Impact of osteoporotic fracture type and subsequent fracture on mortality: the Tromsø Study.

Less is known about the impact of non-hip non-vertebral fractures (NHNV) on early death. This study demonstrated increased risk of dying following hip and NHNV fractures which was further increased by a subsequent fracture. This highlights the importance of early intervention to prevent both initial and subsequent fractures and improve survival. INTRODUCTION: Osteoporotic fractures are a major health concern. Limited evidence exists on their impact on mortality in ageing populations. This study examined the contribution of initial fracture type and subsequent fracture on mortality in a Norwegian population that has one of the highest rates of fractures. METHODS: The Tromsø Study is a prospective population-based cohort in Norway. Women and men aged 50+ years were followed from 1994 to 2010. All incident hip and non-hip non-vertebral (NHNV) fractures were registered. NHNV fractures were classified as either proximal or distal. Information on self-reported co-morbidities, lifestyle factors, general health and education level was collected. Multivariable Cox models were used to quantify mortality risk with incident and subsequent fractures analysed as time-dependent variables. RESULTS: Of 5214 women and 4620 men, 1549 (30%) and 504 (11%) sustained a fracture, followed by 589 (38%) and 254 (51%) deaths over 10,523 and 2821 person-years, respectively. There were 403 (26%) subsequent fractures in women and 68 (13%) in men. Hip fracture was associated with a two-fold increase in mortality risk (HR 2.05, 95% CI 1.73-2.42 in women and 2.49, 95% CI 2.00-3.11 in men). Proximal NHNV fractures were associated with 49% and 81% increased mortality risk in women and men (HR 1.49, 95% CI 1.21-1.84 and 1.81, 95% CI 1.37-2.41), respectively. Distal NHNV fractures were not associated with mortality. Subsequent fracture was associated with 89% and 77% increased mortality risk in women and men (HR 1.89, 95% CI 1.52-2.35 and 1.77, 95% CI 1.16-2.71), respectively. CONCLUSION: Hip, proximal NHNV and subsequent fractures were significantly associated with increased mortality risk in the elderly, highlighting the importance of early intervention.

The association between childhood fractures and adolescence bone outcomes: a population-based study, the Tromsø Study, Fit Futures.

Childhood fracture may predict persistent skeletal fragility, but it may also reflect high physical activity which is beneficial to bone development. We observe a difference in the relationship between previous fracture and bone outcome across physical activity level and sex. Further elaboration on this variation is needed. PURPOSE: Childhood fracture may be an early marker of skeletal fragility, or increased levels of physical activity (PA), which are beneficial for bone mineral accrual. This study investigated the association between a previous history of childhood fracture and adolescent bone mineral outcomes by various PA levels. METHODS: We recruited 469 girls and 492 boys aged 15-18 years to this study. We assessed PA levels by questionnaire and measured areal bone mineral density (aBMD) and bone mineral content (BMC) using dual-energy X-ray absorptiometry (DXA) at arm, femoral neck (FN), total hip (TH), and total body (TB) and calculated bone mineral apparent density (BMAD, g/cm3). Fractures from birth to time of DXA measurements were retrospectively recorded. We analyzed differences among participants with and without fractures using independent sample t test. Multiple linear regression was used to examine the association between fractures and aBMD and BMC measurements according to adolescent PA. RESULTS: Girls with and without a previous history of fracture had similar BMC, aBMD, and BMAD at all sites. In multiple regression analyses stratified by physical activity intensity (PAi), there was a significant negative association between fracture and aBMD-TH and BMC-FN yet only in girls reporting low PAi. There was a significant negative association between forearm fractures, BMAD-FN, and BMAD-arm among vigorously active boys. CONCLUSION: Our findings indicate a negative association between childhood fractures and aBMD/BMC in adolescent girls reporting low PAi. In boys, such an association appears only in vigorously active participants with a history of forearm fractures.

Differential effects of epidermal growth factor (EGF) receptor ligands on receptor binding, downstream signalling pathways and DNA synthesis in hepatocytes.

Hepatocytes are responsive to mitogenic effects of several ligands acting via EGFR. Studying primary cultures of rat hepatocytes, we found that, as compared to EGF, HB-EGF had a markedly higher affinity of the EGFR, while AR and TGFα had lower affinity. HB-EGF was also more potent compared to the other growth factors regarding phosphorylation of EGFR, Shc, ERK1/2 and Akt. All ligands induced phosphorylation of ErbB2, indicating receptor heterodimerization. TGFα, despite having much lower receptor affinity, was about equally potent and efficacious as HB-EGF as a stimulator of DNA synthesis. In contrast, EGF had relatively high affinity but markedly lower efficacy in stimulation of DNA synthesis. The results suggest that amplifying and/or inhibitory mechanisms may modulate the mitogenic responses downstream of the initial signalling steps, and that this may affect the effects of the EGFR ligands differentially.

Inhibition of hepatocyte DNA synthesis by transforming growth factor beta 1 and cyclic AMP: effect immediately before the G1/S border.

Previous studies have shown that both transforming growth factor beta (TGF-beta) and cyclic AMP (cAMP) inhibit hepatocyte DNA synthesis. While cAMP (in addition to being stimulatory in G0/early G1) exerts its inhibition on hepatocytes late in G1, the point where TGF-beta inhibits has not been precisely defined. We have now examined further the inhibitory effects of cAMP and TGF-beta 1 on DNA synthesis in primary rat hepatocyte cultures and, in particular, tried to determine where in the prereplicative period the cells are sensitive to these agents. Although a transient exposure to TGF-beta 1 (but not glucagon) during the first hours of the cell culturing led to inhibition of DNA synthesis, the cells were more sensitive at a point late in G1, where they also were inhibited by cAMP. Thus, exposure to TGF-beta 1, glucagon, or the cAMP analogue 8-chlorophenylthio-cAMP at a time when there was a continuous recruitment of cells to S phase strongly decreased the rate of S-phase entry. For both TGF-beta 1 and cAMP the inhibition was established within 1-2 h, the lag time being indistinguishable for the two agents. No evidence was found for a synergism between TGF-beta 1 and cAMP. Treatment with TGF-beta 1 did not detectably alter basal or glucagon-stimulated cAMP concentrations. The results suggest that in hepatocytes there is a process immediately before the G1/S border which is sensitive to both TGF-beta 1 and cAMP and which appears to represent a major point of inhibition.

Effects of butyrate on epidermal growth factor receptor binding, morphology, and DNA synthesis in cultured rat hepatocytes.

We have examined the effect of butyrate on morphology, DNA synthesis, and epidermal growth factor (EGF) receptor binding in primary cultures of rat hepatocytes. Butyrate added 2 h after plating retarded the flattening and maintained the polyhedral shape of the hepatocytes in culture. Both insulin- and EGF-stimulated DNA syntheses were slightly stimulated by butyrate at 1 mM but strongly inhibited at 5 mM. EGF receptor binding was also strongly affected by butyrate treatment of the hepatocytes. The freshly isolated hepatocytes (prior to plating) and the early-stage cultures (2 h) exhibited two classes of surface EGF receptors with high and low affinity (Kd approximately 0.05 and approximately 0.7 nM, respectively). With increasing time in culture there was a decrease in the total EGF receptor number and a corresponding reduction in the capacity for receptor-mediated EGF internalization. The high-affinity receptor class was more strongly reduced than the low-affinity class and was almost absent after 40 h in culture. Butyrate dose-dependently counteracted the decrease in the number of surface EGF receptors during culturing and preserved the high-affinity binding component. Thus, after 40 h, the cells cultured in the presence of butyrate (5 mM) had an approximately 50% elevation in the total number of receptors and the capacity to endocytose EGF compared to control cells, whereas the binding at low ligand concentration (0.02 nM) was increased 4-fold. The results suggest that butyrate, in addition to affecting morphology and DNA synthesis, also has marked effects on the hepatocyte EGF receptor status.

Increased number of beta-adrenoceptors in hepatocytes from rats treated with 2-acetylaminofluorene.

Treatment of rats with chemical carcinogens, including 2-acetylaminofluorene (2-AAF), leads to a strong increase in the hepatic catecholamine-sensitive adenylate cyclase activity. The present study was undertaken to investigate the mechanism for the development of this increase. We report that hepatocytes isolated from rats which had been fed 2-AAF (0.025% w/w) for 8-12 weeks had an increased number of beta-adrenoceptors, as determined by [3H]dihydroalprenolol binding to whole cells and [125I]iodocyanopindolol binding to washed particles. For both ligands the number of binding sites was about 4-fold higher in hepatocytes from 2-AAF-treated rats than in those from controls. The adenylate cyclase activity of the carcinogen-fed animals showed both a general increase manifested in the basal level (2-fold) and in the activities obtained by stimulation with guanine nucleotides (2-3-fold), cholera toxin (1.5-fold), and glucagon (1.3-fold) and a selective, larger increase in the beta-adrenoceptor-linked activity (7-fold increment of the isoproterenol-sensitive activity). The results indicate that the number of hepatocyte beta-adrenoceptors increases during 2-AAF carcinogenesis. This may, at least in part, explain the rise in catecholamine-sensitive adenylate cyclase activity.

Altered hormone control of cyclic AMP formation in isolated parenchymal liver cells from rats treated with 2-acetylaminofluorene.

The hormone control of cyclic AMP-formation in isolated parenchymal liver cells from rats fed the carcinogen 2-acetylaminofluorene (0.025% for 4-8 weeks) was studied. The cells from the carcinogen-treated animals responded much more strongly to adrenergic agents than cells from control animals, while no significant difference was found for the glucagon effect. Of the adrenergic substances studied, the order of potency was isoprenalin larger than or equal to adrenalin larger than phhenylephrine; stimulation was blocked by propranolol, but not by phentolamine. The effects of supramaximal concentrations of isoprenalin and glucagon were not additive.

Bidirectional concentration-dependent effects of glucagon and dibutyryl cyclic AMP on DNA synthesis in cultured adult rat hepatocytes.

Glucagon and dibutyryl cyclic AMP exerted both stimulatory and inhibitory effects on hepatocyte DNA synthesis when added to primary monolayer cultures in the presence of serum, dexamethasone, insulin and epidermal growth factor. The stimulation occurred at low concentrations of glucagon (1 pM-1 nM) or dibutyryl cyclic AMP (1 nM-1 microM), while the agents inhibited DNA synthesis at higher concentrations (usually glucagon at over 10 nM or dibutyryl cyclic AMP at over 10 microM). The stimulatory effect was stronger at low cell densities (less than 20 X 10(3) hepatocytes/cm2). When the hepatocytes were cultured at higher densities, stimulatory effects were reduced or absent and the inhibition of (hormone-induced) DNA synthesis by a high concentration of glucagon was much more pronounced than at low cell densities. These results indicate dual, bidirectional, effects of cyclic AMP on hepatocyte DNA synthesis.

The mycotoxin alternariol induces DNA damage and modify macrophage phenotype and inflammatory responses.

Alternariol (AOH), a mycotoxin produced by Alternaria fungi, is frequently found as a contaminant in fruit and grain products. Here we examined if AOH could modify macrophage phenotype and inflammatory responses. In RAW 264.7 mouse macrophages AOH changed the cell morphology of from round to star-shaped cells, with increased levels of CD83, CD86, CD11b, MHCII and endocytic activity. TNFα and IL-6 were enhanced at mRNA-level, but only TNFα showed increased secretion. No changes were found in IL-10 or IL-12p40 expression. Primary human macrophages changed the cell morphology from round into elongated shapes with dendrite-like protrusions in response to AOH. The levels of CD83 and CD86 were increased, HLA-DR and CD68 were down-regulated and CD80, CD200R and CD163 remained unchanged. Increased secretion of TNFα and IL-6 were found after AOH exposure, while IL-8, IL-10 and IL-12p70 were not changed. Furthermore, AOH reduced macrophage endocytic activity and autophagosomes. AOH was also found to induce DNA damage, which is suggested to be linked to the morphological and phenotypical changes. Thus, AOH was found to change the morphology and phenotype of the two cell models, but either of them could be characterized as typical M1/M2 macrophages or as dendritic cells (DC).

Evidence for the involvement of Gi2 in activation of extracellular signal-regulated kinases in hepatocytes.

BACKGROUND: Activation of the extracellular signal-regulated kinases ERK1 and ERK2 in hepatocytes by prostaglandin (PG)F2alpha was recently found to be inhibited by pertussis toxin (PTX) suggesting a role for Gi proteins. RESULTS: Targeting the Gi2alpha expression by a specific ribozyme inhibited the PGF2alpha -induced ERK1/2 activation in hepatocytes. On the other hand a non-cleaving form of the Gi2alpha ribozyme did not significantly decrease the ERK1/2 activation. In ribozyme-treated cells the Gi2alpha protein level was reduced, while the Gqalpha level was not affected thus confirming the specificity of the ribozyme. CONCLUSION: The present data suggest an important role of Gi2 in PGF2alpha -induced ERK1/2 signaling in hepatocytes.

n-butyrate and dexamethasone synergistically modulate the surface expression of epidermal growth factor receptors in cultured rat hepatocytes.

n-Butyrate was previously found to increase the epidermal growth factor (EGF) receptor binding in primary cultures of rat hepatocytes. We show here that butyrate and dexamethasone synergistically modulate the surface expression of the EGF receptors. The butyrate-induced enhancement of high-affinity EGF binding was only slight in the absence of glucocorticoid, but was strongly and dose-dependently amplified by dexamethasone. Butyrate counteracted the inhibition by insulin of the dexamethasone-induced increase in EGF binding. The results indicate that the glucocorticoid has a permissive effect on a butyrate-sensitive process that determines the surface expression of the high-affinity class of EGF receptors.

Mechanisms for the emergence of catecholamine-sensitive adenylate cyclase and beta-adrenergic receptors in cultured hepatocytes. Dependence on protein and RNA synthesis and suppression by isoproterenol.

Adult male rat hepatocytes, which normally respond poorly to beta-adrenergic agents, acquire such responsiveness during primary monolayer culture. We here show that the rise in catecholamine-sensitive adenylate cyclase activity in hepatocytes in vitro is closely paralleled by an increase in the ability to bind the beta-adrenoceptor ligand [125I]cyanopindolol. The emergence of beta-adrenergic responsiveness did not require cell attachment or serum. Addition of dexamethasone, insulin, thyroxine or dihydrotestosterone to the cultures, singly or in combination, did not prevent the augmented beta-adrenergic responsiveness. The increase in catecholamine-sensitive adenylate cyclase activity and [125I]cyanopindolol binding could be blocked by cycloheximide or actinomycin D. Exposure of the cultures to isoproterenol at 3-hourly intervals led to a dose-dependent suppression of the rise in isoproterenol-responsive adenylate cyclase and prevented the increase in beta-adrenoceptor binding.

Oral contraceptive use and premenopausal breast cancer in Sweden and Norway: possible effects of different pattern of use.

Different use of oral contraceptives (OCs) in relation to reproductive histories and age might explain the conflicting results found in studies from different countries of OC use and premenopausal breast cancer. A population-based case-control study in Sweden and Norway has been analysed separately for the two countries. The study consists of 317 Swedish and 105 Norwegian cases diagnosed 1984-1985 with 317 Swedish and 210 Norwegian controls. The results for each country separately are consistent with the joint analysis showing increased risk for breast cancer with increased duration of OC use with the exception of that for women with more than 15 years since first use. The relative risk of breast cancer for more than four years of use among Norwegian women was 0.6 (95% confidence internal (Cl), 0.2-1.8). In both countries there was a non-significant higher risk associated with increasing duration of use before first full-term pregnancy. Analyses for surveillance bias show no evidence of bias as explanation for the association between total duration of OC use and breast cancer.

Long-term inhibitory effect of cAMP on beta-adrenoceptor acquisition and nonselective attenuation of adenylyl cyclase in hepatocytes.

Long-term effects of cAMP on the surface expression of beta-adrenoceptors and adenylyl cyclase activity were investigated in primary cultures of rat hepatocytes. beta-Adrenoceptor density and catecholamine-responsive adenylyl cyclase activity increased during culturing in a biphasic manner, with a plateau of 10-20 h duration occurring approximately 10 h after plating. Treatment of hepatocyte cultures with 8-bromo-cAMP during the plateau period did not affect the density of beta-adrenoceptors. In contrast, addition of 8-bromo-cAMP, 8-chlorophenylthio-cAMP, forskolin or glucagon during a period of active recruitment of surface beta-adrenoceptors resulted in a suppression of the acquisition of beta-adrenoceptors. In both experimental situations there was a partial decrease in hormone-stimulated and basal adenylyl cyclase activity. The results suggest that cAMP exerts at least two types of long-term regulation of adenylyl cyclase in hepatocytes: a suppressive effect on beta-adrenoceptor acquisition, and a partial, nonselective decrease in adenylyl cyclase activity not involving beta-adrenoceptor down-regulation.

Regulation of hepatocyte epidermal growth factor receptors by n-butyrate and dimethyl sulfoxide: sensitivity to modulation by the tumor promoter TPA.

n-Butyrate and dimethyl sulfoxide (DMSO) are known to promote differentiated characteristics in certain cells, including hepatocytes. We have previously reported that butyrate up-regulates the surface expression of hepatocyte epidermal growth factor (EGF) receptors and preserves a high-affinity receptor subpopulation. In the present study, culturing of hepatocytes with DMSO dose-dependently (0.5-2%) increased EGF binding and maintained a high-affinity binding component which was otherwise down-regulated during culturing. Although butyrate was more effective than DMSO in most experiments, the two agents caused qualitatively the same alteration in hepatocyte EGF receptor status. The high-affinity component of the EGF binding present in cells treated with butyrate or DMSO was reduced by treatment (10 nM-1 microM, 1 h) with the phorbol ester tumor promoter TPA, an activator of protein kinase C. Butyrate- or DMSO-treated hepatocytes were more susceptible to this response to TPA than were untreated hepatocytes. The present data indicate that in hepatocytes both butyrate and DMSO preserve a high-affinity EGF receptor subpopulation which is otherwise down-regulated during hepatocyte culture, and that this effect particularly comprises receptors that are sensitive to modulation by the tumor promoter TPA.

The regulation of cyclic AMP levels in cultured MH1C1 rat hepatoma cells and in solid tumours derived from MH1C1 cell inoculates.

Several studies have found high cAMP content in hepatomas in vivo, while hepatoma cells in vitro have very low levels. To explore this discrepancy and the regulation of cAMP in hepatomas, we have examined the cell line MH1C1 from Morris hepatoma 7795. These cells in culture contained low intracellular cAMP concentrations (approximately 0.5 pmol/mg protein at confluency), and were unresponsive to glucagon and prostaglandins (PG) E1 and E2. In contrast, solid hepatomas in rats developed from inoculates of MH1C1 had a 40-fold higher basal cAMP concentration and were stimulated by PGE1 and PGE2. Fibroblasts cultured from these tumours also contained high cAMP levels and responded strongly to PGE1. This may suggest that the difference in cAMP regulation between hepatomas in vivo and hepatoma cells in vitro results from the presence of other cells in the solid tumour rather than from selection of low-cAMP cells during the cloning procedure. Low-Km and intermediate-Km cAMP phosphodiesterase activity was high in MH1C1, compared to normal hepatocytes. This might contribute to the low cAMP level. The ability of MH1C1 to form cAMP was not defective, as the level could be increased more than 200-fold by beta-adrenergic activation in the presence of the phosphodiesterase inhibitor methylisobutylxanthine.

Breast cancer and oral contraceptives: patterns of risk among parous and nulliparous women--further analysis of the Swedish-Norwegian material.

A Swedish-Norwegian case-control study comprising 473 women less than 45 years old with newly diagnosed invasive breast cancer diagnosed in 1984-85, and 722 age-matched control women, was reanalyzed to evaluate if nulliparous women who had used oral contraceptives (OCs) were at particular risk for breast cancer. The relative risk for nulliparous women who had used OCs for eight years or more was 4.3 (95% confidence interval, 1.4-13.1), and parous women with the same duration of use had relative risk 1.7 (0.7-4.2) as compared to nulliparous and parous women, respectively, who had never used OCs. Parous women who had used OCs for twelve years or more after their first full-term pregnancy had a relative risk of 3.0 (1.3-7.4). The findings from the study suggest that nulliparous women may be particularly susceptible to the risk for breast cancer with long-term OC use.

Injury to the cornea due to fish bile.

A retrospective study of 207 patients with chemical eye injuries in northern Norway revealed that fish bile was the causal agent in 14%. Fish bile caused superficial corneal erosions in 28 of 29 cases. All but one of the patients were professional fishermen or fish industry workers. In one case delayed medical care led to serious corneal opacity. Provided that there was immediate and abundant rinsing of the affected eye and good medical care, the prognosis of fish bile injuries was fairly good. The mechanisms for corneal damage due to fish bile are not clear.