Induction of c-fos protein-like immunoreactivity in the rat and hamster pineal gland after the onset of darkness

Induction of c-fos protein (FOS) after the onset of darkness was studied immunocytochemically in the rat and hamster pineal gland. The animals were kept on a 12:12 h light-dark cycle. Before the dark period no FOS staining was seen in either rat or hamster pineal cells. Five hours after the onset of darkness 342 +/- 18 pinealocytes/0.2 mm2 (mean +/- SD) displayed FOS-like immunoreactivity in the hamster pineal gland; in the rat pineal gland only 5 +/- 2 pinealocytes/0.2 mm2 showed a faint staining. Two hours later the density of FOS positive cells was decreased to 60 +/- 11/0.2 mm2 in the hamster but increased to 519 +/- 103/0.2 mm2 in the rat pineal gland. Three hours before the beginning of the light period no FOS positive cells were detected in either animal. Both the rat and hamster pineal gland showed a transient and temporally defined expression of c-fos protein in the middle of the dark period. This may be related to a more active functional state of pinealocytes, which is reflected in a peak of melatonin synthesis during the darkness.     

Induction of c-fos protein-like immunoreactivity in the rat and hamster pineal gland after the onset of darkness

Summary Induction of c-fos protein (FOS) after the onset of darkness was studied immunocytochemically in the rat and hamster pineal gland. The animals were kept on a 12:12 h light-dark cycle. Before the dark period no FOS staining was seen in either rat or hamster pineal cells. Five hours after the onset of darkness 342±18 pinealocytes/0.2 mm² (mean±SD) displayed FOS-like immunoreactivity in the hamster pineal gland; in the rat pineal gland only 5±2 pinealocytes/0.2 mm² showed a faint staining. Two hours later the density of FOS positive cells was decreased to 60±11/0.2 mm² in the hamster but increased to 519±103/0.2 mm² in the rat pineal gland. Three hours before the beginning of the light period no FOS positive cells were detected in either animal. Both the rat and hamster pineal gland showed a transient and temporally defined expression of c-fos protein in the middle of the dark period. This may be related to a more active functional state of pinealocytes, which is reflected in a peak of melatonin synthesis during the darkness.     

Evaluation of genotoxicity of N-nitrosodibenzylamine in Chinese hamster V79 cells and in Salmonella

Health concerns have arisen due to the formation of N-nitrosodibenzylamine (NDBzA; CAS No. 5336-53-8) in pork processed in a new type of rubber netting. In view of the potent carcinogenicity of related nitrosamines (e.g. N-nitroso-n-dibutylamine and N-nitrosodiethylamine), NDBzA was evaluated for genotoxicity in vitro in both Chinese hamster V79 cells and in Salmonella. In V79 cells, concentrations up to 25 micrograms/ml were tested with and without activation by rat or hamster hepatocytes. Significant elevation of SCE frequency was seen only at 25 micrograms/ml in the presence of uninduced hamster hepatocytes. Mutation to 6-thioguanine resistance was observed at 25 micrograms/ml, in the absence of hepatocytes and in the presence of induced (Aroclor 1254) or uninduced hamster hepatocytes, but not with rat hepatocytes. With uninduced rat hepatocytes, a small but significant (p less than 0.05) increase in the mutation frequency was seen with 10 micrograms/ml NDBzA. In the Salmonella assay, using a pre-incubation protocol and concentrations up to 1000 micrograms/ml, NDBzA was negative in strain TA98, and in TA100 with rat S9, but was positive at the highest dose in TA100 with hamster S9, and more strongly with Aroclor 1254-induced hamster S9. When activated by uninduced rat or hamster hepatocytes, as opposed to S9, NDBzA was negative with all tester strains. Hamster hepatocytes activated more than rat in the V79 studies, and hamster S9 was more strongly activating in the Salmonella assay. These results indicate that NDBzA is weakly mutagenic to both Salmonella and V79 cells.     

Genotoxicity of 1-nitronaphthalene in Chinese hamster V79 cells

1-Nitronaphthalene (1-NN) has been identified in the U.S. National Toxicology Program as a non-carcinogen showing some evidence of in vitro genotoxicity. We tested this compound in Chinese hamster V79 cells at 20-80 micrograms/ml with two endpoints: sister-chromatid exchange (SCE) and thioguanine resistance (TGR), with 5 repeat experiments. The SCE values in the presence of rat or hamster hepatocytes were consistently above the 95% and usually the 99% upper confidence limits for the corresponding control. Without hepatocyte activation, the control upper confidence limits were not exceeded except in one experiment in which the control SCE value was unusually low. TGR was scored both as proportion of plates with mutant colonies and as number of mutant colonies per plate. In 2 of 5 experiments, these values exceeded control 95% or 99% upper confidence limits; on the other hand, these values were substantially lower than those of the positive controls, dimethylbenz[a]anthracene (2.6 micrograms/ml) with activation and ethyl methanesulfonate (155 microgram/ml), which is direct-acting. For TGR, activation of 1-NN by either rat or hamster hepatocytes produced inconsistent results. Overall we would consider this compound to be a weak genotoxin, to which a cancer bioassay would be expected to be relatively insensitive.     

The immunohistolocalization of carbonic anhydrase III in the submandibular gland of rats and hamsters

Summary Carbonic anhydrase III has been localized using the avidin-biotin-glucose oxidase complex (ABC) method in the submandibular gland of the rat and hamster. This isozyme, which is predominant in skeletal muscle, was observed in intercalated duct, striated duct and excretory duct cells in the rat submandibular glands. In contrast, only some striated duct cells in hamster submandibular glands were stained.     

Induction of endoreduplication in Chinese hamsters V79 cells by cytosine arabinoside

Endoreduplication (ER) could be induced very effectively in Chinese hamster V79 cells exposed to cytosine arabinoside (1-β-D-arabinofuranosylcytosine; Ara-C). Cells were cultured for 48 hours in Ara-C containing medium. ER frequency increases rapidly after Ara-C release. About 60% of metaphase cells were endoreduplicated at 8–10 hours after release from Ara-C (5 μg/ml). Induction of ER also depends on Ara-C concentrations.     

Rates of cholesterol synthesis and low-density lipoprotein uptake in the adrenal glands of the rat, hamster and rabbit in vivo

The absolute rate of cholesterol acquisition from de novo synthesis and from receptor-dependent and receptor-independent low-density lipoprotein (LDL) uptake was determined in the adrenal glands of the rat, hamster and rabbit under in vivo conditions. The rate of incorporation of [3H]water into cholesterol in the adrenal gland was much higher in the hamster (1727 nmol/h per g) and rabbit (853 nmol/h per g) than in the rat (71 nmol/h per g). Assuming that 23 atoms of 3H are incorporated into the cholesterol molecule during its biosynthesis, the absolute rates of cholesterol synthesis were then calculated to equal 59, 29 and 2.4 micrograms/h per g of adrenal gland in the hamster, rabbit and rat, respectively. Rates of LDL-cholesterol uptake were measured using a primed continuous infusion of [14C]sucrose-labeled homologous LDL (total LDL transport) and methylated human LDL (receptor-independent LDL transport). The rate of total LDL-cholesterol uptake in the adrenal gland was much higher in the rabbit (227 micrograms/h per g) than in the rat (18 micrograms/h per g) or hamster (6 micrograms/h per g). In all three species LDL uptake was mediated largely (greater than 93%) by receptor-dependent mechanisms. In terms of total cholesterol acquisition, the hamster adrenal gland derived 10-times more cholesterol from de novo synthesis than from LDL uptake, whereas the converse was true in the rabbit. Rates of de novo synthesis and LDL-cholesterol uptake were both low in the rat adrenal gland, which is known to derive cholesterol mainly from circulating high-density lipoproteins. Thus, the adrenal gland acquires cholesterol for hormone synthesis from at least three different sources and the quantitative importance of these sources varies markedly in different animal species, including man.     

Histochemical methods for characterizing secretory and cell surface sialoglycoconjugates

Paraffin sections of trachea, sublingual gland, and pancreas from rats, mice, and hamsters were stained with peanut agglutinin (PNA) or Dolichos biflorus agglutinin (DBA) conjugated to horseradish peroxidase before or after enzymatic removal of sialic acid. Adjacent sections were oxidized with periodate prior to incubation with sialidase and staining with PNA and DBA. PNA binding demonstrated terminal beta-galactose in secretions, at the basolateral plasmalemma of mouse tracheal serous cells, in or at the surface of zymogen granules, and at the apical and basolateral surface of mouse and hamster pancreatic acinar cells. Sialidase digestion revealed PNA binding, demonstrative of penultimate beta-galactose, in secretions of mucous cells in tracheal and sublingual glands and at the apical glycocalyx of ciliated and secretory cells in the tracheal surface epithelium of all the rodents studied. Sialidase also imparted PNA affinity to endothelium in all three species and to secretions and the basolateral plasmalemma of tracheal serous cells and pancreatic acinar cells in the rat. Periodate oxidation blocked the enzymatic removal of N-acetylneuraminic acid as judged by prevention of staining with the sialidase-PNA procedure. Sites in which periodate prevented sialidase-PNA staining included pancreatic islet cells and at the luminal glycocalyx of ciliated and secretory cells in tracheal surface epithelium in all three rodents, most sublingual mucous cells in the hamster, pancreatic acinar cells in the rat, and endothelium, except that of the rat. Glycoconjugate in other sites remained positive with the periodate-sialidase-PNA sequence. Resistance to periodate was interpreted as evidence for the presence of terminal sialic acid with an O-acetylated polyhydroxyl side chain. DBA binding demonstrated terminal alpha-N-acetylgalactosamine in the secretion of all mucous cells in the hamster trachea and 50-90% of those in the rat, secretion and the basolateral plasmalemma of all glandular serous cells in the mouse trachea, at the apical surface of most secretory cells lining the lumen of the rat and hamster trachea, and cilia of 5-10% of ciliated cells in the rat trachea. Periodate oxidation and sialidase digestion demonstrated N-acetylneuraminic acid and penultimate alpha-N-acetylgalactosamine in cilia in the mouse trachea and sialic acid containing O-acetylated polyhydroxyl side chains subtended by N-acetylgalactosamine in the secretion of all mucous cells in the rat and hamster trachea and of 80-90% of mucous cells in the hamster sublingual gland.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization of vimentin in myoepithelial cells of the rat mammary gland

Summary Myoepithelial cells in the virgin rat mammary gland have been shown to contain vimentin, using a polyclonal antiserum to vimentin purified from hamster fibroblasts. This antiserum has been shown to be specific for vimentin by immunoblotting and ELISA techniques. Similar results were obtained with a monoclonal antibody to vimentin. In the mammary glands of pregnant rats, the staining with vimentin antibodies is much weaker in the myoepithelial cells of the developing alveolar buds than in the main ducts. Similarly, in lactating glands, the staining of myoepithelial cells is much weaker in the secretory alveoli than in lactiferous sinuses. In each case, staining with antivimentin co-localizes with staining with polyclonal antisera to callous keratin (which specifically stain myoepithelial cells in the rat mammary gland).     

Interstitial and parenchymal cells in the pineal gland of the golden hamster

Summary A combined thin-section/freeze-fracture study was performed on the superficial pineal gland of the golden hamster, comparing the parenchymal and interstitial cells of this animal with those previously investigated in rats. In contrast to rats, no gap junctions and gap/tight junction combinations could be found between pineal parenchymal cells of the hamster. Furthermore, the interstitial cells of the hamster pineal gland were found to have large flat cytoplasmic processes, which abut over large areas equipped with tight junctions. In thin sections, profiles of interstitial cell processes were seen to surround groups of pinealocytes. Interstitial cells and their sheet-like, tight junction-sealed processes thus appear to delimit lobule-like compartments of the hamster pineal gland. Because the classification of the interstitial cells is uncertain, the expression of several markers characteristic of mature and immature astrocytes and astrocyte subpopulations has been investigated by indirect immunohistology. Many of the non-neuronal elements in the pineal gland are vimentin-positive glial cells, subpopulations of which express glial fibrillary acidic protein (GFA) and C1 antigen. The astroglial character of these cells is supported by the lack of expression of markers for neuronal, meningeal and endothelial cells. M1 antigen-positive cells have not been detected.Supported by a grant from Deutsche Forschungsgemeinschaft (Scha 185/9-2)     

Aneuploidy induction in human fibroblasts: comparison with results in Syrian hamster fibroblasts

The susceptibility of human fibroblast cells in culture to neoplastic transformation by chemical carcinogens is appreciably lower than that of rodent fibroblasts. We have proposed that a key step in the neoplastic progression of Syrian hamster embryo fibroblasts is the induction of aneuploidy by carcinogens. It is possible that the different sensitivity to neoplastic transformation of Syrian hamster versus human cells is due to a difference in genetic stability following treatment with chemicals inducing aneuploidy. Therefore, we measured the induction of numerical chromosome changes in normal human fibroblasts and Syrian hamster fibroblasts by 4 specific aneuploidogens. Dose- and time-dependent studies were performed. Nondisjunction, resulting in aneuploid cells with a near-diploid chromosome number, in up to 14-28% of the hamster cells was induced by colcemid (0.1 microgram/ml), vincristine (30 ng/ml), diethylstilbestrol (DES) (1 microgram/ml) or 17 beta-estradiol (10 micrograms/ml). In contrast, human cells displayed far fewer aneuploid (near-diploid) cells, i.e., 8% following treatment with colcemid (0.02 micrograms/ml) or vincristine (10 ng/ml) and only 3% following treatment with DES (6 micrograms/ml) or 17 beta-estradiol (20 micrograms/ml). The doses at which the maximum effect was observed are given. Treatment of human cells induced a higher incidence of cells with a near-tetraploid chromosome number, which was similar to the level observed in treated hamster cells except at the highest doses. These results indicate that human cells respond differently from hamster cells to agents that induce aneuploidy. In particular, nondisjunction yielding aneuploid human fibroblasts with a near-diploid chromosome number was less frequent. The magnitude of the observed species differences varied with different chemicals. The difference in aneuploidy induction may contribute, in part, to species differences in susceptibility of fibroblasts to neoplastic transformation.     

Peptidergic cells in the mammalian pineal gland. Morphological indications for a paracrine regulation of the pinealocyte

Several neuropeptides are present in the mammalian pineal gland. Most of these peptides, eg neuropeptide Y, vasoactive intestinal peptide, and peptide histidine isoleucine, are located in nerve fibres innervating the gland. In some mammalian species, neuropeptides are also found in cells scattered in the pineal parenchyma. In the rat, bipolar cells immunoreactive for somatostatin are present, just as cells containing mRNA encoding somatostatin can be detected in the gland by in situ hybridisation. In the pineal gland of the European hamster, many cells are immunoreactive for enkephalin. Ultrastructural cytochemical analysis of these cells reveals a pinealocyte morphology. Processes from the opioidergic pinealocytes terminate in the parenchyma between the non-immunoreactive pinealocytes. Some of the processes contain small clear and large dense core vesicles and end in club shaped swellings which make synapse-like contacts with other pinealocytes. The ultrastructural morphology suggests that the opioidergic cells exert a paracrine regulation on other pinealocytes.     

Analysis of sister chromatid exchanges in the 1st, 2d and 3d mitoses using 5-bromodeoxyuridine and 5-bromodeoxycytidine

The cultures of Chinese hamster cells were treated with different concentrations (2.5, 5.0, 10.0, 20.0, 80.0 and 160.0 microgram/ml) 5-BrdU and 5-BrdC during 12 hours. The cultures were fixed at the 24-th hour. The linear increase of sister chromatid exchanges (SCE) was discovered with the increase of BrdU concentration. No change of SCE frequency was observed at different BrdC concentrations. The reasons for these differences in a concentration effect are discussed.     

Morphometric study of ventricular myocardial cells in the bat (Pipistrellus pipistrellus), hamster (Mesocricetus auratus) and Wistar rat.

A comparative morphometric study of ventricular myocardial cells of the West African insect-eating the Pipistrellus pipistrellus, the hamster and the rat revealed significant differences in volume fractions of mitochondria myofibrils, lipid bodies and T tubules. In the cells of the bat, mitochondria constitute 35% of cytoplasmic volume compared with 29 and 30% in the hamster and rat, respectively. Notably, crista density is much higher in the bat cells (3.58 x 10(5) cm-1) than in those of the hamster (2.59 x 10(5) cm-1) and of the rat (2.48 x 10(5) cm-1). Myofibril concentration is 40% in the bat, 53% in the hamster and 55% in the rat. There is greater numerical density of lipid bodies in the bat (212.27 x 10(9) cm-3) than in the hamster (139.20 x 10(9) cm-3) and in the rat (114.00 x 10(9) cm-3). The volume occupied by T tubules is 0.02 in the bat and hamster, and 0.009 in the rat. These differences suggest structural design for efficient metabolic activity in the bat which, among mammals, has high exercise tolerance.     

Pathogenesis of autoimmunity after xenogeneic thymus transplantation

Thymus transplantation is a promising strategy to induce xenotolerance, but may also induce an autoimmune syndrome (AIS). The pathogenesis of this AIS was explored using nude rats as recipients. Thymus grafts consisted of fetal hamster thymic tissue with or without mixing with fetal rat tissue such as thymus, thyroid, salivary gland, and heart. All hamster thymus recipients died of AIS within 2-3 mo. In most recipients of xenothymus mixed with rat tissues such as thymus, thyroid, and salivary gland, but not heart, AIS was prevented, indicating an insufficient presence of rat epithelial cell Ags within the xenothymus. AIS could be transferred to control nude rats by whole splenocytes or by splenocyte subpopulations such as CD3(+), CD3(-), and B lymphocytes, but not by non-T, non-B cells from AIS animals. This transfer could be suppressed by cotransferring either CD4(+) or CD8(+) lymphocytes from euthymic rats, but not by splenocytes from recipients of syngeneic or xenogeneic thymus mixed with rat tissue, indicating a defective generation of regulatory lymphocytes. As for CD4(+) regulatory cells this defect was probably qualitative, because the percentages of CD4(+)CD25(+) or CD4(+)CD45RC(low) populations were normal after xenothymus transplantation. As for the CD8(+) regulatory cells, the defect was quantitative, as CD8(+) cell levels always remained low. The latter was related to the nonvascularized nature of thymus grafts. In conclusion, AIS after xenothymus transplantation in nude rats is due to a combination of insufficient intrathymic presence of host-type epithelial cell Ags and a defective generation of regulatory T lymphocytes.     

Phenomenon of delayed disruption of telomeric links between chromosomes in polykaryocytes formed from human-Chinese hamster hybrid cells

The clones MOM-8-1 and MOM-8-3 of human--Chinese hamster cell hybrids were used for induction of the phenomenon of delayed disruption of the telomeric links between chromosomes. Colcemide (0.08 microgram/ml) and 5-bromodeoxyuridine (20 micrograms/ml) were present in cell culture for 30 hours. The dicentrics were observed in tetraploid, hypotetraploid and hypodiploid metaphases. No differences between the clones were found. The hybrid cells were the second object in which the phenomenon under discussion was reproduced.     

Derepression and carrier turnover: evidence for two distinct mechanisms of hexose transport regulation in animal cells.

Hexose uptake by hamster cells was increased five to ten fold by either substituting D-fructose for glucose or by completely omitting D-glucose from the culture medium for 24 to 48 hours. Conversely, when cycloheximide was present for 24 hours in media containing glucose, up to 20-fold decreases in hexose uptake were observed. However, these decreases in uptake activity were only observed over a narrow range of cycloheximide concentrations. After extended exposure to low concentrations of cycloheximide (0.05 to 10 mug/ml), the uptake by the fed cells decreased parallel with inhibition of protein synthesis whereas at high concentrations (greater than 50 mug/ml) uptake was increased. Cells deprived of glucose and maintained in the presence of cycloheximide did not show decreases in uptake activity. In separate experiments the high uptake rates of glucose-starved cells could be decreased by addition of glucose-free medium. The reversal was complete in 6 to 8 hours. The analog of glucose, 2-deoxy-D-glucose, did not promote the time-dependent decrease suggesting that the 6-phosphoester of glucose is not an inhibitor of transport. In addition, when cycloheximide is added at the same time as glucose, there is no decrease in uptake for at least 12 hours. We propose that turnover of components of hexose uptake systems could account for part of the control of hexose transport. Moreover, the results indicate that the turnover mechanism becomes inactive during glucose starvation and must be resynthetized following refeeding of the starved cells with glucose.     

DNA damage in isolated hamster and rat pancreas cells by pancreatic carcinogens

N-Nitrosobis(2-oxopropyl)amine (BOP), N-nitrosobis(2-hydroxypropyl)-amine (BHP) and N-nitroso(2-hydroxypropyl-2-oxopropyl)amine (HPOP) are pancreatic carcinogens in the Syrian golden hamster (SGH) but do not cause pancreatic tumors in rats. In this study, the ability of these three compounds to induce DNA damage in isolated pancreas cells from both species was determined by alkaline elution analysis. BOP was highly potent in SGH cells, causing DNA damage at concentrations as low as 0.5 micrograms/ml, and HPOP, although less potent than BOP, also caused considerable damage. Isolated SGH pancreas cells are thus able to metabolize BOP and HPOP to DNA-damaging species. Of the three compounds tested, only HPOP at higher doses (25-100 micrograms/ml) induced DNA damage in isolated rat pancreas cells. BHP did not damage rat or SGH pancreas cell DNA at concentrations up to 100 micrograms/ml, apparently due to lack of uptake of this compound by the cells. The observed insensitivity to DNA damage in rat cells is consistent with the resistance of the rat pancreas to carcinogenesis by these three compounds. The sensitivity of SGH pancreas cells to BOP- and HPOP-induced DNA damage correlates with the high carcinogenicity of these compounds for the SGH pancreas.     

Reduction of benzo(a)pyrene induced chromosomal aberrations by DL-alpha-tocopherol

The antioxidant, DL-alpha-tocopherol (vitamin E), has been demonstrated to significantly reduce the percentage of benzo(a)pyrene (BP) induced chromosomal aberrations in vitro. Chinese hamster lung (Don) and Chinese hamster ovary (CHO) cells were treated with either 1 microgram/ml or 5 micrograms/ml BP for 4 to 28 h; some cultures were treated with S9 mix activated BP. Additional cultures of Don and CHO were treated simultaneously with 100 micrograms/ml of DL-alpha-tocopherol and BP. In CHO cells 1 microgram/ml non-activated BP significantly increased the chromosomal aberration percentage above the control level. Aberrations observed included breaks, gaps, fusions, rings, dicentrics, and polyploids. Chinese hamster Don cells treated with 1 microgram/ml or 5 micrograms/ml S9 mix activated BP contained significant increases in aberration percentages above the control levels. When Don cells were treated simultaneously with activated BP and DL-alpha-tocopherol for 4 h, there was a slight decrease in the total aberration frequency to less than that of cells treated with activated BP only; however, when Don cells were treated with BP and DL-alpha-tocopherol for 28 h, there was a significant reduction in the aberration percentage below that of BP-treated cells alone. Similar results have been obtained with CHO cells treated with nonactivated BP and DL-alpha-tocopherol. The results reported here provide further evidence that antioxidants may prevent the potential mutagenic and carcinogenic effects of certain polycyclic compounds.     

Immunohistochemical localization of oxytocin and vasopressin in the adrenal glands of rat,cow, hamster and guinea pig

Summary The distribution of oxytocin and vasopressin in the adrenals of rat, cow, hamster and guinea pig has been studied by use of immunohistochemical techniques. In all the species studied the adrenal cortex contained both peptides; the staining in the zona glomerulosa being more intense than that in zona fasciculata or zona reticularis. The medulla, however, showed considerable species variation. In the cow, both peptides appear to be present in the adrenergic and noradrenergic cells, though staining was particularly prominent in cortical islands interspersed within the medullary tissue. In the rat, groups of medullary cells positive for both peptides were found, though it was not possible to associate these groups with particular chromaffin cell types. In the hamster oxytocin was present only in adrenaline-containing cells, whereas vasopressin was present in all medullary cells. The guinea pig medulla, which contains only adrenaline-secreting cells, was positive for both peptides. The possibilities that vasopressin and oxytocin have an autocrine or paracrine role in functioning of the adrenal gland is discussed.