Nathan J. Cherrington - US grants

The overall goal of this research is to identify the human genes coordinately regulated by the Antioxidant/Electrophile Responsive Element (ARE/EpRE) and elucidate the mechanism of protein binding and transcriptional activation of the ARE/EpRE. Recently, we identified a human neuroblastoma cell line (IMR-32) that shows dramatic induction of ARE/EpRE reporter constructs and endogenous QR protein by treatment with tert-butylhydroquinone (tBHQ). Others have demonstrated that induction of QR by tBHQ in neuroblastoma cells prior to glutamate treatment significantly decreased glutamate-mediated cytotoxicity suggesting that QR was neuroprotective. Recent studies however have shown that QR is not solely responsible for blocking cytotoxicity since stable overexpression of QR in this same cell line did not confer protection to the cells from dopamine-induced cytotoxicity. We have postulated that a coordinate regulation of multiple genes rather than a single gene product is responsible for the neuroprotection seen with tBHQ pretreatment, and that this coordinate regulation is mediated via the ARE/EpRE. Thus the specific aims of this project are: 1) Identify and characterize differentially expressed genes in IMR-32 human neuroblastoma cells up-regulated by tBHQ. 2) Compare the coordinate regulation of genes leading to neuroprotection by tBHQ, dimethyl fumarate and insulin. 3) Determine whether the transcription factors Nrf1 and Nrf2 are transcriptional activators of the ARE/EpRE in human neuroblastoma cells. Because many types of cell death may involve the formation of oxidants, coordinate regulation of genes identified as containing the ARE/EpRE may lead to a new strategy to combat neurotoxicity.

The goal of this project is to determine the mechanism(s) by which Mrp3 is transcriptionally activated in a hepatoprotective response. Mrp3, which is localized to the sinusoidal membrane of hepatocytes, exports a wide range of I organic anions from the liver, into the blood. Thus, the role of hepatic Mrp3 is to decrease the concentration of potentially toxic molecules in the liver. Mrp3 expression is normally low in liver but is significantly increased after exposure to certain inducers and also during cholestasis, further demonstrating the importance of Mrp3-mediated export as a means of hepatoprotection. Cholestatic liver injury results in a substantial clinical burden leading to an estimated 100,000 deaths per year often listed as multiple organ dysfunction. The current clinical management of chotestasis includes phenobarbital, which decreases hepatotoxicity as a side effect of cholestasis. I have previously demonstrated that treatment with multiple reducers of CYP2B 1/2, (transcriptionally activated by CAR) such as phenobarbital, as well as inducers of NADP(H):quinone oxidoreductase (activated through Nrf2) are also capable of inducing Mrp3, showing coordinate regulation of both the Phase I drug-metabolizing genes and Phase III xenobiotic transporters. It has also been demonstrated that cholestasis results in increased Mrp3 but decreased CYP2B 1/2 levels suggesting distinct mechanisms involved in the regulation of Mrp3. Additionally, other outcomes of cholestasis include an increase in oxidative stress, hyperbilirubinemia, inflammation and cytokine releasc, all of which, individually, effect the regulation of xenobiotic transporters or Phase I drug metabolizing genes in a manner consistent with the effects of cholestasis. Therefore, the following aims have been designed to test the hypothesis that Mrp3 is differentially regulated during periods of both chemical insult and cholestatic stress: 1) Determine the role of the Constitutive Androstane Receptor in transcriptional activation by microsomal enzyme inducers that activate Mrp3 expression. 2) Determine whether the induction of Mrp3 during cholestasis is mediated by either prooxidant or antioxidant activation of Nrf2.3) Determine whether cytokine release, signaling, and NF-kB activation are responsible for the differential regulation of Mrp3 during cholestasis. 4) Define the Mrp3 promoter elements that are responsible for the transcriptional activation during both chemical insult and cholestatic stress. Understanding the mechanisms that control Mrp3-mediated excretion of organic anions can potentially serve the scientific community in our objective to create safe and biologically active drugs that alleviate specific transport deficiencies or up-regulate the excretion of chemicals in patients or exposed individuals.

[unreadable] DESCRIPTION (PROVIDED BY APPLICANT: The goal of this project is to determine whether pharmacologic induction of Mrp3 can be used as a means of hepatoprotection in humans during cholestatic disease states. Mrp3, which is normally expressed at low levels, serves to export a wide range of organic anions from the liver, back to the blood, thereby decreasing their exposure and toxicity to the liver. Mrp3 expression is significantly increased both in response to certain microsomal enzyme inducers as well as during cholestasis in rodents. Human expression of Mrp3 is also increased in certain liver diseases. Mrp3 expression can be pharmacologically increased by certain microsomal enzyme inducers in both mouse and rat. However, the mechanism for the induction of Mrp3 appears to be a complex interaction where transcriptional studies performed in vitro are unable to accurately replicate the induction observed in vivo. Aims 1 and 2 are designed to determine whether human Mrp3 expression can be pharmacologically induced by this same mechanism. These are; 1. Determine the common mechanism for the cholestatic and pharmacologic induction of Mrp3 expression. 2. Determine whether the human Mrp3 gene can be induced in vivo using a unique transcriptional activation assay and human liver slices. Cholestasis is a very complex disease with multiple etiologies that results in the accumulation of bile acids and other organic anions that cause profound hepatocellular damage. If Mrp3 can be pharmacologically induced in humans as a means of eliminating hepatic exposure to the toxic effects of accumulating bile acids and other organic anions, this would represent an important means of hepatoprotection during cholestasis. Aims 3 and 4 have been designed to determine whether Mrp3 induction can be used as a legitimate drug target in the treatment of human liver disease. These are; 3. Determine whether microsomal enzyme inducers that increase expression of Mrp3 are capable of altering the disposition of biliary constituents and protecting against hepatotoxicity during cholestasis. 4. Determine whether the transgenic overexpression of human Mrp3 is hepatoprotective in models of cholestasis. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Since its introduction in 1996, highly active antiretroviral therapy (HAART) has been credited with a marked and sustained reduction in AIDS-related death and disease in the United States. As with any pharmacotherapeutic agent, drug delivery to the site of action is absolutely required to elicit a favorable response. A significant obstacle to complete eradication of the HIV virus from the patient's body is the existence of biological barriers creating sanctuaries where the virus remains free from drug exposure. Two such barriers exist, ensuring the virus'survival and possible development of a resistant phenotype: the blood- brain barrier (BBB) and the blood-testis barrier (BTB). While there are no current methods that fully recapitulate the BBB to study the transport of drugs, we have developed a novel means of studying the intact BTB. The BTB protects the developing germ cells from the effects of potentially disruptive chemical interactions as well as immunological influences. Although this normal, protective function of the BTB is physiologically beneficial for reproduction, it can also provide a sanctuary for the HIV virus during antiretroviral therapy. This sanctuary site for HIV is particularly significant because the presence of HIV virus in the ejaculate of infected men is the major means of transmission to uninfected persons. Therefore, even while a patient is being effectively treated with a cocktail of antiretroviral drugs, the selective exclusion of the drugs at the BTB increases the viral load of the ejaculate and potential for transmission. Indeed, protease inhibitors and non-nucleoside reverse transcriptase inhibitors are actively excluded at the BTB and do not reach therapeutic concentrations in the testis. Alternatively, nucleoside analog drugs (NSAs) offer a unique opportunity to examine the characteristics of the BTB as well as the possibility of utilizing endogenous transporters as a means of circumventing the BTB since a number of NSAs are capable of accumulating in the semen of treated men. As the major component of the blood-testis barrier, Sertoli cells separate the adluminal compartment of the seminiferous tubules (STs) from the rest of the body by the formation of tight junctions below developing germ cells. In order to reach the adluminal space, drugs must first avoid barrier-function transporters before subsequently passing through Sertoli cells either by passive diffusion or carrier-mediated transport. Inward-facing transporter proteins, located at the basolateral and luminal membranes, allow specific compounds that cannot pass by passive diffusion to cross the BTB via transepithelial secretion. Selective movement of compounds across Sertoli cells therefore comprises the physiologic or functional aspect of the BTB. Therefore, we hypothesize that specific transporters are present at the basolateral and luminal membranes of Sertoli cells that allow specific NSA drugs to penetrate the BTB. The following aims have been designed to test this hypothesis: 1. Determine the constitutive expression and cellular localization of the major drug transporter proteins at the blood-testis barrier, as well as penetration of NSAs and PIs into the testis. 2. Determine the ability of selected inward-facing transporters (particularly Ent1, Ent2, Mate1 and Mate2) to support transepithelial secretion of NSAs and PIs. 3. Determine the functional capacity and molecular specificity of isolated Sertoli cells and intact STs to transport NSAs (didanosine, azidothymidine, abacavir, emtricitabine, tenofovir) and PIs (lopinavir and indinavir).The major emphasis of the current proposal is to focus on mechanisms by which therapeutics can successfully utilize endogenous transepithelial transporters to accumulate in the adluminal compartment, even within the context of the functional physiologic barrier. PUBLIC HEALTH RELEVANCE: Even when effectively managed by antiretroviral therapy, patients can transmit HIV virus because the blood-testis barrier provides a sanctuary where the virus can hide from the drugs. This project seeks to determine the molecular mechanisms whereby specific nucleoside analog drugs can cross the blood-testis barrier and the possibility of utilizing endogenous transporter systems as a means of circumventing the blood-testis barrier. This mechanism could be utilized in the development of new drugs to reduce the risk of transmission by infected patients.

DESCRIPTION (provided by applicant): Nonalcoholic fatty liver disease (NAFLD) comprises a spectrum of histopathologies that range from simple steatosis to the more severe steatohepatitis (termed non-alcoholic steatohepatitis or NASH). NAFLD is the most common cause of liver disease in preadolescents and adolescents, and the increased prevalence coincides with the rise in childhood obesity, insulin resistance, and hyperlipidemia. One of the major causes of adverse drug reactions is the inability of the individual patient to handle a standard dose of a prescribed drug. A major goal of individualized medicine is to identify the appropriate dose of a drug that will not elicit an adverse response in that patient. A major factor in determining a safe dose of a drug is the capacity of the patient to metabolize and eliminate that drug from the body. Identifying individuals with an impaired capacity to handle a drug prior to initiating treatment would therefore be instrumental in decreasing the number of adverse drug reactions. For the vast majority of drugs, the liver plays a key role in determining the rate at which drugs are eliminated. Several processes are required for efficient hepatic elimination, including entry into hepatocytes by uptake transporters, Phase I and II biotransformation, and efflux from the liver by drug transporters either into bile or back into the blood. Because the liver plays such a critical role in drug metabolism and disposition, any disease state that disrupts or modifies these functions will alter the fate of numerous drugs within the body. The effect of steatosis and NASH on the expression and activity of the major drug metabolizing enzymes is completely unknown, but could have broad implications in identifying both the patients that are at greater risk of developing adverse drug reactions, and the drugs that are likely to cause adverse events in patients with NASH. Our preliminary results in rodent models and humans with NASH indicate significant changes in the expression of drug metabolizing enzymes and transporters, as well as a functional shift in the disposition of drugs. The two major hypotheses to be addressed are; (1) NASH alters the expression and function of major drug metabolizing enzymes and transporters thereby, increasing the risk of adverse drug reactions in children with NASH and (2) Plasma and/or urine levels of APAP-GLUC can be used as a metabolomic biomarker to identify these patients (with NASH) that may be at risk for adverse drug reactions. Aim 1. Determine whether the in vivo activity of the major CYP enzymes is altered in children with steatosis or NASH. Aim 2. Determine whether the functional disposition of APAP metabolites is altered in patients with fatty liver disease. Aim 3. Determine whether the expression and activity of Phase II and III drug metabolizing enzymes and transporters are altered in human livers diagnosed with steatosis and NASH.

DESCRIPTION (provided by applicant): The Toxicology Graduate Program at the University of Arizona has a long-standing reputation for excellence in training Ph.D. scientist. Many of our graduates are now leaders in academia, industry, and government. Current trainees are now selected through a University-wide competition. The graduate program has evolved from a systems-based toxicology experience to training students to apply state-of-the art techniques to solve mechanisms of environmental toxicity affecting complex diseases in various organ systems. The cutting-edge basic science research programs of 22 Training Grant Faculty members, state-of-the-art technologies developed at the University of Arizona in association with the Southwest Environmental Health Sciences Center and Bio5, and translational approaches undertaken by our NIEHS Superfund Program and US-Mexico Binational Center provide an exceptionally stimulating environment for the training of graduate students and postdoctoral fellows. The interactive research of our Training Grant Faculty and our state-of-the-art Facility Cores extend the training environment from a single laboratory-oriented domain into a multidisciplinary experience strongly supportive of collaborative research. The University provides financial support for first year Ph.D. students, providing a large pool of highly qualified candidates for competitive selection of predoctoral trainees. Predoctoral training is achieved through a combination of coursework, laboratory research, and supplemental enrichment activities. Postdoctoral trainees participate in innovative research programs and are guided to develop professional skills in oral and written communication and in supervision. Over past five years, the curricular changes parallel the evolving expertise of the Training Grant Faculty in utilizing state-of-the-art technology for research projects. We have recruited 3 senior (Professor) and 3 junior (Assistant Professor) faculty into the Training Grant, which significantly enhances the strength in the core of mechanistic based molecular toxicology training. We have opened the Training Grant for University-wide selection to further stimulate interdisciplinary/multidisciplinary approaches in research and training. The request for continuation of NIEHS support is validated by the highly successful nature of our program, the clear demand for our graduates, the strong emphasis we place on leadership skills for our trainees and postdoctoral fellows, the increasing number of students interested in toxicology, substantial institutional commitment, strong and well-funded research programs of our faculty, and the excellence of the training environment.

The Strategic Vision of the Southwest Environmental Health Sciences Center (SWEHSC) is to facilitate and implement innovative research aimed at understanding the mechanisms underlying the modulation of human disease risks due to environmental exposures among populations living in arid environments. The objective is to bring interdisciplinary scientists together to study the environment, genetics, and the resulting toxicology that influence morbidity in our underserved American Indian and Hispanic communities. SWEHSC incorporates state-of-the-art technologies across the environmental health sciences to assess exposures and health risks (Human Population and Exposure Resource, IHSFC) including small molecule detection and quantification (Emerging Contaminants Analytical Resource, IHSFC), subcellular confocal imaging (Cellular Imaging Facility Core), genetic and genomic/epigenomic analyses (Genomics Facility Core), and cutting-edge bioinformatics (Data Science Resource, IHSFC) in exposed communities, and through a strong Community Engagement Core program that focuses on the social and cultural needs and practices of affected people. The themes of the SWEHSC are demonstrated through three Research Focus Groups (RFG). RFG1, Environmental Exposures to Southwest Populations, works with multiple stakeholder groups to assess multiple routes of exposure in arid environments. RFG2, Environmental Lung Disease, must account for the low humidity and high wind velocities that result in complex inhalation exposures. RFG3, Adaptive Responses to Environmental Stress, focuses on the molecular pathways of adaptive responses to environmental stressors such as arsenic and ultraviolet light that result in oxidative stress. The desert Southwest is the only US region that adequately represents much of the world's arid habitats; thus, the accomplishments of the SWEHSC will have broad applicability to other diverse populations. This is essential to our long-term goal of improving the lives of the people in arid environments by developing rational approaches to mitigating their risks of hazardous environmental exposures and by developing intervention strategies to reduce adverse health outcomes.        

PROJECT SUMMARY The testis and epididymis mark the initial portion of the male genital tract (MGT) and are impermeable to most hydrophilic compounds due to tight junctions. This barrier ? the blood- testes barrier (BTB) ? can act as an obstacle for therapies requiring entry into these tissues for full effect; for example contributing to testicular relapse in acute lymphoblastic leukemia (ALL) or acting as a sanctuary site for HIV infection. However, these epithelial barriers are known to express alternative xenobiotic transporters that may be utilized to allow select agents to gain access to the MGT and reduce the frequency of testicular relapse or HIV transmission. Rather than studying why drugs fail to cross the BTB, the major focus of our studies is to characterize the endogenous transport processes that could allow drugs to access the MGT. We hypothesize that the penetration of MGT relevant drugs into the BTB occurs through transport processes that can be modeled computationally, and that therapeutic concentration of these drugs is greatly affected by epididymal transporters and water reabsorption. Our grant focuses on 3 aims: Aim 1: Establish the transepithelial transport mechanisms by which MGT relevant drugs can cross the BTB and develop a predictive model for rational therapeutic design. Aim 2: Determine the impact of epididymal water reabsorption on drug concentrations within the MGT in vivo. Aim 3: Determine whether nucleoside transport is supported in the epididymis and the impact on nucleoside-based drug disposition. Understanding these pathways will be foundational for the development of drugs, such as antiviral, chemotherapy, contraception, and fertility agents, that require access to the MGT in order to achieve full therapeutic effect.

The Administrative Core (AC) provides the necessary infrastructure to integrate the leadership, organization, and strategic vision of the Southwest Environmental Health Sciences Center (SWEHSC) and to implement the SWEHSC mission by facilitating coordinated research, training, and community engagement efforts. Accordingly, the AC coordinates and integrates Center activities, attracts new investigators with outstanding environmental health sciences (EHS) capabilities, develops the capacity of EHS researchers through career development opportunities, facilitates interdisciplinary EHS activities, and promotes cutting-edge research opportunities and the translation of findings to improve medical and public health interventions for the unique Southwest populations and environment. Likewise, the AC facilitates statewide, national, and international SWESHC initiatives, coordinates community outreach to native and indigenous populations, and ensures the integration of the Facility Cores (FCs) and research focus groups. Specifically, the SWEHSC AC supports the Community Engagement Core (CEC) in public outreach efforts to Native American tribes and the unique populations in the Southwest, and ensure that the FCs provide the innovative technologies needed by Center members in an efficient and cost effective manner that fully integrates all of the Center resources. The ultimate goals of the SWEHSC AC are to grow the number of investigators (particularly new investigators) studying environmental health issues, support new and emerging research initiatives that meet the vision and mission of the Center, and to increase the Center's research grant base.

The Southwest Environmental Health Sciences Center (SWEHSC) Pilot Projects program facilitates the implementation of the SWEHSC strategic vision and goal of testing the most innovative ideas relevant to environmental health by fostering opportunities to fill important gaps in the SWEHSC research portfolio. To date, the Pilot Project Program has enabled the successful creation of several scientifically productive collaborations that have emphasized the strengths of the SWEHSC in the conduct of translational studies and aided in securing significant extramural research funds for SWEHSC investigators. To support the SWEHSC strategic vision, the Pilot Projects program provides short-term support for innovative projects that explore new areas of environmental health research or that aim to acquire the preliminary data necessary to pursue long-term extramural funding. As well, the Pilot Projects program supports the Career Development of new investigators and highly successful senior investigators wishing to redirect their research to encompass environmental health.

PROJECT SUMMARY The interrelation between liver disease and kidney function is becoming increasingly researched, as hepatic- derived systemic inflammation can have a profound effect on the physiology of the kidney. This is a vital factor to consider with respect to precision medicine, as these changes can disrupt the proper metabolism and elimination of the 32% of marketed therapeutics that rely upon renal function for excretion. Among the liver diseases that affect renal physiology is nonalcoholic fatty liver disease (NAFLD), characterized by a series of mechanistic events that mediate the transition from simple steatosis to nonalcoholic steatohepatitis (NASH), which include inflammatory events. Our lab has identified NASH-induced phenotypic conversions of several drug metabolizing enzyme and transport proteins that significantly alter the pharmacokinetic profiles of certain drugs and xenobiotics. Interestingly, in a profiling study of various rodent models of NASH, we have also identified NASH-induced phenoconversion of renal transport proteins, a phenomenon that also contributes to altered pharmacokinetics of xenobiotic substrates in vivo. To date, no studies have been published examining human NASH-related phenoconversion of renal drug transporters, and that will be the first item we address in this application. Our central hypothesis is that NASH alters the expression and function of major renal drug transporters, thereby increasing the risk of adverse drug reactions and environmental toxicities in patients with NASH. Our study design seeks not only to identify phenoconversion of specific renal transporters in NASH, but also to pinpoint the associated secretory pathways to better narrow down mechanisms of altered pharmacokinetics for certain therapeutics and environmental contaminants. Our aims are to: 1) Determine the changes in expression and localization of renal transporters in human NASH patients, 2) Determine the functional changes in individual secretion pathways and resulting potential for environmental toxicity in a rodent model of NASH, and 3) Determine the impact of NASH on GFR and select secretion pathways in human patients. By completing these aims, we will be able to identify classes of drugs that present a greater risk of adverse drug events for NASH patients.

Environmental Health Sciences at the University of Arizona has a long-standing reputation for excellence in training Ph.D. and postdoctoral scientists, as evidenced by the fact that many of our graduates are leaders in academia, industry, and government. To this end, our graduate program has evolved from a systems-based toxicology experience to a curriculum in which students are trained to apply state-of-the art techniques to solve mechanisms of environmental toxicity affecting human diseases in various organ systems. The cutting-edge research programs of 30 Training Grant Faculty members are augmented by innovative technologies developed at the University of Arizona in association with the Southwest Environmental Health Sciences and BIO5 Centers. Additionally, translational approaches undertaken by our NIEHS Superfund Program and Dean Carter Binational Center for Environmental Sciences and Toxicology provide an exceptionally stimulating environment for the training of graduate students and postdoctoral fellows. The interactive research of our Training Grant Faculty and our state-of-the-art Facility Cores extend the training environment from a single laboratory-oriented domain to a multidisciplinary experience strongly supportive of collaborative research. Current trainees are now selected through a University-wide competition and the UA Graduate College provides financial support for all first year Ph.D. students through an umbrella recruitment program, providing a large pool of highly qualified candidates for competitive selection of predoctoral trainees. Predoctoral training is achieved through a combination of coursework, laboratory research, and supplemental enrichment activities. Postdoctoral trainees participate in innovative research programs and are guided to develop professional skills in oral and written communication and leadership. Over the past five years, our curricular changes have paralleled the evolving expertise of the Training Grant Faculty. We have recruited eight senior full Professors, three Associate Professors, and seven junior Assistant Professors into the Training Grant, which significantly enhanced our core strengths in mechanistic-based molecular toxicology training. The request for continuation of NIEHS support is justified by the highly successful nature of our program, the clear demand for our graduates, the strong emphasis we place on leadership skills for our trainees and postdoctoral fellows, the increasing number of students interested in toxicology and environmental health, substantial institutional commitment, the strong and well- funded research programs of our faculty, and the excellence of the training environment.