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.

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.

Role of Nrf2 antioxidant defense in mitigating cadmium-induced oxidative stress in the olfactory system of zebrafish.

Exposure to trace metals can disrupt olfactory function in fish leading to a loss of behaviors critical to survival. Cadmium (Cd) is an olfactory toxicant that elicits cellular oxidative stress as a mechanism of toxicity while also inducing protective cellular antioxidant genes via activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway. However, the molecular mechanisms of Cd-induced olfactory injury have not been characterized. In the present study, we investigated the role of the Nrf2-mediated antioxidant defense pathway in protecting against Cd-induced olfactory injury in zebrafish. A dose-dependent induction of Nrf2-regulated antioxidant genes associated with cellular responses to oxidative stress was observed in the olfactory system of adult zebrafish following 24h Cd exposure. Zebrafish larvae exposed to Cd for 3h showed increased glutathione S-transferase pi (gst pi), glutamate-cysteine ligase catalytic subunit (gclc), heme oxygenase 1 (hmox1) and peroxiredoxin 1 (prdx1) mRNA levels indicative of Nrf2 activation, and which were blocked by morpholino-mediated Nrf2 knockdown. The inhibition of antioxidant gene induction in Cd-exposed Nrf2 morphants was associated with disruption of olfactory driven behaviors, increased cell death and loss of olfactory sensory neurons (OSNs). Nrf2 morphants also exhibited a downregulation of OSN-specific genes after Cd exposure. Pre-incubation of embryos with sulforaphane (SFN) partially protected against Cd-induced olfactory tissue damage. Collectively, our results indicate that oxidative stress is an important mechanism of Cd-mediated injury in the zebrafish olfactory system. Moreover, the Nrf2 pathway plays a protective role against cellular oxidative damage and is important in maintaining zebrafish olfactory function.

Copper-induced deregulation of microRNA expression in the zebrafish olfactory system.

Although environmental trace metals, such as copper (Cu), can disrupt normal olfactory function in fish, the underlying molecular mechanisms of metal-induced olfactory injury have not been elucidated. Current research has suggested the involvement of epigenetic modifications. To address this hypothesis, we analyzed microRNA (miRNA) profiles in the olfactory system of Cu-exposed zebrafish. Our data revealed 2, 10, and 28 differentially expressed miRNAs in a dose-response manner corresponding to three increasing Cu concentrations. Numerous deregulated miRNAs were involved in neurogenesis (e.g., let-7, miR-7a, miR-128, and miR-138), indicating a role for Cu-mediated toxicity via interference with neurogenesis processes. Putative gene targets of deregulated miRNAs were identified when interrogating our previously published microarray database, including those involved in cell growth and proliferation, cell death, and cell morphology. Moreover, several miRNAs (e.g., miR-203a, miR-199*, miR-16a, miR-16c, and miR-25) may contribute to decreased mRNA levels of their host genes involved in olfactory signal transduction pathways and other critical neurological processes via a post-transcriptional mechanism. Our findings provide novel insight into the epigenetic regulatory mechanisms of metal-induced neurotoxicity of the fish olfactory system and identify novel miRNA biomarkers of metal exposures.

Species-specific differences and structure-activity relationships in the debromination of PBDE congeners in three fish species.

Previous studies have suggested that there may be species-specific differences in the metabolism of polybrominated diphenyl ethers (PBDEs) among different fish species. In this study, we investigated the in vitro hepatic metabolism of eleven individual PBDE congeners (tri- through decaBDEs) in three different fish species: rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio), and Chinook salmon (O. tschwatcha). In addition, we evaluated the influence of PBDE structural characteristics (i.e., bromine substitution patterns) on metabolism. Six of the eleven congeners we evaluated, BDEs 99, 153, 183, 203, 208, and 209, were metabolically debrominated to lower brominated congeners. All of the congeners that were metabolized contained at least one meta-substituted bromine. Metabolites were not detected for congeners without one meta-substituted bromine (e.g., BDEs 28, 47, and 100). Metabolite formation rates were generally 10 to 100 times faster in carp than in trout and salmon. BDEs 47, 49, 101, 154, and 183 were the major metabolites observed in all three species with the exception of BDE 47, which was only detected in carp. Carp demonstrated a preference toward meta-debromination, while trout and salmon debrominated meta- and para-bromine atoms to an equal extent. We compared glutathione-S-transferase (GST) and deiodinase (DI) activity among all three species as these enzyme systems have been hypothesized to play a role in PBDE debromination in teleosts. Carp exhibited a preference for meta-deiodination of the thyroid hormone thyroxine, which was consistent with the preference for meta-debromination of PBDEs observed in carp.

The role of mitochondrial and oxidative injury in BDE 47 toxicity to human fetal liver hematopoietic stem cells.

The polybrominated diphenyl ethers (PBDEs) are a group of flame retardants whose residues have markedly increased in the environment and in human tissues during the last decade. Of the various congeners, BDE 47 (2,2',4,4'-tetrabromodiphenyl ether) is typically the predominant congener observed in fish and wildlife samples, as well as in human tissues. Several studies indicate in utero transfer of PBDEs during pregnancy with residues accumulating in fetal tissues, and thus the potential for BDE 47-mediated injury in utero is of concern. In this study, we examined the mechanisms of BDE 47-mediated injury to primary human fetal liver hematopoietic stem cells (HSCs), which comprise a large proportion of fetal hepatic cells and play a key role in hematopoiesis during fetal development. Incubation of fetal liver HSCs with BDE 47 led to a loss of mitochondrial membrane potential and the onset of apoptosis. These effects were observed in the low micromolar range of BDE 47 exposures. At higher concentrations, BDE 47 elicited a loss of viability, which was accompanied by the generation of reactive oxygen species and peroxidation of HSC lipids. Preincubation of fetal liver HSCs with N-acetylcysteine, a glutathione (GSH) precursor, caused an increase in cellular GSH concentrations, restored mitochondrial redox status, and ameliorated the toxicity of BDE 47. BDE 47-mediated cytotoxicity or oxidative injury was not evident at the lower concentrations (< 1microM). Collectively, these data support a role for oxidative stress in the cytotoxicity of BDE 47 and indicate that oxidative stress-associated biomarkers may be useful in assessing the sublethal effects of BDE 47 toxicity in other models. However, the fact that BDE 47 undergoes a concentration-dependent accumulation in other primary cells in media that can underestimate cellular concentrations (W. R. Mundy et al., 2004, Toxicol. Sci. 82, 164-169) suggests that the HSC cell injury observed in our study may be of less relevance to human in utero PBDE exposures.

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.

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.

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."

Transfection of HepG2 cells with hGSTA4 provides protection against 4-hydroxynonenal-mediated oxidative injury.

4-Hydroxynonenal (4-HNE) is a mutagenic alpha,beta-unsaturated aldehyde produced during oxidative injury that is conjugated by several glutathione S-transferase (GST) isoforms. The alpha class human GSTA4-4 enzyme (hGSTA4-4) has a particularly high catalytic efficiency toward 4-HNE conjugation. However, hGST4-4 expression is low in most human cells and there are other aldehyde metabolizing enzymes that detoxify 4-HNE. In the current study, we determined the effect of over-expression of hGSTA4 mRNA on the sensitivity of HepG2 cells to 4-HNE injury. HepG2 cells transfected with an hGSTA4 vector construct exhibited high steady-state hGSTA4 mRNA, high GST-4-HNE catalytic activities, but lower basal glutathione (GSH) concentrations relative to insert-free vector (control) cells. Exposure to 4-HNE elicited an increase in GSH concentrations in the control and hGSTA4 cells, although the dose-response of GSH induction differed among the two cell types. Specifically, hGSTA4 cells had significantly higher GSH concentrations when exposed to 5-15 microM 4-HNE, but not at 20 microM 4-HNE, suggesting extensive GSH utilization at high concentrations of 4-HNE. The hGSTA4 cells exhibited a significant growth advantage relative to control cells in the absence of 4-HNE, and a trend towards increased growth at low dose exposures to 4-HNE. However, the hGSTA4 cells did not exhibit a growth advantage relative to control cells at higher 4-HNE exposures associated with increased GSH utilization. As expected, the hGSTA4 cells showed resistance to 4-HNE stimulated lipid peroxidation at all 4-HNE doses. In summary, our data indicates that over-expression of hGSTA4 at levels conferring high GST-4-HNE conjugating activity confers a partial growth advantage to HepG2 cells and protects against 4-HNE oxidative injury. However, the loss of proliferative capacity of hGSTA4 cells challenged with levels of 4-HNE associated with severe oxidative stress indicates a role of other aldehyde metabolizing enzymes, and/or GSH-electrophile transporter proteins, in providing full cellular protection against 4-HNE toxicity.

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.

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.

Several glutathione S-transferase isozymes that protect against oxidative injury are expressed in human liver mitochondria.

The mitochondrial environment is rich in reactive oxygen species (ROS) that may ultimately peroxidize membrane proteins and generate unsaturated aldehydes such as 4-hydroxy-2-nonenal (4HNE). We had previously demonstrated the presence of hGSTA4-4, an efficient catalyst of 4HNE detoxification, in human liver mitochondria to the exclusion of the cytosol. In the present study, GSH-affinity chromatography was used in conjunction with biochemical and proteomic analysis to determine the presence of additional cytosolic glutathione S-transferases (GSTs) in human hepatic mitochondria. HPLC-subunit analysis of GSH affinity-purified liver mitochondrial proteins indicated the presence of several potential mitochondrial GST isoforms. Electrospray ionization-mass spectrometry analysis of eluted mitochondrial GST subunits yielded molecular masses similar to those of hGSTP1, hGSTA1 and hGSTA2. Octagonal matrix-assisted laser desorption/ionization time of flight mass spectrometry and proteomics analysis using MS-FIT confirmed the presence of these three GST subunits in mitochondria, and HPLC analysis indicated that the relative contents of the mitochondrial GST subunits were hGSTA1>hGSTA2>hGSTP1. The mitochondrial localization of the alpha and pi class GST subunits was consistent with immunoblotting analysis of purified mitochondrial GST. Enzymatic studies using GSH-purified mitochondrial GST fractions demonstrated the presence of significant GST activity using the nonspecific GST substrate 1-chloro-2,4-dinitrobenzene (CDNB), as well as 4HNE, delta(5)-androstene-3,17-dione (ADI), and cumene hydroperoxide (CuOOH). Interestingly, the specific mitochondrial GST activities toward 4HNE, a highly toxic alpha,beta-unsaturated aldehyde produced during the breakdown of membrane lipids, exceeded that observed in liver cytosol. These observations are suggestive of a role of GST in protecting against mitochondrial injury during the secondary phase of oxidative stress, or modulation of 4HNE-mediated mitochondrial signaling pathways. However, other properties of mitochondrial GST, such as conjugation of environmental chemicals and binding of lipophilic non-substrate xenobiotics and endogenous compounds, remain to be investigated.

Using salmonid microarrays to understand the dietary modulation of carcinogenesis in rainbow trout.

The highlighted article in this issue by Tilton et al. (2005a) is an innovative approach to evaluate the modulation of estrogen receptor (ER) and aryl hydrocarbon (Ah)-receptor pathways as mechanisms underlying indole-3-carbinol (I3C) tumor promotion in rainbow trout (Onchorhynchus mykiss). I3C and its major in vivo component 3,3'-diindolylmethane (DIM) are potent tumor promoters that appear to target both of the aforementioned receptor pathways. However, the relative importance of I3C modulation of ER and AhR-dependent pathways in the promotion of rainbow trout hepatocarcinogenesis has not been established. Previously, researchers within this group reported that I3C promotes aflatoxin B1 (AFB1)-induced trout hepatocarcinogenesis post-initiation at low concentrations in the diet that induce the expression of vitellogenin, a downstream marker for the activation of ER-dependent pathways in fish. Furthermore, the promotional effects of I3C on AFB1 hepatocarcinogenesis in rainbow trout occur at concentrations that differentially induce vitellogenin, but not CYP1A expression. Interestingly, higher I3C concentrations induce the expression of both CYP1A and vitellogenin. Thus, the relative induction of vitellogenin and CYP1A expression, which are respective markers for activation of fish ER and AhR-mediated gene expression, suggest that these pathways may be important for tumor promotion by dietary I3C in trout. Understanding the complexities of I3C-mediated tumor promotion is essential from several perspectives. For example, there is the obvious need to increase our basic understanding of dietary modulation of carcinogenesis. In addition, I3C also exhibits significant antioxidant and cancer chemoprotective effects under certain experimental conditions and in certain models which have led to its recent marketing as a dietary supplement, as well as its development as a possible chemopreventive agent in humans.

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.