A rapid return to normal: temporal gene expression patterns following cold exposure in the bumble bee Bombus impatiens.

Bumble bees are common in cooler climates and many species likely experience periodic exposure to very cold temperatures, but little is known about the temporal dynamics of cold response mechanisms following chill exposure, especially how persistent effects of cold exposure may facilitate tolerance of future events. To investigate molecular processes involved in the temporal response by bumble bees to acute cold exposure, we compared mRNA transcript abundance in Bombus impatiens workers exposed to 0°C for 75 min (inducing chill coma) and control bees maintained at a constant ambient temperature (28°C). We sequenced the 3' end of mRNA transcripts (TagSeq) to quantify gene expression in thoracic tissue of bees at several time points (0, 10, 30, 120 and 720 min) following cold exposure. Significant differences from control bees were only detectable within 30 min after the treatment, with most occurring at the 10 min recovery time point. Genes associated with gluconeogenesis and glycolysis were most notably upregulated, while genes related to lipid and purine metabolism were downregulated. The observed patterns of expression indicate a rapid recovery after chill coma, suggesting an acute differential transcriptional response during recovery from chill coma and return to baseline expression levels within an hour, with no long-term gene expression markers of this cold exposure. Our work highlights the functions and pathways important for acute cold recovery, provides an estimated time frame for recovery from cold exposure in bumble bees, and suggests that cold hardening may be less important for these heterothermic insects.

Ultraviolet radiation significantly enhances the molecular response to dispersant and sweet crude oil exposure in Nematostella vectensis.

Estuarine organisms are subjected to combinations of anthropogenic and natural stressors, which together can reduce an organisms' ability to respond to either stress or can potentiate or synergize the cellular impacts for individual stressors. Nematostella vectensis (starlet sea anemone) is a useful model for investigating novel and evolutionarily conserved cellular and molecular responses to environmental stress. Using RNA-seq, we assessed global changes in gene expression in Nematostella in response to dispersant and/or sweet crude oil exposure alone or combined with ultraviolet radiation (UV). A total of 110 transcripts were differentially expressed by dispersant and/or crude oil exposure, primarily dominated by the down-regulation of 74 unique transcripts in the dispersant treatment. In contrast, UV exposure alone or combined with dispersant and/or oil resulted in the differential expression of 1133 transcripts, of which 436 were shared between all four treatment combinations. Most significant was the differential expression of 531 transcripts unique to one or more of the combined UV/chemical exposures. Main categories of genes affected by one or more of the treatments included enzymes involved in xenobiotic metabolism and transport, DNA repair enzymes, and general stress response genes conserved among vertebrates and invertebrates. However, the most interesting observation was the induction of several transcripts indicating de novo synthesis of mycosporine-like amino acids and other novel cellular antioxidants. Together, our data suggest that the toxicity of oil and/or dispersant and the complexity of the molecular response are significantly enhanced by UV exposure, which may co-occur for shallow water species like Nematostella.

Spatiotemporal expression and transcriptional regulation of heme oxygenase and biliverdin reductase genes in zebrafish (Danio rerio) suggest novel roles during early developmental periods of heightened oxidative stress.

Heme oxygenase 1 (HMOX1) degrades heme into biliverdin, which is subsequently converted to bilirubin by biliverdin reductase (BVRa or BVRb) in a manner analogous to the classic anti-oxidant glutathione-recycling pathway. To gain a better understanding of the potential antioxidant roles the BVR enzymes may play during development, the spatiotemporal expression and transcriptional regulation of zebrafish hmox1a, bvra and bvrb were characterized under basal conditions and in response to pro-oxidant exposure. All three genes displayed spatiotemporal expression patterns consistent with classic hematopoietic progenitors during development. Transient knockdown of Nrf2a did not attenuate the ability to detect bvra or bvrb by ISH, or alter spatial expression patterns in response to cadmium exposure. While hmox1a:mCherry fluorescence was documented within the intermediate cell mass, a transient location of primitive erythrocyte differentiation, expression was not fully attenuated in Nrf2a morphants, but real-time RT-PCR demonstrated a significant reduction in hmox1a expression. Furthermore, Gata-1 knockdown did not attenuate hmox1a:mCherry fluorescence. However, while there was a complete loss of detection of bvrb expression by ISH at 24hpf, bvra expression was greatly attenuated but still detectable in Gata-1 morphants. In contrast, 96 hpf Gata-1 morphants displayed increased bvra and bvrb expression within hematopoietic tissues. Finally, temporal expression patterns of enzymes involved in the generation and maintenance of NADPH were consistent with known changes in the cellular redox state during early zebrafish development. Together, these data suggest that Gata-1 and Nrf2a play differential roles in regulating the heme degradation enzymes during an early developmental period of heightened cellular stress.

Characterization of heme oxygenase and biliverdin reductase gene expression in zebrafish (Danio rerio): Basal expression and response to pro-oxidant exposures.

While heme is an important cofactor for numerous proteins, it is highly toxic in its unbound form and can perpetuate the formation of reactive oxygen species. Heme oxygenase enzymes (HMOX1 and HMOX2) degrade heme into biliverdin and carbon monoxide, with biliverdin subsequently being converted to bilirubin by biliverdin reductase (BVRa or BVRb). As a result of the teleost-specific genome duplication event, zebrafish have paralogs of hmox1 (hmox1a and hmox1b) and hmox2 (hmox2a and hmox2b). Expression of all four hmox paralogs and two bvr isoforms were measured in adult tissues (gill, brain and liver) and sexually dimorphic differences were observed, most notably in the basal expression of hmox1a, hmox2a, hmox2b and bvrb in liver samples. hmox1a, hmox2a and hmox2b were significantly induced in male liver tissues in response to 96h cadmium exposure (20µM). hmox2a and hmox2b were significantly induced in male brain samples, but only hmox2a was significantly reduced in male gill samples in response to the 96h cadmium exposure. hmox paralogs displayed significantly different levels of basal expression in most adult tissues, as well as during zebrafish development (24 to 120hpf). Furthermore, hmox1a, hmox1b and bvrb were significantly induced in zebrafish eleutheroembryos in response to multiple pro-oxidants (cadmium, hemin and tert-butylhydroquinone). Knockdown of Nrf2a, a transcriptional regulator of hmox1a, was demonstrated to inhibit the Cd-mediated induction of hmox1b and bvrb. These results demonstrate distinct mechanisms of hmox and bvr transcriptional regulation in zebrafish, providing initial evidence of the partitioning of function of the hmox paralogs.

Phylogenetic Analysis of Molluscan Metallothioneins: Evolutionary Insight from Crassostrea virginica.

Mechanisms by which organisms genetically adapt to environmental conditions are of fundamental importance to studies of evolutionary biology and environmental physiology. Natural selection acts on existing genetic variation leading to adaptation through selection of new mutations that confer beneficial advantages to populations. The American oyster, Crassostrea virginica, is an excellent model to investigate interactions between environmental and ecological factors as driving forces for natural selection. A great example of this is represented by the diversity of C. virginica metallothioneins (CvMT), metal-binding proteins involved in homeostasis and tolerance, that have resulted from a series of duplication events to produce the greatest structural diversity of MT proteins found in a single species. We present phylogenetic evidence of two distinct ancestral ß-domain MTs that gave rise to a variety of ßß and αß CvMT proteins, as well as CvMT-II proteins consisting solely of one to four α-domains. Furthermore, we annotate the complete locus containing the paralogous CvMT-I, -II, and -IV genes, providing supporting evidence of a hypothesized series of exon and gene duplication events that gave rise to the various CvMT-I and -II isoforms. We also highlight unique MT expression profiles from four separate C. virginica populations to demonstrate differences in gene diversity and copy number which appear to be enriched in southeastern U.S. oyster populations. These observations contribute to a better understanding of the molecular mechanisms leading to adaptation in organisms that experience substantial environmental stress, with a specific focus on evolutionary adaptations of gene structure.

Comparative physiological, biochemical and molecular thermal stress response profiles for two unionid freshwater mussel species.

Freshwater mussels, aquatic keystone species, are in global decline. Long life spans, sedentary lifestyles, and unique reproductive strategies involving obligate parasitic stages make unionid freshwater mussels particularly sensitive to environmental perturbations resulting from global climate change. A greater understanding of the mechanisms by which closely related species differ in their response to thermal challenge is critical for successful conservation and management practices. As such, both an acute heat shock and a chronic warming simulation were conducted in order to evaluate responses between hypothesized thermally tolerant (Villosa lienosa) and thermally sensitive (Villosa nebulosa) freshwater mussels in response to predicted thermal warming. Multiple biological responses were quantified, including mortality, condition index, growth rates, glycogen and triglyceride content, and candidate gene expression. During acute heat shock, both species upregulated HSP90 and HSP70, although V. lienosa showed consistently greater transcript levels during upregulation. This pattern was consistent during the chronic warming simulation, with V. nebulosa showing greater induction of HSP60 Chronic warming stimulated increases in condition index for V. nebulosa; however, declines in growth rates during a recovery period were observed with no concurrent change in tissue glycogen levels. This contrasts with V. lienosa, where tissue glycogen significantly increased during chronic warming, although no response was observed for condition index or growth rates. These biological differences might indicate disparate thermal stress response mechanisms correlated with metabolic demands and resource utilization, and could thus be a factor influencing current ranges of these two species and their ability to cope with future persistent warming in their native habitats.

Transcriptomic evaluation of the American oyster, Crassostrea virginica, deployed during the Deepwater Horizon oil spill: Evidence of an active hydrocarbon response pathway.

Estuarine organisms were impacted by the Deepwater Horizon oil spill which released ∼5 million barrels of crude oil into the Gulf of Mexico in the spring and summer of 2010. Crassostrea virginica, the American oyster, is a keystone species in these coastal estuaries and is routinely used for environmental monitoring purposes. However, very little is known about their cellular and molecular responses to hydrocarbon exposure. In response to the spill, a monitoring program was initiated by deploying hatchery-reared oysters at three sites along the Alabama and Mississippi coast (Grand Bay, MS, Fort Morgan, AL, and Orange Beach, AL). Oysters were deployed for 2-month periods at five different time points from May 2010 to May 2011. Gill and digestive gland tissues were harvested for gene expression analysis and determination of aliphatic and polycyclic aromatic hydrocarbon (PAH) concentrations. To facilitate identification of stress response genes that may be involved in the hydrocarbon response, a nearly complete transcriptome was assembled using Roche 454 and Illumina high-throughput sequencing from RNA samples obtained from the gill and digestive gland tissues of deployed oysters. This effort resulted in the assembly and annotation of 27,227 transcripts comprised of a large assortment of stress response genes, including members of the aryl hydrocarbon receptor (AHR) pathway, Phase I and II biotransformation enzymes, antioxidant enzymes and xenobiotic transporters. From this assembly several potential biomarkers of hydrocarbon exposure were chosen for expression profiling, including the AHR, two cytochrome P450 1A genes (CYP1A-like 1 and CYP1A-like 2), Cu/Zn superoxide dismutase (CuZnSOD), glutathione S-transferase theta (GST theta) and multidrug resistance protein 3 (MRP3). Higher expression levels of GST theta and MRP3 were observed in gill tissues from all three sites during the summer to early fall 2010 deployments. Linear regression analysis indicated a statistically significant relationship between total PAH levels in digestive gland tissue samples with CYP1A-like 2, CuZnSOD, GST theta and MRP3 induction. These observations provide evidence of a potentially conserved AHR pathway in invertebrates and yield new insight into the development of novel biomarkers for use in environmental monitoring activities.

The transcriptional response to oxidative stress during vertebrate development: effects of tert-butylhydroquinone and 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Oxidative stress is an important mechanism of chemical toxicity, contributing to teratogenesis and to cardiovascular and neurodegenerative diseases. Developing animals may be especially sensitive to chemicals causing oxidative stress. The developmental expression and inducibility of anti-oxidant defenses through activation of NF-E2-related factor 2 (NRF2) affect susceptibility to oxidants, but the embryonic response to oxidants is not well understood. To assess the response to chemically mediated oxidative stress and how it may vary during development, zebrafish embryos, eleutheroembryos, or larvae at 1, 2, 3, 4, 5, and 6 days post fertilization (dpf) were exposed to DMSO (0.1%), tert-butylhydroquinone (tBHQ; 10 µM) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 2 nM) for 6 hr. Transcript abundance was assessed by real-time qRT-PCR and microarray. qRT-PCR showed strong (4- to 5-fold) induction of gstp1 by tBHQ as early as 1 dpf. tBHQ also induced gclc (2 dpf), but not sod1, nqo1, or cyp1a. TCDD induced cyp1a but none of the other genes. Microarray analysis showed that 1477 probes were significantly different among the DMSO-, tBHQ-, and TCDD-treated eleutheroembryos at 4 dpf. There was substantial overlap between genes induced in developing zebrafish and a set of marker genes induced by oxidative stress in mammals. Genes induced by tBHQ in 4-dpf zebrafish included those involved in glutathione synthesis and utilization, signal transduction, and DNA damage/stress response. The strong induction of hsp70 determined by microarray was confirmed by qRT-PCR and by use of transgenic zebrafish expressing enhanced green fluorescent protein (EGFP) under control of the hsp70 promoter. Genes strongly down-regulated by tBHQ included mitfa, providing a molecular explanation for the loss of pigmentation in tBHQ-exposed embryos. These data show that zebrafish embryos are responsive to oxidative stress as early as 1 dpf, that responsiveness varies with development in a gene-specific manner, and that the oxidative stress response is substantially conserved in vertebrate animals.

Inhibition of endogenous MTF-1 signaling in zebrafish embryos identifies novel roles for MTF-1 in development.

The metal responsive element-binding transcription factor-1 (MTF-1) responds to changes in cellular zinc levels caused by zinc exposure or disruption of endogenous zinc homeostasis by heavy metals or oxygen-related stress. Here we report the functional characterization of a complete zebrafish MTF-1 in comparison with the previously identified isoform lacking the highly conserved cysteine-rich motif (Cys-X-Cys-Cys-X-Cys) found in all other vertebrate MTF-1 orthologs. In an effort to develop novel molecular tools, a constitutively nuclear dominant-negative MTF-1 (dnMTF-1) was generated as tool for inhibiting endogenous MTF-1 signaling. The in vivo efficacy of the dnMTF-1 was determined by microinjecting in vitro transcribed dnMTF-1 mRNA into zebrafish embryos (1-2 cell stage) followed by transcriptomic profiling using an Agilent 4x44K array on 28- and 36-hpf embryos. A total of 594 and 560 probes were identified as differentially expressed at 28hpf and 36hpf, respectively, with interesting overlaps between timepoints. The main categories of genes affected by the inhibition of MTF-1 signaling were: nuclear receptors and genes involved in stress signaling, neurogenesis, muscle development and contraction, eye development, and metal homeostasis, including novel observations in iron and heme homeostasis. Finally, we investigate both the transcriptional activator and transcriptional repressor role of MTF-1 in potential novel target genes identified by transcriptomic profiling during early zebrafish development.

Knockdown of a zebrafish aryl hydrocarbon receptor repressor (AHRRa) affects expression of genes related to photoreceptor development and hematopoiesis.

The aryl hydrocarbon receptor repressor (AHRR) is a transcriptional repressor of aryl hydrocarbon receptor (AHR) and hypoxia-inducible factor (HIF) and is regulated by an AHR-dependent mechanism. Zebrafish (Danio rerio) possess two AHRR paralogs; AHRRa regulates constitutive AHR signaling during development, whereas AHRRb regulates polyaromatic hydrocarbon-induced gene expression. However, little is known about the endogenous roles and targets of AHRRs. The objective of this study was to elucidate the role of AHRRs during zebrafish development using a loss-of-function approach followed by gene expression analysis. Zebrafish embryos were microinjected with morpholino oligonucleotides against AHRRa or AHRRb to knockdown AHRR protein expression. At 72 h postfertilization (hpf), microarray analysis revealed that the expression of 279 and 116 genes was altered by knockdown of AHRRa and AHRRb, respectively. In AHRRa-morphant embryos, 97 genes were up-regulated and 182 genes were down-regulated. Among the down-regulated genes were several related to photoreceptor function, including cone-specific genes such as several opsins (opn1sw1, opn1sw2, opn1mw1, and opn1lw2), phosphodiesterases (pde6H and pde6C), retinol binding protein (rbp4l), phosducin, and arrestins. Down-regulation was confirmed by RT-PCR and with samples from an independent experiment. The four genes tested (opn1sw1, pde6H, pde6C, and arr3b) were not inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin. AHRRa knockdown also caused up-regulation of embryonic hemoglobin (hbbe3), suggesting a role for AHRR in regulating hematopoiesis. Knockdown of AHRRb caused up-regulation of 31 genes and down-regulation of 85 genes, without enrichment for any specific biological process. Overall, these results suggest that AHRRs may have important roles in development, in addition to their roles in regulating xenobiotic signaling.

Developmental exposure to valproic acid alters the expression of microRNAs involved in neurodevelopment in zebrafish.

Congenital malformations are a prevalent cause of infant mortality in the United States and their induction has been linked to a variety of factors, including exposure to teratogens. However, the molecular mechanisms of teratogenicity are not fully understood. MicroRNAs are an important group of small, non-coding RNAs that regulate mRNA expression. MicroRNA roles in early embryonic development are well established, and their disruption during development can cause abnormalities. We hypothesized that developmental exposure to teratogens such as valproic acid alters microRNA expression profiles in developing embryos. Valproic acid is an anticonvulsant and mood-stabilizing drug used to treat epilepsy, bipolar disorder and migraines. To examine the effects of valproic acid on microRNA expression during development, we used zebrafish embryos as a model vertebrate developmental system. Zebrafish embryos were continuously exposed to valproic acid (1mM) or vehicle control (ethanol) starting from 4h post-fertilization (hpf) and sampled at 48 and 96hpf to determine the miRNA expression profiles prior to and after the onset of developmental defects. At 96hpf, 95% of the larvae showed skeletal deformities, abnormal swimming behavior, and pericardial effusion. Microarray expression profiling was done using Agilent zebrafish miRNA microarrays. Microarray results revealed changes in miRNA expression at both time points. Thirteen miRNAs were differentially expressed at 48hpf and 22 miRNAs were altered at 96hpf. Among them, six miRNAs (miR-16a, 18c, 122, 132, 457b, and 724) were common to both time points. Bioinformatic target prediction and examination of published literature revealed that these miRNAs target several genes involved in the normal functioning of the central nervous system. These results suggest that the teratogenic effects of valproic acid could involve altered miRNA expression.

Identification and expression of multiple CYP1-like and CYP3-like genes in the bivalve mollusk Mytilus edulis.

Various sequencing projects over the last several years have aided the discovery of previously uncharacterized invertebrate sequences, including new cytochrome P450 genes (CYPs). Here we present data on the identification and characterization of two CYP1-like and three CYP3-like genes from the bivalve mollusk Mytilus edulis, and assess their potential as biomarkers based on their responses to several known vertebrate aryl hydrocarbon receptor (AHR) agonists. Quantitative real-time PCR was used to measure CYP transcript levels in digestive gland, labial palps, adductor muscle, gill, foot, and different regions of the mantle. Levels of both CYP1-like genes were highest in digestive gland, whereas labial palps had the highest expression levels of the three CYP3-like genes followed by digestive gland and outer margin of the mantle. Mussels were exposed by injection to the AHR agonists, ß-naphthoflavone (BNF; 25 µg g(-1)), 3,3',4,4',5-polychlorinated biphenyl (PCB126; 2 µg g(-1)), or 6-formylindolo[3,2-b]carbazole (FICZ; 0.1 µg g(-1)), or to Aroclor 1254 (a mixture of PCBs; 50 µg g(-1)) for 24 h, followed by CYP expression analysis. There was no statistically significant change in expression of either of the CYP1-like genes after exposure to the various AHR agonists. The CYP3-like-1 gene was significantly up-regulated by BNF in gill tissues and the CYP3-like-2 gene was up-regulated in digestive gland by PCB126 and in gill tissue by BNF. These results suggest that distinct mechanisms of CYP gene activation could be present in M. edulis, although the importance of the CYP1-like and CYP3-like genes for xenobiotic and endogenous lipids biotransformation requires additional investigation.

Effects of short-term exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin on microRNA expression in zebrafish embryos.

Although many drugs and environmental chemicals are teratogenic, the mechanisms by which most toxicants disrupt embryonic development are not well understood. MicroRNAs, single-stranded RNA molecules of ~22 nt that regulate protein expression by inhibiting mRNA translation and promoting mRNA sequestration or degradation, are important regulators of a variety of cellular processes including embryonic development and cellular differentiation. Recent studies have demonstrated that exposure to xenobiotics can alter microRNA expression and contribute to the mechanisms by which environmental chemicals disrupt embryonic development. In this study we tested the hypothesis that developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a well-known teratogen, alters microRNA expression during zebrafish development. We exposed zebrafish embryos to DMSO (0.1%) or TCDD (5nM) for 1h at 30hours post fertilization (hpf) and measured microRNA expression using several methods at 36 and 60hpf. TCDD caused strong induction of CYP1A at 36hpf (62-fold) and 60hpf (135-fold) as determined by real-time RT-PCR, verifying the effectiveness of the exposure. MicroRNA expression profiles were determined using microarrays (Agilent and Exiqon), next-generation sequencing (SOLiD), and real-time RT-PCR. The two microarray platforms yielded results that were similar but not identical; both showed significant changes in expression of miR-451, 23a, 23b, 24 and 27e at 60hpf. Multiple analyses were performed on the SOLiD sequences yielding a total of 16 microRNAs as differentially expressed by TCDD in zebrafish embryos. However, miR-27e was the only microRNA to be identified as differentially expressed by all three methods (both microarrays, SOLiD sequencing, and real-time RT-PCR). These results suggest that TCDD exposure causes modest changes in expression of microRNAs, including some (miR-451, 23a, 23b, 24 and 27e) that are critical for hematopoiesis and cardiovascular development.

Nrf2b, novel zebrafish paralog of oxidant-responsive transcription factor NF-E2-related factor 2 (NRF2).

NF-E2-related factor 2 (NRF2; also called NFE2L2) and related NRF family members regulate antioxidant defenses by activating gene expression via antioxidant response elements (AREs), but their roles in embryonic development are not well understood. We report here that zebrafish (Danio rerio), an important developmental model species, possesses six nrf genes, including duplicated nrf1 and nrf2 genes. We cloned a novel zebrafish nrf2 paralog, nrf2b. The predicted Nrf2b protein sequence shares several domains with the original Nrf2 (now Nrf2a) but lacks the Neh4 transactivation domain. Zebrafish-human comparisons demonstrate conserved synteny involving nrf2 and hox genes, indicating that nrf2a and nrf2b are co-orthologs of human NRF2. nrf2a and nrf2b displayed distinct patterns of expression during embryonic development; nrf2b was more highly expressed at all stages. Embryos in which Nrf2a expression had been knocked down with morpholino oligonucleotides were more sensitive to tert-butylhydroperoxide but not tert-butylhydroquinone, whereas knockdown of Nrf2b did not affect sensitivity of embryos to either chemical. Gene expression profiling by microarray identified a specific role for Nrf2b as a negative regulator of several genes, including p53, cyclin G1, and heme oxygenase 1, in embryos. Nrf2a and Nrf2b exhibited different mechanisms of cross-talk with the Ahr2 signaling pathway. Together, these results demonstrate distinct roles for nrf2a and nrf2b, consistent with subfunction partitioning, and identify a novel negative regulatory role for Nrf2b during development. The identification of zebrafish nrf2 co-orthologs will facilitate new understanding of the multiple roles of NRF2 in protecting vertebrate embryos from oxidative damage.

Transcriptomic assessment of resistance to effects of an aryl hydrocarbon receptor (AHR) agonist in embryos of Atlantic killifish (Fundulus heteroclitus) from a marine Superfund site.

BACKGROUND: Populations of Atlantic killifish (Fundulus heteroclitus) have evolved resistance to the embryotoxic effects of polychlorinated biphenyls (PCBs) and other halogenated and nonhalogenated aromatic hydrocarbons that act through an aryl hydrocarbon receptor (AHR)-dependent signaling pathway. The resistance is accompanied by reduced sensitivity to induction of cytochrome P450 1A (CYP1A), a widely used biomarker of aromatic hydrocarbon exposure and effect, but whether the reduced sensitivity is specific to CYP1A or reflects a genome-wide reduction in responsiveness to all AHR-mediated changes in gene expression is unknown. We compared gene expression profiles and the response to 3,3',4,4',5-pentachlorobiphenyl (PCB-126) exposure in embryos (5 and 10 dpf) and larvae (15 dpf) from F. heteroclitus populations inhabiting the New Bedford Harbor, Massachusetts (NBH) Superfund site (PCB-resistant) and a reference site, Scorton Creek, Massachusetts (SC; PCB-sensitive). RESULTS: Analysis using a 7,000-gene cDNA array revealed striking differences in responsiveness to PCB-126 between the populations; the differences occur at all three stages examined. There was a sizeable set of PCB-responsive genes in the sensitive SC population, a much smaller set of PCB-responsive genes in NBH fish, and few similarities in PCB-responsive genes between the two populations. Most of the array results were confirmed, and additional PCB-regulated genes identified, by RNA-Seq (deep pyrosequencing). CONCLUSIONS: The results suggest that NBH fish possess a gene regulatory defect that is not specific to one target gene such as CYP1A but rather lies in a regulatory pathway that controls the transcriptional response of multiple genes to PCB exposure. The results are consistent with genome-wide disruption of AHR-dependent signaling in NBH fish.

Modelling interactions of acid-base balance and respiratory status in the toxicity of metal mixtures in the American oyster Crassostrea virginica.

Heavy metals, such as copper, zinc and cadmium, represent some of the most common and serious pollutants in coastal estuaries. In the present study, we used a combination of linear and artificial neural network (ANN) modelling to detect and explore interactions among low-dose mixtures of these heavy metals and their impacts on fundamental physiological processes in tissues of the Eastern oyster, Crassostrea virginica. Animals were exposed to Cd (0.001-0.400 microM), Zn (0.001-3.059 microM) or Cu (0.002-0.787 microM), either alone or in combination for 1 to 27 days. We measured indicators of acid-base balance (hemolymph pH and total CO(2)), gas exchange (Po(2)), immunocompetence (total hemocyte counts, numbers of invasive bacteria), antioxidant status (glutathione, GSH), oxidative damage (lipid peroxidation; LPx), and metal accumulation in the gill and the hepatopancreas. Linear analysis showed that oxidative membrane damage from tissue accumulation of environmental metals was correlated with impaired acid-base balance in oysters. ANN analysis revealed interactions of metals with hemolymph acid-base chemistry in predicting oxidative damage that were not evident from linear analyses. These results highlight the usefulness of machine learning approaches, such as ANNs, for improving our ability to recognize and understand the effects of sub-acute exposure to contaminant mixtures.

The tryptophan photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) binds multiple AHRs and induces multiple CYP1 genes via AHR2 in zebrafish.

The tryptophan photooxidation product 6-formylindolo[3,2-b]carbazole (FICZ) has been proposed as a physiological ligand for the mammalian aryl hydrocarbon receptor (AHR), which it binds with high-affinity, inducing expression of cytochrome P450 1A1 (CYP1A1). We investigated whether the response to FICZ is evolutionarily conserved in vertebrates by measuring FICZ binding to two zebrafish AHRs (AHR1B and AHR2) and its ability to induce zebrafish CYP1 genes (CYP1A, CYP1B1, CYP1C1, CYP1C2, and CYP1D1) in vivo. Exposure of zebrafish embryos (48 h-post-fertilization; hpf) to 10 nM FICZ for 6h caused strong induction of CYP1A mRNA and a statistically significant but modest induction of CYP1B1 and CYP1C1. Neither CYP1C2 nor CYP1D1 expression was induced by FICZ under the conditions of dose, time or developmental stage examined here. CYP1A induction was significantly greater after 6 h than after 12 h of exposure to FICZ, suggesting a rapid degradation of inducer. The 6-h EC(50) values for induction of CYP1A and CYP1B1 by FICZ were 0.6 and 0.5 nM compared to 72-h EC(50) values of 2.3 and 2.7 nM for PCB126, indicating that in zebrafish embryos FICZ is a more potent inducer than PCB126. FICZ at 10 nM was able to completely displace binding of 2,3,7,8-tetrachloro-1,6[3H]-dibenzo-p-dioxin to in vitro-expressed zebrafish AHR2 and AHR1B. Inhibition of AHR2 translation in zebrafish embryos by an AHR2-specific morpholino antisense oligonucleotide decreased the induction of CYP1A and CYP1B1 by FICZ and by PCB126. Together, these results demonstrate that FICZ is a potent AHR agonist in zebrafish, inducing expression of multiple CYP1 genes largely through AHR2. Evolutionary conservation of the response to FICZ is consistent with a possible role as an endogenous signaling molecule acting through the AHR.

Distinct roles of two zebrafish AHR repressors (AHRRa and AHRRb) in embryonic development and regulating the response to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The aryl hydrocarbon receptor (AHR) repressor (AHRR), an AHR-related basic helix-loop-helix/Per-AHR nuclear translocator-Sim protein, is regulated by an AHR-dependent mechanism and acts as a transcriptional repressor of AHR function. Resulting from a teleost-specific genome duplication, zebrafish have two AHRR genes (AHRRa and AHRRb), but their functions in vivo are not well understood. We used antisense morpholino oligonucleotides (MOs) in zebrafish embryos and a zebrafish liver cell line (ZF-L) to characterize the interaction of AHRRs and AHRs in normal embryonic development, AHR signaling, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. Zebrafish embryos exposed to TCDD (2 and 8nM) during early development showed strong induction of CYP1A, AHRRa, and AHRRb at 48 and 72 hours post-fertilization (hpf). An MO targeting AHR2 inhibited TCDD-induced expression of CYP1A, AHRRa, and AHRRb by 84-95% in 48 hpf embryos, demonstrating a primary role for AHR2 in mediating AHRR induction. Dual MO knockdown of both AHRRs in ZF-L cells enhanced TCDD induction of CYP1A, but not other CYP1 genes. In embryos, dual knockdown of AHRRs, or knockdown of AHRRb alone, enhanced the induction of CYP1A, CYP1B1, and CYP1C1 by TCDD and decreased the constitutive expression of Sox9b. In contrast, knockdown of AHRRa did not affect Sox9b expression or CYP1 inducibility. Embryos microinjected with each of two different MOs targeting AHRRa and exposed to dimethyl sulfoxide (DMSO) displayed developmental phenotypes resembling those typical of TCDD-exposed embryos (pericardial edema and lower jaw malformations). In contrast, no developmental phenotypes were observed in DMSO-exposed AHRRb morphants. These data demonstrate distinct roles of AHRRa and AHRRb in regulating AHR signaling in vivo and suggest that they have undergone subfunction partitioning since the teleost-specific genome duplication.

New cytochrome P450 1B1, 1C2 and 1D1 genes in the killifish Fundulus heteroclitus: Basal expression and response of five killifish CYP1s to the AHR agonist PCB126.

Knowledge of the complement of cytochrome P450 (CYP) genes is essential to understanding detoxification and bioactivation mechanisms for organic contaminants. We cloned three new CYP1 genes, CYP1B1, CYP1C2 and CYP1D1, from the killifish Fundulus heteroclitus, an important model in environmental toxicology. Expression of the new CYP1s along with previously known CYP1A and CYP1C1 was measured by qPCR in eight different organs. Organ distribution was similar for the two CYP1Cs, but otherwise patterns and extent of expression differed among the genes. The AHR agonist 3,3',4,4',5-pentachlorobiphenyl (PCB126) (31 pmol/g fish) induced expression of CYP1A and CYP1B1 in all organs examined, while CYP1C1 was induced in all organs except testis. The largest changes in response to PCB126 were induction of CYP1A in testis (approximately 700-fold) and induction of CYP1C1 in liver (approximately 500-fold). CYP1B1 in liver and gut, CYP1A in brain and CYP1C1 in gill also were induced strongly by PCB126 (> 100-fold). CYP1C1 expression levels were higher than CYP1C2 in almost all tissues and CYP1C2 was much less responsive to PCB126. In contrast to the other genes, CYP1D1 was not induced by PCB126 in any of the organs. The organ-specific response of CYP1s to PCB126 implies differential involvement in effects of halogenated aromatic hydrocarbons in different organs. The suite of inducible CYP1s could enhance the use of F. heteroclitus in assessing aquatic contamination by AHR agonists. Determining basal and induced levels of protein and the substrate specificity for all five CYP1s will be necessary to better understand their roles in chemical effects and physiology.