Triazoles and aromatase: The impact of copper cocktails☆
Graphical abstract
Introduction
Triazoles (belonging to the group of azoles) are systemic fungicides frequently used to crop protection against yeast and mildew. In these target species, azoles inhibit the biosynthesis of steroids (Egbuta et al., 2014; Zarn et al., 2003) mainly by blocking fungal cytochrome P450 monooxygenase sterol 14α-demethylase (CYP51A1, EC 1.14.14.154). The inhibition of fungal CYP51 leads to the depletion of ergosterol, essential in fungal plasma membranes and thus the destabilization of the membrane (Alcazar-Fuoli et al., 2008). For humans, inhibition of CYP51 is not crucial because diet supplies cholesterol (an intermediate).
Many azoles also inhibit aromatase (CYP19A1, EC 1.14.14.14) (Conner et al., 2012). Cyp19 catalyses the conversion of androstenedione, testosterone or 16α-hydroxytestosterone to estrone, 17β-estradiol or 17β,16α-estriol, respectively (Conley et al., 2001; Trösken et al., 2004). The depletion of estrogens is the principle of the use of azoles in the antiestrogen therapy against estrogen-dependent adenocarcinoma and breast cancer cells (Egbuta et al., 2014; Lang et al., 1993).
Since most azoles are not completely specific, they are able to inhibit additional key cytochrome P450 enzymes, e.g. lanosterol-14-demethylase (CYP1) (Beijer et al., 2018), cholesterol side-chain cleavage (CYP11A) steroid 11β-hydroxylase (CYP11B and CYP11C1) and steroid 17α-hydroxylase/17,20 lyase (CYP17) responsible for transformation of steroid hormone (Chen and Ying, 2015).
These effects on highly conserved cytochrome P450 monooxygenases such as CYP51, are similar to those observed in mammals including human (Chen and Ying, 2015; Lepesheva et al., 2003). In healthy non-target organisms, azoles are consequently considered to be potential endocrine disruptors (Taxvig et al., 2008; Trösken et al., 2004; Zarn et al., 2003).
The individual influence of various azoles on the target enzymes has been well described. Because of the treatment strategies, the issue of the possible effect of (tri)azoles in a mixture (cocktail effect) on given enzymes is of great interest (Kjaerstad et al., 2010; Rieke et al., 2014; Seeger et al., 2019; Schmidt et al., 2016).
Triazoles as fungicides are widely used in agriculture, in the field from which they easily spread through the soil; and contaminate groundwater and even reach urban centres. Moreover, these compounds are also used in therapeutic and personal care products (shampoos, dermal creams, and soaps etc.) (Assress et al., 2020). Their removal rate in wastewater treatment plant is rather low. Azole fungicides are generally detected in surface water and sediment of the aquatic environment (Chen and Ying, 2015). In our previous studies it was found that the behaviour of the triazoles and their degradation pathways are also strongly influenced by their reactions with the ions of various elements present (Jaklová Dytrtová et al., 2014; Kovač et al., 2020).
Besides triazoles, especially copper compounds are also widely applied as fungicides for crops (Komárek et al., 2010; Vazquez-Blanco et al., 2020). There has been evidence that triazole fungicides can create complexes with copper ions and change their behaviour in the environment, i.e., their accumulation in soils, degradation, mobility, and stability (Arias et al., 2006; Jakl et al., 2017; Jakl and Jaklová Dytrtová, 2018; Jaklová Dytrtová et al., 2011). Copper as an ion coordinated in many enzymes plays an essential role in the biochemistry of all living organisms. On the other hand, its free ions in cells may be toxic via the catalysis of Fenton (Haber-Weiss) reactions forming reactive oxygen species and hydroxyl radicals. In addition to the oxidative damages, the presence of free Cu ions may lead to the direct impairment of redox-sensitive signalling and to functional changes of activator protein 1 (AP-1), a transcription factor regulating gene expression (Letelier et al., 2009). In the presence of copper ions, the activity of cytochrome P450 monooxygenases could be passivated by their oxidation, by copper ions binding to their amino acids and by other structural changes (Letelier et al., 2009).
This research has focused on three triazolic compounds: tebuconazole, penconazole and cyproconazole, which are often used in crop protection. Tebuconazole is even the world’s most widely used triazolic fungicide (as a seed dressing and foliar spray against rusts, smuts, bunt, powdery mildew, leaf spots, and blights) with the potentially most harming impact on the environment (Li et al., 2019; Sharma et al., 2019). Only a few studies have been focused on penconazole, however its toxic effects were reported on various organisms, including mice and fish (Aksakal and Ciltas, 2018; Meng et al., 2020). Cyproconazole has been widely used in restraining and curing plant diseases and deeply studied since it was shown to have stereoselective toxicity against Chlorella pyrenoidosa and Pelophylax nigromaculatus (black-spotted frog) (He et al., 2019).
All of these compounds have a relatively similar structure (see the Graphical Abstract), which concerns not only the nitrogen heterocycle but also the chlorinated benzene ring on their aliphatic chains. On the contrary, they do not contain other halogens or epoxy chains, which could fundamentally change their reactivity. The main aim here is to bring the information on how the cocktails of these similar triazoles in their different (typically racemic) mixtures and with copper ions influence the activity of aromatase and thus to predict their environmental behaviour and impact.
Section snippets
Chemicals
Tebuconazole (Teb; 1-(4-chlorophenyl)-4,4-dimethyl-3-(1H-1,2,4-triazol-1-ylmethyl)pentan-3-ol), penconazole (Pen; 1-[2-(2,4-dichlorophenyl)pentyl]-1,2,4-triazole) and cyproconazole (Cyp; 2-(4-chlorophenyl)-3-cyclopropyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol) were of analytical grade purity (Pestanal®), and CuCl2 of 99.999% (trace metal basis) purity. All of these chemicals were purchased from Sigma-Aldrich (Czech Republic). The solid standards were used for the preparation of stock solutions. The
Results and discussion
Each individually assessed triazole significantly reduced the aromatase activity (Table 1), in the case of penconazole even to 41%. Their combinations had a similar effect as was expected from hypothetical calculations. On the other hand, the addition of Cu2+ to the triazole mixtures decreased the aromatase activity in contrast to the hypothetical expectations. It is generally assumed that the effect of all triazoles is more or less similar. However, it is visible from our results (Table 1)
Conclusion
The group effects did not differ significantly from the calculated average of the effects of every particular triazoles in a mixture. This means that the reactivity of triazoles in groups is not significantly affected by interactions among them. On the other hand, the presence of copper together with triazole(s) (cocktail effect) considerably increases the inhibitory effect to aromatase activity. The highest inhibitory effect (greater than 60%) has been observed in the mixtures containing
Funding
J.J.D. thanks to the Grantová agentura České republiky (project no. 18-01710S) for the financial support.
CRediT authorship contribution statement
Jana Jaklová Dytrtová: Conceptualization, Methodology, Investigation, Formal analysis, Writing - original draft, Funding acquisition. Kateřina Bělonožníková: Investigation, Formal analysis, Writing - original draft. Michal Jakl: Investigation, Formal analysis, Writing - original draft. Helena Ryšlavá: Conceptualization, Methodology, Writing - original draft.
Declaration of competing interest
The authors declare that there are no competing interests associated with the manuscript.
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This paper has been recommended for acceptance by Klaus Kümmerer.