A sense of place: pink salmon use a magnetic map for orientation.

The use of 'map-like' information from the Earth's magnetic field for orientation has been shown in diverse taxa, but questions remain regarding the function of such maps. We used a 'magnetic displacement' experiment to demonstrate that juvenile pink salmon (Oncorhynchus gorbuscha) use magnetic cues to orient. The experiment was designed to simultaneously explore whether their magnetic map is used to direct fish (i) homeward, (ii) toward the center of their broad oceanic range or (iii) along their oceanic migratory route. The headings adopted by these navigationally naive fish coincided remarkably well with the direction of the juveniles' migration inferred from historical tagging and catch data. This suggests that the large-scale movements of pink salmon across the North Pacific may be driven largely by their innate use of geomagnetic map cues. Key aspects of the oceanic ecology of pink salmon and other marine migrants might therefore be predicted from magnetic displacement experiments.

Elevated CO2 impairs olfactory-mediated neural and behavioral responses and gene expression in ocean-phase coho salmon (Oncorhynchus kisutch).

Elevated concentrations of CO2 in seawater can disrupt numerous sensory systems in marine fish. This is of particular concern for Pacific salmon because they rely on olfaction during all aspects of their life including during their homing migrations from the ocean back to their natal streams. We investigated the effects of elevated seawater CO2 on coho salmon (Oncorhynchus kisutch) olfactory-mediated behavior, neural signaling, and gene expression within the peripheral and central olfactory system. Ocean-phase coho salmon were exposed to three levels of CO2 , ranging from those currently found in ambient marine water to projected future levels. Juvenile coho salmon exposed to elevated CO2 levels for 2 weeks no longer avoided a skin extract odor that elicited avoidance responses in coho salmon maintained in ambient CO2 seawater. Exposure to these elevated CO2 levels did not alter odor signaling in the olfactory epithelium, but did induce significant changes in signaling within the olfactory bulb. RNA-Seq analysis of olfactory tissues revealed extensive disruption in expression of genes involved in neuronal signaling within the olfactory bulb of salmon exposed to elevated CO2 , with lesser impacts on gene expression in the olfactory rosettes. The disruption in olfactory bulb gene pathways included genes associated with GABA signaling and maintenance of ion balance within bulbar neurons. Our results indicate that ocean-phase coho salmon exposed to elevated CO2 can experience significant behavioral impairments likely driven by alteration in higher-order neural signal processing within the olfactory bulb. Our study demonstrates that anadromous fish such as salmon may share a sensitivity to rising CO2 levels with obligate marine species suggesting a more wide-scale ecological impact of ocean acidification.

Kinetics of Glutathione Depletion and Antioxidant Gene Expression as Indicators of Chemical Modes of Action Assessed in Vitro in Mouse Hepatocytes with Enhanced Glutathione Synthesis.

Here we report a vertically integrated in vitro - in silico study that aims to elucidate the molecular initiating events involved in the induction of oxidative stress (OS) by seven diverse chemicals (cumene hydroperoxide, t-butyl hydroperoxide, hydroquinone, t-butyl hydroquinone, bisphenol A, Dinoseb, and perfluorooctanoic acid). To that end, we probe the relationship between chemical properties, cell viability, glutathione (GSH) depletion, and antioxidant gene expression. Concentration-dependent effects on cell viability were assessed by MTT assay in two Hepa-1 derived mouse liver cell lines: a control plasmid vector transfected cell line (Hepa-V), and a cell line with increased glutamate-cysteine ligase (GCL) activity and GSH content (CR17). Changes to intracellular GSH content and mRNA expression levels for the Nrf2-driven antioxidant genes Gclc, Gclm, heme oxygenase-1 ( Hmox1), and NADPH quinone oxidoreductase-1 ( Nqo1) were monitored after sublethal exposure to the chemicals. In silico models of covalent and redox reactivity were used to rationalize differences in activity of quinones and peroxides. Our findings show CR17 cells were generally more resistant to chemical toxicity and showed markedly attenuated induction of OS biomarkers; however, differences in viability effects between the two cell lines were not the same for all chemicals. The results highlight the vital role of GSH in protecting against oxidative stress-inducing chemicals as well as the importance of probing molecular initiating events in order to identify chemicals with lower potential to cause oxidative stress.

Differential copper-induced death and regeneration of olfactory sensory neuron populations and neurobehavioral function in larval zebrafish.

Fish rely heavily on their sense of smell to maintain behaviors essential for survival, such as predator detection and avoidance, prey selection, social behavior, imprinting, and homing to natal streams and spawning sites. Due to its direct contact with the outside environment, the peripheral olfactory system of fish is particularly susceptible to dissolved contaminants. In particular, environmental exposures to copper (Cu) can cause a rapid loss of olfactory function. In this study, confocal imaging of double-transgenic zebrafish larvae with differentially labeled ciliated and microvillous olfactory sensory neurons (OSNs) were used to examine cell death and regeneration following Cu exposure. Changes in cell morphologies were observed at varying degrees within both ciliated and microvillous OSNs, including the presence of round dense cell bodies, cell loss and fragmentation, retraction or loss of axons, disorganized cell arrangements, and loss of cells and fluorescence signal intensity, which are all indicators of cell death after Cu exposure. A marked loss of ciliated OSNs relative to microvillous OSNs occurred after exposure to low Cu concentrations for 3 h, with some regeneration observed after 72 h. At higher Cu concentrations and 24-h exposures, ciliated and microvillous OSNs were damaged with increased severity of injury with longer Cu exposures. Interestingly, microvillous, but not ciliated OSNs, regenerated rapidly within the 72-h time period of recovery after death from Cu exposure, suggesting that microvillous OSNs may be replaced in lieu of ciliated OSNs. An increase in bromodeoxyuridine labeling was observed 24 h after Cu-induced OSN death, suggesting that increased proliferation of the olfactory stem cells replaced the damaged OSNs. Olfactory behavioral analyses supported our imaging studies and revealed both initial loss and restoration of olfactory function after Cu exposures. In summary, our studies indicate that following zebrafish OSN damage by Cu, regeneration of microvillous OSNs may occur exceeding ciliated OSNs, likely via increased proliferation of the cellular reservoir of neuronal OSC precursors. Transgenic zebrafish are a valuable tool to study metal olfactory injury and recovery and to characterize sensitive olfactory neuron populations in fish exposed to environmental pollutants.

Comparative behavioral toxicology with two common larval fish models: Exploring relationships among modes of action and locomotor responses.

Behavioral responses inform toxicology studies by rapidly and sensitively detecting molecular initiation events that propagate to physiological changes in individuals. These behavioral responses can be unique to chemical specific mechanisms and modes of action (MOA) and thus present diagnostic utility. In an initial effort to explore the use of larval fish behavioral response patterns in screening environmental contaminants for toxicity and to identify behavioral responses associated with common chemical specific MOAs, we employed the two most common fish models, the zebrafish and the fathead minnow, to define toxicant induced swimming activity alterations during interchanging photoperiods. Though the fathead minnow (Pimephales promelas) is a common model for aquatic toxicology research and regulatory toxicology practice, this model has received little attention in behavioral studies compared to the zebrafish, a common biomedical model. We specifically compared behavioral responses among 7 different chemicals (1-heptanol, phenol, R-(-)-carvone, citalopram, diazinon, pentylenetetrazole (PTZ), and xylazine) that were selected and classified based on anticipated MOA (nonpolar narcosis, polar narcosis, electrophile, specific mechanism) according to traditional approaches to examine whether these comparative responses differ among chemicals with various structure-based predicted toxicity. Following standardized experimental guidelines, zebrafish embryos and fathead minnow larvae were exposed for 96 h to each compound then were observed using digital behavioral analysis. Behavioral observations included photomotor responses, distance traveled, and stimulatory, refractory and cruising locomotor activity. Though fathead minnow larvae displayed greater behavioral sensitivity to 1-heptanol, phenol and citalopram, zebrafish were more sensitive to diazinon and R-(-)-carvone. Both fish models were equally sensitive to xylazine and PTZ. Further, the pharmaceuticals citalopram and xylazine significantly affected behavior at therapeutic hazard values, and each of the seven chemicals elicited unique behavioral response profiles. Larval fish behaviors appear useful as early tier diagnostics to identify mechanisms and pathways associated with diverse biological activities for chemicals lacking mechanistic data.

Comparative effects of cadmium, zinc, arsenic and chromium on olfactory-mediated neurobehavior and gene expression in larval zebrafish (Danio rerio).

Studies have shown that olfactory-mediated behaviors that are critical to survival can be disrupted by exposure to certain metals. Polluted waterways often contain elevated levels of metals, yet only a subset have been characterized for their potential to cause olfactory toxicity. A larval zebrafish behavioral assay was developed to characterize concentration-response curves for zinc (Zn), hexavalent chromium (Cr), and arsenate (As) olfaction inhibition. Cadmium (Cd), an established olfactory toxicant, was used as a positive control. As expected, following a 24-hour exposure to Cd, we observed a reduced response to taurocholic acid (TCA), a substrate for ciliated olfactory sensory neurons (OSNs), thus validating the behavioral assay. Zn exposure similarly decreased the olfactory response toward TCA, (IC50: 36 µg/L and 76 µg/L, for Cd and Zn, respectively). The response towards a secondary odorant L-cysteine (Cys), a substrate for ciliated and microvillous OSNs, was significantly altered by both Cd and Zn exposure, although the response to Cys was not completely removed in Zn treated larvae, suggesting preferential toxicity towards ciliated OSNs. No significant changes in olfactory responses were observed following Cr and As exposures. Exposures to binary mixtures of Cd and Zn indicated that Zn had a protective effect against Cd toxicity at low Zn concentrations. QuantiGene (QDP) RNA analysis revealed Cd to be a potent inducer of metallothionein 2 (mt2) mRNA in zebrafish larvae, and Zn to be a weak mt2 inducer, suggesting a protective role of mt2 in Cd and Zn olfactory injury. By contrast, QDP analysis of eight other genes important in mitigating the effects of oxidative stress suggested an antioxidant response to Cd, but not Zn, As, and Cr suggesting that oxidative stress was not a primary mechanism of Zn-induced olfactory dysfunction. In summary, our study indicates that Zn inhibits zebrafish olfaction at environmental concentrations and may potentially mitigate Cd induced olfactory dysfunction when present in mixtures. The zebrafish behavioral trough assay incorporating the odorants L-cysteine and TCA is an effective assay to assess the effects of metals on olfactory function.

Adverse metabolic effects in fish exposed to contaminants of emerging concern in the field and laboratory.

Several metabolic parameters were assessed in juvenile Chinook salmon (Oncorhynchus tshawytscha) and staghorn sculpin (Leptocottus armatus) residing in two estuaries receiving wastewater treatment effluent and one reference estuary. We also conducted a laboratory study with fish dosed for 32 days with 16 of the most common contaminants of emerging concern (CECs) detected in feral fish. Several blood chemistry parameters and other indicators of health were measured in fish from the field and laboratory study that were used to assess potential metabolic disruption. The blood chemistry values observed in feral juvenile Chinook salmon were relatively consistent among fish collected from effluent-impacted sites and substantially different compared to reference site fish. These responses were more pronounced in Chinook salmon, which is supported by the disparity in accumulated CECs. The blood chemistry results for juvenile Chinook salmon collected at effluent-impacted sites exhibited a pattern generally consistent with starvation because of similarities to observations from studies of food-deprived fish; however, this response is not consistent with physical starvation but may be contaminant induced. The altered blood chemistry parameters are useful as an early indicator of metabolic stress, even though organismal characteristics (lipid content and condition factor) were not different among sites indicating an early response. Evidence of metabolic disruption was also observed in juvenile Chinook salmon that were exposed in the laboratory to a limited mixture of CECs; however, the plasma parameters were qualitatively different possibly due to exposure route, season, or the suite of CECs. Growth was impaired in the high-dose fish during the dosing phase and the low- and medium-dose fish assayed after 2 weeks of depuration. Overall, these results are consistent with metabolic disruption for fish exposed to CECs, which may result in early mortality or an impaired ability to compete for limited resources.

The Molecular Design Research Network.

Herein, we provide an overview of a research network that is aimed at fostering interdisciplinary collaboration between chemists and toxicologists with the goal of rationally designing safer commercial chemicals. The collaborative is the Molecular Design Research Network (MoDRN) that was created in 2013 with funding from the EPA-National Science Foundation Networks for Sustainable Molecular Design and Synthesis (NSMDS) program. MoDRN is led by 4 universities, Baylor University, University of Washington, The George Washington University, and Yale University. The overarching goal of the network is to enable and empower the design of safer chemicals based on the fourth Principle of Green Chemistry that states, "chemical products should be designed to preserve efficacy of function while minimizing toxicity."

Determining potential adverse effects in marine fish exposed to pharmaceuticals and personal care products with the fish plasma model and whole-body tissue concentrations.

The Fish Plasma Model (FPM) was applied to water exposure and tissue concentrations in fish collected from two wastewater treatment plant impacted estuarine sites. In this study we compared predicted fish plasma concentrations to Cmax values for humans, which represents the maximum plasma concentration for the minimum therapeutic dose. The results of this study show that predictions of plasma concentrations for a variety of pharmaceutical and personal care products (PPCPs) from effluent concentrations resulted in 37 compounds (54%) exceeding the response ratio (RR = Fish [Plasma]/1%Cmaxtotal) of 1 compared to 3 compounds (14%) detected with values generated with estuarine receiving water concentrations. When plasma concentrations were modeled from observed whole-body tissue residues, 16 compounds out of 24 detected for Chinook (67%) and 7 of 14 (50%) for sculpin resulted in an RRtissue value greater than 1, which highlights the importance of this dose metric over that using estuarine water. Because the tissue residue approach resulted in a high percentage of compounds with calculated response ratios exceeding a value of unity, we believe this is a more accurate representation for exposure in the field. Predicting plasma concentrations from tissue residues improves our ability to assess the potential for adverse effects in fish because exposure from all sources is captured. Tissue residues are also more likely to represent steady-state conditions compared to those from water exposure because of the inherent reduction in variability usually observed for field data and the time course for bioaccumulation. We also examined the RR in a toxic unit approach to highlight the importance of considering multiple compounds exhibiting a similar mechanism of action.

Effect of contaminants of emerging concern on liver mitochondrial function in Chinook salmon.

We previously reported the bioaccumulation of contaminants of emerging concern (CECs), including pharmaceuticals and personal care products (PPCPs) and perfluorinated compounds, in field-collected juvenile Chinook salmon from urban estuaries of Puget Sound, WA (Meador et al., 2016). Although the toxicological impacts of CECs on salmon are poorly understood, several of the detected contaminants disrupt mitochondrial function in other species. Here, we sought to determine whether environmental exposures to CECs are associated with hepatic mitochondrial dysfunction in juvenile Chinook. Fish were exposed in the laboratory to a dietary mixture of 16 analytes representative of the predominant CECs detected in our field study. Liver mitochondrial content was reduced in fish exposed to CECs, which occurred concomitantly with a 24-32% reduction in expression of peroxisome proliferator-activated receptor (PPAR) Y coactivator-1a (pgc-1α), a positive transcriptional regulator of mitochondrial biogenesis. The laboratory exposures also caused a 40-70% elevation of state 4 respiration per unit mitochondria, which drove a 29-38% reduction of efficiency of oxidative phosphorylation relative to controls. The mixture-induced elevation of respiration was associated with increased oxidative injury as evidenced by increased mitochondrial protein carbonyls, elevated expression of glutathione (GSH) peroxidase 4 (gpx4), a mitochondrial-associated GSH peroxidase that protects against lipid peroxidation, and reduction of mitochondrial GSH. Juvenile Chinook sampled in a WWTP effluent-impacted estuary with demonstrated releases of CECs showed similar trends toward reduced liver mitochondrial content and elevated respiratory activity per mitochondria (including state 3 and uncoupled respiration). However, respiratory control ratios were greater in fish from the contaminated site relative to fish from a minimally-polluted reference site, which may have been due to differences in the timing of exposure to CECs under laboratory and field conditions. Our results indicate that exposure to CECs can affect both mitochondrial quality and content, and support the analysis of mitochondrial function as an indicator of the sublethal effects of CECs in wild fish.

A targeted gene expression platform allows for rapid analysis of chemical-induced antioxidant mRNA expression in zebrafish larvae.

Chemical-induced oxidative stress and the biochemical pathways that protect against oxidative damage are of particular interest in the field of toxicology. To rapidly identify oxidative stress-responsive gene expression changes in zebrafish, we developed a targeted panel of antioxidant genes using the Affymetrix QuantiGene Plex (QGP) platform. The genes contained in our panel include eight putative Nrf2 (Nfe2l2a)-dependent antioxidant genes (hmox1a, gstp1, gclc, nqo1, prdx1, gpx1a, sod1, sod2), a stress response gene (hsp70), an inducible DNA damage repair gene (gadd45bb), and three reference genes (actb1, gapdh, hprt1). We tested this platform on larval zebrafish exposed to tert-butyl hydroperoxide (tBHP) and cadmium (Cd), two model oxidative stressors with different modes of action, and compared our results with those obtained using the more common quantitative PCR (qPCR) method. Both methods showed that exposure to tBHP and Cd induced expression of prdx1, gstp1, and hmox1a (2- to 12-fold increase via QGP), indicative of an activated Nrf2 response in larval zebrafish. Both compounds also elicited a general stress response as reflected by elevation of hsp70 and gadd45bb, with Cd being the more potent inducer. Transient changes were observed in sod2 and gpx1a expression, whereas nqo1, an Nrf2-responsive gene in mammalian cells, was minimally affected by either tBHP or Cd chemical exposures. Developmental expression analysis of the target genes by QGP revealed marked upregulation of sod2 between 0-96hpf, and to a lesser extent, of sod1 and gstp1. Once optimized, QGP analysis of these experiments was accomplished more rapidly, using far less tissue, and at lower total costs than qPCR analysis. In summary, the QGP platform as applied to higher-throughput zebrafish studies provides a reasonable cost-effective alternative to qPCR or more comprehensive transcriptomics approaches to rapidly assess the potential for chemicals to elicit oxidative stress as a mechanism of chemical toxicity.

Toward the Design of Less Hazardous Chemicals: Exploring Comparative Oxidative Stress in Two Common Animal Models.

Sustainable molecular design of less hazardous chemicals presents a potentially transformative approach to protect public health and the environment. Relationships between molecular descriptors and toxicity thresholds previously identified the octanol-water distribution coefficient, log D, and the HOMO-LUMO energy gap, ΔE, as two useful properties in the identification of reduced aquatic toxicity. To determine whether these two property-based guidelines are applicable to sublethal oxidative stress (OS) responses, two common aquatic in vivo models, the fathead minnow (Pimephales promelas) and zebrafish (Danio rerio), were employed to examine traditional biochemical biomarkers (lipid peroxidation, DNA damage, and total glutathione) and antioxidant gene activation following exposure to eight structurally diverse industrial chemicals (bisphenol A, cumene hydroperoxide, dinoseb, hydroquinone, indene, perfluorooctanoic acid, R-(-)-carvone, and tert-butyl hydroperoxide). Bisphenol A, cumene hydroperoxide, dinoseb, and hydroquinone were consistent inducers of OS. Glutathione was the most consistently affected biomarker, suggesting its utility as a sensitivity response to support the design of less hazardous chemicals. Antioxidant gene expression (changes in nrf2, gclc, gst, and sod) was most significantly (p < 0.05) altered by R-(-)-carvone, cumene hydroperoxide, and bisphenol A. Results from the present study indicate that metabolism of parent chemicals and the role of their metabolites in molecular initiating events should be considered during the design of less hazardous chemicals. Current empirical and computational findings identify the need for future derivation of sustainable molecular design guidelines for electrophilic reactive chemicals (e.g., SN2 nucleophilic substitution and Michael addition reactivity) to reduce OS related adverse outcomes in vivo.

From the Cover: Cadmium Exposure Differentially Alters Odorant-Driven Behaviors and Expression of Olfactory Receptors in Juvenile Coho Salmon (Oncorhynchus kisutch).

Salmon exposed to waterborne metals can experience olfactory impairment leading to disrupted chemosensation. In the current study, we investigated the effects of cadmium (Cd) on salmon olfactory function by modeling an exposure scenario where juvenile salmon transiently migrate through a polluted waterway. Coho were exposed to environmentally relevant concentrations of waterborne Cd (2 and 30 µg/L) for 48 h and (0.3 and 2 µg/L) for 16 days, followed by a 16-day depuration associated with outmigration. Cadmium exposures inhibited behavioral responses towards L-cysteine and conspecific odorants, with effects persisting following the depuration. Behavioral alterations following the 30 µg/L exposure were associated with increased olfactory epithelial gene expression of metallothionein (mt1a) and heme oxygenase (hmox1); reduced expression of olfactory signal transduction (OST) molecules; and reduced expression of mRNAs encoding major coho odorant receptors (ORs). Salmon OR array analysis indicated that Cd preferentially impacted expression of OST and OR markers for ciliated olfactory sensory neurons (OSNs) relative to microvillus OSNs, suggesting a differential sensitivity of these two major OSN populations. Behavioral alterations on exposure to 0.3 and 2 µg/L Cd were associated with increased mt1a, but not with major histological or OR molecular changes, likely indicating disrupted OST as a major mechanism underlying the behavioral dysfunction at the low-level Cd exposures. Laser-ablation mass spectrometry analysis revealed that the OSN injury and behavioral dysfunction was associated with significant Cd bioaccumulation within the olfactory sensory epithelium. In summary, low-level Cd exposures associated with polluted waterways can induce differential and persistent olfactory dysfunction in juvenile coho salmon.

Dual NRF2 paralogs in Coho salmon and their antioxidant response element targets.

The transcription factor NFE2L2 (Nuclear Factor, Erythroid 2-Like 2, or NRF2) plays a key role in maintaining the redox state within cells. Characterization of this pathway has extended to fish, most notably zebrafish (Danio rerio), in which two paralogs of the transcription factor exist: Nrf2a, an activator, and Nrf2b, a negative regulator during embryogenesis. Only one ARE target has been thoroughly delineated in zebrafish, and this deviated from the canonical sequence derived from studies in mammals. In general, the mechanistic pathway has not been characterized in non-model aquatic organisms that are commonly exposed to environmental pollutants. The current study compares the zebrafish paralogs to those found in a non-model teleost, the ecologically important salmonid, Oncorhnychus kisutch (coho salmon). Two salmon paralogs, Nrf2A and -2B, described here were found to possess only slightly greater identity between one another (84% of amino acids) than to the singleton ortholog of the esocid Esox lucius (80-82%), the nearest non-salmonid outgroup. Unlike one of the zebrafish forms, each is a strong activating factor based on sequence homology and in vitro testing. To uncover functional target AREs in coho, promoter flanking sequences were isolated for five genes that protect cells against oxidative stress: heme oxygenase 1, peroxiredoxin 1, glutamate-cysteine ligase, and the glutathione S-transferases pi and rho (hmox1, prdx1, gclc, gstp, and gstr). All except gstr had functional elements and all fit the standard mammalian-derived canonical sequence, unlike the motif found in zebrafish gstp. Expression studies demonstrate the presence of both Nrf2 paralogs in multiple organs, although in differing ratios. Collectively, our findings extend the conservation of Nrf2 and the ARE to salmonids, and should help inform future work in teleosts on mechanisms of redox control, as well as responsiveness of this pathway and its downstream antioxidant gene targets to chemical exposures in the environment.

Contaminants of emerging concern in a large temperate estuary.

This study was designed to assess the occurrence and concentrations of a broad range of contaminants of emerging concern (CECs) from three local estuaries within a large estuarine ecosystem. In addition to effluent from two wastewater treatment plants (WWTP), we sampled water and whole-body juvenile Chinook salmon (Oncorhynchus tshawytscha) and Pacific staghorn sculpin (Leptocottus armatus) in estuaries receiving effluent. We analyzed these matrices for 150 compounds, which included pharmaceuticals, personal care products (PPCPs), and several industrial compounds. Collectively, we detected 81 analytes in effluent, 25 analytes in estuary water, and 42 analytes in fish tissue. A number of compounds, including sertraline, triclosan, estrone, fluoxetine, metformin, and nonylphenol were detected in water and tissue at concentrations that may cause adverse effects in fish. Interestingly, 29 CEC analytes were detected in effluent and fish tissue, but not in estuarine waters, indicating a high potential for bioaccumulation for these compounds. Although concentrations of most detected analytes were present at relatively low concentrations, our analysis revealed that overall CEC inputs to each estuary amount to several kilograms of these compounds per day. This study is unique because we report on CEC concentrations in estuarine waters and whole-body fish, which are both uncommon in the literature. A noteworthy finding was the preferential bioaccumulation of CECs in free-ranging juvenile Chinook salmon relative to staghorn sculpin, a benthic species with relatively high site fidelity.

Olfactory Transcriptional Analysis of Salmon Exposed to Mixtures of Chlorpyrifos and Malathion Reveal Novel Molecular Pathways of Neurobehavioral Injury.

Pacific salmon exposed to sublethal concentrations of organophosphate pesticides (OP) have impaired olfactory function that can lead to loss of behaviors that are essential for survival. These exposures often involve mixtures and can occur at levels below those which inhibit acetylcholinesterase (AChE). In this study, juvenile Coho salmon were exposed for 24 h to either 0.1, 0.5, or 2.5 ppb chlorpyrifos (CPF), 2, 10, or 50 ppb malathion (MAL), or binary mixtures of 0.1 CPF:2 ppb MAL, 0.5 CPF:10 ppb MAL, or 2.5 CPF:10 ppb MAL to mimic single and binary environmental exposures. Microarray analysis of olfactory rosettes from pesticide-exposed salmon revealed differentially expressed genes involved in nervous system function and signaling, aryl hydrocarbon receptor signaling, xenobiotic metabolism, and mitochondrial dysfunction. Coho exposed to OP mixtures exhibited a more pronounced loss in detection of a predatory olfactory cue relative to those exposed to single compounds, whereas respirometry experiments demonstrated that exposure to OPs, individually and in mixtures, reduced maximum respiratory capacity of olfactory rosette mitochondria. The observed molecular, biochemical, and behavioral effects occurred largely in the absence of effects on brain AChE. In summary, our results provide new insights associated with the sublethal neurotoxic effects of OP mixtures relevant to environmental exposures involving molecular and cellular pathways of injury to the salmon olfactory system that underlie neurobehavioral injury.

Differential Gene Expression in Liver, Gill, and Olfactory Rosettes of Coho Salmon (Oncorhynchus kisutch) After Acclimation to Salinity.

Most Pacific salmonids undergo smoltification and transition from freshwater to saltwater, making various adjustments in metabolism, catabolism, osmotic, and ion regulation. The molecular mechanisms underlying this transition are largely unknown. In the present study, we acclimated coho salmon (Oncorhynchus kisutch) to four different salinities and assessed gene expression through microarray analysis of gills, liver, and olfactory rosettes. Gills are involved in osmotic regulation, liver plays a role in energetics, and olfactory rosettes are involved in behavior. Between all salinity treatments, liver had the highest number of differentially expressed genes at 1616, gills had 1074, and olfactory rosettes had 924, using a 1.5-fold cutoff and a false discovery rate of 0.5. Higher responsiveness of liver to metabolic changes after salinity acclimation to provide energy for other osmoregulatory tissues such as the gills may explain the differences in number of differentially expressed genes. Differentially expressed genes were tissue- and salinity-dependent. There were no known genes differentially expressed that were common to all salinity treatments and all tissues. Gene ontology term analysis revealed biological processes, molecular functions, and cellular components that were significantly affected by salinity, a majority of which were tissue-dependent. For liver, oxygen binding and transport terms were highlighted. For gills, muscle, and cytoskeleton-related terms predominated and for olfactory rosettes, immune response-related genes were accentuated. Interaction networks were examined in combination with GO terms and determined similarities between tissues for potential osmosensors, signal transduction cascades, and transcription factors.

Effect of omega-3 fatty acid oxidation products on the cellular and mitochondrial toxicity of BDE 47.

High levels of the flame retardant 2,2',4,4'-tetrabromodiphenyl ether (BDE 47) have been detected in Pacific salmon sampled near urban areas, raising concern over the safety of salmon consumption. However, salmon fillets also contain the antioxidants eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), whose oxidation products induce cellular antioxidant responses. Because oxidative stress is a mechanism of BDE 47 toxicity, we hypothesized that oxidized EPA and DHA can ameliorate the cellular and mitochondrial toxicity of BDE 47. HepG2 cells were treated with a mixture of oxidized EPA and DHA (oxEPA/oxDHA) at a ratio relevant to salmon consumption (1.5/1 oxEPA/oxDHA) followed by exposure to 100 µM BDE 47. Pretreatment with oxEPA/oxDHA for 12 h prior to BDE 47 exposure prevented BDE 47-mediated depletion of glutathione, and increased expression of antioxidant response genes. oxEPA/oxDHA also reduced the level of reactive oxygen species production by BDE 47. The oxEPA/oxDHA antioxidant responses were associated with partial protection against BDE 47-induced loss of viability and also mitochondrial membrane potential. Mitochondrial electron transport system functional analysis revealed extensive inhibition of State 3 respiration and maximum respiratory capacity by BDE 47 were partially reversed by oxEPA/oxDHA. Our findings indicate that the antioxidant effects of oxEPA/oxDHA protect against short exposures to BDE 47, including a protective role of these compounds on maintaining cellular and mitochondrial function.

The role of biomarkers in the assessment of aquatic ecosystem health.

Ensuring the health of aquatic ecosystems and identifying species at risk from the detrimental effects of environmental contaminants can be facilitated by integrating analytical chemical analysis with carefully selected biological endpoints measured in tissues of species of concern. These biological endpoints include molecular, biochemical, and physiological markers (i.e., biomarkers) that when integrated, can clarify issues of contaminant bioavailability, bioaccumulation, and ecological effects while enabling a better understanding of the effects of nonchemical stressors. In the case of contaminant stressors, an understanding of chemical modes of toxicity can be incorporated with diagnostic markers of aquatic animal physiology to help understand the health status of aquatic organisms in the field. Furthermore, new approaches in functional genomics and bioinformatics can help discriminate individual chemicals, or groups of chemicals among complex mixtures that may contribute to adverse biological effects. Although the use of biomarkers is not a new paradigm, such approaches have been underused in the context of ecological risk assessment and natural resource damage assessment. From a regulatory standpoint, these approaches can help better assess the complex effects from coastal development activities to assessing ecosystem integrity pre- and post development or site remediation.

Brief exposure to copper induces apoptosis and alters mediators of olfactory signal transduction in coho salmon.

Pacific salmon are particularly susceptible to copper (Cu)-induced olfactory injuries that can ultimately inhibit neurobehaviors critical to survival. However, the molecular mechanisms underlying Cu-mediated olfactory impairment remain poorly understood. In the present study, we conducted a short-term Cu exposure at levels relevant to urban runoff (5, 25 and 50 ppb) , and investigated the roles of impaired olfactory signal transduction and induced apoptosis as underlying mechanisms of olfactory injury. Increased cell death in the olfactory epithelium was evident in coho receiving 4h exposures to 25 and 50 ppb Cu. Expression of olfactory marker protein (omp), a marker of mature olfactory sensory neurons, also decreased at 50 ppb Cu. Immunohistochemical analysis of coho olfactory epithelium demonstrated a loss of type 3 adenylate cyclase (ACIII) in the apical olfactory epithelium cilia at all levels of Cu exposure, suggesting an inhibitory effect of Cu in olfactory signaling. Accompanying the loss of ACIII in Cu-exposed coho were reduced intracellular cyclic guanosine monophosphate (cGMP) levels in the olfactory rosettes. Collectively, these results support a linkage among the initial steps of olfactory signaling in Cu-induced salmon olfactory injury, and suggesting that monitoring olfactory cGMP levels may aid in the assessment of salmon olfactory injury.