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Azoles additively inhibit cytochrome P450 1 (EROD) and 19 (aromatase) in rainbow trout (Oncorhynchus mykiss)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Environmental toxicology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Environmental toxicology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Environmental toxicology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Environmental toxicology.
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2018 (English)In: Aquatic Toxicology, ISSN 0166-445X, E-ISSN 1879-1514, Vol. 198, p. 73-81Article in journal (Refereed) Published
Abstract [en]

Antifungal azoles are widely used in medicine, agriculture, and material protection and several antifungal azoles have been found in environmental samples. Although these compounds were designed to inhibit fungal enzymes such as lanosterol-14-demethylase (cytochrome P450 (CYP) 51), it is well established that the inhibitory actions of azoles are not specific for fungal CYP isozymes.

We refined a gill filament assay to determine the inhibition of CYP1, measured as reduced 7-ethoxyresorufin-O-deethylase (EROD) activity, in rainbow trout (Oncorhynchus mykiss) gill tissue ex vivo. The advantage of this method is that both induction and inhibition of EROD are performed ex vivo. Among thirteen azoles studied, the five that caused the strongest inhibition of gill EROD activity at a concentration of 5 μM were selected for concentration–response assessment. These compounds (bifonazole, clotrimazole, imazalil, miconazole, and prochloraz) showed IC50 values ranging from 0.1 to 1.5 μM. CYP19 (aromatase) inhibition was measured using microsomes from rainbow trout brains. Concentration-response curves for CYP19 inhibition were determined for letrozole, bifonazole, clotrimazole, imazalil, miconazole and prochloraz, which gave IC50 values ranging from 0.02 to 3.3 μM. It was further found that mixtures of the five most potent azoles reduced both CYP1 and 19 catalytic activity in an additive fashion (IC50 = 0.7 μM and 0.6 μM, in the respective assay). Bifonazole (IC50 = 0.1 μM) is not previously known to inhibit CYP1 activity.

The additive inhibition of CYP1 and CYP19 catalytic activity is an important finding of the present study. We conclude that this additive action of azoles could mediate adverse impacts on CYP regulated physiological functions in environmentally exposed fish.

Place, publisher, year, edition, pages
2018. Vol. 198, p. 73-81
Keywords [en]
Azole fungicide, EROD activity, cytochrome P450 (CYP), CYP1A, CYP19, aromatase, pharmaceutical, contaminant, chemical, fish, rainbow trout, gill
Keywords [sv]
EROD aktivitet, cytokrom P450 (CYP), CYP1A, CYP19, aromatase, läkemedel, azol, fungicid, kemikalier, förorening, fisk, regnbågslax, gäle
National Category
Other Biological Topics
Research subject
Biology with specialization in Environmental Toxicology
Identifiers
URN: urn:nbn:se:uu:diva-249010DOI: 10.1016/j.aquatox.2018.02.016ISI: 000430630100008OAI: oai:DiVA.org:uu-249010DiVA, id: diva2:805578
Funder
Mistra - The Swedish Foundation for Strategic Environmental ResearchAvailable from: 2015-04-15 Created: 2015-04-10 Last updated: 2018-08-07Bibliographically approved
In thesis
1. Azoles and Contaminants in Treated Effluents Interact with CYP1 and CYP19 in Fish:
Open this publication in new window or tab >>Azoles and Contaminants in Treated Effluents Interact with CYP1 and CYP19 in Fish:
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Numerous contaminants are present in mixtures in the aquatic environment. Among these are the azoles, a group of chemicals that includes both pharmaceuticals and pesticides. Azole fungicides are designed to inhibit lanosterol 14-demethylase (cytochrome P450 (CYP) 51), while other azoles are intended to inhibit aromatase (CYP19), i.e. the enzyme catalyzing biosynthesis of estrogens. In fish, a variety of CYP enzymes are involved in biotransformation of waterborne contaminants, and in metabolism of endogenous compounds including steroidal hormones. The induction of CYP1A protein and 7-ethoxyresorufin O-deethylase (EROD) activity are common biomarkers for exposure to aryl hydrocarbon receptor (AhR) agonists in fish. We developed an assay to measure inhibition of CYP1A activity (EROD) in three-spined stickleback and rainbow trout gill tissue ex vivo. Several azole fungicides were found to be potent inhibitors of CYP1A activity. A wastewater effluent containing high concentrations of pharmaceuticals was also shown to inhibit CYP1A activity. Further, several azoles inhibited CYP19 activity in rainbow trout brain microsomes in vitro. Azole mixtures reduced both CYP1A and CYP19 activity monotonically and in an additive way. Given the additive action of the azoles, studies to determine adverse effects of azole mixtures on CYP-regulated physiological functions in fish are needed. Induction of EROD and of gene expression of CYP1 in several organs was observed in an in vivo exposure with the same effluent shown to inhibit EROD. This finding could imply that there was a mixture of AhR agonists and CYP1A inhibitors in the effluent. Finally, wastewater treatment technologies were evaluated using biomarker responses in rainbow trout exposed to effluents of different treatments. The results from chemical analysis together with the biomarker results show that ozone and granulated active carbon treatment removed most pharmaceuticals, as well as AhR agonists and other chemicals present in the regular effluent. This part of the thesis demonstrates that biomarkers in fish such as induction of CYP1 gene expression are applicable to evaluate the efficiency of different treatment technologies for wastewater.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. p. 42
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1255
Keywords
Azole, fungicide, chemical, CYP1A, CYP19, EROD, aromatase, effluent, STP, wastewater, fish, stickleback, rainbow trout
National Category
Natural Sciences Biological Sciences
Research subject
Biology with specialization in Environmental Toxicology
Identifiers
urn:nbn:se:uu:diva-251295 (URN)978-91-554-9248-9 (ISBN)
Public defence
2015-06-04, Zootissalen, EBC, Villavägen 9, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2015-05-13 Created: 2015-04-15 Last updated: 2015-07-07

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Beijer, KristinaJönsson, MariaBrunström, BjörnBrandt, Ingvar

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