Short communication: Tissue-specific transcript expression of P-glycoprotein isoforms abcb1a and abcb1b in rainbow trout (Oncorhynchus mykiss) following induction with clotrimazole.

P-glycoprotein (P-gp) plays a pivotal role in cellular defense, aimed at reducing xenobiotic accumulation. As a member of the ABC family of proteins, expression of this protein confers the multixenobiotic resistant (MXR) phenotype in aquatic organisms, including fish. To identify tissues protected by or contributing to the elimination of xenobiotics via P-gp, tissue-specific P-gp isoforms abcb1a and abcb1b transcript expression were measured in rainbow trout (Oncorhynchus mykiss). Tissues investigated included the proximal and distal intestines, liver, head kidney, gills, gonads, and 5 regions of the brain: olfactory lobe, cerebrum, optic lobe, cerebellum and medulla. Abcb1a transcript was more widely expressed across tissues and generally showed higher transcript expression than abcb1b. Deviation from this trend occurred in the gills, cerebrum and head kidney, where transcript levels were relatively equal between abcb1a and abcb1b. Intestinal tissues had greater abcb1a expression than abcb1b (3 orders of magnitude). Abcb1b was absent from liver tissue indicating that abcb1a is relied upon for hepatic defense. This study suggests that abcb1b acts to protect sensitive organs from compounds in the systemic circulation (brain and gonad), whereas abcb1a acts primarily in an elimination role in organs such as liver and intestine. To determine if P-gp induction alters transcript responses, the antifungal mammalian Pregnane-X-Receptor (PXR) agonist clotrimazole (CTZ) was used. CTZ-treated rainbow trout showed significantly increased abcb1b transcript expression in the optic lobe and distal intestine, providing evidence that trout PXR exhibits a similar substrate base as mammalian PXR, albeit selectively in regions of the brain and intestine.

Physiological disturbances in juvenile sockeye salmon (Oncorhynchus nerka) exposed to the water-soluble fraction of diluted bitumen.

Current and proposed transcontinental pipelines for the transport of diluted bitumen (dilbit) from the Canadian oil sands traverse the coastal watersheds of British Columbia, habitat essential to Pacific salmonids. To determine the potential risks posed to these keystone species, juvenile sockeye (Oncorhynchus nerka; 1+ parr) were acutely (24-96 h) or subchronically (21-42 d) exposed to 4 concentrations of the water-soluble fraction (WSF) of unweathered Cold Lake Blend dilbit (initial total PAC concentrations: 0, 13.7, 34.7 and 124.5 µg/L) in a flow-through system. Dilbit effects on iono-osmoregulation, the physiological stress response, and the immune system were assessed by both biochemical and functional assays. Hydrocarbon bioavailability was evidenced by a significant induction of liver ethoxyresorufin-O-deethylase (EROD) activity in exposed fish. Acute and subchronic exposure significantly reduced gill Na+-K+-ATPase activity and resulted in lower plasma osmolality, Cl-, and Na+ concentrations. Acute exposure to dilbit resulted in a classic physiological stress response, however at 21 d of exposure, plasma cortisol remained elevated while other measured parameters had returned to baseline values. A compromised immune system was demonstrated by a 29.5 % higher mortality in fish challenged with Vibrio (Listonella) anguillarum following dilbit exposure compared to unexposed controls. Exposure of juvenile salmonids to the WSF of dilbit (at TPAC concentrations at the ppb level) resulted in sublethal effects that included a classic physiological stress response, and alterations in iono-osmoregulatory homeostasis and immunological performance.

Time course of hepatic gene expression and plasma vitellogenin protein concentrations in estrone-exposed juvenile rainbow trout (Oncorhynchus mykiss).

Estrone (E1), a natural estrogen hormone found in sewage effluents and surface waters, has known endocrine disrupting effects in fish, thus, it is a contaminant of emerging concern. Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to an environmentally-relevant concentration of E1 (24ng/L E1 [0.1nM]) for 7d and then placed in clean water for a 9d recovery period. RNA sequencing showed transcripts from numerous affected biological processes (e.g. immune, metabolic, apoptosis, clotting, and endocrine) were altered by E1 after 4d of treatment. The time course of E1-inducible responses relating to vitellogenesis was examined daily during the two phases of exposure. Hepatic gene expression alterations evaluated by quantitative polymerase chain reaction (QPCR) were found during the treatment period for vitellogenin (VTG), vitelline envelope proteins (VEPs) α, ß and γ, and estrogen receptor α1 (ERα1) transcripts. ERα1 was the only transcript induced each day during the treatment phase, thus it was a good indicator of E1 exposure. Gradual increases occurred in VEPß and VEPγ transcripts, peaking at d7. VTG transcript was only elevated at d4, making it less sensitive than VEPs to this low-level E1 treatment. Inductions of ERα1, VEPα, VEPß and VEPγ transcripts ceased 1d into the recovery phase. Plasma VTG protein concentrations were not immediately elevated but peaked 7d into the recovery phase. Thus, elevated vitellogenesis-related gene expression and protein production occurred slowly but steadily at this concentration of E1, confirming the sequence of events for transcripts and VTG protein responses to xenoestrogen exposure.

Evaluating the treatment of a synthetic wastewater containing a pharmaceutical and personal care product chemical cocktail: compound removal efficiency and effects on juvenile rainbow trout.

Pharmaceutical and personal care products (PPCPs) can evade degradation in sewage treatment plants (STPs) and can be chronically discharged into the environment, causing concern for aquatic organisms, wildlife, and humans that may be exposed to these bioactive chemicals. The ability of a common STP process, conventional activated sludge (CAS), to remove PPCPs (caffeine, di(2-ethylhexyl)phthalate, estrone, 17α-ethinylestradiol, ibuprofen, naproxen, 4-nonylphenol, tonalide, triclocarban and triclosan) from a synthetic wastewater was evaluated in the present study. The removal of individual PPCPs by the laboratory-scale CAS treatment plant ranged from 40 to 99.6%. While the efficiency of removal for some compounds was high, remaining quantities have the potential to affect aquatic organisms even at low concentrations. Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to influent recreated model wastewater with methanol (IM, solvent control) or with PPCP cocktail (IC), or CAS-treated effluent wastewater with methanol (EM, treated control) or with PPCP cocktail (EC). Alterations in hepatic gene expression (evaluated using a quantitative nuclease protection plex assay) and plasma vitellogenin (VTG) protein concentrations occurred in exposed fish. Although there was partial PPCP removal by CAS treatment, the 20% lower VTG transcript levels and 83% lower plasma VTG protein concentration found in EC-exposed fish compared to IC-exposed fish were not statistically significant. Thus, estrogenic activity found in the influent was retained in the effluent even though typical percent removal levels were achieved raising the issue that greater reduction in contaminant load is required to address hormone active agents.

Alteration of immune function endpoints and differential expression of estrogen receptor isoforms in leukocytes from 17ß-estradiol exposed rainbow trout (Oncorhynchus mykiss).

While the endocrine system is known to modulate immune function in vertebrates, the role of 17ß-estradiol (E2) in cellular immune function of teleosts is poorly understood. The cellular and molecular responses of juvenile rainbow trout (Oncorhynchus mykiss) to E2 treatment were evaluated by exposing fish to 0.47±0.02µg/L E2 (mean±SEM) for either 2 or 7d, with a subsequent 14d recovery period. After 2 and 7d of exposure to E2, hematocrit was significantly lower than in control fish. Lipopolysaccharide-induced lymphocyte proliferation was elevated on day 2 and concanavalin A-induced lymphocyte proliferation was reduced following 7d of E2 exposure. Four estrogen receptor (ER) transcripts were identified in purified trout head kidney leukocytes (HKL) and peripheral blood leukocytes (PBL). While the mRNA abundance of ERß1 and ERß2 was unaffected by treatment, ERα1 was up-regulated in HKL and PBL following 7d of E2 exposure. ERα2 was up-regulated in HKL after 7d of E2 exposure, but down-regulated in PBL after 2 and 7d of treatment. All parameters that were altered during the E2 exposure period returned to baseline levels following the recovery period. This study reports the presence of the full repertoire of ERs in purified HKL for the first time, and demonstrates that ERα transcript abundance in leukocytes can be regulated by waterborne E2 exposure. It also demonstrated that physiologically-relevant concentrations of E2 can modulate several immune functions in salmonids, which may have widespread implications for xenoestrogen-associated immunotoxicity in feral fish populations inhabiting contaminated aquatic environments.

Inorganic nitrogen, sterols and bacterial source tracking as tools to characterize water quality and possible contamination sources in surface water.

The effects of agricultural activities on stream water quality were assessed by nitrogen analysis, further investigated by gas chromatography mass spectrometry (GC-MS) sterol analysis (including chemometric analysis), and characterized by bacterial source tracking (BST). Surface water samples were collected from five sites, throughout the agriculturally-influenced Nathan Creek watershed, British Columbia, Canada and a nearby control site between October 2005 and March 2006. From a total of 48 samples, Canadian Water Quality Guidelines were exceeded nineteen times for nitrate (NO3-; guideline value: 2.94 mg/L N) and four times for un-ionized ammonia (NH3; guideline value 0.019 mg/L N). Gas chromatography mass spectrometry single ion monitoring (GC-MS SIM) analysis of 18 sterols showed that five fecal sterols (coprostanol, episoprostanol, cholesterol, cholestanol, desmosterol) were detected at all sites except the control site (where only cholesterol, cholestanol and desmosterol were detected). Three phytosterols (campesterol, stigmasterol and ß-sitosterol) were also detected at all sites while the hormone estrone was present at one site on two occasions at concentrations of 0.01 and 0.04 µg/L. Chemometric analysis (principal component analysis and cluster analysis) grouped sites based on their similarities in sterol composition. Analysis of ten sterol ratios (seven for identifying human fecal contamination and four for differentiating sources of fecal contamination) showed multiple instances of human and animal contamination for every site but the control site. Application of a Bacteroides-BST method confirmed contamination from ruminant animals, pigs and dogs in varying combinations at all impact sites. Together, these results confirmed the impact of agricultural activities on the Nathan Creek watershed and support a need for better land management practices to protect water quality and aquatic life.

Integrating omic technologies into aquatic ecological risk assessment and environmental monitoring: hurdles, achievements, and future outlook.

BACKGROUND: In this commentary we present the findings from an international consortium on fish toxicogenomics sponsored by the U.K. Natural Environment Research Council (Fish Toxicogenomics-Moving into Regulation and Monitoring, held 21-23 April 2008 at the Pacific Environmental Science Centre, Vancouver, BC, Canada). OBJECTIVES: The consortium from government agencies, academia, and industry addressed three topics: progress in ecotoxicogenomics, regulatory perspectives on roadblocks for practical implementation of toxicogenomics into risk assessment, and dealing with variability in data sets. DISCUSSION: Participants noted that examples of successful application of omic technologies have been identified, but critical studies are needed to relate molecular changes to ecological adverse outcome. Participants made recommendations for the management of technical and biological variation. They also stressed the need for enhanced interdisciplinary training and communication as well as considerable investment into the generation and curation of appropriate reference omic data. CONCLUSIONS: The participants concluded that, although there are hurdles to pass on the road to regulatory acceptance, omics technologies are already useful for elucidating modes of action of toxicants and can contribute to the risk assessment process as part of a weight-of-evidence approach.

Gene expression pattern in the liver during recovery from an acute stressor in rainbow trout.

The physiological response to stressors, including hormonal profiles and associated tissue responsiveness, has been extensively studied with salmonid fish, but less is known about the molecular basis of this adaptive response. As liver is the major target organ for metabolic adjustments, we exploited a selective transcriptomics approach to address molecular response in this tissue during acute stress adaptation in rainbow trout. The stressor consisted of a standardized 3 min handling disturbance of trout, and plasma and liver samples were collected either prior to or 1 and 24 h after stressor exposure. We developed a low density custom cDNA array consisting of 147 rainbow trout genes designed specifically to represent stress-responsive and endocrine-related pathways in fish. The acute stress response and recovery was confirmed by the transient elevation in plasma cortisol concentration at 1 h, which returned to pre-stress levels over a 24 h period. This was accompanied by significant upregulation of 40 genes at 1 h, and 15 genes at 24 h after stressor exposure in trout liver. Many of these genes were involved in energy metabolism, implicating a rapid liver molecular reprogramming as critical for the metabolic adjustments to an acute stressor. Several other transcripts not previously implicated in the stress response process in fish, including genes involved in immune function and protein degradative pathways, were found to be stress-responsive in trout. A large number of these stress-responsive transcripts were also shown previously to be glucocorticoid-responsive in fish. Together, our results suggest a role for stressor-mediated genomic cortisol signaling in the liver molecular programming associated with stress in fish. Overall, the study demonstrates the complex nature of the adaptive stress response at the molecular level and underscores the utility of targeted gene expression studies for identifying stress coping mechanisms.

The bactericidal agent triclosan modulates thyroid hormone-associated gene expression and disrupts postembryonic anuran development.

We investigated whether exposure to environmentally relevant concentrations of the bactericidal agent, triclosan, induces changes in the thyroid hormone-mediated process of metamorphosis of the North American bullfrog, Rana catesbeiana and alters the expression profile of thyroid hormone receptor (TR) alpha and beta, basic transcription element binding protein (BTEB) and proliferating nuclear cell antigen (PCNA) gene transcripts. Premetamorphic tadpoles were immersed in environmentally relevant concentrations of triclosan and injected with 1 x 10(-11)mol/g body weight 3,5,3'-triiodothyronine (T3) or vehicle control. Morphometric measurements and steady-state mRNA levels obtained by quantitative polymerase chain reaction were determined. mRNA abundance was also examined in Xenopus laevis XTC-2 cells treated with triclosan and/or 10nM T3. Tadpoles pretreated with triclosan concentrations as low as 0.15+/-0.03 microg/L for 4 days showed increased hindlimb development and a decrease in total body weight following T3 administration. Triclosan exposure also resulted in decreased T3-mediated TRbeta mRNA expression in the tadpole tail fin and increased levels of PCNA transcript in the brain within 48 h of T3 treatment whereas TRalpha was unaffected [corrected] Triclosan alone altered thyroid hormone receptor alpha transcript levels in the brain of premetamorphic tadpoles and induced a transient weight loss. In XTC-2 cells, exposure to T3 plus nominal concentrations of triclosan as low as 0.03 microg/L for 24h resulted in altered thyroid hormone receptor mRNA expression. Exposure to low levels of triclosan disrupts thyroid hormone-associated gene expression and can alter the rate of thyroid hormone-mediated postembryonic anuran development.