Comparative pulmonary and genotoxic responses to inhaled nickel subsulfide and nickel sulfate in F344 rats.

Inhalation studies with nickel (Ni) subsulfide (Ni3 S2 ) and Ni sulfate hexahydrate (NiSO4 ·6H2 O) investigated differences in mode of action that could explain why the former induced lung tumors in rats and the latter did not. Male rats were exposed to ≤0.22 mg Ni/m3 NiSO4 ·6H2 O or 0.44 mg Ni/m3 Ni3 S2 , 6 h/day, 5 days/week for 3 and 13 weeks; subsets of the rats exposed for 13 weeks were held for an additional 13 weeks. Analyses of bronchoalveolar lavage fluid, isolated cells, and whole lung tissue were conducted to compare the extent and persistence of any induced lung effects. Histological findings were qualitatively identical for both compounds and consistent with lesions reported in earlier studies. After 13 weeks of exposure, the incidence and severity of pulmonary inflammation and epithelial cell hyperplasia were greater among Ni3 S2 -exposed rats, whereas the reverse response was seen for apoptosis. Only Ni3 S2 exposure significantly increased epithelial and non-epithelial cell proliferation after 13 weeks of exposure. Both compounds induced DNA damage in isolated lung cells and DNA hypermethylation of whole lung tissue after 13 weeks of exposure at the highest exposure concentrations. Increases in cell proliferation, DNA damage, and tissue DNA hypermethylation did not persist during the 13-week recovery period. In summary, the highest concentrations of each compound produced marked pulmonary toxicity, but the lowest concentrations produced minimal or no effects. Differences in the proliferative and apoptotic responses between the two compounds may help explain differences in carcinogenicity, whereas the identification of no observed adverse effect concentrations (NOAECs) contributes to the risk characterization for inhalation exposure to nickel compounds.

EAT-18 is an essential auxiliary protein interacting with the non-alpha nAChR subunit EAT-2 to form a functional receptor.

Nematode parasites infect approximately 1.5 billion people globally and are a significant public health concern. There is an accepted need for new, more effective anthelmintic drugs. Nicotinic acetylcholine receptors on parasite nerve and somatic muscle are targets of the cholinomimetic anthelmintics, while glutamate-gated chloride channels in the pharynx of the nematode are affected by the avermectins. Here we describe a novel nicotinic acetylcholine receptor on the nematode pharynx that is a potential new drug target. This homomeric receptor is comprised of five non-α EAT-2 subunits and is not sensitive to existing cholinomimetic anthelmintics. We found that EAT-18, a novel auxiliary subunit protein, is essential for functional expression of the receptor. EAT-18 directly interacts with the mature receptor, and different homologs alter the pharmacological properties. Thus we have described not only a novel potential drug target but also a new type of obligate auxiliary protein for nAChRs.

The role of nuclear quantum effects in the relative stability of hexagonal and cubic ice.

At atmospheric pressure, hexagonal ice (Ih) is thermodynamically stable relative to cubic ice (Ic), although the magnitude and underlying physical origin of this stability difference are not well defined. Pure Ic crystals are not accessible experimentally, and hence computer simulations have often been used to interrogate the relative stabilities of Ih and Ic; however, these simulations are dominated by molecular interaction models that ignore the intramolecular flexibility of individual water molecules, do not describe intermolecular hydrogen-bonding with sufficient accuracy, or ignore the role of nuclear quantum effects (NQEs) such as zero-point energy. Here, we show that when comparing the relative stability of Ih and Ic using a flexible, anharmonic molecular interaction model, while also accurately accounting for NQEs, a new picture emerges: Ih is stabilized relative to Ic as a result of subtle differences in the intramolecular geometries and intermolecular interactions of water molecules which are modulated by NQEs. Our simulations hence suggest that NQEs are a major contributor to the stabilization of Ih under terrestrial conditions and thus contribute to the well-known hexagonal (sixfold) symmetry of ice crystals.

Accelerated path-integral simulations using ring-polymer interpolation.

Imaginary-time path-integral (PI) molecular simulations can be used to calculate exact quantum statistical mechanical properties for complex systems containing many interacting atoms and molecules. The limiting computational factor in a PI simulation is typically the evaluation of the potential energy surface (PES) and forces at each ring-polymer "bead"; for an n-bead ring-polymer, a PI simulation is typically n times greater than the corresponding classical simulation. To address the increased computational effort of PI simulations, several approaches have been developed recently, most notably based on the idea of ring-polymer contraction which exploits either the separation of the PES into short-range and long-range contributions or the availability of a computationally inexpensive PES which can be incorporated to effectively smooth the ring-polymer PES; neither approach is satisfactory in applications to systems modeled by PESs given by on-the-fly ab initio calculations. In this article, we describe a new method, ring-polymer interpolation (RPI), which can be used to accelerate PI simulations without any prior assumptions about the PES. In simulations of liquid water modeled by an empirical PES (or force field) under ambient conditions, where quantum effects are known to play a subtle role in influencing experimental observables such as radial distribution functions, we find that RPI can accurately reproduce the results of fully-converged PI simulations, albeit with far fewer PES evaluations. This approach therefore opens the possibility of large-scale PI simulations using ab initio PESs evaluated on-the-fly without the drawbacks of current methods.

Functional characterization of a novel family of acetylcholine-gated chloride channels in Schistosoma mansoni.

Acetylcholine is the canonical excitatory neurotransmitter of the mammalian neuromuscular system. However, in the trematode parasite Schistosoma mansoni, cholinergic stimulation leads to muscle relaxation and a flaccid paralysis, suggesting an inhibitory mode of action. Information about the pharmacological mechanism of this inhibition is lacking. Here, we used a combination of techniques to assess the role of cholinergic receptors in schistosome motor function. The neuromuscular effects of acetylcholine are typically mediated by gated cation channels of the nicotinic receptor (nAChR) family. Bioinformatics analyses identified numerous nAChR subunits in the S. mansoni genome but, interestingly, nearly half of these subunits carried a motif normally associated with chloride-selectivity. These putative schistosome acetylcholine-gated chloride channels (SmACCs) are evolutionarily divergent from those of nematodes and form a unique clade within the larger family of nAChRs. Pharmacological and RNA interference (RNAi) behavioral screens were used to assess the role of the SmACCs in larval motor function. Treatment with antagonists produced the same effect as RNAi suppression of SmACCs; both led to a hypermotile phenotype consistent with abrogation of an inhibitory neuromuscular mediator. Antibodies were then generated against two of the SmACCs for use in immunolocalization studies. SmACC-1 and SmACC-2 localize to regions of the peripheral nervous system that innervate the body wall muscles, yet neither appears to be expressed directly on the musculature. One gene, SmACC-1, was expressed in HEK-293 cells and characterized using an iodide flux assay. The results indicate that SmACC-1 formed a functional homomeric chloride channel and was activated selectively by a panel of cholinergic agonists. The results described in this study identify a novel clade of nicotinic chloride channels that act as inhibitory modulators of schistosome neuromuscular function. Additionally, the iodide flux assay used to characterize SmACC-1 represents a new high-throughput tool for drug screening against these unique parasite ion channels.

Investigation of acetylcholine receptor diversity in a nematode parasite leads to characterization of tribendimidine- and derquantel-sensitive nAChRs.

Nicotinic acetylcholine receptors (nAChRs) of parasitic nematodes are required for body movement and are targets of important "classical" anthelmintics like levamisole and pyrantel, as well as "novel" anthelmintics like tribendimidine and derquantel. Four biophysical subtypes of nAChR have been observed electrophysiologically in body muscle of the nematode parasite Oesophagostomum dentatum, but their molecular basis was not understood. Additionally, loss of one of these subtypes (G 35 pS) was found to be associated with levamisole resistance. In the present study, we identified and expressed in Xenopus oocytes, four O. dentatum nAChR subunit genes, Ode-unc-38, Ode-unc-63, Ode-unc-29 and Ode-acr-8, to explore the origin of the receptor diversity. When different combinations of subunits were injected in Xenopus oocytes, we reconstituted and characterized four pharmacologically different types of nAChRs with different sensitivities to the cholinergic anthelmintics. Moreover, we demonstrate that the receptor diversity may be affected by the stoichiometric arrangement of the subunits. We show, for the first time, different combinations of subunits from a parasitic nematode that make up receptors sensitive to tribendimidine and derquantel. In addition, we report that the recombinant levamisole-sensitive receptor made up of Ode-UNC-29, Ode-UNC-63, Ode-UNC-38 and Ode-ACR-8 subunits has the same single-channel conductance, 35 pS and 2.4 ms mean open-time properties, as the levamisole-AChR (G35) subtype previously identified in vivo. These data highlight the flexible arrangements of the receptor subunits and their effects on sensitivity and resistance to the cholinergic anthelmintics; pyrantel, tribendimidine and/or derquantel may still be effective on levamisole-resistant worms.

A T-dependent antibody response evaluation in CD-1 mice after an acute whole-body inhalation exposure to nickel (II) chloride hexahydrate.

Nickel (Ni) in ambient air may vary regionally with contributions from both natural processes and anthropogenic activities. Exposure to Ni compounds in ambient air above a certain level is associated with acute adverse effects, such as upper respiratory tract irritation, pneumonitis, and chronic adverse effects, such as respiratory cancer. Inhalation reference exposure standards are enacted in different jurisdictions to minimize exposures to ambient Ni above levels that can elicit adverse effects. This paper reports a guideline-/GLP-compliant study designed for setting inhalation exposure standards to protect from immunological effects associated with acute exposure to Ni. Female CD-1 mice were exposed via whole-body inhalation to aerosolized nickel chloride hexahydrate for 24-hr at nominal (vs. mean analyzed) concentrations of 20 (16), 50 (44) and 100 (81) µg Ni/m3. Host T-cell antibody immunological responses to intravenously-injected sheep red blood cells were then measured ex vivo in an Antibody-Forming Cell (AFC) assay. Exposure to the Ni substance significantly decreased spleen cell levels by 33%, but this was within biological variability for outbred mice. No concurrent decreases in spleen, thymus, or body weights were noted. No immunosuppression was observed with the Ni substance in the context of Total Spleen Activity [IgM AFC/spleen (× 103)] and Specific Activity [IgM AFC/spleen cells (× 106)]. Significant concentration-independent increases in Total Spleen Activity and Specific Activity seen with the nickel chloride hexahydrate were normal and within biological variability for outbred mice. In contrast, cyclophosphamide (positive control) significantly decreased spleen cell numbers, spleen and thymus weights, and abolished Specific Activity and Total Spleen Activity. Based on results here, an NOAEC of 81 µg Ni/m3 for immunosuppressive effects from inhaled nickel chloride hexahydrate was identified. It is hoped this value can be used to derive a reference standard for human exposure to ambient Ni.

Gene (HPRT) and chromosomal (MN) mutations of nickel metal powder in V79 Chinese hamster cells.

Nickel metal is a naturally occurring element used in many industrial and consumer applications. Human epidemiological data and animal cancer bioassays indicate that nickel metal is not likely to be a human carcinogen. Yet, nickel metal is classified as a suspected human carcinogen (CLP) and possibly carcinogenic to humans (IARC). There are no reliable studies on the potential for nickel metal to induce gene and micronucleus (MN) mutations. To fill these datagaps and increase our understanding of the mechanisms underlying the lack of nickel metal carcinogenicity, gene and micronucleus mutation studies were conducted with nickel metal powder (N36F) in V79 Chinese Hamster cells following OECD 476 and 487 guidelines, respectively, under GLP. Gene mutation at the hprt locus was tested, with and without metabolic activation, after 4-h treatment with 0.05-2.5 mM nickel metal powder. Cytokinesis-block MN frequency following exposure to 0.25-1.5 mM nickel metal was tested after 4-h treatment, with and without metabolic activation, followed by a 24-h treatment without metabolic activation. In the gene mutation assay, there were modest increases in hprt mutants observed at some test concentrations, not exceeding 2.2-fold, which were either within the historical control values and/or showed no concentration-response trend. The positive controls showed increases of at least 7-fold. Likewise, no increases in the MN frequency exceeding 1.5-fold were observed with nickel metal, with no concentration-response trends. Taking these results together, it can be concluded that nickel metal is non-mutagenic and does not cause gene nor chromosomal mutations.

Tead1 is required for maintaining adult cardiomyocyte function, and its loss results in lethal dilated cardiomyopathy.

Heart disease remains the leading cause of death worldwide, highlighting a pressing need to identify novel regulators of cardiomyocyte (CM) function that could be therapeutically targeted. The mammalian Hippo/Tead pathway is critical in embryonic cardiac development and perinatal CM proliferation. However, the requirement of Tead1, the transcriptional effector of this pathway, in the adult heart is unknown. Here, we show that tamoxifen-inducible adult CM-specific Tead1 ablation led to lethal acute-onset dilated cardiomyopathy, associated with impairment in excitation-contraction coupling. Mechanistically, we demonstrate Tead1 is a cell-autonomous, direct transcriptional activator of SERCA2a and SR-associated protein phosphatase 1 regulatory subunit, Inhibitor-1 (I-1). Thus, Tead1 deletion led to a decrease in SERCA2a and I-1 transcripts and protein, with a consequent increase in PP1-activity, resulting in accumulation of dephosphorylated phospholamban (Pln) and decreased SERCA2a activity. Global transcriptomal analysis in Tead1-deleted hearts revealed significant changes in mitochondrial and sarcomere-related pathways. Additional studies demonstrated there was a trend for correlation between protein levels of TEAD1 and I-1, and phosphorylation of PLN, in human nonfailing and failing hearts. Furthermore, TEAD1 activity was required to maintain PLN phosphorylation and expression of SERCA2a and I-1 in human induced pluripotent stem cell-derived (iPS-derived) CMs. To our knowledge, taken together, this demonstrates a nonredundant, novel role of Tead1 in maintaining normal adult heart function.

Curiouser and Curiouser: The Macrocyclic Lactone, Abamectin, Is also a Potent Inhibitor of Pyrantel/Tribendimidine Nicotinic Acetylcholine Receptors of Gastro-Intestinal Worms.

Nematode parasites may be controlled with drugs, but their regular application has given rise to concerns about the development of resistance. Drug combinations may be more effective than single drugs and delay the onset of resistance. A combination of the nicotinic antagonist, derquantel, and the macrocyclic lactone, abamectin, has been found to have synergistic anthelmintic effects against gastro-intestinal nematode parasites. We have observed in previous contraction and electrophysiological experiments that derquantel is a potent selective antagonist of nematode parasite muscle nicotinic receptors; and that abamectin is an inhibitor of the same nicotinic receptors. To explore these inhibitory effects further, we expressed muscle nicotinic receptors of the nodular worm, Oesophagostomum dentatum (Ode-UNC-29:Ode-UNC-63:Ode-UNC-38), in Xenopus oocytes under voltage-clamp and tested effects of abamectin on pyrantel and acetylcholine responses. The receptors were antagonized by 0.03 µM abamectin in a non-competitive manner (reduced Rmax, no change in EC50). This antagonism increased when abamectin was increased to 0.1 µM. However, when we increased the concentration of abamectin further to 0.3 µM, 1 µM or 10 µM, we found that the antagonism decreased and was less than with 0.1 µM abamectin. The bi-phasic effects of abamectin suggest that abamectin acts at two allosteric sites: one high affinity negative allosteric (NAM) site causing antagonism, and another lower affinity positive allosteric (PAM) site causing a reduction in antagonism. We also tested the effects of 0.1 µM derquantel alone and in combination with 0.3 µM abamectin. We found that derquantel on these receptors, like abamectin, acted as a non-competitive antagonist, and that the combination of derquantel and abamectin produced greater inhibition. These observations confirm the antagonistic effects of abamectin on nematode nicotinic receptors in addition to GluCl effects, and illustrate more complex effects of macrocyclic lactones that may be exploited in combinations with other anthelmintics.

Pharmacological profile of Ascaris suum ACR-16, a new homomeric nicotinic acetylcholine receptor widely distributed in Ascaris tissues.

BACKGROUND AND PURPOSE: Control of nematode parasite infections relies largely on anthelmintic drugs, several of which act on nicotinic ACh receptors (nAChRs), and there are concerns about the development of resistance. There is an urgent need for development of new compounds to overcome resistance and novel anthelmintic drug targets. We describe the functional expression and pharmacological characterization of a homomeric nAChR, ACR-16, from a nematode parasite. EXPERIMENTAL APPROACH: Using RT-PCR, molecular cloning and two-electrode voltage clamp electrophysiology, we localized acr-16 mRNA in Ascaris suum (Asu) and then cloned and expressed acr-16 cRNA in Xenopus oocytes. Sensitivity of these receptors to cholinergic anthelmintics and a range of nicotinic agonists was tested. KEY RESULTS: Amino acid sequence comparison with vertebrate nAChR subunits revealed ACR-16 to be most closely related to α7 receptors, but with some striking distinctions. acr-16 mRNA was recovered from Asu somatic muscle, pharynx, ovijector, head and intestine. In electrophysiological experiments, the existing cholinergic anthelmintic agonists (morantel, levamisole, methyridine, thenium, bephenium, tribendimidine and pyrantel) did not activate Asu-ACR-16 (except for a small response to oxantel). Other nAChR agonists: nicotine, ACh, cytisine, 3-bromocytisine and epibatidine, produced robust current responses which desensitized at a rate varying with the agonists. Unlike α7, Asu-ACR-16 was insensitive to α-bungarotoxin and did not respond to genistein or other α7 positive allosteric modulators. Asu-ACR-16 had lower calcium permeability than α7 receptors. CONCLUSIONS AND IMPLICATIONS: We suggest that ACR-16 has diverse tissue-dependent functions in nematode parasites and is a suitable drug target for development of novel anthelmintic compounds.

Tribendimidine: mode of action and nAChR subtype selectivity in Ascaris and Oesophagostomum.

The cholinergic class of anthelmintic drugs is used for the control of parasitic nematodes. One of this class of drugs, tribendimidine (a symmetrical diamidine derivative, of amidantel), was developed in China for use in humans in the mid-1980s. It has a broader-spectrum anthelmintic action against soil-transmitted helminthiasis than other cholinergic anthelmintics, and is effective against hookworm, pinworms, roundworms, and Strongyloides and flatworm of humans. Although molecular studies on C. elegans suggest that tribendimidine is a cholinergic agonist that is selective for the same nematode muscle nAChR as levamisole, no direct electrophysiological observations in nematode parasites have been made to test this hypothesis. Also the hypothesis that levamisole and tribendimine act on the same receptor, does not explain why tribendimidine is effective against some nematode parasites when levamisole is not. Here we examine the effects of tribendimidine on the electrophysiology and contraction of Ascaris suum body muscle and show that tribendimidine produces depolarization antagonized by the nicotinic antagonist mecamylamine, and that tribendimidine is an agonist of muscle nAChRs of parasitic nematodes. Further pharmacological characterization of the nAChRs activated by tribendimidine in our Ascaris muscle contraction assay shows that tribendimidine is not selective for the same receptor subtypes as levamisole, and that tribendimidine is more selective for the B-subtype than the L-subtype of nAChR. In addition, larval migration inhibition assays with levamisole-resistant Oesophagostomum dentatum isolates show that tribendimidine is as active on a levamisole-resistant isolate as on a levamisole-sensitive isolate, suggesting that the selectivity for levamisole and tribendimidine is not the same. It is concluded that tribendimidine can activate a different population of nematode parasite nAChRs than levamisole, and is more like bephenium. The different nAChR subtype selectivity of tribendimidine may explain why the spectrum of action of tribendimidine is different to that of other cholinergic anthelmintics like levamisole.

Diethylcarbamazine increases activation of voltage-activated potassium (SLO-1) currents in Ascaris suum and potentiates effects of emodepside.

Diethylcarbamazine is a drug that is used for the treatment of filariasis in humans and animals; it also has effects on intestinal nematodes, but its mechanism of action remains unclear. Emodepside is a resistance-busting anthelmintic approved for treating intestinal parasitic nematodes in animals. The novel mode of action and resistance-breaking properties of emodepside has led to its use against intestinal nematodes of animals, and as a candidate drug for treating filarial parasites. We have previously demonstrated effects of emodepside on SLO-1 K+-like currents in Ascaris suum. Here, we demonstrate that diethylcarbamazine, which has been proposed to work through host mediated effects, has direct effects on a nematode parasite, Ascaris suum. It increases activation of SLO-1 K+ currents and potentiates effects of emodepside. Our results suggest consideration of the combination of emodepside and diethylcarbamazine for therapy, which is predicted to be synergistic. The mode of action of diethylcarbamazine may involve effects on parasite signaling pathways (including nitric oxide) as well as effects mediated by host inflammatory mediators.

Levamisole receptors: a second awakening.

Levamisole and pyrantel are old (1965) but useful anthelmintics that selectively activate nematode acetylcholine ion channel receptors; they are used to treat roundworm infections in humans and animals. Interest in their actions has surged, giving rise to new knowledge and technical advances, including an ability to reconstitute receptors that reveal more details of modes of action/resistance. We now know that the receptors are plastic and may form diverse species-dependent subtypes of receptor with different sensitivities to individual cholinergic anthelmintics. Understanding the biology of the levamisole receptors is expected to inform other studies on anthelmintics (ivermectin and emodepside) that act on ion channels.

Electrophysiological recording from parasitic nematode muscle.

Infection of man and animals with parasitic nematodes is recognized as a significant global problem (McLeod in Int J Parasitol 25(11):1363-1367, 1994; Hotez et al. in N Engl J Med 357(10):1018-1027, 2007). At present control of these infections relies primarily on chemotherapy. There are a limited number of classes of anthelmintic compounds and the majority of these act on ion-channels of the parasite (Martin et al. in Parasitology 113:S137-S156, 1996). In this report, we describe electrophysiological recording techniques as applied to parasitic nematodes. The aim of this report is: (1) to promote the study of ion channels in nematodes to help further the understanding of antinematodal drug action; (2) to describe our recording equipment and experimental protocols; and (3) provide some examples of the information to be gleaned from this approach and how it can increase our understanding of these important pathogens.