Actions and comparative efficacy of phosphatidylcholine formulation and isolated sodium deoxycholate for different cell types.

BACKGROUND: Phosphatidylcholine formulation has been used to dissolve local fat deposits. This study aimed to evaluate and compare the effects of phosphatidylcholine formulation and its vehicle sodium deoxycholate alone on different cell lines to understand better its mechanism of action. METHODS: Cells and media including 3T3-L1 preadipocytes, normal foreskin fibroblasts, neonatal human dermal microvascular endothelial cells (CADMEC), and fetal human skeletal muscle cells (HSkMC) were used. After 24 h, cells were exposed in 3-4, 5-dimethylthiazol-2-yl-2, 3-diphenyl tetrazolium bromide reagent (MTT assays) to increasing dosages of phosphatidylcholine formulation (0.0156-0.5 mg/ml) or an equivalent vehicle, sodium deoxycholate solution, pH 9.0 (0.0066-0.210 mg/ml). Viability was assessed after 1, 2, and 3 days of treatment. Fat tissue (4 x 4 cm) obtained ex vivo from the dorsal fat pads of five rabbits was injected with 2 ml of phosphatidylcholine formulation (50 mg/ml), sodium deoxycholate (21 mg/ml), or normal saline and incubated for 24 h. These were examined histologically to identify cell lysis and morphologic changes. RESULTS: At 0.125- and 0.25-mg/ml doses of phosphatidylcholine solution, CADMEC and HSkMC were more sensitive (P < 0.001, one-way ANOVA) than adipocytes at all time points examined. Phosphatidylcholine formulation at a dose of 0.5 mg/ml and the equivalent vehicle, sodium deoxycholate, at a dose of 0.21-mg/ml both induced nearly 100% fat cell lysis after 24 h, and evidence of cell lysis as early as 6 h after exposure. After incubation of fat tissue for 24 h with phosphatidylcholine formulation, loss of intracellular lipid staining with an increase in extracellular lipids was seen. CONCLUSIONS: Isolated sodium deoxycholate was almost as effective as the phosphatidylcholine formulation, at clinical concentrations, in reducing the viability of mature adipocytes over time. Similar cytotoxic effects of phosphatidylcholine formulation on normal foreskin fibroblasts, endothelial cells, and human skeletal muscle cells also were observed. The data prove that the formulation acts in a nonspecific manner and that its unintentional administration to other tissues causes cell death.

Molecular markers in Paget disease of the breast.

BACKGROUND AND OBJECTIVES: Molecular markers are increasingly being analyzed in tumor specimens because of their relevance to both prognosis and choice of therapy. Paget disease of the breast is an uncommon form of breast cancer, in which molecular markers have not been well characterized. The objective of this study was to investigate the expression of c-erbB-2, p53, Ki-67, Cyclin D1, Bcl-2, estrogen receptors (ER), and progesterone receptors (PR) in mammary Paget disease. METHODS: Archival tumor tissues from 14 patients diagnosed between 1990 and 1999 with Paget disease of the breast were analyzed for these molecular markers by using an automated immunohistochemical assay. Both the intraepidermal Paget cells and the underlying carcinoma were assessed for these markers. RESULTS: The majority of Paget cells were positive for c-erbB-2 (92.9%), Cyclin D1 (100%), and Ki-67 (85.7%), but very few were positive for Bcl-2 (14.3%). p53 was overexpressed in 42.9% of the cases, and only 28.6% were positive for ER and PR. The rate of expression of these biologic markers was similar in both the Paget cells and the underlying intraductal and/or ductal carcinoma cells. CONCLUSIONS: Tumors from patients with Paget disease of the breast were positive for c-erbB-2, Cyclin D1, and Ki-67, molecular markers commonly associated with more aggressive tumor behavior and poorer survival in breast cancer patients. Few of these tumors expressed Bcl-2 or ER and PR, which are generally associated with a better prognosis. Similar expression of these markers in both Paget cells and the underlying carcinoma supports the theory that these cells are the result of an intraepidermal spread of ductal carcinoma.

Interferon alpha2b inhibits the murine melanoma cell line Cloudman S91 in vivo but not in vitro: a model for studying tumor cell-cytokine interactions.

Interferon alpha2b has recently been shown to improve outcome in patients with metastatic malignant melanoma. The high-dose interferon therapy used is however associated with significant systemic adverse effects. These adverse effects are likely related to the multitude of actions of interferon which in addition to its antineoplastic effects also possesses antiviral and immunomodulating properties. Elucidation of the mechanism of the antiproliferative effects of interferon may allow for the development of agents that possess the antineoplastic properties while being devoid of the other effects that make interferon toxic. In the animal model developed for this study tumors in mice receiving interferon alpha2b grew at a slower rate and achieved a small final tumor volume (3040 +/- 690 vs 1400 +/- 314 mm3 for the control and treated groups respectively, P < 0.05). Furthermore the final tumor weight in the treated group was significantly smaller (1.50 +/- 0.21 g vs 2.76 +/- 0.46 g for the treated and control groups respectively; P = 0.036). The (3-[4,5-Dimethylthiazol-2-y]-2,5-diphenyltetrazolium bromide) (MTT) colorimetric assay failed to reveal any direct effects of interferon alpha2b on this murine melanoma cell line. This antiproliferative effect of interferon alpha2b was in addition found to be independent of alterations in the expression of the angiogenic cytokines vascular endothelial growth factor, basic fibroblast growth factor, and transforming growth factor beta.

Characterization of serum dehydroepiandrosterone secretion in golden hamsters.

Dehydroepiandrosterone (DHEA) is an adrenal androgen whose function is poorly understood. Although DHEA and DHEA sulfate (DHEAS) are secreted in relatively high quantities by the human adrenal, the laboratory rat secretes very little, thus hindering experimental studies of the hormone. In this paper, we measured the changes in serum DHEA and DHEAS under various physiological conditions in golden hamsters. Evening serum DHEAS fell from 6.30 +/- 0.78 microg/dl (mean +/- SE) before surgery to 3.03 +/- 0.23 microg/dl 12 days after bilateral adrenalectomy. Hamsters had higher levels of DHEA and DHEAS in the evening than in the morning, but removal of the gonads did not consistently decrease serum DHEA or DHEAS in males or females. Evening levels of DHEA and DHEAS reached a peak around 7 weeks of age and then gradually decreased to about one-third of these levels by one year of age. These results suggest that DHEA and DHEAS are secreted at least in part from the hamster adrenal, that they do not originate from the gonads, and that there is a daily rhythm with peak levels at a time of day just preceding the active phase. In addition, the levels of these hormones decrease with aging.

Dehydroepiandrosterone and exercise in golden hamsters.

Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) are adrenal androgens that have been associated with a sense of well-being in humans. We describe two experiments done to test the hypothesis that an increase in DHEA or DHEAS secretion is associated with the inclination to exercise using a hamster model. In the first experiment, morning blood samples were obtained from adult male golden hamsters at various intervals after being placed in cages with (EX group) or without (SED group) access to running wheels. The EX group had lower DHEA (6, 12, and 14 weeks; p < 0.05) and DHEAS (13 and 16 weeks; p < 0.01) levels than the SED hamsters. In the second experiment, the number of wheel revolutions was monitored in castrated adult male hamsters implanted with Silastic capsules containing no hormone (blank control group), testosterone, or DHEA. The number of wheel revolutions in the group receiving DHEA was not significantly different than the blank control group, whereas testosterone increased wheel running at 4, 5, and 7 weeks (p < 0.05). These results indicate that DHEA and DHEAS levels decrease with exercise in male golden hamsters and that exogenous DHEA does not enhance the tendency to run on wheels.

Does exercise affect female hamster gonadotropins by reducing estradiol negative feedback?

Exercise stimulates reproductive function in hamsters exposed to short-day photoperiod (SDP) in contrast to its inhibitory effects in women and rats. SDP inhibits hamster reproduction in part by increasing the sensitivity of the hypothalamo-pituitary-gonadal axis (HPGA) to the negative feedback of gonadal steroids. To determine whether EX facilitates reproduction in female hamsters by affecting this mechanism, we examined the influence of estradiol (E2) on basal LH and FSH concentrations in exercising and sedentary hamsters maintained on long-day photoperiod (LD 14:10, LDP) or SDP (LD 8:16). In the LDP, serum LH and FSH were unaffected or reduced by exercise in ovariectomized (OVX) nonhormone-replaced hamsters, and LH was increased after tonic E2 replacement compared to sedentary controls. In the SDP, serum LH and FSH were significantly higher in OVX exercising than in sedentary hamsters, whether the exercisers were injected with a high dose of E2 or not. Thus, the effects of exercise on basal gonadotropin secretion in female hamsters appear to depend on the level of estradiol negative feedback (ENF). When this feedback is low (LDP OVX condition), exercise is either ineffective or inhibitory. When the ENF is increased by exposure to SDP and/or by treatment with E2, exercise has a stimulatory effect on basal gonadotropin secretion. Exercise may stimulate hamster gonadotropin secretion by reducing the ENF either by lowering the sensitivity of the HPGA to steroid negative feedback or by other means.

Bromodeoxyuridine improves the cytotoxic effect of cisplatin: a comparison with 5-fluorouracil.

We compared the effects of the radiosensitizers, 5-bromo-2'-deoxyuridine (BUdR) and 5-fluorouracil (5-FU) alone and in combination and cis-diamminedichloroplatinum (cisplatin, DDP) on the growth of B16 amelanotic melanoma (B16a) tumors in mice. In a preliminary study, tumor growth was significantly inhibited in the presence of BUdR and was further reduced with the combination of BudR and DDP. In a second experiment, BUdR was found to be more effective than 5-FU when used in combination with DDP. At the completion of the study, tumor volumes as a percentage of control values in mice treated with a single drug were as follows: 5-FU (50 mg/kg per day for 7 days) 76.5% (P < 0.05), BUdR (100 mg/kg per day for 7 days) 68% (P < 0.05) and DDP (5 mg/kg x 3) 54% (P < 0.01). Combining 5-FU and DDP at these dosages reduced volumes to 38% (P < 0.01), while BUdR + DDP-treated mice had tumor volumes only 28% (P < 0.001) the size of untreated controls. Furthermore, the toxicity, as demonstrated by a decrease in body weight and an increase in mortality, was more severe in mice receiving 5-FU than in those receiving in BUdR. DDP interacts synergistically with either BUdR or 5-FU in its cytotoxic action in vivo. No such relationship could be demonstrated in vitro, suggesting that the pharmacologic activity of these drugs may be responsible for the antitumor activity than direct cytotoxic effects. We propose that BUdR is more effective than 5-FU as a potentiator of DDP in this murine melanoma model.

Decreased adrenal sex steroid levels in the absence of glucocorticoid suppression in postmenopausal asthmatic women.

BACKGROUND AND AIM: Reduction of serum sex steroid levels has been reported to occur after the administration of beta-adrenergic medication. In that beta-adrenergic blockade is a central pathophysiologic feature of asthma, this study was done to explore the possibility of hormonal alteration in asthma. METHODS: Sex steroids obtained from 22 postmenopausal asthmatic and 22 age-matched, postmenopausal, nonasthmatic women were assayed. No subject had received estrogens, progestins, or oral corticosteroids for 120 days before the study. RESULTS: Mean dehydroepiandrosterone sulfate (DHEAS; p < 0.002), dehydroepiandrosterone (DHEA; p < 0.03), estradiol (p < 0.02), and estrone (p < 0.02) levels were lower in asthmatic patients compared with nonasthmatic subjects. Results could not be accounted for by current medication. Patients with asthma demonstrated no decrease in 17-hydroxyprogesterone or cortisol compared with nonasthmatic subjects, limiting findings to the delta 5, and not the delta 4, steroidogenic pathway. In a second phase of the study, DHEAS was measured before and after 3 days of oral beta-agonist stimulation in eight postmenopausal asthmatic women. Serum DHEAS concentration increased in eight of eight subjects, from a mean of 28.6 +/- 19.9 micrograms/dl (mean +/- SD) to 40.7 +/- 24.8 micrograms/dl (p = 0.002). Serum cortisol concentration was unchanged. CONCLUSION: The results indicate that postmenopausal asthmatic women have lower serum levels of adrenally derived sex steroids than their nonasthmatic peers and that this anomaly may be ameliorated by adrenergic stimulation.

Voluntary exercise increases gonadotropin secretion in male Golden hamsters.

To determine the effect of voluntary exercise and food restriction on reproductive hormone secretion, 48 adult male hamsters were placed in cages with (EX) or without (SED) running wheels. One-half of the animals in each exercise group was fed ad libitum, and the other half was food restricted to reduce their body weight to 90 g over 4 wk. After 10 wk, the EX ad libitum-fed group had much larger testes and much higher serum follicle-stimulating hormone and testosterone levels than the other three groups, but these values in the EX food-restricted hamsters were similar to those in the SED food-restricted group. In experiment 2, 20 adult male hamsters were castrated and later implanted with silicone rubber capsules containing testosterone. Two weeks after implantation of the capsules, the serum follicle-stimulating hormone levels were higher in the EX than in the SED group of testosterone-treated hamsters, but not in animals receiving blank capsules. These data suggest that exercise increases gonadotropin secretion by inhibiting the negative feedback of testosterone.

Olfactory bulbectomy counteracts inhibitory effect of food restriction on reproductive function.

Previous studies have shown that bilateral removal of the olfactory bulbs (BX) results in a large increase in gonadotropin secretion in golden hamsters. The principal question addressed by the present study was whether BX would offset the inhibitory effect of food restriction on reproductive function. BX or sham (SH) BX male golden hamsters were fed ad libitum or were restricted to only enough food to maintain them at 70% of the body weight of control groups fed ad libitum. The SH-70% group underwent marked testicular regression after approximately 6-8 wk, but the testes size of the BX-70% hamsters decreased only in proportion to the decrease in body weight. The BX food-restricted group had to be fed more food to maintain the same weight as the SH-70% hamsters, and the BX-70% group also had a higher core body temperature, lower percent body fat, and higher serum free thyroxine levels than SH food-restricted animals. In summary, removal of the olfactory bulbs appears to facilitate tonic gonadotropin secretion, such that food restriction is no longer capable of inducing testicular regression. In addition, the olfactory bulbs may have a strong influence on metabolic function in golden hamsters.

Olfactory bulbectomy induces reproductive recrudescence in golden hamsters on short photoperiod.

Olfactory bulbectomy results in a marked increase in gonadotropin secretion and prevents the reproductive regression associated with short photoperiod when the olfactory bulbectomy is done before exposure to the inhibitory photoperiod. The present study tested whether olfactory bulbectomy would offset the influence of short photoperiod if done after the reproductive system had regressed. Adult golden hamsters Mesocricetus auratus were divided into four groups: early sham (surgery at week-4); early olfactory bulbectomy (surgery at week-4); late sham (surgery at week 14) and late olfactory bulbectomy (surgery at week 14). At t = 0, all golden hamsters were placed in a short photoperiod (L:D 10:14). Early olfactory bulbectomy prevented testicular regression; the late olfactory bulbectomy group recrudesced much earlier than the sham groups. These results indicate that the tonic inhibitory influence of the olfactory bulbs is required for initiation of short photoperiod induced testicular regression and is also essential for the maintenance of the regressed state.

Unilateral but not bilateral olfactory bulbectomy inhibits body weight gain in hamsters.

The relation of the olfactory bulbs and photoperiod to the regulation of body weight was studied in male golden hamsters. Animals underwent sham operation, bilateral olfactory bulbectomy, or unilateral bulbectomy. They were left on long photoperiod for 5 weeks and then were transferred to short photoperiod for 11 weeks. The unilaterally olfactory bulbectomized hamsters gained less weight on long or short photoperiod than the sham operated group, while the bilaterally bulbectomized hamsters gained at least as much weight as the sham group. Thus, we report the novel finding that unilateral but not bilateral olfactory bulbectomy reduces body weight gain in male golden hamsters.

Olfactory bulbectomy lengthens circadian period of locomotor activity in golden hamsters.

Removal of the olfactory bulbs (BX) of rats or mice lengthens the circadian period of locomotor activity. In golden hamsters, BX elevates serum gonadotropin levels of hamsters maintained in long or short photoperiod and prevents the testicular regression associated with short days without altering the secretion or action of pineal melatonin. The present study examined the influence of BX on circadian wheel-running activity in hamsters and tested whether this effect was related to changes in serum testosterone levels. BX lengthened the free-running period of locomotor activity in gonadally intact hamsters by a mean of 21.0 min, and BX had a similar effect in orchidectomized animals with or without testosterone replacement. These results suggest that the olfactory bulbs normally tend to increase the frequency of the hamster's circadian oscillator and that this effect is unrelated to altering gonadal steroid levels.

The olfactory bulbs tonically inhibit serum gonadotropin and prolactin levels in male hamsters on long or short photoperiod.

Abstract Bilateral removal of the olfactory bulbs (BX) inhibits the testicular regression associated with maintenance of golden hamsters on short photoperiod (10L:14D). The present study was done to examine the reproductive endocrine changes following BX of hamsters, to test whether BX increases gonadotropin secretion by enhancing the rate of metabolism of peripheral testosterone, and to determine whether BX inhibits the response to photoperiod by blocking chemosensory signals from conspecifics. BX resulted in a marked increase in tonic serum gonadotropin levels in pre-pubertal (23 days old) and adult hamsters maintained on long photoperiod (14L:10D). Maintenance on 10L14D inhibited gonadotropin secretion in BX hamsters, but this only reduced the previously elevated levels to those of the sham group on stimulatory photoperiod, and the testes therefore remained large. BX hamsters on 10L:14D had a higher post-castration increase in serum luteinizing hormone than sham-operated hamsters. Following testosterone replacement (20 mm Silastic capsules), BX animals had lower serum testosterone and higher serum follicle- stimulating hormone levels than the sham group. BX hamsters had a shorter mean half-time for disappearance of testosterone from serum following removal of the capsule, but some animals in the sham group also metabolized testosterone rapidly. Isolation in cages receiving air filtered to remove pheromonal type molecules did not affect the rate or degree of testicular regression in response to short days. We conclude that the olfactory bulbs tonically inhibit gonadotropin release in golden hamsters on long or short photoperiod. The olfactory bulbs may facilitate the negative feedback of testosterone and may inhibit testosterone metabolism, but there were also steroid-independent effects. The influence of the olfactory bulbs on the hamsters' response to short days is apparently not related to chemosensory information from other hamsters.

The olfactory bulbs enhance reproductive hormone secretion in male rats on long or short photoperiod.

Abstract Unlike seasonally breeding species such as the Syrian hamster, Sprague-Dawley laboratory rats do not normally respond to short photoperiod (6L18D) with reproductive regression. However, removal of the olfactory bulbs (BX) unmasks a photoperiodic response in pre-pubertal rats so that blinding or short photoperiod results in an inhibition of reproductive hormone secretion and/or a delay in pubertal development. This is apparently mediated by pineal melatonin which inhibits gonadotropin and/or prolactin secretion, but the mechanism by which BX facilitates the response to photoperiod is not clear. Experiment I was performed to determine serum levels of reproductive hormones at frequent intervals following BX and/or maintenance on short days. Twenty-three-day old male rats were BX or underwent sham BX (SH). They were thereafter maintained on a 14L:10D (long photoperiod) or 6L:18D photoperiod for the duration of the study. At 6 weeks following surgery, BX rats on either photoperiod had smaller testes than the SH groups. At week 8, the BX group on 6L:18D had smaller testes than the other three groups. There were transient reductions in serum luteinizing hormone and follicle-stimulating hormone in the BX rats on short photoperiod, but there were prolonged effects of BX decreasing prolactin levels in rats on long or short photoperiod. In SH rats, testosterone was elevated for weeks 6 through 10 of the study, and BX blocked this increase. Experiment II was performed to determine whether BX alters testosterone feedback on gonadotropin secretion. Twenty-three-day old male rats were BX or underwent SH operation and were then returned to a room on 14L10D. Six to 8 weeks later, all animals were castrated and placed on 6L18D or returned to 14L:10D. Eight weeks following castration, the rats were implanted with Silastic capsules containing 0, 10, 20 or 40mm testosterone. The post-castration increase in serum luteinizing hormone and follicle-stimulating hormone was lower in the BX than SH rats. In long photoperiod, serum luteinizing hormone and follicle-stimulating hormone were often lower in the BX rats receiving no testosterone replacement or lower doses of testosterone than in the SH group receiving similar capsules. Maintenance on short photoperiod increased the responsiveness to testosterone so that even the rats receiving low doses of testosterone had very low luteinizing hormone and follicle-stimulating hormone levels whether they were SH or BX. In summary, BX rats on long or short photoperiod had lower serum prolactin and testosterone levels than the comparable SH group and BX inhibited the post-castration increase in gonadotropin secretion. The data therefore suggest that the olfactory bulbs tonically enhance reproductive hormone secretion (especially around the time of pubertal development).

Bilateral transection of the lateral olfactory tract but not removal of the vomeronasal organs inhibits short-photoperiod-induced testicular regression in golden hamsters.

It is now known that removal of the olfactory bulbs increases basal gonadotropin secretion and prevents short-photoperiod-induced testicular regression in Syrian hamsters. The experiments described in the present paper were an attempt to determine which neuronal systems associated with the olfactory bulbs are responsible for this influence on the reproductive neuroendocrine axis. In the first experiment, removal of the vomeronasal organ failed to influence gonadotropin secretion or testes weight in hamsters on long or short photoperiod, suggesting that the vomeronasal-accessory olfactory pathway is not individually responsible for the effect of the olfactory bulbs on gonadotropin secretion. In the second experiment, bilateral transection of the lateral olfactory tracts (LOT) did prevent short-photoperiod-induced testicular regression and the associated decrease in gonadotropin secretion. Since the nervus terminalis is confined to the surface of the medical olfactory bulb pathway, the results of LOT transection indicate that the nervus terminalis, which itself contains gonadotropin releasing hormone, does not mediate the influence of the olfactory bulbs on gonadotropin secretion. These results further suggest that the olfactory bulb influence on gonadotropin secretion is due to neural connections to the pyriform cortex, entorhinal cortex or amygdala.

Exercise inhibits reproductive quiescence induced by exogenous melatonin in hamsters.

Voluntary exercise inhibits the reproductive regression associated with a short photoperiod in male or female hamsters. The question addressed by the present study was whether exercise would also attenuate the reproductive regression associated with injection of exogenous melatonin. In male hamsters exercise inhibited the testicular regression, decline in gonadotropin secretion, and reduction in testosterone release associated with two daily injections (15 micrograms) of melatonin in pinealectomized hamsters on long days. After the reproductive system of the sedentary melatonin group had regressed, one-half of these hamsters were placed in cages with exercise wheels. Access to the exercise wheels stimulated testicular recrudescence and restored gonadotropin secretion to levels found in vehicle-injected hamsters. Sedentary female hamsters injected with melatonin tended to go into a state of constant diestrus associated with daily afternoon increases in serum luteinizing hormone, whereas most exercising hamsters injected with melatonin generally continued having regular estrous cycles with proestrus luteinizing-hormone surges. The ability of exercise to inhibit the effect of exogenous melatonin in pinealectomized hamsters suggests that exercise acts, at least in part, by mechanisms other than altering melatonin secretion.

Olfactory bulbs influence testosterone feedback on gonadotropin secretion in male hamsters on long or short photoperiod.

Previous studies have shown that prepubertal olfactory bulbectomy will prevent the testicular regression associated with short photoperiod in golden hamsters. The gonadal regression which normally occurs in hamsters on short photoperiod is known to be due in part to an increased responsiveness of the reproductive neuroendocrine system to the negative feedback actions of testosterone on LH and FSH secretion. The present study tested whether the olfactory bulbs influence the feedback effects of testosterone on gonadotropin secretion. Twenty-four- to 26-day-old male golden hamsters were either olfactory-bulbectomized (BX) or sham-olfactory-bulbectomized. Eight weeks later, all hamsters were castrated, and one half of each group was placed in LD 10:14 (this was called week-8 of the study), while the other half was returned to long photoperiod (LD 14:10). Eight weeks following castration (week 0 of the study), all animals were implanted with silastic capsules containing 0, 4, 8 or 16 mm of testosterone. All hamsters were bled by cardiac puncture at -8, -4, 0, +2, +4, +6 and +8 weeks. The concentration of LH and FSH in these samples was then determined by RIA. BX completely prevented the negative feedback of testosterone on gonadotropin secretion in hamsters on either long or short photoperiod at all levels of testosterone tested in this study. In addition, there were seemingly steroid-independent effects of BX on gonadotropin levels in the castrated hamsters prior to testosterone replacement at weeks -4 and 0.(ABSTRACT TRUNCATED AT 250 WORDS)