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bioRxiv2018

Role of the high-affinity leukotriene B4 receptor signaling in fibrosis after unilateral ureteral obstruction in mice

Leukotriene B4 (LTB4) is a lipid mediator that acts as a potent chemoattractant for inflammatory leukocytes. Kidney fibrosis is caused by migrating inflammatory cells and kidney-resident cells. Here, we examined the role of the high-affinity LTB4 receptor BLT1 during development of kidney fibrosis in wild-type (WT) mice and BLT1 knockout (BLT1-/-) mice with unilateral ureteral obstruction (UUO). We found elevated expression of 5-lipoxygenase (5-LOX), which generates LTB4, in the renal tubules of WT and BLT1-/- UUO mice. Accumulation of immunoreactive type I collagen in UUO kidneys of WT mice increased over time; however, the increase was less prominent in BLT1-/- mice. Accumulation of S100A4-positive fibroblasts also increased temporally in WT UUO kidneys, but was again less pronounced in those of BLT1-/- mice. The same was true of mRNA encoding transforming growth factor-{beta} (TGF)-{beta} and fibroblast growth factor (FGF)-2. Finally, accumulation of F4/80-positive macrophages, which secrete TGF-{beta}, also increased temporally in WT UUO and BLT1-/- kidneys, but to a lesser extent in the latter. Following LTB4 stimulation in vitro, macrophages showed increased expression of mRNA encoding TGF-{beta}/FGF-2 and Col1a1, whereas L929 fibroblasts showed increased expression of mRNA encoding smooth muscle actin (SMA). Bone marrow (BM) transplantation studies revealed that the area positive for type I collagen was significantly smaller in BLT1-/--BM[->]WT UUO kidneys than in WT-BM[->]WT kidneys. Thus, LTB4-BLT1 signaling plays a critical role in fibrosis in UUO kidneys by increasing accumulation of macrophages and fibroblasts. Therefore, blocking BLT1 may prevent renal fibrosis.

pharmacology and toxicology↗

bioRxiv2018

DNMT inhibitors increase methylation of CpGs related to p53 pathway genes in colon, lymphoma, bladder, ovarian, and breast cancer cells

BackgroundDNA methyltransferase inhibitors (DNMTi) decitabine and azacytidine are approved therapies for acute myeloid leukemia and myelodysplastic syndrome. Identification of CpGs violating demethylaion due to DNMTi treatment may help to understand their resistance mechanisms.\n\nMaterials and MethodsTo identify such CpGs, we analysed publicly available 450K methylation data of multiple cancer type cell lines.\n\nResultsWe identified 637 CpGs corresponding to genes enriched for p53 and olfactory receptor pathways with a transient increase in methylation (median {Delta}{beta} = 0.12) after decitabine treatment in HCT116 cells. Azacytidine treatment also increased methylation of identified CpGs in 9 colon, 9 ovarian, 3 breast, and 1 lymphoma cancer cell lines.\n\nConclusionDNMTi treatment increases methylation of subset of CpGs in cancer genome.

pharmacology and toxicology↗

bioRxiv2018

Farmers perceptions on the causes of cassava root bitterness in konzo-affected areas of Mtwara Region, Tanzania

The agronomic factors influencing increased cyanogenic glucoside levels, particularly in bitter cassava varieties during periods without water stress, in areas where konzo (a cassava cyanide related paralytic disorder also called spastic paraparesis) persists, are hardly known. However, through their assessment of bitter taste, farmers may have noticed factors unrelated to water stress and variety type that additionally influence cassava root cyanogenic glucoside content in these environments. Bitterness in cassava is usually associated with high cyanogenic glucoside levels. Using some konzo-affected areas in Mtwara region of Tanzania as a case study, a survey was thus carried out to identify the factors, hitherto overlooked, that may additionally influence cyanogenic glucoside levels in cassava. A total of 120 farmers were interviewed. A number of factors unrelated to water stress and variety type that could be additionally influencing cyanogenic glucoside production in cassava plants were mentioned. The mentioned factors included nutrient poor soils, plant age at harvest, weeds, piecemeal harvesting, and branch pruning; the factors, respectively, constituted 14.2%, 7.5%, 0.8%, 0.8%, and 0.8% of the total responses given. The revealed factors constitute permanent environmental characteristics and commonly used crop management practices by farmers living in konzo-prone Mtwara region of Tanzania that could be additionally resulting in high cyanogenic glucoside levels in cassava, regardless of water stress.

pharmacology and toxicology↗

bioRxiv2018

Positive human health effects of sea spray aerosols: molecular evidence from exposed lung cell lines.

Sea spray aerosols (SSAs) have profound effects on climate and ecosystems. Furthermore, the presence of microbiota and biogenic molecules, produced by among others marine phytoplankton, in SSAs could lead to potential human health effects. Yet the exposure and effects of SSAs on human health remain poorly studied. Here, we exposed human epithelial lung cells to different concentrations of extracts of a natural sea spray aerosol (SSA), a laboratory-generated SSA, the marine algal toxin homoyessotoxin and a chemical mTOR inhibitor. The mTOR inhibitor was included as it has been hypothesized that natural SSAs may influence the mTOR cell signaling pathway. We observed significant effects on the mTOR pathway and PCSK9 in all exposures. Based on these expression patterns, a clear dose response relationship was observed. Our results indicate a potential for positive health effects when lung cells are exposed to environmentally relevant concentrations of natural SSAs, whereas potential negative effects were observed at high levels of the laboratory SSA and the marine algal toxin. Overall, these results provide a substantial molecular evidence base for potential positive health effects of SSAs at environmentally relevant concentrations through the mTOR pathway. The results provided here suggest that SSAs contain biomolecules with significant pharmaceutical potential in targeting PCSK9.

pharmacology and toxicology↗

bioRxiv2018

Dimethylarsenic acid (DMA) accumulation positively correlates with realgar-induced subchronic toxicity in rats

The toxicity of realgar depends largely on different arsenic species accumulation and distribution in the body. Here, after continuous oral administration of different doses of realgar for 90 days and subsequent 60-day withdrawal period, clinical observations, food consumption, body weights, blood biochemistry, hematology, and histomorphological examination of rats were performed. Realgar 40mg{middle dot}kg-1{middle dot}d-1 and 170 mg{middle dot}kg-1{middle dot}d-1 of realgar (which is equivalent to 40-fold and 100-fold the maximum clinical dose, respectively) can cause toxicity in rats, including degreased body weight, peripheral blood neutrality abnormal ratio of granulocytes and lymphocytes, hypercoagulability of the blood, liver and kidney tissue damage, liver and kidney may be the main toxic target organs of realgar. The no observed adverse effect level (NOAEL) dose is 10 mg{middle dot}kg-1. At the same time, the content and distribution of arsenic species in tissues were determined. The content of total arsenic (tAs) and Dimethylarsenic acid (DMA) in the tissues of the realgar group was significantly higher than those of the control group. After 60 days of discontinuation, the DMA content in the realgar group decreased, but it was still higher than that in the control group, and liver and kidney damage occurred during the administration period basically returned to normal. Therefore, the authors speculated that when the DMA content in the tissue exceeds a certain range, liver and kidney toxicity will be induced. However, when the DMA content is lower than the above threshold after drug withdrawal, the liver and kidney lesions can return to normal.

pharmacology and toxicology↗

bioRxiv2018

A serial dilution method for assessment of microplastic toxicity in suspension

The occurrence of microplastic (MP) in the environment is of global concern. MP risk assessment, however, is currently hampered by lacking ecotoxicological methods due to conceptual and practical problems with particle exposure. Natural particles of similar size as MP, e.g., clay and cellulose, occur abundantly in the environment. For MP risk assessment and regulation it must be established whether the addition of MP to these particles represents an additional hazard. We present a novel approach employing a serial dilution of MP and reference particles, in mixtures, which allows the differentiation of MP effects from other particulates. We demonstrate the applicability of the method using an immobilisation test with Daphnia magna exposed to polyethylene terephthalate (MP) and kaolin clay (reference material). In the concentration range of 0.1 to 10000 mg L-1 of total suspended solids (TSS), with MP contributing 0-100 %, the LC50 values for MP-kaolin mixtures were significantly lower compared to the pure kaolin suspension. MP particles were thus more harmful to daphnids than the reference material. The estimated threshold for %MP contribution above which higher mortality was observed was 1 % MP at 36 mg TSS L-1. This approach has a potential for standardisation of MP ecotoxicological testing as well as other particulate material of anthropogenic origin.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC=\"FIGDIR/small/401331_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (40K):\norg.highwire.dtl.DTLVardef@13e1153org.highwire.dtl.DTLVardef@1e37652org.highwire.dtl.DTLVardef@6d3efborg.highwire.dtl.DTLVardef@420d7a_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2018

Aquaporin-4 facilitator TGN-073 demonstrates novel analgesic activity

During pre-clinical development, we tested the novel, internally developed AQP-4 facilitator TGN-073 for its effect in a rodent pain model. Therein, TGN-073 was found to exert a strong analgesic effect. Following a single 200 mg/kg (i.p.) administration of TGN-073, a virtually complete block in the acetic acid writhing test was observed. Subsequent in vitro tests demonstrated that TGN-073 had no binding affinity for the -opioid or NK-1 receptors. Accordingly, we suspect TGN-073 or other AQP-4 facilitators may be developed into potent non-opioid analgesic agents. Given the potential significance of this discovery, we feel it should be openly shared with the scientific community.

pharmacology and toxicology↗

bioRxiv2018

Incorporation of doxorubicin in different polymer nanoparticles and their anti-cancer activity

Nanoparticles are under investigation as carrier systems for anti-cancer drugs. They have been shown to accumulate in cancer tissues through the enhanced permeability and retention (EPR) effect, to reduce toxicity to non-target tissues, and to protect drugs from preliminary inactivation. However, nanoparticle preparations are not commonly compared for their anti-cancer effects at the cellular level. Here, we prepared doxorubicin-loaded nanoparticles based on poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), and PEGylated PLGA (PLGA-PEG) by solvent displacement and emulsion diffusion approaches. The resulting nanoparticles covered a size range between 73 and 246 nm. PLGA-PEG nanoparticle preparation by solvent displacement resulted in the smallest nanoparticles. In PLGA nanoparticles, the drug load could be optimised using solvent displacement at pH7 reaching 53 {micro}g doxorubicin/mg nanoparticle. In addition, these PLGA nanoparticles displayed sustained doxorubicin release kinetics compared to the more burst-like kinetics of the other preparations. In neuroblastoma cells, doxorubicin-loaded PLGA-PEG nanoparticles (presumably due to their small size) and PLGA nanoparticles prepared by solvent displacement at pH7 (presumably due to their high drug load and superior drug release kinetics) exerted the strongest anti-cancer effects. In conclusion, doxorubicin-loaded nanoparticles made by different methods from different materials displayed substantial discrepancies in their anti-cancer activity at the cellular level. Optimised preparation methods resulted in PLGA nanoparticles characterised by increased drug load, controlled drug release, and high anti-cancer efficacy. The design of drug-loaded nanoparticles with optimised anti-cancer activity at the cellular level is an important step in the development of improved nanoparticle preparations for anti-cancer therapy.

pharmacology and toxicology↗

bioRxiv2018

TP53 mutations and drug sensitivity in acute myeloid leukaemia cells with acquired MDM2 inhibitor resistance

BackgroundMDM2 inhibitors are under investigation for the treatment of acute myeloid leukaemia (AML) patients in phase III clinical trials. To study resistance formation to MDM2 inhibitors in AML cells, we here established 45 sub-lines of the AML TP53 wild-type cell lines MV4-11 (15 sub-lines), OCI-AML-2 (10 sub-lines), OCI-AML-3 (12 sub-lines), and SIG-M5 (8 sub-lines) with resistance to the MDM2 inhibitor nutlin-3.\n\nMethods: Nutlin-3-resistant sub-lines were established by continuous exposure to stepwise increasing drug concentrations. The TP53 status was determined by next generation sequencing, cell viability was measured by MTT assay, and p53 was depleted using lentiviral vectors encoding shRNA.\n\nResultsAll MV4-11 sub-lines harboured the same R248W mutation and all OCI-AML-2 sub-lines the same Y220C mutation, indicating the selection of pre-existing TP53-mutant subpopulations. In concordance, rare alleles harbouring the respective mutations could be detected in the parental MV4-11 and OCI-AML-2 cell lines. The OCI-AML-3 and SIG-M5 sub-lines were characterised by varying TP53 mutations or wild type TP53, indicating the induction of de novo TP53 mutations. Doxorubicin, etoposide, gemcitabine, cytarabine, and fludarabine resistance profiles revealed a noticeable heterogeneity among the sub-lines even of the same parental cell lines. Loss-of-p53 function was not generally associated with decreased sensitivity to cytotoxic drugs.\n\nConclusionWe introduce a substantial set of models of acquired MDM2 inhibitor resistance in AML. MDM2 inhibitors select, in dependence on the nature of a given AML cell population, pre-existing TP53-mutant subpopulations or induce de novo TP53 mutations. Although loss-of-p53 function has been associated with chemoresistance in AML, nutlin-3-adapted sub-lines displayed in the majority of experiments similar or increased drug sensitivity compared to the respective parental cells. Hence, chemotherapy may remain an option for AML patients after MDM2 inhibitor therapy failure. Even sub-lines of the same parental cancer cell line displayed considerable heterogeneity in their response to other anti-cancer drugs, indicating the need for the detailed understanding and monitoring of the evolutionary processes in cancer cell populations in response to therapy as part of future individualised treatment protocols.

pharmacology and toxicology↗

bioRxiv2018

Primary Target Prediction of Bioactive Molecules from Chemical Structure

There are various tools for computational target prediction of bioactive molecules from a chemical structure in a machine-readable material but these tools cant distinguish a primary target from other targets. Also, due to the complex nature of bioactive molecules, there has not been a method to predict a target and or a primary target from a chemical structure in a non-digital material (for example printed or hand-written documents) yet. In this study, an attempt to simplify primary target prediction from a chemical structure was resulted in developing an innovative method based on the minimum structure which can be used in both formats of non-digital and machine-readable materials. A minimum structure does not represent a real molecule or a real association of functional groups, but is a part of a molecular structure which is necessary to ensure the primary target prediction of bioactive molecules. Structurally related bioactive molecules with the minimum structure were considered as neighbor molecules of the query molecule. The known primary target of the neighbor molecule is used as a reference for predicting the primary target of the neighbor molecule with an unknown primary target. In results, we confirmed the usefulness of our proposed method for primary target prediction in 548 drugs and pesticides involved in four primary targets by eight minimum structures.

pharmacology and toxicology↗

bioRxiv2018

The Utilization of Pharmacophore-based 3D QSAR Modeling and Virtual Screening in Safety Profiling: a Case Study to Identify Antagonistic Activities against Adenosince Receptor, A2aR, using 1,897 known drugs

ABSTRACTSafety pharmacology screening against a wide range of unintended vital targets using in vitro assays is crucial to understand off-target interactions with drug candidates. With the increasing demand for in vitro assays, ligand-and structure-based virtual screening approaches have been evaluated for potential utilization in safety profiling. Although ligand based approaches have been actively applied in retrospective analysis or prospectively within well-defined chemical space during the early discovery stage (i.e., HTS screening and lead optimization), virtual screening is rarely implemented in later stage of drug discovery (i.e., safety). Here we present a case study to evaluate ligand-based 3D QSAR models built based on in vitro antagonistic activity data against adenosine receptor 2A (A2aR). The resulting models, obtained from 268 chemically diverse compounds, were used to test a set of 1,897 chemically distinct drugs, simulating the real-world challenge of safety screening when presented with novel chemistry and a limited training set. Due to the unique requirements of safety screening versus discovery screening, the limitations of 3D QSAR methods (i.e., chemotypes, dependence on large training set, and prone to false positives) are less critical than early discovery screen. We demonstrated that 3D QSAR modelling can be effectively applied in safety assessment prior to in vitro assays, even with chemotypes that are drastically different from training compounds. It is also worth noting that our model is able to adequately make the mechanistic distinction between agonists and antagonists, which is important to inform subsequent in vivo studies. Overall, we present an in-depth analysis of the appropriate utilization and interpretation of pharmacophore-based 3D QSAR models for safety screening.

pharmacology and toxicology↗

bioRxiv2018

First Whole-Body Three-Dimensional Tomographic Imaging of Alpha Particle Emitting Radium-223

Objective: Dose optimization and pharmacokinetic evaluation of alpha emitting Radium-223 dichloride (223RaCl2) by planar gamma camera or single photon emission computed tomographic (SPECT) imaging are hampered by the low photon abundance and injection activities. Here, we demonstrate SPECT of 223Ra using phantoms and small animal in vivo models. Methods: Line phantoms and mice bearing 223Ra were imaged using a next generation dedicated small animal SPECT by detecting the low energy photon emissions from 223Ra. Localization of the therapeutic agent was verified by whole body and whole limb autoradiography and its effect determined by immunofluorescence. Results: A state-of-the-art commercial small animal SPECT system equipped with a highly sensitive collimator enables collection of sufficient counts for three-dimensional reconstruction. Line sources of 223Ra in both air and in a water scattering phantom gave linear response functions with provide full-width-at-half-maximum of 1.45 mm. Early and late phase imaging of the pharmacokinetics of the radiopharmaceutical were captured. Uptake at sites of active bone remodeling were correlated with DNA damage from the alpha particle emissions. Conclusions: This work demonstrates the capability to noninvasively define the distribution of 223Ra, a recently approved alpha emitting radionuclide. This approach allows quantitative assessment of 223Ra distribution and may provide radiation dose optimization strategies to improve therapeutic response and ultimately to enable personalized treatment planning.

pharmacology and toxicology↗

bioRxiv2018

Esterase Activity is Affected by Genetics, Age, Insecticide Exposure, and Viral Infection in the Honey Bee, Apis mellifera

Non-target impacts of insecticide treatments are a major public and environmental concern, particularly in contemporary beekeeping. Therefore, it is important to understand the physiological mechanisms contributing to insecticide sensitivity in honey bees. In the present studies, we sought to evaluate the role of esterases as the source of variation in insecticide sensitivity. To address this question, the following objectives were completed: 1) Evaluated esterase activity among honey bee stocks, 2) Assessed the correlation of esterase activity with changes in insecticide sensitivity with honey bee age, 3) Established if esterases can be used as a biomarker of insecticide exposure, and 4) Examined the effects of Varroa mite infestation and viral infection on esterase activity.\n\nResults indicated that honey bees have a dynamic esterase capacity that is influenced by genetic stock and age. However, there was no consistent connection of esterase activity with insecticide sensitivity across genetic stocks or with age, suggests other factors are more critical for determining insecticide sensitivity. The trend of increased esterase activity with age in honey bees suggests this physiological transition is consistent with enhanced metabolic rate with age. The esterase inhibition with naled but not phenothrin or clothianidin indicates that reduced esterase activity levels may only be reliable for sublethal doses of organophosphate insecticides. The observation that viral infection, but not Varroa mite infestation, reduced esterase activity shows viruses have extensive physiological impacts. Taken together, these data suggest that honey bee esterase activity toward these model substrates may not correlate well with insecticide sensitivity. Future studies include identification of esterase substrates and inhibitors that are better surrogates of insecticide detoxification in honey bees as well as investigation on the usefulness of esterase activity as a biomarker of pesticide exposure, and viral infection.

pharmacology and toxicology↗

bioRxiv2018

T vector velocity: A new ECG biomarker for identifying drug effects on cardiac ventricular repolarization

We present a new family TrX of ECG biomarkers based on the T vector velocity (TVV) for assessing drug effects on ventricular repolarization. Assuming a link between the TVV and the instantaneous change of the cellular action potentials, drugs accelerating repolarization by blocking inward (depolarizing) ion currents cause a relative increase of the TVV, while drugs delaying repolarization by blocking outward ion currents cause a relative decrease of the TVV.\n\nEvaluating the published data from two FDA funded studies, the TrX effect profiles indicate increasingly delayed electrical activity over the entire repolarization process for drugs solely reducing outward potassium current (dofetilide, moxifloxacin). For drugs eliciting block of the inward sodium or calcium currents (mexiletine, lidocaine), the TrX effect profiles were consistent with accelerated electrical activity in the initial repolarization phase. For multichannel blocking drugs (ranolazine) or drug combinations blocking multiple ion currents (dofetilide + mexiletine, dofetilide + lidocaine), the overall TrX effect profiles indicate a superposition of the individual TrX effect profiles.\n\nThe parameter Tr40c allows separating pure potassium channel blocking drugs from multichannel blocking drugs with an area under the ROC curve (AUC) value of 0.90, CI = [0.88 to 0.92]. This is significantly larger than the performance of J-Tpeakc (0.81, CI = [0.78 to 0.84]) using the published data from the second FDA study. Further performance improvement was achieved by combining the ten parameters Tr10c to Tr100c in a logistic regression model, resulting in an AUC value of 0.94.\n\nThe TVV based approach substantially improves assessment of drug effects on cardiac repolarization, providing a plausible and improved mechanistic link between drug effects on ionic currents and overall ventricular repolarization reflected in the body surface ECG. TVV may contribute to a better assessment of the proarrhythmic risk of drugs beyond QTc prolongation and JTpeakc.

pharmacology and toxicology↗

bioRxiv2018

Effects of Nicotine and THC Vapor Inhalation Administered by An Electronic Nicotine Delivery System (ENDS) in Male Rats

BackgroundElectronic nicotine delivery systems (ENDS, e-cigarettes) are increasingly used for the self-administration of nicotine by various human populations, including previously nonsmoking adolescents. Studies in preclinical models are necessary to evaluate health impacts of ENDS including the development of nicotine addiction, effects of ENDS vehicles, flavorants and co-administered psychoactive substances such as {triangleup}9-tetrahydrocannabinol (THC). This study was conducted to validate a rat model useful for the study of nicotine effects delivered by inhalation of vapor created by ENDS. MethodsMale Sprague-Dawley rats (N=8) were prepared with radiotelemetry devices for the reporting of temperature and activity. Experiments subjected rats to inhalation of vapor generated by an electronic nicotine delivery system (ENDS) adapted for rodents. Inhalation conditions included vapor generated by the propylene glycol (PG) vehicle, Nicotine (1, 10, 30 mg/mL in the PG) and THC (12.5, 25 mg/mL). ResultsNicotine inhalation increased spontaneous locomotion and decreased body temperature of rats. Pretreatment with the nicotinic cholinergic receptor antagonist mecamylamine (2 mg/kg, i.p.) prevented stimulant effects of nicotine vapor inhalation and attenuated the hypothermic response. Combined inhalation of nicotine and THC resulted in apparently independent effects which were either additive (hypothermia) or opposed (activity). ConclusionsThese studies provide evidence that ENDS delivery of nicotine via inhalation results in nicotine-typical effects on spontaneous locomotion and thermoregulation in male rats. Effects were blocked by a nicotinic antagonist, demonstrating mechanistic specificity. This system will therefore support additional studies of the contribution of atomizer/wick design, vehicle constituents and/or flavorants to the effects of nicotine administered by ENDS.

pharmacology and toxicology↗

bioRxiv2018

The in vitro toxicity of nitrile and epithionitrile derivatives of glucosinolates from rutabaga in human and bovine liver cells.

Previous evidence suggests that select nitrile and epithionitrile derivatives of glucosinolates can cause liver disease in cows grazing on brassica forage crops. A toxic incidence in New Zealand in cattle grazing brassica led us to investigate the direct in vitro hepatotoxicity and possible inhibition of the ABCG2 transporter of five nitrile compounds. In this study, we investigated 1-cyano-2-hydroxy-3-butene (CHB, epithionitrile derivative of progoitrin), 1-cyano-2-hydroxy-3,4-epithiobutane (CHEB, nitrile derivative of progoitrin), 3-butenenitrile (nitrile from sinigrin), 4-pentenenitrile (nitrile from gluconapin), and 5-hexenenitrile (nitrile from glucobrassicanapin). Cell viability was assessed following 24- and 72-hr treatments with the 5 different compounds using the MTT assay (HepG2 cells and bovine primary liver cells). Additionally, ABCG2 transporter function was assessed. The results showed that none of the tested compounds caused cytotoxicity at concentrations up to 2 mM for 24hr. Over 72-hr the maximum concentration was 20 M but no reduction in cell viability was observed. No inhibition of the ABCG2 transporter occured at concentrations up to 1 mM. Overall this study suggests that direct or secondary toxicity due to selected nitrile or epithionitrile derivatives of these glucosinolates was not the cause of the toxic event in cattle.

pharmacology and toxicology↗

bioRxiv2018

Honey bee workers reared in a neonicotinoid contaminated in-hive environment

With the currently updated risk assessment of three neonicotinoid pesticides, the European Food Safety Authority has confirmed that different applications of these substances represent a risk to wild and managed bees and their use was therefore severely restricted. However, to close further gaps in knowledge, this experiment covers exposure of honey bee worker brood reared in a neonicotinoid contaminated in-hive environment with focus on the individual. In a worst case scenario, mini-hives were fed chronically with a sublethal concentration of clothianidin (15 {micro}g/kg), which is highly toxic to bees already in small amounts. Freshly hatched workers from these colonies were subsequently marked and introduced into non-contaminated colonies, where their lifespan and behavior was monitored. Nineteen days after exposure, clothianidin treated bees had no reduced lifespan or showed any signs of behavioral impairment when compared to the control, demonstrating that social buffering is not a simple substitution of dead bees by rearing more brood. Our results suggest that the social environment plays a crucial role for the individual in terms of \"superorganism resilience\". These findings are discussed in context with the current use of lower tier test systems in risk assessment and contrary results obtained from laboratory experiments.\n\nHIGHLIGHTSO_LISublethal clothianidin treatment did not affect lifespan nor behavior of workers.\nC_LIO_LIEffects on individual bees reared within a mini-hive are translatable to full-sized colonies.\nC_LIO_LI\"Superorganism resilience\" is not a simple substitution of dead bees by rearing more brood.\nC_LIO_LILaboratory testing in the risk assessment of plant protection products bears severe weaknesses.\nC_LI

pharmacology and toxicology↗

bioRxiv2018

Identification of Kinase Inhibitors that Regulate Nuclear Receptor Nurr1 (NR4A2) Cellular Activity

The ability to regulate the activity NR4A subfamily of nuclear receptors would be potentially useful in the treatment of multiple diseases, but to date, few regulators have been reported. This is likely due to the fact that the NR4A subfamily does not have a typical unoccupied nuclear receptor ligand binding pocket, but rather a pocket that is occupied with hydrophobic side chains of adjacent amino acids. It follows that traditional nuclear receptor assays that seek to identify ligands that bind within the ligand binding pocket would not be successful. We have thus focused on an alternate assay to identify NR4A regulators based on the fact that regulation of NR4A, at least partially, results from phosphorylation/dephosphorylation of the amino terminal region of the protein. We developed a medium throughput cellular assay using a fusion of the amino terminus of Nurr1 (NR4A2) with luciferase reporter and used the assay to screen a large and diverse protein kinase inhibitor set (PKIS). We identified multiple kinase inhibitor compounds from PKIS that significantly increased or decreased the cellular activity of Nurr1. These molecules serve as starting points to discover selective tools for regulation of Nurr1/Nurr1pathway.

pharmacology and toxicology↗