John J Stegeman

Woods Hole Oceanographic Institution, Falmouth, Massachusetts, United States

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Publications (306)893.66 Total impact

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    ABSTRACT: Atlantic killifish (Fundulus heteroclitus) thrive in New Bedford Harbor (NBH), MA, highly contaminated with polychlorinated biphenyls (PCBs). Resident killifish have evolved tolerance to dioxin-like (DL) PCBs, whose toxic effects through the aryl hydrocarbon receptor (AhR) are well studied. In NBH, non-dioxin like PCBs (NDL PCBs), which lack activity toward the AhR, vastly exceed levels of DL congeners yet how killifish counter NDL toxic effects has not been explored. In mammals and fish, NDL PCBs are potent activators of ryanodine receptors (RyR), Ca2+ release channels necessary for a vast array of physiological processes. In the current study we compared the expression and function of RyR related pathways in NBH killifish with killifish from the reference site at Scorton Creek (SC, MA). Relative to the SC fish, adults from NBH displayed increased levels of skeletal muscle RyR1 protein, and increased levels of FK506-binding protein 12 kDa (FKBP12), an accessory protein essential for NDL PCB-triggered changes in RyR channel function. In accordance with increased RyR1 levels, NBH killifish displayed increased maximal ligand binding, increased maximal response to Ca2+ activation and increased maximal response to activation by the NDL PCB congener PCB 95. Compared to SC, NBH embryos and larvae had increased levels of mtor and ryr2 transcripts at multiple stages of development, and generations, while levels of serca2 were decreased at 9 days post-fertilization in the F1 and F2 generations. These findings suggest that there are compensatory and heritable changes in RyR mediated Ca2+ signaling proteins or potential signaling partners in NBH killifish.
    Aquatic Toxicology. 12/2014;
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    ABSTRACT: Killifish survive and reproduce in the New Bedford Harbor (NBH) in Massachusetts (MA), USA, a site severely contaminated with polychlorinated biphenyls (PCBs) for decades. Levels of 22 different PCB congeners were analyzed in liver from killifish collected in 2008. Concentrations of dioxin-like PCBs in liver of NBH killifish were ∼400 times higher, and the levels of non-dioxin-like PCBs ∼3000 times higher than in killifish from a reference site, Scorton Creek (SC), MA. The NBH killifish are known to be resistant to the toxicity of dioxin-like compounds and to have a reduced aryl hydrocarbon receptor (AhR) signaling response. Little is known about the responses of these fish to non-dioxin-like PCBs, which are at extraordinarily high levels in NBH fish. In mammals, some non-dioxin-like PCB congeners act through nuclear receptor 1I2, the pregnane-X-receptor (PXR). To explore this pathway in killifish, a PXR cDNA was sequenced and its molecular phylogenetic relationship to other vertebrate PXRs was determined. Killifish were also collected in 2009 from NBH and SC, and after four months in the laboratory they were injected with a single dose of either the dioxin-like PCB 126 (an AhR agonist) or the non-dioxin-like PCB 153 (a mammalian PXR agonist). Gills and liver were sampled three days after injection and transcript levels of genes encoding PXR, cytochrome P450 3A (CYP3A), P-glycoprotein (Pgp), AhR2 and cytochrome P450 1A (CYP1A) were measured by quantitative PCR. As expected, there was little effect of PCB exposure on mRNA expression of AhR2 or CYP1A in liver and gills of NBH fish. In NBH fish, but not in SC fish, there was increased mRNA expression of hepatic PXR, CYP3A and Pgp upon exposure to either of the two PCB congeners. However, basal PXR and Pgp mRNA levels in liver of NBH fish were significantly lower than in SC fish. A different pattern was seen in gills, where there were no differences in basal mRNA expression of these genes between the two populations. In SC fish, but not in NBH fish, there was increased mRNA expression of branchial PXR and CYP3A upon exposure to PCB126 and of CYP3A upon exposure to PCB153. The results suggest a difference between the two populations in non-AhR transcription factor signaling in liver and gills, and that this could involve killifish PXR. It also implies possible cross-regulatory interactions between that factor (presumably PXR) and AhR2 in liver of these fish.
    Aquatic Toxicology. 12/2014;
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    ABSTRACT: Ligand-activated receptors regulate numerous genes, and mediate effects of a broad set of endogenous and exogenous chemicals in vertebrates. Understanding the roles of these transcription factors in zebrafish (Danio rerio) is important to the use of this non-mammalian model in toxicological, pharmacological and carcinogenesis research. Response to a potential agonist for the pregnane X receptor (Pxr) (pregnenolone PN) was examined in developing zebrafish, to assess involvement of Pxr in regulation of selected genes, including genes in cytochrome P450 subfamilies CYP2 and CYP3. We also examined interaction of Pxr and the aryl hydrocarbon receptor (Ahr) signaling pathways. PN caused a dose-dependent increase in mRNA levels of pxr, ahr2, CYP1A, CYP2AA1, CYP2AA12, CYP3A65 and CYP3C1, most of which peaked at 3 μM PN. The well-known Ahr agonist 3,3',4,4',5-pentachlorobiphenyl (PCB126) also up-regulated expression of pxr, ahr2, CYP1A, CYP2AA12, CYP3A65 and CYP3C1 in a dose-dependent manner. Inhibition of pxr translation by morpholino antisense oligonucleotides (MO) suppressed PN-induced expression of pxr, ahr2, CYP3A65 and CYP3C1 genes. Levels of CYP2AA1 and CYP2AA12 mRNA were increased in the control-MO group exposed to PN; this was prevented by knocking down Pxr. Similarly, Ahr2-MO treatment blocked PCB126-induced mRNA expression of pxr, CYP1A, CYP2AA12, CYP3A65 and CYP3C1. The present study shows self-regulation of pxr by PN in developing zebrafish. Selected zebrafish CYP1, CYP2 (including several CYP2AAs) and CYP3 genes appear to be under the regulation of both Pxr and Ahr2. © The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.
    Toxicological sciences : an official journal of the Society of Toxicology. 11/2014;
  • Saimi Tokunaga, John J Stegeman
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    ABSTRACT: In the course of detecting nuclear transcription factors by electrophoretic mobility shift assay using digoxigenin (DIG)-labeled probes, we encountered a problem with a considerable nonspecific shift band in negative control lanes from which protein extracts were omitted. This nonspecific shift band can interfere with the detection of the desired target protein. Purification of the DIG-labeled probes by removing unincorporated DIG-labeled nucleotides did not resolve the problem. However, the introduction of an additional step of heating at 95°C for 5 min and subsequent re-annealing after DIG-labeled probe synthesis eliminated these nonspecific shift bands and allowed accurate analysis of the target protein.
    Analytical Biochemistry 07/2014; · 2.58 Impact Factor
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    ABSTRACT: 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.
    PLoS ONE 01/2014; 9(11):e113158. · 3.53 Impact Factor
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    ABSTRACT: The cardiovascular system is one of the most characteristic and important targets for developmental toxicity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in fish larvae. However, knowledge of the mechanism of TCDD-induced edema after heterodimerization of aryl hydrocarbon receptor type 2 (AHR2) and AHR nuclear translocator type 1 (ARNT1) is still limited. In the present study, microscopic analysis with a high-speed camera revealed that TCDD increased the size of a small cavity between the heart and body wall in early eleutheroembryos, a toxic effect that we designate as precardiac edema. A concentration–response curve for precardiac edema at 2 days post fertilization (dpf) showed close similarity to that for conventional pericardial edema at 3 dpf. Precardiac edema caused by TCDD was reduced by morpholino knockdown of AHR2 and ARNT1, as well as by an antioxidant (ascorbic acid). A selective inhibitor of cyclooxygenase type 2 (COX2), NS398, also markedly inhibited TCDD-induced precardiac edema. A thromboxane receptor (TP) antagonist, ICI-192,605 almost abolished TCDD-induced precardiac edema and this effect was canceled by U46619, a TP agonist, which was not influential in the action of TCDD by itself. Knockdown of COX2b and thromboxane A synthase 1 (TBXS), but not COX2a, strongly reduced TCDD-induced precardiac edema. Knockdown of COX2b was without effect on mesencephalic circulation failure caused by TCDD. The edema by TCDD was also inhibited by knockdown of c-mpl, a thrombopoietin receptor necessary for thromobocyte production. Finally, induction of COX2b, but not COX2a, by TCDD was seen in eleutheroembryos at 3 dpf. These results suggest a role of the COX2b–thromboxane pathway in precardiac edema formation following TCDD exposure in developing zebrafish.
    Aquatic Toxicology. 01/2014; 154:19–26.
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    ABSTRACT: Sterol 14α-demethylase (cytochrome P450 51, CYP51, P45014DM) is a microsomal enzyme that in eukaryotes catalyzes formation of sterols essential for cell membrane function and as precursors in biosynthesis of steroid hormones. Functional properties of CYP51s are unknown in non-mammalian deuterostomes. PCR-cloning and sequencing and computational analyses (homology modeling and docking) addressed CYP51 in zebrafish Danio rerio, the reef fish sergeant major Abudefduf saxatilis, and the sea urchin Strongylocentrotus purpuratus. Following N-terminal amino acid modification, zebrafish CYP51 was expressed in Escherichia coli, and lanosterol 14α-demethylase activity and azole inhibition of CYP51 activity were characterized using GC/MS. Molecular phylogeny positioned S. purpuratus CYP51 at the base of the deuterostome clade. In zebrafish, CYP51 is expressed in all organs examined, most strongly in intestine. The recombinant protein bound lanosterol and catalyzed 14α-demethylase activity, at 3.2nmol/min/nmol CYP51. The binding of azoles to zebrafish CYP51 gave KS (dissociation constant) values of 0.26μM for ketoconazole and 0.64μM for propiconazole. Displacement of carbon monoxide also indicated zebrafish CYP51 has greater affinity for ketoconazole. Docking to homology models showed that lanosterol docks in fish and sea urchin CYP51s with an orientation essentially the same as in mammalian CYP51. Docking of ketoconazole indicates it would inhibit fish and sea urchin CYP51s. Biochemical and computational analyses are consistent with lanosterol being a substrate for early deuterostome CYP51s. The results expand the phylogenetic view of animal CYP51, with evolutionary, environmental and therapeutic implications.
    Biochimica et Biophysica Acta 12/2013; · 4.66 Impact Factor
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    ABSTRACT: The pregnane X receptor (PXR) (nuclear receptor NR1I2) is a ligand activated transcription factor, mediating responses to diverse xenobiotic and endogenous chemicals. The properties of PXR in fish are not fully understood. Here we report on cloning and characterization of full-length PXR of zebrafish, Danio rerio, and pxr expression in vivo. Initial efforts gave a cDNA encoding a 430 amino acid protein identified as zebrafish pxr by phylogenetic and synteny analysis. The sequence of the cloned Pxr DNA binding domain (DBD) was highly conserved, with 74% identity to human PXR-DBD, while the ligand-binding domain (LBD) of the cloned sequence was only 44% identical to human PXR-LBD. Sequence variation among clones in the initial effort prompted sequencing of multiple clones from a single fish. There were two prominent variants, one sequence with S183, Y218 and H383 and the other with I183, C218 and N383, which we designate as alleles pxr*1 (nr1i2*1) and pxr*2 (nr1i2*2), respectively. In COS-7 cells co-transfected with a PXR-responsive reporter gene, the full-length Pxr*1 (the more common variant) was activated by known PXR agonists clotrimazole and pregnenolone 16α-carbonitrile but to a lesser extent than the full-length human PXR. Activation of full-length Pxr*1 was only 10% of that with the Pxr*1 LBD. Quantitative real time PCR analysis showed prominent expression of pxr in liver and eye, as well as brain and intestine of adult zebrafish. The pxr was expressed in heart and kidney at levels similar to that in intestine. The expression of pxr in liver was weakly induced by ligands for mammalian PXR or constitutive androstane receptor (NR1I3). The results establish a foundation for PXR studies in this vertebrate model. PXR allelic variation and the differences between the full-length PXR and the LBD in reporter assays have implications for assessing the action of PXR ligands in zebrafish.
    Aquatic toxicology (Amsterdam, Netherlands) 09/2013; 142-143C:447-457. · 3.12 Impact Factor
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    ABSTRACT: Embryonic development involves dramatic changes in cell proliferation and differentiation that must be highly coordinated and tightly regulated. Cellular redox balance is critical for cell fate decisions, but it is susceptible to disruption by endogenous and exogenous sources of oxidative stress. The most abundant endogenous non-protein antioxidant defense molecule is the tri-peptide glutathione (g-glutamyl-cysteinylglycine, GSH), but the ontogeny of GSH concentration and redox state during early life stages is poorly understood. Here, we describe the GSH redox dynamics during embryonic and early larval development (0-5 days post-fertilization) in the zebrafish (Danio rerio), a model vertebrate embryo. We measured reduced and oxidized glutathione (GSH, GSSG) using HPLC, and calculated the whole embryo total glutathione (GSHT) concentrations and redox potentials (Eh) over 0-120 hours of zebrafish development (including mature oocytes, fertilization, mid-blastula transition, gastrulation, somitogenesis, pharyngula, pre-hatch embryos, and hatched eleutheroembryos). GSHT concentration doubled between 12 hours post fertilization (hpf) and hatching. The GSH Eh increased, becoming more oxidizing during the first 12h, and then oscillated around -190mV through organogenesis, followed by a rapid change, associated with hatching, to a more negative (more reducing) Eh (-220mV). After hatching, Eh stabilized and remained steady through 120hpf. The dynamic changes in GSH redox status and concentration defined discrete windows of development: primary organogenesis, organ differentiation, and larval growth. We identified the set of zebrafish genes involved in the synthesis, utilization, and recycling of GSH, including several novel paralogs, and measured how expression of these genes changes during development. Ontogenic changes in the expression of GSH-related genes support the hypothesis that GSH redox state is tightly regulated early in development. This study provides a foundation for understanding the redox regulation of developmental signaling and investigating the effects of oxidative stress during embryogenesis.
    Free Radical Biology and Medicine 06/2013; · 5.27 Impact Factor
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    ABSTRACT: The cytochrome P450 (CYP) 2 gene family is the largest and most diverse CYP gene family in vertebrates. In zebrafish, we have identified 10 genes in a new subfamily, CYP2AA, which does not show orthology to any human or other mammalian CYP genes. Here we report evolutionary and structural relationships of the 10 CYP2AA genes and expression of the first two genes, CYP2AA1 and CYP2AA2. Parsimony reconstruction of the tandem duplication pattern for the CYP2AA cluster suggests that CYP2AA1, CYP2AA2 and CYP2AA3 likely arose in the earlier duplication events and thus are most diverged in function from the other CYP2AAs. On the other hand, CYP2AA8 and CYP2AA9 are genes that arose in the latest duplication event, implying functional similarity between these two CYPs. A molecular model of CYP2AA1 showing the sequence conservation across the CYP2AA cluster reveals that the regions with the highest variability within the cluster map into CYP2AA1 near the substrate access channels, suggesting differing substrate specificity. Zebrafish CYP2AA1 transcript was expressed predominantly in intestine, while CYP2AA2 was most highly expressed in kidney, suggesting differing roles in physiology. In liver CYP2AA2 expression but not that of CYP2AA1, was increased by 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) and, to a lesser extent, by phenobarbital (PB). In contrast, pregnenolone 16α-carbonitrile (PCN) increased CYP2AA1, but not CYP2AA2 in liver. The results identify a CYP2 subfamily in zebrafish that includes genes apparently induced by PB-type chemicals and PXR agonists, the first concrete in vivo evidence for a PB-type response in fish.
    Toxicology and Applied Pharmacology 05/2013; · 3.98 Impact Factor
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    ABSTRACT: The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.
    Nature 04/2013; 496(7445):311-316. · 38.60 Impact Factor
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    ABSTRACT: The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.
    Nature 04/2013; 496(7445):311-316. · 38.60 Impact Factor
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    David R Nelson, Jared V Goldstone, John J Stegeman
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    ABSTRACT: The neighbourhoods of cytochrome P450 (CYP) genes in deuterostome genomes, as well as those of the cnidarians Nematostella vectensis and Acropora digitifera and the placozoan Trichoplax adhaerens were examined to find clues concerning the evolution of CYP genes in animals. CYP genes created by the 2R whole genome duplications in chordates have been identified. Both microsynteny and macrosynteny were used to identify genes that coexisted near CYP genes in the animal ancestor. We show that all 11 CYP clans began in a common gene environment. The evidence implies the existence of a single locus, which we term the 'cytochrome P450 genesis locus', where one progenitor CYP gene duplicated to create a tandem set of genes that were precursors of the 11 animal CYP clans: CYP Clans 2, 3, 4, 7, 19, 20, 26, 46, 51, 74 and mitochondrial. These early CYP genes existed side by side before the origin of cnidarians, possibly with a few additional genes interspersed. The Hox gene cluster, WNT genes, an NK gene cluster and at least one ARF gene were close neighbours to this original CYP locus. According to this evolutionary scenario, the CYP74 clan originated from animals and not from land plants nor from a common ancestor of plants and animals. The CYP7 and CYP19 families that are chordate-specific belong to CYP clans that seem to have originated in the CYP genesis locus as well, even though this requires many gene losses to explain their current distribution. The approach to uncovering the CYP genesis locus overcomes confounding effects because of gene conversion, sequence divergence, gene birth and death, and opens the way to understanding the biodiversity of CYP genes, families and subfamilies, which in animals has been obscured by more than 600 Myr of evolution.
    Philosophical Transactions of The Royal Society B Biological Sciences 01/2013; 368(1612):20120474. · 6.23 Impact Factor
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    ABSTRACT: Transcription factors in the CNC-bZIP family (NFE2, NRF1, NRF2 and NRF3) regulate genes with a wide range of functions in response to both physiological and exogenous signals, including those indicating changes in cellular redox status. Given their role in helping to maintain cellular homeostasis, it is imperative to understand the expression, regulation, and function of CNC-bZIP genes during embryonic development. We explored the expression and function of six nrf genes (nfe2, nrf1a, nrf1b, nrf2a, nrf2b, and nrf3) using zebrafish embryos as a model system. Analysis by microarray and quantitative RT-PCR showed that genes in the nrf family were expressed throughout development from oocytes to larvae. The spatial expression of nrf3 suggested a role in regulating the development of the brain, brachia and pectoral fins. Knock-down by morpholino anti-sense oligonucleotides suggested that none of the genes were necessary for embryonic viability, but nfe2 was required for proper cellular organization in the pneumatic duct and subsequent swim bladder function, as well as for proper formation of the otic vesicles. nrf genes were induced by the oxidant tert-butylhydroperoxide, and some of this response was regulated through family members Nrf2a and Nrf2b. Our results provide a foundation for understanding the role of nrf genes in normal development and in regulating the response to oxidative stress in vertebrate embryos.
    PLoS ONE 01/2013; 8(10):e79574. · 3.53 Impact Factor
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    ABSTRACT: 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, β-naphthoflavone (BNF; 25μgg(-1)), 3,3',4,4',5-polychlorinated biphenyl (PCB126; 2μgg(-1)), or 6-formylindolo[3,2-b]carbazole (FICZ; 0.1μgg(-1)), or to Aroclor 1254 (a mixture of PCBs; 50μgg(-1)) for 24h, 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.
    Aquatic toxicology (Amsterdam, Netherlands) 11/2012; 128-129C:101-112. · 3.12 Impact Factor
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    ABSTRACT: The teleost swim bladder is assumed a homolog of the tetrapod lung. Both swim bladder and lung are developmental targets of persistent aryl hydrocarbon receptor (AHR(2)) agonists; in zebrafish (Danio rerio) the swim bladder fails to inflate with exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB126). The mechanism for this effect is unknown, but studies have suggested roles of cytochrome P4501 (CYP1) and cyclooxygenase 2 (Cox-2) in some Ahr-mediated developmental effects in zebrafish. We determined relationships between swim bladder inflation and CYP1 and Cox-2 mRNA expression in PCB126-exposed zebrafish embryos. We also examined effects on β-catenin dependent transcription, histological effects, and Ahr2 dependance of the effect of PCB126 on swim bladder using morpholinos targeting ahr2. One-day-old embryos were exposed to waterborne PCB126 or carrier (DMSO) for 24h and then held in clean water until day 4, a normal time for swim bladder inflation. The effects of PCB126 were concentration-dependent with EC50 values of 1.4 to 2.0nM for induction of the CYP1s, 3.7 and 5.1nM (or higher) for cox-2a and cox-2b induction, and 2.5nM for inhibition of swim bladder inflation. Histological defects included a compaction of the developing bladder. Ahr2-morpholino treatment rescued the effect of PCB126 (5nM) on swim bladder inflation and blocked induction of CYP1A, cox-2a, and cox-2b. With 2nM PCB126 approximately 30% of eleutheroembryos(3) failed to inflate the swim bladder, but there was no difference in CYP1 or cox-2 mRNA expression between those embryos and embryos showing inflated swim bladder. Our results indicate that PCB126 blocks swim bladder inflation via an Ahr2-mediated mechanism. This mechanism seems independent of CYP1 or cox-2 mRNA induction but may involve abnormal development of swim bladder cells.
    Toxicology and Applied Pharmacology 10/2012; · 3.98 Impact Factor
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    ABSTRACT: While deep-sea fish accumulate high levels of persistent organic pollutants (POPs), the toxicity associated with this contamination remains unknown. Indeed, the recurrent collection of moribund individuals precludes experimental studies to investigate POP effects in this fauna. We show that precision-cut liver slices (PCLS), an in vitro tool commonly used in human and rodent toxicology, can overcome such limitation. This technology was applied to individuals of the deep-sea grenadier Coryphaenoides rupestris directly upon retrieval from 530-m depth in Trondheimsfjord (Norway). PCLS remained viable and functional for 15 h when maintained in an appropriate culture media at 4 °C. This allowed experimental exposure of liver slices to the model POP 3-methylcholanthrene (3-MC; 25 μM) at levels of hydrostatic pressure mimicking shallow (0.1 megapascal or MPa) and deep-sea (5-15 MPa; representative of 500-1500 m depth) environments. As in shallow water fish, 3-MC induced the transcription of the detoxification enzyme cytochrome P4501A (CYP1A; a biomarker of exposure to POPs). This induction was diminished at elevated pressure, suggesting a limited responsiveness of C. rupestris toward POPs in its native environment. This very first in vitro toxicological investigation on a deep-sea fish opens the route for understanding pollutants effects in this highly exposed fauna.
    Environmental Science & Technology 08/2012; 46(18):10310-6. · 5.48 Impact Factor
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    ABSTRACT: South American cyprinodontiform fish are potential candidates to be used as model biomarker species of exposure in environmental toxicology. The aim of this study was to identify molecular and biochemical biomarkers of pollution using Poecilia vivipara (Poecilidae) and Jenynsia multidentata (Anablepidae). Partial nucleotide sequences for cytochrome P-450 1A (CYP1A), a classical biomarker of exposure to organic contaminants in fish, were identified in P. vivipara and J. multidentata (approximately 650 nucleotides) using degenerated primers and polymerase chain reaction (PCR). These sequences shared approximately 90% identity in the predicted amino acid sequence with the corresponding CYP1A region of Fundulus heteroclitus. Real-time quantitative PCR (RT-qPCR) analysis confirmed that CYP1A transcription was markedly induced in the liver and gills of J. multidentata (approximately185-fold and 20-fold, respectively) and P. vivipara (122-fold and 739-fold, respectively) 24 h after exposure to 1 μM synthetic CYP1A inducer β-naphthoflavone (BNF). At 24 h after injection with 1 μg/g environmental carcinogenic contaminant benzo[a]pyrene (BaP), a decreased total antioxidant capacity against peroxyl radicals was observed both in liver of J. multidentata and gills of P. vivipara. BaP injection in both fish did not produce changes in lipid peroxide (thiobarbituric acid-reactive substances, TBARS) levels, suggesting an absence of an oxidative stress condition. The newly identified CYP1A may thus serve as general biomarker of exposure to organic contaminant in future studies using P. vivipara and J. multidentata. Data also indicate the importance of species-specific differences in biomarker responses in these South American cyprinodontiform fish, suggesting distinct resistance/susceptibility properties to polycyclic aromatic hydrocarbons.
    Journal of Toxicology and Environmental Health Part A 08/2012; 75(16-17):1023-34. · 1.73 Impact Factor
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    ABSTRACT: In fish there are four cytochrome P450 (CYP1) subfamilies: CYP1A, CYP1B, CYP1C, and CYP1D. Here we cloned Poecilia vivipara CYP1A, with an inferred amino acid sequence 91% identical to CYP1A from the killifish Fundulus heteroclitus, another member of the Cypriniformes, and an important model in ecotoxicology. In addition, we examined the expression of CYP1A, CYP1B1, and CYP1C1 by qPCR in liver, gill, and intestine of adult P. vivipara injected with 3-methylcholanthrene (3-MC) or held in clean water (control group) for 24h. All three tissues examined showed basal expression of the three CYP1 genes. CYP1A was most strongly expressed in the liver, while CYP1B1, and CYP1C1 were most strongly expressed in the gill and intestine respectively. 3-MC induced CYP1A, CYP1B1, and CYP1C1 significantly (20-120-fold) in the three organs, consistent with the regulation of CYP1A, CYP1B1 and CYP1C1 via the aryl hydrocarbon receptor. Validation of CYP1 gene biomarkers in fish collected from a contaminated urban mangrove environment was confirmed with significant induction of CYP1A and CYP1C1 in gills (10-15-fold) and CYP1B1 in liver (23-fold), relative to fish from a control site. The responsiveness of these CYP1 genes indicates P. vivipara is suitable as a model for environmental toxicology studies and environmental assessment in Brazil.
    Aquatic toxicology (Amsterdam, Netherlands) 08/2012; 124-125:106-13. · 3.12 Impact Factor

Publication Stats

7k Citations
893.66 Total Impact Points

Institutions

  • 1979–2014
    • Woods Hole Oceanographic Institution
      • Department of Biology
      Falmouth, Massachusetts, United States
  • 2013
    • The University of Tennessee Health Science Center
      • Department of Microbiology, Immunology and Biochemistry
      Memphis, TN, United States
  • 2011
    • Federal University of Rio de Janeiro
      • Instituto de Biofísica Carlos Chagas Filho (IBCCF)
      Rio de Janeiro, Rio de Janeiro, Brazil
  • 2010
    • Uppsala University
      Uppsala, Uppsala, Sweden
  • 2002–2010
    • Rakuno Gakuen University
      • Department of Veterinary Medicine
      Ebetsu, Hokkaido, Japan
  • 2007
    • Boston University
      • Department of Biomedical Engineering
      Boston, MA, United States
  • 2006
    • United States Geological Survey
      Reston, Virginia, United States
    • Singapore-MIT Alliance
      Cambridge, Massachusetts, United States
    • University of Notre Dame
      South Bend, Indiana, United States
  • 2004–2006
    • Fordham University
      New York City, New York, United States
    • Lincoln College USA
      Lincoln, Illinois, United States
  • 2002–2004
    • Marine Biological Laboratory
      Falmouth, Massachusetts, United States
  • 2001–2002
    • University of Alaska Fairbanks
      • • School of Fisheries and Ocean Sciences (SFOS)
      • • Institute of Arctic Biology
      Fairbanks, AK, United States
    • Procter & Gamble
      Cincinnati, Ohio, United States
    • Michigan State University
      • Department of Fisheries and Wildlife
      East Lansing, MI, United States
  • 1998
    • Eawag: Das Wasserforschungs-Institut des ETH-Bereichs
      • Swiss Federal Institute for Environmental Science and Technology (EAWAG) and Swiss Federal Institute of Technology (ETH)
      Duebendorf, Zurich, Switzerland
    • Tel Aviv University
      • Faculty of Life Sciences
      Tel Aviv, Tel Aviv, Israel
  • 1997
    • Virginia Institute of Marine Science
      Gloucester Point, Virginia, United States
    • University of Wisconsin - Milwaukee
      Milwaukee, Wisconsin, United States
  • 1993
    • Lankenau Institute for Medical Research
      Wynnewood, Oklahoma, United States
  • 1989
    • University of Rhode Island
      • Graduate School of Oceanography
      Kingston, RI, United States