Malignant Myoepithelioma of the Parotid Gland in a Rat.

Myoepitheliomas of the salivary glands have been described in laboratory mice, but not in rats. A 20-week-old Wistar (Han) female rat developed a white to grey firm mass at the left side of the neck. Histologically, the mass was unencapsulated and infiltrated the adjacent tissue. The tumour parenchyma was cell rich without acinar or tubular architecture. The tumour showed a palisading basal cell pattern adjacent to blood vessels. There were areas of necrosis filled with cellular debris. The tumour cells showed strong immunohistochemical labelling for pan-cytokeratin types I and II (AE1/AE3), pan-cytokeratin (cytokeratins 1, 5, 6 and 8), cytokeratin 5, cytokeratin 14, vimentin and podoplanin, and only very slight positivity for cytokeratin 8 in small areas. There was no expression of smooth muscle actin. Based on the histological appearance and the immunohistochemistry, the tumour was diagnosed as a malignant myoepithelioma of the parotid gland originating from the parotid duct.

Evaluation of the fate and pathological response in the lung and pleura of brake dust alone and in combination with added chrysotile compared to crocidolite asbestos following short-term inhalation exposure.

This study was designed to provide an understanding of the biokinetics and potential toxicology in the lung and pleura following inhalation of brake dust following short term exposure in rats. The deposition, translocation and pathological response of brake-dust derived from brake pads manufactured with chrysotile were evaluated in comparison to the amphibole, crocidolite asbestos. Rats were exposed by inhalation 6h/day for 5 days to either brake-dust obtained by sanding of brake-drums manufactured with chrysotile, a mixture of chrysotile and the brake-dust or crocidolite asbestos. The chrysotile fibers were relatively biosoluble whereas the crocidolite asbestos fibers persisted through the life-time of the animal. This was reflected in the lung and the pleura where no significant pathological response was observed at any time point in the brake dust or chrysotile/brake dust exposure groups through 365 days post exposure. In contrast, crocidolite asbestos produced a rapid inflammatory response in the lung parenchyma and the pleura, inducing a significant increase in fibrotic response in both of these compartments. Crocidolite fibers were observed embedded in the diaphragm with activated mesothelial cells immediately after cessation of exposure. While no chrysotile fibers were found in the mediastinal lymph nodes, crocidolite fibers of up to 35 µm were observed. These results provide support that brake-dust derived from chrysotile containing brake drums would not initiate a pathological response in the lung or the pleural cavity following short term inhalation.

Toxicity of a quartz with occluded surfaces in a 90-day intratracheal instillation study in rats.

This 90-day study was aimed at characterizing the differences in biological activity between a crystalline ground reference quartz (DQ12) and a quartz with occluded surfaces (quartz isolate) obtained from a clay deposit formed 110 to 112 million years ago. In different test groups, rats were dosed with the same total mass and quartz level by intratracheal instillation, with a total high dose of 15.2 mg/kg (body weight, bw) or approximately 4.7 mg/rat of each quartz species in a saline suspension. The reference quartz was mixed with titanium dioxide to achieve a positive control mixture, which contained the same quartz content as in the quartz isolate. At 3 days post dosing, both quartz groups showed a significant inflammatory response based on total and differential cell counts from bronchoalveolar lavageate (BAL) analysis. At 28 and 90 days, the quartz isolate values were no longer statistically different from vehicle control group values; however, the positive control group values were approximately 12 and 65 times greater than those of the control group, respectively. After 28 days, histopathological evaluation showed moderate effects in the quartz isolate group compared to the saline control animals. These effects did not progress in severity at 90 days. In contrast, the positive control group exhibited more severe effects than the quartz isolate group and these effects showed a progression to a persistent and self-perpetuating inflammatory state. The toxicological properties of quartz particles can vary significantly dependent on their surface characteristics. Toxicity can range from a high-dose-induced, modest, transient inflammation from quartz with occluded surfaces, to a severe and persistent inflammatory state caused by ground quartz with fractured surfaces.

Internalization, cytotoxicity, apoptosis, and tumor necrosis factor-alpha expression in rat alveolar macrophages exposed to various dusts occurring in the ceramics industry.

In 1997 The International Agency for Research on Cancer classified some exposures to crystalline silica as carcinogenic to humans. Such exposures were acknowledged to be very variable, and even in the same monograph it was admitted that coal dust, containing as much as 20% quartz, could not be classified. Clearly there is a need to develop methods for assessing any risks posed by various silica containing dusts in different workplaces. A European collective research project, SILICERAM, was launched with the aim of assessing the toxicity of various dusts in the ceramics industry and improving worker protection. This study examined the effect of particles, namely, DQ12 quartz, China clay, feldspar, and a sample resembling a typical mixture used in the ceramic industry (a "contrived sample" or CS), on NR8383, a rat alveolar macrophage (AM) cell line. Titanium dioxide and aluminum oxide were also used as negative controls. Confocal microscopy observations showed internalization of DQ12 and CS in NR8383. Cell viability decreased dramatically after a 2-h incubation exposure period with DQ12 (-71%). CS was less toxic than DQ12 at 2 h. China clay and feldspar were slightly cytotoxic to NR8383 cells. DQ12 induced apoptosis, with a smaller effect of CS and China clay. TNFalpha gene expression was analyzed by RT-PCR. DQ12, at a noncytotoxic dose of 10 microg/cm(2), induced a significant expression of TNFalpha (+2 times increase). In contrast, similar doses of CS and China clay did not produce a significant increase, while TiO2 and Al2O3 displayed no effect. Co-treatment with 10 microM aluminum lactate significantly reduced the effects of silica-containing particles on cytotoxicity, apoptosis, and TNFalpha expression.

Effects of nonfibrous particles on ceramic fiber (RCF1) toxicity in rats.

In previous investigations a reference test sample of prepared ceramic fibers called RCF1 induced lung tumors in a 2-yr inhalation study in rats. It was hypothesized that nonfibrous particles in RCF1 may have played a significant role. The objective of the present study was to compare lung retention and biological effects of another sample of ceramic fibers, called RCF1a, to the original RCF1. The main difference between these 2 samples was the content of nonfibrous particles: 25% of the mass of RCF1 versus 2% for RCF1a. These nonfibrous particles were chemically identical to the fibers. Female Wistar rats were exposed 6 h/day, 5 days/wk for 3 wk to either RCF1a or RCF1 fiber aerosol at a concentration of about 125 fibers (>20 microm long)/ml. Because of differences in the nonfibrous particle contents, the average gravimetric aerosol concentration differed between the two samples (RCF1, 51.2 mg/m(3); RCF1a, 25.8 mg/m(3)). The posttreatment observation period was 12 mo. Biological effects measured include the clearance function of alveolar macrophages (clearance of fibers and tracer particles), and inflammation and its persistence during the recovery period. Alveolar clearance of tracer particles ((46)Sc(2)O(3)) was barely retarded after RCF1a exposure (80 days clearance half-time compared to 60 days in controls). After RCF1 exposure, however, a severe retardation of clearance was observed (1200 vs. 66 days). In both groups, differential cell counts on pulmonary lavage showed a significant increase of polymorphonuclear leukocytes (PMNs) (about 15%) and lymphocytes 3 days after the end of exposure. The PMN influx persisted longer after exposure to RCF1 than RCF1a. The conclusion of the study is that the particle fraction of RCF1 significantly enhanced any adverse effects. This clearly demonstrates the importance of the physical characteristics of the test material for the degree of toxic effects to be expected. The presence of nonfibrous particulates can enhance the effects on the lung of a mixture of fibrous and nonfibrous particulates following exposure.

Inhalation tolerance study for p-aramid respirable fiber-shaped particulates (RFP) in rats.

This study was designed to assess the lung clearance function in rats after subchronic exposure to p-aramid respirable fiber-shaped particulates (RFP). Male Wistar rats were exposed 6 hrs/day, 5 days/week for 3 months to 50, 200, and 800 RFP/ml measured by scanning electron microscopy (SEM). Recovery effects were followed up through 9 months postexposure. The retention of RFP (length > 5 microm) was about 25 x 10(6) RFPs per lung in the low dose group after 3 months of exposure. The corresponding values in the medium and high dose groups amounted to overproportionally higher values of 122 x 10(6) and 576 x 10(6) RFPs per lung, respectively. A decrease in the length of the retained RFPs over the 9-month recovery period was observed, indicating a breakage of long fibrils. Alveolar clearance half-times measured by gamma tracers indicated a dust overloading of lungs for the high dose group at 0 and 3 months postexposure. Bronchoalveolar lavage parameters revealed that p-aramid RFPs induced pronounced inflammatory effects in the high and medium dose groups. Histopathologically, slight fibrotic and hyperplastic lesions were observed in the medium and high dose groups directly after the end of exposure. The findings at the 3-month postexposure interval resulted in a reduction of inflammatory changes in the medium and high dose groups compared to the sacrifices upon cessation of exposure. No histopathologic effects were detected in the low dose group. In the high dose group the maximum functionally tolerated dose was exceeded. The No Observed Adverse Effect Level (NOAEL) of RFP was 50 RFP/ml as measured by SEM.

Investigation of Chronic Toxic and Carcinogenic Effects of Gasoline Engine Exhausts Deriving from Fuel without and with Ferrocene Additive.

Chronic toxic and carcinogenic effects of gasoline engine exhaust inhalation were investigated in rats. The exhaust from the combustion of commercial fuel containing 30 ppm ferrocene additive was compared to exhaust from the same fuel without ferrocene. This study was part of a procedure to get a special authorization for the use of ferrocene as gasoline additive according to the German Gasoline Lead Act. To generate the exhausts, pairs of engines of the same type and age were operated on computer-controlled test benches in a combined urban-freeway driving cycle. The engines were equipped with three-way catalysts and lambda sensors. Rats inhaled the exhausts after dilution at ratios of about 1.20 and 1:40 for 18 h/day, 5 days/wk for 12 mo (chronic toxicity study) or for 24 mo followed by 6 mo of clean air (carcinogenicity study). The limiting factor for the exhaust concentration was the relative humidity of the exposure atmosphere. At defined intervals, body weight and food consumption, parameters of clinical chemistry, hematology, bronchoalveolar lavage (BAL), and mechanical lung function were measured, as well as lung clearance and particle retention in the lungs. In the high-dose groups and the controls the complete organ/tissue spectrum was investigated histopathologically, and in the low-dose groups the respiratory tract. Only slight exposure-related effects could be detected, like a loss in the background iron content of the cell pellet of the bronchoalveolar lavage fluid and cytoplasmic inclusions and goblet-cell hyperplasias in the nasal cavity. Between the clean-air controls and the exhaust-exposed groups, no exposure-related differences occurred in body weight development, mortality incidences, or any of the clinical investigations. Ninety-two to 94% of the animals developed age-related tumors, predominantly in the mammary glands, uterus, adrenals, thyroid, and pituitary. In the respiratory tract a total of five tumors was found: one in the controls and four in the low-dose groups. No physical, chemical, or toxicological differences between the exhausts from fuel without or with ferrocene were demonstrated.

Bioavailability of fine dispersed platinum as emitted from automotive catalytic converters: a model study.

Automobile exhaust catalytic converters emit fine dispersed elemental platinum, Pt (0), in the nanometer range coated on larger aluminium oxide carrier particles. A pre-requisite for a potential systemic toxic effect of the emitted platinum is its bioavailability which was investigated using laboratory animals. To this end, a model substance was synthesised which consisted of aluminium oxide particles < or = 5 microns onto which platinum particles > or = 4 nm were deposited by a calcination process. These particles closely resemble those emitted from automobile exhaust converters. This model substance was applied to female Lewis rats in two doses by intratracheal instillation; the animals were killed after 1, 7, 28 and 90 days. In addition, the model substance was also applied during a 90-day inhalation study. After microwave digestion of the tissues, the platinum was determined in all organs and body fluids by inductively coupled plasma/mass spectrometry (ICP/MS). Platinum was found in the blood, urine and faeces and all important organs (liver, spleen, kidneys, adrenals, stomach, femur). Based on the platinum content determined in the body fluids and all organs (except the lung and the faeces) it was calculated that up to 16% of the platinum was retained in the lung 1 day after intratracheal instillation and up to 30% of the fine dispersed platinum deposited on an average during 90 days inhalation in the lung was bioavailable. Using size exclusion chromatography (SEC) in combination with ICP/MS, it was shown that > or = 90% of the bioavailable platinum was bound to high molecular weight compounds (approximately 80-800 kDa), most likely proteins.

The carcinogenic potency of carbon particles with and without PAH after repeated intratracheal administration in the rat.

The role of carcinogenic PAH in soot- and carbon black-related lung tumour induction in rats was investigated after intratracheal administration of carbon blacks (CB) and two types of diesel soot (DS), either as original or as toluene extracted particles. The total particle dose per animal was 15 mg subdivided into 16-17 weekly applications. There was one vehicle control and two groups were treated with a total dose of either 30 or 15 mg pure BaP as positive control. The main tumour results were: (a) original DS induced a higher tumour rate than extracted DS; (b) the carcinogenic potency of extracted CB probably depends on the size of the primary carbon particles and on the specific surface area of the particles; (c) extracted DS covered with 11 micrograms BaP per mg carbon particles caused a lower lung tumour rate than original DS containing only 0.9 ng BaP per mg, but a variety of other PAH and NO2-PAH; (d) a total dose of 15 mg pure BaP caused a lung tumour rate very similar to that of 30 mg extracted DS, 15 mg original DS or 15 mg Printex 90T CB extracted or covered with approximately 29.5 micrograms BaP per mg CB.

Phagocytosis and chemotaxis of rat alveolar macrophages after a combined or separate exposure to ozone and carbon black.

Male Wistar rats were treated by ozone or carbon black (CB) alone as well as in combination. Intratracheal instillation with various amounts of CB was followed either by an acute 7-day or subchronic 2-month ozone exposure (0.5 ppm). Two functional parameters were investigated in alveolar macrophages from bronchoalveolar lavagates, the phagocytotic capacity and the chemotactic migration capability. In the phagocytosis assay, the percentage of phagocytizing macrophages decreased significantly in the CB-exposed groups whereas the ozone groups remained close to or at the control level after 7 days and 2 months of exposure, respectively. The number of ingested particles per macrophage and the formation of superoxide anion radicals were not changed after a 7-day exposure to ozone compared to the control group but were increased after a 2-month ozone exposure. However, a reduction was found in the CB groups. A stimulating effect of ozone was observed in the combined groups. Chemotactic migration was generally retarded in the CB-treated groups. From the results it can be concluded that ozone is able to stimulate the phagocytotic and chemotactic activity of alveolar macrophages whereas CB impairs these functions.

Irreversible pulmonary changes induced in rat lung by dust overload.

The objective of this study was to investigate whether the effects of dust overload are reversible upon cessation of subchronic exposure to test toner. Female rats were exposed 6 hr/day, 5 days/week for 3 months to a test toner at 0, 10, and 40 mg/m3. The retained quantity of test toner in the lungs at the end of exposure was 0.4 and 3.0 mg for the low and high exposure groups, respectively. Fifteen months later, the corresponding values were 0.12 and 2.65 mg in the lungs. Alveolar clearance of tracer aerosols as well as cytologic and enzymatic parameters in the bronchoalveolar fluid was investigated at the end of exposure and subsequently up to 15 months later. The alveolar clearance of 59Fe2O3, 51Cr-polystyrene, and 85Sr-polystyrene tracer aerosols was slightly retarded at the low and substantially impaired at the high exposure level. At the low exposure level, there was some recovery in the clearance behavior up to 6 months after exposure. In contrast, at the high exposure level there was no indication of a reversal of the impaired clearance. For the beta-glucuronidase activity and the number of polymorphonuclear cells, the pattern of the effects was similar to the effects on the half-time tracer particle clearance. In conclusion, the dust overload at a lung burden of 3 mg test toner in rats was persistent for at least 15 months after termination of exposure.

Pulmonary response to toner upon chronic inhalation exposure in rats.

A chronic inhalation study of a test toner was conducted by exposure of groups of F-344 rats for 6 hr/day, 5 days/week for 24 months. The test toner was a special Xerox 9000 type xerographic toner, enriched in respirable-sized particles compared to commercial toner, such that it was about 35% respirable according to the ACGIH criteria. The target test aerosol exposure concentrations were 0, 1.0 (low), 4.0 (medium), and 16.0 (high) mg/m3. Titanium dioxide (5 mg/m3) and crystalline silicon dioxide (1 mg/m3), used as negative and positive controls for fibrogenicity, were also evaluated. Inhalation of the test toner or the control materials showed no signs of overt toxicity. Body weight, clinical chemistry values, food consumption, and organ weights were normal in the toner- and TiO2-exposed groups, except for a 40% increase in lung weight in the toner high-exposure group. All of the changes in the toner-exposed groups were restricted to the lungs or associated lymph nodes. A chronic inflammatory response was evident from the bronchoalveolar lavage parameters for the toner high-exposure group. The incidence of primary lung tumors was comparable among the three toner-exposed groups and the TiO2-exposed, and air-only controls, as well as consistent with historical background levels. A mild to moderate degree of lung fibrosis was observed in 92% of the rats in the toner high-exposure group, and a minimal to mild degree of fibrosis was noted in 22% of the animals in the toner middle-exposure group. The pulmonary changes in the toner high-exposure group were smaller in magnitude than those found in the crystalline silica-exposed group. The comparative fibrogenic potency of TiO2, toner, and SiO2 was estimated to be 1:5:418 using a dosimetric model and assuming a common mechanistic basis. There were no pulmonary changes of any type at the toner low-exposure level, which is most relevant in regard to potential human exposures. The lung alterations in the toner high-exposure group are interpreted in terms of "lung overloading," a generic response of the respiratory system to saturation of its detoxification capacity. The maximum tolerated dose (MTD) criterion was met at the toner high (16 mg/m3)-exposure level.

Lung clearance and retention of toner, utilizing a tracer technique, during chronic inhalation exposure in rats.

Male and female F-344 rats were exposed to 6 hr/day, 5 days/week for up to 24 months to a special test toner at 0, 1, 4, and 16 mg/m3, TiO2 at 5 mg/m3, or SiO2 at 1 mg/m3 by the inhalation route. 59Fe-labeled iron oxide and 85Sr-labeled polystyrene particles were periodically inhaled by the nose-only route and used to measure alveolar clearance rates during the course of the study. This method was used to describe a maximum functionally tolerated dose (MFTD). Pulmonary retention of toner and control materials (TiO2 and SiO2) was measured after 3, 9, 15, 21, and 24 months of exposure. The quantity of all three materials retained in the lungs and lung-associated lymph nodes increased with exposure duration and level. The final pulmonary burdens of toner at the three exposure levels were 0.22, 1.73, and 15.6 mg/lung, respectively. Alveolar clearance of both tracers was substantially impaired at the toner high-exposure level, and moderately slowed at the toner middle-exposure level. The excessive quantity of toner retained and the substantially retarded clearance in the toner high-exposure group are indicative of "lung overloading." Alveolar clearance of 85Sr-polystyrene particles was slightly slowed in the TiO2-exposed group and substantially impaired in the SiO2-exposed group. The alveolar clearance of the unexposed rats decreased about 30% during the study, a change ascribed to aging. For a general description of the toxicokinetics of the various dusts, a semiempirical kinetic model was developed, which could generally be useful for the extrapolation of lung retention of insoluble particles from a subchronic to a chronic inhalation study. Both the maximum tolerated dose (MTD) and the MFTD were exceeded at the toner high-exposure level during the study in rats.

Reversibility of clearance impairment after subchronic test toner inhalation.

In a chronic study of a test toner in F-344 rats an impairment of alveolar clearance and increased toner retention were observed at the high and, to a lesser degree, at the middle exposure level throughout the study. The objectives of the present study were: 1. To investigate the nature and the time course of test toner and surrogate tracer alveolar clearance and toner retention behaviour following a subchronic exposure interval. 2. To investigate the dependence of the response upon the pulmonary burden of the test toner.

Reversibility of biochemical and cytological alterations in broncho-alveolar lavagate upon cessation of dust exposure.

The objectives of the present study were: 1. To investigate the nature and time course of the biochemical and cytological changes following acute and subchronic exposure intervals. 2. To investigate the dependence of the response upon the pulmonary burden of the test material. Alveolar clearance measurements were performed in parallel and are presented in a separate poster (1).