Project: Research project

Project Details


The proposal is based on the hypothesis that the 2- and 4-hydroxylated
metabolites of estrogens (2.and 4.OH.CEs) can contribute to the genesis of
breast cancer by mechanisms involving oxidative DNA damage. The hypothesis
is based on the following evidence. First, catecholestrogens (CEs) can be
metabolized to quinone estrogens (QEs), that can form depurinating adducts,
and can also generate reactive oxygen species (ROS) during redox cycling
between CEs and QEs. Second, forms of P450 that can catalyze NADPH-
dependent.2.and 4-hydroxylation of estrogens (Es) are expressed in normal
human mammary ductal epithelial cells that are the cells of origin of
breast cancers. Implicit in the hypothesis is that Es and xenobiotics can
potentiate each others actions, since forms of P450 that catalyze metabolic
activation of Es also catalyze metabolic activation of environmental
procarcinogens. Three specific aims are: 1) To complete characterizing
forms of P450 expressed in human ductal epithelial cells that can catalyze
CE formation by NADPH-and organic hydroperoxide-dependent mechanisms,
respectively. The major remaining task in this regard is identifying
form(s) of P450 that mediate organic hydroperoxide-dependent pathway of 2-
and 4-OH-CEs formation . This is required since this pathway is postulated
to e a major source of excess QEs and ROS. Identification will be
accomplished using the strategy of RACE (rapid amplification of cDNA ends),
using primers based on conserved sequences in the P450 superfamily; 2) To
identify additional gene products and markers of cell damage for monitoring
levels of oxidative stress in human breast parenchyma in histoculture
system to be established under Specific Aim 3: a) three phase II enzymes
that inactivate CEs and QEs, and that are inducible by electrophiles; b)
Ref-1, a redox-responsive protein that also functions to repair oxidative
DNA damage and; c) 4-hydroxynonenal, a major toxic reactive aldehyde
scission product of lipid peroxidation; 3) To characterize a histoculture
system of human breast parenchyma appropriate for critical testing of the
CE/OE hypothesis. Human breast parenchyma, maintained on GelFoam, will be
characterized with respect to: a) expression of enzymes mediating CE
formation via NADPH-and OHP-dependent mechanisms; b) expression of
putative markers of oxidative stress, identified under Specific Aim 2, and;
c) putative markers of oxidative damage to DNA or adducts of QEs. Once the
system is characterized the histoculture system will be used to test the
hypothesis by determining the effect on these markers of adding Es to the
histoculture, in the absence or presence of other potential sources of ROS.
Effective start/end date4/5/971/31/98


  • National Cancer Institute

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