The biochemical role of
estrogens in the development of
estrogen-dependent
breast cancer remains to be elucidated, and the involvement of
estrogens in
tumor initiation remains controversial. Reports regarding
estrogen-mediated DNA damage include the induction of
8-oxo-2'-deoxyguanosine (8-oxo-dG) in vitro and in vivo, indicating a role for oxidative stress in
tumor initiation and/or progression. However,
DNA isolation, cellular DNA repair, and high
antioxidant status have made the measurement of
8-oxo-dG in vivo and in cell culture somewhat challenging. In this regard, a potentiation in DNA damage can be achieved by depleting cellular stores of
glutathione. We chose to deplete
glutathione in the
estrogen receptor (ER)-positive MCF-7
breast cancer cell line with a
gamma-glutamylcysteine transpeptidase enzyme inhibitor buthionine sulphoximine (BSO) for the purpose of studying
estrogen-induced DNA damage. Treatment of GSH-depleted MCF-7 cells with 10 microM 2-OH-E2 or 4-OH-E2 for 30 min resulted in a statistically significant increase in 8-oxo-dG/10(5) dG of 127 and 160%, respectively. A potentiation in
catechol estrogen-induced DNA damage was observed with the addition of
copper(II) chloride for both 2-OH-E2 and 4-OH-E2 by 165 and 200%, respectively. In addition, 100 nM and 1.0 microM
estradiol increased DNA damage in a dose-response-like fashion by 145 and 189%, respectively. The depletion of GSH by BSO may prove to be an advantageous technique for the study of DNA damage in cells otherwise resistant to oxidative stress and/or
alkylating agents and has proven useful in the study of
estrogen-induced oxidative DNA damage in a highly reproducible and sensitive manner.