Cytoplasmic localization and the choice of ligand determine aggregate formation by androgen receptor with amplified polyglutamine stretch.

Polyglutamine tract expansion in androgen receptor is a recognized cause of spinal and bulbar muscular atrophy (SBMA), an X-linked motor neuronopathy. Similar mutations have been identified in proteins associated with other neurodegenerative diseases. Recent studies have shown that amplified polyglutamine repeat stretches form cellular aggregates that may be markers for these neurodegenerative diseases. Here we describe conditions that lead to aggregate formation by androgen receptor with polyglutamine stretch amplification. In transfection experiments, the mutant, compared with the wild-type receptor, was delayed in its cytoplasmic-nuclear translocation and formed large cytoplasmic aggregates in the presence of androgen. The cytoplasmic environment appears crucial for this aggregation, since retention of both the wild-type and mutant receptors in this cellular compartment by the deletion of their nuclear localization signals resulted in massive aggregation. Conversely, rapid nuclear transport of both receptors brought about by deletion of their ligand binding domains did not result in aggregate formation. However, androgen antagonists that altered the conformation of the ligand binding domain and promoted varying rates of cytoplasmic-nuclear translocation all inhibited aggregate formation. This demonstrates that in addition to the cytoplasmic localization, a distinct contribution of the ligand binding domain of the receptor is necessary for the aggregation. The finding that antiandrogens inhibit aggregate formation may provide the basis for in vivo determination of the role of these structures in SBMA.

Environmental benefits and concerns on safety: communicating latest results on nanotechnology safety research-the project DaNa2.0.

The use of nanotechnology and advanced materials promises to revolutionise many areas of technology and improve our daily life. In that respect, many positive effects on the environment are expected, either directly, by developing new technologies for remediation, filtering techniques or energy generation, or indirectly, by e.g. saving resources due to lower consumption of raw materials, or lower energy and fuel consumption due to reduced weight of vehicles. However, such beneficial effects of new technologies are often confronted by concerns regarding the safety of novel substances or materials. During the past 10 years, great effort has been put into research on potential hazards of nanomaterials towards environmental organisms. As the methodology for reliable assessment of nanomaterials was immature, many studies reporting contradictory results have been published, hindering both risk assessment for nanomaterials, as well as the knowledge communication to all involved stakeholders. Thus, DaNa2.0 serves as a platform to implement trusted knowledge on nanomaterials for an objective discussion.

Killing tumour cells by alkylphosphocholines: evidence for involvement of CD95.

Many lipids act as cellular messengers and lead to a variety of different cellular responses. Out of the group of these compounds the ceramides are able to induce apoptosis, and some synthetic lipids can mimic this effect. Apoptosis is an important mechanism whereby chemotherapeutics exhibit their anti-oncogenic activity. Although, some lipid analogues were used in clinical trials, they exert severe side effects and their mechanism of action is widely unknown. We present here a new class of synthetic alkylphosphocholines (APC) that induce programmed cell death in leukaemia cells. The signs of apoptosis arise after 1 h of incubation with these compounds as shown by phosphatidylserine externalisation followed by caspase activation and DNA fragmentation. We demonstrate that the molecular target of these lipids is upstream of caspases and Bcl-2. Experiments with FADD dominant negative cells reveal that induction of apoptosis occurs on the level of CD95 and that these compounds can now be optimised for their capacity to activate the apoptosis-inducing receptor CD95.

High throughput screening method for identification of new lipofection reagents.

Lipofection, the transfer of genetic material into cells by means of cationic lipids, is of growing interest for in vitro and in vivo approaches. In order to identify ideal lipofection reagents in a HTS, we have developed an automated lipofection method for the transfer of reporter genes into cells and for determination of the lipofection results. The method has specifically been designed and optimized for 96-well microtiter plates and can successfully be carried out by a pipetting robot with accessory equipment. It consists of two separate parts: (1) pretransfection (preparation of liposomes, formation of lipoplexes, and lipoplex transfer to the cells) and (2) posttransfection (determination of the reporter enzyme activity and the protein content of the transfected cells). Individual steps of the lipofection method were specifically optimized - for example, lipoplex formation and incubation time as well as cell lysis, cell cultivating, and the reporter gene assay. The HTS method facilitates characterization of the transfection properties (efficiency and cytotoxicity) of large numbers of (cationic) lipids in various adherent cell types.

Effects of organometals on cellular signaling. I. Influence of metabolic inhibitors on metal-induced arachidonic acid liberation.

Organic lead and tin compounds stimulate an increase of free arachidonic acid (AA) in HL-60 cells. This fatty acid is involved in numerous health problems and physiological mechanisms. Three major pathways result in a liberation of AA from membrane phospholipids and there is evidence that G-proteins serve as couplers within all three pathways. Therefore we investigated the influence of pertussis toxin (PT) on the organometallic-induced AA liberation. The effect of all studied compounds (organotin and organo-lead) was diminished by PT. We conclude that the organometals activate PLA2 to some extent via a PT-sensitive pathway. The ionophor A 23187 (1-10 microM) led to an increase of free AA by raising the intracellular Ca2+ level. One of the postulated ways of AA release is via Ca2+ channel activation; phospholipases are Ca2+ dependent. Thus, we examined the necessity of free intracellular Ca2+ for the organometallic effect. The Ca2+ chelator EGTA inhibited the increase of free AA induced by organometals. This is true also for verapamil, a Ca2+ channel blocker. Quinacrine, which is thought to be an inhibitor of phospholipase A2 (PLA2), prevented the AA liberation from membrane phospholipids induced by organometals. This could be due to the inhibition of PLA2, but it could also be the result of an inhibited Ca2+ influx.

Effects of organometals on cellular signaling. II. Inhibition of reincorporation of free arachidonic acid and influence on paf-acether synthesis by triethyllead.

Organometal compounds affect many enzymes, especially those containing SH-groups as acyl- and acetyltransferases involved in lysophospholipid reacylation. In HL-60 cells, organotin and -lead compounds stimulate phospholipase A2 activity, contributing thus to increase the level of lysophospholipids. In the present study, we have tested whether paf-acether (paf) biosynthesis was affected by treatment with triethyllead (Et3PbCl) in HL-60 cells. Et3PbCl inhibits the incorporation of exogenous arachidonic acid in the presence of high (> or = 50 microM) but not low concentrations (< or = 1 microM). High concentrations of the lead compound are unable to induce paf formation by itself, however, lower concentrations (< or = 10 microM) acted synergistically with TPA or fMLP to stimulate paf formation. Whereas unstimulated cells produced 0.4 pmole paf/2 x 10(6) cells, the stimulation with low fMLP (0.1 microM) resulted in the synthesis of 1.7 pmole and with low TPA (2 ng/ml) in 0.5 pmole paf. Preincubation of the cells with 10 microM Et3PbCl for 20 to 30 min increased the amount of paf formed by these cells to 3.3 pmole after treatment with 0.1 microM fMLP and 1.5 pmole after TPA. Furthermore, the results showed an inhibition of acetyltransferase (the key enzyme of paf synthesis) by the high and not by low concentrations of the lead compound. We conclude that low concentrations of Et3PbCl (< or = 10 microM) may act as a synergistic inducer of paf synthesis initiated via a receptor-coupled stimulation.

The antiproliferative alkylphospholipid S-1-O-phosphocholine-2-N-acetyl-octadecane induces apoptosis in leukemia cell lines.

Lipids are involved in a multitude of important cellular functions. They act as signaling molecules and can even provoke apoptosis. In this context we investigated the efficacy of synthetic alkylphosphocholines (APCs) as potential anti-cancer membrane-affecting drugs. Leading to novel therapeutic strategies for cancer treatment, the new agents interact with the cell membrane and do not affect the DNA. The data presented here show a cell death-inducing capacity for 1-O-phosphocholine-2[S]-O-acetyl-octadecane and 1-O-phosphocholin-2[S]-N-acetyl-octadecane in Jurkat T cells as well as in BJAB cells. The activation of caspases is generally required for the induction of apoptosis as shown by experiments with specific caspase inhibitors. The results point on the one hand to the formation of a functional DISC after APC-treatment as indicated by the clustering of receptor molecules and on the other hand to the dependency on the instrinsic apoptotic machinery and the downstream of mitochondria-activated apoptosome.

The structure of organometals determines cytotoxicity and alteration of calcium homeostasis in HL-60 cells.

There is increasing concern about the degradation and metabolisation as well as the biochemical mechanisms of action of organometallic compounds. They are known to be immunotoxic and/or neurotoxic. Because of their different toxic capacities, the development of a reliable correlation between molecular parameters and biochemical effects, which could be helpful in risk assessment, was an aim of this study. The tested organolead and -tin compounds decrease the viability of human cells in culture in a time- and concentration-dependent manner. Parabolic QSAR(1)(1) The abbreviations used are: TMT, trimethyltin chloride; TET, triethyltin bromide; TPT, tripropyltin chloride; TBT, tri- n-butyltin chloride; DBT, di- n-butyltin dichloride; TEL, triethyllead chloride; DEL, diethyllead dichloride; TML, trimethyllead chloride; TPhL, triphenyllead chloride; QSAR, quantitative structure-activity relationships; TSA, total surface area; MW(ion), ionic molecular weight; fMLP, N-formyl-L-methionyl-L-leucyl-L-phenylalanine; fluo-3, fluo-3 free acid; fluo-3 AM, fluo-3 acetoxymethyl ester; Me(2)SO, dimethyl sulfoxide; PLA(2), phospholipase A(2) (EC 3.1.1.4); FCS, fetal calf serum; HEPES, 4-(2-hydroxy-ethyl)-1-piperazineethanesulfonic acid; EGTA, [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid; [Ca(2+)](i), cytosolic free Ca(2+) concentration models yield an adequate correlation between toxicity expressed as LC(50) and structural parameters like ionic molecular weight (MW(ion)) or total surface area (TSA). Two main chemical attributes of the organometals are probably responsible for such a parabolic relationship: the hydrophobic side chain and the polar metal atom. Furthermore, all tested organometal compounds evoke a persistent increase of the cytosolic free calcium concentration [Ca(2+)](i). This effect is mainly due to an influx from the extracellular space. Further results suggest that Ca(2+) enters the cell via opened calcium channels. Based on the essential role of Ca(2+) within cellular signalling, the perturbation of calcium homeostasis appears to be an important event in final cell killing by organometals and it is most likely that other biochemical mechanisms, e.g. activation of phospholipase A(2), are possibly mediated by an increase of [Ca(2+)](i).

A new coculture-system of bronchial epithelial and endothelial cells as a model for studying ozone effects on airway tissue.

In order to study the inflammatory potential of ozone on the airway tissue, we developed an in vitro model system in which human bronchial epithelial cells (BEAS 2B) and human umbilical vein endothelial cells (ECV304) were able to communicate with each other. The BEAS 2B cells were grown on filter supports which were inserted into six-well culture dishes. Endothelial cells were cultivated on the bottom of the basolateral compartment. The upper epithelial cells were exposed to 0.15 ppm ozone for 90 min. Supernatants were collected after 1, 4 and 24 h and were quantified for IL-6 and IL-8 secretion. At the same time points we measured the expression of ICAM-1 on the umbilical vein endothelial cells. Exposure of the coculture-system to air or ozone induced the production of IL-6 as well as IL-8 that exceeded the sum of the amounts produced by the two cell types when exposed separately. At 24 h after ozone exposure the IL-6 and IL-8 levels were significantly elevated compared with the air treated cells. Concerning the ICAM-1 expression on ECV304 cells we found elevated ICAM-1 levels on cells which had been cocultured with BEAS 2B cells compared with cells cultured alone. This might be a hint for the secretion of a soluble factor that acts as a mediator in amplifying the response of epithelial cells.

In vitro effects of nitrogen dioxide on the release of nitric oxide by bovine alveolar macrophages.

Bovine alveolar macrophages (BAM) were assessed for nitric oxide-production after exposure to synthetic air or NO2 [0.2 ppm] for 2 h and subsequent treatment with lipopolysaccharide (LPS) [1 microg/ml]. Nitric oxide (NO) release, measured as nitrite in the supernatant, was quantified at different time points. LPS-stimulated BAM showed elevated nitrite levels after 24 h. Pre-treatment with NO2 resulted in higher nitrite levels in comparison to air exposed BAM. Some of the BAM preparations resulted in pre-activated macrophages that produce high amounts of NO without LPS treatment. Within these cells LPS treatment reduced the nitrite levels in comparison to untreated control incubations from the same preparation. These pre-activated BAM exhibited no uniform effect after NO2-exposure. In some experiments nitrite levels of pre-activated control BAM were decreased, while in others no influence could be observed. NO-production by pre-activated BAM or after LPS-treatment could be inhibited by N(G)-monomethyl-L-arginine (L-NMMA) [1 mM]. In order to investigate, whether NO2-exposure alters inducible NO-synthase mRNA expression, we now perform reverse transcription polymerase chain reaction (RT-PCR) of iNOS mRNA. This method will be a good tool to elucidate whether NO2-exposure can modulate iNOS mRNA expression.

Formation of reactive oxygen species in rat epithelial cells upon stimulation with fly ash.

Fly ash was used as a model for ambient particulate matter which is under suspicion to cause adverse pulmonary health effects. The fly ash was pre-sized and contained only particles < 20 microm including an ultrafine fraction (< 100 nm) that contributed 31% to the particle number. In our study, we investigated the influence of fly ash on the promotion of early inflammatory reactions like the formation of reactive oxygen species (ROS) in rat lung epithelial cells (RLE-6TN). Furthermore, we determined the formation of nitric oxide (NO). The cells show a clear dose-response relationship concerning the formation of ROS with regard to the mass of particles applied. Lipopolysaccharide (LPS) added as a co-stimulus did not increase the formation of ROS induced by fly ash. Furthermore, in LPS (0.1 microg/ml) and tumour necrosis factor-alpha (TNF-alpha; 1 ng/ml) pre-treated cells no increase in reactive oxygen species comparable to fly ash alone is observable. In presence of the metal chelator, desferrioxamine (DFO), ROS formation can be significantly reduced. Neither fly ash nor LPS induced a significant NO release in RLE-6TN cells.

C60 fullerene: a powerful antioxidant or a damaging agent? The importance of an in-depth material characterization prior to toxicity assays.

Since the discovery of fullerenes in 1985, these carbon nanospheres have attracted attention regarding their physico/chemical properties. Despite little knowledge about their impact on the environment and human health, the production of fullerenes has already reached an industrial scale. However, the toxicity of C(60) is still controversially discussed. The aim of this study was to clarify the biological effects of tetrahydrofuran (THF) suspended C(60) fullerene in comparison to water stirred C(60) fullerene suspensions. Beyond that, we analyzed the effects on the Crustacea Daphnia magna an indicator for ecotoxicological effects and the human lung epithelial cell line A549 as a simplified model for the respiratory tract. We could demonstrate that water-soluble side products which were formed in THF nC(60) suspension were responsible for the observed acute toxic effects, whereas fullerenes themselves had no negative effect regardless of the preparative route on either A549 cell in vitro or D. magna in vivo.

Oops they did it again! Carbon nanotubes hoax scientists in viability assays.

New materials of emerging technological importance are single-walled carbon nanotubes (SWCNTs). Because SWCNTs will be used in commercial products in huge amounts, their effects on human health and the environment have been addressed in several studies. Inhalation studies in vivo and submerse applications in vitro have been described with diverging results. Why some indicate a strong cytotoxicity and some do not is what we report on here. Data from A549 cells incubated with carbon nanotubes fake a strong cytotoxic effect within the MTT assay after 24 h that reaches roughly 50%, whereas the same treatment with SWCNTs, but detection with WST-1, reveals no cytotoxicity. LDH, FACS-assisted mitochondrial membrane potential determination, and Annexin-V/PI staining also reveal no cytotocicity. SWCNTs appear to interact with some tetrazolium salts such as MTT but not with others (such as WST-1, INT, XTT). This interference does not seem to affect the enzymatic reaction but lies rather in the insoluble nature of MTT-formazan. Our findings strongly suggest verifying cytotoxicity data with at least two or more independent test systems for this new class of materials (nanomaterials). Moreover, we intensely recommend standardizing nanotoxicological assays with regard to the material used: there is a clear need for reference materials. MTT-formazan crystals formed in the MTT reaction are lumped with nanotubes and offer a potential mechanism to guide bioremediation and clearance for SWCNTs from "contaminated" tissue. SWCNTs are good supporting materials for tissue growth, as attachment of focal adhesions and connections to the cytoskeleton suggest.

Stimulation of Phospholipase A(2) by Toxic Main Group Heavy Metals: Partly Dependent on G-proteins?

Organometals induce platelet aggregation and inorganic metal ions such as Cd(2+) or Pb(2+) sensitise human blood platelets to aggregating agents and this action is associated with the liberation of arachidonic acid and eicosanoid formation. The same mechanism is observed using human leukaemia cells (HL-60) when treated with MeHgCl or Et(3)PbCl. The fatty acid liberation within human platelets and HL-60 cells could only be inhibited with phospholipase A(2) inhibitors of different specificity.Preincubation of the cells with pertussis toxin reduces the activation induced by Et(3)PbCl to a great extent. The non-catalytic B subunit, that only mediates the binding of the toxin to the cell membranes, has no effect at all. When summarised, these results suggest that one possible mechanism for the stimulation of phospholipase A(2) by Et(3)PbCl functions via a G-protein dependent pathway.

Directed shift of fatty acids from phospholipids to triacylglycerols in HL-60 cells induced by nanomolar concentrations of triethyl lead chloride: involvement of a pertussis toxin-sensitive pathway.

Triethyl lead chloride (Et3PbCl) was found to induce a shift of fatty acids from membrane phospholipids to triacylglycerols in the human promyelocytic leukemia cell line HL-60. High concentrations of Et3PbCl (greater than 10 microM) caused a substantial liberation of [14C]arachidonic acid within 10 to 20 min in dimethyl sulfoxide-differentiated cells, comparable to the effect of the calcium ionophore A23187 (10 microM). Following liberation of arachidonic acid, its metabolites could be detected. Prolongation of the incubation time and reduction of Et3PbCl concentration resulted in a shift of fatty acids from phospholipids to triacylglycerols. Deacylation of phospholipids and reacylation into phospholipids and triacylglycerols were in equilibrium when the cells were treated with Et3PbCl at concentrations of less than or equal to 10 microM for 5 hr or less than or equal to 1 microM for 24 hr; no increase of free fatty acids could be observed, and the loss of fatty acids within the phospholipids was equivalent to the increase of fatty acid content within the triacylglycerols. Moreover, under these conditions, no loss of viability was seen after 24 hr, as compared with untreated differentiated cells. This concentration- and time-dependent effect of Et3PbCl might be due to a stimulated liberation of fatty acids via phospholipase A2, because this stimulation could be totally prevented by the phospholipase inhibitors quinacrine and p-bromophenacylbromide. Additionally, pretreatment of differentiated HL-60 cells with pertussis toxin resulted in a drastic reduction of [14C]arachidonic acid liberation when cells were stimulated with Et3PbCl. These results suggest the involvement of a pertussis toxin-sensitive GTP-binding protein and of a signal transduction mechanism during stimulated fatty acid release; release does not seem to be via a direct stimulation of phospholipase activity by the lead compound.

Inhibition by pesticides of prostaglandin formation in blood platelets.

Aggregation of human platelets was investigated after pre-incubation of platelet rich plasma with various pesticides of the carbamate type. Whereas N-methyl-carbamates (insecticides) inhibited the arachidonic acid induced aggregation, N-phenyl-carbamates (herbicides) had no effect. The influence of the different carbamates on aggregation coincided with their inhibition of thromboxane B2-formation. - DDE, a metabolite of the insecticide DDT, affected aggregation to a similar extent as N-methyl-carbamates. DDT, however, had no inhibitory activity.

The stimulation of arachidonic acid metabolism by organic lead and tin compounds in human HL-60 leukemia cells.

The liberation of fatty acids, above all arachidonic acid, in human blood cells is involved in numerous health problems or physiological mechanisms. The activity of cellular phospholipases leads to lipid metabolites such as eicosanoids, platelet activating factor, diacylglycerol, and inositolphosphates that are capable of mediating such pathological symptoms. The results presented here demonstrate that organic heavy metal compounds induce arachidonic acid liberation or its rearrangement within the lipid classes of HL-60 cells before a loss in viability can be detected. Four of the compounds tested, triethyllead (Et3Pb+), diethyllead (Et2Pb2+), trimethyllead (Me3Pb+), and trimethyltin (Me3Sn+), show a threshold concentration at which the viability of the cells is drastically decreased after 60 to 180 min incubation, whereas dibutyltin (But2Sn2+) induces a constant increase of cell death during the whole incubation time. In the case of threshold concentrations, the compounds stimulate a loss of arachidonic acid within the phospholipids and an increase of free fatty acid and eicosanoids before cell death could be detected. An important fact is the rearrangement of arachidonic acid within the lipid classes of these cells induced by metal concentrations that were not able to kill the cells within the given time. Primarily affected is phosphatidylethanolamine which loses arachidonic acid and, to a minor extent, phosphatidylcholine. Portions of the liberated fatty acid were then metabolized and/or shifted into neutral lipids and other phospholipids. All compounds tested show comparable effects, although at different concentrations. The toxicities of the compounds can be ordered as follows: Et3Pb+ greater than or equal to Et2Pb2+ greater than But2Sn2+ greater than or equal to Me3Pb+ much greater than Me3Sn+ greater than or equal to Pb2+. The cellular shape change following incubation with metal compounds is a further strong indication of a change in the membrane lipids. The cells lose their characteristic microvilli and/or blebs and become round without a loss in viability.

Mechanism of inhibition of cyclo-oxygenase in human blood platelets by carbamate insecticides.

Carbamates are a widely used class of insecticides and herbicides. They were tested for their ability to affect human blood platelet aggregation and arachidonic acid metabolism in platelets. (1) The herbicides of the carbamate type have no, or only little, influence up to a concentration of 100 microM; the carbamate insecticides, however, inhibit both aggregation and arachidonic acid metabolism in a dose- and time-dependent manner. (2) Carbaryl, the most effective compound, inhibits platelet aggregation and cyclo-oxygenase activity completely at 10 microM. The liberation of arachidonic acid from phospholipids and the lipoxygenase pathway are not affected, whereas the products of the cyclo-oxygenase pathway are drastically decreased. (3) By using [14C]carbaryl labelled in the carbamyl or in the ring moiety, it could be proved that the carbamyl residue binds covalently to platelet proteins. In contrast with acetylsalicylic acid, which acetylates only one protein, carbaryl carbamylates a multitude of platelet proteins. (4) One of the carbamylated proteins was found to be the platelet cyclo-oxygenase, indicating that carbaryl resembles in this respect acetylsalicylic acid, which is known to inhibit this enzyme specifically by acetylation.