Nuclear receptor 4A1 (NR4A1) antagonists target paraspeckle component 1 (PSPC1) in cancer cells.

Paraspeckles compound 1 (PSPC1) is a multifunctional protein that plays an important role in cancer cells, where PSPC1 is a master regulator of pro-oncogenic responses that includes activation of TGFß (TGFß1), TGFß-dependent EMT, and metastasis. The pro-oncogenic activities of PSPC1 closely resembled those observed for the orphan nuclear receptor 4A1 (NR4A1, Nur77) and knockdown of NR4A1 decreased expression of PSPC1 in MDA-MB-231 breast, H1299 lung, and SNU449 liver cancer cells. Similar results were observed in these same cell lines after treatment with bisindole-derived (CDIMs) NR4A1 antagonists. Moreover, PSPC1-dependent regulation of TGFß, genes associated with cancer stem cells and epithelial to mesenchymal transition (EMT) were also downregulated after NR4A1 silencing or treatment of breast, lung, and liver cancer cells with CDIM/NR4A1 antagonists. Results of chromatin immunoprecipitation (ChIP) assays suggest that NR4A1 regulates PSPC1 through interaction with an NBRE sequence in the PSPC1 gene promoter. These results coupled with in vivo studies showing that NR4A1 antagonists inhibit breast tumor growth and downregulate PSPC1 in tumors indicate that the pro-oncogenic nuclear PSPC1 factor can be targeted by CDIM/NR4A1 antagonists.

Methane supplementation improves graft function in experimental heart transplantation.

BACKGROUND: Maintenance of cell viability during cold storage is a key issue in organ transplantation. Methane (CH4) bioactivity has recently been recognized in ischemia/reperfusion conditions; we therefore hypothesized that cold storage in CH4-enriched preservation solution can provide an increased defense against organ dysfunction during experimental heart transplantation (HTX). METHODS: The hearts of donor Lewis rats were stored for 60 minutes in cold histidine-tryptophan-ketoglutarate (Custodiol [CS]) or CH4-saturated CS solution (CS-CH4) (n = 12 each). Standard heterotopic HTX was performed, and 60 minutes later, the left ventricular (LV) pressure-volume relationships LV systolic pressure (LVSP), systolic pressure increment (dP/dtmax), diastolic pressure decrement, and coronary blood flow (CBF) were measured. Tissue samples were taken to detect proinflammatory parameters, structural damage (by light microscopy), endoplasmic reticulum (ER) stress, and apoptosis markers (CCAAT/enhancer binding protein [C/EBP] homologous protein, GRP78, glycogen synthase kinase-3ß, very low-density lipoprotein receptor, caspase 3 and 9, B-cell lymphoma 2, and bcl-2-like protein 4), whereas mitochondrial functional changes were analyzed by high-resolution respirometry. RESULTS: LVSP and dP/dtmax increased significantly at the largest pre-load volumes in CS-CH4 grafts as compared with the CS group (114.5 ± 16.6 mm Hg vs 82.8 ± 4.6 mm Hg and 3,133 ± 430 mm Hg/s vs 1,739 ± 169 mm Hg/s, respectively); the diastolic function and CBF (2.4 ± 0.4 ml/min/g vs 1.3 ± 0.3 ml/min/g) also improved. Mitochondrial oxidative phosphorylation capacity was more preserved (58.5 ± 9.4 pmol/s/ml vs 27.7 ± 6.6 pmol/s/ml), and cytochrome c release was reduced in CS-CH4 storage. Signs of HTX-caused myocardial damage, level of ER stress, and the transcription of proapoptotic proteins were significantly lower in CS-CH4 grafts. CONCLUSION: The addition of CH4 during 1 hour of cold storage improved early in vitro graft function and reduced mitochondrial dysfunction and activation of inflammation. Evidence shows that CH4 reduced ER stress-linked proapoptotic signaling.

Antibacterial and transfection activities of nebulized formulations incorporating long n-alkyl chain silver N-heterocyclic carbene complexes.

The development of new antibacterial molecules is essential in view of the emergence of pathogenic strains resistant to multiple antibiotics. Among the infectious pathologies, pulmonary infections are particularly difficult to treat due to the complexity of lung anatomy and the presence of natural barriers such as mucus. At present, the aerosol delivery of antibacterial compounds is still poorly employed. Furthermore, the presence of bacteria in lungs negatively affects aerosolized Cystic Fibrosis gene therapy efficiency. A multi-functional formulation (antibacterial and transfection activities) could increase the therapeutic effect. This work reports the synthesis of new N-heterocyclic carbene silver complexes (Ag-NHC) featuring a lipid chain and the evaluation of their antibacterial potency, especially when delivered following aerosolization. When formulated alone in water, these Ag-NHC displayed remarkable antibacterial activities against some Staphyloccocus aureus strains and Pseudomonas aeruginosa clinical strains. Moreover, combined with cationic lipid and DNA (ternary combination), they could be used to deliver therapeutic genes via aerosolization in infected lungs. Altogether, the data reported herein support n-alkyl chain Ag-NHC as a possible alternative to conventional antibiotics for treating respiratory infections and to combat the emergence of multi-resistant bacteria.

Methane alleviates sepsis-induced injury by inhibiting pyroptosis and apoptosis: in vivo and in vitro experiments.

Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. Methane has been reported to have anti-oxidative, anti-apoptotic and anti-inflammatory properties. We investigated the potential protective effects of methane on sepsis-induced injury and determined the related mechanisms. C57BL/6 mice received laparotomy with cecal ligation and puncture (CLP) to create a septic model, followed by methane-rich saline (MRS) treatment after CLP. MRS treatment improved the 5-day survival rate and organ functions and alleviated pathological damage of the mice, as well as reduced excessive inflammatory mediators, such as tumor necrosis factor-α and interleukin-6. MRS treatment also decreased the levels of oxidative stress index proteins, decreased the apoptosis of cells and inhibited nod-liker receptor protein (NLRP)3-mediated pyroptosis in the lung and intestine. In in vitro experiments, RAW264.7 and primary peritoneal macrophages were treated with lipopolysaccharide (LPS) plus adenosine-triphosphate (ATP) to induce inflammation and pyroptosis. Consistent with the in vivo results, methane-rich medium (MRM) treatment also reduced the levels of excessive inflammatory mediators, and decreased the levels of ROS, inhibited apoptosis and pyroptosis. Our results indicate that methane offers a protective effect for septic mice via its anti-inflammation, anti-oxidation, anti-pyroptosis and anti-apoptosis properties.

Methane inhalation reduces the systemic inflammatory response in a large animal model of extracorporeal circulation.

OBJECTIVES: Extracorporeal circulation induces cellular and humoral inflammatory reactions, thus possibly leading to detrimental secondary inflammatory responses. Previous data have demonstrated the bioactive potential of methane and confirmed its anti-inflammatory effects in model experiments. Our goal was to investigate the in vivo consequences of exogenous methane administration on extracorporeal circulation-induced inflammation. METHODS: Two groups of anaesthetized Vietnamese minipigs (non-treated and methane treated, n = 5 each) were included. Standard central cannulation was performed, and extracorporeal circulation was maintained for 120 min without cardiac arrest or ischaemia, followed by an additional 120-min observation period with haemodynamic monitoring. In the methane-treated group, 2.5% v/v methane-normoxic air mixture was added to the oxygenator sweep gas. Blood samples through the central venous line and tissue biopsies from the heart, ileum and kidney were taken at the end point to determine the whole blood superoxide production (chemiluminometry) and the activity of xanthine-oxidoreductase and myeloperoxidase, with substrate-specific reactions. RESULTS: Methane treatment resulted in significantly higher renal blood flow during the extracorporeal circulation period compared to the non-treated group (63.9 ± 16.4 vs 29.0 ± 9.3 ml/min). Whole blood superoxide production (548 ± 179 vs 1283 ± 193 Relative Light Unit (RLU)), ileal myeloperoxidase (2.23 ± 0.2 vs 3.26 ± 0.6 mU/(mg protein)) and cardiac (1.5 ± 0.6 vs 4.7 ± 2.5 pmol/min/mg), ileal (2.2 ± 0.6 vs 7.0 ± 3.4 pmol/min/mg) and renal (1.2 ± 0.8 vs 13.3 ± 8.0 pmol/min/mg) xanthine-oxidoreductase activity were significantly lower in the treated group. CONCLUSIONS: The addition of bioactive gases, such as methane, through the oxygenator of the extracorporeal circuit represents a novel strategy to influence the inflammatory effects of extracorporeal perfusion in cardiac surgical procedures.

Analgesic Effect of Methane Rich Saline in a Rat Model of Chronic Inflammatory Pain.

How oxidative stress contributes to neuro-inflammation and chronic pain is documented, and methane is reported to protect against ischemia-reperfusion injury in the nervous system via anti-inflammatory and antioxidant properties. We studied whether methane in the form of methane rich saline (MS) has analgesic effects in a monoarthritis (MA) rat model of chronic inflammatory pain. Single and repeated injections of MS (i.p.) reduced MA-induced mechanical allodynia and multiple methane treatments blocked activation of glial cells, decreased IL-1ß and TNF-α production and MMP-2 activity, and upregulated IL-10 expression in the spinal cord on day 10 post-MA. Furthermore, MS reduced infiltrating T cells and expression of IFN-γ and suppressed MA-induced oxidative stress (MDA and 8-OHDG), and increased superoxide dismutase and catalase activity. Thus, MS may offer anti-inflammatory and antioxidant effects to reduce chronic inflammatory pain.

Methane attenuates retinal ischemia/reperfusion injury via anti-oxidative and anti-apoptotic pathways.

Retinal ischemia/reperfusion injury (IRI) may cause incurable visual impairment due to neural regeneration limits. Methane was shown to exert a protective effect against IRI in many organs. This study aims to explore the possible protective effects of methane-rich saline against retinal IRI in rat. Retinal IRI was performed on the right eyes of male Sprague-Dawley rats, which were immediately injected intraperitoneally with methane-saturated saline (25ml/kg). At one week after surgery, the number of retinal ganglion cells (RGCs), total retinal thickness, visual function were measured by hematoxylin and eosin staining, FluoroGold anterograde labeling and flash visual evoked potentials. The levels of 8-hydroxy-2-deoxyguanosine (8-OHdG), 4-Hydroxy-2-nonenal (4-HNE), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), caspase-3, caspase-9, B cell lymphoma/leukemia-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in retinas were assessed by immunofluorescence staining, enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. As expected, methane treatment significantly improved the retinal IRI-induced RGC loss, total retinal layer thinning and visual dysfunction. Moreover, methane treatment significantly reduced the levels of oxidative stress biomarkers (8-OHdG, 4-HNE, MDA) and increased the antioxidant enzyme activities (SOD, CAT, GPx) in the retinas with IRI. Meanwhile, methane treatment significantly increased the anti-apoptotic gene (Bcl-2) expression and decreased the pro-apoptotic gene (Bax) expression, accompanied by the suppression of caspase-3 and caspase-9 activity. Thus, these data demonstrated that methane can exert a neuroprotective role against retinal IRI through anti-oxidative and anti-apoptotic pathways.

Methane-rich saline protects against concanavalin A-induced autoimmune hepatitis in mice through anti-inflammatory and anti-oxidative pathways.

Methane is a common gas which has been reported to play a protective role in organ injury and presents an anti-inflammatory property. However, its effects on Concanavalin A (Con A)-induced autoimmune hepatitis (AIH) remain unknown. Thus, the aim of this study was to investigate the effects of methane on Con A-induced autoimmune hepatitis in mice and its underlying mechanism. Autoimmune hepatitis was induced by Con A (15 mg/kg) in healthy C57BL/6 mice and methane-rich saline (MS) (20 ml/kg) was intraperitoneally injected 30 min after the challenge with Con A. We found that methane treatment significantly reduced the elevated serum aminotransferase levels and ameliorated liver pathological damage. Furthermore, methane treatment obviously suppressed the secretion of proinflammatory cytokines including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) and increased anti-inflammatory cytokine interleukin-10 (IL-10). Moreover, we found that the levels of malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) were highly increased while the activities of superoxide dismutase (SOD) and catalase (CAT) were decreased in liver with the injection of Con A, which was reversed by methane. Also, the data demonstrated that the phosphorylated IκB, NF-κB and P38 MAPK in liver were significantly down-regulated by methane. These results suggested that methane protected liver against Con A-induced injury through anti-inflammatory and anti-oxidative pathways.

Methane Attenuates Hepatic Ischemia/Reperfusion Injury in Rats Through Antiapoptotic, Anti-Inflammatory, and Antioxidative Actions.

Hepatic ischemia/reperfusion (I/R) injury, which occurs in various diseases, introduces severe tissue damage and liver dysfunction. However, no promising therapies for such a significant condition currently exist. Methane has been suggested to exert a protective effect against intestinal I/R injury. In this study, we introduced methane to treat hepatic I/R injury to show its promising protective effect. Also, intraperitoneal injection with methane-rich saline, which could have potential clinical applications, was applied as a new method. Partial liver warm ischemia was applied in Sprague-Dawley rats for 60 min followed by succedent reperfusion. In the test for effective dosage, methane-rich saline was administrated intraperitoneally to the rats at doses of 1, 5, 20, or 40 mL/kg at onset of reperfusion. In the test for protective effect, rats received methane-rich saline intraperitoneally at a dose of 10 mL/kg before the initiation of reperfusion. We found that methane-rich saline significantly decreased serum alanine aminotransferase, aspartate aminotransferase activity, and the occurrence of necrosis. Moreover, methane-rich saline reduced the amount of caspase-3 and the number of apoptotic cells. In addition, methane-rich saline increased the level of superoxide dismutase and decreased the level of malondialdehyde and 8-hydroxyguanosine. Furthermore, research indicated that methane-rich saline markedly decreased gene expression and content of tumor necrosis factor-α and interleukin-6. Also, reduced CD68-positive cells showed decreased inflammatory cells in the liver. Our results suggest that methane protects the liver against I/R injury through antiapoptotic, antioxidative, and anti-inflammatory actions.

Retention and excretion of inhaled 3H and 14C radiolabeled methane in rats.

A radiological concern for workers at heavy water reactor nuclear facilities is the hazard presented by tritium (H) and C. Radioactive methane is one of many potential H and C containing chemicals to which Nuclear Energy Workers (NEWs) may be exposed. Current dosimetric models for H- and C-methane, recommended by the International Commission on Radiological Protection (ICRP), are based on the assumption that 1% of methane is absorbed following its inhalation. Of this 1%, all H is converted immediately to tritiated water and C is converted immediately to CO2 (50%) and organically bound carbon (50%). In the study, rats were exposed to methane standards (H-methane and C-methane) mixed with breathing air to give a final concentration of 0.27% methane and resulting in final activity concentrations of 4.2 GBq m and 0.88 GBq m for H and C, respectively. This corresponds to exposure estimates of 580 kBq g and 120 kBq g. Simultaneous exposure to H- and C-methane allowed for the direct comparison of the retention of these radionuclides and removed uncertainties concerning their relative uptake and retention. The results demonstrate that the total methane uptake from the inhaled dose was threefold less than the 1% methane uptake predicted by the ICRP dosimetric models for H- and C-methane, with the H concentration being substantially higher than anticipated in the liver. This study provided data suggesting that current ICRP dosimetric methane models overestimate the fraction of H- and C-methane that is absorbed following inhalation and assisted in providing information to better understand the metabolism of inhaled H and C radiolabeled methane.

Photodynamic properties and photoinactivation of Candida albicans mediated by brominated derivatives of triarylmethane and phenothiazinium dyes.

The photodynamic activity of brominated derivatives of New Fuchsin and Azure B was studied in solution and in cell suspensions of Candida albicans. The spectroscopic and photodynamic properties of these photosensitizers were compared with those of Crystal Violet and Azure B, which represent active photosensitizer related to each family of compounds. Triarylmethane derivatives absorb intensely with a band centered at ∼ 570 nm, while the phenothiazinium dyes at ∼ 650 nm. Photooxidation of 9,10-dimethylanthracene was observed using phenothiazinium compounds indicating the formation of singlet molecular oxygen, while it was not detected using triarylmethane agents. However, triarylmethane dyes were able to photooxidize l-tryptophan. In yeast cell suspensions, the photosensitized inactivation of C. albicans increases with photosensitizer concentration, causing a ∼ 5 log decrease of cell survival, when the cultures are treated with 20 µM of Crystal Violet and irradiated for 60 min. Under these conditions, the photodynamic activity of 50 µM Azure B induced a ∼ 3 log decrease of cell survival. Studies of photodynamic action mechanism indicated that photoinactivation of C. albicans cells induced by triarylmethane compounds involves mainly type I photoprocess. Although, phenothiazinium derivatives produce singlet molecular oxygen, a contribution of other reactive oxygen species cannot be discarded in the photoinactivation of C. albicans.

[Characterization of the antiinflammatory properties of methane inhalation during ischaemia-reperfusion]. / Metánbelélegzés gyulladáscsökkento hatásának vizsgálata ischaemia-reperfusio alatt.

INTRODUCTION: Gastrointestinal methane generation has been demonstrated in various conditions, but it is not known whether it has any impact on the mammalian physiology or pathophysiology. Our aim was to characterize the effects of exogenous methane on the process of inflammatory events induced by reoxygenation in a canine model of ischemia-reperfusion. MATERIALS AND METHODS: Sodium pentobarbital-anesthetized inbred beagle dogs (n = 18) were randomly assigned to sham-operated or ischemia-reperfusion (I/R) groups. I/R was induced by occluding the superior mesenteric artery for 1 h, and the subsequent reperfusion was monitored for 3 h. For 5 min before reperfusion, the animals were mechanically ventilated with normoxic artificial air with or without 2.5% methane. The macrohemodynamics and small intestinal pCO2 gap changes were recorded and tissue superoxide and nitrotyrosine levels and myeloperoxidase activity changes were determined in intestinal biopsy samples. Structural mucosal damage was measured via light microscopy and HE staining. RESULTS: Methane inhalation positively influenced the macrohemodynamic changes, significantly reduced the intestinal pCO2 gap changes and the magnitude of the tissue damage after reperfusion. Further, the intestinal myeloperoxidase activity, the superoxide and nitrotyrosine levels were reduced. CONCLUSIONS: These data demonstrate the anti-inflammatory profile of methane. The study provides evidence that exogenous methane modulates leukocyte activation and affects key events of I/R-induced oxidative and nitrosative stress.

The anti-inflammatory effects of methane.

OBJECTIVE: Gastrointestinal methane generation has been demonstrated in various stress conditions, but it is not known whether nonasphyxiating amounts have any impact on the mammalian pathophysiology. We set out to characterize the effects of exogenous methane administration on the process of inflammatory events arising after reoxygenation in a large animal model of ischemia-reperfusion. DESIGN: A randomized, controlled in vivo animal study. SETTING: A university research laboratory. SUBJECTS: Inbred beagle dogs (12.7 6 2 kg). INTERVENTIONS: Sodium pentobarbital-anesthetized animals were randomly assigned to sham-operated or ischemia-reperfusion groups, where superior mesenteric artery occlusion was maintained for 1 hr and the subsequent reperfusion was monitored for 3 hrs. For 5 mins before reperfusion, the animals were mechanically ventilated with normoxic artificial air with or without 2.5% methane. Biological responses to methane-oxygen respirations were defined in pilot rat studies and assay systems were used with xanthine oxidase and activated canine granulocytes to test the in vitro bioactivity potential of different gas concentrations. MEASUREMENTS AND MAIN RESULTS: The macrohemodynamics and small intestinal pCO(2) gap changes were recorded and peripheral blood samples were taken for plasma nitrite/nitrate and myeloperoxidase analyses. Tissue superoxide and nitrotyrosine levels and myeloperoxidase activity changes were determined in intestinal biopsy samples; structural mucosal damage was measured by hematoxylin and eosin staining. Methane inhalation did not influence the macrohemodynamics but significantly reduced the magnitude of the tissue damage and the intestinal pCO(2) gap changes after reperfusion. Furthermore, the plasma and mucosal myeloperoxidase activity and the intestinal superoxide and nitrotyrosine levels were reduced, whereas the plasma nitrite/nitrate concentrations were increased. Additionally, methane effectively and specifically inhibited leukocyte activation in vitro. CONCLUSIONS: These data demonstrate the anti-inflammatory profile of methane. The study provides evidence that exogenous methane modulates leukocyte activation and affects key events of ischemia-reperfusion-induced oxidative and nitrosative stress and is therefore of potential therapeutic interest in inflammatory pathologies.

In vivo activity of thiophene-containing trisubstituted methanes against acute and persistent infection of non-tubercular Mycobacterium fortuitum in a murine infection model.

OBJECTIVES: Mycobacterium fortuitum causes opportunist non-tubercular infection in humans. Chronic infection of M. fortuitum has been clinically documented and requires prolonged chemotherapy. The objectives of this study were to characterize acute and persistent infection of M. fortuitum in a murine infection model and to screen thiophene-containing trisubstituted methanes active against both acute and persistent infection. METHODS: A murine infection model of M. fortuitum was used. Bacillary count, bioluminescence, disease symptoms, host immune response, drug susceptibility and mortality were measured. Reactivation of persistent bacilli was induced by dexamethasone. Trisubstituted methanes containing thiophene rings were synthesized and screened in vitro by agar dilution and BACTEC assay and in mice. Cytotoxicity was tested with Vero monkey kidney cells using a resazurin assay. RESULTS: The acute infection in mice was marked by a 3 log rise in viable counts, the appearance of disease symptoms and a rise in the Th1 immune response. Bacilli were susceptible to fluoroquinolones. This was followed by persistent infection, in which disappearance of disease symptoms, a decline in Th1 response and non-susceptibility to fluoroquinolones was observed. When the mice were immunocompromised on day 40 post-infection (persistent state) by dexamethasone, a rise in viable counts, symptoms and susceptibility to fluoroquinolones and a prominent Th1 response reappeared. Two lead compounds were found that cleared the mice of bacilli in acute infection and caused a 2.29-2.99 log reduction in cfu of persistent bacilli. CONCLUSIONS: The study established acute and persistent infection in mice and identified two promising anti-M. fortuitum compounds with a selectivity index >10.

Formulation-based approach to support early drug discovery and development efforts: a case study with enteric microencapsulation dosage form development for a triarylmethane derivative TRAM-34; a novel potential immunosuppressant.

BACKGROUND: Enteric microencapsulation of the potential immunosuppressant TRAM-34 was investigated as a means of enhancing oral drug delivery and minimizing or eliminating hydrolysis of pyrazole-substituted triarylmethane to the respective alcohol. METHOD: TRAM-34 was successfully enteric microencapsulated by a coacervation method using the pH-sensitive Eudragit L 100 polymer. In this study, we utilized water-miscible organic solvents such as acetone and ethanol, which are considered safe class 3 solvents according to the ICH guideline. We deemed such an approach suitable for safe scale up and for enteric coating application to other compounds of a similar lipophilicity. RESULTS: The resulting microparticles were spherical and uniform with an average particle size of 460 microm at 15% theoretical loading. The encapsulation efficiency was 90 +/- 1.9% and the percentage yield was found to be 91.5 +/- 0.3%. The oral administration in rhesus macaques of TRAM-34-loaded enteric-coated microparticles illustrated six times enhancement in its oral bioavailability. However, the TRAM-34 plasma concentration was less than the therapeutic effective level. CONCLUSION: The low oral bioavailability, even after enteric coating, could be attributed to the compound's inherent absorption characteristics and high lipophilicity.

Methanogenic toxicity in anaerobic digesters treating municipal wastewater.

In upflow anaerobic sludge blanket (UASB) digesters treating raw sewage at low temperatures, the sludge progressively lost methanogenic activity, indicating the possibility of methanogenic activity inhibition caused by wastewater constituents. To check this fact, batch and semi-continuous methanogenic toxicity assays were carried out with raw and centrifuged sewage. Permanent methanogenic toxicity on anaerobic sludge of approximately 50% was found when the sludge exposure to wastewater was renewed in a semi-continuous way. A stronger methanogenic inhibition of about 70-100% was observed when an active anaerobic sludge was exposed to mixed liquor from the UASB digester treating municipal wastewater. Suspended solids removal from sewage slightly reduced methanogenic toxicity. Effective concentration of municipal wastewater that caused a 50% reduction in methanogenic activity was estimated to be in the range of 150-200 mg CODl(-1). As methanogenic inhibition appeared to be related to remaining COD, higher methanogenic toxicity in digesters operating with low conversion efficiency will be expected.

Potentiation of erythroid abnormalities following macrophage depletion in aged rats.

OBJECTIVES: The effects of prolonged macrophage depletion on haematological parameters were investigated in aged rats and compared with those in young ones. METHODS: Four weekly i.v. injections of dichloromethylene diphosphonate-containing liposomes (Cl2MDP-CL) were employed to achieve a prolonged depletion of bone marrow (BM) and spleen macrophages. The number of BM macrophages was then assessed by flow cytometry, whereas the spleen clearance function was judged by the elimination of oxidised red blood cells (RBC). Haematological parameters and signs of RBC ageing (reduced MCV, increased density and augmented 4.1a/4.1b membrane protein ratio) were determined. Finally, the recovery from phlebotomy-induced acute anaemia was investigated. RESULTS: Following the Cl2MDP-CL treatment, in comparison with young rats, the aged animals showed: (i) reduced numbers of BM macrophages; (ii) greater impairment of spleen clearance function; (iii) similar anaemic condition and signs of RBC ageing; (iv) greater increase in white blood cell (WBC) numbers (mainly neutrophils). In addition, whereas aged control rats showed a recovery from phlebotomy-induced acute anaemia which was similar to that of the untreated young animals, in the aged-treated rats, a significantly diminished/delayed restoration of RBC, Hb and reticulocyte to normal values was observed, accompanied by a significantly higher increase in WBC numbers than in the other groups of animals. CONCLUSION: Haematological abnormalities because of Cl2MDP-CL-induced macrophage depletion are potentiated in aged rats in which the BM regenerative potential of the erythroid lineage as well as the clearance function of the spleen appear compromised. Thus, in aged rats, macrophage dysfunction is likely to interfere with erythroid homeostasis particularly during haemopoietic stress.

Methane, a gas produced by enteric bacteria, slows intestinal transit and augments small intestinal contractile activity.

The presence of methane on lactulose breath test among irritable bowel syndrome (IBS) subjects is highly associated with the constipation-predominant form. Therefore, we set out to determine whether methane gas can alter small intestinal motor function. In dogs, small intestinal fistulae were created to permit measurement of intestinal transit. Using a radiolabel, we evaluated transit during infusion of room air and subsequently methane. In this model, small intestinal infusion of methane produced a slowing of transit in all dogs by an average of 59%. In a second experiment, guinea pig ileum was pinned into an organ bath for the study of contractile activity in response to brush strokes applied to the mucosa. The force of contraction was measured both orad and aborad to the stimulus. The experiment was repeated while the bath was gassed with methane. Contractile activities orad and aborad to the stimulus were significantly augmented by methane compared with room air (P < 0.05). In a third experiment, humans with IBS who had undergone a small bowel motility study were compared such that subjects who produced methane on lactulose breath test were compared with those producing hydrogen. The motility index was significantly higher in methane-producing IBS patients (1,851 +/- 861) compared with hydrogen producers (1,199 +/- 301) (P < 0.05). Therefore, methane, a gaseous by-product of intestinal bacteria, slows small intestinal transit and appears to do so by augmenting small bowel contractile activity.

Biokinetics of inhaled radioactive methane in rats: a pilot study.

Current dosimetric models for radioactive methane assume 1% of inhaled methane is absorbed, all 3H activity is converted immediately to [3H]H2O, and 14C activity is converted immediately to [14C]CO2 (50%) and organically bound carbon (50%). Results of a pilot study using rats to test these models suggest the models overestimate uptake but underestimate organic fixation of 3H and 14C, especially in liver. Also, the biokinetic properties of organically bound 3H and 14C in liver were markedly different from other tissues. Preliminary dose estimates based on observed uptake and organic fixation of label suggest current methane models likely overestimate radiation doses from radioactive methane by 3- to 10-fold.

Contraceptive progestins. Various 11-substituents combined with four 17-substituents: 17alpha-ethynyl, five- and six-membered spiromethylene ethers or six-membered spiromethylene lactones.

Norethisterone (NET) is a 19-nortestosterone derivative with progestagenic and some androgenic activity, which was used in the first generation of contraceptives. NET was succeeded by levonorgestrel (LNG) and later on by desogestrel (DSG) and gestodene (GSD). Although these latter two progestins had increased potency, there was still androgenicity with gestodene and to a lesser extent with desogestrel. New progestins were synthesized in order to further enhance progestagenic and to reduce androgenic activity. Four different chemical moieties were introduced in position 17 of 19-nortestosterone, viz. 17alpha-ethynyl, five- and six-membered spiromethylene ethers, and a six-membered-spiromethylene lactone. In combination with these structures seven different substituents were added at position 11, i.e. methylene, methyl, ethyl, ethenyl, ethynyl, 2-propenyl and 1-propynyl. All substituents except for methylene occupied the 11beta-position. All these 32 compounds were synthesized and analysed in vitro and in vivo against etonogestrel (ETG, 3-keto-desogestrel), the biologically active metabolite of desogestrel. Their relative binding potency to progesterone (PR), androgen (AR) and estrogen (ER) receptors were determined in cell lysates of human breast tumor MCF-7 cells and to glucocorticoid (GR) receptors in that of human leukemic IM-9 cells. Moreover, their relative agonistic activities were assessed in Chinese hamster ovary cell-based transactivation assays. All in vivo activities were determined in McPhail (progestagenic), ovulation inhibition (progestagenic and estrogenic), Hershberger (androgenic), hormone screening (glucocorticoid and estrogen) and Allen-Doisy (estrogenic) tests after oral and for the McPhail test also after subcutaneous administration. The progestagenic binding and transactivation potencies of all compounds in the three 17-spiro series were higher than those of the corresponding analogues in the 17alpha-ethynyl series. None of the compounds showed estrogenic or clear androgenic binding and transactivation potential except for a six-membered-spiromethylene lactone with a propynyl group. This compound showed strong androgenic binding. The glucocorticoid binding and transactivation were very low for the compounds with the 17alpha-ethynyl and the five-membered-spiromethylene ether groups, whereas both six-membered-spiro series showed, clearly with methyl and ethynyl substituents, and less pronounced with methylene and ethenyl, higher binding and transactivation values. For the 17alpha-ethynyl series, the McPhail test showed high potencies with methylene, methyl and ethenyl substituents after oral treatment or with propenyl after subcutaneous administration. The introduction of the spiro substituents in position 17 led to high potencies for other 11-substituents as well. Besides methyl, also ethyl, ethynyl and propynyl were potent substituents. With ovulation inhibition tests, the ethyl, ethenyl and ethynyl substituents were the more potent compounds in all four series. However, compounds with methyl or ethynyl additions appeared to be glucocorticoidal in the hormone screening test irrespective of the 17-substituent, while with the three spiro series even methylene and ethenyl groups became active. Androgenicity was only observed at dose levels at or above 5 mg/kg, which is 2.5-fold weaker than ETG. Moreover, estrogenicity appeared negligible with the three spiro series, while with the 17alpha-ethynyl series methyl, ethyl, ethenyl and ethynyl substituents, a very high estrogenic potential was assessed. Based on the high efficacy and low side-effects, the following compounds show a high selectivity: 17alpha-ethynyl with ethyl, ethenyl and 2-propenyl substituents, six-membered spiromethylene ether with ethyl and six-membered-spiromethylene lactone with ethyl, 2-propenyl or 1-propynyl substituents. (ABSTRACT TRUNCATED)