{"id":7115,"date":"2013-08-27T19:12:54","date_gmt":"2013-08-27T19:12:54","guid":{"rendered":"https:\/\/web.uri.edu\/pharmacy\/?page_id=7115"},"modified":"2013-08-27T19:12:54","modified_gmt":"2013-08-27T19:12:54","slug":"projects","status":"publish","type":"page","link":"https:\/\/web.uri.edu\/pharmacy\/research\/king\/projects\/","title":{"rendered":"Research Projects"},"content":{"rendered":"

Metabolism & Enzymology Research<\/strong><\/a><\/h1>\n

Roberta King, Ph.D.<\/a><\/h3>\n

Located in Lab Module 470 on Level 4 of Avedisian Hall<\/a><\/strong><\/p>\n

Overview<\/a> Research<\/a> Publications<\/a> Research Team<\/a> Contact<\/a><\/div>\n

Research Projects<\/strong><\/h3>\n

Project 1:\u00a0Reversible inhibitors of human sulfotransferases <\/strong>
\nSulfotransferase\u00a0enzymes catalyze the conjugation of endogenous compounds including monoamine neurotransmitters, steroid hormones and thyroid hormones. They also sulfonate a\u00a0wide variety of xenobiotic substrates including many therapeutic drugs, dietary\u00a0constituents, and environmental compounds. Depending on the substrate, the\u00a0result of sulfonate conjugation leads to one of three results: (1) decrease in biological\/therapeutic activity, (2) increase in biological\/therapeutic\u00a0activity, or (3) increase in cytotoxicity or genotoxicity via covalent adduct\u00a0formation from a reactive metabolite. Thus, inhibition of sulfotransferase activity can lead to either beneficial or adverse effects depending on the substrate and isoform involved. Since sulfotransferases are involved in so many\u00a0different biological processes, it is imperative to understand mechanisms modulating the activity of each sulfotransferase isoform.
\nThe specific aims of this project are to (1) identify inhibitors of the important human\u00a0cytosolic sulfotransferases by screening selected environmental and dietary compounds, and (2) use a structure-guided design approach to develop inhibitors which are highly specific for each sulfotransferase member. We select compounds for screening based on structural relationships with known sulfotransferase ligands. Since inhibitors identified by screening are of varying specificity and\u00a0may affect other enzyme systems or steroid receptors, there is also a critical need for a structure-based design approach to elucidate the structural features\u00a0necessary for potent and specific binding to the substrate\/cofactor binding site for each major sulfotransferase isoform. A portion of aim 1 of this project is\u00a0complete and recently submitted for publication. Aim 2 is the subject of current research and proposals pending.<\/p>\n

    \n
  • Presentations: 1 invited oral (2002), 2 posters with published abstracts\u00a0(2000, 2001), 1 additional poster (2001), and many student presentations<\/span><\/li>\n
  • Publications: 1 total, Current Drug Metabolism,<\/em> submitted September 21, 2005<\/span><\/li>\n
  • 1 M.S. thesis.<\/span><\/li>\n
  • Funding: 5 awards, total of $155,980; 1 pending<\/span><\/li>\n
  • Personnel: Anasuya Ghosh, Jinfang Wu,\u00a0Nicholas Rue<\/span><\/li>\n
  • Status of project: continuing<\/span><\/li>\n<\/ul>\n
    \n
    \n<\/div>\n

    Project 2:\u00a0\u00a0<\/strong>Biotransformation of indole to indoxyl sulfate <\/strong>
    \nIndoxyl sulfate, a\u00a0sulfate conjugate of indoxyl, is a known circulating uremic toxin promoting the progression of glomerular necrosis and renal failure, and serum concentration of this metabolite is markedly increased in uremic patients. Indoxyl sulfate is mainly produced after bacterial decomposition of dietary tryptophan in the intestines. The initial product of tryptophan metabolism in the intestines is indole, which is subsequently hydroxylated to indoxyl and sulfonated to form\u00a0indoxyl sulfate. It had been speculated that the initial tryptophan metabolite indole is absorbed, hydroxylated by cytochrome P-450 (P450, CYP) enzymes, conjugated by sulfotransferases (SULT), and excreted by the kidneys. The goal of\u00a0this study was to determine which isoforms of cytochrome P450 and sulfotransferase were responsible for human metabolism of indole to indoxyl sulfate.
    \nIn brief, we found that human and rat cytochrome P450 enzymes catalyze oxidation of indole to indoxyl and that CYP2E1 isoform is responsible for indoxyl formation in rat liver microsomes. Since the human CYP2E1 is very similar catalytically to the rat CYP2E1, we proposed that\u00a0human CYP2E1 is the isoform responsible for indoxyl formation in humans. We also\u00a0found that human and rat phenol sulfotransferases catalyze sulfonation of indoxyl to indoxyl sulfate and that SULT1A1 isoform is responsible for the sulfonation activity in human liver cytosol. Since both CYP2E1 and SULT1A1\u00a0exhibit wide individual variation in activity, these results support a mechanism for individual variation in formation of a toxic metabolite and may causes differences in susceptibility to the toxic effects of indole and indoxyl sulfate\u00a0formed from dietary tryptophan.<\/p>\n

      \n
    • Publications: 2 total, European\u00a0Journal of Drug Metabolism and Pharmacokinetics,<\/em> 2000 and 2001.<\/span><\/li>\n
    • Funding: 1 award, $3,000\u00a0total<\/span><\/li>\n
    • Personnel: Erden Banoglu<\/span><\/li>\n
    • Status of project:\u00a0complete<\/span><\/li>\n<\/ul>\n
      \n
      \n<\/div>\n

      Project\u00a03: Metabolism of the heterocyclic amine mutagen 2-amino-alpha-carboline <\/strong>
      \n2-Amino-alpha-carboline (AaC) is one of the\u00a0five major heterocyclic aromatic amines (HAAs) present in the diet and is formed\u00a0by pyrolysis of tryptophan. HAAs are naturally occurring genotoxic carcinogens\u00a0produced by cooking meat and other protein-containing foods. They are not present in uncooked meat, but are readily produced under normal household\u00a0cooking conditions. In addition to dietary exposure, AaC is found in combustion smokes of wood and cigarettes, in automobile exhaust, and in municipal water\u00a0sources. Human exposure to HAAs including AaC is low but chronic. AaC is clearly\u00a0mutagenic and carcinogenic in model systems. For example, AaC induced\u00a0preneoplastic lesions in the livers of male F344 rats, mutations in the colon of transgenic Big Blue mice, and lymphomas and liver tumors in CDF1 mice. However,\u00a0despite many years of study, association of heterocyclic amine exposure with\u00a0human carcinogenesis remains suspected but unproven.
      \nThe aim of this study is to use model\u00a0systems (tissue homogenates, purified enzymes, cultured cells, rodents in vivo)\u00a0to develop methods for efficient detection, identification and quantification of\u00a0metabolites and DNA adducts of the heterocyclic amine 2-amino-alpha-carboline.\u00a0Our goal is to develop plausible biomarkers for human molecular epidemiological\u00a0and exposure studies for AaC. These are necessary to clarify whether AaC is a\u00a0human carcinogen at the low but chronic human exposures.
      \nOur results show that AaC is highly\u00a0metabolized by oxidation and conjugation to stable, excreted metabolites.\u00a0Approximately 14 metabolites were observed in the rat bile from the in vivo\u00a0study, and initial structure determination indicated oxidation and extensive\u00a0\u00a0 conjugation. Further structure elucidation was conducted on a similar number of\u00a0\u00a0 metabolites formed and excreted by rat hepatocytes and human hepG2 liver tumor\u00a0\u00a0 cells. We found three sites of aromatic ring hydroxylation, one more than\u00a0observed in the microsomal studies we published previously. Four major\u00a0\u00a0 metabolites were formed in both cell systems, three of them identical: AaC-6-sulfate, AaC-3-sulfate, and N-acetyl-AaC. In human liver tumor cells\u00a0\u00a0 (hepG2) the fourth major metabolite was a double conjugate, N-acetyl-AaC-6-sulfate. In rat hepatocytes, the fourth major metabolite was one of the AaC-N-glucuronides. We confirmed expected sites of AaC metabolism, but also observed unexpected metabolites. The unexpected metabolites include extensive N\u2011acetylated conjugates and a total of three different N-glucuronides.\u00a0Also noteworthy are metabolites that were not detected: no direct N-sulfonation to form the sulfamate, and very little O\u2011glucuronidation even by the rat\u00a0hepatocytes which are known to have active glucuronidation systems.
      \nThese results combined with our\u00a0earlier publications on the reactive, DNA-adduct-forming, metabolites of AaC\u00a0indicate that both bioactivation and detoxification share the same metabolic\u00a0pathways\u2014oxidation, acetylation, and sulfonation. We have not yet confirmed the\u00a0isoforms responsible for transformation to the stable metabolites, but based on\u00a0other HAAs it is likely to be CYP1A2, SULT1A1, NAT1, and NAT2. These are the same enzymes responsible for the DNA-reactive metabolites. This may have the end\u00a0result of \u2018protecting\u2019 those individuals who have relatively high levels of\u00a0these enzymes\u2014increased bioactivation would be balanced by increased detoxification.<\/p>\n

        \n
      • Presentations: 2 invited oral (2001,\u00a0\u00a02004), 2 posters with published abstracts (2002, 2004), 2 additional posters (2002, 2004), and many student presentations<\/span><\/li>\n
      • Publications: 3 total (1999, 2000,\u00a0\u00a02000), 2 in preparation<\/span><\/li>\n
      • 1 M.S. thesis.<\/span><\/li>\n
      • Funding: 4 awards, total of\u00a0\u00a0$140,980<\/span><\/li>\n
      • Personnel: Gautam Jha, Zhixin\u00a0\u00a0 Yuan<\/span><\/li>\n
      • Status of project: complete<\/span><\/li>\n<\/ul>\n
        \n
        \n<\/div>\n

        Project\u00a0\u00a04: Effect of sulfotransferase inhibitors on estradiol homeostasis and actions <\/strong>
        \nIncreased estrogen exposure is clearly related to mammary\u00a0\u00a0 cancer development, but mechanisms controlling mammary tissue estradiol\u00a0\u00a0 homeostasis are only partially understood. Several enzymes are known or proposed\u00a0\u00a0 to be involved in the control of local estradiol biosynthesis and degradation including aromatase, sulfatase, 17b-hydroxysteroid dehydrogenase, and estrogen\u00a0sulfotransferase (EST). However, little is known about the role of\u00a0\u00a0 sulfotransferase toward estradiol homeostasis in normal or transformed human mammary cells. Because of the abundance of environmental and dietary compounds\u00a0\u00a0 known to be relatively potent inhibitors of EST, we believe it is imperative to\u00a0understand the (potentially negative) cellular effect of these inhibitors on estradiol concentrations and downstream actions.
        \nOur long-term goals are to establish\u00a0the relative contribution of EST toward regulation of breast cellular estrogen\u00a0activities in cancer initiation and progression, and to establish the mechanisms\u00a0of EST suppression and stimulation in breast cancer. The central hypothesis of\u00a0this study is that EST inhibition\/suppression will directly alter endogenous cellular estradiol concentrations and mimic higher estradiol exposure in the\u00a0\u00a0 cell model systems. As models, we are studying the effect of EST inhibition on\u00a0transformed (MCF-7) and non-transformed (human mammary epithelial, HME) cell\u00a0systems. These two systems will establish the approach toward both initiation\u00a0(HME) and progression (MCF-7). Our approach is to modulate EST activity in the\u00a0cell systems and, directly and indirectly, measure the effect on local estradiol\u00a0concentration and estrogenic activities.
        \nThis project is innovative in that it is the first to directly relate change in local estradiol concentration with activity of estradiol metabolic enzymes in human mammary cell systems. Moreover,\u00a0it focuses on pharmacological inhibitors of human estrogen sulfotransferase.\u00a0Many environmental and dietary compounds are known to be relatively potent inhibitors of EST, but no one has shown whether these compounds actually alter cellular estradiol concentrations or affect estrogenic activity. The rationale\u00a0for this work is that fuller understanding of the effect of EST inhibition on normal and transformed human mammary cell systems may establish EST inhibition\u00a0as an additional mechanism for mammary cancer initiation or progression.<\/p>\n

          \n
        • Presentations: regional and student presentations<\/span><\/li>\n
        • Publications: 1 in\u00a0preparation<\/span><\/li>\n
        • 1 M.S. thesis.<\/span><\/li>\n
        • Funding: 3 awards, total of\u00a0\u00a0 $34,150<\/span><\/li>\n
        • Personnel: Jinfang Wu, Roseanne\u00a0\u00a0 Meyer<\/span><\/li>\n
        • Status of project: continuing<\/span><\/li>\n<\/ul>\n
          \n
          \n<\/div>\n

          Collaborative\u00a0Project 5: Metabolism of benzo[ghi]fluoranthrene: determination of microsomal\u00a0metabolites (with Bongsup P. Cho)<\/strong>
          \nTwo undergraduate students have\u00a0worked on this project during one summer each. This compound is a carcinogenic PAH under study by Bongsup Cho. The goal of this work was to profile the rat\u00a0liver microsomal metabolites of benzo[ghi]fluoranthrene. The first student found\u00a0\u00a0 the metabolites formed by microsomal oxidation systems were not the same as the synthetic oxidation products. (Their HPLC retention time and UV\/Vis spectra were different.) This result is very interesting and of potential scientific impact. A second student scaled-up the microsomal incubate enough to allow preparation of enough compound for structure determination. His samples are awaiting further purification and structure analysis.<\/p>\n

            \n
          • Presentations: student presentations<\/span><\/li>\n
          • Publications: none<\/span><\/li>\n
          • Funding: none<\/span><\/li>\n
          • Personnel: Siobhan O\u2019Brian (1\u00a0summer), Steve Rougas (1 summer)<\/span><\/li>\n
          • Status of project: inactive<\/span><\/li>\n<\/ul>\n
            \n
            \n<\/div>\n

            Collaborative Project 6: Metabolism of linolenic acid in salmon liver hepatocytes (with Chong\u00a0Lee, Nutrition)<\/strong>
            \nThe goal of this project is to determine how various treatments affect relative formation of known linolenic acid metabolites (DHA, E) in salmon. I am supervising Mary Anne Eaton (graduate student in Chong Lee\u2019s lab) in hepatocyte isolation, incubation with radiolabeled substrate, and subsequent analysis and quantitation of metabolites. This project is similar in technique and approach to my own work on metabolism of heterocyclic aromatic amine carcinogens.<\/p>\n

              \n
            • Presentations: none<\/span><\/li>\n
            • Publications: none<\/span><\/li>\n
            • Funding: by Chong Lee only<\/span><\/li>\n
            • Personnel: Mary Anne Eaton (graduate\u00a0\u00a0 student in Nutrition)<\/span><\/li>\n
            • Status of project:\u00a0continuing<\/span><\/li>\n<\/ul>\n","protected":false},"excerpt":{"rendered":"

              Metabolism & Enzymology Research Roberta King, Ph.D. Located in Lab Module 470 on Level 4 of Avedisian Hall Overview Research Publications Research Team Contact Research Projects Project 1:\u00a0Reversible inhibitors of human sulfotransferases Sulfotransferase\u00a0enzymes catalyze the conjugation of endogenous compounds including monoamine neurotransmitters, steroid hormones and thyroid hormones. They also sulfonate a\u00a0wide variety of xenobiotic substrates […]<\/p>\n","protected":false},"author":639,"featured_media":0,"parent":7111,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"page-twocol.php","meta":{"_acf_changed":false,"footnotes":"","_links_to":"","_links_to_target":""},"class_list":["post-7115","page","type-page","status-publish","hentry"],"acf":[],"_links":{"self":[{"href":"https:\/\/web.uri.edu\/pharmacy\/wp-json\/wp\/v2\/pages\/7115","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/web.uri.edu\/pharmacy\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/web.uri.edu\/pharmacy\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/web.uri.edu\/pharmacy\/wp-json\/wp\/v2\/users\/639"}],"replies":[{"embeddable":true,"href":"https:\/\/web.uri.edu\/pharmacy\/wp-json\/wp\/v2\/comments?post=7115"}],"version-history":[{"count":0,"href":"https:\/\/web.uri.edu\/pharmacy\/wp-json\/wp\/v2\/pages\/7115\/revisions"}],"up":[{"embeddable":true,"href":"https:\/\/web.uri.edu\/pharmacy\/wp-json\/wp\/v2\/pages\/7111"}],"wp:attachment":[{"href":"https:\/\/web.uri.edu\/pharmacy\/wp-json\/wp\/v2\/media?parent=7115"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}