Pathology of ocular irritation with acetone, cyclohexanol, parafluoroaniline, and formaldehyde in the rabbit low-volume eye test.

The ocular irritation responses to 11 different surfactants and two concentrations of acetic acid and sodium hydroxide have been shown to depend on the extent of initial injury, despite marked differences in the processes leading to tissue damage. The purpose of these studies was to determine the extent to which this fundamental relationship applies to other nonsurfactants. Ten microl of acetone (ACT). cyclohexanol (CY), parafluoroaniline (PF), or 37% formaldehyde (FA) was directly applied to the cornea of the right eye of each rabbit. Eyes and eyelids were macroscopically scored for signs of irritation beginning 3 hours after dosing and periodically until recovery or 35 days. Tissues were obtained for light microscopic examination after 3 hours and on days 1, 3, and 35. Initial corneal injury was characterized quantitatively at 3 hours and I day using in vivo confocal microscopy (CM) and by postmortem quantitation of dead corneal epithelial cells and keratocytes using a Live Dead Assay (L/D, Molecular Probes) and scanning laser CM. Corneal changes over time were characterized quantitatively using in vivo CM performed at 3 hours and 1, 3, 7, 14, and 35 days. The changes with ACT were consistent with mild irritation. Corneal injury was limited to the epithelium and superficial stroma, with the mean normalized depth of injury (NDI) being less than 10% with the majority of regions showing no stromal injury. Changes with CY and PF were consistent with moderate to severe irritation, and FA caused severe irritation. Specifically, corneal injury by CY and PF tended to involve the epithelium and anterior stroma, with the mean NDI being 10.4% to 23.8%, while injury with FA involved the epithelium, deep stroma, and at times the endothelium. Interestingly, with FA significantly less injury was observed at 3 hours with a dramatic increase in injury observed at 1 day and thereafter. 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. We believe this approach can be applied to developing alternative assays based on injury to ex vivo eyes or injury to an in vitro corneal equivalent system.

Use of in vivo confocal microscopy to understand the pathology of accidental ocular irritation.

In vivo confocal microscopy (CM) provides a unique ability to section optically through living, intact tissues and organs to characterize qualitatively and quantitatively pathological changes in 4 dimensions (x, y, and z, and time). It involves the capture of real-time images without the need for excision, fixation and processing. In vivo CM principally has been used for evaluation of eyes in patients and laboratory animals but has potential application to studies of other tissues/organs. In vivo CM is being used in human ophthalmology clinics. It has been used as a research tool for quantitative, in situ measurement of corneal wound contraction, fibroblast migration, corneal endothelial cell migration, corneal epithelial cell size and desquamation following contact lens wear and surgery, and the assessment of corneal surface toxicity following application of commonly used ophthalmic preservatives. In vivo CM allows us to (a) characterize changes to a light microscopic (i.e., cellular) level; (b) quantify changes objectively: (c) conduct studies of injury and repair in the same animal and directly correlate microscopic changes to clinical observations over time as this technique is used in the living animal; and (d) conduct comparative studies in humans. Here we present a brief overview of in vivo CM and how we are using it to provide noninvasive, in situ qualitative and quantitative histopathologic characterization of accidental ocular irritation. Our intent is to provide an awareness of this relatively new methodology and one practical application of its use in research. The goal of our work is to provide objective, quantitative data for use in developing and validating mechanistically based in vitro replacement tests.

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.

Confounded carcinogenicity study of sodium fluoride in CD-1 mice.

To determine its carcinogenic potential, sodium fluoride (NaF) was fed to CD-1 mice for up to 97 weeks. Mice given NaF at a dose of 4, 10, or 25 mg/kg of body weight per day added to a low-fluoride diet were compared to controls given either an unsupplemented low-fluoride diet or laboratory chow. Nonneoplastic changes consistent with those previously recognized from fluoride toxicity were observed in teeth, bones, and joints. Unexpectedly, osteomas occurred in all groups. The incidence of osteomas was similar in groups given the low-fluoride control diet, laboratory chow, or NaF doses of 4 or 10 mg/kg per day. The incidence of osteomas in these groups was increased over that historically experienced at the laboratory and reported in the literature for CD-1 mice. The incidence of osteomas in the mice given 25 mg NaF/kg per day added to a low-fluoride diet was increased over that in the other groups. Osteomas were first observed at Week 55. No malignant bone tumors were observed during the course of the study. The locations, multiplicity, and morphologic features of the osteomas in all groups were similar to those associated with virus-induced bone tumors. Electron microscopic examination revealed abundant retrovirus particles in all osteomas examined from control and test mice. It was concluded that the study was confounded by a retrovirus which contributed to the induction of the osteomas. Because the study was confounded, it cannot be considered a valid bioassay to be used for risk assessment.

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.

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.

Cytokine and growth factor release by alveolar macrophages: potential biomarkers of pulmonary toxicity.

Studies comparing pulmonary responses to crystalline silica (SiO2) and titanium dioxide (0.3 microns diameter, TiO2-F) demonstrated a positive correlation between alveolar macrophage (AM) release of interleukin-1 (IL-1), tumor necrosis factor (TNF) and fibronectin and, pulmonary granuloma formation, inflammation and fibrosis, respectively. AM IL-1 release was associated with the development of pulmonary granulomas after SiO2 exposure. AM release of TNF positively correlated with the degree of neutrophil recruitment after SiO2 or TiO2-F exposure. A persistent increase in AM fibronectin release consistently correlated with the development of pulmonary fibrosis after SiO2 or TiO2-F exposure. Studies comparing pulmonary responses to ultrafine TiO2 (TiO2-D; particle diameter, 0.02 microns) with TiO2-F demonstrate that ultrafine particles have a relatively greater toxicity on a mass/lung basis. Exposure to TiO2-D resulted in a persistent increase in AM TNF and fibronectin release which was associated with neutrophil recruitment and fibrosis, respectively. TiO2-D did not stimulate AM IL-1 release and this was consistent with the absence of a granulomatous response to TiO2-D. In light of the known bioactivities of IL-1, TNF and fibronectin, these correlative findings suggest that these mediators play significant roles in pulmonary responses to mineral dust exposure and may serve as potential early biomarkers of pulmonary toxicity.

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.

Two-year carcinogenicity study of sodium fluoride in rats.

To determine the carcinogenic potential of sodium fluoride (NaF), we fed Sprague-Dawley rats a diet containing NaF for up to 99 weeks. Rats receiving NaF at a dose of 4, 10, or 25 mg/kg per day added to a low-fluoride diet were compared with controls receiving either a low-fluoride diet or laboratory chow. Each treatment group consisted of 70 rats of each sex. A 30% decrement in weight gain occurred at an NaF dose of 25 mg/kg per day. Evidence of fluoride toxicity was seen in the teeth, bones, and stomach, and the incidence and severity of these changes were related to the dose of NaF and the duration of exposure. Despite clear evidence of toxicity, NaF did not alter the incidence of preneoplastic and neoplastic lesions at any site in rats of either sex. Results from this study indicate that NaF is not carcinogenic in Sprague-Dawley rats.

Pulmonary response to silica or titanium dioxide: inflammatory cells, alveolar macrophage-derived cytokines, and histopathology.

We investigated the effects of silica (SiO2) and titanium dioxide (TiO2) on the pulmonary recruitment of inflammatory cells and the ability of alveolar macrophages (AMs) to release the pro-inflammatory cytokines, interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF). Rats were intratracheally instilled with 5 to 100 mg/kg of the materials, and bronchoalveolar lavage cell populations and AM cytokine release were characterized on days 1, 7, 14, and 28. Both dusts elicited dose-related increases in neutrophils, lymphocytes, and AMs; however, this response was more pronounced and persistent with SiO2. SiO2 at greater than or equal to 50 mg/kg increased AM release of IL-1 and TNF at all time points; lower SiO2 doses had either a transient or no effect on AM-derived cytokines. TiO2 did not result in AM IL-1 release and increased TNF release transiently at doses greater than or equal to 50 mg/kg. Both dusts primed AMs to release increased levels of IL-1 and TNF upon in vitro stimulation with lipopolysaccharide. Histopathology (day 28) demonstrated dose-related interstitial inflammation associated with SiO2 exposure, an effect that was less severe with TiO2. SiO2 doses of greater than or equal to 50 mg/kg elicited a granulomatous response. Development of granulomatous inflammation only at SiO2 doses for which persistent AM IL-1 release occurred suggests involvement of this cytokine in the formation of SiO2-induced granulomas. The ability of SiO2 to activate AM release of IL-1 and TNF in a more pronounced and persistent manner than TiO2 is likely responsible, at least in part, for the greater inflammation and pneumotoxicity associated with SiO2.

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.

Absence of papillomavirus in skin tumors induced in SENCAR mice by a two-stage carcinogenesis protocol.

SENCAR mice are unusually sensitive to induction of papillomas and squamous cell carcinomas by initiation with 7,12-dimethylbenzanthracene (DMBA) and promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). Tumors induced by this protocol were tested for the presence of papillomavirus by immunohistochemistry, Southern blot, reverse Southern blot and dot blot hybridization techniques. Papillomavirus antigens were not detected in any of 235 tumors or 142 non-tumor-bearing skin samples analyzed. Southern blots and dot blots, using a mixed probe of cloned rodent papillomavirus DNA from the multimammate rat, Mastomys natalensis, and the European harvest mouse, Micromys minutus, did not reveal the presence of either episomal or integrated papillomavirus genomes in total cellular DNA extracts from the tumors or non-tumor-bearing skin. To circumvent the possibility that insufficient cross-homology existed to detect a papillomavirus genome with the mixed probe used, DNAs extracted from six papillomas were labeled and each used to probe reference blots that contained 25 cloned papillomavirus genomes excised from their vectors. No evidence for the presence of a papillomavirus genome was detected by this method. Therefore, it is unlikely that papillomaviruses play a role in the induction of tumors in SENCAR mice by two-stage carcinogenesis protocols.

Assessment of the influence of NaF or 1,25-(OH)2 vitamin D3 on the proliferative bone effect of an avian osteopetrosis virus, MAV-2(O).

1,25-(OH)2 vitamin D3 (D3) and sodium fluoride (NaF) were given to chicken embryos and newly hatched chickens infected with a slow onset strain of avian osteopetrosis-inducing virus [MAV-2(O)] to determine if either agent influenced MAV-2(O)-induced proliferation of bone. Embryos were administered MAV-2(O) and treated with: 1) up to 240 micrograms NaF or up to 100 ng D3 as embryos; 2) up to 1.8 g NaF/kg or up to 9.5 micrograms D3/kg after hatching: or 3) 240 micrograms NaF as embryos and up to 1.8 g NaF/kg after hatching. Administration of MAV-2(O) alone resulted in expansion of the cortical diameter of bone. Coadministration of NaF or D3 with MAV-2(O) did not influence the change in cortical diameter seen with MAV-2(O) alone at 18 days of incubation, and 3 and 6 weeks after hatching. Increased osteoid relative to bone (hyperosteoidosis), with NaF and MAV-2(O) compared to MAV-2(O) alone, and NaF compared to untreated controls reflected delayed mineralization of osteoid, a known fluoride effect. We conclude that the administration of NaF or D3 did not influence the incidence, severity or time of onset of the MAV-2(O)-induced proliferative changes of bone.

Comparison of the responses of male rats to dietary sodium saccharin exposure initiated during nursing with responses to exposure initiated at weaning.

In an attempt to define the role of exposure to sodium saccharin (NaS) during early life on the subsequent development of bladder tumours, we compared the responses of male rat pups to exposure to 5% dietary NaS initiated at parturition with those to exposure initiated at weaning. We also compared the effects of exposure from parturition to NaS given in a low-carbohydrate (L-CHO) diet with those of NaS in rat chow. NaS ingestion by the dam was associated with low saccharin concentrations in the pups' urine and had no effect on the caecal or bladder mass in the suckling pups. In the 10 wk after weaning, the rats ingesting NaS in chow showed decreased weight gain and increases in feed consumption, mass of caecal contents and tissue, urine output, bladder mass, relative water consumption (g water consumed/g feed consumed) and bladder hyperplasia. Except for bladder hyperplasia these effects were generally greater in the rats exposed to NaS from parturition than in those exposed only from weaning. The animals exposed to NaS in the L-CHO diet had the highest level of urinary saccharin but showed no bladder hyperplasia. The significance of these findings to the role of pre-weaning saccharin exposure in bladder tumorigenesis is discussed, and it is concluded that the effects on urinary parameters and the bladders of rats exposed to NaS during suckling and weaning may be secondary to the effects of NaS on the gastro-intestinal tract.

Effect of inherent urine output on the response of male rats to 7.5% dietary sodium saccharin.

Young male rats were preselected as high urine (57 g/kg body weight) or low urine (35 g/kg) voiders and were fed a diet containing 7.5% sodium saccharin (NaS) for 10 wk. Urine output was found to be a stable characteristic and high urine output was associated with increased water and feed consumption and increased weight gain. Rats responded in a very similar fashion to 7.5% dietary NaS regardless of their inherent urine output. NaS ingestion was associated with increases in water consumption, caecal mass and urine volume. Among rats that had ingested 7.5% dietary NaS for 10 wk there was a high incidence (12/20) of bladder epithelial hyperplasia. The results are discussed with regard to the concept that increased urine output is an important factor in NaS-induced bladder tumours.

Trypsin inhibitor ingestion-induced urinary indican excretion and pancreatic acinar cell hypertrophy.

Sodium saccharin (NaSacc) has been shown to be a protease inhibitor and to induce an increase in urinary indican, which is a product that is dependent on microbial metabolism of tryptophan. These findings suggest that urinary indican might provide a noninvasive marker of increased pancreatic acinar cell size associated with plant trypsin inhibitor ingestion. The results demonstrate the 7.5% of dietary NaSacc, which increases urinary indican, also increases relative pancreas mass (g/kg body weight), and that these effects are not induced by intravenous infusion of NaSacc. Dietary soybean trypsin inhibitor in the dose range of 17-713 mg/100 g diet was associated with parallel dose-dependent increases in urinary indican and pancreatic acinar cell size (assessed histologically). These findings suggest that measurement of relative urinary indican excretion (microgram/g diet ingested) can provide a noninvasive marker of increased pancreatic acinar cell size in rats that ingest compounds which inhibit digestive proteases.