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Due to the detection frequencies and measured concentrations in surface water, the type I pyrethroid insecticide, bifenthrin, has been of particular concern within the Sacramento-San Joaquin Delta in California. Concentrations have been detected above levels previously reported to impair neuroendocrine function and induce neurotoxicity to several species of salmonids. Metabolomic and transcriptomic studies indicated impairment of cellular signaling within the brain of exposed animals and potential alteration of lipid metabolism. To better understand the potential impacts of bifenthrin on brain lipids, juvenile rainbow trout (Oncorhynchus mykiss) were exposed to mean bifenthrin concentrations of 28 or 48 ng/L for 14 days, and non-targeted lipidomic profiling in the brain was conducted. Brain tissue sections were also assessed for histopathological insult following bifenthrin treatment. Bifenthrin-exposed trout had a concentration-dependent decrease in the relative abundance of triglycerides (TGs) with levels of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) significantly altered following 48 ng/L bifenthrin exposure. An increased incidence of histopathological lesions, such as focal hemorrhages and congestion of blood vessels, was noted in the brains of bifenthrin-treated animals, suggesting an association between altered lipid metabolism and neuronal cell structure and integrity.

Low doses of antibiotics are ubiquitous in the marine environment and may exert negative effects on non-target aquatic organisms. Using primary macrophages of common carp, we investigated the mechanisms of action following exposure to several common antibiotics; cefotaxime, enrofloxacin, tetracycline, sulfamonomethoxine, and their mixtures, and explored the immunomodulatory effects associated with the nuclear factor-κB (NF-κB) signaling pathway. A KEGG pathway analysis was conducted using the sixty-six differentially expressed genes found in all treatments, and showed that exposure to 100 μg/L of antibiotics could affect regulation of the NF-κB signaling pathway, suggesting that activation of NF-κB is a common response in all four classes of antibiotics. In addition, the four antibiotics induced nf-κb and NF-κB-associated cytokines expression, as verified by qPCR, however, these induction responses by four antibiotics were minor when compared to the same concentration of LPS treatment (100 μg/L). Antagonists of NF-κB blocked many of the immune effects of the antibiotics, providing evidence that NF-κB pathways mediate the actions of all four antibiotics. Moreover, exposure to environmentally relevant, low levels (0.01-100 μg/L) of antibiotics induced a NF-κB-mediated immune response, including endogenous generation of ROS, activity of antioxidant enzymes, as well as expression of cytokine and apoptosis. Moreover, exposure to mixtures of antibiotics presented greater effects on most tested immunological parameters than exposure to a single antibiotic, suggesting additive effects from multiple antibiotics in the environment. This study demonstrates that exposure of fish primary macrophages to low doses of antibiotics activates the NF-kB pathway.

Crude oil and polycyclic aromatic hydrocarbon (PAH) exposure in early life stage fish has been well-characterized to induce phenotypic malformations such as altered heart development and other morphological impacts. The effects of chemical oil dispersants on toxicity are more controversial. To better understand how chemical dispersion of oil can impact toxicity in pelagic fish, embryos of mahi-mahi (Coryphaena hippurus) were exposed to three concentrations of the chemical dispersant Corexit 9500A, or Corexit 9500A-oil mixtures (chemically enhanced water accommodated fractions: CEWAF) of Deepwater Horizon crude oil for 48 h. RNA sequencing, gene ontology enrichment, and phenotypic measurements were conducted to assess toxicity. Exposure to Corexit 9500A altered expression of less than 50 genes at all concentrations (2.5, 5, and 10 mg/L nominal concentration) and did not induce acute mortality or phenotypic malformations, corroborating other studies showing minimal effects of Corexit 9500A on developing mahi-mahi embryos. CEWAF preparations contained environmentally relevant ∑PAH concentrations ranging from 1.4 to 3.1 μg/L and similarly did not alter larval morphology. Differentially expressed genes and significantly altered pathways related to cardiotoxicity, visual impairments, and Ca2+ homeostasis reinforced previous work that expression of genes associated with the heart and eye are highly sensitive molecular endpoints in oil-exposed early life stage fish. Differential expression and gene ontology pathways were similar across the three CEWAF treatments, indicating that increased chemical dispersion did not alter molecular outcomes within the range tested here. In addition, significant sublethal molecular responses occurred in the absence of observable phenotypic changes to the heart, indicating that effects of oil on early life stage fish may not be completely dependent on cardiac function.

Tebuconazole is a widely used fungicide that has been detected in water ecosystems, of which the concentrations may affect the endocrine function of aquatic organisms. At present study, tissue-specific bioaccumulation of tebuconazole was found in ovary of adult zebrafish, indicating a potential risk of endocrine disruption. In order to evaluate the potential endocrine disrupting effects, three life stages (2 hpf (hours post-fertilization) -60 dpf (days post-fertilization), Stage I; 60-120 dpf, Stage II; 180-208 dpf, Stage III) of zebrafish (Danio rerio) were chronically exposed to tebuconazole at the concentrations ranging from 0.05 mg/L to 1.84 mg/L. Result showed that exposed to tebuconazole could lead to a male-biased sex differentiation in juvenile zebrafish and significant decrease of the percentage of germ cells in sexually-mature zebrafish. Egg production was significantly inhibited by 57.8% and 19.2% after Stage II- and Stage III-exposures, respectively. The contents of 17β-estradiol in gonad decreased by 63.5% when exposed to 0.20 mg/L tebuconazole at Stage II and by 49.5% after exposed to 0.18 mg/L tebuconazole at Stage III, respectively. For all stages exposure, reductions in 17β-estradiol/testosterone ratio were observed, indicating an imbalance in steroids synthesis. Additionally, tebuconazole reduced the expression of cyp19a, which was consistent with the decrease of E2 level. In overall, the present findings indicated that, playing as an anti-estrogen-like chemical, tebuconazole inhibited the expression of Cyp19, thereby impairing steroid hormones biosynthesis, leading to a diminished fecundity of zebrafish.

BackgroundMahi-mahi (Coryphaena hippurus) is a commercially and ecologically important fish species that is widely distributed in tropical and subtropical waters. Biological attributes and reproductive capacities of mahi-mahi make it a tractable model for experimental studies. In this study, life development of cultured mahi-mahi from the zygote stage to adult has been described.ResultsA comprehensive developmental table has been created reporting development as primarily detailed observations of morphology. Additionally, physiological, behavioral, and molecular landmarks have been described to significantly contribute in the understanding of mahi life development.ConclusionRemarkably, despite the vast difference in adult size, many developmental landmarks of mahi map quite closely onto the development and growth of Zebrafish and other warm-water, active Teleost fishes.

Bifenthrin (BF) is a pyrethroid insecticide widely used in urban and agricultural applications. Previous studies in embryos of zebrafish have shown that BF can affect estradiol biosynthesis and the dopaminergic system. To examine the role of the estrogen receptor (ER) in the endocrine effects of BF, embryos were exposed for 96 h to a mixture of 0.15 and 1.5 μg/L BF and an ER agonist (17α-ethynylestradiol - EE2) at 0.09 μg/L. Transcripts related to estrogenic (vitellogenin VTG) and dopaminergic (tyrosine hydroxylase (TH), dopamine receptor 1 (DR1), monoamine oxidase (MAO), and catechol-O-methyltransferase b (COMTb)) signaling pathways were investigated by qRT-PCR. Dopamine (DA) and its metabolites (homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC)) were also measured. There was a significant increase in VTG, DR1, MAO and COMTb mRNA levels and HVA-DA ratios within all zebrafish embryos exposed to EE2, including EE2 alone, 0.15 μg/L BF + EE2 and 1.5 μg/L BF + EE2. A significant decrease in homogenate concentrations of DA was observed within all zebrafish embryos exposed to EE2, which included EE2 alone, 0.15 μg/L BF + EE2 and 1.5 μg/L BF + EE2. Co-exposure of BF with EE2 failed to diminish estrogenic or dopaminergic signaling in embryos. Additionally, embryos with diminished ERα expression by morpholino injection were exposed to 0.15 μg/L BF, 1.5 μg/L BF and 0.09 μg/L EE2, with subsequent gene expression measurements. ERα knockdown did not prevent the effects of BF, indicating ERα may have a limited role in the estrogenic and dopaminergic effects caused by BF in zebrafish embryos.

Hydrophobic legacy contaminants like dichlorodiphenyltrichloroethane (DDT) and polychlorinated biphenyls (PCBs) were banned almost half a century ago. While their residues still remain in many environmental compartments, they have undergone extensive aging and likely have lower bioaccessibility (the available fraction) compared to fresh residues. However, risk assessment relies heavily on the use of total chemical concentration, rather than accounting for age-diminished bioaccessibility, likely leading to overestimated risks. In this study, we used 24 h Tenax desorption to measure the potential bioaccessibility of DDTs and PCBs in two sediment cores taken from the Palos Verdes Shelf Superfund site in the Pacific Ocean. The total concentrations of DDTs and PCBs from the core located at the sewage outfall (8C) were as high as 41,000-15,700 μg/kg (dry weight, dw) and 530-2600 μg/kg dw, respectively, while those from a location 7 km northeast of the outfall (3C) were 2-3 orders of magnitude lower. Bioaccessibility estimated by 24-h Tenax-aided desorption (F24h) decreased in the order of DDD > DDE > DDT for DDT derivatives, and PCB 52 > PCB 70 > PCB 153 for PCB congeners, showing a negative correlation with their log Kow. Due to the extensive aging, F24h values were

Compared to the total chemical concentration, bioavailability is a better measurement of risks of hydrophobic organic contaminants (HOCs) to biota in contaminated soil or sediment. Many different bioavailability estimation methods have been introduced to assess the effectiveness of remediation treatments. However, to date the different methods have rarely been evaluated against each other, leading to confusions in method selection. In this study, four different bioavailability estimation methods, including solid phase microextraction (SPME) and polyethylene passive sampling (PE) aiming to detect free chemical concentration (Cfree), and Tenax desorption and isotope dilution method (IDM) aiming to measure chemical accessibility, were used in parallel to estimate in bioavailability of DDT residues (DDXs) in a historically contaminated soil after addition of different black carbon sorbents. Bioaccumulation into earthworm (Eisenia fetida) was measured concurrently for verification. Activated carbon or biochar amendment at 0.2-2% decreased earthworm bioaccumulation of DDXs by 83.9-99.4%, while multi-walled carbon nanotubes had a limited effect (4.3-20.7%). While all methods correctly predicted changes in DDX bioavailability after black carbon amendment, passive samplers offered more accurate predictions. Predicted levels of DDXs in earthworm lipid using the estimated bioavailability and empirical BCFs matched closely with the experimentally derived tissue concentrations. However, Tenax and IDM overestimated bioavailability when the available DDX levels were low. Our findings suggested that both passive samplers and bioaccessibility methods can be used in assessing remediation efficiency, presenting flexibility in method selection. While accessibility-oriented methods offer better sensitivity and shorter sampling time, passive samplers may be more advantageous because of their better performance and computability for in situ deployment.

Synthetic hormones in wastewater effluents released into the aquatic environments may interfere with the normal endocrine systems of fish in receiving streams. Norgestrel (NGT) is a synthetic progestin widely used in oral contraceptives and frequently detected in wastewater effluents. In this study, adult female mosquitofish (Gambusia affinis) were exposed to three environmentally relevant concentrations of norgestrel (NGT) (i.e., 3.6, 35.8, and 368.0 ng L-1) for 42 d, fin morphology, histology of the ovary, and reproductive behaviors were evaluated. The results showed that NGT at all three concentrations caused an increased frequency of atretic follicular cells in ovaries and impaired mating behaviors exhibited by males toward the NGT-exposed females. In mosquitofish exposed to NGT at 35.8 and 368 ng L-1, the anal fin of females had an increased length ratio of ray4/ray 6, an increased width of ray 3, and increased number of segments in ray 3. The histopathological analysis showed that exposure to NGT increased the incidence of spermatogenesis in ovaries. Mating behavior was impaired 58.4%, 65.7%, and 76.4% (P

Diuron is one of the most used herbicide in the world, and its field application has been particularly increased in Brazil due to the expansion of sugarcane crops. Diuron has often been detected in freshwater ecosystems and it can be biodegraded into three main metabolites in the environment, the 3,4-dichloroaniline (DCA), 3,4-dichlorophenylurea (DCPU) and 3,4-dichlorophenyl-N-methylurea (DCPMU). Negative effects under aquatic biota are still not well established for diuron, especially when considering its presence in mixture with its different metabolites. In this study, we evaluated the effects of diuron alone or in combination with its metabolites, DCPMU, DCPU and 3,4-DCA on biochemical stress responses and biotransformation activity of the fish Oreochromis niloticus. Results showed that diuron and its metabolites caused significant but dispersed alterations in oxidative stress markers and biotransformation enzymes, except for ethoxyresorufin-O-deethylase (EROD) activity, that presented a dose-dependent increase after exposure to either diuron or its metabolites. Glutathione S-transferase (GST) activity was significant lower in gills after exposure to diuron metabolites, but not diuron. Diuron, DCPMU and DCA also decreased the multixenobiotic resistance (MXR) activity. Lipid peroxidation levels were increased in gill after exposure to all compounds, indicating that the original compound and diuron metabolites can induce oxidative stress in fish. The integration of all biochemical responses by the Integrated Biomarker Response (IBR) model indicated that all compounds caused significant alterations in O. niloticus, but DCPMU caused the higher alterations in both liver and gill. Our findings imply that diuron and its metabolites may impair the physiological response related to biotransformation and antioxidant activity in fish at field concentrations. Such alterations could interfere with the ability of aquatic animals to adapt to environments contaminated by agriculture.

Calculating risk from seafood exposure to persistent organic pollutants continues to be problematic as estimates of exposure from diet require extensive monitoring of fish species and limited assessments of bioavailability from sediments where the contaminants tend to reside. Previous studies in our laboratory utilized a laboratory-based isotope dilution method (IDM) to estimate the bioavailability of DDT [1,1,1-trichloro-2, 2-bis(p-chloro-phenyl)ethane] and its metabolites from sediment to biota from a superfund site on the shelf of the Palos Verdes (PVS) Peninsula in California (USA). Using a biota-sediment accumulation factor (BSAF) derived from IDM and biomagnification factors (BMF) calculated from previous studies as well as seafood-consumption data specific to anglers in the PVS area, we estimated cancer and non-cancer risks for anglers and nursing infants representing sensitive groups. Predicted cancer risks from consumption of White croaker (Genyonemus lineatus) to the 50th and 95th percentile to all shore mode anglers were, respectively, 2×10-7 and 7×10-7, which were similar to field studies using fish concentrations of all DDT isomers and their environmental degradates (ΣDDT) from collected animals. The calculated non-cancer hazard quotient values for the 50th and 95th percentile shore mode anglers consuming White croaker from this study (0.008 and 0.023, respectively) were also of similar magnitude as those obtained from studies based on samples obtained solely from fish. For nursing infants, similar results were also observed. These results indicate that estimates of bioavailability using IDM from sediment could be used accurately to determine risk to ΣDDT in humans from fish consumption.

The impacts of Deepwater Horizon (DWH) oil on morphology and function during embryonic development have been documented for a number of fish species, including the economically and ecologically important pelagic species, mahi-mahi (Coryphaena hippurus). However, further investigations on molecular events and pathways responsible for developmental toxicity have been largely restricted due to the limited molecular data available for this species. We sought to establish the de novo transcriptomic database from the embryos and larvae of mahi-mahi exposed to water accommodated fractions (HEWAFs) of two DWH oil types (weathered and source oil), in an effort to advance our understanding of the molecular aspects involved during specific toxicity responses. By high throughput sequencing (HTS), we obtained the first de novo transcriptome of mahi-mahi, with 60,842 assembled transcripts and 30,518 BLAST hits. Among them, 2,345 genes were significantly regulated in 96hpf larvae after exposure to weathered oil. With comparative analysis to a reference-transcriptome-guided approach on gene ontology and tox-pathways, we confirmed the novel approach effective for exploring tox-pathways in non-model species, and also identified a list of co-expressed genes as potential biomarkers which will provide information for the construction of an Adverse Outcome Pathway which could be useful in Ecological Risk Assessments.

The impacts of Deepwater Horizon (DWH) oil on morphology and function during embryonic development have been documented for a number of fish species, including the economically and ecologically important pelagic species, mahi-mahi (Coryphaena hippurus). However, further investigations on molecular events and pathways responsible for developmental toxicity have been largely restricted due to the limited molecular data available for this species. We sought to establish the de novo transcriptomic database from the embryos and larvae of mahi-mahi exposed to water accommodated fractions (HEWAFs) of two DWH oil types (weathered and source oil), in an effort to advance our understanding of the molecular aspects involved during specific toxicity responses. By high throughput sequencing (HTS), we obtained the first de novo transcriptome of mahi-mahi, with 60,842 assembled transcripts and 30,518 BLAST hits. Among them, 2,345 genes were significantly regulated in 96hpf larvae after exposure to weathered oil. With comparative analysis to a reference-transcriptome-guided approach on gene ontology and tox-pathways, we confirmed the novel approach effective for exploring tox-pathways in non-model species, and also identified a list of co-expressed genes as potential biomarkers which will provide information for the construction of an Adverse Outcome Pathway which could be useful in Ecological Risk Assessments.

Mahi-mahi (Coryphaena hippurus) is a commercially and ecologically important species of fish occurring in tropical and temperate waters worldwide. Understanding early life events is crucial for predicting effects of environmental stress, which is largely restricted by a lack of genetic resources regarding expression of early developmental genes and regulation of pathways. The need for anchoring developmental stages to transcriptional activities is highlighted by increasing evidence on the impacts of recurrent worldwide oil spills in this sensitive species during early development. By means of high throughput sequencing, we characterized the developmental transcriptome of mahi-mahi at three critical developmental stages, from pharyngula embryonic stage (24 hpf) to 48 hpf yolk-sac larva (transition 1), and to 96 hpf free-swimming larva (transition 2). With comparative analysis by multiple bioinformatic tools, a larger number of significantly altered genes and more diverse gene ontology terms were observed during transition 2 than transition 1. Cellular and tissue development terms were more significantly enriched in transition 1, while metabolism related terms were more enriched in transition 2, indicating a switch progressing from general embryonic development to metabolism during the two transitions. Special focus was given on the most significant common canonical pathways (e.g. calcium signaling, glutamate receptor signaling, cAMP response element-binding protein signaling, cardiac β-adrenergic signaling, etc.) and expression of developmental genes (e.g. collagens, myosin, notch, glutamate metabotropic receptor etc.), which were associated with morphological changes of nervous, muscular, and cardiovascular system. These data will provide an important basis for understanding embryonic development and identifying molecular mechanisms of abnormal development in fish species.

Measurements of an objects fundamental physical properties like mass, volume, and density can offer valuable insights into the composition and state of the object. However, many important biological samples reside in a liquid environment where it is difficult to accurately measure their physical properties. We show that by using a simple piece of glass tubing and some inexpensive off-the-shelf electronics, we can create a sensor that can measure the mass, volume, and density of microgram-sized biological samples in their native liquid environment. As a proof-of-concept, we use this sensor to measure mass changes in zebrafish embryos reacting to toxicant exposure, density changes in seeds undergoing rehydration and germination, and degradation rates of biomaterials used in medical implants. Since all objects have these physical properties, this sensor has immediate applications in a wide variety of different fields including developmental biology, toxicology, materials science, plant science, and many others.

High concentrations of DDT and metabolites (ΣDDT) have been detected in sediment and the demersal flatfish hornyhead turbot (Pleuronichtys verticalis) collected from Palos Verdes (PV), California, USA, a site contaminated with over 100 metric tons of DDT throughout 1960s-70s. This study was conducted to assess the transfer of ΣDDT from PV-sediment into polychaetes (Neanthes arenaceodentata) and hornyhead turbot, and to investigate if the responses in turbots from two different laboratory exposures mimic those in turbots caught in PV (PV-turbot). Turbot fed PV-sediment-contaminated polychaete for 7 days had liver concentrations of ΣDDT similar to PV-turbot. After 28 days, ΣDDT also accumulated in livers of turbot gavaged with a ΣDDT mixture. In vitro cell bioassays indicated significant increases of 17β-estradiol equivalents (EEQ) in turbot bile extracts as compared to the control in the 7-day study. These responses corresponded to those measured in PV-fish. Glucocorticoid receptor (GR), anti-androgen receptor (anti-AR), estrogen receptor (ER) or aryl hydrocarbon receptor (AhR) activities were also observed in extracts of PV-sediment, and PV-sediment-exposed worm. Anti-AR, AhR and GR activities were significantly higher in PV-sediment than reference sediment (San Diego, SD). Higher transcripts of hepatic VTG, ERα and ERβ were found in PV-turbot than SD-turbot, but were unaltered in fish exposed to sediment-contaminated worms for the 7-day study. In contrast, liver extracts from the 28-day treatment of ΣDDT showed lower EEQ but similar hepatic VTG and ERβ transcripts relative to those of PV-turbot. These data indicated that trophic transfer of sediment-associated DDT in 7-day exposures corresponded to field measurements of DDT residues and in vitro ER bioactivities, but failed to mimic in vivo biological effects observed in field fish. In contrast, treatment with ΣDDT alone for 28 days mimicked in vivo biological effects of DDTs in PV fish, but did not correspond to liver concentrations or in vitro bioactivities.

Anthropogenic disturbance of seleniferous soils can lead to selenium contamination of waterways. Although selenium is an essential micronutrient, bioaccumulation and maternal transfer of proteinaceous selenomethionine (SeMet) can result in embryo toxicity. Furthermore, as the climate changes, the salinity of spawning grounds in water-restrained estuaries is increasing. Although a small increase in salinity may not directly impact adult fish, it may alter the detoxification strategies of developing organisms. Previous research indicates that hypersalinity may potentiate SeMet embryo toxicity at an early developmental stage. However, embryonic development is a complex, spatiotemporal process with a constantly shifting cellular microenvironment. To generate thresholds and an adverse outcome pathway for the interactions between selenium and salinity, we sought to identify windows of susceptibility for lethality and deformities in the Japanese medaka (Oryzias latipes). Embryos were treated in freshwater or saltwater for 24 h with 0.5 µM, 5 µM, and 50 µM SeMet at 6 different developmental stages (9, 17, 25, 29, 34, and 38). Survival, hatch, deformities (total, type, and severity), and days to hatch were quantified. Selenium embryo tissue measurements were performed. Selenomethionine exposures of 5 µM and 50 µM significantly decreased survival and hatch at all stages. However, SeMet uptake was stage-dependent and increased with stage. Stage 17 (early neurulation) was identified as the most susceptible stage for lethality and deformities. Selenomethionine in saltwater caused significantly greater toxicity than freshwater at stage 25 (early organogenesis), suggesting a role for liver and osmoregulatory organogenesis in toxicity.

Some species of butterflyfish have had preyed upon corals for millions of years, yet the mechanism of butterflyfish specialized coral feeding strategy remains poorly understood. Certain butterflyfish have the ability to feed on allelochemically rich soft corals, e.g. Sinularia maxima. Cytochrome P450 (CYP) is the predominant enzyme system responsible for the detoxification of dietary allelochemicals. CYP2-like and CYP3A-like content have been associated with butterflyfish that preferentially consumes allelochemically rich soft corals. To investigate the role of butterflyfish CYP2 and CYP3A enzymes in dietary preference, we conducted oral feeding experiments using homogenates of S. maxima and a toxin isolated from the coral in four species of butterflyfish with different feeding strategies. After oral exposure to the S. maxima toxin 5-episinulaptolide (5ESL), which is not normally encountered in the Hawaiian butterflyfish diet, an endemic specialist, Chaetodon multicinctus experienced 100% mortality compared to a generalist, Chaetodon auriga, which had significantly more (3-6 fold higher) CYP3A-like basal content and catalytic activity. The specialist, Chaetodon unimaculatus, which preferentially feed on S. maxima in Guam, but not in Hawaii, had 100% survival, a significant induction of 8-12 fold CYP3A-like content, and an increased ability (2-fold) to metabolize 5ESL over other species. Computer modeling data of CYP3A4 with 5ESL were consistent with microsomal transformation of 5ESL to a C15-16 epoxide from livers of C. unimaculatus. Epoxide formation correlated with CYP3A-like content, catalytic activity, induction, and NADPH-dependent metabolism of 5ESL. These results suggest a potentially important role for the CYP3A family in butterflyfish-coral diet selection through allelochemical detoxification.

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.

Physiological development requires precise spatiotemporal regulation of cellular and molecular processes. Disruption of these key events can generate developmental toxicity in the form of teratogenesis or mortality. The mechanism behind many developmental toxicants remains unknown. While recent work has focused on the unfolded protein response (UPR), oxidative stress, and apoptosis in the pathogenesis of disease, few studies have addressed their relationship in developmental toxicity. Redox regulation, UPR, and apoptosis are essential for physiological development and can be disturbed by a variety of endogenous and exogenous toxicants to generate lethality and diverse malformations. This review examines the current knowledge of the role of oxidative stress, UPR, and apoptosis in physiological development as well as in developmental toxicity, focusing on studies and advances in vertebrates model systems.

Many cases of severe environmental contamination arise from historical episodes, where recalcitrant contaminants have resided in the environment for a prolonged time, leading to potentially decreased bioavailability. Use of bioavailable concentrations over bulk chemical levels improves risk assessment and may play a critical role in determining the need for remediation or assessing the effectiveness of risk mitigation operations. In this study, we applied the principle of isotope dilution to quantify bioaccessibility of legacy contaminants DDT and PCBs in marine sediments from a Superfund site. After addition of 13C or deuterated analogues to a sediment sample, the isotope dilution reached a steady state within 24 h of mixing. At the steady state, the accessible fraction (E) derived by the isotope dilution method (IDM) ranged from 0.28 to 0.89 and was substantially smaller than 1 for most compounds, indicating reduced availability of the extensively aged residues. A strong linear relationship (R2=0.86) was found between E and the sum of rapid (Fr) and slow (Fs) desorption fractions determined by sequential Tenax desorption. The IDM-derived accessible concentration (Ce) was further shown to correlate closely with tissue residue in the marine benthic polychaete Neanthes arenaceodentata exposed in the same sediments. As shown in this study, the IDM approach involves only a few simple steps and may be readily adopted in laboratories equipped with mass spectrometers. This novel method is expected to be especially useful for historically contaminated sediments or soils, for which contaminant bioavailability may have changed significantly due to aging and other sequestration processes.

Selenium (Se) is an essential micronutrient that can cause embryotoxicty at levels 7-30 times above essential concentrations. Exposure to hypersaline conditions and 50 μM selenomethionine (SeMet) decreased embryo hatch and depleted glutathione in Japanese medaka embryos without affecting Se accumulation. To better understand the impacts of nonchemical stressors on developmental toxicity of Se in fish, several adverse outcome pathways were evaluated in the Japanese medaka (Oryzias latipes). We treated medaka embryos at 12 h post fertilization with 50 μM SeMet for 12 hours in freshwater or in 13 ppth hypersalinity and evaluated the contributions of oxidative stress, the unfolded protein response and apoptosis to reduced hatch. Exposure to SeMet and hypersalinity decreased embryo hatch to 3.7% ± 1.95, and induced teratogenesis in 100% ± 0 of hatched embryos. In contrast, treatments of freshwater, saltwater, and SeMet in freshwater resulted in 89.8% ± 3.91-86.7% ± 3.87 hatch, and no significant increase in deformities. We found no significant differences in lipid peroxidation, indicating that oxidative stress may not be responsible for the observed toxicity in embryos at this time point (24 h). Although significant changes in apoptosis were not observed, we witnessed up to 100 fold increases in transcripts of the endoplasmic reticulum (ER) chaperone, immunoglobulin binding protein (BiP) and trends toward increasing downstream signals, activating transcription factor 4 (ATF4) and ATF6 indicating potential contributions of the unfolded protein response to the effects of SeMet and hypersaline conditions. These data indicate that multiple adverse outcome pathways may be responsible for the developmental toxicity of Se and salinity, and these pathways may be time dependent.

Phase I biotransformation enzymes are critically important in the disposition of xenobiotics within biota and are regulated by multiple environmental cues, particularly in anadromous fish species. Given the importance of these enzyme systems in xenobiotic/endogenous chemical bioactivation and detoxification, the current study was designed to better characterize the expression of Phase I biotransformation enzymes in coho salmon (Oncorhynchus kisutch) and the effects of salinity acclimation on those enzymes. Livers, gills, and olfactory tissues were collected from coho salmon (O. kisutch) after they had undergone acclimation from freshwater to various salinity regimes of seawater (8, 16 and 32 g/L). Using immunoblot techniques coupled with testosterone hydroxylase catalytic activities, 4 orthologs of cytochrome P450 (CYP1A, CYP2K1, CYP2M1, and CYP3A27) were measured in each tissue. Also, the expression of 2 transcripts of flavin-containing monooxygenases (FMO A and B) and associated activities were measured. With the exception of CYP1A, which was down-regulated in liver, protein expression of the other 3 enzymes was induced at higher salinity, with the greatest increase observed in CYP2M1 from olfactory tissues. In liver and gills, 6β- and 16β-hydroxylation of testosterone was also significantly increased after hypersaline acclimation. Similarly, FMO A was up-regulated in all 3 tissues in a salinity-dependent pattern, whereas FMO B mRNA was down-regulated. FMO-catalyzed benzydamine N-oxygenase and methyl p-tolyl sulfoxidation were significantly induced in liver and gills by hypersalinity, but was either unchanged or not detected in olfactory tissues. These data demonstrate that environmental conditions may significantly alter the toxicity of environmental chemicals in salmon during freshwater/saltwater acclimation.

The pyrethroid insecticide bifenthrin is frequently detected at ng/L concentrations in tributaries of the San Francisco Bay Delta. The estuary is also experiencing increasing salinity through climate change and water redirection. To evaluate the impacts of hypersaline conditions on bifenthrin toxicity in anadromous salmonids of the San Francisco Bay Delta (CA, USA), a 14-d laboratory exposure was performed using 2 strains of Oncorhynchus mykiss (rainbow trout and steelhead) acclimated to freshwater and to 8 g/L and 17 g/L salinity. The fish were then exposed to nominal concentrations of 0 µg/L, 0.1 µg/L, and 1.5 µg/L bifenthrin. Rainbow trout exhibited significant mortality following exposure to 1.5 µg/L (1.07 ± 0.35 µg/L measured) bifenthrin in freshwater. Elevated levels of Na⁺ /K⁺ adenosine triphosphatase α1A mRNA subunit expression was observed in the gill of rainbow trout acclimated to hypersaline conditions relative to freshwater animals. No significant difference was noted in Na⁺ /K⁺ adenosine triphosphatase subunit levels in brains of either strain in freshwater or hypersaline conditions. Likewise, significant differences were not observed in plasma vitellogenin or steroid hormone concentrations in either strain whether maintained in freshwater or saltwater. Saltwater acclimation significantly reduced nicotinamide adenine dinucleotide phosphate-catalyzed biotransformation of bifenthrin in liver microsomes of rainbow trout but not of steelhead. The present study showed that, relative to steelhead, rainbow trout have different responses to bifenthrin acute toxicity as well as different rates of hepatic bifenthrin biotransformation and regulation of Na⁺ /K⁺ adenosine triphosphatase subunits in gills. These data indicate that significant differences exist between the strains and that animal life history may have important effects on the susceptibility of each strain to environmental contaminants.

Sentinel fish hornyhead turbot (Pleuronichthysverticalis) captured near wastewater outfalls are used for monitoring exposure to industrial and agricultural chemicals of ~ 20 million people living in coastal Southern California. Although analyses of hormones in blood and organ morphology and histology are useful for assessing contaminant exposure, there is a need for quantitative and sensitive molecular measurements, since contaminants of emerging concern are known to produce subtle effects. We developed a second generation multi-species microarray with expanded content and sensitivity to investigate endocrine disruption in turbot captured near wastewater outfalls in San Diego, Orange County and Los Angeles California. Analysis of expression of genes involved in hormone [e.g., estrogen, androgen, thyroid] responses and xenobiotic metabolism in turbot livers was correlated with a series of phenotypic end points. Molecular analyses of turbot livers uncovered altered expression of vitellogenin and zona pellucida protein, indicating exposure to one or more estrogenic chemicals, as well as, alterations in cytochrome P450 (CYP) 1A, CYP3A and glutathione S-transferase-α indicating induction of the detoxification response. Molecular responses indicative of exposure to endocrine disruptors were observed in field-caught hornyhead turbot captured in Southern California demonstrating the utility of molecular methods for monitoring environmental chemicals in wastewater outfalls. Moreover, this approach can be adapted to monitor other sites for contaminants of emerging concern in other fish species for which there are few available gene sequences.

BackgroundEndocrine disruptors include plasticizers, pesticides, detergents, and pharmaceuticals. Turbot and other flatfish are used to characterize the presence of chemicals in the marine environment. Unfortunately, there are relatively few genes of turbot and other flatfish in GenBank, which limits the use of molecular tools such as microarrays and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to study disruption of endocrine responses in sentinel fish captured by regulatory agencies.ObjectivesWe fabricated a multigene cross-species microarray as a diagnostic tool to screen the effects of environmental chemicals in fish, for which there is minimal genomic information. The array included genes that are involved in the actions of adrenal and sex steroids, thyroid hormone, and xenobiotic responses. This microarray will provide a sensitive tool for screening for the presence of chemicals with adverse effects on endocrine responses in coastal fish species.MethodsWe used a custom multispecies microarray to study gene expression in wild hornyhead turbot (Pleuronichthys verticalis) collected from polluted and clean coastal waters and in laboratory male zebrafish (Danio rerio) after exposure to estradiol and 4-nonylphenol. We measured gene-specific expression in turbot liver by qRT-PCR and correlated it to microarray data.ResultsMicroarray and qRT-PCR analyses of livers from turbot collected from polluted areas revealed altered gene expression profiles compared with those from nonaffected areas.ConclusionsThe agreement between the array data and qRT-PCR analyses validates this multispecies microarray. The microarray measurement of gene expression in zebrafish, which are phylogenetically distant from turbot, indicates that this multispecies microarray will be useful for measuring endocrine responses in other fish.