Testosterone treatment of HSDD in naturally menopausal women: the ADORE study.

OBJECTIVE: To evaluate the efficacy and safety of a transdermal testosterone patch (TTP, 300 microg/day) in naturally menopausal women with hypoactive sexual desire disorder (HSDD). METHODS: A total of 272 naturally menopausal women, predominantly not using hormone therapy, were randomized in this 6-month, placebo-controlled, double-blind, multicenter study to receive twice weekly either TTP or an identical placebo. Efficacy endpoints measured were the 4-week frequency of satisfying sexual episodes (SSE) using the Sexual Activity Log, the sexual desire domain of the Profile of Female Sexual Function and distress by the Personal Distress Scale. Safety was assessed by adverse events, laboratory parameters and hormone levels. RESULTS: The TTP group demonstrated significant improvements in SSE (p = 0.0089) as well as in sexual desire (p = 0.0007) and reduced personal distress (p = 0.0024) versus placebo at 6 months (intent-to-treat analysis, n = 247). The results were significant for all three endpoints in the subgroup (n = 199) not using hormone therapy. Similar numbers of women treated with placebo and TTP discontinued (n = 39, 27.5% vs. n = 26, 20%), reported adverse events (including application site reactions) (n = 101, 71.1% vs. n = 81, 62.3%) and withdrew due to adverse events (n = 20, 14.1% vs. n = 9, 6.9%). No clinically relevant changes were noted in laboratory parameters. Serum free and total testosterone levels increased from baseline in the TTP group (geometric means 5.65 pg/ml and 67.8 ng/dl, respectively, at week 24) within the physiological range; no changes were seen in estradiol and sex hormone binding globulin levels. CONCLUSIONS: TTP was effective in treating HSDD and improving sexual function in this study of naturally menopausal women with and without concurrent hormone therapy.

The treatment of non-union of the clavicle with the AO mini external fixator.

Osteosynthesis of non-union of fractures of the clavicle were achieved using bone graft and the AO mini external fixator. The technique was used in nine patients, and no neurovascular or pleural complications occurred. The average time before the patient was considered for this method was 11 months. The external fixator was removed at an average of eight weeks, with solid union in all cases. The outcome was excellent and all patients returned to pre-injury level of normal activity. This method provides safe and relatively simple treatment for this difficult non-union.

Nuclear dependence of sulfur mustard-mediated cell death.

Although sulfur mustard (SM) has been reported to be a DNA alkylating agent, it is not clear how much of the cytotoxicity of this agent is secondary to DNA damage. To test the hypothesis that the presence of a nucleus is required for the toxicity of sulfur mustard, enucleated endothelial cytoplasts were treated with SM. Using a combination of biochemical and microscopic assays, we demonstrate that some aspects of SM-induced cell death may be dependent on the presence of a nucleus, while others may not be. For example, it was found that cytoskeletal changes, such as loss of stress fibers and rounding, proceed in response to sulfur mustard treatment even in the absence of a nucleus. However, significant further increases in caspase activity and the associated phosphatidylserine translocation were not observed in cytoplasts treated with 500 microM SM for 6 h (following a 20-h recovery at the end of cytoplast preparation). In contrast, cytoplasts treated with chelerythrine, an agent previously reported to induce rapid apoptosis, demonstrated increases in caspase activity in cytoplasts comparable to that observed in the nucleated cells. This indicates that sulfur mustard-induced alkylation of nuclear DNA may be an important stimulus for activation of caspases in nucleated cells. Interestingly, the baseline caspase activity in cytoplasts was greater than in nucleated cells. Analysis of the time course of caspase activation in untreated adherent cytoplasts indicated that the activity increases initially and then stabilizes by 8 h to a low level that was comparable to the level observed at 26 h in untreated cytoplasts. This indicates that cytoplasts are able to tolerate stable low levels of caspase activity and not proceed immediately into the execution phase of apoptosis. The cytoplast model may be quite useful in the toxicological assessment of agents that are thought to exert their toxicity through DNA damage.

N-acetylcysteine and endothelial cell injury by sulfur mustard.

Understanding the underlying mechanisms of cell injury and death induced by the chemical warfare vesicant sulfur mustard (HD) will be extremely helpful in the development of effective countermeasures to this weapon of terror. We have found recently that HD induces both apoptosis and necrosis in endothelial cells (Toxicol. Appl. Pharmacol. 1996; 141: 568-583). Pretreatment of the endothelial cells for 20 h with the redox-active agent N-acetyl-L-cysteine (NAC) selectively prevented apoptotic death induced by HD. In this study, we tested the hypotheses that pretreatment with NAC acts through two different pathways to minimize endothelial injury by HD: NAC pretreatment acts via a glutathione (GSH)-dependent pathway; and NAC pretreatment acts to suppress HD-induced activation of the nuclear transcription factor NFkappaB. We used a fluorescence microscopic assay of apoptotic nuclear features to assess viability and electrophoretic mobility shift assays (EMSAs) to assess the activity of NFkappaB following exposure to HD. The cells were treated with 0-10 mM GSH for 1 h prior to and during exposure to 0 or 500 microM HD for 5-6 h. Cells were also treated with 50 mM NAC or 200 microM buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, alone or in combination overnight prior to exposure to 0 or 500 microM HD for 5-6 h. Externally applied GSH up to a concentration of 5 mM had no toxic effect on the cells. Mild toxicity was associated with 10 mM GSH alone. There was a dose-related enhancement of viability when 2.5 and 5 mM GSH were present during the HD exposure. Pretreatment with BSO alone had no discernible toxicity. However, pretreatment with this inhibitor of GSH synthesis potentiated the toxicity of HD. Pretreatment with 50 mM NAC, as previously reported, provided substantial protection. Combining pretreatment with both BSO and NAC eliminated the protective effect of NAC pretreatment alone on HD injury. These observations are highly suggestive that NAC enhances endothelial survival via GSH-dependent effects and confirms and extends the work of others with different models that externally supplied GSH alone may be a fairly effective countermeasure against HD injury of endothelium. We next examined the hypothesis that HD may activate the nuclear transcription factor NFkappaB by performing EMSAs with nuclear extracts of endothelial cells following exposure to 0, 250 or 500 microM HD. This demonstrated an up to 2.5-fold increase (scanning densitometry) in activation of NFkappaB binding to its consensus sequence induced by 500 microM HD after 5 h of HD exposure. Paradoxically, treatment of the endothelial cells alone with 50 mM NAC activated NFkappaB, although HD-induced activation of NFkappaB was partially suppressed by NAC at 5 h. Factor NFkappaB is an important transcription factor for a number of cytokine genes (e.g. tumor necrosis factor, TNF), which can be activated following stress in endothelial cells. Taken together, these observations suggest that the protective effects of NAC may be mediated by enhanced GSH synthesis. The increased GSH may act to scavenge HD and also prevent oxidative activation of NFkappaB. Under some conditions, NAC may act as an oxidizing agent and thus increase NFkappaB activity. The NFkappaB-dependent gene expression may be important in inducing endothelial cell death as well as in generating a local inflammatory reaction associated with the release of endothelial-derived cytokines.

Inhibition of mono-ADP-ribosyltransferase activity during the execution phase of apoptosis prevents apoptotic body formation.

The objective of this study was to understand factors responsible for apoptotic body formation and release during apoptosis. We have found that inhibition of mono-ADP ribosylation after ultraviolet (UV) light induction of apoptosis in HL-60 cells does not block caspase-3 activation, gelsolin cleavage, or endonucleolytic DNA fragmentation. However, the cytoskeletal features of apoptosis leading to apoptotic body formation and release were inhibited by meta-iodobenzylguanidine (MIBG) and novobiocin, potent inhibitors of arginine-specific mono-ADP-ribosyltransferases (mono-ADPRTs). Suppression of mono-ADP ribosylation as late as 120 min following UV irradiation blocked the depolymerization of actin and release of apoptotic bodies. This suggested that the cytoskeletal changes of apoptosis may be decoupled from the caspase cascade and that there may be a biochemical event either distal to or independent of caspase-3 that regulates apoptotic body formation. To test the hypothesis that ADP ribosylation of actin may occur with the induction of apoptosis, an in vivo assay of mono-ADPRT activity using an antibody against ADP-ribosylarginine was used. An approximately 64% increase in the ADP ribosylation of actin was observed at 2 h following exposure to UV light. When MIBG or novobiocin was present, the ADP ribosylation of actin was only 14-18% above the levels observed in control nonirradiated cells. The current study is the first to demonstrate a relationship between ADP-ribosylation of actin and the formation of apoptotic bodies.

Activation of poly [ADP-Ribose] polymerase in endothelial cells and keratinocytes: role in an in vitro model of sulfur mustard-mediated vesication.

Although endothelial cells and keratinocytes appear to be the primary cellular targets of sulfur mustard (SM), the role of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) in SM-induced vesication has not been clearly defined. PARP is thought to play a crucial role in DNA repair mechanisms following exposure to alkylating agents like SM. Using a combination of fluorescence microscopy and biochemical assays, we tested the hypothesis that SM causes activation of PARP in endothelial cells and keratinocytes with subsequent loss of nicotinamide adenine dinucleotide (NAD) and depletion of adenosine triphosphate (ATP) levels. To determine if PARP activation accounts for SM-induced vesication, keratinocyte adherence and permeability of endothelial monolayers were measured as in vitro correlates of vesication. As early as 2 to 3 h after exposure to SM concentrations as low as 250 microM, dramatic changes were induced in keratinocyte morphology and microfilament architecture. Exposure to 500 microM SM induced a fourfold increase in PARP activity in endothelial cells, and a two- to threefold increase in keratinocytes. SM induced a dose-related loss of NAD+ in both endothelial cells and keratinocytes. ATP levels fell to approximately 50% of control levels in response to SM concentrations >/=500 microM. SM concentrations >/=250 microM significantly reduced keratinocyte adherence as early as 3 h after exposure. Endothelial monolayer permeability increased substantially with concentrations of SM >250 microM. These observations support the hypothesis that the pathogenic events necessary for SM-induced vesication (i.e., capillary leak and loss of keratinocyte adherence) at higher vesicating doses of SM (>/=500 microM) may depend on NAD loss with PARP activation and subsequent ATP-dependent effects on microfilament architecture. Vesication developing as a result of exposure to lower concentrations of SM presumably occurs by mechanisms that do not depend on loss of cellular ATP (e.g., apoptosis and direct SM-mediated damage to integrins and the basement membrane).