Enrique Cadenas - US grants

Long-term Objectives-To test the hypothesis that cellular two-electron transfer systems-involving DT-diaphorase catalysis and glutathione nucleophilic addition-play a central role in the bioreductive activation of quinonoid compounds and are associated with their cytotoxicity and carcinogenicity as well as with the activation of chemotherapeutic agents. This hypothesis appears tenable since [I] our recent results indicate that the two-electron transfer to quinones-as accomplished by DT-diaphorase and thiol nucleophilic addition-cannot be regarded as a detoxification system, but as a process accompanied in most instances by high production of oxygen radicals. [II] Thiol reactivity is a salient feature of many classes of therapeutic drugs, hence, it should be considered as matter of course when evaluating the metabolic pathways of quinonoid compounds. [III] The cellular role of DT-diaphorase-whose activity is enhanced in transformed cells and preneosplatic nodules-is still controversial and it has been implicated in both the resistance against certain toxins and the metabolic activation of carcinogens. Specific aims - The aims of this research are directed to an understanding of the molecular mechanisms inherent in the two-electron bioreduction of quinones and their implications for quinone cytotoxicity. The proposed research represents a systematic and comprehensive approach to gather such information and it involves three major specific aims: [1] To determine the effect of substitution patterns on quinone reactivity and cytotoxicity within the framework of biological two-electron transfer systems. [2] To determine the role of DT-diaphorase in the initial reduction and subsequent redox transitions of quinone bioalkylating agents. [3] To determine the toxic effects of quinones on cultured cell lines in terms of [a] the significance of two-electron transfer processes in the activation of quinones, [b] the factors regulating cellular induction of DT-diaphorase and whether or not this induction is accompanied by a parallel induction of superoxide dismutase, and [c] how cells with an induced DT-diaphorase activity regulate quinone metabolism as a function of the physico-chemical properties of the quinonoid compound. Experimental design and methods - [1] Various compounds from the p-benzo- and 1,4-naphthoquinone series, selected on the basis of their substitution pattern, reduction potential, and capability to become reactive electrophiles, will be studied in relation to two-electron transfer processes: DT-diaphorase catalysis and thiol nucleophilic addition. [2] The inborn redox properties of p-benzoquinone and naphthoquinone bioalkylating agents will be assessed in experimental models involving the above-described two-electron transfer systems. [3] Quinone cytotoxicity will be evaluated with C3H/10T1/2 cells -a mouse embryofibroblast line suitable for these investigations- as a function of the induced levels of DT-diaphorase and superoxide dismutase and the chemical reactivity of the quinonoid compounds. Cytotoxicity assays will be performed by measuring reduction in plating efficiency of treated cells. Spector- and fluorometric methods will be used to measure enzyme activity, active oxygen species, glutathione, and glutathione disulfide. ESR with or without spin trapping technique will be used for the detection of oxygen-derived- and thiyl radicals, and semiquinones. HPLC methods with different detection modes will be used as follows: [a] electrochemical detection: determination quinones, hydroquinones, quinone epoxides, quinone-thioether derivatives, and hydroxyl radical. [b] UV detection: determination of glutathione disulfide and glutathione sulfonate.

The objective of this proposal is to test the hypothesis that the reaction of myoglobin with hydrogen peroxide or other biological oxidants leading to the formation of the high oxidation state of this hemoprotein, ferrylmyoglobin, is an early functional change with toxicological implications for muscle tissue. We propose that the chemical reactivity with which ferrylmyoglobin is endowed is a basis for ensuing tissue oxidative damage. This hypothesis appears tenable since [I] ferrylmyoglobin occurs in cells and perfused heart, [II] myoglobin is oxidized by physiological peroxides in myocytes, and [III] ferrylmyoglobin is reduced efficiently in vitro by several antioxidants. This proposal is to study chemical mechanisms inherent in the formation of the high oxidation state of myoglobin, its recovery to a functional hemoprotein, and its implications for myocardial toxicity from studies with myocytes and mitochondria. Specific Aim 1 is to provide an understanding of the redox transitions encompassed by the two-electron oxidation of myoglobin to ferrylmyoglobin as well as to characterize the individual chemical reactivities of the electrophilic centers in ferrylmyoglobin. Specific Aim 2 is concerned with the recovery of ferrylmyoglobin to a functional hemoprotein by different electron donors by one-electron transfer processes as well as the kinetic and environmental factors that govern these reactions. The studies devised in Specific Aim 3 are directed at evaluating the role of mitochondrially generated hydrogen peroxide on myoglobin oxidation and the implications of these reactions for muscle oxidative injury. [l] The mechanistic and catalytic aspects implied in myoglobin oxidation will be evaluated by experimental models involving one- and two-electron oxidation of the hemoprotein. [2] The electron-transfer reactions leading to myoglobin recovery will be assessed with a range of electron donors with different physico-chemical properties. [3] The biological implications of the reaction between myoglobin and hydrogen peroxide will be determined with isolated heart mitochondria and myocytes and assessed in terms of the reactivity of ferrylmyoglobin towards membrane antioxidants and cytoskeletal proteins. Absorption spectroscopy will be used to monitor the different oxidation states of myoglobin and oxidative reactions involving protein-heme and drug-heme covalent bindings. ESR with the spin trapping technique will be used to detect oxygen-derived radicals and thiyl radicals. Direct ESR with will be used to identify free radicals derived from electron donors, such as ascorbate, vitamin E, and ubiquinol. Direct ESR assisted by flow experiments will be used to identify protein- derived radicals. HPLC methods with different detection modes will be used as follows: [a] UV detection: determination of glutathione and oxidation products of vitamin E, ubiquinol, and water-soluble electron products. [b] Electrochemical detection: identification and red ox properties of oxidation products of different electron donors. [c] Fluorometric detection: determination of vitamin E. SDS-PAGE analysis and Western blot analysis will be used to study cross-linking of cytoskeletal proteins with myoglobin.

DESCRIPTION: (adapted from Investigator's abstract) The hypothesis to be tested is that sustained production of free radicals by mitochondria leads to critical mitochondrial DNA damage and dysfunction that are amplified to lethal cellular events by initiating apoptotic pathways through cyctochrome c release and c-jun N-terminal kinase activation. The four specific aims are: (1) To determine the effects of age on the regulation of mitochondrial functions by metabolic state, oxygen, and nitric oxide. (2) To identify the mechanisms and consequences of mitochondrial DNA (mtDNA) oxidative damage leading to specific mtDNA mutations. (3) To elucidate the consequences of mtDNA mutations at the cellular level within the framework of mitochondrial changes signaling for apoptosis. (4) To examine the role of the thiol/disulfide status in mtDNA damage and further consequences at mitochondrial and cellular levels. The focus of this research proposal is to identify the critical molecular events that are involved in the sequence from mitochondrial dysfunction to apoptosis by assessing various stimulatory and protective pathways that can initiate or delay the onset of cell death.

Long-term goal. The long-term goal of the proposed study is to understand the role of mitochondria and oxidative stress in the dopaminergic cell death, associated with the Parkinson's disease. Hypothesis. The hypothesis to be tested is that (a) an increase in the steady-state levels of nitric oxide stimulates both dopamine autoxidation and mitochondrial oxidant production; (b) the ensuing increase in the oxidative load of the dopaminergic cell leads to specific mitochondrial dysfunctions. Specific Aims. The testing of the biochemical pathway envisioned by this hypothesis will constitute the specific aims of this study, which include: (1) Determine how nitric oxide regulates autoxidation of dopamine, a process which generates reactive oxygen species and nitrogen-centered oxidants. (2) Determine the effects of nitric oxide and dopamine on mitochondrial functional integrity in intact cells. (3) Identify the mitochondrial proteins that become oxidized by reactive species derived from dopamine autoxidation. (4) Explore the mechanisms by which oxidative/nitrosative damage to mitochondria may be attenuated. Collectively, these studies will attempt to scrutinize the plausibility of a biochemical model explaining the deleterious alterations occurring during Parkinson's disease. Significance. Specifically, the results in this study will help elucidate the mechanisms by which nitric oxide induces autoxidation of dopamine and leads to the impairment of mitochondrial functions. Understanding of the nature of the putative mechanisms is deemed to be an indispensable step in developing therapeutic interventions.

DESCRIPTION (provided by applicant): Two key concepts serve to underscore the significance of this research proposal: First, mitochondria generate messengers, such as H2O2 and nitric oxide ('NO), which are involved in the regulation of redox-sensitive cell signaling through the mitogen-activated protein kinase (MAPK;e.g., JNK) pathway. Second, mitochondria are the recipients of the effects of cytosolic signaling molecules, such as JNK, which is translocated to mitochondria under stress conditions and aging and elicits profound metabolic effects in the organelle through the activation of phosphorylation cascades. The interaction between these two processes is essential for the coordination of the cell functional responses and we hypothesize that impairment of this interaction or communication is critical for the development of the loss of function inherent in aging. Long-term goal - The long-term goal of the proposed studies is to elucidate the interactive role of redoxdependent mitochondria-cytosol interactions in the initiation and progression of the aging process. Hypothesis- The hypothesis to be tested is that impairment of mitochondria-cytosol interactions -constituted of mitochondrial H2O2 and 'NO, metabolites such as pyruvate, and MAPK signaling pathways- is critical for the development of the oxidative and nitrosative cellular damage and loss of function inherent in aging. Specific Aims - The validty of the hypotheses will be tested through four specific aims, which incorporate studies on: : (1) JNK-mediated regulation of mitochondrial functions. (2) Downstream signaling effects of JNK in mitochondria. (3) Modulation of JNK-mediated changes of mitochondrial functions by aging and caloric restriction, and (4) JNK-signaling on mitochondria in a PC12 cell model. The first three specific aims will be carried out with mitochondria isolated from the brain of rats from different age groups, whereas the fourth specific aim is directed at assessing the mitochondrion / JNK interactions in a cellular setting under the control of metabolic-, redox-, and apoptotic stimuli. Significance - This research contributes to the elucidation of the processes involved in the metabolic network that controls cellular energy levels and the redox environment and its impairment during aging by assessing the regulatory mechanism that coordinate mitochondrial functions with the rest of the cell. These mechanisms play a critical role in the progression of the aging process and may precede or modulate age-related oxidative- and nitrosative damages.

[unreadable] DESCRIPTION (provided by applicant): We request partial funding for the 13th Biennial Meeting of the Society for Free Radical Research International (SFRRI) to be held on August 15-19, 2006, at the congress Center of Davos, Switzerland. Funds will defray the cost of travel, lodging, and registration fees of invited speakers from USA so that other funds can be directed toward supporting the participation of students, fellows, and young investigators. The meeting will last for four days. The first half day will host two invited keynote lectures. The following three days will have two invited keynote lectures each day, one delivered in the morning and one in the afternoon, coordinated with three parallel sessions both in the morning and afternoon. The last half day will have only three parallel sessions in the morning. The themes of the 21 symposia are: 1. Nitric Oxide and Nitrolipids; 2. Proteasome; 3. Carotenoids, Reactive Oxygen Species, and Human Disease; 4. Signaling by Oxygen; 5. Redox Regulation of Cell Function; 6. Inflammation; 7. Acute Brain Injury and Neurodegeneration; 8. Antioxidant Network Strategies; 9. Diabetes and Metabolic Syndrome; 10. Aging; 11. The Antioxidant Dilemma; 12. Mitochondria and reactive oxygen species; 13. Immune Response; 14. DNA Damage and Repair; 15. Proteomics and Free Radicals; 16. Vitamin E, Phytoestrogens, Polyphenols, and Gene Expression; 17. Selenoproteins; 18. Young Investigator Seminars; 19. Redox Signaling in Plants; 20. Nitric oxide and Mitochondria; 21. Tissue Remodeling. Each session will have 6 speakers, 4 of whom have been selected by designated experts who will serve as chairpersons of the session and two will be selected from the abstracts submitted by Society members and non-members. The sessions will address a broad range of research activities on the diverse roles that reactive oxygen and nitrogen species play in biology and medicine. The scientific data presented will have an impact on our understanding of aging as well as cardiovascular, neoplastic, inflammatory, and neurodegenerative diseases, and will provide new insights into the mechanisms by which antioxidants and therapeutics act on these diseases. From 12:30-1:30 pm on days two, three, and four, a Lunchtime Free Radical School will be held, featuring experts in the field addressing fundamental aspects of free radical chemistry and biology to students, fellows, and young investigators. Poster sessions will be displayed in half-day shifts. Posters will be displayed and discussed (between 5:30-6:30 pm) on computer screens (in several separated pages). In summary, the congress provides a forum for exchange of the most recent basic and applied research in free radical chemistry, biology, and medicine as well as an opportunity for scientists to network and plan new collaborative engagements engagements. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The 7th Gordon Research Conference (GRC) on Oxidative Stress and Disease has been assembled around the theme The Metabolic - Inflammatory Axis in Brain Aging and Alzheimer's Disease and is designed from an interdisciplinary perspective to provide discovery, translational, and clinical scientists with the latest findings on the role of oxidative stress in energy metabolism and inflammatory responses in brain aging and neurodegeneration, with emphasis on Alzheimer's disease. The GRCs are internationally recognized for the high-quality and cutting-edge nature of their meetings. The 2013 GRC on Oxidative Stress and Disease, The Metabolic-Inflammatory Axis in Brain Aging and Alzheimer's Disease, will be held on 14-19 April 2013 at the Les Diablerets Conference Center, Switzerland. The scientific program focuses on bioenergetic deficits and activation of inflammatory responses, their redox (oxidative stress) regulation, and their role in synaptic plasticity and cognition in healthy brain aging and Alzheimer's. Dynamic interactions among these systems are examined in terms of their causative or in-tandem occurrence and how does the systemic environment, e.g., insulin resistance, diabetes, and inflammation, impact on brain function. The significance of addressing 'healthy' brain aging and Alzheimer's disease is established by the escalation of Alzheimer's, which currently (2012) impacts more than 5.4 million Americans of all ages, and is projected to rise by 30% by 2025. This 2013 GRC on Oxidative Stress and Disease brings together an international cadre of experts with a broad range of interests and approaches to healthy brain aging and to Alzheimer's disease; this provides a unique forum for in-depth discussions on cellular mechanisms and their forward translation into interventions to prevent, delay, or treat the memory loss and cognitive slowing associated with brain aging and exacerbated in Alzheimer's disease. This GRC is also an example of reverse translation inasmuch as clinical scientists using non-invasive neuroimaging techniques will share their findings with basic scientists. In addition, a Gordon Research Seminar (GRS) will be organized by junior investigators at the post-doctoral level. The GRS will be held the day before the GRC (13 April 2013) and is oriented towards junior investigators and aimed at (a) providing them with solid background on common mechanisms involved in brain aging and neurodegeneration in order to maximize the understanding of the science that will be discussed in the subsequent GRC; (b) promoting communications between young investigators that nurture potential collaborations, and creating a collegial mentorship from senior members for career and academic development. We fully anticipate that the scientific discussions, research talks, poster sessions and other close interactions among the participants of the GRC and the GRS will significantly advance their understanding of the mechanisms inherent in healthy brain aging or its progression into Alzheimer's disease and set the foundations for collaborative investigations aimed at promoting new therapeutic interventions for these disorders.

PROJECT SUMMARY-ANALYTIC CORE C The mission of our Perimenopause in Brain Aging and Alzheimer's Disease Program Project (P3) is to discover biological transformations in brain that occur during the perimenopausal transition that lead to endophenotypes predictive of risk for Alzheimer's disease (AD). Our goals are to identify the mechanisms by which these transformations occur and to translate these discoveries into strategies to prevent conversion to an at-Alzheimer's-risk phenotype. To achieve our Perimenopausal Program Project mission, we have developed a set of integrated program-wide specific aims that span mechanistic discovery to clinical neuroimaging in ApoE genotyped perimenopausal and postmenopausal women to a global population through the ENIGMA network of neuroimages, clinical data and ApoE genetics. The mission of Analytic Core is to provide standardized steroid hormone regimens, sample archiving, processing and distribution, as well as analytic support across the Perimenopausal Program Project. The mission of Analytic Core is to provide standardized steroid hormone regimens, sample archiving, processing and distribution, as well as analytic support across the Program Project. To achieve this mission, the Analytic Core will provide two levels of support. The first level of support entails standardized steroid hormone regimens and sample management. The second level of support entails sample analysis and data interpretation on four major studies: LC-MS/MS steroid analysis, ApoE genotyping, custom array gene expression analysis, and bioinformatic gene functional analysis. Analytic Core was an instrumental and essential resource for the previous Progesterone Program Project and the currently ongoing Perimenopause Program Project. Through the infrastructure and analytic methods it has developed, Analytic Core will continue to play a crucial role in achieving the renewed Perimenopausal Program Project's mission and aims.

PROJECT SUMMARY-ANIMAL CORE B The mission of our Perimenopause in Brain Aging and Alzheimer's Disease Program Project (P3) is to discover biological transformations in brain that occur during the perimenopausal transition that lead to endophenotypes predictive of risk for Alzheimer's disease (AD). Our goals are to identify the mechanisms by which these transformations occur and to translate these discoveries into strategies to prevent conversion to an at-Alzheimer's-risk phenotype. The proposed program of research builds on our discovery of the perimenopausal bioenergetic transition in brain and its predictive association with cognitive decline in postmenopausal women to investigate the impact of the APOE4 allele on this uniquely female aging experience. Outcomes of our mechanistic to clinical to global population program of research could provide insights into the increased burden of the ApoE4 gene and risk of AD in ApoE4 positive women. The mission of Animal Core (Core B) is to ensure the success of the Perimenopause Program Project through provision of animals as needed to Projects 1?2. To achieve its mission, the Animal Core will extend previously developed rodent models of perimenopause and menopause to humanized ApoE4 and ApoE3 rodent mice and rat models; maintain and track animals from acquisition or birth, determination of cycling status and perimenopausal transition, randomized study enrollment, experimental manipulation, to tissue collection across the entire Perimenopause Program Project; and obtain blinded tissue samples for analyses by Analytic Core and/or Projects.