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Azoles and Contaminants in Treated Effluents Interact with CYP1 and CYP19 in Fish:
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Environmental toxicology.
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 [en]
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: urn:nbn:se:uu:diva-251295ISBN: 978-91-554-9248-9 (print)OAI: oai:DiVA.org:uu-251295DiVA: diva2:805580
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
List of papers
1. CYP1A inhibition in fish gill filaments: a novel assay applied on pharmaceuticals and other chemicals
Open this publication in new window or tab >>CYP1A inhibition in fish gill filaments: a novel assay applied on pharmaceuticals and other chemicals
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.

Keyword
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:nbn:se:uu:diva-120904 (URN)10.1016/j.aquatox.2009.10.018 (DOI)000274978500009 ()19913926 (PubMedID)
Available from: 2010-03-17 Created: 2010-03-17 Last updated: 2015-07-07Bibliographically approved
2. Azoles inhibit cytochrome P450 enzymes in rainbow trout involved in biotransformation and steroid hormone synthesis additively
Open this publication in new window or tab >>Azoles inhibit cytochrome P450 enzymes in rainbow trout involved in biotransformation and steroid hormone synthesis additively
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2015 (English)Manuscript (preprint) (Other academic)
Keyword
Azole fungicide, EROD activity, cytochrome P450 (CYP), CYP1A, CYP19, aromatase, pharmaceutical, contaminant, chemical, fish, rainbow trout, gill, 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:nbn:se:uu:diva-249010 (URN)
Available from: 2015-04-15 Created: 2015-04-10 Last updated: 2017-06-30Bibliographically approved
3. Effluent from drug manufacturing affects cytochrome P450 1 regulation and function in fish
Open this publication in new window or tab >>Effluent from drug manufacturing affects cytochrome P450 1 regulation and function in fish
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2013 (English)In: Chemosphere, ISSN 0045-6535, E-ISSN 1879-1298, Vol. 90, no 3, 1149-1157 p.Article in journal (Refereed) Published
Abstract [en]

We have previously reported very high concentrations of pharmaceuticals in the effluent from a treatment plant receiving wastewater from about 90 bulk drug manufacturers near Hyderabad, India. The main objective of the present study was to examine how high dilutions of this effluent affect mRNA expression of cytochrome P450 (CYP) 1 family genes and ethoxyresorufin O-deethylase (EROD) activity in exposed wildlife, using the three-spined stickleback (Gasterosteus aculeatus) as a model. In gill filaments exposed to diluted effluent ex vivo, EROD activity was strongly inhibited in a concentration-dependent manner. In a subsequent in vivo study, groups of fish were exposed (24. h) to three concentrations of effluent, 0.8%, 1.6% or 3.2%. In this experiment, EROD in gills was induced 27-, 52- or 60-fold, respectively. Accordingly, CYP1A mRNA was markedly up-regulated in gill, liver and brain of fish exposed to all three effluent concentrations. Expression of mRNA for CYP1B1 and CYP1C1 was induced in gills at all concentrations while effects on these genes in liver and brain were weak or absent. The results of a time course study suggested that most CYP1-inducing substances in the effluent were readily metabolised or excreted, because the induced EROD activity and mRNA expression decreased when the fish were transferred to clean water. Considering that CYP1 enzymes play important roles in biotransformation of endogenous and foreign compounds, the observed dual effect of the effluent on CYP1 catalytic activity and mRNA expression suggests that multiple physiological functions could be affected in exposed wildlife.

Keyword
CYP1, EROD, Gills, Pharmaceuticals, Three-spined stickleback, Treated wastewater, Drug products, Effluent treatment, Fish, Gene expression, Wastewater treatment, Effluents, cytochrome P450, cytochrome P450 1, cytochrome P450 1A, cytochrome P450 1B1, cytochrome P450 1C1, cytochrome P450 1C2, ethoxyresorufin deethylase, industrial effluent, messenger RNA, tap water, unclassified drug, biotransformation, concentration (composition), drug, ecological modeling, effluent, enzyme activity, manufacturing, metabolism, pollution exposure, teleost, wastewater, water treatment, animal experiment, animal tissue, article, brain, controlled study, enzyme induction, enzyme inhibition, female, Gasterosteus aculeatus, gene, genetic transcription, gill, liver, mortality, nonhuman, spiggin gene, upregulation, vitellogenin gene, waste water treatment plant, Andhra Pradesh, Hyderabad [Andhra Pradesh], India
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-192012 (URN)10.1016/j.chemosphere.2012.09.023 (DOI)000312978700035 ()
Note

De två första författarna delar förstaförfattarskapet.

Available from: 2013-01-24 Created: 2013-01-15 Last updated: 2017-12-06Bibliographically approved
4. Reduction of pharmaceuticals and other contaminants in sewage treatment effluents by active carbon filtration and ozonation: Evaluation using biomarker responses in fish and chemical analysis
Open this publication in new window or tab >>Reduction of pharmaceuticals and other contaminants in sewage treatment effluents by active carbon filtration and ozonation: Evaluation using biomarker responses in fish and chemical analysis
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(English)Manuscript (preprint) (Other academic)
Keyword
effluent, STP, wastewater, active carbon, ozonation, rainbow trout
National Category
Other Biological Topics
Research subject
Biology with specialization in Environmental Toxicology
Identifiers
urn:nbn:se:uu:diva-251294 (URN)
Available from: 2015-04-15 Created: 2015-04-15 Last updated: 2015-07-07

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