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Basal and Pollutant-induced Expression of CYP1A, 1B and 1C isoforms in Fish: Implications for Biomonitoring
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Environmental toxicology. (Ingvar Brandt)
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Aquatic wildlife are exposed to contaminants in their natural habitats, and toxic pollutants may induce toxicity in sensitive target tissues and cells. There is therefore a need to establish biomarkers to determine exposure to certain classes of contaminants and the subsequent biological responses. In the present work the whole suite of cytochrome P450 1 (CYP1) genes expressed in fish were examined with regard to their inducibility and potential use as biomarkers. Complementary DNA of the CYP1A, 1B and 1C transcripts in rainbow trout and three-spined stickleback were cloned and characterized by quantitative mRNA expression analysis. All CYP1 transcripts could be induced by two selected aryl hydrocarbon receptor (AhR) agoinsts (indigo and PCB 126) in both species, suggesting that all genes were regulated by the AhR. CYP1 mRNA expression profiles induced by PCB 126 and indigo varied over time showing that PCB 126 gave rise to a high and persistent induction in gills and liver while induction by indigo was transient in both organs. The uptake and kinetics of 14C-indigo was studied by autoradiography in rainbow trout. A rapid uptake of 14C-indigo from the water and a subsequent elimination in bile and intestinal contents was observed, explaining the transient CYP1 induction. A high accumulation of 14C-indigo in the gills was completely blocked the CYP1 inhibitor ellipticine, suggesting a CYP1-dependent uptake. High dilutions of a sewage treatment plant effluent containing a complex mixture of pharmaceuticals were investigated. The same water sample both induced CYP1A mRNA expression and inhibited catalytic activity of CYP1A. A field study revealed different induction signatures of the CYP1 genes examined at various locations in Uppsala water environments. As concluded by the temporal and spatial responses obtained, the results of this work suggest that the CYP1 gene panel could be used for biomonitoring of environmental contaminants acting on the CYP system. Further field studies will be required to evaluate this possibility.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. , 52 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1044
National Category
Other Biological Topics
Identifiers
URN: urn:nbn:se:uu:diva-198658ISBN: 978-91-554-8670-9 (print)OAI: oai:DiVA.org:uu-198658DiVA: diva2:617132
Public defence
2013-05-28, Lindahlsalen, Norbyvägen 18A, Uppsala, 09:15 (English)
Supervisors
Available from: 2013-05-06 Created: 2013-04-22 Last updated: 2013-08-30Bibliographically approved
List of papers
1. Induction patterns of new CYP1 genes in environmentally exposed rainbow trout
Open this publication in new window or tab >>Induction patterns of new CYP1 genes in environmentally exposed rainbow trout
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2010 (English)In: Aquatic Toxicology, ISSN 0166-445X, E-ISSN 1879-1514, Vol. 98, no 4, 311-321 p.Article in journal (Refereed) Published
Abstract [en]

The cytochrome P4501 (CYP1) gene family comprises four subfamilies in fish: CYP1A, CYP1B, CYP1C, and CYP1D. Only two CYP1 genes, CYP1A1 and CYP1A3, are so far known in rainbow trout (Oncorhynchus mykiss). The present study aimed to identify other CYP1 subfamily genes in rainbow trout, to establish methods for quantitative mRNA expression analysis of these genes, and to determine their basal and induced mRNA expression in gills and liver. Another goal was to examine their mRNA expression in environmentally exposed fish. We cloned four new transcripts, denoted rbCYP1B1, rbCYP1C1, rbCYP1C2, and rbCYP1C3. Levels of these and the previously known rbCYP1A transcripts were determined by real-time PCR in unexposed fish, fish exposed to the potent aryl hydrocarbon receptor (AhR) agonist 3,3',4,4',5-pentachlorobiphenyl (PCB126), and fish caged in various waters in the Uppsala region (Sweden). The mRNA expression patterns observed in unexposed rainbow trout (basal levels) were markedly similar to those reported for orthologous genes in other species. All six transcripts were induced by PCB126 in gills and liver, suggesting all genes to be AhR regulated. The caged fish showed clear rbCYP1 induction in gills at all monitoring sites (up to 70-fold the basal level), whereas the liver responses were weak; induction (up to 5-fold) was recorded only at the Uppsala municipal sewage treatment plant outlet. Gill filament EROD activity was induced at all caging sites. Most interestingly, the rbCYP1 gene response patterns in gills differed among caging sites and among subfamilies. The EROD induction seemed to only reflect induction of rbCYP1A transcription. Response patterns of multiple CYP1 genes in gills and liver could provide an improved monitoring strategy. Such patterns could be used to characterize complex mixtures of AhR agonists and antagonists in aquatic environments.

National Category
Biological Sciences
Research subject
Ecotoxicology
Identifiers
urn:nbn:se:uu:diva-130469 (URN)10.1016/j.aquatox.2010.03.003 (DOI)000278982800001 ()20371123 (PubMedID)
Available from: 2010-09-08 Created: 2010-09-08 Last updated: 2017-12-12Bibliographically approved
2. Cytochrome P450 1A, 1B, and 1C mRNA induction patterns in three-spined stickleback exposed to a transient and a persistent inducer
Open this publication in new window or tab >>Cytochrome P450 1A, 1B, and 1C mRNA induction patterns in three-spined stickleback exposed to a transient and a persistent inducer
2011 (English)In: Comparative Biochemistry and Physiology - Part C: Toxicology & Pharmacology, ISSN 1532-0456, E-ISSN 1878-1659, Vol. 154, no 1, 42-55 p.Article in journal (Refereed) Published
Abstract [en]

Cytochrome P450 1 (CYP1) mRNA induction patterns in three-spined stickleback (Gasterosteus aculeatus) were explored for use in environmental monitoring of aryl hydrocarbon receptor (AHR) agonists. The cDNAs of stickleback CYP1A, CYP1B1, CYP1C1, and CYP1C2 were cloned and their basal and induced expression patterns were determined in the brain, gill, liver and kidney. Also, their induction time courses were compared after waterborne exposure to a transient (indigo) or a persistent (3,3',4,4',5-pentacholorbiphenyl PCB 126) AHR agonist. The cloned stickleback CYP1s exhibited a high amino acid sequence identity compared with their zebrafish orthologs and their constitutive tissue distribution patterns largely agreed with those reported in other species. PCB 126 (100 nM) induced different CYP1 expression patterns in the four tissues, suggesting tissue-specific regulation. Both indigo (1 nM) and PCB 126 (10 nM) induced a strong CYP1 expression in gills. However, while PCB 126 gave rise to a high and persistent induction in gills and liver, induction by indigo was transient in both organs. The number of putative dioxin response elements found in each CYP1 gene promoter roughly reflected the induction levels of the genes. The high responsiveness of CYP1A,CYP1B1, and CYP1C1 observed in several organs suggests that three-spined stickleback is suitable for monitoring of pollution with AHR agonists.

Keyword
Three-spined stickleback, Cytochrome P450 1A, Cytochrome P450 1B, Cytochrome P450 1C, CYP1A gene, CYP1B gene, CYP1C gene, Gill EROD activity, Indigo, PCB 126, Biomarkers
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-155388 (URN)10.1016/j.cbpc.2011.02.005 (DOI)000291178700007 ()
Available from: 2011-06-22 Created: 2011-06-22 Last updated: 2017-12-11Bibliographically 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. Disposition of indigo in rainbow trout:  Relationships between tissue binding kinetics and CYP gene induction
Open this publication in new window or tab >>Disposition of indigo in rainbow trout:  Relationships between tissue binding kinetics and CYP gene induction
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-198644 (URN)
Available from: 2013-04-22 Created: 2013-04-22 Last updated: 2013-04-29

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