Direct Assessment of Cumulative Aryl Hydrocarbon Receptor Agonist Activity in Sera from Experimentally Exposed Mice and Environmentally Exposed Humans

Background: Aryl hydrocarbon receptor (AhR) ligands adversely affect many biological processes. However, assessment of the significance of human exposures is hampered by an incomplete understanding of how complex mixtures affect AhR activation/inactivation. Objectives: These studies used biological readouts to provide a broader context for estimating human risk than that obtained with serum extraction and gas chromatography/mass spectroscopy (GC/MS)-based assays alone. Methods: AhR agonist activity was quantified in sera from dioxin-treated mice, commercial human sources, and polychlorinated biphenyl (PCB)–exposed Faroe Islanders using an AhR-driven reporter cell line. To validate relationships between serum AhR agonist levels and biological outcomes, AhR agonist activity in mouse sera correlated with toxic end points. AhR agonist activity in unmanipulated (“neat”) human sera was compared with these biologically relevant doses and with GC/MS-assayed PCB levels. Results: Mouse serum AhR agonist activity correlated with injected dioxin dose, thymic atrophy, and heptomegaly, validating the use of neat serum to assess AhR agonist activity. AhR agonist activity in sera from Faroe Islanders varied widely, was associated with the frequency of recent pilot whale dinners, but did not correlate with levels of PCBs quantified by GC/MS. Surprisingly, significant “baseline” AhR activity was found in commercial human sera. Conclusions: An AhR reporter assay revealed cumulative levels of AhR activation potential in neat serum, whereas extraction may preclude detection of important non-dioxin-like biological activity. Significant levels of AhR agonist activity in commercial sera and in Faroe Islander sera, compared with that from experimentally exposed mice, suggest human exposures that are biologically relevant in both populations

Environmental Effects of Sediment Transport Alteration and Impacts on Protected Species: Edgartown Tidal Energy Project

The Islands of MarthaâÃÂÃÂs Vineyard and Nantucket are separated from the Massachusetts mainland by Vineyard and Nantucket Sounds; water between the two islands flows through Muskeget Channel. The towns of Edgartown (on MarthaâÃÂÃÂs Vineyard) and Nantucket recognize that they are vulnerable to power supply interruptions due to their position at the end of the power grid, and due to sea level rise and other consequences of climate change. The tidal energy flowing through Muskeget Channel has been identified by the Electric Power Research Institute as the strongest tidal resource in Massachusetts waters. The Town of Edgartown proposes to develop an initial 5 MW (nameplate) tidal energy project in Muskeget Channel. The project will consist of 14 tidal turbines with 13 providing electricity to Edgartown and one operated by the University of Massachusetts at Dartmouth for research and development. Each turbine will be 90 feet long and 50 feet high. The electricity will be brought to shore by a submarine cable buried 8 feet below the seabed surface which will landfall in Edgartown either on Chappaquiddack or at Katama. Muskeget Channel is located between MarthaâÃÂÃÂs Vineyard and Nantucket. Its depth ranges between 40 and 160 feet in the deepest portion. It has strong currents where water is transferred between Nantucket Sound and the Atlantic Ocean continental shelf to the south. This makes it a treacherous passage for navigation. Current users of the channel are commercial and recreational fishing, and cruising boats. The US Coast Guard has indicated that the largest vessel passing through the channel is a commercial scallop dragger with a draft of about 10 feet. The tidal resource in the channel has been measured by the University of Massachusetts-Dartmouth and the peak velocity flow is approximately 5 knots. The technology proposed is the helical Gorlov-type turbine positioned with a horizontal axis that is positively buoyant in the water column and held down by anchors. This is the same technology proposed by Ocean Renewable Power Company in the Western Passage and Cobscook Bay near Eastport Maine. The blades rotate in two directions capturing the tides energy both during flood and ebb tides. The turbines will be anchored to the bottom and suspended in the water column. Initial depth of the turbines is expected to be about 25 feet below the surface to avoid impacting navigation while also capturing the strongest currents. The Town of Edgartown was initially granted a Preliminary Permit by the Federal Energy Regulatory Commission (FERC) on March 1, 2008, and has recently received a second permit valid through August 2014. The Preliminary Permit gives Edgartown the exclusive right to apply for a power generation license for power generated from the hydrokinetic energy in the water flowing in this area. Edgartown filed a Draft Pilot License Application with FERC on February 1, 2010 and an Expanded Environmental Notification Form with the Massachusetts Environmental Policy Act (MEPA) Office at the same time. It expects to file a Final License Application in late 2013. Harris Miller Miller & Hanson (HMMH) of Burlington Massachusetts is acting as the Project Manager for the Town of Edgartown and collaborating with other partners of the project including the University of Massachusetts - Dartmouth's Marine Renewable Energy Center and the Massachusetts Clean Energy Center. HMMH was awarded a grant under the Department of Energy's Advanced Water Program to conduct marine science and hydrokinetic site-specific environmental studies for projects actively seeking a FERC License. HMMH, on behalf of the Town, is managing this comprehensive study of the marine environment in Muskeget Channel and potential impacts of the tidal project on indicator species and habitats. The University of Massachusetts School of Marine Science and Technology (SMAST) conducted oceanographic studies of tidal currents, tide level, benthic habitat, and potential for biofouling and foundation scouring. Woods Hole Oceanographic Institution, cooperating with SMAST, developed an oceanographic model to predict changes in sediment transport as a result of the proposed tidal energy project. Provincetown Center for Coastal Studies prepared background material on protected species - including whales, seals, and sea turtles - in the project area and implemented an initial tagging program to record location specific information on seals and sea turtles. HMMH communicated research plans and findings with local stakeholder groups, state and federal resource agency staff, and the ocean power industry. The information is being used to prepare environmental permit applications and obtain approvals for project construction

Environmental chemical-induced bone marrow B cell apoptosis: Death receptor-independent activation of a caspase-3 to caspase-8 pathway

ABSTRACT Programmed cell death is a critical process in B lymphocyte development. Premature apoptosis in developing B cells could affect the repertoire and number of mature B cells produced. Of particular concern is the ability of environmentally ubiquitous polycyclic aromatic hydrocarbons (PAH) to induce B cell apoptosis within the bone marrow microenvironment in a clonally nonspecific way. Here, models of bone marrow B cell development were used to assess the role of the "extrinsic" apoptosis pathway in PAH-induced apoptosis and to compare PAH-induced apoptosis with that induced during clonal deletion. A

Growth of a human mammary tumor cell line is blocked by galangin, a naturally occurring bioflavonoid, and is accompanied by down-regulation of cyclins D3, E, and A

INTRODUCTION: This study was designed to determine if and how a non-toxic, naturally occurring bioflavonoid, galangin, affects proliferation of human mammary tumor cells. Our previous studies demonstrated that, in other cell types, galangin is a potent inhibitor of the aryl hydrocarbon receptor (AhR), an environmental carcinogen-responsive transcription factor implicated in mammary tumor initiation and growth control. Because some current breast cancer therapeutics are ineffective in estrogen receptor (ER) negative tumors and since the AhR may be involved in breast cancer proliferation, the effects of galangin on the proliferation of an ER(-), AhR(high )line, Hs578T, were studied. METHODS: AhR expression and function in the presence or absence of galangin, a second AhR inhibitor, α-naphthoflavone (α-NF), an AhR agonist, indole-3-carbinol, and a transfected AhR repressor-encoding plasmid (FhAhRR) were studied in Hs578T cells by western blotting for nuclear (for instance, constitutively activated) AhR and by transfection of an AhR-driven reporter construct, pGudLuc. The effects of these agents on cell proliferation were studied by (3)H-thymidine incorporation and by flow cytometry. The effects on cyclins implicated in mammary tumorigenesis were evaluated by western blotting. RESULTS: Hs578T cells were shown to express high levels of constitutively active AhR. Constitutive and environmental chemical-induced AhR activity was profoundly suppressed by galangin as was cell proliferation. However, the failure of α-NF or FhAhRR transfection to block proliferation indicated that galangin-mediated AhR inhibition was either insufficient or unrelated to its ability to significantly block cell proliferation at therapeutically relevant doses (IC(50 )= 11 μM). Galangin inhibited transition of cells from the G(0)/G(1 )to the S phases of cell growth, likely through the nearly total elimination of cyclin D3. Expression of cyclins A and E was also suppressed. CONCLUSION: Galangin is a strong inhibitor of Hs578T cell proliferation that likely mediates this effect through a relatively unique mechanism, suppression of cyclin D3, and not through the AhR. The results suggest that this non-toxic bioflavonoid may be useful as a chemotherapeutic, particularly in combination with agents that target other components of the tumor cell cycle and in situations where estrogen receptor-specific therapeutics are ineffective

Identification and developmental expression of the full complement of Cytochrome P450 genes in Zebrafish

© The Authors, 2010. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in BMC Genomics 11 (2010): 643, doi:10.1186/1471-2164-11-643.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.This work was supported by NIH grants R01ES015912 and P42ES007381 (Superfund Basic Research Program at Boston University) (to JJS). MEJ was a Guest Investigator at the Woods Hole Oceanographic Institution (WHOI) and was supported by grants from the Swedish research council Formas and Carl Trygger's foundation. AK was a Post-doctoral Fellow at WHOI, and was supported by a fellowship from the Japanese Society for Promotion of Science (JSPS). JZ and TP were Guest Students at the WHOI and were supported by a CAPES Ph.D. Fellowship and CNPq Ph.D. Sandwich Fellowship (JZ), and by a CNPq Ph.D. Fellowship (TP), from Brazil

Regulation of constitutive and inducible AHR signaling : complex interactions involving the AHR repressorstar

Author Posting. © Elsevier B.V., 2009. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Biochemical Pharmacology 77(2009): 485-497, doi:10.1016/j.bcp.2008.09.016.The AHR is well known for regulating responses to an array of environmental chemicals. A growing body of evidence supports the hypothesis that the AHR also plays perhaps an even more important role in modulating critical aspects of cell function including cell growth, death, and migration. As these and other important AHR activities continue to be elucidated, it becomes apparent that attention now must be directed towards the mechanisms through which the AHR itself is regulated. Here, we review what is known of and what biological outcomes have been attributed to the AHR repressor (AHRR), an evolutionarily conserved bHLH-PAS protein that inhibits both xenobiotic-induced and constitutively active AHR transcriptional activity in multiple species. We discuss the structure and evolution of the AHRR and the dominant paradigm of a xenobiotic-inducible negative feedback loop comprised of AHR-mediated transcriptional up-regulation of AHRR and the subsequent AHRR-mediated suppression of AHR activity. We highlight the role of the AHRR in limiting AHR activity in the absence of xenobiotic AHR ligands and the important contribution of constitutively repressive AHRR to cancer biology. In this context, we also suggest a new hypothesis proposing that, under some circumstances, constitutively active AHR may repress AHRR transcription, resulting in unbridled AHR activity. We also review the predominant hypotheses on the molecular mechanisms through which AHRR inhibits AHR as well as novel mechanisms through which the AHRR may exert AHR-independent effects. Collectively, this discussion emphasizes the importance of this understudied bHLH-PAS protein in tissue development, normal cell biology, xenobiotic responsiveness, and AHR-regulated malignancy.Supported by P01-ES11624 (D.H.S.), ArtBeCAUSE (D.H.S.), R01ES006272 (M.E.H.), P42ES007381 (M.E.H. and D.H.S.

Occurrence and Distribution of Diverse Populations of Magnetic Protists in a Chemically Stratified Coastal Salt Pond

Chemical stratification occurs in the water columns and sediments of many aquatic habitats resulting in vertical chemical and redox gradients. Various types of microorganisms are often associated with specific depths and chemical parameters in these situations. For example, magnetite-producing magnetotactic bacteria are known to form horizontal “plates” of cells at the oxic/anoxic transition zone (OATZ) of such environments. Here, we report the presence of populations of diverse magnetic protists in a seasonally chemically stratified, coastal salt pond. The protistan types included several biflagellates, a dinoflagellate, and a ciliate that were each associated with specific depths and thus, specific chemical, microbiological and redox conditions in the water column. Most cells contained crystals of magnetite usually arranged in chains similar to the magnetosomes in the magnetotactic bacteria. The origin of the particles is unknown. There was no evidence of the presence of endosymbiotic magnetotactic bacteria within the protists nor were the protists observed to be engulfing magnetotactic bacteria despite the fact that high numbers of the latter were present at the same depths as the protists. However, this does not exclude ingestion of magnetotactic bacteria as a source of the magnetic particles while others may biomineralize magnetite. Because protists play an important role in the availability of iron for phytoplankton in marine waters by ingesting and reducing colloidal iron particles, the protists described here could play a significant role in iron cycling in chemically stratified anoxic basins, either through endogenous production of magnetic iron oxide and/or iron sulfide particles, or by ingestion of magnetotactic bacteria and the subsequent reduction and dissolution of iron oxide and/or sulfide particles in their magnetosomes