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Controlling the size and charge of nanometer-sized objects is of upmost importance for their interactions with cells. We herein present the synthesis of poly(2-oxazoline) based nanogels comprising a hydrophilic shell and an amine containing core compartment. Amine groups were cross-linked using glutaraldehyde resulting in imine based nanogels. As a drug model, amino fluorescein was covalently immobilized within the core, quenching excessive aldehyde functions. By varying the amount of cross-linker, the zeta potential and, hence, the cellular uptake could be adjusted. The fluorescence of the nanogels was found to be dependent on the cross-linking density. Finally, the hemocompatibility of the described systems was studied by hemolysis and erythrocyte aggregation assays. While cellular uptake was shown to be dependent on the zeta potential of the nanogel, no harmful effects to red blood cells was observed, rendering the present system as an interesting toolbox for the production of nanomaterials with a defined biological interaction profile.

The current investigation examines whether the fungicide vinclozolin, which has an anti-androgenic mode of action, is capable of disrupting endocrine homeostasis at very low doses. The data generated clarify whether a non-monotonic dose-response relationship exists to enhance the current debate about the regulation of endocrine disruptors. Moreover, it is part of a series of investigations assessing the dose-response relationship of single and combined administration of anti-androgenic substances. A pre-postnatal in vivo study design was chosen which was compliant with regulatory testing protocols. The test design was improved by additional endpoints addressing hormone levels, morphology and histopathological examinations. Doses were chosen to represent an effect level (20 mg/kg bw/d), the current NOAEL (4 mg/kg bw/d), and a dose close to the "ADI" (0.005 mg/kg bw/d) for the detection of a possible non-monotonic dose-response curve. Anti-androgenic changes were observable at the effect level but not at lower exposures. Nipple/areola counts appeared to be the most sensitive measure of effect, followed by male sex organ weights at sexual maturation, and finally gross and histopathological findings. The results indicate the absence of evidence for effects at low or very low dose levels. A non-monotonic dose-response relationship was not evident.

Fenoxaprop-p-ethyl (FPE) was studied for possible ecotoxicity on two representative toxigenic cyanobacteria including Microcystis aeruginosa and Microcystis viridis. Growth curves, chlorophyll a content, protein content, microcystin levels, oxidative stress, and apoptosis rates were measured for the two cyanobacteria after exposure to different concentrations of FPE. Results showed that the changes in chlorophyll a content and protein content were consistent with cell density, and M. viridis was more sensitive than M. aeruginosa to FPE. The results of oxidative stress indicated that FPE induced the generation of malondialdehyde (MDA) and enhanced the activities of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in these two cyanobacteria. To further explore the toxicity of FPE, apoptosis rates and toxin levels were measured for the two cyanobacteria. Different degrees of apoptosis rates were observed in the two cyanobacteria, and the apoptosis rates increased with the increase concentration of FPE. The intracellular and extracellular MC-LR were both affect by FPE. The presence of FPE in aquatic ecosystem may stimulate the synthesis and release of MC-LR, which may cause serious water pollution and pose threats to human health. These results may be useful for the ecotoxicity assessment of FPE and guiding the rational use of pesticides in agriculture.

Nitro-containing compounds such as nitrofuran and nitroimidazole are drugs used for the treatment of infectious diseases. However, many of these nitro-containing drugs are positive in the bacterial reverse mutation assay (Ames test). The recently approved anti-multidrug-resistant tuberculosis (MDR-TB) drug, delamanid (Deltyba™; OPC-67683), a derivative of 4-nitroimidazole, was negative for mutagenicity in the Ames assay. In Salmonella typhimurium, mutagenicity of nitro compounds has been closely associated with the ability of nitroreductase to metabolize (degradation)these compounds. To explore the lack of mutagenicity for delamanid, we examined the initial metabolic rate and mutagenic-specific activity of a series of nitro compounds in S. typhimurium TA100. The order of maximum mutagenic-activity was nitrofuran > 2-nitroimidazole > 5-nitroimidazole ≥ 4-nitroimidazole, which is very similar to the order of initial metabolic rate, i.e., the Pearson's correlation coefficient (r = 0.85) showed a correlation between metabolic rate and mutagenic-activity. No metabolism of delamanid was detected even after 60 h of treatment. In addition, delamanid was not reduced by two human nitroreductases. These facts may explain the absence of genotoxicity of delamanid in both in vitro and in vivo tests., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Given the proven utility of natriuretic peptides as serum biomarkers of cardiovascular maladaptation and dysfunction in humans and the high cross-species sequence conservation of atrial natriuretic peptides, natriuretic peptides have the potential to serve as translational biomarkers for the identification of cardiotoxic compounds during multiple phases of drug development. This work evaluated and compared the response of N-terminal proatrial natriuretic peptide (NT-proANP) and N-terminal probrain natriuretic peptide (NT-proBNP) in rats during exercise-induced and drug-induced increases in cardiac mass after chronic swimming or daily oral dosing with a peroxisome proliferator-activated receptor γ agonist. Male Sprague-Dawley rats aged 8 to 10 weeks were assigned to control, active control, swimming, or drug-induced cardiac hypertrophy groups. While the relative heart weights from both the swimming and drug-induced cardiac hypertrophy groups were increased 15% after 28 days of dosing, the serum NT-proANP and NT-proBNP values were only increased in association with cardiac hypertrophy caused by compound administration. Serum natriuretic peptide concentrations did not change in response to adaptive physiologic cardiac hypertrophy induced by a 28-day swimming protocol. These data support the use of natriuretic peptides as fluid biomarkers for the distinction between physiological and drug-induced cardiac hypertrophy.

Exposure to environmental endocrine disruptors may interfere with nervous system's activity. Fungicides such as tebuconazole, triadimefon, and vinclozolin have antifungal activities and are used to prevent fungal infections in agricultural plants. In the present study, we studied effects of tebuconazole, triadimefon, and vinclozolin on rat's neurosteroidogenic 5 α -reductase 1 (5 α -Red1), 3 α -hydroxysteroid dehydrogenase (3 α -HSD), and retinol dehydrogenase 2 (RDH2). Rat's 5 α -Red1, 3 α -HSD, and RDH2 were cloned and expressed in COS-1 cells, and effects of these fungicides on them were measured. Tebuconazole and triadimefon competitively inhibited 5 α -Red1, with IC 50 values of 8.670 ± 0.771 × 10 -6  M and 17.390 ± 0.079 × 10 -6  M, respectively, while vinclozolin did not inhibit the enzyme at 100 × 10 -6  M. Triadimefon competitively inhibited 3 α -HSD, with IC 50 value of 26.493 ± 0.076 × 10 -6  M. Tebuconazole and vinclozolin weakly inhibited 3 α -HSD, with IC 50 values about 100 × 10 -6  M, while vinclozolin did not inhibit the enzyme even at 100 × 10 -6  M. Tebuconazole and triadimefon weakly inhibited RDH2 with IC 50 values over 100 × 10 -6  M and vinclozolin did not inhibit this enzyme at 100 × 10 -6  M. Docking study showed that tebuconazole, triadimefon, and vinclozolin bound to the steroid-binding pocket of 3 α -HSD. In conclusion, triadimefon potently inhibited rat's neurosteroidogenic enzymes, 5 α -Red1 and 3 α -HSD.

Background: γ-Aminobutyric acid (GABA) receptors play a central role in fast inhibitory neurotransmission in insects. Several classes of insecticides targeting insect GABA-gated chloride channels have been developed. The important resistant to dieldrin GABA receptor subunit (RDL) has been used to investigate insecticide sites of action using radioligands, electrophysiology and site-directed mutagenesis. Although this important subunit readily forms robust functional homomeric receptors when expressed, alternative splicing and RNA A-to-I editing can generate diverse forms of the receptor., Methods: We have reviewed studies on native and recombinant insect GABA-gated chloride channels, their interactions with ligands acting at orthosteric and allosteric sites and their interactions with insecticides. Since some GABA receptor modulators act on L-glutamate-gated chloride channels, some comparisons are included., Results: The actions on GABA-gated chloride channels of polychlorocycloalkanes, cyclodienes, macrocyclic lactones, phenylpyrazoles, isoxazolines, and metadiamides are described and the mechanisms of action of members of these insecticide classes are addressed. Mutations that lead to resistance are discussed as they can be important in developing field diagnostic tests. Toxicity issues relating to insecticides targeting GABA-gated chloride channels are also addressed. An overview of all major insecticide classes targeting insect GABA-gated chloride channels has enhanced our understanding of these important receptors and their insecticide binding sites. However, the subunit composition of native GABA receptors remains unknown and studies to clarify this are needed. Also, the precise sites of action of the recently introduced isoxazolines and meta-diamides will be of interest to pursue., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The stereoselective cytotoxicity of new chiral acaricide etoxazole and its dissipation in citrus and soil were investigated for the first time. Enantioselective toxicity and oxidative stress of etoxazole toward MCF-7 cells was conducted. The phenomenon of dose- and form-dependent cytotoxicity was demonstrated by MTT and LDH assays, ROS generation, and SOD and CAT activity alternation. Cytotoxicity ranks were found to be consistent with oxidative damage as (R)- > Rac- > (S)-etoxazole. Moreover, the results of enantioselective degradation showed that (S)-etoxazole degraded faster than its antipode (R)-etoxazole. The gradual raise of EF values indicated the achievement of enantioselective degradation in citrus and soil, leaving the enrichment of (R)-etoxazole isomer. Significant differences of environmental behavior and cytotoxicity of etoxazole enantiomers were found in this study which provided valuable insight into the mechanism of potential toxicity and warranted more careful assessment of this pesticide before its agricultural application.

The study was performed to detect the effects of anti-androgenic compounds on the reproduction. In this paper alterations observed in the marine calanoid copepod Acartia tonsa exposed to environmental concentrations of cyproterone acetate (CPA), linuron (LIN), vinclozolin (VIN), and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE) for 21 days covering a full life cycle are described. Histological alterations were studied with a focus on reproductive organs, gonad and accessory sexual glands. Exposure to ≥1.2 µg L(-1) CPA caused degeneration of spermatocytes and deformation of the spermatophore in males. In a single male exposed to 33 µg L(-1) CPA, an ovotestis was observed. In CPA exposed females, enhancement of oogenesis, increase in apoptosis and a decrease in proliferation occurred. Exposure of males to ≥12 µg L(-1) LIN caused degenerative effects in spermatogonia, spermatocytes and spermatids, and at 4.7 µg L(-1) LIN, the spermatophore wall displayed an irregular formation. In LIN exposed females, no such structural alterations were found; however, the proliferation index was reduced at 29 µg L(-1) LIN. At an exposure concentration of ≥100 µg L(-1) VIN, distinct areas in male gonad were stimulated, whereas others displayed a disturbed spermatogenesis and a deformed spermatophore wall. In VIN exposed female A. tonsa, no effects were observed. Male A. tonsa exposed to p,p'-DDE displayed an impairment of spermatogenesis in all stages with increased degrees of apoptosis. In p,p'-DDE-exposed females, a statistical significant increase of the proliferation index and an intensification of oogenesis were observed at 0.0088 µg L(-1).

Endocrine-disrupting chemicals (EDCs) that interfere with the steroid axis can affect amelogenesis, leading to enamel hypomineralization similar to that of molar incisor hypomineralization, a recently described enamel disease. We investigated the sex steroid receptors that may mediate the effects of EDCs during rat amelogenesis. The expression of androgen receptor (AR), estrogen receptor (ER)-α, and progesterone receptor was dependent on the stage of ameloblast differentiation, whereas ERβ remained undetectable. AR was the only receptor selectively expressed in ameloblasts involved in final enamel mineralization. AR nuclear translocation and induction of androgen-responsive element-containing promoter activity upon T treatment, demonstrated ameloblast responsiveness to androgens. T regulated the expression of genes involved in enamel mineralization such as KLK4, amelotin, SLC26A4, and SLC5A8 but not the expression of genes encoding matrix proteins, which determine enamel thickness. Vinclozolin and to a lesser extent bisphenol A, two antiandrogenic EDCs that cause enamel defects, counteracted the actions of T. In conclusion, we show, for the first time, the following: 1) ameloblasts express AR; 2) the androgen signaling pathway is involved in the enamel mineralization process; and 3) EDCs with antiandrogenic effects inhibit AR activity and preferentially affect amelogenesis in male rats. Their action, through the AR pathway, may specifically and irreversibly affect enamel, potentially leading to the use of dental defects as a biomarker of exposure to environmental pollutants. These results are consistent with the steroid hormones affecting ameloblasts, raising the issue of the hormonal influence on amelogenesis and possible sexual dimorphism in enamel quality.

Type 2 diabetes took insulin resistance as the main clinical manifestation. PPARs have been reported to be the therapeutic targets of metabolic disorders, such as obesity, hypertension, diabetes, and cardiovascular disease. Previously, PPARγ agonist rosiglitazone was restricted in clinic due to cardiomyocytes infarction, weight gain, and other serious side-effects, which were mainly due to the single and selective PPARγ agonism. In recent years, multitarget-directed PPAR agonists with synergistic reaction as well as fewer side-effect have been the hot topic in designing promising agents. In this review, we updated and generalized the development of PPARγ partial agonists, PPARγ antagonists, PPARα/γ dual agonists, PPARδ partial agonists, PPARδ antagonists, PPARα/δ dual agonists, PPARγ/δ dual agonists, and PPARα/γ/δ pan-agonists published in recent decade. Most of these molecules were modified from known structures or came from high-throughput screening. Among these molecules, some were expected to be promising drugs against metabolic disorders, while others seemed to provide new insight for designing novel PPAR agents., (© 2016 John Wiley & Sons A/S.)

Methoxychlor (MXC) and vinclozolin (VIN) are well-recognized endocrine disrupting chemicals known to alter epigenetic regulations and transgenerational inheritance; however, non-endocrine disruption endpoints are also important. Thus, we determined the effects of MXC and VIN on the dysregulation of gap junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) in WB-F344 rat liver epithelial cells. Both chemicals induced a rapid dysregulation of GJIC at non-cytotoxic doses, with 30 min EC50 values for GJIC inhibition being 10 µM for MXC and 126 µM for VIN. MXC inhibited GJIC for at least 24 h, while VIN effects were transient and GJIC recovered after 4 h. VIN induced rapid hyperphosphorylation and internalization of gap junction protein connexin43, and both chemicals also activated MAPK ERK1/2 and p38. Effects on GJIC were not prevented by MEK1/2 inhibitor, but by an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), resveratrol, and in the case of VIN, also, by a p38 inhibitor. Estrogen (ER) and androgen receptor (AR) modulators (estradiol, ICI 182,780, HPTE, testosterone, flutamide, VIN M2) did not attenuate MXC or VIN effects on GJIC. Our data also indicate that the effects were elicited by the parental compounds of MXC and VIN. Our study provides new evidence that MXC and VIN dysregulate GJIC via mechanisms involving rapid activation of PC-PLC occurring independently of ER- or AR-dependent genomic signaling. Such alterations of rapid intercellular and intracellular signaling events involved in regulations of gene expression, tissue development, function and homeostasis, could also contribute to transgenerational epigenetic effects of endocrine disruptors., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Many studies have investigated exposure biomarkers for high dose radiation. However, no systematic study on which biomarkers can be used in dose estimation through premature chromosome condensation (PCC) analysis has been conducted. The present study aims to screen the high-dose radiation exposure indicator in calyculin A-induced PCC. The dose response of multiple biological endpoints, including G2/A-PCC (G2/M and M/A-PCC) index, PCC ring (PCC-R), ratio of the longest/shortest length (L/L ratio), and length and width ratio of the longest chromosome (L/B ratio), were investigated in calyculin A-induced G2/A-PCC spreads in human peripheral blood lymphocytes exposed to 0-20Gy (dose-rate of 1Gy/min) cobalt-60 gamma-rays. The G2/A-PCC index was decreased with enhanced absorbed doses of 4-20Gy gamma-rays. The G2/A PCC-R at 0-12Gy gamma-rays conformed to Poisson distribution. Three types of PCC-R were scored according to their shape and their solidity or hollowness. The frequencies of hollow PCC-R and PCC-R including or excluding solid ring in G2/A-PCC spreads were enhanced with increased doses. The length and width of the longest chromosome, as well as the length of the shortest chromosome in each G2/M-PCC or M/A-PCC spread, were measured. All L/L or L/B ratios in G2/M-PCC or M/A-PCC spread increased with enhanced doses. A blind test with two new irradiated doses was conducted to validate which biomarker could be used in dose estimation. Results showed that hollow PCC-R and PCC-R including solid ring can be utilized for accurate dose estimation, and that hollow PCC-R was optimal for practical application., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Vinclozolin is a fungicide used in agriculture that can reach aquatic ecosystems and affect the organisms living there. Its effects have been intensively studied in vertebrates, where it acts as an antiandrogen, but there is a lack of information about its mechanistic effects on invertebrates. In this work, we analyzed the response of genes related to the endocrine system, the stress response, and the detoxification mechanisms of Chironomus riparius fourth instar larvae after 24h and 48h exposures to 20 (69.9nM), 200 (699nM), and 2000μg/L (6.99μM) of Vinclozolin. Survival analysis showed that this compound has low toxicity, as it was not lethal for this organism at the concentrations used. However, this fungicide was shown to modify the transcriptional activity of the ecdysone response pathway genes EcR, E74, and Kr-h1 by increasing their mRNA levels. While no changes were observed in disembodied, a gene related with the ecdysone synthesis metabolic pathway, Cyp18A1, which is involved in the inactivation of the active form of ecdysone, was upregulated. Additionally, the expression of two genes related to other hormones, FOXO and MAPR, did not show any changes when Vinclozolin was present. The analysis of stress response genes showed significant changes in the mRNA levels of Hsp70, Hsp24, and Gp93, indicating that Vinclozolin activates the cellular stress mechanisms. Finally, the expressions of the genes Cyp4G and GstD3, which encode enzymes involved in phase I and phase II detoxification, respectively, were analyzed. It was found that their mRNA levels were altered by Vinclozolin, suggesting their involvement in the degradation of this compound. For the first time, these results show evidence that Vinclozolin can modulate gene expression, leading to possible significant endocrine alterations of the insect endocrine system. These results also offer new clues about the mode of action of this compound in invertebrates., (Copyright © 2016 Elsevier B.V. All rights reserved.)

American sloughgrass is a troublesome annual grass weed in winter wheat field rotated with rice in China. The overreliance on acetyl-coenzyme A carboxylase (ACCase) inhibiting herbicides has resulted in resistance evolution in this weed. In this study, the cross-resistance patterns to fenoxaprop-p-ethyl, clodinafop-propargyl, fluazifop-p-butyl, haloxyfop-p-methyl, sethoxydim, clethodim and pinoxaden were established using purified plants individually homozygous for specific mutant ACCase alleles. Results indicated that 1781Leu allele endows high-level resistance to APPs, CHDs and pinoxaden while confers moderate resistance to haloxyfop-p-methyl. The 2027Cys and 2041Asn alleles endow high-level resistance to APPs and pinoxaden and lower level resistance to CHDs. The 2078Gly allele confers high-level resistance to all herbicides tested in this study, however, moderate resistance to sethoxydim. The 2096Ala very likely endows high-level resistance to fluazifop-p-butyl, haloxyfop-p-methyl and moderate resistance to sethoxydim. In addition, one undefined resistance mechanism was involved in population SD-04., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A range of environmental factors, including chemicals, can affect epigenetic processes in organisms leading to variations in phenotype. Thus, epigenetics displays an important environmentally responsive element. The transgenerational impact of environmental stressors on DNA methylation and phenotype was the focus of this study. The influence of two known DNA methylation-changing agents, the phytoestrogen genistein and the fungicide vinclozolin, on the overall DNA methylation level in the Asian tiger mosquito Aedes albopictus was investigated. The experiment comprised four generations in a full life-cycle design with an exposed parental generation and three consecutive non-exposed offspring generations. Application of the methylation agents to the parental generation of the study led to an alteration of the global DNA methylation level of the exposed individuals and those in two subsequent generations. The phenotypic variability of the offspring generations was assessed by examining their insecticide sensitivity. Here, a significant decrease in sensitivity (p<0.01) towards the model insecticide imidacloprid revealed alterations of the mosquito's phenotype in two subsequent generations. Thus, the evaluation of A. albopictus from an epigenetic perspective can contribute important information to the study of the high adaptability of this invasive disease vector to new environments, and its underlying mechanisms., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Exposure to endocrine disruptors during critical periods of development can impact the sustainability of wild fish populations. Anti-androgenic compounds have received less attention, but are capable of modulating gonad differentiation and maturation, and impairing reproduction in fish. The fungicide vinclozolin (VZ) has been shown to impair reproduction in adult fish, but less is known about its effects following exposure earlier in development. Here we show that waterborne exposure to 400μg VZ/L during critical periods of sex differentiation (21-35 days post fertilization) permanently shifts sex ratios towards females, and alters the maturation of the gonad. Both fecundity and fertility were reduced, even when oogenesis and spermatogenesis recover and sperm motility is not altered. These results demonstrate the need to better understand the impacts of early exposure to anti-androgenic compounds on fish., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The embryonic stem cell test (EST) is a promising system to detect embryotoxicity in vitro. Recent studies have pointed out some limitations of the EST and suggest that the applicability domain of the EST and its prediction model have to be better defined. Here, eight substances of known reproductive toxicity were tested in the EST under blind conditions. We applied the prediction model to the data of the EST after classifying the substances according to the published criteria. In addition, a simplified classification of the EST results into two classes as an approach to hazard assessment was compared to the European Union Classification, Labelling and Packaging (CLP) Regulation labels of the substances. With one exception, substances that are labeled as reproductive toxicants according to the CLP Regulation were detected as embryotoxic in the EST while substances without label were found to be non-embryotoxic according to the EST.

The present study describes the optimization of a series of novel benzoxazole-piperidine (piperazine) derivatives combining high dopamine D2 and serotonin 5-HT1A, 5-HT2A receptor affinities. Of these derivatives, the pharmacological features of compound 29 exhibited high affinities for the DA D2, 5-HT1A and 5-HT2A receptors, but low affinities for the 5-HT2C and histamine H1 receptors and human ether-a-go-go-related gene (hERG) channels. Furthermore, compound 29 reduced apomorphine-induced climbing and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced head twitching without observable catalepsy, even at the highest dose tested. Thus, compound 29 is a promising candidate as a multi-target antipsychotic treatment., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Shortawn foxtail (Alopecurus aequalis) is a troublesome grass weed infesting winter wheat and oilseed rape productions in China. Fenoxaprop-p-ethyl and mesosulfuron-methyl failed to control shortawn foxtail of AHSX-1 population collected from a wheat field in Shou County, Anhui province. Molecular analyses revealed that Asp2078Gly mutation of ACCase and Trp574Leu mutation of AHAS were present in plants of the AHSX-1 population. The homozygous plants were isolated and cultured until seed maturity. Whole-plant herbicide bioassays were conducted in the greenhouse using the purified seeds of F1 generation. Dose-response experiments showed that the AHSX-1 population has evolved a very high level resistance to fenoxaprop-p-ethyl (RI = 275) and mesosulfuron-methyl (RI = 788). To determine the sensitivity to other herbicides, assays were conducted at the single recommended rate of each herbicide. Based on the results, the AHSX-1 population was considered to be highly resistant to clodinafop-propargyl, pyroxsulam and flucarbazone-sodium, moderately or highly resistant to quizalofop-p-ethyl, clethodim, sethoxydim and pinoxaden, and susceptible to isoproturon and chlorotoluron. This is the first report of Asp2078Gly mutation in shortawn foxtail and the two robust dCAPS markers designed could quickly detect Asp2078 and Trp574 mutations in ACCase and AHAS gene of shortawn foxtail, respectively., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Recent studies suggest that disturbing androgen-signaling pathways in porcine ovarian follicles may cause granulosa cell (GC) death. For this reason, we investigated which apoptotic pathway is initiated after GC exposure to an environmental antiandrogen, vinclozolin (Vnz), in vitro. Immunocytochemistry, Western blots, and fluorometric assays were used to quantify caspase-3 and -9 expression and activity. To elucidate the specific mechanism of Vnz action and toxicity, GCs were assessed for viability, cytotoxicity, and apoptotic activity using the ApoTox-Glo Triplex Assay. To further determine the mechanism of GC death induced by Vnz, we used the Apoptosis Antibody Array Kit. In response to Vnz stimulus, we found an increased level of caspase-3 protein expression (P ≤ 0.001) and an increase in caspase-3 proteolytic activity (P ≤ 0.001), confirming that Vnz is a potent proapoptotic factor. The strong immunoreaction of caspase-9 after Vnz treatment (P ≤ 0.001) suggests that intrinsic mitochondrial apoptosis pathway was activated during GC death. On the other hand, caspase-8, being a part of the extrinsic receptor pathway, was also activated (P ≤ 0.001). Therefore, it is possible that Vnz induces porcine granulosal apoptosis also through a parallel pathway. Activation of these two pathways was confirmed by the Apoptosis Antibody Array Kit. In conclusion, it is possible that the intrinsic signaling pathway may not act as an initial trigger for GC apoptosis but might contribute to the amplification and propagation of apoptotic cell death in the granulosa layer after treatment with this antiandrogen. Moreover, Vnz disturbs the physiological process of programmed cell death. Consequently, this could explain why atretic follicles are rapidly removed and suggests that normal function of the ovarian follicle may be destroyed., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Liver damage occurred in some patients who took troglitazone (TGZ) for type II diabetes. The 2,4-thiazolidinedione (TZD) ring in TGZ's structure has been implicated in its hepatotoxicity. To further examine the potential role of a TZD ring in toxicity we used HepG2 cells to evaluate two series of compounds containing different cyclic imides. N-phenyl analogues comprised 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT); 3-(3,5-dichlorophenyl)-2,4-oxazolidinedione (DCPO) and N-(3,5-dichlorophenyl)succinimide (NDPS). Benzylic compounds, which closely resemble TGZ, included 5-(3,5-dichlorophenylmethyl)-2,4-thiazolidinedione (DCPMT); 5-(4-methoxyphenylmethyl)-2,4-thiazolidinedione (MPMT); 5-(4-methoxyphenylmethylene)-2,4-thiazolidinedione (MPMT-I); 5-(4-methoxyphenylmethyl)-2,4-oxazolidinedione (MPMO); 3-(4-methoxyphenylmethyl)succinimide (MPMS) and 3-(4-methoxyphenylmethylene)succinimide (MPMS-I). Cytotoxicity was assessed using the MTS assay after incubating the compounds (0-250μM) with HepG2 cells for 24h. Only certain TZD derivatives (TGZ, DCPT, DCPMT and MPMT-I) markedly decreased cell viability, whereas MPMT had low toxicity. In contrast, analogues without a TZD ring (DCPO, NDPS, MPMO, MPMS and MPMS-I) were not cytotoxic. These findings suggest that a TZD ring may be an important determinant of toxicity, although different structural features, chemical stability, cellular uptake or metabolism, etc., may also be involved. A simple clustering approach, using chemical fingerprints, assigned each compound to one of three classes (each containing one active compound and close homologues), and provided a framework for rationalizing the activity in terms of structure., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

We present a quantitative in vitro-in vivo extrapolation framework enabling the estimation of the external dermal exposure dose from in vitro experimental data relevant to a toxicity pathway of interest. The framework adapts elements of the biological pathway altering dose (BPAD) method [Judson et al. Chem Res Toxicol 2011;24:451] to the case of dermal exposure. Dermal doses of four toxicants equivalent to concentrations characterizing their effect on estrogen receptor α or androgen receptor activity in chemical-activated luciferase expression (CALUX) assays are estimated. The analysis shows that dermal BPADs, calculated from one in vitro concentration, can differ by up to a factor of 55, due to the impact applied dose and dermal exposure scenarios can have on skin permeation kinetics. These features should therefore be taken into account in risk assessment of dermally applied chemicals., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In recent years, the spread of new psychoactive substances has dramatically increased in term of availability of both number of compounds and chemical families. In November 2012, 4,4'-Dimethylaminorex (4,4'-DMAR), a novel synthetic stimulant, was first detected in the Netherlands. Between June 2013 and June 2014, thirty-one deaths associated with the consumption of this new drug have been registered across Europe. In this MiniReview, we have summarized the chemical, pharmacological and toxicological information about this new legal high., (© 2015 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Endocrine disruptors may play substantial roles in the high incidence of breast cancer. We previously described how early exposure to the mixture of phytoestrogen genistein (G) and the anti-androgen vinclozolin (V) affects peripubertal mammary development. This study evaluates the carcinogenic potential of exposure to V alone or associated with G from conception until weaning in Wistar rats. Dams were exposed to V, G or GV during pregnancy/lactation. At PND50 offspring were treated with DMBA[7,12-dimethylbenz(a)anthracene]. V or GV maternal exposure decreased number of DMBA-induced mammary tumors in the offspring, without significant modifications in tumor incidence, multiplicity and latency. G exposure decreased number of tumors, incidence and multiplicity. Unexpectedly, GV exposure increased tumor volume (p=0.04 vs controls) and epithelial proliferation (p=0.001 vs controls; p=0.005 vs G,V only). All tumors were in situ carcinomas. Concluding, maternal gestation/lactation exposure to a vinclozolin and genistein mixture significantly increases offspring tumor growth without changes in carcinogenesis susceptibility., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: 4-Methyl-5-(4-methylphenyl)-4,5-dihydrooxazol-2-amine (4,4'-dimethylaminorex (4,4'-DMAR)) is a derivative of the controlled substances aminorex and 4-methylaminorex marketed as a potential novel psychoactive substance. This paper brings together the published scientific and 'grey' literature to understand 4,4'-DMAR as a novel psychoactive substance., Methods: Searches of the published scientific and 'grey' literature, using the keywords '4-methyl-euphoria', '4-methyl-U4Euh', '4-M-4-MAR', '4,4-dimethylaminorex', '4,4'-DMAR', 'para-methyl-4-aminorex' and 'Serotoni', were undertaken to identify information on the availability, prevalence of use and desired/unwanted effects of 4,4'-DMAR., Results: No studies have reported the prevalence of use of 4,4'-DMAR. Internet snapshot studies in April and May 2014 showed availability of 4,4'-DMAR from one and two Internet suppliers respectively. Price decreased with increasing purchase amounts from €12/g for a 1-g purchase to €2.20/g for a 100-g purchase in April 2014. Internet discussion fora suggest that the desired and unwanted effects of 4,4'-DMAR are similar to those seen with other sympathomimetic drugs such as 3,4-methylenedioxy-methamphetamine and mephedrone, although the duration of unwanted effects and 'comedown' appear to be longer. Unwanted effects were reported at doses of 5-200 mg, which overlaps with the reported doses (10-200 mg) associated with desired effects. 4,4'-DMAR has been detected, along with other drugs, in 27 deaths in Europe; the contribution of the 4,4'-DMAR in these deaths has not been established., Conclusions: Currently, there appears to limited availability of 4,4'-DMAR; therefore, use is likely to be low. Its desired and unwanted effects appear similar to other sympathomimetic recreational drugs such as methylenedioxy-methamphetamine or mephedrone., (Copyright © 2015 John Wiley & Sons, Ltd.)

Vinclozolin (VZ) is a dicarboximide fungicide widely used on fruits, vegetables and wines, effective against fungi plagues. In this study we characterized the effects of VZ using a 4-day reproductive chronic assay with the freshwater rotifer Brachionus calyciflorus. The assay included observations of several features of asexual and sexual reproduction. Our results indicate that VZ: (a) increased asexual and sexual reproduction, (b) caused severe abnormality in females and (c) these abnormalities were inherited by sexual and asexual reproduction. At 1.2 mg/L three abnormal females were found out of 457 total females (0.66 %). This low percentage is consistent and reproducible according to further analysis, where we increased the number of replicates and total females exposed to 1.2 mg/L of VZ, and found 18 abnormal females out of 2868 total females (0.63 % abnormality). Interestingly, abnormal females found at 5.6 mg/L VZ exposure, were able to show mating behavior. Our results suggest that VZ behaves as a strong endocrine disruptor whose effects show the characteristic inverted-U-shape exposure concentration response curve regarding the intrinsic population increase and the percentage of abnormalities as endpoints.

In mammals, germ cell differentiation is initiated in the Primordial Germ Cells (PGCs) during fetal development. Prenatal exposure to environmental toxicants such as endocrine disruptors may alter PGC differentiation, development of the male germline and induce transgenerational epigenetic disorders. The anti-androgenic compound vinclozolin represents a paradigmatic example of molecule causing transgenerational effects on germ cells. We performed prenatal exposure to vinclozolin in mice and analyzed the phenotypic and molecular changes in three successive generations. A reduction in the number of embryonic PGCs and increased rate of apoptotic cells along with decrease of fertility rate in adult males were observed in F1 to F3 generations. Blimp1 is a crucial regulator of PGC differentiation. We show that prenatal exposure to vinclozolin deregulates specific microRNAs in PGCs, such as miR-23b and miR-21, inducing disequilibrium in the Lin28/let-7/Blimp1 pathway in three successive generations of males. As determined by global maps of cytosine methylation, we found no evidence for prominent changes in DNA methylation in PGCs or mature sperm. Our data suggest that embryonic exposure to environmental endocrine disruptors induces transgenerational epigenetic deregulation of expression of microRNAs affecting key regulatory pathways of germ cells differentiation.

We prepared statistical copolymers composed of 2-methyl-2-oxazoline (MeOx) in combination with 2-butenyl-2-oxazoline (BuOx) or 2-decenyl-2-oxazoline (DecOx) as a basis for polymer analogous introduction of 1,2-aminothiol moieties at the side chain. MeOx provides hydrophilicity as well as cyto- and hemocompatibility, whereas the alkene groups of BuOx and DecOx serve for functionalization with a thiofunctional thiazolidine by UV-mediated thiol-ene reaction. After deprotection the cysteine content in functionalized poly(2-oxazoline) (POx) is quantified by NMR and a modified trinitrobenzenesulfonic acid assay. The luminescent cell viability assay shows no negative influence of cysteine-functionalized POx (cys-POx) concerning cell viability and cell number. cys-POx was used for multiple chemically orthogonal couplings with thioester-terminated peptides through native chemical ligation (NCL), which was performed and confirmed by NMR and MALDI-ToF measurements.

Objectives: Currently used fibre-reinforced composite (FRC) intracanal posts possess low flexural strength which usually causes post fracture when restoring teeth with extensive loss. To improve the flexural strength of FRC, we aimed to apply a high-performance fibre, poly p-phenylene-2, 6-benzobisoxazole (PBO), to FRCs to develop a new intracanal post material., Methods: To improve the interfacial adhesion strength, the PBO fibre was treated with coupling agent (Z-6040), argon plasma, or a combination of above two methods. The effects of the surface modifications on PBO fibre were characterised by determining the single fibre tensile strength and interfacial shear strength (IFSS). The mechanical properties of PBO FRCs were characterised by flexural strength and flexural modulus. The cytotoxicity of PBO FRC was evaluated by the MTT assay., Results: Fibres treated with a combination of Z-6040 and argon plasma possessed a significantly higher IFSS than untreated fibres. Fibre treated with the combination of Z-6040-argon-plasma FRC had the best flexural strength (531.51 ± 26.43MPa) among all treated fibre FRCs and had sufficient flexural strength and appropriate flexural moduli to be used as intracanal post material. Furthermore, an in vitro cytotoxicity assay confirmed that PBO FRCs possessed an acceptable level of cytotoxicity., Conclusions: In summary, our study verified the feasibility of using PBO FRC composites as new intracanal post material. Although the mechanical property of PBO FRC still has room for improvement, our study provides a new avenue for intracanal post material development in the future., Clinical Significance: To our knowledge, this is the first study to verify the feasibility of using PBO FRC composites as new intracanal post material. Our study provided a new option for intracanal post material development., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Calyculin C, a minor derivative of the calyculins, has an additional methyl group on C32 of calyculin A. A recent biosynthetic study of calyculins revealed that an end product of calyculin biosynthesis is the pyrophosphate form, phosphocalyculin A. However, the pyrophosphate counterpart derived from calyculin C had not been reported. We isolated phosphocalyculin C as a minor pyrophosphate derivative, by a detailed investigation of an extract from the sponge Discodermia calyx. The treatment of phosphocalyculin C with the D. calyx cell-free extract significantly enhanced its cytotoxicity, providing molecular evidence for its role as the protoxin of calyculin C., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The fungicide vinclozolin (VZ) is in use globally and known to disrupt reproductive function in male. The present study tested the hypothesis that VZ disrupts testicular function in goldfish (Carassius auratus) by affecting brain-pituitary-testis axis. Goldfish were exposed to 100, 400 and 800 μg/L VZ and 5 μg/L 17β-estradiol (E2) for comparison. In VZ treated goldfish, 11-ketotesteosterone (11-KT) secretion was changed depending on dose and duration period of treatment. Following 7 days of exposure, 11-KT was decreased in goldfish exposed to 800 μg/L VZ, while it was increased in goldfish exposed to 100 μg/L VZ after 30 days of exposure. Circulating E2 level was unchanged in VZ treated goldfish, however the E2/11-KT ratio was increased in a concentration-related manner. In E2 treated goldfish, circulatory 11-KT and E2 levels were decreased and increased, respectively, which resulted in an increase in the E2/11-KT ratio. Exposure to VZ at 100 μg/L caused a significant increase in the circulatory luteinizing hormone (LH) after 30 days. In E2 treated fish circulatory LH was decreased, significantly. Transcripts of genes encoding gonadotropin-releasing hormone and androgen receptor in the brain, and those of genes encoding LH and follicle-stimulating hormone receptors, StAR, CYP17, and 3β-HSD in the testis changed in VZ-treated goldfish depending on concentration and period of treatment. mRNA of genes encoding vitellogenin and estrogen receptor in the liver and cytochrome P450 aromatase in the brain were increased in E2-treated goldfish. The results suggest that VZ-induced changes in 11-KT were due to disruption in brain-pituitary-testis axis and provide integrated characterization of VZ-related reproductive disorders in male fish., (Copyright © 2014 Elsevier B.V. All rights reserved.)

How an individual responds to the environment depends upon both personal life history as well as inherited genetic and epigenetic factors from ancestors. Using a 2-hit, 3 generations apart model, we tested how F3 descendants of rats given in utero exposure to the environmental endocrine-disrupting chemical (EDC) vinclozolin reacted to stress during adolescence in their own lives, focusing on sexually dimorphic phenotypic outcomes. In adulthood, male and female F3 vinclozolin- or vehicle-lineage rats, stressed or nonstressed, were behaviorally characterized on a battery of tests and then euthanized. Serum was used for hormone assays, and brains were used for quantitative PCR and transcriptome analyses. Results showed that the effects of ancestral exposure to vinclozolin converged with stress experienced during adolescence in a sexually dimorphic manner. Debilitating effects were seen at all levels of the phenotype, including physiology, behavior, brain metabolism, gene expression, and genome-wide transcriptome modifications in specific brain nuclei. Additionally, females were significantly more vulnerable than males to transgenerational effects of vinclozolin on anxiety but not sociality tests. This fundamental transformation occurs in a manner not predicted by the ancestral exposure or the proximate effects of stress during adolescence, an interaction we refer to as synchronicity.

Objective: Many pesticides including pyrethroids and azole fungicides are suspected to have an endocrine disrupting property. At present, the joint activity of compound mixtures is only marginally known. Here we tested the hypothesis that the antiandrogenic activity of mixtures of azole fungicides can be predicted by the concentration addition (CA) model., Methods: The antiandrogenic activity was assessed in MDA-kb2 cells. Following assessing single compounds activities mixtures of azole fungicides and vinclozolin were investigated. Interactions were analyzed by direct comparison between experimental and estimated dose-response curves assuming CA, followed by an analysis by the isobole method and the toxic unit approach., Results: The antiandrogenic activity of pyrethroids deltamethrin, cypermethrin, fenvalerate and permethrin was weak, while the azole fungicides tebuconazole, propiconazole, epoxiconazole, econazole and vinclozolin exhibited strong antiandrogenic activity. Ten binary and one ternary mixture combinations of five antiandrogenic fungicides were assessed at equi-effective concentrations of EC25 and EC50. Isoboles indicated that about 50% of the binary mixtures were additive and 50% synergistic. Synergism was even more frequently indicated by the toxic unit approach., Conclusion: Our data lead to the conclusion that interactions in mixtures follow the CA model. However, a surprisingly high percentage of synergistic interactions occurred. Therefore, the mixture activity of antiandrogenic azole fungicides is at least additive., Practice: Mixtures should also be considered for additive antiandrogenic activity in hazard and risk assessment., Implications: Our evaluation provides an appropriate "proof of concept", but whether it equally translates to in vivo effects should further be investigated., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A selective α2C -adrenoceptor (AR) agonist was developed for the treatment of neuropathic pain. The objective was to dissociate analgesic activity from cardiovascular and sedative side effects commonly observed with nonselective agents. A 2-amino-oxazoline derivative (compound A), identified as a dual α2C -AR agonist/α2A -AR antagonist in in vitro-binding assays, exhibited in vivo efficacy in rodent pain models. Its safety profile was compared with that of clonidine in six different in vivo models. Contrary to clonidine, compound A did not induce hypotension in pentobarbital-anesthetized rats, in conscious spontaneous hypertensive rats, or in telemetered dogs. Both agents induced similar dose-dependent decreases in heart rate in dogs and rats. In anesthetized pithed rats, clonidine showed dose-dependent hypertension and inhibited electrical nerve stimulation-induced tachycardia at doses close to its efficacious doses in the mouse formalin test, while compound A had much weaker vasoconstrictive and antichronotropic effects. Finally, in a mouse Irwin test, no sedation was observed with compound A at 30-fold its ED50 in the mouse formalin test, while sedative effects of clonidine started from three-fold its ED50 . These data confirm the advantageous safety profile of the new dual α2C -AR agonist/α2A -AR antagonist agent vs. the nonselective agonist clonidine., (© 2013 Société Française de Pharmacologie et de Thérapeutique. Published by John Wiley & Sons Ltd.)

During the second half of 2013, a total of 26 deaths involving para-methyl-4-methylaminorex (4,4'-DMAR) were reported to the European Monitoring Centre for Drugs and Drug Addiction. While aminorex and 4-methylaminorex (4-MAR) are known psychostimulants, nothing is known about the comparatively new para-methyl analog. Analytical characterization of two independent samples obtained from online vendors confirmed the presence of the (±)-cis isomer that also appeared to be associated with at least 18 of the 26 deaths. Extensive characterizations included crystal structure analysis, single, tandem, and high-resolution mass spectrometry, liquid and gas chromatography, and nuclear magnetic resonance spectroscopy. For the work described here, both the (±)-cis and (±)-trans racemates were also synthesized, confirming that the differentiation between these two forms was straight-forward. Monoamine transporter activity was studied using rat brain synaptosomes which included the comparison with d-amphetamine, aminorex and (±)-cis-4-MAR. (±)-cis-4,4'-DMAR was a potent, efficacious substrate-type releaser at transporters for dopamine, norepinephrine and serotonin with EC50 values of 8.6 ± 1.1 nM (DAT), 26.9 ± 5.9 nM (NET) and 18.5 ± 2.8 nM (SERT), respectively. The potency of (±)-cis-4,4'-DMAR at DAT and NET rivalled that of other psychomotor stimulant drugs like d-amphetamine and aminorex. However, (±)-cis-4,4'-DMAR had much more potent actions at SERT and activity at SERT varied more than 100-fold across the four drugs. The potent releasing activity of (±)-cis-4,4'-DMAR at all three monoamine transporters predicts a potential for serious side-effects such as psychotic symptoms, agitation, hyperthermia and cardiovascular stimulation, especially after high-dose exposure or following combination with other psychostimulants., (Copyright © 2014 John Wiley & Sons, Ltd.)

The aim of this study was to investigate whether the androgens testosterone and dihydrotestosterone (DHT) and the antiandrogenic fungicide vinclozolin (Vnz) exert proapoptotic effects on porcine granulosa cells (GCs), and to examine the roles of these compounds in follicular atresia. Granulosa cells isolated from pig follicles were cultured for 24 hours, and then exposed to 0.1 μM testosterone, 0.1 μM DHT, 14 μM Vnz, or the equivalent concentrations of testosterone and Vnz or DHT and Vnz for a further 24 hours. Apoptosis and necrosis of the GCs were determined via Hoechst staining and flow cytometry analyses of annexin V-stained cells. Whole porcine follicles were also exposed to the same compounds and combinations of compounds for 24 hours. The sections were stained with hematoxylin and eosin for morphologic assessments, and a Terminal deoxynucleotidyl Transferase Biotyn-dUTP Nick-End Labeling (TUNEL) assay was performed to determine the number of apoptotic cells. The progesterone and estradiol concentrations secreted into the culture media by isolated GCs and follicles were also measured. Exposure to the androgens resulted in an increased number of apoptotic GCs both in vitro and in the organotypic model. Vinclozolin exposure increased and decreased the number of necrotic and apoptotic GCs, respectively. Furthermore, compared with control follicles, those exposed to testosterone, DHT, or Vnz displayed enhanced atresia, and coadministration of Vnz attenuated the promotive effect of these androgens on atresia. Estradiol secretion was stimulated by the combination of testosterone and Vnz, whereas exposure to Vnz alone reduced it. Progesterone production declined after the combined addition of androgens and the antiandrogen. In summary, Vnz caused massive necrosis of GCs in vitro and induced apoptosis of GCs in whole follicles. The androgens testosterone and DHT enhanced these effects. The results presented here suggest that selective destruction of porcine follicles is a serious consequence of exposure to Vnz, and may lead to premature ovarian failure in affected animals., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Various testing methods for the detection of the endocrine disruptive activities of chemicals have been developed in freshwater fish species. However, a few relatively easier specific methods for detecting anti-androgenic activities are available for fish. The aim of this study was to verify the papillary process in Japanese medaka (Oryzias latipes) as an indicator of the anti-androgenic activity of chemicals. Japanese medaka were exposed to two types of anti-androgenic compounds, vinclozolin and flutamide, using two short-term assays; one was conformed to the existing short-term reproduction assay using adult fish (adult test) and the other was a test based on the same methods but using juvenile fish at the beginning of exposure (juvenile test). Significant decreases in male papillary processes were observed in the juvenile test treated with the highest concentration of both antiandrogens (640 µg l(-1) vinclozolin and 1000 µg l(-1) flutamide); however, no significant effects were observed in the adult test. Consequently, our results indicate that papillary processes in Japanese medaka can be used as the end-point for screening the anti-androgenic activity of chemicals using juvenile fish for a specific period based on the existing short-term reproduction assay., (Copyright © 2013 John Wiley & Sons, Ltd.)

Several endocrine disrupting compounds (EDC) elicit skeletal dysgenesis at pharmacological doses. We have investigated the impact of doses below the "No Observed Adverse Effect" (NOAEL) for vinclozolin (V), an anti-androgenic fungicide, alone or associated with xenoestrogens (Genistein, G and bisphenol-A, BPA). V, G, BPA and their combinations were administered orally to female Wistar rats during gestation and lactation. F1 and F2 offspring were investigated for skeletal anomalies at post-natal days 30, 110 (d30, d110). Skeletal development was monitored by measuring caudal vertebrae and long bones dimensions by X-ray micro-CT-scan. A significant increase in Inter Transverse Apophysis (ITA) distance at the upper head of caudal vertebrae, associated with a reduction in vertebral body height was observed in treated F1 females, but not males. Histometrical analysis of vertebral body growth plate cartilage was performed on serial sections of caudal vertebrae. F1 females but not males showed a diminution in growth plate thickness, with greater impact on the hypertrophic zone. All effects were maximal at d30. Effects on ITA width persisted until d110 while effects on growth plate disappeared. These effects were essentially vinclozolin or BPA-dependent. F2 animals were not affected. Our data suggest that vinclozolin and xenoestrogens act as cartilage developmental disruptors. We suggest that present NOAEL values for these compounds, and EDC at large, might be reconsidered using gestational exposure models. Finally, micro CT-scan appears a valuable non-invasive technique to detect EDC effects on live fauna., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

American sloughgrass (Beckmannia syzigachne) is a troublesome weed in winter wheat field rotated with rice in China. Fenoxaprop-p-ethyl and pinoxaden were observed failing to control American sloughgrass in the same filed in Lujiang county in 2011 and 2012, respectively. Whole-plant bioassay was conducted to determine the resistance to fenoxaprop-p-ethyl, pinoxaden and other herbicides in American sloughgrass. Dose-response experiment indicated that Lujiang population was highly resistant to fenoxaprop-p-ethyl (199.8-fold), pinoxaden (76.2-fold), clodinafop-propargyl (334.1-fold) and sethoxydim (15.9-fold); moderately resistant to clethodim (6.3-fold), susceptible to mesosulfuron-methyl, flucarbazone-sodium, pyroxsulam and isoproturon. Partial gene of CT domain was cloned and sequenced to confirm the molecular mechanism of resistance to ACCase-inhibiting herbicides. A Trp2027Cys mutation was found in Lujiang population according to the sequencing result. This mutation is the molecular mechanism of resistance to fenoxaprop-p-ethyl in Lujiang population. Furthermore the Trp2027Cys mutation very likely results in cross resistance to clodinafop-propargyl and pinoxaden in Lujiang population. 103 mutant homozygotes were detected from the 108 plants tested using a rapid dCAPS method developed in this paper. This is the first report of pinoxaden resistance and a mutation at position of 2027 for American sloughgrass., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Siderophores are small iron-binding molecules secreted by bacteria to scavenge iron. Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis, produces the siderophores mycobactin and carboxymycobactin. Complexes of the mycobacterial membrane proteins MmpS4 and MmpS5 with the transporters MmpL4 and MmpL5 are required for siderophore export and virulence in Mtb. Here we show that, surprisingly, mycobactin or carboxymycobactin did not rescue the low-iron growth defect of the export mutant but severely impaired growth. Exogenous siderophores were taken up by the export mutant, and siderophore-delivered iron was used, but the deferrated siderophores accumulated intracellularly, indicating a blockade of siderophore recycling. This hypothesis was confirmed by the observation that radiolabeled carboxymycobactin was taken up and secreted again by Mtb. Addition of iron salts to an Mtb siderophore biosynthesis mutant stimulated more growth in the presence of a limiting amount of siderophores than iron-loaded siderophores alone. Thus, recycling enables Mtb to acquire iron at lower metabolic cost because Mtb cannot use iron salts without siderophores. Exogenous siderophores were bactericidal for the export mutant in submicromolar quantities. High-resolution mass spectrometry revealed that endogenous carboxymycobactin also accumulated in the export mutant. Toxic siderophore accumulation is prevented by a drug that inhibits siderophore biosynthesis. Intracellular accumulation of siderophores was toxic despite the use of an alternative iron source such as hemin, suggesting an additional inhibitory mechanism independent of iron availability. This study indicates that targeting siderophore export/recycling would deliver a one-two punch to Mtb: restricting access to iron and causing toxic intracellular siderophore accumulation.

An experiment was conducted under laboratory conditions to investigate the effect of combined application of three systemic herbicides, viz., fenoxaprop, pendimethalin and paraquat at 50 g, 1.0 kg and 1.0 kg a.i. ha(-1), respectively, on the changes of microbial biomass C, N and P in relation to transformations and availability of some plant nutrients in an alluvial soil (Typic Orchraqualf) of West Bengal, India. Application of the herbicides, in general, significantly increased microbial biomass C, N and P, resulting in greater availability of C, N and P in soil. The microbial biomass C was highly induced (28.4 %) when fenoxaprop was applied with pendimethalin, while fenoxaprop along with paraquat exerted maximum stimulation towards microbial biomass N (19.9 %) and biomass P (16.2 %). Application of fenoxaprop along with pendimethalin retained the highest amount of organic C (17.4 %), exchangeable NH4(+) (29 %) and available P (19.6 %), while fenoxaprop with paraquat augmented total N and soluble NO3(-) by 21.4 % and 25.2 %, respectively in soil.

We have previously reported the discovery of cytotoxic and pro-apoptotic hit compound 1,1-dimethylethyl (S)- 2,2-dimethyl-4-[(3-nitrophenoxy)methyl]-3-oxazolidinecarboxylate 1 against leukemia cells. In the present work we describe the synthesis of 25 derivatives of this hit varying the substituent at ring or stereochemistry of the oxazolidine ring and evaluated them against human cancer cells lines. Six compounds exerted significant activity against HL60 promyelocytic leukemia cells with IC50 in low micromolar range (4-18 μM) and three compounds displayed activity against MDA-MB231 breast cancer cells (25-37 μM). In vitro cytotoxicity on normal cells PBMC (human peripheral blood mononuclear cells) was also evaluated. Compounds 7e (p-NO2, S) and 7m (p-COOCH3, S) showed good antiproliferative activity against HL60 (4 and 5 μM) and MDA-MB231 (37 and 25 μM) without affecting lymphocyte proliferation in PBMC, indicating low toxicity to normal cells. Besides, compound 7e induced DNA fragmentation on about 100% of HL60 cells at 50 μM. In this case, it was more potent than 7m and lead 1. This indicated that compound 7e has a great pro-apoptotic potential.

Imprinting is an epigenetic form of gene regulation that mediates a parent-of-origin-dependent expression of the alleles of a number of genes. Imprinting, which occurs at specific sites within or surrounding the gene, called differentially methylated domains, consists in a methylation of CpGs. The appropriate transmission of genomics imprints is essential for the control of embryonic development and fetal growth. A number of endocrine disruptors (EDs) affect male reproductive tract development and spermatogenesis. It was postulated that the genetic effects of EDs might be induced by alterations in gene imprinting. We tested two EDs: methoxychlor and vinclozolin. Their administration during gestation induced in the offspring a decrease in sperm counts and significant modifications in the methylation pattern of a selection of paternally and maternally expressed canonical imprinted genes. The observation that imprinting was largely untouched in somatic cells suggests that EDs exert their damaging effects via the process of reprogramming that is unique to gamete development. Interestingly, the effects were transgenerational, although disappearing gradually from F1 to F3. A systematic analysis showed a heterogeneity in the CpG sensitivity to EDs. We propose that the deleterious effects of EDs on the male reproductive system are mediated by imprinting defects in the sperm. The reported effects of EDs on human male spermatogenesis might be mediated by analogous imprinting alterations., (© 2014 Elsevier Inc. All rights reserved.)

In the present review, we first summarize the main benefits, limitations and pitfalls of conventional in vivo approaches to assessing male reproductive structures and functions in rodents in cases of endocrine active substance (EAS) exposure from the postulate that they may provide data that can be extrapolated to humans. Then, we briefly present some integrated approaches in rodents we have recently developed at the organism level. We particularly focus on the possible effects and modes of action (MOA) of these substances at low doses and in mixtures, real-life conditions and at the organ level, deciphering the precise effects and MOA on the fetal testis. It can be considered that the in vivo experimental EAS exposure of rodents remains the first choice for studies and is a necessary tool (together with the epidemiological approach) for understanding the reproductive effects and MOA of EASs, provided the pitfalls and limitations of the rodent models are known and considered. We also provide some evidence that classical rodent models may be refined for studying the multiple consequences of EAS exposure, not only on the reproductive axis but also on various hormonally regulated organs and tissues, among which several are implicated in the complex process of mammalian reproduction. Such models constitute an interesting way of approaching human exposure conditions. Finally, we show that organotypic culture models are powerful complementary tools, especially when focusing on the MOA. All these approaches have contributed in a combinatorial manner to a better understanding of the impact of EAS exposure on human reproduction.

Two new cytotoxic dilactones, bisisorhizopodin (1) and isorhizopodin (2), together with known divalent actin depolymerizer rhizopodin (3), were isolated from the culture broth of a myxobacterium Myxococcus stipitatus. Spectroscopic analyses established that 1 and 2 are doubly and singly acyl-migrated isomers of 3, respectively, and comparison of their cytotoxicity revealed gradual decrease in the activity as the size of the ring contracted. Because the side chains of macrolide toxins uniformly block the contact between the actin protomers, the present result demonstrates substantial contribution of structurally diverse rings to the affinity of macrolide toxins for its target protein.

Vinclozolin, a dicarboximide fungicide, is an endocrine disrupting chemical that competes with an androgenic endocrine disruptor compound. Most research has focused on the epigenetic effect of vinclozolin in humans. In terms of ecotoxicology, understanding the effect of vinclozolin on non-target organisms is important. The expression profile of a comprehensive set of genes in the amphipod Hyalella azteca exposed to vinclozolin was examined. The expressed sequence tags in low-dose vinclozolin-treated and -untreated amphipods were isolated and identified by suppression subtractive hybridization. DNA dot blotting was used to confirm the results and establish a subtracted cDNA library for comparing all differentially expressed sequences with and without vinclozolin treatment. In total, 494 differentially expressed genes, including hemocyanin, heatshock protein, cytochrome, cytochrome oxidase and NADH dehydrogenase were detected. Hemocyanin was the most abundant gene. DNA dot blotting revealed 55 genes with significant differential expression. These genes included larval serum protein 1 alpha, E3 ubiquitin-protein ligase, mitochondrial cytochrome c oxidase, mitochondrial protein, proteasome inhibitor, hemocyanin, zinc-finger-containing protein, mitochondrial NADH-ubiquinone oxidoreductase and epididymal sperm-binding protein. Vinclozolin appears to upregulate stress-related genes and hemocyanin, related to immunity. Moreover, vinclozolin downregulated NADH dehydrogenase, related to respiration. Thus, even a non-lethal concentration of vinclozolin still has an effect at the genetic level in H. azteca and presents a potential risk, especially as it would affect non-target organism hormone metabolism.

In the late 1980s vinclozolin was tested for prenatal developmental toxicity in rats for registration purposes in USA. At 1000mg/kgbw, 95% of all fetuses were female upon visual inspection (ano-genital distance determination). Anti-androgenic effects (AA) were also noted in a subsequent 2-generation study. These findings triggered mechanistic investigations at BASF and at US-EPA. Results published by the latter were the starting point of the endocrine disruption (ED) discussion in the 1990s. AA effects of vinclozolin are mediated by two metabolites, which have an antagonistic effect on the androgen receptor. Currently, determination of ED has become a major end-point in toxicology testing and the US-EPA has set up an elaborated testing paradigm to fulfill this requirement. Future screening for ED can be improved making use of new technologies. ED modes of action can be determined by three alternative (3R) methods. Steroid synthesis in H295R cells (1), androgen-receptor binding in modified yeast (2) and metabolomics (3). Using vinclozolin as a case study, results indicate: (1) an effect on steroid synthesis in vitro, (2) an antagonistic effect on the androgen receptor and (3) that the metabolome profile of vinclozolin is similar to that of other receptor mediated anti-androgens (e.g. flutamide)., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

A number of environmental factors (e.g. toxicants) have been shown to promote the epigenetic transgenerational inheritance of disease and phenotypic variation. Transgenerational inheritance requires the germline transmission of altered epigenetic information between generations in the absence of direct environmental exposures. The primary periods for epigenetic programming of the germ line are those associated with primordial germ cell development and subsequent fetal germline development. The current study examined the actions of an agricultural fungicide vinclozolin on gestating female (F0 generation) progeny in regards to the primordial germ cell (PGC) epigenetic reprogramming of the F3 generation (i.e. great-grandchildren). The F3 generation germline transcriptome and epigenome (DNA methylation) were altered transgenerationally. Interestingly, disruptions in DNA methylation patterns and altered transcriptomes were distinct between germ cells at the onset of gonadal sex determination at embryonic day 13 (E13) and after cord formation in the testis at embryonic day 16 (E16). A larger number of DNA methylation abnormalities (epimutations) and transcriptional alterations were observed in the E13 germ cells than in the E16 germ cells. These observations indicate that altered transgenerational epigenetic reprogramming and function of the male germline is a component of vinclozolin induced epigenetic transgenerational inheritance of disease. Insights into the molecular control of germline transmitted epigenetic inheritance are provided.