Bromocriptine inhibits adipogenesis and lipogenesis by agonistic action on α2-adrenergic receptor in 3T3-L1 adipocyte cells.

The primary goals of the present study were to investigate the inhibitory effects of bromocriptine (BC) on adipogenesis and lipogenesis in 3T3-L1 adipocyte cells as well as to elucidate its molecular mechanism of action. Adipogenic and lipogenic capacity of BC-treated cells was evaluated by oil red-O staining, triglyceride content assay, real-time RT-PCR and immunoblotting. To determine the mechanism responsible for the anti-obesity effect of BC, we applied two methods. Firstly, we knocked down dopamine D2 receptor (D2R) up to 50% using siRNA. Secondly, we blocked the activity of α2-adrenergic receptor (α2-AR) by yohimbine treatment and monitored its effects on adipogenic and lipogenic events in 3T3-L1 cells. BC decreased the expression levels of adipogenic activators, including Pparα, Pparγ, and Cebpα, as well as major lipogenic target genes, including Me1, Acc1, 6Pgd, Fasn, and Prkaa1. Moreover, BC markedly reduced intracellular nitric oxide formation in a dose-dependent manner and expression of pro-inflammatory genes, Tnfα and Il6, which reflects attenuated pro-inflammatory responses. Further, upon treatment with BC, D2R-deficient cells displayed a significant decrease in lipogenic activity compared to control cells, whereas yohimbine-treated cells exhibited no reduction in lipogenic activity. BC can effectively attenuate adipogenesis and lipogenesis in 3T3-L1 cells by downregulating the expression of lipogenic genes and proteins. Our current experimental data collectively establish that the anti-obesity effects of BC are not D2R-dependent but result from the action of α2-AR in 3T3-L1 adipocytes.

Differential stimulation pathways of progesterone secretion from newly formed corpora lutea in rats treated with ethylene glycol monomethyl ether, sulpiride, or atrazine.

Ethylene glycol monomethyl ether (EGME), sulpiride, and atrazine are known ovarian toxicants, which increase progesterone (P4) secretion and induce luteal cell hypertrophy following repeated administration. The aim of this study was to define the pathways by which these compounds exerted their effects on the ovary and hypothalamic-pituitary-gonadal (HPG) axis. In the ovary, changes in the steroidogenic activity of new and old corpora lutea (CL) were addressed. EGME (300 mg/kg), sulpiride (100 mg/kg), or atrazine (300 mg/kg) were orally given daily for four times from proestrus to diestrus in normal cycling rats. Treatment with all chemicals significantly increased serum P4 levels, and EGME as well as sulpiride induced increases in prolactin (PRL) levels. In new CL, at both the gene and the protein levels, all three chemicals upregulated the following steroidogenic factors: scavenger receptor class B type I, steroidogenic acute regulatory protein, P450 cholesterol side-chain cleavage, and 3ß-hydroxysteroid dehydrogenase (HSD) and downregulated the luteolytic gene, 20α-HSD. Coadministration of EGME and bromocriptine, a D2 agonist, completely inhibited PRL but not P4 secretion. Additionally, steroidogenic factor expression levels were upregulated, and 20α-HSD level was downregulated in new CL. These results suggest that EGME both directly and indirectly stimulates P4 production in luteal cells, whereas sulpiride elevates P4 through activation of PRL secretion in the pituitary. Atrazine may directly activate new CL by stimulating steroidogenic factor expressions. The present study suggests that multiple pathways mediate the effects of EGME, sulpiride, and atrazine on the HPG axis and luteal P4 production in female rats in vivo.

Collaborative work on evaluation of ovarian toxicity. 15) Two- or four-week repeated-dose studies and fertility study of bromocriptine in female rats.

The main focus of this study is to determine the optimal administration period concerning toxic effects on ovarian morphological changes in a repeated-dose toxicity study. To assess morphological and functional changes induced in the ovary by bromocriptine, the compound was administered to female rats at dose levels of 0, 0.08, 0.4 and 2 mg/kg for the 2- or 4-week repeated-dose toxicity study, and for the female fertility study from 2 weeks prior to mating to day 7 of gestation. In the 2-week repeated-dose toxicity study, increase of ovarian weights was observed at 2 mg/kg. In the 4-week repeated-dose toxicity study, ovarian weights were increased at 0.4 and 2 mg/kg. The number of corpora luteum was increased in the 0.4 and 2 mg/kg groups of the 2- and 4-week repeated-dose toxicity studies by histopathological examination of the ovaries. Bromocriptine did not affect estrous cyclicity in 2- and 4-week repeated dosing. In the female fertility study, although animals in any groups mated successfully, no females in 0.4 and 2 mg/kg groups were pregnant. There were no adverse effects on reproductive performance in the 0.08 mg/kg group. Based on these findings, the histopathological changes in the ovary are considered important parameters for evaluation of drugs including ovarian damage. We conclude that a 2-week administration period is sufficient to detect ovarian toxicity of bromocriptine in a repeated-dose toxicity study.

Developmental atrazine exposure suppresses immune function in male, but not female Sprague-Dawley rats.

Each year, 75 million pounds of the broadleaf herbicide atrazine (ATR) are applied to crops in the United States. Despite limited solubility, ATR is common in ground and surface water, making it of regulatory concern. ATR suppresses the immunomodulatory hormones prolactin (PRL) and the thyroid hormones (THs), with developmental exposure to ATR permanently disrupting PRL regulation. We hypothesized that ATR may cause developmental immunotoxicity through its disruption of PRL or THs. To test this hypothesis, pregnant Sprague-Dawley (SD) rats were exposed to 35-mg ATR/kg/d from gestational day (GD) 10 through postnatal day (PND) 23. Separate groups were exposed to bromocryptine (BCR) at 0.2 mg/kg/2x/day to induce hypoprolactinemia or to propylthiouracil (PTU) at 2 mg/kg/day to induce hypothyroidism. After the offspring reached immunologic maturity (at least 7 weeks old), the following immune functions were evaluated: natural killer (NK) cell function; delayed-type hypersensitivity (DTH) responses; phagocytic activity of peritoneal macrophages; and antibody response to sheep erythrocytes (SRBC). ATR decreased the primary antibody and DTH responses in male offspring only. Neither PTU nor BCR caused immunosuppression in any measured variable, although PTU increased phagocytosis by peritoneal macrophages. These results demonstrate that developmental exposure to ATR produced gender-specific changes in immune function in adult rats and suggest that immune changes associated with ATR are not mediated through the suppression of PRL or THs.

Blunted central bromocriptine-induced tachycardia in conscious, malnourished rats.

Bromocriptine-induced tachycardia, persisting after adrenalectomy, is mediated by central dopamine D2 receptor stimulation through activation of the sympathetic outflow to the heart. The present study investigated the effects of malnutrition during pregnancy on bromocriptine-induced tachycardia in adult conscious rats. Malnourished rats were obtained by feeding dams a multideficient diet (providing 8% protein) during mating and pregnancy. Birth weight was significantly reduced in malnourished rats when compared to control rats born to dams fed standard commercially diet (23% protein) during mating and pregnancy. Baseline mean aortic pressure and heart rate in malnourished rats were comparable to those of well-nourished rats. Tachycardia (33+/-9 beats/min.), but not the hypotensive response to intravenous bromocriptine (150 microg/kg) was significantly reduced in malnourished rats, compared with control rats (70+/-10 beats/min.). In malnourished rats, pretreatment with intravenous domperidone (500 microg/kg) blocked the bromocriptine-induced hypotension, without affecting the tachycardia. Neither cardiac vagal (40+/-6 beats/min.) nor sympathetic tone (76+/-6 beats/min.) was significantly altered by multideficient diet-induced malnutrition (51+/-6 and 67+/-10 beats/min., respectively). In isolated perfused heart preparations from malnourished rats, positive inotropic response to isoproterenol (10-8 to 10-4 M) was not significantly different compared to that in control rats. In summary, malnutrition during foetal life blunted the bromocriptine-induced tachycardia, an effect that could be related to central dopamine D2 receptor desensitization rather than to impairment of autonomic regulation of the heart or cardiac beta-adrenoceptor desensitization.

Evaluation of the male pubertal assay's ability to detect thyroid inhibitors and dopaminergic agents.

The male pubertal onset assay is under consideration as an alternate Tier I screening assay to detect potential endocrine active chemicals (EACs) acting through a variety of steroid hormone and thyroid hormone receptor-mediated and non-receptor-mediated mechanisms. This study focused on the assay's ability to detect several non-receptor-mediated EACs. Weanling male CD rats (21 days old) were dosed for 30 d by gavage with vehicle (0.5% METHOCEL) or the following EAC classes (mg/kg/d): a potent thyroid agent (6-propylthiouracil, PTU, 240), a weak thyroid agent (phenobarbital, PB, 50 or 100), a dopamine antagonist (haloperidol, HALO, 2 or 4), or a dopamine agonist (bromocryptine, BRC, 10 or 50). In vehicle-treated males, preputial separation (PPS) occurred at 44.4 +/- 2.0 days of age. Age at PPS was delayed with PTU and 50 BRC, treatments that also delayed growth. Absolute testes and/or epididymal weights were decreased by PTU and 100 PB. BRC (50) and PB (100) decreased absolute prostate and seminal vesicle weights. Relative thyroid weights were altered by HALO, PTU, and PB, agents that significantly decreased serum T(4) levels. PTU increased serum thyroid-stimulating hormone (TSH) by 8.5 times and markedly altered thyroid histology, whereas HALO and PB did not significantly increase TSH and had marginal effects on thyroid histology. Thus, this assay detected both strong (PTU) and weak (PB) thyroid agents as well as the dopamine agonist BRC; however, its ability to detect dopamine antagonists remains unproven. These results confirm that thyroid weight measurements, although not required in the current male pubertal assay protocol, may add valuable information for interpretation of thyroid effects. Due to the apical nature of the male pubertal assay end points, additional work will be required to establish definitive criteria for a positive result in this assay.

Accumulation of membranes and photoreceptor degeneration in mouse retina.

Ultrastructural response of photoreceptors to 10 days of treatment with reserpine and bromocryptine, and to 10 days of illumination, was studied. Treatment with isoproterenol was also applied in some experiments with continuous illumination. Treatment with bromocryptine and reserpine induced elongation of rod outer segments and accumulation of membranes in the subretinal space. Increased incidence of apoptosis but not of autophagocytosis was observed in both cases. Continuous illumination with or without treatment with isoproterenol was found to induce intracellular accumulation of membranes, autophagocytosis and apoptosis. Incidence of apoptosis was slightly decreased in the case of isoproterenol application. We conclude that intracellular or extracellular accumulation of membranes can function as the apoptotic trigger in photoreceptors. Triggering of apoptosis was not dependent on autophagocytosis. Possible role of membrane peroxidation in photoreceptor cell death is discussed.

Nitric oxide (NO) pathway and locomotor hyperactivity towards dopaminomimetics in rats.

The effects of the NO donor molsidomine and the inhibitors of the NO synthase (NOS) 7-nitroindazole (7-NI) and NG-nitro-L-arginine methyl ester (L-NAME) on the motor hyperactivity induced by indirect (amphetamine and cocaine) or direct (SKF 38393 and bromocriptine) dopamine (DA) agonists were studied in rats. The hyperactivity induced by 0.5 mg/kg of amphetamine or 5 mg/kg of cocaine was potentiated in a dose-dependent manner by molsidomine (30-100 mg/kg), but attenuated by 7-NI (3-30 mg/kg) or L-NAME (3-30 mg/kg). The NOS inhibitors also inhibited the locomotor hyperactivity evoked by 15 mg/kg of SKF 38393 (a DA D1 agonist) or 5 mg/kg of bromocriptine (a DA D2 agonist). On the other hand, the hyperactivity induced by those direct DA receptor agonists was potentiated by molsidomine. The present findings provide further evidence for an interaction at the behavioral level between NO and the DA-mediated effects of amphetamine and cocaine; moreover, they seem to indicate that both DA D1 and DA D2 receptor subtypes are under such influence.

Usefulness of recombinant human prolactin for treatment of poor puerperal lactation in a rat model.

Recombinant human prolactin (r-hPRL) was produced by a line of murine C127 cells transfected with human PRL gene. To assess the biological efficacy of r-hPRL in vivo, we studied its influence on milk secretion using a rat model in which lactation was reduced by bromocriptine treatment. Puerperal rats were injected daily for 9 days after delivery with bromocriptine or bromocriptine plus r-hPRL, and lactational performance was assessed by weighing the pups. The concentrations of rat and human PRL in rat serum were measured by specific radioimmunoassays and the mammary glands were examined on postpartum day 10. Daily injection of bromocriptine (0.1 mg/rat) significantly reduced the endogenous level of rat PRL and impaired the weight gain of the pups. Administration of r-hPRL increased the serum level of human PRL. Daily injections of r-hPRL (50 micrograms/rat, twice a day) restored lactational performance and significantly increased the weight of the pups. The detrimental effect of bromocriptine on the mammary glands, assessed by both weight and histological appearance, was reversed by administration of r-hPRL. These results demonstrate that r-hPRL is biologically active in vivo and replacement therapy of r-hPRL is effective in improving the lactational performance in bromocriptine-treated rats, and also that r-hPRL may be useful for the treatment of women with poor lactation.

Assessment of genotoxicity of two anti-parkinsonian drugs (selegiline hydrochloride and bromocriptine mesylate) in vivo in mouse bone marrow cells.

Selegiline hydrochloride (1-deprenyl) and bromocriptine mesylate (2-bromo-alpha-ergocryptine) are two drugs that have shown considerable promise in the treatment of Parkinson's disease. The in vivo mouse bone marrow micronucleus assay was used to examine their clastogenic and cytotoxic potential in human clinical dose range. Our results indicate that both drugs failed to induce significant number of micronuclei and were not cytotoxic at any of the doses tested, in vivo in mouse bone marrow cells, at doses as high as 16-times the clinical dose used in humans.

Use of bromoergocryptine in the validation of protocols for the assessment of mechanisms of early pregnancy loss in the rat.

Validated protocols for evaluating maternally mediated mechanisms of early pregnancy failure in rodents are needed for use in the risk assessment process. To supplement previous efforts in the validation of a panel of protocols assembled for this purpose, bromoergocryptine (BEC) was used as a model compound because it is known to inhibit pituitary prolactin secretion. BEC was tested using the early pregnancy protocol (EPP), the decidual cell response technique (DCR), the pre- vs postimplantation protocol (PPP), and embryo transport rate analysis (ETRA). These protocols evaluate the effects of chemicals on multiple endpoints following exposure during (a) the first 8 days of pregnancy, (b) early pseudopregnancy accompanied by decidual induction, (c) the pre- and postimplantation intervals of early pregnancy, or (d) the period of embryo transport. In the EPP, dosing with BEC during Days 1-8 of pregnancy reduced the number of implantation sites found on Day 9 as well as serum progesterone. The DCR technique revealed a dose-dependent inhibition of decidual growth concomitant with decreased serum progesterone as a result of BEC treatment. A modified DCR technique using hormone-supplemented ovariectomized rats demonstrated that BEC did not impair decidual growth in the presence of adequate progestogenic support. Pre- vs postimplantation dosing indicated that implantation is vulnerable to BEC effects at least through Day 4. BEC had no effect on embryo transport rate. Data from these protocols identified BEC as having adverse maternal effects during early pregnancy. While the pituitary was not identified by these protocols as the site of BEC's primary action, the protocols did identify a reduction in serum progesterone and impaired uterine function as toxicological mechanisms mediating the reduced fertility seen following BEC exposure.

Effects of drugs on pituitary fine structure in laboratory animals.

Although an increasing number of chemicals are reported to affect endocrine glands, only a few studies are dealing with their toxic effect on pituitary. The drugs can induce lesions acting directly on endocrine cells or indirectly by interfering with the regulation of their endocrine activities. Some drugs stimulate pituitary cell proliferation leading to hyperplasia and tumor formation; other chemicals have an inhibitory effect on adenohypophysial cells; and only one drug, hexadimethrine bromide, has been found to induce pituitary necrosis. Although complex toxicologic studies have been carried out on many chemicals, the mechanism of action of most drugs is not completely elucidated and further studies are necessary to establish structure function correlations.

Effects of bromocriptine and perphenazine on prolactin and progesterone concentrations in pregnant pony mares during late gestation.

Pregnant pony mares in Group A (n = 4) received i.m. injections at 07:00 and 17:00 h of 0.8 mg bromocriptine/kg body weight 0.75 per day beginning on Day 295 of gestation and continuing until parturition. Group B (n = 4) was treated similarly, but perphenazine was administered orally at 0.375 mg/kg body weight twice a day beginning on Day 305 of gestation and continuing until parturition. Mares in Group C (n = 3) received i.m. injections of saline. Mean plasma prolactin and progesterone concentrations were greater (P less than 0.05) for mares in Group C than in Groups A and B from 295 to 309 days of gestation. From 305 days of gestation, plasma prolactin and progesterone concentrations were greater (P less than 0.05) in Group B and C than in Group A mares. Progesterone and prolactin concentrations increased over this period for Group B and Group C mares, but remained constant in Group A mares. From 10 days pre partum through foaling, mares in Group A had lower progesterone (P less than 0.05) and prolactin (P less than 0.01) concentrations than Group B and C mares. All mares in Group A were agalactic at foaling, while all mares in Groups B and C had normal milk secretion. Gestation was longer (P less than 0.05) in Group A than in Group C mares. In Group A, 2 mares retained the placenta for greater than 3 h, 3 mares had dystocia and all 4 mares had thickened, haemorrhagic placentae.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of bromocriptine treatment on immune responses and 3-methylcholanthrene-induced tumorigenesis in rats.

Seven-week-old male Sprague-Dawley rats were given a single injection of 1.5 mg of 3-methylcholanthrene (3MC) to induce in situ fibrosarcomas. The rats were also treated with the dopamine agonist bromocriptine (BCR) from two days prior to 14 days after 3MC treatment and again for 14 consecutive days beginning at week 5. Tumor incidence was markedly increased and latency decreased in BCR-3MC rats compared to 3MC controls. Natural killer (NK) cell cytotoxicity responses and production of interleukin 2 (IL2) was enhanced at two weeks in rats treated with only BCR. Natural killer cell activity was suppressed at two weeks in rats treated with only 3MC. This effect was reversed by BCR treatment. Rats treated with 3MC and BCR had suppressed NK cell responses and production of IL2 and interferon-gamma (IFN) at 12 weeks. In another study, rats injected with 1, 3 or 5 mg/kg BCR for 14 consecutive days had increased NK cell activity and IL2 production at all doses and increased IFN production at the two high doses. Antibody (IgG) responses to an injected antigen and delayed-type hypersensitivity reactions were not affected by BCR treatment. Animals treated with the two high doses of BCR had decreased serum prolactin (PRL) levels. Serum growth hormone (GH) concentrations were markedly increased in the group treated with 3 mg/kg BCR. These data suggest that BCR enhances 3MC-induced tumorigenesis. The mechanism of this effect is apparently not mediated by suppression of the immune system since BCR-treated rats had selectively enhanced immune function. Enhancement of immune responses by BCR has not been previously reported.

Evaluation of the teratogenic potential of the dopamine agonist bromocriptine in rats.

Single subcutaneous injections of bromocryptine (BCR; 2 mg/kg) to rats on days 5, 7 or 10 of gestation caused a lowering of the serum prolactin (PRL) concentration and, on days up to and including day 8 of gestation, abortion of entire litters. Coincident with the end of the PRL-dependent phase of gestation (approximately day 9), BCR was no longer abortifacient, allowing higher doses of the drug to be administered from this time without the risk of abortion. Treatment at 50 or 100 mg/kg from day 10 to 16 of pregnancy, despite affecting the dams (increased water consumption), had no adverse effects on pregnancy or fetal development.

The influence of dopamine agonists and antagonists on gastric lesions in mice.

The dopamine agonist bromocriptine and L-dopa significantly inhibited whereas dopamine antagonist haloperidol aggravated the gastric lesions induced by pylorus ligation in mice as found earlier for rats. Furthermore, the successful use of a dopamine antagonist alone for the induction of gastric lesions also in mice was demonstrated, since the gastric lesions were induced by a single dose of haloperidol without any additional noxious treatment. Bromocriptine successfully inhibited both the gastric lesion-potentiating as well as the gastric lesion-inducing effect of haloperidol.

On some central effects of elymoclavine.

The pharmacological investigation of the ergot alkaloid of the group of clavines elymoclavine isolated from Claviceps sp. cp. II showed the following: LD50 for mice for 24 hours was 350 (228 divided by 535) mg/kg and for rats--145 (81 divided by 258) mg/kg; elymoclavine induced a dose-dependent stereotypy in rats and mice which was antagonized by haloperidol and pimozide; it prevented the development of haloperidol catalepsy in rats and produced rotations contralateral to the striatal lesions with 6-OHDA which were antagonized by pimozide and partly by cyproheptadine. Elymoclavine increased the exploratory activity of rats in open field as this effect was antagonized by haloperidol and was essentially influenced by many substances acting on different transmitter systems (NA, DA, GABA). Elymoclavine inhibited the picroroxin and electroshock convulsive seizures but potentiated the pentylenetetrazol ones in mice as these effects were differently influenced by pimozide, haloperidol, 5-HT, atropine and phentolamine. The observed effects of elymoclavine are mainly due to its DAergic agonistic action. It seems, however, that influences on other transmitter receptors also underlie the mechanism of action of this ergot alkaloid.

Sister-chromatid exchanges and cell kinetics in human and rabbit lymphocytes exposed in vivo and in vitro to 2-bromo-alpha-ergocryptine.

The possible mutagenic and DNA-synthesis inhibitory effects of 2-bromo-alpha-ergocryptine, a new semi-synthetic ergot alkaloid, was studied in human and rabbit lymphocytes exposed to it in vivo and in vitro. The analysis of SCE was mainly used to evaluate potential mutagenicity, and the mitotic and DNA-synthesis inhibition was explored by examining the proportions of first-, second- and third-division metaphases in the corresponding lymphocyte cultures. The results obtained show that 2-bromo-alpha-ergocryptine does not induce SCE in the cell systems tested, or structural chromosome aberrations in human lymphocytes in vivo. On the other hand, a marked mitotic inhibitory effect and associated cell kinetic changes could be clearly attributed to the drug, probably related to its cytotoxicity.

The lactation-blocking drug bromocriptine and its application to studies of weaning and behavioral development.

An experimental method for blocking maternal lactation is reviewed and the possible application of this technique for experimentally manipulating weaning is considered. Maternal milk production can be inhibited using the prolactin-suppressing drug bromocriptine. The suitability of bromocriptine for use in behavioural experiments is considered. The pharmacology of bromocriptine (CB 154) is briefly outlined and a compilation of the reported lactation-inhibiting doses for various species is presented. The possible endocrine and behavioral side-effects and the toxicity of the drug are discussed. It is concluded that, in most species studied so far, the drug is relatively free from significant side-effects at the low doses needed to suppress lactation. Guidelines for the practical use of bromocriptine are suggested. Finally, some ideas about the possible application of the drug to the study of behavioral development and parent-offspring relationships are discussed.