Inhalation TTC values: A new integrative grouping approach considering structural, toxicological and mechanistic features.

The present publication describes an integrative grouping concept to derive threshold values for inhalation exposure. The classification scheme starts with differences in toxicological potency and develops criteria to group compounds into two potency classes, namely toxic (T-group) or low toxic (L-group). The TTC concept for inhalation exposure is based on the TTC RepDose data set, consisting of 296 organic compounds with 608 repeated-dose inhalation studies. Initially, 21 structural features (SFs) were identified as being characteristic for compounds of either high or low NOEC values (Schüürmann et al., 2016). In subsequent analyses these SF groups were further refined by taking into account structural homogeneity, type of toxicological effect observed, differences in absorption, metabolism and mechanism of action (MoA), to better define their structural and toxicological boundaries. Differentiation of a local or systemic mode of action did not improve the classification scheme. Finally, 28 groups were discriminated: 19 T-groups and 9 L-groups. Clearly distinct thresholds were derived for the T- and L-toxicity groups, being 2 × 10(-5) ppm (2 µg/person/day) and 0.05 ppm (4260 µg/person/day), respectively. The derived thresholds and the classification are compared to the initial mainly structure driven grouping (Schüürmann et al., 2016) and to the Cramer classification.

Mutational analyses of c-FLIPR, the only murine short FLIP isoform, reveal requirements for DISC recruitment.

Cellular FLICE-inhibitory protein (c-FLIP) proteins are known as potent inhibitors of death receptor-mediated apoptosis by interfering with caspase-8 activation at the death-inducing signaling complex (DISC). Among the three human isoforms, c-FLIP(long), c-FLIP(short) and c-FLIP(R), the latter isoform is poorly characterized. We report here the characterization of murine c-FLIP(R) and show that it is the only short c-FLIP isoform expressed in mice. By generating several mutants, we demonstrate that both death effector domains (DEDs) are required for DISC binding and the antiapoptotic function of c-FLIP(R). Surprisingly, the C-terminal tail is important for both protein stability and DISC recruitment. Three-dimensional modeling of c-FLIP(R) revealed a substantial similarity of the overall structures and potential interaction motifs with the viral FLIP MC159. We found, however, that c-FLIP(R) uses different structural motifs for its DISC recruitment. Whereas MC159 interferes with interaction and self-oligomerization of the DISC component FADD by its extensive hydrophilic surface, a narrow hydrophobic patch of c-FLIP(R) on the surface of DED2 is crucial for DISC association. Thus, despite the presence of similar tandem DEDs, viral and cellular FLIPs inhibit apoptosis by remarkably divergent mechanisms.

Disseminated toxoplasmosis in a patient with non-Hodgkin lymphoma.

Toxoplasmosis is a well-recognized opportunistic disease in HIV-infected individuals that is caused by the reactivation of a previous infection, primarily in the central nervous system, during profound immunodeficiency. Toxoplasmosis has been described more rarely in patients with cancer and chemotherapy. We report a case of a patient with a history of chemotherapy for non-Hodgkin lymphoma who developed pain and progressive paresthesia of the right arm 6 weeks after remission. Relapsing lymphoma was suspected, and steroid and radiation treatment were initiated, but the patient died 5 days later due to multiple organ failure. Autopsy revealed disseminated toxoplasmosis. This case illustrates that toxoplasmosis should be suspected in patients with neoplastic disease, especially lymphomas, who present with unexplained neurologic, pulmonary, or febrile symptoms during or after chemotherapy.

Exposure and ecotoxicological risk assessment of mixtures of top prescribed pharmaceuticals in Swedish freshwaters.

Surface water concentrations of 54 pharmaceuticals were predicted for seven major Swedish rivers and the Stockholm City area basins using the STREAM-EU model. These surface water concentrations were used to predict the ecotoxicological impact resulting from the exposure of aquatic organisms to this mixture of 54 pharmaceuticals. STREAM-EU model results indicated that <10 substances were present at median annual water concentrations greater than 10 ng/L with highest concentrations occurring mostly in the more densely populated area of the capital city, Stockholm. There was considerable spatial and temporal variability in the model predictions (1-3 orders of magnitude) due to natural variability (e.g. hydrology, temperature), variations in emissions and uncertainty sources. Local mixture ecotoxicological pressures based on acute EC50 data as well as on chronic NOEC data, expressed as multi-substance potentially affected fraction of species (msPAF), were quantified in 114 separate locations in the waterbodies. It was estimated that 5% of the exposed aquatic species would experience exposure at or above their acute EC50 concentrations (so-called acute hazardous concentration for 5% of species, or aHC5) at only 7% of the locations analyzed (8 out of 114 locations). For the evaluation based on chronic NOEC concentrations, the chronic HC5 (cHC5) is exceeded at 27% of the locations. The acute mixture toxic pressure was estimated to be predominantly caused by only three substances in all waterbodies: Furosemide, Tramadol and Ibuprofen. A similar evaluation of chronic toxic pressure evaluation logically demonstrates that more substances play a significant role in causing a higher chronic toxic pressure at more sites as compared to the acute toxic pressure evaluation. In addition to the three substances contributing most to acute effects, the chronic effects are predominantly caused by another five substances: paracetamol, diclofenac, ethinylestradiol, erythromycin and ciprofloxacin. This study provides regulatory authorities and companies responsible for water quality valuable information for targeting remediation measures and monitoring on a substance and location basis.

Proline-rich sequence recognition domains (PRD): ligands, function and inhibition.

Low-affinity protein-protein interactions (PPI) between domains of modular proteins and short, solvent-exposed peptide sequences within their binding partners play an essential role in intracellular signaling. An important class of PPIs comprises proline-rich motifs (PRM) that are specifically recognized by PRM-binding domains (PRD). Aromatic side chains of the PRDs define the binding pockets that often recognize individual proline residues, while flanking sequences mediate specificity. Several of these PRM:PRD interactions are associated with cellular malfunction, cancer or infectious diseases. Thus, the design of PRM:PRD inhibitors by using structure-based molecular modeling as well as peptidomimetic approaches and high-throughput screening strategies is of great pharmacological interest. In this chapter we describe the molecular basis of PRM:PRD interactions, highlight their functional role in certain cellular processes and give an overview of recent strategies of inhibitor design.

Model-predicted occurrence of multiple pharmaceuticals in Swedish surface waters and their flushing to the Baltic Sea.

An exposure assessment for multiple pharmaceuticals in Swedish surface waters was made using the STREAM-EU model. Results indicate that Metformin (27 ton/y), Paracetamol (6.9 ton/y) and Ibuprofen (2.33 ton/y) were the drugs with higher amounts reaching the Baltic Sea in 2011. 35 of the studied substances had more than 1 kg/y of predicted flush to the sea. Exposure potential given by the ratio amount of the drug exported to the sea/amount emitted to the environment was higher than 50% for 7 drugs (Piperacillin, Lorazepam, Metformin, Hydroxycarbamide, Hydrochlorothiazide, Furosemide and Cetirizine), implying that a high proportion of them will reach the sea, and below 10% for 27 drugs, implying high catchment attenuation. Exposure potentials were found to be dependent of persistency and hydrophobicity of the drugs. Chemicals with Log D > 2 had exposure potentials <10% regardless of their persistence. Chemicals with Log D  <  -2 had exposure potentials >35% with higher ratios typically achieved for longer half-lives. For Stockholm urban area, 17 of the 54 pharmaceuticals studied had calculated concentrations higher than 10 ng/L. Model agreement with monitored values had an r2 = 0.62 for predicted concentrations and an r2 = 0.95 for predicted disposed amounts to sea.

Variation in predicted internal concentrations in relation to PBPK model complexity for rainbow trout.

The present study is motivated by the increasing demand to consider internal partitioning into tissues instead of exposure concentrations for the environmental toxicity assessment. To this end, physiologically based pharmacokinetic (PBPK) models can be applied. We evaluated the variation in accuracy of PBPK model outcomes depending on tissue constituents modeled as sorptive phases and chemical distribution tendencies addressed by molecular descriptors. The model performance was examined using data from 150 experiments for 28 chemicals collected from US EPA databases. The simplest PBPK model is based on the "Kow-lipid content" approach as being traditional for environmental toxicology. The most elaborated one considers five biological sorptive phases (polar and non-polar lipids, water, albumin and the remaining proteins) and makes use of LSER (linear solvation energy relationship) parameters to describe the compound partitioning behavior. The "Kow-lipid content"-based PBPK model shows more than one order of magnitude difference in predicted and measured values for 37% of the studied exposure experiments while for the most elaborated model this happens only for 7%. It is shown that further improvements could be achieved by introducing corrections for metabolic biotransformation and compound transmission hindrance through a cellular membrane. The analysis of the interface distribution tendencies shows that polar tissue constituents, namely water, polar lipids and proteins, play an important role in the accumulation behavior of polar compounds with H-bond donating functional groups. For compounds without H-bond donating fragments preferable accumulation phases are storage lipids and water depending on compound polarity.

Evaluation of route-to-route extrapolation factors based on assessment of repeated dose toxicity studies compiled in the database RepDose®.

The majority of repeated dose toxicity studies are available for the oral route. For risk assessment, however, data are needed from the relevant exposure route, i.e. inhalation or dermal. Instead of conducting additional animal studies, route-to-route (R2R) extrapolation may be performed. To explore uncertainties associated with this approach, we derived extrapolation factors (EF) based on no/lowest effect levels (NOELs/LOELs) in the Fraunhofer RepDose® database. For R2R extrapolation oral-to-inhalation 246 study pairs on 110 chemicals were analyzed. Systemic effects triggered the LOELs in the underlying inhalation studies in 49.2%, local effects in 21.9% and both local and systemic effects in 30.9% of the data pairs. For systemic effects in inhalation studies an EF of 2.2 (95% confidence interval: 1.2-3.1) was derived, for local effects, the EF was 4.4 (95% confidence interval: 2.0-8.6), and the EF without distinguishing local or systemic effects (any EF) was 3.2 (95%, confidence interval: 1.7-5.0). Calculation with LOELs instead of NOELs, exposure duration and intrinsic properties of the chemical (toxicity or physicochemical properties) did not influence the EF significantly. For R2R extrapolation oral-to-dermal 46 study pairs on 28 chemicals were analyzed. An overall EF of 0.4 (95%, confidence interval: 0.2-0.9) was obtained. Here, we found a significant difference of EFs for low and high toxic chemicals. Overall, we conclude that reliable systemic NOELs/LOELs can be obtained for inhalation studies via R2R extrapolation from oral studies. Based on the data for any EF we propose to use an EF of 3, which covers also the uncertainty that unexpected local effects may occur in an inhalation study. For the dermal route, our dataset was too small to allow general conclusions, but the results so far do suggest that the current ECHA guidance is conservative when assuming that dermal absorption is as high as oral absorption.

Solution structures of the YAP65 WW domain and the variant L30 K in complex with the peptides GTPPPPYTVG, N-(n-octyl)-GPPPY and PLPPY and the application of peptide libraries reveal a minimal binding epitope.

The single mutation L30 K in the Hu-Yap65 WW domain increased the stability of the complex with the peptide GTPPPPYTVG (K(d)=40(+/-5) microM). Here we report the refined solution structure of this complex by NMR spectroscopy and further derived structure-activity relationships by using ligand peptide libraries with truncated sequences and a substitution analysis that yielded acetyl-PPPPY as the smallest high-affinity binding peptide (K(d)=60 microM). The structures of two new complexes with weaker binding ligands chosen based on these results (N-(n-octyl)-GPPPYNH(2) and Ac-PLPPY) comprising the wild-type WW domain of Hu-Yap65 were determined. Comparison of the structures of the three complexes were useful for identifying the molecular basis of high-affinity: hydrophobic and specific interactions between the side-chains of Y28 and W39 and P5' and P4', respectively, and hydrogen bonds between T37 (donnor) and P5' (acceptor) and between W39 (donnor) and T2' (acceptor) stabilize the complex.The structure of the complex L30 K Hu-Yap65 WW domain/GTPPPPYTVG is compared to the published crystal structure of the dystrophin WW domain bound to a segment of the beta-dystroglycan protein and to the solution structure of the first Nedd4 WW domain and its prolin-rich ligand, suggesting that WW sequences bind proline-rich peptides in an evolutionary conserved fashion. The position equivalent to T22 in the Hu-Yap65 WW domain sequence is seen as responsible for differentiation in the binding mode among the WW domains of group I.

Residues within transmembrane helices 2 and 5 of the human gonadotropin-releasing hormone receptor contribute to agonist and antagonist binding.

To understand the ligand binding properties of the human GnRH receptor (hGnRH-R), 24 site-specific mutants within transmembrane helices (TMH) 1, 2, and 5 and the extracellular loop 2 (E2) were generated. These mutants were analyzed by using a functional reporter gene assay, monitoring receptor signaling via adenylate cyclase to a cAMP-responsive element fused to Photinus pyralis luciferase. The functional behavior of 14 receptor mutants, capable of G-protein coupling and signaling, was studied in detail with different well described agonistic and antagonistic peptide ligands. Furthermore, the binding constants were determined in displacement binding experiments with the antagonist [125I]Cetrorelix. The substitution of residues K36, Q204, W205, H207, Q208, F20, F213, F216, and S217 for alanine had no or only a marginal effect on ligand binding and signaling. In contrast, substitution of N87, Eg9, D9, R179, W206, Y211, F214, and T215 for alanine resulted in receptor proteins neither capable of ligand binding nor signal transduction. Within those mutants affecting ligand binding and signaling to various degrees, W101A, N102A, and N212Q differentiate between agonists and antagonists. Thus, in addition to N102 already described, the residues W101 in TMH2 and N212 in TMH5 are important for the architecture of the ligand-binding pocket. Based on the experimental data, three-dimensional models for binding of the superagonist D-Trp6-GnRH (Triptorelin) and the antagonist Cetrorelix to the hGnRH-R are proposed. Both decapeptidic ligands are bound to the receptor in a bent conformation with distinct interactions within the binding pocket formed by all TMHs, E2, and E3. The antagonist Cetrorelix with bulky hydrophobic N-terminal amino acids interacts with quite different receptor residues, a hint at the failure to induce an active, G protein-coupling receptor conformation.

White paper on the promotion of an integrated risk assessment concept in European regulatory frameworks for chemicals.

The vision of a sustainable and safe use of chemicals to protect human health, preserve the environment and maintain the ecosystem requires innovative and more holistic approaches to risk assessment (RA) in order to better inform decision making. Integrated risk assessment (IRA) has been proposed as a solution to current scientific, societal and policy needs. It is defined as the mutual exploitation of environmental risk assessment (ERA) for human health risk assessment (HHRA) and vice versa in order to coherently and more efficiently characterize an overall risk to humans and the environment for better informing the risk analysis process. Extrapolating between species which are relevant for HHRA and ERA requires a detailed understanding of pathways of toxicity/modes of action (MoA) for the various toxicological endpoints. Significant scientific advances, changes in chemical legislation, and increasing environmental consciousness have created a favourable scientific and regulatory environment to develop and promote the concept and vision of IRA. An initial proof of concept is needed to foster the incorporation of IRA approaches into different chemical sectorial regulations and demonstrate their reliability for regulatory purposes. More familiarity and confidence with IRA will ultimately contribute to an overall reduction in in vivo toxicity testing requirements. However, significant progress will only be made if long-term support for MoA-related research is secured. In the short term, further exchange and harmonization of RA terminology, models and methodologies across chemical categories and regulatory agencies will support these efforts. Since societal values, public perceptions and cultural factors are of increasing importance for the acceptance of risk analysis and successful implementation of risk mitigation measures, the integration of socio-economic analysis and socio-behavioural considerations into the risk analysis process may help to produce a more effective risk evaluation and consideration of the risks and benefits associated with the use of chemicals.

Proper receptor signalling in a mutant catfish gonadotropin-releasing hormone receptor lacking the highly conserved Asp(90) residue.

The negatively charged side chain of an Asp residue in transmembrane domain 2 is likely to play an important role in receptor signalling since it is highly conserved in the whole family of G protein-coupled receptors, except in mammalian gonadotropin-releasing hormone (GnRH) receptors. In this paper we show that the conserved Asp(90) of the catfish GnRH receptor can be substituted by a neutral Asn(90) without abolishing receptor signalling if another negatively charged Glu(93) is introduced in a proximal region of the receptor interior, thereby mimicking the Glu(90)-Lys(121) salt bridge of mammalian GnRH receptors.

Histaprodifens: synthesis, pharmacological in vitro evaluation, and molecular modeling of a new class of highly active and selective histamine H(1)-receptor agonists.

A new class of histamine analogues characterized by a 3, 3-diphenylpropyl substituent at the 2-position of the imidazole nucleus has been prepared outgoing from 4,4-diphenylbutyronitrile (4b) via cyclization of the corresponding methyl imidate 5b with 2-oxo-4-phthalimido-1-butyl acetate or 2-oxo-1,4-butandiol in liquid ammonia, followed by standard reactions. The title compounds displayed partial agonism on contractile H(1) receptors of the guinea-pig ileum and endothelium-denuded aorta, respectively, except 10 (histaprodifen; 2-[2-(3, 3-diphenylpropyl)-1H-imidazol-4-yl]ethanamine) which was a full agonist in the ileum assay. While 10 was equipotent with histamine (1), methylhistaprodifen (13) and dimethylhistaprodifen (14) exceeded the functional potency of 1 by a factor of 3-5 (13) and 2-3 (14). Compounds 10 and 13-17 relaxed precontracted rat aortic rings (intact endothelium) with relative potencies of 3.3- up to 28-fold (compared with 1), displaying partial agonism as well. Agonist effects were sensitive to blockade by the selective H(1)-receptor antagonist mepyramine (pA(2) approximately 9 (guinea-pig) and pA(2) approximately 8 (rat aorta)). The affinity of 10 and 13-17 for guinea-pig H(1) receptors increased 20- to 100-fold compared with 1. Two lower homologues of 10 were weak partial H(1)-receptor agonists while two higher homologues of 10 were silent antagonists endowed with micromolar affinity for rat and guinea-pig H(1) receptors. In functional selectivity experiments, 10, 13, and 14 did not stimulate H(2), H(3), and several other neurotransmitter receptors. They displayed only low to moderate affinity for these sites (pA(2) < 6). For a better understanding of structure-activity relationships, the interaction of 1 and 10, 13 and 14 within the transmembrane (TM) domains of the human histamine H(1) receptor were studied using molecular dynamics simulations. Remarkable differences were found between the binding modes of 10, 13, and 14 and that of 1. The imidazole ring of 10, 13, and 14 was placed 'upside down' compared with 1, making the interaction of the N(pi)-atom with Tyr431 possible. This new orientation was mainly caused by the space filling substitution at the 2-position of the imidazole ring and influenced the location of the protonated N(alpha)-atom which was positioned more between TM III and TM VI. This orientation can explain both the increased relative potency and the maximum effect of 10, 13, and 14 compared with 1. Compound 13 (methylhistaprodifen; N(alpha)-methyl-2-[2-(3, 3-diphenylpropyl)-1H-imidazol-4-yl]ethanamine) is the most potent histamine H(1)-receptor agonist reported so far in the literature and may become a valuable tool for the study of physiological and pathophysiological H(1)-receptor-mediated effects.

The acoustic behaviour of the bushcricket Tettigonia cantans II. Transmission of airborne-sound and vibration signals in the biotope.

The airborne-sound and the vibratory signals produced by stridulating Tettigonia cantans males, and the transmission of these signals in the natural biotope were investigated. The song of T. cantans is composed of repeated uniform syllables with a rate of ca. 30/sec. Intensity approaches 100 dB SPL, 10 cm away from the animal. The spectrum shows three dominant frequency ranges around 8, 16 and 32 kHz. Airborne transmission of the song in such vegetation layers as are found in the biotopes of T. cantans shows an excess attenuation which increases with frequency. The relative intensities of the frequency components of the song vary as a result of the kind of vegetation, the positions of emitter and receiver, and the separation distance. These relative differences in intensity may be useful during the phonotactic approach to conspecific partners, providing a measure of the distance from the sound source. Stridulating males also produce vibratory signals in the plants they sit on. The spectrum of these signals includes frequencies up to 8 kHz, the first dominant frequency of the song: low frequency components are induced in the plants via the legs and abdomen of the animal. The vibratory signals are transmitted mainly in the form of bending waves. Near the animal, amplitude modulation corresponds to that of the song. At greater distances, reflections and frequency-dependent propagation velocities, cause distortions of this time pattern. Transmission depends greatly on the mechanical properties of the particular plant, attenuation values of 20-50 dB/m being found. Nevertheless, in most cases, vibratory signals may be perceived up to 1.5 - 2 metres away from a stridulating male.

The acoustic behaviour of the bushcricket Tettigonia cantans III. Coprocessing of auditory and vibratory information in the central nervous system.

All the acoustic units in the ventral-nerve cord respond to both sound and vibration. Most of them show improved coding abilities when stimulated simultaneously with conspecific songs and vibration signals. This is also true for habituating neurons. Stridulating tettigoniids produce both airborne sound and substrate borne vibration and their simultaneous processing in the central nervous system may therefore lead to a better localization of a nearly sound source in the biotope.

The responses of ventral cord neurons of Decticus verrucivorus (L) to sound and vibration stimuli.

The real time analysis of the song of D. verrucivorus recorded in the sun and shade shows that changes occur predominantly in the time parameters and not in the frequency content. Single unit recordings in the ventral nerve cord of D. verrucivorus show that all the acoustic units respond to both sound and vibration. However, on the basis of their response characteristics they may be classified as vibration (V), vibration and sound (VS) and sound (S) neurons. The responses of some of the units depend upon the degree of habituation. Single parameter processing was not observed; the characteristic frequencies of these units range across the whole of the frequency band investigated, and distinct intensity response fields were observed. Some of the V neurons were more sensitive than the receptors, and some units responded well to the species song when both sound and vibration were presented simultaneously. The source of the vibratory input is shown to be predominantly from the ipsilateral foreleg. Many of the units run together in an 'acoustic bundle'; some run through fibres, passing from the posterior thoracic ganglia to the cervical connectives. In many cases the primary fibres projecting to these central units can be predicted from their response characteristics. An hypothesis of the mechanisms underlying conspecific song recognition at the ventral cord level is presented.

A simple method to obtain an approximate solution of the Free-Wilson model.

A method is proposed which yields an approximate solution of the Free-Wilson model very rapidly without using a computer. Although the resulting group contributions are numerically somewhat different from the exact Free-Wilson solution they correctly reflect the relative order of the substituents with respect to their effect on biological activity within each position as well as the relative importance of different positions. Thus, the approximate results can well be used to select the most promising candidates for further synthesis and testing.

Chemonucleolysis. Development, experience, perspectives.

Concerning the so-called minimal invasive procedures currently available for the treatment of lumbar disc prolapse (percutaneous endoscopic discectomy, APLD, laser decompression, chemonucleolysis), the intradiscal application of Chymopapain represents the method with the longest period of clinical use and experience. Long-term studies have shown good clinical results. When considering of the indication and the few contraindications - particularly allergic diathesis - chemonucleolysis provides a low-risk, efficient, minimally invasive therapy that closes the therapeutic window between conservative and open surgical treatment.

[Papilla carcinoma and adenoma--diagnosis and therapy]. / Das Papillenkarzinom und -adenom--Diagnostik und Therapie.

26 peripapillary adenomas and 64 papillary carcinomas are the background for a discussion of the adenoma-carcinoma-sequence in these tumours. An early diagnosis of the adenomas means in this context secondary prophylaxis of papillary carcinomas. Nowadays, the greatest difficulties consist in the differentiation of adenomyomatosis-like processes of the papilla and in the certainty that an adenoma has not changed to a carcinoma. In patients who cannot tolerate a greater surgical procedure the endoscopic papillotomy, possibly in combination with a prosthesis, is an appropriate measure.