Introduction

The Fyris River flows through the city of Uppsala, located in southern Sweden. The river also flows through agricultural lands and is the recipient of Uppsala sewage treatment plant (STP). These factors probably lead to contamination of the water in the Fyris River. To investigate if contaminants present in the aquatic environment have an effect on the aquatic ecosystem, studies of different biomarkers can be performed.

Expression of the gene CYP1A1 acts as a biomarker indicating contamination of AhR (Aryl hydrocarbon receptor) agonist (Jönsson et al. 2002), while levels of vitellogenin (Vtg) act as a biomarker indicating contaminations of estrogenic compounds (Biales et al. 2007).

The organic pollutants that bind strongly to the AhR, and act as AhR agonists, are dioxins and dioxin-like compounds such as some PCBs. When the compound binds to the receptor, it translocates into the cell nucleus and upregulates transcription of CYP1A-like enzymes. These enzymes can be measured as EROD activity (7-ethoxyresorufin O-deethylase) and thereby indicate exposure to and toxic effects of dioxin-like compounds (Braune et al. 2011, Mimura and Fujii-Kuriyama 2003).

Expression of vtg in male fish is used as an indicator of exposure to estrogenic compounds, as the messenger RNA vtg measured is found to correspond to the exposure to estrogenic compounds. Vtg is an egg yolk precursor normally present in female organisms (Biales et al. 2007). The expression of CYP1A and vtg can be measured by fluorescence and by using real-time PCR (Biales et al. 2007).

By placing juvenile trout in cages at different exposure sites and comparing the results of different gene expressions with control fish kept in tanks with tap water under controlled conditions, a semi-field study of known biological responses to toxic compounds can be performed. The advantages of such a study are many: for example, unexposed fish placed at exposure sites have not been able to build up a resistance to environmental pollutants and might therefore be a good indicator of the health of the aquatic ecosystem.

Samples of liver and gill tissue from the exposed and non-exposed fish are taken in order to investigate uptake of pollutants from the surrounding water. Water is transported through the gills of the fish for oxygen uptake and excretion of unwanted compounds. The substances present in the water will follow the pathway of the water through the gills, which can result in an uptake of pollutants (Jönsson et al. 2002). Samples of gill and liver can act as complements to each other as the pollutants present in liver tissue can be metabolites of pollutants biotransformed in the gill tissue (Jönsson et al. 2002).

The purpose of this study  is to investigate effects of possible environmental pollutants in fish. By measuring gene expressions induced by certain well-known substances in fish tissues, conclusions concerning the aquatic ecosystem health could be made.

Materials and methods

Juvenile rainbow trout (Onchorynchus mykiss) were collected from fish tanks at EBC, Uppsala University and placed in cages at three different locations in the Fyris River water system: 1.) Jumkil River, 2.) near Fyrishov and 3.) at the water outlet from Uppsala (STP) (Figure 1). For unexposed controls, rainbow trout held in tap water were used. The experiment was carried out the 14-15th of November in 2011. The fish were transported to the sites in a box with 20 liter of tap water and an aeration device. Transportation from the exposure sites was carried out similarly but with water from the exposure sites. Twelve fishes were caged at each exposure site for 24 h and four fishes from each site were sampled. The pH and temperature was measured at each site (Table 1.).

Figure 1. Sites where the cages were placed. 1.) Jumkil River, on the road between Åkerby and Börje Church. 2.) At the landing of canoes on Fyrishov camping site. 3.) At the water outlet from Uppsala municipal sewage treatment plant.

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Table 1. Temperature and pH at the different sampling sites, 1.) Jumkil River (a reference site), 2.) Fyrishov, 3.) Uppsala municipal sewage treatment plant (STP) and 4.) tap water control.

	pH	Temperature (oC)
Jumkil River (1)	8	4.4
Fyrishov (2)	7.6	4.4
STP (3)	6.3	14.8
Tap water (4)	8	9.7

Sampling

The fishes were anesthetized in water containing benzocaine (125 mg/l). The head was then removed by a cut behind the opercula . Thereafter the fish was dissected , and a small piece of liver and gill filaments from two gill arches were each placed in a labeled eppendorf tube. The tubes were thereafter quickly placed in liquid nitrogen. These samples were then stored at -80°C. The remaining gill arches were stored in ice cold HEPES-Cortland (HC) (Jönsson et al. 2002).

Gill EROD assay

The assay was performed as described in Jönsson et al. (2002). From four fishes per site, twenty gill filaments (10 per well) of approximately 2 mm were collected from each fish and incubated with a reaction buffer (7-ethoxyresorufin and dicumarol and HC buffer). Samples were taken after 30 and 50 minutes and were transferred to a 96-well plate. The fluorescence of each well was measured at 590 nm after excitation at 544 nm. The EROD activity was then calculated.

RNA isolation

The RNA isolation was performed as described in the instruction manual for the AurumTM Total RNA Fatty and Fibrous Tissue Kit (Home page of BIO-RAD). The amount of tissue used was filaments from two gill arches and a 4 x 4 x 4 mm piece of liver per fish. Four fishes were sampled from each site.

cDNA synthesis

The cDNA synthesis was performed as described in the instruction manual for the AurumTM Total RNA Fatty and Fibrous Tissue Kit (BIO-RAD). The volume of total RNA solution needed to obtain 1000ng of RNA was calculated for each sample. Thereafter the specific calculated volume was diluted with Nuclease free water up to 15μl in a PCR tube. A master mix with 5x iScript reaction mix and iScript reverse transcriptase was prepared and added to each sample. The samples were then incubated in a PCR machine.

Real time PCR analysis

The cDNA synthesis was performed as described in the instruction manual for the AurumTM Total RNA Fatty and Fibrous Tissue Kit (Home page of BIO-RAD). The genes analyzed were CYP1A1, vtg and EF1α. EF1α was used as an internal control gene and CYP1A1 and vtg as biomarkers. For each gene, a Master Mix was prepared with iQ SYBR Green Supermix, forward primer, reverse primer (primers specific for each transcript) and sterile water. Thereafter, the cDNA samples were diluted and pipetted to three PCR tubes per sample. The Master mixes were then added to the samples (one master mix per PCR tube and sample). Thereafter, 20μl from each PCR tube was pipetted into three real time PCR tubes. The tubes were placed in the real time PCR machine. The calculations were made according to the method and formulas presented in Schmittgen and Livak (2008).

Results

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The EROD activity (mean + SD) was highest in fish caged at the outlet of Uppsala STP with a value of 0.06 + 0.02 pmol resorufin per filament per min. The lowest value of EROD activity was for the tap water control, 0.008 + 0.004 pmol resorufin per filament per min (Figure 2).

Figure 2. EROD activity (mean + SD) in gill filaments from four juvenile rainbow trouts (Onchorynchus mykiss) per location, caged at three different sites for 24 hours in the Fyris River system; Jumkil River (reference), outside Fyrishov, the outlet from Uppsala municipal sewage treatment plant (STP) and a tap water control.

The hepatic relative expression of vtg was 1.7 +1.3 –fold higher in fish caged outside Fyrishov than in unexposed control. Fish caged outside Uppsala STP had a gill relative expression of vtg 1.7 + 2.9–fold higher values than the unexposed control (Figure 3).

Figure 3. Relative expression of vitellogenin (VG) (mean +SD) in liver and gill tissue from four juvenile rainbow trouts (Onchorynchus mykiss) per location caged for 24 hours at three different sites in the Fyris River system; Jumkil River (reference), outside Fyrishov, the outlet from Uppsala municipal sewage treatment plant (STP) and a tap water control.

The relative expression of CYP1A1 in gill tissue was elevated in fish from all exposure sites. The highest relative expression was in the fish caged outside of Fyrishov (10.1 + 5.8). The relative expression of CYP1A1 in fish liver tissue from all exposure sites was similar and did not differ substantially from that of the unexposed control (Figure 4).

Figure 4. Relative expression of CYP1A (mean + SD) in liver and gill tissue from four juvenile rainbow trouts (Onchorynchus mykiss) per location caged for 24 hours at three different sites in the Fyris River system; Jumkil River (reference), outside Fyrishov, the outlet from Uppsala municipal sewage treatment plant (STP) and a tap water control.

Discussion

The results from this semi-field study indicate that the Fyris River water system is contaminated with AhR agonists as well as estrogenic compounds. Some fish caged at the exposure sites have shown consistent elevated gene expression. A factor in common for these areas is the expected pollutions based on where they are situated. However , from this experiment, identification of the different compounds causing the measured effect (Figure 2, 3, 4) is not possible.

As seen in figure 2, the mean EROD activity was highest in the fish caged at the outlet of Uppsala STP. This has been found in previous experiments for example the study conducted by Jönsson et al. (2002). The contaminants might not be able to break down in the STP and will therefore follow the effluent out into the river.

The expression of vtg in gill and liver tissue from fish caged at the different exposure sites relative to the expression of fish held in tap water was not expected. As seen in figure 3, the lowest values obtained were for the fish from the reference site, Jumkil River. Also, the relative expression in gills from the fish caged at Fyrishov was lower than that from the unexposed tap water control. The relative hepatic expression from the fish caged at the effluent from Uppsala STP was also lower than that of the control. Based on the results from this study, it is not possible to conclude the cause of the differences between the sampling sites. These differences may be explained by inter-individual variation but according to Biales et al. (2007), the vtg transcriptional assay is not sensitive to such variations. Variations between samples could possibly be due to variations in oxygen saturation in the water during transport of live fish, due to differences of aeration devices. Low concentrations of oxygen lead to an increased filtration of water over the gills, which in turn could lead to a higher exposure and uptake of contaminants as shown in Jönsson et al. (2002).

The gill CYP1A1 expression was quite high in the fish from all the exposure sites while the expression in the liver showed no difference from the control (Figure 4). This indicates that the gills play a crucial role in the enzymatic detoxification preventing the parent compound from reaching the liver. It is known that gill epithelial cells metabolize contaminants, acting as a barrier between the water and the blood and thereby protecting the rest of the organism (Carlsson et al. 1999).

Literature

Biales A.D, Bencic D.C, Flick R. W, Lazorchak J, Lattier D.L. 2007. Quantification and associated variability of induced vitellogenin gene transcripts in fathead minnow (Pimpephales promelas) by quantitative real-time polymerase chain reaction assay. Environmental toxicology and chemistry 26: 287–296.

BIO-RAD Instruction Manual. AurumTM Total RNA Fatty and Fibrous Tissue Kit. Catalog #732-6830. WWW-document: https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10001298A.PDF. Page viewed: 24 Novermber 2011

Braune BM, Trudeau S, Jeffrey DA, Mallory ML. 2011. Biomarker responses associated with halogenated organic contaminants in northern fulmars (Fulmarus glacialis) breeding in the Canadian Arctic. Environmental Pollution 159: 2891–2898.

Carlsson C, Pärt P, Brunström B. 1999. 7-Ethoxyresorufin O-deethylase induction in cultured gill epithelial cells from rainbow trout. Aquatic Toxicology 47: 117–128.

Jönsson EM, Brandt I, Brunström B. 2002. Gill filament-based EROD assay for monitoring waterborne dioxin-like polllutants in fish. Environmental Science and Technology 36: 3340–3344.

Mimura J, Fujii-Kuriyama Y. 2003. Functional role of AhR in the expression of toxic effects by TCDD. Biochimica et Biophysica Acta (BBA) – General subjects 1619: 263–268.

Schmittgen TD, Livak KJ. 2008. Analyzing real-time PCR data by the comparative C(T) method. Nature Protocols 3: 1101–1108.