No abstract

Epidemiologic data indicate that cigarette smoking is associated with an important anti-estrogenic effect, and increased hepatic metabolism has been suggested as a possible mechanism. We examined the hypothesis that cigarette smoking in women induces an increase in estradiol 2-hydroxylation. This irreversible metabolic pathway yields 2-hydroxyestrogens, which possess minimal peripheral estrogenic activity and are cleared rapidly from the circulation. We found a significant increase in estradiol 2-hydroxylation in premenopausal women who smoked at least 15 cigarettes per day. The extent of the reaction (mean +/- SEM) was 53.6 +/- 2.2 percent among 14 smokers and 35.1 +/- 1.8 percent among 13 nonsmoking controls--an increase of approximately 50 percent (P less than 0.001). The extent of 2-hydroxylation among five smokers did not vary during the follicular and luteal phases of their menstrual cycles. In addition, urinary excretion of estriol relative to estrone was significantly decreased among smokers (P less than 0.01), providing evidence that the smoking-induced increase in 2-hydroxylation diminishes the competing metabolic pathway involving 16 alpha-hydroxylation. This study demonstrates that smoking exerts a powerful inducing effect on the 2-hydroxylation pathway of estradiol metabolism, which is likely to lead to decreased bioavailability at estrogen target tissues. Elucidation of the mechanism responsible for smoking-induced changes in 2-hydroxylation may be useful in the development of strategies to reduce the risk of hormone-dependent tumors.

The three dominant oxidative biotransformations of estradiol were examined in 10 normal women and 33 females with breast cancer by using a recently devised radiometric method. Estradiol tracers, labeled with 3H specifically in the 17a, C-2, or 16a position, were used to measure both the rate and extent of 17P-ol oxidation (the initial metabolic step) and the subsequent 2-and 16a-oxidative reactions. The mean ± SEM values for the extent of estradiol metabolism at these three specific sites were 76.9 ± 5.3%, 31.1 ± 4.0%, and 9.3 ± 0.8%, respectively, in normal subjects. Corresponding data in patients with breast cancer-i.e., 73.0 ± 4.2%, 32.7 ± 2.7%, and 14.9 ± 1.5%-revealed a significantly greater extent of 16a-hydroxylation in the latter population. Because the 16a-hydroxylated compounds (including estriol) are themselves potent estrogens, these changes may have important hyperestrogenic consequences that could have a bearing on the etiology of the disease.Endocrine factors have been implicated in the initiation or promotion, or both, of mammary tumorigenesis based on data collected from several sources. These include both experimental studies using animal models and epidemiological and clinical investigations in human subjects (1). Thus, certain features of the menstrual history and age at parity, which can lengthen the period ofexposure to estrogens secreted by the ovaries, appear to be associated with an increased risk for the disease (2, 3). On the other hand, oophorectomy prior to age 35 can lessen the risk of breast cancer (3). Because an augmentation in estrogen secretion could be implicated in the etiology of the human disease, numerous studies have been conducted to detect such an increase in women with, or at high risk to develop, breast cancer (4). Although some investigators have reported significant differences in urinary or plasma estrogen levels, or both, between these subjects and normal individuals (5-7), these findings have not been consistent and have been challenged by others (8, 9). An alternative and possibly more viable hypothesis that an alteration in estrogen metabolism is associated with breast cancer (4, 10) has also been the subject of extensive investigations.The metabolism of estradiol, which is primarily oxidative, consists ofan initial oxidation ofthe 17/3-hydroxy group to yield estrone. This steroid is subsequently metabolized mainly through either of two alternate hydroxylative pathways; namely, hydroxylation at the C-2 or the 16a position (11). These hydroxylations are of particular interest in that they constitute competing reactions whose products are themselves active compounds characterized by markedly different biological properties. The 16a-hydroxyestrogens-estriol and 16a-hydroxyestrone-demonstrate uterotropic activity comparable to that of the parent hormone, estradiol (12, 13). On the other hand, the principal 2-hydroxyestrogens-2-hydroxyestrone and 2-methoxyestrone exhibit virtually no peripheral estrogenic effects but appear to play a regulatory role in neur...

The interactions of 16a-hydroxyestrone (16a-OHE1), a metabolite of estradiol (E2), with estrogen receptors (ERs) were compared in this study to the classic E2-receptor mechanism in human breast cancer cells MCF-7 in culture. When MCF-7 cells were incubated with radioinert 16a-OHE1 or its 3H-labeled form for 4 weeks, the estrogen bound extensively and irreversibly in a time-dependent fashion to nuclear protein species that correspond to the ER. Here weshow that the interactions of 16a-OHE, with the ER are different from those of E2 with the receptor. Dissociation of tritiated E2-ER or 16ci-OHE,-ER complexes, salt extraction, DNase and proteinase K digestion, and ethanol treatment demonstrated that the binding of 16a-OHE, to the ER corresponds to two different forms: a classical noncovalent interaction similar to that of E2, and a covalent adduct formation between the metabolite and the ER. These complexes localized preferentially in nuclear matrix components as revealed by cell fractionation and probing with a monoclonal anti-ER antibody.[3H]16a-OHEj-ER complexes analyzed by polyacrylamide gel electrophoresis demonstrated a radiolabeled band at %66 kDa that was absent when the exposure of cells was done in the presence of E2 in competition and that was also absent in PH]E2incubations. The present results when considered together with our previous findings of elevated activities of estrogen 16a-hydroxylase, the enzyme responsible for the formation of 16a-OHE1, in breast cancer patients and in women at enhanced risk for the disease, suggest that covalent modification of the ER may be one mechanism of malignant transformation in estrogen target tissues.The pathway of receptor-mediated estrogen actions in target tissues has been studied extensively (1)(2)(3)(4)(5). Evidence that target tissues for estradiol (E2) respond variably to its metabolites raises questions whether the estrogen receptor (ER) bound to these metabolites might exert unusual functions at the nuclear level. We have previously shown that the natural estrogen metabolite 16a-hydroxyestrone (16a-OHE1), whose role in biological processes has not been established, binds covalently to primary amino groups of proteins by a nonenzymatic process. In vitro and in vivo covalent interactions of 16a-OHE1 with proteins occur between the steroid carbonyl and the E-amino group of lysine residues in many human tissues (6-9). Our group has been studying the role of steroid metabolites and their covalent interactions in the induction of human breast cancer, since we have found that the activity of estrogen 16a-hydroxylase, the enzyme involved in the formation of 16a-OHE,, is increased in a population of women with breast cancer and in women at risk for the disease (10, 11).The objective of the present study has been to test directly the hypothesis that 16a-OHE, binds covalently to nuclear regulatory proteins, specifically the ER, in estrogen target cells. We now report that covalent binding of 16a-OHE1 to specific nuclear proteins is extensive and that ERs at ...

In this report, we describe our findings on the relationship between estradiol 16a-hydroxylation and mammary tumor incidence. A close correlation between the two has been demonstrated with 16-hydroxylation being elevated in strains with a high incidence of tumors, such as RIII and C3H, and low in strains with a low incidence of cancer, such as C57BL. The extent of reaction is highly reproducible and unaffected by age or presence of overt mammary tumors. Studies on the inheritance of estradiol 16a-hydroxylase showed that it is inherited as an autosomal dominant and is not correlated with estradiol 2-hydroxylase or androgen and progestin 16a-hydroxylases. In addition, the reaction was shown to be markedly enhanced by the presence of murine mammary tumor virus and diminished in the absence of the virus. These studies establish a relationship between genetics, hormonal factors, and murine mammary tumor virus, the three key factors in mammary tumorigenesis. disease, unless the increase was a consequence of the cancer itself. An animal model with spontaneous estrogen-mediated mammary tumors in which the extent of estrogen 16a-hydroxylation could be readily studied before and after tumor development would assist in choosing between the alternative hypotheses offered above. In addition, such a model would facilitate studies on the mechanisms by which this reaction is regulated and whether it affects the initiation or promotion of the disease. In this report, we describe our studies on estrogen metabolism in the murine mammary tumor model, which show that a close relationship between the extent of tumor incidence and 16a-hydroxylation of estradiol exists. Furthermore, we find that the presence of exogenous mouse mammary tumor virus (MMTV) increases 16a-hydroxylation of estradiol, providing a possible link between the viral and hormonal elements of mammary tumorigenesis in the mouse.Epidemiological evidence dating back to the observations of Beatson (1) provides a strong argument that endogenous estrogens participate in the etiology and progress of human breast cancer (2). Because of this, much effort has been directed to the identification of differences in the secretion and/or metabolism of estrogens in women with breast cancer, but the results obtained have been contradictory and the existence of such differences remains equivocal (3). A possible reason for this failure to discern a consistent difference is that breast cancer is a disease with a long latent period (4) and, consequently, the hormonal status at the time of diagnosis may not reflect that existing at the time of disease initiation. Clearly, secretion of estrogens is highly dependent on age and menopausal status and we have therefore sought to find a measure of estrogen metabolism that is not affected by age or menopausal stage and that will reflect metabolic status at the time of disease initiation. Using a radiometric procedure for measuring the enzymatic activity of the major pathways of the oxidative metabolism of estradiol, 17,3-ol oxidation, a...