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Highlights: [•] Vicenin 2 is a potent inhibitor of α-glucosidase, PTP1B, and RLAR. [•] Vicenin 2 effectively inhibited the formation of advanced glycation end products. [•] Vicenin 2 suppressed glycation-induced protein oxidation. [•] Vicenin 2 also suppressed the formation of amyloid cross-β structures. [•] Vicenin 2 might be useful for the treatment of diabetes and its complications. [Copyright &y& Elsevier]

Abstract: In the present work the scavenging capacity of coffee brews against ROS (ROO , H2O2, HO and HOCl) and RNS (NO and ONOO−) was evaluated and correlated with their bioactive compounds by multivariate analysis. It is the first time that the complete antioxidant capacity profile of coffee brews against different ROS and RNS is evaluated and that the capacity of coffee brews to scavenge NO is reported. The coffee brews were able to scavenge all the reactive species tested and possibly NO2 and CO3 −. The capacity of the coffee brews to scavenge ROO (2523 to 3673μmol TE/g), HO (IC50 =2.24 to 4.38μg/mL), NO (IC50 =3.07 to 5.67μg/mL) and ONOO− (IC50 =1.29 to 2.88μg/mL) was positively correlated to the contents of chlorogenic acids, chlorogenic acid lactones and p-coumaric acid, while the scavenging capacity against H2O2 (IC50 =336 to 531μg/mL) was positively correlated to the content of browned compounds, mainly melanoidins. The coffee brews showed to be potent HOCl scavengers (IC50 =5.12 to 11.20μg/mL), which was correlated to the contents of caffeic acid, 5-hydroxymethylfurfural and browned compounds. These results reinforce the hypothesis that the scavenging capacity against ROS/RNS is one of the mechanisms that can explain the association between the consumption of coffee brews and the decreased risk of chronic-degenerative diseases. [Copyright &y& Elsevier]

Highlights: [•] We prepared the α-amino alcohols from the corresponding α-amino acids. [•] Synthesis of the 4-ethoxy-4-iminobutanenitrile hydrochloride from succinonitrile. [•] Synthesis of new chiral oxazolines. [•] Investigation of biological activities. [•] We studied the antioxidant, antimicrobial and analgesic activities. [ABSTRACT FROM AUTHOR]

Abstract: Rhizoma Curcumae is a common Chinese dietary spice used in South Asia and China for thousands of years. As the main extract, Rhizoma Curcumae oil has attracted a great interest due to its newly raised therapeutic activities including its pharmacological effects upon central nervous system such as neuroprotection, cognitive enhancement, and anti-seizure efficacy; however the molecular mechanisms and the target identification remain to be established. Here we characterize an inhibitory effect of curcumol, a major bioactive component of Rhizoma Curcumae oil, on the excitability of hippocampal neurons in culture, the basal locomotor activity of freely moving animals, and the chemically induced seizure activity in vivo. Electrophysiological recording showed that acute application of curcumol significantly facilitated the γ-aminobutyric acid (GABA)-activated current in cultured mouse hippocampal neurons and in human embryonic kidney cells expressing α1- or α5-containing A type GABA (GABAA) receptors in a concentration-dependent manner. Measurement of tonic and miniature inhibitory postsynaptic GABAergic currents in hippocampal slices indicated that curcumol enhanced both forms of inhibition. In both pentylenetetrazole and kainate seizure models, curcumol suppressed epileptic activity in mice by prolonging the latency to clonic and tonic seizures and reducing the mortality as well as the susceptibility to seizure, presumably by facilitating the activation of GABAA receptors. Taken together, our results identified curcumol as a novel anti-seizure agent which inhibited neuronal excitability through enhancing GABAergic inhibition. [Copyright &y& Elsevier]

Highlights: [•] Mn exposure induces HtrA2 upregulation in rat striatum. [•] Upregulated HtrA2 was correlated with XIAP reduction and marked neuronal apoptosis. [•] HtrA2 inhibitor UCF-101 attenuated Mn-triggered XIAP downregulation. [•] UCF-101 partially protects neuronal cells from Mn-induced apoptosis. [ABSTRACT FROM AUTHOR]

Highlights: [•] Dimebon protects hippocampal slices subjected to oxygen and glucose deprivation. [•] Dimebon reduces reactive oxygen species. [•] It prevents activation of NFκB and iNOS induction. [•] It stabilizes Ψm and blocks intracellular calcium transients induced by glutamate. [ABSTRACT FROM AUTHOR]

Abstract: Thiol redox state (TRS) refers to the balance between reduced thiols and their corresponding disulfides and is mainly reflected by the ratio of reduced and oxidized glutathione (GSH/GSSG). A decrease in GSH/GSSG, which reflects a state of thiol oxidative stress, as well as thiol modifications such as S-glutathionylation, has been shown to have important implications in a variety of cardiovascular diseases. Therefore, research models for inducing thiol oxidative stress are important tools for studying the pathophysiology of these disease states as well as examining the impact of pharmacological interventions on thiol pathways. The purpose of this study was to evaluate the use of a dithiocarbamate derivative, 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA), as a pharmacological model of thiol oxidative stress by examining the extent of thiol modifications induced in H9c2 rat cardiomyocytes and its impact on cellular functions. The extent of thiol oxidative stress produced by 2-AAPA was also compared to other models of oxidative stress including hydrogen peroxide (H2O2), diamide, buthionine sulfoximine, and N,N׳-bis(2-chloroethyl)-N-nitroso-urea. Results indicated that 2-AAPA effectively inhibited glutathione reductase and thioredoxin reductase activities and decreased the GSH/GSSG ratio by causing a significant accumulation of GSSG. 2-AAPA also increased the formation of protein disulfides as well as S-glutathionylation. The alteration in TRS led to a loss of mitochondrial membrane potential, release of cytochrome c, and increase in reactive oxygen species production. Compared to other models, 2-AAPA is more potent at creating a state of thiol oxidative stress with lower cytotoxicity, higher specificity, and more pharmacological relevance, and could be utilized as a research tool to study TRS-related normal and abnormal biochemical processes in cardiovascular diseases. [Copyright &y& Elsevier]

Highlights: [•] 17β-HSD3 is involved in the formation of androgenic hormone testosterone (T). [•] RM-532-105 is an androsterone derivative inhibiting 17β-HSD3. [•] RM-532-105 inhibits the formation of T in homogenized and whole HEK-293 cells overexpressing 17β-HSD3. [•] RM-532-105 (10mg/kg, s.c.) reached a plasma concentration of 250ng/mL at 7h in rat. [Copyright &y& Elsevier]

Abstract: let-7 microRNA (miRNA) is implicated in various biological processes, and its downregulation essentially linked to human malignancy. Regulation of gene expression of the let-7 family is critically linked to RNA-binding proteins. For instance, Lin28B and its paralog, Lin28A, inhibit the pre-let-7 precursor from being processed to mature miRNA by recruiting terminal uridyltransferase, TUT4, which adds oligomeric U at the 3′ end, suggesting that deregulation of Lin28B, together with Lin28A, may alter various biological processes through modulation of let-7 expression. Here, we showed that the Lin28B protein level is regulated via ubiquitin-mediated proteasomal degradation, and identified the ubiquitin ligase as human TRIM-NHL domain-containing TRIM71. In cells, TRIM71 negatively regulates Lin28B protein stability by catalyzing polyubiquitination. Compared with its paralog, Lin28A, a C-terminal unique ~50 amino acid stretch of Lin28B is essential for TRIM71 interactions and subsequent polyubiquitination. Moreover, the N-terminal RING finger motif of TRIM71 is critical for protein–protein interactions and polyubiquitination of Lin28B, and consequent let-7 expression. Consistent with the let-7 stimulatory role of TRIM71 via Lin28B polyubiquitination, specific knockdown of TRIM71 led to downregulation of let-7 expression. Expression of one of the known let-7 targets, HMGA2, was derepressed after knockdown of TRIM71. We additionally showed that enhanced expression of let-7 is part of a feedback loop that targets TRIM71 3′UTR, which contains two conserved let-7 target sites. Our findings collectively reveal critical aspects of regulatory complexity of let-7 biogenesis at the posttranscriptional level. [Copyright &y& Elsevier]

Abstract: Asthma is a chronic inflammatory disorder that causes contraction in the smooth muscle of the airway and blocking of airflow. Reversal the contractile process is a strategy for the search of new drugs that could be used for the treatment of asthma. This work reports the semisynthesis, ex vivo relaxing evaluation and SAR studies of a series of 18 coumarins. The results pointed that the ether derivatives 1–3, 7–9 and 13–15 showed the best activity (E max = 100%), where compound 2 (42 μM) was the most potent, being 4-times more active than theophylline (positive control). The ether homologation (methyl, ethyl and propyl) in position 7 or positions 6 and 7 of coumarins lead to relaxing effect, meanwhile formation of esters generated less active compounds than ethers. The SAR analysis showed that it is necessary the presence of two small ether groups and the methyl group at position 4 (site 3) encourage biological activity through soft hydrophobic changes in the molecule, without drastically affecting the cLogP. [Copyright &y& Elsevier]

Abstract: Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a multi-functional serine/threonine protein kinase that controls a range of cellular functions, including proliferation. The biological properties of CaMKII are regulated by multi-site phosphorylation and targeting via interactions with specific proteins. To investigate the role specific CaMKII phosphorylation sites play in controlling cell proliferation and cell cycle progression, we examined phosphorylation of CaMKII at two sites (T253 and T286) at various stages of the cell cycle, and also examined the effects of overexpression of wild-type (WT), T286D phosphomimic, T253D phosphomimic and T253V phosphonull forms of CaMKIIα in MDA-MB-231 breast cancer and SHSY5Y neuroblastoma cells on cellular proliferation and cell cycle progression. We demonstrate herein that whilst there is no change in total CaMKII expression or T286 phosphorylation throughout the cell cycle, a marked dephosphorylation of CaMKII at T253 occurs during the G2 and/or M phases. Additionally, we show by molecular inhibition, as well as pharmacological activation, that protein phosphatase 2A (PP2A) is the phosphatase responsible for this dephosphorylation. Furthermore, we show that inducible overexpression of WT, T286D and T253V forms of CaMKIIα in MDA-MB-231 and SHSY5Y cells increases cellular proliferation, with no alteration in cell cycle profiles. By contrast, overexpression of a T253D phosphomimic form of CaMKIIα significantly decreases proliferation, and cells accumulate in mitosis, specifically in metaphase. Taken together, these results strongly suggest that the dephosphorylation of CaMKII at T253 is involved in controlling the cell cycle, specifically the metaphase–anaphase transition. [Copyright &y& Elsevier]

Abstract: MicroRNAs (miRNAs) play an essential role in regulating cell differentiation either by inhibiting mRNA translation or by inducing its degradation. However, the role of miRNAs in odontoblastic cell differentaion is largely unknown. In the present study, we demonstrate that the expression of miR-27 was significantly increased during MDPC-23 odontoblastic cell differentiation. Furthermore, the up-regulation of miR-27 promotes the differentiation of MDPC-23 odontoblastic cells and accelerates mineralization without cell proliferation. In addition, our results of target gene prediction revealed that the mRNA of adenomatous polyposis coli (APC) associated with Wnt/β-catenin signaling pathway has miR-27 binding site in the its 3′ UTR and is suppressed by miR-27. Subsequentially, the down-regulated APC by miR-27 triggered the activation of Wnt/β-catenin signaling through accumulation of β-catenin in the nucleus. Our data suggest that miR-27 promotes MDPC-23 odontoblastic cell differentiation by targeting APC and activating Wnt/β-catenin signaling. Therefore, miR-27 might be considered a critical candidate as an odontoblastic differentiation molecular target for the development of miRNA based therapeutic agents in the dental medicine. [Copyright &y& Elsevier]

Highlights: [•] We applied a proteomics approach to study the effects of BDE-209 and/or BDE-47 on proteins from NSC and explored mechanisms. [•] We found 39 differential expression protein spots in NSC after BDE treatment. [•] We identified nineteen proteins, in which cofilin-1 and vimentin expression were decreased. [•] We provide a useful basis to the study underlying PBDE-mediated neurotoxicity. [ABSTRACT FROM AUTHOR]

Highlights: [•] Oxidative stress and Ca2+ influx play an important role in pain. [•] Effects of H. perforatum (HP) on Ca2+ and apoptosis in the DRG were investigated. [•] Ca2+ influx through TRPM2 channel in the DRG neuron is high. [•] HP modulated oxidative stress and Ca2+ influx in the DRG. [•] The study is the first to compare effects of HP on TRPM2 channels in the DRG. [Copyright &y& Elsevier]

Highlights: [•] Melatonin receptors and HIOMT are present in the olfactory bulb. [•] OB cells have transcriptional responses to melatonin application. [•] A subset of OB cells has electrical responses to melatonin application. [ABSTRACT FROM AUTHOR]

Abstract: To examine the role of intracellular labile iron pool (LIP), ferritin (Ft), and antioxidant defence in cellular resistance to oxidative stress on chronic adaptation, a new H2O2-resistant Jurkat T cell line “HJ16” was developed by gradual adaptation of parental “J16” cells to high concentrations of H2O2. Compared to J16 cells, HJ16 cells exhibited much higher resistance to H2O2-induced oxidative damage and necrotic cell death (up to 3mM) and had enhanced antioxidant defence in the form of significantly higher intracellular glutathione and mitochondrial ferritin (FtMt) levels as well as higher glutathione-peroxidase (GPx) activity. In contrast, the level of the Ft H-subunit (FtH) in the H2O2-adapted cell line was found to be 7-fold lower than in the parental J16 cell line. While H2O2 concentrations higher than 0.1mM fully depleted the glutathione content of J16 cells, in HJ16 cells the same treatments decreased the cellular glutathione content to only half of the original value. In HJ16 cells, H2O2 concentrations higher than 0.1mM increased the level of FtMt up to 4-fold of their control values but had no effect on the FtMt levels in J16 cells. Furthermore, while the basal cytosolic level of LIP was similar in both cell lines, H2O2 treatment substantially increased the cytosolic LIP levels in J16 but not in HJ16 cells. H2O2 treatment also substantially decreased the FtH levels in J16 cells (up to 70% of the control value). In contrast in HJ16 cells, FtH levels were not affected by H2O2 treatment. These results indicate that chronic adaptation of J16 cells to high concentrations of H2O2 has provoked a series of novel and specific cellular adaptive responses that contribute to higher resistance of HJ16 cells to oxidative damage and cell death. These include increased cellular antioxidant defence in the form of higher glutathione and FtMt levels, higher GPx activity, and lower FtH levels. Further adaptive responses include the significantly reduced cellular response to oxidant-mediated glutathione depletion, FtH modulation, and labile iron release and a significant increase in FtMt levels following H2O2 treatment. [Copyright &y& Elsevier]

Well preserved frozen biospecimens are ideal for evaluating the genome, transcriptome, and proteome. While papers reviewing individual aspects of frozen biospecimens are available, we present a current overview of experimental data regarding procurement, storage, and quality assurance that can inform the handling of frozen biospecimens. Frozen biospecimen degradation can be influenced by factors independent of the collection methodology including tissue type, premortem agonal changes, and warm ischemia time during surgery. Rapid stabilization of tissues by snap freezing immediately can mitigate artifactually altered gene expression and, less appreciated, protein phosphorylation profiles. Collection protocols may be adjusted for specific tissue types as cellular ischemia tolerance varies widely. If data is not available for a particular tissue type, a practical goal is snap freezing within 20min. Tolerance for freeze–thaw events is also tissue type dependent. Tissue storage at −80°C can preserve DNA and protein for years but RNA can show degradation at 5years. For −80°C freezers, aliquots frozen in RNAlater or similar RNA stabilizing solutions are a consideration. It remains unresolved as to whether storage at −150°C provides significant advantages relative to that at −80°C. Histologic quality assurance of tissue biospecimens is typically performed at the time of surgery but should also be conducted on the aliquot to be distributed because of tissue heterogeneity. Biobanking protocols for blood and its components are highly dependent on intended use and multiple collection tube types may be needed. Additional quality assurance testing should be dictated by the anticipated downstream applications. [ABSTRACT FROM AUTHOR]

Abstract: Recent reports suggest that N-methyl-d-aspartate receptor (NMDAR) blockade by MK-801 decreases tumor growth. Thus, we investigated whether other ionotropic glutamate receptor (iGluR) antagonists were also able to modulate the proliferation of melanoma cells. On the other hand, the antiestrogen tamoxifen (TAM) decreases the proliferation of melanoma cells, and is included in combined therapies for melanoma. As the efficacy of TAM is limited by its metabolism, we investigated the effects of the NMDAR antagonist MK-801 in combination with TAM and its active metabolites, 4-hydroxytamoxifen (OHTAM) and endoxifen (EDX). The NMDAR blockers MK-801 and memantine decreased mouse melanoma K1735-M2 cell proliferation. In contrast, the NMDAR competitive antagonist APV and the AMPA and kainate receptor antagonist NBQX did not affect cell proliferation, suggesting that among the iGluR antagonists only the NMDAR channel blockers inhibit melanoma cell proliferation. The combination of antiestrogens with MK-801 potentiated their individual effects on cell biomass due to diminished cell proliferation, since it decreased the cell number and DNA synthesis without increasing cell death. Importantly, TAM metabolites combined with MK-801 promoted cell cycle arrest in G1. Therefore, the data obtained suggest that the activity of MK-801 and antiestrogens in K1735-M2 cells is greatly enhanced when used in combination. [Copyright &y& Elsevier]

Abstract: The main purpose of this research was to prepare and to characterize ferulic acid-loaded nanostructured lipid carrier (FA-NLC) to evaluate the cytotoxic effect on human glioblastoma cancer U87MG cells. First of all, the influence of different materials on mean size and homogeneity of NLC prepared by a low energy organic solvent-free method was investigated. Technological characterization (encapsulation efficiency, mean particle size, homogeneity and in vitro release profile) was performed on the selected NLC in comparison to others lipid carriers, nanoemulsion and SLN. Furthermore, the thermal behavior of NLC and SLN was investigated using Differential Scanning Calorimetry (DSC) in order to evaluate their structure. Biological studies (MTT bioassay and caspase-3 cleavage) on the selected NLC showed no cytotoxic effects of the unloaded tested NLC. Besides, the effectiveness of FA-loaded NLC was higher compared to the free drug. Cells treated with FA or FA-loaded NLC showed a greater effect compared to idebenone (IDE) or IDE-loaded NLC, respectively. These results strongly support that FA-loaded NLC could be potentially used for the treatment of glioblastoma. [Copyright &y& Elsevier]

Highlights: [•] Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA. [•] Anthraquinones quinizarin and danthron lengthen linear DNA. [•] Anthraquinones quinizarin and danthron possess middle binding affinity to DNA. [•] Anthraquinones quinizarin and danthron interact with DNA by intercalating mode. [Copyright &y& Elsevier]

Abstract: Microtubules (MTs) are essential components of the cytoskeleton that play critical roles in neurodevelopment and adaptive central nervous system functioning. MTs are essential to growth cone advance and ultrastructural events integral to synaptic plasticity; these functions figure significantly into current pathophysiologic conceptualizations of schizophrenia. To date, no study has directly investigated MT dynamics in humans with schizophrenia. We therefore compared the stability of MTs in olfactory neuroepithelial (OE) cells between schizophrenia cases and matched nonpsychiatric comparison subjects. For this purpose, we applied nocodazole (Nz) to cultured OE cells obtained from tissue biopsies from seven living schizophrenia patients and seven matched comparison subjects; all schizophrenia cases were on antipsychotic medications. Nz allows MT depolymerization to be followed but prevents repolymerization, so that in living cells treated for varying time intervals, the MTs that are stable for a given treatment interval remain. Our readout of MT stability was the time at which fewer than 10 MTs per cell could be distinguished by anti-β-tubulin immunofluorescence. The percentage of cells with ≥10 intact MTs at specified intervals following Nz treatment was estimated by systematic uniform random sampling with Visiopharm software. These analyses showed that the mean percentages of OE cells with intact MTs were significantly greater for schizophrenia cases than for the matched comparison subjects at 10, 15, and 30min following Nz treatment indicating increased MT stability in OE cells from schizophrenia patients (p=0.0007 at 10min; p=0.0008 at 15min; p=0.036 at 30min). In conclusion, we have demonstrated increased MT stability in nearly all cultures of OE cells from individuals with schizophrenia, who received several antipsychotic treatments, versus comparison subjects matched for age and sex. While we cannot rule out a possible confounding effect of antipsychotic medications, these findings may reflect analogous neurobiological events in at least a subset of immature neurons or other cell types during gestation, or newly generated cells destined for the olfactory bulb or hippocampus, suggesting a mechanism that underlies findings of postmortem and neuroimaging investigations of schizophrenia. Future studies aimed at replicating these findings, including samples of medication-naïve subjects with schizophrenia, and reconciling the results with other studies, will be necessary. Although the observed abnormalities may suggest one of a number of putative pathophysiologic anomalies in schizophrenia, this work may ultimately have implications for an improved understanding of pathogenic processes related to this disorder. [Copyright &y& Elsevier]

Highlights: [•] Chenopodium ambrosioides oil showed potential antileishmanial activity. [•] Chenopodium oil caused better selectivity compared with its major components. [•] Products cause a breakdown of mitochondrial membrane potential and redox indexes. [•] Oil showed effect against Plasmodium falciparum and Trypanosoma brucei. [ABSTRACT FROM AUTHOR]

Highlights: [•] The binge-like pattern of alcohol exposure is associated with the development of a depression-like symptom. [•] Chronic binge drinking resulted in decreased hippocampal neurogenesis and BDNF expression during the withdrawal period. [•] Augmenting BDNF restored neurogenesis and abolished the depression-like behaviors caused by chronic binge alcohol. [Copyright &y& Elsevier]

Abstract: Human topoisomerase 1B, the unique target of the natural anticancer compound camptothecin, catalyzes the unwinding of supercoiled DNA by introducing transient single strand nicks and providing covalent protein–DNA adducts. The functional properties and the drug reactivity of the single Arg634Ala mutant have been investigated in comparison to the wild type enzyme. The mutant is characterized by an identical relaxation and cleavage rate but it displays resistance to camptothecin as indicated by a viability assay of the yeast cells transformed with the mutated protein. The mutant also displays a very fast religation rate that is only partially reduced by the presence of the drug, suggesting that this is the main reason for its resistance. A comparative analysis of the structural–dynamical properties of the native and mutant proteins by molecular dynamics simulation indicates that mutation of Arg634 brings to a loss of motion correlation between the different domains and in particular between the linker and the C-terminal domain, containing the catalytic tyrosine residue. These results indicate that the loss of motion correlation and the drug resistance are two strongly correlated events. [Copyright &y& Elsevier]

Abstract: In the last years, there has been a revival of total reflection X-ray fluorescence spectrometry (TXRF), which was firstly applied for analytical purposes in the late 80s. The aim of this work is to discuss and compare the current approaches for sample pretreatment including in situ microdigestion, slurry preparation, acid digestion, extraction, etc. prior to TXRF analysis. Advantages and drawbacks inherent to each of those procedures are considered. A comprehensive revision in the period January 2008–July 2013 about different sample preparation strategies prior to TXRF analysis apart from early pioneering reports dealing with sample pretreatment are included in the review. Non-conventional sample pretreatment approaches such as microflow online preconcentration, lab-on-a-chip, etc., are also discussed. Finally, future prospects in sample preparation prior to TXRF analysis are outlined. [Copyright &y& Elsevier]

Abstract: Oral delivery of camptothecin has a treatment advantage but is limited by low bioavailability and gastrointestinal toxicity. Poly(amido amine) or PAMAM dendrimers have shown promise as intestinal penetration enhancers, drug solubilizers and drug carriers for oral delivery in vitro and in situ. There have been very limited studies in vivo to evaluate PAMAM dendrimers for oral drug delivery. In this study, camptothecin (5mg/kg) was formulated and co-delivered with cationic, amine-terminated PAMAM dendrimer generation 4.0 (G4.0) (100 and 300mg/kg) and anionic, carboxylate-terminated PAMAM generation 3.5 (G3.5) (300 and 1000mg/kg) in CD-1 mice. Camptothecin associated to a higher extent with G4.0 than G3.5 in the formulation, attributed to an electrostatic interaction on the surface of G4.0. Both PAMAM G4.0 and G3.5 increased camptothecin solubilization in simulated gastric fluid and caused a 2–3 fold increase in oral absorption of camptothecin when delivered at 2h. PAMAM G4.0 and G3.5 did not increase mannitol transport suggesting that the oral absorption of camptothecin was not due to tight junction modulation. Histologic observations of the epithelial layer of small intestinal segments of the gastrointestinal tract (GIT) at 4h post dosing supported no evidence of toxicity at the evaluated doses of PAMAM dendrimers. This study demonstrates that both cationic (G.4) and anionic (G3.5) PAMAM dendrimers were effective in enhancing the oral absorption of camptothecin. Results suggest that drug inclusion in PAMAM interior controlled solubilization in simulated gastric and intestinal fluids, and increased oral bioavailability. [Copyright &y& Elsevier]

Highlights: [•] Ospemifene induced beneficial vaginal effects in ovariectomized (OVX) rats. [•] In comparison with estrogen, similarities and differences in the vaginal effects of ospemifene were noted in OVX rats. [•] Ospemifene effectively treated VVA in postmenopausal women in Phase 3 studies. [•] Thus, the OVX rat model accurately predicted SERM activity in postmenopausal VVA. [Copyright &y& Elsevier]

Highlights: [•] TNF-α inhibits AT1 receptor expression in DRG neurons via β-catenin signaling. [•] TNF-α inhibits AT1 receptor expression in DRG neurons via p38 MAPK/GSK-3β. [•] Angiotensin II inhibits TNF-α expression via the AT1 receptor in DRG neurons. [•] Angiotensin II induces TNF-α expression via the AT2 receptor in DRG neurons. [ABSTRACT FROM AUTHOR]

Highlights: [•] BP decreased cell viability of LNCaP cells in a dose- and time-dependent manner. [•] The induction of G0/G1 phase arrest and apoptosis caused BP-induced cytotoxicity. [•] FAS-dependent, mitochondrial, and ER stress pathway mediate apoptosis. [•] These results were revealed by proteomic and functional studies. [ABSTRACT FROM AUTHOR]

Highlights: [•] Comparison of assay performance in plasma, CSF and dried blood spots (DBS). [•] Assay performance in DBS is similar to that of acetaminophen in plasma samples. [•] Automated DBS extraction met acceptance criteria, however, was inferior to manual extractions. [•] Matching DBS and plasma samples (n =167) from a clinical trial showed excellent correlation. [•] This comparison also showed a 26.6% positive bias of the DBS when compared to plasma. [Copyright &y& Elsevier]

Abstract: Introduction: Neferine, a kind of isoquinoline alkaloid, extracted from the seed embryo of Nelumbo nucifera Gaertn, has long been recognized in traditional medicine as a medicinal plant with various usages. Neferine has many biological activities, including anti-hypertensive, anti-arrhythmic, negative inotropic effect and relaxation on vascular smooth muscle. Although previous studies have reported its antithrombotic effect, the mechanisms by which it exerts antithrombotic effect have not been thoroughly studied. Method: Washed mice platelets and mice platelet-rich-plasma (PRP) were used to investigate the effects of neferine on platelet aggregation, secretion induced by various agonists and dissociation of agonist-formed platelet aggregates. Bioflux plates coated with collagen were used to investigate the effect of neferine on platelet adhesion and thrombosis in vitro. With collagen-epinephrine-induced acute pulmonary thrombus formation mouse model, the effect of neferine on thrombosis in vivo was also examined. Results: Neferine, significantly and dose-dependently, inhibited collagen-, thrombin-, U46619-induced platelet aggregation in mice washed platelets, or ADP-induced platelet aggregation in PRP. Neferine treatment decreased platelet dense granule secretion initiated by collagen, thrombin and U46619. Also, Neferine dramatically and dose-dependently promoted the dissociation of platelet aggregates pre-formed by various agonists including collagen, thrombin, U46619 or ADP. Neferine can significantly reduce the area of mice platelets adhesion to the collagen and inhibit thrombosis in vitro. In collagen-epinephrine-induced acute pulmonary thrombus mouse model, neferine, at 6mg/kg, significantly attenuated thrombus formation. Conclusions: Neferine remarkably prevents thrombus formation by inhibiting platelet activation, adhesion and aggregation, as well as promoting disassembly of pre-formed platelet aggregates. The inhibitory effects of neferine on platelet activation might be relevant in cases involving aberrant platelet activation where neferine could be used as an anti-platelet and antithrombotic agent. [Copyright &y& Elsevier]

Highlights: [•] Capsaicin triggers glutamate and dopamine release in the striatum of young rats only. [•] TRPV1R function is not enhanced under chemical and genetic ablation of CB1Rs. [•] Our data reveal a possible neurodevelopmental role for the TRPV1R in the rodent brain. [ABSTRACT FROM AUTHOR]

Abstract: Previous single-molecule fluorescence resonance energy transfer (smFRET) studies in which the second and sixth ankyrin repeats (ARs) of IκBα were labeled with FRET pairs showed slow fluctuations as if the IκBα AR domain was unfolding in its native state. To systematically probe where these slow dynamic fluctuations occur, we now present data from smFRET studies wherein FRET labels were placed at ARs 1 and 4 (mutant named AR 1–4), at ARs 2 and 5 (AR 2–5), and at ARs 3 and 6 (AR 3–6). The results presented here reveal that AR 6 most readily detaches/unfolds from the AR domain, undergoing substantial fluctuations at room temperature. AR 6 has fewer stabilizing consensus residues than the other IκBα ARs, probably contributing to the ease with which AR 6 “loses grip”. AR 5 shows almost no fluctuations at room temperature, but a significant fraction of molecules shows fluctuations at 37°C. Introduction of stabilizing mutations that are known to fold AR 6 dampen the fluctuations of AR 5, indicating that the AR 5 fluctuations are likely due to weakened inter-repeat stabilization from AR 6. AR 1 also fluctuates somewhat at room temperature, suggesting that fluctuations are a general behavior of ARs at ends of AR domains. Remarkably, AR 1 still fluctuates in the bound state, but mainly between 0.6 and 0.9 FRET efficiency, whereas in the free IκBα, the fluctuations extend to <0.5 FRET efficiency. Overall, our results provide a more complete picture of the energy landscape of the native state dynamics of an AR domain. [Copyright &y& Elsevier]

Highlights: [•] 1,8-Dibenzoyl-rifampicin (DiBenzRif) was obtained by dibenzoylation of rifampicin. [•] DiBenzRif inhibited P-glycoprotein (P-gp) activity in RBE4 cells. [•] DiBenzRif decreased ATP intracellular levels and inhibited P-gp ATPase activity. [•] DiBenzRif increased membrane fluidity and enhanced rhodamine123 accumulation in liposomes. [•] DiBenzRif may be useful to enhance the access of drugs to the brain. [Copyright &y& Elsevier]

Abstract: The development of tumor-selective drugs with low systemic toxicity has always been a major challenge in cancer treatment. Our group previously identified the 7,8-dihydroxy-4-methylcoumarin (DHMC) as a potential chemotherapeutic agent due to its potent, selective anti-proliferative and apoptosis-inducing effects on several cancer cell lines over peripheral blood mononuclear cells. However, there are still no published reports that can explain such selectivity of action. Herein, we addressed this question by using the U-937 promonocytic leukemia cell line, which can be forced to differentiate into a monocyte-like phenotype in vitro. U-937 cells differentiation is dependent on the nuclear expression of p21Cip1/WAF1, a protein that is absent in immature U-937 cells but present in both the nucleus and the cytoplasm of normal DHMC-resistant monocytes. Considering that induction of differentiation rendered U-937 cells resistant to DHMC, we evaluated the possible causal role of cytoplasmic p21Cip1/WAF1 in the onset of such resistance by employing U-937 cells stably transfected with a ZnCl2-inducible p21Cip1/WAF1 variant lacking the nuclear localization signal (U-937/CB6-ΔNLS-p21 cells). Expression of cytoplasmic p21Cip1/WAF1 did not induce differentiation of the cells but turned them resistant to DHMC through inhibition of JNK, a crucial mediator of DHMC-induced apoptosis in U-937 cells. Sub-acute toxicity evaluation of DHMC in Balb/c mice indicated that DHMC administered intraperitoneally at doses up to 100mg/kg induced no systemic damage. Collectively, our results explain for the first time the selective cytotoxicity of DHMC for tumor cells over normal monocytes, and encourage further in vivo studies on this compound as potential anti-leukemic agent. [Copyright &y& Elsevier]

Highlights: [•] Leptin and ghrelin protect hippocampal neurons from Aβ oligomers-induced toxicity. [•] Leptin and ghrelin prevent oxidative stress and mitochondrial dysfunction. [•] Leptin and ghrelin revert GSK3β activation induced by Aβ oligomers. [•] Neuroprotection by leptin and ghrelin occurs in a receptor-mediated manner. [ABSTRACT FROM AUTHOR]

Highlights: [•] We investigate the role of PPARδ in a model of Parkinson’s disease. [•] PPARδ is upregulated after the neurotoxin MPTP. [•] PPARδ antagonism enhances MPP+ toxicity which is reversible by PPARδ agonism. [•] PPARδ agonism protects against MPTP-toxicity. [ABSTRACT FROM AUTHOR]

Abstract: Cholinergically differentiated SH-SY5Y neuroblastoma cells were treated with a pathophysiologically relevant, low (300nM), and a high (3μM) dose of amyloid beta 1-42 (Abeta) or 42-1 (revAbeta). At early (1 and 4h) and late (24h) time points, the pro- and anti-apoptotic factors – caspase-3 and p53, and B-cell lymphoma 2 protein (Bcl-2), respectively – were assessed together with lactate dehydrogenase (LDH) release as measure of cell viability and ATP levels as marker of mitochondrial activity. The low peptide dose significantly increased Bcl-2 and, time-delayed, caspase-3 and ATP levels, but barely impacted on LDH release, while the high concentration remarkably depressed Bcl-2 levels, depleted ATP and led to increased LDH release. We also monitored acetylcholinesterase (AChE) enzymatic activity and splice variant levels (tailed and readthrough AChE; AChE-T and AChE-R), and assessed choline acetyltransferase (ChAT) and high-affinity choline uptake (HACU). The low Abeta concentration drastically upregulated AChE-R and increased both ChAT and HACU, while the high dose caused cholinergic toxicity. We believe this study offers the first insight into the highly concentration-dependent effects of Abeta on cholinergic dynamics. In particular, it highlights the rescuing role of AChE-R as being, together with mitochondrial activity, involved in cholinergic adaptation to low doses of Abeta. [Copyright &y& Elsevier]

Abstract: Protein oxidation, irreversible modification, and inactivation may play key roles in various neurodegenerative disorders. Therefore, we studied the effects of the potentially in vivo occurring nitric oxide-related species on two different markers of protein oxidation: protein carbonyl generation on bovine serum albumine (BSA) and loss of activity of a cysteine-dependent protease, papain, in vitro by using Angeli''s salt, papanonoate, SIN-1, and S-nitrosoglutathione (GSNO) as donors of nitroxyl, nitric oxide, peroxynitrite, and nitrosonium ions, respectively. Angeli''s salt, SIN-1, and papanonoate (0–1000 μM) all generated a concentration-dependent increase in carbonyl formation on BSA (107, 60, and 45%, respectively). GSNO did not affect carbonyl formation. Papain was inhibited by Angeli''s salt, SIN-1, papanonoate, and GSNO with IC50 values of 0.62, 2.3, 54, and 80 μM, respectively. Angeli''s salt (3.16 μM)-induced papain inactivation was only partially reversible, while the effects of GSNO (316 μM) and papanonoate (316 μM) were reversible upon addition of excess DTT. The Angeli''s salt-mediated DTT-irreversible inhibition of papain was prevented by GSNO or papanonoate pretreatment, hypothetically through mixed disulfide formation or S-nitrosylation of the catalytically critical thiol group of papain. These results, for the first time, compare the generation of carbonyls in proteins by Angeli''s salt, papanonoate, and SIN-1. Furthermore, these results suggest that S-nitrosothiols may have a novel function in protecting critical thiols from irreversible oxidative damage. [Copyright &y& Elsevier]

Abstract: There is major interest in designing inhibitors for matrix metalloproteinase 2 (MMP-2, gelatinase A) since this enzyme is known to be involved in pathological processes such as tumor invasion or rheumatoid arthritis. The majority of MMP-2 inhibitor candidate drugs block the active site of MMP-2 by binding to its catalytic Zn2+ ion through a chelating (hydroxamate, sulphonate etc.) group. Despite the general interest in designing MMP-2 inhibitors, the results with many of the drug candidates were disappointing, their failure was usually explained by cross-reactions with other MMPs. One way to enhance MMP-2 selectivity is to design inhibitors that interact with both the active site and exosites such as the fibronectin type II (FN2) domains of the enzyme. In the present work, we have examined the inhibitory potential and MMP-2 selectivity of hydroxamates of three groups of peptides known to bind to the collagen-binding FN2 domains of MMP-2. The first type of peptides consisted of collagen-like (Pro-Pro-Gly)n repeats, peptides of the second group were identified from a random 15-mer phage display library based on their binding to immobilized FN2 domains of MMP-2. A hydroxamate of peptide p33–42, known to bind to the third FN2 domain of MMP-2 has also been tested. Our studies have shown that these compounds inhibited MMP-2 with IC50 values of 10–100 μM. The fact that their inhibitory potential was nearly identical for MMP-2del, a recombinant version of MMP-2 that lacks the FN2 domains, suggests that inhibition is not mediated by their binding to FN2 domains. It seems likely that the failure to exploit interaction with the FN2 domains is due to the fact that the FN2 domains and the catalytic domain of MMP-2 tumble independently, therefore only a tiny fraction of the conformational isomers can bind peptide hydroxamates via both the active site and the FN2 domain(s). [Copyright &y& Elsevier]

Five organochlorine pesticides, namely, chlordane, dieldrin, aldrin, endrin, and endosulfan, activate human retinoic acid receptor (RAR)-mediated gene transcription via a retinoic acid response element (RARE). Transactivation studies were performed with stable RARα, β, or γ reporter cell lines in which the RAR DNA-binding domain (DBD) was replaced by that of estrogen receptor α (ERα)?. Five of the organochlorine pesticides tested activated RARβ and RARγ but not RARα; their half-maximal luciferase activity (EC50) was determined. Furthermore, that activity was RAR-specific and organochlorine pesticides did not activate the retinoid X receptor (RXR) pathway. However, competitive binding experiments with [3H]-CD367, a pan-RAR agonist, showed that only chlordane could bind RARβ and RARγ, albeit with low affinity. In addition, organochlorine pesticides strongly induce cytochrome P450RAI1 (P450RAI1), a key factor of retinoic acid level regulation in many tissues and whose expression and activity are strongly induced by retinoic acid. This study shows that organochlorine pesticides can activate two RAR homologues, with low-binding affinity. Although the agonistic potential of organochlorine pesticides is lower than that of (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl] benzoic acid (TTNPB), they are able to induce RAR-mediated gene transcription as P450RAI1 and may disrupt the retinoid signaling pathway. Because these chemicals are extremely persistent and tend to accumulate in biological tissues, these results support the hypothesis that the increase in teratogenicity observed in some developing countries could be due to prolonged exposure to organochlorine pesticides ubiquitously present in the environment. [Copyright &y& Elsevier]

The combination of the topoisomerase I (Topo I) inhibitor CPT-11 with the anti-epidermal growth factor receptor (EGFR) agent Gefitinib (Iressa™, ZD1839) represents a promising medical approach for colorectal cancer patients. In this report, we provide pre-clinical evidences for their optimal combination schedule in HT-29 and LoVo human colon cancer cell lines. We analyzed the different effects that three different combination schedules of SN-38 (the active CPT-11 metabolite) and Gefitinib (Gefitinib before; Gefitinib simultaneously; Gefitinib after SN-38) have on cell growth, cell cycle, apoptosis, and expression/phosphorylation of EGFR, Topo I and some steps of the signal transduction pathway. We first determined the IC50 of each drug choosing the 5 days exposure for Gefitinib (0.6 and 3.8 μM for LoVo and HT-29 cells, respectively) and 1 day exposure for SN-38 (0.31 and 0.5 μM for LoVo and HT-29 cells, respectively). The different drug combination schedules were tested in various concentrations by using equiactive concentrations of the two drugs. The cytotoxicity of Gefitinib and SN-38 combination was schedule- and concentration-dependent but not cell line-specific. The most synergistic schedule was Gefitinib given after SN-38, with combination indexes (CI) of 0.007 and 0.454 in HT-29 and LoVo, respectively. Analysis of bio-molecular targets showed that Gefitinib was able to modulate SN-38 ability to inhibit Topo I, to accumulate cells in S-phase, and to induce apoptosis. Interestingly, SN-38 was able to activate EGFR and its signal transduction pathway. Confirming preliminary clinical experience of Gefitinib with other cytotoxic drugs, it seems that Gefitinib after SN-38 represents the best cytotoxic combination schedule but the biomolecular basis for this synergism remain to be completely elucidated. [Copyright &y& Elsevier]

The Syrian hamster embryo (SHE) cell transformation assay was used to test 28 chemical substances for their ability to induce morphologically transformed colonies. The purpose was to determine how well the assay method could be transferred from an experienced laboratory by including 18 chemicals previously evaluated and 10 new chemicals. Technical training was obtained in the experienced lab prior to testing. The assay was conducted at pH 6.7, a treatment period of 7 days was used, and single experiments were performed for each chemical. With this limited testing, 78% concordance with rodent bioassay results was obtained, and this high concordance would have increased if small, but statistically negative responses from single trials were overturned by positive data from repeat trials. Similarly, the results were highly concordant (90%) with the experienced lab results; only 2 chemical evaluations were discordant, and the use of repeat experiments would likely have eliminated these apparent disagreements. Thus, with appropriate training, the pH 6.7 SHE assay was successfully and reliably transferred. [Copyright &y& Elsevier]

2-Arachidonoylglycerol (2-AG) is an endogenous cannabinoid that binds to CB1 and CB2 cannabinoid receptors, inducing cannabimimetic effects. However, the cannabimimetic effects of 2-AG are weak in vivo due to its rapid enzymatic hydrolysis. The enzymatic hydrolysis of 2-AG has been proposed to mainly occur by monoglyceride lipase (monoacylglycerol lipase). Fatty acid amide hydrolase (FAAH), the enzyme responsible for the hydrolysis of N-arachidonoylethanolamide (AEA), is also able to hydrolyse 2-AG. In the present study, we investigated the hydrolysis of endocannabinoids in rat cerebellar membranes and observed that enzymatic activity towards 2-AG was 50-fold higher than that towards AEA. Furthermore, various inhibitors for 2-AG hydrolase activity were studied in rat cerebellar membranes. 2-AG hydrolysis was inhibited by methyl arachidonylfluorophosphonate, hexadecylsulphonyl fluoride and phenylmethylsulphonyl fluoride with ic50 values of 2.2 nM, 241 nM and 155 μM, respectively. Potent FAAH inhibitors, such as OL-53 and URB597, did not inhibit the hydrolysis of 2-AG, suggesting that 2-AG is inactivated in rat cerebellar membranes by an enzyme distinct of FAAH. The observation that the hydrolysis of 1(3)-AG and 2-AG occurred at equal rates supports the role of MGL in 2-AG inactivation. This enzyme assay provides a useful method for future inhibition studies of 2-AG degrading enzyme(s) in brain membrane preparation having considerably higher MGL-like activity when compared to FAAH activity. [Copyright &y& Elsevier]

6-Substituted purine and 9-β-d-ribofuranosyl purine analogues were synthesized and their biological activities were evaluated. CD Spectra and thermal melting studies showed that compounds 8, 9, 10 could interact with RNA and DNA in solution. Compound 8 and 10 may bind with RNA single strand and interfere the formation of RNA duplex. Among of these compounds, compound 8 showed middle inhibition on the growth of HeLa cells (70.21%) and HL-60 cells (70.85%) at 10 μM. Comparing to the structures of these synthetic compounds, it may indicate that the sugar moiety and the 6-amino side chain of nucleoside 8 play an important role in the biological activities. [Copyright &y& Elsevier]

Costunolide is an active sesquiterpene lactone of medicinal herbs with anti-inflammatory and potential anti-cancer activity. Nevertheless, the pharmacological pathways of costunolide have not yet been fully elucidated. In this study we showed that costunolide exerts a dose-dependent antiproliferative activity in the human breast cancer MCF-7 cells. In addition, light microscopy observations indicated that costunolide affected nuclear organization and reorganized microtubule architecture. The antiproliferative and antimicrotubular effects of costunolide were not influenced by paclitaxel, well-known microtubule-stabilizing anticancer agent. The microtubule-interacting activity of costunolide was confirmed by in vitro studies on purified microtubular protein. In fact, costunolide demonstrated polymerizing ability, by inducing the formation of well organized microtubule polymers. Our data suggest an interaction of costunolide with microtubules, which may represent a new intracellular target for this drug. [Copyright &y& Elsevier]

Although much progress has been made in understanding synapse formation, little is known about the mechanisms underlying synaptic maintenance and loss. The formation of agrin-induced AChR clusters on cultured myotubes requires both activation of the receptor tyrosine kinase MuSK and intracellular calcium fluxes. Here, we provide evidence that such AChR clusters are maintained by agrin/MuSK-induced intracellular calcium fluxes. Clamping intracellular calcium fluxes after AChR clusters have formed leads to rapid MuSK and AChR tyrosine dephosphorylation and cluster dispersal, even in the continued presence of agrin. Both the dephosphorylation and the dispersal are inhibited by the tyrosine phosphatase inhibitor pervanadate. In contrast, clamping intracellular calcium at the time of initial agrin stimulation has no effect on agrin-induced MuSK or AChR phosphorylation, but blocks AChR cluster formation. These findings suggest an avenue by which postsynaptic stability can be regulated by modification of intracellular signaling pathways that are distinct from those used during synapse formation. [Copyright &y& Elsevier]

The aim of the present work was to analyse the interaction between Na+,K+-ATPase and one of our recent synthesized coumestans, namely the original molecule 2-methoxy-3,8,9-trihydroxy coumestan (PCALC36). Rat brain (mainly α2 and α3 Na+,K+-ATPase isoforms) and kidney (α1 isoform) fractions enriched with Na+,K+-ATPase were utilized to compare the inhibition promoted by PCALC36 with that of classical inhibitors like ouabain and vanadate. Analysis of inhibition curves revealed that unlike ouabain, which was about a thousand times more potent to inhibit brain isoforms than kidney isoform, PCALC36 had a similar affinity for brain (ic50=4.33±0.90 μM) and kidney (ic50=11.04±0.86 μM) isoforms. The inhibitory effect of PCALC36 was not antagonized by 1–10 mM K+, as observed with ouabain. Whereas vanadate was more potent in ionic conditions promoting the E2 conformation of the enzyme, the inhibitory effect of PCALC36 was equal in ionic conditions favouring either the E1 or E2 conformations. Equilibrium binding assays with [3H]ouabain revealed that the addition of 2–10 μM PCALC36 did not change the Kd of ouabain but decreased its maximal binding (Bmax) in a concentration-dependent manner (from 76.6 to 44.0 pmol/mg protein). This inhibitory effect of PCALC36 was not reverted after an extensive washing procedure indicating that it forms a very stable complex with Na+,K+-ATPase. We conclude that PCALC36, a new molecule with a non-steroidal skeleton, inhibits the Na+,K+-ATPase by a mechanism of action different from the cardiac glycosides and could thus serve as a structural paradigm to develop new inotropic drugs. [Copyright &y& Elsevier]

9-Bromo-5-morpholino-tetrazolo[1,5-c]quinazoline (BMTQ) acted cytotoxically on murine leukemia cell line L1210 and human colon carcinoma cells Caco-2. We found the two highest concentrations of BMTQ (149.2 and 74.6 μM) induced an acute cytotoxic effect, however other tested concentrations (<74.6 μM) manifested a concentration/dependent and time/dependent cytotoxic effect. The sensitivity of murine leukemia cells L1210 and human colon carcinoma cells Caco-2 was expressed in the same order. The cytotoxicity of BMTQ was not accompanied by changes of the cell cycle profile. Following the cytotoxicity-related effects of BMTQ we observed the induction of ssDNA breaks after BMTQ treatment. All the concentrations of BMTQ increased the level of ssDNA breaks 1.3–2.9 times (after 2 h of treatment) and 1.6–2.8 times (after 4 h of treatment) in Caco-2 cells compared to the control. No apoptotic DNA fragmentation induced by BMTQ in Caco-2 cells was recorded. [Copyright &y& Elsevier]

Oral administration of lindane (2.5, 5, 10 and 15 mg/kg, body weight) for 5 days was found to produce a dose-dependent increase in the activity of P450 dependent 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-dealkylase (PROD) and N-nitrosodimethylamine demethylase (NDMA-d) in rat brain and liver. A significant increase in the hepatic and brain P450 monooxygenases was also observed when the duration of exposure of low dose (2.5 mg/kg) of lindane was increased from 5 days to 15 or 21 days. As observed with different doses, the magnitude of induction in the activity of P450 monooxygenases was several fold higher in liver microsomes when compared with the brain. Western blotting studies have indicated that the increase in the P450 enzymes could be due to the increase in the expression of P450 1A1/1A2, 2B1/2B2 and 2E1 isoenzymes. In vitro studies using organic inhibitors specific for individual P450 isoenzymes and antibody inhibition experiments have further demonstrated that the increase in the activity of PROD, EROD and NDMA-d are due to the increase in the levels of P450 2B1/2B2, 1A1/1A2 and 2E1 isoenzymes, respectively. Induction studies have further shown that while pretreatment of 3-methylcholanthrene (MC), an inducer of P4501A1/1A2, did not produce any significant effect in the incidence of lindane induced convulsions, pretreatment with phenobarbital (PB), an inducer of P450 2B1/2B2 or ethanol, an inducer of P450 2E1 catalysed reactions, significantly increased the incidence of lindane induced convulsions. Similarly, when the P450-mediated metabolism of lindane was blocked by cobalt chloride incidence of convulsions was increased in animals treated with lindane indicating that lindane per se or its metabolites formed by PB or ethanol inducible P450 isoenzymes are involved in its neurobehavioral toxicity. [Copyright &y& Elsevier]