Curtis D. Klaassen - US grants

Cadmium is an environmental pollutant which has been shown to be toxic to a number of tissues in mammalian systems. These studies will make clear what dose is necessary to produce injury to the various tissues, which tissue is most sensitive to cadmium, and if there is a marked difference in the toxic sign between animals that receive one high, single dose and those that receive multiple, low doses over a long time. Information will be gained on the importance of metallothionein on the toxicity of cadmium. Does metallothionein alter the distribution of cadmium? Do metallothionein and cadmium levels in the tissues continue to rise with prolonged administration at different doses, or is a plateau achieved? What is the mechanism by which cadmium is excreted into the bile? Can it be enhanced by treatment with microsomal enzyme inducers? Will interruption of the enterohepatic circulation increase the elimination of cadmium from the body? If we can answer these very basic questions, then we should be able to treat cadmium poisoning on a more informed basis.

The objective of the proposed studies is to gain further insight into the mechanisms by which the liver removes various environmental chemicals and drugs from the blood and excretes them into the bile. The mechanism or mechanisms by which xenobiotics enter the liver will be examined by using the isolated hepatocyte technique. Further insight into the mechanisms which stimulate and regulate the development of hepatic excretory function will be examined by measuring hepatic uptake processes in rats of various ages and determining if substrates for the hepatic excretory mechanism will stimulate its development. The mechanisms responsible for the first-pass effect of xenobiotics will be examined and the importance of the first-pass effect on toxicity will be examined. Thus, by investigating the hepatic uptake and excretory mechanisms by a number of techniques, a deeper insight into the basic mechanisms of detoxification of foreign chemicals by biliary excretion will be gained, an area in which development has lagged relative to detoxification by metabolism and renal excretion.

Cadmium (Cd) is an environmental pollutant which is toxic to a number of tissues. The long-term goal of our research is to better understand the mechanisms of toxicity and how metallothionein can alter its toxicity, so that we can both protect and treat humans from Cd toxicity. The metal is highly cumulative with a biological half-life of 10-40 years in humans. Over half of the total body burden is concentrated in liver and kidney, and of this, most is bound to metallothionein (MT). The present proposal has three specific aims. The first (1) is to examine the function of MT. Establishing the functions of MT remain elusive because there has been no experimental method of increasing MT without producing other effects. For example, metals, such as zinc, have been used to induce MT, but in addition to increasing MT, these metals also produce a myriad of other biological effects. Thus, it has been difficult to ascertain a direct cause and effect role for MT. The investigators recently have obtained transgenic mice that normally possess MT levels about 40-times higher than their non-transgenic counterparts. These animals provide a unique model for studying the protective role of MT without the introduction of agents that also alter other important systems. They plan to use these mice to determine MT's role in protecting against Cd-induced hepatotoxicity, cisplatin-induced nephrotoxicity, and oxidative stress produced by adriamycin, paraquat, and X-rays. In the (2) second aim, they plan to investigate the cellular regulation of MT in rat and human liver cells. They have shown that basal levels of MT are extremely important in determining the susceptibility of cells to the toxic effects of Cd. Human liver has about 50-100 times more MT than rat liver. They plan to determine the biochemical and molecular reasons for the differences between human and rats. That is, whether there is a difference in degradation and/or synthesis of MT, and if there is an increased synthesis, are the mRNA levels higher. If mRNA levels are higher, they will determine if this is due to increased synthesis and/or slower degradation of MT mRNA in humans than in rats. They will also test the hypothesis that the high basal level of MT gene expression in humans is due to species-specific trans-acting factor(s) not present in rats. In the (3) third aim, they plan to use renal proximal tubule cell culture systems to investigate the mechanism of CdMT nephrotoxicity, the main target organ of Cd toxicity in humans. From the proposed experiments using transgenic animals they will determine with certainty the functions of MT in protecting us from toxicants, using molecular biology techniques they will determine the mechanism by which humans have much more MT than rats, and using tissue culture techniques, determine the mechanisms of CdMT-induced nephrotoxicity.

This award will support a seminar on "Metallothionein in Biology and Medicine," organized by Prof. Curtis D. Klaassen of the University of Kansas Medical Center, and Prof. Kazuo Suzuki of the National Institute for Environmental Studies, Tsukuba, Japan. Participants will meet in Honolulu, Hawaii, in December 1989 to exchange recent research results and promote future joint research projects. The seminar will deal with the general biological and chemical properties of metallothionein; its physiological and nutritional aspects; transport of metallothionein within the body; diseases derived from metallothionein; methods of analysis of metallothionein; and use of metallothionein in medicine. Metallothionein is a protein which is easily taken into tissues in the presence of cadmium. Since current work on metallothionein grew out of cadmium contamination of the Japanese environment and subsequent physiological disorders, the Japanese have become expert in this field. This seminar may improve the understanding of metallothionein: while the dynamics of metallothionein uptake and transport has been to some extent well-delineated over the past few years, its function is still unknown. The multidisciplinary approach evidenced by the diversity of those invited may lead to a breakthrough in understanding the function of metallothionein, and therefore its good and bad effects.

Two major groups of molecules are sulfated in the body; small molecules such as drugs, hormones and environmental chemicals, as well as large molecules such as proteins and glycosaminoglycans. The sulfate conjugation reaction is catalyzed by sulfotransferases utilizing adenosine 3' -phosphate 5' -phosphosulfate (PAPS) as the sulfate donor. Brachymorphic mice have a defect in PAPS synthesis, and thus undersulfate glycosaminoglycans which results in joint and skeletal defects. The sulfation of xenobiotics is characterized as being a high-affinity, how- capacity pathway. The low-capacity sulfation of xenobiotics in rats is due to a limited availability of PAPS, which in turn is limited by the availability of sulfate. Molybdate also decreases hepatic levels of PAPS and sulfate. Molybdenum is known to produce joint problems, which can be prevented by sulfate and methionine. Because joints are largely cartilage and a major constituent of cartilage is sulfated glycosaminoglycans, we propose to test the hypothesis that joint problems produced by molybdate are due to an undersulfation of the glycosaminoglycans. There is also evidence in the literature that drugs that are sulfated decrease the sulfation of glycosaminoglycans and produce teratological effects in the cartilage-skeletal system. Six specific aims will be addressed in the proposed research. We plan to test the hypotheses that: (1) PAPS concentrations can be decreased by molybdate, (2) PAPS depletion by molybdate is a useful tool for determining the importance of sulfation in toxicology, (3) molybdate interferes with glycosaminoglycan sulfation, (4) chlorinated phenols inhibit glycosaminoglycan synthesis by inhibiting sulfation, (5) chemicals that are sulfated decrease glycosaminoglycan sulfation, (6) and chemical interference of glycosaminoglycan sulfation is not unique to rats. Methods to be employed are enzymatic quantitation of PAPS, HPLC- ion chromatographic analysis of sulfate and use of isotopes to quantitate glycosaminoglycan synthesis. Scientifically these investigations are intriguing because they may reveal novel mechanisms for toxic effects of chemicals via impairment of the sulfation of glycosaminoglycans. They are also of potential medical significance, because sulfation of the glycosaminoglycans in cartilage is essential for the proper functioning of joints, and joint ailments are the number one cause of debilitation in the aging American population. Therefore, the long-term goal of these studies is to determine if and how chemicals interfere with cartilage formation and thus joint disease.

The overall purpose of this meeting is to bring together the leading scientists in the United States, Japan, and Europe to discuss their most recent research results on the role of "Metallothionein in Biology and Medicine". It is appropriate that scientists from the US and Japan organize this meeting as many of the leading scientists working on metallothionein are from these two nations. The work on metallothionein grew out of the environmental contamination in Japan with cadmium resulting in the disease known as Itai-Itai. As a result of this exposure, most research has been performed in the United States and Japan to further understand the toxicity of cadmium. Great progress has been made in understanding the structure of this protein. Not only has the amino acid sequence of metallothionein been determined, but the molecular biology and the characterization of binding of metals to metallothionein has been extensively delineated the last few years, however the function f this protein has been illusive. Therefore, the objective of the meeting is to bring together scientists researching the function of metallothionein as well as few experts on the chemistry and molecular biology of metallothionein (who do not often have the opportunity to interact because they come from various disciplines) to have a scientific meeting where new concepts, new approaches, and hopefully a breakthrough in understanding the function of metallothionein will be obtained.

DESCRIPTION: The 4th International Metallothionen meeting will be held from September 17-20, 1997, at the Westin Crown Center in Kansas City, Missouri. The goal of the meeting is to provide a forum for both American and foreign investigators of different disciplines who are actively engaged in the cutting edge of metallothionen research. Metallothioneins are low molecular weight, cysteine-rich, metal-binding proteins which have been proposed to play an important role in metal toxicology and in scavenging of free radicals. Because this field has attracted scientists from widely diverse disciplines (i.e., cell and molecular biology, analytical chemistry, pathology, biochemistry, physiology, pharmacology, internal medicine and environmental toxicology) it is pertinent that such investigators are provided a forum to analyze, critically evaluate, and discuss the latest developments, unresolved problems, and current theories in metallothionen biology. Since the Third International Meeting in Japan, many interesting and new aspects of Metallothionen research have been stimulated and is rapidly expanding. The symposium will consist of platform lectures and posters to disseminate the latest advances, both in data and theory, in the field. Significant time for discussions will be included. The proceedings will be published in book form.

DESCRIPTION (Adapted from the Investigator's Abstract): The ultimate goal of this project is to assess the significance of endocrine disruptors that increase thyroxine (T4) glucuronidation on thyroid carcinogenesis, because many endocrine disruptors are suspected to be thyroid tumor promoters. The mechanism by which endocrine disruptors promote thyroid tumors has been proposed to result from alterations in the hypothalamus-pituitary-thyroid axis. Endocrine disruptors alter the hypothalamus-pituitary-thyroid axis by increasing T4 glucuronidation and elimination, which reduces serum T4 as a compensatory feedback mechanism, thyroid stimulating hormone (TSH) will be released from the pituitary, which will stimulate the thyroid and result in thyroid cell proliferation and neoplasia. However, the preliminary studies suggest that a number of endocrine disruptors (3MC and PCB) interfere with the normal hypothalamus-pituitary-thyroid axis because these endocrine disruptors do not increase serum TSH. Therefore, the central hypothesis of this application is that endocrine disruptors that increase T4 glucuronidation are thyroid tumor promoters only when they increase serum TSH. To test this hypothesis, there are five specific aims: (1) This aim is to test the hypothesis that endocrine disruptors, which increase serum TSH, produce thyroid follicular cell proliferation via proliferating cell nuclear antigen (PCNA) immunocytochemistry. In aim (2), the hypothesis that the tumor promoting effects of endocrine disruptors are not correlated with the decrease in serum T4, but with the increase in serum TSH will be tested in a 25-week bioassay. Rats will be given the thyroid initiating agent, N-bis(2-hydroxypropyl)nitrosamine (DHPN), followed by exposure to endocrine disruptors. This study will provide critical information on the relationship between thyroid hormone imbalance, TSH secretion, and thyroid tumor promotion of rats treated with endocrine disruptors. Aim (3) is to test the hypothesis that endocrine disruptors decrease plasma T4 pharmacokinetically by increasing its glucuronidation. The pharmacokinetics of T4, as well as T3, will be determined to understand the mechanism(s) by which endocrine disruptors decrease serum T4 in the final aim (4), the mechanism by which 3MC and PCBs "blunt" the TSH response to reduced serum T4 will be examined, testing physiologic, pathologic or thyroid receptor binding mechanisms. If the overall hypothesis is true, then it has important implications in toxicology, for many endocrine disruptors have been shown to reduce serum T4. However, their effect on TSH is, at best, variable. The expectation is that increases in serum TSH, rather than reductions in serum T4 is a better indicator for thyroid tumorigenicity resulting from exposure to endocrine disruptors. If the investigators demonstrate that TSH mediates endocrine disruptor thyroid tumor promoting activity, then as long as the promotional mechanism is prevented (increase in serum TSH), cancer would be prevented.

DESCRIPTION (provided by applicant): Cellular uptake is a fundamental phenomenon required for endogenous compounds and xenobiotics, such as drugs and environmental pollutants, to elicit physiological, pharmacological, and toxicological events within the cell. One might expect that the mechanism(s) by which xenobiotics are transported into hepatic parenchymal cells will affect hepatocellular biotransformation and biliary excretion. There are numerous transport mechanisms putatively responsible for hepatic sinusoidal uptake of organic molecules. These mechanisms include sodium-independent transport of a broad range of organic anions mediated by organic anion transporting polypeptides (Oatps). The Oatp sinusoidal transporters constitute an important organic anion transport system that we postulate will be regulated by classical enzyme inducing chemicals and bile acids. Despite much progress made in cloning and identifying Oatps, there is only a limited understanding of the regulation and function of Oatps. This deficiency, coupled with the emergence of the mouse genome sequence and the availability of numerous knockout mouse models, provides us an unprecedented opportunity to study and understand the regulation of Oatp gene expression. In parallel, our newly developed Oatp4-null mouse (the first and only Oatp-null mouse) gives our laboratory a unique tool to examine functions of this important Oatp in vivo. Therefore, the current proposal represents our plans to: (1) determine the molecular regulatory mechanisms responsible for both constitutive and altered expression of the Oatp gene family in liver, and (2) characterize the in vivo function of the liver-specific transporter Oatp4. The data generated regarding the expression and regulation of Oatps, as well as functional data from our Oatp4-null mouse will greatly advance our knowledge concerning the importance of Oatps in physiology, pharmacology, and toxicology, and ultimately not only aid the scientific community in predicting drug efficacy and safety in humans, but allow the development of liver-specific drug delivery.

DESCRIPTION ( Adapted from the applicant's abstract): Biotransformation and detoxication of xenobiotics, pharmaceuticals, and chemicals is intrinsically dependent on the organism's capacity to excrete these compounds. The excretory systems can be upregulated by chemotherapeutic agents in the phenomenon of multidrug resistance observed in many cancers. Chemotherapy-induced drug resistance is due to the overexpression of proteins which transport chemicals out of cancer cells. These proteins include P-glycoprotein and multidrug-resistance proteins (Mrp) which are encoded by the multiple drug resistance gene (MDR) and the mrp genes, respectively. In normal liver, localization of transport proteins in the canalicular membrane of hepatic parenchymal cells is physiologically important for export/excretion of chemicals into bile. One member of the mrp gene family, Mrp2 (formerly cMOAT), is an ATP-dependent canalicular transporter protein responsible for the excretion of various organic anions, including glutathione, sulfate, and glucuronide conjugates of chemicals and xenobiotics. These transport processes, of which Mrp2 is a critical component, are now considered the "phase III" process of drug/chemical hepatic metabolism. The possibility that this third phase of biotransformation can be regulated in coordination with both phase I and II systems in response to chemical stimuli is intriguing and would suggest that all three phases of biotransformation are coupled to increase the efficiency of hepatocellular metabolism/detoxication of chemicals. This laboratory has reported on the ability of chemicals to increase hepatobiliary function, which we postulate to be intrinsically related to Mrp2 expression and function. The first two aims will test the hypothesis that these observations are due to altered phase III metabolism, more specifically altered Mrp2 regulation. This laboratory has published an extensive amount of research showing that marked increases in toxicity of chemicals in newborn animals occur as a result of chemical-induced enhanced hepatobiliary function. Aim 3 of this application will test the hypothesis that chemical-elicited maturation of neonatal hepatobiliary function is due to enhancement of Mrp2 function and/or expression. Further, this laboratory has reported that chemical-chemical interactions can cause a transition shift in the vector of hepatic excretion to decrease hepatobiliary excretion and increase hepatovascular excretion. Aim 4 will test the hypothesis that this transition in hepatic excretion occurs due to the chemical-induced differential expression of Mrp2 on the hepatobiliary membrane and Mrp3 on the sinusoidal membrane. The last Aim tests an entirely new concept in drug metabolism and control of proteins involved in biliary excretion, that is, that Mrp2 is regulated by an orphan nuclear receptor, the pregnane X receptor. Elucidation of mechanisms that control Mrp2-mediated excretion of drugs will continue to enlighten the scientific and medical communities as to the importance of xenobiotic transport processes in relation to creating safe and biologically active drugs that alleviate specific transport deficiencies and protecting the public from chemical exposure.

The ultimate goal of this project is to assess the significance of endocrine disruptors that increase triiodothyronine (T3) hepatic uptake and glucuronidation on thyroid carcinogenesis. Many thyroid endocrine disruptors are suspected thyroid tumor promoters, which promote thyroid tumors by altering thyroid hormone homeostasis. It was previously proposed that endocrine disruptors alter the hypothalamus- pituitary-thyroid axis by increasing T4 glucuronidation and elimination, which reduces serum T4. As a compensatory feedback mechanism, thyroid stimulating hormone (TSH) is released from the pituitary, which stimulates the thyroid and results in thyroid cell proliferation and neoplasia. However, we have found that induction of T3 glucuronidation, rather than T4, is better correlated with increases in TSH of rats treated with thyroid endocrine disruptors. Therefore, we propose to test the importance of increased metabolism of T3 (ie., hepatic uptake and glucuronidation of T3) in mediating increases in TSH of endocrine disruptor-treated rats. In this application, we will test the hypothesis that thyroid hormone disruptors that specifically increase hepatic uptake and glucuronidation of T3 and increase serum TSH, are thyroid tumor promoters. We propose that the molecular mechanism by which these thyroid hormone disruptors increase hepatic uptake and glucuronidation of T3 is mediated through the ligand-activated pregnane-X-receptor (PXR), which increases the transcription and eventual protein levels of hepatic T3 sinusoidal transporters, as well as increases the glucuronosyltransferase(s) that glucuronidates T3. This results in an increase in the transport of T3 into hepatocytes and T3 glucuronidation, resulting in reduced blood levels of T3, reduced negative feedback effect at the hypothalamus and pituitary, increased serum TSH, stimulation of thyroid follicular cell proliferation, and ultimately thyroid tumor promotion. These studies will provide critical information on the relationship between thyroid hormone imbalance, TSH secretion, and thyroid tumor promotion of rats treated with thyroid endocrine disruptors. If the overall hypothesis is true, then it has important implications in toxicology and risk assessment, for many endocrine disruptors have been shown to disrupt thyroid hormone homeostasis. Also, if our hypothesis is true, that a specific glucuronosyltransferase is responsible for the glucuronidation of T3 that initiates the increase in serum TSH, then a biomarker for this type of endocrine disruption could be developed. If our molecular hypothesis is true, that the initial interaction that produces thyroid tumors is through an interaction of the endocrine disruptor with the PXR, a screen for potential thyroid-tumor promoters could be developed.

[unreadable] DESCRIPTION (provided by applicant): Elucidating the mechanisms by which chemicals and drugs are taken up into and eliminated from cells can enhance the understanding of drug bioavailability and can aid in prediction of tissue-specific distribution and toxicity of drugs. Organic anion transporting polypeptide (Oatp) and multidrug resistance-associated protein (Mrp) transporters mediate uptake and efflux, respectively, of a wide variety of substrates in liver. Coordinate regulation of uptake and efflux transporters may be a mechanism by which cells protect themselves from chemicals, for example, by simultaneously decreasing entrance and enhancing elimination of chemicals. Perflourodecanoic acid (PFDA) is a ten-carbon fluorinated fatty acid and a component of numerous commercial products. PFDA is a peroxisome proliferator that also produces a broad spectrum of toxicity in rodents. Our own experiments have demonstrated that PFDA causes unique and dramatic changes in hepatic expression of Oatp and Mrp transporters. These PFDA-mediated changes in transporter expression are likely to have physiological implications by affecting hepatic uptake and elimination of both endogenous and xenobiotic substrates. These novel effects of PFDA on transporter gene expression thus merit further investigation. Therefore, PFDA can be used as a powerful tool to elucidate mechanisms of transporter regulation. To further our knowledge of transporter regulation by PFDA, we propose the following studies: 1) examine alterations in transporter gene expression as a function of both dose and time, 2) determine the effects of these changes in gene expression on xenobiotic distribution, and 3) examine the contribution of several key transcription factors in the regulation of transporters by PFDA. The studies proposed will increase our understanding of the unique and dramatic effects of PFDA on transporter genes and on the consequences of altering transporter gene expression. More generally, these investigations will elucidate mechanisms of transporter gene regulation, information that is important in predicting possible therapeutic benefits of transporter modulation, as well as possible drug-drug interactions in humans. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]

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DESCRIPTION (provided by applicant): Multidrug resistance-associated proteins (Mrps) play a key role in hepatic detoxication by transporting Phase-ll conjugates and other organic compounds out of hepatocytes. Similarly, upregulation of Mrps in tumor cells confers resistance to chemotherapeutic drugs by transporting these cytotoxic compounds out of cells. The regulatory mechanisms governing Mrp expression in normal and diseased liver, and in tumor cells, are not understood. Thus, the overall goal of this application is to study the molecular mechanisms of transcriptional regulation of Mrps. Nuclear factor E2 related-factor 2 (Nrf2) is emerging as a critical transcription factor in regulation of both constitutive and inducible expression of Phase-ll enzymes. Because Mrps play a key role in the efflux of Phase-ll conjugates, we hypothesize that Mrps are coordinately regulated with Phase-ll enzymes by Nrf2. We recently examined Mrp expression in three different models: 1) mice treated with monofunctional inducers that selectively upregulate Phase-ll enzymes, 2) bile-duct ligation, a surgical model of extrahepatic cholestasis, and 3) mice with targeted disruption of glutathione synthesis. In each model, induction of Mrps and classical Nrf2 target genes was observed. Moreover, using in-silico analysis, we identified putative Nrf2-responsive sequences, known as electrophile response elements (EpREs), in the 5'flanking regions of the Mrp2, 3, and 4 genes. We hypothesize that activation and subsequent binding of Nrf2 to these EpREs results in increased expression of Mrp2, 3, and 4. Thus we propose Mrps belong to the battery of Nrf2-regulated detoxication genes. To test this hypothesis, we will determine: 1) the role of Nrf2 in Mrp induction in mice, taking advantage of Nrf2-null mice, 2) Nrf2 activation and subsequent translocation to the nucleus, 3) critical response elements by in vitro and in vivo reporter gene assay in combination with promoter deletion analysis, 4) Nrf2 binding to EpREs identified in Mrp 5'flanking regions, and 5) specificity of Nrf2 binding to Mrp promoter regions. Data from the experiments in this proposal will provide novel insight into the transcriptional regulation of Mrps. Elucidation of the role of Nrf2 in the regulation of the efflux transport process will have significant ramifications in toxicology, xenobiotics disposition, drug-drug interaction, and cancer chemoprevention.

DESCRIPTION (provided by applicant): Five years of funding are requested to develop a Center of Biomedical Research Excellence at Kansas University Medical Center with a focus on Nuclear Receptors and their Role in Liver Health and Disease. Five talented new faculty who share this research interest were selected with the goal of helping them become funded, independent investigators. A PI, co-PI, an internal advisory committee of experienced senior faculty and an external advisory committee of prominent scientists have been assembled to mentor them to this goal. The Center will also help provide infrastructure and equipment to supplement the research environment. Ultimately, through the achievement of these initial goals, the final long-range goal is the submission of a program project grant application centered on the theme of nuclear receptors and related topics. An important feature of this Proposal is that for each initial junior faculty member, two co-mentors have been assigned and individual mentoring plans and timetables have been developed. Central features of the mentoring plans include ongoing critical evaluation of the research project by the mentors and IAC, semiannual conferences with EAC members, and special training on statistics, manuscript and grant writing, and teaching. The initial group will receive support for up to three years. Within this time period each member will be expected to compete successfully for independent NIH R01-type research support. Once funded externally, they will financially rotate off the grant to make room for addition of new junior faculty members. Over five years, the grant will support 10-15 independent research investigators. Another important feature of the Proposal is to establish five new Core capabilities to provide additional research support for the Center's faculty. These cores include an administrative Core as well as Cores in the areas of molecular biology, null-mice development and husbandry, phenotyping capabilities, and bioanalytical support. In summary, the outstanding combination of scientific talent, existing research environment, and requested new core facilities ensure that this proposed Center will foster the development of a thematic multi-disciplinary research center, to enhance the ability of new investigators to compete independently for complementary NIH and other external peer-reviewed support, and to strengthen existing biomedical research infrastructure at KUMC.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have spent a considerable amount of time this last year planning joint projects that we hope will evolve into one or more program projects. We started out by having meetings to decide topics that we might develop into program projects. Because the metabolic syndrome is a major public health problem, and NASH (non-alcoholic steahepatitis) is a component of this syndrome, we decided to examine factors that might contribute to this phenomenon. Therefore, we have designed four experiments to investigate the circadian rhythm, various genetic backgrounds (strains), various diets, and ages (old) of mice. We have collected the tissues of three of these experiments.

DESCRIPTION (provided by applicant): Current therapeutic interventions for treating liver diseases are very limited;identification of a therapeutic target is a prerequisite for developing new drugs to treat liver diseases. Oxidative stress and inflammation play key roles in the pathogenesis of liver diseases. Nuclear factor 2-related factor 2 (Nrf2), a transcription factor expressed in both hepatocytes and immune cells, is a key factor in anti-oxidative and anti-inflammatory responses. In quiescent hepatocytes, Nrf2 is sequestered in cytosol by kelch-like ECH-associated protein 1 (Keap1);in Keap1-knockdown mice, nuclear translocation of Nrf2 increases, resulting in activation of Nrf2 target genes. The importance of Nrf2 in cytoprotection is recognized. Nevertheless, the role of Nrf2 in liver pathophysiology has just recently gained attention. Our long-term goal is to identify a novel drug target and develop a therapeutic/preventive strategy for liver diseases. The objective of this R01 grant proposal is to investigate in-depth the role of Nrf2 in protecting against liver pathophysiology and understand its underlying molecular mechanisms. Based upon our strong preliminary data, our central hypothesis is that Nrf2 is a master regulator of liver protection through its multifaceted roles in biotransformation/disposition, antioxidation, tissue- repair, and anti-inflammation in multiple cell types in liver. Aim 1 will identify direct targets of Nrf2 regulation using ChIP-Sequencing and determine Nrf2-dependent mRNA induction in the Keap1-knockdown mouse. Aim 2 will test the hypothesis that Nrf2 activation protects against chemical, dietary, and genetic models of liver disease. We will use 3 genetic models of mice with differential Nrf2 activity, namely Nrf2-null, wild-type, and Nrf2-enhanced mice, to test liver disease susceptibility in 13 different models. Aim 3 will develop novel and specific Nrf2 activator lead candidates by high throughput screening in collaboration with the Office of Therapeutics, Discovery, and Development. Our proposed study is novel, because it utilizes 3 mouse lines with different Nrf2 activities to investigate the opposing effects of Nrf2 deficiency and Nrf2 activation on liver diseases induced via diverse mechanisms of toxicity. This proposal utilizes innovative techniques including ChIP-sequencing, multiplex mRNA analysis, and nuclear translocation high throughput screening in testing the 3 aims. Our proposed study is significant, because results from our experiments will provide solid evidence regarding the importance of Nrf2 deficiency and Nrf2 activation in liver pathophysiology and its underlying molecular mechanisms, setting the scientific foundation for developing Nrf2 activators as novel drugs to treat/prevent various liver diseases, such as chemical-induced liver injury, inflammatory liver diseases, steatohepatitis, and liver fibrosis/cirrhosis. PUBLIC HEALTH RELEVANCE: Liver disease is a common ailment, however in contrast to most other diseases, there are very few drugs for liver failure. We hypothesize that Nrf2, an essential transcription factor, is a drug target that can be used to prevent and treat a wide-range of liver ailments. This study will determine whether this drug target will be effective against a wide-spectrum of animal models of liver disease.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Administrative Core of the Program will be responsible for theoverall oversight, organization, and management of the scientific aspects of the COBRE grant. In addition to the scientific oversight, the Core will also provide a number of support services to Program-associated investigators, including fiscal/budgetary management, preparation of progress reports, scheduling of research meetings for project-associated investigators, coordinating travel arrangements and meetings of the external science advisory board, and organizing a regular scientific seminar program, liver club, "school of hard knocks," amid various work sessions as described in the mentoring section.

DESCRIPTION (provided by applicant): The long-term goal is to understand the molecular mechanism of hepatic uptake and systemic disposition of endogenous signaling molecules and xenobiotics. Organic anion transporting polypeptides (Oatps) transport a broad range of organic anions. The liver-specific transporter Oatp1b2 (in rodents) has hum an orthologs OATP1B1 and OATP1B3, which are responsible for hepatic uptake of a large number of chemicals. Studies of Oatp1b2-null mice clearly illustrate an essential role of Oatp1b2 in hepatic uptake of certain xenobiotics. Our recent data suggest that Oatp1b2-null mice have decreased hepatic uptake and increased circulating levels of certain important endogenous molecules, such as unconjugated hydrophilic bile acids, bilirubin, a peptide hormone cholecystokinin-8, and prostaglandin E2 (PGE2). Our preliminary studies indicate that Oatp1b2-null mice have unexpected difficulty in maintaining their body temperature during surgical experiments, but are protected from lipopolysaccharide-induced hypothermia and hyperalgesia. The objective of this proposal is to use Oatp1b2-null and heterozygous mice as well as in vitro cellular and liver uptake systems to elucidate the physiological function and gene-dosage effects of Oatp1b2 in hepatic uptake and systemic disposition of endogenous chemicals and xenobiotics. The central hypothesis is that Oatp1b2 is essential in hepatic uptake of unconjugated hydrophilic bile acids, cholecystokinin-8, and PGE2, which have thermoregulatory and immunomodulatory activities. Increases in circulating levels of these signaling molecules in Oatp1b2-null mice lead to alterations in inflammatory responses and thermoregulation. Results from studies in 5 specific aims will elucidate: 1) the importance of Oatp1b2 in hepatic uptake of bile acids;2) how alterations of pH and bile acids influence Oatp1b2-dependent and -independent hepatocyte uptake of unconjugated bilirubin;3) gene- dosage effects of Oatp1b2 on hepatic uptake and systemic disposition of peptide hormones, prostaglandins, organic dyes, and a peptide-analog anti-diabetic drug;4) the importance of Oatp1b2 in hepatic uptake and systemic disposition of PGE2 and its impact on febrile response;and 5) the mechanism of protection of Oatp1b2-null mice from lipopolysaccharide-induced syndrome. This study is novel, because it will elucidate the mechanisms of the exciting preliminary findings that loss of Oatp1b2 results in not only greatly decreased hepatic uptake of certain xenobiotics, but also marked alteration of circulating endogenous signaling molecules and physiological changes during surgical conditions and inflammatory stresses. This study is significant, because results from this study will provide novel knowledge regarding how the loss/decrease of a liver-specific importer Oatp1b2 (OATP1B1/1B3 in humans) decreases hepatic uptake and increases circulating essential endogenous chemicals (bilirubin, bile acids, peptide hormones, prostaglandins) and xenobiotics, resulting in altered physiology and pharmacokinetics/toxicokinetics.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We had a number of meetings to decide topics that we might develop into program projects. Because the metabolic syndrome is a major public health problem, and NASH (non-alcoholic steahepatitis) is a component of this syndrome, we decided to examine factors that might contribute to this phenomenon. Therefore, we have designed four experiments to investigate the circadian rhythm, various genetic backgrounds (strains), various diets, and ages (old) of mice. We have collected the tissues of three of these experiments.

DESCRIPTION (provided by applicant): Despite recent rapid progress in understanding the expression patterns and regulatory mechanisms of drug processing genes, namely drug metabolizing enzymes and transporters in adults, little is known about these in the pediatric period. The long-term goal is to understand mechanisms of ontogenic regulation of drug processing genes, so that efficacious and safe drug treatments can be achieved in children. Several factors are known to be essential for normal development, including hepatocyte nuclear factor 11 (HNF11), farnesoid X receptor (FXR), growth hormone (GH) signaling, and epigenetic influences. HNF11, a master regulator of early liver development, regulates hepatic expression of a large battery of drug processing genes. Initiation of bile-acid signaling pathways, mediated largely via the FXR, is a hallmark of perinatal liver development. GH is essential for postnatal hepatic gene expression and maturation. The accessibility of transcription factors to the target genes is largely determined by the methylation/acetylation status of histones and DNA sequences. Preliminary studies illustrate that in developing mouse livers, drug processing genes and transcription factors are expressed in distinct dynamic patterns and correlate with epigenetic signatures. The objective of this proposal is to elucidate the regulatory mechanisms of ontogenic expression of drug processing genes in mice. The rationale of this proposal is that its successful completion will generate basic knowledge that will serve as the foundation for further understanding pediatric pharmacology in humans. The central hypothesis is: developmental regulation of drug processing genes is a sequential event regulated by hormones, which activate transcription factors to modify epigenetic signatures and regulate gene expression. This hypothesis will be tested in 2 aims. Aim 1 will determine the ontogenic expression patterns of drug processing genes and the correlation with transcription factors and epigenetic signatures. The relative mRNA expression of major phase I/II enzymes and drug transporters in male mouse livers versus intestine and kidney will be examined, and correlated with expression of transcription factors and chromosome modifications (genome-wide DNA methylation and histone modifications). Aim 2 will elucidate roles of transcription factors and GH in determining ontogenic hepatic expression of drug processing genes in HNF11-null, FXR-null, and GH deficiency (lit/lit) mice using the same working strategy. This study is novel, because it will use a genome-wide approach to elucidate how alterations of hormones and transcription factors modulate epigenetic signatures and hepatic ontogenic expression of drug processing genes. This study is significant, because little is known about the regulation of hepatic drug processing genes in pediatric stages. Results from this study will: 1) provide basic knowledge on the ontogenic expression patterns of drug processing genes and nuclear receptors in liver, kidney, and intestine;and 2) help to understand how perinatal alterations in hormones and nuclear receptors, via modulating epigenetic signatures, affect stage-specific and long-term expression of drug processing genes. PUBLIC HEALTH RELEVANCE: The proposed studies are of importance and an under-investigated area of regulatory mechanisms of the developmental expression patterns of genes important in the absorption, distribution, metabolism, and excretion of therapeutic drugs. Thus, the findings are expected to be applicable to the improvement of efficacy and safety of pediatric pharmacology.

? DESCRIPTION (provided by applicant): Developmental exposure to the flame-retardant polybrominated diphenyl ethers (PBDEs) has attracted growing concerns recently, because these highly persistent environmental toxicants are accumulated much more in infants through breast milk, and produce multiple detrimental effects. Although a growing body of research has been done regarding the toxicities of PBDEs themselves, little is known about the potential involvement of PBDEs in modulating the pharmacokinetics of drugs in newborns and children, who are at a much higher risk of adverse drug reactions. More importantly, there is no information regarding whether developmental exposure to PBDEs produces long lasting modifications of drug metabolism beyond childhood. We and others have identified that PBDEs are novel activators of the major xenobiotic-sensing nuclear receptors pregnane X receptor (PXR) and constitutive and rostane receptor (CAR). Neonatal activation of CAR results in epigenetic memory on histone methylation signatures and permanent change of drug metabolism in mouse liver, whereas PXR also regulates distinct epigenetic modifiers. Thus the objective of this research is to utilize multidisciplinary approaches to strategically investigate he epigenetic mechanisms of PBDEs in modulating the transcriptional activities of PXR and CAR and drug-processing capacities during and beyond the neonatal period on a genome-wide scale. Our central hypothesis is: neonatal exposure to PBDEs activates CAR and/or PXR, which in turn reprograms the ontogeny of critical chromatin epigenetic modifiers (such as DNA and histone methylation as well as histone acetylation), leading to epigenetic memory and altered ontogeny of drug-processing genes (DPGs), and long-term alterations in the pharmacokinetics and toxicokinetics beyond childhood. We will test this hypothesis in 3 specific aims: Aim 1 will use xeno-sensor null mice and second- generation sequencing to determine the roles of PXR and CAR in modulating the chromatin epigenetic signatures and expression of DPGs following neonatal exposure to PBDEs; Aim 2 will determine the effect of silencing key chromatin epigenetic modifiers on the expression of PXR- and CAR-target genes in PBDE- treated primary hepatocytes; Aim 3 will determine the role of neonatal PBDE exposure in modulating xeno- sensor activities, chromatin epigenetic signatures, expression of DPGs, and pharmacokinetics of drugs in xeno-sensor humanized mice and human hepatocytes. We will also examine the species-differences in PXR and CAR in response to PBDE exposure. Overall, the proposed work will unveil for the first time critical epigenetic and PXR/CAR-mediated mechanisms underlying PBDE-mediated regulation of drug metabolizing enzymes and transporters in newborns, and more importantly, determine the potential long-term effects caused by neonatal PBDE exposure on epigenetic memory and drug-processing capacities in adults. Our study is a paradigm shift in pediatric pharmacology, and will provide the first mechanistic investigations of PBDE-mediated modulation of drug metabolism and transport in newborns on a genome-wide scale.