Gamma-hydroxybutyrate (GHB), 1,4-butanediol (1,4BD), and gamma-butyrolactone (GBL) intoxication: A state-of-the-art review.

γ-hydroxybutyrate (GHB) is synthesized endogenously from γ-aminobutyric acid (GABA) or exogenously from 1,4-butanediol (butane-1,4-diol; 1,4-BD) or γ-butyrolactone (GBL). GBL, and 1,4-BD are rapidly converted to GHB. The gastric absorption time, volume of distribution, and half-life of GHB are between 5 and 45 min, 0.49 ± 0.9 L/kg, and between 20 and 60 min, respectively. GHB and its analogues have a dose-dependent effect on the activation of GHB receptor, GABA-B, and GABA localized to the central nervous system. After ingestion, most patients present transient neurological disorders (lethal dose: 60 mg/kg). Chronic GHB consumption is associated with disorders of use and a withdrawal syndrome when the consumption is discontinued. GHB, GBL, and 1,4-BD are classified as narcotics but only the use of GHB is controlled internationally. They are used for drug facilitated (sexual) assault, recreational purposes, slamsex, and chemsex. To confirm an exogenous intake or administration of GHB, GBL, or 1-4-BD, the pre-analytical conservation is crucial. The antemortem cutoff doses for detection are 5 and 5-15 mg/L, with detection windows of 6 and 10 h in the blood and urine, respectively Control of GHB is essential to limit the number of users, abuse, associated risks, and death related to their consumption.

N-3-oxododecanoyl homoserine lactone exacerbates endothelial cell death by inducing receptor-interacting protein kinase 1-dependent apoptosis.

Endothelial dysfunction is associated with the initiation of sepsis-associated organ failure. Bacterial quorum-sensing molecules act as pathogen-associated molecular patterns; however, the effects of quorum-sensing molecules on endothelial cells remain less understood. This study investigated the molecular mechanisms of quorum-sensing molecule-induced cell death and their interaction with lipopolysaccharide (LPS) in human umbilical vein endothelial cells. Endothelial cells were treated with N-3-oxododecanoyl homoserine lactone (3OC12-HSL) and LPS derived from Pseudomonas aeruginosa. Treatment with 3OC12-HSL reduced cell viability in a dose-dependent manner, and cotreatment with 3OC12-HSL and LPS enhanced cell death. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assay revealed an increase in apoptotic cell death following 3OC12-HSL treatment; furthermore, cotreatment with 3OC12-HSL and LPS enhanced apoptosis. Western blotting revealed that treatment with 3OC12-HSL activated the receptor-interacting protein kinase 1 (RIPK1) pathway, leading to an increase in the levels of cleaved caspase 8 and 3. In addition, we found that treatment with necrostatin-1, an RIPK1 inhibitor, reduced cell death and ameliorated the activation of the RIPK1-dependent apoptotic pathway in 3OC12-HSL-treated cells. In conclusion, 3OC12-HSL induced endothelial cell apoptosis via the activation of the RIPK1 pathway, independent of LPS toxicity. Inhibition of RIPK1 may act as a therapeutic option for preserving endothelial cell integrity in patients with sepsis by disrupting the mechanism by which quorum-sensing molecules mediate their toxicity.

Diffusible Signaling Factor, a Quorum-Sensing Molecule, Interferes with and Is Toxic Towards Bdellovibrio bacteriovorus 109J.

Bdellovibrio bacteriovorus 109J is a predatory bacterium which lives by predating on other Gram-negative bacteria to obtain the nutrients it needs for replication and survival. Here, we evaluated the effects two classes of bacterial signaling molecules (acyl homoserine lactones (AHLs) and diffusible signaling factor (DSF)) have on B. bacteriovorus 109J behavior and viability. While AHLs had a non-significant impact on predation rates, DSF considerably delayed predation and bdelloplast lysis. Subsequent experiments showed that 50 µM DSF also reduced the motility of attack-phase B. bacteriovorus 109J cells by 50% (38.2 ± 14.9 vs. 17 ± 8.9 µm/s). Transcriptomic analyses found that DSF caused genome-wide changes in B. bacteriovorus 109J gene expression patterns during both the attack and intraperiplasmic phases, including the significant downregulation of the flagellum assembly genes and numerous serine protease genes. While the former accounts for the reduced speeds observed, the latter was confirmed experimentally with 50 µM DSF completely blocking protease secretion from attack-phase cells. Additional experiments found that 30% of the total cellular ATP was released into the supernatant when B. bacteriovorus 109J was exposed to 200 µM DSF, implying that this QS molecule negatively impacts membrane integrity.

A toxicogenomics approach reveals characteristics supporting the honey bee (Apis mellifera L.) safety profile of the butenolide insecticide flupyradifurone.

Flupyradifurone, a novel butenolide insecticide, selectively targets insect nicotinic acetylcholine receptors (nAChRs), comparable to structurally different insecticidal chemotypes such as neonicotinoids and sulfoximines. However, flupyradifurone was shown in acute toxicity tests to be several orders of magnitude less toxic to western honey bee (Apis mellifera L.) than many other insecticides targeting insect nAChRs. The underlying reasons for this difference in toxicity remains unknown and were investigated here. Pharmacokinetic studies after contact application of [14C]flupyradifurone to honey bees revealed slow uptake, with internalized compound degraded into a few metabolites that are all practically non-toxic to honey bees in both oral and contact bioassays. Furthermore, receptor binding studies revealed a lack of high-affinity binding of these metabolites to honey bee nAChRs. Screening of a library of 27 heterologously expressed honey bee cytochrome P450 enzymes (P450s) identified three P450s involved in the detoxification of flupyradifurone: CYP6AQ1, CYP9Q2 and CYP9Q3. Transgenic Drosophila lines ectopically expressing CYP9Q2 and CYP9Q3 were significantly less susceptible to flupyradifurone when compared to control flies, confirming the importance of these P450s for flupyradifurone metabolism in honey bees. Biochemical assays using the fluorescent probe substrate 7-benzyloxymethoxy-4-(trifluoromethyl)-coumarin (BOMFC) indicated a weak, non-competitive inhibition of BOMFC metabolism by flupyradifurone. In contrast, the azole fungicides prochloraz and propiconazole were strong nanomolar inhibitors of these flupyradifurone metabolizing P450s, explaining their highly synergistic effects in combination with flupyradifurone as demonstrated in acute laboratory contact toxicity tests of adult bees. Interestingly, the azole fungicide prothioconazole is only slightly synergistic in combination with flupyradifurone - an observation supported by molecular P450 inhibition assays. Such molecular assays have value in the prediction of potential risks posed to bees by flupyradifurone mixture partners under applied conditions. Quantitative PCR confirmed the expression of the identified P450 genes in all honey bee life-stages, with highest expression levels observed in late larvae and adults, suggesting honey bees have the capacity to metabolize flupyradifurone across all life-stages. These findings provide a biochemical explanation for the low intrinsic toxicity of flupyradifurone to honey bees and offer a new, more holistic approach to support bee pollinator risk assessment by molecular means.

Flupyradifurone reduces nectar consumption and foraging but does not alter honey bee recruitment dancing.

Foraging is essential for honey bee colony fitness and is enhanced by the waggle dance, a recruitment behavior in which bees can communicate food location and quality. We tested if the consumption of nectar (sucrose solution) with a field-realistic concentration of 4 ppm flupyradifurone (FPF) could alter foraging behavior and recruitment dancing in Apis mellifera. Foragers were repelled by FPF. They visited the FPF feeder less often and spent less time imbibing sucrose solution (2.5 M, 65% w/w) with FPF. As a result, bees feeding on the FPF treatment consumed 16% less nectar. However, FPF did not affect dancing: there were no effects on unloading wait time, the number of dance bouts per nest visit, or the number of dance circuits performed per dance bout. FPF could therefore deter bees from foraging on contaminated nectar. However, the willingness of bees to recruit nestmates for nectar with FPF is concerning. Recruitment can rapidly amplify the number of foragers and could overcome the decrease in consumption of FPF-contaminated nectar, resulting in a net inflow of pesticide to the colony. FPF also significantly altered the expression of 116 genes, some of which may be relevant for the olfactory learning deficits induced by FPF and the toxicity of FPF.

Caspase-1-dependent mechanism mediating the harmful impacts of the quorum-sensing molecule N-(3-oxo-dodecanoyl)-l-homoserine lactone on the intestinal cells.

N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), a quorum-sensing (QS) molecule produced by Gram-negative bacteria in the gastrointestinal tract, adversly impacts host cells. Our previous study demonstrated that 3-oxo-C12-HSL induced a decrease in cell viability via cell apoptosis and eventually disrupted mucin synthesis from LS174T goblet cells. However, the molecular mechanism underlying cell apoptosis and whether pyroptosis was involved in this process are still unknown. In this study, we emphasized on the caspases signal pathway and sterile inflammation to reveal the harmful effects of 3-oxo-C12-HSL on LS174T goblet cells. Our data showed that 3-oxo-C12-HSL is a major inducer of oxidative stress indicated by a high level of intracellular reactive oxygen species (ROS). However, TQ416, an inhibitor of paraoxonase 2, can effectively block oxidative stress. A higher ROS level is the trigger for activating the caspase-1 and 3 cascade signal pathways. Blockade of ROS synthesis and caspase-1 and 3 cascades can obviously rescue the viability of LS174T cells after 3-oxo-C12-HSL treatment. We also found that paralleled with a higher level of ROS and caspases activation, an abnormal expression of proinflammatory cytokines was induced by 3-oxo-C12-HSL treatment; however, the blockage of TLRs-NF-κB pathway cannot restore cell viability and secretary function. These data collectively indicate that 3-oxo-C12-HSL exposure induces damages to cell viability and secretary function of LS174T goblet cells, which is mediated by oxidative stress, cell apoptosis, and sterile inflammation. Overall, the data in this study will provide a better understanding of the harmful impacts of some QS molecules on host cells and their underlying mechanism.

Lethal and sublethal synergistic effects of a new systemic pesticide, flupyradifurone (Sivanto®), on honeybees.

The honeybee ( Apis mellifera L.) is an important pollinator and a model for pesticide effects on insect pollinators. The effects of agricultural pesticides on honeybee health have therefore raised concern. Bees can be exposed to multiple pesticides that may interact synergistically, amplifying their side effects. Attention has focused on neonicotinoid pesticides, but flupyradifurone (FPF) is a novel butenolide insecticide that is also systemic and a nicotinic acetylcholine receptor (nAChR) agonist. We therefore tested the lethal and sublethal toxic effects of FPF over different seasons and worker types, and the interaction of FPF with a common SBI fungicide, propiconazole. We provide the first demonstration of adverse synergistic effects on bee survival and behaviour (poor coordination, hyperactivity, apathy) even at FPF field-realistic doses (worst-case scenarios). Pesticide effects were significantly influenced by worker type and season. Foragers were consistently more susceptible to the pesticides (4-fold greater effect) than in-hive bees, and both worker types were more strongly affected by FPF in summer as compared with spring. Because risk assessment (RA) requires relatively limited tests that only marginally address bee behaviour and do not consider the influence of bee age and season, our results raise concerns about the safety of approved pesticides, including FPF. We suggest that pesticide RA also test for common chemical mixture synergies on behaviour and survival.

Treatment of γ-Hydroxybutyric Acid and γ-Butyrolactone Overdose with Two Potent Monocarboxylate Transporter 1 Inhibitors, AZD3965 and AR-C155858.

The illicit use of γ-hydroxybutyric acid (GHB), and its prodrug, γ-butyrolactone (GBL), results in severe adverse effects including sedation, coma, respiratory depression, and death. Current treatment of GHB/GBL overdose is limited to supportive care. Recent reports indicate that GHB-related deaths are on the rise; a specific treatment may reduce lethality associated with GHB/GBL. Pretreatment with inhibitors of monocarboxylate transporter 1 (MCT1), a transporter that mediates many of the processes involved in the absorption, distribution (including brain uptake), and elimination of GHB/GBL, has been shown to prevent GHB-induced respiratory depression by increasing the renal clearance of GHB. To identify whether MCT1 inhibition is an effective treatment of GHB overdose, the impact of two MCT1 inhibitors, (S)-5-(4-hydroxy-4-methylisoxazolidine-2-carbonyl)-1-isopropyl-3-methyl-6-((3-methyl-5-(trifluoromethyl)-1H-pyrazol-4-yl)methyl)thieno[2,3-day]pyrimidine-2,4(1H,3H)-dione (AZD3965) and 6-[(3,5-dimethyl-1H-pyrazol-4-yl)methyl]-5-[[(4S)-4-hydroxy-2-isoxazolidinyl]carbonyl]-3-methyl-1-(2-methylpropyl)thieno[2,3-day]pyrimidine2,4(1H,3H)-dione (AR-C155858), on the toxicokinetics and toxicodynamics of GHB/GBL was assessed when the administration of the inhibitor was delayed 60 and 120 minutes (post-treatment) after administration of GHB/GBL. AR-C155858 and AZD3965 reduced the toxicodynamic effects of GHB when GHB was administered intravenously, orally, or orally as the prodrug GBL. The impact of these inhibitors on GHB toxicokinetics was dependent on the route of GHB administration and the delay between GHB/GBL administration and administration of the MCT1 inhibitor. The reduction in GHB plasma exposure did not explain the observed effect of MCT1 inhibition on GHB-induced respiratory depression. The efficacy of MCT1 inhibition on GHB toxicodynamics is likely driven by the pronounced reduction in GHB brain concentrations. Overall, this study indicates that inhibition of MCT1 is an effective treatment of GHB/GBL overdose.

Acute and chronic toxicity of neonicotinoid and butenolide insecticides to the freshwater amphipod, Hyalella azteca.

Neonicotinoids are the most widely used insecticides in the world. They are preferentially toxic to insects while displaying a low toxicity toward vertebrates, and this selective toxicity has resulted in the rapid and ubiquitous use of these compounds. However, neonicotinoids have been detected in agricultural surface waters and are known to cause adverse effects in non-target aquatic organisms. A wide range of toxicity has been reported for aquatic crustaceans, but most of the studies focus on the acute effects of imidacloprid, and few data are available regarding chronic effects of other neonicotinoids or neonicotinoid replacements (e.g., butenolides). The objective of this study was to assess the acute and chronic toxicity of six neonicotinoids (imidacloprid, thiamethoxam, acetamiprid, clothianidin, thiacloprid, and dinotefuran) and one butenolide (flupyradifurone) to the freshwater amphipod Hyalella azteca. Chronic (28-d), water-only, static-renewal tests were conducted. Survival was assessed weekly, and growth was measured at the end of the exposure. Effects of neonicotinoids varied depending on the compound. Acute (7-d) LC50s were 4.0, 4.7, 60, 68, 230, and 290 µg/L for clothianidin, acetamiprid, dinotefuran, thiacloprid, imidacloprid, and thiamethoxam, respectively. Chronic (28-d) survival and growth were reduced at similar concentrations to acute (7-d) survival for thiamethoxam, acetamiprid, clothianidin, and dinotefuran. However, chronic survival and growth of amphipods exposed to imidacloprid and thiacloprid were reduced at lower concentrations than acute survival, with respective 28-d LC50s of 90 and 44 µg/L, and EC50s of 4 and 3 µg/L. Flupyradifurone was intermediate in toxicity compared to the neonicotinoids: 7-d LC50, 28-d LC50, and 28-d EC50 were 26, 20, and 16 µg/L, respectively. The concentrations of imidacloprid and clothianidin reported for North American surface waters fall within the effect ranges observed in this study, indicating the potential for these compounds to cause adverse effects to indigenous populations of H. azteca.

The novel pesticide flupyradifurone (Sivanto) affects honeybee motor abilities.

Honeybees and other pollinators are threatened by changing landscapes and pesticides resulting from intensified agriculture. In 2018 the European Union prohibited the outdoor use of three neonicotinoid insecticides due to concerns about pollinators. A new pesticide by the name of "Sivanto" was recently released by Bayer AG. Its active ingredient flupyradifurone binds to the nicotinic acetylcholine receptor (AchR) in the honeybee brain, similar to neonicotinoids. Nevertheless, flupyradifurone is assumed to be harmless for honeybees and can even be applied on flowering crops. So far, only little has been known about sublethal effects of flupyradifurone on honeybees. Intact motor functions are decisive for numerous behaviors including foraging and dancing. We therefore selected a motor assay to investigate in how far sublethal doses of this pesticide affect behavior in young summer and long-lived winter honeybees. Our results demonstrate that flupyradifurone (830 µmol/l) can evoke motor disabilities and disturb normal motor behavior after a single oral administration (1.2 µg/bee). These effects are stronger in long-lived winter bees than in young summer bees. After offering an equal amount of pesticide (1.0-1.75 µg) continuously over 24 h with food the observed effects are slighter. For comparisons we repeated our experiments with the neonicotinoid imidacloprid. Intriguingly, the alterations in behavior induced by this pesticide (4 ng/bee) were different and longer-lasting compared to flupyradifurone, even though both substances bind to nicotinic acetylcholine receptors.

Cytogenetic alterations induced by flupyradifurone, a new butenolide insecticide, in human lymphocytes.

Flupyradifurone (FPD), a member of the new class of butenolide insecticides, acts on nicotinic acetylcholine receptors. Studies on genotoxic and carcinogenic effects of FPD are very limited. This is the first study to investigate the cytotoxic and genotoxic effects of FPD and its metabolites on human lymphocyte cultures with or without a metabolic activation system (S9 mix) using chromosomal aberration (CA) and micronucleus (MN) tests. The cultures were treated with 85, 170, and 340 µg/ml of FPD in the presence (3 h treatment) and absence (48 h treatment) of S9 mix. Dimethyl sulfoxide (DMSO) was used as a solvent control. Statistically significant decreases were detected at the medium and highest concentrations for 48 h treatments while decreases in mitotic index (MI) in the presence of the S9 mix were found statistically significant at all FPD concentrations tested when compared with the solvent control. FPD also decreased the nuclear division index (NDI) at the highest concentration (340 µg/ml) in the absence of S9 mix. When compared with the solvent control, increases in CA frequencies were significant at the medium and highest concentrations. Significantly increased MN frequency was only found at the highest FPD concentration in cultures without S9 mix compared with the solvent control while increases in the MN frequencies in the presence of S9 mix were statistically significant at all FPD concentrations. The results of the present study indicate that FPD and its metabolites can show cytotoxic and genotoxic effects in human lymphocytes. More genotoxicity studies are necessary to make a possible risk assessment in humans.

Synthesis and QSAR study of novel α-methylene-γ-butyrolactone derivatives as antifungal agents.

Thirty-six new α-benzylidene-γ-lactone compounds based α-methylene-γ-butyrolactone substructure were prepared and characterized by spectroscopic analysis. All compounds were evaluated for antifungal activities in vitro against six plant pathogenic fungi and the half maximal inhibitory concentration (IC50) against Botrytis cinerea and Colletotrichum lagenarium were investigated. Compounds 5c-3 and 5c-5 with the halogen atom exhibited excellent fungicidal activity against B. cinerea (IC50=22.91, 18.89µM). The structure-activity relationships (SARs) analysis indicated that the derivatives with electron-withdrawing substituents at the meta- or para-positions improves the activity. Via the heuristic method, the generated quantitative structure-activity relationship (QSAR) model (R2=0.961) revealed a strong correlation of antifungal activity against B. cinerea with molecular structures of these compounds. Meanwhile, the cytotoxicity of 20 representative derivatives was tested in the human tumor cells line (HepG2) and the hepatic L02 cells line, the result indicated that the synthesized compounds showed significant inhibitory activity and limited selectivity. Compound 5c-5 has the highest fungicidal activity with IC50=18.89µM (against B. cinerea.) but low cytotoxicity with IC50=35.4µM (against HepG2 cell line) and IC50=68.8µM (against Hepatic L02 cell line). These encouraging results can be providing an alternative, promising use of α-benzylidene-γ-lactone through the design and exploration of eco-friendly fungicides with low toxicity and high efficiency.

Rebound burst firing in the reticular thalamus is not essential for pharmacological absence seizures in mice.

Intrinsic burst and rhythmic burst discharges (RBDs) are elicited by activation of T-type Ca(2+) channels in the thalamic reticular nucleus (TRN). TRN bursts are believed to be critical for generation and maintenance of thalamocortical oscillations, leading to the spike-and-wave discharges (SWDs), which are the hallmarks of absence seizures. We observed that the RBDs were completely abolished, whereas tonic firing was significantly increased, in TRN neurons from mice in which the gene for the T-type Ca(2+) channel, CaV3.3, was deleted (CaV3.3(-/-)). Contrary to expectations, there was an increased susceptibility to drug-induced SWDs both in CaV3.3(-/-) mice and in mice in which the CaV3.3 gene was silenced predominantly in the TRN. CaV3.3(-/-) mice also showed enhanced inhibitory synaptic drive onto TC neurons. Finally, a double knockout of both CaV3.3 and CaV3.2, which showed complete elimination of burst firing and RBDs in TRN neurons, also displayed enhanced drug-induced SWDs and absence seizures. On the other hand, tonic firing in the TRN was increased in these mice, suggesting that increased tonic firing in the TRN may be sufficient for drug-induced SWD generation in the absence of burst firing. These results call into question the role of burst firing in TRN neurons in the genesis of SWDs, calling for a rethinking of the mechanism for absence seizure induction.

Determination of two potential toxicity metabolites derived from the disruption of the pksCT gene in Monascus aurantiacus Li As3.4384.

BACKGROUND: We previously demonstrated that disruption of the pksCT gene of Monascus led to a greater than 98% decrease in its citrinin production capacity in Monascus (PHDS26). Two potentially toxic compounds, monascopyridine A (MPA) and monascopyridine B (MPB), were found in the fermentation products of the pksCT gene-disrupted Monascus. Moreover, a rapid and reliable high-performance liquid chromatography method was developed for the simultaneous determination of MPA and MPB. We studied the effects of various extraction parameters and designed an orthogonal experiment to investigate the importance of each factor. RESULTS: The optimal extraction conditions were: methanol concentration, 90%; extraction temperature, 40 °C; extraction time, 10 min; two extraction cycles; and a solid-liquid ratio of 1:25. Under the optimal chromatographic conditions, good linearity was reached over the concentration ranges 0.5-200 µg mL-1 and 0.5-300 µg mL-1 for MPA and MPB, respectively, and the corresponding determination coefficients were 0.9999 and 0.9997. The percentage relative standard deviation values of within-day and between-day precision for MPA were 2.0% and 2.1%, respectively; the corresponding values for MPB were 4.8% and 4.6%. The average recovery for MPA and MPB was 99.9% and 94%, respectively. CONCLUSION: Maximum MPA and MPB yields (2073.7 and 1961.7 µg g-1 , respectively) were observed after 16 days of cultivation. © 2017 Society of Chemical Industry.

A proteomic approach for the identification of immunotoxic properties of Tulipalin A.

The immune system is permanently exposed to several environmental influences that can have adverse effects on immune cells or organs leading to immunosuppression or inappropriate immunostimulation, called direct immunotoxicity. The natural compound Tulipalin A (TUPA), a lactone with α-methylene-γ-butyrolactone moiety, can influence the immune system and lead to allergic contact dermatitis. This in vitro study focused on effects of TUPA using two immune cell lines (Jurkat T cells and THP-1 monocytes). To evaluate the immunotoxic potential of the compound, a proteomic approach applying 2D gel electrophoresis and MALDI-TOF/TOF-MS in combination with metabolomic analysis was used after exposure of the cells to IC10 of TUPA. THP-1 cells showed a strong robustness to TUPA treatment since only five proteins were altered. In contrast, in Jurkat T cells an increase in the abundance of 66 proteins and a decrease of six proteins was determined. These intracellular proteins were mapped to biological processes. Especially an accumulation of chaperones and an influence on the purine synthesis were observed. The changes in purine synthesis were confirmed by metabolomic analysis. In conclusion, the data indicate possible target processes of low doses of TUPA in Jurkat T cells and provides knowledge of how TUPA affects the functionality of immune cells.

Matrix Effect of Human Reconstructed Epidermis on the Chemoselectivity of a Skin Sensitizing α-Methylene-γ-Butyrolactone: Consequences for the Development of in Chemico Alternative Methods.

Adoption of new legislations and social pressure are pushing toward the development of alternative methods to the use of animals for the assessment of most toxicological end-points including skin sensitization. To that respect, much efforts have been put in the first step of the adverse outcome pathway focusing on chemical interactions taking place between sensitizing chemicals or haptens and epidermal proteins. However, these in chemico approaches have been so far only based on the use of model nucleophiles, amino acids, peptides, or proteins in water/buffer solution and focused mainly on thiol reactivity. These studies even if bringing a valuable set of information are very far from reflecting chemical interactions that may happen between a xenobiotic and nucleophiles present in a complex heterogeneous tissue such as the epidermis. Recently, we have shown that using a high-resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR) technique it was possible to characterize chemical interactions taking place between a skin sensitizer and nucleophilic amino acids present in a 3-D reconstructed human epidermis (RHE). We have now compared the chemical reactivity and chemoselectivity of a sensitizing α-methylene-γ-butyrolactone toward human serum albumin used as a model protein and RHE. Using this technique, we showed that amino acid modifications by this hapten was different according to the model used and that in RHE histidine residues seem to have an important role in the formation of adducts. Obviously, the role of histidine in the induction of skin sensitization has been so far neglected and should probably be taken into account for the refinement of in chemico approaches for the detection and potency classification of skin sensitizers.

Small molecule screen yields inhibitors of Pseudomonas homoserine lactone-induced host responses.

Pseudomonas aeruginosa infections are commonly associated with cystic fibrosis, pneumonias, neutropenia and burns. The P. aeruginosa quorum sensing molecule N-(3-oxo-dodecanoyl) homoserine lactone (C12) cause multiple deleterious host responses, including repression of NF-κB transcriptional activity and apoptosis. Inhibition of C12-mediated host responses is predicted to reduce P. aeruginosa virulence. We report here a novel, host-targeted approach for potential adjunctive anti-Pseudomonal therapy based on inhibition of C12-mediated host responses. A high-throughput screen was developed to identify C12 inhibitors that restore NF-κB activity in C12-treated, lipopolysaccharide (LPS)-stimulated cells. Triazolo[4,3-a]quinolines with nanomolar potency were identified as C12-inhibitors that restore NF-κB-dependent luciferase expression in LPS- and TNF-stimulated cell lines. In primary macrophages and fibroblasts, triazolo[4,3-a]quinolines inhibited C12 action to restore cytokine secretion in LPS-stimulated cells. Serendipitously, in the absence of an inflammatory stimulus, triazolo[4,3-a]quinolines prevented C12-mediated responses, including cytotoxicity, elevation of cytoplasmic calcium, and p38 MAPK phosphorylation. In vivo efficacy was demonstrated in a murine model of dermal inflammation involving intradermalC12 administration. The discovery of triazolo[4,3-a]quinolines provides a pharmacological tool to investigate C12-mediated host responses, and a potential host-targeted anti-Pseudomonal therapy.

Synthesis and cytotoxicity evaluation of 4-amino-4-dehydroxylarctigenin derivatives in glucose-starved A549 tumor cells.

The natural product arctigenin (ATG) demonstrated preferential cytotoxicity to cancer cells under glucose starvation. A series of 4-amino-4-dehydroxylarctigenin derivatives based on lead compound ATG were designed and synthesized by bioisosteric modifications. Their cytotoxicities were evaluated in glucose-starved A549 tumor cells and the results indicated that the 4-amino-4-dehydroxylarctigenin showed more potent cytotoxicity than arctigenin, and the further substituent group on 4-amino would result in the cytotoxicities decreased significantly. 4-Substituted-arctigenin could selectively target on glucose-starved A549 tumor cells which provide an alternative strategy for anticancer drug development with minimal normal tissue toxicity.

Hepatic proteomic responses in marine medaka (Oryzias melastigma) chronically exposed to antifouling compound butenolide [5-octylfuran-2(5H)-one] or 4,5-dichloro-2-N-octyl-4-isothiazolin-3-one (DCOIT).

The pollution of antifoulant SeaNine 211, with 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT) as active ingredient, in coastal environment raises concerns on its adverse effects, including endocrine disruption and impairment of reproductive function in marine organisms. In the present study, we investigated the hepatic protein expression profiles of both male and female marine medaka (Oryzias melastigma) exposed to low concentrations of DCOIT at 2.55 µg/L (0.009 µM) or butenolide, a promising antifouling agent, at 2.31 µg/L (0.012 µM) for 28 days. The results showed that proteins involved in phase I (CYP450 enzyme) metabolism, phase II (UDPGT and GST) conjugation as well as mobilization of retinoid storage, an effective nonenzymatic antioxidant, were consistently up-regulated, possibly facilitating the accelerated detoxification of butenolide. Increased synthesis of bile acid would promote the immediate excretion of butenolide metabolites. Activation of fatty acid ß-oxidation and ATP synthesis were consistent with elevated energy consumption for butenolide degradation and excretion. However, DCOIT did not significantly affect the detoxification system of male medaka, but induced a marked increase of vitellogenin (VTG) by 2.3-fold in the liver of male medaka, suggesting that there is estrogenic activity of DCOIT in endocrine disruption. Overall, this study identified the molecular mechanisms and provided sensitive biomarkers characteristic of butenolide and DCOIT in the liver of marine medaka. The low concentrations of butenolide and DCOIT used in the exposure regimes highlight the needs for systematic evaluation of their environmental risk. In addition, the potent estrogenic activity of DCOIT should be considered in the continued applications of SeaNine 211.

RE12 derivatives displaying Vaccinia H1-related phosphatase (VHR) inhibition in the presence of detergent and their anti-proliferative activity against HeLa cells.

New derivatives of Vaccinia H1-related phosphatase (VHR) inhibitor RE12 (5) were designed by replacing the long straight alkyl chain with other hydrophobic functionalities containing two aromatic rings, with the aim of obtaining potent, cell-active inhibitors. We established a direct coupling reaction between tetronic acid derivative and thioimidate to prepare the RE derivatives 6a-6i efficiently. These compounds all showed VHR-inhibitory activity in the presence of 0.001% NP-40, whereas RE12 (5) was inactive under this condition, even at 100 µM. Further structure-activity studies focused on terminal substitution afforded trifluoromethyl derivative 6k (RE176) and nitro derivative 6l (RE177). The IC50 value of 6l in the presence of NP-40 was almost equivalent to that of RE12 (5) in its absence. Compound 6k (RE176) potently inhibited proliferation of HeLa cells.