Dual effect of metformin on growth inhibition and oestradiol production in breast cancer cells.

Evidence has been accumulating for a role for metformin in reducing breast cancer risk in post-menopausal women. It inhibits growth of breast cancer cells via several mechanisms, primarily the AMPK/mTOR signalling pathway. Another possible protective mechanism may be the ability of metformin to inhibit aromatase activity. In the present study, we investigated the effects of metformin on the basal growth of MCF-7 cells, after oestradiol (E2) stimulation and after the inhibition of mTOR by rapamycin. Secondly, we investigated the effects of metformin on the activity of a number of steroidogenic enzymes and the mRNA expression of aromatase and steroid sulphatase (STS). High doses of metformin significantly inhibited both basal and oestrogen-stimulated cell division. Low-dose rapamycin (10-10 M) did not inhibit growth, but the addition of metformin induced a significant reduction in growth. High-dose rapamycin (10-8 M) inhibited growth, and this was further attenuated by the addition of metformin. Exposure to low (10-7 M) and high (10-4 M) doses of metformin for 7-10 days significantly reduced the conversion of androstenedione (ANDRO) and testosterone (TESTO) (both requiring aromatase), but not the conversion of oestrone or oestrone sulphate (ES) via 17ß-hydroxysteroid dehydrogenase/sulphatase to E2. This attenuation was via a downregulation in the expression of total aromatase mRNA and promoter II, whilst the expression of sulphatase was unaffected by metformin. In conclusion, plasma levels of metformin have a dual therapeutic action, first by directly inhibiting cell proliferation which can be augmented by rapamycin analogues, and secondly, by inhibiting aromatase activity and reducing the local conversion of androgens to E2.

Prothrombin complex concentrates used alone in urgent reversal of warfarin anticoagulation.

BACKGROUND: Prothrombinex-VF (a three-factor prothrombin complex concentrate) contains little factor VII. Therefore, the Warfarin Reversal Consensus Guidelines from 2004 published by The Australasian Society of Haemostasis and Thrombosis recommend that it be administered with fresh frozen plasma to reverse warfarin anticoagulation. AIM: To evaluate the efficacy and safety of Prothrombinex-VF used alone in warfarin reversal. METHODS: Adult patients requiring urgent reversal of warfarin anticoagulation were defined as having achieved complete (target international normalized ratio (INR) <1.4) or partial reversal (target INR 1.4-2.0) of their anticoagulation. Prothrombinex-VF was given at doses of between 25 and 50 IU/kg based on the intent of reversal and an INR was obtained 30min post infusion. RESULTS: A total of 50 patients (mean age 72years, range 32-85years) was included. The median initial INR in the complete reversal arm (n= 35) was 3.5 (range 1.7-20) with 91% achieving the target INR (mean 1.1, range 0.9-1.4). In the partial reversal arm (n= 15) the mean initial INR was 5.6 (range 2.5-12) with 93% achieving the target INR (mean 1.6, range 1.4-2.2). There were no adverse effects attributed to Prothrombinex-VF. CONCLUSIONS: Prothrombinex-VF is very effective and safe when used alone to reverse warfarin anticoagulation. The supplementary use of fresh frozen plasma in these patients is not required. A review of the current Warfarin Reversal Consensus Guidelines is needed.

The Sea Empress oil spill (Wales, UK): effects on Common Scoter Melanitta nigra in Carmarthen Bay and status ten years later.

Carmarthen Bay, UK, regularly supports internationally important numbers (>16,000) of non-breeding Common Scoters Melanitta nigra. The spill of 72,000 tonnes of crude oil from the Sea Empress in 1996 affected birds both through direct mortality and likely pollution of key food resources. Numbers were greatly reduced following the spill, whilst changes in the distribution of birds within Carmarthen Bay suggested that potentially sub-optimal foraging zones were used. However, ten years after the incident, numbers of Common Scoter were no different to those recorded immediately before the spill. Compared to some other spills, rapid revival is evident. Numbers increased to pre-spill levels within three winters and distributional changes suggested a concurrent return to previously contaminated feeding areas, implying that the ecosystem had regenerated sufficiently to support its top predator. The importance of prolonged, standardised monitoring of bird numbers and distribution as indicators of ecological recovery from environmental damage is emphasised.

Genome sequence of the enterobacterial phytopathogen Erwinia carotovora subsp. atroseptica and characterization of virulence factors.

The bacterial family Enterobacteriaceae is notable for its well studied human pathogens, including Salmonella, Yersinia, Shigella, and Escherichia spp. However, it also contains several plant pathogens. We report the genome sequence of a plant pathogenic enterobacterium, Erwinia carotovora subsp. atroseptica (Eca) strain SCRI1043, the causative agent of soft rot and blackleg potato diseases. Approximately 33% of Eca genes are not shared with sequenced enterobacterial human pathogens, including some predicted to facilitate unexpected metabolic traits, such as nitrogen fixation and opine catabolism. This proportion of genes also contains an overrepresentation of pathogenicity determinants, including possible horizontally acquired gene clusters for putative type IV secretion and polyketide phytotoxin synthesis. To investigate whether these gene clusters play a role in the disease process, an arrayed set of insertional mutants was generated, and mutations were identified. Plant bioassays showed that these mutants were significantly reduced in virulence, demonstrating both the presence of novel pathogenicity determinants in Eca, and the impact of functional genomics in expanding our understanding of phytopathogenicity in the Enterobacteriaceae.

Phytoestrogens inhibit aromatase but not 17beta-hydroxysteroid dehydrogenase (HSD) type 1 in human granulosa-luteal cells: evidence for FSH induction of 17beta-HSD.

BACKGROUND: Studies using purified enzyme preparations, placental microsomes or cell lines have shown that certain phytoestrogens can inhibit the enzymes that convert androgens to estrogens, namely aromatase and 17beta-hydroxysteroid dehydrogenase (HSD) type 1 and type 5. The study aim was to investigate the effects of selected phytoestrogens on aromatase and 17beta-HSD type 1 activity in primary cultures of human granulosa-luteal (GL) cells. METHODS AND RESULTS: GL cells, cultured for 48 h in medium containing 5% fetal calf serum and for a further 24 h in serum-free medium with or without hFSH or hCG, were exposed to steroid substrates during the last 1-4 h of the experiment. The production of progesterone in the presence of pregnenolone or estradiol synthesis from androstenedione, estrone or testosterone showed dose- and time-dependent increases. Whilst hCG priming had no effect on progesterone production, FSH priming induced mean 68 and 56% increases in the production of estradiol from androstenedione (A-dione) and estrone respectively, but had no significant effect on the metabolism of testosterone to estradiol. None of the phytoestrogens investigated had any acute effects on enzyme activity. In contrast, when GL cells were exposed to the compounds for 24 h prior to exposure to steroid substrates for 4 h, 10 micro mol/l apigenin and zearalenone significantly inhibited aromatase activity, whilst biochanin A and quercetin had no effect. None of the phytoestrogens inhibited FSH-induced 17beta-HSD type 1 activity, and only quercetin significantly inhibited progesterone production. CONCLUSIONS: The inability of phytoestrogens to acutely inhibit steroidogenic enzymes in human GL cells (as has been shown in cell-free models) suggests that they are either rapidly metabolized to relatively inactive compounds or that the high enzyme activity in human GL cells masks any inhibitory effects of the compounds at the concentration tested.

Evidence that nitric oxide inhibits steroidogenesis in cultured rat granulosa cells.

1. A few studies have shown that nitric oxide may exert cytotoxic and/or steroidogenic effects on cultured ovarian cells but the source of this factor within the ovary remains equivocal. 2. In this study we have investigated the effects of nitric oxide on progesterone secretion, cell viability and cell morphology of cultured rat granulosa/lutein cells and examined whether granulosa cells are an important source of nitric oxide. 3. Only very low or undetectable levels of nitrites were measured in granulosa/lutein-cell-only cultures, although there was a small but significant increase in nitrite release observed in granulosa/ lutein cells obtained from oestrous rats compared with those obtained from proestrous rats. 4. There was a concentration-dependent inhibition of progesterone synthesis in the presence of the nitric oxide donors sodium nitroprusside and S-nitroso-N-acetyl-penicillamine which corresponded with an increased concentration of nitrite accumulation in the culture medium. 5. High concentrations of nitrites were measured in the medium of granulosa/lutein cells co-cultured with peritoneal macrophages and progesterone synthesis was inhibited. This effect of the macrophages was partially reversed by inhibitors of nitric oxide synthesis, aminoguanidine, NG-methyl-L-arginine and NG-L-arginine-methyl-ester, and the reversal of inhibition was inversely proportional to the concentration of nitrites measured in the medium. Dose-response curves for the three drugs on the inhibition of nitrite accumulation in macrophage cultures were obtained. 6. The nitric oxide scavenger c-PTIO [2-(4-carboxy-phenyl) -4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide.potassium salt] partially reversed the effects of S-nitroso-N-acetyl-penicillamine and macrophages on progesterone synthesis in granulosa/ lutein cells. 7. With the exception of the high dose of sodium nitroprusside, there was no evidence that any of the drugs reduced cellular viability, as assessed by measurement of cellular dehydrogenases and Trypan Blue exclusion, although high concentrations of nitrite in the culture medium derived either from the nitric oxide donors or macrophages were associated with a loss of morphological luteinization. 8. Based on the available evidence, we suggest that nitric oxide can inhibit steroidogenesis and that in vivo the source of nitric oxide may be from macrophages, which invade the ovary during the periovulatory period, and/or from endothelial cells of ovarian blood vessels.

Increased responsiveness of the hypothalamic-pituitary axis to steroid feedback effects in ovariectomized rats treated neonatally with monosodium L-glutamate.

Chronic ovariectomized rats treated neonatally with MSG showed reduced circulating concentrations of LH coupled with elevated hypothalamic LHRH stores. Despite the apparent loss of LHRH secretion, the small pituitary glands showed an increased density of LHRH receptors and normal responsiveness to the releasing hormone. The positive feedback effects of progesterone on LH release in oestrogen-primed animals was greatly exaggerated reflecting the build-up of hypothalamic LHRH stores without loss of pituitary responsiveness to LHRH.

Effects of 6-hydroxydopamine treatment of rats on the in-vitro release of LHRH and pituitary responsiveness to LHRH.

Injection of 150 micrograms 6-hydroxydopamine (6-OHDA) into the third ventricle of rats depleted the mean hypothalamic concentration of noradrenaline by 71% whereas the mean dopamine concentration was only reduced by an insignificant 7%. Isolated perfused pituitary glands taken from intact 6-OHDA-treated rats showed a markedly increased LH response to pulses of LHRH although there was no significant difference in the circulating levels of LH. Ovarian cyclicity was disrupted and the hypothalamic content of LHRH was significantly reduced. Hypothalamic synaptosomes prepared from intact 6-OHDA-treated animals consistently released less LHRH than did controls although the difference was not significant. Noradrenaline, dopamine and adrenaline did not significantly affect LHRH release from experimental or control synaptosome preparations. In ovariectomized rats 6-OHDA treatment did not inhibit the positive feedback effects of progesterone administration to oestrogen-primed animals, although the negative feedback effects of the priming oestrogen treatment was augmented. The results indicate that depletion of hypothalamic noradrenaline causes subtle changes in the endogenous release of LHRH and may alter the negative feedback effects of steroids on the hypothalamic-pituitary axis.

Characterization of the inhibitory effects of hyperprolactinaemia on the mechanism controlling LH secretion in chronically ovariectomized rats.

The time-course of the inhibitory effect of hyperprolactinaemia on LH secretion was delineated. Hyperprolactinaemia was induced in ovariectomized rats with injections of domperidone or ovine prolactin and circulating LH levels were measured from 1 h to 9 days after the treatment. Inhibition of LH secretion occurred within 2-4 h after treatment, and was maintained (provided that serum prolactin remained elevated) for a period of 6 days only. Thereafter LH levels increased to become insignificantly different from control levels on Day 9. A reduction in pituitary responsiveness was not associated with the acute or sub-chronic inhibition of LH secretion, although a significant fall in responsiveness was observed simultaneously with the return of serum LH levels to control values. No changes in hypothalamic LH-RH content was found. It is concluded that an impairment of pituitary function is not responsible for the inhibitory action of prolactin on LH secretion.

Safety evaluation of cimetidine: 54 month interim report on long-term study in dogs.

Cimetidine [N"-cyano-N-methyl-N'-2[(5-methylimidazol-4-yl)methylthio]ethyl guanidine] was administered orally to eight male and four female beagle dogs at a dose level of 144 mg per kg bodyweight per day. Four males and two females received placebo tablets. Dosing began in March 1976. During the first 26 months of dosing the animals became obese--the control animals more so than the cimetidine-treated. Since month 27 feeding time was restricted and there has been an overall weight loss. Some control animals are still obese. One control male and one dosed female have been killed because they developed convulsions. No treatment-related effects on haematology, clinical biochemistry, urinalysis, electrocardiography or clinical condition have been seen. Three out of the five surviving control animals but none of the 11 surviving cimetidine-dosed animals have developed cataracts. Biopsies of gastric mucosa taken during endoscopy in months 41, 47 and 53 have shown no changes attributable to cimetidine treatment.

Neural connexions between the medial forebrain bundle, the preoptic area and the basal hypothalamus in the rat: an electrophysiological study.

1. Electrophysiological experiments have been performed on intact cycling female rats to investigate the neural connexions that exist between the medial forebrain bundle, the anterior hypothalamic region, which included the preoptic area, and the basal hypothalamus. Recordings have been made from a total of 351 neurones in the anterior hypothalamus of which 216 were responsive to stimulation of either or both the medial forebrain bundle and basal hypothalamus (arcuate and ventromedial nuclei).2. Forty-six of these cells were responsive to a stimulus applied both to the medial forebrain bundle and the basal hypothalamus with a variety of response combinations. The majority of neurones were orthodromically activated by stimulation in both sites. Inhibition by stimulation of the medial forebrain bundle coupled with orthodromic excitation from the basal hypothalamus, or the reverse situation, was also encountered frequently.3. A few cells were antidromically invaded by the stimulation of the medial forebrain bundle and these received orthodromic or inhibitory inputs from the basal hypothalamus, although one unit outside the anterior hypothalamus was antidromically activated by both stimuli.4. Ninety per cent of all the doubly responsive units that could be antidromically activated by stimulation of the basal hypothalamus received an orthodromic input from the medial forebrain bundle, and no cells in the anterior hypothalamus that projected to the basal hypothalamus were found to receive an inhibitory input from the medial forebrain bundle.5. These results provide electrophysiological evidence for inhibitory and excitatory inputs from the medial forebrain bundle to the preoptic and anterior hypothalamic cells that either project to, or receive connexions from, the basal hypothalamus. Neurones in the preoptic area which project to the basal hypothalamus are implicated in the control of anterior pituitary function, particularly gonadotrophin secretion. These experiments, coupled with functional studies, suggest that there is an excitatory input from the medial forebrain bundle to these preoptic and anterior hypothalamic cells which may modulate adenohypophyseal secretions.

Prostaglandins and the release of LHRH from hypothalamic synaptosomes. Effects of indomethacin pretreatment.

The possible role of prostaglandins (PGs) in the hypothalamic control of ovulation has been investigated by studying the effects of indomethacin (ID) and PGs on the release of luteinizing hormone-releasing hormone (LHRH) from hypothalamic synaptosomes. Female rats were chronically pretreated with ID on diestrus 2 (D2) and proestrus (P), or on diestrus 1 (D1) and D2. Basal LHRH release from hypothalamic synaptosomes prepared from rats pretreated on D2/P was reduced to 48% after a 20-min incubation period, compared with sham-treated animals. ID pretreatment on D1/D2 caused a similar but less marked reduction in LHRH release (to 66% of controls at 20 min). Extraction of the LHRH remaining within the synaptosomal pellet showed that ID pretreatment also reduced the synaptosome content of LHRH. (15S)-15-methyl PGE2 (15-E2) (10(-4) and 10(-6) M) added to the incubating medium stimulated the release of LHRH BY APPROXIMATELY 40%, but this effect was only observed in synaptosomes prepared from ID-treated rats. PGE2 (10(-4) and 10(-6) M) had no significant effect in either sham-treated or ID-treated groups.

Responses and pharmacological properties of preoptic/anterior hypothalamic neurones following medial forebrain bundle stimulation.

1. The responses of neurones in the anterior hypothalamic and preoptic areas (POA/AHA) to stimulation of the medial forebrain bundle (MFB) have been studied in urethane anaesthetized female rats. Extracellular unit recordings have been made from 150 neurones which were responsive to a single stimulus applied to the MFB at the level of the mammillary nucleus. 2. Forty-five per cent of these cells were orthodromically activated with latencies ranging from 7.5 to 100 msec. However, the majority of cells responded with latencies of less than 40 msec. 3. Marked inhibition of spontaneous activity was observed in 41.5% of the units. Response latencies of up to 40 msec were observed in these cells, the inhibitory periods lasting up to 150 msec. 4. A small proportion of cells (13.5%) were antidromically activated and the average conduction velocity of these neurones in the POA/AHA with axons passing down to the mid-brain was estimated to be 0.3 m sec-1. It is suggested that they represent part of the descending MFB. 5. The experiments did not show any discrete topographical organization of cells in the POA/AHA which could be driven by MFB stimulation although the units tended to be located in more lateral rather than medial areas. 6. The responses to iontophoretically applied dopamine (DA) or noradrenaline (NA) was tested on sixty-one cells. These amines suppressed the activity of the majority of both orthodromically activated and inhibited units; the remaining cells were unresponsive. 7. These results provide electrophysiological evidence for both a direct and indirect input of MFB fibres to cells in the POA/AHA and that these inputs can be either excitatory or inhibitory. The data also indicate that a small number of fibres in the descending MFB originate from cells in the POA/AHA. 8. The sensitivity of these units to NA and DA suggests an inhibitory aminergic input, although this evidence is as yet indirect. 9. These connexions of the MFB, with neurones in the POA/AHA may be part of the neural circuits important for extra-hypothalamic modulation of gonadotrophin secretion.

Electrophysiological and pharmacological studies of amygdalo-hypothalamic connections in the rat.

Electrophysiological techniques have been used to investigate the neural connections between the amygdala and the rostral hypothalamus of female rats. Recordings have been made from 120 neurones in the preoptic/anterior hypothalamic area (POA/AHA) which could be driven by electrical stimulation of the amygdala. 89% of the neurones were activated orthodromically and, of these, 2 groups have been distinguished according to the latency of the responses. The remainder of identified cells were antidromically activated by stimulation of the amygdala. The response of these identified preoptic cells to the iontophoretic application of dopamine (DA) and noradrenaline (NA) has also been investigated. DA inhibited the spontaneous or glutamate-induced activity in 18 of 21 identified cells so tested and NA inhibited all 25 units; excitation was never observed. The role of the amygdala and of aminergic pathways in the control of ovulation is discussed.