James K. Maurer

The comparison of a fibrogenic and two nonfibrogenic dusts by bronchoalveolar lavage

Analysis of bronchoalveolar lavage fluid (BALF) appears to be a sensitive approach to characterizing an acute inflammatory response within the lung. More work, however, is needed to determine if analyses of BALF endpoints can predict chronic responses (i.e., fibrosis). The objective of the present study was to compare the dose and temporal pulmonary response of a known fibrogenic agent, silica, and two known nonfibrogenic agents, aluminum oxide and titanium dioxide. Animals were instilled with silica (0, 0.2, 1.0, or 5.0 mg/100 g body wt), titanium dioxide (1.0 or 5 mg/100 g body wt), aluminium oxide (1.0 or 5.0 mg/100 g body wt) or saline. Animals (n = 5/group) were terminated 1, 7, 14, 28, and 63 days following instillation, and the BALF was characterized by biochemical and cellular assays. Histopathological changes were determined at 60 days after exposure. The biochemical results demonstrated BALF levels of lactate dehydrogenase (LDH), beta-glucuronidase (BG), N-acetylglucosaminidase (NAG), and total protein (TP) increased in a dose-related fashion at the earlier time points for all test materials, with the magnitude of change being greatest for silica. The temporal response for these parameters was significantly different for the two classes of materials. With time, the response for the fibrogenic dust steadily increased, while the levels for the nonfibrogenic dusts decreased toward normal values during the 2-month study period. Of the cellular changes, total cell numbers, neutrophils, and lymphocyte numbers were the most sensitive markers of the pulmonary response. As shown with the biochemical parameters, the cellular response to silica increased with time while that of the nuisance dusts did not. It was also found that, similar to inhalation studies, high doses of a nuisance dust may result in toxicity/inflammation. This toxicity at high dose levels emphasizes the importance of choosing relevant doses when comparing potentially fibrogenic and nonfibrogenic dusts. In conclusion, the persistent and progressive changes seen in the biochemical (LDH, TP, BG, NAG) and cellular parameters (total cells, neutrophils and lymphocytes) following silica administration correlated with the fibrotic response which occurred after exposure to this material. The less dramatic and transient changes seen with aluminum oxide and titanium dioxide correlated with the inert nature of these nuisance dusts. The results of this study indicate evaluation of BALF may provide a means to predict the chronic pulmonary response to a material.

Respiratory tract responses to dust: Relationships between dust burden, lung injury, alveolar macrophage fibronectin release, and the development of pulmonary fibrosis

A multidisciplinary approach was used to investigate the responses of the respiratory tract to silica (SiO2) or titanium dioxide (TiO2). Rats were intratracheally instilled with 5-100 mg/kg of dust and bronchoalveolar lavage fluid (BALF) lactate dehydrogenase (LDH) and total protein (TP) and ex vivo alveolar macrophage (AM) fibronectin release assessed on Days 7, 14, and 28 after exposure. Lung dust burdens were determined on Days 1, 7, and 28 after instillation. Both dusts elicited dose-related increases in BALF LDH and TP, a response which was more pronounced and progressive with SiO2. All doses of SiO2 elicited persistent increases in AM fibronectin release. TiO2 stimulated persistent increases in AM fibronectin release at greater than or equal to 50 mg/kg, with transient or no effect at less than or equal to 10 mg/kg. Increased SiO2 retention was observed for all doses and TiO2 retention was increased only at doses greater than or equal to 50 mg/kg. In vitro exposure of naive AM to SiO2 or TiO2 did not stimulate AM fibronectin release. Histopathology demonstrated fibrosis at all SiO2 doses; only TiO2 doses greater than or equal to 50 mg/kg resulted in fibrosis. These results reveal an association between increased dust retention, lung injury, activation of AM fibronectin release, and the development of fibrosis. The magnitude and temporal pattern of responses clearly differentiated SiO2 from TiO2. The correlation of BALF markers of lung injury and increased AM fibronectin release with the development of fibrosis supports the use of these parameters as predictive biomarkers of dust-induced interstitial lung disease.

Stimulation of rat alveolar macrophage fibronectin release in a cadmium chloride model of lung injury and fibrosis

Rats were exposed to saline or cadmium chloride (CdCl2) at 25, 100, or 400 micrograms/kg body weight by intratracheal instillation. At 3, 7, 14, and 28 days after exposure five animals/treatment were euthanized, the lungs were lavaged, and bronchoalveolar lavage fluid (BALF) was analyzed for lactate dehydrogenase (LDH), total protein, N-acetylglucosamindase (NAG), and cell number, type, and viability. Lung hydroxyproline concentration was characterized as a marker of lung collagen. Alveolar macrophages (AM) obtained in BALF were cultured and the release of fibronectin and TNF was determined. Lung tissue was examined microscopically at 28 and 90 days after exposure. Exposure to CdCl2 resulted in lung injury and inflammation demonstrated by increases in BALF LDH, total protein, NAG, and inflammatory cells. AM TNF release was not significantly changed by CdCl2 treatment. All doses of CdCl2 stimulated AM fibronectin secretion, a response which persisted throughout the 28-day postexposure period examined. Pulmonary fibrosis was demonstrated biochemically and/or histologically (trichrome staining tissue) at all CdCl2 dose levels. The association of CdCl2-induced AM fibronectin release with lung fibrosis confirms and extends previous observations relating AM-derived fibronectin to the development of interstitial lung disease and provides further evidence that the persistent increase in AM fibronectin release represents an early indicator of fibrosis.

Pulmonary response to inhaled silica or titanium dioxide

The pulmonary response to mineral dust inhalation was investigated by characterizing markers of lung injury and inflammation, macrophage activation, dust clearance, and histopathology. Rats were exposed (6 hr/day x 5 days) to air or 50 mg/m3 crystalline silica (SiO2) or titanium dioxide (TiO2). At 7, 14, 28, and 63 days after exposure, bronchoalveolar lavage fluid (BALF) was analyzed for lactate dehydrogenase (LDH), total protein, and N-acetylglucosaminidase, as well as cell number, type, and viability. Alveolar macrophages (AM) obtained in BALF were cultured with or without LPS and release of interleukin-1 (IL-1) and fibronectin was determined. Histopathology was conducted at 28 and 63 days. The exposure protocol resulted in 1.8-1.9 mg of mineral dust being deposited in the pulmonary region. Clearance of SiO2 was significantly less than TiO2. SiO2 increased BALF neutrophils (Days 14, 28, and 63), total protein (Days 28 and 63), and LDH and lymphocytes (Day 63). SiO2 increased AM-derived fibronectin release (Day 63) and LPS-induced IL-1 release (all time points), but not spontaneous release of IL-1. TiO2 did not change BALF biochemical or cellular parameters or AM secretory activity. Histopathology revealed minimal interstitial inflammation with SiO2 and no significant response in control or TiO2 rats. These results demonstrate the pulmonary response to inhaled SiO2 can be differentiated from the relatively innocuous TiO2 by changes in BALF markers of injury and inflammation further supporting the use of BALF analysis to make relative assessments of pulmonary toxicity. The stimulation of macrophage fibronectin release by the fibrogenic dust SiO2 and not TiO2 is consistent with a role for this glycoprotein in lung injury and repair. Last, the early and persistent effect of SiO2 on LPS-induced AM IL-1 release indicates this response may represent a sensitive early marker of dust-induced changes in the AM population.

Application of In Vivo Confocal Microscopy to the Understanding of Surfactant-Induced Ocular Irritation*

The purpose of this study was to assess the ability of in vivo confocal microscopy (CM) to provide noninvasively derived histopathologic correlates of surfactant-induced eye irritation from which specific pathologic mechanisms can be identified and later evaluated in alternative in vitro models. Rats and rabbits, divided into groups of 5, received 10 μl of an anionic or cationic surfactant in one eye with the other eye used as a control. At specified times, eyes were examined and scored for ocular irritancy using a penlight and slit-lamp. Subsequently, corneas were evaluated by in vivo CM to evaluate epithelial layer thickness and surface epithelial cell area, corneal thickness, depth of necrosis, inflammation, fibrosis, and endothelial injury. At 3 hr, the anionic surfactant produced slight irritation with peak scores of 12.4 and 8.0 out of a possible 110 in the rats and rabbits, respectively. In vivo CM revealed changes limited to the corneal epithelium that decreased in thickness to 78% in rats and 81% in rabbits at 3 hr. This decrease in the thickness correlated with a significant decrease in surface epithelial cell area from 2,061 ± 395 μm2 to 567 ± 330 μm2 in the rats and 1,523 ± 185 μm2 to 934 ± 71 μm2 in the rabbits (p < 0.005 and 0.005, respectively). The cationic surfactant produced severe irritation in both the rats and rabbits with peak scores of 85.4 and 80.2 occurring at day 2, respectively. In vivo CM in the rats showed complete loss of corneal epithelium, lysis of keratocytes, and loss of corneal endothelium. In the rabbits, injury appeared limited to the anterior cornea with complete loss of epithelium and loss of keratocytes extending to 52% of the corneal thickness. These findings establish the application of noninvasive, in vivo CM to qualitatively and quantitatively characterize the pathobiology of ocular irritation in situ. This information will be important in the development and evaluation of mechanistically based in vitro alternatives for ocular irritancy testing.

Alveolar macrophage cytokine and growth factor production in a rat model of crocidolite‐induced pulmonary inflammation and fibrosis

The present study was undertaken to further define the role of alveolar macrophages (AM) in the pulmonary response to crocidolite fibers. Briefly, groups of 4 male F344 rats were intratracheally instilled with saline or saline suspensions of crocidolite at 2 or 20 mg/kg body weight. Animals were sacrificed 3, 7, 14, and 28 d after exposure and the lung response was characterized by analysis of bronchoalveolar lavage fluid (BALF) for markers of lung injury and inflammation. AM obtained in BALF were cultured and their production of the pro-inflammatory cytokines, tumor necrosis factor alpha (TNF alpha), and interleukin-1 (IL-1) were characterized along with fibronectin, a protein known to stimulate fibroblast migration and proliferation. Lung hydroxyproline content was determined 28 d after exposure and lung histopathology was characterized on d 28 and 90 after exposure. Crocidolite instillation resulted in transient dose-related pulmonary inflammation as evidenced by increased numbers of BALF neutrophils at the low dose and neutrophils, macrophages, and lymphocytes at the high dose. Cytotoxicity and increased permeability were demonstrated by increased levels of BALF lactate dehydrogenase (LDH) and total protein, respectively. AM TNF alpha and IL-1 production were increased only at the high crocidolite dose. This cytokine response was greatest at d 3 and decreased thereafter. AM TNF alpha and IL-1 release were positively correlated with the increased BALF neutrophils. In contrast to TNF alpha and IL-1, AM fibronectin release was increased at both the low and high doses, with the magnitude of response increasing over time. Consistent with previous acute asbestos inhalation studies, histopathology revealed inflammation localized at the level of the terminal bronchioles and alveolar ducts. Fibrosis was demonstrated at both doses by increased trichrome staining of lung tissue sections. Only the high dose resulted in a detectable increase in lung hydroxyproline. Given the bioactivities of TNF alpha, IL-1, and fibronectin, their increased production after crocidolite exposure indicates they contribute to the pulmonary inflammation and fibrosis occurring with this mineral fiber. In addition, the correlation of increased AM TNF alpha and IL-1 production with increased BALF neutrophils supports a role for these cytokines in crocidolite-induced inflammatory cell recruitment. Lastly, association of a persistent increase in AM fibronectin production with an eventual increase in lung collagen deposition extends the growing database indicating this response is a predictive marker of pulmonary fibrosis.

Ocular Irritation: Microscopic Changes Occurring Over Time in the Rat with Surfactants of Known Irritancy

The pathology of surfactant-induced ocular irritation, especially in the context of accidental human exposures and animal tests used to assess a surfactants potential ocular irritation, is not well understood. The purpose of this study was to characterize the microscopic changes in rats at 3 hr and on days 1, 2, 3, 4, 7, 14, and 35 following treatment with anionic, cationic, and nonionic surfactants of differing irritancy. The right eye of each rat was treated by placing 10 μl of a surfactant directly on the cornea. Untreated left eyes served as the controls. At each time point, eyes and eyelids were macroscopically examined and collected for microscopic examination. Macroscopically, the differing levels of irritation were characterized by differences in incidence and magnitude of scores, reflecting involvement of the cornea, conjunctiva, and iris, as well as by the incidence of neovascularization and time to recovery. Microscopically, differences in the area and depth of injury paralleled the differences seen grossly and the relative irritancies of the various surfactants. All surfactants affected the corneal and conjunctival epithelium. All surfactants, except the slightly irritating anionic surfactant, caused corneal stromal changes, with this involvement being proportional to their overall level of irritation. Corneal endothelial cell effects principally occurred with only the severely irritating cationic surfactant. Over time, responses to surfactants of differing irritancy were qualitatively and quantitatively different, and these differences correlated with the extent of initial injury. Qualitative differences in response included presence of keratocyte regeneration, corneal neovascularization, and conjunctivalization of the corneal epithelium with all of the surfactants except the slight irritant. Quantitative differences in response occurred in the extent of epithelial regeneration, edema, and inflammation for surfactants of slight to severe irritancy, and with neovascularization, keratocyte regeneration, and conjunctivalization for surfactants of mild to severe irritancy. These results suggest that by defining initial area and depth of injury associated with an ocular irritant, it may be possible to predict the subsequent response and final outcome. Such an approach would be applicable to the development of mechanistically based in vitro assays.

Light Microscopic Comparison of Surfactant-Induced Eye Irritation in Rabbits and Rats at Three Hours and Recovery/Day 35*1

Limited information exists on the pathologic changes occurring with surfactant-induced ocular irritation in the context of accidental human exposures and animal tests used to assess for such irritation. The purpose of this study was to begin to characterize the pathologic changes that occur with surfactants in the context of standard animal tests and compare the response in rats to that in rabbits. Representative anionic, cationic, and nonionic surfactants causing slight to severe ocular irritation were directly applied to the corneas of rabbits and rats at a dose of 10 μl. Eyes and eyelids of each animal were macroscopically examined for signs of irritation beginning 3 hr after dosing and periodically until recovery or day 35. Eyes and eyelids from animals in each group were collected for microscopic examination after 3 hr and at recovery or day 35. Microscopically, all of the surfactants caused erosion, denudation, and/or necrosis of the conjunctival and corneal epithelium in rabbits and rats. Necrosis of keratocytes was observed in rabbits and rats treated with the severely irritating cationic surfactant and in rats treated with anionic surfactants that were mildly irritating and moderately irritating. Corneal endothelial changes were observed in rabbits and rats with only the cationic surfactant. Changes in eyes of rabbits and rats that had not recovered by day 35 included decreased prominence of goblet cells, conjunctivalization of the corneal epithelium, neovascularization and fibrosis of the cornea, and presence of devitalized stroma. Overall, the changes in rabbits and rats were similar and suggest that the rat may be used as a surrogate for the rabbit in studies to understand better mechanisms of surfactant-induced eye irritation.

Chronic toxicity and carcinogenicity studies of olestra in Fischer 344 rats.

Two 2-year feeding studies were carried out in Fischer 344 rats with olestra, a mixture of the hexa-, hepta- and octaesters of sucrose formed with long-chain fatty acids. Olestra was fed at 0, 0.99, 4.76 or 9.09% (w/w) of the diet in the first study, and at 0 or 9.09% (w/w) of the diet in the second. Daily observations, feed consumption and body weights, ophthalmoscopic examinations, organ weights, serum chemistry, haematology, urinalysis and histopathological evaluations revealed no evidence of any adverse effects associated with olestra ingestion. Relative to controls, there was a higher incidence of basophilic liver foci in olestra-fed female rats at 12 months. At 24 months, foci were observed in most animals in all groups but were more numerous in olestra-fed females. The foci were not associated with hepatic tumours, alterations in liver function, or increases in liver weight and therefore not considered to represent a toxic response to olestra. Isolated statistically significant differences in mortality, mononuclear cell leukaemia, and pituitary adenomas were observed but were not considered to be related to olestra ingestion since they were not reproducible across the two studies, generally not dose responsive, not consistent between sexes, and the incidences were within the ranges for historical and contemporary laboratory controls. The results of the two studies show that olestra was not toxic or carcinogenic when fed to rats at up to 9% of the diet for 24 months.

Pathology of Ocular Irritation with Bleaching Agents in the Rabbit Low-Volume Eye Test

Despite differences in the processes leading to tissue damage, the ocular irritation response to various surfactants, two concentrations of an acid and an alkali, and an acetone, alcohol, aromatic amine, and aldehyde has been shown to depend on the extent of initial injury. The purpose of this study was to assess the extent to which this fundamental relationship exists for bleaching agents in the rabbit low-volume eye test. Ten μl of sodium perborate monohydrate (NaBO 3), sodium hypochlorite (NaOCl), 10% hydrogen peroxide (H2O 2), and 15% H2O2 was applied directly to the cornea of the right eye of each rabbit. Macroscopic assessments for irritation were made 3 hours after dosing and periodically until 35 days. Light microscopic examinations were conducted on tissues obtained at 3 hr and on 1, 3, and 35 days. In vivo confocal microscopy (CM) and measurements of dead corneal epithelial cells and keratocytes at 3 hours and 1 day were used to characterize quantitatively initial corneal injury, while in vivo CM performed at 3 hours and 1, 3, 7, 14, and 35 days was used to characterize quantitatively the corneal changes over time. The changes with NaBO3 and NaOCl were consistent with mild irritancy. For both, corneal injury was limited to the epithelium and superficial stroma. The changes with 10% H2O2 and 15% H2O2 were consistent with severe irritation. Both concentrations affected the epithelium and deep stroma, with 15% H2O 2 also at times affecting the endothelium. However, unlike other irritants previously studied, with 10% H2O2 and 15% H2O 2 there was an incongruity between the extent of epithelial and stromal injury, with stromal injury being more extensive than epithelial injury. A similar, although less dramatic, effect was observed with NaBO3. Additionally, there was still significant keratocyte loss at 35 days with 10% H2O2 and 15% H2O2 even though the eyes at times were considered to be macroscopically normal. These observations highlight the need to include both epithelial and stromal components in an ex vivo or in vitro alternative assay. In conclusion, these results continue to support and extend our hypothesis that ocular irritation is principally defined by the extent of initial injury despite clear differences in the means by which irritants cause tissue damage. Importantly, we have identified unique differences in the ocular injury and responses occurring with bleaching agents that are important to consider in the development and validation of alternative ocular irritation tests to characterize a broad range of materials differing in type and irritancy.