated with a number of immunological effects including increased levels of serum immunoglobulins (in particular IgG), increased prevalence of autoantibodies, and autoimmune disease. Recent studies using rodent models have shown that experimental silicosis is associated with a T-helper (TH)1 pattern of T-cell activation in the lungs and lung-associated lymph nodes after silica inhalation, which are also the sites of increased IgG production. We therefore hypothesized that the subclass distribution of IgG production occurring in experimental silicosis would suggest TH1 activation as the primary stimulus for IgG production. Using an ELISPOT assay, we found increased IgG-secreting spot-forming cells of all IgG subclasses in lung-associated lymph nodes taken from silica-exposed rats 3 to 4 months after aerosol exposure to silica. Neither TH1- nor TH2-dependent IgG subclass-secreting cells were selectively enhanced. Our findings suggest that TH1 activation alone does not account for increased production of IgG in experimental silicosis.

Comparative pulmonary toxicity of 6 abrasive blasting agents.

Inhalation of silica dust is associated with pulmonary fibrosis. Therefore, substitute abrasive materials have been suggested for use in abrasive blasting operations. To date, toxicological evaluation of most substitute abrasives has been incomplete. Therefore, the objective of this study was to compare the pulmonary toxicity of a set of substitute abrasives (garnet, staurolite, coal slag, specular hematite, and treated sand) to that of blasting sand. Rats were exposed to blasting sand or an abrasive substitute by intratracheal instillation and pulmonary responses to exposure were monitored 4 weeks postexposure. Pulmonary damage was monitored as lactate dehydrogenase (LDH) in the acellular lavage fluid. Pulmonary inflammation was evaluated from the yield of polymorphonuclear leukocytes (PMN) obtained by bronchoalveolar lavage. The activity of alveolar macrophages was determined by measuring zymosan-stimulated chemiluminescence. Blasting sand caused lung damage and showed histologic evidence for inflammation and fibrosis. Garnet, staurolite, and treated sand exhibited toxicity and inflammation that were similar to blasting sand, while coal slag caused greater pulmonary damage and inflammation than blasting sand. In contrast, specular hematite did not significantly elevate LDH or PMN levels and did not stimulate macrophage activity 4 weeks postexposure.

Immunoglobulin responses to experimental silicosis.

Silicosis is a crippling fibrotic lung disease induced by inhalation of crystalline silica. One feature of silicosis is systemic and pulmonary immune dysfunction characterized in part by elevations in serum and bronchoalveolar lavage (BAL) immunoglobulins. A major specific aim of the current report was to demonstrate that an experimental model of silicosis previously well characterized for the development of pulmonary inflammation and fibrosis would also exhibit increased levels of serum and BAL IgG and IgM similar to those of human silicosis. We also sought to document the anatomic compartments responsible for these immunoglobulin responses. To address these specific aims, we compared levels of IgG and IgM in serum and BAL from rats with experimental silicosis induced by inhalation of silica with levels of these immunoglobulins in titanium dioxide (TiO(2))- and sham (air)-exposed controls. The ability of mononuclear cell populations from lung, lung-associated lymph node, and spleen to produce IgG and IgM ex vivo were also compared. We found that experimental silicosis was associated with elevated IgG and IgM levels in blood and BAL relative to the control groups. Our findings also suggested that draining lung-associated lymph nodes (LALN) were the most important sites for increased IgG and IgM production in experimental silicosis, with lungs contributing to a lesser degree. Increased production in the LALN appeared related to marked expansion in total numbers, but not relative proportion, of B lymphocytes.

A characterization of amiodarone-induced pulmonary toxicity in F344 rats and identification of surfactant protein-D as a potential biomarker for the development of the toxicity.

Amiodarone (AD) is gaining support as a first-line antiarrhythmic drug despite its potentially fatal pulmonary toxicity involving inflammation and fibrosis. The goals of this study were to characterize a rat model of AD-induced pulmonary toxicity (AIPT) and identify a serum biomarker to aid in the diagnosis of the onset of pulmonary toxicity. Male F344 rats were instilled intratracheally with AD (6.25 mg/kg with a 3.125 mg/ml solution) in sterile water or the sterile water vehicle on days 0 and 2, a protocol that led to the development of pulmonary fibrosis on day 28 in the AD-treated animals. Animals were killed on days 3, 5, 6, 7, or 10 and bronchoalveolar lavage (BAL) was performed. Recovery of alveolar macrophages and eosinophils was increased on days 3 and 5, while neutrophil recovery and albumin levels in the first BAL fraction were significantly elevated only on day 3. BAL cells recovered from AD-treated rats at day 3 produced more phorbol myristate acetate-stimulated luminol-dependent chemiluminescence (LDCL) over 20 min than BAL cells from control rats. Experiments using specific inhibitors implicated superoxide and nitric oxide in at least part of the LDCL response. Serum levels of surfactant protein-D (SP-D), a surfactant-associated protein, were increased concurrently with the inflammatory response in the lungs. These findings indicate that this model exhibits transient pulmonary inflammation and damage, with the potential for elevated oxidant production in the lungs and subsequent pulmonary fibrosis. Also, SP-D is proposed as a specific biomarker to monitor the onset of AIPT in this model.

Ovalbumin aeroallergen exposure-response in Brown Norway rats.

A major route of exposure to allergens is through the respiratory tract. Comparatively few animal studies have used aerosolized high-molecular-weight allergens for sensitization, and in these studies, proper characterization of the aeroallergen exposure was usually missing. The purpose of this study was to profile the exposure-response relationship in Brown Norway rats (BNR) to well-characterized ovalbumin (OVA) aerosols. Rats were exposed 30 min/wk x 6 wk to respirable OVA aerosols from <1 mg/m(3) to 64 mg/m(3) air. Ovalbumin-specific circulating immunoglobulin (Ig)E, IgG, and IgA were measured throughout the study period. Rats were sacrificed 1 day after the last exposure. Pulmonary tissue was processed for histopathological and histochemical analysis. Tracheas were isolated, perfused, and assessed for in vitro responsiveness to methacholine. Serum concentrations of OVA-specific antibodies increased with both exposure concentration and number of exposures. The number of BNR with measurable titers also increased with both dose and time. Pulmonary inflammatory changes were noted only in BNR exposed to higher OVA concentrations (15 and 64 mg/m(3) air). Increased tracheal reactivity to methacholine was not found in any of the sensitized BNR. In summary, sustained aeroallergen concentration-dependent changes in specific antibody responses and pulmonary inflammation have been demonstrated.

Transforming and carcinogenic potential of cadmium chloride in BALB/c-3T3 cells.

A large number of workers are potentially exposed to cadmium during mining and processing. Therefore, there is a concern regarding the potential carcinogenic hazards of cadmium to exposed workers. Studies have been performed to determine if cadmium chloride (CdCl(2)) can induce morphological cell transformation, DNA from CdCl(2)-induced transformed cells can transform other mammalian cells, and the transformed cells induced by CdCl(2) can form tumors in nude mice. BALB/c-3T3 cells were treated with different concentrations of CdCl(2) for 72 h. The frequency of transformed foci from each treatment was determined after cells were cultured for 4 to 5 weeks. DNAs from five CdCl(2)-induced transformed cell lines were isolated and gene transfection assay was performed using NIH-3T3 cells. Non-transformed BALB/c-3T3 cells and cells from 10 transformed cell lines induced by CdCl(2) were injected into both axillary regions of nude mice. Mice were screened once a week for the appearance and size of tumors. CdCl(2) caused a statistically significant, concentration-related increase in the transformation frequency. DNA from all five CdCl(2)-induced transformed cell lines tested was found to induce varying degrees of transfection-mediated transformation in NIH-3T3 cells. All 10 CdCl(2)-induced transformed cell lines formed fibrosarcomas in nude mice within 39 days of inoculation. Within this time period, no tumors were found in nude mice injected with non-transformed BALB/c-3T3 cells. These results indicate that CdCl(2) is capable of inducing morphological cell transformation and that the transformed cells induced by CdCl(2) are potentially tumorigenic.

Use of tetrandrine to differentiate between mechanisms involved in silica-versus bleomycin-induced fibrosis.

Animals exposed to silica or bleomycin (BLM) develop pulmonary fibrosis. Tetrandrine (TT) has been shown to inhibit stimulant-induced macrophage respiratory burst and effectively reduce silica-induced lung injury. The present study employed TT as a probe to assess the differences in mechanisms involved in silica- and BLM-induced pulmonary responses. Rats received a single intratracheal instillation of silica (40 mg/rat, sacrificed 4 wk postexposure) or BLM (1 mg/kg or approximately 0.25 mg/rat, sacrificed up to 2 wk postexposure). TT was administered orally at 18 mg/kg, 3 times/wk for desired time periods beginning 5 d before silica or BLM exposure. Both the silica and BLM exposures resulted in a significant increase in lung weight, total protein, lactate dehydrogenase (LDH), and phospholipids (PL) content in the acellular fluid from the first lavage, and hydroxyproline content in the lung tissue. Alveolar macrophages (AM) isolated from rats exposed to silica or BLM exhibited significant increases in secretion of interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-alpha), and transforming growth factor beta (TGF-beta). TT treatment significantly lowered the silica- or BLM-induced increase in lung weight, while marginally reducing the release of IL-1 and TNF-alpha by AM. TT, however, markedly inhibited the silica-induced increase in the acellular protein, LDH and PL, hydroxyproline content, and the production of TGF-beta by AM but had no marked effect on these same parameters in BLM-exposed rats. Histological examination of rats exposed to BLM for 14 d showed pulmonary inflammation and fibrosis. TT treatment had only a small effect on limiting the extent of these lesions and did not significantly affect their severity. In summary, data indicate that many inflammatory and fibrotic effects of in vivo silica exposure are substantially attenuated by TT, whereas the stimulation by BLM is only marginally affected by this drug. Since TT acts to attenuate AM-mediated reactions, these results suggest that AM may play a pivotal role in silica-induced fibrotic development and may be less involved in the pathogenesis of BLM-induced fibrosis.

Acute inflammatory reaction in rats after intratracheal instillation of material collected from a nylon flocking plant.

Several cases of interstitial lung disease have been diagnosed among workers at a nylon flock plant, but the etiologic agent for the disease outbreak was unknown. The results of a medical survey and industrial hygiene study indicated that the dust present in the plant may be responsible. Thus, airborne dust collected at the plant was examined for its inflammatory potential in rat lungs. The endpoints measured were: (1) breathing rates, (2) differential cell counts of bronchoalveolar lavage cells, (3) alveolar macrophage (AM) chemiluminescence, (4) albumin concentration and matrix metalloprotease activities in the acellular fluid from the initial bronchoalveolar lavage, and (5) pulmonary histopathology. In the first study, rats received a single dose of the airborne dust sample (10 mg/kg body weight) by intratracheal (IT) instillation. At 1 d post-IT, all inflammatory endpoints were significantly increased versus controls, but by 29 d post-IT they did not differ significantly from controls. Histopathology demonstrated mild to moderate, multifocal, suppurative pneumonia, usually centered around bronchioles, at 1 d post-IT. At 29 d post-IT, pulmonary inflammation was minimal to mild and characterized by alveolar histocytosis usually restricted to the immediate area of retained bire-fringent fibers. In subsequent experiments, airborne dust was extracted with water and the dust (washed airborne dust) and water extract (soluble fraction) were separated by centrifugation for further study. Nylon tow dust was prepared in the laboratory by milling uncut nylon strands (called tow) that had not been treated with the finish or dyes that are commonly used in the flock plants. Rats were administered a single dose of a dust sample (10 mg/kg body weight) or the soluble fraction (1.3 ml/kg body weight) by IT administration and the same endpoints were measured at 1 d post-IT. The dust samples caused significant increases in all of the inflammatory endpoints; however, the soluble fraction was much less active. Histological analysis of the lungs 1 d post-IT confirmed lung inflammation was occurring and tended to center around bronchioles. The results suggest that: (1) nylon flocking generates particles of respirable size that can interact with AM in the lung and can be detected in the lung 29 d after exposure, (2) the dust samples examined cause an inflammatory response, (3) water-extractable agent(s) from airborne dust contribute only minimally to the inflammatory response, and (4) the acute inflammatory response to these dusts is substantial when compared to other pathologic occupational dusts previously examined in our laboratory.

Toxicity of inhaled 91YCl3 in dogs.

This study was conducted in dogs to determine the toxicity of inhaled 91YCl3, which is of interest because 91Y is a fission-product radionuclide that is abundant in a reactor inventory after sustained operation. Yttrium-91 has a short half-life, 59 days, and decays with the emission of beta particles and low-yield gamma rays. The study was conducted in 58 beagle dogs with equal numbers of males and females. Forty-six dogs inhaled the 91YCl3 aerosol, while 12 served as controls. Four exposure levels were used. To determine the long-term retained burden (LTRB) of 91Y, each dog was periodically whole-body counted and its excreta were analyzed radiochemically. Over time, the 91Y transferred from the lung primarily to the skeleton and liver. The dogs were observed over their life spans for biological effects. Fatal hematological dyscrasia occurred from 12 to 33 days after exposure in the dogs with the highest LTRBs. Bone-associated tumors of the nasal and oral mucosae occurred in 5 dogs from 2000 to 5800 days after they inhaled the 91YCl3 aerosols. Five dogs died with malignant lung tumors and 2 dogs with malignant liver tumors. The results of this study were compared to those from similar studies in beagles that inhaled 90SrCl2 or 144CeCl3 or were injected with 137CsCl. The comparison showed that the biological effects in each study were clearly dependent on the cumulative doses to critical organs.

Preneoplastic potential of morphologically distinct transformed foci induced by 3-methylcholanthrene.

Individual variability of scoring foci positive for transformation presents a difficult problem in assessing the transformation assay. In this study, an attempt was made to identify five morphologically distinct types of transformed foci based on size (2-3, 3-4, and > or = 4 mm in diameter), invasiveness (smooth vs. invading margins), and other properties (piling vs. spread) induced by 3-methylcholanthrene in Balb/c-3T3 cells. The transformed focal cells were used in in vitro studies including anchorage-independent analysis, focal reconstruction, gene transfection using NIH-3T3 host cells, and Southern blotting to assess amplification of five proto-oncogenes (K-ras, H-ras, c-fos, c-jun, c-myc) and a tumor suppressor (p53) gene. Results showed that 1) there was a significant increase in anchorage-independent growth of all five types of foci ranging from 7-12%; 2) all five morphological types of transformed foci showed 8-15% focal reconstruction; 3) DNA from all five types of transformed foci induced transformation in NIH-3T3 cells at a level significantly above the control DNA; 4) gene amplification studies indicated amplification in both K-ras and H-ras proto-oncogenes; however, c-fos, c-jun, and c-myc did not show DNA amplification. The tumor suppressor gene (p53) was activated and the increase was up to 3-fold over the normal Balb/c-3T3 DNA. These findings are consistent with our hypothesis that all five morphologically different foci have preneoplastic potential and that any foci of size > or = 2 mm regardless of invasiveness and piling should be scored as positive during the transformation assay.

Adjuvant effect of respiratory irritation on pulmonary allergic sensitization: time and site dependency.

It has been suggested that airway irritation, by acting as an adjuvant, as well as producing damage, may be an important factor related to asthma. The present study examined the window of time following acute upper and lower airway irritant exposure to determine the period of increased risk of immunological sensitization. Brown Norway rats were exposed to 87 ppm NO2 or 1000 ppm NH3 for 1 hr. A 30-min ovalbumin (OVA) exposure of 18.14 microg/liter air was given at various times based upon the time course of irritant associated inflammatory response (either immediately prior to or 1 or 7 days after the irritant exposure). OVA-only, NO2-only or NH3-only controls, and saline controls were also studied. Weekly booster exposures of OVA (or saline) were given. Circulating OVA-specific IgE, IgA, and IgG levels were quantified periodically during the 6 weeks of the study. Bronchoalveolar lavage (BAL) was also performed to examine the inflammatory response to allergic and irritant challenge. Significant increases in OVA-specific IgE, IgG, and IgA antibody titers were seen in rats given the sensitizing OVA exposure within 1 day of the NO2, but not NH3 exposures. Enhancement of cellular infiltrate in BAL was noted in groups given the sensitizing OVA exposure within 1 day of the NO2 or NH3. It is concluded that the inflammatory and immunological response to antigen exposure can be modified by the site of respiratory tract irritation and the relative times of irritant and antigen exposure.

Acute lung injury induced by a commercial leather conditioner.

Following a formulation change, a leather conditioner was involved in a 1992 nationwide outbreak of respiratory illness. We investigated the composition and toxicity of the conditioner produced before (previous product) and after (new product) the disease outbreak. The new product induced tachypnea, pulmonary edema, pulmonary hemorrhage, and sporadic deaths in exposed guinea pigs and rats. Ultrastructurally, these changes were associate with direct pulmonary cytotoxicity characterized by necrosis of alveolar type I cells and alveolar septal interstitial edema. Chemical analyses suggested major alterations in the fluorohydrocarbon constituents in the new formulation of the leather conditioner. While these alterations could not be specifically identified, they appeared to include changes from fluoralkanes to fluoroalkenes, fluorophenyl, and/or fluoroalcohol compounds. Changes in solvent composition were consistent with traces of 2-butoxyethanol and isomers of dipropylene glycol methyl ether, and additional C10-C12 alkanes. In this study, we demonstrated the toxicity of the new product in laboratory animals. Some of the altered constituents of the new product have been identified and are potential candidates for additional investigations to identify specific etiologic agents.