Project: Research project

Project Details


The hypothesis that some types of damage to DNA may be involved in the
aging process will be investigated using a model system, adrenocortical
cells in culture. Some of the damage to DNA in aging may result from the
toxic side-effects of oxygen. Adrenocortical cells form a useful system
for investigating the role of this oxidative damage in aging. Both human
and bovine adrenocortical cells are well-studied systems for investigating
cell culture senescence. Protocols to increase or decrease the amount of
oxidative damage occurring in cultured adrenocortical cells will be
developed, by changing the oxygen concentration at which the cells are
grown; by modifying the glutathione content of the cells; by varying the
tocopherol and selenium status of the cells, by depletion and selective
readdition; by addition of other biological or synthetic antioxidants; by
irradiation; and by addition of peroxides or superoxide-generating
systems. The effectiveness of these protocols will be assessed by release
of 45Ca; measurement of changes in superoxide dismutase, catalase, and
glutathione peroxidase, the principal cellular enzymatic defenses against
oxidative damage; assessment of cellular levels of ubiquinone, which is
sensitive to oxidative damage; and fluorescence microscopic determination
of lipofuscin, a measure of an oxidative damage product in the living
cell. Using protocols that yield varying degrees of oxidative damage, the
amount of damage to DNA will be measured, by unscheduled DNA synthesis, a
measure of DNA repair; by poly(ADP-ribose) synthesis, a response to DNA
damage; by sister chromatid exchange rate; and by frequency of chromosome
aberrations. These assays measure viable cell responses to damage to DNA
and have been shown to respond to oxidative damage. Such protocols will
then be tested for effects on replicative potential (life span) and cloning
efficiency, to determine if the replicative life span is associated with
the level or type of oxidative damage incurred by the cells and with the
level or type of DNA damage. Some of these protocols will be used to
examine losses of differentiated functions during culture senescence.
Functions to be measured are ACTH receptor content; ACTH- and
PGE1-stimulated cyclic AMP production; mitogenic response to angiotensin
II; restimulated levels of steroidogenic cytochrome P-450 enzymes
(11-hydroxylase, cholesterol side-chain cleavage, 21-hydroxylase, and
17-hydroxylase); P-450 enzymes involved in benzo[a]pyrene metabolism;
3beta-hydroxysteroid dehydrogenase; and synthesis of prostaglandins.
Differentiated functions may be affected directly by oxidative damage, or
secondary to DNA damage.
Effective start/end date8/1/853/31/98


  • National Institutes of Health: $74,000.00
  • National Institutes of Health: $245,516.00
  • National Institutes of Health: $5,000.00
  • National Institutes of Health: $5,000.00


  • Medicine(all)


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