When rat heart preparations were perfused with quinolinic acid at a slow constant rate, a decrease in contractility was observed. A higher rate of perfusion resulted in a biphasic response, thus both a positive inotropic effect and then a decrease in heart contractility were visible. Using a polarographic method, the association constant of quinolinic acid with calcium ions (Ka) was found to be equal to 220. By combining the values from heart perfusion experiments with the calculated ones of free calcium ions, a linear correlation was obtained between the decreases of contractility and of calcium ions (r = 0.94).
The shape of urothelial cell nuclei in 27 urinary cytological preparations has been quantified by means of Fourier analysis. Fourier amplitudes were calculated as parameters of the nuclear shape. The T-test and a discriminance analysis showed significant differences in nuclear shape between preparations with malignant urothelial cells and nonmalignant cells with reactive changes. Only one preparation was reclassified false-negatively. No preparation was reclassified false-positively. No significant differences were found between normal cells with and without reactive changes. Thus it can be concluded that nuclear shape is an important criterion for the diagnosis of urothelial carcinomas in urinary cytological preparations. Fourier analysis allows the complete reproduction of the convex shape of a nucleus and it seems to be useful as an additional quantitative procedure in the diagnosis of malignant tumours.
Since ethane-1,2-dimethanesulphonate (EDS) causes Leyding cell destruction which may elicit an immune reponse the lymphoid tissues of sexually mature male rats were examined after an intraperitoneal injection of EDS. The changes observed in the testicular regional lymph nodes confirm an immune response while those observed in the thymus may indicate involvement in the immunological response or be a toxic reaction to EDS.
Periportal (pp) or perivenous (pv) liver parenchymal cells from female adult Uje: WIST rats were isolated after retro- or antegrade digitonin infusion followed by collagenase perfusion in the opposite direction. The morphological results revealed a distinct acinar-related destruction of the pv- or pp-zone by digitonin. The remaining cells of the respective other zone showed a good structural maintenance. After subsequent conventional collagenase perfusion the yield, viability and structural integrity of the isolated hepatocytes were high. The zonal cell separation was indicated by significant differences in the pp marker glucose-6-phosphatase and the pv marker glutamine synthetase found in the isolated pp or pv cell populations. Under our experimental conditions including the use of female rats, the alanine aminotransferase and glutamate dehydrogenase as well as ethylmorphine N-demethylase and ethoxycoumarin O-deethylase activities were evenly distributed in both preparations. Under stimulating conditions the capacity for urea synthesis was similar in both pv and pp cells.
The purpose of the present study was to establish the effect of the carcinogen 1,2 dimethylhydrazine on the activities of the jejunal glucosidases and to assess the possible modifying effect of different diets. Two control groups of Wistar albino rats were used - fed standard pellet diet and fed the same diet + 1,2 dimethylhydrazine treatment. Six experimental groups treated with 1,2 dimethylhydrazine were provided. One of them was fed standard diet, containing 30% of wheaten bran and the other 5 groups received high-lipid diets, containing 30% of different fats. The rats were injected subcutaneously once a week for 12 weeks with 20 mg 1,2 dimethylhydrazine/kg b.m. and left for 12 weeks in order to develop a tumor growth. The activities of 5 glucosidases (lactase, maltase, sucrase, palatinase and cellobiase) were determined in homogenates from jejunal mucosa taken near by the tumors and in homogenates from the tumors themselves. An expressed decrease of the jejunal glucosidase activities near the tumors and in the tumors was established. The animals fed 30% wheaten bran diet did not develop tumorigenesis and showed comparatively slight decrease of the enzyme activities. In general, the high-fat regimens did not exert such a preventive effect.
A Wilms' tumor cell line (HFWT) was established after tissue culture of the Wilms' tumor which had developed in the left kidney of a five-month-old boy. The HFWT line has the following cyto-biological characteristics: 1. The cells have a spindle, round or polygonal shape with neoplastic and pleomorphic features that grew in multilayers without contact inhibition. 2. The cells show a stable proliferation, giving 95 passages within 4 years. 3. The chromosomes show a wide aneuploidy distribution, and the modal number was found in diploid range. The stem cells have a normal karyotyping, 46, XY. 4. Heterotransplantation into nude mice can be easily made, and anaplastic Wilms' tumor resembling the original tumor forms. 5. The principal tumor markers produced by the cells in large amounts in the conditioned culture media were carbohydrate antigen 125 (CA 125) and tissue polypeptide antigen (TPA). 6. A good correlation was found between enlargement of the tumor formed by heterotransplantation into nude mice and the levels of CA 125 and TPA in the serum of the nude mouse. No report on the study of tumor markers widely used in clinical treatment of Wilms' tumor has been available to us, but CA 125 and TPA are considered to be useful in the diagnosis and treatment of Wilms' tumor.
After balloon injury of the carotid artery in the rat, gap junctional vesicles were frequently detected in neointimal smooth muscle cells. They represent spherical structures confined by pentalamellar membrane containing a fine filamentous and granular material inside. Since gap junctional vesicles are not typical for the medial smooth muscle cells their occurrence in the intimai cells may be related to their secretory function and/or proliferative capacity.
These studies have examined aortic atherogenesis in cholesterol-fed rabbits and have correlated the effects of daltroban to the pathomechanism of the vessel wall lesions. After feeding a 0.5% cholesterol-enriched diet for 96 d atherosclerotic alterations were seen, which exhibited a proximo-distal pattern, to which the branching of the aorta contributed considerably. Depending on their localization and size a varying cellular constitution of the plaques was obvious. Large plaques, which were mainly seen in the aortic arch and the proximal descending thoracic aorta, consisted of numerous proliferating cells, masses of fibrillar ground substance, clusters of foam cells, and rarely contained cholesterol crystals and necroses. Emerging plaques mainly found in distal thoracic and abdominal aorta imposed as fatty streaks. Daltroban treatment, used in a clinically relevant doses of 10 mg/kg b. wt. per day, reduced extension and protrusional area of plaques to about 40%, which was evaluated using a newly developed computerized morphormetric method, in association with significant reductions in free cholesterol content within the aorta. The results suggest that daltroban inhibits the progression of atherosclerosis in cholesterol-fed rabbits. This effect may be related to its antagonistic interaction with the thromboxane A2 receptor and also to an inhibition of the cholesterol metabolism.
Fat-storing cells and other non-parenchymal cells (endothelial and Kupffer cells) were isolated from rat liver by a combined pronase-collagenase procedure and subsequent Visotrast-370 density gradient centrifugation. The lactate dehydrogenase isoenzyme pattern of fat-storing cells was found different from that of other non-parenchymal liver cells. Fat-storing cells contain LDH-4 as the main isoenzyme and do not contain LDH-1, whereas the other non-parenchymal cells have all five lactate dehydrogenase isoenzymes, among which LDH-5 is dominating. All non-parenchymal liver cell populations contain the M-type pyruvate kinase. The alkaline phosphatase of fat-storing cells has the same electrophoretic mobility as that of the other non-parenchymal cells.
After in vitro-induced neoplastic transformation by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), altered actin organization in pulmonary epithelial cells was examined. A certain correlation was found between anchorage independency (AI), tumorigenicity in nude mice and altered actin organization. DNase I inhibition assay demonstrated significant loss of detergent-insoluble F-actin (stress fibers, p<0.02) and soluble F-actin (single filaments, p<0.0001) after AI transformation in comparison with untransformed cells, while the level of G-actin increased significantly (p<0.0001). At the same time, fluorescence and electron microscopy also revealed that after AI transformation there was a striking loss of stress fibers usually accompanied by reorganization of at least some of the lost stress fibers into F-actin aggregations. After s.c. implantation of AI-transformed cells into nude mice followed by recultivation of the developed tumors, DNase I-inhibition assay showed a significant increase (p<0.001) in the level of detergent-insoluble F-actin as compared with untransformed cells, but no significant difference in the amount of G-actin. However, most of this increased detergent-insoluble F-actin was in the form of aggregations as revealed by immunofluorescence and electron microscopy. This growth behaviour-dependent alteration in actin organization occuring after exposure to MNNG may be causally related to the progressive development of neoplastic phenotypes, although the biological significance of actin aggregation formation remains unclear. The results have also pointed out the importance of parallel investigations into both the biochemical and morphological statuses of actin, particularly when it may be regarded as an indicator of neoplastic transformation and malignancy.
Rats having received drinking water enriched with zinc (zinc acetate, 22.8 mmol/l) developed significantly more pulmonal metastases after an i.v. injection of 5 x 10(5) cultivated cells of a benzpyrene-induced sarcoma than receiving normal drinking water. Zinc ions seem to promote the emigration, implantation and outgrowth of circulating tumour cells.
Transplantation of pancreaticoduodenal allografts in the PVG.RT 1c --> PVG.RT 1u high responder rat strain combination led to the evolution of haemorrhagic pancreatic necrosis over a rejection course of 8d. A cellular infiltrate consisting mainly of monocytes and macrophages was detectable in the stroma while acinar cells showed degranulation and a progressive decrease in number. From day 4 on apoptotic bodies could be identified ultrastructurally within the exocrine tissue. Apoptosis during pancreas rejection, previously undescribed, contributes to acinar cell loss after transplantation in this model.
Porcine pancreatic elastase (PPE) causes irreversible secretory cell metaplasia (SCM) in the intrapulmonary bronchi of hamsters. To determine whether this lesion can be induced in another rodent species. PPE was transorally instilled into the lungs of anesthetized rats. Saline-treated rats served as controls. Animals were killed 1 week, 3 weeks and 3 months after enzyme treatment and the lungs were inflation-fixed with 4% formalin/1% glutaraldehyde in 0.1 M Na cacodylate buffer at pH 7.4. Transverse sections from 3 intrapulmonary airway levels were embedded in paraffin and in glycol methacrylate. The secretory cell index (SCI) was determined from PAS-stained paraffin sections using a standardized scale of 0 to 4. There were no significant differences between SCI values of PPE-treated rats and saline-treated controls at any time point. Secretory cells in methacrylate sections were subclassified into S1, S2 and S3 cells on the basis of increasing amounts of PAS-positive intracellular granules. At one week, there was no effect of PPE on airway level I secretory cells, but significant increases in numbers of S2 and S3 cells were seen in level II and in S3 cells in level III. At 3 weeks, the number of S1 cells were increased in level I and S2 and S3 cells were increased in level III: level II secretory cells were unaffected. Level III secretory cells had returned to normal by 3 months. There were no significant differences between treatment groups in the total number of secretory cells (S1 + S2 + S3) at any airway level or time point examined. We conclude that a single intratracheal instillation of PPE causes mild SCM in rat intrapulmonary bronchi: recovery is complete by 3 months.
After subcutaneous injections of doses of 5 x 2 and 1 x 10 mg cobalt (II) oxide/kg/week (total dose 1,000 mg/kg), respectively 5 out of 10 and 4 out of 10 rats in a lifetime study developed local malignant tumours (controls 0/10). After intraperitoneal injections of 3 x 200 mg cobalt (II) oxide/kg 14 out of 20 rats developed malignant intraperitoneal tumours (controls 1/20). With a Co/Al/Cr spinel the same treatment produced malignant intraperitoneal tumours in only 2 out of 20 rats. After intratracheal instillation of cobalt (II) oxide in single doses of 2 mg/kg (total dose 78 mg/kg) and 10 mg/kg (total dose 390 mg/kg) there were 2 benign pulmonary tumours among 100 rats in the low-dose group and 2 benign and 4 malignant pulmonary tumours among 100 rats in among 100 rats in the high-dose group. The Co/Al/Cr spinel was tested intratracheally only in the high dose (total dose 390 mg/kg); among the 100 rats this produced 3 malignant pulmonary tumours. Alternating intratracheal instillation of cobalt (II) oxide (total dose 470 mg/kg) and benzo[a]pyrene (total dose 200 mg/kg) led to 9 malignant pulmonary tumours in 20 rats, whereas benzo[a]pyrene alone caused only 1 malignant pulmonary tumour in 20 rats. The results suggest that cobalt (II) oxide (76.7% Co) has a weakly carcinogenic effect. The Co/Al/Cr spinel investigated is still less active, and even in very sensitive tests (i.p. and i.t. administration) shows no statistically significant carcinogenic effect. This smaller effect may possibly be explained by its lower cobalt content (24.0%) or its much lower solubility (only less than 10% of the solubility of CoO in 0.1 n HCl).
Sequential changes in rhodamine or fluorescein isothiocyanate-conjugated lectin binding of proximal and distal colonic crypts were studied during and after the administration of the 1,2 dimethylhydrazine (DMH). Five adult its unexposed to DMH or vehicle served as baseline controls. Tissue from normal appearing colon and tumor tissue was incubated with Ulex europaeus agglutinin 1 (UEA), Arachis hypogaea (PNA), Dolichos biflorus (DBA) and Griffonia simplicafolia 1 (GSA1). Distinct regional differences were noted in the baseline controls. UEA, PNA and DBA binding were absent in the distal colonic crypt cells. In the proximal colon minimal UEA and PNA binding was noted in the lower crypt whereas DBA binding was intense. GSA1 binding was diffuse in the upper and lower crypt of both regions. During carcinogenesis a progressive increase in PNA binding was noted in normal appearing colonic crypts from both regions. A progressive increase in PNA binding in proximal and distal colonic tumors was noted over time. Similar to normal tissue, DBA bound markedly to proximal colon tumors but was absent in most distal colonic tumors. UEA stained all proximal tumors intensely at all time points. In distal colonic tumors, UEA staining was diminished at 30 weeks compared to tumors analyzed at 16, 22 and 26 weeks. Mucin depletion was also a feature of tumor tissue compared to adjacent normal and hyperplastic glands. This study documents the region specific changes in lectin binding in normal and neoplastic colonic crypts induced by DMH.
Chondrocyte cultures may serve as a model in investigating changes of the cartilage metabolism. Adherent chondrocytes in vitro maintain polygonal morphology at high cell density in the primary and secondary culture. Collagen type II is only clearly detected in multilayered or nodular areas. The differentiation of the chondrocytes is also indicated by a low HA concentration of the cultural medium. It depends on high cell density, a low number of subcultures and their duration. However, the medium GAG of chondrocyte cultures does not exactly mirror the state of cell differentiation but can partly be used to check it. Subcultures of chondrocytes on small cover slides (minicultures) are used to determine proteoglycan synthesis and degradation for 48 h each. Both synthesis and degradation of cell-associated GAG or proteoglycans, resp., follow similar complex kinetics. The half lives of sulfated GAG or proteoglycans are initially 10 h (T-1 for O-6 h of chase), later 39 h or 95 h (T-2 for 6-48 h of chase). Conditioned medium of casein-elicited rat peritoneal macrophages reduce the sulfate incorporation into chondrocyte proteoglycans and their degradation rates increase. In the additional presence of E. coli endotoxin (0.5 microgram/ml) the synthesis of proteoglycans is only little affected; the degradation rate is stronger increased. To peritoneal macrophages of rats manifold pretreated with BCG and perhaps desensitized, LPS is added in vitro. Conditioned medium of these MP does not affect the chondrocyte proteoglycan synthesis but enhances the degradation rates in a concentration-dependent manner. Thus it can be demonstrated that chondrocyte monolayer miniscale cultures may serve to elucidate changes in the proteoglycan synthesis and different degradative steps.
There are only few reports on the correlation between bacterial products and the GAG pattern of cartilage. Mycobacteria bovis (BCG) were applied to chondrocyte monolayer cultures for one week. The following parameters did change: cell proliferation increased, glycosaminoglycan synthesis and secretion decreased, hyaluronic acid in secreted and cell-associated glycosaminoglycans increased, a correlation between the degree of these changes and the degree of cell differentiation seems to exist. The contact of bacteria like BCG to chondrocytes may change the cellular metabolism. On the tissue level this may injure articular cartilage and thus support the concept of predamaged cartilage that is readily susceptible to further degradation.
Serially transplanted experimental tumors of the central and peripheral nervous system can be used as models to investigate open questions in human neurooncology. Altered susceptibility of higher passages to chemotherapy might be correlated with chromosome number and DNA-content variations which would be partly expressed as changes in proliferation behaviour. Karyotypes therefore were analysed in the 73rd to 90th generations of transplanted experimental gliomas. Wide variation of chromosome number was observed; 2 major types of distribution occurred, the one presenting with, the other without stemlines. Large chromosomes # 1 and # 4 were often monosomic, while small chromosomes of ## 8 to 20 were increased up to the fivefold. Lines with prominent and few markers were observed. On the whole, cells of the proliferating pool of the tumor had to be considered as hypotetraploid. Comparison of chromosome numbers and DNA content gave good correlation; differences between the 2 were explained by the fact that only the number of chromosomes was taken into account, regardless of whether small or large chromosomes were lacking or in excess. When intracerebrally transplanted tumors had been previously treated by administration of BCNU, the DNA content was altered, indicating an increased share of diploid cells in the proliferation pool. Results are at variance with earlier findings in tissue cultures of directly induced malignant gliomas and neurinomas in rats. The findings in transplanted tumors can be interpreted as a result of increased malignancy in transplantation tumors, documented by rapid growth in the animal and dedifferentiated histologic morphology.
Monoclonal and polyclonal antibodies specific for methylation adducts have been applied in an immunohistochemical study of DNA damage in rat and hamster liver following exposure to dimethylnitrosamine. The approach was validated, for frozen and paraffin-embedded sections, by comparison with biochemical data on adduct levels in whole tissues or specific cell populations in the same experimental systems. The potential application of this method to human exposure assessment is discussed.
