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Record Count: 4
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DESCRIPTION (provided by applicant):
Arsenic is a well-documented human carcinogen. Our goal is to address the central hypothesis that phosphorylation of histones and their upstream kinases play an important functional role in arsenic-induced cell transformation and carcinogenesis. Specific Aim 1 is to study the role of histone phosphorylation in arsenic-induced cell transformation; Specific Aim 2 is to investigate and identify the histone kinases that phosphorylate histone H3 and H2B at different amino acid residues; Specific Aim 3 is to study the crystal structure of histone kinase RSK2, perform in- silico screening and design RSK2 inhibitors for suppressing arsenic-induced histone phosphorylation and cell transformation; and Specific Aim 4 is to study the role of RSK2 in arsenic/ultraviolet A (UVA)-induced skin carcinogenesis and determine RSK2's potential as a target for chemoprevention of cancer. The strategy for Specific Aim 1 is to use point mutations at key phosphorylation sites of histone H3 and H2B siRNA gene knockdown and overexpressing stable cell lines to test the role of H3 and H2B in soft agar cell transformation assays. For Specific Aim 2, we will use in vitro kinase assays, specific mutations, LTQ Orbitrap hybrid mass spectrometer analysis and RSK2 knockout cells as well as inhibitors of RSK2. In Specific Aim 3, we will use x-ray crystallography to determine the structure of RSK2. Then we will use a super computer to screen a database of 2.5 million chemicals to find inhibitors for RSK2 to be tested in an in vitro kinase assay. In Specific Aim 4, we will test the effect of the RSK2 inhibitor kaempferol and RSK2 knockout mice in UVA/arsenic-induced mouse skin carcinogenesis. Such knowledge will facilitate the design of more effective and specific strategies with fewer side effects for chemoprevention of arsenic- induced cancer. PUBLIC HEALTH RELEVANCE: Environmental arsenic contamination is a major problem in many parts of the world and is a well-documented human carcinogen. By using state-of-the-art technology such as x-ray crystallography, super computer based molecular modeling and drug screen, cellular and molecular biology and gene knockout mice, we will study the novel mechanism involved in histone phosphorylation on arsenic-induced cell transformation to cancer and the carcinogenesis process. These studies will facilitate the development of more effective agents with fewer side effects for chemoprevention against environmental carcinogens such as arsenic- induced cancer.
DESCRIPTION (provided by applicant): Furan is an important industrial compound that is also present in the environment. It is both hepatoxic and carcinogenic in mice and rats. Based on these results and the potential for human exposure, furan has been listed as a possible human carcinogen. The mechanism of tumor induction by furan is unknown. The available experimental evidence suggests that both nongenotoxic and genotoxic mechanisms contribute to the overall carcinogenic outcome. It is clear that the toxicity and carcinogenicity is initiated by cytochrome P450 catalyzed oxidation of furan to an a,(3-unsaturated dialdehyde, c/s-2-butene-1,4-dial. However, it is possible that further metabolism of c/s-2-butene-1,4-dial generates other reactive metabolites that contribute to the toxic effects of furan. Our long-range goal is to determine the mechanism of furan-induced carcino- genesis in rodents so that we can develop effective tools to determine if humans exposed to furan are sus- ceptible to its toxicological properties. The objective of this application is to determine if furan is a genotoxic carcinogen in vivo and to better define the metabolic processes that lead to its activation or detoxification. The central hypothesis is that both cytotoxic and genotoxic pathways contribute to the overall carcinogenic effects of furan with the involvement of multiple metabolic pathways. We formulated this hypothesis on the basis of strong preliminary data that suggest that the oxidation of furan to c/s-2-butene-1,4-dial initiates the process. While this reactive metabolite can alkylate both DNA and protein in vitro, it is also likely to be further metabolized to other toxic compounds in vivo. We plan to test our hypothesis by pursuing the following specific aims: 1) Determine the mutagenic activity of furan in Big Blue rodents; 2) Characterize the in vivo metabolic pathways of furan; 3) Compare the pathways of furan metabolism in human and rodent hepatocytes and determine the enzymes involved in each pathway. Collectively, these specific aims will provide important data for testing mechanisms of furan toxicity and tumorigenesis. With this information, we can develop appropriate model systems to assess th6 role of genotoxicity and cytotoxicity in the carcino- genic properties of furan. In addition, these studies will aid in the development of appropriate biomarkers (urinary metabolites, protein and/or DNA adducts) that can be used to determine if these toxicologically important reactions are occurring in humans exposed to furan.
This is a revised application from a well established investigator that seeks to further determine the mechanism of furan toxicity and mutagenicity. The strengths of this proposal are the strong background of the applicant in organic metabolite elucidation and DNA damage mechanisms, the clear and logical approach laid out in the specific aims and the responsiveness to the previous critiques. The weaknesses of this application are minimal. Some concerns are the potential for a null result in specific aims 1 and, particularly in aim 3 which may negate the use of a rodent model for determination of furan toxicity and mutagenicity in humans.
DESCRIPTION (provided by applicant): According to the U.S. Dept. of Health and Human Services, acetaldehyde is "reasonably anticipated to be a human carcinogen". When administered to laboratory animals by inhalation, acetaldehyde produces nasal and laryngeal carcinomas. Acetaldehyde occurs widely in the human environment, is a major constituent of cigarette smoke, and is the main metabolite of ethanol. Levels of acetaldehyde in the environment may increase with the introduction of ethanol-containing fuels. DNA adducts are critical in the carcinogenic process. In this program, we have identified DNA adducts of acetaldehyde including the major adduct N2- ethylidene-dGuo (adduct 1), the 1,A/2-propano-dGuo adducts 3 which are also formed from crotonaldehyde, and a related interstrand cross-link (adduct 4). We have developed highly sensitive mass spectrometric methods to quantify adducts 1 and 3 in human tissues. We have demonstrated that adduct 1 can be quantified in low microgram amounts of DNA, that it is an endogenous DNA adduct, and that its levels are influenced by cigarette smoking. We have also shown that adduct 3 is present in human lung DNA and has miscoding potential in human cells. In this renewal application, we propose to continue our studies on acetaldehyde DNA adducts to test our overall hypothesis that they are involved as causes of human cancer, particularly of the lung, and head and neck. Our goal is to investigate the occurrence in humans and the biological significance of acetaldehyde DNA adducts. Our specific aims are: 1. Quantify levels of adducts 1, 3, and the related acrolein-derived adduct 5 in human lung DNA from current smokers (confirmed by urinary cotinine) and compare levels of these adducts to those derived from benzo[a]pyrene (BaP) and tobacco-specific nitrosamines in the same tissues. 2a. Determine the influence of polymorphisms in alcohol dehydrogenase (ADH1C) and aldehyde dehydrogenase (ALDH2) genes on levels of adduct 1 in leukocyte DNA of non-smokers who consume alcohol. b. Quantify levels of adduct 1 in leukocyte DNA of non-smoking, non-drinking Chinese women who regularly engage in wok cooking compared to those who do not. 3. Investigate the genotoxic properties of adduct 1 in cells, and by studies in A/J mice which compare adduct formation and tumorigenicity of compounds that do or do not generate acetaldehyde. 4. Investigate the mechanism of translesion synthesis across adduct 3 and "half-excised" interstrand cross-links. We have shown that these adducts are bypassed by mammalian DNA polymerases in cells. Here we propose to identify those polymerases and characterize the translesion synthesis by in vitro and cellular approaches. These studies will provide critical data on the occurrence and biological significance of acetaldehyde DNA adducts and their possible role in human cancer.
Crisp Terms/Key Words: smoking, DNA damage, aldehyde dehydrogenase, alcohol dehydrogenase, chemical carcinogen, chemical carcinogenesis, respiratory neoplasm, head /neck neoplasm, tobacco abuse, benzopyrene, mass spectrometry, crosslink, leukocyte, laboratory mouse, acetaldehyde, ethanol, clinical research, DNA directed DNA polymerase, human subject, adduct, nitrosamine
DESCRIPTION (provided by applicant): This is a competing continuation application for the grant ES10751 entitled "Molecular Epidemiology of Parkinson's Disease". Key steps in the pathogenesis of Parkinson's disease (PD) may include increased soluble alpha-synuclein and its aggregation and fibrillization. These events are attenuated by protein degradation and fibril sequestration. The linkage-derived genes alpha-synuclein (SNCA), ubiquitin carboxy-terminal hydrolase L1 (UCHL1), microtubule-associated protein tau (MAPT), and parkin (PRKN) confer PD susceptibility, possibly via their effects on this pathogenesis pathway. We postulate that functional variability in these four genes and their joint effects modify PD severity outcomes, and that age, gender, and environmental factors also contribute to susceptibility and outcomes. We propose renewal of our grant ES10751 with the following 3 specific aims: A1. To refine the association between linkage-derived susceptibility genes and PD. Human, primate, and rodent cross-species sequence conservation will be used to highlight functional domains within the SNCA, UCHL1, MAPT, and PRKN loci. Additional variants, including single nucleotide polymorphisms (SNPs), will be identified within these regions (VISTA or "vSNPs"). Linkage disequilibrium will be used to identify haplotype-tagging variants ("ht-SNPs"). Family-based and case-control association analyses will be performed in an expanded Mayo sample of 1,500 matched pairs of PD cases and unaffected siblings or unrelated population controls. A2. To study interactions of these genes, and their interactions with environmental and gender-related factors. Using data from aim 1, we will apply recursive partitioning and conditional logistic regression approaches to investigate the joint effects of SNCA, UCHL1, MAPT, and PRKN on PD. We will also determine how these genes interact with environmental risk factors including pesticides, tobacco, coffee, and alcohol. In addition, we will assess how gender and endogenous or exogenous estrogen modify these interactions in women. A3. To study genetic factors influencing PD outcomes. In an established cohort of 1,000 Mayo PD patients, we will perform longitudinal assessments and conduct survival and regression analyses to determine whether polymorphism in the SNCA, UCHL1, MAPT, and PRKN genes correlates with disease severity outcomes. In particular, we will consider death, nursing home placement, incident dementia, and Hoehn and Yahr stage. We will also consider activities of daily living, complications of therapy, and quality of life. The first two aims will refine our understanding of molecular targets for neuroprotective therapies, and the third aim will predict their efficacy. Our findings will contribute to early disease detection (biomarkers) and primary prevention strategies. They will also accelerate the development of new treatments, reduce research and development costs, and identify subgroups of patients most likely to benefit.
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