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.

Relationship between estradiol 16 alpha-hydroxylation and human papillomavirus infection in cervical cell transformation.

The ovarian steroid hormone estradiol and its metabolite estrone were examined in 45 normal women and 127 premenopausal women with precancerous cervical lesions. Interviews, colposcopy and cervical scrapings were performed. The mean +/- SD values for estradiol and estrone were 0.07 +/- 0.08 ng/ml and 0.06 +/- 0.02 ng/ml, respectively in normal subjects. Corresponding data in patients with cervical intraepithelial neoplasia alone or in association with human papillomavirus (HPV) infection were 0.074 +/- 0.03 ng/ml and 0.076 +/- 0.03 ng/ml or 0.080 +/- 0.03 ng/ml and 0.148 +/- 0.02 ng/ml, respectively, which revealed a significantly greater extent of estrogenic action in the latter population (p < 0.05). We considered that the presence of HPV infection probably increased 16 alpha-hydroxylation of estradiol, providing a possible link between the viral and hormonal elements, possibly having a bearing on the etiology of the disease.

Conjugated estrogens--the natural SERMs.

Tissue selective and metabolite replacement therapy may become a new aspect in hormone replacement therapy (HRT). In addition to the naturally secreted hormones, there are also the later formed metabolites that exert a characteristic pharmacological profile. This mechanism is well known in thyroid replacement therapy, when triiodothyronine, the metabolite of thyroxine, is added to substitution therapy. The same is true for testosterone replacement therapy, when dihydrotestosterone is used for replacement. Also in menopausal HRT these aspects will gain tremendous importance. Progesterone metabolites have a strong clinical potency as neurosteroids, and estradiol metabolites are important factors in angiogenesis and angiostasis. Conjugated estrogens consist of different metabolites such as 16-hydroxy-equilin, which has no angiogenetic effect compared with 16-hydroxy-estrone. Estrone sulfate, the main component in conjugated estrogens, can be activated into estrone and 17 beta-estradiol in a tissue specific manner. This aspect will become of interest in clinical practice with HRT.

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.

Catechol estrogen production in rat microsomes after treatment with indole-3-carbinol, ascorbigen, or beta-naphthaflavone: a comparison of stable isotope dilution gas chromatography-mass spectrometry and radiometric methods.

Compounds like indole-3-carbinol (I3C) have been shown to increase catechol estrogen formation and reduce mammary tumor incidence in mice. These compounds may exert a protective effect for breast cancer development by decreasing the overall estrogen pool available for the formation of 16 alpha-hydroxyestrone (16 alpha-OHE1), a metabolite that retains significant estrogenic activity, may be mutagenic and could represent a potential carcinogenic intermediate of estradiol degradation. I3C and ascorbigen originate from the breakdown of glucobrassicin. We have compared the inductive effects of I3C with ascorbigen and beta-naphthaflavone (Bnf) in microsomes from rats pretreated with these compounds using isotope dilution GC-MS and a radiometric method. Incubated microsomes from rats pretreated with I3C and ascorbigen yielded high levels of 2-hydroxyestradiol (2-OHE2) that were comparable to levels induced by Bnf and were significantly above control group levels (p < 0.005). Absolute values determined by the radiometric method were approximately 40% lower than 2-OHE2 concentrations determined by GC-MS, although the relative changes in each group were the same. These differences may be attributed to the radiolabel becoming trapped in microsomal intermediates in the sequence leading to tritium entering the aqueous compartment. Both ascorbigen- and Bnf-treated animals exhibited significant increases in 2-hydroxyestrone (2-OHE1) (p < 0.05). The ability of ascorbigen to induce estradiol C-2 hydroxylation has not been previously reported. Based on these data, we speculate that ascorbigen will act as an anticarcinogenic agent and will inhibit or reduce the incidence of mammary tumor formation.

In vitro synthesis of 16 alpha-hydroxyestrone by female rat liver microsomes: its possible role in the etiology of breast cancer.

Liver homogenates from female rat strains (Sprague-Dawley, Wistar and Fisher) were incubated in a NADPH regenerating medium in the presence of labelled and unlabelled estrone. Liver microsomes isolated from male rats and female mice were used as positive controls. Using HPLC and paper chromatography, under the experimental conditions used it was found that liver homogenates from female rats were able to convert estrone to various metabolites such as 16 alpha-hydroxyestrone. In a mutagenicity assay (Ames test), with 16 alpha-hydroxyesterone as test substance, two strains (TA98 and TA1538) of the five strains tested showed a 2-3-fold increase in the number of his+ revertants relative to the control values. Estrone did not cause any mutagens in the test used. It is concluded that female rats are able to synthesize 16 alpha-hydroxyestron in vitro. Whether this compound is risk factor for breast cancer remains unclear.

High-performance liquid chromatographic determination of aromatization of 16 alpha-hydroxylated androgens with human placental microsomes.

A sensitive nonradiometric assay of aromatization of 16 alpha-hydroxylated androgens, 16 alpha-hydroxy-4-androstene-3,17-dione (16 alpha-OHA), and 16 alpha-hydroxytestosterone (16 alpha-OHT), has been developed using reverse-phase high-performance liquid chromatography with voltametric detector. The estrogens produced by human placental microsomes, estriol (E3) and 16 alpha-hydroxyestrone (16 alpha-OHE1), were simultaneously detected in quantities as low as 1-2 ng using 3-methoxy-1,3,5(10)-estratriene-2, 16 alpha,17 beta-triol as an internal standard. E3 was the only estrogen detected from the incubate of 16 alpha-OHT with the microsomes and NADPH, while 16 alpha-OHA gave 16 alpha-OHE1 and E3 under the same conditions. Apparent Km and Vmax of the microsomal aromatase for 16 alpha-OHA and 16 alpha-OHT were 2.56 microM and 71.4 pmol/min/mg and 13.33 microM and 15.4 pmol/min/mg, respectively.

Formation of 2- and 4-hydroxyestrogens by brain, pituitary, and liver of the human fetus.

When [6,7-3H]estradiol was incubated with tissue homogenates of the brain, the pituitary, and the liver of two human female fetuses, a number of radioactive metabolites more "polar" than the incubated substrate were detected. Among these, the identification of two types of catecholestrogens, i.e. the 2- and 4-hydroxyestrogens, was of major interest. Compared on the basis of wet weight of tissues (250 mg), the conversion of estradiol to 2-hydroxyestrogens (2-hydroxyestradiol and 2-hydroxyestrone) was 0.8% in the frontal cortex, 1.0% in the hypothalamus, 2.1% in the pituitary, and 7.8% in the liver. For the first time, the formation of 4-hydroxyestrogens was demonstrated. The percentages of incubated estradiol hydroxylated at C-atom 4 (4-hydroxyestradiol and 4-hydroxyestrone) were 0.5 in the cortex, 0.4% in the hypothalamus, .1% in the pituitary, and 0.5% in the liver. The results show that fetal brain and pituitary tissue can hydroxylate estradiol in positions 2 and 4 to a similar extent, whereas in the liver, about 15 times more 2-hydroxy than 4-hydroxy compounds are formed. Moreover, the 2-hydroxylating capacity of the liver is definitely greater than that of the brain, whereas the 4-hydroxylating capacity is about the same as that of the brain.

Introduction of a 16 alpha-hydroxyl function into estrone by Streptomyces roseochromogenes.

Streptomyces roseochromogenes (NRRL B-1233) converted estrone predominantly into its 16 alpha-hydroxyl derivative. Chemical and spectroscopic (UV, IR, NMR, MS) methods were used in establishing the structure and strereochemistry of the oxidation product. The product was assigned as 16 alpha-hydroxyestrone (yield, 17%). No other oxidation product was detected in this experiment. An interrelationship between cell growth and 16 alpha-hydroxy-estrone formation was observed. Also, 16 alpha-hydroxylation of estrone was observed in resting cells. 16 alpha-Hydroxylase showed good activity at 3.7 X 10(-4)M of estrone concentration and was completely inhibited by 1.1 X 10(-3)M. These results indicate the presence of a constitutive 16 alpha-hydroxylase in the organism investigated.