1985 |
Trudell, James Robert |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Human Metabolism of Halothane Mechanisms of Toxicity
There is growing concern about chronic exposure of operating room personnel and acute exposure of patients to inhalation anesthetics. Recent epidemiological and long-term rat testing data have implicated the inhalation anesthetics in a variety of toxic reactions including liver necrosis, cancer, teratogenicity, and bone marrow defects. Many anesthetics have structural similarities to known toxic compounds such as vinyl chloride and 1, 2-dichloro-2-bromopropane. We have chosen to study halothane as a representative of the halogenated hydrocarbon inhalation anesthetics because of our previous experience with its urinary, volatile, and cellular-bound metabolites. In our previous studies we have demonstrated that human metabolism of halothane is different from that in rats and monkeys. Therefore, we propose to study metabolism in humans directly. We have developed the methodology to purify human cytochromes P-450 to homegeneity and to reconstitute these cytochromes P-450 along with human cytochrome P-450 NADPH-reductase into a phospholipid vesicle system that is capable of carrying out metabolism like that in liver chromosomes. Because of the well-defined nature of this system, we will be able to define exact pathways of anesthetic metabolism as well as to differentiate the pathways dictated by the various cytochromes P-450 produced by several enzyme-inducing agents. We will use this human liver preparation to study production of volatile metabolites as well as binding of highly reactive intermediates to proteins and phospholipids of the liver cell. Studies in rats have associated production of toxic metabolites and liver necrosis with pretreatment by certain enzyme-inducing drugs followed by metabolism of anesthetics under anaerobic conditions. We will attempt to correlate toxicity in animals and humans with specific metabolic pathways occurring in the liver. A goal will be to define those conditions to which patients and operating room personnel should not be subjected in order to prevent significant toxic metabolism.
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0.958 |
1987 — 1990 |
Trudell, James Robert |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Human Metabolism of Halothane-Mechanisms of Toxicity
The long term goal of this grant is to understand the mechanism of toxicity of both acute hepatic necrosis and chronic sub-clinical impairment of liver function that result from anesthetic metabolism in humans. We will carry out experiments in two complementary experimental systems: Monolayers of hepatocytes in primary culture will allow study of the effects of metabolism on the integrated systems of the cell as well as attack of hepatocytes by macrophages activated by metabolically-produced chemo attractants or antigens. A synthetic model of a liver cell formed by reconstituting human cytochrome p-450 and NADPH cytochrome P-450 reductase into phospholipid vesicles will allow study of individual steps of anesthetic metabolism, formation of chemo-attractant leukotrienes, and antigenic metabolites on the cell surface. The following experiments with halothane will also contribute to the understanding of how other halogenated hydrocarbons cause liver disease and cancer. 1) We wil continue our studies of the toxicity of halothane metabolism in monolayers of hepatocytes under conditions that mimic those that exacerbate halothane toxicity in vivo, especially the effect of hypoxia. 2) We will use reconstituted human cytochromes P-450 to continue our studies of production of arachidonic acid hydroperoxides during metabolism of halothane and their conversion by specific isozymes of human cytochrome P-450 to the potent chemoattractant leukotriene B-4 (LTB-4) 3) We will measure production and release of LTB-4 from monolayers of hepatocytes in response to injury by halothane metabolism as a function of oxygen concentration. 4) We will isolate both leukocytes and the resident liver macrophages, Kupffer cells, and measure their lysis of hepatocytes as a consequence of activation by leukotrienes produced during halothane metabolism. 5) We will use reconstituted human cytochrome P-450 to study production of N-trifluoroacetyl-phosphatidylethanolamide, a known metabolite of halothane that we suggest may act as an antigen if exposed on the hepatocyte plasma membrane. We will prepare antibodies to this antigen. 6) We will allow monolayers of hepatocytes to metabolize halothane to produce the proposed antigen on the plasma membrane surface, add the specific antibodies, and measure immune complex mediated lysis of hepatocytes by leukocytes or Kupffer cells.
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0.958 |
1991 — 1993 |
Trudell, James Robert |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Human Metabolism of Halothane--Markers of Toxic Exposure
The long term goal of this grant is to understand the mechanism of toxicity of both acute hepatic necrosis and chronic sub-clinical impairment of liver function that result from anesthetic metabolism in humans. We will carry out experiments in two complementary experimental systems: Monolayers of hepatocytes in primary culture will allow study of the effects of metabolism on the integrated systems of the cell as well as attack of hepatocytes by macrophages activated by metabolically-produced chemo attractants or antigens. A synthetic model of a liver cell formed by reconstituting human cytochrome p-450 and NADPH cytochrome P-450 reductase into phospholipid vesicles will allow study of individual steps of anesthetic metabolism, formation of chemo-attractant leukotrienes, and antigenic metabolites on the cell surface. The following experiments with halothane will also contribute to the understanding of how other halogenated hydrocarbons cause liver disease and cancer. 1) We wil continue our studies of the toxicity of halothane metabolism in monolayers of hepatocytes under conditions that mimic those that exacerbate halothane toxicity in vivo, especially the effect of hypoxia. 2) We will use reconstituted human cytochromes P-450 to continue our studies of production of arachidonic acid hydroperoxides during metabolism of halothane and their conversion by specific isozymes of human cytochrome P-450 to the potent chemoattractant leukotriene B-4 (LTB-4) 3) We will measure production and release of LTB-4 from monolayers of hepatocytes in response to injury by halothane metabolism as a function of oxygen concentration. 4) We will isolate both leukocytes and the resident liver macrophages, Kupffer cells, and measure their lysis of hepatocytes as a consequence of activation by leukotrienes produced during halothane metabolism. 5) We will use reconstituted human cytochrome P-450 to study production of N-trifluoroacetyl-phosphatidylethanolamide, a known metabolite of halothane that we suggest may act as an antigen if exposed on the hepatocyte plasma membrane. We will prepare antibodies to this antigen. 6) We will allow monolayers of hepatocytes to metabolize halothane to produce the proposed antigen on the plasma membrane surface, add the specific antibodies, and measure immune complex mediated lysis of hepatocytes by leukocytes or Kupffer cells.
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0.958 |
1993 — 1994 |
Trudell, James Robert |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Antibody-Mediated Hepatotoxicity of Ethanol
The principle metabolite of ethanol, acetaldehyde, has been shown to react with amino groups on proteins and phospholipids to yield secondary N-ethylamine adducts. In the case of protein adducts formed during metabolism of ethanol, these amines have been shown to act as neoantigens and generate an immune response. Previous studies have shown a correlation between the presence in the serum of antibodies that bind to these adducts and alcohol-related hepatotoxicity. We have recently shown that some of the polyclonal IgG antibodies raised in rabbits against protein adducts of acetaldehyde cross-react with synthetic acetaldehyde-phosphatidylethanolamine adducts (N-ethyl-PE). If N-ethyl-PE adducts are formed in vivo during metabolism of alcohol, then binding of cross-reactive antibodies to these adducts exposed on the surface of hepatocytes could contribute to alcohol-related hepatotoxicity. The aims of this proposal are to determine if acetaldehyde adducts of phospholipids appear on the surface of alcohol-exposed hepatocytes and if binding of antibodies to them results in lysis of hepatocytes as a consequence of activation of neutrophils or complement. A sequence of steps is described that will determine whether acetaldehyde adducts of phospholipids are formed in vivo, measure the binding of antibodies to hepatocytes that have N-ethyl-phosphatidylethanolamine incorporated into their surface using flow cytometry, and measure the immune-mediated cytotoxicity that could result from binding of neutrophils or complement to antibodies on the hepatocyte surface. These studies may be significant in two ways: First, measurement of the appearance of acetaldehyde-phospholipid adducts may add an important additional component to existing assays for markers of alcohol exposure. Second, the additional haptenic epitopes provided by acetaldehyde-phospholipid adducts on the hepatocyte surface may be a contributing factor in binding of neutrophils or complement that results in immune-mediated hepatotoxicity.
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0.958 |
1999 — 2002 |
Trudell, James Robert |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Molecular Models of Inhaled Anesthetic Binding Site @ University of California San Francisco
binding sites; inhalation anesthesia; membrane channels; drug interactions; chemical models; model design /development; glycine receptors; site directed mutagenesis; membrane proteins; GABA receptor; nicotinic receptors; chloride channels;
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0.911 |
2002 — 2004 |
Trudell, James Robert |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Dimensions and Polarity of Anesthetic Binding Sites
DESCRIPTION (provided by applicant): Our long-term goal is to improve the design and administration of volatile anesthetics by learning the molecular mechanisms of anesthesia. Our short-term goal is to understand how volatile anesthetic potency is altered by site-directed mutations in the transmembrane domains of ligand-gated ion channels. Our hypothesis is that cavities within transmembrane domains provide a common motif for volatile anesthetic binding sites within the superfamily of GABA, glycine, nicotinic acetyicholine, and 5-NT receptors. We suggest that specific amino acid residues define the dimensions and polarity of these binding sites and thereby determine the relative efficacy of volatile anesthetics. This hypothesis will be tested in two Specific Aims: Aim 1. We will test the hypothesis that variations in the dimensions of cavities within transmembrane subunits determine the relative potency of anesthetics within the superfamily of GABA, glycine, and nicotinic acetyicholine receptors. Mutation of two critical amino acid residues in transmembrane segments of the glycine alpha 1 receptor (S267 and A288) modulates the potentiation of agonists by volatile anesthetics. The volume of these residues is the best predictor of anesthetic potency. We will build molecular models of the transmembrane domains of these subunits and predict additional residues that may define the dimensions of these putative cavities. Aim 2. We will test the hypothesis that variations in the polarity of cavities within transmembrane subunits determine the relative potency of volatile anesthetics. Although the volume of amino acid side-chains has a dominant effect, the distinct in vivo and in vitro pharmacology of pairs of anesthetic isomers demonstrate that the polarity and shape of binding sites is important. We will use molecular modeling to rationalize existing data and predict new site-directed mutations for study by our collaborators in an iterative series of experiments. In summary, our initial computational models with two transmembrane alpha helices have been of value in rationalizing and predicting the effect of site-directed mutations. Building a more complete 3-dimensional model of an anesthetic binding site will allow us to define those molecular properties that confer distinct pharmacologies on volatile anesthetics.
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0.958 |
2002 — 2009 |
Trudell, James Robert |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Properties of Specific Alcohol Binding Sites
DESCRIPTION (provided by applicant): Alcohol abuse and alcoholism are major health problems. It is likely that a solution to these problems will require an understanding of the effects of alcohol on specific ion channels and the kinases that modulate them. Specific binding sites for alcohols have recently been described in the transmembrane domain of the superfamily of glycine, GABA, nicotinic acetylcholine, and 5-HT3 receptors. Our hypothesis is that alcohols bind within cavities that are bounded by transmembrane segments of these receptors. Our goal is to define the properties of those sites that regulate binding and efficacy of alcohols. These binding sites may provide a common motif for binding of alcohols within other classes of ion channels. We will build computational models of binding sites and design specific site-directed mutations to test this hypothesis. These mutations will be expressed and tested by our collaborators, Drs. R. Adron Harris and S. John Mihic, in separately funded experiments. Specifically: Aim 1. We will define specific amino acid residues that determine the "cutoff" length of long-chain alcohols. We have previously shown that mutation of S267 in transmembrane segment 2 (TM2) and A288 in TM3 of the glycine alphal receptor can change the alcohol "cutoff' from heptanol to dodecanol. We will develop computational molecular models that allow us to suggest mutations that will determine additional residues in TM1 and TM4 that may also form "walls" of the putative binding cavities. We will refine our models by iterations in which we optimize the structure of an initial model, use it to predict mutations, test if the model is consistent with the resulting experimental data, and then modify the model in a way that would better fit the data. Aim 2. We will determine the structural requirements of alcohols for potentiation of agonist potency by providing models in which a series of alcohol analogs are covalently linked to site-directed cysteine mutations in the putative binding cavities. Since we will know that a single alcohol analog is bound to the putative site, we can distinguish binding from efficacy. Aim 3. We will define the proximity of amino acid residues important for alcohol potentiation of agonists by building models that predict double site-directed cysteine mutations that are appropriate for cross-linking. We will predict pairs of residues that could be linked by direct disulfide formation or with bi-functional methanethiosulfonate reagents with 1-5 carbon spacers. In summary, these computational studies will provide new knowledge about determinants of alcohol binding and efficacy.
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0.958 |
2013 — 2017 |
Trudell, James Robert |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Defining Alcohol Binding Sites in Ligand-Gated Ion Channels
DESCRIPTION (provided by applicant): Alcohol abuse and alcoholism are significant health problems that affect over 17 million people and cost nearly $200 billion annually. It is likely tha a solution to these problems will result from a better understanding of alcohol effects on neuronal ion channels and the proteins that modulate them. The goal of the proposed research will be a significant step towards understanding the properties of alcohol-binding sites at an atomic level. We believe this knowledge will be essential for future design and selection of drugs that could reduce craving or addiction induced by alcohol. Specifically, we will study the sites fo alcohol binding in ligand- gated ion channels (LGICs), which include GABAaRs and GlyRs. Our homology modeling and experimental methods will provide 3-dimensional visualization of GABAaRs to increase our understanding of alcohol's action. This innovative approach combines cutting-edge computational and neuroscience techniques with molecular biology. We expect our results will have a significant impact on the broader class of alcohol- binding sites in other important receptors of the nervous system. Our Approach focuses on three aspects of alcohol-binding sites via three Specific Aims: Where are alcohol-binding sites; intra-subunit versus inter- subunit in LGICs (Aim 1), which specific residues or segments in LGICs mediate the effect of alcohol binding at these sites (Aims 1 and 2), and how do these sites modulate ligand binding (Aims 2 and 3). In Aim 1, Trudell and Bertaccini will build computational models of alcohol-binding sites in GABA receptors and design site-directed mutations to test the models. Harris and Howard, under a subcontract to the University of Texas, Austin, will test the function of these mutated receptors. We will iteratively refine the models the Trudell group will use the models to predict the effects of mutations; the Harris group will test if the models are consistent with experimental data; and the Trudell group will then modify the models to fit the new data. They will address this controversial question: What is the most important alcohol effect site in GABAaR? Is it Intra-subunit or Inter-subunit? They will also test the hypothesis that the GABAaR TM3 helix must rotate during activation in order to incorporate all recent experimental data. In Aim 2, the Trudell and Harris laboratories will recreate the alcohol-binding site from GABAaRs in the homologous but natively EtOH insensitive ion channel, GLIC, by determining which residues are specific to EtOH binding in GABAR and mutating these into their corresponding homologous positions within GLIC. In Aim 3, Trudell and Bertaccini will use three docking programs to investigate binding of alcohol analogs. Our investigators have proven accomplishment in alcohol research and possess the resources necessary to accomplish our Aims. Our proposal is responsive to both the NIAAA initiative in computational neuroscience and the NIH Roadmap: Bioinformatics and Computational Biology. These significant studies will provide essential knowledge needed to design alcohol-binding antagonists which could revolutionize treatment for alcohol abuse and dependence.
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0.958 |