Stegeman John J.

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Stegeman

First Name

John J.

ORCID

0000-0001-8204-5357

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Cytochrome P4501A biomarker indication of the timeline of chronic exposure of Barrow’s goldeneyes to residual Exxon Valdez oil

( 2010-11) Esler, Daniel ; Ballachey, Brenda E. ; Trust, Kimberly A. ; Iverson, Samuel A. ; Reed, John A. ; Miles, A. Keith ; Henderson, John D. ; Woodin, Bruce R. ; Stegeman, John J. ; McAdie, Malcolm ; Mulcahy, Daniel M. ; Wilson, Barry W.

We examined hepatic EROD activity, as an indicator of CYP1A induction, in Barrow's goldeneyes captured in areas oiled during the 1989 Exxon Valdez spill and those from nearby unoiled areas. We found that average EROD activity differed between areas during 2005, although the magnitude of the difference was reduced relative to a previous study from 1996/97, and we found that areas did not differ by 2009. Similarly, we found that the proportion of individuals captured from oiled areas with elevated EROD activity ( 2 times unoiled average) declined from 41% in winter 1996/97 to 10% in 2005 and 15% in 2009. This work adds to a body of literature describing the timelines over which vertebrates were exposed to residual Exxon Valdez oil and indicates that, for Barrow's goldeneyes in Prince William Sound, exposure persisted for many years with evidence of substantially reduced exposure by 2 decades after the spill.
Isolation and phylogeny of novel cytochrome P450 genes from tunicates (Ciona spp.) : a CYP3 line in early deuterostomes?

( 2006-04-04) Verslycke, Tim A. ; Goldstone, Jared V. ; Stegeman, John J.

Cytochromes P450 (CYPs) form a gene superfamily involved in the biotransformation of numerous endogenous and exogenous natural and synthetic compounds. In humans, CYP3A4 is regarded as one of the most important CYPs due to its abundance in liver and its capacity to metabolize more than 50% of all clinically used drugs. It has been suggested that all CYP3s arose from a common ancestral gene lineage that diverged between 800 and 1100 million years ago, before the deuterostome-protostome split. While CYP3s are well known in mammals and have been described in lower vertebrates, they have not been reported in non-vertebrate deuterostomes. Members of the genus Ciona belong to the tunicates, whose lineage is thought to be the most basal among the chordates, and from which the vertebrate line diverged. Here we describe the cloning, exon-intron structure, phylogeny, and estimated expression of four novel genes from Ciona intestinalis. We also describe the gene structure and phylogeny of homologous genes in Ciona savignyi. Comparing these genes with other members of the CYP clan 3, show that the Ciona sequences bear remarkable similarity to vertebrate CYP3A genes, and may be an early deuterostome CYP3 line.
PCBs : exposures, effects, remediation, and regulation with special emphasis on PCBs in schools

( 2015-11) Hunt, Gary ; Stegeman, John J. ; Robertson, Larry

The Eighth International PCB Workshop: PCBs in Schools was held in Woods Hole, MA, October 5-9, 2014, and was attended by more than 130 scientists and other interested persons, including citizen’s groups and concerned parents. The program included a wide range of thematic areas. Presentations addressed essential questions and progress toward understanding mechanisms of PCB toxication and risks of PCB exposure. Presentations were also held illuminating several key PCB contamination problems.
Resistance to Cyp3a induction by polychlorinated biphenyls, including non-dioxin-like PCB153, in gills of killifish (Fundulus heteroclitus) from New Bedford Harbor

(Elsevier, 2021-01-08) Celander, Malin C. ; Goldstone, Jared V. ; Brun, Nadja R. ; Clark, Bryan W. ; Jayaraman, Saro ; Nacci, Diane E. ; Stegeman, John J.

Previous reports suggested that non-dioxin-like (NDL) PCB153 effects on cytochrome P450 3A (Cyp3a) expression in Atlantic killifish (Fundulus heteroclitus) gills differed between F0 generation fish from a PCB site (New Bedford Harbor; NBH) and a reference site (Scorton Creek; SC). Here, we examined effects of PCB153, dioxin-like (DL) PCB126, or a mixture of both, on Cyp3a56 mRNA in killifish generations removed from the wild, without environmental PCB exposures. PCB126 effects in liver and gills differed between populations, as expected. Gill Cyp3a56 was not affected by either congener in NBH F2 generation fish, but was induced by PCB153 in SC F1 fish, with females showing a greater response. PCB153 did not affect Cyp3a56 in liver of either population. Results suggest a heritable resistance to NDL-PCBs in killifish from NBH, in addition to that reported for DL PCBs. Induction of Cyp3a56 in gills may be a biomarker of exposure to NDL PCBs in fish populations that are not resistant to PCBs.
Environmental health and the coastal zone

(National Institute of Environmental Health Sciences, 2002-11) Stegeman, John J. ; Solow, Andrew R.

Reconciling coastal development and the maintenance of a quality environment represent an enormous management challenge to both public and private interests. Wise management of coastal areas will require an understanding of the nature of dynamic physical, chemical, and biological interactions in the coastal zone, knowledge of how changes in other components of the Earth system affect coastal zones and their role in global cycles, and insight into how to best use these areas as coastal populations increase. Maintaining the integrity and health of the coastal zone is essential to the quality of marine biological resources and, ultimately, of human life.
Chemical impacts in fish and shellfish from Cape Cod and Massachusetts Bays

(Barnstable County Department of Health and Environment, 1998) Moore, Michael J. ; Smolowitz, Roxanna M. ; Leavitt, Dale F. ; Stegeman, John J.

Mununichogs, soft shell clams, and blue mussels from some or all of 10 sites in Boston Harbor and Massachusetts and Cape Cod Bays were examined histologically: a suite of pathological changes previously known to be associated with chemical contamination were found in animals from the more contaminated sites. In particular, liver tumors were evident in 14% of the adult mununichogs from the Island End River, a tributary of the Mystic River in Boston Harbor. Additionally, a number of pathologies previously shown to be associated with chemical exposure were seen in the two bivalve species at a number of contaminated sites. Induction of cytochrome P45() IA (CYPIA) was also seen in muntntichogs from the more contaminated sites: CYPIA induction is a biochemical change associated with exposure to dioxin and other planar halogenated and aromatic hydrocarbons. These findings suggest that there are measurable biochemical and pathological changes in intertidal fish and shellfish from the more contaminated parts of the Massachusetts Bays system. These types of changes were less evident in the two reference sites in Cape Cod Bay.
The new vertebrate CYP1C family : cloning of new subfamily members and phylogenetic analysis

( 2005-03-11) Godard, Celine A. J. ; Goldstone, Jared V. ; Said, Maya R. ; Dickerson, Richard L. ; Woodin, Bruce R. ; Stegeman, John J.

Two novel CYP1 genes from teleost fish constituting a new subfamily have been cloned. These paralogous sequences are designated CYP1C1 and CYP1C2. Both genes were initially obtained from untreated scup Stenotomus chrysops tissues by RT-PCR and RACE. Scup CYP1C1 and CYP1C2 code for 524 and 525 amino acids, respectively, and share 80-81% identity at the nucleotide and amino acid levels. Orthologues of CYP1C1 and CYP1C2 were identified in genome databases for other fish species, and both CYP1B1 and CYP1C1 were cloned from zebrafish (Danio rerio). Phylogenetic analysis shows that CYP1Cs and CYP1Bs constitute a sister clade to the CYP1As. Analysis of sequence domains likely to have functional significance suggests the two CYP1Cs in scup may have catalytic functions and/or substrate specificity that differ from each other and from those of mammalian CYP1Bs or CYP1As. RT-PCR results indicate that CYP1C1 and CYP1C2 are variously expressed in several scup organs.
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

( 2009-04-30) Zanette, Juliano ; Jenny, Matthew J. ; Goldstone, Jared V. ; Woodin, Bruce R. ; Watka, Lauren A. ; Bainy, Afonso C. D. ; Stegeman, John J.

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 (~700-fold) and induction of CYP1C1 in liver (~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.
Centers for Oceans and Human Health : contributions to an emerging discipline

(BioMed Central, 2008-11-07) Laws, Edward A. ; Fleming, Lora E. ; Stegeman, John J.

The oceans are the dominant feature of the planet and are fundamentally linked to human history and to human health. Concerns about the impact of the oceans on human health can be traced to ancient times. Jewish law prohibited the consumption of shellfish, probably reflecting the fact that filter-feeding bivalves can accumulate pathogens and toxins. The Portuguese explorer Pedro Fernandes de Queirós described symptoms associated with ciguatera fish poisoning after eating Caribbean sea bream in 1606, and several of British explorer James Cook's crew experienced similar symptoms after eating fish off the coast of Vanuatu in 1774 [1]. Roughly 1,200 people died from the consumption of fish and shellfish contaminated with methyl mercury in Minamata (Japan) during the 20th century; an even larger number were affected by chronic long-term neurotoxicological impacts [2]. A tsunami caused by an undersea earthquake on December 26, 2004 killed more than 225,000 people in eleven countries bordering the Indian Ocean; and more than 1,400 people died within a single day when the storm surge generated by Hurricane Katrina overwhelmed the New Orleans levee system on August 29, 2005 [3]. Looking ahead, the International Panel on Climate Change has projected a sea level rise of as much as 88 cm during the 21st century as a result of global warming [4], with major implications for the welfare and sustainability of coastal communities.
Concerning P450 evolution: structural analyses support bacterial origin of sterol 14α-demethylases

(Oxford University Press, 2020-10-08) Lamb, David C. ; Hargrove, Tatiana Y. ; Zhao, Bin ; Wawrzak, Zdzislaw ; Goldstone, Jared V. ; Nes, William David ; Kelly, Steven L. ; Waterman, Michael R. ; Stegeman, John J. ; Lepesheva, Galina I.

Sterol biosynthesis, primarily associated with eukaryotic kingdoms of life, occurs as an abbreviated pathway in the bacterium Methylococcus capsulatus. Sterol 14α-demethylation is an essential step in this pathway and is catalyzed by cytochrome P450 51 (CYP51). In M. capsulatus, the enzyme consists of the P450 domain naturally fused to a ferredoxin domain at the C-terminus (CYP51fx). The structure of M. capsulatus CYP51fx was solved to 2.7 Å resolution and is the first structure of a bacterial sterol biosynthetic enzyme. The structure contained one P450 molecule per asymmetric unit with no electron density seen for ferredoxin. We connect this with the requirement of P450 substrate binding in order to activate productive ferredoxin binding. Further, the structure of the P450 domain with bound detergent (which replaced the substrate upon crystallization) was solved to 2.4 Å resolution. Comparison of these two structures to the CYP51s from human, fungi, and protozoa reveals strict conservation of the overall protein architecture. However, the structure of an “orphan” P450 from nonsterol-producing Mycobacterium tuberculosis that also has CYP51 activity reveals marked differences, suggesting that loss of function in vivo might have led to alterations in the structural constraints. Our results are consistent with the idea that eukaryotic and bacterial CYP51s evolved from a common cenancestor and that early eukaryotes may have recruited CYP51 from a bacterial source. The idea is supported by bioinformatic analysis, revealing the presence of CYP51 genes in >1,000 bacteria from nine different phyla, >50 of them being natural CYP51fx fusion proteins.
Metabolic arsenal of giant viruses: host hijack or self-use?

(eLife Sciences Publications, 2022-07-08) Brahim Belhaouari, Djamal ; Pires De Souza, Gabriel Augusto ; Lamb, David C. ; Kelly, Steven L. ; Goldstone, Jared V. ; Stegeman, John J. ; Colson, Philippe ; La Scola, Bernard ; Aherfi, Sarah

Viruses generally are defined as lacking the fundamental properties of living organisms in that they do not harbor an energy metabolism system or protein synthesis machinery. However, the discovery of giant viruses of amoeba has fundamentally challenged this view because of their exceptional genome properties, particle sizes and encoding of the enzyme machinery for some steps of protein synthesis. Although giant viruses are not able to replicate autonomously and still require a host for their multiplication, numerous metabolic genes involved in energy production have been recently detected in giant virus genomes from many environments. These findings have further blurred the boundaries that separate viruses and living organisms. Herein, we summarize information concerning genes and proteins involved in cellular metabolic pathways and their orthologues that have, surprisingly, been discovered in giant viruses. The remarkable diversity of metabolic genes described in giant viruses include genes encoding enzymes involved in glycolysis, gluconeogenesis, tricarboxylic acid cycle, photosynthesis, and β-oxidation. These viral genes are thought to have been acquired from diverse biological sources through lateral gene transfer early in the evolution of Nucleo-Cytoplasmic Large DNA Viruses, or in some cases more recently. It was assumed that viruses are capable of hijacking host metabolic networks. But the giant virus auxiliary metabolic genes also may represent another form of host metabolism manipulation, by expanding the catalytic capabilities of the host cells especially in harsh environments, providing the infected host cells with a selective evolutionary advantage compared to non-infected cells and hence favoring the viral replication. However, the mechanism of these genes' functionality remains unclear to date.
Announcing the Minderoo – Monaco Commission on plastics and human health

(Ubiquity Press, 2022-08-25) Landrigan, Philip J. ; Raps, Hervé ; Symeonides, Christos ; Chiles, Thomas ; Cropper, Maureen ; Enck, Judith ; Hahn, Mark E. ; Hixson, Richard ; Kumar, Pushpam ; Mustapha, Adetoun ; Park, Yongjoon ; Spring, Margaret ; Stegeman, John J. ; Thompson, Richard C. ; Wang, Zhanyun ; Wolff, Megan ; Yousuf, Aroub ; Dunlop, Sarah

Plastic is the signature material of our age. In the 75 years since large-scale production began in the aftermath of World War II, plastic has transformed our world, supported many of the most significant advances of modern civilization, and enabled breakthroughs in virtually every field of human endeavor. But plastic also poses great and growing dangers to human health and the environment, harms that fall disproportionately on the world’s poorest and most vulnerable populations. The extent and magnitude of these dangers are only beginning to be understood.
Characterization of a virally encoded flavodoxin that can drive bacterial cytochrome P450 monooxygenase activity

(MDPI, 2022-08-11) Lamb, David C. ; Goldstone, Jared V. ; Zhao, Bin ; Lei, Li ; Mullins, Jonathan G. L. ; Allen, Michael J. ; Kelly, Steven L. ; Stegeman, John J.

Flavodoxins are small electron transport proteins that are involved in a myriad of photosynthetic and non-photosynthetic metabolic pathways in Bacteria (including cyanobacteria), Archaea and some algae. The sequenced genome of 0305φ8-36, a large bacteriophage that infects the soil bacterium Bacillus thuringiensis, was predicted to encode a putative flavodoxin redox protein. Here we confirm that 0305φ8-36 phage encodes a FMN-containing flavodoxin polypeptide and we report the expression, purification and enzymatic characterization of the recombinant protein. Purified 0305φ8-36 flavodoxin has near-identical spectral properties to control, purified Escherichia coli flavodoxin. Using in vitro assays we show that 0305φ8-36 flavodoxin can be reconstituted with E. coli flavodoxin reductase and support regio- and stereospecific cytochrome P450 CYP170A1 allyl-oxidation of epi-isozizaene to the sesquiterpene antibiotic product albaflavenone, found in the soil bacterium Streptomyces coelicolor. In vivo, 0305φ8-36 flavodoxin is predicted to mediate the 2-electron reduction of the β subunit of phage-encoded ribonucleotide reductase to catalyse the conversion of ribonucleotides to deoxyribonucleotides during viral replication. Our results demonstrate that this phage flavodoxin has the potential to manipulate and drive bacterial P450 cellular metabolism, which may affect both the host biological fitness and the communal microbiome. Such a scenario may also be applicable in other viral-host symbiotic/parasitic relationships.
Nrf2 and Nrf2-related proteins in development and developmental toxicity : insights from studies in zebrafish (Danio rerio)

( 2015-06-15) Hahn, Mark E. ; Timme-Laragy, Alicia R. ; Karchner, Sibel I. ; Stegeman, John J.

Oxidative stress is an important mechanism of chemical toxicity, contributing to developmental toxicity and teratogenesis as well as to cardiovascular and neurodegenerative diseases and diabetic embryopathy. Developing animals are especially sensitive to effects of chemicals that disrupt the balance of processes generating reactive species and oxidative stress, and those anti-oxidant defenses that protect against oxidative stress. The expression and inducibility of anti-oxidant defenses through activation of NFE2-related factor 2 (Nrf2) and related proteins is an essential process affecting the susceptibility to oxidants, but the complex interactions of Nrf2 in determining embryonic response to oxidants and oxidative stress are only beginning to be understood. The zebrafish (Danio rerio) is an established model in developmental biology and now also in developmental toxicology and redox signaling. Here we review the regulation of genes involved in protection against oxidative stress in developing vertebrates, with a focus on Nrf2 and related cap’n’collar (CNC)-basic-leucine zipper (bZIP) transcription factors. Vertebrate animals including zebrafish share Nfe2, Nrf1, Nrf2, and Nrf3 as well as a core set of genes that respond to oxidative stress, contributing to the value of zebrafish as a model system with which to investigate the mechanisms involved in regulation of redox signaling and the response to oxidative stress during embryolarval development. Moreover, studies in zebrafish have revealed nrf and keap1 gene duplications that provide an opportunity to dissect multiple functions of vertebrate NRF genes, including multiple sensing mechanisms involved in chemical-specific effects.
Systemic effects of Arctic pollutants in beluga whales indicated by CYP1A1 expression

(National Institute of Environmental Health Sciences, 2005-07-14) Wilson, Joanna Y. ; Cooke, Suzy R. ; Moore, Michael J. ; Martineau, Daniel ; Mikaelian, Igor ; Metner, Donald A. ; Lockhart, W. Lyle ; Stegeman, John J.

Cytochrome P450 1A1 (CYP1A1) is induced by exposure to polycyclic aromatic hydrocarbons (PAHs) and planar halogenated aromatic hydrocarbons (PHAHs) such as non-ortho polychlorinated biphenyls (PCBs). In this study, we examined CYP1A1 protein expression immunohistochemically in multiple organs of beluga whales from two locations in the Arctic and from the St. Lawrence estuary. These beluga populations have some of the lowest (Arctic sites) and highest (St. Lawrence estuary) concentrations of PCBs in blubber of all cetaceans. Samples from these populations might be expected to have different contaminant-induced responses, reflecting their different exposure histories. The pattern and extent of CYP1A1 staining in whales from all three locations were similar to those seen in animal models in which CYP1A has been highly induced, indicating a high-level expression in these whales. CYP1A1 induction has been related to toxic effects of PHAHs or PAHs in some species. In St. Lawrence beluga, the high level of CYP1A1 expression coupled with high levels of contaminants (including CYP1A1 substrates, e.g., PAH procarcinogens potentially activated by CYP1A1) indicates that CYP1A1 could be involved in the development of neoplastic lesions seen in the St. Lawrence beluga population. The systemic high-level expression of CYP1A1 in Arctic beluga suggests that effects of PAHs or PHAHs may be expected in Arctic populations, as well. The high-level expression of CYP1A1 in the Arctic beluga suggests that this species is highly sensitive to CYP1A1 induction by aryl hydrocarbon receptor agonists.
Altered gene expression associated with epizootic shell disease in the American lobster, Homarus americanus

( 2010-07-21) Tarrant, Ann M. ; Stegeman, John J. ; Verslycke, Tim A.

Epizootic shell disease is a poorly understood condition that has significantly affected the American lobster fishery in New England (northeastern US) since the 1990s. Here we present the results of a study to identify changes in gene expression in lobsters exhibiting symptoms of epizootic shell disease. Suppressive subtractive hybridization (SSH) was used to compare gene expression between cDNA pools from diseased (symptomatic) and apparently healthy (asymptomatic) lobsters. Subsequently, quantitative real‐time polymerase chain reaction (qPCR) was used to measure expression of nine genes that were differentially‐expressed in the SSH analysis, in seven tissues (muscle, gill, heart, hepatopancreas, brain, branchiostegite, gonad) dissected from individual symptomatic and asymptomatic lobsters. Expression of arginine kinase (involved in cellular energetics) was significantly decreased in muscle of symptomatic lobsters. Expression of hemocyanin (a respiratory hemolymph protein involved in oxygen transport) was highest in hepatopancreas and showed highly variable expression with a trend toward higher expression in asymptomatic individuals. α2‐Macroglobulin (involved in the innate immune system) was most highly expressed in the ovary, particularly of symptomatic lobsters. The ESTs produced through this study add to the fledgling field of crustacean genomics and revealed three genes that could be further evaluated in lobsters of varying shell disease severity, molt stage, and reproductive condition, for possible implication in epizootic shell disease.
Identification and developmental expression of the full complement of Cytochrome P450 genes in Zebrafish

(BioMed Central, 2010-11-18) Goldstone, Jared V. ; McArthur, Andrew G. ; Kubota, Akira ; Zanette, Juliano ; Parente, Thiago ; Jonsson, Maria E. ; Nelson, David R. ; Stegeman, John J.

Increasing use of zebrafish in drug discovery and mechanistic toxicology demands knowledge of cytochrome P450 (CYP) gene regulation and function. CYP enzymes catalyze oxidative transformation leading to activation or inactivation of many endogenous and exogenous chemicals, with consequences for normal physiology and disease processes. Many CYPs potentially have roles in developmental specification, and many chemicals that cause developmental abnormalities are substrates for CYPs. Here we identify and annotate the full suite of CYP genes in zebrafish, compare these to the human CYP gene complement, and determine the expression of CYP genes during normal development. Zebrafish have a total of 94 CYP genes, distributed among 18 gene families found also in mammals. There are 32 genes in CYP families 5 to 51, most of which are direct orthologs of human CYPs that are involved in endogenous functions including synthesis or inactivation of regulatory molecules. The high degree of sequence similarity suggests conservation of enzyme activities for these CYPs, confirmed in reports for some steroidogenic enzymes (e.g. CYP19, aromatase; CYP11A, P450scc; CYP17, steroid 17a-hydroxylase), and the CYP26 retinoic acid hydroxylases. Complexity is much greater in gene families 1, 2, and 3, which include CYPs prominent in metabolism of drugs and pollutants, as well as of endogenous substrates. There are orthologous relationships for some CYP1 s and some CYP3 s between zebrafish and human. In contrast, zebrafish have 47 CYP2 genes, compared to 16 in human, with only two (CYP2R1 and CYP2U1) recognized as orthologous based on sequence. Analysis of shared synteny identified CYP2 gene clusters evolutionarily related to mammalian CYP2 s, as well as unique clusters. Transcript profiling by microarray and quantitative PCR revealed that the majority of zebrafish CYP genes are expressed in embryos, with waves of expression of different sets of genes over the course of development. Transcripts of some CYP occur also in oocytes. The results provide a foundation for the use of zebrafish as a model in toxicological, pharmacological and chemical disease research.
Biological effects of 6-formylindolo[3,2-b]carbazole (FICZ) in vivo are enhanced by loss of CYP1A function in an Ahr2-dependent manner

(Elsevier, 2016-04-22) Wincent, Emma ; Kubota, Akira ; Timme-Laragy, Alicia R. ; Jonsson, Maria E. ; Hahn, Mark E. ; Stegeman, John J.

6-Formylindolo[3,2-b]carbazole (FICZ) is a potent aryl hydrocarbon receptor (AHR) agonist that is efficiently metabolized by AHR-regulated cytochrome P4501 enzymes. FICZ is a proposed physiological AHR ligand that induces its own degradation as part of a regulatory negative feedback loop. In vitro studies in cells show that CYP1 inhibition in the presence of FICZ results in enhanced AHR activation, suggesting that FICZ accumulates in the cell when its metabolism is blocked. We used zebrafish (Danio rerio) embryos to investigate the in vivo effects of FICZ when CYP1A is knocked down or inhibited. Embryos were injected with morpholino antisense oligonucleotides targeting CYP1A (CYP1A-MO), Ahr2, or a combination of both. FICZ exposure of non-injected embryos or embryos injected with control morpholino had little effect. In CYP1A-MO-injected embryos, however, FICZ dramatically increased mortality, incidence and severity of pericardial edema and circulation failure, reduced hatching frequency, blocked swim bladder inflation, and strongly potentiated expression of Ahr2-regulated genes. These effects were substantially reduced in embryos with a combined knockdown of Ahr2 and CYP1A, indicating that the toxicity was mediated at least partly by Ahr2. Co-exposure to the CYP1 inhibitor alpha-naphthoflavone (αNF) and FICZ had similar effects as the combination of CYP1A-MO and FICZ. HPLC analysis of FICZ-exposed embryos showed increased levels of FICZ after concomitant CYP1A-MO injection or αNF co-exposure. Together, these results show that a functioning CYP1/AHR feedback loop is crucial for regulation of AHR signaling by a potential physiological ligand in vivo and further highlights the role of CYP1 enzymes in regulating biological effects of FICZ.
Developmental expression of the Nfe2-related factor (Nrf) transcription factor family in the zebrafish, Danio rerio

(Public Library of Science, 2013-10-24) Williams, Larissa M. ; Timme-Laragy, Alicia R. ; Goldstone, Jared V. ; McArthur, Andrew G. ; Stegeman, John J. ; Smolowitz, Roxanna M. ; Hahn, Mark E.

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.
Cytochrome P450 induced differentially in endothelial cells cultured from different organs of Anguilla rostrata

( 2004-12-15) Garrick, Rita Anne ; Woodin, Bruce R. ; Stegeman, John J.

Endothelial cells are a structural barrier and an active regulator of many bodily processes. CYP1A activity is induced in the endothelium of teleosts and mammals exposed to lipophilic xenobiotics, such as polycyclic aromatic hydrocarbons, and can have significant consequences for endothelial functions. We exposed cultures of characterized endothelial cells from the heart, kidney and rete mirabile of the eel, Anguilla rostrata, to AhR agonists. In heart endothelial cells the maximum response (based on EROD activity) to TCDD, 113 pmol/mg-min, was at 1 nM TCDD and the peak response to βNF, 135 pmol/mg-min, was at 3 μM βNF. The maximum response to TCDD in the kidney endothelial cells is 12 pmol/mg-min at 0.3 nM TCDD. The rete mirabile capillary endothelial cells responded minimally or not at all to exposure to TCDD and βNF. Both the heart and kidney endothelial cells (but not the rete mirabile capillary cells) have a low level of EROD activity (12.7 and 5.2 pmol/mg-min respectively) in untreated or DMSO-treated cells. The robust response of the heart endothelial cells to induction and the lack of response in the rete mirabile capillary endothelial cells indicate that these cells are a good resource to use to investigate the physiological consequences of AhR agonist exposure and CYP1A induction in different areas of the vasculature.

Stegeman John J.

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