Hahn Mark E.

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Hahn

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Mark E.

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0000-0003-4358-2082

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Divergent forms of pyroplastic: lessons learned from the M/V X-Press Pearl ship fire

(American Chemical Society, 2022-07-29) James, Bryan D. ; de Vos, Asha ; Aluwihare, Lihini I. ; Youngs, Sarah ; Ward, Collin P. ; Michel, Anna P. M. ; Hahn, Mark E. ; Reddy, Christopher M.

In late May 2021, the M/V X-Press Pearl container ship caught fire while anchored 18 km off the coast of Colombo, Sri Lanka and spilled upward of 70 billion pieces of plastic or “nurdles” (∼1680 tons), littering the country’s coastline. Exposure to combustion, heat, chemicals, and petroleum products led to an apparent continuum of changes from no obvious effects to pieces consistent with previous reports of melted and burned plastic (pyroplastic) found on beaches. At the middle of this continuum, nurdles were discolored but appeared to retain their prefire morphology, resembling nurdles that had been weathered in the environment. We performed a detailed investigation of the physical and surface properties of discolored nurdles collected on a beach 5 days after the ship caught fire and within 24 h of their arrival onshore. The color was the most striking trait of the plastic: white for nurdles with minimal alteration from the accident, orange for nurdles containing antioxidant degradation products formed by exposure to heat, and gray for partially combusted nurdles. Our color analyses indicate that this fraction of the plastic released from the ship was not a continuum but instead diverged into distinct groups. Fire left the gray nurdles scorched, with entrained particles and pools of melted plastic, and covered in soot, representing partial pyroplastics, a new subtype of pyroplastic. Cross sections showed that the heat- and fire-induced changes were superficial, leaving the surfaces more hydrophilic but the interior relatively untouched. These results provide timely and actionable information to responders to reevaluate cleanup end points, monitor the recurrence of these spilled nurdles, gauge short- and long-term effects of the spilled nurdles to the local ecosystem, and manage the recovery of the spill. These findings underscore partially combusted plastic (pyroplastic) as a type of plastic pollution that has yet to be fully explored despite the frequency at which plastic is burned globally.
The landscape of extreme genomic variation in the highly adaptable Atlantic killifish

(Oxford University Press, 2017-03-01) Reid, Noah M. ; Jackson, Craig E. ; Gilbert, Don ; Minx, Patrick ; Montague, Michael J. ; Hampton, Thomas H. ; Helfrich, Lily W. ; King, Benjamin L. ; Nacci, Diane E. ; Aluru, Neelakanteswar ; Karchner, Sibel I. ; Colbourne, John K. ; Hahn, Mark E. ; Shaw, Joseph R. ; Oleksiak, Marjorie F. ; Crawford, Douglas L. ; Warren, Wesley C. ; Whitehead, Andrew

Understanding and predicting the fate of populations in changing environments require knowledge about the mechanisms that support phenotypic plasticity and the adaptive value and evolutionary fate of genetic variation within populations. Atlantic killifish (Fundulus heteroclitus) exhibit extensive phenotypic plasticity that supports large population sizes in highly fluctuating estuarine environments. Populations have also evolved diverse local adaptations. To yield insights into the genomic variation that supports their adaptability, we sequenced a reference genome and 48 additional whole genomes from a wild population. Evolution of genes associated with cell cycle regulation and apoptosis is accelerated along the killifish lineage, which is likely tied to adaptations for life in highly variable estuarine environments. Genome-wide standing genetic variation, including nucleotide diversity and copy number variation, is extremely high. The highest diversity genes are those associated with immune function and olfaction, whereas genes under greatest evolutionary constraint are those associated with neurological, developmental, and cytoskeletal functions. Reduced genetic variation is detected for tight junction proteins, which in killifish regulate paracellular permeability that supports their extreme physiological flexibility. Low-diversity genes engage in more regulatory interactions than high-diversity genes, consistent with the influence of pleiotropic constraint on molecular evolution. High genetic variation is crucial for continued persistence of species given the pace of contemporary environmental change. Killifish populations harbor among the highest levels of nucleotide diversity yet reported for a vertebrate species, and thus may serve as a useful model system for studying evolutionary potential in variable and changing environments.
Evolutionary concepts can benefit both fundamental research and applied research in toxicology (A comment on brady et al. 2017).

(John Wiley & Sons Ltd, 2018-11-05) Hahn, Mark E.
When evolution is the solution to pollution : key principles, and lessons from rapid repeated adaptation of killifish (Fundulus heteroclitus) populations

(John Wiley & Sons, 2017-04-26) Whitehead, Andrew ; Clark, Bryan W. ; Reid, Noah M. ; Hahn, Mark E. ; Nacci, Diane E.

For most species, evolutionary adaptation is not expected to be sufficiently rapid to buffer the effects of human-mediated environmental changes, including environmental pollution. Here we review how key features of populations, the characteristics of environmental pollution, and the genetic architecture underlying adaptive traits, may interact to shape the likelihood of evolutionary rescue from pollution. Large populations of Atlantic killifish (Fundulus heteroclitus) persist in some of the most contaminated estuaries of the United States, and killifish studies have provided some of the first insights into the types of genomic changes that enable rapid evolutionary rescue from complexly degraded environments. We describe how selection by industrial pollutants and other stressors has acted on multiple populations of killifish and posit that extreme nucleotide diversity uniquely positions this species for successful evolutionary adaptation. Mechanistic studies have identified some of the genetic underpinnings of adaptation to a well-studied class of toxic pollutants; however, multiple genetic regions under selection in wild populations seem to reflect more complex responses to diverse native stressors and/or compensatory responses to primary adaptation. The discovery of these pollution-adapted killifish populations suggests that the evolutionary influence of anthropogenic stressors as selective agents occurs widely. Yet adaptation to chemical pollution in terrestrial and aquatic vertebrate wildlife may rarely be a successful “solution to pollution” because potentially adaptive phenotypes may be complex and incur fitness costs, and therefore be unlikely to evolve quickly enough, especially in species with small population sizes.
Sequence and functional characterization of hypoxia inducible factors, HIF1α, HIF2αa, and HIF3α, from the estuarine fish, Fundulus heteroclitus

( 2016-11) Townley, Ian K. ; Karchner, Sibel I. ; Skripnikova, Elena ; Wiese, Thomas E. ; Hahn, Mark E. ; Rees, Bernard B.

The hypoxia inducible factor (HIF) family of transcription factors plays central roles in the development, physiology, pathology, and environmental adaptation of animals. Because many aquatic habitats are characterized by episodes of low dissolved oxygen, fish represent ideal models to study the roles of HIF in the response to aquatic hypoxia. The estuarine fish Fundulus heteroclitus occurs in habitats prone to hypoxia, it responds to low oxygen via behavioral, physiological, and molecular changes, and one member of the HIF family, HIF2α, has been previously described. Herein, cDNA sequencing, phylogenetic analyses, and genomic approaches were used to determine other members of the HIFα family from F. heteroclitus and their relationships to HIFα subunits from other vertebrates. In vitro and cellular approaches demonstrated that full-length forms of HIF1α, 2α, and 3α independently formed complexes with the β subunit (ARNT) to bind to hypoxia response elements and activate reporter gene expression. Quantitative PCR showed that HIFα mRNA abundance varied among organs of normoxic fish in an isoform-specific fashion. Analysis of the F. heteroclitus genome revealed a locus encoding a second HIF2α, HIF2αb, a predicted protein lacking oxygen sensing and transactivation domains. Finally, sequence analyses demonstrated polymorphism in the coding sequence of each F. heteroclitus HIFα subunit, suggesting that genetic variation in these transcription factors may play a role in the variation in hypoxia responses among individuals or populations.
Molecular evolution of two vertebrate aryl hydrocarbon (dioxin) receptors (AHR1 and AHR2) and the PAS family

(National Academy of Sciences, 1997-12) Hahn, Mark E. ; Karchner, Sibel I. ; Shapiro, Miriam A. ; Perera, Samanthi A.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause altered gene expression and toxicity. The AHR belongs to the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) family of transcriptional regulatory proteins, whose members play key roles in development, circadian rhythmicity, and environmental homeostasis; however, the normal cellular function of the AHR is not yet known. As part of a phylogenetic approach to understanding the function and evolutionary origin of the AHR, we sequenced the PAS homology domain of AHRs from several species of early vertebrates and performed phylogenetic analyses of these AHR amino acid sequences in relation to mammalian AHRs and 24 other members of the PAS family. AHR sequences were identified in a teleost (the killifish Fundulus heteroclitus), two elasmobranch species (the skate Raja erinacea and the dogfish Mustelus canis), and a jawless fish (the lamprey Petromyzon marinus). Two putative AHR genes, designated AHR1 and AHR2, were found both in Fundulus and Mustelus. Phylogenetic analyses indicate that the AHR2 genes in these two species are orthologous, suggesting that an AHR gene duplication occurred early in vertebrate evolution and that multiple AHR genes may be present in other vertebrates. Database searches and phylogenetic analyses identified four putative PAS proteins in the nematode Caenorhabditis elegans, including possible AHR and ARNT homologs. Phylogenetic analysis of the PAS gene family reveals distinct clades containing both invertebrate and vertebrate PAS family members; the latter include paralogous sequences that we propose have arisen by gene duplication early in vertebrate evolution. Overall, our analyses indicate that the AHR is a phylogenetically ancient protein present in all living vertebrate groups (with a possible invertebrate homolog), thus providing an evolutionary perspective to the study of dioxin toxicity and AHR function.
Developmental exposure to domoic acid disrupts startle response behavior and circuitry in zebrafish

(Oxford University Press, 2021-06-07) Panlilio, Jennifer M. ; Jones, Ian T. ; Salanga, Matthew C. ; Aluru, Neelakanteswar ; Hahn, Mark E.

Harmful algal blooms produce potent neurotoxins that accumulate in seafood and are hazardous to human health. Developmental exposure to the harmful algal bloom toxin, domoic acid (DomA), has behavioral consequences well into adulthood, but the cellular and molecular mechanisms of DomA developmental neurotoxicity are largely unknown. To assess these, we exposed zebrafish embryos to DomA during the previously identified window of susceptibility and used the well-known startle response circuit as a tool to identify specific neuronal components that are targeted by exposure to DomA. Exposure to DomA reduced startle responsiveness to both auditory/vibrational and electrical stimuli, and even at the highest stimulus intensities tested, led to a dramatic reduction of one type of startle (short-latency c-starts). Furthermore, DomA-exposed larvae had altered kinematics for both types of startle responses tested, exhibiting shallower bend angles and slower maximal angular velocities. Using vital dye staining, immunolabeling, and live imaging of transgenic lines, we determined that although the sensory inputs were intact, the reticulospinal neurons required for short-latency c-starts were absent in most DomA-exposed larvae. Furthermore, axon tracing revealed that DomA-treated larvae also showed significantly reduced primary motor neuron axon collaterals. Overall, these results show that developmental exposure to DomA targets large reticulospinal neurons and motor neuron axon collaterals, resulting in measurable deficits in startle behavior. They further provide a framework for using the startle response circuit to identify specific neural populations disrupted by toxins or toxicants and to link these disruptions to functional consequences for neural circuit function and behavior.
Proteomic identification, cDNA cloning and enzymatic activity of glutathione S-transferases from the generalist marine gastropod, Cyphoma gibbosum

( 2008-06) Whalen, Kristen E. ; Morin, Dexter ; Lin, Ching Yu ; Tjeerdema, Ronald S. ; Goldstone, Jared V. ; Hahn, Mark E.

Glutathione S-transferases (GST) were characterized from the digestive gland of Cyphoma gibbosum (Mollusca; Gastropoda), to investigate the possible role of these detoxification enzymes in conferring resistance to allelochemicals present in its gorgonian coral diet. We identified the collection of expressed cytosolic Cyphoma GST classes using a proteomic approach involving affinity chromatography, HPLC and nanospray liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two major GST subunits were identified as putative mu-class GSTs; while one minor GST subunit was identified as a putative theta-class GST, apparently the first theta-class GST identified from a mollusc. Two Cyphoma GST cDNAs (CgGSTM1 and CgGSTM2) were isolated by RT-PCR using primers derived from peptide sequences. Phylogenetic analyses established both cDNAs as mu-class GSTs and revealed a mollusc-specific subclass of the GST-mu clade. These results provide new insights into metazoan GST diversity and the biochemical mechanisms used by marine organisms to cope with their chemically defended prey.
Generalized concentration addition predicts joint effects of aryl hydrocarbon receptor agonists with partial agonists and cCompetitive antagonists

(National Institute of Environmental Health Sciences, 2009-12-22) Howard, Gregory J. ; Schlezinger, Jennifer J. ; Hahn, Mark E. ; Webster, Thomas F.

Background: Predicting the expected outcome of a combination exposure is critical to risk -assessment. The toxic equivalency factor (TEF) approach used for analyzing joint effects of dioxin-like chemicals is a special case of the method of concentration addition. However, the TEF method assumes that individual agents are full aryl hydrocarbon receptor (AhR) agonists with parallel dose–response curves, whereas many mixtures include partial agonists. Objectives: We assessed the ability of generalized concentration addition (GCA) to predict effects of combinations of full AhR agonists with partial agonists or competitive antagonists. Methods: We measured activation of AhR-dependent gene expression in H1G1.1c3 cells after application of binary combinations of AhR ligands. A full agonist (2,3,7,8-tetrachlorodibenzo-p--dioxin or 2,3,7,8-tetrachlorodibenzofuran) was combined with either a full agonist (3,3´,4,4´,5-penta-chloro-biphenyl), a partial agonist (2,3,3´,4,4´-pentachlorobiphenyl or galangin), or an antagonist (3,3´-diindolylmethane). Combination effects were modeled by the TEF and GCA approaches, and goodness of fit of the modeled response surface to the experimental data was assessed using a nonparametric statistical test. Results: The GCA and TEF models fit the experimental data equally well for a mixture of two full agonists. In all other cases, GCA fit the experimental data significantly better than the TEF model. Conclusions: The TEF model overpredicts effects of AhR ligands at the highest concentration combinations. At lower concentrations, the difference between GCA and TEF approaches depends on the efficacy of the partial agonist. GCA represents a more accurate definition of additivity for mixtures that include partial agonist or competitive antagonist ligands.
Cytochrome P450 diversity and induction by gorgonian allelochemicals in the marine gastropod Cyphoma gibbosum

(BioMed Central, 2010-12-01) Whalen, Kristen E. ; Starczak, Victoria R. ; Nelson, David R. ; Goldstone, Jared V. ; Hahn, Mark E.

Intense consumer pressure strongly affects the structural organization and function of marine ecosystems, while also having a profound effect on the phenotype of both predator and prey. Allelochemicals produced by prey often render their tissues unpalatable or toxic to a majority of potential consumers, yet some marine consumers have evolved resistance to host chemical defenses. A key challenge facing marine ecologists seeking to explain the vast differences in consumer tolerance of dietary allelochemicals is understanding the biochemical and molecular mechanisms underlying diet choice. The ability of marine consumers to tolerate toxin-laden prey may involve the cooperative action of biotransformation enzymes, including the inducible cytochrome P450s (CYPs), which have received little attention in marine invertebrates despite the importance of allelochemicals in their evolution. Here, we investigated the diversity, transcriptional response, and enzymatic activity of CYPs possibly involved in allelochemical detoxification in the generalist gastropod Cyphoma gibbosum, which feeds exclusively on chemically defended gorgonians. Twelve new genes in CYP family 4 were identified from the digestive gland of C. gibbosum. Laboratory-based feeding studies demonstrated a 2.7- to 5.1-fold induction of Cyphoma CYP4BK and CYP4BL transcripts following dietary exposure to the gorgonian Plexaura homomalla, which contains high concentrations of anti-predatory prostaglandins. Phylogenetic analysis revealed that C. gibbosum CYP4BK and CYP4BL were most closely related to vertebrate CYP4A and CYP4F, which metabolize pathophysiologically important fatty acids, including prostaglandins. Experiments involving heterologous expression of selected allelochemically-responsive C. gibbosum CYP4s indicated a possible role of one or more CYP4BL forms in eicosanoid metabolism. Sequence analysis further demonstrated that Cyphoma CYP4BK/4BL and vertebrate CYP4A/4F forms share identical amino acid residues at key positions within fatty acid substrate recognition sites. These results demonstrate differential regulation of CYP transcripts in a marine consumer feeding on an allelochemical-rich diet, and significantly advance our understanding of both the adaptive molecular mechanisms that marine consumers use to cope with environmental chemical pressures and the evolutionary history of allelochemical-metabolizing enzymes in the CYP superfamily.
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.
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.
Biochemical warfare on the reef : the role of glutathione transferases in consumer tolerance of dietary prostaglandins

(Public Library of Science, 2010-01-06) Whalen, Kristen E. ; Lane, Amy L. ; Kubanek, Julia ; Hahn, Mark E.

Despite the profound variation among marine consumers in tolerance for allelochemically-rich foods, few studies have examined the biochemical adaptations underlying diet choice. Here we examine the role of glutathione S-transferases (GSTs) in the detoxification of dietary allelochemicals in the digestive gland of the predatory gastropod Cyphoma gibbosum, a generalist consumer of gorgonian corals. Controlled laboratory feeding experiments were used to investigate the influence of gorgonian diet on Cyphoma GST activity and isoform expression. Gorgonian extracts and semi-purified fractions were also screened to identify inhibitors and possible substrates of Cyphoma GSTs. In addition, we investigated the inhibitory properties of prostaglandins (PGs) structurally similar to antipredatory PGs found in high concentrations in the Caribbean gorgonian Plexaura homomalla. Cyphoma GST subunit composition was invariant and activity was constitutively high regardless of gorgonian diet. Bioassay-guided fractionation of gorgonian extracts revealed that moderately hydrophobic fractions from all eight gorgonian species examined contained putative GST substrates/inhibitors. LC-MS and NMR spectral analysis of the most inhibitory fraction from P. homomalla subsequently identified prostaglandin A2 (PGA2) as the dominant component. A similar screening of commercially available prostaglandins in series A, E, and F revealed that those prostaglandins most abundant in gorgonian tissues (e.g., PGA2) were also the most potent inhibitors. In vivo estimates of PGA2 concentration in digestive gland tissues calculated from snail grazing rates revealed that Cyphoma GSTs would be saturated with respect to PGA2 and operating at or near physiological capacity. The high, constitutive activity of Cyphoma GSTs is likely necessitated by the ubiquitous presence of GST substrates and/or inhibitors in this consumer's gorgonian diet. This generalist's GSTs may operate as ‘all-purpose’ detoxification enzymes, capable of conjugating or sequestering a broad range of lipophilic gorgonian compounds, thereby allowing this predator to exploit a range of chemically-defended prey, resulting in a competitive dietary advantage for this species.
Amino acid sequence of the ligand-binding domain of the aryl hydrocarbon receptor 1 predicts sensitivity of wild birds to effects of dioxin-like compounds

( 2012-07-17) Farmahin, Reza ; Manning, Gillian E. ; Crump, Doug ; Wu, Dongmei ; Mundy, Lukas J. ; Jones, Stephanie P. ; Hahn, Mark E. ; Karchner, Sibel I. ; Giesy, John P. ; Bursian, Steven J. ; Zwiernik, Matthew J. ; Fredricks, Timothy B. ; Kennedy, Sean W.

The sensitivity of avian species to the toxic effects of dioxin-like compounds (DLCs) varies up to 1000-fold among species and this variability has been associated with inter-species differences in aryl hydrocarbon receptor 1 ligand binding domain (AHR1 LBD) sequence. We previously showed that LD50 values, based on in ovo exposures to DLCs, were significantly correlated with in vitro EC50 values obtained with a luciferase reporter gene (LRG) assay that measures AHR1-mediated induction of cytochrome P4501A in COS-7 cells transfected with avian AHR1 constructs. Those findings suggest that the AHR1 LBD sequence and the LRG assay can be used to predict avian species sensitivity to DLCs. In the present study, the AHR1 LBD sequences of 86 avian species were studied and differences at amino acid sites 256, 257, 297, 324, 337 and 380 were identified. Site-directed mutagenesis, the LRG assay and homology modeling highlighted the importance of each amino acid site in AHR1 sensitivity to 2,3,8,8-tetrachlorodibenzo-p-dioxin and other DLCs. The results of the study revealed that: (1) only amino acids at sites 324 and 380 affect the sensitivity of AHR1 expression constructs of 86 avian species to DLCs and (2) in vitro luciferase activity in AHR1 constructs containing only the LBD of the species of interest is significantly correlated (r2 = 0.93, p<0.0001) with in ovo toxicity data for those species. These results indicate promise for the use of AHR1 LBD amino acid sequences independently, or combined with the LRG assay, to predict avian species sensitivity to DLCs.
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.
Development of the morpholino gene knockdown technique in Fundulus heteroclitus : a tool for studying molecular mechanisms in an established environmental model

( 2008-02-13) Matson, Cole W. ; Clark, Bryan W. ; Jenny, Matthew J. ; Fleming, Carrie R. ; Hahn, Mark E. ; Di Giulio, Richard T.

A significant challenge in environmental toxicology is that many genetic and genomic tools available in laboratory models are not developed for commonly used environmental models. The Atlantic killifish (Fundulus heteroclitus) is one of the most studied teleost environmental models, yet few genetic or genomic tools have been developed for use in this species. The advancement of genetic and evolutionary toxicology will require that many of the tools developed in laboratory models be transferred into species more applicable to environmental toxicology. Antisense morpholino oligonucleotide (MO) gene knockdown technology has been widely utilized to study development in zebrafish and has been proven to be a powerful tool in toxicological investigations through direct manipulation of molecular pathways. To expand the utility of killifish as an environmental model, MO gene knockdown technology was adapted for use in Fundulus. Morpholino microinjection methods were altered to overcome the significant differences between these two species. Morpholino efficacy and functional duration were evaluated with molecular and phenotypic methods. A cytochrome P450-1A (CYP1A) MO was used to confirm effectiveness of the methodology. For CYP1A MO-injected embryos, a 70% reduction in CYP1A activity, a 86% reduction in total CYP1A protein, a significant increase in β-naphthoflavone-induced teratogenicity, and estimates of functional duration (50% reduction in activity 10 dpf, and 86% reduction in total protein 12 dpf) conclusively demonstrated that MO technologies can be used effectively in killifish and will likely be just as informative as they have been in zebrafish.
The Ah receptor: adaptive metabolism, ligand diversity, and the xenokine model

(American Chemical Society, 2020-04-07) Avilla, Mele N. ; Malecki, Kristen M. C. ; Hahn, Mark E. ; Wilson, Rachel H. ; Bradfield, Christopher A.

The Ah receptor (AHR) has been studied for almost five decades. Yet, we still have many important questions about its role in normal physiology and development. Moreover, we still do not fully understand how this protein mediates the adverse effects of a variety of environmental pollutants, such as the polycyclic aromatic hydrocarbons (PAHs), the chlorinated dibenzo-p-dioxins (“dioxins”), and many polyhalogenated biphenyls. To provide a platform for future research, we provide the historical underpinnings of our current state of knowledge about AHR signal transduction, identify a few areas of needed research, and then develop concepts such as adaptive metabolism, ligand structural diversity, and the importance of proligands in receptor activation. We finish with a discussion of the cognate physiological role of the AHR, our perspective on why this receptor is so highly conserved, and how we might think about its cognate ligands in the future.
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.
Species-specific relative ahr1 binding affinities of 2,3,4,7,8-pentachlorodibenzofuran explain avian species differences in its relative potency

( 2013-12) Farmahin, Reza ; Jones, Stephanie P. ; Crump, Doug ; Hahn, Mark E. ; Giesy, John P. ; Zwiernik, Matthew J. ; Bursian, Steven J. ; Kennedy, Sean W.

Results of recent studies showed that 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are equipotent in domestic chicken (Gallus gallus domesticus) while PeCDF is more potent than TCDD in ring-necked pheasant (Phasianus colchicus) and Japanese quail (Coturnix japonica). To elucidate the mechanism(s) underlying these differences in relative potency of PeCDF among avian species, we tested the hypothesis that this is due to species-specific differential binding affinity of PeCDF to the aryl hydrocarbon receptor 1 (AHR1). Here, we modified a cell-based binding assay that allowed us to measure the binding affinity of dioxin-like compounds (DLCs) to avian AHR1 expressed in COS-7 (fibroblast-like cells). The results of the binding assay show that PeCDF and TCDD bind with equal affinity to chicken AHR1, but PeCDF binds with greater affinity than TCDD to pheasant (3-fold) and Japanese quail (5-fold) AHR1. The current report introduces a COS-7 whole-cell binding assay and provides a mechanistic explanation for differential relative potencies of PeCDF among species of birds.
Mechanistic research in aquatic toxicology : perspectives and future directions

( 2011-05-31) Hahn, Mark E.

On the thirtieth anniversary of the journal, I provide a perspective on some of the questions and opportunities for new understanding that will interest aquatic toxicologists during the next thirty years. I focus on mechanisms of toxicity involving transcription factors, signalling pathways, and gene networks involved in toxic and adaptive responses in aquatic animals. Prominent questions address the value of a toxicity pathways approach in aquatic systems, issues involving extrapolation among species, identification of susceptibility genes and useful biomarkers of adverse effect, new emerging contaminants, the importance of epigenetic mechanisms, effects of multiple stressors, evolutionary toxicology, and the relative roles of technical and conceptual limitations to our understanding of chemical effects on aquatic systems.

Hahn Mark E.

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