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.
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