1887

There is a clear association between exposure to estrogens and initiation of cancer in various tissues. The estrogens estrone (E1) and estradiol (E2) are metabolized to reactive catechol estrogen quinones [E1(E2)-2,3-Q and E1(E2)-3,4-Q], which react with DNA to form depurinating N7Gua and N3Ade adducts. Abasic sites that are formed by depurination can induce mutations and initiate cancer. Hence, these quinones have been implicated as the ultimate metabolites responsible for estrogen carcinogenesis. NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase) is involved in phase II detoxification by catalyzing the two-electron reduction of quinones to hydroquinones. It has often been suggested to be involved in cancer prevention by detoxifying electrophilic quinones. In fact, our recent studies show that breast tissue from women without breast cancer has higher expression of the NQO1 enzyme than does breast tissue from women with breast cancer. Thus, in women with breast cancer, estrogen metabolism may be related to NQO1. Earlier studies have investigated the role of NQO1 in estrogen metabolism. It was concluded that E1-3,4-Q is spontaneously reduced to its catechol (4-OHE1) by the cofactor NADH alone, even without the presence of NQO1. This was interpreted as E1-3,4-Q not being a substrate for NQO1 (Nutter et. al. Chem. Res. Toxicol. 7:609, 1994). We have initiated the present work to find out whether NQO1 is involved in deactivation of estrogen quinones. An LC/MS/MS-based assay was developed to study the NQO1-catalyzed reduction of E2-3,4-Q. This assay estimates the formation of 4-OHE2 by LC/MS/MS, in the reaction mixture as a function of enzyme activity. In agreement with earlier studies, we have found that E2-3,4-Q is rapidly reduced by NADH without enzyme activity. However, for the first time, using this assay, we are able to show that NQO1 catalyzes the reduction of E2-3,4-Q when the cofactor tetrahydrofolic acid (THF), which has a lower reducing potential, is used. The assay mixture consisted of 25 mM Tris buffer, pH 6.2, BSA (0.7 mg/ml), NQO1 (0.2 μg/ml) and 50 μM E2-3,4-Q. The reaction was initiated by addition of THF (500 μM) to the assay mixture. Control assays were carried out without enzyme or cofactor. LC/MS/MS analysis of the extracted assay mixture shows the formation of 4-OHE2. The reduction of E2-3,4-Q in the assay was increased by 2.2 fold compared to control. THF also spontaneously reduced E2-3,4-Q to some extent. The involvement of the enzyme in the reduction of E2-3,4-Q was further supported by inhibitor studies. When dicumarol, a potent inhibitor of NQO1, was added to the assay mixture, the formation of 4-OHE2 was reduced by 35% compared to the control without inhibitor. The enzyme has a maximum activity at pH 6.2. These data suggest that E2-3,4-Q is a substrate and NQO1 might play a role in deactivation of estrogen quinones. (Supported by DoD grant DAMD17-03-1-0229 & U.S.P.H.S. grant P01 CA49210)

[Proc Amer Assoc Cancer Res, Volume 47, 2006]