Current Research Activities
Our laboratory has diverse interests that range from basic research in cancer biology to applied research in chemical risk assessment. A partial list of the research interests and links to more detailed descriptions of the projects are below.
- Function of the aryl hydrocarbon receptor in breast cancer
- Use of genomic and metabonomic data for chemical hazard prediction
- Analysis of genomic dose-response data for application to chemical risk assessment
- Application of cell-based functional genomic screens to identify regulators of specific signaling pathways
- Development of novel biomarkers of chemical or drug exposure and effect
- Interpretation of high-throughput toxicity screening data
- Mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin B-cell immunotoxicity
The current statistics associated with breast cancer continue to show a relatively high recurrence rate together with a poor survival for aggressive metastatic disease. These findings reflect, in part, the pharmaceutical intractability of processes involved in the metastatic process and highlight the need to identify additional drug targets for the treatment of late-stage disease. Molecular characterization of the AhR across vertebrate and invertebrate species demonstrated that the receptor is highly conserved and plays a significant role in tissue development. In the mouse, targeted disruption of the AhR results in altered immune function, ovarian follicle development, seminal vesicle maintenance, vascular remodeling, and mammary development.
The cancer-related effects of AhR activation have been primarily characterized using xenobiotic ligands. Similar to the nuclear hormone receptors, these studies have identified context- and tissue-specific effects that include tumor promotion in certain tissues and a decreased tumor incidence in others. In humans, occupational exposures demonstrated an increased risk for all combined cancers and for lung cancer. Although controversial, some epidemiological studies have shown a significant decrease in breast and endometrial cancers. The tissue-related differences in the tumor response in humans have also been observed in rodent studies. In the most recent rodent cancer bioassay, an increased tumor incidence was observed in the liver, lung, and oral mucosa, while a significant decrease in tumors was observed in the mammary gland, pituitary, and thyroid. The studies in our lab aim to understand whether the inhibition of mammary tumorigenesis is relevant in humans and the mechanism that underlies this inhibition.
Hall, J.M., Barhoover, M.A., Kazmin, D., McDonnell, D.P., Greenlee, W.F., and Thomas, R.S. (2010). Activation of the aryl-hydrocarbon receptor inhibits invasive and metastatic features of human breast cancer cells and promotes breast cancer cell differentiation. Mol. Endocrinol. 24(2):359-69. (link)
Barhoover, M.A., Hall, J.M., Greenlee, W.F., and Thomas R.S. (2010). The AHR regulates cell cycle progression in human breast cancer cells via a functional interaction with CDK4. Mol. Pharmacol. 77(2):195-201. (link)
The primary goal of toxicology and safety testing is to identify agents that have the potential to cause adverse effects in humans. Unfortunately, many of these tests have not changed significantly in the past 30 years and most are inefficient, costly, and rely heavily on the use of animals. The rodent cancer bioassay is one of these safety tests and was originally established as a screen to identify potential carcinogens that would be further analyzed in human epidemiological studies. Today, the rodent cancer bioassay has evolved into the primary means to determine the carcinogenic potential of a chemical and generate quantitative information on the dose-response behavior for chemical risk assessments.
The experimental design for rodent cancer bioassays involves exposing mice and rats of both sexes for a period of two years. Several dosage levels are chosen with the high dose corresponding to the maximum tolerated dose (MTD). Approximately 50 animals per sex per dose level are used in each study. Due to the resource-intensive nature of these studies, each bioassay costs $2 to $4 million and takes over three years to complete. Over the past 30 years, only 1,468 chemicals have been tested in a rodent cancer bioassay. By comparison, approximately 9,000 chemicals are used by industry in quantities greater than 10,000 lbs and nearly 90,000 chemicals have been inventoried by the U.S. Environmental Protection Agency as part of the Toxic Substances Control Act. Given the disparity between the number of chemicals tested in a rodent cancer bioassay and the number of chemicals used by industry, a more efficient and economical system of identifying chemical carcinogens needs to be developed. The studies in our lab aim to develop more efficient and economical alternatives to chronic rodent toxicity studies through the use of genomic and metabonomic technologies.
Thomas, R.S., Bao, W., Chu, T.M., Bessarabova, M., Nikolskaya, T., Nikolsky, Y., Andersen, M.E., and Wolfinger, R.D. (2009). Use of short-term transcriptional profiles to assess the long-term cancer-related safety of environmental and industrial chemicals. Toxicol. Sci. 112(2):311-321. (link)
Thomas, R.S., Pluta, L., Yang, L., and Halsey, T.A. (2007) Application of genomic biomarkers to predict increased lung tumor incidence in two-year rodent cancer bioassays. Toxicol. Sci. 97(1): 55-64. (link)
Thomas, R.S., O’Connell, T.M., Pluta, L., Wolfinger, R.D., Yang, L. and Page, T.J. (2007). A comparison of transcriptomic and metabonomic technologies for identifying biomarkers predictive of two-year rodent cancer bioassays. Toxicol. Sci. 96(1): 40-46. (link)
Shi, L., Campbell, G., Jones, W.D., Campagne, F., Wen, Z., Walker, S.J., Su, Z., Chu, T.M., Goodsaid, F.M., Pusztai, L., Shaughnessy, J.D. Jr, Oberthuer, A., Thomas, R.S., et al. (2010). The MicroArray Quality Control (MAQC)-II study of common practices for the development and validation of microarray-based predictive models. Nat Biotechnol. 28(8):827-38. (link)
The quantitative assessment of health risks associated with chemical exposure is a time- and resource-intensive process that requires years to complete. For a given chemical, all relevant biochemical, cellular, animal, and human studies are collected and evaluated in the context of the four steps of the risk assessment process. These steps include hazard identification, dose-response assessment, exposure assessment, and risk characterization. Upon completion, the results for industrial and environmental chemicals are published by regulatory agencies such as the U.S. Environmental Protection Agency's (EPA) Integrated Risk Information System (IRIS) or by the Office of Pesticides Programs (OPP) for pesticide chemicals. If a chemical does not meet minimal data requirements, no risk assessment values are published. Given the nature of the studies that are currently required to complete a full risk assessment, only 553 substances have been published in the IRIS database and 613 pesticides have been evaluated by the OPP from 1991 through 2008.
There are multiple reasons to increase the efficiency in the risk assessment process and incorporate modern technology in evaluating the potential health effects of chemicals. First, chemicals without published risk assessment values are not considered quantitatively in the overall hazard index calculation when evaluating contaminated sites where multiple chemicals exist. Therefore, these chemicals are treated as not posing any hazard leading to a potential underestimation of hazard at any given contaminated site. Second, the economic costs, time, and animal numbers required to perform the traditional toxicity tests that underlie current risk assessments limit the number of chemicals that can be evaluated. The resulting studies cannot keep pace with the number of chemicals introduced into commerce or released into the environment. These difficulties lead to a lack of data on which to base risk assessment values. Finally, the use of traditional high dose animal studies and a simple linear extrapolation to environmental doses does not incorporate potential dose-dependent changes in mechanism that occur for many chemicals. This may reduce the biological relevance of the endpoints used in the risk assessment. The studies in our lab aim to apply transcriptomic dose-response data to chemical risk assessment and develop the analysis methods that would underlie this application.
Thomas, R.S., Clewell, H.J., Allen, B.C., Yang, L., Healy, E., and Andersen, M.E. (2012). Integrating Pathway-Based Transcriptomic Data into Quantitative Chemical Risk Assessment: A Five Chemical Case Study. Mut Res. (In Press).
Thomas, R.S., Clewell, H.J., Allen, B.C., Wesselkamper, S.C., Wang, N.C.Y., Lambert, J.C., Hess-Wilson, J.K., Zhao, Q.J., and Andersen, M.E. (2011). Application of transcriptional benchmark dose values in quantitative cancer and noncancer risk assessment. Toxicol Sci. 120(1):194-205. (link)
Andersen, M.E., Clewell, H.J., Bermudez, E., Dodd, D.E., Willson, G.A., Campbell, J.L. and Thomas, R.S. (2010).Formaldehyde: Integrating dosimetry, cytotoxicity and genomics to understand dose-dependent transitions for an endogenous compound. Toxicol Sci. 118(2):716-31. (link)
Andersen, M.E., Clewell, H.J. 3rd, Bermudez, E., Willson, G.A., and Thomas R.S. (2008). Genomic signatures and dose dependent transitions in nasal epithelial responses to inhaled formaldehyde in the rat. Toxicol. Sci. 105(2):368-383. (link)
Thomas, R.S., Allen, B.C., Nong, A., Yang, L., Bermudez, E., Clewell, H.J., and Andersen, M.E. (2007). A method to integrate benchmark dose estimates with genomic data to assess the functional effects of chemical exposure. Toxicol. Sci. 98(1): 240-248. (link)
Yang, L., Allen, B.C., and Thomas, R.S. (2007). BMDExpress: A software tool for the benchmark dose analyses of genomic data. BMC Genomics 8:387. (link)
Application of Cell-Based Functional Genomic Screens to Identify Regulators of Specific Signaling Pathways
With the draft of the human genome completed in 2001 and experimental organisms such as the mouse and rat either completed or near completion, a large volume of sequence information has been generated with only limited understanding of the contents. Greater than 40% of the predicted human genes do not have a putative molecular function from experimental data or homology with known genes. Closing the knowledge gap by assigning functions to these genes and placing them contextually into signaling pathways will be critical for understanding cellular responses to toxic agents and disease. To accomplish this goal, large-scale genetic screens have been utilized to link specific genetic alterations to phenotypic end points and are classified into two primary types— forward and reverse screens. In forward screens, a specific biological end point is measured in all of the genetically manipulated organisms without prior knowledge of the genetic changes. In those organisms with altered phenotypes, the genetic changes are characterized, allowing the association between phenotype and genotype. In reverse screens, the sequence of events is inverted, and the genetic changes are known up-front in the process followed by phenotypic measurements in each organism. Although most genetic screens in the literature have utilized forward genetic designs (e.g., MNU mutagenesis screens), continued improvements in high-throughput technologies and robotics have allowed reverse genetic screens to be performed on increasingly larger scales and with more sophisticated designs.
A number of research groups including ours have begun to apply large-scale reverse genetic screens to systematically identify genes that play a functional role in specific disease pathways and assign putative molecular roles to previously uncharacterized genes. In these screens, cell-based assays are constructed with various cellular end points or reporter genes that indicate activation of a specific pathway. Full-length cDNAs or siRNAs are introduced into the cells to provide a gain-of-function or loss-of-function form of genetic manipulation, respectively. The genes identified using these approaches are assumed to play a functional role in the specific pathway of interest. Contextual organization of the genes can be performed by screening the full-length cDNAs in combination with the siRNAs. The studies in our lab aim to apply functional genomic screens to key toxicity pathways in order to understand their organization and what genes play a direct or indirect role.
Halsey, T.A., Yang, L., Walker, J.R., Hogenesch, J.B., and Thomas, R.S. (2007). A functional map of NFκB signaling identifies novel modulators and multiple system controls. Genome Biol. 8(6):R104. (link)
Boellmann, F. and Thomas, R.S. (2010). The identification of protein kinase C iota as a regulator of the Mammalian heat shock response using functional genomic screens. PLoS One 5(7):e11850. (link)
There are two types of biomarkers. Biomarkers of effect are measurable characteristics that can indicate a potential or established health impairment or disease. Biomarkers of exposure are measurable characteristics of the amount of exposure or internal dose, to or by a specific chemical. Cellular release of RNA molecules into the circulation has been shown to occur through multiple mechanisms. Among passive processes, the release of cellular mRNA and miRNA has been shown following necrotic cell death. The RNA molecules enter circulation and are either associated with cellular debris or in naked form. Among active processes, mRNA and miRNA molecules have been identified within membrane-encapsulated vesicles released by cells. These include exosomes, shedding vesicles, and apoptotic blebs. Exosomes are small vesicles (40 - 100 nm) that are formed by inward budding of endosomal membranes. The vesicles are packaged within larger intracellular multivesicular bodies that release their contents to the extracellular environment through exocytosis. Shedding vesicles (<200 nm) are released from live cells through direct budding from the plasma membrane while apoptotic blebs (100 - >1000 nm) bud directly from the plasma membrane upon cell death. After release from the cell, exosomes, shedding vesicles, and apoptotic blebs circulate in the extracellular space where most are broken down within minutes due to the display of phosphatidylserine on the external side of the membrane. A fraction of the vesicles move by diffusion into the circulation and appear in biological fluids. The function of exosomes and shedding vesicles are believed to be cell-to-cell communication and platforms for multi-signaling processes. While exosomes and shedding vesicles are released in healthy individuals, many pathological conditions and cellular perturbations stimulate further release of the particles.
Circulating RNAs have been shown to be useful biomarkers for multiple clinical endpoints including mortality in acute trauma patients, the diagnosis of pre-eclampsia, the diagnosis and monitoring of diabetic retinopathy and neuropathy, and as diagnostic or prognostic markers for multiple cancers. The use of circulating mRNA as a biomarker offers several advantages. First, it can be readily obtained from patients. Second, amplification technologies such as PCR allow highly sensitive and quantitative detection of specific mRNAs. Third, identification of targets of toxicity can be achieved using tissue-specific transcripts. Finally, microarray technologies can be exploited to broadly survey transcriptional changes in biological processes and signaling pathways and develop high-dimensional transcriptional profiles to discriminate among disease states or treatments. The studies in our lab aim to develop circulating mRNAs as biomarkers of exposure and effect with an initial focus on hepatotoxicity.
Wetmore, B.A., Brees, D.J., Singh, R., Watkins, P.B., Andersen, M.E., Loy, J. and Thomas, R.S. (2010). Quantiative analyses and transcriptomic profiling of circulating messenger RNAs as biomarkers of rat liver injury. Hepatology 51(6):2127-2139. (link)
The current paradigm for testing agricultural and industrial chemicals for potential human health effects is inefficient, expensive, and relies heavily on experimental animals. To address the large number of chemicals for toxicity testing and improve chemical risk management, the U.S. Environmental Protection Agency (EPA) developed a high-throughput testing program called ToxCast to screen chemicals and prioritize limited testing resources toward those representing the greatest potential risk to human health. In the first phase of the ToxCast program, hundreds of in vitro assays were used to screen a library of agricultural and industrial chemicals to identify cellular pathways and processes perturbed by these chemicals. However, the use of in vitro assay potencies alone for prioritizing chemicals for testing may over- or under-estimate the potential risk of these chemicals due to differences in bioavailability, clearance, and exposure in vivo. In these high-throughput screening activities, most of the initial effort has focused on characterizing the biological activity of agricultural and industrial chemicals across multiple cellular pathways and processes. Less attention has been paid to determining the relationship between concentrations of the chemical active in vitro and expected concentrations in human populations. Pharmacokinetic properties and human exposure characteristics are equally important as the biological activity in determining a chemical's risk to human health. The studies in our lab aim to develop methods and approaches for interpreting high-throughput toxicity screening data.
Wetmore, B.A., Wambaugh, J.F., Ferguson, S.S., Sochaski, M.A., Rotroff, D.M., Freeman, K., Clewell, H.J. 3rd, Dix, D.J., Andersen, M.E., Houck, K.A., Allen, B., Judson, R.S., Singh, R., Kavlock, R.J., Richard, A.M., and Thomas, R.S. (2012). Integration of Dosimetry, Exposure and High-Throughput Screening Data in Chemical Toxicity Assessment.Toxicol Sci.125(1):157-74. (link)
Judson, R.S., Kavlock, R.J., Setzer, R.W., Cohen-Hubal, E.A., Martin, M.T., Knudsen, T.B., Houck, K.A., Thomas, R.S., Wetmore, B.A., and Dix ,D.J. (2011). Estimating toxicity-related biological pathway altering doses for high-throughput chemical risk assessment. Chem Res Toxicol. 24(4):451-462. (link)
Rotroff, D.M., Wetmore, B.A., Dix, D.J., Ferguson, S.S., Clewell, H.J., Houck, K.A., Lecluyse, E.L., Andersen, M.E., Judson, R.S., Smith, C.M., Sochaski, M.A., Kavlock, R.J., Boellmann, F., Martin, M.T., Reif, D.M., Wambaugh, J.F., and Thomas, R.S. (2010). Incorporating human dosimetry and exposure into high-throughput in vitro toxicity screening. Toxicol Sci. (In Press). (link)
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and certain polychlorinated biphenyls, polychlorinated dibenzofurans are environmental contaminants that posses long half-lives and impart a wide range of toxicological effects on mammalian systems. Among these toxicological effects, immune suppression is one of the earliest and most sensitive toxicological endpoints. Although TCDD affects multiple aspects of the immune system, cell-type fractionation reconstitution studies of heterogeneous leukocyte preparations have shown that B cells are the cellular target principally responsible for suppression of primary humoral immune responses. Moreover, addition of TCDD directly to cultured naïve spleen cells, purified naïve splenic B cells, or B cell lines (e.g., CH12.LX cells) suppressed IgM responses, demonstrating that TCDD directly targets B cells.
The regulation of plasma cell differentiation follows a common biological paradigm, in which a phenotypic change is regulated through a cascading series of events that include critical transcriptional control points. In B cells, the early transcriptional regulators are contained within two reciprocal negative feedback loops. In an inactivated state, two key transcriptional repressors, B cell lymphoma 6 (Bcl6) and Paired box protein 5 (Pax5), are actively transcribed. Bcl6 and Pax5 both repress key genes involved in B cell to plasma cell differentiation, while Bcl6 also represses the transcription of an activator of B cell differentiation, PR domain zinc finger protein 1 (Prdm1). Activation of B cells via LPS or the B-cell receptor increases the expression of Prdm1 which then represses transcription of Bcl6 and Pax5 allowing differentiation to proceed. These three transcription factors, Prdm1, Bcl6, and Pax5, constitute an important control point in B-cell differentiation and ultimately regulate the secondary and tertiary waves of transcriptional events.
The AHR is a ligand activated transcription factor and a member of the basic-helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) superfamily. In its non-ligand bound state, the AHR resides in the cytoplasm bound to a dimer of HSP90, AIP, and PTGES3. Following ligand binding, the AHR dissociates from its chaperone proteins, translocates to the nucleus and heterodimerizes with the aryl hydrocarborn receptor nuclear translocator (ARNT). The AHR/ARNT complex binds to xenobiotic response elements (XREs) to activate transcription of target genes. In the context of B-cell function, activation of the AHR by TCDD has been shown to impair differentiation at multiple points. Previous studies have shown that AHR activation suppresses the activity of the activator protein -1 (AP-1) transcriptional complex which is an upstream activator of Prdm1. In addition, the AHR directly supresses IgH expression and indirectly supresses Igκ and IgJ expression leading to decreased assembly and secretion of the IgM complex. The studies in our lab aim understand the mechanism of TCDD-induced B-cell immunotoxicity using an integrated genomics approach.
De Abrew, K.N., Phadnis, A.S., Crawford, R.B., Kaminski, N.E., and Thomas R.S. (2011). Regulation of Bach2 by the aryl-hydrocarbon receptor as a mechanism for suppression of B-cell differentiation by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol Appl Pharmacol. 252(2):150-158. (link)
De Abrew, K.N., Kaminski, N.E., and Thomas R.S. (2010). An integrated genomic analysis of aryl hydrocarbon receptor-mediated inhibition of B-cell differentiation. Toxicol Sci. 118(2):454-69. (link)
Zhang, Q., Bhattacharya, S., Kline, D.E., Crawford, R.B., Conolly, R.B., Thomas, R.S., Kaminski, N.E. and Andersen, M.E. (2010). Stochastic modeling of B lymphocyte terminal differentiation and its suppression by dioxin. BMC Systems Biology 4(1):40. (link)
Bhattacharya, S., Conolly, R.B., Kaminski, N.E., Thomas, R.S., Andersen, M.E., and Zhang, Q. (2010). A Bistable Switch Underlying B Cell Differentiation and Its Disruption by the Environmental Contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Sci. 115(1):51-65. (link)