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CYP1A inhibition in fish gill filaments: a novel assay applied on pharmaceuticals and other chemicals
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Environmental Toxicology. (Ingvar Brandt)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Environmental Toxicology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Environmental Toxicology. (Björn Brunström)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Environmental Toxicology. (Ingvar Brandt)
2010 (English)In: Aquatic Toxicology, ISSN 0166-445X, E-ISSN 1879-1514, Vol. 96, no 2, 145-150 p.Article in journal (Refereed) Published
Abstract [en]

The gill filament 7-ethoxyresorufin O-deethylase (EROD) assay was originally developed as a biomarker for cytochrome P4501A (CYP1A) induction by Ah-receptor agonists in water. In this study, the assay was adapted to measure inhibition of CYP1A activity in fish gill filaments ex vivo. The experiments were carried out using gill arch filaments from beta-naphthoflavone (betaNF)-exposed three-spined stickleback (Gasterosteus aculeatus). Candidate CYP1A inhibitors were added to the assay buffer. Nine selected pharmaceuticals and five known or suspected CYP1A-modulating chemicals were examined with regard to their ability to reduce EROD activity in gill filaments. Ellipticine, a well characterized CYP1A inhibitor, was the most effective inhibitor of the compounds tested. At a concentration in the assay buffer of 1 microM the antifungal azoles ketoconazole, miconazole and bitertanol, and the plant flavonoid acacetin reduced gill EROD activity by more than 50%, implying IC50 values below 1 microM. These compounds have previously been shown to inhibit EROD activity in liver microsomes from fish and mammals at similar concentrations. The proton pump inhibitor omeprazole reduced the gill EROD activity by 39% at 10 microM. It is concluded that the modified gill filament EROD assay is useful to screen for waterborne pollutants that inhibit catalytic CYP1A activity in fish gills.

Place, publisher, year, edition, pages
2010. Vol. 96, no 2, 145-150 p.
Keyword [en]
Gill filament assay, CYP inhibition, Pharmaceuticals, Antifungal azoles, Three-spined stickleback, EROD activity
National Category
Pharmacology and Toxicology Biological Sciences
Research subject
Ecotoxicology
Identifiers
URN: urn:nbn:se:uu:diva-120904DOI: 10.1016/j.aquatox.2009.10.018ISI: 000274978500009PubMedID: 19913926OAI: oai:DiVA.org:uu-120904DiVA: diva2:304128
Available from: 2010-03-17 Created: 2010-03-17 Last updated: 2015-07-07Bibliographically 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. 42 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1255
Keyword
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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