The long-range goal of this research project is to study the relationship between oxidative stress and the [Ah] gene battery. It is becoming increasingly clear that we """"""""age"""""""" because our genes undergo more and more damage by reactive oxygen metabolites (ROMs) as a function of time. When genes are damaged, several escape mechanisms""""""""--including programmed cell death (apoptosis)--occur with increasing frequency. During the past 5 years, an oxidative stress signal transduction pathway has been defined, comprising more than 15 steps and initiated by ROMs by way of physical agents (ionizing and UV irradiation) as well as the metabolism of both endogenous and foreign chemicals. This laboratory has taken a genetic approach to study the role of oxidative stress in gene regulation and cell death. The 14CoS/14CoS mouse contains a 3,800-kb deletion on chromosome 7 and dies during the first 24 h post partum. We found that this mouse exhibits a constitutive oxidative stress response, in which expression of the NAD(P)H:menadione oxidoreductase (Nmol) and other [Ah] Phase II genes is increased. Recent work in other laboratories has shown that homozygous disruption of the fumarylacetoacetate hydrolase (Fah) gene--located in the 3,800-kb deleted region--completely mimics the 14CoS/14CoS mouse, due to ROMs generated by blockade of the tyrosine degradation pathway. Now that we understand more about the 14CoS/14CoS mouse, we can investigate directly the role of oxidative stress in aging through construction of transgenic mouse lines having defects in the control of redox homeostasis. In the next 5 years, we therefore propose to: [1] develop a conventional, as well as an inducible, knockout transgenic mouse having a homozygous disruption in the Fah gene, which will allow us to study ROM pharmacokinetics and cell type- and organ-specific responses of aging secondary to endogenous ROMs; [2] characterize in cells in culture, as well as in the intact animal, the mechanism(s) by which the intracellular levels of reduced glutathione (GSH) are regulated; and [3] develop a conventional, as well as an inducible, knockout transgenic mouse having a homozygous disruption in the gamma glutamylcysteine synthase (Gcs) gene. The GCS enzyme controls GSH production and thus affects at least two critical, distinct steps in the less than 15-step oxidative stress pathway. These studies will greatly enhance our understanding of the cellular responses, and consequences of the role of endogenous ROM-mediated oxidative stress, during the aging process.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
5R01AG009235-09
Application #
2748501
Study Section
Metabolic Pathology Study Section (MEP)
Project Start
1990-09-01
Project End
2000-07-31
Budget Start
1998-08-01
Budget End
2000-07-31
Support Year
9
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Cincinnati
Department
Public Health & Prev Medicine
Type
Schools of Medicine
DUNS #
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
Dalton, Timothy P; Chen, Ying; Schneider, Scott N et al. (2004) Genetically altered mice to evaluate glutathione homeostasis in health and disease. Free Radic Biol Med 37:1511-26
Dieter, Matthew Z; Freshwater, Sarah L; Miller, Marian L et al. (2003) Pharmacological rescue of the 14CoS/14CoS mouse: hepatocyte apoptosis is likely caused by endogenous oxidative stress. Free Radic Biol Med 35:351-67
Yang, Yi; Dieter, Matthew Z; Chen, Ying et al. (2002) Initial characterization of the glutamate-cysteine ligase modifier subunit Gclm(-/-) knockout mouse. Novel model system for a severely compromised oxidative stress response. J Biol Chem 277:49446-52
Solis, Willy A; Childs, Nicole L; Weedon, Michael N et al. (2002) Retrovirally expressed metal response element-binding transcription factor-1 normalizes metallothionein-1 gene expression and protects cells against zinc, but not cadmium, toxicity. Toxicol Appl Pharmacol 178:93-101
Solis, Willy A; Dalton, Timothy P; Dieter, Matthew Z et al. (2002) Glutamate-cysteine ligase modifier subunit: mouse Gclm gene structure and regulation by agents that cause oxidative stress. Biochem Pharmacol 63:1739-54
Dieter, M Z; Freshwater, S L; Solis, W A et al. (2001) Tyrphostin [correction of Tryphostin] AG879, a tyrosine kinase inhibitor: prevention of transcriptional activation of the electrophile and the aromatic hydrocarbon response elements. Biochem Pharmacol 61:215-25
Nebert, D W; Roe, A L; Dieter, M Z et al. (2000) Role of the aromatic hydrocarbon receptor and [Ah] gene battery in the oxidative stress response, cell cycle control, and apoptosis. Biochem Pharmacol 59:65-85
Dalton, T P; Solis, W A; Nebert, D W et al. (2000) Characterization of the MTF-1 transcription factor from zebrafish and trout cells. Comp Biochem Physiol B Biochem Mol Biol 126:325-35
Dalton, T P; Dieter, M Z; Yang, Y et al. (2000) Knockout of the mouse glutamate cysteine ligase catalytic subunit (Gclc) gene: embryonic lethal when homozygous, and proposed model for moderate glutathione deficiency when heterozygous. Biochem Biophys Res Commun 279:324-9
Vasiliou, V; Buetler, T; Eaton, D L et al. (2000) Comparison of oxidative stress response parameters in newborn mouse liver versus simian virus 40 (SV40)-transformed hepatocyte cell lines. Biochem Pharmacol 59:703-12

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