Mechanism of metabolic activation and DNA adduct formation by the human carcinogen diethylstilbestrol: the defining link to natural estrogens

Abstract

Diethylstilbestrol (DES) is a human carcinogen, based on sufficient epidemiological evidence. DES is mainly metabolized to its catechol, 3'-hydroxyDES (3'-OH-DES), which can further oxidize to DES-3',4'-quinone (DES-3',4'-Q). Similarly to estradiol-3,4-quinone, the reaction of DES-3',4'-Q with DNA would form the depurinating 3'-OH-DES-6'-N3Ade and 3'-OH-DES-6'-N7Gua adducts. To prove this hypothesis, synthesis of DES-3',4'-Q by oxidation of 3'-OH-DES with Ag(2)O was tried; this failed due to instantaneous formation of a spiro-quinone. Oxidation of 3'-OH-DES by lactoperoxidase or tyrosinase in the presence of DNA led to the formation of 3'-OH-DES-6'-N3Ade and 3'-OH-DES-6'-N7Gua adducts. These adducts were tentatively identified by LC-MS/MS as 3'-OH-DES-6'-N3Ade, m/z = 418 [M+H](+), and 3'-OH-DES-6'-N7Gua, m/z = 434 [M+H](+). Demonstration of their structures derived from their oxidation by MnO(2) to the DES quinone adducts and subsequent tautomerization to the dienestrol (DIES) catechol adducts, which are identical to the standard 3'-OH-DIES-6'-N3Ade, m/z = 416 [M+H](+), and 3'-OH-DIES-6'-N7Gua, m/z = 432 [M+H](+), adducts. The reaction of DIES-3',4'-Q or lactoperoxidase-activated 3'-OH-DIES with DNA did not produce any depurinating adducts, due to the dienic chain being perpendicular to the phenyl planes, which impedes the intercalation of DIES into the DNA. Enzymic oxidation of 3'-OH-DES suggests that the catechol of DES intercalates into DNA and is then oxidized to its quinone to yield N3Ade and N7Gua adducts. These results suggest that the common denominator of tumor initiation by the synthetic estrogen DES and the natural estrogen estradiol is formation of their catechol quinones, which react with DNA to afford the depurinating N3Ade and N7Gua adducts.

Figure 1

Structures of natural and synthetic estrogens.

Figure 2

Formation of spiro-quinone by oxidation of 3′-OH-DES.

Figure 3

Oxidation of 3′-OH-Z,Z-DIES to Z,Z-DIES-3′,4′-Q and reaction of the quinone with Ade or dG to form N3Ade or N7Gua adducts.

Figure 4

Oxidation of 3′-OH-E,E-DIES to E,E-DIES-3′,4′-Q and reaction of the quinone with Ade or dG to form N3Ade or N7Gua adducts.

Figure 5

Oxidation of 3′-OH-4″-OCH₃-DES to 4″-OCH₃-DES-3,4-Q and reaction of the quinone with Ade or dG to form N3Ade or N7Gua adducts.

Figure 6

Formation of depurinating N3Ade and N7Gua adducts of DES following enzymic activation of 3′-OH-DES in the presence of DNA. The DES adducts were oxidized to the corresponding DIES adducts.

Figure 7

Unified mechanism of metabolic activation and reaction with DNA of the natural estrogens and the synthetic estrogens HES and DES.