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The red imported fire ant (RIFA), Solenopsis invicta , is an invasive pest that causes damage to agricultural and ecological environments worldwide. Fluralaner is a new isoxazoline pesticide with the potential to become a control agent against RIFA. However, it is not clear whether S. invicta responds the same way to fluralaner at different reproductive stages. The present study firstly evaluated the toxicity of fluralaner to S. invicta at different developmental stages, finding that fourth instar larvae (LD 50 , 1744.23 mg/kg) and worker ants (LD 50 , 8.62 mg/kg) were differently susceptible to fluralaner, while the mortality rate of fourth instar larvae was significantly lower at the same concentration of 10 mg/L (5.56 ± 3.14%) than that of worker ants (62.22 ± 3.14%), demonstrating a greater tolerance to fluralaner. Subsequently, the metabolic responses of worker and larval ants to fluralaner stress (10 mg/L) were investigated using non-targeted metabolomics, which indicated that the amount of differential metabolites and the KEGG metabolic pathways enriched were different between workers and larvae when exposed to the same dose (10 mg/L) of fluralaner. Differential metabolites of larvae and worker ants under fluralaner stress were mainly concentrated in organic acids and their derivatives, lipids and lipid-like molecules, nucleosides, nucleotides, and analogues, combined with the enriched metabolic pathways, revealed that the differential metabolic responses of larvae and worker ants were mainly in energy metabolism, detoxification metabolism, and neurotransmitter ligands. Workers consumed more substrates in the arginine synthesis pathway (l-glutamic acid, l-aspartic acid, and fumaric acid) to provide energy for the detoxification (glutathione) of pesticides when exposed to fluralaner stress, and the high accumulation of l-aspartic acid induced excitotoxicity in the worker ants. Larval ants consumed more arachidonic acid to synthesize PG D2, and changes in the metabolism of antioxidants such as catechins, hesperidin, and l-ascorbic acid suggested that larvae were more capable of scavenging the ROS response than worker ants. The results of non-targeted metabolomics successfully revealed differences in the sensitivity of larvae and workers to fluralaner agents, providing insights into the fluralaner control of Solenopsis invicta .

Small drug-like molecules that can block the function of serotonin 5-HT 2A receptors have garnered considerable attention due to their ability to inhibit platelet aggregation and the possible prevention of atherosclerotic lesions. Although clinical data provided compelling evidence for the efficacy of this approach in the prevention of various cardiovascular conditions, the chemical space of 5-HT 2A receptor antagonists is limited to ketanserin and sarpogrelate. To expand the portfolio of novel chemical motifs with potential antiplatelet activity, we evaluated the antiplatelet activity of a series of 6-fluorobenzo[d]isoxazole derivatives that possess a high affinity for 5-HT 2A receptor. Here we describe in vitro studies showing that 6-fluorobenzo[d]isoxazole derivatives exert promising antiplatelet activity in three various in vitro models of platelet aggregation, as well as limit serotonin-induced vasoconstriction. Compound AZ928 showed in vitro activity greater than the clinically approved drug sarpogrelate. In addition to promising antiplatelet activity, the novel series was characterized by a favorable safety profile. Our findings show that the novel series exerts promising antiplatelet efficacy while being deprived of potential side effects, such as hemolytic activity, which render these compounds as potential substances for further investigation in the field of cardiovascular research., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Fluralaner, a systemic pesticide, was originally registered with the US Food and Drug Administration in 2014 under the trade name Bravecto for flea treatment for pets. As a GABA antagonist, the footprint of fluralaner has expended beyond medical and veterinary pests in recent years. In this study, we examined the acute toxicity of fluralaner against three pests of Henosepilachna vigintioctopunctata, Megalurothrips usitatus, and Phyllotreta striolata in the Solanaceae, Fabaceae, and Cruciferae families, respectively, and the sublethal impact of fluralaner on Propylaea japonica, a widely distributed predatory ladybeetle. Based on LC 50 , fluralaner was effective against H. vigintioctopunctata (0.098 mg a.i. L -1 for the second instar larvae), M. usitatus (0.134 mg a.i. L -1 for adult females), and P. striolata (0.595 mg a.i. L -1 for adults). For P. japonica, however, fluralaner was substantially less effective (1.177 mg a.i. L -1 for the third instar larvae). Furthermore, the LC 10 and LC 30 of P. japonica were also consistently higher than the LC 50 of the three pests. In addition, we did not observe any significant impacts of fluralaner at LC 10 and LC 30 on the life history traits, including body weight, developmental time, pre-oviposition period, and fecundity of P. japonica. Based on our results from acute toxicities and sublethal impacts, fluralaner is effective against vegetable pests, while potentially friendly to P. japonica when employed as a biological control agent., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Lysophosphatidic acid receptors (LPARs) are G-protein-coupled receptors involved in many physiological functions in the central nervous system. However, the role of the LPARs in multiple sclerosis (MS) has not been clearly defined yet., Methods: Here, we investigated the roles of LPARs in myelin oligodendrocyte glycoprotein peptides-induced experimental autoimmune encephalomyelitis (EAE), an animal model of MS., Results: Pre-inhibition with LPAR1-3 antagonist Ki16425 deteriorated motor disability of EAE low . Specifically, LPAR1-3 antagonist (intraperitoneal) deteriorated symptoms of EAE low associated with increased demyelination, chemokine expression, cellular infiltration, and immune cell activation (microglia and macrophage) in spinal cords of mice compared to the sham group. This LPAR1-3 antagonist also increased the infiltration of CD4 + /IFN-γ + (Th1) and CD4 + /IL-17 + (Th17) cells into spinal cords of EAE low mice along with upregulated mRNA expression of IFN-γ and IL-17 and impaired blood-brain barrier (BBB) in the spinal cord. The underlying mechanism for negative effects of LPAR1-3 antagonist was associated with the overproduction of reactive oxygen species (ROS)-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) 2 and NOX3. Interestingly, LPAR1/2 agonist 1-oleoyl-LPA (LPA 18:1) (intraperitoneal) ameliorated symptoms of EAE high and improved representative pathological features of spinal cords of EAE high mice., Conclusions: Our findings strongly suggest that some agents that can stimulate LPARs might have potential therapeutic implications for autoimmune demyelinating diseases such as MS., (© 2021. The Author(s).)

Background: Ocular adverse events are common dose-limiting toxicities in cancer patients treated with HSP90 inhibitors, such as AUY922; however, the pathology and molecular mechanisms that mediate AUY922-induced retinal toxicity remain undescribed., Methods: The impact of AUY922 on mouse retinas and cell lines was comprehensively investigated using isobaric tags for relative and absolute quantitation (iTRAQ)‑based proteomic profiling and pathway enrichment analysis, immunohistochemistry and immunofluorescence staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, MTT assay, colony formation assay, and western blot analysis. The effect of AUY922 on the Transient Receptor Potential cation channel subfamily M member 1 (TRPM1)-HSP90 chaperone complex was characterized by coimmunoprecipitation. TRPM1-regulated gene expression was analyzed by RNAseq analysis and gene set enrichment analysis (GSEA). The role of TRPM1 was assessed using both loss-of-function and gain-of-function approaches., Results: Here, we show that the treatment with AUY922 induced retinal damage and cell apoptosis, dysregulated the photoreceptor and retinal pigment epithelium (RPE) layers, and reduced TRPM1 expression. Proteomic profiling and functional annotation of differentially expressed proteins reveals that those related to stress responses, protein folding processes, regulation of apoptosis, cell cycle and growth, reactive oxygen species (ROS) response, cell junction assembly and adhesion regulation, and proton transmembrane transport were significantly enriched in AUY922-treated cells. We found that AUY922 triggered caspase-3-dependent cell apoptosis, increased ROS production and inhibited cell growth. We determined that TRPM1 is a bona fide HSP90 client and characterized that AUY922 may reduce TRPM1 expression by disrupting the CDC37-HSP90 chaperone complex. Additionally, GSEA revealed that TRPM1-regulated genes were associated with retinal morphogenesis in camera-type eyes and the JAK-STAT cascade. Finally, gain-of-function and loss-of-function analyses validated the finding that TRPM1 mediated the cell apoptosis, ROS production and growth inhibition induced by AUY922., Conclusions: Our study demonstrates the pathology of AUY922-induced retinal toxicity in vivo. TRPM1 is an HSP90 client, regulates photoreceptor morphology and function, and mediates AUY922-induced cytotoxicity., (© 2021. The Author(s).)

Filth flies cause billions of dollars of losses annually to the animal production industry. Fluralaner is a relatively new pesticide currently sold for control of fleas, ticks, and mites on companion animals and poultry. We examined the efficacy of fluralaner against three species of filth flies. Insecticide-susceptible horn flies and stable flies were tested topically. Fluralaner outperformed permethrin by > 2-fold for the horn flies but underperformed permethrin by > 45-fold for stable flies at 24 h. House flies were tested topically with fluralaner in comparison to permethrin at 48 h and orally with fluralaner in comparison to imidacloprid at 24 h. Topical fluralaner was 6- to 28-fold as toxic as permethrin in four pyrethroid-resistant strains and not significantly less toxic than permethrin in a susceptible strain and a mildly pyrethroid-resistant strain. There was slight cross-resistance between topically applied fluralaner and permethrin in all five insecticide-resistant strains tested. Oral fluralaner was more toxic than imidacloprid in all four house fly strains tested, 9- to 118-fold as toxic. Oral cross-resistance between imidacloprid and fluralaner was not detected, but imidacloprid resistance was not high in any of the tested strains. Fluralaner shows promise for control of horn flies and house flies.

A commercial formulation, 37% dispersible oil suspension (DOS) (fomesafen, clomazone, and clethodim), is being registered in China to control annual or perennial weeds in soybean fields. In this paper, a liquid chromatography tandem mass spectrometry method with QuEChERS (quick, easy, cheap, effective, rugged, and safe) sample preparation was developed for the simultaneous determination of fomesafen, clomazone, clethodim, and its two metabolites (CSO and CSO 2 ) in soybean, green soybean, and soybean straw samples. The mean recoveries of our developed method for the five analytes in three matrices were ranged from 71% to 116% with relative standard deviations (RSDs) less than 12.6%. The limits of quantification (LOQs) were 0.01 mg/kg in soybean, 0.01 mg/kg in green soybean, and 0.02 mg/kg in soybean straw while the limits of detection (LODs) ranged from 0.018 to 0.125 μg/kg for these five analytes. The highest final residual amount of CSO 2 in green soybean samples (0.015 mg/kg) appeared in Anhui, and the highest in soybean straw samples was 0.029 mg/kg in Guangxi, whilst the terminal residues of fomesafen, clomazone, clethodim and CSO were lower than LOQs (0.01 mg/kg) in all samples. Furthermore, these terminal residues were all lower than the maximum residue limits (MRLs) set by China (0.1 mg/kg for fomesafen and clethodim, 0.05 mg/kg for clomazone) at harvest. Additional chronic dietary risk was evaluated using a risk quotients (RQs) method based on Chinese dietary habits. The chronic dietary exposure risk quotients were 4.3 for fomesafen, 0.12 for clomazone, and 19.3 for clethodim, respectively, which were significantly lower than 100. These results demonstrated that the dietary exposure risk of fomesafen, clomazone, and clethodim used in soybean according to good agricultural practices (GAP) was acceptable and would not pose an unacceptable health risk to Chinese consumers. These results not only offer insight with respect to the analytes, but also contribute to environmental protection and food safety.

Glyphosate, the most widely used herbicide worldwide, targets the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme in the shikimate pathway found in plants and some microorganisms. While the potential for glyphosate to induce a broad range of biological effects in exposed organisms has been demonstrated, the global molecular mechanisms of toxicity and potential effects in bacterial symbionts remain unclear, in particular for ecologically important marine species such as bivalve molluscs. Here, the effects of glyphosate (GLY), its degradation product aminomethylphosphonic acid (AMPA), and a mixture of both (MIX) on the mussel M. galloprovincialis were assessed in a controlled experiment. For the first time, next generation sequencing (RNA-seq and 16S rRNA amplicon sequencing) was used to evaluate such effects at the molecular level in both the host and its respective microbiota. The results suggest that the variable capacity of bacterial species to proliferate in the presence of these compounds and the impairment of host physiological homeostasis due to AMPA and GLY toxicity may cause significant perturbations to the digestive gland microbiota, as well as elicit the spread of potential opportunistic pathogens such as Vibrio spp.. The consequent host-immune system activation identified at the molecular and cellular level could be aimed at controlling changes occurring in the composition of symbiotic microbial communities. Overall, our data raise further concerns about the potential adverse effects of glyphosate and AMPA in marine species, suggesting that both the effects of direct toxicity and the ensuing changes occurring in the host-microbial community must be taken into consideration to determine the overall ecotoxicological hazard of these compounds., Competing Interests: Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Contamination of water sources due to herbicide is of great concern. Clomazone is a pesticide with a high contamination potential which could possibility lixiviate to water streams. Changes caused by residual herbicide include flora modifications which are generally detrimental for some species. The lack of morphological studies performed in aquatic plants exposed to herbicide-contaminated environments has encouraged the development of our research. For the first time, we present a study that aimed to evaluate leaf injuries visible to the naked eye as well as microscopical effects which may be caused by clomazone on Pistia stratiotes. Pistia stratiotes was subjected to five concentrations of clomazone. Our analysis showed leaf injuries, especially after 15 days of clomazone application. Hormesis was observed when the water lettuce was subjected to the lower concentrations. Total leaf area showed increase following by reduction while injured until reaching the highest concentration. Although the concentrations of clomazone tested in our study are not lethal to water lettuce, such herbicide have still caused morphoanatomical damages on leaves which advocates for the use of P. stratiotes as a bioindicator of the presence of herbicides such as clomazone in water.

Background: Leishmania infantum is the parasite responsible for the disease in humans known as zoonotic visceral leishmaniasis (ZVL). Dogs are considered the main domestic reservoir of ZVL and sand flies are the proven vectors. The use of systemic insecticides in dogs has been studied as an alternative strategy to control ZVL in endemic areas. One systemic insecticide in dogs, fluralaner, has a proven anti-sand fly effect in membrane-fed studies. However, the efficacy and duration on sand flies directly feeding from dogs treated with fluralaner remains unknown., Methods: Direct feeding bioassays were performed on 10 beagle dogs that had been randomly assigned to two groups: one with five dogs orally treated with Bravecto® (fluralaner) and other five as a control. About 30 females of Phlebotomus papatasi were allowed to directly feed from dogs at seven days before the administration of the treatment and Days 3, 17, 31, 45 and 73 post-treatment. Sand fly mortality after feeding was observed every 24 h for 5 days. The Kaplan-Meyer method, Henderson-Tilton formula and a negative binomial mixed model were used to respectively calculate: (i) mortality and its 95% confidence interval (CI); (ii) efficacy of the insecticide at killing sand flies in 24 h; and (iii) differences in the risk of sand fly death at 24 h after feeding., Results: Control sand fly mortality 24 h after feeding was always ≤ 20% and mortality in the fluralaner group ranged from 2% (95% CI: 0-4%) 7 days before treatment to 100% at 3 days post-treatment. Fluralaner efficacy was 100, 93, 94 and 75% at Days 3, 17, 31 and 45, respectively (P < 0.0001). The increase in the risk of sand fly death was 32.9 (95% CI: 4-263), 76 (95% CI: 8-705), 95.8 (95% CI: 9-1029) and 10.6 times (95% CI: 1.43-79) on Days 3, 17, 31 and 45, respectively CONCLUSIONS: The efficacy of fluralaner, orally administered to dogs, against sand-flies was above 90% for 31 days. Fluralaner administered to dogs should be further evaluated as a control strategy in ZVL endemic areas.

Glyphosate is the most heavily applied among pesticides in the world, and thus human exposure to this substance continues to increase. WHO changed classification of glyphosate to probably cancerogenic to humans, thus there is urgent need to assess in detail genotoxic mechanism of its action. We have assessed the effect of glyphosate, its formulation (Roundup 360 PLUS) and its main metabolite (aminomethylphosphonic acid, AMPA) in the concentration range from 1 to 1000 μM on DNA damage in human peripheral blood mononuclear cells (PBMCs). The cells were incubated for 24 h. The compounds studied and formulation induced DNA single and double strand-breaks and caused purines and pyrimidines oxidation. None of compounds examined was capable of creating adducts with DNA, while those substances increased ROS (including • OH) level in PBMCs. Roundup 360 PLUS caused damage to DNA even at 5 μM, while glyphosate and particularly AMPA induced DNA lesions from the concentration of 250 μM and 500 μM, respectively. DNA damage induced by glyphosate and its derivatives increased in order: AMPA, glyphosate, Roundup 360 PLUS. We may conclude that observed changes were not associated with direct interaction of xenobiotics studied with DNA, but the most probably they occurred through ROS-mediated effects., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The photodegradation and phytotoxicity of the pharmaceutical antibiotic, sulphamethoxazole (SMX) and the azo-dye reactive-red-194 (RR194) under visible-light irradiation of TiO 2 nanoparticles modified by silver and zirconium was investigated. The results indicated that sulphamethoxazole and its toxic degradation by product, 3-amino-5-methylisoxazole and RR-194 could be degraded efficiently by the co-doped Zr/Ag-TiO 2 catalyst. PL studies and ROS generation results suggested that the effective charge separation was carried out while irradiation of the modified TiO 2 nanoparticles. Phytotoxicity tests demonstrated lower percentage of germination in P. vulgaris (40%), V. radiata (30%) and P. lunatus (30%) of the seeds treated with 50 ppm of SMX, compared to the seeds treated with the degradation products (100%). The results with 50 ppm of RR-194 also showed lower percentage of germination in P. vulgaris (40%), V. radiata (50%) and P. lunatus (30%) compared to the degradation products (100%). Furthermore, significant increase in root and shoot development was observed in the seeds treated with the degraded products when compared with SMX and RR-194. Overall, this study contributes to further understanding the photodegradation mechanisms, degradation products and environmental fate of SMX and RR-194 in water which helps in the evaluation and mitigation of the environmental risk of SMX and RR-194 for water reuse and crop irrigation., (Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.)

This work describes the synthesis of a new series of isoxazole derivatives, their immunosuppressive properties, and the mechanism of action of a representative compound. A new series of N ′-substituted derivatives of 5-amino- N ,3-dimethyl-1,2-oxazole-4-carbohydrazide ( MM1 ⁻ MM10 ) was synthesized in reaction of 5-amino- N ,3-dimethyl-1,2-oxazole-4-carbohydrazide with relevant carbonyl compounds. The isoxazole derivatives were tested in several in vitro models using human cells. The compounds inhibited phytohemagglutinin A (PHA)-induced proliferation of peripheral blood mononuclear cells (PBMCs) to various degrees. The toxicity of the compounds with regard to a reference A549 cell line was also differential. 5-amino- N ′-(2,4-dihydroxyphenyl)methylidene- N ,3-dimethyl-1,2-oxazole-4-carbohydrazide ( MM3 ) compound was selected for further investigation because of its lack of toxicity and because it had the strongest antiproliferative activity. The compound was shown to inhibit lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF α) production in human whole blood cell cultures. In the model of Jurkat cells, MM3 elicited strong increases in the expression of caspases, Fas, and NF-κB1, indicating that a proapoptotic action may account for its immunosuppressive action in the studied models.

There is a need for robust in vitro models to sensitively capture skeletal muscle adverse toxicities early in the research and development of novel xenobiotics. To this end, an in vitro rat skeletal muscle model (L6) was used to study the translation of transcriptomics data generated from an in vivo rat model. Novel sulfonyl isoxazoline herbicides were associated with skeletal muscle toxicity in an in vivo rat model. Gene expression pathway analysis on skeletal muscle tissues taken from in vivo repeat dose studies identified enriched pathways associated with mitochondrial dysfunction, oxidative stress, energy metabolism, protein regulation and cell cycle. Mitochondrial dysfunction and oxidative stress were further explored using in vitro L6 metabolic models. These models demonstrated that the sulfonyl isoxazoline compounds induced mitochondrial dysfunction, mitochondrial superoxide production and apoptosis. These in vitro findings accurately concurred with the in vivo transcriptomics data, thereby confirming the ability of the L6 skeletal muscle models to identify relevant in vivo mechanisms of xenobiotic-induced toxicity. Moreover, these results highlight the sensitivity of the L6 galactose media model to study mitochondrial perturbation associated with skeletal muscle toxicity; this model may be utilised to rank the potency of novel xenobiotics upon further validation., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

The ability to recover to original states after disturbances makes macroinvertebrates useful tools for assessing the impacts of pesticides. Many studies showed that direct exposure to pesticides decreases macroinvertebrate richness and alters their composition. The main objective of this study was to assess recovery patterns in macroinvertebrate communities after pesticide application in irrigated rice fields. We analyzed short-term temporal dynamics of macroinvertebrate communities after application of the herbicides bispyribac-sodium and clomazone and the insecticide chlorantraniliprole, over the rice-growing season in southern Brazil. We selected three conventional rice fields and the recovery of macroinvertebrate communities was also compared with three adjacent natural ponds. The study was developed from November 2011 to February 2012 (rice-growing season). Five macroinvertebrate collections were carried out 3, 7, 14, 38, and 60 days after pesticide application (November 25). Rice fields showed lower richness and abundance than ponds in the period immediately after pesticide application, and recovery rates in the richness of macroinvertebrate communities were more conspicuous as pesticide residuals dissipated from the fields. Macroinvertebrate community structure in rice fields also became more similar to natural ponds as pesticide traces were scarcer. However, macroinvertebrate abundance patterns were not related to pesticide concentrations in the fields. Our results supported the general hypothesis on the negative effects of pesticide application on macroinvertebrate community in irrigated rice fields, although other environmental features (e.g., length of the flooded period) also contributed to explain temporal dynamics in the macroinvertebrate communities from irrigated rice fields.

Fluralaner is a novel isoxazoline insecticide which shows high insecticidal activity against parasitic, sanitary and agricultural pests, but there is little information about the effect of fluralaner on non-target organisms. This study reports the acute toxicity, bioconcentration, elimination and antioxidant response of fluralaner in zebrafish. All LC 50 values of fluralaner to zebrafish were higher than 10 mg L -1 at 24, 48, 72 and 96 h. To study the bioconcentration and elimination, the zebrafish were exposed to sub-lethal concentrations of fluralaner (2.00 and 0.20 mg L -1 ) for 15 d and then held 6 d in clean water. The results showed medium BCF of fluralaner with values of 12.06 (48 h) and 21.34 (144 h) after exposure to 2.00 and 0.20 mg L -1 fluralaner, respectively. In the elimination process, a concentration of only 0.113 mg kg -1 was found in zebrafish on the 6th day after removal to clean water. After exposure in 2.00 mg L -1 fluralaner, the enzyme activities of SOD, CAT, and GST, GSH-PX, CarE and content of MDA were measured. Only CAT and CarE activities were significantly regulated and the others stayed at a stable level compared to the control group. Meanwhile, transcriptional expression of CYP1C2, CYP1D1, CYP11A were significantly down-regulated at 12 h exposed to 2.00 mg L -1 of fluralaner. Except CYP1D1, others CYPs were up-regulated at different time during exposure periods. Fluralaner and its formulated product (BRAVECTO ® ) are of low toxicity to zebrafish and are rapidly concentrated in zebrafish and eliminated after exposure in clean water. Antioxidant defense and metabolic systems were involved in the fluralaner-induced toxicity. Among them, the activities of CAT and CarE, and most mRNA expression level of CYPs showed fast response to the sub-lethal concentration of fluralaner, which could be used as a biomarker relevant to the toxicity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Temperature is an important factor influencing the toxicity of chemicals in aquatic environments. Neotropical tadpoles experience large temperature fluctuations in their habitats and many species are distributed in areas impacted by agriculture. This study evaluated the effects caused by the exposure to clomazone (Gamit ® ) at different temperatures (28, 32 and 36 °C) on biochemical stress responses and esterase activities in Physalaemus nattereri and Rhinella schneideri tadpoles. Results evidenced that temperature modulates the effects of clomazone on biochemical response of tadpoles. Antioxidant enzymes, including catalase, superoxide dismutase (SOD), and glucose-6-phosphate dehydrogenase had their activities increased by clomazone in P. nattereri treated at higher temperatures. The biotransformation enzyme glutathione-S-transferase (GST) was also induced by clomazone at 32 and 36 °C. In R. schneideri, clomazone failed to alter antioxidant enzymes at 28 °C, but SOD and GST were increased by clomazone at higher temperatures after three days. All enzymes had their activities returned to the control levels after eight days in R. schneideri. Lipid peroxidation was induced in both species exposed to clomazone at 32 and 36 °C, but not at 28 °C. Acetylcholinesterase was not sensitive to clomazone and temperature, while most treatments impaired carboxylesterase activity. Integrated biomarker response (IBR) was notably induced by temperature in both species, and a synergic effect of temperature and clomazone was mostly observed after three days of exposure. These findings imply that tadpoles from tropical areas may present differential responses in their physiological mechanism linked to antioxidant defense to deal with temperature fluctuations and agrochemicals presence in their habitats., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Herbicides are the most widely used group of pesticides but after reaching water bodies they are able to cause adverse effects on non-target organisms. Different formulations using the same active ingredient are frequently available, which raises the issue of potential influence of different formulation types on herbicide toxicity. The present study evaluated the toxicity and teratogenic effects of the active ingredient clomazone and its two formulations (Rampa ® EC and GAT Cenit 36 CS, both containing 360g a.i./l of clomazone) on zebrafish embryos. The crucial difference between the two formulation types is the way of active substance release. This investigation is the first report on zebrafish embryotoxicity of both clomazone and its formulations. The technical active ingredient and formulations caused mortality and diverse teratogenic effects, showing different levels of toxicity. The LC 50 values for the technical ingredient, Rampa ® EC and GAT Cenit 36 CS were 61.4, 9.6 and 92.5mg a.i./l, respectively. Spontaneous movements in 22 hpf embryos decreased under exposure to both the technical ingredient and formulations. A significant number of underdeveloped embryos was detected after exposure to clomazone and Rampa ® EC, while no underdevelopment was noted in embryos exposed to GAT Cenit 36 CS. Exposure to the technical ingredient and formulations led also to a series of morphological changes and interfered with the growth of zebrafish embryos. The EC 50 based on detection of edemas, spine and tail tip deformations and gas bladder absence (120hpf) was 12.1, 10.1 and 24.1mg/l for technical clomazone, Rampa ® EC and GAT Cenit 36 CS, while teratogenicity index (TI) based on LC 50 /EC 50 ratio was 5.1, 1 and 3.8, respectively. The data in this study showed that the emulsifiable concentrate formulation (Rampa ® EC) caused statistically significantly higher toxicity, and the aqueous capsule suspension (GAT Cenit 36 CS) lower toxicity than technical clomazone. It indicates that different formulations with the same active ingredient may have different environmental impacts, which is why risk assessment based only on active ingredient toxicity might not be sufficient in terms of preventing formulation effects on the environment., (Copyright © 2017 Elsevier B.V. All rights reserved.)

HSPC1 is a critical protein in cancer development and progression, including colorectal cancer (CRC). However, clinical trial data reporting the effectiveness of HSPC1 inhibitors on several cancer types has not been as successful as predicted. Furthermore, some N-terminal inhibitors appear to be much more successful than others despite similar underlying mechanisms. This study involved the application of three N-terminal HSPC1 inhibitors, 17-DMAG, NVP-AUY922 and NVP-HSP990 on CRC cells. The effects on client protein levels over time were examined. HSPC1 inhibitors were also applied in combination with chemotherapeutic agents commonly used in CRC treatment (5-fluorouracil, oxaliplatin and irinotecan). As HSPA1A and HSPB1 have anti-apoptotic activity, gene-silencing techniques were employed to investigate the significance of these proteins in HSPC1 inhibitor and chemotherapeutic agent resistance. When comparing the action of the three HSPC1 inhibitors, there are distinct differences in the time course of important client protein degradation events. The differences between HSPC1 inhibitors were also reflected in combination treatment-17-DMAG was more effective compared with NVP-AUY922 in potentiating the cytotoxic effects of 5-fluorouracil, oxaliplatin and irinotecan. This study concludes that there are distinct differences between N-terminal HSPC1 inhibitors, despite their common mode of action. Although treatment with each of the inhibitors results in significant induction of the anti-apoptotic proteins HSPA1A and HSPB1, sensitivity to HSPC1 inhibitors is not improved by gene silencing of HSPA1A or HSPB1. HSPC1 inhibitors potentiate the cytotoxic effects of chemotherapeutic agents in CRC, and this approach is readily available to enter clinical trials. From a translational point of view, there may be great variability in sensitivity to the inhibitors between individual patients.

The use of agrochemicals in agriculture is intense and most of them could be carried out to aquatic environment. Nevertheless, there are only few studies that assess the effects of these xenobiotics on amphibians. Clomazone is an herbicide widely used in rice fields, where amphibian species live. Thus, those species may be threatened by non-target exposure. However, nanoparticles are being developed to be used as a carrier system for the agrochemicals. Such nanoparticles release the herbicide in a modified way, and are considered to be more efficient and less harmful to the environment. The aim of this study was to comparatively evaluate the effect of clomazone in its free form and associated with nanoparticles, in the liver of bullfrog tadpoles (Lithobates catesbeianus) when submitted to acute exposure for 96 h. According to semi-quantitative analysis, there was an increase in the frequency of melanomacrophage centres, in the accumulation of eosinophils and in lipidosis in the liver of experimental groups exposed to clomazone - in its free form and associated with nanoparticles - in comparison with the control group, and the nanotoxicity of chitosan-alginate nanoparticles. The increase of melanomacrophage centres in all exposed groups was significant (P < 0.0001) in comparison to control group. Therefore, the results of this research have shown that exposure to sublethal doses of the herbicide and nanoparticles triggered hepatic responses. Moreover, these results provided important data about the effect of the clomazone herbicide and organic nanoparticles, which act as carriers of agrochemicals, on the bullfrog tadpole liver., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Aim: Leflunomide is an immunosuppressive agent marketed as a disease-modifying antirheumatic drug. But it causes severe side effects, including fatal hepatitis and liver failure. In this study we investigated the contributions of hepatic metabolism and transport of leflunomide and its major metabolite teriflunomide to leflunomide induced hepatotoxicity in vitro and in vivo., Methods: The metabolism and toxicity of leflunomide and teriflunomide were evaluated in primary rat hepatocytes in vitro. Hepatic cytochrome P450 reductase null (HRN) mice were used to examine the PK profiling and hepatotoxicity of leflunomide in vivo. The expression and function of sodium/bile acid cotransporter (NTCP) were assessed in rat and human hepatocytes and NTCP-transfected HEK293 cells. After Male Sprague-Dawley (SD) rats were administered teriflunomide (1,6, 12 mg · kg(-1) · d(-1), ig) for 4 weeks, their blood samples were analyzed., Results: A nonspecific CYPs inhibitor aminobenzotriazole (ABT, 1 mmol/L) decreased the IC50 value of leflunomide in rat hepatocytes from 409 to 216 μmol/L, whereas another nonspecific CYPs inhibitor proadifen (SKF, 30 μmol/L) increased the cellular accumulation of leflunomide to 3.68-fold at 4 h. After oral dosing (15 mg/kg), the plasma exposure (AUC0-t) of leflunomide increased to 3-fold in HRN mice compared with wild type mice. Administration of leflunomide (25 mg·kg(-1) · d(-1)) for 7 d significantly increased serum ALT and AST levels in HRN mice; when the dose was increased to 50 mg·kg(-1) · d(-1), all HRN mice died on d 6. Teriflunomide significantly decreased the expression of NTCP in human hepatocytes, as well as the function of NTCP in rat hepatocytes and NTCP-transfected HEK293 cells. Four-week administration of teriflunomide significantly increased serum total bilirubin and direct bilirubin levels in female rats, but not in male rats., Conclusion: Hepatic CYPs play a critical role in detoxification process of leflunomide, whereas the major metabolite teriflunomide suppresses the expression and function of NTCP, leading to potential cholestasis.

Drug-induced liver injury (DILI) is one of the serious and frequent drug-related adverse events. This adverse event is a main reason for regulatory action pertaining to drugs, including restrictions in clinical indications and withdrawal from clinical trials or the marketplace. Idiosyncratic DILI especially has become a major clinical concern because of its unpredictable nature, frequent hospitalization, need for liver transplantation and high mortality. The estimation of the potential for compounds to induce idiosyncratic DILI is very difficult in non-clinical studies because the precise mechanism of idiosyncratic DILI is still unknown. Recently, many in vitro assays which indicate a possibility of the prediction of the idiosyncratic DILI have been reported. Among these, some in vitro assays focus on the effects of compounds on mitochondrial function and the apoptotic effects of compounds on human hepatocytes. In this study, we measured oxygen consumption rate (OCR) and caspase-3/7 activity as an endpoint of mitochondrial dysfunction and apoptosis, respectively, with human hepatocytes after treatment with compounds causing idiosyncratic DILI (troglitazone, leflunomide, ranitidine and diclofenac). Troglitazone and leflunomide decreased the OCR but did not affect caspase-3/7 activity. Ranitidine increased caspase-3/7 activity but did not affect the OCR. Diclofenac decreased the OCR and increased caspase-3/7 activity. Acetaminophen and ethanol, which are also hepatotoxicants but do not induce idiosyncratic DILI, did not affect the OCR or caspase-3/7 activity. These results indicate that a combination assay of mitochondrial dysfunction and apoptosis is useful for the estimation of potential risk of compounds to induce idiosyncratic DILI.

Background: Cyclooxygenase-2 selective inhibitors have been developed to alleviate pain and inflammation; however, the use of a selective cyclooxygenase-2 inhibitor is associated with mild edema, hypertension, and cardiovascular risk., Aim: To evaluate, in an experimental model in normotensive rats, the effect of treatment with parecoxib in comparison with diclofenac and aspirin and L-NAME, a non-selective nitric oxide synthetase, on mean arterial blood pressure, and cyclooxygenase-1 and -2 messenger RNA and protein expression in aortic tissue., Methods: Rats were treated for seven days with parecoxib (10 mg/kg/day), diclofenac (3.2 mg/kg/day), aspirin (10 mg/kg/day), or L-NAME (10 mg/kg/day). Mean arterial blood pressure was evaluated in rat tail; cyclooxygenase-1 and -2 were evaluated by reverse transcription-polymerase chain reaction and Western blot analysis in aortic tissue., Results: Parecoxib and L-NAME, but not aspirin and diclofenac, increased mean arterial blood pressure by about 50% (p < 0.05) without changes in cardiac frequency. Messenger RNA cyclooxygenase-1 expression in aortic tissue was not modified with any drug (p < 0.05). L-NAME and parecoxib treatment decreased messenger RNA cyclooxygenase-2 and cyclooxygenase-2 (p < 0.05). While cyclooxygenase-1 protein decreased with the three drugs tested but not with L-NAME (p < 0.05), the cyclooxygenase-2 protein decreased only with aspirin and parecoxib (p < 0.05)., Conclusion: Parecoxib increases the blood pressure of normotensive rats by the suppression of COX-2 gene expression, which apparently induced cardiovascular control.

Background: Fluralaner is a novel isoxazoline eliciting both acaricidal and insecticidal activity through potent blockage of GABA- and glutamate-gated chloride channels. The aim of the study was to investigate the susceptibility of juvenile stages of common tick species exposed to fluralaner through either contact (Rhipicephalus sanguineus) or contact and feeding routes (Ornithodoros moubata)., Methods: Fluralaner acaricidal activity through both contact and feeding exposure was measured in vitro using two separate testing protocols. Acaricidal contact activity against Rhipicephalus sanguineus life stages was assessed using three minute immersion in fluralaner concentrations between 50 and 0.05 μg/mL (larvae) or between 1000 and 0.2 μg/mL (nymphs and adults). Contact and feeding activity against Ornithodoros moubata nymphs was assessed using fluralaner concentrations between 1000 to 10(-4) μg/mL (contact test) and 0.1 to 10(-10) μg/mL (feeding test). Activity was assessed 48 hours after exposure and all tests included vehicle and untreated negative control groups., Results: Fluralaner lethal concentrations (LC₅₀, LC₉₀/₉₅) were defined as concentrations with either 50%, 90% or 95% killing effect in the tested sample population. After contact exposure of R. sanguineus life stages lethal concentrations were (μg/mL): larvae - LC₅₀ 0.7, LC₉₀ 2.4; nymphs - LC₅₀ 1.4, LC₉₀ 2.6; and adults - LC₅₀ 278, LC₉₀ 1973. After exposure of O. moubata nymphs to fluralaner lethal concentrations were (μg/mL): contact exposure - LC₅₀ 720, LC₉₅ 1133; and feeding exposure- LC₅₀ 0.00007, LC₉₅ 0.09., Conclusions: Fluralaner demonstrates potent in vitro acaricidal activity against all life stages of the brown dog tick, R.sanguineus. The testing of fluralaner contact and feeding routes using O. moubata nymphs demonstrates a high acaricidal activity in both exposure routes.

Drug-induced liver injury has been observed in patients treated with the endothelin receptor antagonists sitaxentan and bosentan, but not following treatment with ambrisentan. The aim of our studies was to assess the possible role of multiple contributory mechanisms in this clinically relevant toxicity. Inhibition of the bile salt export pump (BSEP) and multidrug resistance-associated protein 2 was quantified using membrane vesicle assays. Inhibition of mitochondrial respiration in human liver-derived HuH-7 cells was determined using a Seahorse XF(e96) analyzer. Cytochrome P450 (P450)-independent and P450-mediated cell toxicity was assessed using transfected SV40-T-antigen-immortalized human liver epithelial (THLE) cell lines. Exposure-adjusted assay ratios were calculated by dividing the maximum human drug plasma concentrations by the IC50 or EC50 values obtained in vitro. Covalent binding (CVB) of radiolabeled drugs to human hepatocytes was quantified, and CVB body burdens were calculated by adjusting CVB values for fractional drug turnover in vitro and daily therapeutic dose. Sitaxentan exhibited positive exposure-adjusted signals in all five in vitro assays and a high CVB body burden. Bosentan exhibited a positive exposure-adjusted signal in one assay (BSEP inhibition) and a moderate CVB body burden. Ambrisentan exhibited no positive exposure-adjusted assay signals and a low CVB body burden. These data indicate that multiple mechanisms contribute to the rare, but potentially severe liver injury caused by sitaxentan in humans; provide a plausible rationale for the markedly lower propensity of bosentan to cause liver injury; and highlight the relative safety of ambrisentan., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

The study investigated the capacity of diphenyl diselenide [(PhSe)2] (3.0mg/kg), on reduce the oxidative damage in liver, gills and muscle of carp and silver catfish exposed to clomazone (192h). Silver catfish exposed to clomazone showed increased thiobarbituric acid-reactive substance (TBARS) in liver and muscle and protein carbonyl in liver and gills. Furthermore, clomazone in silver catfish decrease non-protein thiols (NPSH) in liver and gills and glutathione peroxidase and ascorbic acid in liver. (PhSe)2 reversed the effects caused by clomazone in silver catfish, preventing increases in TBARS and protein carbonyl. Moreover, NPSH and ascorbic acid were increased by values near control. The results suggest that (PhSe)2 attenuated the oxidative damage induced by clomazone in silver catfish. The clomazone no caused an apparent situation of oxidative stress in carp, showing that this species is more resistant to this toxicant. Altogether, the containing (PhSe)2 diet helps fish to increase antioxidants defenses., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The photocatalytic degradation of the herbicide clomazone (0.05mM) in aqueous suspensions of TiO2 Degussa P25 was examined as a function of the different operational parameters. The optimum concentration of the catalyst was found to be 0.50mgmL(-1) under UV light at the pH 10.3. In the first stage of the reaction, the photocatalytic degradation of clomazone followed the pseudo-first order kinetics, with and the heterogeneous catalysis proceeding via OH radicals. The results also showed that the disappearance of clomazone led to the formation of a number of organic intermediates and ionic byproducts, whereas its complete mineralization occurred after about 55min. Tentative photodegradation pathways were proposed and discussed. A comparison of the evolution of toxicity that was evaluated in vitro in rat hepatoma (H-4-II-E) and human fetal lung (MRC-5) cell lines with the degradation kinetics indicates that the irradiation contributed to the decrease of the toxicity of the mixture that is no longer dominated by the parent compound. The study also encompassed the effect of the quality of natural water on the rate of removal of clomazone., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The indiscriminate use of herbicides has led to the contamination of water bodies, possibly affecting the health of aquatic biota. Therefore, to evaluate the possible effects of the clomazone-based herbicide (Gamit(®) 500) on the fish Prochilodus lineatus, juveniles were exposed for 96h to three concentrations (1, 5 and 10mgL(-1)) of clomazone, and an analysis was made of their hematological parameters: hemoglobin (Hb); hematocrit (Hct); red blood cell (RBC) count; mean corpuscular volume (MCV); mean corpuscular hemoglobin (MCH); mean corpuscular hemoglobin concentration (MCHC) and biochemical parameters: glutathione S-transferase (GST); catalase (CAT); glutathione peroxidase (GPx) and acetylcholinesterase (AChE). Hct presented a significant decrease at the concentration of 10mgL(-1), while the parameters Hb, HCM and MCHC presented a significant decrease at the two higher concentrations, indicating an anemic condition. The RBC increased significantly at the lowest concentration, possibly due to the release of new red blood cells into the bloodstream in response to splenic contraction, which may occur as an adaptive response to the stressor agent. P. lineatus presented activation of the biotransformation pathway, indicated by augmented hepatic activity of the enzyme GST and hepatic activation of the antioxidant enzyme CAT at the higher concentrations. Liver GPx was significantly inhibited at the higher concentrations, which may indicate the efficient action of CAT in the elimination of H2O2 or its competition with GST for the same substrate (GSH). AChE activity in brain and muscle was inhibited at the higher concentrations, indicating the neurotoxic effects of the herbicide in the fish. The hematological and biochemical alterations led to the conclusion that the herbicide clomazone has toxic effects on the species P. lineatus, and that its presence in the environment may jeopardize the health of these animals., (Copyright © 2013 Elsevier B.V. All rights reserved.)

E1210 is a new antifungal compound with a novel mechanism of action and broad spectrum of antifungal activity. We investigated the in vitro antifungal activities of E1210 compared to those of fluconazole, itraconazole, voriconazole, amphotericin B, and micafungin against clinical fungal isolates. E1210 showed potent activities against most Candida spp. (MIC(90) of ≤0.008 to 0.06 μg/ml), except for Candida krusei (MICs of 2 to >32 μg/ml). E1210 showed equally potent activities against fluconazole-resistant and fluconazole-susceptible Candida strains. E1210 also had potent activities against various filamentous fungi, including Aspergillus fumigatus (MIC(90) of 0.13 μg/ml). E1210 was also active against Fusarium solani and some black molds. Of note, E1210 showed the greatest activities against Pseudallescheria boydii (MICs of 0.03 to 0.13 μg/ml), Scedosporium prolificans (MIC of 0.03 μg/ml), and Paecilomyces lilacinus (MICs of 0.06 μg/ml) among the compounds tested. The antifungal action of E1210 was fungistatic, but E1210 showed no trailing growth of Candida albicans, which has often been observed with fluconazole. In a cytotoxicity assay using human HK-2 cells, E1210 showed toxicity as low as that of fluconazole. Based on these results, E1210 is likely to be a promising antifungal agent for the treatment of invasive fungal infections.

Background and Purpose: alpha7-Nicotinic acetylcholine receptors (alpha7 nAChRs) play an important role in cognitive function. Positive allosteric modulators (PAMs) amplify effects of alpha7 nAChR agonist and could provide an approach for treatment of cognitive deficits in neuropsychiatric diseases. PAMs can either predominantly affect the apparent peak current response (type I) or increase both the apparent peak current response and duration of channel opening, due to prolonged desensitization (type II). The delay of receptor desensitization by type II PAMs raises the possibility of Ca2+-induced toxicity through prolonged activation of alpha7 nAChRs. The present study addresses whether type I and II PAMs exhibit different cytotoxicity profiles., Experimental Approach: The present studies evaluated cytotoxic effects of type I PAM [N-(4-chlorophenyl)]-alpha-[(4-chlorophenyl)-aminomethylene]-3-methyl-5-isoxazoleacet-amide (CCMI) and type II PAM 1-[5-chloro-2,4-dimethoxy-phenyl]-3-[5-methyl-isoxazol-3-yl]-urea (PNU-120596), or 4-[5-(4chloro-phenyl)-2-methyl-3-propionyl-pyrrol-1-yl]-benzenesulphonamide (A-867744). The studies used cultures of PC12 cells and primary cultures of rat cortical neuronal cells., Key Results: Our results showed that neither type I nor type II PAMs had any detrimental effect on cell integrity or cell viability. In particular, type II PAMs did not affect neuron number and neurite outgrowth under conditions when alpha7 nAChR activity was measured by Ca2+ influx and extracellular signal-regulated kinases 1 and 2 phosphorylation, following exposure to alpha7 nAChR agonists., Conclusions and Implications: This study demonstrated that both type I and type II alpha7 nAChR selective PAMs, although exhibiting differential electrophysiological profiles, did not exert cytotoxic effects in cells endogenously expressing alpha7 nAChRs.

This study aims to verify the effects of the clomazone concentration used in rice fields on acetylcholinesterase (AChE), thiobarbituric acid reactive substances (TBARS), protein carbonyl and catalase activity in tissues of piava (Leporinus obtusidens). LC(50)-96h was 5.0 mg L(-1) and the fish were exposed to 1/10 of LC(50)-96 h: 0.5 mg L(-1) of clomazone for 96 and 192h. The same parameters were also assayed after a recovery period of 192 h in clean water. AChE activity was reduced only in the brain and heart of fish exposed for 96 h. AChE activity was decreased in the brain, muscle and heart tissues after 192 h of exposure. After 192 h of recovery period, AChE activity remained diminished in brain and muscle and showed a decrease in eye. However, after 192 h of recovery, AChE activity in heart was recovered. Fish showed increased TBARS levels in brain at all experimental periods. TBARS levels decreased in liver and muscle tissues after 192 h of exposure. The increase in muscle TBARS persisted in fish transferred to clean water. Protein carbonyl in the liver was increased in all periods studied including the recovery period. Catalase activity was reduced during all periods. The present study demonstrates the occurrence of disorders in AChE, TBARS, protein carbonyl and catalase activity in piava. The results also show changes in fish after exposure to an environmentally relevant concentration of clomazone. Most effects observed persisted after the recovery period. Thus, these parameters may be used to monitor clomazone toxicity in fish.

With the eventual goal of making zonisamide (ZNS), a relatively new antiepileptic drug, available for the treatment of epilepsy in cats, the pharmacokinetics after a single oral administration at 10mg/kg and the toxicity after 9-week daily administration of 20mg/kg/day of ZNS were studied in healthy cats. Pharmacokinetic parameters obtained with a single administration of ZNS at 10mg/day were as follows: Cmax=13.1microg/ml; Tmax=4.0h; T(1/2)=33.0h; areas under the curves (AUCs)=720.3microg/mlh (values represent the medians). The study with daily administrations revealed that the toxicity of ZNS was comparatively low in cats, suggesting that it may be an available drug for cats. However, half of the cats that were administered 20mg/kg/day daily showed adverse reactions such as anorexia, diarrhoea, vomiting, somnolence and locomotor ataxia.

Purpose: Aim of this study was to prove the efficacy and safety of the new malononitrilamide immunosuppressive FK778 in prolonging clear graft survival following allogeneic orthotopic keratoplasty in rats., Methods: Sixty-seven penetrating keratoplasties were performed using Fisher and Lewis rats as donors and recipients, respectively: group 1 (n=11), allogeneic control without therapy; group 2 (n=12), syngeneic control; group 3 (n=11), mycophenolate mofetil (MMF) 40 mg/kg bodyweight; group 4 (n=12), FK778 5 mg/kg bodyweight; group 5 (n=12), FK778 10 mg/kg bodyweight; and group 6 (n=9), FK778 20 mg/kg bodyweight. Four animals in each group were killed for immunohistological evaluation on day 14. Therapy was administered orally for 18 days. The grafts were evaluated every three days by means of a scoring system including opacity, oedema, and vascularization. Time to rejection was analysed with the Kaplan-Meier survival analysis and compared with the log-rank test. The densities of infiltrating immune cells were compared statistically using the non-parametric Mann-Whitney test., Results: Mean rejection-free graft survival was 11.4 days in group 1 (allogeneic control), 100 days (total follow-up time) in group 2 (syngeneic control), 24.0 days in group 3 (MMF 40 mg/kg), 15.7 days in group 4 (FK778 5 mg/kg), 19.1 days in group 5 (FK778 10 mg/kg), and 25.4 days in group 6 (FK778 20 mg/kg) (P<0.005)., Conclusions: Systemic immunosuppression with FK778 prolongs graft survival in the rat keratoplasty model. FK778's efficacy is comparable with that of MMF in preventing immunologic graft rejection.

The effects of the herbicide, clomazone, on acetylcholinesterase (AChE), catalase and TBARS formation in teleost fish (Rhamdia quelen) were studied. The fish were exposed to 0.5 or 1.0 mg L(-1) of clomazone for 12, 24, 48, 96 and 192 h. After 192 h of exposure period, fish were transferred to clean water and kept in the same for 192 h to study the recovery response. Same parameters as that of exposure period were assayed after 96 and 192 h of recovery period. Specific AChE activity was reduced in the brain and muscle after treatments, reaching a maximum inhibition of 47% in the brain and 45% in the muscle after 12h of exposure. Fish exposed to clomazone increased TBARS production in the liver for all exposure periods. The brain presented elevated TBARS levels after 12, 24 and 48 h, but after 96 and 192 h, these levels decreased. The decrease of TBARS levels persisted in brain tissue after 96 h of recovery and returned to the control value after 192 h in clean water. Catalase activity was reduced for all periods of exposure. Histological analysis showed vacuolation in the liver after herbicide exposure. Some of the alterations observed were completely restored after recovery period.

The effects of clomazone (0.5 and 1.0 mg/L) according to nominal concentrations used in paddy rice fields (0.4-0.7 mg/L) on protein and carbohydrate metabolism and haematological parameters were evaluated in silver catfish (Rhamdia quelen) after 12, 24, 48, 96 and 192 h of exposure with a recovery period of 96 and 192 h. Liver glycogen increased significantly (P<0.05) in all periods and concentrations tested. The maximum glycogen increase reaches 250% after 12h of exposure. Muscle glycogen reduced significantly after 24, 48, 96 and 192 h for both clomazone concentrations (P<0.05). Significantly elevated plasma glucose values (P<0.05) and variation in glucose in the liver and muscle of exposed fish were observed. Muscle lactate levels increased after 12, 24 and 48 h of clomazone exposure (22-67%), but reduced in the liver (P<0.05). Protein levels were enhanced in the liver and white muscle, except at 96 and 192 h of exposure, whereas it increased in the plasma in the period from 48 to 96 h (P<0.05). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were significantly elevated in the plasma (P<0.05). In the liver, ALT increased after 24 h, while AST activity was enhanced only after 12 h of exposure. Hematocrit contents were reduced after 96 and 192 h of exposure. Most of the metabolic disorders observed did not persist after the recovery period, except for the liver AST and ALT activity. Clomazone concentrations used in this study appear safe to fish, Rhamdia quelen, because overall parameters can be recovered after 96 and 192 h in clean water. ALT and AST activity may be an early biomarker of clomazone toxicity.

We sought to determine whether valdecoxib is as effective as diclofenac in treating acute ankle sprain. Patients (n=202) with acute first- and second-degree ankle sprain were randomized to valdecoxib (40 mg twice daily on day 1 followed by 40 mg once daily on days 2-7) or diclofenac (75 mg twice daily). The primary efficacy end-point was the Patient's Assessment of Ankle Pain visual analogue scale (VAS, 0-100 mm) value on day 4. Valdecoxib was as efficacious as diclofenac in treating the signs and symptoms of acute ankle sprain. The mean VAS reduction in ankle pain on day 4 was not different between groups; the two-sided 95% confidence interval for the between-group difference was within the prespecified limit for non-inferiority (10 mm). There were no significant differences between groups for all secondary efficacy end-points. The two treatments were similarly effective and well tolerated for treatment of acute ankle sprain.

Fingerlings of the silver catfish (Rhamdia quelen) were exposed to three herbicides widely used in rice culture in south Brazil: clomazone, quinclorac, and metsulfuron methyl. LC50 was determined and acetylcholinesterase (AChE) activity was evaluated in brain and muscle tissue of fish exposed to different herbicide concentrations after 96h (short term). The LC50 value (nominal concentration) was 7.32 mg/L for clomazone and 395 mg/L for quinclorac, but was not obtained for metsulfuron-methyl since all fingerlings survived the highest concentration of 1200 mg/L. Brain and muscle AChE activity in unexposed fish were 17.9 and 9.08 micromol/min/g protein, respectively. Clomazone significantly inhibited AChE activity in both tissues, achieving maximal inhibition of about 83% in brain and 89% in muscle tissue. In contrast, quinclorac and metsulfuron methyl caused increases in enzyme activity in the brain (98 and 179%, respectively) and inhibitions in muscle tissue (88 and 56%, respectively). This study demonstrated short-term effects of exposure to environmentally relevant concentrations of rice field herbicides on AChE activity in brain and muscle tissue of silver catfish.

SU101 (leflunomide, N-[4-(trifluoromethyl)-phenyl] 5-methylisoxazole-4-carboxamide), an inhibitor of platelet-derived growth factor receptor signaling, has shown promising clinical activity in Phase I and II studies. Currently, SU101 in combination with cytotoxic agents is in late-stage clinical development for the treatment of cancers. In previous reports, efficacy in vivo versus varied tumor xenografts was observed. As part of the preclinical development of SU101 as a cancer therapy, the combination of SU101 with cytotoxic agents was studied in athymic mice bearing small, established, s.c. human tumor cell xenografts of glioblastoma (SF763T cells), lung (Calu-6 cells), or head and neck (KB cells) origin. In the SF763T model, the combination of SU101 with carmustine resulted in a statistically significant growth inhibition of 74% compared with the vehicle control; this combination was more effective than either agent alone. In the Calu-6 model, the combination of SU101, cisplatin, and etoposide resulted in a growth inhibition of 75% that was statistically greater than that of the vehicle-treated control group and groups treated with one or two agents. In the KB model, the combination of SU101, 5-fluorouracil, and cisplatin resulted in a statistically significant growth inhibition of 69% compared with the vehicle control. Treatment with one or two agents did not significantly inhibit growth in this model. Importantly, in addition to enhanced efficacy resulting from combination therapies, the combination treatments tested were well tolerated, as evidenced by lack of mortality. These data suggest that SU101 in combination with cytotoxic agents may provide clinical benefit and warrant further clinical investigation.

Many reports have cited coexpression of platelet-derived growth factor (PDGF) and its receptors by tumor cells or cells supporting tumor growth, suggesting both autocrine and paracrine mechanisms for PDGF-mediated tumor growth. We found that a small organic molecule, N-[4-(trifluoromethyl)phenyl] 5-methylisoxazole-4-carboxamide (SU101, leflunomide), inhibited PDGF-mediated signaling events, including receptor tyrosine phosphorylation, DNA synthesis, cell cycle progression, and cell proliferation. SU101 inhibited PDGF-stimulated tyrosine phosphorylation of PDGF receptor (PDGFR) beta in C6 (rat glioma) and NIH3T3 cells engineered to overexpress human PDGFRbeta (3T3-PDGFRbeta). SU101 blocked both PDGF- and epidermal growth factor (EGF)-stimulated DNA synthesis. Previously, this compound was shown to inhibit pyrimidine biosynthesis by interfering with the enzymatic activity of dihydroorotate dehydrogenase. In the current study, EGF-stimulated DNA synthesis was restored by the addition of saturating quantities of uridine, whereas PDGF-induced DNA synthesis was not, suggesting that the compound demonstrated some selectivity for the PDGFR pathway that was independent of pyrimidine biosynthesis. Selectivity was further demonstrated by the ability of the compound to block the entry of PDGF-stimulated cells into the S phase of the cell cycle, without affecting cell cycle progression of EGF-stimulated cells. In cell growth assays, SU101 selectively inhibited the growth of PDGFRbeta-expressing cell lines more efficiently than it inhibited the growth of PDGFRbeta-negative cell lines. SU101 inhibited the s.c., i.p., and intracerebral growth of a panel of cell lines including cells from glioma, ovarian, and prostate origin. In contrast, SU101 failed to inhibit the in vitro or s.c. growth of A431 and KB tumor cells, both of which express EGF receptor but not PDGFRbeta. SU101 also inhibited the growth of D1B and L1210 (murine leukemia) cells in syngeneic immunocompetent mice, without causing adverse effects on the immune response of the animals. In an i.p. model of tumor growth in syngeneic immunocompetent mice, SU101 prevented tumor growth and induced long-term survivors in animals implanted with 7TD1 (murine B-cell hybridoma) tumor cells. Because PDGFRbeta was detected on most of the tumor cell lines in which in vivo growth was inhibited by SU101, these data suggest that SU101 is an effective inhibitor of PDGF-driven tumor growth in vivo.

The therapeutic efficacy of an experimental antiviral agent, WIN 54954, was evaluated in a mouse model in which infection by coxsackievirus B4 (CVB4) strain E2 was followed by diabetes mellitus. Male CD1 mice (age, 5 weeks) were inoculated with 10(4) PFU of CVB4. WIN 54954 was administered orally via gavage tube in a dose of either 5 or 50 mg/kg of body weight per day. Treatment was initiated on the day of inoculation and was continued for 10 days. Control animals received the xanthan gum carrier only. At 3 days postinoculation (p.i.), the mean titer of virus in the pancreas was found to be significantly lower in both the high-dose (P < 0.001) and low-dose (P < 0.05) treatment groups compared with that in the controls. Furthermore, islet histologic abnormalities were significantly less common in the high-dose group (P < 0.02) than in the controls. At 7 weeks p.i., both fasting and 1-h postprandial glucose levels in blood were significantly lower for both the high-dose (P < 0.001) and the low-dose (P < 0.01) treatment groups than in controls. The proportion of mice with persistent viral RNA in the pancreas at this time, as detected by polymerase chain reaction, was significantly reduced in the high-dose treatment group (4 of 11 mice) compared with that in the controls (7 of 8 mice). When mice received 50 mg of WIN 54954 per kg daily beginning at either 48 or 72 h postinoculation, the titers in the pancreas were again significantly reduced at 3 days p.i. compared with those in the controls (P < 0.01 and P < 0.05, respectively). Thus, WIN 54954 effectively reduces virus replication and islet histologic changes acutely and decreases, at 7 weeks, both the metabolic alteration associated with diabetes mellitus and the incidence of detectable viral RNA in the pancreas.

Anticonvulsant properties of compounds that enhance GABA-mediated inhibition through modulatory sites on the GABAA receptor [phenobarbital (PB), clonazepam (CZP), alpha-ethyl-alpha-methyl-gamma-thiobutyrolactone (alpha-EMTBL)] were compared with anticonvulsant effects of compounds believed to be antagonists at these modulatory sites (Ro15-1788 and alpha-isopropyl-alpha-methyl-gamma-butyrolactone gamma-IMGBL)] and to 4,5,6,7-tetrahydroisoxazolo-[4,5-c]-pyridin-3-ol (THIP, GABAA receptor agonist), (+/-) baclofen (GABAB receptor agonist), and gamma-vinyl GABA, a compound that increases endogenous GABA. The compounds were tested for their ability to block experimental seizures caused by maximal electroshock, pentylenetetrazol, picrotoxin, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), bicuculline (BIC), aminophylline, strychnine, and t-butyl-bicyclophosphorothionate (TBPS) in mice. CZP blocked all but strychnine seizures. PB was also highly effective, blocking all but TBPS seizures. alpha-EMTBL, representing a new class of experimental anticonvulsant drugs, prevented all seizures except strychnine (STR)- and aminophylline-induced seizures. The antagonists are effective only against one convulsant stimulus. Ro15-1788 and alpha-IMGBL prevented only DMCM- and pentylenetetrazol (PTZ)-induced seizures, respectively. THIP and gamma-vinyl GABA both blocked only BIC and picrotoxin seizures. Baclofen had no anticonvulsant activity. These data demonstrate that compounds that increase neuronal inhibition by potentiating the action of GABA have a broader spectrum of anticonvulsant action than either antagonists or GABAmimetic agents or compounds that increase endogenous GABA.

Acivicin is an investigational amino acid antitumor antibiotic currently being evaluated in Phase II clinical trials. In humans acivicin causes reversible, dose-limiting central nervous system (CNS) effects including somnolence, ataxia, personality changes, and hallucinations. We have observed and reported previously that acivicin-treated cats exhibit symptoms (ataxia, sedation, somnolence) resembling CNS toxicity reported in humans. We hypothesized that if acivicin uptake into brain were mediated by a saturable transport system common to endogenous amino acids, drug uptake and CNS toxicity might be blocked by elevation of normal amino acid concentrations in circulating plasma. To test this hypothesis, cats received constant-rate i.v. infusions of either saline or Aminosyn, 10% (a commercially available mixture of 16 amino acids not containing glutamine, glutamate, aspartate, or cysteine) for 4 h prior to and 18 h subsequent to administration of acivicin at a dose producing marked behavioral changes in control cats. Presence or absence of ataxia and sedation were noted at intervals after acivicin treatment. Results showed that Aminosyn infusion prevented CNS symptoms in six of eight cats. Subsequent experiments showed that acivicin levels in brain tissue of Aminosyn-treated cats were 13% of the drug levels in saline-infused cats. Acivicin levels in most peripheral tissues were also decreased significantly by Aminosyn infusion but not to the extent observed in brain. Decreased brain uptake was shown to be due to a combination of amino acid blockade of drug transport into that organ and of increased total body clearance of drug. Concomitant Aminosyn treatment did not alter the efficacy of acivicin in mice bearing L1210 leukemia or MX-1 human mammary carcinoma. Further studies demonstrated that a solution containing only four large neutral amino acids (leucine, isoleucine, phenylalanine, and valine) could also protect cats from acivicin-induced CNS toxicity, apparently without increasing acivicin total body clearance. However, a mixture of several other amino acids contained in Aminosyn (alanine, arginine, tyrosine, histidine, proline, serine, and glycine) failed to prevent CNS toxicity. We conclude that cotreatment with Aminosyn or a mixture of large neutral amino acids could protect cancer patients from acivicin-induced CNS toxicity without ablating antitumor efficacy.

Mofezolac (N-22) is a new developed analgesic and anti-inflammatory agent. A teratogenicity study of N-22 was carried out in New Zealand White rabbits to examine the effect on the dams and the teratogenic potentiality. N-22 was administered orally at the dose levels of 12.5, 50 and 200 mg/kg during the organogenesis from day 6 to day 18 of gestation. All pregnant females were sacrificed on day 28 of gestation and their fetuses were examined. There were no growth retardation and teratogenic effects on fetuses from the dams administered N-22, although the administration of 200 mg/kg produced a decrease in food consumption of the dams concomitant with the embryocidal effects as shown by an increase in the early resorption rate. Thus, non-toxic dose level of N-22 on the dams and fetuses was considered to be 50 mg/kg.

Fifty two-week oral toxicity study of mofezolac (N-22), a new developed analgesic and anti-inflammatory agent, was carried out in Wistar rats with dose levels of 5, 20, 60 and 120 mg/kg/day, and the 5-week withdrawal was followed for recovery study. Hematuria, blanching of the skin and suppression of body weight gain were observed in females given 120 mg/kg, and 9 of these prostrated and died from week 20 to week 52, or were euthanized when moribund. These symptoms were not seen in males at any dose levels. In 120 mg/kg group, increased positive cases of fecal occult blood were observed during the administration period, and the pathological examination revealed gastrointestinal lesions such as erosion, ulcer, hemorrhage and mucosal regeneration in the small intestine. Renal disorder was also involved mainly in females given 120 mg/kg, as shown by increase in urine volume with declined osmotic pressure and specific gravity, serum urea nitrogen, creatinine, inorganic phosphorus, and other related parameters. In addition to enlargement, rough surface and scar formation, dilated tubular lumen and papillary ducts of the kidney were observed as a main lesion. Incidental findings with those disorders, observed mainly in females given 120 mg/kg, included increase in leucocytes with high neutrophils ratio, and enlargement of the spleen, adrenals and mesenteric lymph node. There were some of the similar gastrointestinal and renal changes mainly in females given 60 mg/kg. Anemic findings were noted in both sexes of 120 mg/kg and in females of 60 mg/kg group, and mainly females given 120 mg/kg showed increase in platelets, reticulocytes and fibrinogen, shortening of blood coagulation time, as well as extramedullary or accented hematopoiesis of the liver, spleen and bone marrow. Other serum biochemical changes observed mainly in females given 120 mg/kg were decrease or decreasing trend in total protein, A/G ratio, and transaminase activity. Fine structure of the liver from females given 20 mg/kg or more revealed cisternal dilatation of smooth-surfaced endoplasmic reticulum of hepatocytes, and the increased liver weight was observed in females given 120 mg/kg. Accordingly, non-effective dose level of N-22 in 52-week chronic toxicity study was estimated to be 20 mg/kg for males and 5 mg/kg for females.

1. The reverse mutation test was carried out on mofezolac (N-22) at dose range of 50-5000 micrograms/plate using Salmonella typhimurium strains, TA100, TA98, TA1535 and TA1537, and Escherichia coli strain WP2uvrA. In all tester strains no significant increases were observed in the number of revertant colonies as compared with solvent control in the absence or presence of mammalian metabolic activation system. 2. The chromosomal aberration test on N-22 was carried out using cultured Chinese hamster lung cells (CHL). The cells were treated with N-22 at the doses of 37.5, 75.0, 150 and 300 micrograms/ml without S9 Mix and at the doses of 150, 300, 450 and 600 micrograms/ml with S9 Mix. No significant differences were found in the incidence of structural-and numeral-aberrations of chromosomes as compared with the solvent control in the system without S9 Mix. However, in the system with S9 Mix, structural aberration (11.5%) and numeral aberration (14.2%) of chromosomes were observed in the groups of dosing 600 micrograms/ml with dose dependency. 3. These results indicate that N-22 has clastogenic activity by the metabolic activation.

Mofezolac (N-22) is a newly developed analgesic and anti-inflammatory agent. The acute toxicities of N-22 were investigated in ICR mice and Wistar rats in oral (p.o.), intraperitoneal (i.p.) and subcutaneous (s.c.) routes. LD50 values of N-22 in mice were 1528 mg/kg (p.o.), 275 mg/kg (i.p.) and 612 mg/kg (s.c.) for males, and 1740 mg/kg (p.o.), 321 mg/kg (i.p.) and 545 mg/kg (s.c.) for females. Those in rats were 920 mg/kg (p.o.), 378 mg/kg (i.p.) and 572 mg/kg (s.c.) for males, and 887 mg/kg (p.o.), 342 mg/kg (i.p.) and 510 mg/kg (s.c.) for females. The sex difference was not clearly observed in mice and rats, but the species difference was observed in p.o. routes. As an initial toxic sign, the hypoactivity was observed in mice and rats of all routes, subsequently, paleness of skin, anemic conjunctiva, emaciation, stupor and/or coma were observed in mice and rats of p.o. and i.p. routes. In rats of those routes, tonic and/or asphyxial convulsion and dyspnea were also observed. In pathological examination of mice and rats, gastrointestinal disorders were observed in p.o. and i.p. routes, and changes of subcutaneous tissue at the injection site were observed in s.c. route.

peri- and postnatal study of mofezolac (N-22), a new developed analgesic and anti-inflammatory agent, was carried out with Wistar rats. N-22 at dose levels of 0, 25, 50 and 100 mg/kg/day were administered orally to pregnant and subsequent delivered dams from day 17 of gestation through day 21 of lactation. Body weight gains of dams treated with 50 and 100 mg/kg/day were depressed during perinatal period. Prolongation of pregnancy period, low performance of pup care with decrease in body weights and food consumptions were observed in 100 mg/kg/day group. Decrease in number and birth index, increase in number of stillborns and tendency to decrease in viability index on day 4 were found in 100 mg/kg/day group. Other parameters of development, behavior or reproductive capability of F1 animals showed no changes related to administration of N-22. It was suggested that no effect dose levels of N-22 were 25 mg/kg/day for dams viewpoint of general toxicity and that was 50 mg/kg/day for dams on reproductive performance and for offspring on development.

Mofezolac (N-22), a newly developed analgesic and anti-inflammatory agent, at dose levels of 0, 10, 30 and 90 mg/kg/day were administered orally to males from pre-mating to mating period and to females from pre-mating to early gestation period. Effects on reproductive performance of both sexes, especially reproductive capability, and development of offspring were examined. 1. In male parents, no changes in body weight and food consumption were found in all male groups, but the increase in gastric mucosal lesions such as ulcers were observed in 30 and 90 mg/kg/day groups. 2. In female parents, the decrease in body weight and food consumption of 90 mg/kg/day group during early gestation period were found, but at necropsy no changes were shown in all female groups. 3. Reproductive capability, mating and pregnancy performance were not affected. 4. No effects of N-22 on fetuses were observed. 5. The suggestions were as follows: No effect dose levels (NOELs) for male and female general-toxicologically were 10 and 30 mg/kg/day, respectively. NOELs for reproductive capability and for fetal development were 90 mg/kg/day.

A three-month oral subacute toxicity study of mofezolac (N-22), a non-steroidal anti-inflammatory agent, was performed using dose levels of 6, 20, 60 and 200 mg/kg in rats, and recovery was also assessed one month after withdrawal. 1. Toxic signs caused by N-22 administration, observed only in the 200 mg/kg group, were as follows: soiling around the mouth and/or nose, piloerection, anemia, diarrhea, emaciation and decreased spontaneous locomotor activity. Nine males and thirteen females in the 200 mg/kg group excreted bloody diarrhea and died of general exhaustion between weeks four and thirteen of study. 2. In the 200 mg/kg group, decrease in food consumption and suppression of body weight gain were noted in males from about week four and in females from about week six after initiation of administration, and increase in water consumption was noted in males from about week seven. 3. Urinary examination revealed a decline in urinary pH in males of the 20 mg/kg and above groups and elevation of urobilinogen levels in males of the 60 and 200 mg/kg groups. 4. Hematological examination showed decreases in erythrocyte count (RBC), hematocrit value (Ht) and hemoglobin concentration (Hb) and increase in reticulocyte rate in both sexes of the 200 mg/kg group and an increase in neutrophil rate in males of the 200 mg/kg group. 5. Biochemical examination demonstrated a decrease in chloride (Cl-) in males receiving the 20 mg/kg or above doses and a decrease in calcium (Ca++) in males of the 60 and 200 mg/kg groups. Moreover, there were decreases in cholinesterase (ChE) activity, total protein (TP) and albumin (Alb) values, as well as increases in blood urea nitrogen (BUN), uric acid (UA) and potassium (K+) in both sexes of the 200 mg/kg group, along with elevations in GOT and lactate dehydrogenase (LDH) activities in females of the 200 mg/kg group. 6. The absolute and/or relative organ weights for liver, kidneys, spleen and adrenals were increased in the 200 mg/kg group. 7. On pathological examination, perforating ulceration in the jejunum and ileum, turbid ascites, adhesion and inflammatory changes in capsules of the abdominal organs, splenomegaly, mesenteric lymph node hyperplasia and inflammatory changes in the thoracic cavity were observed in dead animals of the 200 mg/kg group. Similar pathological changes were observed in a few survival cases of the 200 mg/kg group. 8. After a one month recovery period, the above-mentioned changes had mostly recovered, indicating that they were reversible.(ABSTRACT TRUNCATED AT 250 WORDS)