Enantiomer stability and combined toxicity of duloxetine and econazole on Daphnia magna using real concentrations determined by capillary electrophoresis.

Enantiomer stability was investigated in this work for the first time for duloxetine and econazole in individual solutions and their mixtures under the standardized ecotoxicity test experimental conditions for Daphnia magna and abiotic conditions. Real (and not nominal) enantiomer concentrations were employed for calculations since their determination was achieved by Capillary Electrophoresis. Relevant differences were found in stability profiles for both drugs in any case. Toxicity was evaluated for the first time in this work for mixtures of duloxetine and econazole on Daphnia magna. Dose-effect parameters were calculated at different exposure times (24, 48, and 72 h) showing a significant inhibition of daphnids mobility when increasing the incubation time. Combination index values enabled to obtain the type and level of interaction of drugs with the organism. A strong synergism was observed at 48 h exposure time and any effect level, which demonstrated the high toxicity of the drug mixture compared with the individual drug solutions. These results were corroborated when evaluating the oxidative stress using fluorescence images.

Stability and toxicity studies for duloxetine and econazole on Spirodela polyrhiza using chiral capillary electrophoresis.

Stability and toxicity studies for duloxetine and econazole were achieved using individual solutions and their mixtures. Stability of drugs racemates and enantiomers was investigated under abiotic and biotic conditions. Toxicity was evaluated for the first time on Spirodela polyrhiza. EC50 values were calculated for each individual drug and for their binary mixture. Real (not nominal) concentrations determined by Capillary Electrophoresis were employed in the calculations of toxicity parameters. The use of a 25 mM phosphate buffer (pH 3.0) with 1.5% S-ß-CD as chiral selector at a temperature of 30 °C and a separation voltage of -20 kV enabled the simultaneous enantiomeric separation of duloxetine and econazole in 7.5 min with enantiomeric resolutions of 7.9 and 6.5, respectively. For individual solutions, decay percentages under abiotic conditions were higher for duloxetine (80%) than for econazole (60%), while in presence of Spirodela polyrhiza they increased for duloxetine but not for econazole. Econazole showed the highest decay percentages under abiotic or biotic conditions (100%) in binary mixtures. EC50 values for duloxetine and econazole enabled to include both drugs within the group of very toxic compounds although econazole showed a higher toxicity than duloxetine and the binary mixture.

Tebuconazole and Econazole Act Synergistically in Mediating Mitochondrial Stress, Energy Imbalance, and Sequential Activation of Autophagy and Apoptosis in Mouse Sertoli TM4 Cells: Possible Role of AMPK/ULK1 Axis.

Tebuconazole and Econazole are triazole and imidazole fungicides currently used worldwide. Although their reproductive toxicity in mammals has been described, their effect on male reproductive systems has been poorly investigated. As humans may be exposed to different azole compounds simultaneously, the combinational in vitro toxicity of Tebuconazole and Econazole (MIX) in mouse Sertoli TM4 cells was investigated. This study demonstrates that Tebuconazole (40 µM) and Econazole (20 µM) act synergistically in mediating decrease of mitochondrial membrane potential (ΔΨm) and changes in mitochondrial morphology. These events were associated with ATP depletion, cell cycle arrest, and sequential activation of autophagy and apoptosis. Remarkable differences on other parameters such as AMP/ATP ratio and adenylate energy charge were observed. Pharmacological inhibition of autophagy by bafilomycin A1 leads to enhanced MIX-induced apoptosis suggesting an adaptive cytoprotective function for MIX-modulated autophagy. Finally, a possible role of AMPK/ULK1 axis in mediating adaptive signalling cascades in response to energy stress was hypothesized. Consistently, ULK1 Ser 555 phosphorylation occurred in response to AMPK (Thr 172) activation. In conclusion, Tebuconazole and Econazole combination, at concentrations relevant for dermal and clinical exposure, induces a severe mitochondrial stress in SCs. Consequently, a prolonged exposure may affect the ability of the cells to re-establish homeostasis and trigger apoptosis.

Molecular mechanisms of econazole-induced toxicity on human colon cancer cells: G0/G1 cell cycle arrest and caspase 8-independent apoptotic signaling pathways.

Econazole (Eco), a potent broad-spectrum anti-fungal agent, has been used in the treatment of superficial mycosis. Eco is a store-operated Ca2+ channel antagonist which induces cytotoxic cell death of leukemia. However, little is known about its cytotoxic effect upon solid tumor cells. The purpose of this study is to investigate both the in vitro and in vivo molecular mechanisms of Eco-induced toxicity on colon cancer cells. We used COLO 205 cell line and nude mice xenograft model to investigate the cytotoxic effect of Eco. We demonstrated that lower doses Eco (5-20 microM) arrested human colon cancer cells at the G0/G1 phase of the cell cycle. The protein levels of p53, p21/Cip1, and p27/Kip1 were significantly elevated while CDK2 and CDK4 kinase activity were significantly suppressed by Eco treatment in COLO 205 cells. At higher doses (40-60 microM), Eco induced COLO 205 cells apoptosis evidenced by ladder formation in DNA fragmentation assay and sub-G1 peak in flow cytometry analysis. Western blot analysis showed that caspases 3, 9 but not 8 were activated by high dose Eco treatment to the COLO 205 cells accompanied with cytochrome c and apoptosis-inducing factor (AIF) translocation. Significant anti-tumorigenesis effect was further demonstrated in vivo by treating nude mice bearing COLO 205 tumor xenografts with Eco 50 mg/kg intraperitoneally. Our findings highlight the molecular mechanisms underlying the Eco-induced toxicity on colon cancer cells.

Antioxidative natural product protect against econazole-induced liver injuries.

The study objective of this research is in order to investigate the hepatoprotective and therapeutic effects of propolis ethanol extract (PEE) on acute econazole-induced liver injury. Positive control of various concentrations of PEE on liver function and the dose-response relationship of liver injury induced by various doses of econazole were firstly observed from biochemical assay of serum level of aspartate transaminase (SGOT) and serum alanine transaminase (SGPT) and histopathological microscopic examination. The hepatoprotective effects of various concentration of PEE on liver damage induced by hepatotoxic dose (300 mg/kg) of econazole were observed by the obvious decrement of SGOT and SGPT level and further confirmed by hepatohistological microscopic examination. The inhibitory effects of PEE on FeCl(2)-induced (in vitro) or econazole-induced (in vivo) lipid peroxidation were investigated from the measurement of the formed malonic dialdehyde (MDA) level in the rat liver homogenate. The IC(50) (microM) of various concentrations of PEE in the superoxide scavenging activity in econazole (300 mg/kg)-damaged rat liver homogenate were assessed by cytochrome c reduction method and compared with that of (+)-alpha-tocopherol. It could be postulated that the hepatoprotective effect of PEE may be, at least in part, due to their inhibitory ability on membrane lipid peroxidation and free radical formation or due to their free radical scavenging ability.

Cyclodextrin inclusion complexes of antimycotics intended to act in the oral cavity--drug supersaturation, toxicity on TR146 cells and release from a delivery system.

The dissolution rate, the toxicity and the release from chewing gum of miconazole and econazole cyclodextrin products and complexes were investigated. The dissolution rate studies showed that an amorphous miconazole hydroxypropyl-beta-cyclodextrin product gave drug supersaturation, whereas drug supersaturation was not present during dissolution rate testing of an econazole hydroxypropyl-beta-cyclodextrin product. The miconazole hydroxypropyl-beta-cyclodextrin product and genuine cyclodextrin inclusion complexes of miconazole, econazole and clotrimazole were toxic on a human TR146 buccal cell culture model. The toxicity was probably due to drug supersaturation, thereby increasing the bioavailability of the antimycotics. The econazole hydroxypropyl-beta-cyclodextrin product and physical mixtures of miconazole or econazole and beta-cyclodextrin did not give supersaturation and were not as toxic as the above-mentioned compounds. Neat econazole and miconazole, a genuine econazole beta-cyclodextrin complex and the miconazole hydroxypropyl-beta-cyclodextrin product were incorporated in chewing gum. The miconazole hydroxypropyl-beta-cyclodextrin gum had a much higher drug release in vitro than the neat miconazole gum. The genuine econazole beta-cyclodextrin complex only increased the drug release moderately when compared with the release from the neat econazole gum. The release studies were performed on a mastication device.

Induction of micronuclei in V79 Chinese hamster cells by hydroquinone and econazole nitrate.

Two suspect aneugens (hydroquinone and econazole nitrate) were examined for their ability to induce micronuclei in a number of V79 Chinese hamster cell lines which express rat cytochrome P-450 cDNAs. Hydroquinone elevated micronucleated cell frequencies in a dose-dependent manner in cell lines V79, XEM2 (expresses CYP1A1) and SD1 (expresses CYP2B1). Econazole nitrate was an effective inducer of micronuclei over a narrow dose range in cell lines V79, XEM2 and XEMd-MZ (expresses CYP1A2). The different cell lines showed similar responses to the test agents, indicating that hydroquinone is not a substrate for biotransformation by rat CYP1A1 or CYP2B1, nor is econazole nitrate biotransformed by rat CYP1A1 or CYP1A2.

Further evaluation of a modified micronucleus assay with V79 cells for detection of aneugenic effects.

In an earlier publication, we reported on the development of a modified micronucleus assay with V79 cells enabling preferential detection of aneugen-induced micronuclei (Seelbach et al., 1993). Here we present a further evaluation of the modified micronucleus assay based on the investigation of seven further suspected aneugens. Five compounds gave positive results: cadmium chloride, chloral hydrate, hydroquinone, thimerosal and vinblastine. Econazole and pyrimethamine were negative. Up to now, our experience has shown that data produced by the modified V79/micronucleus assay are quite reliable: the variation of spontaneous micronucleus frequencies was low (0.8-1.7%) and the reproducibility of the data was good.

Detection of aneuploidy-inducing carcinogens in the Syrian hamster embryo (SHE) cell transformation assay.

As evidenced by the recent report of the Commission of the European Communities (CEEC) project (Detection of Aneugenic Chemicals-CEEC project, 1993), there currently is a great deal of effort towards developing and validating assays to detect aneuploidy-inducing chemicals. In this report, we describe the utility of the Syrian hamster embryo (SHE) cell transformation assay for detecting carcinogens with known or suspected aneuploidy-inducing activity. The following carcinogens were tested: asbestos, benomyl, cadmium chloride, chloral hydrate, diethylstilbestrol dipropionate, and griseofulvin. Thiabendazole, a noncarcinogen, was also tested. Chemicals of unknown or inconclusive carcinogenicity data, colcemid, diazepam, econazole nitrate, and pyrimethamine were also evaluated. All of the above chemicals except thiabendazole induced a significant increase in morphological transformation (MT) in SHE cells. Based on these results as well as those published in the literature previously, the SHE cell transformation assay appears to have utility for detecting carcinogens with known or suspected aneuploidy-inducing ability.

Induction of mitotic aneuploidy using Chinese hamster primary embryonic cells. Test results of 10 chemicals.

Using primary Chinese hamster embryonic cells, 10 known or suspected aneugens supplied as a part of the EC 4th Environmental Research and Development Programme were evaluated by the technique described by Dulout and Natarajan (1987). The chemicals included cadmium chloride, chloral hydrate, colchicine, diazepam, econazole, hydroquinone, pyrimethamine, thiabendazole, thimerosal and vincristine. All chemicals except pyrimethamine gave clearly positive effect at most of the doses tested. The ease with which the assay is performed and reproducible results that are obtained with the suspected compounds indicate that this in vitro test using primary embryonic fibroblasts is a promising one for routine screening.

Are econazole, miconazole and clotrimazole mutagenic to bacteria?

The fungistatic drugs econazole, miconazole and clotrimazole were investigated as to mutagenic properties in the fluctuation test with Klebsiella pneumoniae and Escherichia coli K12 as test organisms and in the plate-incorporation test, with and without metabolic activation, with Salmonella typhimurium strains TA98 and TA100. No mutagenic activity of the 3 compounds on these microorganisms was found.

The size of cytokinesis-blocked micronuclei in human peripheral blood lymphocytes as a measure of aneuploidy induction by Set A compounds in the EEC trial.

We have investigated the potential value of estimating number and size of micronuclei in cytokinesis-blocked human peripheral blood lymphocytes as a measure of the aneuploidy-inducing ability of 5 chemicals in the EEC trial. Colchicine induced metaphase arrest, high numbers of cells with micronuclei and also of cells with completely fragmented nuclei. The majority of micronuclei were very significantly larger in relation to the main nucleus than a comparable set taken from untreated control cultures. In the same assay system, diazepam weakly increased the incidence of MN in a dose-related fashion. There was a slight increase in MN but only at toxic doses when cells were treated with econidazole, chloral hydrate or hydroquinone. For each of these 4 drugs, statistical treatment of the data confirmed a difference in size distribution of the MN as compared to those found in negative control cultures. Although this approach has potential, it may not be optimal to detect weak aneuploidy-inducing agents.

An overview of the results of testing of known or suspected aneugens using mammalian cells in vitro.

Ten known or suspected aneugens were analyzed in a variety of in vitro mammalian cell cultures using different endpoints which included: micronuclei, kinetochore-positive micronuclei in binucleated cells, changes in the number of chromosomes or aberrations of mitosis and division. Human lymphocytes, human diploid fibroblasts and Chinese hamster transformed cells were used as target cells. The relative merits of different in vitro test systems employed are briefly discussed here.

In vivo studies on chemically induced aneuploidy in mouse somatic and germinal cells.

Within the context of a coordinated program to study aneuploidy induction sponsored by the European Community, nine chemicals were tested in mouse bone marrow and spermatocytes after intraperitoneal injection. In somatic cells, cell progression delay, hyperploidy, polyploidy induction and induction of micronucleated polychromatic erythrocyte (MnPCE) were studied. In germ cells hyperploidy induction was evaluated. The chemicals selected were: colchicine (COL), econazole (EZ), hydroquinone (HQ), thiabendazole (TB), diazepam (DZ), chloral hydrate (CH), cadmium chloride (CD), pyrimethamine (PY) and thimerosal (TM). Using literature data on c-mitotic effects in bone marrow as a reference, the same doses were tested in somatic and germ cells in order to compare the effects induced. Bone marrow cells were sampled 18 or 24 h after treatment. Germ cells were sampled 6, 8 or 18 h after treatment. Effects of COL and HQ in bone marrow have been reported elsewhere. Somatic effects were induced by CH (hyperploidy and cell cycle lengthening), TB (MnPCEs and cell cycle lengthening) and by PY (MnPCEs). EZ, DZ, CD and TM did not induce any kind of somatic effects. An increase in the incidence of hyperploid spermatocytes was induced by COL, at three dose levels, and by one dose of HQ and TB. All the other chemicals did not induce germinal aneuploidy at any dose or time tested. The hyperploidy control frequency ranged between 0.4 and 1.0% in somatic cells and from 0.3 to 0.9% in germ cells. In both somatic and germ cells, the maximum yield of induced hyperploidy did not exceed 3.5%. The time period of target cell sensitivity is probably restricted and this, associated with the heterogeneity and the asynchrony of cellular maturation processes, may account for our data. Under these circumstances, the negative data should be interpreted with some caution, particularly in germ cells, where additional indicators of chemical-cell interaction and cell cycle effects were not provided by standardized approaches. The possibility of increasing the size of analyzed cell samples could be considered in the light of automatic scoring procedures.

Synopsis of the in vivo results obtained with the 10 known or suspected aneugens tested in the CEC collaborative study.

The synopsis of the in vivo test results in the first collaborative CEC Aneuploidy Project with 10 selected chemicals, colchicine (COL), econazole (EZ), chloral hydrate (CH), hydroquinone (HQ), diazepam (DZ), thiabendazole (TB), cadmium chloride (CD), thimerosal (TM), pyrimethamine (PY) and vinblastine (VBL), allowed several conclusions. (1) The spindle poisons, COL and VBL, were positive in all bone marrow and germ cell tests; (2) the clastogen HQ also induced aneuploidy in somatic and germinal cells; (3) the other seven compounds gave contradictory results either between laboratories or between test systems which require further experimental clarification; (4) CREST labeling or in situ hybridization for centromere identification showed about 70% fluorescent signals in micronuclei induced by COL or VBL but only about 15% in HQ induced micronuclei; (5) the tests for induction of a delay in cell division progression can be recommended as a prescreen for possible aneugens; (6) all test methods applied in these experiments require standardization with respect to sample size, sampling times and statistical treatment of the data. A second CEC Aneuploidy Programme has started recently to answer some of the questions raised by the first study regarding tissue and sex specificities.

Effects of potential aneuploidy inducing agents on microtubule assembly in vitro.

The present study was carried out with the 10 known or suspected spindle poisons of the Commission of the European Communities program to study aneuploidy induction. We have investigated these substances on the assembly of isolated bovine microtubules at 10, 100 and 1000 microM and studied morphology by electron microscopy. The substances could be grouped into two categories, strong and weak inhibitors. Colchicine, vinblastine and thimerosal were strong inhibitors; cadmium chloride, thiabendazole, chloral hydrate, hydroquinone, diazepam and econazole were weak inhibitors, the latter three causing aberrant forms visible on electron microscopy. Pyrimethamine did not inhibit the assembly of microtubules, but produced aberrant forms.

A comparison of two in vitro mammalian cell cytogenetic assays for the detection of mitotic aneuploidy using 10 known or suspected aneugens.

Two in vitro cytogenetic assays were evaluated for their ability to detect aneugenic and polyploidy-inducing agents using a battery of 10 known or suspected aneugens supplied as part of the EEC 4th Environmental Research and Development Programme. The compounds tested were colchicine, vinblastine, chloral hydrate, thiabendazole, hydroquinone, thimerosal, cadmium chloride, econazole nitrate, pyrimethamine and diazepam. The cell division aberration assay employed a differential chromosome/spindle staining procedure to detect perturbations of the mitotic division apparatus. This assay was carried out in two pulmonary-derived Chinese hamster cell lines; the immortal DON:Wg3h culture and a low passage LUC2 culture. The second assay involved quantification of metaphase chromosomes, for which only the LUC2 cell line was used, due to the stability of its diploid karyotype. All the chemicals induced spindle disturbances in the immortal line. In addition, all the compounds except cadmium chloride yielded positive results in the LUC2 culture, although many were not as potent. In the low passage line, 8 of the compounds (colchicine, vinblastine, chloral hydrate, thiabendazole, thimerosal, econazole nitrate, pyrimethamine and diazepam) induced aneuploidy and/or tetraploidy. Cadmium chloride was negative in the chromosome enumeration assay and hydroquinone yielded inconclusive results. The study of cell division aberrations was much less time-consuming and technically complex than the counting of metaphase chromosomes. In addition, it provided a degree of mechanistic understanding of the mode of action of some aneugenic and polyploidy-producing agents. However, the enumeration of chromosomes provides a more definitive data set for the evaluation of a chemical's aneugenic potential.

The detection and assessment of the aneugenic potential of environmental chemicals: the European Community Aneuploidy Project.

Within the framework of its' Environment Research and Development Programme, the European Communities (EC) Directorate General (DG) XII has supported a research project aimed at developing and validating assay systems for the detection and evaluation of chemicals capable of inducing numerical chromosome changes such as aneuploidy and polyploidy. A range of test chemicals were selected, which include a core set comprising; colchicine, econazole nitrate, chloral hydrate, hydroquinone, diazepam, thiabendazole, cadmium chloride, thimerosol, pyrimethamine and vinblastine sulphate. These test chemicals were used to evaluate the ability of test systems ranging from tubulin polymerisation, fungal cultures, cultured mammalian cells and intact rodents to detect chemical aneugens and to assess the significance of such activity to exposed human populations.

C-mitosis and numerical chromosome aberration analyses in human lymphocytes: 10 known or suspected spindle poisons.

As a part of a coordinated EEC project to validate suitable assays for chemically induced genomic mutations, numerical chromosomal aberrations and spindle effects were studied in human lymphocyte cultures exposed to cadmium chloride, chloral hydrate, colchicine, diazepam, econazole, hydroquinone, pyrimethamine, thiabendazole, thimerosal and vinblastine. Chromosome number analysis was carried out after treatment for 48 and 72 h; spindle effects, i.e., increases in the mitotic indices and c-mitoses, were analyzed in cultures treated 5 h before fixation. Dose-related numerical chromosomal aberrations are induced by colchicine and vinblastine, the only chemicals that also induce c-mitotic effects in a wide range of doses. Hyperdiploidy is induced by chloral hydrate, cadmium chloride and thimerosal without dose-effect relationship; chloral hydrate and thimerosal affect spindle functions while only a weak spindle effect is produced by cadmium chloride. Tetraploid and/or endoreduplicated cells are induced without dose-effect relationship by hydroquinone, thiabendazole and thimerosal, all of them able to produce c-mitotic effects. Diazepam and econazole induce only hypodiploidy; pyrimethamine does not induce numerical chromosomal aberrations.

The cytochalasin-B micronucleus/kinetochore assay in vitro: studies with 10 suspected aneugens.

An in vitro micronucleus assay in low passage Chinese hamster Luc2 cells capable of detecting numerical and structural chromosome changes was developed. Chromosome loss was inferred by indirect visualisation of human CREST antikinetochore antibodies bound to centromeres in chemically-induced micronuclei of cytochalasin-B arrested binucleated cells. The assay was used to evaluate 10 chemicals which had been selected for their known or suspected effects upon various components of the cell-division apparatus. These chemicals were colchicine (COL), vinblastine (VBL), thiabendazole (TBZ), chloral hydrate (CH), thimerosal (TM), diazepam (DZ), pyrimethamine (PYR), hydroquinone (HQ), cadmium chloride (CdCl2) and econazole nitrate (EZ). Mitomycin-C (MMC) was used as a positive control for the induction of micronuclei. 8 of the core chemicals induced micronuclei in Chinese hamster Luc2 cells. 4 of the chemicals (COL, VBL, TBZ, CH) increased levels of micronuclei which were positive for kinetochore antibody labelling and hence chromosome loss. 3 of the chemicals (DZ, PYR, HQ) and the positive control (MMC) increased the levels of Mn which were negative for kinetochore antibody labelling. The results with TM were equivocal and EN was negative. The results of these studies suggest that the cytochalasin-B Mn/k assay is a cost-effective, simple and rapid alternative to classical cytogenetic assays for the detection of chemically induced aneuploidy.