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Quantitative structure-activity relationships (QSARs) have been used to predict mixture toxicity. However, current research faces gaps in achieving accurate predictions of the mixture toxicity of azole fungicides. To address this gap, the application of machine learning (ML) algorithms has emerged as an effective strategy. In this study, we applied 12 algorithms, namely, k-nearest neighbor (KNN), kernel k-nearest neighbors (KKNN), support vector machine (SVM), random forest (RF), stochastic gradient boosting (GBM), cubist, bagged multivariate adaptive regression splines (Bagged MARS), eXtreme gradient boosting (XGBoost), boosted generalized linear model (GLMBoost), boosted generalized additive model (GAMBoost), bayesian regularized neural networks (BRNN), and recursive partitioning and regression trees (CART) to build ML models for 225 mixture toxicity of azole fungicides towards Auxenochlorella pyrenoidosa. A total of 36 single ML models and 12 consensus models were developed. The results indicated that models employing concentration addition (CA), independent action (IA), and molecular descriptors (MD) as variables demonstrated superior predictive abilities. The consensus model combining SVM and RF algorithms (labeled as CM0) demonstrated the highest level of accuracy in fitting the data, with a coefficient of determination of 0.980. Additionally, it showed strong predictive abilities when tested with external data, achieving an external R 2 value of 0.945 and a Concordance Correlation Coefficient of 0.967. This study provides a positive contribution to the ecological risk assessment of a mixture of azole fungicides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Assessing the interactions between environmental pollutants and these mixtures is of paramount significance in understanding their negative effects on aquatic ecosystems. However, existing research often lacks comprehensive investigations into the physiological and biochemical mechanisms underlying these interactions. This study aimed to reveal the toxic mechanisms of cyproconazole (CYP), imazalil (IMA), and prochloraz (PRO) and corresponding these mixtures on Auxenochlorella pyrenoidosa by analyzing the interactions at physiological and biochemical levels. Higher concentrations of CYP, IMA, and PRO and these mixtures resulted in a reduction in chlorophyll (Chl) content and increased total protein (TP) suppression, and malondialdehyde (MDA) content exhibited a negative correlation with algal growth. The activity of catalase (CAT) and superoxide dismutase (SOD) decreased with increasing azole fungicides and their mixture concentrations, correlating positively with growth inhibition. Azole fungicides induced dose-dependent apoptosis in A. pyrenoidosa, with higher apoptosis rates indicative of greater pollutant toxicity. The results revealed concentration-dependent toxicity effects, with antagonistic interactions at low concentrations and synergistic effects at high concentrations within the CYP-IMA mixtures. These interactions were closely linked to the interactions observed in Chl-a, carotenoid (Car), CAT, and cellular apoptosis. The antagonistic effects of CYP-PRO mixtures on A. pyrenoidosa growth inhibition can be attributed to the antagonism observed in Chl-a, Chl-b, Car, TP, CAT, SOD, and cellular apoptosis. This study emphasized the importance of gaining a comprehensive understanding of the physiological and biochemical interactions within algal cells, which may help understand the potential mechanism of toxic interaction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this study., (Copyright © 2024. Published by Elsevier B.V.)

Climbazole is an azole biocide that has been widely used in formulations of personal care products. Climbazole can cause developmental toxicity and endocrine disruption as well as gut disturbance in aquatic organisms. However, the mechanisms behind gut toxicity induced by climbazole still remain largely unclear in fish. Here, we evaluate the gut effects by exposing grass carp (Ctenopharyngodon idella) to climbazole at levels ranging from 0.2 to 20 μg/L for 42 days by evaluating gene transcription and expression, biochemical analyses, correlation network analysis, and molecular docking. Results showed that climbazole exposure increased cyp1a mRNA expression and ROS level in the three treatment groups. Climbazole also inhibited Nrf2 and Keap1 transcripts as well as proteins, and suppressed the transcript levels of their subordinate antioxidant molecules (cat, sod, and ho-1), increasing oxidative stress. Additionally, climbazole enhanced NF-κB and iκBα transcripts and proteins, and the transcripts of NF-κB downstream pro-inflammatory factors (tnfα, and il-1β/6/8), leading to inflammation. Climbazole increased pro-apoptosis-related genes (fadd, bad1, and caspase3), and decreased anti-apoptosis-associated genes (bcl2, and bcl-xl), suggesting a direct reaction to apoptosis. The molecular docking data showed that climbazole could form stable hydrogen bonds with CYP1A. Mechanistically, our findings suggested that climbazole can induce inflammation and oxidative stress through CYP450s/ROS/Nrf2/NF-κB pathways, resulting in cell apoptosis in the gut of grass carp., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Early life exposure to endocrine disrupting chemicals (EDCs) has been suggested to adversely affect reproductive health in humans and wildlife. Here, we characterize endocrine and adverse effects on the reproductive system after juvenile exposure to propiconazole (PROP) or imazalil (IMZ), two common azole fungicides with complex endocrine modes of action. Using the frog Xenopus tropicalis, two short-term (2-weeks) studies were conducted. I: Juveniles (2 weeks post metamorphosis (PM)) were exposed to 0, 17 or 178 µg PROP/L. II: Juveniles (6 weeks PM) were exposed to 0, 1, 12 or 154 µg IMZ/L. Histological analysis of the gonads revealed an increase in the number of dark spermatogonial stem cells (SSCs)/testis area, and in the ratio secondary spermatogonia: dark SSCs were increased in all IMZ groups compared to control. Key genes in gametogenesis, retinoic acid and sex steroid pathways were also analysed in the gonads. Testicular levels of 3β-hsd, ddx4 were increased and cyp19 and id4 levels were decreased in the IMZ groups. In PROP exposed males, increased testicular aldh1a2 levels were detected, but no histological effects observed. Although no effects on ovarian histology were detected, ovarian levels of esr1, rsbn1 were increased in PROP groups, and esr1 levels were decreased in IMZ groups. In conclusion, juvenile azole exposure disrupted testicular expression of key genes in retinoic acid (PROP) and sex steroid pathways and in gametogenesis (IMZ). Our results further show that exposure to environmental concentrations of IMZ disrupted spermatogenesis in the juvenile testis, which is a cause for concern as it may lead to impaired fertility. Testicular levels of id4, ddx4 and the id4:ddx4 ratio were associated with the number of dark SSCs and secondary spermatogonia suggesting that they may serve as a molecular markers for disrupted spermatogenesis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

It is acknowledged that azole fungicides may release into the environment and pose potential toxic risks. The combined toxicity interactions of azole fungicide mixtures, however, are still not fully understood. The combined toxicities and its toxic interactions of 225 binary mixtures and 126 multi-component mixtures on Chlorella pyrenoidosa were performed in this study. The results demonstrated that the negative logarithm 50% effect concentration (pEC 50 ) of 10 azole fungicides to Chlorella pyrenoidosa at 96 h ranged from 4.23 (triadimefon) to 7.22 (ketoconazole), while the pEC 50 values of the 351 mixtures ranged from 3.91 to 7.44. The high toxicities were found for the mixtures containing epoxiconazole. According to the results of the model deviation ratio (MDR) calculated from the concentration addition (MDR CA ), 243 out of 351 (69.23%) mixtures presented additive effect at the 10% effect, while the 23.08% and 7.69% of mixtures presented synergistic and antagonistic effects, respectively. At the 30% effect, 47.29%, 29.34%, and 23.36% of mixtures presented additive effects, synergism, and antagonism, respectively. At the 50% effect, 44.16%, 34.76%, and 21.08% of mixtures presented additive effects, synergism, and antagonism, respectively. Thus, the toxicity interactions at low concentration (10% effect) were dominated by additive effect (69.23%), whereas 55.84% of mixtures induced synergism and antagonism at high concentration (50% effect). Climbazole and imazalil were the most frequency of components presented in the additive mixtures. Epoxiconazole was the key component induced the synergistic effects, while clotrimazole was the key component in the antagonistic mixtures., (© 2023 Wiley Periodicals LLC.)

Azole fungicides is one of the major fungicides in agricultural field. In this study, toxic effects of diniconazole (DIN), metconazole (MET), and tebuconazole (TEB) to radish leaves and roots were investigated using targeted metabolomics with gas chromatography-mass spectrometry (GC-MS/MS). Especially, the changes of functional chemicals, including phytosterols and glucosinolates evaluated. Radish leaves and roots were harvested after 7 days and 14 days from last exposure. In multivariate analysis, the experimental groups showed clear separation in PCA and PLS-DA score plots. Phytosterols and glucosinolates were significantly changed by azole fungicide. Six metabolic pathways which are affected by fungicides were selected and showed similar patterns regardless of the type of azole fungicide used. As a result, azole fungicide induces the defense mechanisms of plants and affects both primary and secondary metabolism., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

We selected azole pesticides products that are managed by setting maximum residue limits (MRLs) in the Republic of Korea and describe the estrogen receptor (ER) α-related negative effect to endocrine system using in vitro Organization for Economic Cooperation and Development performance-based test guideline. No azoles were found to be an ERα agonist. Conversely, three azoles (bitertanol, cafenstrole, and tebufenpyrad) were determined to be ERα antagonists. In addition, the ERα antagonistic activities of bitertanol, cafenstrole, and tebufenpyrad were not significantly perturbed in the existence of phase I (hydroxylation, dealkylation, oxidation or reduction) and phase II (conjugation). Regarding the mechanism underlying their ERα-mediated endocrine disrupting potentials, ERα proteins cannot be translocated to the nucleus by suppressing the dimerization of ERα in the cytoplasm by bitertanol, cafenstrole, and tebufenpyrad. These data indicated that azole pesticide products show the capability to interfere the ERα-related human endocrine system. Furthermore, we identified the mechanism of ERα-mediated endocrine disrupting by azole insecticide products through this study., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The antimicrobial activity and biological efficiency of silver nanoparticles (AgNps) have been widely described and can be modeled through stabilizing and reducing agents, especially if they exhibit biocidal properties, which can enhance bioactivity against pathogens. The selective action of AgNps remains a major concern. In this regard, the use of plant extracts for the green synthesis of nanoparticles offers advantages because it improves the toxicity of Nps for microorganisms and is harmless to normal cells. However, biological evaluations of the activity of AgNps synthesized using different reducing agents are determined independently, and comparisons are frequently overlooked. Thus, we investigated and compared the antifungal and cytotoxic effects of two ecological AgNps synthesized from Moringa oleifera aqueous leaf extract (AgNp-M) and glucose (AgNp-G) against azole-resistant clinical isolates of Candida spp. and nontumor mammalian cells. Synthesized AgNps exhibited an antifungal effect on planktonic cells of drug-resistant C. albicans and C. tropicalis (MIC 0.21-52.6 µg/mL). The toxicity was influenced by size. However, the use of M. oleifera extracts allows us to obtain AgNps that are highly selective and nongenotoxic to Vero cells due to modifications of the shape and surface. Therefore, these results suggest that AgNp-M has antimicrobial potential and deserves further investigation for biomedical applications.

Objectives: In this study, we examined the toxicities, including poisoning and overdoses, with polyene, azole, flucytosine and echinocandin antifungals reported to the Swiss National Poison Centre., Methods: An observational cross-sectional study on antifungals was performed based on reports between 1995 and 2016 to Tox Info Suisse. Patient demographic and clinical characteristics were summarised among all reported calls, stratified by age group. In secondary analyses, we evaluated cases with clinical follow-up information., Results: In total, 149 cases were reported to the National Poison Centre during the study period, of which 49 (32.9%) were male and 91 (61.1%) were female, and 95 (63.8%) were adults and 54 (36.2%) were children (age ≤16 years). The most frequently reported drug class was azoles (136; 91.3%). In 31 cases (20.8%) reported by treating physicians, further clinical follow-up information was available. Nearly one-half of these patients were asymptomatic (15/31; 48.4%). In 11 patients (35.5%) among those with symptoms, the symptoms of toxicity were categorised with a strong causality to the respective antifungal. Clinical findings caused by triazoles were effects in the gastrointestinal tract, hallucinations and predelirium state. Clinical findings caused by polyenes were mostly minor symptoms with infusion-related effects or hypokalaemia. The severity was categorised as minor in 6 (54.5%) of 11 cases and as moderate in 5 cases (45.5%)., Conclusion: Despite high administered doses, no severe or fatal cases occurred within the study period. Although various toxicities can occur with antifungal administration and overdoses, they showed a favourable safety profile., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Human aromatase, also called CYP19A1, plays a major role in the conversion of androgens into estrogens. Inhibition of aromatase is an important target for estrogen receptor (ER)-responsive breast cancer therapy. Use of azole compounds as aromatase inhibitors is widespread despite their low selectivity. A toxicological evaluation of commonly used azole-based drugs and agrochemicals with respect to CYP19A1 is currently requested by the European Union- Registration, Evaluation, Authorization and Restriction of Chemicals (EU-REACH) regulations due to their potential as endocrine disruptors. In this connection, identification of structural alerts (SAs) is an effective strategy for the toxicological assessment and safe drug design. The present study describes the identification of SAs of azole-based chemicals as guiding experts to predict the aromatase activity. Total 21 SAs associated with aromatase activity were extracted from dataset of 326 azole-based drugs/chemicals obtained from Tox21 library. A cross-validated classification model having high accuracy (error rate 5%) was proposed which can precisely classify azole chemicals into active/inactive toward aromatase. In addition, mechanistic details and toxicological properties (agonism/antagonism) of azoles with respect to aromatase were explored by comparing active and inactive chemicals using structure-activity relationships (SAR). Lastly, few structural alerts were applied to form chemical categories for read-across applications., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Azole fungicides are widely used chemicals in agriculture and medicine. Their antifungal activity involves inhibition of steroid biosynthesis via inhibition of several cytochrome p450 enzymes. Evidence is accumulating in fish species to suggest azole fungicides perturb multiple hormone signaling pathways. The objective of this review was to comprehensively review data for azole-mediated impacts on the teleost endocrine system. We emphasize aspects of azole-induced endocrine disruption in several fish species, with special focus on the hypothalamic-pituitary-gonadal (HPG), hypothalamus-pituitary-thyroid (HPT) and hypothalamic-pituitary-adrenal (HPA) axis. Histopathological, physiological, and molecular data suggest azole fungicides at environmentally relevant concentrations and above are endocrine disruptors in fish. Endocrine disruption has been well documented for some azoles (e.g., difenconazole, fadrozole, ketoconazole, tebuconazole, triadimefon), but there are little data for others (e.g., cyproconazole, expoxiconazole, imidazole, metoconazole, nocodazole) in fish, revealing a knowledge gap in our understanding of azole toxicity. Based upon literature, computational analyses of transcriptome responses revealed progesterone-mediated oocyte maturation, insulin signaling pathway, adrenergic signaling, and metabolism of angiotensinogen may be processes disrupted by azoles. However, hormonal regulation of the sympathetic nervous system and the cardiovascular system in response to azole exposure has yet to be investigated in fish. Recommendations for studies moving forward include focus on non-steroid endocrine pathways, mechanisms of neuroendocrine disruption, and transgenerational effects of azoles on fish. This critical review identifies knowledge gaps and future directions for environmental studies focused on the effects of azoles in aquatic species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The increasing consumption of azole antifungal agents leads to their uncontrolled release into the environment. Therefore, it is crucial to remove their residues from natural ecosystems. This study aimed to examine the biological and chemical degradation of four typical azole fungicides: fluconazole (Fc), clotrimazole (Cl), climbazole (Cb), and epoxiconazole (Ep). The biodegradation was investigated using activated sludge and two novel Gram-negative bacterial strains. The chemical degradation experiments aimed to assess the efficiency of fungicides removal through UV treatment, the Fenton reaction, and a combination of these methods. Transformation products of Cb, Ep, and Cl photocatalytic removal were identified by mass spectrometry. In addition, the AlamarBlue® Assay and the MTT Assay allowed careful evaluation of the toxicity of azole derivatives and their transformation products towards newly isolated strains, Stenotrophomonas maltophilia AsPCl2.3 and Pseudomonas monteilii LB2. Among all azole fungicides, Cb was the most susceptible to biological removal while Fc, Ep, and Cl were basically resistant to biodegradation. Cl and Ep showed a significant biosorption on the activated sludge. Under optimized photolysis conditions, the removal efficiency of Cl, Cb, and Ep was significantly higher than that of biodegradation. The Fenton reaction supported by the UV-irradiation offered the best results of fungicides elimination. After 1 min of the experiment, Cl was almost completely removed while Cb and Ep removal rates reached an average of 60%. The proposed main degradation route of azole fungicides during UV-irradiation includes halogen atoms substitution by hydroxyl moieties. The final degradation product was imidazole or triazole. Azole fungicides and their transformation products differently affected the metabolic activity of Gram-negative bacteria. Cl and Cb intermediates showed lower toxicity than parent compounds. The findings help better understand the environmental impact of azole fungicides, their degradation, and toxicity. They also stress the need for reducing their uncontrolled release to the environment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Infections due to triazole-resistant Aspergillus fumigatus are increasingly reported worldwide and are associated with treatment failure and mortality. The principal class of azole-resistant isolates is characterized by tandem repeats of 34 bp or 46 bp within the promoter region of the cyp51A gene. Loop-mediated isothermal amplification (LAMP) is a widely used nucleic acid amplification system that is fast and specific. Here we describe a LAMP assay method to detect the 46 bp tandem repeat insertion in the cyp51A gene promoter region based on novel LAMP primer sets. It also differentiated strains with TR 46 tandem repeats from those with TR 34 tandem repeats. These results showed this TR 46 -LAMP method is specific, rapid, and provides crucial insights to develop novel antifungal therapeutic strategies against severe fungal infections due to A. fumigatus with TR 46 tandem repeats., (© 2021. The Author(s).)

Adverse outcome pathway (AOP) is a conceptual framework that links a molecular initiating event (MIE) via intermediate key events (KEs) with adverse effects (adverse outcomes, AO) relevant for risk assessment, through defined KE relationships (KERs). The aim of the present work is to describe a linear AOP, supported by experimental data, for skeletal craniofacial defects as the AO. This AO was selected in view of its relative high incidence in humans and the suspected relation to chemical exposure. We focused on inhibition of CYP26, a retinoic acid (RA) metabolizing enzyme, as MIE, based on robust previously published data. Conazoles were selected as representative stressors. Intermediate KEs are RA disbalance, aberrant HOX gene expression, disrupted specification, migration, and differentiation of neural crest cells, and branchial arch dysmorphology. We described the biological basis of the postulated events and conducted weight of evidence (WoE) assessments. The biological plausibility and the overall empirical evidence were assessed as high and moderate, respectively, the latter taking into consideration the moderate evidence for concordance of dose-response and temporal relationships. Finally, the essentiality assessment of the KEs, considered as high, supported the robustness of the presented AOP. This AOP, which appears of relevance to humans, thus contributes to mechanistic underpinning of selected test methods, thereby supporting their application in integrated new approach test methodologies and strategies and application in a regulatory context., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Novel N-methylated ebselenamine antioxidants were prepared from the corresponding diselenides with iodomethane. All ebselenamines showed excellent chain-breaking and glutathione peroxidase (GPx)-like activities. They could also inhibit lipid peroxidation much more efficiently than α-tocopherol. They could also mimic the functions of the GPx-enzymes nearly two times better than ebselen in the coupled reductase assay. Also, they were found to scavenge the ROS produced at low concentration (10 μM) with low toxicity effects and could have therapeutic potential against autoxidation. It is anticipated that these compounds could potentially be used against several diseases caused by autoxidation, and thus provide protection from cell death to mammals.

Azole fungicides (benzimidazoles, triazoles and imidazoles) are among the most widely used agrochemicals in the world. Unfortunately, azole fungicides are increasingly recognized for playing the role of endocrine disruptors in non-target organisms. Previously, the fecundity of ants with semi-claustral colony founding was found to be severely decreased in response to field-realistic concentrations of azole fungicides. However, during claustral colony founding, the ant queens do not feed and could therefore be protected against effects of agrochemicals applied during the colony founding. In the present study, we hypothesized that claustral colony founding is associated with a lower risk of oral exposure of ant queens to azole fungicides. We exposed queens of a common farmland ant species with claustral colony founding, Lasius niger, to four azole fungicides (epoxiconazole, flusilazole, prochloraz and thiophanate-methyl) that are commonly used in foliar applications and analyzed the differences in fecundity between fungicide-treated groups and the control water-treated group. We found that oral exposure to all four tested formulations of azole fungicides decreased the fecundity of L. niger queens. The decreases in fecundity ranged from 30.5% (epoxiconazole) to 40.3% (prochloraz), although the concentrations of fungicides used were several times lower than the minimum effective concentrations used to eliminate the target fungi by foliar applications of examined fungicides on various crops. Ants with both claustral and semi-claustral colony founding are highly vulnerable to field-realistic concentrations of azole fungicides that are sprayed in foliar applications. Azole fungicides substantially decrease the fitness of ant queens and may explain part of the recently observed decreases in farmland insect abundance and diversity., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Azoles are used in agriculture and medicine to combat fungal infections. We have previously examined the endocrine disrupting properties of the agricultural azole fungicides triticonazole and flusilazole. Triticonazole displayed strong androgen receptor (AR) antagonism in vitro, whereas in utero exposure resulted in anti-androgenic effects in vivo evidenced by shorter anogenital distance (AGD) in fetal male rats. Flusilazole displayed strong AR antagonism, but less potent than triticonazole, and disrupted steroidogenesis in vitro, whereas in utero exposure disrupted fetal male plasma hormone levels. To elaborate on how these azole fungicides can disrupt male reproductive development by different mechanisms, and to investigate whether feminization effects such as short AGD in males can also be detected at the transcript level in fetal testes, we profiled fetal testis transcriptomes after in utero exposure to triticonazole and flusilazole by 3'Digital Gene Expression (3'DGE). The analysis revealed few transcriptional changes after exposure to either compound at gestation day 17 and 21. This suggests that the observed influence of flusilazole on hormone production may be by directly targeting steroidogenic enzyme activity in the testis at the protein level, whereas observations of shorter AGD by triticonazole may primarily be due to disturbed androgen signaling in androgen-sensitive tissues. Expression of Calb2 and Gsta2 was altered by flusilazole but not triticonazole and may pinpoint novel pathways of disrupted testicular steroid synthesis. Our findings have wider implication for how we integrate omics data in chemical testing frameworks, including selection of non-animal test methods and building of Adverse Outcome Pathways for regulatory purposes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Currently, few studies have investigated the joint toxicity mechanism of azole fungicides at different exposure times and mixed at the relevant environmental concentrations. In this study, three common azole fungicides, namely, myclobutanil (MYC), propiconazole (PRO), and tebuconazole (TCZ), were used in studying the toxic mechanisms of a single substance and its ternary mixture exposed to ambient concentrations of Chlorella pyrenoidosa. Superoxide dismutase (SOD), catalase (CAT), chlorophyll a (Chla), and total protein (TP), were used as physiological indexes. Results showed that three azole fungicides and ternary mixture presented obvious time-dependent toxicities at high concentrations. MYC induced a hormetic effect on algal growth, whereas PRO and TCZ inhibit algal growth in the entire range of the tested concentrations. The toxicities of the three azole fungicides at 7 days followed the order PRO > TCZ > MYC. Three azole fungicides and their ternary mixture induced different levels of SOD and CAT activities in algae at high concentrations. The ternary mixture showed additive effects after 4 and 7 days exposure, but no effect was observed at actual environmental concentrations. The toxic mechanisms may be related to the continuous accumulation of reactive oxygen species, which not only affected protein structures and compositions but also damaged thylakoid membranes, hindered the synthesis of proteins and chlorophyll a, and eventually inhibited algal growth. These findings increase the understanding of the ecotoxicity of azole fungicides and use of azole fungicides in agricultural production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Azoles are considered as one of the most efficient fungicides for the treatment of humans, animals, and plant fungal pathogens. They are of significant clinical importance as antifungal drugs and are widely used in personal care products, ultraviolet stabilizers, and in aircraft for its anti-corrosive properties. The prevalence of azole compounds in the natural environment and its accumulation in fish raises questions about its impact on aquatic organisms., Objectives: The objective of this paper is to review the scientific studies on the effects of azole compounds in fish and to discuss future opportunities for the risk evaluation., Methods: A systematic literature search was conducted on Web of Science, PubMed, and ScienceDirect to locate peer-reviewed scientific articles on occurrence, environmental fate, and toxicological impact of azole fungicides on fish., Results: Studies included in this review provide ample evidence that azole compounds are not only commonly detected in the natural environment but also cause several detrimental effects on fish. Future studies with environmentally relevant concentrations of azole alone or in combination with other commonly occurring contaminants in a multigenerational study could provide a better understanding., Conclusion: Based on current knowledge and studies reporting adverse biological effects of azole on fish, considerable attention is required for better management and effective ecological risk assessment of these emerging contaminants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Lithium, commonly used to treat bipolar disorder, potentiates the ability of the muscarinic agonist pilocarpine to induce seizures in rodents. As this potentiation by lithium is reversed by the administration of myo-inositol, the potentiation may be mediated by inhibition of inositol monophosphatase (IMPase), a known target of lithium. Recently, we demonstrated that ebselen is a 'lithium mimetic' in regard to behaviours in both mice and man. Ebselen inhibits IMPase in vitro and lowers myo-inositol in vivo in the brains of mice and men, making ebselen the only known inhibitor of IMPase, other than lithium, that penetrates the blood-brain barrier. Our objective was to determine the effects of ebselen on sensitization to pilocarpine-induced seizures and neural activity. We administered ebselen at different doses and time intervals to mice, followed by injection of a sub-seizure dose of pilocarpine. We assessed seizure and neural activity by a subjective seizure rating scale, by monitoring tremors, and by induction of the immediate early gene c-fos. In contrast to lithium, ebselen did not potentiate the ability of pilocarpine to induce seizures. Unexpectedly, ebselen inhibited pilocarpine-induced tremor as well as pilocarpine-induced increases in c-fos mRNA levels. Both lithium and ebselen inhibit a common target, IMPase, but only lithium potentiates pilocarpine-induced seizures, consistent with their polypharmacology at diverse molecular targets. We conclude that ebselen does not potentiate pilocarpine-induced seizures and instead, reduces pilocarpine-mediated neural activation. This lack of potentiation of muscarinic sensitization may be one reason for the lack of side-effects observed with ebselen treatment clinically., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Environmental risk assessment associated with aquatic and terrestrial contamination is mostly based on predicted or measured environmental concentrations of a limited list of chemicals in a restricted number of environmental compartments. High resolution mass spectrometry (HRMS) can provide a more comprehensive picture of exposure to harmful chemicals, particularly through the retrospective analysis of digitally stored HRMS data. Using this methodology, our study characterized the contamination of various environmental compartments including 154 surface water, 46 urban effluent, 67 sediment, 15 soil, 34 groundwater, 24 biofilm, 41 gammarid and 49 fish samples at 95 sites widely distributed over the Swiss Plateau. As a proof-of-concept, we focused our investigation on antifungal azoles, a class of chemicals of emerging concern due to their endocrine disrupting effects on aquatic organisms and humans. Our results demonstrated the occurrence of antifungal azoles and some of their (bio)transformation products in all the analyzed compartments (0.1-100 ng/L or ng/g d.w.). Comparison of actual and predicted concentrations showed the partial suitability of level 1 fugacity modelling in predicting the exposure to azoles. Risk quotient calculations additionally revealed risk of exposure especially if some of the investigated rivers and streams are used for drinking water production. The case study clearly shows that the retrospective analysis of HRMS/MS data can improve the current knowledge on exposure and the related risks to chemicals of emerging concern and can be effectively employed in the future for such purposes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Azoles are emerging contaminants that are resistant to biodegradation during wastewater treatment. Their presence has been widely reported in wastewater effluents and receiving waters. In this work, the potential inhibition of nitrification process by six different azole compounds in wastewater treatment plants was investigated in batch bioassays. The azoles studied included three diazoles: pyrazole (Pz); 1-methylpyrazole (MePz); 3,5-dimethylpyrazole (DMePz); and three triazoles: 1,2,4-triazole (Tz); benzotriazole (BTz); and 5-methyl benzotriazole (MeBTz). The concentration of azoles causing 50% inhibition (IC 50 ) increased (azoles became less inhibitory) in the following order (mg L -1 ): BTz (1.99) < MeBTz (2.18) < Pz (2.69) < Tz (3.53) < DMePz (17.3) < MePz (49.6). No clear structure-inhibitory relationships were found using Log P and pKa as structural properties. The toxicity of any given azole may be related to the role of substituent groups on disabling/enabling binding to the active sites of metallo-enzymes in nitrifying microorganisms. This is exemplified by the low toxicity of MePz, which has a cyclic N blocked by a methyl group. The observed inhibition caused to nitrifying bacteria is more severe than their cytotoxicity to other target organisms (e.g., methanogens and heterotrophic bacteria), suggesting a specific inhibition to the copper-containing enzyme, ammonium monooxygenase, in ammonia oxidizing nitrifying microorganisms., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The occurrence of azole antifungals in the environment presents one of the emerging concerns due to their ecotoxicological threat as well as their potential contribution to the evolution of drug resistant fungi in the environment. In this study, the occurrence of eight commonly prescribed azole antifungal drugs was seasonally determined in influent and effluent water samples from three wastewater treatment plants and a drinking water treatment plant in South Africa. In addition, the risk quotient (RQ) method was employed to investigate the potential ecological and human health risks associated with their presence in the wastewater and/or drinking water. Clotrimazole, econazole, fluconazole, itraconazole, ketoconazole and miconazole were detected at least once in the water samples, while posaconazole and voriconazole were not detected in any of the samples for all seasons at which the samples were collected. Fluconazole was detected at higher frequency (about 96%) with a concentration up to 9959.0 ng L -1 . Clotrimazole had the second highest frequency of detection (about 33%) with a concentration up to 143.3 ng L -1 . Statistically significant temporal variation in clotrimazole (p < 0.05) and spatial variation in fluconazole (p < 0.05) were observed. In general, the preliminary ecological risk assessment based on risk quotient (RQ) calculation indicated that there is currently no high risk against aquatic organisms (Algae, Daphnia and Fish) related to the azole antifungals. Meanwhile, human health risk assessment demonstrated that fluconazole represented high risk in drinking water. Furthermore, risk estimates showed a potential for the detected concentrations of fluconazole and itraconazole in water samples to pose moderate to high risk for development of antifungal drug resistance. Some of the azole antifungal drugs are ubiquitous in the wastewater and future monitoring and validation studies should be conducted for those drugs that seem to pose human health and ecological risks., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Ketoconazole (KTZ) and itraconazole (ITZ) are antifungal agents that have a broad spectrum of activity against fungal pathogens. However, the therapeutic indications of many antifungal drugs, including those of the azole group, are restricted due to possible hepatotoxicity. We performed toxicogenomic analyses using in vivo and in vitro models to investigate the molecular mechanisms underlying the hepatotoxicity of two azole antifungal drugs. C57BL/6 male mice were treated daily with KTZ or ITZ, sacrificed at days 1 or 7, and the serum biochemistry and histopathology results showed that the KTZ-treated mice exhibited hepatotoxicity. Primary hepatocytes from C57BL/6 mice also exposed to KTZ or ITZ, and the cytotoxic effects of KTZ and ITZ were evaluated; KTZ exerted a greater cytotoxic effect than ITZ. The gene expression profiles in the livers of the 7-day-treated group and primary hepatocytes of the 24-h-treated group for both KTZ and ITZ were comparatively analyzed. Differentially expressed genes were selected based on the fold-changes and statistical significance, and the biological functions were analyzed using ingenuity pathways analysis. The results revealed that genes related to cholesterol synthesis were overexpressed in the liver in the KTZ-treated group, whereas expression of those related to acute phase injury was significantly altered in the ITZ-treated group. Causal gene analyses suggested that sterol regulatory element-binding transcription factors are key regulators that activate the transcription of target genes associated with the hepatotoxicity induced by oral KTZ. These findings enhance our understanding of the molecular mechanisms underlying the hepatotoxicity of azole drugs.

This work investigated the phenotypic behavior of Candida parapsilosis species complex in response to exposure to agricultural azoles and fluconazole. Three fluconazole-susceptible strains of C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis were used. Initial minimum inhibitory concentrations (iMICs) for agricultural and clinical azoles were determined by broth microdilution. Then, the strains were exposed to tebuconazole, tetraconazole and fluconazole for 15 days, at concentrations that were two-folded daily, starting at one-eighth the iMIC (iMIC/8) up to 64 times iMIC (64xiMIC). After 15-day-exposure, antifungal susceptibility, biofilm formation, CDR, MDR and ERG expression were evaluated. The three cryptic species developed tolerance to the antifungals they were exposed and presented reduction (P < 0.05) in fluconazole susceptibility. In addition, C. parapsilosis sensu stricto and C. metapsilosis also presented reduced susceptibility to voriconazole, after fluconazole exposure. Azole exposure decreased (P < 0.05) biofilm production by C. parapsilosis sensu stricto and C. orthopsilosis and increased (P < 0.05) the expression of ERG11 in all tested strains. The results show that exposure to agricultural azoles and fluconazole induces changes in the phenotypic behavior and gene expression by the three cryptic species of C. parapsilosis complex, highlighting the importance of environmental determinants for the development of antifungal resistance., (Copyright © 2018. Published by Elsevier B.V.)

The ability of azole fungicides to inhibit cytochrome P450 dependent metabolism is proposed to be the main mechanism for their synergizing effect on pyrethroid insecticide toxicity in aquatic invertebrates. This study investigates the correlation between inhibition strength and synergistic potential of azole fungicides in the crustacean Daphnia magna and the insect larvae Chironomus riparius. Inhibition strength was measured in vivo toward the cytochrome P450 catalysed conversion of 7-ethoxycoumarin to 7-hydroxycoumarin (ECOD). Synergistic potentials were determined as the ratio between predicted and observed toxicity of mixtures based on the model of concentration addition (CA) and independent action (IA). Azoles (n = 9-11) enhanced the toxicity of α-cypermethrin in D. magna (Synergy ratios CA: 0.8 - 16; IA: 1.1 - 22) and inhibited cytochrome P450 activity by different degrees (IC50: 0.0023 - 36 μM for D. magna and 0.08 - 24 μM for C. riparius). Inhibition strengths were strongly correlated in the two organisms (r: 0.937 p: 0.019 for triazoles and r: 0.903 p: 0.097 for imidazoles). Lipophilicity governed the inhibition strength of triazoles in both species (r > 0.9, p < 0.05). No correlation was observed between inhibition strengths and synergistic potentials. Several reasons for the apparent lack of correlation were discussed., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Facial malformations represent one of the most frequent abnormality in humans. The adverse outcome pathway involved in facial defects seems to be related to retinoic acid (RA) pathway imbalance. Environmental agents inducing craniofacial malformations in experimental models include pesticides (especially azole fungicides). By using the in vitro alternative method postimplantation rat whole embryo culture (WEC), we evaluated the intrinsic embryotoxic activity of some azole antifungals (cyproconazole, CYPRO; triadimefon, FON; flusilazole, FLUSI; and prochloraz, PCZ), in comparison to RA. All the tested molecules induced in a dose-related manner specific defects of the craniofacial structures (fused branchial arches), similar to those induced by RA. Collected data were modelled using PROAST 65.5 software to characterise the relative potency factors (RPFs) versus RA. In comparison to RA, all the evaluated azoles were less potent, showing among them a similar potency. Our data suggest a possible azole-related RA signalling perturbation to be further investigated. Moreover, the present results indicate the approach used in this work to be an interesting tool applicable to the hazard evaluation of novel compounds or the assessment of combined exposure to azoles or other dismorphogens., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Based on the advantages of azole molecules and fluoroquinolone drugs, we designed and synthesized 34 clinafloxacin-azole conjugates using fragment-based drug design and drug combination principles. The in vitro activities of the synthesized conjugates against Mycobacterium tuberculosis (H37Rv), Hela cell as well as Gram-positive and Gram-negative bacteria were assayed. The bioassay results revealed that most of the target molecules had anti-tuberculosis (anti-TB) activity, of which 14 compounds had very strong anti-TB activity [minimum inhibitory concentration (MIC) < 2 μM]. In addition, the compounds with strong activity towards H37Rv had weak activity towards Gram-negative and Gram-positive bacteria, showing obvious selectivity towards H37Rv. Predicted toxicity data indicated that 27 molecules were less toxic or equivalent to that of the original drug (clinafloxacin). Especially, it is demonstrated that compound TM2l exhibited the strongest anti-TB activity (MIC = 0.29 μM), low antibacterial activity, negligible toxicity, and good drug-likeness values, which can be considered as an ideal lead molecule for future optimization., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Azole fungicides, designed to halt fungal growth by specific inhibition of fungal cytochrome P450 (CYP51), inhibit cytochrome P450s involved in the metabolism of xenobiotics in several non-target organisms thus raising environmental concern. The present study investigates the degree by which inhibition strengths of azoles toward cytochrome P450 in rat liver, the insect Chironomus riparius larvae and the snail Lymnaea stagnalis can be extrapolated from estimated in silico affinities. Azoles' affinities toward human cytochrome P450 isoforms involved in xenobiotic metabolism (CYP3A4, CYP2C9 and CYP2D6) as well as fungal CYP51 were estimated with a ligand-protein docking model based on the ChemScore scoring function. Estimated affinities toward the selected enzymatic structures correlated strongly with measured inhibition strengths in rat liver (ChemScore vs. logIC 50 among cytochrome P450 isoforms: -0.662 < r < -0.891, n = 17 azoles), while weaker correlations were found for C. riparius larvae (-0.167 < r < -0.733, n = 9) and L. stagnalis (-0.084 < r < -0.648, n = 8). Inhibition strengths toward C. riparius and rat liver activities were found to be highly correlated to each other (r: 0.857) while no significant relationship was found between either of the species and L. stagnalis. The inhibition of cytochrome P450 due to azole fungicides could be estimated in vitro and to a lesser extent in silico for C. riparius but not for L. stagnalis, possibly due to different enzymatic susceptibility toward azole inhibition among the species., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Most β-d-xylosides with hydrophobic aglycones are nontoxic primers for glycosaminoglycan assembly in animal cells. However, when Ebselen was conjugated to d-xylose, d-glucose, d-galactose, and d-lactose (8A-D), only Ebselen β-d-xyloside (8A) showed significant cytotoxicity in human cancer cells. The following facts indicated that the aglycone Ebselen and β-d-xyloside primed glycosaminoglycans co-contributed to the observed cytotoxicity: 1. Ebselen induced S phase cell cycle arrest, whereas 8A induced G2/M cell cycle arrest; 2. 8A augmented early and late phase cancer cell apoptosis significantly compared to that of Ebselen and 8B-D; 3. Both 8A and phenyl-β-d-xyloside primed glycosaminoglycans with similar disaccharide compositions in CHO-pgsA745 cells; 4. Glycosaminoglycans could be detected inside of cells only when treated with 8A, indicating Ebselen contributed to the unique property of intracellular localization of the primed glycosaminoglycans. Thus, 8A represents a lead compound for the development of novel antitumor strategy by targeting glycosaminoglycans.

In the present study, we show the value of combining toxico-dynamic and -kinetic in vitro approaches for embryotoxicity testing of azoles. Both the whole embryo culture (WEC) and the embryonic stem cells test (EST) predicted the in vivo potency ranking of twelve tested azoles with moderate accuracy. Combining these results with relative placental transfer rates (Papp values) as determined in the BeWo cell culture model, increased the predictability of both WEC and EST, with R 2 values increasing from 0.51 to 0.87 and from 0.35 to 0.60, respectively. The comparison of these in vitro systems correlated well (R 2  = 0.67), correctly identifying the in vivo strong and weak embryotoxicants. Evaluating also specific gene responses related with the retinoic acid and sterol biosynthesis pathways, which represent the toxicological and fungicidal mode of action of azoles respectively in the WEC and EST, we observed that the differential regulation of Dhrs3 and Msmo1 reached higher magnitudes in both systems compared to Cyp26a1 and Cyp51. Establishing sensitive biomarkers across the in vitro systems for studying the underlying mechanism of action of chemicals, such as azoles, is valuable for comparing alternative in vitro models and for improving insight in the mechanism of developmental toxicity of chemicals., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

Single active substances of pesticides are thoroughly examined for their toxicity before approval. In this context, the liver is frequently found to be the main target organ. Since consumers are generally exposed to multiple residues of different active substances via the diet, it is important to analyse combinations of active substances for potential mixture effects. For the (tri-)azoles, a group of agricultural fungicides and antifungal drugs, combination effects on the liver are likely because of a similar mode of action. Hepatotoxic effects of mixtures of two triazoles (cyproconazole and epoxiconazole) and an imidazole (prochloraz) were investigated in a 28-day feeding study in rats at three dose levels ranging from a typical toxicological reference value to a clear effect dose. Test parameters included organ weights, clinical chemistry, histopathology and morphometry. In addition, molecular parameters were investigated by means of pathway-focused gene expression arrays, quantitative real-time PCR and enzyme activity assays. Effects were compared to those caused by the individual substances as observed at the same dose levels in a previous study. Mixture effects were substantiated by increases in relative and absolute liver weights, histopathological findings and alterations in clinical chemistry parameters at the top dose level. On the molecular level also at lower dose levels, additive effects could be observed for the induction of several cytochrome P 450 enzymes (Cyp1a1, Cyp2b1, Cyp3a2), transporters (Abcb1a, Abcc3) and of genes encoding for enzymes involved in fatty acid or phospholipid metabolism (Ppargc1a, Sc4 mol). In most cases, treatment with mixtures caused a more pronounced effect as compared to the individual substances. However, the assumption of dose additivity was in general sufficiently conservative to cover mixture effects observed under the conditions of the present study.

The widespread use of copper nanomaterials (CuNMs) as antibacterial and antifouling agents in consumer products increases the risk for metal contamination and adverse effects in aquatic environments. Information gaps exist on the potential toxicity of CuNMs in marine environments. We exposed field-collected marine meio- and macrobenthic communities to sediments spiked with micronized copper azole (MCA) using a novel method that brings intact benthic cores into the laboratory and exposes the organisms via surface application of sediments. Treatments included field and laboratory controls, 3 spiked sediments: low-MCA (51.9 mg/kg sediment), high-MCA (519 mg/kg sediment), and CuSO 4 (519 mg/kg sediment). In addition, single-species acute testing was performed with both MCA and CuSO 4. Our results indicate that meio- and macrofaunal assemblages exposed to High-MCA and CuSO 4 treatments differed significantly from both the laboratory control and the low-MCA treatments. Differences in macrofauna were driven by decreases in 3 Podocopa ostracod species, the bivalve Gemma gemma, and the polychaetes Exogone verugera and Prionospio heterobranchia relative to the laboratory control. Differences in the meiofaunal community are largely driven by nematodes. The benthic community test results were more sensitive than the single-species test results. Findings of this investigation indicate that CuNMs represent a source of risk to marine benthic communities comparable to that of dissolved Cu. Environ Toxicol Chem 2018;37:362-375. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America., (Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.)

We evaluated the effect of six azoles on embryonic development in the rat whole embryo culture (WEC). Using the total morphological scoring system (TMS), we calculated the ID 10 concentration (effective dose for 10% decrease in TMS). For evaluating gene specific responses, we combined previously and newly collected transcriptomics data of rat WEC exposed to a total of twelve azoles at their ID 10 for 4h. Results revealed shared expressions responses in genes involved in the retinoic acid (RA) and sterol biosynthesis pathways, which are respectively representatives of developmental toxicity and targeted fungicidal action of the azoles. Azoles with more pronounced effects on the regulation of RA-associated genes were generally characterized as more potent embryotoxicants. Overall, compounds with strong sterol biosynthesis related responses and low RA related responses were considered as more favourable candidates, as they specifically regulated genes related to a desired target response. Among the identified sterol associated genes, we detected that methylsterol monooxygenase 1 (Msmo1) was more sensitively induced compared to Cyp51, a classical biomarker of this pathway. Therefore, we suggest that Msmo1 could be a better biomarker for screening the fungicidal value of azoles. In summary, we conclude that the embryonic regulation of RA and sterol metabolic pathways could be indicators for ranking azoles as embryotoxicants and determining their drug efficacy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Consumers are exposed to low concentrations of a variety of pesticide residues in or on food. Some of them might interfere with the endocrine system. While each individual active substance has been extensively tested for toxicity and safety, potential combination effects possibly resulting from combined exposure to different pesticides have seldomly been tested so far, especially in vivo. Since the adrenal gland is a key endocrine organ, we investigated if and how substances of a group of fungicides presumed to interfere with the biosynthesis of steroid hormones affect this organ when applied individually and in combination in a broad dose range. A 28-day feeding study was conducted in Wistar rats by using three (tri)azole fungicides considered to potentially affect the endocrine system (cyproconazole, epoxiconazole and prochloraz) individually at five dose levels, ranging from 0.9ppm to 2400ppm, and in combination at three dose levels. The parameters analysed included classical toxicology (pathology, histopathology, clinical chemistry) and molecular toxicology endpoints (gene expression arrays and quantitative real time PCR e.g. of Star, HSD3β, Cyp11a1, Cyp11b1, Cyp11b2, Cyp 21, ApoE), as well as hormone analysis. A dose-dependent decrease in the adrenal gland weight of rats treated with epoxiconazole alone, which was accompanied by an atrophy of the adrenal gland as well as by an increase in the serum cholesterol level and which only became statistically significant at the top dose levels, was observed. These effects were attenuated in the combination experiments, although the same epoxiconazole concentration was used., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In mammals, the pregnane X receptor (PXR) is a transcription factor with a key role in regulating expression of several genes involved in drug biotransformation. PXR is present in fish and some genes known to be under its control can be up-regulated by mammalian PXR ligands. Despite this, direct involvement of PXR in drug biotransformation in fish has yet to be established. Here, the full length PXR sequence was cloned from carp (Cyprinus carpio) and used in a luciferase reporter assay to elucidate its role in xenobiotic metabolism in fish. A reporter assay for human PXR (hPXR) was also established to compare transactivation between human and carp (cPXR) isoforms. Rifampicin activated hPXR as expected, but not cPXR. Conversely, clotrimazole (CTZ) activated both isoforms and was more potent on cPXR, with an EC50 within the range of concentrations of CTZ measured in the aquatic environment. Responses to other azoles tested were similar between both isoforms. A range of pharmaceuticals tested either failed to activate, or were very weakly active, on the cPXR or hPXR. Overall, these results indicate that the cPXR may differ from the hPXR in its responses and/or sensitivity to induction by different environmental chemicals, with implications for risk assessment because of species differences., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Differential gene expression analysis in the rat whole embryo culture (WEC) assay provides mechanistic insight into the embryotoxicity of test compounds. In our study, we hypothesized that comparative analysis of the transcriptomes of rat embryos exposed to six azoles (flusilazole, triadimefon, ketoconazole, miconazole, difenoconazole and prothioconazole) could lead to a better mechanism-based understanding of their embryotoxicity and pharmacological action. For evaluating embryotoxicity, we applied the total morphological scoring system (TMS) in embryos exposed for 48h. The compounds tested showed embryotoxicity in a dose-response fashion. Functional analysis of differential gene expression after 4h exposure at the ID 10 (effective dose for 10% decreased TMS), revealed the sterol biosynthesis pathway and embryonic development genes, dominated by genes in the retinoic acid (RA) pathway, albeit in a differential way. Flusilazole, ketoconazole and triadimefon were the most potent compounds affecting the RA pathway, while in terms of regulation of sterol function, difenoconazole and ketoconazole showed the most pronounced effects. Dose-dependent analysis of the effects of flusilazole revealed that the RA pathway related genes were already differentially expressed at low dose levels while the sterol pathway showed strong regulation at higher embryotoxic doses, suggesting that this pathway is less predictive for the observed embryotoxicity. A similar analysis at the 24-hour time point indicated an additional time-dependent difference in the aforementioned pathways regulated by flusilazole. In summary, the rat WEC assay in combination with transcriptomics could add a mechanistic insight into the embryotoxic potency ranking and pharmacological mode of action of the tested compounds., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Macrolide antibiotics and azole fungicides are detected widely in the aquatic environment as a result of their increased use in humans and animal livestock disease and their incomplete removal by wastewater treatment plants. In most cases, ecotoxicological tests are performed by using individual chemical substances, but because of the coexistence of a number of chemicals in the environment, organisms are exposed to many chemicals simultaneously. Therefore, it is important to evaluate effects of chemical interactions, adding to potential hazards of individual chemical. Here, we investigated the synergetic effects of combined chemicals (the azole fungicide ketoconazole and either of two macrolide antibiotics, erythromycin and clarithromycin) in growth inhibition testing using Pseudokirchneriella subcapitata according to OECD Test guideline 201. Combination index plots, isobolograms, and curve-shift analyses revealed that the combination of macrolide antibiotic and ketoconazole at various ratios resulted in strong synergism that enhanced growth inhibition of P. subcapitata, suggesting the necessity of investigating potential hazard of combined chemicals for regulatory purposes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

An efficient scheme to synthesize novel ring-A fused heterocyclic derivatives of betulin was developed. The starting reaction of this synthesis was one-pot selective bacterial oxidation of betulin to betulone used as the key compound to synthesize the substituted azoles such as C(2)-C(3)-fused 1,2,3-triazoles, oxazoles and 1,2,4-triazine, as well as C(1)-C(2)-fused isoxazoles. The semi-synthetic compounds were screened for their cytotoxic activity against human cancer cell lines A549, HCT 116, HEp-2, MS and RD TE32 with use of the photometric MTT assays. Among the tested compounds, N-acetyltriazole of betulin (10) displayed impressive cytotoxic activity with IC 50 2.3-7.5 μM against HCT 116, HEp-2, MS and RD TE32 cell lines as well as 3-methyl-4-oxido-1,2,4-triazine-derivative of betulonic acid (12) that was active against HCT 116 and HEp-2 cell lines with IC 50 1.4 and 1.5 μM, respectively. Comparative experiments showed triazole (10) to have a lower cytotoxicity to normal epithelial cells, in comparison with compound (12). In accord with the in vivo acute toxicity test, the LD 50 of triazole (10) exceeded 600 mg/kg. The ability of the most potent active triazole (10) to trigger apoptotic cell death was explored in the Annexin V-FITC test and by analyzing of caspase activity and morphological alterations in mitochondria and nuclei of HCT 116 cells., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Tango ® Super is a two-compound fungicide formulation widely employed in grain protection. However, details of Tango ® Super effects on cell cultures have not been fully investigated. In this study, bovine lymphocytes were exposed to a concentration range 0.5; 1.5; 3; 6; and 15 μg mL -1 for 4 h to assess the cytotoxicity and genotoxicity of the fungicide. Our experiments revealed that this fungicide treatment reduced cell viability, decreased cell proliferation and provoked apoptotic cell death. Cell cycle analysis showed predominant accumulation of cells in the G 0 /G 1 phase of the cell cycle. The fungicide was able to induce mitochondrial superoxide production accompanied by elevated levels of carbonylated proteins and changes in the lipid membrane composition. The fungicide did not induce micronuclei production, but stimulated both DNA double-strand breaks and the formation of p53 binding protein, which is accumulated during the DNA repair process at the site of double-strand breaks. Based on the obtained data we suppose that the fungicide-induced DNA damage is the result of oxidative stress, which may contribute to higher occurrence of apoptotic cell death. Because ergosterol biosynthesis-inhibiting fungicides are widely used in agriculture to ensure higher crop yields and may cause health impairment of animals and humans, there is a need for further testing to elucidate their potential genotoxic effects using in vivo and/or in vitro systems., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Drug-drug interactions (DDIs) via cytochrome P450 (P450) induction are one clinical problem leading to increased risk of adverse effects and the need for dosage adjustments and additional therapeutic monitoring. In silico models for predicting P450 induction are useful for avoiding DDI risk. In this study, we have established regression models for CYP3A4 and CYP2B6 induction in human hepatocytes using several physicochemical parameters for a set of azole compounds with different P450 induction as characteristics as model compounds. To obtain a well-correlated regression model, the compounds for CYP3A4 or CYP2B6 induction were independently selected from the tested azole compounds using principal component analysis with fold-induction data. Both of the multiple linear regression models obtained for CYP3A4 and CYP2B6 induction are represented by different sets of physicochemical parameters. The adjusted coefficients of determination for these models were of 0.8 and 0.9, respectively. The fold-induction of the validation compounds, another set of 12 azole-containing compounds, were predicted within twofold limits for both CYP3A4 and CYP2B6. The concordance for the prediction of CYP3A4 induction was 87% with another validation set, 23 marketed drugs. However, the prediction of CYP2B6 induction tended to be overestimated for these marketed drugs. The regression models show that lipophilicity mostly contributes to CYP3A4 induction, whereas not only the lipophilicity but also the molecular polarity is important for CYP2B6 induction. Our regression models, especially that for CYP3A4 induction, might provide useful methods to avoid potent CYP3A4 or CYP2B6 inducers during the lead optimization stage without performing induction assays in human hepatocytes., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is an organoselenium radical scavenger compound, which has strong antioxidant and anti-inflammatory effects. Because of its properties, it may be protective against injury to the nervous tissue. However, evidence suggests that its glutathione peroxidase activity could underlie certain deleterious actions on cell physiology. In this study we have analyzed the effect of ebselen on rat hippocampal astrocytes in culture. Cellular oxidative status, cytosolic free-Ca(2+) concentration ([Ca(2+)]c), setting of endoplasmic reticulum stress and phosphorylation of glial fibrillary acidic protein and major mitogen-activated protein kinases were analyzed. Our results show that ebselen induced a concentration-dependent increase in the generation of reactive oxygen species in the mitochondria. We observed a concentration-dependent increase in global cysteine oxidation and in the level of malondialdehyde in the presence of ebselen. We also detected increases in catalase, glutathione S-transferase and glutathione reductase activity. Ebselen also evoked a concentration-dependent increase in [Ca(2+)]c. Moreover, we observed a concentration-dependent increase in the phosphorylation of the unfolded protein response markers, eukaryotic translation initiation factor 2α and X-box binding protein 1. Finally, ebselen also induced an increase in the phosphorylation of glial fibrillary acidic protein, SAPK/JNK, p38 MAPK and p44/42 MAPK. Our results provide strong evidence that implicate endoplasmic reticulum stress and activation of crucial mitogen-activated protein kinases in an oxidative damage of cells in the presence of ebselen. The compound thus might exert deleterious actions on astrocyte physiology that could compromise their function., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Two 28-day feeding studies were performed in male rats to investigate combination effects of azole fungicides in a broad dose range. Following separate administration of cyproconazole, epoxiconazole, prochloraz, propiconazole, and tebuconazole at five dose levels, the first three compounds were selected to be administered in two different mixtures at three dose levels including very low doses. Here we present the data obtained by clinical observations, pathology, histopathology, clinical chemistry and haematology. The liver was the common main target organ of all compounds and their mixtures. In addition, epoxiconazole exhibited an effect on the adrenals. Furthermore, food consumption and efficiency and body weight (gain) were affected. Adverse effects of the combinations were observed at dose levels at which the individual substances caused similar effects. No evidence of adverse effects was found at dose levels below the previously established NOAELs. Our findings indicate that the concept of dose additivity appears sufficiently protective for risk assessment of the fungicides examined. Besides toxicological testing, tissue residues of the azole compounds in liver, testis and kidney were determined revealing remarkable differences following administration of the single substances and of the mixtures., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Scientific community is striving to understand the role of heterocycles and fused heterocycles in drug discovery programme due to its impact on multi-drug resistance (MDR) of anticancer drugs. Architecting of various scaffolds for cancer treatment has become gradually increased in many years. Till now there is no treatment which is so proficient that it can cure the cancer from the roots. Hence, it is very necessary to design novel anticancer agents with minimum side effects. Synthesis of hybrids from natural leads is one of the rationale approaches in medicinal chemistry. It remains a big challenge to invent new efficient drugs to beat cancer. The design and synthesis of fused molecules as anticancer agents is one of the great innovations of modern era. In drug discovery archetype, a variety of heterocycles have been considered for the development of novel lead compounds. This article presents some recent advancements in the field of anticancer heterocyclic agents all around the world and also attracted the structure activity relationship along with the structure of the most promising molecules along with IC50 values against various human cancer cell lines.

Without a doubt, our current antimicrobials are losing the battle in the fight against newly-emerged multidrug-resistant pathogens. There is a pressing, unmet need for novel antimicrobials and novel approaches to develop them; however, it is becoming increasingly difficult and costly to develop new antimicrobials. One strategy to reduce the time and cost associated with antimicrobial innovation is drug repurposing, which is to find new applications outside the scope of the original medical indication of the drug. Ebselen, an organoselenium clinical molecule, possesses potent antimicrobial activity against clinical multidrug-resistant Gram-positive pathogens, including Staphylococcus, Streptococcus, and Enterococcus, but not against Gram-negative pathogens. Moreover, the activity of ebselen against Gram-positive pathogens exceeded those activities determined for vancomycin and linezolid, drugs of choice for treatment of Enterococcus and Staphylococcus infections. The minimum inhibitory concentrations of ebselen at which 90% of clinical isolates of Enterococcus and Staphylococcus were inhibited (MIC90) were found to be 0.5 and 0.25 mg/L, respectively. Ebselen showed significant clearance of intracellular methicillin-resistant S. aureus (MRSA) in comparison to vancomycin and linezolid. We demonstrated that ebselen inhibits the bacterial translation process without affecting mitochondrial biogenesis. Additionally, ebselen was found to exhibit excellent activity in vivo in a Caenorhabditis elegans MRSA-infected whole animal model. Finally, ebselen showed synergistic activities with conventional antimicrobials against MRSA. Taken together, our results demonstrate that ebselen, with its potent antimicrobial activity and safety profiles, can be potentially used to treat multidrug resistant Gram-positive bacterial infections alone or in combination with other antibiotics and should be further clinically evaluated.

The cytochrome P450 1A (CYP1A) biomarker response was studied in the Poeciliopsis lucida hepatocellular carcinoma (PLHC-1) cell line, which represents a good model for studies on aryl hydrocarbon receptor (AhR) - CYP1A signaling. The PLHC-1 cells were exposed to the prototypical CYP1A inducer and AhR agonist β-naphthoflavone (BNF) in combination with different azoles. Two imidazoles (clotrimazole and prochloraz) and two benzimidazoles (nocodazole and omeprazole) were used. Exposure to clotrimazole, prochloraz and nocodazole resulted in 2-4 fold induction of the CYP1A-mediated ethoxyresorufin-O-deethylase (EROD) activities at 24 and 48h, whereas exposure to the omeprazole for 48h had no effect on the EROD activity. Clotrimazole, nocodazole and prochloraz also acted as inhibitors of EROD activities in situ in PLHC-1 cells (IC50=1.3-7.7μM), whereas omeprazole had no effect on this activity (IC50=72μM). Exposure to 10μM prochloraz resulted in 3-fold induction of CYP1A mRNA and exposure to 10μM nocodazole resulted in 16-fold induction of CYP1A mRNA levels at 24h compared to controls. In the mixture experiments, more-than-additive mixture effects between BNF and the azoles clotrimazole, prochloraz and nocodazole on EROD activities were evident, with nocodazole showing the strongest mixture effect. The presence of nocodazole increased the response to BNF up to 200-fold on CYP1A mRNA and up to 16-fold on EROD activities and prolonged the effect of BNF exposure on EROD activities by 24h or longer. This suggests that azoles that are inhibitors and/or competing substrates for the CYP1A enzymes can cause increased sensitivity to exposures to chemicals that depend on CYP1A metabolism for their elimination in situations of mixed chemical exposures. The results also suggest that the EROD biomarker response can be significantly affected in azole-contaminated areas. The responsiveness of the EROD biomarker to BNF exposure was studied in PLHC-1 that had been pre-treated with nocodazole for 5 or 24h at concentrations that are known to disassemble microtubules at 24h in these cells. Pre-treatment of PLHC-1 cells with nocodazole for either 5 or 24h had no effect on the responsiveness to BNF exposure, which implies that the EROD activity can be induced in cells with disassembled microtubules., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Endocrine-mediated effects in fish populations have been widely documented. Most attention has been focused on feminization caused by estrogenic substances, but this paper reviews evidence for the effects of a group of fungicides and pharmaceuticals, the azoles, which have been reported to cause masculinization in fish. The paper considers information from laboratory studies on the effects of azole compounds on fish endocrinology, and on the potential existence of such effects in wild fish. The occurrence of some azoles in effluents and surface waters has also been briefly reviewed. Under laboratory conditions, many azoles are able to cause masculinization or defeminization in fish by inhibition of the P450 enzyme aromatase (CYP19). However, in no case where such effects have been observed in the field has a link been established with this group of substances. In most instances, other more convincing explanations have been proposed. Peak concentrations of some azoles in surface waters can approach those which, under continuous long-term exposure in the laboratory, might lead to some aromatase inhibition. However, available data on exposure and effects provide reassurance that the concentrations of azoles found in surface waters are too low to cause adverse effects in fish by interference with their endocrine system. Compared to the widespread observations of feminization and estrogenic effects in (male) fish, there are relatively few papers describing masculinization or defeminization in (female) wild fish populations, suggesting that this is quite a rare phenomenon. The significance of this result is emphasized by the fact that fish are among the best studied organisms in the environment.

The potential effects of pesticides and their metabolites on the endocrine system are of major concern to wildlife and human health. In this context, the azole pesticides have earned special attention due to their cytochrome P450 aromatase inhibition potential. Cytochrome P450 aromatase (CYP19) catalyses the conversion of androstenedione and testosterone into oestrone and oestradiol, respectively. Thus, aromatase modulates the oestrogenic balance essential not only for females, but also for male physiology, including gonadal function. Its inhibition affects reproductive organs, fertility and sexual behaviour in humans and wildlife species. Several studies have shown that azole pesticides are able to inhibit human and fish aromatases but the information on birds is lacking. Consequently, it appeared to be of interest to estimate the aromatase inhibition of azoles in three different avian species, namely Gallus gallus, Coturnix coturnix japonica and Taeniopygia guttata. In the absence of the crystal structure of the aromatase enzyme in these bird species, homology models for the individual avian species were constructed using the crystal structure of human aromatase (hAr) (pdb: 3EQM) that showed high sequence similarity for G. gallus (82.0%), T. guttata (81.9%) and C. japonica (81.2%). A homology model with Oncorhynchus mykiss (81.9%) was also designed for comparison purpose. The homology-modelled aromatase for each avian and fish species and crystal structure of human aromatase were selected for docking 46 structurally diverse azoles and related compounds. We showed that the docking behaviour of the chemicals on the different aromatases was broadly the same. We also demonstrated that there was an acceptable level of correlation between the binding score values and the available aromatase inhibition data. This means that the homology models derived on bird and fish species can be used to approximate the potential inhibitory effects of azoles on their aromatase.