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Evaluation of Biomarker Responses in Fish: with Special Emphasis on Gill EROD Activity
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Environmental Toxicology.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Many chemicals present in the aquatic environment can interfere with physiological functions in fish. Exposure to chemicals can be revealed by the use of biomarkers. Induction of 7-ethoxyresorufin O-deethylase (EROD) activity is a commonly used biomarker for exposure to CYP1A inducers such as dioxins and polyaromatic hyrdrocarbons. Vitellogenin is a frequently used biomarker for estrogenic compounds in various fish species whereas a biomarker for androgens, spiggin, is only found in sticklebacks. The main objectives of this thesis were to evaluate gill EROD activity as a biomarker and the three-spined stickleback as a model species in ecotoxicological studies.

EROD activities were measured in gill, liver and kidney in rainbow trout (Oncorhynchus mykiss) caged in urban areas in Sweden. EROD induction was most pronounced in the gill. Also in fish caged at reference sites, with an expected low level of known CYP1A inducers, a marked gill EROD induction was found. One suggested inducer in rural waters is humic substances (HS). To evaluate the EROD-inducing capacity of HS, three-spined sticklebacks (Gasterosteus aculeatus) were exposed to HS of natural or synthetic origin. Both kinds of HS caused significant EROD induction. Gill EROD activities were also induced in sticklebacks exposed to ethynylestradiol (EE2) and β-naphthoflavone (βNF), alone and in combinations. Production of vitellogenin was induced in sticklebacks exposed to ≥50 ng EE2/l and a significant decrease in spiggin production was observed in individuals exposed to 170 ng EE2/l.

Results from this thesis further strengthen the contention that gill EROD activity is a very sensitive biomarker for CYP1A inducers and that the stickleback is a suitable biomonitoring species, especially for exposure to CYP1A inducers. The finding that not only classical CYP1A inducers but also HS and high EE2 concentrations stimulate gill EROD activity is of significance for the interpretation of biomonitoring data.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2007. , p. 59
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 345
Keywords [en]
Biology, three-spined stickleback, biomarker, EROD, ethynylestradiol, gill, vitellogenin, spiggin, CYP1A inducers, humic substances, sperm quality, biomonitoring
Keywords [sv]
Biologi
Identifiers
URN: urn:nbn:se:uu:diva-8222ISBN: 978-91-554-6975-7 (print)OAI: oai:DiVA.org:uu-8222DiVA, id: diva2:170757
Public defence
2007-10-19, Lindahlsalen, EBC, Norbyvägen 18A, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2007-09-28 Created: 2007-09-28 Last updated: 2011-01-27Bibliographically approved
List of papers
1. Gill EROD in monitoring of CYP1A inducers in fish: A study in rainbow trout (Oncorhynchus mykiss) caged in Stockholm and Uppsala waters
Open this publication in new window or tab >>Gill EROD in monitoring of CYP1A inducers in fish: A study in rainbow trout (Oncorhynchus mykiss) caged in Stockholm and Uppsala waters
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2007 (English)In: Aquatic Toxicology, ISSN 0166-445X, E-ISSN 1879-1514, Vol. 85, no 1, p. 1-8Article in journal (Refereed) Published
Abstract [en]

The gill filament 7-ethoxyresorufin O-deethylase (EROD) assay was evaluated as a monitoring tool for waterborne cytochrome P4501 A (CYP1A) inducers using rainbow trout (Oncorhynchus mykiss) caged in urban area waters in Sweden. To compare the CYP1A induction response in different tissues, EROD activity was also analyzed in liver and kidney microsomes. Immunohistochemistry was used to localize CYP1A protein in gill and kidney. In two separate experiments fish were caged at sites with fairly high expected polyaromatic hydrocarbon (PAH) contamination. In the first experiment, gill EROD activities were analyzed in fish exposed for 1-21 days in a river running through Uppsala. The reference site was upstream of Uppsala. In the second, gill, liver and kidney EROD activities were analyzed in fish exposed for 1-5 days in fresh or brackish waters of Stockholm and in a reference lake 60 km north of Stockholm. Fish exposed for 5 days followed by 2 days of recovery in tap water in the laboratory were also examined. The gill consistently showed a higher EROD induction compared with the liver and the kidney. After I day of caging, gill EROD activity was markedly induced (6-17-fold) at all sites examined. Induction in gill was pronounced (5-7-fold) also in fish caged at the reference sites. In the 21-day exposure study gill EROD activity remained highly induced throughout the experiment (26-fold at most) and the induced CYP1A protein was exclusively confined to the gill secondary lamellae. In the 5-day exposure experiment, EROD activity peaked after I day and then declined in both gill and liver, while CYP1A immunostaining in the gill remained intense over the 5-day period. In the kidney, CYP1A staining was weak or absent. We conclude that gill EROD activity is a more sensitive biomarker of exposure to waterborne CYP1A inducers than EROD activity in liver and kidney.

Keywords
fish; gill; CYP1A; EROD; monitoring
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-95923 (URN)10.1016/j.aquatox.2007.07.013 (DOI)000250181300001 ()
Available from: 2007-05-09 Created: 2007-05-09 Last updated: 2017-12-14Bibliographically approved
2. Impact of humic substances on EROD activity in gill and liver of three-spined sticklebacks (Gasterosteus aculeatus)
Open this publication in new window or tab >>Impact of humic substances on EROD activity in gill and liver of three-spined sticklebacks (Gasterosteus aculeatus)
2010 (English)In: Chemosphere, ISSN 0045-6535, E-ISSN 1879-1298, Vol. 81, no 2, p. 156-160Article in journal (Refereed) Published
Abstract [en]

Humic substances (HS) are ubiquitous in the environment and have been found to influence physiological functions of aquatic organisms. In the present study, three-spined sticklebacks (Gasterosteus aculeatus) were exposed to HS of different origins to evaluate effects on the  7-ethoxyresorufin O-deethylase (EROD) activity catalyzed by cytochrome   P4501A (CYP1A) in the liver and the gill. To that end, three-spined   sticklebacks were exposed for 48 h to different concentrations of synthetic humic acid (AHA), Nordic reservoir natural organic matter  (N.R.-NOM) and water from six lakes with different concentrations of   HS. EROD activity was significantly induced (3-6-fold) in the gills of   fish exposed to water from all lakes except the lake with the lowest   concentration of HS. All tested concentrations of AHA and N.R.-NOM   significantly induced gill EROD activity and the induction was   dose-dependent. AHA, but neither N.R.-NOM nor lake water, induced EROD activity in the liver. In addition, fish were exposed to the potent  CYP1A inducers benzo(a)pyrene (BaP) and PCB126 in combination with AHA.   Presence of AHA had no significant effect on EROD induction by BaP or   PCB126. The components in HS responsible for EROD induction remain to be identified. Our finding that HS of both natural and synthetic origin induce EROD activity in the gill is of significance for the   interpretation of biomonitoring data on EROD activity as well as for the choice of suitable reference waters.

Keywords
Humic substances, Three-spined stickleback, EROD activity, Natural organic matter
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-96246 (URN)10.1016/j.chemosphere.2010.06.073 (DOI)000282155400003 ()20797764 (PubMedID)
Available from: 2007-09-28 Created: 2007-09-28 Last updated: 2017-12-14Bibliographically approved
3. Effects of 17α-ethynylestradiol on EROD activity, spiggin and vitellogenin in three-spined stickleback (Gasterosteus aculeatus)
Open this publication in new window or tab >>Effects of 17α-ethynylestradiol on EROD activity, spiggin and vitellogenin in three-spined stickleback (Gasterosteus aculeatus)
2007 (English)In: Aquatic Toxicology, ISSN 0166-445X, E-ISSN 1879-1514, Vol. 83, no 1, p. 33-42Article in journal (Refereed) Published
Abstract [en]

The three-spined stickleback (Gasterosteus aculeatus) has quantifiable biomarkers of exposure to estrogens (vitellogenin), androgens (spiggin) and aryl hydrocarbon receptor (AhR) agonists (EROD activity) and is therefore a promising test species for biomonitoring of reprotoxic chemicals in aquatic environments. In this study we evaluated the effects of 17α-ethynylestradiol (EE2) on EROD activity, induction of vitellogenin and spiggin, hepatosomatic index (HSI), ovarian somatic index (OSI) and nephrosomatic index (NSI). Adult male and female three-spined sticklebacks were exposed to concentrations of 0–170 ng EE2/l (measured concentrations) in a flow-through system for 21 days. Exposure to 170 ng EE2/l resulted in a significant 8- and 9-fold induction of gill EROD activity in males and females, respectively. In livers, EROD activity expressed in relation to microsomal protein content was suppressed due to a significant increase in microsomal protein content. Hepatic EROD activity per se expressed as picomol/min was not affected by exposure to EE2. The lowest observed effect concentration for induction of vitellogenin in males was 53.7 ng EE2/l. In females, vitellogenin levels were significantly higher in those exposed to170 ng EE2/l compared to controls. Spiggin production was significantly inhibited and NSI lower in males exposed to 170 ng EE2/l. In both females and males LSI was significantly higher in fish exposed to 170 ng EE2/l than in controls. In females exposed to 170 ng EE2/l, OSI was significantly lower and NSI higher than controls. The observed results from this study show that a synthetic estrogen can affect the well-known biomarker of exposure for dioxin-like compounds, EROD activity, and further that this response can differ between tissues. These findings are important for interpretation of biomonitoring data.

Keywords
EROD activity, Spiggin, Vitellogenin, Three-spined stickleback, 17α-ethynylestradiol
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-96247 (URN)10.1016/j.aquatox.2007.03.008 (DOI)000246952400004 ()17445917 (PubMedID)
Available from: 2007-09-28 Created: 2007-09-28 Last updated: 2017-12-14Bibliographically approved
4. A chemometrical approach to study interactions between ethynylestradiol and an AhR-agonist in stickleback (Gasterosteus aculeatus)
Open this publication in new window or tab >>A chemometrical approach to study interactions between ethynylestradiol and an AhR-agonist in stickleback (Gasterosteus aculeatus)
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2010 (English)In: Journal of Chemometrics, ISSN 0886-9383, E-ISSN 1099-128X, Vol. 24, no 11-12, p. 768-778Article in journal (Refereed) Published
Abstract [en]

Quantifiable responses in fish, such as induction of certain proteins, can be used as indicators of chemical contamination of waterways. In order to evaluate differences in ethoxyresorufin-O-deethylase (EROD) induction capacity of the gill and the liver and effects on organs and biomarker proteins, e.g. gill and liver EROD, hepatosomatic index (HSI), nephrosomatic index (NSI), gonadosomatic index (GSI), spiggin, vitellogenin and sperm motility were analysed in male three-spined sticklebacks (Gasterosteus aculeatus) exposed for 21 days to β-naphthoflavone (βNF) alone (Exp 1) or in combination with 17α-ethynylestradiol (EE2) (Exp 2). The sperm motility variables were studied using computer-assisted sperm analysis (CASA).

Exp 1: Gill EROD activity was significantly induced in fish exposed to ≥1.2 µg/l and hepatic EROD activity in fish exposed to ≥6 µg/l. No significant effect of ßNF on the production of spiggin or vitellogenin or on sperm variables was found.

Exp 2: A significant additative effect of EE2 + βNF was shown for gill EROD. A significant antagonistic effect of the two compounds was found on NSI where an increased EE2 concentration led to an increase in NSI while an increased concentration of βNF led to a decreased NSI. Interestingly, the results showed that exposure to intermediate concentrations of EE2 and ßNF led to a significant increase in the sperm variables. In the aquatic environment mixtures of numerous chemicals with oestrogenic activity are present, so if the capacity to induce gill EROD activity is a general property of oestrogen-acting chemicals, our findings are important.

Keywords
Doehlert design, gill EROD activity, hepatic EROD activity, sperm motility
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-136127 (URN)10.1002/cem.1368 (DOI)000286291500016 ()
Available from: 2010-12-10 Created: 2010-12-10 Last updated: 2017-12-11Bibliographically approved

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