Eva Albertsson

Oxidative stress, evident in antioxidant defences and damage products, in rainbow trout caged outside a sewage treatment plant

Sewage treatment plants (STPs) are common sources of a wide variety of chemicals entering into the aquatic environment. We have investigated the effects of STP effluent on oxidative stress parameters in rainbow trout via measurements of several enzymatic antioxidants as well as the molecular antioxidant glutathione (GSH). In addition, we have measured levels of oxidative damage, i.e. protein carbonyls and lipid peroxides. Our findings indicate that STP effluent does contain prooxidants that affect fish. Oxidative damage was a more consistent indicator of exposure to prooxidants than antioxidant enzymes. Protein carbonyls and lipid peroxides were both elevated (1.3- and 1.8-fold increases, respectively) in fish caged at the STP site compared with reference site as was the amount of oxidized GSH (GSSG), which showed a 3-fold increase. Antioxidant enzymes showed no clear-cut response. Additionally, older pollutants that have been present in down stream sediment for decades had no effects on the parameters measured here.

Does waterborne citalopram affect the aggressive and sexual behaviour of rainbow trout and guppy

Citalopram is one of several selective serotonin reuptake inhibitors (SSRIs) commonly found in treated sewage effluents. Accordingly, there are concerns about possible adverse effects of SSRIs on aquatic organisms, particularly behavioural effects similar to those associated with SSRI use in humans. Rainbow trout fry and adult male guppies were therefore exposed to waterborne citalopram, ranging from environmentally relevant to high concentrations (1, 10, 100 μg/L) for 3-7 days. Under these experimental conditions citalopram does not appear to cause significant effects on aggression in rainbow trout fry or on sexual behaviour in male guppies. This may be explained by a relatively low uptake of citalopram from water to fish.

Hepatic transcriptome profiling indicates differential mRNA expression of apoptosis and immune related genes in eelpout (Zoarces viviparus) caught at Göteborg harbor, Sweden.

The physiology and reproductive performance of eelpout (Zoarces viviparus) have been monitored along the Swedish coast for more than three decades. In this study, transcriptomic profiling was applied for the first time as an exploratory tool to search for new potential candidate biomarkers and to investigate possible stress responses in fish collected from a chronically polluted area. An oligonucleotide microarray with more than 15,000 sequences was used to assess differentially expressed hepatic mRNA levels in female eelpout collected from the contaminated area at Göteborg harbor compared to fish from a national reference site, Fjällbacka. Genes involved in apoptosis and DNA damage (e.g., SMAC/diablo homolog and DDIT4/DNA-damage-inducible protein transcript 4) had higher mRNA expression levels in eelpout from the harbor compared to the reference site, whereas mRNA expression of genes involved in the innate immune system (e.g., complement components and hepcidin) and protein transport/folding (e.g., signal recognition particle and protein disulfide-isomerase) were expressed at lower levels. Gene Ontology enrichment analysis revealed that genes involved biological processes associated with protein folding, immune responses and complement activation were differentially expressed in the harbor eelpout compared to the reference site. The differential mRNA expression of selected genes involved in apoptosis/DNA damage and in the innate immune system was verified by quantitative PCR, using the same fish in addition to eelpout captured four years later. Thus, our approach has identified new potential biomarkers of pollutant exposure and has generated hypotheses on disturbed physiological processes in eelpout. Despite a higher mRNA expression of genes related to apoptosis (e.g., diablo homolog) in eelpout captured in the harbor there were no significant differences in the number of TUNEL-positive apoptotic cells between sites. The mRNA level of genes involved in apoptosis/DNA damage and the status of the innate immune system in fish species captured in polluted environments should be studied in more detail to lay the groundwork for future biomonitoring studies.

Induction of hepatic carbonyl reductase/20β-hydroxysteroid dehydrogenase mRNA in rainbow trout downstream from sewage treatment works—Possible roles of aryl hydrocarbon receptor agonists and oxidative stress

Carbonyl reductase/20beta-hydroxysteroid dehydrogenase (CR/20beta-HSD) serves both as a key enzyme in the gonadal synthesis of maturing-inducing hormone in salmonids, and as an enzyme protecting against certain reactive oxygen species. We have previously shown that mRNA of the hepatic CR/20beta-HSD B isoform is increased in rainbow trout caged downstream from a Swedish sewage treatment plant. Here, we report an increase of both the A as well as B form in fish kept downstream from a second sewage treatment plant. The two mRNAs were also induced in fish hepatoma cells in vitro after exposure to effluent extract. This indicates that the effects observed in vivo could be a direct effect on the liver, i.e. the mRNA induction does not require a signal from any other organ. When fish were exposed in vivo to several effluents treated with more advanced methods (ozone, moving bed biofilm reactor or membrane bioreactor) the expression of hepatic mRNA CR/20beta-HSD A and B was significantly reduced. Their abundance did not parallel the reduction of estrogen-responsive transcripts, in agreement with our previous observations that ethinylestradiol is not a potent inducer. Treatment with norethisterone, methyltestosterone or hydrocortisone in vivo did not induce the hepatic CR/20beta-HSD A and B mRNA expression. In contrast, both isoforms were markedly induced by the aryl hydrocarbon receptor agonist beta-naphthoflavone as well as by the pro-oxidant herbicide paraquat. We hypothesize that the induction of CR/20beta-HSD A and B by sewage effluents could be due to anthropogenic contaminants stimulating the aryl hydrocarbon receptor and/or causing oxidative stress.

A gene to organism approach—assessing the impact of environmental pollution in eelpout (Zoarces viviparus) females and larvae

Abstract A broad biomarker approach was applied to study the effects of marine pollution along the Swedish west coast using the teleost eelpout (Zoarces viviparus) as the sentinel species. Measurements were performed on different biological levels, from the molecular to the organismal, including measurements of messenger RNA (mRNA), proteins, cellular and tissue changes, and reproductive success. Results revealed that eelpout captured in Stenungsund had significantly higher hepatic ethoxyresorufin O‐deethylase activity, high levels of both cytochrome P4501A and diablo homolog mRNA, and high prevalence of dead larvae and nuclear damage in erythrocytes. Eelpout collected in Göteborg harbor displayed extensive macrovesicular steatosis, whereby the majority of hepatocytes were affected throughout the liver, which could indicate an effect on lipid metabolism. Results also indicate that eelpouts collected at polluted sites might have an affected immune system, with lower mRNA expression of genes involved in the innate immune system and a higher number of lymphocytes. Biomarker assessment also was performed on livers dissected from unborn eelpout larvae collected from the ovary of the females. No significant differences were noted, which might indicate that the larvae to some extent are protected from effects of environmental pollutants. In conclusion, usage of the selected set of biological markers, covering responses from gene to organism, has demonstrated site‐specific biomarker patterns that provided a broad and comprehensive picture of the impact of environmental stressors. Environ Toxicol Chem 2015;34:1511–1523.

Biomarker responses in eelpouts from four coastal areas in Sweden, Denmark and Germany

To increase our understanding of possible chemical impacts on coastal fish populations in the Baltic Sea, Kattegat and Skagerrak, the viviparous eelpout (Zoarces viviparus) was used as sentinel species in two major sampling campaigns (spring and autumn) in 16 different coastal sites. Condition factor (CF), liver somatic index (LSI), gonad somatic index (GSI) were measured and the activity of the hepatic enzymes ethoxyresorufin-O-deethylase (EROD), glutathione reductase GR), glutathione S-transferase (GST), catalase (CAT) and muscular activity of acetylcholinesterase (AChE) were assessed. PAH metabolites in bile were also analyzed. The most notable finding in the data set was the low EROD activity in eelpouts collected at the relatively polluted region in Germany compared to the other regions, which could be due to an inhibition of the CYP1A-system or to adaptation to chronic exposure of pollutants in this area. Additionally, low AChE activity was noted in the German region in the autumn campaign and low AChE activity detected in the Danish region in the spring campaign. These differences suggest possible season-specific differences in the use and release of AChE-inhibiting chemicals in the Danish and German regions. Clustering of biomarkers on site level indicated a relationship between CF and GSI and suggested that sites with a high CF contained eelpout that put a larger effort into their larvae development. Clustering of the oxidative stress markers GR, GST and CAT on the individual level reflected a possible coordinated regulation of these enzymes. Overall, the results support the importance of taking into account general regional differences and seasonal variation in biomarker activity when monitoring and assessing the effects of pollution. Despite the expected seasonal variation for most of the measured endpoint, several markers (GSI, EROD and CF) vary similarly between all selected sites in both spring and autumn. This suggests that the differences between sites for these endpoints are independent of season.

Carbonyl reductase mRNA abundance and enzymatic activity as potential biomarkers of oxidative stress in marine fish

Carbonyl reductase (CBR) is an enzyme involved in protection from oxidative stress. In rainbow trout (Oncorhynchus mykiss), the hepatic mRNA abundance of the two isoforms (A and B) is increased after exposure to treated sewage effluents, as well as after exposure with β-naphthoflavone (β-NF) and the pro-oxidant paraquat. In this study, we show that the same chemicals similarly increase the single known hepatic CBR mRNA level and CBR catalytic activity in the coastal living eelpout (Zoarces viviparus). Hepatic CBR mRNA abundance and catalytic activity were also compared between eelpout collected at contaminated and reference sites on the Swedish west coast, but no differences were observed. In conclusion, CBR is a potential biomarker candidate for monitoring the exposure and effects of AhR agonists and/or pro-oxidants in the marine environment, but more research is needed to investigate temporal regulation as well as dose dependency for different chemicals. The mRNA and enzymatic assays presented in this study provide two additional tools for researchers interested in expanding their biomarker battery.