Azole Fungicides Research Articles

Dissection of the Activity of Agricultural Fungicides against Clinical Aspergillus Isolates with and without Environmentally and Medically Induced Azole Resistance.

Azole resistance is an emerging problem in patients with aspergillosis. The role of fungicides for resistance development and occurrence is not fully elucidated. EUCAST reference MICs of 17 fungicides (11 azoles and 6 others), five azole fungicide metabolites and four medical triazoles were examined against two reference and 28 clinical isolates of A. fumigatus, A. flavus and A. terreus with (n = 12) and without (n = 16) resistance mutations. Eight/11 azole fungicides were active against wild-type A. fumigatus, A. flavus and A. terreus, including four (metconazole, prothioconazole-desthio, prochloraz and imazalil) with low MIC50 (≤2 mg/L) against all three species and epoxiconazole, propiconazole, tebuconazole and difenoconazole also against wild-type A. terreus. Mefentrifluconazole, azole metabolites and non-azole fungicides MICs were >16 mg/L against A. fumigatus although partial growth inhibition was found with mefentrifluconazole. Moreover, mefentrifluconazole and axozystrobin were active against wild-type A. terreus. Increased MICs (≥3 dilutions) were found for TR34/L98H, TR34(3)/L98H, TR46/Y121F/T289A and G432S compared to wild-type A. fumigatus for epoxiconazole, propiconazole, tebuconazole, difenoconazole, prochloraz, imazalil and metconazole (except G432S), and for prothioconazole-desthio against TR46/Y121F/T289A, specifically. Increased MICs were found in A. fumigatus harbouring G54R, M220K and M220R alterations for five, one and one azole fungicides, respectively, compared to MICs against wild-type A. fumigatus. Similarly, increased MICs wer found for A. terreus with G51A, M217I and Y491H alterations for five, six and two azole fungicides, respectively. Azole fungicides showed activity against wild-type A. fumigatus, A. terreus and A. flavus, but not against all mutant isolates, suggesting the environmental route of azole resistance may have a role for all three species.

Prevalence of Azole-Resistant Aspergillus fumigatus is Highly Associated with Azole Fungicide Residues in the Fields.

Triazole resistance in Aspergillus fumigatus is a growing public health concern. In addition to its emergence in the therapy of invasive aspergillosis by triazole medicines, it has been frequently detected in agricultural fields all over the world. Here, we explore the potential link between residues of azole fungicides with similar chemical structure to triazole medicines in soil and the emergence of resistant A. fumigatus (RAF) through 855 500 km2 monitoring survey in Eastern China covering 6 provinces. In total, 67.3%, 15.2%, 12.3%, 2.9%, 1.5%, 0.4%, and 0.3% of the soil samples contained these five fungicides (tebuconazole, difenoconazole, propiconazole, hexaconazole, and prochloraz) of 0-100, 100-200, 200-400, 400-600, 600-800, 800-1000, and >1000 ng/g, respectively. The fractions of samples containing RAF isolates were 2.4%, 5.2%, 6.4%, 7.7%, 7.4%, 14.3%, and 20.0% of the samples with total azole fungicide residues of 0-100, 100-200, 200-400, 400-600, 600-800, 800-1000, and >1000 ng/g, respectively. We find that the prevalence of RAFs is positively (P < 0.0001) correlated with residual levels of azole fungicides in soils. Our results suggest that the use of azole fungicides in agriculture should be minimized and the intervals between treatments expanded to reduce the selective pressure toward the development of resistance in A. fumigatus in agricultural fields.

Pesticide risk assessment at the molecular level using honey bee cytochrome P450 enzymes: A complementary approach

Honey bee (Apis mellifera) first-tier pesticide risk assessment is largely based on standardized laboratory toxicity bioassays after both acute and chronic exposure. Recent research on honey bee cytochrome P450 monooxygenases (P450s) uncovered CYP9Q3 as the molecular determinant mediating neonicotinoid insecticide selectivity and explaining why certain neonicotinoids such as thiacloprid show > 1000-fold lower acute toxicity than others (e.g. imidacloprid). Here this knowledge is leveraged for mechanistic risk assessment at the molecular level using a fluorescence-based high-throughput in vitro assay, predicting the interaction of diverse pesticidal chemotypes, including azole fungicides, with recombinantly expressed honey bee CYP9Q enzymes, known to metabolize thiacloprid, acetamiprid and tau-fluvalinate. Some azole fungicides were shown to be synergistic in combination with certain insecticides, including neonicotinoids and pyrethroids, whereas others such as prothioconazole were not. We demonstrate that biochemical CYP9Q2/CYP9Q3 inhibition data of azoles revealed a striking correlation with their synergistic potential at the organismal level, and even allow to explain combined toxicity effects observed for tank mixtures under field conditions. Our novel toxicogenomics-based approach is designed to complement existing methods for pesticide risk assessment with unprecedented screening capacity, by utilizing honey bee P450 enzymes known to confer pesticide selectivity, in order to biochemically address issues of ecotoxicological concern.

Towards understanding transfluthrin efficacy in a pyrethroid-resistant strain of the malaria vector Anopheles funestus with special reference to cytochrome P450-mediated detoxification

Malaria vector control interventions rely heavily on the application of insecticides against anopheline mosquitoes, in particular the fast-acting pyrethroids that target insect voltage-gated sodium channels (VGSC). Frequent applications of pyrethroids have resulted in resistance development in the major malaria vectors including Anopheles funestus, where resistance is primarily metabolic and driven by the overexpression of microsomal cytochrome P450 monooxygenases (P450s). Here we examined the pattern of cross-resistance of the pyrethroid-resistant An. funestus strain FUMOZ-R towards transfluthrin and multi-halogenated benzyl derivatives, permethrin, cypermethrin and deltamethrin in comparison to the susceptible reference strain FANG. Transfluthrin and two multi-fluorinated derivatives exhibited micromolar potency - comparable to permethrin - to functionally expressed dipteran VGSC in a cell-based cation influx assay. The activity of transfluthrin and its derivatives on VGSC was strongly correlated with their contact efficacy against strain FUMOZ-R, although no such correlation was obtained for the other pyrethroids due to their rapid detoxification by the resistant strain. The low resistance levels for transfluthrin and derivatives in strain FUMOZ-R were only weakly synergized by known P450 inhibitors such as piperonyl butoxide (PBO), triflumizole and 1-aminobenzotriazole (1-ABT). In contrast, deltamethrin toxicity in FUMOZ-R was synergized > 100-fold by all three P450 inhibitors. The biochemical profiling of a range of fluorescent resorufin and coumarin compounds against FANG and FUMOZ-R microsomes identified 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC) as a highly sensitive probe substrate for P450 activity. BOMFC was used to develop a fluorescence-based high-throughput screening assay to measure the P450 inhibitory action of potential synergists. Azole fungicides prochloraz and triflumizole were identified as extremely potent nanomolar inhibitors of microsomal P450s, strongly synergizing deltamethrin toxicity in An. funestus. Overall, the present study contributed to the understanding of transfluthrin efficacy at the molecular and organismal level and identified azole compounds with potential to synergize pyrethroid efficacy in malaria vectors.

Claustral colony founding does not prevent sensitivity to the detrimental effects of azole fungicides on the fecundity of ants

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.

Biodegradation of typical azole fungicides in activated sludge under aerobic conditions

Widespread use of azole fungicides and low removal efficiency in wastewater treatment plants (WWTPs) have led to the elevated concentration of azole fungicides in receiving environment. However, there was limited research about the removal mechanism of azole fungicides in the biological treatment of WWTPs. Imidazole fungicide climbazole and triazole fungicide fluconazole were selected to investigate the biodegradation mechanism of azole fungicides in activated sludge under aerobic conditions. Climbazole was found to be adsorbed to solid sludge and resulted in quick biodegradation. The degradation of climbazole in the aerobic activated sludge system was fitted well by the first-order kinetic model with a half-life of 5.3 days, while fluconazole tended to stay in liquid and had only about 30% of loss within 77 days incubation. Ten biotransformation products of climbazole were identified by high resolution mass spectrometry using suspect and non-target screening method. But no biodegradation products of fluconazole were identified due to its limited removal. The possible biodegradation pathways for climbazole were proposed based on the products identification and pathway prediction system, and involves oxidative dehalogenation, side chain oxidation and azole ring loss. The findings from this study suggest that it should be a concern for the persistence of fluconazole in the environment.

Effects of Agricultural Fungicide Use on Aspergillus fumigatus Abundance, Antifungal Susceptibility, and Population Structure.

Antibiotic resistance is an increasing threat to human health. In the case of Aspergillus fumigatus, which is both an environmental saprobe and an opportunistic human fungal pathogen, resistance is suggested to arise from fungicide use in agriculture, as the azoles used for plant protection share the same molecular target as the frontline antifungals used clinically. However, limiting azole fungicide use on crop fields to preserve their activity for clinical use could threaten the global food supply via a reduction in yield. In this study, we clarify the link between azole fungicide use on crop fields and resistance in a prototypical human pathogen through systematic soil sampling on farms in Germany and surveying fields before and after fungicide application. We observed a reduction in the abundance of A. fumigatus on fields following fungicide treatment in 2017, a finding that was not observed on an organic control field with only natural plant protection agents applied. However, this finding was less pronounced during our 2018 sampling, indicating that the impact of fungicides on A. fumigatus population size is variable and influenced by additional factors. The overall resistance frequency among agricultural isolates is low, with only 1 to 3% of isolates from 2016 to 2018 displaying resistance to medical azoles. Isolates collected after the growing season and azole exposure show a subtle but consistent decrease in susceptibility to medical and agricultural azoles. Whole-genome sequencing indicates that, despite the alterations in antifungal susceptibility, fungicide application does not significantly affect the population structure and genetic diversity of A. fumigatus in fields. Given the low observed resistance rate among agricultural isolates as well the lack of genomic impact following azole application, we do not find evidence that azole use on crops is significantly driving resistance in A. fumigatus in this context.IMPORTANCE Antibiotic resistance is an increasing threat to human health. In the case of Aspergillus fumigatus, which is an environmental fungus that also causes life-threatening infections in humans, antimicrobial resistance is suggested to arise from fungicide use in agriculture, as the chemicals used for plant protection are almost identical to the antifungals used clinically. However, removing azole fungicides from crop fields threatens the global food supply via a reduction in yield. In this study, we survey crop fields before and after fungicide application. We find a low overall azole resistance rate among agricultural isolates, as well as a lack of genomic and population impact following fungicide application, leading us to conclude azole use on crops does not significantly contribute to resistance in A. fumigatus.

Antifungal Drug Repurposing.

Control of fungal pathogens is increasingly problematic due to the limited number of effective drugs available for antifungal therapy. Conventional antifungal drugs could also trigger human cytotoxicity associated with the kidneys and liver, including the generation of reactive oxygen species. Moreover, increased incidences of fungal resistance to the classes of azoles, such as fluconazole, itraconazole, voriconazole, or posaconazole, or echinocandins, including caspofungin, anidulafungin, or micafungin, have been documented. Of note, certain azole fungicides such as propiconazole or tebuconazole that are applied to agricultural fields have the same mechanism of antifungal action as clinical azole drugs. Such long-term application of azole fungicides to crop fields provides environmental selection pressure for the emergence of pan-azole-resistant fungal strains such as Aspergillus fumigatus having TR34/L98H mutations, specifically, a 34 bp insertion into the cytochrome P450 51A (CYP51A) gene promoter region and a leucine-to-histidine substitution at codon 98 of CYP51A. Altogether, the emerging resistance of pathogens to currently available antifungal drugs and insufficiency in the discovery of new therapeutics engender the urgent need for the development of new antifungals and/or alternative therapies for effective control of fungal pathogens. We discuss the current needs for the discovery of new clinical antifungal drugs and the recent drug repurposing endeavors as alternative methods for fungal pathogen control.

Use of extracts from Prosopis nigra in the control of Cercospora kikuchii and Septoria glycines

The antifungal activity of organic extracts from leaves, bark and hearthwood of the timber tree Prosopis nigra was investigated against fungi responsible for soybean late season diseases. Nine extracts were assayed by the disc diffusion test toward Septoria glycines and Cercospora kikuchii. The methanol extract of leaves was the most active with the lowest inhibitory dose of 200 μg disc−1 on both fungi and diameters of growth inhibition zones of 18.5 mm (C. kikuchii) and 20.5 mm (S. glycines). Tryptamine was the main antifungal constituent of the extract. The methanol extract and tryptamine synergized the effect of difenoconazole. Tryptamine also inhibited cercosporin produced by C. kikuchii in 36–96 % when applied at 25−100 μg and 70 % when provided in mixture with the fungicide. Preventive applications of the extract and the mixture on soybean plants strongly reduced disease severity produced by both fungal pathogens. Our results indicate that the leaf methanol extract of P. nigra deserves further research as an additive of azole fungicides applied for the control of C. kikuchii and S. glycines.

Transcriptome analysis of fetal rat testis following intrauterine exposure to the azole fungicides triticonazole and flusilazole reveals subtle changes despite adverse endocrine effects

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.

Global transcriptomic responses orchestrate difenoconazole resistance in Penicillium spp. causing blue mold of stored apple fruit

BackgroundBlue mold is a globally important and economically impactful postharvest disease of apples caused by multiple Penicillium spp. There are currently four postharvest fungicides registered for blue mold control, and some isolates have developed resistance manifesting in decay on fungicide-treated fruit during storage. To date, mechanisms of fungicide resistance have not been explored in this fungus using a transcriptomic approach.ResultsWe have conducted a comparative transcriptomic study by exposing naturally-occurring difenoconazole (DIF) resistant (G10) and sensitive (P11) blue mold isolates to technical grade difenoconazole, an azole fungicide in the commercial postharvest product Academy (Syngenta Crop Protection, LLC). Dynamic changes in gene expression patterns were observed encompassing candidates involved in active efflux and transcriptional regulators between the resistant and sensitive isolates. Unlike other systems, 3 isoforms of cytochrome P450 monoxygenase (CYP51A-C) were discovered and expressed in both sensitive and resistant strains upon difenoconazole treatment. Active efflux pumps were coordinately regulated in the resistant isolate and were shown to mediate the global resistance response as their inhibition reversed the difenoconazole-resistant phenotype in vitro.ConclusionsOur data support the observation that global transcriptional changes modulate difenoconazole resistance in Penicillium spp. While the dogma of CYP51 overexpression is supported in the resistant isolate, our studies shed light on additional new mechanisms of difenoconazole resistance on a global scale in Penicillium spp. These new findings broaden our fundamental understanding of azole fungicide resistance in fungi, which has identified multiple genetic targets, that can be used for the detection, management, and abatement of difenoconazole-resistant blue mold isolates during long-term storage of apples.

Algorithm to assess the presence of Aspergillus fumigatus resistant strains: The case of Norwegian sawmills

ABSTRACT Association between selection pressure caused by the use of azole fungicides in sawmills and the development of fungal resistance has been described. The aim of this study was to implement an algorithm to assess the presence of Aspergillus section Fumigati resistant strains in sawmills. Eighty-six full-shift inhalable dust samples were collected from eleven industrial sawmills in Norway. Different culture media were used and molecular identification to species level in Aspergillus section Fumigati was done by calmodulin sequencing and TR34/L98H and TR46/Y121F/T289A mutations were screened by real-time PCR assay and confirmed by cyp51A sequencing. Six Fumigati isolates were identified as A. fumigatus sensu stricto and two of these grew on azole-supplemented media and were further analyzed by real-time PCR. One was confirmed to be a TR34/L98H mutant. The obtained results reinforce the need to assess the presence of A. fumigatus sensu stricto resistant isolates at other workplaces with fungicide pressure.

Toxic mechanism of three azole fungicides and their mixture to green alga Chlorella pyrenoidosa

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.

Can parasites adapt to pollutants? A multigenerational experiment with a Daphnia × Metschnikowia model system exposed to the fungicide tebuconazole

There is increasing evidence about negative effects of fungicides on non-target organisms, including parasitic species, which are key elements in food webs. Previous experiments showed that environmentally relevant concentrations of fungicide tebuconazole are toxic to the microparasite Metschnikowia bicuspidata, a yeast species that infects the planktonic crustacean Daphnia spp. However, due to their short-term nature, this and other experimental studies were not able to test if parasites could potentially adapt to these contaminants. Here, we tested if M. bicuspidata parasite can adapt to tebuconazole selective pressure. Infected D. magna lineages were reared under control conditions (no tebuconazole) and environmentally realistic tebuconazole concentrations, for four generations, and their performance was compared in a follow-up reciprocal assay. Additionally, we assessed whether the observed effects were transient (phenotypic) or permanent (genetic), by reassessing parasite fitness after the removal of selective pressure. Parasite fitness was negatively affected throughout the multigenerational exposure to the fungicide: prevalence of infection and spore load decreased, whereas host longevity increased, in comparison to control (naive) parasite lineages. In a follow-up reciprocal assay, tebuconazole-conditioned (TEB) lineages performed worse than naive parasite lineages, both in treatments without and with tebuconazole, confirming the cumulative negative effect of tebuconazole. The underperformance of TEB lineages was rapidly reversed after removing the influence of the selective pressure (tebuconazole), demonstrating that the costs of prolonged exposure to tebuconazole were phenotypic and transient. The microparasitic yeast M. bicuspidata did not reveal potential for rapid evolution to an anthropogenic selective pressure; instead, the long-term exposure to tebuconazole was hazardous to this non-target species. These findings highlight the potential environmental risks of azole fungicides on non-target parasitic fungi. The underperformance of these microbes and their inability to adapt to such stressors can interfere with the key processes where they intervene. Further research is needed to rank fungicides based on the hazard to non-target fungi (parasites, but also symbionts and decomposers), towards more effective management and protective legislation.

Medical Tablets Paracetamol And Mertronidazole Affects Wheat Seed Germination And Seedlings Growth As Plant Growth Regulators

Triazole derivatives are regularly used as systemic fungicides for seed dressing or as plant growth regulator, but it can cause numerous changes in plant morphological and physiological characteristics. An experiment included the local Iraqi bread wheat variety Tammuz-2 and the introduced Turkish ones Adana-99 were exposed to two concentrations (500 and 1000ppm) of the antibiotic medicinal tablets paracetamol and metronidazole, to illustrate the biological activity in terms of seeds germination and early seedlings growth traits; as they are structurally resemble the well-known azole fungicides and growth retardant derivatives. The results obviously revealed that these drugs exhibited growth regulatory activity in terms of their toxic effects in reducing of germination % from 84.5% to 69.75% for Tammuz-2 with paracetamol at 1000ppm, and from 84.50% to 76.00% with metronidazole at the same concentration. Similarly the germination% of Adana -99 was also dropped from 91.5 % to 77% and 82.5% for paracetamol and metronidazole at 1000ppm, respectively, this reduction in germination % was accompanied with increment in abnormal seedlings%. The deleterious impact on Adana-99 shoot length was exaggerated with metronidazole at 1000ppm which recorded (5.75 cm) in comparison to control (10.11cm). Concerning with the length of the longest rootlet, drastic reduction was noticed for both varieties with metronidazole at both concentrations 500 and 1000ppm, but the lowest was 1.47 cm at 1000ppm for metronidazole in comparison to the control 11.21cm for Tammuz-2 variety. The effect of drugs on number of rootlets resembled rootlets length as it was reduced by metronidazole, whereas the highest concentration 1000ppm only significantly reduced seedlings fresh weight. Although there was discrepancy in seedlings dry weight as metronidazole particularly with high concentration caused drastic suppression in rootlets growth, but it resulted in higher dry weight, the interpretation for such ambiguity was that the seeds were not removed from the seedlings and it was contributed in whole seedlings weight and could be also due to the better food reserved in the germinated seeds. It was worth to declare that paracetamol treated seeds colored dark black, which was attributed to the tyrosinase enzyme activity present in seeds pericarp which oxidize phenol into quinines and subsequently converted to dark colored melanin by polymerization and interaction with protein. The results ascertained the side effect of these drugs as they possess regulatory behavior; therefore awareness required utilizing such pharmaceutical compounds to avoid unanticipated risk of soil contamination and agricultural ecosystems pollution

Reaction of Wheat Cultivars to Fusarium Crown And Root Rot under Field Condition at Duhok-kurdistan Region, Iraq

Triazole derivatives are regularly used as systemic fungicides for seed dressing or as plant growth regulator, but it can cause numerous changes in plant morphological and physiological characteristics. An experiment included the local Iraqi bread wheat variety Tammuz-2 and the introduced Turkish ones Adana-99 were exposed to two concentrations (500 and 1000ppm) of the antibiotic medicinal tablets paracetamol and metronidazole, to illustrate the biological activity in terms of seeds germination and early seedlings growth traits; as they are structurally resemble the well-known azole fungicides and growth retardant derivatives. The results obviously revealed that these drugs exhibited growth regulatory activity in terms of their toxic effects in reducing of germination % from 84.5% to 69.75% for Tammuz-2 with paracetamol at 1000ppm, and from 84.50% to 76.00% with metronidazole at the same concentration. Similarly the germination% of Adana -99 was also dropped from 91.5 % to 77% and 82.5% for paracetamol and metronidazole at 1000ppm, respectively, this reduction in germination % was accompanied with increment in abnormal seedlings%. The deleterious impact on Adana-99 shoot length was exaggerated with metronidazole at 1000ppm which recorded (5.75 cm) in comparison to control (10.11cm). Concerning with the length of the longest rootlet, drastic reduction was noticed for both varieties with metronidazole at both concentrations 500 and 1000ppm, but the lowest was 1.47 cm at 1000ppm for metronidazole in comparison to the control 11.21cm for Tammuz-2 variety. The effect of drugs on number of rootlets resembled rootlets length as it was reduced by metronidazole, whereas the highest concentration 1000ppm only significantly reduced seedlings fresh weight. Although there was discrepancy in seedlings dry weight as metronidazole particularly with high concentration caused drastic suppression in rootlets growth, but it resulted in higher dry weight, the interpretation for such ambiguity was that the seeds were not removed from the seedlings and it was contributed in whole seedlings weight and could be also due to the better food reserved in the germinated seeds. It was worth to declare that paracetamol treated seeds colored dark black, which was attributed to the tyrosinase enzyme activity present in seeds pericarp which oxidize phenol into quinines and subsequently converted to dark colored melanin by polymerization and interaction with protein. The results ascertained the side effect of these drugs as they possess regulatory behavior; therefore awareness required utilizing such pharmaceutical compounds to avoid unanticipated risk of soil contamination and agricultural ecosystems pollution

The Connection of Azole Fungicides with Xeno-Sensing Nuclear Receptors, Drug Metabolism and Hepatotoxicity.

Azole fungicides, especially triazole compounds, are widely used in agriculture and as pharmaceuticals. For a considerable number of agricultural azole fungicides, the liver has been identified as the main target organ of toxicity. A number of previous studies points towards an important role of nuclear receptors such as the constitutive androstane receptor (CAR), the pregnane-X-receptor (PXR), or the aryl hydrocarbon receptor (AHR), within the molecular pathways leading to hepatotoxicity of these compounds. Nuclear receptor-mediated hepatic effects may comprise rather adaptive changes such as the induction of drug-metabolizing enzymes, to hepatocellular hypertrophy, histopathologically detectable fatty acid changes, proliferation of hepatocytes, and the promotion of liver tumors. Here, we present a comprehensive review of the current knowledge of the interaction of major agricultural azole-class fungicides with the three nuclear receptors CAR, PXR, and AHR in vivo and in vitro. Nuclear receptor activation profiles of the azoles are presented and related to histopathological findings from classic toxicity studies. Important issues such as species differences and multi-receptor agonism and the consequences for data interpretation and risk assessment are discussed.

Effects of Azole Fungicides on Secreted Metabolomes of Botrytis cinerea.

Botrytis cinerea, gray mold, is one of the most notorious phytopathgens, causing serious economic loss in the agricultural industry. The phytotoxic effects are mainly derived from secreted virulence proteins and terpenoid-type secondary metabolites. Azole fungicides are commonly used to manage the disease. However, their biochemical effects other than sterol biosynthesis were not documented, especially toxic secreted metabolites. In this study, six azole fungicides were treated with in vitro and in vivo conditions. Comprehensive profiles of primary and secondary metabolites in culture media were evaluated to assess the fungal metabolomes under pesticide-stressed conditions. The results indicated that extensive metabolic differentiation was induced by azole fungicides. Epoxiconazole clearly reduced the extracellular phytotoxin concentrations, while the level of indole-3-acetic acid was increased. In addition, significant differentiation of primary metabolism could be deduced from secreted metabolite profiles, including the tricarboxylic acid cycle and aromatic amino acid catabolism. Cellular lipid profiles, including fatty acids and sterol, have been altered drastically by azoles, which indicate extensive changes of cellular lipid metabolism. These system-wide metabolic alterations resulted in reduced plant damages, proven by the in vivo assay with tomato. Overall, azole fungicides induced significant changes of endo- and exometabolomes and could reduce the fungal infection. The experimental results will provide a more detailed understanding of physiological changes of phytopathogens under pesticide treatment and information for new pesticide development.

Development of an adverse outcome pathway for cranio-facial malformations: A contribution from in silico simulations and in vitro data

Mixtures of substances sharing the same molecular initiating event (MIE) are supposed to induce additive effects. The proposed MIE for azole fungicides is CYP26 inhibition with retinoic acid (RA) local increase, triggering key events leading to craniofacial defects. Valproic acid (VPA) is supposed to imbalance RA-regulated gene expression trough histone deacetylases (HDACs) inhibition. The aim was to evaluate effects of molecules sharing the same MIE (azoles) and of such having (hypothetically) different MIEs but which are eventually involved in the same adverse outcome pathway (AOP). An in silico approach (molecular docking) investigated the suggested MIEs. Teratogenicity was evaluated in vitro (WEC). Abnormalities were modelled by PROAST software. The common target was the branchial apparatus. In silico results confirmed azole-related CYP26 inhibition and a weak general VPA inhibition on the tested HDACs. Unexpectedly, VPA showed also a weak, but not marginal, capability to enter the CYP 26A1 and CYP 26C1 catalytic sites, suggesting a possible role of VPA in decreasing RA catabolism, acting as an additional MIE. Our findings suggest a new more complex picture. Consequently two different AOPs, leading to the same AO, can be described. VPA MIEs (HDAC and CYP26 inhibition) impinge on the two converging AOPs.

Dummy molecularly imprinted microspheres prepared by Pickering emulsion polymerization for matrix solid-phase dispersion extraction of three azole fungicides from fish samples

Dummy molecularly imprinted microspheres (DMIMs) were synthesized by Pickering emulsion polymerization and used as the matrix solid-phase dispersion extraction (MSPD) sorbent for sample pre-treatment of azole fungicides in fish samples. Alpha-(2,4-Dichlorophenyl)-1H-imidazole-1-ethanol (DCE) was used as the fragment dummy template for the imprinting of climbazole (CBZ), clotrimazole (CMZ) and miconazole (MNZ). The morphology of the microspheres were characterized by scanning electron microscopy (SEM) and nitrogen adsorption measurements, narrow diameter distribution (20–50 μm) with regular spherical shape and high surface area (SBET = 408.91 ± 6.72 m2 g–1) were achieved. Good class-selectivity of the DMIMs was found for CBZ, CMZ and MNZ by static adsorption experiments. The imprinted microspheres as MSPD sorbent was then evaluated for the extraction and purification of CBZ, CMZ and MNZ in fish samples. The extracted azole fungicides were detected by HPLC-DAD analysis at 225 nm. MSPD conditions including elution, mass ratio of sample/sorbent and washing were carefully evaluated. The optimized MSPD method have good recoveries (89.2–101.5%) and reproducibility (RSDs 1.6–4.8%, n = 5) for fish samples spiked at 0.5, 2.5 and 12.5 μg g–1. The limits of detection (LODs) were 0.045, 0.036 and 0.033 μg g–1 for CBZ, CMZ and MNZ, respectively. The results show that this method has a good application prospect for the pretreatment of azole fungicides in fish samples.