Activation and irreversible binding of regiospecifically labeled catechol estrogen by rat liver microsomes: evidence for differential cytochrome P-450 catalyzed oxidations

Estradiol and 2-hydroxyestradiol labeled with 3H at different positions in rings A or B were incubated with male rat liver microsomes, and their oxidative transformation was followed by the transfer of 3H into 3H2O. 14C-labeled estrogen or catechol estrogen was used to determine the fraction that becomes bound covalently to microsomal protein. The further metabolism of 2-hydroxyestradiol involves activation of the steroid at C-4 and, to a much lesser extent at C-1, by a cytochrome P-450 mediated reaction as indicated by the effects of NADPH, spermine, SKF-525A, and CO in the microsomal system. Glutathione promoted the loss of 3H from C-4 of either estradiol or 2-hydroxyestradiol but had less effect on this reaction at C-1 and inhibited it at C-6,7. It also abolished the irreversible binding of 14C-labeled estradiol and 2-hydroxyestradiol to microsomal protein. NADPH was needed specifically for glutathione to exert its effect both on the transfer of 3H into 3H2O and on the formation of water-soluble products from catechol estrogen by rat liver microsomes. It could not be replaced by NADP, NAD, or NADH. Ascorbic acid inhibited these enzymatic reactions but did not affect significantly the initial 2-hydroxylation of estradiol. Evidence is also provided for the further hydroxylation of 2-hydroxyestradiol at C-6 (or C-7). These results indicate that cytochrome P-450 activates catechol estrogens by an electron abstraction process.     

Differential effect of Cu2+ and Zn2+ on the formation and further metabolism of catechol estrogen by rat liver microsomes

The action of a number of different divalent metal ions on the rat liver microsomal release of 3H2O from estradiol and 2-hydroxyestradiol labeled with 3H at C-2 or C-4 was investigated. Cu2+ at low concentration (10 microM) produced a marked and specific inhibition of the 2-hydroxylation of estradiol with virtually no effect on the further oxidative activation of catechol estrogen. In contrast, Zn2+ inhibited the interaction of 2-hydroxyestradiol with microsomal protein as measured by the release of 3H from C-4 of the labeled steroids but did not influence 2-hydroxylation, except at high concentration. Other metal ions tested produced little or no change. Cu2+ inhibited the irreversible binding of estradiol to protein but activated this reaction with the catechol estrogen as substrate. The action of both Cu2+ and Zn2+ was reversed by glutathione. The differential effect of these metal ions on estrogen metabolism gives additional support for two different mechanisms in the cytochrome P-450-catalyzed formation of catechol estrogens and their further activation to form protein conjugates.     

The characteristic binding of catechol estrogens to estrogen receptors in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors

The binding of catechol estrogens (2-hydroxyestrone, 4-hydroxyestrone, 2-hydroxyestradiol, and 4-hydroxyestradiol) to estrogen receptors in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumor cytosols was investigated. Cytosol estrogen receptors exhibited high affinities (Ka = 1.12-1.88 X 10(8) M-1) for all catechol estrogens as well as estradiol. The receptor level of catechol estrogens (46.1-97.5 fmol/mg protein) was 1.6-3.0 times higher than that of estradiol; especially the binding of 4-hydroxyestrone to estrogen receptors was the highest of all catechol estrogens and estradiol. In judging the receptor level of more than 20 fmol/mg protein to be positive, the binding of catechol estrogens to estrogen receptors was approximately correlated with that of estradiol. The positive receptor level of catechol estrogens was found in a half of tumor cytosols which showed the negative receptor level of estradiol. These results suggested that characteristic estrogen receptors indicating high affinities for catechol estrogens might be present in rat mammary tumor cytosols.     

4-Hydroxyestradiol is conjugated with thiols primarily at C-2: evidence from regiospecific displacement of tritium by rat liver microsomes or tyrosinase

4-Hydroxyestradiol bearing a 3H label specifically at C-2 was prepared chemically and incubated with male rat liver microsomes or mushroom tyrosinase. A very high proportion (80-90%) of the 3H was displaced from the labeled steroid when either glutathione or N-acetylcysteine was present, and tyrosinase was shown not to require NADPH as cofactor for this reaction. In either case, only negligible amounts (less than 3%) of the 3H radioactivity were found associated with water-soluble adducts in contrast to 3H-labeled 2-hydroxyestradiol, which gave rise to about 25% of such products. The effect of ascorbic acid on the microsomal reaction with regiospecifically labeled estradiol, 2-hydroxyestradiol, and 4-hydroxyestradiol was also investigated, and the results are discussed in terms of the reactivity at different carbon atoms in ring A of the catechol estrogens. All the evidence points to conjugation of 4-hydroxyestradiol with glutathione or N-acetylcysteine at C-2 but not C-1 of this highly reactive catechol estrogen. Measuring the displacement of 3H as 3H2O from specific positions in the steroid ring provides a useful and sensitive method to assess the formation of adducts in cases where their isolation and characterization is particularly difficult.     

The metabolic fate of [H]estradiol in relation to dietary intake of boron in ovariectomized rats

It has been reported that boron (B) deprivation reversibly lowers plasma estradiol levels in postmenopausal women. In order to establish whether this reflects disturbances in the estrogen catabolic pathway and in particular in catechol estrogen metabolism, the influence of dietary B on the catabolism of [³H]estradiol-17β has been studied in ovariectomized rats. Rats were given diets containing <0.1 or 40 mg B.kg⁻¹, ovariectomized and then infused with [³H]estradiol-17β using osmotic pumps. Analysis of urine samples for conjugated, catechol and non-catechol estrogens did not reveal any effects of B on the recovery or the metabolic fate of tritium from the infused estradiol. These results do not therefore support the proposal that B influences estrogen catabolism by interacting with catechol estrogens.     

Formation of the depurinating N3adenine and N7guanine adducts by reaction of DNA with hexestrol-3',4'-quinone or enzyme-activated 3'-hydroxyhexestrol. Implications for a unifying mechanism of tumor initiation by natural and synthetic estrogens

The nonsteroidal synthetic estrogen hexestrol (HES), which is diethylstilbestrol hydrogenated at the C-3-C-4 double bond, is carcinogenic. Its major metabolite is the catechol, 3'-OH-HES, which can be metabolically converted to the catechol quinone, HES-3',4'-Q. Study of HES was undertaken with the scope to substantiate evidence that natural catechol estrogen-3,4-quinones are endogenous carcinogenic metabolites. HES-3',4'-Q was previously shown to react with deoxyguanosine to form the depurinating adduct 3'-OH-HES-6'-N7Gua by 1,4-Michael addition [Jan S-T, Devanesan PD, Stack DE, Ramanathan R, Byun J, Gross ML, et al. Metabolic activation and formation of DNAadducts of hexestrol,a synthetic nonsteroidal carcinogenic estrogen. Chem Res Toxicol 1998;11:412-9.]. We report here formation of the depurinating adduct 3'-OH-HES-6'-N3Ade by reaction of HES-3',4'-Q with Ade by 1,4-Michael addition. The structure of the N3Ade adduct was established by NMR and MS. We also report here formation of the depurinating 3'-OH-HES-6'-N7Gua and 3'-OH-HES-6'-N3Ade adducts by reaction of HES-3',4'-Q with DNA or by activation of 3'-OH-HES by tyrosinase, lactoperoxidase, prostaglandin H synthase or 3-methylcholanthrene-induced rat liver microsomes in the presence of DNA. The N3Ade adduct was released instantaneously from DNA, whereas the N7Gua adduct was released with a half-life of approximately 3 h. Much lower (<1%) levels of unidentified stable adducts were detected in the DNA from these reactions. These results are similar to those obtained by reaction of endogenous catechol estrogen-3,4-quinones with DNA. The similarities extend to the instantaneously-depurinating N3Ade adducts and relatively slowly-depurinating N7Gua adducts. The endogenous estrogens, estrone and estradiol, their 4-catechol estrogens and HES are carcinogenic in the kidney of Syrian golden hamsters. These results suggest that estrone (estradiol)-3,4-quinones and HES-3',4'-Q are the ultimate carcinogenic metabolites of the natural and synthetic estrogens, respectively. Reaction of the electrophilic quinones by 1,4-Michael addition with DNA at the nucleophilic N-3 of Ade and N-7 of Gua is suggested to be the major critical step in tumor initiation by these compounds.     

Determination of estradiol 2- and 4-hydroxylase activities by gas chromatography with electron-capture detection

A highly sensitive assay has been developed for measuring the rate of formation of 2-hydroxyestradiol and 4-hydroxyestradiol from estradiol by microsomal preparations. Catechol estrogens were converted to heptafluorobutyryl esters, which were separated by capillary column gas chromatography and quantified using electron-capture detection. 2-Hydroxyestradiol 17-acetate was used as an internal standard. The identity of catechol estrogen derivatives was verified by gas chromatography—mass spectrometry using negative-ion chemical ionization. Estrogens were identified by negative molecular ions and/or by characteristic fragments. This procedure permits quantification of catechol estrogens at the subpicogram level. The assay was validated by comparing estrogen 2- and 4-hydroxylase activities in microsomes from hamster and rat liver with values reported previously.     

Slow loss of deoxyribose from the N7deoxyguanosine adducts of estradiol-3,4-quinone and hexestrol-3',4'-quinone. Implications for mutagenic activity

A variety of evidence has been obtained that estrogens are weak tumor initiators. A major step in the multi-stage process leading to tumor initiation involves metabolic formation of 4-catechol estrogens from estradiol (E2) and/or estrone and further oxidation of the catechol estrogens to the corresponding catechol estrogen quinones. The electrophilic catechol quinones react with DNA mostly at the N-3 of adenine (Ade) and N-7 of guanine (Gua) by 1,4-Michael addition to form depurinating adducts. The N3Ade adducts depurinate instantaneously, whereas the N7Gua adducts depurinate with a half-life of several hours. Only the apurinic sites generated in the DNA by the rapidly depurinating N3Ade adducts appear to produce mutations by error-prone repair. Analogously to the catechol estrogen-3,4-quinones, the synthetic nonsteroidal estrogen hexestrol-3',4'-quinone (HES-3',4'-Q) reacts with DNA at the N-3 of Ade and N-7 of Gua to form depurinating adducts. We report here an additional similarity between the natural estrogen E2 and the synthetic estrogen HES, namely, the slow loss of deoxyribose from the N7deoxyguanosine (N7dG) adducts formed by reaction of E2-3,4-Q or HES-3',4'-Q with dG. The half-life of the loss of deoxyribose from the N7dG adducts to form the corresponding 4-OHE2-1-N7Gua and 3'-OH-HES-6'-N7Gua is 6 or 8 h, respectively. The slow cleavage of this glycosyl bond in DNA seems to limit the ability of these adducts to induce mutations.     

Prostaglandin H synthase catalyzes regiospecific release of tritium from labeled estradiol

Prostaglandin H synthase (PHS) from ram seminal vesicle microsomes was found to catalyze the release of tritium (3H) from estradiol (E2) regiospecifically labeled in position C-2 or C-4 of ring A but not from positions C-17 alpha, C-16 alpha, or C-6,7. Formation of 3H2O from ring A of E2 is dependent upon native enzyme supplemented with either arachidonic acid, eicosapentaenoic acid, or hydrogen peroxide and proceeds very rapidly as do other cooxidation reactions catalyzed by PHS-peroxidase. The 3H-loss from ring A of E2 reflecting oxidative displacement of this isotope by PHS increases linearly up to 100 microM under our conditions (8-45 nmol/mg x 5 min). Loss of tritium in various blanks is negligible by comparison. Indomethacin (0.07 and 0.2 mM) inhibited the PHS-dependent release of 3H2O from estradiol but less efficiently than it inhibited DES-cooxidation measured in parallel incubations under similar conditions. Addition of EDTA (0.5 mM) had no effect on the regiospecific transfer of 3H from E2 or on DES-oxidation; ascorbic acid (0.5 mM) or NADH (0.33 mM) clearly inhibited both reactions and to a similar extent. These data suggest that estradiol-2/4-hydroxylation can be catalyzed by PHS in vitro probably via its peroxidase activity and point to PHS as an enzyme that could contribute to catechol estrogen formation in vitro by tissue preparations in the presence of unsaturated fatty acids or peroxides.     

Influence of indole-3-carbinol on the hepatic microsomal formation of catechol estrogens.

The oral administration of indole-3-carbinol (IC), present in cabbage and other members of the Cruciferae family, to female rats almost doubled their ability to convert estradiol to catechol estrogens in the liver. This was determined by the release of 3H from C-2 of the estrogen and also by isolation of the 14C-labeled catechol derivative after incubation with hepatic microsomal fractions. The yield of 4-hydroxyestradiol was also elevated and these effects were similar to those produced by 3-methylcholanthrene (MC), a well-characterized cytochrome P450 inducer. Further evidence for the involvement of a mixed-function oxidase was provided by a 70% to 80% decrease in the yield of 3H2O and water-soluble radioactivity by SKF-525A (0.1 mM) when added to the microsomal fractions isolated from the livers of control or IC-treated rats. In addition, NADPH could not be replaced by NADH in these experiments. Pretreatment with ethionine prevented the increase in estradiol metabolism brought about by oral administration of IC. Both IC and MC inhibited catechol estrogen formation when added directly to the liver microsomal system, confirming earlier findings that in vivo inducers can act as in vitro inhibitors. However, IC was less inhibitory than MC, supporting the theory that IC is converted to a more active product in the stomach. Thus, IC may be conferring protection against estrogen-dependent neoplasia by increasing the hepatic oxidation of estradiol, thereby lowering the amount of available active estrogen.     

Unbalanced metabolism of endogenous estrogens in the etiology and prevention of human cancer

Among the numerous small molecules in the body, the very few aromatic ones include the estrogens and dopamine. In relation to cancer initiation, the estrogens should be considered as chemicals, not as hormones. Metabolism of estrogens is characterized by two major pathways. One is hydroxylation to form the 2- and 4-catechol estrogens, and the second is hydroxylation at the 16α position. In the catechol pathway, the metabolism involves further oxidation to semiquinones and quinones, including formation of the catechol estrogen-3,4-quinones, the major carcinogenic metabolites of estrogens. These electrophilic compounds react with DNA to form the depurinating adducts 4-OHE(1)(E(2))-1-N3Ade and 4-OHE(1)(E(2))-1-N7Gua. The apurinic sites obtained by this reaction generate the mutations that may lead to the initiation of cancer. Oxidation of catechol estrogens to their quinones is normally in homeostasis, which minimizes formation of the quinones and their reaction with DNA. When the homeostasis is disrupted, excessive amounts of catechol estrogen quinones are formed and the resulting increase in depurinating DNA adducts can lead to initiation of cancer. Substantial evidence demonstrates the mutagenicity of the estrogen metabolites and their ability to induce transformation of mouse and human breast epithelial cells, and tumors in laboratory animals. Furthermore, women at high risk for breast cancer or diagnosed with the disease, men with prostate cancer, and men with non-Hodgkin lymphoma all have relatively high levels of estrogen-DNA adducts, compared to matched control subjects. Specific antioxidants, such as N-acetylcysteine and resveratrol, can block the oxidation of catechol estrogens to their quinones and their reaction with DNA. As a result, the initiation of cancer can be prevented.     

Inhibition by bromoestrogens of the effects of estradiol on apomorphine-induced climbing behavior

The chronic administration of estrogens to mice or rats will result in antidopaminergic effects. Apomorphine-induced climbing behavior in mice, the result of direct stimulation of dopamine receptors in the striatal and mesolimbic regions, is a simple animal model for examining these antidopaminergic effects of estrogens. Bromoestrogens, inhibitors of catechol estrogen formation, have been utilized in order to examine the role of estrogen metabolism in dopaminergic antagonism. Mice were pretreated for 3 days with 2-bromoestradiol, 4-bromoestradiol, or 2,4-dibromoestradiol dibenzoates alone or in combination with estradiol benzoate prior to apomorphine administration. The haloestrogens did not alter the climbing-induced responses elicited by apomorphine, whereas estradiol benzoate clearly attentuated the actions of apomorphine. Furthermore, the bromoestradiol dibenzoates were effective in reversing the effects of estradiol benzoate when the two steroids (estradiol benzoate and a bromoestrogen dibenzoate) were administered simultaneously during pretreatment. Thus, the bromoestrogens are able to inhibit the antidopaminergic effects of estradiol exhibited in the apomorphine-induced mouse climbing model.     

Diaryl dimers of estradiol and of estrone may be formed as major metabolites by mouse mammary glands

The formation of a novel estrogen metabolite by mammary tissues was investigated. Polar and nonpolar metabolites of endogenous estrogens are formed in liver and other tissues. Polar products such as the catechol estrogens are implicated in tumorigenesis in breast tissue, whereas a nonpolar metabolite, 2-methoxyestradiol, may be protective. Diaryl ether dimers, as a novel form, have been reported as nonpolar products from liver microsomes. We have noted major amounts of nonpolar metabolites in other tissues that were neither 2-methoxyestrogens nor estrogen fatty acid esters. The possible formation of such novel metabolites by breast tissues from adult nulliparous mice with [³H]-labeled estrogens as substrates was considered. Steroids were recovered from media by solid-phase extraction and profiles were obtained from HPLC (acetonitrile:water). Saponification was done with an internal standard of estradiol stearate. Major amounts of nonpolar metabolites were formed in all instances, with one or two principal peaks. Alkaline hydrolysis had no effect on the nonpolar product(s) but released estradiol from its stearate. Strong acid treatment also had no effect as shown by HPLC. Thus, it is suggested that diaryl dimers of estrogens may be formed as major metabolites by mouse mammary glands.     

Raloxifene and desmethylarzoxifene block estrogen-induced malignant transformation of human breast epithelial cells

There is association between exposure to estrogens and the development and progression of hormone-dependent gynecological cancers. Chemical carcinogenesis by catechol estrogens derived from oxidative metabolism is thought to contribute to breast cancer, yet exact mechanisms remain elusive. Malignant transformation was studied in MCF-10A human mammary epithelial cells, since estrogens are not proliferative in this cell line. The human and equine estrogen components of estrogen replacement therapy (ERT) and their catechol metabolites were studied, along with the influence of co-administration of selective estrogen receptor modulators (SERMs), raloxifene and desmethyl-arzoxifene (DMA), and histone deacetylase inhibitors. Transformation was induced by human estrogens, and selectively by the 4-OH catechol metabolite, and to a lesser extent by an equine estrogen metabolite. The observed estrogen-induced upregulation of CYP450 1B1 in estrogen receptor negative MCF-10A cells, was compatible with a causal role for 4-OH catechol estrogens, as was attenuated transformation by CYP450 inhibitors. Estrogen-induced malignant transformation was blocked by SERMs correlating with a reduction in formation of nucleobase catechol estrogen (NCE) adducts and formation of 8-oxo-dG. NCE adducts can be formed consequent to DNA abasic site formation, but NCE adducts were also observed on incubation of estrogen quinones with free nucleotides. These results suggest that NCE adducts may be a biomarker for cellular electrophilic stress, which together with 8-oxo-dG as a biomarker of oxidative stress correlate with malignant transformation induced by estrogen oxidative metabolites. The observed attenuation of transformation by SERMs correlated with these biomarkers and may also be of clinical significance in breast cancer chemoprevention.     

Active nonaromatic intermediates in the conversion of steroidal estrogens into catechol estrogens

A mechanism is proposed for mixed-function oxidase-catalyzed formation of the catechol estrogens 2-hydroxy- and 4-hydroxyestradiol from estradiol. This mechanism involves nonaromatic epoxyenones as intermediates. The isomeric 1 alpha,2 alpha-epoxy-17 beta-hydroxyestr-4-en-3-one and 1 beta,2 beta-epoxy-17 beta-hydroxyestr-4-en-3-one (the latter as its 17-acetate) were synthesized from 17 beta-hydroxy-5 alpha-estran-3-one. The isomeric 4 alpha,5 alpha-epoxy-17 beta-hydroxyestr-1-en-3-one and 4 beta,5 beta-epoxy-17 beta-hydroxyestr-1-en-3-one were prepared from 19-nortestosterone. From incubations of [6,7-3H]estradiol with microsomes from MCF-7 human breast cancer cells, which principally catalyze the formation of 2-hydroxyestradiol from estradiol, we were able to isolate a 3H-labeled product with the chromatographic properties of 1 beta, 2 beta-epoxy-17 beta-hydroxyestr-4-en-3-one (as its 17-acetate). The soluble protein fraction of homogenates of rat liver, which is devoid of estrogen 2-/4-hydroxylase activity, has been shown to catalyze the formation of 2- and 4-hydroxyestradiol from the 1 alpha,2 alpha-epoxide and from the 4 alpha,5 alpha- and 4 beta,5 beta-epoxides, respectively. We suggest that these results taken together strongly support a role for epoxyenones as intermediates in the formation of catechol estrogens.     

Is bisphenol A a weak carcinogen like the natural estrogens and diethylstilbestrol?

Bisphenol A (BPA) displays weak estrogenic properties and could be a weak carcinogen by a mechanism similar to that of estrone (E(1)), estradiol (E(2)) and the synthetic estrogen diethylstilbestrol, a human carcinogen. A wide variety of scientific evidence supports the hypothesis that certain estrogen metabolites, predominantly catechol estrogen-3,4-quinones, react with DNA to cause mutations that can lead to the initiation of cancer. One of the major pathways of estrogen metabolism leads to the 4-catechol estrogens, 4-OHE(1)(E(2)), which are oxidized to their quinones, E(1)(E(2))-3,4-Q. The quinones react with DNA to form predominantly the depurinating adducts 4-OHE(1)(E(2))-1-N3Ade and 4-OHE(1)(E(2))-1-N7Gua. This process constitutes the predominant pathway in the initiation of cancer by estrogens. One pathway of BPA metabolism is hydroxylation of one of its symmetric benzene rings to form its catechol, 3-OHBPA. Subsequent oxidation to BPA-3,4-quinone would lead to reaction with DNA to form predominantly the depurinating adducts 3-OHBPA-6-N3Ade and 3-OHBPA-6-N7Gua. The resulting apurinic sites in the DNA could generate mutations in critical genes that can initiate human cancers. The catechol of BPA may also alter expression of estrogen-activating and deactivating enzymes, and/or compete with methoxylation of 4-OHE(1)(E(2)) by catechol-O-methyltransferase, thereby unbalancing the metabolism of estrogens to increase formation of E(1)(E(2))-3,4-Q and the depurinating estrogen-DNA adducts leading to cancer initiation. Thus, exposure to BPA could increase the risk of developing cancer by direct and/or indirect mechanisms. Knowledge of these mechanisms would allow us to begin to understand how BPA may act as a weak carcinogen and would be useful for regulating its use.     

Estrogen carcinogenesis in Syrian hamster tissues: role of metabolism

Evidence for a role of estrogen metabolism in hormonal carcinogenesis was obtained with the Syrian hamster as an in vivo model system. Both natural and synthetic estrogens are capable of inducing a high incidence of renal carcinomas in this species. A high incidence of hepatocellular carcinomas can also be induced in the hamster with synthetic estrogens such as ethinyl estradiol or diethylstilbestrol, provided alpha-naphthoflavone (ANF) is present in the diet. Although steroid receptor-mediated hormonal events appear to be intimately involved in the process of in vivo cell transformation of both tissues, certain observations strongly suggest that nonhormonal events are also important. Despite their potent estrogenic activity at the doses used, ethinyl estradiol and alpha-zearalanol induce relatively low renal tumor incidences after 9.0 and 10.0 months of continuous treatment, respectively. A role for the metabolism of estrogens to reactive intermediates is also suggested by studies showing estrogen-induced renal tumorigenesis can be partially inhibited by concomitant administration of ANF or ascorbic acid. Consistent with this is the general correlation between the amount of catechol estrogen formed by a compound, as mediated by estrogen 2-/4-hydroxylase, and renal carcinogenicity data. Recently, additional supporting evidence has been obtained from studies involving the irreversible binding of reactive metabolites of steroidal or stilbene estrogens to hamster liver microsomal proteins.     

Resveratrol and N-acetylcysteine block the cancer-initiating step in MCF-10F cells

Substantial evidence suggests that catechol estrogen-3,4-quinones react with DNA to form predominantly the depurinating adducts 4-hydroxyestrone (estradiol)-1-N3Ade [4-OHE(1)(E(2))-1-N3Ade] and 4-OHE(1)(E(2))-1-N7Gua. Apurinic sites resulting from these adducts generate critical mutations that can initiate cancer. The paradigm of cancer initiation is based on an imbalance in estrogen metabolism between activating pathways that lead to estrogen-DNA adducts and deactivating pathways that lead to estrogen metabolites and conjugates. This imbalance can be improved to minimize formation of adducts by using antioxidants, such as resveratrol (Resv) and N-acetylcysteine (NAcCys). To compare the ability of Resv and NAcCys to block formation of estrogen-DNA adducts, we used the human breast epithelial cell line MCF-10F treated with 4-OHE(2). Resv and NAcCys directed the metabolism of 4-OHE(2) toward protective pathways. NAcCys reacted with the quinones and reduced the semiquinones to catechols. This pathway was also carried out by Resv. In addition, Resv induced the protective enzyme quinone reductase, which reduces E(1)(E(2))-3,4-quinones to 4-OHE(1)(E(2)). Resv was more effective at increasing the amount of 4-OCH(3)E(1)(E(2)) than NAcCys. Inhibition of estrogen-DNA adduct formation was similar at lower doses, but at higher doses Resv was about 50% more effective than NAcCys. Their combined effects were additive. Therefore, these two antioxidants provide an excellent combination to protect catechol estrogens from oxidation to catechol quinones.     

Peroxidase-catalyzed displacement of tritium from regiospecifically labeled estradiol and 2-hydroxyestradiol

Estradiol and 2-hydroxyestradiol with 3H at different positions in rings A, B or D were incubated with lactoperoxidase without added H2O2 and their oxidative transformation was followed by transfer of 3H into 3H2O. With estradiol, 3H loss from different positions in the aromatic ring was almost equal and also occurred to a lesser extent from the alicyclic portion of the molecule. Glutathione had less effect on the formation of 3H2O for the aromatic ring of estradiol than from that of the catechol estrogen where it increased the yield 6-fold. The rate of 3H loss was also very much greater from tritiated 2-hydroxyestradiol than from estradiol and NADPH was inhibitory with both steroids. Conditions for the release of 3H from estradiol and 2-hydroxyestradiol by peroxidase as well as the effect of some biochemical inhibitors were also investigated. The possible contribution of peroxidative formation of 3H2O during the radiometric assay for catechol estrogen biosynthesis by tissue monooxygenases is discussed.