Young Investigator Award Winners
236th ACS National Meeting, Philadelphia, PA, August 17-21, 2008
Abstracts
20. Selective enrichment and analysis of nitrated proteins from iNOS activated mouse macrophages. K. E. Schlicht, J. S. Wishnok, S. R. Tannenbaum
Activated macrophages can generate nitric oxide (NO·) and superoxide (O2·-). These reactive species can rapidly combine to form peroxynitrite anion (ONOO-), a highly reactive and potent molecule capable of lipid oxidation, DNA damage, and protein modification. A major product of protein nitration by ONOO- is 3-nitrotyrosine, an important biomarker of nitric oxide/peroxynitrite-related damage that has been associated with inflammatory diseases, neurological disorders, and cancer. Identification of individual sites of nitration has proven difficult with current methods. Therefore, we developed an enrichment method to isolate nitrated proteins and determine sites of nitration on individual peptides in mouse macrophage RAW 264.7 cells. Nitrated macrophage proteins were selectively labeled with a biotin tag, isolated using a novel affinity-capture method, and identified. Using this selective proteomics-based approach, we can identify the specific protein targets of nitration from complex biological samples to better understand the link between nitric oxide/peroxynitrite damage and disease.
22. The contribution of key enzyme-substrate interactions to the cytochrome P450 catalyzed dehydrogenation of tamoxifen metabolites and the role of P450 conformational dynamics during tamoxifen metabolism. K. Shahrokh, G. S. Yost, T. E. Cheatham III
The conformational and electronic factors that contribute to the formation of reactive electrophilic intermediates during drug metabolism by hepatic cytochrome P450 (P450) enzymes are of great interest for the in silico prediction of drug metabolism. To gain insight into the contributions of substrate-enzyme interactions during P450 catalysis we are using a combination of theory and experiment to study the P450 isozyme-specific metabolism of a commonly used anti-cancer drug: tamoxifen. Here we present the results of HF/ 6-31G* and B3LYP/6-31G* level calculations for key tamoxifen metabolites that are formed during P450-catalyzed oxygenation and dehydrogenation reactions. We have also performed molecular dynamics simulations of major classes of hepatic P450 involved in tamoxifen metabolism to determine the contribution of predicted P450 conformational dynamics on docking studies of tamoxifen and its metabolites. These results provide encouraging insights into the role of electronic and conformational constraints in the selectivity for P450-catalyzed oxygenation versus dehydrogenation reaction mechanisms.
42. Analysis of hydroxybenzo[a]pyrenes in urine by liquid chromatography-tandem mass spectroscopy: Applications as a response biomarker for polycyclic aromatic hydrocarbon (PAH) exposure. S. Sen, S. L. Gelhaus, I. A. Blair
Tobacco smoke, a leading cause of death in the US, contains a complex mixture of components including polycyclic aromatic hydrocarbons (PAHs). PAHs have been cited as causative agents in lung, skin and bladder cancer. Tobacco smoke exposure is currently measured by detection of the pyrene metabolite, 1-hydroxypyrene (1-OH-P) in urine and plasma. Unfortunately, 1-OH-P is not the best biomarker of tobacco carcinogen exposure because pyrene is non-carcinogenic. The current study focuses on the detection of benzo[a]pyrene (B[a]P), a model carcinogenic PAH, and its metabolites as biomarkers of tobacco exposure. These metabolites, 1-, 3-, 9-hydroxy-benzo[a]pyrene ?(OH-B[a]P) are found at very low levels in urine; therefore, extremely sensitive methods are required. Here we report the detection of the B[a]P metabolites, 1-OH-B[a]P, 3-OH-B[a]P and 9-OH-B[a]P, in urine utilizing the highly sensitive and highly specific stable isotope dilution LC-electron capture APCI/MRM/MS methodology based on the transition m/z ?267 (M-PFB) to m/z 239 (M-PFB-CO) and relevant transitions from the C-13 ?internal standards.
60. Human AKRs display quinone reductase activity with PAH quinones and equilenin-o-quinone . C. A. Shultz, J. L. Bolton, R. G. Harvey, T. M. Penning
Human aldo-keto reductases (AKRs) AKR1A1, 1B1, 1B10, 1C1-4, and 7A2 display substantial quinone reductase (QR) activity with non-K-region polycyclic aromatic hydrocarbon (PAH) o-quinones. AKRs reduce o-quinones to catechols, which auto-oxidize back to o-quinones, generating reactive oxygen species. We compared benzo[a]pyrene-7,8-dione, benzo[a]pyrene-1,6-dione, and benzo[a]pyrene-3,6-dione as QR substrates. The extended benzo[a]pyrene -quinones gave depressed rates of QR activity suggesting that AKRs display preferential activity with o-quinones produced from PAH-trans-dihydrodiols. We examined the HRT metabolite equilenin-o-quinone (3,4-EQ) as a QR substrate. AKR1B1, 1C1-3, and 7A2 catalyzed the reduction of 3,4-EQ at rates 14-500 fold greater than the non-enzymatic rate depending on the isoform. In endometrial cancer AKR1C1 and AKR1C3 expression is elevated (Mol. Cell. Endocrinol. 248 (2006) 126) and may contribute to a depressed Progesterone : 17?-Estradiol ratio. QR of AKRs may increase oxidative stress inflicted by PAH quinones and 3,4-EQ, which can contribute to chemical and hormonal carcinogenesis. (Supported by RO1 CA39504, P30-ES013508).
63. Identification and quantification of candidate protein biomarkers of preterm birth by liquid chromatography/tandem mass spectrometry. S. J. Shah, E. Ciccimaro, K. Yu, S. I. Parry, I. A. Blair
Preterm birth (PTB), a leading contributor of perinatal mortality with poorly defined etiology, is influenced by complex genetic-environmental interactions, including race, toxins, smoking and diet. Early identification of at-risk women can alleviate this problem, and hence the need for novel biomarkers. Our study utilizes stable isotope labeling by amino acids in cell culture (SILAC) to develop labeled proteome standard which models human cervical-vaginal fluid (CVF) proteins. Secreted proteins from human endocervical (End1) and vaginal (VK2) cells labeled with [13C615N2]-Lysine and [13C615N1]-Leucine were characterized by three-dimensional liquid chromatography/tandem mass spectrometry (3D-LC-MS/MS). An LC-multiple reaction monitoring/MS (LC-MRM/MS) assay was developed to quantitate twenty promising biomarker candidates for PTB. This unique method provides for relative quantification of twenty proteins simultaneously. This assay has been applied to investigate human CVF samples for PTB, and study the effect of specific environmental toxins and smoking on PTB to identify novel biomarkers. Supported by NIH Grant P30ES013508.
70. N2-(1-Carboxyethyl)deoxyguanosine: Potential mutagenic and prognostic implications of glycated DNA . D. Tamae, J. Termini
Methylglyoxal is an electrophilic alpha-oxoaldehyde that is formed by the enzymatic and non-enzymatic conversion of triose and triose-phosphates. Under certain pathological conditions such as diabetes, aging, and cancer, the increased cellular concentration of MG can lead to reactions with protein, lipid, and DNA, resulting in advanced glycation end-products (AGEs). The exquisitely sensitive and highly specific technique of HPLC-ESI-MS/MS was used to both identify and quantitate both stereoisomers of the DNA adduct, N2-(1-carboxyethyl)-deoxyguanosine (CEdG). Utilizing a stable isotope dilution method, this study represents the first time this DNA lesion has been quantitated in biological samples. Levels of CEdG were measured in normal and cancerous human breast tissue (12 and 4 adducts per 107 dG, respectively) and free CEdG from the urine of non-diabetic and diabetic rats (76.8 and 277.2 pg/ml/24hr, respectively). Separately, the chemical synthesis of CEdG in the triphosphate form and in an oligonucleotide has allowed for steady state kinetic studies using model replicative polymerases. Using Klenow fragment, the level of misincorporation of dATP opposite the lesion was favored by an order of magnitude relative to the “correct” insertion of dCTP.