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A novel series of 17beta-hydroxysteroid dehydrogenase type 3 (17beta-HSD3) inhibitors has been identified. These inhibitors, based on a dibenzazocine core, exhibited picomolar to low nanomolar inhibition of 17beta-HSD3 in cell-free enzymatic as well as in cell-based transcriptional reporter assays.
Petrosal ganglion neurons are depolarized and fire action potentials in response to acetylcholine and nicotine. However, little is known about the subtype(s) of nicotinic acetylcholine receptors involved, although alpha4 and alpha7 subunits have been identified in petrosal ganglion neurons. Cytisine, an alkaloid unrelated to nicotine, and its bromo derivatives are agonists exhibiting different affinities, potencies and efficacies at nicotinic acetylcholine receptors containing alpha4 or alpha7 subunits. To characterize the receptors involved, we studied the effects of these agonists and the nicotinic acetylcholine receptor antagonists hexamethonium and alpha-bungarotoxin in isolated petrosal ganglion neurons. Petrosal ganglia were excised from anesthetized cats and cultured for up to 16 days. Using patch-clamp technique, we recorded whole-cell currents evoked by 5-10 s applications of acetylcholine, cytisine or its bromo derivatives. Agonists and antagonists were applied by gravity from a pipette near the neuron surface. Neurons responded to acetylcholine, cytisine, 3-bromocytisine and 5-bromocytisine with fast inward currents that desensitized during application of the stimuli and were reversibly blocked by 1 microM hexamethonium or 10 nM alpha-bungarotoxin. The order of potency of the agonists was 3-bromocytisine >> acetylcholine approximately = cytisine >> 5-bromocytisine, suggesting that homomeric alpha7 neuronal nicotinic receptors predominate in cat petrosal ganglion neurons in culture.
The connection between smoking and depression, the antidepressant actions of nicotine and the targeting of nicotinic acetylcholine receptors (nAChRs) by monoamine re-uptake inhibitors all point to a potential role of nAChRs in the etiology and/or symptomatology of depression. In the current study, we evaluated nAChR subtypes in brain regions of rats subjected to olfactory bulbectomy (OBX), a standard animal model that recapitulates many of the behavioral and neurochemical alterations thought to underlie human depression. Comparisons were made both to sham-operated controls and unoperated animals. OBX led to upregulation of cerebrocortical alpha4beta2 nAChRs and downregulation of striatal alpha7 nAChRs as compared to either the sham-operated or unoperated groups. Striatal alpha4beta2 nAChRs were also downregulated but the sham surgery by itself produced a partial effect, masking the contribution of the OBX lesion. In agreement with earlier studies, we also found downregulation of muscarinic AChRs (both m1 and m2 subtypes) in the striatum when comparing the OBX group to sham-operated controls, but because sham surgery evoked mAChR upregulation, the effect was not apparent when the OBX animals were contrasted to the unoperated group. Accordingly, caution needs to be exercised in interpreting studies of cholinergic function in the OBX model that do not include unoperated animals as an additional comparison group. Our results reinforce a relationship between depression and nAChR expression and point to the need for parallel studies in human depression that might lead to the design of novel therapies targeting specific nAChR subtypes.
The autoinhibitory control of electrically evoked release of [3H]-dopamine and the properties of that induced by nicotinic receptor (nAChR) stimulation were studied in slices of the human neocortex. In both models [3H]-dopamine release was action potential-induced and exocytotic. The selective dopamine D2 receptor agonist (-)-quinpirole reduced electrically evoked release of [3H]-dopamine, yielding IC50 and I(max) values of 23 nM and 76%, respectively. Also, the effects of several other subtype-selective dopamine receptor ligands confirmed that the terminal dopamine autoreceptor belongs to the D2 subtype. The autoinhibitory feedback control was slightly operative under stimulation conditions of 90 pulses and 3 Hz, with a biophase concentration of endogenous dopamine of 3.6 nM, and was enhanced under blockade of dopamine reuptake. [3H]-dopamine release evoked in an identical manner in mouse neocortical slices was not inhibited by (-)-quinpirole, suggesting the absence of dopamine autoreceptors in this tissue and underlining an important species difference. Also, nAChR stimulation-induced release of [3H]-dopamine revealed a species difference: [3H]-dopamine release was evoked in human, but not in rat neocortical slices. The nAChRs inducing [3H]-dopamine release most probably belong to the alpha3/beta2subtype, according to the potencies and efficacies of subtype-selective nAChR ligands. Part of these receptors may be located on glutamatergic neurons.
A new energetic substance hexanitrohexaazaisowurtzitane (or CL-20) was tested for its toxicities to various ecological receptors. CL-20 (epsilon-polymorph) was amended to soil or deionized water to construct concentration gradients. Results of Microtox (15-min contact) and 96-h algae growth inhibition tests indicate that CL-20 showed no adverse effects on the bioluminescence of marine bacteria Vibrio fischeri and the cell density of freshwater green algae Selenastrum capricornutum respectively, up to its water solubility (ca. 3.6 mg l(-1)). CL-20 and its possible biotransformation products did not inhibit seed germination and early seedling (16-19 d) growth of alfalfa (Medicago sativa) and perennial ryegrass (Lolium perenne) up to 10,000 mg kg(-1) in a Sassafras sandy loam soil (SSL). Indigenous soil microorganisms in SSL and a garden soil were exposed to CL-20 for one or two weeks before dehydrogenase activity (DHA) or potential nitrification activity (PNA) were assayed. Results indicate that up to 10,000 mg kg(-1) soil of CL-20 had no statistically significant effects on microbial communities measured as DHA or on the ammonium oxidizing bacteria determined as PNA in both soils. Data indicates that CL-20 was not acutely toxic to the species or microbial communities tested and that further studies are required to address the potential long-term environmental impact of CL-20 and its possible degradation products.
Environmental investigations have been conducted at 23 military firing ranges in the United States and Canada. The specific training facilities most frequently evaluated were hand grenade, antitank rocket, and artillery ranges. Energetic compounds (explosives and propellants) were determined and linked to the type of munition used and the major mechanisms of deposition.
Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), and 2,4,6-trinitrotoluene (TNT) are explosives that are frequently found as environmental contaminants on military installations. Hydrogen has been shown to support the anaerobic transformation of these explosives. We investigated ethanol and propylene glycol as electron donors for providing syntrophically produced H2 for stimulating the anaerobic biodegradation of explosives in contaminated soil. The study was conducted using anoxic microcosms constructed with slurries of the contaminated soil and groundwater. The addition of 5mM ethanol and propylene glycol enhanced the biodegradation of RDX and HMX relative to the control bottles. Ethanol was depleted within about 20 days, resulting in the transient formation of hydrogen, acetate, and methane. The hydrogen headspace concentration increased from 8 ppm to 1838 ppm before decreasing to background concentrations. Propylene glycol was completely degraded after 15 days, forming hydrogen, propionate, and acetate as end-products. The hydrogen headspace concentrations increased from 56 ppm to 628 ppm before decreasing to background concentrations. No methane formation was observed during the incubation period of 48 days. Our findings indicate the addition of ethanol and propylene to the aquifer slurries increased the hydrogen concentrations and enhanced the biotransformation of RDX and HMX in the explosive-contaminated soil.
We report the ability of nickel-based catalysts to degrade explosives compounds in aqueous solution. Several nickel catalysts completely degraded the explosives, although rates varied. Nearly all of the organic explosive compounds tested, including 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), were rapidly degraded to below detection limits by a powdered nickel on an alumina-silicate support (Aldrich nickel catalyst). Perchlorate degradation was minimal (<25%). Degradation of TNT by Aldrich nickel catalyst resulted in apparent first-order kinetics. Significant gaseous 14C was released and collected in an alkaline solution (most likely carbon dioxide) from [14C]RDX and [14C]HMX, indicating heterocyclic ring cleavage. Significant gaseous 14C was not produced from [14C]TNT, but spectrophotometric evidence indicated loss of aromaticity. Degradation occurred in low ionic strength solutions, groundwater, and from pH 3 to pH 9. Degradation of TNT, RDX, and HMX was maintained in flow-through columns of Aldrich nickel catalyst mixed with sand down to a hydraulic retention time of 4h. These data indicate that nickel-based catalysts may be an effective means for remediation of energetics-contaminated groundwater.
CL-20 is a polycyclic energetic nitramine, which may soon replace the monocyclic nitramines RDX and HMX, because of its superior explosive performance. Therefore, to predict its environmental fate, analytical and physico-chemical data must be made available. An HPLC technique was thus developed to measure CL-20 in soil samples based on the US Environmental Protection Agency method 8330. We found that the soil water content and aging (21 days) had no effect on the recoveries (>92%) of CL-20, provided that the extracts were kept acidic (pH 3). The aqueous solubility of CL-20 was poor (3.6 mg l(-1) at 25 degrees C) and increased with temperature to reach 18.5 mg l(-1) at 60 degrees C. The octanol-water partition coefficient of CL-20 (log KOW = 1.92) was higher than that of RDX (log KOW = 0.90) and HMX (log KOW = 0.16), indicating its higher affinity to organic matter. Finally, CL-20 was found to decompose in non-acidified water upon contact with glass containers to give NO2- (2 equiv.), N2O (2 equiv.), and HCOO- (2 equiv.). The experimental findings suggest that CL-20 should be less persistent in the environment than RDX and HMX.
An Agilent 3DCE capillary electrophoresis system using sulfobutylether-beta-cyclodextrin (SB-beta-CD)-ammonium acetate separation buffer pH 6.9 was coupled to a Bruker Esquire 3000+ quadrupole ion trap mass detector via a commercially available electrospray ionization interface with acetonitrile sheath flow. The CE-MS system was applied in negative ionization mode for the resolution and detection of nitroaromatic and polar cyclic or caged nitramine energetic materials including TNT [2,4,6-trinitrotoluene, formula mass (FW) 227.13], TNB (1,3,5-trinitrobenzene, FW 213.12), RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine, FW 222.26) HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine, FW 296.16), and CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane, FW 438.19). The CE-MS system conformed to the high-performance liquid chromatography with ultraviolet absorbance detection (HPLC-UV) and HPLC-MS reference methods for the identification of energetic contaminants and their degradation products in soil and marine sediment samples.
Explosives are released into the environment at production and processing facilities, as well as through field use. These compounds may be toxic at relatively low concentrations to a number of ecological receptors. A toxicity assessment was carried out on soils from an explosive-contaminated site at a Canadian Forces Area Training Center. Toxicity studies on soil organisms using endpoints such as microbial processes (potential nitrification activity, dehydrogenase activity, substrate-induced respiration, basal respiration), plant seedling and growth (Lactuca sativa and Hordeum vulgare), and earthworm (Eisenia andrei) growth and reproduction were carried out. Results showed that 1,3,5,7-tetranitro-1,3,5,7-tetrazacyclooctane (HMX) was the principal polynitro-organic compound measured in soils. Soils from the contaminated site decreased microbial processes and earthworm reproduction; whereas plant growth was not significantly reduced. Toxicity to aquatic organisms and genotoxicity were also assessed on soil elutriates using Microtox (Vibrio fischeri), growth inhibition of algae (Selenastrum capricornutum), and SOS Chromotest (Escherichia coli). Results indicated that soil elutriates were generally not toxic to bacteria (Microtox) and algae. However, genotoxicity was found in a number of soil elutriate samples. Thus, the explosive-contaminated soils from the antitank firing range may represent a hazard for the soil organisms. Nevertheless, the global toxicity might have partially resulted from HMX as well as from other (not identified) contaminants such as heavy metals.
Effects of cholinergic drugs on human alpha4beta2 nicotinic acetylcholine receptors expressed in Xenopus oocytes have been investigated in electrophysiological and ligand binding experiments. Atropine, scopolamine, physostigmine, and tacrine combine potentiation of ion current induced by low concentrations of acetylcholine with inhibition of ion current evoked by high concentrations of acetylcholine. Rivastigmine, galanthamine, and dichlorvos cause only inhibition of ion current evoked by low concentrations of acetylcholine. Binding experiments show that the potentiating cholinergic drugs atropine, scopolamine, and physostigmine are competitive ligands of human alpha4beta2 nicotinic acetylcholine receptors. Conversely, the inhibitory cholinergic drugs galanthamine and rivastigmine are non-competitive. The non-competitive drugs are not allosteric, since they do not affect the saturation curve of the radioligand [3H]cytisine. Effects of potentiating cholinergic drugs on nicotinic acetylcholine receptors are consistent with and predicted by a model comprising competitive drug effects at two equivalent agonist recognition sites on the nicotinic acetylcholine receptor combined with non-competitive ion channel block.
An alpha7 nicotinic acetylcholine receptor sequence was cloned from Rhesus monkey (Macaca mulatta). This clone differs from the mature human alpha7 nicotinic acetylcholine receptor in only four amino acids, two of which are in the extracellular domain. The monkey alpha7 nicotinic receptor was characterized in regard to its functional responses to acetylcholine, choline, cytisine, and the experimental alpha7-selective agonists 4OH-GTS-21, TC-1698, and AR-R17779. For all of these agonists, the EC(50) for activation of monkey receptors was uniformly higher than for human receptors. In contrast, the potencies of mecamylamine and MLA for inhibiting monkey and human alpha7 were comparable. Acetylcholine and 4OH-GTS-21 were used to probe the significance of the single point differences in the extracellular domain. Mutants with the two different amino acids in the extracellular domain of the monkey receptor changed to the corresponding sequence of the human receptor had responses to these agonists that were not significantly different in EC(50) from wild-type human alpha7 nicotinic receptors. Monkey alpha7 nicotinic receptors have a serine at residue 171, while the human receptors have an asparagine at this site. Monkey S171N mutants were more like human alpha7 nicotinic receptors, while mutations at the other site (K186R) had relatively little effect. These experiments point toward the basic utility of the monkey receptor as a model for the human alpha7 nicotinic receptor, albeit with the caveat that these receptors will vary in their agonist concentration dependency. They also point to the potential importance of a newly identified sequence element for modeling the specific amino acids involved with receptor activation.
Neuronal nicotinic acetylcholine receptors subserve predominantly modulatory roles in the brain, making them attractive therapeutic targets. Natural products provide key leads in the quest for nicotinic receptor subtype-selective compounds. Cytisine, found in Leguminosae spp., binds with high affinity to alpha4beta2* nicotinic receptors. We have compared the effect of C3 and C5 halogenation of cytisine and methylcytisine (MCy) on their interaction with native rat nicotinic receptors. 3-Bromocytisine (3-BrCy) and 3-iodocytisine (3-ICy) exhibited increased binding affinity (especially at alpha7 nicotinic receptors; Ki approximately 0.1 microM) and functional potency, whereas C5-halogenation was detrimental. 3-BrCy and 3-ICy were more potent than cytisine at evoking [3H]dopamine release from striatal slices (EC50 approximately 11 nM), [3H]noradrenaline release from hippocampal slices (EC50 approximately 250 nM), increases in intracellular Ca2+ in PC12 cells and inward currents in Xenopus oocytes expressing human alpha3beta4 nicotinic receptor (EC50 approximately 2 microM). These compounds were also more efficacious than cytisine. C3-halogenation of cytisine is proposed to stabilize the open conformation of the nicotinic receptor but does not enhance subtype selectivity.
An extensive investigation at the Camp Edwards, Massachusetts Military Reservation (MMR) demonstrates that assessment of groundwater and soil contamination at military ranges can be limited primarily to explosive-related compounds such as RDX, HMX, perchlorate, TNT and their transformation products. A modified analytical method is recommended to expand the list of explosives and to improve the detection limits. Analyses of metals, VOCs, SVOCs, and TICs are unnecessary. Soil samples may require the analyses of PAHs and PCNs for burn areas. Camp Edwards, as one of the few military ranges that have been exhaustively investigated for contaminants, is an ideal point of departure for evaluating other ranges. The permeable site soils promote leaching of contaminants and inhibit biotic and abiotic transformations. Moreover, the site has experienced an unusual extent of activities in its more than ninety years of active use. The recommendations in this report are based on data obtained for more than 200 analytes from more than 15,000 environmental samples.
alpha7-Nicotinic acetylcholine receptors (alpha7-nAChR) have been implicated in a range of cognitive deficits in schizophrenia. Therefore we examined alpha7-nAChR knockout (KO), heterozygote (HT) and wildtype (WT) littermate mice in the 5-CSR (a rodent model of sustained attention) and odour span (a novel mouse working memory paradigm) tasks, and related performance to nAChR density. Whilst there was no difference between groups in baseline 5-CSR task performance, alpha7-nAChR KO's exhibited significantly higher omission levels compared to WT mice on increasing the attentional load, with HT mice performing at an intermediate level. Furthermore, alpha7-nAChR KO mice were significantly impaired in the odour span task when compared to WT mice, in a pattern consistent with impaired attention. These behavioural deficits were associated with the loss of alpha7-nAChRs, as alpha4beta2-nAChR density was unaltered in these mice. Thus these studies intimate that the attentional impairment in alpha7-nAChR transgenic mice maybe core to other deficits in cognition.
The effect of a synthetic cyclic disulfide compound, SA3443, on neovascularization was investigated. In vitro, enzyme-linked immunosorbent assay and RT-PCR demonstrated that SA3443 suppressed the expression of the hypoxia-induced vascular endothelial growth factor (VEGF) at both protein and mRNA levels in ARPE-19 cells. In vivo, the administration of SA3443 to mice with laser-induced choroidal neovascularization (CNV) suppressed the leakage from the lesions and reduced their size. Furthermore, the expression level of VEGF protein was significantly reduced by the administration of SA3443. Taken together, our results demonstrate that SA3443 suppresses VEGF production and reduces vascular leakage and the growth of mouse experimental CNV.
Several H2-producing fermentative anaerobic bacteria including Clostridium, Klebsiella and Fusobacteria degraded octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) (36 microM) to formaldehyde (HCHO) and nitrous oxide (N2O) with rates ranging from 5 to 190 nmol h(-1)g [dry weight] of cells(-1). Among these strains, C. bifermentans strain HAW-1 grew and transformed HMX rapidly with the detection of the two key intermediates the mononitroso product and methylenedinitramine. Its cellular extract alone did not seem to degrade HMX appreciably, but degraded much faster in the presence of H2, NADH or NADPH. The disappearance of HMX was concurrent with the release of nitrite without the formation of the nitroso derivative(s). Results suggest that two types of enzymes were involved in HMX metabolism: one for denitration and the second for reduction to the nitroso derivative(s).