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.

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.

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.

A parenteral econazole formulation using a novel micelle-to-liposome transfer method: in vitro characterization and tumor growth delay in a breast cancer xenograft model.

PURPOSE: The purpose of this study was to develop a parenteral liposomal formulation of econazole, a poorly water-soluble compound not previously available in an intravenous form. We are investigating econazole as an anticancer agent based on its unique mechanism of action to which cancer cells are preferentially sensitive. An intravenous formulation of econazole was desired for preclinical toxicity and efficacy studies of econazole. METHODS: Liposomal econazole was prepared using a novel micelle exchange technique to incorporate the drug into the lipid bilayer of pre-formed liposomes using a poly(ethylene) glycol-linked phospholipid, distearoyl phosphatidylethanolamine (DSPE-PEG). This method allowed for stable and efficient drug incorporation into DPPC and DMPC liposomes at a final drug:lipid ratio of 0.05 (w/w) and increased solubility in saline from <0.1 to 5 mg/ml. RESULTS: Stability over 14 days at 4 degrees C in buffer was demonstrated as well as in vitro plasma stability at 37 degrees C. Plasma elimination studies of micelle-loaded liposomal econazole showed a half-life of approximately 35 min and plasma AUC of 281 microg/ml min. In MCF-7 human breast cancer xenografts in Rag2M mice. Liposomal econazole did not induce significant hepatoxicity, renal toxity or weight loss compared to empty liposomes. Tumor growth was slightly delayed in liposomal econazole-treated mice, with approximately 10-day lag time to reach 300 mm(3) compared to vehicle controls. CONCLUSIONS: The micelle transfer method provided an efficient means of preparing liposomal econazole suitable for intravenous administration. Liposomal econazole was successfully administered to tumor bearing mice at 50 mg/kg, and no significant toxicities attributable to econazole were observed.

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.

Comparison of the toxicity of fluconazole and other azole antifungal drugs to murine and human granulocyte-macrophage progenitor cells in vitro.

We studied the inhibitory effects on colony formation by granulocyte-macrophage colony forming units (cfu-gm) of eight azole antifungal agents in vitro. All agents, except fluconazole, inhibited colony formation dose-dependently with 50% inhibitory concentrations (IC50) in the range of 0.78-49 micromol/L in cultures of murine and human bone marrow. For human cells, the IC50 values were 0.553 mg/L for itraconazole, 1.24 mg/L for saperconazole, 2.58 mg/L for clotrimazole, 5.33 mg/L for miconazole, 6.17 mg/L for econazole, 6.27 mg/L for ketoconazole and 8.38 mg/L for oxiconazole. The IC50 of itraconazole for human cfu-gm in vitro was similar to the plasma level of this drug recommended for systemic antifungal therapy (>0.5 mg/L) thus indicating the potential clinical relevance of our data. The IC50 of ketoconazole for human cfu-gm in vitro may be exceeded by plasma levels produced in vivo by high (> or =400 mg) doses, whereas fluconazole failed to reduce colony formation by 50% even at 100 mg/L, a concentration not reached in vivo even after extremely high doses (2000 mg/day). To most of the drugs studied, murine progenitor cells seemed to be less sensitive than the human ones. There was, however, a close correlation between the murine and human log IC50 values of the drugs (r2 = 0.964, P< 0.001), suggesting that cultures of murine bone marrow may be suitable to predict the in-vitro toxicity of azole antifungals to human cfu-gm.

Cyclodextrin inclusion complexes of miconazole and econazole--isolation, toxicity on human cells, and confirmation of a new interpretation of the drug supersaturation phenomenon.

Parameters that influence the precipitation of the beta-cyclodextrin (beta-CD) inclusion complexes of the antimycotics miconazole and econazole were investigated. The mechanistic reason for the superior antimycotic activity of the miconazole inclusion complex was studied. The toxicity of the complex was estimated. The temperature, the buffer strength, and the effect of the addition of hydrotropic agents on the CD solubility diagrams for the antimycotics were estimated. The miconazole and the CD dissolution rate for the complex was measured. The hemolytic activity of the miconazole inclusion complex, the physical mixture, miconazole, and the nitrate salt were compared. The toxicity on TR146 oral cell layers was measured. Lowering the temperature meant that both complexes precipitated at lower CD concentrations. Addition of hydrotropic agents and variation of the buffer strength affected the solubility diagrams. The dissolution medium was supersaturated with miconazole. The supersaturation was not disclosed by the traditional method to analyze for drug supersaturation. The miconazole complex was more toxic to erythrocytes than the physical mixture. On the other hand, the toxic effects of the two products on the TR146 cell layers were similar. Lowering the temperature eased the isolation of genuine CD inclusion complexes of miconazole and econazole. The miconazole supersaturation is likely to be the reason for the superior antimycotic activity of the complex. The complex and the physical mixture had about the same toxicity on TR146 cell layers.

Colcemid and econazole do not show aneugenicity in male mouse germ cells.

Colcemid (COM) and econazole (EZ) were tested for induction of mitotic arrest and C-mitotic effects in mouse bone marrow cells and for meiotic delay and induction of hyperploidy in mouse spermatocytes after single injection. Doses of 1 and 3 mg/kg COM were used and bone marrow and spermatocytes were sampled at 2, 6, 10, 14 and 18 h. For EZ a dose of 120 mg/kg and intervals of 6, 10, 14 and 18 h were chosen. At 2 h after COM treatment of bone marrow cells the mitotic index and C-mitotic effect were highest, then both decreased from 6 to 18 h. At 18 h the mitotic indices decreased to or significantly below the control level at doses of 1 and 3 mg/kg respectively, whereas the corresponding frequencies of C-mitotic cells were still significantly higher than in the controls. EZ also induced mitotic arrest and C-mitotic effects in mouse bone marrow cells. In contrast to COM, the effects of EZ were highest at 18 h after treatment. COM and EZ caused disturbances in progression from the first to second meiotic division, however, after COM treatment the ratios of MMII to MMI were significantly below the control. In contrast, after EZ treatment the ratios were significantly higher than in the controls. Such differences may result from the different mechanisms by which COM and EZ act on cell cycle progression. COM and EZ did not induce non-disjunction under the experimental conditions. However, EZ induced structural chromosomal aberrations.

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.

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.

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.

An evaluation of the use of in vitro tubulin polymerisation, fungal and wheat assays to detect the activity of potential chemical aneugens.

The test chemicals included in the EC Aneuploidy Project were evaluated for their ability to induce aneuploidy or aneuploidy related endpoints in assays using in vitro tubulin polymerisation, fungi and wheat. The results obtained demonstrated considerable qualitative and quantitative differences between the responses of the assays to the 10 test chemicals. Fungal assays failed to respond to the potent mammalian spindle poisons colchicine and vinblastine and only three chemicals were positive in all three fungal test systems i.e. chloral hydrate, thimerosol and thiabendazole. The in vitro tubulin polymerisation assays produced unambiguous positive results with three chemicals i.e. colchicine, thimerosol and vinblastine sulphate. The hexaploid wheat assay produced a positive response with 8 of the test chemicals i.e. colchicine, econazole, thimerosol, pyrimethamine, thiabendazole, cadmium chloride, vinblastine and diazepam. However, the wheat assay was relatively insensitive to the potent spindle poison colchicine.

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.