An o-quinone form of estrogen produces free radicals in human breast cancer cells: Correlation with DNA damage

Abstract

The o-quinone forms of 2,3- and 3,4-catechol estrogens have been implicated in the carcinogenicity of these hormones. The concomitant production of reactive oxygen species during reduction of the o-quinone estrogens has been inferred to play a mechanistic role in their mutagenic potential. Conclusive evidence documenting the production of hydrogen peroxide, the hydroxyl radical, and the estrone 3,4-semiquinone in estrone 3,4-quinone (3,4-EQ)-treated human breast cancer subcellular fractions was demonstrated in the absence of exogenously added catalysts. Subcellular fractions of MCF-7 cells treated with 3,4-EQ and NADPH, including nuclei, mitochondria, and microsomes, were shown to support significant amounts of hydrogen peroxide production. Hydrogen peroxide production in 3,4-EQ-treated cellular fractions and the chromosomal DNA damage induced in 3,4-EQ-treated MCF-7 cells were abolished by the addition of catalase. A significant and potentially physiologically relevant spontaneous reduction of 3,4-EQ by NADPH resulting in hydrogen peroxide production was demonstrated. The results unequivocally demonstrate that free radicals are produced during the metabolism of estrone 3,4-quinone in human cells.

Comparison of estrogen-derived ortho-quinone and para-quinol concerning induction of oxidative stress

“…The origin of this excess H 2 O 2 in tissue is principally O 2 •− (see Fig. 1) and involves the enzyme superoxide dismutase (SOD) [4,6,12]. The prooxidant effect of 3,4-E1Q was especially profound in the uterus homogenate of ovariectomized animals.…”

“…H 2 O 2 production in tissues was determined using the method described Nutter et al [12] with some modifications [10]. Briefly, brain, liver, retina and uterus homogenates (0.2% w/v in 0.1M PBS, pH 7.4, final volume of 100 μL) were incubated without and with E2 or the estrogen derivatives (100 μM), NADPH (200 μM) and sodium azide (20 μM) at 37°C for 30 minutes in a shaking water bath.…”

“…In order to prove this hypothesis, we incubated different rat tissue homogenates (brain, liver, uterus and retina) with para-quinol obtained from E2, and measured hydrogen peroxide (H 2 O 2 ) production compared to untreated control as an indicator of excess O 2 •− formation. In addition to E2, H 2 O 2 production induced by estra-1,5(10)-diene-3,4,17-trione (3,4-E1Q, an estrogen-derived ortho-quinone well-known as a potent prooxidant [12] and an endogenous tumor initiator [8]) was measured as a reference. We have also chosen 3,4-E1Q as a positive control in our experiments, because the isomeric estrogen-2,3-quinones are short-lived, which apparently reduces their prooxidant effects compared to the considerably longer-lived estrogen-3,4-quinones [7].…”

Mechanistic investigations on the antioxidant action of a neuroprotective estrogen derivative

“…Prooxidant activity of 1 and 2 in OVX brain homogenate was evaluated according to a previously published method based on the production of H 2 O 2 production at 37 °C [33]. Briefly, in brain homogenate (0.2% w/v in 0.1M PBS, pH 7.4, final volume of 100 µL) 100 µM test compounds 1, 2 or 3,4-ortho-quinone of estrone as positive control, NAD(P)H (200 µM) and sodium azide (20 µM) at 37°C for 30 minutes in a shaking water bath.…”

“…Such redox cycling is believed to be responsible, in part, for the carcinogenicity of estrogens [45]. The profoundly prooxidant estrone-3,4-quinone [33] was used as positive control in this experimental paradigm. The measured hydrogen peroxide production (given in nmol·h −1 · mg protein −1 ) in OVX rat brain homogenate clearly showed that the para-quinol to phenol (2→1) conversion does not produce oxidative stress compared to that of the control, while estrone-3,4-quinone produced approximately a 5-fold increase in H 2 O 2 production (Fig.…”