REGULATION OF XENOBIOTIC-METABOLIZING CYP3A

Richard C Zangar, Senior Research Scientist
Battelle Pacific Northwest Laboratories
box 999, 902 Battelle Blvd
richland, Wa 99352

Grant 5R01DK054812-03 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: ALTX

Abstract: Cytochrome P450 3A (CYP3A) levels are important determinants of aflatoxin and acetaminophen toxicity and the uptake and elimination of numerous therapeutic agents. CYP3A protein has been reported to be stabilized by some CYP3A substrates. The PI reported that several structurally and functionally distinct agents that are not known CYP3A substrates alter CYP3A protein stability in primary cultured rat hepatocytes. The changes in CYP3A stability are consistent with effects of these agents on phospholipase C or D (PLC or PLD, respectively). Studies in microsomes have indicated that the PLD metabolite, phosphatidic acid (PA), is important in determining CYP3A stability. We propose to test this hypothesis that CYP3A protein stability is dependent upon microsomal PA levels and to further elucidate the molecular mechanism by which phospholipases and other enzymes that regulate PA levels contribute to CYP3A protein stability. The specific aims are 1) To further establish the role of PLD and PLC in the regulation of CYP3A protein stability. 2) To determine if agents that alter CYP3A stability in primary cultured hepatocytes also function in in vitro incubations. 3.) To determine if PA analogs with different fatty acid moieties have distinct effects on CYP3A protein stability in microsomal samples. 4) To determine whether agents that regulate rat CYP3A stability act in a like manner in human primary cultured hepatocytes and microsomes. These studies will provide information that can be used to predict drug interactions by providing an understanding of the underlying molecular mechanisms involved in the post-translational regulation of CYP3A, and will test a new hypothesis regarding the interactions of phospholipase activities and a key drug-metabolizing enzyme

Keywords: acid, cytochrome P450, drug metabolism, liver metabolism, protein structure enzyme activity, microsome, nitrobenzene, phenanthroline, phospholipase C, phospholipase D, protein isoform, proteolysis human tissue, laboratory rat, tissue /cell culture

Project start date: 1999-09-30

Project end date: 2003-08-31

5R01DK054812-03 (2001): $212901

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REGULATION OF XENOBIOTIC-METABOLIZING CYP3A

Richard C Zangar, Senior Research Scientist
Battelle Pacific Northwest Laboratories
box 999, 902 Battelle Blvd
richland, Wa 99352

Grant 5R01DK054812-02 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: ALTX

Abstract: Cytochrome P450 3A (CYP3A) levels are important determinants of aflatoxin and acetaminophen toxicity and the uptake and elimination of numerous therapeutic agents. CYP3A protein has been reported to be stabilized by some CYP3A substrates. The PI reported that several structurally and functionally distinct agents that are not known CYP3A substrates alter CYP3A protein stability in primary cultured rat hepatocytes. The changes in CYP3A stability are consistent with effects of these agents on phospholipase C or D (PLC or PLD, respectively). Studies in microsomes have indicated that the PLD metabolite, phosphatidic acid (PA), is important in determining CYP3A stability. We propose to test this hypothesis that CYP3A protein stability is dependent upon microsomal PA levels and to further elucidate the molecular mechanism by which phospholipases and other enzymes that regulate PA levels contribute to CYP3A protein stability. The specific aims are 1) To further establish the role of PLD and PLC in the regulation of CYP3A protein stability. 2) To determine if agents that alter CYP3A stability in primary cultured hepatocytes also function in in vitro incubations. 3.) To determine if PA analogs with different fatty acid moieties have distinct effects on CYP3A protein stability in microsomal samples. 4) To determine whether agents that regulate rat CYP3A stability act in a like manner in human primary cultured hepatocytes and microsomes. These studies will provide information that can be used to predict drug interactions by providing an understanding of the underlying molecular mechanisms involved in the post-translational regulation of CYP3A, and will test a new hypothesis regarding the interactions of phospholipase activities and a key drug-metabolizing enzyme

Keywords: acid, cytochrome P450, drug metabolism, liver metabolism, protein structure enzyme activity, microsome, nitrobenzene, phenanthroline, phospholipase C, phospholipase D, protein isoform, proteolysis human tissue, laboratory rat, tissue /cell culture

Project start date: 1999-09-30

Project end date: 2003-08-31

5R01DK054812-02 (2000): $207803

REGULATION OF XENOBIOTIC-METABOLIZING CYP3A

Richard C Zangar, Senior Research Scientist
Battelle Pacific Northwest Laboratories
box 999, 902 Battelle Blvd
richland, Wa 99352

Grant 1R01DK054812-01A1 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: ALTX

Abstract: Cytochrome P450 3A (CYP3A) levels are important determinants of aflatoxin and acetaminophen toxicity and the uptake and elimination of numerous therapeutic agents. CYP3A protein has been reported to be stabilized by some CYP3A substrates. The PI reported that several structurally and functionally distinct agents that are not known CYP3A substrates alter CYP3A protein stability in primary cultured rat hepatocytes. The changes in CYP3A stability are consistent with effects of these agents on phospholipase C or D (PLC or PLD, respectively). Studies in microsomes have indicated that the PLD metabolite, phosphatidic acid (PA), is important in determining CYP3A stability. We propose to test this hypothesis that CYP3A protein stability is dependent upon microsomal PA levels and to further elucidate the molecular mechanism by which phospholipases and other enzymes that regulate PA levels contribute to CYP3A protein stability. The specific aims are 1) To further establish the role of PLD and PLC in the regulation of CYP3A protein stability. 2) To determine if agents that alter CYP3A stability in primary cultured hepatocytes also function in in vitro incubations. 3.) To determine if PA analogs with different fatty acid moieties have distinct effects on CYP3A protein stability in microsomal samples. 4) To determine whether agents that regulate rat CYP3A stability act in a like manner in human primary cultured hepatocytes and microsomes. These studies will provide information that can be used to predict drug interactions by providing an understanding of the underlying molecular mechanisms involved in the post-translational regulation of CYP3A, and will test a new hypothesis regarding the interactions of phospholipase activities and a key drug-metabolizing enzyme

Keywords: acid, cytochrome P450, drug metabolism, liver metabolism, protein structure enzyme activity, microsome, nitrobenzene, phenanthroline, phospholipase C, phospholipase D, protein isoform, proteolysis human tissue, laboratory rat, tissue /cell culture

Project start date: 1999-09-30

Project end date: 2003-08-31

1R01DK054812-01A1 (1999): $200902

Proteomic Identification Of NAF Biomarkers

Richard C Zangar, Senior Research Scientist
Battelle Pacific Northwest Laboratories Box 999, 902 Battelle Blvd Richland, Wa 99352

Grant 5R33CA097526-02 from National Cancer Institute, IRG: ZCA1

Abstract: Breast cancer is responsible for ~43,000 deaths per year, a number which could be reduced dramatically by early detection of the disease. Currently, detection of breast cancer relies primarily on physical examination and conventional mammography. While these procedures have improved early detection of breast cancer and thereby decreased mortality, they still result in relatively high rates of false-positive and false-negative diagnoses. In addition, breast cancer prognosis is based on histological examination and is inadequate for assessing micrometastases. Due to this inability to accurately predict the risk of recurrence, 50% or more of breast cancer patients are unnecessarily treated with adjuvant therapy. A more useful and accurate evaluation of breast cancer could be obtained if these current, observation-based methods were supplemented with a molecular assessment. A number of serum proteins are reported to be altered in individuals with breast cancer when compared with healthy individuals. There is not, however, any known circulating protein that is suitable to define the stage of breast cancer or as a general marker for breast cancer, especially in the early stages of this disease. Mammary ductal cells are the cellular origin for 70% to 80% of breast cancer cases. Nipple aspirate fluid (NAF), which is obtained from non-lactating women, contains proteins secreted directly by these ductal cells. As such, we hypothesize that NAF is a concentrated and selective source of protein biomarkers for breast cancer. Proteomic approaches offer an unbiased way to evaluate NAF as a source of biomarkers and is sufficiently sensitive for small NAF volumes (10 to 50 (l). Therefore, the goals of this proposal are to characterize the proteome of NAF and to undertake preliminary analyses to identify potential protein biomarkers. To accomplish these goals, we propose to undertaken the following specific aims Aim #1. Characterize the Proteome of Pooled NAFs Using Mass Spectrometry. Aim #2. Identify Potential Protein Biomarkers in Individual NAF Samples from Women with Early-Stage Breast Cancer using Isotopic Labeling and Mass Spectrometry. Upon completion of these aims, we will have provided the first detailed characterization of the NAF proteome and identified a subset of NAF proteins that are potential markers of breast disease.

Keywords: biomarker, breast neoplasm, proteomics, breast neoplasm /cancer diagnosis, cancer risk, early diagnosis, women s health, clinical research, female, human middle age (35-64), human subject, mass spectrometry, questionnaire

Project start date: 2004-06-01

Project end date: 2007-05-31

5R33CA097526-02 (2005): $406349

1R33CA097526-01A2 (2004): $411390

High-Throughput Evaluation Of Breast Cancer Markers

Richard C Zangar, Senior Research Scientist
Battelle Pacific Northwest Laboratories

Grant 5U01CA117378-04 from National Cancer Institute, IRG: ZCA1

Abstract: Breast cancer is one of the most prevalent cancer in the United States, resulting in the death of ~43,000 women per year. Current methods for early detection (e.g., mammography and breast exam) of this disease rely on physical means to detect a tumor and are unreliable. Since a number of blood proteins have been reported to be altered in women with breast cancer, a more useful and accurate evaluation of breast cancer could potentially be obtained by an analysis of these proteins. Since breast cancer is a multifaceted disease, it seems likely that analysis of more than one protein will be needed to detect all forms of this disease. In addition, the normal levels of many cancer markers will be affected by age, reproductive history, menopausal status and other epidemiological factors. Therefore, we hypothesize that it will be necessary to use a profile of markers in order to accurately detect breast cancer and that the accuracy of this profile will be improved by accounting for predictable effects of epidemiological factors. In order to test this hypothesis, we will undertake a "phase 1" biomarker discovery analysis using sophisticated proteomics methodologies and leveraging the results from several independently funded studies. Based on results of this first study and other information, we will undertake a "phase 2" analysis of 50 proteins in ~1000 plasma samples using ELISA microarray technology. Finally, we will undertaken an extensive "phase 3" retrospective study with the goal of determining whether a selected subset of plasma markers can be used to predict the presence of breast cancer in a population of high-risk women prior to detection of that disease by conventional methods. Therefore, the proposed research will effectively utilize new technologies to significantly accelerate the pace of biomarker research. The final result of these analyses will be an extensive characterization of a whole profile of protein levels. We will use sufficient numbers of samples to draw statistically valid conclusions about the ability of this biomarker profile to detect the presence of early disease and whether incorporation of epidemiological factors can improve the accuracy of this analysis

Project start date: 2005-09-26

Project end date: 2010-07-31

5U01CA117378-03 (2007): $434557

5U01CA117378-02 (2006): $414718

1U01CA117378-01 (2005): $284056

Bioinformatics For Protein Microarrays

Richard C Zangar, Senior Research Scientist
Battelle Pacific Northwest Laboratories

Grant 5R01EB006177-03 from National Institute Of Biomedical Imaging And Bioengineering, IRG: BDMA

Abstract: Antibody micro arrays are an emerging technology that has the potential to efficiently and quantitatively analyze many proteins in thousands of samples. Therefore, this technology can provide a badly needed mechanism for determining the clinical usefulness of individual or profiles of potential biomarkers. At present, though, antibody micro array analysis lacks the guiding principles, standard procedures, honed laboratory practices, foundational statistics and supporting software to produce quality results on a large scale. Therefore, we have started to develop both the protocols and supporting software ("ProMAT") specifically for analyzing protein micro array data. The goal of this proposal is to advance antibody micro array to the point that it can be used as a routine tool for clinical biomarker validation. To accomplish this goal, we plan to establish novel protocols for the use of internal and external standards, develop the statistical foundation for evaluating data quality at all stages of data collection and analysis, and develop a sophisticated bioinformatics tool for rapid data analysis, including advanced quality control features. This system will also aid in identifying sources of data variability and will allow for data normalization. We will use an iterative process that cycles through resolving assay requirements, deriving statistical methods and composing prototype software, honing laboratory practices and assay procedures, and evaluating progress through repetitive experimentation that focuses on assessing data quality and reproducibility. At the completion of this work, we will have uncovered guiding principles and requirements for conducting antibody micro array experiments and developed an integrated system for rapidly generating the high quality data required to evaluate the clinical potential of biomarker profiles

Keywords: bioinformatics, biomarker, protein aging, antibody, base, biological model, computer program /software, computer system design /evaluation, conditioning, data collection, data quality /integrity, environment, enzyme linked immunosorbent assay, experience, gene, human, information system, microarray technology, model, motivation, pharmacokinetics, proteomics, sectioning, sex, sound, statistics /biometry, therapy

Project start date: 2007-02-01

Project end date: 2010-01-31

1R01EB006177-01A1 (2007): $316487

CORE--ELISA MICROARRAY FACILITY

Richard C Zangar, Senior Research Scientist
Battelle Memorial Institute

Grant 5U54ES016015-029002 from National Institute Of Environmental Health Sciences, IRG: ZES1

High-Throughput Evaluation Of Breast Cancer Markers

Richard C Zangar, Senior Research Scientist
Battelle Pacific Northwest Laboratories

Grant 3U01CA117378-04S1 from National Cancer Institute, IRG: ZCA1

Project start date: 2005-09-26

Project end date: 2010-07-31

REGULATION OF CYP2E1 EXPRESSION BY TOXICANTS

Richard C Zangar, Senior Research Scientist
Wayne State University
sponsored Program Administration
detroit, Mi 48202

Grant 1F32ES005657-01 from National Institute Of Environmental Health Sciences, IRG: BIOL

1F32ES005657-01 (1994): $28600