Moderate alcohol consumption during pregnancy increases potency of two different drugs (the antifungal fluconazole and the antiepileptic valproate) in inducing craniofacial defects: prediction by the in vitro rat whole embryo culture.

The prenatal exposure to ethanol (Eth), fluconazole (FLUCO) and sodium valproate (VPA) is related to effects on development, producing characteristic syndromic pictures. Among embryotoxic effects described for the three molecules, the alteration on craniofacial morphogenesis is a common feature in humans and animal models, including rodent embryos developed in vitro. The aim of the present work is to evaluate the developmental effects of low Eth serum concentration (17 mM, corresponding to the legal limit to drive in UK, USA, Canada, and many other countries) in mixture with increasing realistic concentrations of the antifungal drug FLUCO (62.5-500 µM) or with increasing realistic concentrations of the antiepileptic drug VPA (31.25-250 µM). Groups exposed to Eth alone (17-127.5 mM), FLUCO alone (62.5-500 µM) or VPA alone (31.25-750 µM) were also included. The chosen alternative animal model was the post-implantation rat whole embryo culture (WEC). E9.5 embryos were exposed in vitro to the test molecules during the whole test period (48 h, corresponding to the developmental stages characteristics of any vertebrate, for human embryos post-fertilization days 23-31). Data were statistically analyzed and processed for modelling applying the benchmark dose (BMD) and relative potency factor (RPF) approaches. Concentration-related effects on facial outcomes were observed in all experimental groups, with a significant enhancement in the groups co-exposed with Eth in comparison to the single exposures. Data obtained by the present work suggest an additional alert for the assumption of even low levels of alcohol in pregnant women during FLUCO or VPA therapy.

Enantioselective toxic effects of mefentrifluconazole in the liver of adult zebrafish (Danio rerio) based on transcription level and metabolomic profile.

Mefentrifluconazole, a new type of chiral triazole fungicide, is widely applied to control a variety of fungal diseases in crops. However, the toxicological effects of mefentrifluconazole on aquatic organisms are unknown, especially at the enantiomer level. In the present study, zebrafish were selected as a typical model for mefentrifluconazole enantiomer exposure. Metabolomic and transcription analyses were performed with 0.01 and 0.10 mg/L mefentrifluconazole and its enantiomers (i.e., rac-mfz/(-)-mfz/(+)-mfz) at 28 days. The 1H nuclear magnetic resonance (NMR)-based metabolomics analysis showed that 9, 10 and 4 metabolites were changed significantly in the rac-mfz, (+)-mfz and (-)-mfz treatment groups compared with the control group, respectively. The differential metabolites were related to energy metabolism, lipid metabolism and amino acid metabolism. The qRT-PCR analysis revealed that the expression of lipid metabolism-, apoptosis- and CYP-related genes in the livers of female zebrafish in rac-mfz and (+)-mfz was 1.61-108.92 times and 2.37-551.34 times higher than that in (-)-mfz, respectively. The results above indicate that exposure to mefentrifluconazole induced enantioselective liver toxicity in zebrafish. Our study underlined the importance of distinguishing different enantiomers, which will contribute to environmental protection.

Evaluation of in vitro activity of five antimicrobial agents on Acanthamoeba isolates and their toxicity on human corneal epithelium.

BACKGROUND: Acanthamoeba keratitis (AK) is an important cause of ocular morbidity in both contact lens wearers and non wearers. Medical management comprises prolonged empiric treatment with multiple drugs, leading to adverse effects and suboptimal cure. The present study evaluated the efficiency and safety of common antimicrobial agents used in treatment of AK. METHODS: Six Acanthamoeba isolates (four AK, two water samples) were axenized and subjected to in vitro susceptibility testing against chlorhexidine, pentamidine isethionate, polymyxin B, miltefosine, and fluconazole to check for trophocidal and cysticidal activity. The safety profile was analysed by observing the cytotoxicity of the highest cidal concentration toward human corneal epithelial cell (HCEC) line. RESULTS: Chlorhexidine had the lowest cidal concentration against both cysts and trophozoites (range 4.16-25 µg/ml) followed by pentamidine isethionate (range 25-166.7 µg/ml). Both agents were nontoxic to HCEC. Polymyxin B (range 25-200 µg/ml) and fluconazole (range 64-512 µg/ml) had relatively higher minimum inhibitory concentrations (MIC); fluconazole was nontoxic even at 1024 µg/ml, but cytotoxicity was observed at 400 µg/ml with polymyxin B. Miltefosine was not effective against cysts at tested concentrations. A. castellanii were more susceptible to all agents (except pentamidine isethionate) than A. lenticulata. Clinical isolates were less susceptible to polymyxin B and fluconazole than environmental isolates, reverse was true for miltefosine. CONCLUSION: Chlorhexidine and pentamidine isethionate were the most effective and safe agents against both trophozoites and cysts forms of our Acanthamoeba isolates. Fluconazole had higher MIC but was nontoxic. Polymyxin B was effective at high MIC but therapeutic dose was found toxic. Miltefosine, at tested concentrations, could not inhibit cysts of Acanthamoeba. Clinical isolates had higher MICs for polymyxin B and fluconazole.

Antifungal Treatment Aggravates Sepsis through the Elimination of Intestinal Fungi.

Prophylactic antifungal therapy is widely adopted clinically for critical patients and effective in reducing the morbidity of invasive fungal infection and improves outcomes of those diagnosed patients; however, it is not associated with higher overall survival. As intestinal commensal fungi play a fundamental role in the host immune response in health and disease, we propose that antifungal therapy may eliminate intestinal fungi and aggravate another critical syndrome, sepsis. Here, with murine sepsis model, we found that antifungal therapy with fluconazole dismissed intestinal fungal burden and aggravated endotoxin-induced but no gram-positive bacteria-induced sepsis. Nevertheless, antifungal therapy did not exert its detrimental effect on germ-free mice. Moreover, colonizing more commensal fungi in the mouse intestine or administration of fungal cell wall component mannan protected the mice from endotoxin-induced sepsis. On the molecular level, we demonstrated that antifungal therapy aggravated endotoxin sepsis through promoting Gasdermin D cleavage in the distal small intestine. Intestinal colonization with commensal fungi inhibited Gasdermin D cleavage in response to lipopolysaccharide challenge. These findings show that intestinal fungi inhibit Gasdermin D-mediated pyroptosis and protect the mice from endotoxin-induced sepsis. This study demonstrates the protective role of intestinal fungi in the pathogenesis of endotoxin-induced sepsis in the laboratory. It will undoubtedly prompt us to study the relationship between antifungal therapy and sepsis in critical patients who are susceptible to endotoxin-induced sepsis in the future.

Effects of nanoplastic on toxicity of azole fungicides (ketoconazole and fluconazole) in zebrafish embryos.

The ubiquity of nanoplastics (NPs) raises concerns about their interactions and combined toxicity with other common contaminants. Although azoles are present throughout the natural environment, their interactions with NP are not well known. We investigated the effects of polystyrene (PS) NP on the toxicity of ketoconazole (KCZ) and fluconazole (FCZ) in zebrafish embryos using the developmental toxicity, oxidative-stress-related biochemical parameters, and expression of genes related to neurotoxicity (ache), cardiotoxicity (gata4, bmp4), inflammation (il1b), oxidative stress (sod1, sod2, cyp1a), and apoptosis (bax, bcl2). Co-exposure to NP (1 mg/L) and KCZ/FCZ (1 mg/L) for 96 h reduced the hatching rate, survival rate, and heart rate and increased the malformation rate and catalase activity. The bax/bcl2 ratio, an apoptosis indicator, was higher after NP, KCZ, or FCZ treatment. However, the bax/bcl2 ratio after exposure to NP + KCZ or NP + FCZ was much higher than that after single exposure. Overall, the results indicated that NP aggravated the toxicity of azole by significantly increasing the reactive oxygen species, lipid peroxidation and altering the expression of oxidative-stress- and apoptosis-related genes. The interactive toxicity of PS NP with KCZ/FCZ reported in this study emphasises the need for caution in the release of azole fungicides in the environment.

Ocular toxicity assessment of nanoemulsion in-situ gel formulation of fluconazole.

PURPOSE: Fluconazole is an effective anti-fungal drug. Due to the limitations of fluconazole, such as poor water solubility and consequently low ocular bioavailability, an optimized fluconazole nanoemulsion in-situ gel formulation (temperature-sensitive) was developed. METHODS AND MATERIALS: To verify formulation's safety for ophthalmic use, preparation was tested for potential ocular toxicity using a cell viability assay on retinal cells. The hen's egg test-chorioallantoic membrane (HET-CAM), as a borderline test between in vivo and in vitro techniques, was chosen for investigating the irritation potential of the formulation. HET-CAM test was done by adding the formulation directly to the CAM surface and monitoring the vessels visually in terms of irritation reactions. Eye tolerance was determined using the modified Draize test. RESULTS: Viability assay on retinal cells displayed that fluconazole nanoemulsion in-situ gel formulation was non-toxic and can be safely used in the eye at concentrations of 0.1% and 0.5%. HET-CAM and Draize tests revealed that optimized formulation of fluconazole did not result in any irritation and was considered non-irritant and well-tolerated for ocular use. CONCLUSION: Regarding to the findings of the three mentioned methods, fluconazole nanoemulsion in-situ gel formulation is harmless and as a proper and safe alternative, can be considered for ocular delivery of fluconazole in the future.

Antibiotic-Based Conjugates Containing Antimicrobial HLopt2 Peptide: Design, Synthesis, Antimicrobial and Cytotoxic Activities.

Recent studies have shown that modified human lactoferrin 20-31 fragment, named HLopt2, possesses antibacterial and antifungal activity. Thus, we decided to synthesize and evaluate the biological activity of a series of conjugates based on this peptide and one of the antimicrobials with proven antibacterial (ciprofloxacin, CIP, and levofloxacin, LVX) or antifungal (fluconazole, FLC) activity. The drugs were covalently connected to the peptide via amide, methylenecarbonyl moieties, or a disulfide bridge. The antibacterial and antifungal activities were evaluated under Clinical and Laboratory Standard Institute (CLSI) recommended conditions or in a low-salt brain-heart infusion diluted medium (BHI1/100). Results showed that conjugation of the peptide with the drug increased its antimicrobial activity up to 4-fold. Under CLSI-recommended conditions, all the compounds revealed rather low efficiency. Among conjugates, the highest antibacterial activity was recorded for the CIP-Cys-S-S-HLopt2-NH2 (III). In BHI1/100, which had lower differentiating properties, all of the conjugates revealed low MIC and MMC (minimum inhibitory and microbicidal concentrations) values. The disulfide bridge used as a linker in the most active conjugate (III) upon incubation with S. aureus cells is reduced, releasing constituent peptide and CIP-Cys. In addition, we showed that its fluorescently labeled analogue and constituent peptide are able to be internalized into both C. albicans and S. aureus cells. Moreover, the invaluable advantage of the presented conjugates was their low toxicity to mammalian cells and very low hemolytic activity. The current research can form a solid basis for further in vivo studies and drug development.

The classic azole antifungal drugs are highly potent endocrine disruptors in vitro inhibiting steroidogenic CYP enzymes at concentrations lower than therapeutic Cmax.

Azole antifungal drugs are used worldwide to treat a variety of fungal infections such as vulvovaginal candidiasis, particularly in pregnant women who are at increased risk. The aim of this study was to mechanistically investigate the endocrine disrupting potential of four commonly used azole antifungal drugs; clotrimazole, miconazole, ketoconazole and fluconazole in vitro using the H295R cell assay and two recombinant, CYP17A1 and CYP19A1 (aromatase), assays. Steroids were quantified using LC-MS/MS. In both recombinant assays, all four azoles inhibited the CYP enzymes investigated, at therapeutically relevant concentrations. However, responses were much more complex in the H295R cell line. Clotrimazole inhibited steroid production in a dose-dependent manner with IC50 values for CYP17A1 and CYP19A1 in the range 0.017-0.184 µM. Miconazole and ketoconazole increased all steroids on the hydroxylase axis (IC50 MIC: 0.042-0.082 µM, KET: 0.041-1.2 µM), leading to accumulation of progestagens and corticosteroids and suppression of androgens and estrogens, indicating inhibition of CYP17A1, in particular lyase activity. However, ketoconazole suppressed all steroids at higher concentrations, resulting in bell-shaped curves for all steroids on the hydroxylase axis. Fluconazole was found to inhibit CYP17A1-lyase activity, causing suppression of androgens (IC50 = 114-209 µM) and estrogens (IC50 = 28 µM). The results indicate that these four azole drugs are highly potent in vitro and, based on plasma Cmax values, may exert endocrine disrupting effects at therapeutically relevant concentrations. This raises concern for endocrine related effects in patients using azole antifungal drugs, particularly when taken during sensitive periods like pregnancy.

Innovative selection approach for a new antifungal agent mefentrifluconazole (Revysol®) and the impact upon its toxicity profile.

Mefentrifluconazole (trade name: Revysol®) is an agrochemical active ingredient from the new sub-class of isopropanol-triazole fungicides, with high selective fungicide activity. A full program of toxicity testing conducted according to OECD guidelines has shown mefentrifluconazole (MFZ) to be non-genotoxic and non-carcinogenic. Repeated dose studies in rats, mice and dogs identified the liver as the main target organ. Prenatal developmental toxicity studies in rats and rabbits did not indicate treatment-related embryofetal toxicity or teratogenicity up to the highest dose levels tested. In a two-generation dietary study in rats, the high dose level resulted in reduced food consumption and body weight gain throughout the dosing-period. Mating performance and fertility, estrous cycles, gestation length and pre-and post-natal survival of offspring were essentially unaffected and there was no evidence of masculinization of female pups or feminization of male pups. The screening strategy that led to the selection of MFZ was aimed to identify candidates with both high fungicidal activity and minimal likelihood of adverse side effects thought to arise from aromatase inhibition. The success of the selection strategy has been illustrated for MFZ by the absence in toxicity studies of effects that would indicate an endocrine disrupting potential.

Vitamin B combination reduces fluconazole toxicity in Wistar rats.

BACKGROUND: The major adverse effect associated with systemic administration of Fluconazole (FLZ), is hepatic toxicity. FLZ is most commonly used antifungal drug in treatment of invasive fungal infections. METHODS: FLZ toxicity was challenged by individual and in combination of three vitamins (B1, B2 B3). Animals were divided nine groups with six animals in each group. FLZ, at a dose of 50 mg/kg b.w, was orally administered for 90 days in experimental animals. Vitamins as individual or in combination was administered concomitantly to challenge or alleviate the toxicity of FLZ. They were sacrificed at the end of protocol for biochemical and histopathology analysis. Focus was made to observe the role of these micro nutrient's (vitamins) on liver for alteration in of pathological and physiological effects by FLZ in the Wistar albino rats. RESULTS: Combination of vitamin B1 + B2 + B3 in FLZ induced toxicity was able to restore the level of alkaline phosphatase (ALP) near to normal but with high level of ALP in B1 Control group. Aspartate aminotransferase (AST) was restored to normal in FLZ + B1, FLZ + B2 and FLZ + B1 + B2 + B3 groups and vice versa in FLZ + B3 group animals. Further the level of alanine aminotransferase (ALT) was restored to normal in FLZ + B3 animals. There were no significant changes found in total bilirubin (TBI), and direct bilirubin (DBI) as compare to normal control. Histopathological studies on animals' studies validated the serological results in normalizing the cellular architecture of liver. CONCLUSIONS: Restoration of altered biochemical parameters and cellular architecture of hepatocytes by different combination of these vitamins proves the chemo preventive potential of these micro nutrients' in FLZ toxicity. Graphical abstract Vitamin B combination attenuates fluconazole toxicity.

Fluconazole induces genotoxicity in cultured human peripheral blood mononuclear cells via immunomodulation of TNF-α, IL-6, and IL-10: new challenges for safe therapeutic regimens.

Context: Fluconazole (FNZ) is a drug used in antifungal therapy. However, the minimum FNZ dose to interfering with immune responses or inducing DNA damage is still unknown. Objective: This study investigated the toxicological profile of FNZ on cultured human peripheral blood mononuclear cells (PBMCs) treated with different concentrations of this azole. Materials and methods: Cultured PBMCs were exposed to FNZ (6, 12, 30, 60 and 120 µg/mL) and the toxicological profile was assessed by the following parameters: cytotoxic and nuclear division index (necrotic, apoptotic and viable cells), DNA damage (alkaline comet test), mutagenic potential (micronucleus test), cytokine modulation (IL-1, IL-6, IL-10, TNF-α, IFN-γ), and predictive toxicity (Osiris® and LAZAR® programs). Results: Our results demonstrated that FNZ induced cellular DNA damage and mutagenicity at concentrations above the plasma peak (>30 µg/mL) and 6 µg/mL, respectively, which was associated with increased TNF-α, and decrease IL-6 and IL-10 concentrations. These effects may be related to increased apoptosis and cytotoxic nuclear division index in the cultured PBMCs. In silico results indicated potential mutagenic, tumorigenic, irritant, and carcinogenic effects, which were partially confirmed by the above assays. Discussion and conclusions: Together, these findings suggest the need to rationalize the use of FNZ, especially if it is used for long periods or with concomitant pathologies requiring azole therapy that may increase FNZ's plasma concentration.

Gel in core carbosomes as novel ophthalmic vehicles with enhanced corneal permeation and residence.

Carbopol is a good bio-adhesive polymer that increases the residence time in the eye. However, the effect of blinking and lacrimation still reduce the amount of polymer and the incorporated drug available for bioadhesion. Gel-core liposomes are advanced systems offering benefits making it a good tool for improved ocular drug delivery and residence time. Incorporation of carbopol in gel-core liposomes and their potential in ocular delivery have not so far been investigated. Fluconazole (FLZ) was selected as a challenging important ocular antifungal suffering from poor corneal permeation and short residence time. In this study, gel-core carbosomes have been elaborated as novel carbopol-based ophthalmic vehicles to solve ocular delivery obstacles of FLZ and to sustain its effect. Full in vitro appraisal was performed considering gel-core structure, entrapment efficiency, particle size and stability of the vesicles as quality attributes. Structure elucidation of the nanocarrier was performed using optical, polarizing and transmission electron microscopy before and after Triton-X100 addition. Ex-vivo ocular permeation and in vivo performance were investigated on male albino rabbits. Optimized formulation (CBS5) showed gel-core structure, nanosize (339.00 ±â€¯5.50 nm) and not defined before (62.00% ±â€¯1.73) entrapment efficiency. Cumulative amount of CBS5 permeated ex-vivo after 6 h, was 2.43 and 3.43 folds higher than that of conventional liposomes and FLZ suspension, respectively. In-vivo corneal permeation of CBS5 showed significantly higher AUC0-24 h (487.12 ±â€¯74.80) compared to that of FLZ suspension (204.34 ±â€¯7.46) with longer residence time in the eye lasts for more than 18 h. In conclusion, novel gel-core carbosomes could successfully be used as a promising delivery system for chronic ocular diseases.

The Ascidian Embryo Teratogenicity assay in Ciona intestinalis as a new teratological screening to test the mixture effect of the co-exposure to ethanol and fluconazole.

The aim of this work was to evaluate the Ascidian Embryo Teratogenicity assay (AET) as new alternative invertebrate model to test the developmental effects of the co-exposure to ethanol and fluconazole. Ciona intestinalis embryos were exposed to the azolic fungicide fluconazole, (FLUCO, 7.8-250µM), to ethanol (Eth, 0.01-0.5%) and to their mixture (0.01% Eth+FLUCO 7.8-250µM) from neurula to larval stage. At the end of the exposure period, larvae were morphologically evaluated and benchmark analysis performed by using the PROAST modelling software. Both compounds were teratogenic in a concentration-related manner, particularly affecting the pigmented organs. The co-exposure to Eth enhanced the effects of FLUCO, the additive hypothesis was not rejected by the modelling. The results demonstrated that AET could be considered a good vertebrate-free alternative model for toxicological investigation in embryos.

Novel fluconazole derivatives with promising antifungal activity.

The fungistatic nature and toxicity concern associated with the azole drugs currently on the market have resulted in an increased demand for new azole antifungal agents for which these problematic characteristics do not exist. The extensive use of azoles has resulted in fungal strains capable of resisting the action of these drugs. Herein, we report the synthesis and antifungal activity of novel fluconazole (FLC) analogues with alkyl-, aryl-, cycloalkyl-, and dialkyl-amino substituents. We evaluated their antifungal activity by MIC determination and time-kill assay as well as their safety profile by hemolytic activity against murine erythrocytes as well as cytotoxicity against mammalian cells. The best compounds from our study exhibited broad-spectrum activity against most of the fungal strains tested, with excellent MIC values against a number of clinical isolates. The most promising compounds were found to be less hemolytic than the least hemolytic FDA-approved azole antifungal agent voriconazole (VOR). Finally, we demonstrated that the synthetic alkyl-amino FLC analogues displayed chain-dependent fungal membrane disruption as well as inhibition of ergosterol biosynthesis as possible mechanisms of action.

Parasex Generates Phenotypic Diversity de Novo and Impacts Drug Resistance and Virulence in Candida albicans.

Candida albicans is a diploid fungus that is a frequent cause of mucosal and systemic infections in humans. This species exhibits an unusual parasexual cycle in which mating produces tetraploid cells that undergo a nonmeiotic program of concerted chromosome loss to return to a diploid or aneuploid state. In this work, we used a multipronged approach to examine the capacity of parasex to generate diversity in C. albicans First, we compared the phenotypic properties of 32 genotyped progeny and observed wide-ranging differences in fitness, filamentation, biofilm formation, and virulence. Strikingly, one parasexual isolate displayed increased virulence relative to parental strains using a Galleria mellonella model of infection, establishing that parasex has the potential to enhance pathogenic traits. Next, we examined parasexual progeny derived from homothallic, same-sex mating events, and reveal that parasex can generate diversity de novo from identical parental strains. Finally, we generated pools of parasexual progeny and examined resistance of these pools to environmental stresses. Parasexual progeny were generally less fit than control strains across most test conditions, but showed an increased ability to grow in the presence of the antifungal drug fluconazole (FL). FL-resistant progeny were aneuploid isolates, often being diploid strains trisomic for both Chr3 and Chr6. Passaging of these aneuploid strains frequently led to loss of the supernumerary chromosomes and a concomitant decrease in drug resistance. These experiments establish that parasex generates extensive phenotypic diversity de novo, and that this process has important consequences for both virulence and drug resistance in C. albicans populations.

Synthesis, antifungal activity of caffeic acid derivative esters, and their synergism with fluconazole and nystatin against Candida spp.

We tested the antifungal potential of caffeic acid and 8 of its derivative esters against Candidaalbicans ATCC 90028 and 9 clinical isolatesand carried out a synergism assay with fluconazole and nystatin. Propyl caffeate (C3) showed the best antifungal activity against the tested strains. When in combination, C3 markedly reduced the MIC of fluconazole and nystatin with synergistic effect up to 64-fold. Finally, C3 showed a high IC50 value and selective indexagainst oral keratinocytes, demonstrating low toxicity against this cell type and selectivity for yeast cells. Further research should confirm its antifungal potential for development of combined therapy to treat C. albicans infections.

Phytotoxicity of wastewater-born micropollutants--Characterisation of three antimycotics and a cationic surfactant.

Sewage sludge applied to soil may be a valuable fertiliser but can also introduce poorly degradable and highly adsorptive wastewater-born residues of pharmaceuticals and personal care products (PPCPs) to the soil, posing a potential risk to the receiving environment. Three azole antimycotics (climbazole, ketoconazole and fluconazole), and one quaternary ammonium compound (benzyldimethyldodecylammonium chloride, BDDA) that are frequently detected in municipal sewage sludge and/or treated wastewater were therefore characterised in their toxicity toward terrestrial (Brassica napus) and aquatic (Lemna minor) plants. Fluconazole and climbazole showed the greatest toxicity to B. napus, while toxicity of ketoconazole and BDDA was by one to two orders of magnitude lower. Sludge amendment to soil at an agriculturally realistic rate of 5 t/ha significantly reduced the bioconcentration of BDDA in B. napus shoots compared to tests without sludge amendment, although not significantly reducing phytotoxicity. Ketoconazole, fluconazole and BDDA proved to be very toxic to L. minor with median effective concentrations ranging from 55.7 µg/L to 969 µg/L. In aquatic as well as terrestrial plants, the investigated azoles exhibited growth-retarding symptoms presumably related to an interference with phytohormone synthesis as known for structurally similar fungicides used in agriculture. While all four substances exhibited considerable phytotoxicity, the effective concentrations were at least one order of magnitude higher than concentrations measured in sewage sludge and effluent. Based on preliminary hazard quotients, BDDA and climbazole appeared to be of greater environmental concern than the two pharmaceuticals fluconazole and ketoconazole.

Uptake of azoles by lamb's lettuce (Valerianella locusta L.) grown in hydroponic conditions.

An uptake and translocation study of azole compounds was performed in lamb's lettuce (Valerianella locusta L.) grown in nutrient solution fortified with different azoles. Three azoles, (clotrimazole, fluconazole and propiconazole), which have different physico-chemical properties and are ubiquitous in the aquatic environment, were the compounds selected. An analytical method, based on matrix solid phase dispersion (MSPD) followed by LC-MS/MS determination, was developed to quantify these compounds in aqueous solution and in roots and leaves. The physicochemical properties of azoles are the main factors governing the uptake and plant accumulation. These azoles were detected in leaves indicating their transport within lamb's lettuce. Translocation from nutrient solution to the aerial part of lamb's lettuce was found to be highly dependent on the hydrophobicity of the azole. Clotrimazole accumulates in roots causing necrosis in roots and leaves, whereas fluconazole was the azole with the highest concentration in leaves without causing apparent phytotoxicity symptoms. The assessment of the levels of these azoles in leaves indicates that the risk for human health is negligible.

In vivo toxicity of a new antifungal agent 2,4-dithiophenoxy-1-iodo-4-bromo benzene: a follow up on our in vitro study.

Triazole fungicide fluconazole has become the most widely used antifungal agent in the world, mainly because of its ability to penetrate well into body fluids and tissues. However, it has been reported to interact with many drugs and because of its common use, the risk of resistance to fluconazole increases. This calls for new anti-fungal drugs that would be able to replace it. In 2006, a new thialo benzene derivative - 2,4-dithiophenoxy-1-iodo-4-bromo benzene (C18H12S2IBr) - was synthesised with a carbon backbone similar to fluconazole, and, according to the early in vitro tests, much greater efficiency. Followed an in vitro test of its cytotoxicity, in which the new drug showed promising results as an alternative to fluconazole. The aim of this study was take the next step and test C18H12S2IBr toxicity in vivo. We opted for a four-week test on Wistar rats, in which the new antifungal agent was orally applied at doses two and a half and five times lower than those of fluconazole. There were no changes in daily food and water consumption, but weight gain in female rats and relative organ weights changed in the treated groups, pointing to sex-related differences in drug metabolism and effects. Fluconazole significantly increased leukocytes and lowered neutrophils whereas C18H12S2IBr did not, while other haematological changes in respect to the vehicle control were similar between the treated groups. Differences in cytochrome c in the liver and kidney suggested greater apoptotic effect of the new drug, but interpretation remains inconclusive, considering that other key indicators (biochemistry and histopathology) do not support greater toxicity. Considering that C18H12S2IBr is more active at lower concentrations and has comparable toxic effects to fluconazole in rats, this new compound shows some promise in the treatment of fungal infections. Future, more detailed animal studies are needed, that will include drug interactions and molecular toxicity pathways. If the results are promising, clinical studies should follow.

Involvement of constitutive androstane receptor in liver hypertrophy and liver tumor development induced by triazole fungicides.

We clarified the involvement of constitutive androstane receptor (CAR) in triazole-induced liver hypertrophy and tumorigenesis using CAR-knockout (CARKO) mice. Seven-week-old male CARKO and wild-type (WT) mice were treated with 200 ppm cyproconazole (Cypro), 1500 ppm tebuconazole (Teb), or 200 ppm fluconazole (Flu) in the diet for 27 weeks after initiation by diethylnitrosamine (DEN). At weeks 4 (without DEN) and 13 (with DEN), WT mice in all treatment groups and CARKO mice in Teb group revealed liver hypertrophy with mainly Cyp2b10 and following Cyp3a11 inductions in the liver. Teb also induced Cyp4a10 in both genotypes. Cypro induced slight and duration-dependent liver hypertrophy in CARKO mice. At week 27, Cypro and Teb significantly increased eosinophilic altered foci and/or adenomas in WT mice. These proliferating lesions were clearly reduced in CARKO mice administered both compounds. The eosinophilic adenomas caused by Flu decreased in CARKO mice. The present study indicates that CAR is the main mediator of liver hypertrophy induced by Cypro and Flu, but not Teb. In contrast, CAR played a crucial role in liver tumor development induced by all three triazoles.