Core--Biomarker

Ian A Blair, A.n. Richards Prof. Of Pharmacology
University Of Pennsylvania
3451 Walnut Street
philadelphia, Pa 19104

Grant 5P50HL083799-039001 from National Heart, Lung, And Blood Institute, IRG: ZHL1

Abstract: The Core will provide proteomics and metabolomics services to the projects in the SCCOR. Methodology for proteomics analysis will be based on 2D-differential gel electrophoresis (DIGE) coupled with in-gel digestion and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF), MALDI/TOF/TOF, or liquid chromatography/tandem mass spectrometry (LC/MS/MS). Protein spots will be imaged using differential in gel electrophoresis methodology so that proteins can be quantified. All spots will be excised from the gel but identification of differentially expressed spots will be the major goal of analyses that are performed. The other spots will be stored at -70 degrees C in case other targets are discovered during subsequent research. The proteins present in gel cores will be subjected to in-gel digestion with subsequent extraction of the peptide fragments for analysis by MS. In-gel digestion will normally be conducted with trypsin. Samples will be de-salted prior to MS analyses using commercially available micro columns. The samples will then be spotted on MALDI-TOF targets or injected directly on an LC/MS system. MALDI/MS will be performed to obtain peptide maps for protein characterization. MALDI/MS/MS and LC/MS/MS will be performed for structural confirmation. The Core will also provide methodology for serum and cellular proteomics to conduct multidimensional LC/MS and LC/MS/MS of protease digests. Stable isotope dilution methodology that has been developed at the University of Pennsylvania will be used for high specificity protein quantitation. The Core will also provide diverse analytical services based primarily on liquid chromatography/tandem mass spectrometry (LC/MS/MS) methodology to quantify metabolomic biomarkers. These assays, which are based upon the use of stable isotope dilution methodology have been developed over the last several years in the Blair laboratory and are now available as routine assays to the investigators within the program. Assays that will be available include etheno-DNA-adducts, hepta-etheno-DNA-adducts, oxidized-DNA-adducts, glutathione adducts, chiral bioactive lipids, and protein adducts. The Core will also provide methodology for the analysis of nitric oxide, develop assays for drugs used in the SCCOR (as required), and maintain a laboratory information management system

Keywords: biomarker, biomedical facility, cardiovascular injury, chemical structure function, lipid, lipid metabolism, metabolomics, proteomics information system, nitric oxide, protein quantitation /detection electrospray ionization mass spectrometry, gas chromatography mass spectrometry, liquid chromatography mass spectrometry, mass spectrometry, two dimensional gel electrophoresis

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Dosimetry Of Lipid-Derived Amino Acid Adducts By LC/MS

Ian A Blair, A.n. Richards Prof. Of Pharmacology
University Of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104

Grant 5R01CA095586-05 from National Cancer Institute, IRG: ZRG1

Abstract: We recently identified the genotoxic bifunctional electrophiles, 4,5-epoxy-2-decenal, 4-oxo-2-nonenal, and 4- hydroxy-2-nonenal together with the potential genotoxin 4- hydroperoxy-2-nonenal as major products of vitamin C-mediated decomposition of lipid hydroperoxides. Surprisingly, vitamin C was more than twice as efficient as transition metal ions at inducing this decomposition. It has long been thought that lipid hydroperoxides play a role in degenerative diseases of aging such as cancer and cardiovascular disease. We have now made the exciting observation that 4-oxo-2-nonenal efficiently forms adducts with the amino acid arginine. Previous studies have demonstrated 4-hydroxy-2-nonenal can induce covalent modifications with model amino acids. However, the efficiency of this process is much lower than we observed with 4-oxo-2-nonenal The ability to quantify covalent modifications to plasma amino acids, plasma proteins, and arginine-rich histones will provide a dosimeter of exposure to lipid hydroperoxide-derived genotoxins that would complement data obtained with DNA-adducts. Furthermore, quantitation of covalent modifications to arginine and arginine-rich histones could provide additional insight into cancer risk. Plasma arginine is the precursor for the generation a nitric oxide a known mediator of tumorigenesis. Lipid hydroperoxide-mediated covalent modification of free plasma arginine would prevent deiminase-mediated conversion to citrulline and nitric oxide. This would limit endogenous substrate availability and could also act as an endogenous inhibitor. Endogenous inhibitors have been proposed but none have not been detected in sufficiently large amounts to account for the L-arginine paradox. This paradox arises from the observation that infusions of L-arginine can cause additional nitric oxides release even though there is apparently sufficient free intracellular arginine to saturate endogenous nitric oxide synthases. Covalent modifications to histone arginines would also affect transcription and cellular proliferation. We propose to focus initially on quantifying the covalent modifications that are induced by the interaction of lipid hydroperoxide-derived bifunctional electrophiles with free arginine. Studies will then be conducted with hemoglobin, albumin, and histones in vitro. Structural analysis will be performed using conventional protease digests coupled with liquid chromatography and tandem mass spectrometry. If necessary, specific covalent modifications will be characterized by chemical synthesis. The relative importance of these lesions will then be assessed when the bifunctional electrophiles are generated by vitamin C-mediated homolytic decomposition of lipid hydroperoxides. Such studies have been performed in the past using transition metal ion-mediated decomposition of lipid hydroperoxides. Unfortunately, transition metal ions also catalyze Huber-Weiss reactions, which results in the formation of reactive oxygen species. The use of transition metal ion-free buffers and vitamin C overcomes this problem. Therefore, it will be possible for the first time to study lipid hydroperoxide-mediated protein damage without the complication of simultaneous oxidative damage. The changes in histone function resulting from vitamin C-induced lipid hydroperoxide decomposition will be assessed in relevant in vitro models. Finally, free arginine, albumin and hemoglobin will be isolated from plasma of patients with leukemia. Lipid hydroperoxide- derived covalent modifications in arginine and proteins from these populations will then be quantified by stable isotope dilution liquid chromatography/tandem mass spectrometry and compared with arginine and proteins from normal subjects. These studies will serve as a precursor to future dosimetry studies in large populations and will provide insight into the role of lipid peroxidation as a mediator of carcinogenesis.

Keywords: adduct, aminoacid, intermolecular interaction, lipid peroxide, posttranslational modification, radiation dosage, DNA damage, arginine, ascorbate, carcinogenesis, covalent bond, hemoglobin, histone, leukemia, molecular oncology, oxidative stress, plasma, serum albumin, children, clinical research, electrospray ionization mass spectrometry, human tissue, liquid chromatography, liquid chromatography mass spectrometry, nutrition aspect of cancer, nutrition related tag

Project start date: 2002-04-01

Project end date: 2008-03-31

5R01CA095586-05 (2006): $275500

5R01CA095586-04 (2005): $282130

5R01CA095586-03 (2004): $282130

1R01CA095586-01 (2002): $282130

Training Program In Cancer Pharmacology

Ian A Blair, A.n. Richards Prof. Of Pharmacology
Pharmacologyuniversity Of Pennsylvania
3451 Walnut Street
philadelphia, Pa 19104

Grant 5R25CA101871-05 from National Cancer Institute, IRG: NCI

Abstract: Training Grant in Cancer Pharmacology. The purpose of the multidisciplinary Cancer Pharmacology training program is to provide predoctoral and postdoctoral students with a training environment that does not currently exist at the University of Pennsylvania. Drs. Blair (Program Director), Penning (Program Co-Director) and Assoian (Program Co-Director) have provided the sustained leadership required to develop a multidisciplinary program in molecular mechanisms of multi-stage carcinogenesis. There is now a real need for similar sustained leadership to be applied to the recruitment and training of both predoctoral and postdoctoral scientists in the multidisciplinary environment required for the field of cancer pharmacology. Major advances in the treatment of cancer patients in the next decade will result from multidisciplinary approaches in understanding of how malignant cells work at the molecular level and in designing novel therapeutic agents to disrupt these processes. Therefore, the proposed training program will help fill the current deficit of individuals qualified to develop the next generation of chemotherapeutic agents. The ultimate goal of the program is to provide training in cancer pharmacology that goes from laboratory to bedside and back again. Specifically, trainees will learn how cancer pharmacology can be used to identify new targets, how small molecules are synthesized and tested against these targets, how they are used in Phase I trials, how Phase I studies are developed into full clinical trials, and how epidemiology and pharmacogenetics are utilized to assess efficacy and lead to the discovery of new targets. Trainees will also receive specific training in how to project quantitative measures of drug effect from proof of concept in model systems into the rational selection of dosing in humans. These goals will be accomplished through the courses that are offered and the multidisciplinary research experiences that will be available from the faculty. The 26 faculty mentors with diverse complementary expertise are grouped into three programs A. Pharmacology Discovery and Development. B. Cell and Molecular Cancer Pharmacology. C. Clinical Cancer Pharmacology. The trainees will have research mentors from two of these programs. The mentors on this proposed program have a very strong record of accomplishment as cancer researchers. They are Principal Investigators or Project Leaders on 68 NIH grants of which 39 (57%) are from NCI (27 RO-1s, 8 PO-1 Projects, 1 PO-1 Scientific Core, 1 P30 Scientific Core, 1 UO-1, 1 T-32). Apart from training grants, 11 of the mentors have NIH-funded (primarily through NCI) collaborative research grants. The mentors serve on Editorial Boards of 23 Journals and two of them are Journal Editors. Over the past 10-years, the mentors have trained 105 predoctoral students and 250 postdoctoral fellows; they currently have 52 predoctoral and 76 postdoctoral trainees in their laboratories. The mentors have clearly demonstrated that they have the scientific expertise and training experience to develop a new multidisciplinary training program in Cancer Pharmacology. Mentors have superb access to different patient populations through their association with 11 NCI-funded clinical programs. Furthermore, one of the mentors is Director of the General Clinical Research Center at the University of Pennsylvania (Dr. FitzGerald) and one is acting Director of the General Clinical Research Center at Children´s Hospital of Philadelphia (Dr. Adamson). Both of these mentors have research programs with a focus on translational therapeutics and so they provide another outstanding training resource

Keywords: drug discovery /isolation, drug screening /evaluation, education evaluation /planning, health science manpower, health science research, neoplasm /cancer chemotherapy, neoplasm /cancer pharmacology, postgraduate education career, curriculum, experimental design, health science profession

Project start date: 2003-07-24

Project end date: 2009-06-30

5R25CA101871-05 (2007): $487787

5R25CA101871-04 (2006): $495274

5R25CA101871-03 (2005): $520264

5R25CA101871-02 (2004): $370668

1R25CA101871-01 (2003): $210587

CORE - HUMORAL AND CELLULAR MEDIATORS OF LUNG INJURY

Ian A Blair
Vanderbilt University Medical Center Nashville, Tn 372036869

Grant 5P50HL019153-139004 from National Heart, Lung, And Blood Institute, IRG:

Abstract: This project includes the quantitative analysis of eicosanoids and "platelet activating factor" (PAF) from in vitro and in vivo experimental preparations. The accurate purification and quantification of PAF and eicosanoids released in vivo into sheep lung lymph and bronchoalveolar lavage fluid following exposure of animals to endotoxin, hyperoxia or thoracic radiation are necessary to Section III, Projects A, B and C. Included within this project are the development of highly sophisticated analytic techniques which will allow us to accurately quantify eicosanoids, PAF and new mediators as they are identified. These studies will help to define the role of lipid mediators in a variety of lung injuries and help to formulate logical and efficacious methods for the future study and therapy of diffuse lung injury.

Keywords: BIOMEDICAL FACILITIES, FATTY ACIDS, EICOSANOIDS, RESPIRATORY DISORDERS, RESPIRATORY INSUFFICIENCY-FAILURE, ADULT RESPIRATORY DISTRESS SYNDROME, BLOOD PLATELETS, PLATELET ACTIVATING FACTOR, FATTY ACIDS, EICOSANOIDS, LEUKOTRIENES, ANIMALS, CHORDATES, MAMMALS, UNGULATES, SHEEP, CHEMISTRY, ANALYTICAL METHODS, SPECTROMETRY, MASS, CHEMISTRY, ANALYTICAL METHODS, SPECTROMETRY, MASS FRAGMENTOMETRY, ISOTOPE LABELED, STABLE ISOTOPES, PHYSICAL SEPARATION, CHROMATOGRAPHY, HIGH PERFORMANCE LIQUID CHROMATOGRAPHY, PHYSICAL SEPARATION, CHROMATOGRAPHY, THIN LAYER

Molecular Dosimetry Of Endogenous Genotoxins

Ian A Blair, A.n. Richards Prof. Of Pharmacology
University Of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104

Grant 5R01CA091016-05 from National Cancer Institute, IRG: ZRG1

Abstract: The role that endogenously produced chemicals may play in the etiology of cancer has become increasingly important over the last decade. This is largely due to epidemiological data, which show that apart from tobacco smoke ad sunlight, exposure to genotoxic environmental carcinogens that are derived from lipid hydroperoxides. The formation of lipid hydroperoxides is a complex process, which involves a number of different racial intermediates. However lipid hydroperoxides are also readily formed as a consequence of LOX-mediated oxidation of endogenous polyunsaturated acids (PUFAs). Therefore, there are both enzymatic and non-enzymatic pathways by which lipid hydroperoxides can be formed. In view of their potential role as genotoxins, it is important to understand how structural modifications to DNA can occur from reactions with lipid hydroperoxides. Their genotoxic properties are thought to result from generation of bifunctional electrophiles such as malondialdehyde and 4- hydroxy-2-nonenal. These bifunctional electrophiles then react at electron rich sites of the DNA bases to form DNA-adducts. Evidence has been obtained for the presence of both malondialdehyde- and 4-hydroxy- nonenal-derived DNA-adducts in vivo. Transition metal ions are known to enhance the formation of bifunctional electrophiles through a homolytic process. The presence of transition metal cations bound to the polar sugar residues of DNA can potentially enhance the breakdown of lipid hydroperoxides to genotoxic bifunctional electrophiles. We have recently studied the decomposition of 13-hydroperoxylinoleic acid (a prototypic w- 6 PUFA hydroperoxide) in the presence of the DNA bases. From the structures of the resulting DNA-adducts, we proposed that the covalent modifications arose through the generation of 4-oxo-2-nonenal from 13- HPODE. We have also demonstrated that the same adducts were formed when the DNA-bases were treated with synthetic 4-oxo-2-nonenal. This lead us to speculate that 4-oxo-2-nonenal was the major breakdown product of lipid hydroperoxides rather than 4-hydroxy-2-nonenal. We have obtained definitive evidence that 4-oxo-2-nonenal is indeed a major breakdown product of lipid hydroperoxides. In addition, we have documented the unexpected formation of 4-hydroperoxy-2-nonenal as an additional lipid peroxidation breakdown product. We now propose to elucidate the mechanisms by which these bifunctional electophiles are formed and to define the relative importance of their formation as a consequence of lipid peroxidation. We plan to develop highly sensitive quantitative methodology for the analysis of these adducts in vivo using a novel mass spectrometry technique that we have discovered recently. We plan to examine the relative importance of DNA-adduct and RNA-adduct formation in a model of oxidative stress.

Keywords: breast neoplasm, chemical carcinogen, chemical carcinogenesis, lipid peroxide, mutagen, DNA damage, adduct, biomarker, neoplasm /cancer genetics, oxidative stress, MCF7 cell, mass spectrometry, urinalysis

Project start date: 2001-05-18

Project end date: 2006-08-31

5R01CA091016-05 (2005): $263110

5R01CA091016-04 (2004): $263110

1R01CA091016-01 (2001): $263110

CORE--ANALYTICAL LABORATORY

Ian A Blair, A.n. Richards Prof. Of Pharmacology
Vanderbilt University Medical Center Nashville, Tn 372036869

Grant 2P50DK039261-119002 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG:

Abstract: The purpose of this Core is to provide support to Center investigators for the structural and quantitative analysis of cyclooxygenase metabolites. The Core will provide state-of-the-art sensitive and specific assay methodology based on capillary column gas chromatography/electron capture negative ion chemical ionization mass spectrometry for quantitative analyses. Structural analyses will be carried out on one of the nine mass spectrometers that are available in the Core. Confirmation of structures will involve the use of NMR spectroscopy (if possible) and total synthesis of novel metabolites. In addition to using existing methods we propose to develop new methods for eicosanoid analysis based on capillary HPLC in combination with electrospray mass spectrometry. It is anticipated that this will provide a new powerful way to carry out quantitative analyses. In addition, we propose to explore the use of a new four-sector tandem mass spectrometer for carrying out the structural elucidation of novel eicosanoids. Of particular interest will be the coupling of high resolution electrospray ionization with the high energy collision regime that is available on the four sector instrument. Samples will be introduced into the electrospray source using an ABI-based capillary HPLC system that was recently constructed in our laboratory. The instrumentation used in these studies will also be available to individual investigators should they want to carry out their own analyses. Investigators will be trained in the operation of instrumentation by the mass spectrometry engineer. Advice on the use of appropriate ionization techniques and column selection will be provided by personnel in the Core. The Core will also provide a resource for the synthesis of eicosanoid metabolites and stable isotope standards for use in stable isotope dilution assays.

Keywords: analytical chemistry, biomedical facility, eicosanoid, electrospray ionization mass spectrometry, gas chromatography, gas chromatography mass spectrometry, high performance liquid chromatography, mass spectrometry

Project start date: 1997-08-01

Project end date: 1998-07-31

CORE--ANALYTICAL FACILITY

Ian A Blair
Institution:

Grant 5P01GM031304-149001 from National Institute Of General Medical Sciences, IRG:

Abstract: The purpose of the Analytical Core is to provide support to the various Projects for the structural and quantitative analysis of drugs and metabolites. The Core will provide sensitive and specific assay methodology based on chiral HPLC, GC/MS and LC/MS. Structural analyses will be carried out on one of the nine mass spectrometers that will be available in the Core. Confirmation of structures will involve the use of NMR spectroscopy and total synthesis of novel metabolites. In addition to using existing methods it is proposed to develop new methods for drug and metabolite analysis based on capillary HPLC in combination with electrospray mass spectrometry. It is anticipated that this will also provide a new powerful way to carry out quantitative analyses. We also propose to explore the use of a new four-sector tandem mass spectrometer for carrying out the structural elucidation of metabolites. This instrument will be fitted with a photodiode array detector and so will be extremely sensitive. Of particular interest will be the coupling of high resolution electrospray ionization with the high energy collision regime that is available on the four sector instrument. Samples will be introduced into the electrospray source using an ABI-based capillary HPLC system that was recently constructed in our laboratory. The Analytical Core will also provide a resource for the synthesis of drug enantiomers, their metabolites, and stable isotope standards for use in stable isotope dilution assays.

Keywords: analytical chemistry, biomedical facility, drug metabolism, mass spectrometry, chemical synthesis, electrospray ionization mass spectrometry, gas chromatography, gas chromatography mass spectrometry, high performance liquid chromatography, radioassay

CORE--ANALYTICAL CORE

Ian A Blair
Vanderbilt University Medical Center Nashville, Tn 372036869

Grant 5P01GM031304-129001 from National Institute Of General Medical Sciences, IRG:

Abstract: The purpose of the Analytical Core is to provide support to the various Projects for the structural and quantitative analysis of drugs and metabolites. The Core will provide sensitive and specific assay methodology based on chiral HPLC, GC/MS and LC/MS. Structural analyses will be carried out on one of the nine mass spectrometers that will be available in the Core. Confirmation of structures will involve the use of NMR spectroscopy and total synthesis of novel metabolites. In addition to using existing methods it is proposed to develop new methods for drug and metabolite analysis based on capillary HPLC in combination with electrospray mass spectrometry. It is anticipated that this will also provide a new powerful way to carry out quantitative analyses. We also propose to explore the use of a new four-sector tandem mass spectrometer for carrying out the structural elucidation of metabolites. This instrument will be fitted with a photodiode array detector and so will be extremely sensitive. Of particular interest will be the coupling of high resolution electrospray ionization with the high energy collision regime that is available on the four sector instrument. Samples will be introduced into the electrospray source using an ABI-based capillary HPLC system that was recently constructed in our laboratory. The Analytical Core will also provide a resource for the synthesis of drug enantiomers, their metabolites, and stable isotope standards for use in stable isotope dilution assays.

Keywords: analytical chemistry, biomedical facility, drug metabolism, mass spectrometry, chemical synthesis, electrospray ionization mass spectrometry, gas chromatography, gas chromatography mass spectrometry, high performance liquid chromatography, radioassay

CORE--CANCER PHARMACOLOGY BIOANALYTICAL FACILITY

Ian A Blair, A.n. Richards Prof. Of Pharmacology
University Of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104

Grant 5P30CA016520-279030 from National Cancer Institute, IRG: NCI

Abstract: The proposed Cancer Pharmacology Bioanalytical Facility will provide expertise in analytical pharmacology to University of Pennsylvania Cancer Center members. Directing the Cancer Pharmacology Bioanalytical Facility will be Ian Blair, PhD. Dr. Blair is Professor of Pharmacology and has extensive expertise in the supervision of bioanalytical facilities. He planned and developed the well-recognized Cancer Center Bioanalytical Facility at Vanderbilt University. Most of the important modern mas spectral ionization techniques have already been acquired and a critical mass of expert technical personnel has been assembled using School of Medicine, department and Cancer Center funds. The state-of-the-art instrumentation, coupled with the expertise of the Facility Director and staff, will create an outstanding environment in which to conduct bioanalytical, pharmacokinetic and pharmacological studies when new therapeutic entities are used in human subjects. This core is an essential resource for studies. The Cancer Pharmacology, Bioanalytical Facility will immediately be used by a number of Cancer Center investigators, particularly those in the Breast Cancer, Chemoprevention, Neuro-Oncology, Melanoma and Pediatric Oncology Research Programs. In addition, the Cancer Pharmacology Bioanalytical Facility will act as a resource for cancer-related drug disposition studies that will be conducted in the future by the new Pediatric Pharmacology Research Unit at the Children s Hospital of Philadelphia (Lange, PI, NCI U01). Usage by Cancer Center members with peer reviewed funding or trials approved by the Clinical Trials Scientific Review and Monitoring Committee is expected to be 50% of overall usage, and total Cancer Center member usage is expected to be 70% of overall usage.

Keywords: analytical method, antineoplastic, biomedical facility, biomedical resource, neoplasm /cancer pharmacology, liquid chromatography mass spectrometry

Core--Proteomics

Ian A Blair, A.n. Richards Prof. Of Pharmacology
University Of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104

Grant 1P50HL070128-019002 from National Heart, Lung, And Blood Institute, IRG: ZHL1

Abstract: The Core will serve all projects in this Program. The use of 2D gels and mass spectrometry (MS) represents the most efficient way to perform proteomic analyses. It is now possible to rapidly characterize 1,000-3,000 of the cellular proteins and to compare protein expression after specific manipulations of the cells. The Core will provide methodology for proteomics analysis based on 2-D gel electrophoresis coupled with in-gel digestion and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF) or liquid chromatography/tandem mass spectrometry (LC/MS/MS). Protein spots will be imaged and stored so that they can be compared with a single standard image. All spots will be excised from the gel but identification of differentially expressed spots will be the major goal of analyses that are performed. The other spots will be stored at -70 degree C in case other targets are discovered during subsequent research. The proteins present in gel cores will be subjected to in-gel digestion with subsequent extraction of the peptide fragments for analysis by MS. In-gel digestion will normally be conducted with trypsin using 96-wellplates. Samples will be de-salted prior to MS analyses using commercially available micro columns. The samples will then be spotted on MALDI-TOF targets or injected directly on an LC/MS system. In a typical proteome analysis, MALDI-TOF/MS will be conducted on peptide digests of the excised gel spots to rapidly obtain partial sequences. Mass measurements will be conducted with accuracy in the 5-10 ppm range by using internal calibration of protease fragments present in the incubation. This method will be particularly useful for proteins that appear in databases as it is possible to identify a protein with as few as 3-5 peptide fragments. For unknown peptides, this approach has only limited applicability. It will be necessary to use LC/MS/MS for the full structural characterization of unknown proteins. MS/MS analyses will also be conducted when necessary using partially purified peptide fragments in combination with nanospray infusions. Sequencing of unknown peptides will be performed using computer programs such as Sequest, MASCOT, peptidesearch, PROWL, and protein. The Sequest system works particularly well with marginal MS/MS data but does not allow searching on the internet. MASCOT will therefore serve as an ideal adjunct for web searching. This Core is an absolutely vital component of the PPG.

Keywords: biomedical facility, proteomics, computer assisted sequence analysis, gel electrophoresis, liquid chromatography mass spectrometry, matrix assisted laser desorption ionization

Project start date: 2002-05-01

Project end date: 2007-03-31

TRANSCELLULAR METABOLISM OF CYTOCHROME P-450 METABOLITES OF ARACHIDONIC ACID

Ian A Blair, A.n. Richards Prof. Of Pharmacology
Institution:

Grant 5P01DK038226-110008 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG:

Abstract: The biological activities of P-450 products of arachidonic acid metabolism are often mediated by the cyclooxygenase enzyme. This could occur through at least two separate mechanisms. The P-450 products could release biologically active cyclooxygenase products (perhaps through a receptor mediated process) and these could then be responsible for the observed activity. Alternatively, the P-450 metabolites themselves could undergo cyclooxygenase-mediated transcellular metabolism to provide compounds with different biological activities. There is evidence from our own laboratory and from several other laboratories that this latter mechanism may be the most dominant for both epoxygenase and omega/omega-1 oxidation products of arachidonic acid metabolism. There is evidence that metabolism of some of the P-450 metabolites can also be carried out by lipoxygenase enzymes. The major goals of Project 3 are to further refine our understanding of transcellular metabolism of P-450 arachidonic acid metabolites by cyclooxygenase and lipoxygenase, to determine whether glucuronidation of the metabolites in normal physiological processes and to ascertain whether they are involved in pathophysiological processes. Structural characterization of urinary metabolites will be carried out primarily by gas chromatography/electron ionization mass spectrometry. Quantitative studies will involve the development of analytical methodology based on stable isotope dilution gas chromatography/electron capture negative ion chemical ionization mass spectrometry. The transcellular metabolites will be analyzed in urine from animal models and from human subjects to provide a measure of the renal biosynthesis of these compounds. the contribution of these metabolites to the overall eicosanoid excretion rate will then be assessed. In vitro model systems to examine the generation of transcellular metabolites will be developed and these model systems will be used to delineate the mechanism by which the individual transcellular metabolites are formed. We will also focus on possible participation of transcellular metabolites in signal transduction mechanisms. In particular, the possibility that some of the transcellular metabolites are incorporated into specific phospholipid pools will be explored. This could lead to the generation of diacylglycerols with enhanced activity in phosphoinositide signalling pathways.

Keywords: arachidonate, cytochrome P450, eicosanoid metabolism, kidney function, lipoxygenase, prostaglandin synthase, biological signal transduction, chemical structure function, glucuronide, hypertension, lipid biosynthesis, microsome, phospholipid, gas chromatography mass spectrometry, human tissue, laboratory rat, tissue /cell culture

Core--Bioanalytical

Ian A Blair
University Of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104

Grant 1P01CA092537-01A19001 from National Cancer Institute, IRG: NCI

Abstract: The Bioanalytical Core will serve all projects in this Program. It will consist of an Analytical component (directed by Dr. Blair-Center for Cancer Pharmacology, University of Pennsylvania), and a Synthetic component (directed by Dr. Harvey-The Ben May Institute, University of Chicago). The major roles of the Analytical component are 1) to assess the purity of PAHmetabolites used by all Project Investigators (Projects 1-3), and this is pertinent to the use of anti- and syn-diol epoxides which hydrolyze to tetraols; 2) to quantitate PAH-metabolites formed in cell culture experiments, once structural validation of the analyte has been achieved; 3) to quantitate concentrations of PAH-o-quinones used in cell culture experiments, once structural validation of the analyte has been achieved; 4) to quantitate covalent modifications to DNA by PAH-metabolites and reactive oxygen species in Project 1 once methods development and validation has been achieved in Project 2; and 5) with support from the Administrative Core A to act as a liaison between the NCI Chemical Carcinogen Reference Standard Repository and Project Investigators to maintain stocks of individual PAH-metabolites and PAH-synthons. The analytical component will be reliant on a PAH-Database and a Program Database maintained in the Administrative Core-A these will monitor stocks of PAH-metabolites and perform statistical analysis of PAH-DNA adducts. The Analytical component of Core B contains a triple-quadruple mass spectrometer and an ion-trap mass spectrometer. The Synthetic component of Core B will develop efficient synthetic strategies to 1) the major stable benzo[a]pyrene diol-epoxide (BPDE) adducts [(+) and (-)-anti-BPDE-N6-dAdo (trans-isomers); (+) and (-)-anti-BPDE-N6 dAdo (cis-isomers), (+) and (-) anti-BPDE-N2-dGuo (trans-isomers) (+)-and (-)-anti-BPDE-N2-dGuo (cisisomers)], major radical-cation BP depurination adducts [N7- and C8-guanine and N7-adenine adducts of BP], and the stable and depurinating adducts of BP-2 7,8-dione [N -dGuo, N6-dAdo, N7-guanine and N7-adenine]. These reference standards will be used in Projects 1 and 2 to identify unknowns; and 2) to develop synthetic strategies to the stable and depurinating adducts of BA-3,4- dione [N2-dGuo, N6-dAdo, N7-guanine and N7-adenine] and the stable and depurinating adducts of DMBA-3,4-dione [N2-dGuo, N6-dAdo, N7-guanine and N7- adenine] for their detection in cell culture paradigms in collaboration with Project 1. Via Core A, we will inform the Repository of the availability of novel PAH-DNA adducts for distribution to the PAH-research community. This will establish a symbiotic relationship between Core B and the Repository. Thus, Core B is a vital component to the success of all Projects of the PPG and may impact PAH-DNA adduct research in general.

Keywords: analytical chemistry, biomedical facility, carbopolycyclic compound, chemical synthesis, chemical information system, chemical registry /resource, chemical standardization, toxin metabolism

Project start date: 2002-08-30

Project end date: 2007-07-31

CORE--ANALYTICAL CORE

Ian A Blair, A.n. Richards Prof. Of Pharmacology
Vanderbilt University Medical Center Nashville, Tn 372036869

Grant 5P50GM015431-339001 from National Institute Of General Medical Sciences, IRG:

Abstract: The Analytical Core will provide support to Center investigators for the structural and quantitative analysis of eicosanoids. Assay methodology based on capillary column gas chromatography/electron capture negative ion chemical ionization mass spectrometry will be available for high sensitivity quantitative analyses. Lower sensitivity assays will be carried out using gas chromatography/electron ionization mass spectrometry. Structural analyses will be carried out using one of the nine mass spectrometers that are available in the Core. Ionization techniques that are available include, electron ionization, liquid secondary ion, electrospray ionization and desorption chemical ionization. We propose to develop new methods for the routine analysis of eicosanoids based on microbore LC in combination with electrospray and tandem mass spectrometry. Ultimately, it is anticipated that this methodology could replace gas chromatography/mass spectrometry as the method of choice for quantitative analyses of eicosanoids. In addition, we propose to explore the use of tandem mass spectrometry for carrying out the structural elucidation of novel eicosanoids. The single quadrupole instruments maintained by Core personnel will also be available to individual Center investigators should they want to carry out their own analyses. Investigators will be trained in instrument operation when necessary. Advice on the use of appropriate ionization techniques and column selection will be provided by personnel in the Core. The Core will also provide a resource for the synthesis of eicosanoid metabolites and stable isotope standards for use in stable isotope dilution assays. Structural confirmation of new metabolites will be carried out by chemical synthesis.

Keywords: analytical chemistry, analytical method, biomedical facility, chemical structure, chemical synthesis, eicosanoid, method development, eicosanoid metabolism, electrospray ionization mass spectrometry, gas chromatography mass spectrometry, high energy particle, high performance liquid chromatography, liquid chromatography, mass spectrometry, stable isotope

Project start date: 2000-07-01

Project end date: 2001-06-30

ANALYTICAL CORE

Ian A Blair
Vanderbilt University Medical Center Nashville, Tn 372036869

Grant 2P50GM015431-219001 from National Institute Of General Medical Sciences, IRG:

Abstract: The Analytical Core provides support to Center Investigators for the application of modern methods of synthesis and analysis to their projects. The Core also functions as a resource in the development of new analytical procedures, with particular emphasis on the use of mass spectrometry. Methods that are available for structural analysis include fast atom bombardment mass spectrometry, desorption chemical ionization, high resolution mass spectrometry and linked scanning techniques and high resolution gas chromatography mass spectrometry. Quantitative analysis of trace amounts of complex lipid-derived mediators can be carried out using highly sensitive methods based on stable isotope dilution negative ion chemical ionization mass spectrometry. The application of these diverse methodologies will greatly enhance the ability of the Center Investigators to carry out their proposed projects. It is envisaged that during the course of their proposed studies new exciting directions of research will emerge. The Analytical Core will provide expertise that will help to facilitate such possibilities. Furthermore, the presence of a well equipped synthetic laboratory will allow the synthesis of otherwise unavailable compounds to be carried out. The Analytical Core has in the past developed analytical methods, carried out structural studies and has enabled Center Investigators to access highly sophisticated instrumentation. In addition, it has provided Center Investigators with a number of otherwise inaccessible synthetic standards. It is envisaged that the Core will continue to function in these roles in the future.

Keywords: BIOMEDICAL FACILITIES, CHEMICAL SYNTHESIS, DESIGN AND PRODUCTION, CHEMISTRY, ANALYTICAL METHODS, FATTY ACIDS, EICOSANOIDS, LEUKOTRIENES, BIOMEDICAL ENGINEERING, INSTRUMENTATION NOT CLINICALLY ORIENTED, CHEMICAL STRUCTURE, ISOMERS, STEREOISOMERS, CHEMISTRY, ANALYTICAL METHODS, SPECTROMETRY, MASS, CHEMISTRY, ANALYTICAL METHODS, SPECTROMETRY, MASS FRAGMENTOMETRY, DIAGNOSTIC TESTS, DESIGN, DEVELOPMENT AND EVALUATION OF DIAGNOSTIC TESTS, DRUGS, PHARMACOLOGY, BIOCHEMICAL, FATTY ACIDS METABOLISM, FATTY ACIDS METABOLISM, EICOSANOIDS METABOLISM, FATTY ACIDS, EICOSANOIDS, ARACHIDONIC ACID, FATTY ACIDS, EICOSANOIDS, PROSTAGLANDINS, PHYSICAL SEPARATION, CHROMATOGRAPHY, HIGH PERFORMANCE LIQUID CHROMATOGRAPHY, ISOTOPE LABELED, STABLE ISOTOPES

Molecular Dosimetry Of Endogeneous Genotoxins

Ian A Blair, A.n. Richards Prof. Of Pharmacology
Pharmacologyuniversity Of Pennsylvania

Grant 5R01CA091016-08 from National Cancer Institute, IRG: EBT

Abstract: The analysis of DNA-adducts is important for molecular dosimetry studies, and for monitoring treatment options because they can provide insight into the amount of genotoxin that that has reached the DNA in the tissue under study. No specific biomarkers of endogenous lipid hydroperoxide-mediated DNA damage in vivo are available in spite of an intensive research effort spanning several decades. Our studies over the last six years have identified three hepta-etheno-DNA-adducts that can only arise from endogenously formed lipid hydroperoxides. We have now shown that hepta-etheno-2´-deoxyguanosine hepta-etheno-21- deoxy-cytidine adducts are formed in the DNA of rat intestinal epithelial cells that stably express COX-2 and that the adducts are present in mammalian tissue DNA. We have also discovered a new class of cyclic glutathione (GSH)-adducts derived from endogenous lipid hydroperoxides. Lipid hydroperoxides undergo homolytic decomposition to the highly reactive bifunctional electrophile 4-oxo-2(E)-nal (ONE). Lipid hydroperoxide-derived ONE is responsible for the formation of both hepta-etheno- adducts and the novel cyclic GSH-adducts. Our recent discovery that electron capture atmospheric chemical ionization mass spectrometry can be used to analyze chiral lipids with high sensitivity and specificity makes it possible to analyze the lipid hydroperoxide precursors of DNA- and glutathione- adducts. Therefore, we are poised to make some significant advances in understanding the role of oxidative stress in the etiology of cancer. We propose to address the following hypotheses 1. Endogenous GSH-adducts and their metabolites represent a new class of biomarkers that will complement isoprostanes as biomarkers of oxidative stress. 2. Endogenous DNA-, RNA-, and cyclic GSH-adducts adducts can arise from cyclooxygenase-mediated pathways. 3. Endogenous DNA-, RNA-, and cyclic GSH-adducts can arise from 5-lipoxygenase but not 15-lipoxygenase. 4. Hepta- etheno-DNA- and RNA-adducts, together with metabolites of endogenous cyclic GSH-adducts are dosimeters of colon cancer. The proposed research will be conducted under four specific aims. Aim 1. To examine the enzymatic formation and metabolism of endogenous lipid hydroperoxide-derived GSH- adducts. Aim 2. To compare ROS- and COX-2-mediated formation of endogenous DNA-, RNA-, and GSH-adducts with lipid hydroperoxide formation in cellular models. Aim 3. To compare ROS- and LOX- mediated formation of endogenous DNA-, RNA-, and GSH-adducts with lipid hydroperoxide formation in cellular models. Aim 4. To quantify endogenous lipid hydroperoxide-derived DNA-adducts, RNA- adducts, GSH-adducts as potential cancer biomarkers in animal models of colon cancer and in human colon tissue

Keywords: breast neoplasm, chemical carcinogen, chemical carcinogenesis, lipid peroxide, mutagen DNA damage, adduct, biomarker, neoplasm /cancer genetics, oxidative stress MCF7 cell, mass spectrometry, urinalysis

Project start date: 2001-05-18

Project end date: 2011-07-31