Effective synthesis of a hexacyclic-steroid derivative from 4-hydroxyestrone.

Several studies have been reported for the preparation of hexacyclic-steroid derivatives; however, some reagents are expensive and require special conditions for handling. In this way, the objective of this study was to synthesize a hexacyclic-steroid derivative from 4-hydroxyestrone. The chemical structure was evaluated through both 1H NMR and 13C NMR spectroscopic analysis. The results showed good performance of the hexacyclic-steroid derivative. In conclusion in this study, an easy method for the preparation of the hexacyclic-steroid derivative is reported.

16α-Hydroxyestrone: Mass Spectrometry-Based Methodologies for the Identification of Covalent Adducts Formed with Blood Proteins.

Elevated levels of the estrone metabolite, 16α-hydroxyestrone (16αOHE1), have been linked with multiple diseases. As an electrophilic reactive metabolite, covalent binding to proteins is thought to constitute one of the possible mechanisms in the onset of deleterious health outcomes associated with 16αOHE1. Whereas mass spectrometry (MS)-based methodologies are currently considered the best suited to monitor the formation of protein covalent adducts, the application of these approaches for the identification of covalent adducts of 16αOHE1 is yet to be provided. In the present study, with the ultimate goal of determining the most adequate methodology for searching for 16αOHE1-derived covalent adducts, we explored multiple liquid chromatography-electrospray ionization tandem high-resolution mass spectrometry (LC-ESI-HRMS/MS)-based approaches to investigate the nature and specific locations of the covalent adducts produced in human hemoglobin (Hb) and human serum albumin (HSA) modified in vitro with 16αOHE1. The application of a "bottom up" proteomics approach, involving the nanoLC-ESI-HRMS/MS analysis of tryptic peptides, allowed the identification of multiple sites of 16αOHE1 adduction in Hb and HSA. As expected, the majority of the adducted peptides occurred in lysine residues following stabilization of the Schiff base formed with 16αOHE1 by reduction or via Heyns rearrangement, yielding the stable α-hydroxyamine and ketoamine adducts, respectively. Noteworthy is the fact that a serine residue was also identified to be covalently modified with 16αOHE1, which to our knowledge constitutes a first-hand report of a keto electrophile as target of hydroxyl-based nucleophilic amino acids. The N-alkyl Edman degradation resulted to be unsuitable for the identification of 16αOHE1adducts formed with the N-terminal valine of Hb, most probably due to stereochemical restraints of the tested derivatizing agents (fluorescein isothiocyanate and phenyl isothiocyanate) on assessing these bulky covalent adducts. Nonetheless, the digestion of adducted proteins to amino acids resulted in the detection of 16αOHE1-derived keto and α-hydroxyamine Lys adducts. The simplicity of this methodology might be beneficial for clinical studies, with the possibility of offering quantitative information with the preparation of synthetic standards of these adducts. The results obtained are crucial not only for the identification and quantification of biomarkers of exposure to 16αOHE1 but also for clarifying the role of protein binding in the onset of diseases associated with elevated levels of this reactive metabolite.

Detection of 16α-Hydroxyestrone-histone 1 Adduct as High-Affinity Antigen for Rheumatoid Arthritis Autoantibodies.

Increased concentrations of 16α-hydroxyestrone (16α-OHE1) have been observed in rheumatoid arthritis (RA), but the underlying mechanism of this remains elusive. Here we aimed to identify the role played by 16α-OHE1 in RA. In 40 RA patients, the specificities of antibodies from the sera of these patients were checked by direct binding, inhibition ELISA, and quantitative precipitation titration. Competition ELISA was also used for the estimation of 16α-OHE1 in the serum of different RA patients. RA IgG from a patient's sera showed strong recognition to 16α-OHE1-H1 (histone 1) adduct in comparison to control subjects (p < 0.001), as the formation of this adduct brings out various biochemical changes that might generate neo-epitopes, which have been well-recognized by these antibodies. The affinity of RA antibodies for 16α-OHE1-H1 (1.10 × 10- 7 M) was high, as detected by the Langmuir plot. Comparing RA patients to the controls, no significant differences were detected in the level of 16α-OHE1 or 2-hydroxyestrone/16α-OHE1 ratio. 16α-OHE1-H1 might have an antigenic role and function as a high-affinity antigen for RA autoantibodies and, therefore, could be used as a biomarker for this disease.

A stable epoxide as a potential endogenous estrogen metabolite: Possible significance in breast cancer?

Epoxides as reactive intermediates of estrogen metabolism have been considered to be potential precursors of the 2- and 4-hydroxy, catechol estrogens and even to be mutagenic/carcinogenic agents themselves. The labile nature of the intermediates has made proof of their existence difficult in natural biological conditions. In our studies on estrogen metabolism in vitro, in various tissues from several laboratory and domestic species, there was chromatographic evidence of formation of a stable estrogen metabolite that could be seen after incubation with radiolabeled estrone, but not with unlabeled substrate. Investigation with acid treatment of the metabolite yielded material detected as 6-hydroxy-estrone-suggesting the presence of an additional oxygen atom in the molecule. An identification of the "unknown compound" has not yet been made but, with this evidence, the properties revealed so far can best be met by assuming the presence of 5,6-epoxy-estrone. The recent favorable reports on the role of 5α,6α-epoxy-cholesterol in breast cancer has led to the hypothesis that the formation of a similar, stable epoxide of an estrogen could potentially be a compound of interest. If a metabolic pathway from estrone to 6-hydroxy-estrone through a stable epoxide has indeed been observed, it would suggest that identifying and screening for the enzymes responsible for its production, as opposed to those generating the catecholestrogens, could provide valuable information in relation to breast cancer. The balance in production of estrogen epoxides could be a key factor in determining normal health or risk of tumor development.

Redox cycling of catechol estrogens generating apurinic/apyrimidinic sites and 8-oxo-deoxyguanosine via reactive oxygen species differentiates equine and human estrogens.

Metabolic activation of estrogens to catechols and further oxidation to highly reactive o-quinones generates DNA damage including apurinic/apyrimidinic (AP) sites. 4-Hydroxyequilenin (4-OHEN) is the major catechol metabolite of equine estrogens present in estrogen replacement formulations, known to cause DNA strand breaks, oxidized bases, and stable and depurinating adducts. However, the direct formation of AP sites by 4-OHEN has not been characterized. In the present study, the induction of AP sites in vitro by 4-OHEN and the endogenous catechol estrogen metabolite, 4-hydroxyestrone (4-OHE), was examined by an aldehyde reactive probe assay. Both 4-OHEN and 4-OHE can significantly enhance the levels of AP sites in calf thymus DNA in the presence of the redox cycling agents, copper ion and NADPH. The B-ring unsaturated catechol 4-OHEN induced AP sites without added copper, whereas 4-OHE required copper. AP sites were also generated much more rapidly by 4-OHEN. For both catechol estrogens, the levels of AP sites correlated linearly with 8-oxo-dG levels, implying that depuriniation resulted from reactive oxygen species (ROS) rather than depurination of estrogen-DNA adducts. ROS modulators such as catalase, which scavenges hydrogen peroxide and a Cu(I) chelator, blocked the formation of AP sites. In MCF-7 breast cancer cells, 4-OHEN significantly enhanced the formation of AP sites with added NADH. In contrast, no significant induction of AP sites was detected in 4-OHE-treated cells. The greater redox activity of the equine catechol estrogen produces rapid oxidative DNA damage via ROS, which is enhanced by redox cycling agents and interestingly by NADPH-dependent quinone oxidoreductase.

The 4-hydroxyestrone: Electron emission, formation of secondary metabolites and mechanisms of carcinogenesis.

4-Hydroxyestrone (4-OHE(1)), a typical cancer-inducing metabolite, originating from 17beta-estradiol (17beta-E2), was chosen as a model for the studies. The aim was to get a deeper insight in the mechanisms of its ability to initiate cancer. It was found, that 4-OHE(1) can eject electrons (e(aq)(-)), when excited in the singlet state by monochromatic UV-light (lambda=254 nm) in polar media (water:ethanol=40:60 vol.%). The quantum yield Q(e(aq)(-)), determined for various 4-OHE(1) concentrations, is found to be as high as that previously observed for 17beta-E2. It decreases with increasing substrate concentration, but it is enhanced at higher temperature. The ability of 4-OHE(1) to eject as well as to consume and to transfer electrons to other biological systems, classifies it as an electron mediator, similar to 17beta-E2. The 4-OHE(1) transients resulting of the electron emission process are leading to the formation of secondary metabolites. Surprisingly, it was established that the secondary metabolites possess likewise the ability to eject as well as to consume electrons. Hence, they behave similar like 17beta-E2. However, the structure of the secondary formed metabolites, which determinates their biological properties and carcinogenity, depends on the nature of the available reaction partners involved in their formation. A probable reaction mechanism explaining the subject matter is discussed.

Potential biomarker for early risk assessment of prostate cancer.

BACKGROUND: Catechol estrogen quinones (CEQ) derived from 4-hydroxyestrone (4-OHE1) and 4-hydroxyestradiol (4-OHE2) react with DNA to form depurinating--N7Gua and--N3Ade adducts. This damage leads to mutations that can initiate breast and prostate cancer. To determine whether this damage occurs in humans, urine samples from men with prostate cancer and benign urological conditions, and healthy controls were analyzed. The objective was determining whether any of the cancer patients had formed the depurinating 4-OHE1(E2)-1-N3Ade adducts. METHODS: The adducts were extracted from samples by using affinity columns equipped with a monoclonal antibody developed for detecting 4-OHE1(E2)-1-N3Ade adducts. Eluted extracts were separated by capillary electrophoresis with field-amplified sample stacking and/or ultraperformance liquid chromatography. Absorption/luminescence spectroscopies and mass spectrometry were used to identify the adducts. RESULTS: 4-OHE1-1-N3Ade was detected at higher levels in samples from subjects with prostate cancer (n = 7) and benign urological conditions (n = 4) compared to healthy males (n = 5). CONCLUSION: This is the first demonstration that CEQ-derived DNA adducts are present in urine samples from subjects with prostate cancer.

In vitro aromatic bioactivation of the weak estrogen E(2)alpha and genesis of DNA adducts.

Specific A-ring hydroxylated metabolites of 17beta-estrogens are known to be endogenous pro-carcinogens, more particularly the 4-hydroxylated forms of estrogens produced by cytochrome P4501B1. In this study, we investigated whether estradiol-17alpha, the main hepatic residue of estradiol-17beta in cattle treated for anabolic purposes with estradiol containing implants, could be significantly metabolized by human cells, and whether its aromatic metabolites could induce the formation of DNA adducts as estradiol-17beta and estrone do. First, using a human kidney adenocarcinoma cell line, which expresses specifically the cytochrome P4501B1, we showed that estradiol-17alpha is bioactivated into a mixture of 2- and 4-catechol estrogens leading to the corresponding methoxyestrogens unambiguously identified by LC-APCI-MS/MS. We then demonstrated that the 2- and 4-hydroxylated derivatives of estradiol-17alpha incubated under oxidative conditions with calf thymus DNA gave stable DNA adducts and abasic sites, respectively. From these results, we can consider that human cells expressing CYP1B1-dependent hydroxylation activities metabolize estradiol-17alpha at the same magnitude as estradiol-17beta and estrone, and that in oxidative conditions, the resulting aromatic metabolites can lead to the formation of both stable and unstable DNA adducts.

Structural and stereoisomer effects of model estrogen quinone-derived DNA adducts: N6-(2-hydroxyestron-6(alpha,beta)-yl)-2'-deoxyadenosine and N2-(2-hydroxyestron-6(alpha,beta)-yl)-2'-deoxyguanosine.

An extensive conformational analysis has been carried out for two diastereoisomeric pairs of model estrogen quinone-derived DNA adducts, N6-(2-hydroxyestron-6(alpha,beta)-yl)-2'-deoxyadenosine (2-OHE1-6(alpha,beta)-N6-dA) and N2-(2-hydroxyestron-6(alpha,beta)-yl)-2'-deoxyguanosine (2-OHE1-6(alpha,beta)-N2-dG), in a B-DNA duplex and at a primer-template junction in a pol alpha family DNA polymerase. In vitro primer extension studies in pol alpha [Terashima, I., et al. (1998) Biochemistry 37, 13807-13815] have shown that the adenine adducts can incorporate dT, together with a small proportion of the incorrect base dC opposite the lesion, and they block less strongly than the guanine adducts. We have carried out conformational searches with energy minimization for four DNA duplexes containing 2-OHE1-6alpha-N6-dA, 2-OHE1-6beta-N6-dA, 2-OHE1-6alpha-N2-dG, or 2-OHE1-6beta-N2-dG. Our searches revealed that the four-ring nonplanar 2-hydroxyestrone (2-OHE1) moiety strongly prefers to reside in the major groove of the adenine adducts or the minor groove of the guanine adducts in a B-DNA duplex, with stereochemistry-dependent orientational differences in each case. No low energy conformations involving intercalation of the 2-OHE1 moiety were located in the searches. This stems from the largely nonplanar, nonaromatic nature of the 2-OHE1 ring system and implies that the proclivity for such bulky, nonplanar adducts to reside at the DNA helix exterior is a plausible conformational feature of other structurally similar estrogen quinone-derived DNA adducts, independent of base sequence context. In addition, the adenine adduct isomers, located in the major groove, manifest serious disturbance to the Watson-Crick base pairs at and near the lesion site, suggesting repair susceptibility. Possible structures of these adducts in a pol alpha family polymerase were also investigated through molecular modeling. The results rationalized the experimental in vitro primer extension studies. In addition, poor accommodation of the beta-stereoisomers within the polymerase was noted, suggesting that these stereoisomers would be more prone to cause blockage. Stereochemistry-dependent differences in adduct orientation could be expected to produce different biochemical effects, as has been observed in adducts derived from polycyclic aromatic hydrocarbons.

Spectral characterization of catechol estrogen quinone (CEQ)-derived DNA adducts and their identification in human breast tissue extract.

Estrogens, including the natural hormones estrone (E(1)) and estradiol (E(2)), are thought to be involved in tumor induction. Catechol estrogen quinones (CEQ) derived from 4-hydroxyestrone (4-OHE(1)) and 4-hydroxyestradiol (4-OHE(2)) react with DNA and form depurinating N7Gua and N3Ade adducts that might be responsible for tumor initiation (Cavalieri, E. L., et al. (2000) J. Natl. Cancer Inst. Monogr. 27, 75). Current detection limits for the CEQ-derived DNA adducts by high-performance liquid chromatography with multichannel electrochemical detection are in the picomole range. To improve the limit of detection (LOD) for CEQ-derived DNA adducts, spectrophotometric monitoring was investigated. Spectroscopic studies of 4-OHE(1)-1-N3Ade, 4-OHE(1)-1-N7Gua, 4-OHE(2)-1-N3Ade, and 4-OHE(2)-1-N7Gua adduct standards were performed at 77 and 300 K. Upon laser excitation at 257 nm, the 4-OHE(1)- and 4-OHE(2)-derived N7Gua and N3Ade adducts are strongly phosphorescent at T = 77 K. No phosphorescence was observed at 300 K. Both N3Ade and N7Gua adduct types have weak phosphorescence origin bands near 383 and 385 nm, respectively. The corresponding phosphorescence lifetimes are 1.11 +/- 0.05 and 0.37 +/- 0.05 s. The LOD, based on phosphorescence measurements, is in the low femtomole range. The concentration LOD is approximately 10(-9) M, i.e., similar to that recently obtained for CEQ-derived N-acetylcysteine conjugates (Jankowiak, R., et al. (2003) Chem. Res. Toxicol. 16, 304). The LOD in capillary electrophoresis (CE) with field-amplified sample stacking and absorbance detection is about 3 x 10(-8) M. To verify whether CEQ-derived DNA adducts are formed in humans or not, tissue extracts from two breast cancer patients were analyzed by CE interfaced with room temperature absorption and low temperature (laser-excited) phosphorescence spectroscopies. For the first time, formation of CEQ-derived DNA adducts is shown in humans. For example, the level of 4-OHE(1)-1-N3Ade in the breast tissue extract from a patient with breast carcinoma (8.40 +/- 0.05 pmol/g of tissue) is larger by a factor of about 30 than that in the breast tissue sample from a woman without breast cancer (0.25 +/- 0.05 pmol/g of tissue). In contrast, similar amounts of 4-OHE(2)-1-N3Ade were observed in both types of tissue. Although more breast tissue samples from women with and without breast cancer need to be studied, these results suggest that the N3Ade adducts could serve as biomarkers to predict the risk of breast cancer.

Estradiol metabolism and malignant disease.

Endogenous estradiol metabolism results in metabolic products that are still capable of exerting various biological, partially estrogen-antagonistic actions. This indicates that the effects of estradiol in carcinogenesis may depend on individual variations of metabolic breakdown of estradiol. The aim of this paper is to review and discuss the available data relating to stimulatory and inhibitory properties of estradiol metabolites on carcinogenesis. Results of main D-ring metabolites and main A-ring metabolites are presented. There are indications that the endogenous production of growth influencing estradiol metabolites may be elevated in neoplasias. Some results in this respect are available for stimulating tumor growth for the D-ring metabolite 16-hydroxyestrone and the A-ring metabolites 4-hydroxyestrone and 4-hydroxyestradiol. Inhibitory effects exist for the A-ring metabolite 2-methoxyestradiol (2-ME). So far, only a few metabolites have been studied closely for their influence on carcinogenesis. There is also a dearth of data on the intracellular metabolism of estradiol in neoplastic tissues. Knowledge of the metabolites may reveal new approaches to diagnosis and treatment of malignant diseases. 2-ME has already shown actions in pharmacological dosages which led already to a first trial to prove its suitability for treating human breast cancer.

In vitro generation of peroxynitrite by 2- and 4-hydroxyestrogens in the presence of nitric oxide.

Estrogen metabolism is altered in most, if not all, breast cancer tumors. These alterations primarily lead to the formation of the catechol estrogen metabolites, 2- and 4-hydroxyestrogens, which can generate superoxide anion radicals (O(2)(*)(-)) through the redox cycling of semiquinone/quinone derivatives. In breast cancer cells, the activity of nitric oxide synthase is also frequently elevated, resulting in an increased level of exposure to nitric oxide ((*)NO). Since (*)NO rapidly reacts with O(2)(*)(-) to produce the peroxynitrite anion (ONOO(-)), this study was undertaken to determine whether ONOO(-) can be generated when 2- and 4-hydroxyestrogens are incubated in vitro with (*)NO donor compounds. Using dihydrorhodamine 123 as a specific probe for ONOO(-) formation, a ratio of 100 microM dipropylenetriamine NONOate (DPTA/NO) to 10 microM 4-hydroxyestradiol (4-OHE(2)) gave an optimal ONOO(-) production of 11.9 +/- 1.9 microM (mean +/- SD). Quantification of ONOO(-) was not modified by mannitol, supporting the idea that the hydroxyl radical was not involved. This production of ONOO(-) required the presence of the catechol structure of estrogen metabolites since all methoxyestrogens that were tested were inactive. Hydroxyestrogen metabolites derived from estradiol showed the same efficiency in producing ONOO(-) as those originating from estrone. With DPTA/NO, the 4-hydroxyestrogens generated 30-40% more ONOO(-) than the 2-hydroxyestrogens. Optimal production of ONOO(-) was assessed with DPTA/NO and diethylenetriamine NONOate (initial (*)NO generation rates of 0.76 and 0.08 microM min(-1), respectively). With faster (*)NO-releasing compounds, such as diethylamine NONOate and spermine NONOate, lower levels of ONOO(-) were detected. These data suggest that once the optimal concentration of (*)NO was obtained, the reaction between (*)NO and 4-OHE(2) was saturated. The excess of (*)NO would probably react with aqueous oxygen to form nitrite (NO(2)(-)). Since the third-order reaction rate for the reaction between 2(*)NO and O(2) is 2 x 10(6) M(-2) s(-1), it can therefore be suggested that the reaction between (*)NO and 4-OHE(2) occurs at a faster rate.

Mutagenic properties of estrogen quinone-derived DNA adducts in simian kidney cells.

DNA damage caused by catechol estrogens has been shown to play an etiologic role in tumor formation. Catechol estrogens are reactive to DNA and form several DNA adducts via their quinone forms. To explore the mutagenic properties of 2-hydroxyestrogen-derived DNA adducts in mammalian cells, N(2)-(2-hydroxyestrogen-6-yl)-2'-deoxyguanosine and N(6)-(2-hydroxyestrogen-6-yl)-2'-deoxyadenosine adducts induced by quinones of 2-hydroxyestrone, 2-hydroxyestradiol, or 2-hydroxyestriol were incorporated site-specifically into the oligodeoxynucleotides ((5)(')TCCTCCTCXCCTCTC, where X is dG, dA, 2-OHE-N(2)-dG, or 2-OHE-N(6)-dA). The modified oligodeoxynucleotides were inserted into single-stranded phagemid vectors followed by transfection into simian kidney (COS-7) cells. Preferential incorporation of dCMP, the correct base, was observed opposite all 2-OHE-N(2)-dG adducts. Only targeted G --> T transversions were detected; the highest mutation frequency (18.2%) was observed opposite the 2-OHE(2)-N(2)-dG adduct, followed by 2-OHE(1)-N(2)-dG (4.4%) and 2-OHE(3)-N(2)-dG (1.3%). When 2-OHE-N(6)-dA adducts were used, preferential incorporation of dTMP, the correct base, was observed. Targeted mutations representing A --> T transversions were detected, accompanied by small numbers of A --> G transitions. The highest mutation frequencies were observed with 2-OHE(1)-N(6)-dA and 2-OHE(3)-N(6)-dA (14.5 and 14.1%, respectively), while 2-OHE(2)-N(6)-dA exhibited a mutation frequency of only 6.0%. No mutations were detected with vectors containing unmodified oligodeoxynucleotides. Thus, 2-OHE quinone-derived DNA adducts are mutagenic, generating primarily G --> T and A --> T mutations in mammalian cells. The mutational frequency varied depending on the nature of the 2-OHE moiety.

Effect of metal ions on the stable adduct formation of 16alpha-hydroxyestrone with a primary amine via the Heyns rearrangement.

16alpha-Hydroxyestrone (16alpha-OHE1), one of the major estrogen metabolites in humans that may plays a role in cell transformation, has been found to form stable adducts with nuclear proteins. The mechanism for the formation of a stable covalent adduct of 16alpha-OHE1 with protein has been postulated via the Heyns rearrangement after Schiff base formation. The Heyns rearrangement on the steroidal D-ring alpha-hydroxyimine was investigated using 17-(2-methoxyethylimino)estra-1,3,5(10)-triene-3,16alpha-dio l as a model intermediate. Rates of the Heyns rearrangement and hydrolysis of the steroidal a-hydroxyimine were determined by a high-performance liquid chromatography (HPLC) simultaneously. The Heyns rearrangement was demonstrated to be optimum at pH 6.2 and the reaction rate at physiological pH, 7.3-7.5, was more than 90% of that at the optimum pH. On the other hand, modulator(s) to the reactions were also examined. According to our previous finding of the proton-mediated mechanism of the Heyns rearrangement, the effects of cationic metal ions on the reactions were examined with 29 metal chlorides. Five metal ions, Pt4+, Cu2+, Ni2+, Co2+, and Mn2+, suppressed the formation of Heyns product significantly while Fe2+, Y3+, Gd3+, and Er3+ slightly increased it. The suppression rate was synergistically enhanced by the combination of Pt4+ with Co2+, Cu2+, or Ni2+. These results suggest the five metal ions, Pt4+, Cu2+, Ni2+, Co2+, and Mn2+, reduce the formation of the Heyns product in vivo and, therefore, would be useful tools to clarify the implication of the stable adduct formation of 16alpha-OHE1 with protein.

Enzymic and chemical O-methylation of a 4-hydroxyestrone N-acetylcysteine conjugate.

4-Hydroxyestrone N-acetylcysteine conjugate (4-OHE1-2SR) is considered to be an important compound for monitoring the in vivo formation of catechol estrogen quinones, an intermediary in estrogen carcinogenicity. This article describes the selective synthesis of isomeric monomethyl ethers of 4-OHE1-2SR utilizing the formation of a seven-membered ring lactone by dehydration with acetic anhydride. Using these authentic specimens, enzymic and chemical O-methylation were examined. Enzymic O-methylation, using a rat liver cytosolic fraction, of 4-OHE1-2SR gave its 3-methyl ether as the sole product, while preferential O-methylation of 4-hydroxyestrone (4-OHE1) at the C-4 position was confirmed under the same conditions. Methylation of 4-OHE1-2SR with diazomethane gave initially carboxylate methylation, then the corresponding 3-methyl ether almost exclusively, while methylation of 4-OHE1 also gave its 3-methyl ether preferentially. However, much more rapid formation of the 3-methyl ether was observed with 4-OHE1-2SR than with 4-OHE1 itself. These results show that the hydroxy group at the C-3 position of 4-OHE1-2SR is more reactive than that at the C-4 position, both chemically and enzymatically.

Translesional synthesis on DNA templates containing an estrogen quinone-derived adduct: N2-(2-hydroxyestron-6-yl)-2'-deoxyguanosine and N6-(2-hydroxyestron-6-yl)-2'-deoxyadenosine.

Miscoding properties induced by estrogen quinone-derived DNA adducts were analyzed using an in vitro experimental system to quantify base substitutions and deletions. Site-specifically modified oligodeoxynucleotides containing a single N2-(2-hydroxyestron-6-yl)-2'-deoxyguanosine (2-OHE1-N2-dG) or N6-(2-hydroxyestron-6-yl)-2'-deoxyadenosine (2-OHE1-N6-dA) were prepared postsynthetically and used as templates in primer extension reactions catalyzed by mammalian DNA polymerases (pol) alpha, beta, and delta. The 2-OHE1-N2-dG adduct blocked primer extension reactions more strongly than 2-OHE1-N6-dA. Using pol alpha and delta, 2-OHE1-N2-dG promoted incorporation of dCMP (6.3 and 3.1%, respectively), the correct base, opposite the lesion: when pol delta was used, misincorporation of dTMP (0.52%) was detected. 2-OHE1-N6-dA also promoted incorporation of dTMP, the correct base, opposite the lesion, accompanied by misincorporation of dCTP (0.54% for pol alpha and 3.2% for pol delta) and one-base deletion (0.3-0.5%). Using pol beta, no miscoding was detected. The miscoding occurred only when replicative DNA polymerases were used. Kinetic data were consistent with those obtained from the analysis of fully extended products formed by pol alpha or pol beta. These results indicate that endogenous estrogen quinone-derived DNA adducts have miscoding potential: G --> A and A --> G transitions and deletions are predicted in mammalian cells.

New synthesis of delta 6-estrogens.

An efficient approach to synthesize delta 6-estrogens is described. The key steps in the synthesis are the introduction of a hydroxyl group at the C-6 position of a suitably protected estrogen using a superbase and subsequent dehydration with Martin sulfurane reagent or methyltriphenoxyphosphonium iodide. The two-step synthetic procedure readily gave the delta 6-estrogens in high yield.

Oxidative transformation of 2-hydroxyestrone. Stability and reactivity of 2,3-estrone quinone and its relationship to estrogen carcinogenicity.

The carcinogenicity of estrogens in rodents and man has been attributed to either alkylation of cellular macromolecules and/or redox-cycling, generation of active radicals, and DNA damage. Metabolic activation of estradiol leading to the formation of catechol estrogens is believed to be a prerequisite for its genotoxic effects. 4-Hydroxyestradiol, although not 2-hydroxyestradiol, is a potent inducer of tumors in hamsters. Previous studies have shown that 3,4-estrone quinone can redox-cycle and is capable of inducing exclusively single strand DNA breaks in MCF-7 breast cancer cells, as well as react with various nucleophiles (thiol, imidazole, amino, phenolate, and acetoxy) to give Michael addition products. These results support the possible involvement of 3,4-catechol/quinone estrogens in estrogen's carcinogenicity. To explain the decreased carcinogenicity of 2-hydroxyestrogens, the reactions of 2,3-estrone quinone (2,3-EQ) with nucleophiles were investigated. Reactions of 4-methylimidazole with 2,3-EQ gave a complex mixture of products leadng to the formation of the catechol, C-O dimerization product, and a 1,6-Michael addition product identified as the 1-(4-methylimidazolo)-2-hydroxyestrone. Reactions of 2,3-EQ under mildly basic conditions with either ethyl phenolate or acetate gave several products which were characterized as the C-O and C-C dimers, catechol, and 3,5-dihydroxy-1(10), 3-estradiene-2, 17-dione. No Michael addition products were detected under these experimental conditions. The same products were also observed during the synthesis of 2,3-EQ, which led us to postulate that the lack of carcinogenicity of 2-hydroxyestrogens may be related to the increased reactivity and decreased stability of the quinone under physiological conditions. These results are contrasted with those obtained with 3,4-EQ which is much more stable and therefore could diffuse from the site of formation to the target tissue. These results along with rapid methylation and clearance may be very likely explanations for the decreased carcinogenicity of 2-hydroxyestrogens.

Synthesis of N-acetylcysteine conjugates of catechol estrogens.

The synthesis of N-acetylcysteine conjugates of 2-hydroxyestrone (2-OHE1) and 4-hydroxyestrone (4-OHE1) is described. The reaction of estrone 2,3-quinone with N-acetylcysteine provided 2-OHE1 and its C-4 and C-1 thioether conjugates in a ratio of 1:1, while estrone 3,4-quinone with N-acetylcysteine gave 4-OHE1 and its C-2 thioether conjugate as a sole product. Their structures were characterized by inspection of NMR spectra, chemical derivatization (methylation and acetylation), and comparison with the reactivity of 4-bromoestrone 2,3-quinone or 2-bromoestrone 3,4-quinone toward N-acetylcysteine.

Reaction of lysine with estrone 3,4-o-quinone.

Reaction of lysine with estrone 3,4-o-quinone gave a complex mixture of products. Six compounds were isolated and identified using spectroscopic techniques. Among the reaction products isolated were 4-hydroxyestrone (2), 3-aminoisoestrone (3), 3-(N-pentyl-5-amino)-isoestrone (4), 1-lysylestrone 3,4-o-iminoquinone (5), and two dimeric products of 3,4-catechol estrone (6 and 7).