DIOXIN-LIKE CHEMICALS AND ENVIRONMENTAL ENDOCRINE DISRUPTING CHEMICALS: MOLECULAR MECHANISMS AND BIOANALYTICAL DETECTION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0209018
• Project No.: CA-D-ETX-7575-H
• Project Start Date: Oct 1, 2011
• Project End Date: Sep 30, 2016

Project Director
Denison, M.

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
Environmental Toxicology

Non Technical Summary
Humans and animals are exposed to contaminants from a wide variety of sources including air, water, soil, food and commercial and consumer products and these contaminants exist as a complex mixture of chemicals whose individual components and concentration can vary dramatically, complicating their detection and hazard assessment. While there are many mechanisms by which these chemicals can produce their adverse effects, their ability to modulate the function of intracellular receptor-mediated signaling pathways is one mechanism by which diverse chemicals can produce a common set of responses. Decreased reproductive success and feminization of males of several wildlife species, combined with reported reductions in sperm counts in men and significant increases in hormone-dependent breast and prostate cancer, are responses that may result from exposure to chemicals that adversely affect steroid hormone action. Numerous environmental contaminants that act as endocrine disrupting chemicals (EDCs) have been identified and shown to dramatically affect the function of such nuclear hormone receptors. Although the effects of many of these chemicals on human health remains controversial, since clear evidence exists for their adverse effects in many wildlife populations, at sufficient levels of EDCs would also produce toxic effects in human. This project will focus on the molecular mechanisms by which EDCs disrupt normal physiological processes, particularly receptors that mediate the action of dioxin-like chemicals and those that mediate the action of estrogen (specifically the Ah (dioxin) receptor (AhR) and estrogen receptor, respectively). A continuing emphasis will be on the analysis of the AhR-dependent molecular mechanisms by which dioxin-like chemicals produce their biological and toxicological effects. These chemicals represent a group of highly toxic and persistent environmental contaminants present in relatively high concentrations not only in the state of California, but also throughout the US and the rest of the world and they represent a significant risk to human and animal health. Therefore, increasing our understanding of the basic molecular mechanism by which these chemicals can bind to and activate the AhR will not only allow us to understand the early signaling events produced by these chemicals, but to potentially identify targets for the development of protective measures against HAH toxicity. Additionally, we will continue our application of recombinant DNA technologies to develop, improve and evaluate/validate several simple AhR- and ER-based cell bioassay systems for use in biomonitoring, detection and characterization of dioxins and EDCs in environmental, agricultural, biological, food and feed samples and to identify novel chemicals or classes of chemicals that can modulate the activity of their respective receptors. The identification of natural and/or synthetic chemicals that can affect or interfere with endocrine action is an important first step in exposure assessment and evaluation of the potential risks/effects of materials that contain these chemicals.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
314	3499	1150	10%
314	7010	1040	10%
314	7299	1000	10%
403	7010	1040	10%
711	3499	1150	10%
711	5230	1150	10%
723	6099	1150	30%
723	7299	1000	10%

Knowledge Area
403 - Waste Disposal, Recycling, and Reuse; 711 - Ensure Food Products Free of Harmful Chemicals, Including Residues from Agricultural and Other Sources; 314 - Toxic Chemicals, Poisonous Plants, Naturally Occurring Toxins, and Other Hazards Affecting Animals; 723 - Hazards to Human Health and Safety;

Subject Of Investigation
3499 - Dairy cattle, general/other; 7010 - Biological Cell Systems; 5230 - Feed and feed additives; 6099 - People and communities, general/other; 7299 - Research equipment and methods, general/other;

Field Of Science
1040 - Molecular biology; 1000 - Biochemistry and biophysics; 1150 - Toxicology;

Keywords

dioxin, estrogen, toxicity, molecular mechanisms, endocrine

disruptors, bioassays, bioanalytical, screening assays

Goals / Objectives
The overall objectives of this project are to gain further insights into the molecular mechanisms of toxicity of dioxin-like chemicals and endocrine disruptor chemicals. Additionally, we will take advantage of advances in the Ah (dioxin) and estrogen receptor (AhR and ER, respectively)-dependent mechanisms of action of these chemicals to develop high-throughput recombinant cell-based screening bioassays for rapid and sensitive detection of dioxins and related chemicals (i.e. polychlorinated dioxins, furans and biphenyls, as well as carcinogenic polycyclic aromatic hydrocarbons) as well as endocrine disruptors (particularly that of xenoestrogens) that can be used for screening of environmental, biological and food/feed samples. To accomplish these goals, we propose three specific aims. 1) Determine structure-function relationships of the AhR ligand binding domain in order to understand both the molecular mechanisms by which structurally diverse ligands bind to and activate the AhR and to develop novel ligand-selective AhRs by site-directed mutagenesis that can be used to develop chemical specific bioassay systems. 2) Develop a series of novel recombinant amplified AhR- and ER-based cell bioassay systems for detection of ultra low levels of endocrine disruptor chemicals that exert their action through these nuclear receptor signaling pathways. 3) Utilize the newly developed bioassay systems for screening of extracts of environmental, biological and food samples as well as pure chemicals to identify those that contain chemicals that can affect the AhR an ER signaling pathways. Not only do we expect to gain further insights into the basic molecular mechanisms by which dioxin- and estrogen-like chemicals can produce adverse health effects, but we will develop, optimize and utilize produce novel enhanced cell bioassays particularly useful for detection of low level contamination by these two classes of contaminant.

Project Methods
In Aim 1 we will use information derived from our homology model of the AhR ligand binding domain to generate a large series of targeted mutations of amino acids within the AhR ligand binding domain and to use these mutant AhRs to identify those that are selectively activated by dioxin-like chemicals as well as other classes of AhR ligands. Not only will these basic mechanistic studies provide insights into how these toxic chemicals can bind to and activate the AhR, the protein that mediates their toxicity, but we hope to be able to develop chemical-specific AhRs that can be used to generate chemical-selective bioassay systems. Stable co-transfection of these chemical selective AhRs into AhR deficient cells along with an AhR-responsive DRE-luciferase reporter gene to generate chemical-specific cell bioassays and these will be optimized and characterized as we have done in previous studies. If we are successful, samples for bioassay analysis would not only require minimal clean-up (making the analysis rapid and very inexpensive), but it would generate a chemical specific bioassay with new applications for both field and laboratory use. In Aim 2 we will improve existing bioassays for dioxin- and estrogen-receptor-based cell bioassays to create novel assays with significantly lower limits of detection and greater overall response. We will examine the effect of increasing the number of responsive elements (i.e. DNA binding sites for the AhR and ER) in a given reporter gene plasmid (preliminary results suggest that this will enhance our bioassay response) and we will also examine the effect of amplification of the number of responsive plasmids per cell. Both of these approaches can easily be accomplished using one of several well-documented approaches. TCDD or hormone responsiveness of each stable cell line generated using the above approaches will be examined in dose response experiments as we have carried out in our previous studies and we will identify those which have a significantly lower limit of TCDD or estrogen detection and/or a significantly greater overall induction response. The availability of these novel cell bioassays will greatly facilitate large scale screening studies with large sample numbers but limited sample size and/or ultra low levels of dioxin-like chemicals and endocrine disruptors. Finally, in Aim 3 we will optimize and validate these novel bioassays for the detection of their respective ligands using pure chemicals, chemical mixtures and unknown chemical mixtures. The optimized bioassay systems will also be adapted for use in high throughput screening analysis of extracts from various environmental, biological, food and feed samples and commercial/consumer products obtained from ongoing studies. Positive extracts will be subjected to bioassay-directed fractionation and instrumental analysis to identify the responsible chemical(s). These approaches will allow us to rapidly screen extracts of diverse matrices for the presence of dioxin-like chemicals and endocrine disruptors and should allow us to identify new chemicals or classes of chemicals that can affect these receptor systems.

Progress 10/01/11 to 09/30/16

Outputs
Target Audience:Our target audiences throughout this project have been varied and included: 1. the medical/veterinary/regulatory scientific community in order to understand the molecular basis of disease associated with dioxin-like chemicals and endocrine disruptors; 2. the regulatory community and lay public in order to provide additional validation and application information regarding use of bioanalytical methods for detection and relative quantitation of dioxin-like and endocrine disrupting chemicals and the presence of these chemicals in the food supply; and 3. the regulatory scientific community to alert them to potential risks associated with the presence of dioxin-like chemicals and endocrine disruptors in materials to which humans and animals are exposed and how to estimate exposure and risk associated with these chemicals. We reached theses audiences with our message not only via our published research papers, research presentations at scientific conferences and governmental sponsored meetings and workshops, but also through several webinar presentations to the USEPA, NIEHS, several California State Regulatory agencies (Division of Toxic Substances Control (DTSC), CalEPA and the California Waterboard ), international water agencies/regulatory groups in Australia, Malaysia and the European Union, as well as to several nongovernmental organizations (NGOs) in the US concerned about endocrine disruptor chemicals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided members of our laboratory with numerous the opportunities to interact and collaborate with numerous other US and international researchers and their personnel on several aspects of this project. We have helped these researchers in establishing our cell bioassay systems in their laboratories for use in the detection and characterization of agonists/antagonists of a variety of matrices. In addition, we have collaborated with researchers at several US federal agencies (NOAA, NIEHS, EPA and others) to utilize our recombinant cell bioassays for screening analysis of real-world environmental, biological, food and feed samples. This has provided our laboratory person with firsthand interactions with research in these federal labs and an opportunity to appreciate the complexities of real-world sample analysis and its regulatory implications. In addition, we have provided our students and postdocs with the opportunity to attend and present their research at relevant scientific conferences. This has provided them with an opportunity to interact and discuss their research with a wide range of experts in this area. Their participation not only allowed them to find out about the state of the art in their research area, but it has also given them new perspectives on their work and its significance in a national and global perspective. How have the results been disseminated to communities of interest?As we have described in detail in each of our previous progress reports how we have disseminated the results of our studies, their significance and implications for health effects. Our results have been widely disseminated through a variety of avenues, including: 1) research presentations at many national and international scientific conferences, meetings and workshops; 2) Research seminar presentation at Universities and governmental organizations; 3) Participation and research presentations at meetings and workshops on water reuse and bioassay applications for detection of endocrine disruptors; and. 4) Research and Q&A presentations on online Webinars for the USEPA, nongovernmental organizations (Center for Environmental Health) and general public. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Over the past 5 years, we made significant progress on all three aims proposed in this project. These have been described in detail in the annual reports and our major accomplishments in each of these aims are highlighted below. Analyze the ligand binding modes in the homology model of the AhR LBD Through the combined use of the biochemical, molecular and computational approaches, we developed and improved the first homology model of the Ah receptor (AhR) ligand binding domain (LBD) and characterized its interactions with structurally diverse ligands. These studies not only clearly demonstrated the ability of structurally diverse ligands to differentially bind with amino acids within the AhR ligand binding pocket, but we are now able to predict which chemicals can bind AhR with high affinity. In fact, we have demonstrated by site-directed mutagenesis that we can eliminate the binding of some ligands with no affect on the ability of others to bind to and activate the AhR. This provides us with potential avenues in which to develop ligand-specific AhRs that can be used to make chemical selective bioassays. Additionally, we demonstrated significant species differences in AhR ligand binding specificity that has implications for the development of therapeutic agents for the human AhR. The results of these studies have been published in many papers and the AhR LBD models, our extensive mutagenesis of the LBD and binding/functional analysis of the AhR remains the most comprehensive analysis and is being used by many laboratories worldwide. The results of our studies have opened significant new avenues into the role that the AhR and AhR ligands play in modulating endogenous physiological processes and toxicity. Analyze the functional diversity of endocrine disruptive AhR ligands Our studies over the past five years have clearly demonstrated both the structural and functional diversity of AhR ligands and differential effects of these ligands in different species. Our screening of a combinatorial library of more than 300,000 chemicals identified more than 9,000 that were potent and efficacious activators of the AhR. The structural diversity of these chemicals and the ability to bind to and activate the AhR and AhR signal transduction as well as their ability to produce adverse endocrine disruptive effects (inhibition of estrogen receptor function and inhibition of cell proliferation), strongly suggests that adverse endocrine effects could be produced by a wide range of AhR-active chemicals. We also extensively characterized the ability of both agonists and antagonists to bind to and activate/inhibit AhR functionality. We observed that AhR antagonists interact with amino acids within the AhR LBD that are distinctly different from those with agonist activity. Interesting, site directed mutagenesis allows us to interconvert agonists into antagonists and vice versa, providing insights into how these two distinct classes of ligands affect AhR functionality. These studies also identified a novel AhR antagonist, CH223191, that could block the ability of toxic AhR ligands (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related dioxin-like chemicals) from binding to the AhR, but had no significant effect on nontoxic AhR ligands (beta-naphthoflavone, indirubin, flavonoids and polycyclic aromatic hydrocarbons). This compound provides an avenue in which to develop a potential "antidote" or "blocker" of the effects of toxic dioxin-like chemicals that could be used following accidental exposure or contamination. In addition to studies of AhR antagonists, we identified numerous new classes of natural and synthetic AhR agonists as well as the ability of a yeast species (Malassezia furfur) that is present in the skin of most humans to produce a collection many of the most potent nonhalogenated AhR agonists. In fact, extremely high concentrations of these ligands were shown to be produced by this yeast in skin samples from humans with several skin diseases, suggesting that these chemicals may contribute to the adverse health effect. These results have led to long term collaborative studies with colleagues at the University of Athens to examine these compounds, their potential adverse human health effects and the utility of our AhR antagonists as potential therapeutic agents to block the adverse effects of these compounds. Further application of CALUX bioassays Throughout this project, we continued to improve and expand the applications and use of our recombinant cell-based CALUX bioassays for the detection of activator/inhibitors of AhR and estrogen receptor (ER) signaling pathways. We have further improved the sensitivity of our AhR CALUX bioassay by amplification methods, increasing the minimal detection limit of the bioassay by 10- to 100-fold. Our development of a human-specific CALUX line has also proven to be very useful for the screening and identification of novel AhR activators that have potential utility as therapeutic agents, given the recent demonstration of a critical role for the AhR in human health. Using our species-specific CALUX assays, we have carried out rapid high-throughput screening analysis to identify novel species-specific AhR ligands, with one ligand 10-times more potent than dioxin in human cells, but 100-times less toxic than dioxin in rodent cells. Use of these new cell lines will facilitate the identification of chemicals that may represent a unique species-specific risk. We previously developed an estrogen-responsive recombinant human breast carcinoma (VM7Luc4E2) cell line that has been officially accepted by the US Environmental Protection Agency (USEPA) and the Organization for Economic Co-operation and Development (OECD) as a bioanalytical method for the detection estrogenic agonists/antagonists. Although the VM7Luc4E2 cells have had great utility, cells appear to contain only one of the two known ER isoforms, ERalpha but not ERbeta, and the differential ligand selectivity of these two distinct ERs indicates that the currently accepted screening method only detects a subset of total estrogenic chemicals. Accordingly, we generated a new recombinant cell line that expresses both forms of the ER and these cells (VM7LucERbc9) providing us and the greater scientific community with a new and improved cell line that is a better predictor of the ability of a chemical/extract to stimulate/inhibit ER signaling. The utility of this new cell line was confirmed through our screening analysis of pesticides and industrial chemicals that identified chemicals that preferentially stimulated ER?eta-dependent reporter gene expression. The previous ER cell line would have not detected these ERbeta-specific ligands and would have underestimated their potential endocrine disruptor potential. In addition to our ER- and AhR-based cell bioassays, we developed a new cell bioassay for the detection of chemicals that affect signaling by the glucocorticoid and progesterone receptors and this new cell line will expand the range of endocrine disruption mechanisms that can be assessed. Our improved bioassays and the validation results from our lab and those of our colleagues using these CALUX bioassays to screening extracts from a variety of different matrices continues to greatly expanding the utility and applications of our AhR-based bioassays and this contributes to greater acceptance of this bioassays worldwide.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Neale, P., Ait-Aissa, S., Brack, W., Creusot, N., Denison, M.S., Deutschmann, B., Hilscherova, K., Hollert, H., Krauss, M., Novak, J., Schulze, T., Seiler, T.-B., Serra, H., Shao, Y. and Escher, B. (2015) Linking in vitro effects and detected organic micropollutants in surface water using mixture toxicity modeling, Environ. Sci. Tech. 49, 14614-14624. PMID: 26516785
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Croes, K., Van den Heuvel, R., Van den Bril, B., Staelens, J., Denison, M.S., Van Langenhove, K., Vandermarken, T. and Elskens, M. (2016) Assessment of estrogenic and androgenic activity in PM10 air samples from an urban, industrial and rural area in Flanders (Belgium) using the CALUX bioassay. Environ Res. 150, 66-72. PMID: 27257826
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Khedidji, S., Croes, K., Yassaa, N., Ladji, R., Denison, M.S., Baeyens, W. and Elskens, M. (2016) Assessment of dioxin-like activity in PM10 air samples from an industrial location in Algeria, using the DRE-CALUX bioassay, Environ. Sci. Poll. Res. doi:10.1007/s11356-015-5841-4. PMID: 26611627
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Bak, S.-M., Iida, M., Soshilov, A.A., Denison, M.S., Iwata, H. and Kim, E.-Y. (2016) Auto-induction mechanism of aryl hydrocarbon receptor 2 (AHR2) gene by TCDD-activated AHR1 and AHR2 in the red seabream (Pagrus major), Arch. Toxicol. 91(1):301-312. PMID: 27188387
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Cole, B., Brander, S., Jeffries, K., Hasenbein, S., He, G., Denison, M.S., Fangue, N. and Connon, R. (2016) Changes in Menidia beryllina gene expression and in vitro hormone receptor activation following exposure to estuarine waters near treated wastewater outfalls, Arch. Environ. Contam. Toxicol. 71, 210-223. PMID: 27155869
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Corrada, D., Soshilov, A.A., Denison, M.S. and Bonati, L. (2016) Deciphering Dimerization Modes of PAS Domains: Computational and Experimental Analyses of the AhR:ARNT Complex Reveal New Insights Into the Mechanisms of AhR Transformation, PLoS Comp. Biol. 12(6), e1004981. PMC4905635
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Joshi, G., Schmidt, R., Scow, K., Denison, M.S. and Hristova, K. (2016) Effect of benzene and ethylbenzene on the expression of methyl-tert-butyl ether degradation genes of methylibium petroleiphilum PM1, Microbiology 162, 1563-1571. PMID: 27450417
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Breznan D, Karthikeyan S, Phaneuf M, Kumarathasan P, Cakmak S, Denison MS, Brook JR, Vincent R. (2016) Development of an integrated approach for comparison of in vitro and in vivo responses to particulate matter. Part. Fibre Toxicol. 13(1), 41. PMC27520027
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Cheng, Y., Jin, Y.-H., Davidson, L., Chapkin, R.S., Jayaraman, A., Tamamis, P., Orr, A., Allred, C., Denison, M.S., Soshilov, A, Weaver, E. and Safe, SA. (2016) Microbial-derived 1,4-dihydroxy-2-naphthoic acid and related compounds as aryl hydrocarbon receptor agonists/antagonists: Structure-activity relationships and receptor modeling, Toxicol. Sci. 155, 458-473. PMID: 27837168
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Vandermarken, T., Boonen, I., Van Langenhove, K., Goeyens, L., Denison, M.S., Croes, K. and Elskens, M. (2916) In vitro reporter gene reactivity of PCDD/Fs and DL-PCBs in wood ask from domestic heating systems (Belgium) using the H1L7.5c1 CALUX cell bioassay, Organohalogen Compounds 78, 1107-1110.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Giani Tagliabue, S., Soshilov, A.A., Denison, M.S. and Bonati, L. (2016) Binding specificity of diverse AhR ligands interpreted by molecular modeling, Organohalogen Compounds 78, 877-878.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Corrada, D., Soshilov, A.A., Denison, M.S. and Bonati, L. (2016) Deciphering the AHR:ARNT dimerization process: how to assembly the functional puzzle of interacting interfaces. Organohalogen Compounds 78, 880-882.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Malisch, R., Denison, M.S., Fiedler, H., F�rst, P., Hoogenboom, R., Schaechtele, A. and Dieter Schrenk (2016) Are PCDD/PCDF standard solutions used in cell bioassay- or instrumental analysis-based dioxin analysis potentially acutely toxic to lab personnel?, Organohalogen Compounds 78,824-825.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Haedrich, J., Stumpf, C. and Denison, MS. (2016) Pushing the limits: bioassay performance at low levels of contamination in food samples based on third generation 20 DRE h4l7.5 rat hepatoma cells, Organohalogen Compounds 78, 810-812.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Van Langenhove, K., Reyns, T., Vandermarken, T., Servais, P., Denison, M.S., Van Loco, J. and Elskens, M. (2016) Endocrine activity in waste and river waters from the Brussels region, Belgium using the BG1Luc4E2 CALUX bioassay, Organohalogen Compounds 78,785-788.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Mehinto, A., Lao, W., Vandervort, D., Mazor, R., He, G., Denison, M.S., Vliet, S., Volz, D and Maruya, K. (2016) high throughput bioanalytical screening of inland waters of southern California, Organohalogen Compounds 78, 835-837.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Target audiences still continue to include: 1. the medical/veterinary/regulatory scientific community in order to understand the molecular basis of disease associated with dioxin-like chemicals and endocrine disruptors, 2. the medical/regulatory scientific community in order to provide additional validation and application information regarding use of bioanalytical methods for detection and relative quantitation of dioxin-like and endocrine disrupting chemicals and the presence of these chemicals in the food supply, and 3. the regulatory scientific community to alert them to potential risks associated with the presence of dioxin-like chemicals and endocrine disruptors in materials to which humans and animals are exposed and how to estimate exposure and risk associated with these chemicals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project continued to provide members of our laboratory the opportunities to interact and collaborate with US and international researchers on several aspects of this project. We continued our collaborations with Dr. Laura Bonati (University of Milano-Bicocca, Italy), Dr. Magnus Engwal (Obreo University, Sweden), Kim Croes (Free University of Brussels, Belgium,) and Dr. Prokopios Magiatis (University of Athens, Greece) in the characterization of the AhR and development, optimization, validation and application of our CALUX cell lines for detection of endocrine active chemicals. These researchers continue to use our cell bioassay systems for the detection and characterization of agonists/antagonists of these nuclear receptor-based bioassays in their own laboratories. In addition, new collaborations have been established with researchers at the US National Oceanographic & Atmospheric Administration in which we are analyzing extracts of sediment samples for dioxin-like and estrogenic chemicals. Dr. Soshilov and Zhao attended the Annual Society of Toxicology meeting in San Diego, where they presented the results of their studies and had the opportunity to interact and discuss their research with a wide range of experts in this area. Their participation not only allowed them to not only find out about the state of the art in their research area,but it has also gave both of them a new perspective on their work and its significance. Additionally, their interactions with scientists at this meeting has led to several additional research collaborations. How have the results been disseminated to communities of interest? The utilization and development of new and improved recombinant CALUX cell lines for detection of estrogenic and dioxin-like (AhR-active) chemicals have resulted in the detection of these chemicals in a wide variety of matrices to which humans and animals are exposed. Additionally, they have allowed us to identify and characterize a number of novel natural and synthetic chemicals that can activate the ER or AhR. Further characterization of these chemicals and extracts will allow assessment of their potential to produce adverse endocrine disrupting and dioxin-like activity in vivo. The availability of the new enhanced CALUX lines provide us with a way to now begin to screen for the presence of DLCs in very small sample volumes or where the levels are extremely low (i.e. ppq range). We have continue to provide our recombinant cell bioassay systems to many US and international laboratories free of charge and the results of these collaborations are not only increasing both the diversity in matrices and chemicals examined, but also leading to new applications and expanded validation of the assays. Results have been disseminated through a number of presentations including: 1) Scientific Conferences: two presentations on our research at the 35th International Symposium on Halogenated Environmental Organic Pollutants and POPs, Sao Paolo, Brazil; two research presentations at the annual Society of Toxicology Annual Meeting, San Diego, CA; one research presentation at the ToxCast Data Summit, Research Triangle Park, NC; one research presentation at the 7th International Workshop on Biomonitoring of Atmospheric Pollution (BIOMAP), Lisbon, Portugal; one research presentation at the 15th EUCHEMS International Conference on Chemistry and the Environment, Leipzig, Germany; one research presentation at the American Society of Nephrology, San Diego, CA; one research presentation at the Recent Advances in Food Analysis, Prague, Czech Republic; one research presentation at SETAC Australasia, Nelson, New Zealand; one research presentation at SETAC, Salt Lake City; 2) Research seminar presentation at Universities: one presentation at the Department of Biomedical and Pharmaceutical Sciences, Center for Environmental Health Sciences, University of Montana, Missoula and one presentation at the Interdisciplinary Environmental Toxicology Program, University of Illinois, Champaign-Urbana, IL (2015); 3) Research presentations and participation at meetings and workshops on water reuse and bioassay applications for detection of endocrine disruptors, including: The National Research Centre for Environmental Toxicology, University of Queensland, Brisbane, Australia, the Smart Water Research Centre, Griffith University, Southport, Australia, and the Southern California Coastal Water Research Project (SCCWRP), Costa Mesa, CA (2015). What do you plan to do during the next reporting period to accomplish the goals?We plan to continue the development, optimization and utilization of our novel cell bioassay systems for detection and relative quantitation of endocrine disrupting chemicals, with a focus on screening of chemicals from biological and food matrices and isolation and identification of the biologically active receptor ligands. In addition, we plan to continue the modeling and mutagenesis of the AhR ligand binding domain and given that the human AhR appaers to exhibit ligand selectivity distinctly different from that of other species, we will direct greater focus on the human AhR, with the ultimate goal of understanding the mechanism by which structurally diverse and human selective ligands can bind to and activate the AhR. This information to develop in silico approaches for high throughput screening methods for detection of AhR agonists and antagonists.

Impacts
What was accomplished under these goals? This past year, we made progress in several areas and the accomplishments are detailed below: Analyze the ligand binding modes in the homology model of the AhR LBD Through the use of the computational approach we developed in the first years of this project, we characterized the interactions of different ligands with the AhR in order to both verify if the approach was able both to predict which chemicals can bind AhR with high affinity and to examine species-specific differences in AhR ligand binding specificity. To this aim, the improved holo homology model of the AhR LBD we previously developed on the basis of the X-ray structures of the human HIF-2α PAS B domain co-crystallized with AhR agonists, was employed. Moreover, the docking approach previously proposed to predict the binding modes of AhR ligands into the modeled LBD was applied to a large test set of chemicals (i.e. ligands) including classical ligands with known experimental IC50 values for the AhR. On the basis of these studies, the computational protocol was revised and some steps were modified and a manuscript is currently in preparation describing the results of these studies. The importance of a flexible docking stage, where ligand docking (i.e. ligand binding) is performed to multiple conformations of the modeled AhR LBD in order to take into account a certain degree of protein flexibility (i.e. "ensemble docking") was confirmed on the basis of the good performances obtained in correctly classifying the "positive" samples. The choice of selecting more than one docking pose for each ligand in each receptor conformation was rejected because it does not improve the accuracy of the results. In addition, tests of the effects of including a refinement stage on the obtained ligand-protein binding geometry demonstrated that the computational cost was not balanced out by a better classification accuracy. Accordingly, the improved docking strategy is computational efficient and can be used for virtual screening of large numbers of putative ligands. Moreover, as a result of these studies, a tool for analyzing the binding poses and highlighting the molecular determinants of ligand binding was proposed and developed. It includes: the analysis of the contributions of each residue internal to the binding cavity to the binding free energy (ΔGbinding) to extract the characteristic "interaction profiles" of high- and low-affine ligands; the decomposition of the ΔGbinding of each residue in the single physico-chemical terms contributing to the stabilizing intermolecular interactions. The above computational protocol has been utilized to analyze the binding ability of a group of structurally diverse AhR agonists to the LBD of AhRs from different mammalian and fish AhRs and the results confirmed the ability of the method to not only correctly identify the high affinity ligands but also to highlight the molecular determinants of their AhR binding affinity Analyze the functional diversity of endocrine disruptive AhR ligands During the last grant cycle our studies also focused on investigating the mechanism of activation of the AhR by partial agonist/antagonists, such as α-naphthoflavone (ANF) and full antagonists, such as CH223191. ANF and similar compounds are unique receptor ligands (compared to what is known about agonists (partial and full) and antagonists of steroid hormone receptors) and they act as full antagonists at low concentration but at higher concentrations they act as full AhR agonists. This phenomenon is not observed with hormone receptor ligands. Studies of ligand-selective AhR antagonism were performed using a combination of experimental approaches including site directed mutagenesis, cross-species and cross-experimental system comparisons, and homology modeling. Comparisons of ANF agonist and antagonist activity for the C57BL mouse AhR across multiple experimental systems (i.e., hepatic cytosol, hepa-1 cells, transient transfections of C57 AhR in COS-1 cells and in vitro expressed C57 AhR/ARNT) strongly suggest that ANF agonism and ANF antagonism may be two independent processes that are mediated by overlapping yet distinctly different binding sites within the AhR LBD. This was confirmed by extensive LBD mutagenesis which revealed distinct clusters of LBD amino acids mediating the antagonist response of ANF, CH223191 and other antagonists, but also the concentration-dependent agonism of ANF. Specifically, the latter region also included amino acid residues that were computationally and experimentally implicated in dimerization of the AhR with ARNT (results from Aim 1), as well as the residue Leu248, which was found to be responsible for a dramatic difference in ANF agonist properties observed between the AhRs of C57BL and C3H mouse (which have distinct but highly responsive AhR alleles). Taken together, the studies carried out in this Aim over the past year led to the development of a novel hypothesis to explain the mechanism of agonism and antagonism by ANF. While a review of the literature has reported on the distinct activities of AhR agonists/antagonists like ANF and other compounds, the mechanism has never been explained and was assumed to be similar to that of steroid hormone receptor partial agonists/antagonists, which our results have demonstrated is not the case. In addition to studies of AhR antagonists, we identified a number of new classes of AhR agonists (naturally-occurring brominated indoles) as well as the ability of a yeast species present in the skin of most humans to produce a collection of some of the most potent nonhalogenated AhR agonists. In fact, extremely high concentrations of these ligands were shown to be produced by Malassezia furfur yeast in skin samples from humans with several skin diseases, suggesting that these chemicals may contribute to the adverse health effect. Studies are continuing in collaboration with colleagues at the University of Athens to examine these compounds and their potential adverse human health effects. Further application of CALUX bioassays We previously developed an estrogen-responsive recombinant human ovarian (BG1Luc4E2) cell line recently accepted by the US Environmental Protection Agency (USEPA) and Organization for Economic Co-operation and Development (OECD) as an officially accepted bioanalytical method to detect agonists/antagonists. Unfortunately, these cells appear to contain only one of the two known ER isoforms, ER? but not ER?, and the differential ligand selectivity of these ERs indicates that the currently accepted screening method only detects a subset of total estrogenic chemicals. To improve the estrogen screening bioassay, BG1Luc4E2 cells were stably transfected with an ER? expression plasmid and positive clones identified using ER?-selective ligands (genistein and Br-ER?-041). A highly responsive clone (BG1LucERbc9) was identified that exhibited greater sensitivity and responsiveness to ER?-selective ligands than BG1Luc4E2 cells, and quantitative reverse-transcription polymerase chain reaction confirmed the presence of ER? expression in these cells. Screening of pesticides and industrial chemicals identified chemicals that preferentially stimulated ER?-dependent reporter gene expression. Together, the results of these studies not only demonstrate the utility of this new dual-ER recombinant cell line for detecting a broader range of estrogenic chemicals than the current BG1Luc4E2 cell line, but screening with both cell lines allows identification of ER? and ER?-selective chemicals.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Vandermarken, T., De Galan, S., Croes, K., Van Langenhove, K., Vercammen, J., Sanctorum, H., Denison, M.S., Goeyens, L., Elskens, M., and Baeyens, W. (2015) Characterisation and implementation of the ERE-CALUX bioassay on indoor dust samples of kindergartens to assess estrogenic potencies, J. Steroid Biochem. Molec. Biol. 155 (Pt B), 182-189.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Maayah, Z.H., Ghebeh, H.B., Alhaider, A.A., El-Kadi, A.O., Soshilov, A.A., Denison, M.S., Ansari, M.A. and Korashy, H.M. (2015) Metformin inhibits 2,7-dimethylbenz[a]anthracene-induced breast carcinogenesis and adduct formation in human breast cells by inhibiting the cytochrome P4501A1/aryl hydrocarbon receptor signaling pathway, Toxicol. Appl. Pharmacol. 284, 217-226.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: DeGroot, D.E., Franks, D.G., Higa, T., Tanaka, J., Hahn, M.E. and Denison, M.S. (2015) Naturally-occurring marine brominated indoles are aryl hydrocarbon receptor ligands/agonists, Chem. Res. Toxicol. 28, 1176-1185.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Brennan, J. C., Bassal, A., He, G. and Denison, M.S. (2015) Development of a recombinant human ovarian (BG1) cell line containing estrogen receptor ? and ? for improved detection of estrogenic/antiestrogenic chemicals, Environ. Toxicol. Chem. 28, 1176-1185.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Brennan, J. C., He, G., Tsutsumi, T., Zhao, J., Wirth, E., Fulton, M. H. and Denison, M.S. (2015) Development of species-specific Ah receptor-responsive third generation CALUX cell lines with increased sensitivity and responsiveness, Environ. Sci. Tech. 49(19):11903-11912.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Croes, K., Debaille, P., Van den Bril, B., Staelens, J., Vandermarken, T., Van Langenhove, K., Denison, M.S., Leermakers, M. and Elskens, M. (2015) Assessment of estrogenic activity in PM10 air samples with the ERE-CALUX bioassay: method optimization and implementation at an urban location in Flanders (Belgium), Chemosphere 144, 392-398.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Bittner, G.D., Denison, M.S., Yang, C.S., Stoner, M.A. and He, G. (2014) Chemicals having estrogenic activity can be released from some bisphenol A-free, hard and clear, thermoplastic resins, Environ. Health 14 (1), 103.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Mexia, N., Gaitanis, G., Velegraki, A., Soshilov, A., Denison, M.S. and Magiatis, P. (2015) Pityriazepin and other potent AhR ligands isolated from Malassezia furfur yeast, Arch. Biochem. Biophys. 571, 16-20.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Caron-Beaudoin, E., Denison, M. S. and Sanderson, J. T. (2016) Effects of neonicotinoids on promoter-specific expression and activity of aromatase in human adrenocortical carcinoma (H295R) and umbilical vein endothelial (HUVEC) cells, Toxicol. Sci., 149, 134-144.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Target audiences include: 1. the medical/veterinary/regulatory scientific community in order to understand the molecular basis of disease associated with dioxin-like chemicals and endocrine disruptors, 2. the medical/regulatory scientific community in order to provide additional validation and application information regarding use of bioanalytical methods for detection and relative quantitation of dioxin-like and endocrine disrupting chemicals and the presence of these chemicals, and 3. the regulatory scientific community to alert them to potential risks associated with the presence of dioxin-like chemicals and endocrine disruptors in materials to which humans and animals are exposed and how to estimate exposure and risk associated with these chemicals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project provided members of our laboratory the opportunities to interact and collaborate with international researchers on several aspects of this project. Dr. Magnus Engwal (Obreo University, Sweden) and Dr. Prokopios magiatis (University of Athens, Greece) were provided with opportunities for direct hands-on experimental training on the cell bioassay systems developed and used in these studies. They will take these materials and their new expertise and apply it to their ongoing research studies. Dr. Soshilov attended the 34th International Symposium on Halogenated Environmental Organic Pollutants and POPs in Madrid, Spain, where he had the opportunity to interact and discuss his research with international scientist. This helped him not only find out about the state of the art in his research area,but it has also given him a new perspective on his work. Additionally, the meeting has provided him with several new collaborators. How have the results been disseminated to communities of interest? 1) five presentations on our research at the 34th International Symposium on Halogenated Environmental Organic Pollutants and POPs, Madrid, Spain; 2) three research presentations at the annual Society of Toxicology Annual Meeting, Phoenix, Arizona and one research presentation at the Society of Environmental Toxicology and Chemistry Meeting, Vancouver; 3) Research presentation at the US EPA, San Francisco (2014) on the application of our dioxin and estrogen bioassays for chemical detection and site characterization, participation in two online webinar (Business Ethics Network (BEN) sponsored Webinar (2014) on estrogenic chemicals in plastics and the Superfund Research Program-US EPA joint CLU-IN webinar (2014) on application of cell bioassays for toxicant detection; 4) description of our recombinant estrogenic cell bioassay and its application for the detection of estrogenic activity in commercial plastic on two public web sites (Center for Environmental Health (http://www.ceh.org/campaigns/plastics/ceh-action/a-warning-for-parents-bpa-free-may-not-equal-safe/) and Mother Jones magazine (http://www.motherjones.com/environment/2014/03/tritan-certichem-eastman-bpa-free-plastic-safe). What do you plan to do during the next reporting period to accomplish the goals? We plan to continue the development, optimization and utilization of our novel cell bioassay systems for detection and relative quantitation of endocrine disrupting chemicals, with a focus on screening of chemicals from biological and food matrices and isolation and identification of the biologically active receptor ligands. In addition, we plan to continue the modeling and mutagenesis of the AhR ligand binding domain with a greater focus on the human AhR, with the ultimate goal of understanding the mechanism by which chemicals can bind to and activate the AhR. This information to develop in silico approaches for high throughput screening methods for detection of AhR agonists and antagonists.

Impacts
What was accomplished under these goals? Significant progress was made in several areas and are detailed below. Analyze the ligand binding modes in the homology model of the AhR LBD. The availability of a reliable molecular docking approach based on the AhR LBD homology model would allow us to characterize the interactions of different ligands with the AhR, to predict which chemicals can bind AhR, and to examine species-specific differences in AhR ligand binding. Accordingly, an improved holo model of the AhR LBD was developed on the basis of the X-ray structures of the human HIF-2α PAS B domain co-crystallized with AhR agonists, and a docking approach was specifically devised to predict the binding modes of AhR ligands within the modeled LBD. This approach included, for each ligand: 1) a flexible ligand docking stage where docking analysis to multiple conformations of the modeled receptor is carried out in order to take into account a certain degree of protein flexibility ("ensemble docking"); 2) a structural refinement of several top scoring poses; and, 3) a rescoring stage, based on the MM-GBSA method, aimed at both selecting the best pose and obtaining an estimate of the binding free energy. We further improved this strategy to obtain a computationally efficient method for virtual screening of large numbers of putative ligands and analyzed different computational protocols derived from the initial proposal. We tested the effects on the docking accuracy using a single AhR model instead of a conformational ensemble and of including only the best docking pose or several top scoring poses for each ligand in the refinement and rescoring steps. The chemical ligand test set included a group of about 70 polychlorinated/polybrominated dibenzo-p-dioxins (PC/BDDs), polychlorinated dibenzofurans (PCDFs), and polycyclic aromatic hydrocarbons (PAHs), each with known experimental IC50 values for binding to the rat AhR. The accuracy of the different protocols (i.e. the ability to classify chemicals as either active or inactive ligands) was assessed by receiver operating characteristic (ROC) curves. The use of the ensemble docking approach and the choice of retaining only the top scoring pose allowed us to obtain the best accuracy in ligand characterization/analysis. This protocol was also used to analyze the binding ability of some structurally diverse AhR agonists, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), PCB126, 3-methylcholanthrene (3MC), indole, flavone, β-naphthoflavone (BNF), and several anti-inflammatory drugs, to mammalian and fish AhRs. The results confirm the ability of the method to correctly identify high affinity ligands but also highlight the molecular determinants of their binding affinity. In fact, the analysis of contributions of the residues internal to the binding cavity to the binding free energy, calculated for each binding pose, allowed extraction of the characteristic interactions of the ligands and to obtain atomistic details about the most relevant intermolecular interactions that stabilize the obtained poses. The proposed computational protocol established and tested during the past year represents a significant step forward in our attempts to develop a reliable approach for analyzing binding of structurally diverse AhR agonists and antagonists to the AhR. Analyze the molecular mechanisms contributing to functional diversity of AhR ligands We also continued to refine our homology model of the AhR and have demonstrated that the diversity in AhR ligand chemical structure results from differences in how the different ligands interact with specific amino acids within the binding pocket (suggestive of multiple binding conformations). This was confirmed by our ability to use site-directed mutagenesis to eliminate the binding of some ligands but not others. Consistent with these results, high throughput screening analysis carried out using our species-specific CALUX assays have identified novel species-specific AhR ligands, with one natural ligand (indirubin) being 10-times more potent than dioxin in human cells, but 100-times less potent than dioxin in rodent cells. In addition, to examine details about the mechanisms responsible for the promiscuity of AhR ligand binding, we determined the effects of mutations of specific amino acids within the AhR LBD of the mouse AhR on its functional activity (i.e. ligand binding and ligand-dependent DNA binding). Specifically, the nature of amino acids in the positions 318 (Phe in the wild type sequence) and 319 (Ile in the wild type sequence) of the mouse AhR were found to have a profound and ligand-selective effect on the binding properties of 12 structurally diverse AhR ligands. We separately generated all possible mutations in these amino acid positions, and found that the resulting sets of mutants (particularly, mutants of Ile319) could be used to differentiate among the distinct mechanisms of ligand binding and ligand-dependent transformation/DNA binding and classify these 12 AhR ligands into four distinct groups based on their ability to interact and activate specific mutant AhRs. In addition, some mutations resulted in an AhR that could only bind and be activated by a single ligand (an AhR containing I319K could only be activated by FICZ). Group 1 ligands consisted of several structurally diverse ligands (i.e. FICZ, BNF and leflunomide); Group 2 ligands included PAHs such as 3MC, benzo(a)pyrene and dibenz(a,h)anthracene; Group 3 ligands included the halogenated aromatic hydrocarbons (i.e. TCDD, TCDF and the dioxin-like PCB 126); and Group 4 also included other structurally diverse ligands (i.e. YH439, THS-017 and indirubin). These results indicate that different groups of AhR ligands, even with similarities in structure (such as PAHs or HAHs), can indeed bind with different amino acid determinants, and therefore likely possess distinct mechanisms of binding/activation of the AhR. Further analysis of these mutant AhRs revealed several unique mutant/ligand combinations that could alter the agonist properties of several AhR ligands. For example, several mutations of Phe318 resulted in the classical AhR agonists BNF and 3-MC, to exhibit partial AhR antagonist behavior. The results of these and additional analyses are suggestive of two distinct binding sites within the AhR that contribute to the agonist and/or antagonist activity of AhR ligands. Further application of CALUX bioassays. We have continued to develop and utilize our recombinant cell-based CALUX bioassays for screening of chemicals and extracts in order to detect and identify natural and synthetic activator/inhibitors of the AhR or steroid hormone receptors (i.e. estrogen and androgen receptors). Over the past year we have identified a variety of natural products that can interact with the AhR and either activate or inhibit its activity. In contrast to toxic AhR activators (i.e. dioxin and related dioxin-like chemicals), these compounds affect the AhR signaling pathway only transiently and thus would likely be non-toxic. We have also screened extracts of more than 38 different commercial children's plastic sippy cups and plates for the presence of both activators/inhibitors of the AhR and estrogen receptor with the goal of identifying materials that may leach endocrine active chemicals into children's food and drink. While ethanol (100%) extracts of many products had substantial levels of estrogenic activity, for some products, water:ethanol (90:10) extracts were more active and 100% water extract of two of these products had even more estrogenic activity. We are attempting to identify the responsible water-soluble estrogenic chemical(s) as it could be easily transferred into food and drinks to which children would be exposed. (7767 characters and spaces)

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: DeGroot, D.E. and Denison, M.S. (2013) Nucleotide specificity of DNA binding of aryl hydrocarbon receptor:ARNT complex is unaffected by ligand structure, Toxicol. Sci. 137, 102-113. PMC3924043
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Anwar-Mohamed, A., Elshenawy, O.H., Soshilov, A.A., Denison, M.S., Le, X.C., Klotz, L.-O., El-Kadi, A.O.S. (2013) Methylated pentavalent arsenic metabolites are bifunctional inducers as they induce cytochrome P4501A1 (CYP1A1) and NAD(P)H:quinone oxidoreducatase (NQO1) through AhR and Nrf2 dependent mechanisms, Free Radical Biol. Med. 67C, 171-187. PMID: 24161444
• Type: Journal Articles Status: Published Year Published: 2014 Citation: DeGroot, D.E., Hayashi, A. and Denison, M.S. (2014) Lack of ligand-selective binding of the aryl hydrocarbon receptor to putative DNA binding sites regulating expression of Bax and Paraoxonase 1 genes, Arch. Biochem. Biophys. 541, 13-20. PMC3875388
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Addeck, A., Croes, K., Van Langenhove, K., Denison, M.S., Elhamalawy, A., Elskens, M. and Baeyens, W. (2014) Time-integrated monitoring of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) in urban and industrial wastewaters using a ceramic toximeter and the CALUX bioassay, Chemosphere 94, 27-35. PMID: 24075528
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Addeck, A., Croes, K., Van Langenhove, K., Denison, M.S., Afify, A.S., Gao, Y., Elskens, M. and Baeyens, W. (2014) Time-integrated monitoring of dioxin-like polychlorinated biphenyls (dl-PCBs) in aquatic environments using the ceramic toximeter and the CALUX bioassay. Talanta 120, 413-418. PMID: 24468390
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Vogel, C.F.A., Khan, E.M., Chang, W.L.W., Wu, D., Haarmann-Stemmann, T., Hoffmann, A. and Denison, M.S. (2014) Cross-talk between aryl hydrocarbon receptor and the inflammatory response: a Role for NF-B, J. Biol. Chem. 289, 1866-1875. PMC3894361
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Soshilov, A.A. and Denison, M.S. (2014) Ligand promiscuity of aryl hydrocarbon receptor agonists and antagonists revealed by site directed mutagenesis, Molec. Cell. Biol. 34, 1707-1719. PMC3993610
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Bessede, A., Gargaro, M., Pallotta, M.T., Matino, D., Servillo, G., Brunacci, C., Bicciato, S., Mazza, E.M.C., Macchiarulo, A., Vacca, C., Iannitti, R., Tissi, L., Volpi, C., Belladonna, M.L., Orabona, C., Bianchi, R., Lanz, T., Platten, M., Della Fazia, M.A., Piobbico, D., Zelante, T., Funakoshi, H., Nakamura, T., Gilot, D., Denison, M.S., Guillemin, G.J., DuHadaway, J.D., Prendergast, G.C., Metz, R., Geffard, M., Boon, L., Pirro, M., Iorio, A., Veyret, A., Romani, Grohmann, U., Fallarino, F. and Puccetti, P. (2014) Aryl hydrocarbon receptor control of a disease tolerance defense pathway, Nature 511, 184-190. PMC4098076
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Ghorbanzadeh, M., van Ede, K.I., Larsson, M.N., van Duursen, M.B.M., Poellinger, L., Lucke, S., Machala, M., Pencikova, K., Vondracek, J., van den Berg, M., Denison, M.S., Ringsted, T. and Andersson, P.L. (2014) In vitro and in silico derived relative effect potencies of Ah-receptor mediated effects by PCDD/Fs and PCBs in rat, mouse and guinea pig CALUX cell lines, Chem. Res. Toxicol. 27, 1120-1132. PMID: 24901989
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Novotna, A., Korhonova, M., Bartonkova, I., Soshilov, A.A., Denison, M.S., Kolar, M. and Dvorak, Z. (2014) Enantiospecific effects of ketoconazole on aryl hydrocarbon receptor, PLoS One 9(7) e101832. PMC4084896

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Target audiences include: 1. the medical/veterinary/regulatory scientific community in order to understand the molecular basis of disease associated with dioxin-like chemicals and endocrine disruptors, 2. the medical/regulatory scientific community in order to provide additional validation and application information regarding use of bioanalytical methods for detection and relative quantitation of dioxin-like and endocrine disrupting chemicals and the presence of these chemicals, and 3. the regulatory scientific community to alert them to potential risks associated with the presence of dioxin-like chemicals and endocrine disruptors in materials to which humans and animals are exposed and how to estimate exposure and risk associated with these chemicals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project provided training in the development, application and utilization of cell bioassays for toxicant detection to Dr. Magnus Engwal (Obreo University, Sweden) and Dr. Prokopios Magiatis (University of Athens, Greece). How have the results been disseminated to communities of interest? Dissemenation of results for 2013 have included: 1) two presentations on our research at the 33rdInternational Symposium on Halogenated Environmental Organic Pollutants and POPs, Daegu, South Korea (9/13), 2) one research presentation at the annual Society of Toxicology Annual Meeting, San Antonio (3/2013), 3) two research presentations at the California Breast Cancer Research Program Meeting, Costa Mesa, CA (6/2013)(3/2013)2) one research presentations at the annual Society of Toxicology Annual Meeting, San Antonio (3/2013), 4) one research presentation at the Department of Environmental and Molecular Toxicology, Oregon State University (11/2013), 5) one research presentation in the Department of Environmental Toxicology, University of California, Davis (10/2013), 6) online descriptions of our recombinant estrogenic and dioxin cell bioassays, their validation and regulatory acceptance in the US (USEPA and ICCVAM), Europe (OECD) and 30 other countries (OECD) described on 8 online sites including the NIH-NIEHS website, in the NIEHS e-Factor newsletter and other online news and information sites. What do you plan to do during the next reporting period to accomplish the goals? We plan to continue the development, optimization and validation of our novel cell bioassay systems for detection and relative quantitation of endocrine disrupting chemicals and to utilize these bioassays for characterization of extracts from a variety of biological, environmental and food samples. In addition, we plan to continue the modeling and mutagenesis of the AhR ligand binding domain with the goal of understanding the mechanism by which chemicals can bind to and activate the AhR and utilize this information to develop docking programs to be able to predict the ability of chemicals to bind to and activate the AhR.

Impacts
What was accomplished under these goals? During the past year, we continued to improve and expand the applications and use of our recombinant cell-based CALUX bioassays for the detection of activator/inhibitors of the Ah (dioxin) receptor (AhR) and estrogen receptor signaling pathways. We have also developed a new cell bioassay for the detection of chemicals that affect signaling by the glucocorticoid and progesterone receptors. These new assays will expand the range of endocrine disruption mechanisms that can be assessed. We have also continued refine our homology model of the AhR and have demonstrated that the diversity in AhR ligand chemical structure results from differences in how the different ligands interact with amino acids within the binding pocket. In fact, we have demonstrated by site-directed mutagenesis that we can eliminate the binding of some ligands with no affect on the ability of others to bind to and activate the AhR. This provides us with potential avenues in which to develop ligand-specific AhRs that can be used to make chemical selective bioassays. We have further improved the sensitivity of our AhR CALUX bioassay by amplification methods, increasing the minimal detection limit of the bioassay by 10- to 100-fold. Our development of a human-specific CALUX line has also proven to be very useful for the screening and identification of novel AhR activators that have potential utility as therapeutic agents, given the recent demonstration of a critical role for the AhR in human health. Using our species-specific CALUX assays, we have carried out rapid high-throughput screening analysis to identify novel species-specific AhR ligands, with one ligand 10-times more potent than dioxin in human cells, but 100-times less toxic than dioxin in rodent cells. Use of these new cell lines will facilitate the identification of chemicals that may represent a unique species-specific risk. Our continued improvement in these bioassays and the generation of additional validation results from our lab and those of our colleagues using these CALUX bioassays to screening extracts from a variety of different matrices is greatly expanding the utility and applications of our AhR-based bioassays and contributing to greater acceptance of this bioassays worldwide. Additionally, the approval of our estrogen receptor CALUX bioassay (referred to as the BG1Luc-ER-TA assay) by the Organization for Economic Cooperation and Development (OECD test guidelines TG455 and TG457) and its acceptance as an alternative bioassay in the USEPA endocrine disruptor screening program will also expand its use internationally.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Zhao, B., Bohonowych, J.E.S., Timme-Laragy, A., Jung, D., Affatato, A.A., Rice, R.H., Di Giulio, R.T. and Denison, M.S. (2013) Common commercial and consumer products contain activators of the aryl hydrocarbon (dioxin) receptor, PloS One 8 (2), e56860.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Kamenickova, A., Anzenbacherova, E., Pavek, P., Soshilov, A.A., Denison, M.S., Anzenbacher, P. and Dvorak, Z. (2013) Pelargonidin activates the AhR and induces CYP1A1 in primary human hepatocytes and human cancer cell lines HepG2 and LS174T, Toxicol. Lett. 218, 253-259.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Odio, C., Holzman, S., Denison, M.S., Fraccalvieri, D., Bonati, L., Franks, D., Hahn, M.E. and Powell, W. (2013) Specific ligand binding domain residues confer low dioxin responsiveness to AHR1? of Xenopus laevis, Biochem. 52, 1746-1754.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Magiatis, P., Pappas, P., Gaitanis, G., Mexia, N., Melliou, E., Galanou, M., Vlachos, C., Stathopoulou, K., Skaltsounis, A.L., Marselos, M., Velegraki, A., Denison, M.S. and Bassukas, I.D. (2013) Human symbiotic yeasts overproduce a collection of exceptionally potent activators of the Ah (dioxin) receptor in diseased skin, J. Invest. Dermatol. 133, 2023-2030.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Van den Berg, M., Denison, M.S., Birnbaum, L.S., DeVito, M., Fiedler, H., Falandysz, J., Rose, M., Schrenk, D., Safe, S., Tohyama, C., Tritscher, A., Tysklind, M. and R.E. Peterson, R.E. (2013) Polybrominated dibenzo-p-dioxins (PBDDs), dibenzofurans (PBDFs) and biphenyls (PBBs) - Inclusion in the toxicity equivalency factor concept for dioxin-like compounds, Toxicol. Sci. 133, 197-208.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Croes, K., Colles, A., Koppen, G., De Galan, S., Vandermarken, T., Govarts, E., Bruckers, L., Nelen, V., Schoeters, G., Van Larebeke, N., Denison, M.S., Mampaey, M. and Baeyens, W. (2013) Determination of PCDD/Fs, PBDD/Fs and dioxin-like PCBs in human milk from mothers residing in the rural areas in Flanders, using the CALUX bioassay and GC-HRMS, Talanta 113, 99-105.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Hu, Q., He, G., Zhao, J., Soshilov, A., Denison, M.S., Zhang, A., Yin, H., Fraccalvieri, D., Bonati, L., Xie, Q. and Zhao, B. (2013) Ginsenosides are novel naturally-occurring aryl hydrocarbon receptor ligands, PLoS One, 8, e66258.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Kamenickova, A., Anzenbacherova, E., Pavek, P., Soshilov, A.A., Denison, M.S., Zapletalova, M., Anzenbacher, P. and Dvorak, Z. (2013) Effects of anthocyanins on the AhR-CYP1A1 signaling pathway in human hepatocytes and human cancer cell lines, Tox. Lett. 221, 1-8.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Brennan, J.C., Denison, M.S., Holstege, D.M., Magiatis, P., Dallas, J.L., Gutierrez, E.G., Soshilov, A.A., deRopp, J.S. and Millam, J.R. (2013) 2,3-cis-2R,3R-(-)-epiafzelechin-3-O-p-coumarate, a novel flavan-3-ol isolated from Fallopia convolvulus seed, is an estrogen receptor agonist in human cell lines, BMC Complementary and Alternative Medicine (Section: Basic Research) 13, 133.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Brander, S.M., Connon, R.E., He, G, Hobbs, J.A., Smalling, K.L., The, S.J., White, J.W., Werner, I., Denison, M.S. and Cherr, G.N. (2013) From omics to otoliths: responses of an estuarine fish to endocrine disrupting compounds across biological scales, PLoS One 8 (9) e74251.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: DeGroot, D.E. and Denison, M.S. (2013) Nucleotide specificity of DNA binding of aryl hydrocarbon receptor:ARNT complex is unaffected by ligand structure, Toxicol. Sci. 137, 102-113.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Addeck, A., Croes, K., Van Langenhove, K., Denison, M.S., Elhamalawy, A., Elskens, M. and Baeyens, W. (2013) Time-integrated monitoring of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) in urban and industrial wastewaters using a ceramic toximeter and the CALUX bioassay, Chemosphere 94, 27-35.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Anwar-Mohamed, A., Elshenawy, O.H., Soshilov, A.A., Denison, M.S., Le, X.C., Klotz, L.-O., El-Kadi, A.O.S. (2013) Methylated pentavalent arsenic metabolites are bifunctional inducers as they induce cytochrome P4501A1 (CYP1A1) and NAD(P)H:quinone oxidoreducatase (NQO1) through AhR and Nrf2 dependent mechanisms, Free Radical Biol. Med.67C, 171-187.
• Type: Book Chapters Status: Published Year Published: 2013 Citation: He, G., Zhao, J., Brennan, J.C., Affatato, A.A., Zhao, B., Rice, R.H. and Denison, M.S. (2013) Cell-based assays for identification of aryl hydrocarbon (Ah) receptor activators. In: Optimization of Drug Discovery: In Vitro Methods, Second Edition (Yan, A. and Caldwell, G.W., Eds.), pp. 221-235, Humana Press, New York, NY.
• Type: Book Chapters Status: Published Year Published: 2013 Citation: Soshilov, A.A. and Denison, M.S. (2013) DNA binding (gel retardation assay) Analysis for identification of aryl hydrocarbon (Ah) receptor agonists and antagonists. In: Optimization of Drug Discovery: In Vitro Methods, Second Edition (Yan, A. and Caldwell, G.W., Eds.), pp. 207-219, Humana Press, New York, NY.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Outputs for 2012 have included: 1) five presentations on our research at the 32th International Symposium on Halogenated Environmental Organic Pollutants and POPs, Cairns, Australia (9/12), 2) two research presentations at the annual Society of Toxicology Annual Meeting, San Francisco (3/2012), 3) description of our recombinant estrogenic cell bioassay and its validation and regulatory acceptance in the US (USEPA and ICCVAM), Europe (OECD) and 30 other countries (OECD) described on the NIH-NIEHS website and in the NIEHS e-Factor newsletter. PARTICIPANTS: Participants in research on this project include: researchers at UC Davis (Dr. Guochun He, Dr. Anatoly Soshilov, Dr. Jing Zhao, Dr. Gary Cherr), and two graduate students (Danica DeGroot, Jennifer Brennan), University of Athens (Dr. Prokopios Magiatis), University of Milan, Italy (Dr. Laura Bonati), Vrije Universiteit, Brussels (Dr. Willy Baeryns), Woods Hole Oceanographic Institute (Dr. Mark Hahn and Dr. Sibel Karchner), University of Nottingham, UK (Dr. Richard Wall), University of Alberta, Canada (Dr. Ayman O.S. El-Kadi), Yong Loo Lin School of Medicine, Singapore (Dr. Eu-Leong Yong), University of Cagliari, Italy (Dr. Gianfranco Balboni), and the Chinese Academy of Sciences, Beijing (Dr. Bin Zhao). TARGET AUDIENCES: Target audiences include: 1. the medical/veterinary/regulatory scientific community in order to understand the molecular basis of disease associated with dioxin-like chemicals and endocrine disruptors, 2. the medical/regulatory scientific community in order to provide additional validation and application information regarding use of bioanalytical methods for detection and relative quantitation of dioxin-like and endocrine disrupting chemicals and the presence of these chemicals, and 3. the regulatory scientific community to alert them to potential risks associated with the presence of dioxin-like chemicals and endocrine disruptors in materials to which humans and animals are exposed and how to estimate exposure and risk associated with these chemicals. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We have continued to improve and expand the applications and use of our recombinant cell-based CALUX bioassays for detection of activators and inhibitors of Ah receptor and estrogen receptor signal transduction pathways. We have also continued define the structure of the AhR ligand binding domain (LBD) by homology modeling and site-directed mutagenesis with the long-term goal of understanding how liand binding leads to AhR activation and to generate ligand-selective AhRs for new bioassay development. We have further refined, improved and validated out homology model of the ligand binding domain of the Ah "dioxin" receptor (AhR) and have extended this modeling to AhRs from a wide variety of mammalian, fish and bird to allow comparative analysis. We have also developed a docking protocol suitable to predict and characterize the binding modes of diverse AhR ligands within the AhR ligand binding pocket. While an initial validation of the docking protocol for virtual screening ~140 ligands confirmed the reliability of the method to predict binding geometries, additional studies are needed to predict affinity of binding. Chemical screening approaches using novel amplified AhR-responsive recombinant cell lines have led to the identification of novel species-specific AhR ligands. In addition, we have developed a novel stable and fully-functional mouse:human AhR chimera containing the human AhR LBD provides for the first time a unique model system to begin to dissect the residues critical for human AhR ligand binding and ligand-dependent AhR functional activity. We also continued to optimize, validate and utilize our endocrine disruptor bioassays and not only demonstrated that two common pyrethoid insecticides were estrogenic, but identified substantial estrogenic activity in water samples from the Upper Mokelumne and Calavaras Rivers in the Sierra Mountains, the identity and source of the estrogenic activity is unknown. Our continued improvement in the sensitivity of the CALUX bioassays, increased validation data from screening results, increased diversity in the matrices and materials examined and the diversity in available bioassays, is greatly expanding our bioassay capabilities and also leading to greater acceptance of these bioassays. In fact, our estrogen receptor CALUX bioassay (referred to as the BG1Luc-ER-TA assay) was recently officially adopted by the international Organization for Economic Cooperation and Development (OECD) in OECD test guidelines TG455 and TG457 and the assay now may be used for official regulatory purposes in the 34 member countries, including the US. The USEPA agreed to include the BG1Luc-ER-TA assay as an accepted method in its endocrine disruptor screening program. (2744 characters and spaces)

Publications

• Denison, M.S., Soshilov, A.A., He, G., DeGroot, D.E. and Zhao, B. (2011) Exactly the same but different: promiscuity and diversity in the molecular mechanisms of action of the aryl hydrocarbon (dioxin) receptor, Toxicol. Sci. 124, 1-22. PMC3196658
• DeGroot, D., He, G., Fraccalvieri, D., Bonati, L., Pandini, A. and Denison, M.S. (2011) AhR Ligands: Promiscuity in Binding and Diversity in Response. In: The Ah Receptor in Biology and Toxicology, Edited by Pohjanvirta, R., pp. 63-79
• John Wiley & Sons, Inc. Hoboken, NJ. Wang, Y., Yang, D., Chang, A., Chan, W.K., Zhao, B., Denison, M.S. and Xue, L. (2012) Synthesis of a ligand-quencher conjugate for the ligand binding study of the aryl hydrocarbon receptor using a FRET assay, Med. Chem. Res. 21, 711-721.
• Addeck, A., Croes, K., Van Langenhove, K., Denison, M.S., Elskens, M. and Willy Baeyens (2012) Dioxin analysis in water by using a passive sampler and CALUX bioassay, Talanta 88, 73-78. PMID: 22265472
• Tiong, C.T., Chen, C., Zhang, S.J., Li, J., Soshilov, A., Denison, M.S., Lee Soon-U, L., Tam, V.H., Wong, S.P., Xu, E., Yong, E.-L. (2011) A novel prenylflavone restricts breast cancer cell growth through AhR-mediated destabilization of ERα protein, Carcinogenesis, 33, 1089-1097.
• Brander, S.M., He, G., Smalling, K.L., Denison, M.S. and Cherr, G.N. (2012) The in vivo estrogenic and in vitro anti-estrogenic activity of permethrin and bifenthrin, Environ. Toxicol. Chem. 31, 2848-2855. Fraccalvieri, D., Soshilov, A.A., Karchner, S.I., Franks, D.G., Pandini, A., Bonati, L., Hahn, M.E. and Denison, M.S. (2013) Comparative analysis of homology models of the Ah receptor ligand binding domain: Verification of structure-function predictions by site-directed mutagenesis of a non-functional AhR. Biochem. 52, 714-725.
• Wall, R.J., He, G., Denison, M.S., Congiu, C., Onnis, V., Fernandes, A., Bell, D.R., Rose, M., Rowlands, J.C. and Balboni, G. (2012) Novel 2-amino-isoflavones exhibit aryl hydrocarbon receptor agonist or antagonist activity in a species/cell-specific contact, Toxicol. 88, 881-887.
• Croes, K., Vandermarken, T., Van Langenhove, K., Elskens, M., Desmedt, M., Roekens, E., Denison, M.S., Van Larebeke, N. and Baeyens, W. (2012) Analysis of PCDD/Fs and dioxin-like PCBs in atmospheric deposition samples from the Flemish measurement network: correlation between the CALUX bioassay and GC-HRMS, Chemosphere 88, 881-887.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Outputs for 2011 have included: 1) six presentations on our research at the 31th International Symposium on Halogenated Environmental Organic Pollutants and POPs, Brussels, Belgium (9/11), 2) three research presentations at the annual Society of Toxicology Annual Meeting, Baltimore (3/2011), 3) description of our recombinant cell bioassay system that is being used in Vietnam and published on the Than Nien Dailey News web site, and 4) description of our improved recombinant cell bioassay system for dioxin-like chemicals and its application for screening of environmental, biological and food samples for these contaminants and published on the NIEHS Superfund web site. PARTICIPANTS: Participants in research on this project include: researchers at UC Davis (Dr. Guochun He, Dr. Anatoly Soshilov, Dr. Jing Zhao), and two graduate students (Danica DeGroot, Jennifer Brennan), University of Athens (Dr. Prokopios Magiatis), University of Milan, Italy (Dr. Laura Bonati) and the Chinese Academy of Sciences, Beijing (Dr. Bin Zhao). TARGET AUDIENCES: Target audiences include: 1. the medical/veterinary/regulatory scientific community in order to understand the molecular basis of disease associated with dioxin-like chemicals and endocrine disruptors, 2. the medical/regulatory scientific community in order to provide additional validation and application information regarding use of bioanalytical methods for detection and relative quantitation of dioxin-like and endocrine disrupting chemicals and the presence of these chemicals, and 3. the regulatory scientific community to alert them to potential risks associated with the presence of dioxin-like chemicals and endocrine disruptors in materials to which humans and animals are exposed and how to estimate exposure and risk associated with these chemicals. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We completed and published the results of our analyses of the ability of a diverse spectrum of chemicals to bind to and activate/inhibit the AhR and AhR-dependent gene expression. These studies demonstrated that ligand promiscuity extends to AhRs from several species and suggested at least two distinct binding patterns for AhR agonists within the AhR LBD: dioxin-like and polychlorinated biphenyl (PCB)-like. Screening of flavonoids, indirubins and more than 150 current therapeutic drugs identified several exceptionally potent novel AhR agonists and not only further supported the structural promiscuity of AhR ligands, but identified a novel ligand-selective AhR antagonist (CH223191) that preferentially inhibits the ability of TCDD and related HAHs to bind to and activate the AhR, but has little inhibitory effect on other ligands. The preferentially inhibition of HAH ligands are consistent with differential interactions of HAH and nonHAH ligands within the AhR ligand binding domain (LBD) and suggest that this could contribute to differences in the toxic/biological potency and effects of these chemicals. Preliminary molecular docking calculations were performed to simulate TCDD binding to the modelled mAhR LBD and to some mutant mAhRs that lacked both TCDD and TCDD- inducible DNA binding. The results were fully consistent with the hypothesis made on the basis of the homology model and our mutagenesis studies in that the binding geometries fully consistent with the location of the internal cavity in the modelled AhR LBD and these findings have improved our understanding of the molecular determinants of TCDD binding. Given the key regulatory role of hsp90 in ligand-dependent AhR functionality, we analyzed the interactions of hsp90 with the AhR LBD to understand how ligand binding stimulates Hsp90 dissociation. Using a series of AhR variants with a modified PAS B domain, AhR PASA-PASB fragments of various lengths and deletions within the AhR LBD, we demonstrated (i) an inhibitory effect on transformation concomitant with Hsp90 binding in the PAS B domain, (ii) an ability of the PASA-PASB fragment of the AhR to reproduce key steps in the transformation process and (iii) a ligand-dependent conformational change in the PAS A domain consistent with increased PAS A exposure during AhR transformation. Our results suggest that ligand-dependent interdomain destabilization of the AhR-Hsp90 interaction is most likely due to the interaction of exposed PAS B hydrophobic regions with other AhR domains. We proposed a new mechanism of AhR transformation by which ligand binding results in a protein conformational change leading to an opening up of the AhR structure in the PAS A and inter-PAS regions, allowing Arnt to first bind to the PAS A domain, progressively displacing AhR bound Hsp90 from PAS B and ultimately from the AhR through competitive binding to overlapping binding sites on the AhR. This finding also confirms that a dynamic description of all the conformational states available to the PAS B domain, and not just the ligand-bound conformation that is described by our homology model, is needed to gain insights into mechanisms of AhR activation.

Publications

• He, G., Tsutsumi, T., Zhao, B., Baston, D.S., Zhao, J., Heath-Pagliuso, S. and Denison, M.S. (2011) Third generation Ah receptor-responsive luciferase reporter plasmids: amplification of dioxin responsive elements dramatically increases CALUX bioassay sensitivity and responsiveness, Toxicol. Sci. 123, 511-522.
• Korashy, H., Anwar-Mohamed, A., Soshilov, A., Denison, M.S. and El-Kadi, A. (2011) The p38 MAPK inhibitor SB203580 induces cytochrome P450 1A1 gene expression in murine and human hepatoma cell lines through ligand-dependent aryl hydrocarbon receptor activation, Chem. Res. Toxicol. Chem. Res. Toxicol. 19, 1540-1548.
• Van Langenhove, K., Croes, K., Denison, M.S., Elskens, M. and Baeyens, W. (2011) The CALUX Bio-assay: Analytical comparison between mouse hepatoma cell lines with a low (H1L6.1c3) and high (H1L7.5c1) number of dioxin response elements. Talanta 85, 2039-2046.
• Elskens, M., Baston, D.S., Stumpf, C., Haedrich, J., Keupers, I., Croes, K., Denison, M.S., Baeyens, W. and Goeyens, L. (2011) CALUX measurements: statistical inferences for the dose response curve, Talanta 85, 1966-1973.
• Croes, K., Van Langenhove, K., Den Hond, E., Bruckers, L., Colles, A., Koppen, G., Loots, I., Nelen, V., Schoeters, G., Nawrot, T., Can Larebeke, N., Denison, M.S. and Baeyens, W. (2011) Quantification of PCDD/Fs and dioxin-like PCBs in small amounts of human serum using the sensitive H1L7.5c1 mouse hepatoma cell line: validation and analysis of human serum samples from adolescents for the Flemish Human Biomonitoring campaign, Talanta 85, 2484-2491.
• Soshilov, A. and Denison, M.S. (2011) Ligand displaces heat shock protein 90 from overlapping binding sites within the aryl hydrocarbon receptor ligand-binding domain, J. Biol Chem. 286, 35275-35282.
• Motto, I., Bordogna, A., Soshilov, A., Denison, M.S. and Bonati, L. (2011) A new aryl hydrocarbon receptor homology model targeted to improve docking reliability, J. Chem. Info. Modeling 51, 2868-2881.
• El-Gendy, M., Soshilov, A., Denison, M.S. and El-Kadi, A.O.S. (2011) Transcriptional and posttranscriptional inhibition of dioxin-mediated induction of CYP1A1 by harmine and harmol, Toxicol. Lett. 208, 51-61.
• Croes, K., Van Langerhove, K., Elskens, M., Desmedt, M., Roekens, E. Kotz, A., Denison, M.S. and Baeyens, W. (2010) Analysis of PCDD/Fs and dioxin-like PCBs in atmospheric deposition samples from the Flemish measurement network: optimization and validation of a new CALUX bioassay method, Chemosphere, 82, 718-724. Baston, D.S. and Denison, M.S. (2010) Considerations for potency equivalent calculations in the Ah receptor-based CALUX bioassay: Normalization of superinduction results for improved sample potency estimation, Talanta, 83, 1415-1421.
• Lee Y.C., Oslund, K.L., Thai, P., Velichko, S., Fujisawa, T., Duong, T., Denison, M.S., Wu, R. (2011) TCDD induced MUC5AC expression: Aryl hydrocarbon receptor-independent/EGFR-dependent MAPK signaling pathway, Amer. J. Respir. Cell Molec. Biol. 45, 270-276.
• He, G., Zhao, B. and Denison, M.S. (2011) Identification of benzothiazole derivatives and polycyclic aromatic hydrocarbons as aryl hydrocarbon receptor agonists present in tire extracts, Environ. Toxicol. Chem. 30, 1915-1925.
• Aung, H.H., Lame, M.W., Gohil, K., He, G., Denison, M.S., Rutledge, J.C. and Wilson, D. W. (2011) Comparative gene responses to collected ambient particles in vitro: endothelial responses, Physiol. Genomics 43, 917-929.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Outputs for 2010 have included: 1) Research seminar to the Human and Ecological Risk Office, Brownsfields and Environmental Restoration Program, California Department of Toxic Substances Control, Sacramento (6/10), describing dioxin action and dioxin bioassays 2) Research seminar to the Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR (7/10) describing our work on translation of toxicological mechanisms into bioassays for chemical detection and site characterization: dioxins, HAHs and endocrine disruptors, 3) five presentations on our research at the 30th International Symposium on Halogenated Environmental Organic Pollutants and POPs, San Antonio, Texas (9/09), 4) a research presentation at the annual Society of Toxicology Annual Meeting, Baltimore (3/09) on our dioxin bioassay systems. PARTICIPANTS: Participants in research on this project include: researchers at UC Davis (Dr. Anatoly Soshilov, Dr. Jing Zhao), two graduate students (Danica DeGroot and Jennifer Brennan) and researchers at the University of Milan, Italy (Dr. Laura Bonati) and the Chinese Academy of Sciences, Beijing (Dr. Bin Zhao). TARGET AUDIENCES: Target audiences include: 1. the medical/veterinary/regulatory scientific community in order to understand the molecular basis of disease associated with dioxin-like chemicals and endocrine disruptors, 2. the medical/regulatory scientific community in order to provide additional validation and application information regarding use of bioanalytical methods for detection and relative quantitation of dioxin-like and endocrine disrupting chemicals, and 3. the regulatory scientific community to alert them to potential risks associated with the presence of dioxin-like chemicals and endocrine disruptors in materials to which humans and animals are exposed and how to estimate exposure and risk associated with these chemicals. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The aryl hydrocarbon "dioxin" receptor (AhR) is a promiscuous ligand-dependent transcription factor that regulates both endogenous biological effects as well as mediating the toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related halogenated aromatic hydrocarbons (HAHs). Our recent analyses have provided fairly convincing evidence that while structurally distinct AhR agonists produce a similar activation effect on the AhR, they appear to differentially interact with amino acids within the AhR ligand binding pocket. Mechanism-based common reactivity pattern (COREPA) modeling of structurally diverse AhR ligands revealed two distinct classes of chemicals that can bind to the AhR based on differences in their specific interactions with the AhR. We have also identified and characterized the ability of CH223191 to selectively antagonize the ability of toxic HAHs to bind to and activate the AhR, with little or no effect on PAHs and other nontoxic AhR ligands. These results provide additional evidence for differences in the ability of structurally diverse chemicals to bind to the AhR and suggest that differences in how these toxic and non-toxic AhR ligands specifically interact with the AhR could contribute to or determine their differential ability to produce beneficial and/or adverse effects in exposed animals. CH223191 is also the first ligand-selective AhR identified to date and given its selectivity, it is a potential therapeutic agent (or lead compound to generate a therapeutic agent) that can block the toxicological and biological effects of toxic HAHs, while not inhibiting the ability of non-toxic/beneficial AhR ligands to bind to and activate the AhR. We have also improved and expanded our recombinant cell-based bioassay systems for detection of AhR agonists and these improved third generation (G3) cell bioassays have a 10-50-fold increased sensitivity and produce more than a 100- to 1,000-fold greater induction response to AhR agonists. Utilizing a recombinant human cell G3 bioassay we developed during the past year we have begun high-throughput screening for chemicals that can bind to and activate the human AhR, given its unique characteristics and the role of the AhR in potential clinical/therapeutic effects. It has been used to identify a novel human-specific AhR antagonist that can promote the expansion of human hematopoietic stem cells and thus has significant therapeutic potential. In addition, our human G3 cell bioassay has been used for high-throughput screening of >300,000 compounds from an NIH chemical library to identify AhR agonists (as part of a NIH X01 grant) and initial screening has identified >100 novel compounds that appear to be specific for the AhR (they failed to activate another promiscuous ligand-activated receptor (i.e. the pregnane X receptor (PXR)). These compounds are being further characterized for their ability to specifically activate the AhR and AhR-responses.

Publications

• Goeyens. L,, Hoogenboom, R., Eppe, G., Malagocki, P., Vanderperren, H., Scippo M-L., Windal, I., Baeyens, W., Denison, M.S. and Hadrich, J. (2010) Discrepancies between bio-analytical and chemo-analytical results have a non-negligible message, Organohalogen Compounds 72, 964-967.
• Baston, D.S. and Denison, M.S. (2011) Considerations for potency equivalent calculations in the Ah receptor-based CALUX bioassay: normalization of superinduction results for improved sample potency estimation, Talanta 83, 1415-1421.
• Petko, P.I., Rowlands, J.C., Budinsky, R., Zhao, B., Denison, M.S. and Mekenyan, O. (2010) Mechanism based common reactivity pattern (COREPA) modeling of AhR binding affinity, SAR QSAR Environ. Res. 21, 187-214.
• Zhao, B., Baston, D.S., Khan, E., Sorrentino, C. and Denison, M.S. (2010) Enhancing the response of CALUX and CAFLUX cell bioassays for quantitative detection of dioxin-like compounds, Science China Chemistry (Science in China Series B) 53, 1010-1016.
• Boitano, A.E., Wang, J., Romeo, R., Bouchez, L.C., Parker, AE., Sutton, S.E., Walker, J.R., Flaveny, C.A., Perdew, G.H., Denison, M.S., Schultz, P.G. and Cooke, M.P. (2010) Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells, Science, 329, 1345-1348.
• Kennedy, K., Macova, M., Bartkow, M.E., Hawker, D.W., Zhao, B., Denison, M.S. and Mueller, J.F. (2010) Effect based monitoring of seasonal ambient air exposures in Australia sampled by PUF passive air samplers, Atmosph. Poll. Res. 1, 50-58.
• Zhao, B., DeGroot, D., Hayashi, A., He, G. and Denison, M.S. (2010) CH223191 is a ligand-selective antagonist of the Ah (dioxin) Receptor, Toxicol. Sci. 117, 393-403.
• Croes, K., Van Langerhove, K., Elskens, M., Desmedt, M., Roekens, E. Kotz, A., Denison, M.S. and Baeyens, W. (2010) Analysis of PCDD/Fs and dioxin-like PCBs in atmospheric deposition samples from the Flemish measurement network: optimization and validation of a new CALUX bioassay method, Chemosphere, 82, 718-724.
• Van Langenhove, K., Croes, K., Denison, M.S., Baston, D.S., Elskens, M., Baeyens, W. (2010) Analytical characteristics of the H1L6.1 and the H1L7.5 mouse hepatoma cell lines, Organohalogen Compounds m72, 1092-1096.

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Outputs for 2009 have included: 1) an interview on KCBS public radio news program (San Francisco) about the USEPAs decision to analyze chemical components of pesticides mixtures for endocrine disruptor activity (4/09), 2) five presentations on our research at the 29th International Symposium on Halogenated Environmental Organic Pollutants and POPs, Beijing, China (9/2009) including one of four Plenary lectures, 3) a research presentation at the annual Society of Toxicology Annual Meeting, Baltimore (3/2009). PARTICIPANTS: Participants in research on this project include: researchers at UC Davis (Dr. Bin Zhao, Dr. Anatoly Soshilov, Dr. Jing Zhao, and three graduate students - Danica DeGroot, David Baston and Ai Hayashi) and a researcher at the University of Milan, Italy - Dr. Laura Bonati). TARGET AUDIENCES: Target audiences include: 1. the medical/veterinary/regulatory scientific community in order to understand the molecular basis of disease associated with dioxin-like chemicals and endocrine disruptors, 2. the medical/regulatory scientific community in order to provide additional validation and application information regarding use of bioanalytical methods for detection and relative quantitation of dioxin-like and endocrine disrupting chemicals, and 2. the regulatory scientific community to alert them to potential risks associated with the presence of dioxin-like chemicals and endocrine disruptors in materials to which humans and animals are exposed. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We have further characterized the ability of structurally diverse chemicals to bind to and activate the AhR and AhR signaling pathway. We have demonstrated that ability of toxic HAHs (i.e. dioxin) to bind to the AhR in a manner distinctly different than that of nontoxic AhR agonists (i.e. flavonoids, indirubins and PAHs). These differences in AhR binding specificity could contribute to the well-established differential ability of AhR ligands to produce beneficial and/or adverse effects in animals exposed to these structurally diverse chemicals. CH223191 has been identified as a novel ligand-dependent AhR antagonist that can specifically block the ability of toxic HAHs, but not PAHs and other nontoxic AhR ligands, to interact with the AhR and activate AhR-dependent gene expression (the initial step in the mechanism of action of these chemicals). Accordingly, CH223191 and/or a derivative of this compound have great potential utility as a therapeutic agent to block the biological and toxicological effects of HAHs. We have further refined our three-dimensional structural homology model of the AhR ligand binding domain by site-directed mutagenesis and have extended this modelling to AhRs form 20 different species. These analyses have further refined the ligand (TCDD) binding fingerprint of conserved residues within the ligand binding cavity necessary for high affinity ligand binding for all of these species. Initial molecular docking simulations were carried out using TCDD and initial refinements in these simulations have further improved our model and allowed us to identify additionally residues that appear to be specifically involved in AhR ligand binding. We have improved our recombinant cell-based bioassay systems for detection of AhR agonists, particularly that of dioxin and related chemicals. While current bioassay systems can detect dioxin and other AhR ligands in sample extracts with a high degree of accuracy, these bioassay are not sensitive enough to allow them to be used for screening of food for contamination by dioxin and related HAHs (at least a 10-fold increase in sensitivity is needed). We have generated a series of species-specific recombinant cell lines that contain a novel luciferase reporter plasmid in which AhR binding sites have been amplified. The resulting cell lines respond to dioxin and other AhR agonists with induction of luciferase activity with a response that is dramatically greater than that of existing bioassays and these cells have a lower limit of sensitivity at least 10-50-fold lower, a range sufficient to allow them to be used for food screening purposes. These new third generation (G3) cell bioassays are currently being evaluated and subjected to validation by several research laboratories interested in establishing these cells as an accepted governmental screening method for dioxins and related HAHs in food.

Publications

• Palumbo, A.J., Denison, M.S., Doroshov, S.I. and Tjeerdema, R.S. (2009) Reduction of vitellogenin synthesis by an aryl hydrocarbon receptor agonist in the white sturgeon (Acipenser transmontamus), Environmental Toxicology & Chemistry 17, 1.
• Pandini, A., Soshilov, A.A., Song, Y., Zhao, J., Bonati, L. and Denison, M.S. (2009) Detection of the TCDD binding fingerprint within the Ah receptor ligand binding domain by structurally driven mutagenesis and functional analysis, Biochemistry 48, 5972-5983.
• Kennedy, K., Macova, M., Leusch, F., Bartkow, M.E., Hawker, D.W., Zhao, B., Denison, M.S. and Mueller, J.F. (2009) Assessing indoor air exposures using passive sampling with bioanalytical methods for estrogenicity and aryl hydrocarbon receptor activity, Analytical Bioanalytical Chemistry 394, 1413-1421.
• Jaikanlaya, C., Settachan, D., Denison, M.S., Ruchirawat, M. and van den Berg, M. (2009) PCB contamination in seafood species at the Eastern Coast of Thailand, Chemosphere 72, 239-249.
• Kanematsu, M., Hayashi, A., Denison, M.S. and Young, T.M. (2009) Characterization and potential environmental risks of leachate from shredded rubber mulches, Chemosphere 76, 952-958.
• Morisseau, C., Merzlikin, O. Lin, A., He, G. Feng, W., Padilla, I., Denison, M.S., Pessah, I.N. and Hammock, B.H. (2009) Toxicology in the fast lane: Application of high-throughput bioassays to detect modulation of key enzymes and receptors, Environmental Health Perspectives 117, 1867-1872.
• Zhao, B., DeGroot, D., He, G., Hayashi, A., and Denison, M.S. (2009) CH223191 is a ligand-specific antagonist of the Ah receptor. Organohalogen Compounds. 71, 2303-2308.
• He, G., Zhao, B. and Denison, M.S. (2009) Ah receptor agonists present in commercial and consumer products: identification and characterization of novel agonists present in tire extracts. Organohalogen Compounds. 71, 2950-2954.
• Denison, M.S., Zhao, B. and Bonati, L. (2009) Biological and toxicological consequences of Ah receptor activation: Just how complicated can one receptor get Organohalogen Compounds. 71, 3162-3165.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: The AhR regulates the expression of a battery of genes in a wide range of species and tissues and is responsible for mediating the toxic and biological effects of TCDD and related halogenated aromatic hydrocarbons (HAHs) and polycyclic aromatic hydrocarbons (PAHs). Differences in the toxic potency of HAHs and PAHs, the latter not producing AhR-dependent toxicity, suggested that these chemicals may differentially bind to and activate the AhR. We have demonstrated that the prototypical HAH (TCDD) and PAH (beta-naphthoflavone (BNF)) can differentially interact with the binding pocket of Ah receptor. This was accomplished by our identification of a novel chemical (MSD237) from high-throughput screening analysis that could inhibit the ability of TCDD, but not BNF, block to bind to AhR and activate AhR-dependent signal transduction pathway. Like that of other AhR ligands, the binding of MSD237 to the AhR ligand binding pocket was essentially irreversible. These results provide insights into the structural diversity of AhR ligands as these ligands may differentially interact with the ligand binding pocket and suggest that differential interactions of HAHs and PAHs with the AhR may contribute to differences in their ability to produce AhR-dependent toxicity. In addition, the use of MSD237 as a potential therapeutic agent to block the biological and toxicological effects of HAHs is being examined. We have also further developed our three-dimensional homology model of the mouse AhR (mAhR) PAS B ligand binding domain by extending this analysis using comparative structural modelling studies of the ligand binding domains of six additional high affinity mammalian AhRs. These results, coupled with site directed mutagenesis and AhR functional analysis, has allowed detection of the "TCDD-binding fingerprint" of conserved residues within the ligand binding cavity necessary for high affinity TCDD binding and TCDD-dependent DNA binding. The essential role of selected residues was further evaluated using molecular docking simulations of TCDD to both wild-type and mutant mAhRs. Further refinement of the docking simulation with TCDD and other known AhR ligands will facilitate the development of a model that can be used for screening purposes to identify new AhR ligands. Taken together, our results allow for a more complete understanding of the molecular determinants of TCDD binding and provides a basis for future studies directed toward rationalizing the observed species differences in AhR sensitivity to TCDD and understanding the mechanistic basis for the dramatic diversity in AhR ligand structure. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We have further developed a three-dimensional structural homology model of the AhR ligand binding domain and extended this modelling to six additional high affinity mammalian AhRs. These analyses have allowed determination of the ligand (TCDD)-binding fingerprint of conserved residues within the ligand binding cavity necessary for high affinity ligand binding. Molecular docking simulations of TCDD to both wild-type and mutant mAhRs were also performed and refinement of these simulations will allow development of a model for use in silico high-throughput screening of chemicals and chemical libraries to identify new AhR ligands. We have also identified and characterized a novel chemical (MSD237) that is a ligand-specific antagonist/inhibitor of the AhR. We found that MSD237 specifically inhibits the ability of TCDD and related toxic HAHs (2,3,7,8-tetrachlorodibenzofuran and 3,3',4,4',5-pentachlorobiphenyl) to bind to and activate the AhR, but that it was ineffective in inhibiting the ability of BNF or 10 other flavonoids to activate the AhR. Indirubin and PAHs which can activate the AhR signaling pathway were also similarly unaffected. MSD237 has potential utility as a therapeutic agent to block the biological and toxicological effects of HAHs and as a novel ligand for examination of the molecular mechanisms of action of dioxin and related chemicals.

Publications

• Ahn, K.C., Zhao, B., Chen, J., Cherednichenko, G., Sanmarti, E., Denison, M.S., Lasley, B., Pessah, I.N., Kultz, D., Chang, D.P., Gee, S.J. and Hammock, B.D. (2008) In vitro biological activities of the antimicrobials triclocarban, its analogs, and triclosan in bioassay screens: receptor-based bioassay screens, Environ. Health Perspect. 116: 1203-1210.
• Bohonowych, J.E.S., Zhao, B., Timme-Laragy, A., Jung, D., Di Giulio, R.T. and Denison, M.S. (2008) Newspapers and newspaper ink contain agonists for the Ah receptor, Toxicol. Sci. 98, 99-110.
• Lawrence, B.P., Denison, M.S., Novak, H., Vorderstrasse, B.A., Harrer, N., Neruda, W., Reichel, C. and Woisetschlager, M. (2008) Activation of the aryl hydrocarbon receptor is essential for mediating the anti-inflammatory effects of a novel low molecular weight compound, Blood, 112, 1158-1165.
• Rochester, J.R., Klasing, K.C., Stevenson, L., Denison, M.S., Berry, W. and Millam, J.R. (2008) Dietary red clover (Trifolium pratense) induces oviduct growth and decreases ovary and testes growth in Japanese quail chick, Reprod. Toxicol. 27: 63-71.
• Soshilov, A.A. and Denison, M.S. (2008) Role of the Per/Arnt/Sim domains in ligand-dependent transformation of the aryl hydrocarbon receptor, J. Biol. Chem. 283, 32995-32305.
• LaMerrill, M., Baston, D.S., Denison, M.S., Birnbaum, L.S., Pomp, D. and Threadgill, D.W. (2009) Am. J. Physiol. Endocrinol. Metab. 296, E203-210.

Progress 01/01/07 to 12/31/07

Outputs
We have continued to optimize our recombinant and in vitro bioassay systems for environmental contaminants (i.e. dioxins and related chemicals) and endocrine disruptors (xenoestrogens) and have conducted numerous validation studies to demonstrate their robustness, validity and precision in the detection of these toxic chemicals. These bioassays have been used to both screen extracts of environmental and food samples for the presence of these chemicals/chemical activities. We have optimized and validated the dioxin cell bioassay system and this assay has met all of the quality control criteria (including double blind validation analysis) to allow it to receive certification by the US EPA as an acceptable screening method for detection and relative quantitation of dioxin and related dioxin-like chemicals in soil, sediment and ash samples. Additional application and development of the assay for use in screening of atmospheric dioxin-like chemicals has also been carried out. We have also improved the quantitative analysis aspect of the bioassay through development of a validated mathematical model developed for prediction of dioxin toxic equivalents in sediment based on our cell bioassay data. This new methodology has application to screening an analysis. Additionally, we have used the dioxin cell bioassay to determine whether a variety of pure chemicals or formulations (i.e. human therapeutic drugs) have the ability to activate these toxicant bioassay systems. We have found that a significant number of commercially available human therapeutic drugs can activate the dioxin receptor signaling pathway, with several of which were highly potent. Additional screening has demonstrated the ability of triclosan and triclocarban, two of the most widely used antimicrobial agents in consumer and health care products, to enhance the effect of the reproductive hormones estrogen and testosterone, thus designating these chemicals as endocrine disruptors. These results have significant implications with regards to the effect of these products on humans and animals.

Impacts
Mechanistically-based cell and in vitro bioassays developed in this project have been applied to the detection of dioxin- and estrogen-like chemicals in environmental samples. The dioxin cell bioassay system has been validated for detection of these chemicals in environmental matrices and the US Environmental Protection Agency has recently approved the use of this bioassay system as an officially accepted method for detection of dioxin and related chemicals. The estrogenic cell and dioxin cell bioassays are also being optimized and validated for use in detection of these and related chemicals in food, feed and agricultural samples.

Publications

• Kennedy, KE, Tang, J, Bartkow, M, Hawker, D, Denison, M, Mueller, JF (2007) Screening for aryl hydrocarbon receptor activity in ambient air using passive sampling (SPMDS) and CAFLUX, Organohalogen Compounds 69, 812-816.
• Denison, MS, Baston, DS, Sorrentino, C, He, G, Hayashi, A and Zhao, B (2007) Bioanalytical approaches for the detection and relative quantitation of halogenated dibenzo-p-dioxins and related chemicals, Organohalogen Compounds 69, 14-19.
• Pandini, A., Denison, M.S., Song, Y., Soshilov, A. and Bonati, L. (2007) Structural and functional characterization of the AhR ligand binding domain by homology modelling and mutational analysis, Biochemistry 23, 696-708.
• Hu, W., Sorrentino, C., Denison, M.S., Kolaja, K. and Fielden, M. (2007) Induction of Cyp1a1 is a Non-Specific Biomarker of Aryl Hydrocarbon Receptor Activation: Results of Large Scale Screening of Pharmaceuticals and Toxicants In Vivo and In Vitro, Molec. Pharmacol. 71, 1475-1486.
• Bohonowych, J.E. and Denison, M.S. (2007) Persistent binding of ligands to the aryl hydrocarbon receptor, Toxicol. Sci. 98, 99-110. Brown, D. J., Orelien, J., Gordo, J. D., Chu, A. C., Chu, M. D., Murata, H. J., Kayama, F., Denison, M. S. and Clark, G. C. (2007) Mathematical model developed for environmental samples: prediction of GC/MS dioxin TEQ from XDS-CALUX bioassay data, Environ. Toxicol. Chem. 41, 4354-4360.
• Nording, M., Persson, Y., Spinnel, E., Baston, D., Denison, M.S. and Haglund, P. (2007) Analysis of dioxins in contaminated soils using CALUX and CAFLUX bioassays, an immunoassay and gas chromatography - high resolution mass spectrometry, Environ. Toxicol. Chem. 26, 1122-1129.
• Cahill, T.M., Groskova, D., Sanborn, J.R., Denison, M.S. and Charles, J. (2007) Atmospheric concentrations of polybrominated diphenyl ethers at near-source site, Environ. Sci. Technol. 41, 6370-6377.
• Gordon, JD, Denison, MS, Chu, MS, Chu, AC, Matherly, C and Clark, GC (2007) Microplate edge effects in range finding studies using the Lumi-Cell ER bioassay: increasing assay throughput, Organohalogen Compounds 69, 1249-1252.