Pleistophora infestation in fathead minnows, Pimephales promelas (Rafinesque).

Pleistophora infestation was observed in adult fathead minnows, Pimephales promelas, held under laboratory conditions. Fish were clinically healthy, and presented no gross findings at necropsy. Histopathology revealed parasitic stages only in the ovaries. Spores within sporophorous vesicles were mainly encountered in late vitellogenic oocytes and were ultrastructurally identified as a microsporidian parasite. Heavily parasitized oocytes underwent degeneration followed by the release of spores into the ovarian interstitium. Degenerating oocytes and interstitial spores caused ovarian inflammation. Male fish showed no parasites in the testes. Parasitic infestation was compared with body length, body weight, gonadal weight, gonadosomatic index and plasma vitellogenin levels, and revealed no statistically significant differences between non-parasitized and parasitized females. The isolated holding conditions of the fish and the presence of parasitic stages in the ovaries suggested that an infestation with Pleistophora ovariaeSummerfelt, 1964 was more probable than that with Pleistophora mirandellae (Vaney & Conte, 1901).

Pathology and treatment of Eimeria zuernii coccidiosis in calves: investigations in an infection model.

Two studies were conducted in the Eimeria zuernii infection model in order to investigate the pathology of E. zuernii coccidiosis and the efficacy of toltrazuril (Baycox 5% suspension) in this infection. For this purpose, a total of 30 calves were infected experimentally with E. zuernii oocysts and faecal samples taken regularly from the rectum and examined for faecal consistency and oocyst excretion. Six of the calves underwent pathological examination at various points in time after infection. Significant macroscopic and microscopic changes were demonstrated and parasitic stages were identified in the intestinal mucosa of infected calves during the late prepatent and patent period. Inflammatory reactions revealed by light microscopy were confirmed by electron microscopical investigations. Treatment of calves with toltrazuril during the late prepatent period resulted in significantly lower frequencies of diarrhoea and levels of oocyst excretion, and weight gain was significantly higher than in shamtreated animals.

Pleural mesothelioma in a young cat.

A mesothelioma was seen as an incidental finding in the thoracic cavity of an eleven-month old female cat. At necropsy, the pale nodular lesions were spread over the ventral parts of the lungs and their corresponding parts of the diaphragm and the thoracic wall. Microscopically, the tumor consisted of mesenchymal tissue with large amounts of collagenous fibers covered by a mainly unilayered, polymorphic, partly vacuolated line of cells with large nuclei. Mitotic figures were rare. Based on morphological appearance the lesion was classified as an early mesothelioma.

DNA from bovine papillomavirus type 2 induces warts in a xenograft model.

Bovine papillomavirus (BPV) type 2 DNA was inoculated into calf scrotal skin before grafting onto severe combined immunodeficient mice. Inoculation with viral DNA isolated from a bovine wart induced fibropapillomas that exhibited all the morphological features of a BPV infection in cattle. The production of capsid protein and infectious BPV2 particles was demonstrated by immunohistochemistry and a transformed cell focus assay. In contrast, the injection of molecularly cloned viral genomic DNA led to the induction of papilloma-like lesions in the epidermis, but a fibroma was not formed. In addition, only early genes were expressed and infectious virus particles could not be detected. A restriction enzyme accessibility assay suggested that the methylation status of the molecularly cloned BPV2 DNA was different from that of native viral DNA. A possible correlation between methylation status and tumour phenotype is discussed.

Rat model of lung fibrosis: comparison of functional, biochemical, and histopathological changes 4 months after single irradiation of the right hemithorax.

This study investigated changes in lung function, hydroxyproline (OH-pro) content of lung tissue and histopathology in anesthetized, spontaneously breathing rats after a single, selective irradiation of the right hemithorax with a single dose of 20 Gy. The objective of this animal model was to examine as to whether non-invasive lung function measurements (LFM) could be used to analyze the magnitude of the irradiation-related pneumonitis and its long-term sequel occurring in the right lung in the presence of a normal left lung. Four months after irradiation, the OH-pro content in the irradiated right lung was determined and compared with the non-irradiated contralateral left lung, as well as lungs from non-irradiated sham controls. LFM revealed significantly depressed flow-volume curves and reduced quasistatic compliance, suggesting a marked diminution of elastic recoil of the lung. Total lung capacity (TLC) was significantly decreased, while the residual volume (RV) and functional residual capacity (FRC) remained almost unchanged. One of the most predominant dysfunction of the lung was a severe maldistribution of ventilation shown by the single-breath N(2)-wash-out test. Single-breath carbon monoxide diffusing capacity (Dlco) was significantly decreased. The content of OH-pro, a marker of increased collagen, was significantly increased in the irradiated right lung but was indistinguishable from sham controls in the non-irradiated left lung. Histopathological examinations provided evidence of both inflammatory and fibrotic lesions in the irradiated lobes, including bronchiolo-alveolar hyperplasia. No changes were observed in the non-irradiated left lung. In summary, effects observed in the irradiated right lung were largely consistent with effects described in other animal models of human interstitial pulmonary fibrosis. Non-invasive LFM were considered to be particularly sensitive to study the overall extent of changes, however, the interpretation of findings appears to be complicated by the lobar heterogeneity of tissue- and flow-related functional end points.

Two-week inhalation toxicity of polymeric diphenylmethane-4, 4'-diisocyanate (PMDI) in rats: analysis of biochemical and morphological markers of early pulmonary response.

The pulmonary response of Wistar rats to respirable polymeric diphenylmethane-4,4'-diisocyanate (PMDI) aerosol was examined in a 2-wk repeated nose-only inhalation exposure study. Exposure concentrations were 1.1, 3.3, and 13.7 mg PMDI/m(3) (6 h/day, 15 exposures). The level of 13.7 mg/m(3) was actually a combination of an initial target concentration of 10 mg/m(3) in wk 1, which was raised to 16 mg/m(3) in wk 2, due to a lack of signs suggestive of pulmonary irritation. An acute sensory irritation study on rats served as basis for selection of these concentrations. Shortly after the 2-wk exposure period, rats were subjected to pulmonary function and arterial blood gas measurements. Lungs were examined by light and transmission electron microscopy, and labeling indices in terminal bronchioles were measured. Bronchoalveolar lavage (BAL) was performed to assess various indicators of pulmonary inflammation, including neutrophil and macrophage numbers, protein, lactate dehydrogenase (LDH), gamma-glutamyltranspeptidase (gamma-GT), alkaline phosphatase (APh), acid phosphatase (ACPh), and beta-N-acetylglucosaminidase (beta-NAG). Phosphatidylcholine in BAL fluid and BAL cells was determined as aggregated endpoint suggestive of changes in pulmonary surfactant. Rats exposed to 3.3 and 13.7 mg/m(3) experienced concentration-dependent signs of respiratory tract irritation. Determination of arterial blood gases, lung mechanics, and carbon monoxide diffusing capacity did not demonstrate specific effects. Analysis of BAL fluid and BAL cells revealed changes indicative of marked inflammatory response and/or cytotoxicity in rats exposed to 13.7 mg/m(3), and the changes were characterized by statistically significantly increased activities of LDH, beta-NAG, and protein. Phospholipid concentrations were increased in rats exposed to 1.1 mg/m(3) and above (elevated levels of lipid material in alveolar macrophages demonstrated by polychrome stain) and 3.3 mg/m(3) and above (increased intracellular ACPh activity and intracellular phospholipids). In these groups, gamma-GT was statistically significantly increased. These findings suggest that changes in phospholipid homeostasis appear to occur at lower levels than those eliciting inflammation and cytotoxicity. Light and transmission electron microscopy suggest that exposure to 3.3 and 13. 7 mg/m(3) resulted in focal inflammatory lesions and an accumulation of refractile, yellowish-brownish material in alveolar macrophages with concomitant activation of type II pneumocytes. In the terminal bronchioles a concentration-dependent increase of bromodeoxyuridine (BrdU)-labeled epithelial cells was observed in all PMDI exposure groups. In summary, it appears that respirable PMDI aerosol interacts with pulmonary surfactant, which, in turn, may stimulate type II pneumocytes to increase their production of surfactant and to proliferate.

Altered p53 expression in early stages of chemically induced rodent hepatocarcinogenesis.

Most available data on the involvement of p53 in rodent carcinogenesis are based on results of the end point of chemically or virally induced carcinogenesis, i.e., tumors. To investigate the role of altered p53 expression in early stages of rodent hepatocarcinogenesis in a systematic way, we treated male Wistar rats for 6 wk, for 13 wk, and for 6 wk followed by a 7-wk recovery period with chemicals classified as genotoxic (200 ppm acetylaminofluorene [AAF], 100 ppm N-nitrosomorpholine [MMN], 200 ppm benzo(a)pyrene), as tumor promoters and carcinogenic in experimental animals (5 ppm ethinylestradiol, 500 ppm phenobarbitone, 3,000 ppm clofibric acid), as carcinogenic in animal experiments (600 ppm thioacetamide), as noncarcinogenic (200 ppm thyroxine), and as tumor promoters in experimental animals (20,000 ppm tryptophan, 120,000 ppm fructose). Immunohistochemical assessment of altered p53 expression on liver sections with polyclonal serum (CM5) resulted in positive staining in 17/21 benzo(a)pyrene-, 1/18 thioacetamide-, 2/21 clofibric acid-, 2/21 phenobarbitone-, 7/19 ethinylestradiol-, 1/21 tryptophan-, 3/19 thyroxine-, and 1/21 fructose-treated rats and in 2/19 controls. These data support earlier results obtained from analogous investigations with a high incidence of altered p53 expression after NNM and AAF treatment. Thus, altered p53 expression appears to be an early and frequent event in rodent carcinogenesis induced by genotoxic chemicals in contrast to most epigenetically acting chemicals.

Photo hen's egg test: a model for phototoxicity.

The aim of this investigation was to establish a new model for phototoxicity which is more advanced than the widely used cultures of yeasts, bacteria or cells of various origin, and at the same time to avoid animal testing. We studied the extraembryonal vasculature of the incubated hen's egg. This model was originally introduced by toxicologists as an alternative to the rabbit's eye irritation test (Draize test). In the photo hen's egg test, substances are applied to the embryo's yolk-sac blood vessel system at a non-toxic concentration and are irradiated with 5 J/cm2 ultraviolet A (UVA) (320-400 nm). Promethazine, haematoporphyrin, ciprofloxacin and 8-methoxypsoralen were tested in this system. Death of the embryo, membrane discoloration and haemorrhage are parameters for phototoxic damage, which were recorded during an observation period of 24 h. These well-known phototoxic substances induced pronounced damage of the yolk-sac membrane and blood vessels which was not found in the controls (test substance alone, UVA alone or untreated) using a 2 x 2 factorial test design. The photo hen's egg test serves as a valid screening model for substances supposed to be photosensitizers owing to a phototoxic mechanism.

Accumulation of p53 protein in chemically induced oval cells during early stages of rodent hepatocarcinogenesis.

Up to now the possible involvement of p53 in rodent cancerogenesis has been based on results of the endpoint of chemically or virally induced carcinogenesis-tumors. To address the role of altered p53 expression in different stages of the multi-step process of rodent carcinogenesis in a systematic way we fed potent chemical carcinogens to male rats for 6, for 12 and for 6 weeks followed by a 6 week recovery period. Assessment of alterations of p53 expression was performed by immunoperoxidase staining with a polyclonal antiserum on frozen liver sections. Positive p53-immunostaining was localized to treatment-induced proliferating oval cells on liver sections of 21/21 2-acetylaminofluorene- (AAF) and 19/21 N-nitrosomorpholine (NNM)-treated rats irrespective of application scheme as well as to foci of hepatocytes in 1/21 NNM-treated animals and in 3/21 AAF-treated animals after 6 weeks of treatment only. The induction of oval cell proliferation by AAF was more pronounced than by NNM, and for NNM appeared to be dependent on application scheme, with a similar lower abundance of oval cells after a 6 week treatment with and without recovery as compared to a 12 week treatment. These results are discussed with respect to the role of p53 in human and rodent carcinogenesis on the one hand, and the disputed function of oval cells as facultative liver stem and tumour progenitor cells on the other.

[Comparative pathology in risk assessment]. / Die vergleichende Pathologie in der Risikobewertung.

Comparative pathological studies are carried out in drug research using disease models and indicator systems in order to assess the effectiveness and the tolerance of test substances. Basis of these experiments in risk assessment is the exposure of humans in the environment, in occupation or as patients. According to the problem, the evaluation of the studies is done using qualitative morphological and/or quantitative morphometric methods. The assessment of the results obtained in experimental studies has to be done from the point of view to extrapolate from animals to humans. In this context, the problem of animals experimentation is pointed out. Finally, aspects of risk minimization and risk acceptance are discussed briefly.

Short-term and long-term effects of serial bronchoalveolar lavages in a nonhuman primate model.

Bronchoalveolar lavage (BAL) has gained widespread use as a tool for investigating human lung diseases. In certain cases, it can be useful to obtain BAL material in a serial manner. There is convincing evidence from experimental and clinical studies that BAL can cause influx of neutrophils into the bronchoalveolar space. However, conflicting data have been reported on whether this side effect of BAL also affects previously nonlavaged lung areas. In addition, there is little information available on whether multiple repetitive BAL procedures cause damage to lung tissue. To reexamine the short-term effects of serial BAL procedures, the left lung of 10 cynomolgus monkeys was lavaged with five 20-ml aliquots of saline four times at 24-h intervals (Group A). 72 h after the initial BAL, the right lung was lavaged as a control. The percentage of neutrophils increased significantly (p < 0.05), with the greatest effect seen at 48 h (30.7 +/- 5.8 versus 0.8 +/- 0.3%, mean +/- SEM). No significant changes were observed in the control BAL of the right lung at 72 h. A multidisciplinary approach was used to assess the long-term effects of multiple BAL procedures. BAL was performed 14 times over 26 mo at 2-mo intervals (Group B, n = 5). The right lung was lavaged as a control 25 mo after the initial BAL. In addition to standard cellular BAL parameters, the concentrations of fibronectin, procollagen III amino-terminal peptide-related antigen, total phospholipids, and lactate dehydrogenase activity were measured.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary alveolar proteinosis in rats after administration of quartz: its possible role in morphogenesis of lung cancer.

In rats, primary peripheral lung tumors composed predominantly of alveolar type II cells have been induced by inhalation of alpha quartz. In our retrospective study on proliferation markers we evaluated lung specimens of 140 Wistar rats from larger experiments, which had been exposed to Dörentrup quartz (DQ12) by inhalation (10 mg/m3, 56 Weeks, 5 days/week, 7 h/day: n = 27) or intratracheal instillations (5 mg: n = 38; 20 mg: n = 10; 50 mg: n = 28; 15 x 3 mg: n = 12). In the last group 8/12 animals developed lung tumors. Animals were sacrificed 1-32 months after administration. For identification of an increased proliferation of alveolar type II cells the DNA content was monitored by microscopic (static) cytophotometry in histological slides. The argyrophil (AgNOR) method for the demonstration of nucleolar organizer regions (NOR) was used as second marker of type II cell proliferation. Measurements made 24 months after inhalation of DQ12 showed a slight increase of pneumocytic proliferation with 1.64 +/- 0.14 AgNOR/nucleus compared to the controls (1.23 +/- 0.04 mean AgNOR/nucleus). After intratracheal instillation of DQ12 a significant increase of AgNOR was found, e.g. 5 mg: 1.93 +/- 0.23 AgNOR/nucleus (6 months) and 1.96 +/- 0.19 (12 months); 50 mg: 1.77 +/- 15 (6 months) and 2.18 +/- 0.05 (12 months); 15 x 3 mg (+2 ml 2% polyvinylpyridine N-oxide s.c.): 1.81 +/- 0.13 AgNOR/nucleus (27-32 months). With the aid of the 2 c deviation index, i.e. the mean square deviation from the diploid DNA value, it was possible also to identify the pathologically increased proliferation of type II cells after intratracheal instillation of quartz: 0.02 +/- 0.01-0.06 +/- 0.04 c2 (controls); 0.07 +/- 0.04 c2 (5 mg/12 months); 0.12 +/- 0.08 c2 (15 x 3 mg/>27 months) and 0.68 +/- 0.48 c2 (50 mg/12 months). Only in the last group were nearly triploid values detected. Summarizing our results, intratracheal instillation and inhalation of quartz in rats regularly induces alveolar proteinosis and interstitial fibrosis in combination with a dose- and time-dependent increase of the type II cell proliferation rate. As mitogenesis increases carcinogenesis, alveolar proteinosis with increased pneumocytic proliferative activity might be a prerequisite for enhanced tumor development.

The early chick embryo as a model to evaluate cardiovascular effects of adrenaline and nicotine.

During the early stages of development, chick embryos offer a good model for vascular and circulatory studies. The present experiments were carried out with nicotine and adrenaline as validation substances, to test whether this system can be applied to evaluate functional cardiovascular effects. Nicotine was injected into the yolk before incubation and administered topically on different days of incubation. Adrenaline was injected intravascularly to evaluate functional effects (heart rate and heart arrhythmia). Mortality rates following nicotine and adrenaline application were determined. An injection of nicotine prior to incubation resulted in a dose-related increase in undeveloped chick embryos, and the topical application led to hyperaemia and haemorrhages. The body weight of the embryos was reduced due to the repeated nicotine application. After iv injection of adrenaline, heart rate increase and the occurrence of heart arrhythmia showed dose-response effects and the mortality rate was three-fold higher as compared with the controls. These results show that, following the application of adrenaline and nicotine, effects on the early chick embryo are similar to those seen in other test systems. In addition to its use in toxicology, this model is suitable for the evaluation of functional cardiovascular effects. The system does not require living animals, and uses stages of development during which the nervous system is still immature.

Inhalation of amorphous silica: morphological and morphometric evaluation of lung associated lymph nodes in rats.

Male Wistar rats inhaled amorphous silica (quartz glass VP 203-006) for 12 months. Animals were sacrificed after 4, 8, and 12 months of inhalation and after a post-inhalation period of additional 12 months. Morphological as well as morphometric changes of lung associated lymph nodes are studied in comparison with findings due to crystalline quartz (DQ-12) and with a control group without dust exposure. Qualitatively, the lymph node morphology of the dust exposed groups is the same. During the first months mainly small quartz-typical reaction areas occur. From 12 months severe fibroses are detectable. Relative organ weights and the total lymph node area (LNA) are increased in all dust exposed groups. After inhalation of the amorphous quarz glass, the changes seem to appear with a certain delay. But, at the end of the experiment, the morphometric parameters (quartz-typical areas (QTA) and QTA/LNA-ratio) are more enlarged after the amorphous quartz glass than after crystalline quartz DQ-12. The results show that amorphous as well as crystalline silica can lead to severe lymph node fibrosis. This indicates that occupational exposure limits for amorphous silica are generally justified.

[Comparative studies of the effect of quartz glass and quartz DQ-12 in inhalation tests in rats]. / Vergleichende Untersuchungen zur Wirkung von Quarzglas und Quarz DQ-12 im Inhalationsversuch bei Ratten.

Fibrogenic effects of amorphous quartz dusts are discussed more and more during recent years. In order to study alterations due to amorphous silica (quartz glass VP 203-006) in comparison with crystalline quartz (DQ-12), an inhalation experiment in rats was carried out. Male Wistar rats were separated in two dust exposed groups (n = 35) and one control group (n = 30). The experiment was carried out in inhalation chambers with a slowly rotating animal cage for 12 months, 7 h per day, and 5 days per week. The dust concentration was 10 mg/m3. After 4 and after 8 months of inhalation, 5 animals of each group were sacrificed. After 12 months 15 rats of the dust exposed groups and 10 controls were euthanized. The remaining animals were kept for another 12 months post-inhalation period. Regarding the macroscopical appearance of the lungs, the relative organ weights and the histomorphological reaction pattern, marked dust depending differences are obvious. In the lungs of DQ-12-exposed animals diffuse structural changes occur, including fibrosis and severe reaction of macrophages. Histology of lungs from quartz glass exposed animals reveals only a slight and focally arranged cellular reaction with a few collagenous fibers. However, in both dust exposed groups the mediastinal lymph nodes are extremely enlarged with severe fibroses. Additionally, the following blood parameters were determined: lysozyme, ACE, GOT, GPT, and AP. The most pronounced changes are detectable in lysozyme and GOT after DQ-12 exposure. After quartz glass exposure, the levels of these parameters are similar to the controls. These results show that the amorphous quartz tested in this experiment (quartz-glass VP 203-006) has to be considered as a compound with certain biological effects. The establishing of occupational standards seems to be justified. But, assessing the effects, the different physical and/or chemical properties of various amorphous quartz dusts have to be considered.