Estrogen metabolism in primary kidney cell cultures from Syrian hamsters

Estrogen metabolism was evaluated in freshly isolated kidney and liver microsomes and in primary kidney cell cultures from Syrian hamsters, a potential experimental model for examining the possible role(s) of estrogens in tumor initiation and development. Initial velocity studies of the conversion of estradiol to 2-hydroxyestradiol, as determined by the 3H2O release assay with the substrate [2-3H]estradiol, resulted in similar apparent Kms of estrogen 2-hydroxylase of 2.85 and 6.25 microM for liver and renal microsomes, respectively. The apparent Vmax for freshly prepared liver microsomes was 0.13 nmol.mg-1.min-1, while that for renal microsomes was 0.040 nmol.mg-1.min-1. Evaluation of estrogen metabolism was also performed in primary cell cultures of hamster kidney cells, consisting of 75% epithelial cells. [6,7-3H]Estradiol (10 microM) was incubated for 0, 24 and 48 h in primary kidney cell cultures, and the organic soluble metabolites analyzed by reverse-phase HPLC. The cultures from untreated, castrated hamsters metabolize [3H]estradiol to yield small quantities of estrone and significant amounts of polar metabolites, while no catechol estrogens were isolated. Estrogen metabolism by diethylstilbestrol-treated (DES-treated) hamster kidney cell cultures also provided small quantities of estrone and no evidence of catechol estrogens. Additionally, larger amounts of additional polar metabolites were isolated in the cultures from DES-treated hamsters. Finally, levels of estrogen 2-hydroxylase were detected in these cultures using the 3H2O release assay. Thus, the short-term primary kidney cell cultures from the Syrian hamster are capable of metabolizing estrogens. Furthermore, the enzymatic processes appear to be available for the conversion of any catechol estrogens formed into more polar metabolites. These investigations in intact cells, capable of performing all biochemical processes, complement both in vivo and subcellular biochemical studies and may aid in elucidating the roles of estrogens and estrogen metabolism in the initiation and development of estrogen-induced, estrogen-dependent kidney tumors in the Syrian hamster.     

Differential effects of glucocorticoids on energy homeostasis in Syrian hamsters

Syrian hamsters, like many humans, increase food intake and body adiposity in response to stress. We hypothesized that glucocorticoids (cortisol and corticosterone) mediate these stress-induced effects on energy homeostasis. Because Syrian hamsters are dual secretors of cortisol and corticosterone, differential effects of each glucocorticoid on energy homeostasis were investigated. First, adrenal intact hamsters were injected with varying physiological concentrations of cortisol, corticosterone, or vehicle to emulate our previously published defeat regimens (i.e., 1 injection/day for 5 days). Neither food intake nor body weight was altered following glucocorticoid injections. Therefore, we investigated the effect of sustained glucocorticoid exposure on energy homeostasis. This was accomplished by implanting hamsters with supraphysiological steady-state pellets of cortisol, corticosterone, or cholesterol as a control. Cortisol, but not corticosterone, significantly decreased food intake, body mass, and lean and fat tissue compared with controls. Despite decreases in body mass and adiposity, cortisol significantly increased circulating free fatty acids, triglyceride, cholesterol, and hepatic triglyceride concentrations. Although corticosterone did not induce alterations in any of the aforementioned metabolic end points, Syrian hamsters were responsive to the effects of corticosterone since glucocorticoids both induced thymic involution and decreased adrenal mass. These findings indicate that cortisol is the more potent glucocorticoid in energy homeostasis in Syrian hamsters. However, the data suggest that cortisol alone does not mediate stress-induced increases in food intake or body mass in this species.     

Cytostatic action of the peritoneal cells of Syrian hamsters on transformed cells

The cytostatic effect (CSE) of intact Syrian hamster peritoneal cells (PC) was determined by their capability to inhibit 3H-thymidine incorporation in target cells of HETR, which were placed in 1 X 10(4) or 4 X 10(4) cells per well together with 4 tenfold differing concentrations of PC (10(2)-10(5]. The optimum of CSE was seen with the use of maximal doses of PC and HETR in reaction. Maximal level of CSE with all effector-target cell rations was observed between 23-28 hours of contact. These data permit to suggest the role of HETR cells in activation of PC, as well as the transfer of cytostatic state in dense cell shift mediated by cell-cell contacts. The role of humoral cytostatic factor is also not excluded.     

Differential hormonal control of aggression and sexual behavior in female Syrian hamsters

These experiments were designed to test the effects of chronic estradiol treatment on aggression and sexual behavior in female hamsters. Isolated female hamsters were ovariectomized and tested for their behavioral responses to a group-housed, ovariectomized female hamster (aggression test) and a group-housed, intact male hamster (sexual behavior test). Following these baseline tests, the experimental females were implanted sc with Silastic capsules containing different concentrations of estradiol (100, 25, 10, or 0%) diluted with cholesterol and retested 3, 7, 10, and 14 days after implantation. High levels of aggression were observed on the baseline test, with no changes in aggression toward an intruder female observed for any implant group on subsequent tests. Despite these high levels of aggression toward another female, most of the estradiol-treated females (80% at 14 days) were sexually responsive in the presence of a male. There was no effect of Silastic estradiol concentration on sexual behavior, even though a range of serum estradiol levels (39–105 pg/ml) resulted. Lordosis latencies decreased and lordosis durations increased over the extent of estradiol treatment. Seventeen days after Silastic implantation, all females were injected with progesterone and retested. Estradiol-treated females showed an extreme reduction in aggression toward a stimulus female, as well as a further stimulation of sexual behavior after progesterone treatment. High levels of aggression in cholesterol-treated females (0% estradiol) were maintained even after progesterone injection, and these females never displayed any sexual responsivity. These results suggest that sexual behavior in the female hamster is sensitive to estradiol alone, whereas the inhibition of aggression requires the combination of estradiol plus progesterone.     

Electrophysiological studies on the cultured cells obtained from transplantable pancreatic carcinoma in Syrian golden hamsters

A pancreatic ductal carcinoma was established as a transplantable tumor line in an inbred strain of Syrian golden hamsters. Intracellular recordings of membrane potentials and input resistance were made from cultured cells obtained from the transplanted tumors using indwelling glass microelectrode. The mean value of the resting membrane potential was -46.5 +/- 1.8 mV (S.E.) (n = 13), while the mean resting input resistance was 21.2 +/- 4.3 M omega (S.E.) (N = 13). Dibutyryl cyclic AMP (2 X 10(-3)M) caused a marked hyperpolarization of about 30 mV accompanied by a reduction of input resistance. The transplantable tumor and its cultured cell line developed in this study have demonstrated their effectiveness as a reliable experimental model for use in pancreatic cancer research.     

Isolation and identification of normal killers from Syrian hamsters

Natural killers (NK cells) possessing cytotoxic activity were isolated from the blood, spleen and bone marrow of Syrian hamsters in discontinuous Percoll density gradient. These cells were morphologically identified as granular lymphocytes (GL). The largest amount of GL was isolated from the fraction containing 52-55% of Percoll. Cytotoxic activity of cells in this fraction was the highest, as compared to nonfractionated control or cells in other Percoll fractions.     

Mutagenic effect of orally given AF-2 on embryonic cells in pregnant Syrian hamsters

Pregnant hamsters were given various doses of AF-2 by stomach tube; then the cells of their embryos were isolated and cultured in normal medium. Chromosome preparations were made within 24 h after the start of primary culture, and examined for chromosomal aberrations. Marked chromosomal abnormalities were observed in cells of embryos of animals treated with AF-2 at over 20 mg/kg. Samples of surviving cells were also cultured in normal medium for 48 h, and then selected in medium containing 8AG or 6TG. This treatment with AF-2 caused marked dose-dependent induction of 8AG- or 6TG-resistant mutations: mutant colonies were even obtained after a single treatment with 2 mg of AF-2 per kg. These results show that this is a sensitive and useful mammalian system for detecting environmental mutagens.     

Ontogeny of endocrine cells in the respiratory system of Syrian golden hamsters

The ontogeny of protein gene product 9.5 (PGP 9.5), serotonin (5-HT), calcitonin gene-related peptide (CGRP), and calcitonin (CT) immunoreactivity was evaluated in small-granule endocrine cells of hamster laryngotracheal epithelium from fetal day 11 to adulthood. Two centrifugal (proximal-to-distal) patterns of differentiation occur. The first pattern begins during fetal life. Endocrine cells, single and clustered in groups (presumptive-or protoneuroepithelial bodies, pNEBs), initially colocalize immunostaining for PGP 9.5, 5-HT, and CGRP in the larynx and proximal 2/3 of the trachea on day 12 and spread to the caudal trachea on day 13.5-HT disappears fleetingly during the 24 h preceding birth; other-wise immunoreactivity for all three substances persists into adulthood. The clusters of endocrine cells survive beyond birth but are so diluted by expansion of the nonendocrine epithelium as to become inconspicuous. Since innervation was not actually observed, these clusters may persist as pNEBs, without developing connections to afferent or efferent nerve fibers. The second pattern concerns single small-granule cells stainable for CGRP but not for 5-HT. These cells first appear in the larynx and cartilaginous part of the cranial trachea on postnatal day 3, and in the middle and caudal trachea, on day 5. The cells increase in number on day 7. In adults, they predominate among endocrine cells of the cartilaginous region. A subset of these cells begins to co-express CT proximally on postnatal day 10, reaching the caudal end of the trachea by 3 weeks. A few elements of the older 5-HT-positive population may also become immunoreactive for CT in juvenile hamsters.     

Ontogeny of endocrine cells in the respiratory system of Syrian golden hamsters

The ontogeny of protein gene product 9.5 (PGP 9.5), serotonin (5–HT), calcitonin gene-related peptide (CGRP), and calcitonin (CT) immunoreactivity was evaluated in small-granule endocrine cells of hamster laryngotracheal epithelium from fetal day 11 to adulthood. Two centrifugal (proximal-to-distal) patterns of differentiation occur. The first pattern begins during fetal life. Endocrine cells, single and clustered in groups (presumptive- or protoneuroepithelial bodies, pNEBs), initially co-localize immunostaining for PGP 9.5, 5–HT, and CGRP in the larynx and proximal 2/3 of the trachea on day 12 and spread to the caudal trachea on day 13. 5–HT disappears fleetingly during the 24 h preceding birth; otherwise immunoreactivity for all three substances persists into adulthood. The clusters of endocrine cells survive beyond birth but are so diluted by expansion of the nonendocrine epithelium as to become inconspicuous. Since innervation was not actually observed, these clusters may persist as pNEBs, without developing connections to afferent or efferent nerve fibers. The second pattern concerns single small-granule cells stainable for CGRP but not for 5–HT. These cells first appear in the larynx and cartilaginous part of the cranial trachea on postnatal day 3, and in the middle and caudal trachea, on day 5. The cells increase in number on day 7. In adults, they predominate among endocrine cells of the cartilaginous region. A subset of these cells begins to co-express CT proximally on postnatal day 10, reaching the caudal end of the trachea by 3 weeks. A few elements of the older 5–HT-positive population may also become immunoreactive for CT in juvenile hamsters.     

Differential effects of nitrogen mustard in vivo on the cancer and host cells in MCa-11 tumours

We analysed the effects of nitrogen mustard (HN₂) on the growth, cell cycle distributions, and ratios of tumour cells to host cells for MCa-11 tumours grown in vivo. Treatment of tumour-bearing BALB/c mice with 3 mg/kg of HN₂ produced a significant slowing of MCa-11 tumour growth. Seventy-two hours after treatment in vivo with either 3 or 4 mg/kg of HN₂, the host cells in the treated tumours showed a significantly decreased G₀/G₁ peak and an increased G₂/M peak (P < 0.01), whereas the cancer cells in the treated tumours showed significant increases in the G₀/G₁ peak coupled with relatively decreased proportions of S and G₂/M tumour cells (P < 0.001). The ratio of the total number of cancer cells to the total number of host cells in the tumours was significantly increased 72 h after HN₂ administration (P<0.01). Thirty-two days after treatment with HN₂, the cell cycle distributions of the host and tumour cells in the treatment and control tumours had returned to being identical, but the ratio of the total number of cancer cells to the total number of host cells remained increased in the treated tumours (P<0.01). These results show that the administration in vivo of HN₂ can lead to entirely different cell cycle effects for the host and cancer cells in the same tumour, and that the partial growth arrest of MCa-11 tumours from HN₂ treatment may be due in part to the preferential destruction of host cells rather than solely to a direct cytotoxic effect on the cancer cells.     

Differential action of 7 beta-hydroxycholesterol on myocardial cells and fibroblasts. Obtaining primary cultures of pure myocardial cells

Newborn rat myocardial cells, grown in primary cultures, beat synchronously. Addition of 7 beta-hydroxycholesterol, at a 2.5 microM concentration, impairs this synchrony and may even stop any contraction. The associated fibroblasts no longer adhere to the support, and can be washed away by fresh culture medium. This restores the synchronous beatings of the myocardial cells, the viability of which is then even improved while they grow in the absence of fibroblasts.     

Chronic clorgyline treatment of Syrian hamsters: an analysis of effects on the circadian pacemaker

Clorgyline, a type A monoamine oxidase inhibitor with antidepressant properties when administered to depressed patients, is often associated with disturbances of the human sleep-wake cycle. In order to assess its effects on the mammalian circadian system, this drug was administered chronically to Syrian hamsters. It was found to affect the hamster circadian system in four specific ways. Clorgyline increased the intrinsic period of wheel-running activity, altered the phase response curve to brief light pulses, altered the reduced waveform of running activity in animals maintained in light-dark cycles or constant darkness, and increased the activity-rest ratio in animals maintained in constant darkness. Our data support the interpretation that clorgyline exhibits direct or indirect input to the circadian pacemaker and alters the processing of photic information to the pacemaker.     

The effects of serotonin agonists on the hypothalamic regulation of sexual receptivity in Syrian hamsters

One brain region that has been implicated in the regulation of lordosis is the medial preoptic-anterior hypothalamic continuum (MPOA-AH). Previous studies have suggested that this zone may be part of the circuit mediating the effects of serotonin (5-HT) on sexual receptivity. In the present experiments, we investigated the role of 5-HT(1a/7) and 5-HT(2) receptor subtypes in the MPOA-AH in the control of lordosis. In two experiments, either 5-HT(1a/7) or 5-HT(2) agonists were injected unilaterally into the MPOA-AH of ovariectomized, hormonally primed female hamsters. In the first experiment, microinjections of the 5-HT(1a/7) agonist 8-hydroxy-2,9-(di-n-propylamino)tetralin resulted in an attenuation of the lordosis posture by causing a decrease in the mean lordosis duration and an increase in the number of lordosis episodes over the entire testing period. In the second experiment, microinjections of the 5-HT(2b/2c) agonist m-chlorophenylpiperazine did not result in any changes in sexual receptivity. However, microinjections of the 5-HT(2) agonist (2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl facilitated lordosis by increasing the mean lordosis duration and decreasing the number of lordosis episodes in the first 5 min of the testing period. These data indicate that serotonin may act in the MPOA-AH via 5-HT(1a/7) receptors to attenuate and 5-HT(2) receptors to facilitate sexual receptivity.     

Effect of photoperiod on vasopressin-induced aggression in Syrian hamsters

Syrian hamsters are photoperiodic and become sexually quiescent when exposed to short "winter-like" photoperiods. In short photoperiods, male hamsters display significantly higher levels of aggression than males housed in long photoperiods. Arginine-vasopressin (AVP) within the anterior hypothalamus (AH) has been reported to modulate aggression in hamsters housed in long photoperiods. Previous studies have shown that AVP can facilitate aggression and its effects appear to be mediated by AVP V(1a) receptors (V(1a)R). In the present study, we investigated whether the increased levels of aggression observed after exposure to short photoperiod were the result of an increased responsiveness to AVP within the AH. Injections of AVP into the AH significantly increased aggression in hamsters housed in a long photoperiod, but had no effect in hamsters housed in a short photoperiod. In addition, injection of a V(1a)R antagonist into the AH significantly inhibited aggression in hamsters housed in long photoperiod, but had no effect in hamsters housed in a short photoperiod. These findings indicate that AVP within the AH increases aggression in hamsters housed in long photoperiods, but not in hamsters housed in short photoperiods.     

Characterization of an antiviral agent from primary murine fibroblast cultures: murine tissue culture CVI

We have previously described a class of virus inhibitors which are produced spontaneously by many types of cells in culture and present in a number of physiological fluids. These inhibitors are differentiated from all other known naturally occurring antiviral substances in regard to their (i) lack of species specificity, (ii) broad antiviral activity (iii) absence of high affinity binding by the inhibitor to the virus, (iv) mechanism of the action of the inhibitor is through inhibition of viral attachment, and (v) extreme thermal stability. In this report, we show that this class of inhibitors can be divided into two distinct subclasses. The first category includes the inhibitor spontaneously produced by cells in culture, originally described as contact-blocking viral inhibitor (CVI), and has a polypeptide component associated with its antiviral activity. The second category includes the inhibitors detected in body fluids and tissue extracts and has no essential peptide structure. Further characterization of CVI with respect to molecular size and stability to heat and a number of chemical reagents and enzymes indicate that the antiviral activity of CVI is associated with a large molecule (90s or approximately 4 million daltons), is stable at 100(8)C, and is resistant to the action of RNase, DNase, sulfhydral reagents, protein denaturants, and extraction by organic solvents.