Organ explant culture of adult Syrian golden hamster pancreas

Summary An organ explant culture system has been developed for long term maintenance of adult pancreatic tissue from the Syrian golden hamster. Gastric and duodenal lobe explants of up to 0.5 cm² size were placed in tissue culture dishes (60 mm²) on Gelfoam sponge rafts to which was added 5 ml of CMRL medium 1066 supplemented with heat inactivated newborn bovine serum,l-glutamine hydrocortisone, insulin, and antibiotics. Dishes were placed in a controlled atmosphere chamber, which was gassed with 45% O₂ 50% N₂, and 5% CO₂ and incubated at 36.5°C. Viability of the tissues was determined by light and electron microscopy as well as by [³]thymidine incorporation. Explants were viable for up to 70 d. Zymogen granule-containing cells characteristic of acinar cells and mucuscontaining cells characteristic of ductal cells were present throughout this period. However, endocrine cells were only present for the 1st wk in culture. This work was supported in part by National Cancer Institute Grant CA-19197-06 through the National Pancreatic Cancer Project and is UMP contribution No. 950.     

Oxidative metabolites of diethylstilbestrol in the fetal Syrian golden hamster

14C-Diethylstilbestrol was administered orally, intraperitoneally, and intrafetally to 15-day pregnant hamsters at a dose of 20 mg/kg body weight, and the radioactivity was determined in the fetus, placenta, and maternal liver after 6 hours. Significant amounts of radioactivity were found in these tissues in every case, indicating maternal-fetal and fetal-maternal transfer of diethylstilbestrol. Part of the radioactivity found in the tissues could not be extracted even after excessive washing. This implied the presence of reactive metabolites. In the fetal and placental extracts, eight oxidative metabolites of diethylstilbestrol were identified by mass fragmentography as hydroxy- and methoxy-derivatives of diethylstilbestrol, pseudodiethylstilbestrol, and dienestrol. The presence of oxidative metabolites in the hamster fetus and the covalent binding to tissue macromolecules are possibly associated with the fetotoxic effects of diethylstilbestrol.     

Body surface area of the golden Syrian hamster.

The body surface areas (BSAs) of 30 Syrian hamsters were actually measured. From these areas, the Mass Coefficients (K values) for the Dubois and Dubois equation (5.31) and for the Meeh-Rubner equation (11.89), were computed. These values were independent of weight and sex. To verify the applicability of the Mass Coefficients, the BSAs of another 20 animals were calculated and compared with the actually measured BSAs. The difference between measured and calculated BSAs was not significant. Therefore, these values can be used with their respective equations to compute BSA in Golden Syrian hamsters.     

Dietary protein level and skeletal development in the golden Syrian hamster

The hamster was used as a model for investigating the effect of low, moderate, and high protein intake (12, 18, and 36% casein) on bone mineral content. Animals fed the low level of protein between 3 and 8 months of age had a reduction in the weight of all skeletal components measured, with the exception of the diaphyseal portion of the long bones. Diaphyseal weight and calcium remained significantly lower when expressed as a percentage of body weight. However, urinary calcium excretion was not lower than that of animals consuming an adequate protein intake. Ingesting a high protein diet resulted in a significant increase in urinary calcium excretion, and a reduced amount of both mineral and organic material in the diaphyses. We conclude that long-term consumption of a high protein ration led to the development of a mild osteoporotic condition in the hamster which was limited to the diaphyseal portions of the long bones.     

Development of intracytoplasmic lumina in diethylnitrosamine-induced tracheal papillomas of Syrian golden hamster

Tracheal papillomas in the Syrian golden hamster were induced with diethylnitrosamine (DEN). Intracytoplasmic lumina filled with mucus were studied by light and electron microscopy. The sequence of lumen genesis is described ultrastructurally. Mucus substances in the center of intracytoplasmic lumen are presumed to be condensations of cell coat macromolecules. The intracytoplasmic lumen is discussed as a common feature of the adenoid component in epithelial neoplasms.     

Expression dynamics of transforming phenotypes in X-irradiated Syrian golden hamster embryo cells

We investigated the dynamics of expression for morphological transformation, for anchorage-independently growing (Anch-) cells and for mutation at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus in X-irradiated Syrian golden hamster embryo (SHE) cells. No Anch- cells were detected with 0-14 days of post-irradiation incubation before selection. No mutants at the HGPRT locus were detected with 0-5 days of post-irradiation incubation before selection. The maximum number of mutants for all doses was found after post-irradiation incubation for 8 days. On the other hand, the highest frequency of morphological transformants for all doses was detected with 0 days of post-irradiation incubation. The frequency of induction of morphological transformants increased with increasing dose. Then morphological transformants abruptly decreased with increasing lengths of post-irradiation incubation and no morphological transformants were detected with 14 days of post-irradiation incubation before selection (less than 10(-4)). A large fraction of morphological transformants (more than 86%) was cloned with feeder cells and expressed more extensive phenotypes of malignant transformation, such as the acquisition of anchorage-independent growth, immortality in vitro and tumorigenicity during further subculturing.     

The pathogenesis of retinoic acid-induced vertebral abnormalities in golden Syrian hamster fetuses

Administration of single doses of retinoic acid to hamsters on days 7, 8, and 9 of pregnancy resulted in missing, irregular, and abnormally fused centers of ossification in the vertebrae of fetuses recovered near term. A study of early events in embryonic tissues following maternal treatment with 60 mg/kg of the teratogen on day 8 revealed a variety of changes which could be linked to the development of the bony defects. By 12 hours following treatment, the mean number of somites in teratogen-exposed embryos was significantly reduced in comparison to controls. Within 24 hours of maternal treatment, lesions were observed in the aortae of the retinoic acid-exposed embryos. The vessels were consistently damaged caudally with dissection of aortic contents into the adjacent unsegmented mesoderm. Kinking of the neural tube, notochordal irregularities, and a loss of intercellular relationships in the paraxial mesoderm accompanied the vascular lesions. By 36 hours following treatment, abnormalities were evident in the appearance of the caudal somites, and at later stages these appeared to translate into defects in the sclerotomes and subsequently, the vertebrae. The observations suggest that vascular damage plays a significant role in the induction of the vertebral defects by disrupting somitogenesis. Moreover, the results support the hypothesis that retinoic acid produces abnormalities in the vertebral skeleton by a mechanism different from that which has been suggested to operate in the induction of defects in the limb skeleton.     

Transplacental action of sodium nitrite on embryonic cells of Syrian golden hamster

Hamster embryos were treated with various doses of NaNO2 in utero, by its oral administration to the mothers, and then the embryonic cells were examined for micronucleus formation, chromosomal aberrations, morphological or malignant transformation and drug-resistant mutations. For induction of resistant mutations, the cells were cultured in normal medium for 72 h, and then selected in media containing 8-azaguanine (10 or 20 microgram/ml) or 1 mM ouabain. This treatment with NaNO2 caused marked dose-dependent induction of 8-azaguanine- and ouabain-resistant mutations. Cultured embryonic fibroblasts in the resting state also showed a marked dose-dependent increase in micronucleus formation but not an increase in chromosomal aberrations. This treatment also caused morphological and neoplastic transformation of the cells. Transplacental oral treatment with DMN, as a positive control, caused changes of similar extent in biological effects of embryonic fibroblasts, and in addition it caused chromosomal aberrations in metaphase plates. On the contrary, transplacental oral application of NaNO2 did not induce any biological change in cultured embryonic fibroblasts.     

Effect of 4-hydroxyandrostenedione on 19-hydroxylation of deoxycorticosterone in the golden Syrian hamster

4-Hydroxyandrostenedione, a known inhibitor of ovarian and peripheral aromatization of androgen (testosterone and androstenedione) to form estrogen, was studied for its inhibitory effect on the 19-hydroxylating enzyme system of the adrenal for the conversion of deoxycorticosterone to 19-hydroxydeoxycorticosterone. In vitro incubation of Golden Syrian hamster adrenal homogenates with tritiated deoxycorticosterone demonstrated (80-85%) reduction in label incorporated into 19-hydroxydeoxycorticosterone in the presence of 4-hydroxyandrostenedione.     

An effective combination of anaesthetics for 6-h experimentation in the golden Syrian hamster

The anaesthetics described for use in hamsters to date are suitable for the performance of short-term experimentation. However, an anaesthetic regimen was required which would provide a stable preparation for 6 h and hence, a suitable combination was developed. In the first set of experiments, the effect of anaesthetics (chloralose, urethane, and pentobarbital) were examined alone and in combination on arterial blood measurements. In the second set of experiments the effect of the combination of anaesthetics on arterial blood measurements and minute ventilation was examined for up to 6 h. Chloralose, urethane and pentobarbital when used alone in the hamster were considered inadequate for our needs. Chloralose did not produce adequate surgical anaesthesia whereas urethane and pentobarbital resulted in marked respiratory depression. Urethane also produced a trend towards metabolic acidosis. In contrast, the combination of agents resulted in surgical anaesthesia and the arterial blood measurements were adequate. Further, the use of the combination of anaesthetics in hamsters resulted in a stable preparation where arterial blood measurements and minute ventilation were maintained in a good range for up to 6 h. The combination of chloralose, urethane and sodium pentobarbital in hamsters should prove useful in long-term non-recovery experimentation which requires early surgical intervention, minimal respiratory depression and an even depth of anaesthesia.     

Down-regulation of membrana granulosa cell gap junctions is correlated with irreversible commitment to resume meiosis in golden Syrian hamster oocytes

One of the currently popular hypotheses for the regulation of meiotic resumption in mammalian oocytes proposes that the preovulatory surge of luteinizing hormone causes down-regulation of follicular gap junctions, which in turn disrupts transfer of a meiotic arrester from the somatic cells into the oocyte. The present study has investigated this hypothesis by examining the integrity of membrana granulosa cell gap junctions during the period of irreversible commitment to maturation of golden Syrian hamster oocytes in vivo. Our results have revealed a significant progressive decrease in the fractional area of cell surface occupied by gap junction membrane with increasing percentage of oocytes irreversibly committed to mature (1.946% and 0.921% fractional gap junction area at 0% and 100% oocytes irreversibly committed to mature, respectively, P less than 0.05). This net loss of membrana granulosa cell gap junctions from the cell surface was accompanied by a significant decrease in density of gap junction particles, whether they were arranged in rectilinear or non-rectilinear packing patterns. Furthermore, the number of gap junction particles per unit area of surface membrane scanned also underwent a significant progressive decrease with increasing percentage of oocytes irreversibly committed to mature. These data with the hamster are consistent with the hypothesis that down-regulation of membrana granulosa cell gap junctions may be of central importance in the regulation of gonadotropic stimulation of meiotic resumption in mammalian oocytes.     

Establishment and characterization of a new,spontaneously immortalized,pancreatic ductal cell line from the Syrian golden hamster

Spontaneously immortal pancreatic cell lines are not available. By use of a defined culture medium, such a line (TAKA-1) was established from the Syrian golden hamster. Cytological, cytogenetic, molecular biological, enzymatic and receptor patterns as well as antigenicity were studied and were compared with those of the normal hamster pancreatic ductal cells in vivo. TAKA-1 cells grew exponentially in a monolayer on collagen gel in a defined medium but did not proliferate in soft agar. Ultrastructurally, the cells closely resembled the normal hamster pancreatic ductal cells. Similarities and dissimilarities were found between the normal ductal cells and TAKA-1 cells. Similarities included the presence of cytokeratin, carbonic anhydrase and some tumor-associated antigens. However, unlike the normal ductal cells, TAKA-1 cells expressed blood group A angigen and anti-vimentin, showed affinity to selected lectins, and an abnormality of chromosome 3, which is suggested to be associated with immortality. Moreover, unlike the hamster pancreatic ductal cancer cells but like the normal hamster pancreatic ductal cells, TAKA-1 cells did not have a c-Ki-ras mutation. EGF, TGF- and secretin, but not CCK or GRP, bound to the TAKA-1 cells. TAKA-1 cells produced TGF-, and their growth was stimulated by exogenous EGF in serum-free medium. This cell line presents a suitable model for biologic and pathologic study of the hamster pancreatic ductal cells in vitro.     

Insulin release from BK virus-induced and in vitro maintained insulinoma cells of Syrian golden hamster

The pancreatic tumor cells (In 111) derived from BK virus-induced insulinoma of Syrian golden hamsters were maintained in culture for several passages and were studied for their insulin secretory ability under various stimulatory conditions. Insulin release was not increased by D-glucose stimulation (27.8 mM), while dibutyryl cyclic AMP (1 mM), theophylline (1 mM), 3-isobutyl-l-methylxanthine (0.1 mM) and elevation of medium calcium from 0.5 to 2.7 mM stimulated insulin release 2.5- to 4-fold. There was a concomitant increase of medium cyclic AMP with addition of theophylline. Streptozotocin (2 mM) treatment for 48 hours significantly reduced insulin release, while alloxan (2 mM), had no inhibitory effect on insulin release. The results indicate that while in vitro-maintained islet tumor cells, In 111, have a cyclic AMP-mediated process involved in insulin secretion analogous to normal beta cells, these cells lack the ability to recognize glucose as an insulin secretagogue probably due to a defect in the cell membrane, though the possibility of alteration in glucose metabolism cannot be fully excluded.