Temporal changes in stress and tissue-specific metabolic responses to municipal wastewater effluent exposure in rainbow trout.

Sub-chronic exposure to municipal wastewater effluent (MWWE) in situ was recently shown to impact the acute response to a secondary stressor in rainbow trout (Oncorhynchus mykiss). However, little is known about whether MWWE exposure in itself is stressful to the animal. To address this, we carried out a laboratory study to examine the organismal and cellular stress responses and tissue-specific metabolic capacity in trout exposed to MWWE. Juvenile rainbow trout were exposed to 0, 20 and 90% MWWE (from a tertiary wastewater treatment plant), that was replenished every 2d, for 14 d. Fish were sampled 2, 8 or 14 d post-exposure. Plasma cortisol, glucose and lactate levels were measured as indicators of organismal stress response, while inducible heat shock protein 70 (hsp70), constitutive heat shock protein 70 (hsc70) and hsp90 expression in the liver were used as markers of cellular stress response. Impact of MWWE on cortisol signaling was ascertained by determining glucocorticoid receptor protein (GR) expression in the liver, brain and, heart, and metabolic capacity was evaluated by measuring liver glycogen content and tissue-specific activities of key enzymes in intermediary metabolism. Plasma glucose and lactate levels were unaffected by exposure to MWWEs, whereas cortisol showed a transient increase in the 20% group at 8d. Liver hsc70 and hsp90, but not hsp70 expression, were higher in the 90% MWWE group after 8d. There was a temporal change in GR expression in the liver and heart, but not brain of trout exposed to MWWE. Liver glycogen content and activities liver gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), and alanine aminotransferase (AlaAT) were significantly affected by MWWE exposure. The glycolytic enzymes pyruvate kinase (PK) and hexokinase (HK) activities were significantly higher temporally by MWWE exposure in the gill and heart, but not in the liver and brain. Overall, a 14 d exposure to MWWE evokes a cellular stress response and perturbs the cortisol stress response in rainbow trout. The tissue-specific temporal changes in the metabolic capacity suggest enhanced energy demand in fish exposed to MWWE, which may eventually lead to reduced fitness.

Tissue-specific metabolic changes in response to an acute handling disturbance in juvenile rainbow trout exposed to municipal wastewater effluent.

The objective of this study was to evaluate the effects of municipal wastewater effluent (MWWE) exposure on aspects of both organismal and cellular stress response in rainbow trout (Oncorhynchus mykiss). Juvenile rainbow trout were exposed for 14 d (2-d static renewal) to tertiary-treated MWWE at concentrations of 0%, 20% and 90%. Following the MWWE exposure, fish were subjected to an acute handling stress and sampled at 1, 4 and 24 h post-stressor, to evaluate the fish performance to additional stressors. Organismal stress response evaluation included measuring plasma cortisol, glucose and lactate concentrations, and tissue metabolic capacity, including gluconeogenic (liver) and glycolytic enzyme activities in the liver, brain, heart and gill. No significant differences between treatments were seen in plasma cortisol, glucose or lactate concentrations after 14 d exposure to MWWE. However, MWWE exposure significantly affected plasma cortisol and glucose response to the acute secondary stressor. Acute handling disturbance enhanced liver gluconeogenic capacity in the control group, but this response was altered in the MWWE exposed groups. MWWE exposure did not affect the acute stressor-mediated enhancement of brain or gill glycolytic capacity, but significantly reduced the glycolytic capacity of liver and heart in response to a secondary stressor compared to the control group. Altogether, chronic exposure to MWWE impacts the metabolic performances to a secondary stressor challenge and this includes disruptions in tissue-specific gluconeogenic and glycolytic capacities in rainbow trout.

Exposure to municipal wastewater effluent impacts stress performance in rainbow trout.

The objective of the study was to examine the impact of municipal wastewater effluents on the functioning of the cortisol stress axis in rainbow trout (Oncorhynchus mykiss). Juvenile rainbow trout were caged upstream (reference) and downstream (100% and 10% effluent) of a tertiary-treated municipal wastewater treatment plant outfall and sampled at 14d later (0 time samples). A second set of fish were then subjected to a 5 min handling disturbance and sampled at 1 and 24h post-stressor exposure. Plasma cortisol, glucose and lactate concentrations, liver and brain glucocorticoid receptor (GR) protein levels, head kidney mRNA abundances of corticosteroidogenesis genes, including steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage (P450scc), 11ß-hydroxylase and melanocortin 2 receptor (MC2R), and key liver metabolic enzyme activities, were measured. Exposure to effluent for 14d significantly elevated plasma cortisol and lactate levels in 100% effluent group compared to the reference and 10% effluent sites. There was a significantly higher StAR mRNA abundance in the effluent groups compared to the upstream control. GR protein levels in the liver, but not the brain, were significantly higher in the 100% effluent group compared to the upstream control group. Chronic exposure to 100% effluent for 14d significantly lowered liver hexokinase and glucokinase activities, but did not affect glycogen content or the activities of phosphoenolpyruvate carboxykinase, pyruvate kinase, lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase compared to the other two groups. Subjecting these fish to a secondary acute stressor elicited a physiological stress response, including significant transient elevation in plasma cortisol, glucose and lactate levels at 1h which dropped to pre-stress levels at 24h after stressor exposure, in the control and 10% effluent groups, but this conserved stress response was impaired in the 100% effluent group. The 100% effluent group fish also had significantly higher StAR and P450scc mRNA abundances at 1h post-stress, while transcript abundances of all the major corticosteroidogenesis genes were suppressed at 24h post-stressor compared to the control and 10% effluent groups. Considered together, exposure to full-strength MWWE for 14d elicits a chronic stress response in rainbow trout, and perturbs the conserved adaptive response to an acute stressor. Our results reveal that the impact of tertiary-treated MWWE on stress performance in rainbow trout is abolished by 90% effluent dilution.

Hepatic transcriptomics and protein expression in rainbow trout exposed to municipal wastewater effluent.

Municipal wastewater effluents (MWWEs) represent one of the largest point sources of contamination, but few studies have addressed the impact on fish populations. We tested the hypothesis that MWWEs disrupt multiple stress-related pathways by examining expression of genes and proteins in rainbow trout (Oncorhynchus mykiss). A caging study was undertaken by placing juvenile trout for 14 d either at an upstream control or 100%, 50%, and 10% MWWE sites. A custom-made low-density rainbow trout cDNA microarray was utilized for transcriptomics, and select gene expression was confirmed with quantitative real-time PCR. MWWE exposure significantly elevated plasma cortisol, glucose, and vitellogenin levels, and altered the expression of a number of hepatic genes. Notably, expression of stress-related genes, hormone receptors, glucose transporter 2, and genes related to immune function were altered. The gene and protein expression of glucocorticoid receptor, heat shock proteins 70 and 90, and cytochrome P4501A1 were also impacted by MWWE exposure. Our results demonstrate that tertiary-treated MWWEs elicit an organismal and cellular stress response in trout and may lead to an enhanced energy demand in the exposed fish. The disruption in multiple stress-related pathways suggests that tertiary-treated MWWEs exposure may reduce fish performance to subsequent stressors.

Biomonitoring of perfluorochemicals and toxicity to the downstream fish community of Etobicoke Creek following deployment of aqueous film-forming foam.

On August 2, 2005, Air France Flight 358 descended on Lester B. Pearson International Airport (Toronto, ON, Canada) during adverse weather conditions and overran the runway, leading to an onboard fire which destroyed the aircraft. Large quantities (48000L) of aqueous film-forming foam (AFFF) were applied to the burning fuselage within meters of Etobicoke Creek. Local authorities could not confirm the identity of the AFFF formulation applied, but chemical analyses of fish livers collected 9 days post-AFFF application indicated that no perfluorinated acids (PFAs) were elevated at the site of application or downstream. This, and higher concentrations of a fluorotelomer sulfonate in fish liver collected downstream, suggests that an AFFF containing telomerized polyfluorinated material was likely used. However, as an urbanized stream within a heavily developed commercial, industrial, and residential watershed, background levels of perfluorinated compounds in Etobicoke Creek were considerable at all sites. Enlarged fish livers adjacent the AFFF-application site, commensurate with depressed peroxisomal beta-oxidation and hepatic oxidative stress, demonstrate some short-term impact of the AFFF on exposed fish within 9 days of its release. Most fish biochemical responses had recovered to normal values by 120 days, although there was some indication that AFFF-associated contamination shifted further downstream over this interval. This study suggests contemporary AFFFs exert relatively low toxicity on fish communities under realistic exposure scenarios.

Adrenocorticotropic hormone suppresses gonadotropin-stimulated estradiol release from zebrafish ovarian follicles.

While stress is known to impact reproductive performance, the pathways involved are not entirely understood. Corticosteroid effects on the functioning of the hypothalamus-pituitary-gonadal axis are thought to be a key aspect of stress-mediated reproductive dysfunction. A vital component of the stress response is the pituitary secretion of adrenocorticotropic hormone (ACTH), which binds to the melanocortin 2 receptor (MC2R) in the adrenal glands and activates cortisol biosynthesis. We recently reported MC2R mRNA abundance in fish gonads leading to the hypothesis that ACTH may be directly involved in gonadal steroid modulation. Using zebrafish (Danio rerio) ovarian follicles, we tested the hypothesis that acute ACTH stimulation modulates cortisol and estradiol (E(2)) secretion. ACTH neither affected cortisol nor unstimulated E(2) release from ovarian follicles. However, ACTH suppressed human chorionic gonadotropin (hCG)-stimulated E(2) secretion in a dose-related manner, with a maximum decrease of 62% observed at 1 I.U. ACTH mL(-1). This effect of ACTH on E(2) release was not observed in the presence of either 8-bromo-cAMP or forskolin, suggesting that the mechanism(s) involved in steroid attenuation was upstream of adenylyl cyclase activation. Overall, our results suggest that a stress-induced rise in plasma ACTH levels may initiate a rapid down-regulation of acute stimulated E(2) biosynthesis in the zebrafish ovary, underscoring a novel physiological role for this pituitary peptide in modulating reproductive activity.

Characterization of the mRNA expression of StAR and steroidogenic enzymes in zebrafish ovarian follicles.

The objective of this study was to investigate the levels of expression of steroid biosynthetic enzymes and steroidogenic acute regulatory protein (StAR) at different stages of ovarian follicular development in zebrafish (Danio rerio), and to investigate the sites within the steroid biosynthetic pathway that may be regulated by gonadotropins. Ovarian follicles of sexually mature fish were separated into primary, previtellogenic, vitellogenic, and mature stages and the expression of StAR, P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450 hydroxylase/lyase (P450c17), 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), 17beta-hydroxysteroid dehydrogenase type 3 (17beta-HSD3), and P450 aromatase (P450aromA) was determined by Real time RT-PCR. The expression of all genes changed significantly as follicles grew, with a decrease in the expression of StAR, P450scc, 3beta-HSD and P450c17 with maturation, and an increase in the expression of 17beta-HSD3 during vitellogenesis and 17beta-HSD1 and P450aromA during previtellogenesis. In vitro incubation of vitellogenic follicles demonstrated that the expression of StAR, 17beta-HSD3, and P450aromA increased in response to hCG, and decreased in the absence of hCG. In contrast, the expression of P450scc, 3beta-HSD, P450c17, and 17beta-HSD1 remained constant between treatments and over time. Testosterone and estradiol production in the culture medium was stimulated by human chorionic gonadotropin (hCG). These experiments aid in the characterization of the roles and regulation of steroids throughout ovarian development, and suggest that gonadotropins play a key role in the regulation of StAR, 17beta-HSD3, and P450aromA in zebrafish.