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A stable epoxide of estrone: Evidence for formation of a ‘new’ estrogen metabolite
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A stable epoxide of estrone: Evidence for formation of a ‘new’ estrogen metabolite James I. Raeside* , Heather L. Christie Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, Canada
A R T I C L E I N F O
Article history: Received 18 August 2016 Received in revised form 12 October 2016 Accepted 24 October 2016 Available online xxx Keywords: Estrone Epoxide Stable metabolite Radioactive steroids Tissue incubations 6a-Hydroxy-estrone
A B S T R A C T
Oxidative metabolism of estrogens is an important feature in liver and some non-hepatic tissues. In initial studies on estrogen metabolism in tissues from the reproductive tract of the stallion, where testicular estrogen secretion is remarkably high, a prominent radiolabeled product from [3H]-estrone (E1) was noted on chromatography; it had a retention time (Rt) between 17b-estradiol (E2) and E1. Unexpectedly, when non-radiolabeled E1 was the substrate no UV absorption at 280 nm was seen at the Rt for the [3H]labeled product—suggesting a non-aromatic ring A. The following efforts were made to reveal more about the nature of the “unknown” compound. Reduction and acetylation showed, separately, the presence of a single keto and hydroxyl group. Exposure to acid gave a single radiolabeled peak with Rt of 6a-hydroxyE1—suggesting the presence of a third molecule of oxygen. Mass spectrometry with limited material was inconclusive but supportive for a formula of C18H22O3. Thus, an epoxide involving the aromatic ring of E1 is suggested as a labile intermediate in the formation of the “unknown” metabolite. Estrogen epoxides as labile, reactive intermediates have been considered as potential precursors of the 2- and 4-hydroxy catechol estrogens with implications in breast cancer [Soloway, 2007]. Because of the association of the “unknown” metabolite with 6a-hydroxy-E1, the structural form proposed for the stable epoxide is that for 5a,6a-epoxy-estrone. This represents an alternative to the production of the 2- and 4-hydroxycatechol estrogens. The broad range in normal tissues where the “unknown” compound was shown to be a persistent metabolite (e.g. mouse mammary glands, ovary, uterus, brain, muscle, equine conceptus, stallion and domestic boar reproductive tracts) suggests more general biological implications. ã 2016 Elsevier Ltd. All rights reserved.
1. Introduction Steroid epoxides in biological systems were the subject of an early review [1]. They have been postulated as being participants in, or products of, selective biological oxidations. Attention was first drawn to them as possible intermediates in the biosynthesis of steroids when squalene epoxide was shown to undergo cyclization leading to the ultimate formation of cholesterol. Their biosynthesis and hydrolysis can occur by enzymatic or non-enzymatic processes—for example, cholesterol-5a,6a-epoxide is formed by ultraviolet light irradiation of skin. Although this epoxide of cholesterol was shown to be a stable compound it is generally conceded that steroid epoxides as oxidative metabolites in nature are quite labile. As a consequence, few have been detected and isolated under relatively physiological conditions. The formation of
* Corresponding author at: Department of Biomedical Sciences, University of Guelph, N1G 2W1, Guelph, ON, Canada. E-mail address: [email protected] (J.I. Raeside).
stable 5,6-epoxides of pregnenolone by liver microsomal preparations is at least one other exception [2]. The position of epoxides in the metabolism of estrogens has generated interest largely in relation to cancer. In analogy with the need for metabolic activation of some polycyclic aromatic hydrocarbons (PAHs) to become carcinogenic [3], a cytochrome P-450mediated oxidative metabolism of the primary estrogens, 17bestradiol (E2) and estrone (E1), seems necessary for carcinogenicity [4]. Some uncertainty remains as to the actual nature of the oxidized estrogen products that account for the initiation of tumor formation and support. It was proposed that epoxidation of E2 and E1 may be the underlying mechanism for inducing the carcinogenicity of these estrogens [5]. In addition, it has been shown that after reacting with dimethyldioxirane (DMDO), a versatile epoxide-forming oxidant, E2 was able to bind DNA forming DNA adducts [6]. However, an alternative pathway to DNA adducts has been the subject of extensive reviews, namely, the role of catechol estrogen quinones as initiators of breast and other human cancers [7,8]. After the initial oxidation to the catechol estrogens (CEs, 2hydroxy-E2/E1 and 4-hydroxy-E2/E1) by cytochrome P450
http://dx.doi.org/10.1016/j.jsbmb.2016.10.007 0960-0760/ã 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: J.I. Raeside, H.L. Christie, A stable epoxide of estrone: Evidence for formation of a ‘new’ estrogen metabolite, J. Steroid Biochem. Mol. Biol. (2016), http://dx.doi.org/10.1016/j.jsbmb.2016.10.007
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enzymes, further oxidation gave rise to the semiquinones/ quinones that were the proximate carcinogens. The oxidative steps in the hydroxylation of E2 and E1 leading to the CEs themselves may well include epoxide formation as the corresponding epoxy-enone intermediates [9]. In fact, the facile conversion of the nonaromatic 1,2- and the 4,5-epoxyenones of E2/E1 to the catechol estrogens, 2- and 4-OH-E2/E1, respectively, was later demonstrated [10]. Although the chemical synthesis of these epoxy-enones was reported over twenty years ago [11], it remains to be determined whether any of the epoxy-estrogens are found in mammalian systems especially in the context of breast and ovarian cancers [12]. In studies on estrogen metabolism in domestic animals we have consistently encountered a metabolite of E1 which remained unidentified. One feature stood out, namely, it appeared as a metabolite of radiolabeled E1 ([3H]-E1) but was not detected (UV 280 nm) when unlabeled E1 was the substrate in incubations with a number of non-hepatic tissues. Its presence varied from being a relatively prominent to a barely detectable, radioactive peak on chromatography. From the literature the occurrence of an epoxide seemed unlikely; however, as the investigation of its nature progressed a point was reached where we now propose that the metabolite is the 5a,6a-epoxide of E1. The following is a brief account of the work leading to this conclusion. A few examples are also given to illustrate the relatively widespread formation of this metabolite and, thereby, to draw further attention to its possible biological significance. Similar results have also been obtained in more limited studies with [3H]-E2. 2. Materials and methods 2.1. Chemicals and reagents The radioactive steroids (NEN) obtained from Perkin–Elmer (Shelton, CT, USA) were [2,4,6,7-3H]-17b-estradiol (71.0 Ci/mmol) and [2,4,6,7-3H]-estrone (74.1 Ci/mmol). Nonradioactive steroids were purchased from Steraloids Inc (Newport, RI, USA). 6aHydroxy-estrone (6a-OH-E1) was made by Dalton Chemical Laboratories Inc., Toronto, ON, Canada. Solvents from Caledon Laboratories, Ltd (Georgetown, ON, Canada) were glass-distilled and reagent grade; acetonitrile (HPLC grade 190) was used for HPLC. Sep-Pak C18 cartridges were purchased from Waters Scientific (Mississauga, ON, Canada). Medium 199 and supplements were supplied by Sigma. All other chemicals were analytical grade from Fisher Scientific (Toronto, ON, Canada). 2.2. Collection and preparation of tissues 2.2.1. Stallion At castration of 8 stallions (3–17 years-old) the testes were placed on ice and taken to the laboratory where the epididymis and vas deferens were removed. Portions of the vas and mid regions of the caput, corpus and cauda epididymides were stripped of adventitious tissues, minced separately and transferred to a test tube containing physiological buffered saline (PBS). Three washes removed spermatozoa by suspension and gravity sedimentation. In each experiment replicates (2–4) of each tissue (250 mg) were dispensed for incubation within 3 h of the surgery. 2.2.2. Domestic boar (Sus scrofa) Reproductive tracts (n >10) were removed at slaughter from pubertal Yorkshire pigs (6 month-old) and at euthanasia, by intravenous injection of pentobarbital, from older breeding boars. Vas and epididymal tissues were prepared as above (2.2.1.). Prostate and seminal vesicles were also recovered from the older animals (n = 6) and dissected free of extraneous tissues before
mincing finely with scissors. Equal amounts of tissue (100–300 mg, wet weight) from each source were dispensed for incubation. 2.2.3. Early equine conceptus Mares from the research herd at the University of Guelph were used to recover conceptuses (Days 24–26), under conditions approved by the University Animal Care Committee. Conceptuses (n = 10) were collected by transcervical uterine lavage, as described previously [13]. The embryo proper (15–30 mg, wet weight) in PBS was dissected free of its extraembryonic membranes (yolk sac wall, allantois and amnion) under a dissecting microscope, using ophthalmic scissors and forceps. The trophoblast was divided into bilaminar (10–30 mg) and trilaminar (40–70 mg) components by dissection; these were incubated separately, without mincing, as was the case for the allantois (15–40 mg). 2.2.4. Mouse tissues Mammary glands were dissected from mature nulliparous female mice, 4–6 months-old (n = 20). In all experiments, the tissues were pooled for each animal, minced with scissors and washed by decanting in PBS. About 85–300 mg wet weight of tissue were dispensed for incubation. Additional tissues were collected from two mice (uterus, ovaries, brain, skeletal muscle and liver), washed and minced as above for the mammary gland. The amounts of tissues taken for incubation were (approximate wet weight, mg): uterus, 45–100; ovary, 10–40; brain, 100; muscle, 85–180; liver, 100. 2.2.5. Tissue incubation Tissues were incubated for 2–4 h in culture medium (TC-199, 5 ml for >300 mg; 2 ml for <300 mg) in small glass flasks in a shaking water-bath at 37 C under 5% CO2 in air, after either [3H]E1/E2 (1 106 cpm; or 2 106 cpm for >300 mg) or non-radioactive E1 (50 or 100 mg) were added as substrates. Within 2 h from time of collection, incubations were done in duplicate for radiolabeled substrates and 2–6 replicates for non-radiolabeled E1. After incubation and removal of the media, the tissues were rinsed twice with 200 ml of fresh medium which was then added to the previously collected media. When the larger amounts of tissues were involved, the contents of the flasks were transferred to tubes for centrifugation to recover the media; in which case the tissues were washed twice with centrifugation in 1 ml TC-199 and the washings pooled with the spent media. Media and tissues were stored in glass vials at 20 C until processed. 2.2.6. Analytical procedures Steroids were recovered from the medium by solid-phase extraction (SPE, Waters C18 Sep-Pak column) as described previously [14]. Briefly, the incubation medium was diluted with water to a volume of 5 ml, applied to a primed column and the unconjugated and conjugated steroids were eluted with 5 ml of diethyl ether and 5 ml of methanol, successively. The amount of radioactive material in each SPE fraction was determined by liquid scintillation counting (LSC) of an aliquot in 4 ml of Ecolite scintillation cocktail (MP Biochemicals, Solon, OH). The eluates were taken to dryness under nitrogen at <45 C. The dried residue was dissolved in 100 ml of acetonitrile:water (1:3) for injection on HPLC. Chromatographic profiles of steroids from SPE were generated on a Waters HPLC System using a Nova-Pak C18 column with UV absorbance monitored at 280, 254 and 210 nm. Four solvent systems were used: (1) a binary solvent gradient (Waters # 8) of acetonitrile: water (28:72 to 90:10); (2) an isocratic solvent system of 30% acetonitrile in water; (3) an isocratic solvent system of 40% acetonitrile in water; and (4) a binary solvent gradient (Waters # 8) of acetonitrile: water (20:80 to 90:10). All systems were run for
Please cite this article in press as: J.I. Raeside, H.L. Christie, A stable epoxide of estrone: Evidence for formation of a ‘new’ estrogen metabolite, J. Steroid Biochem. Mol. Biol. (2016), http://dx.doi.org/10.1016/j.jsbmb.2016.10.007
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40 min at a flow-rate of 700 ml/min. Fraction collections (LKB RediFrac, Pharmacia) were set for 0.5 or 1 min intervals for all systems with an aliquot (usually one-tenth) from each fraction taken for LSC. Identification of a radiolabeled metabolite was based initially on coincidence in elution with an authentic reference steroid run as internal standard. An intrinsic lag-time in fractional collection of the radiolabeled material resulted in a slight delay in recording the retention time (Rt) from that observed for the UV absorption of the associated reference standard.
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activities of the resultant metabolites that could activate their own unique receptors or effectors [18]. Our investigations were directed initially to estrogen metabolism in the reproductive tract of the stallion where the enigma of remarkably high production of estrogens by the testes was first noted by Zondek in 1934 [19]. The estrogen chosen was E1 because of its presence in much higher concentrations than E2 in peripheral blood, albeit as sulfates [20], where E1S is about a hundred times higher than testosterone [21]. 3.1. E1 metabolism in the equine reproductive tract
2.2.7. Chemical reactions Reduction: A modification of the method of Diczfalusy and Anne-Marie v. Münstermann, 1959, was used [15]. To dried residues from HPLC fractions in 1 ml of 90% aqueous MeOH, 5 mg of KBH4 was added and left overnight at room temperature. Water (2 ml) was added, tube shaken and left for 1 h before extraction of steroids with 3 2 ml diethylether, which was washed with 2 2 ml water and taken to dryness at <45 C under nitrogen. Acetylation: Dried residue in stoppered glass test tube was dissolved in pyridine (0.2 ml), an equal volume of acetic anhydride was added and left overnight at room temperature. Reagents were then removed under nitrogen. Water (5 ml) was added to the residue for SPE. Acid treatment: After exposure of the dried residue to 2N H2SO4 (1 ml) at 50 C for 2 h, water (1 ml) was added before neutralzing with 1N NaOH. Extraction was done with 2 5 ml of diethylether, which was washed with 2 2 ml water and the contents taken to dryness under nitrogen. 3. Results and discussion Oxidative metabolism of estrogens takes place mainly in the liver, but many examples of extra-hepatic metabolism have been reported since the early demonstration of 2-hydroxylation of E2 by the rat brain [16,17]. Importantly, a functional role for estrogen metabolism in target tissues may be seen to reside in the biological
Although estrogen sulfates comprise about 40–90% of the metabolites formed they have not yet been examined in this study because the use of solvolysis, as a preliminary step, would be detrimental (see Section 3.3.3). The HPLC profiles of [3H]-E1 metabolites in the unconjugated fraction from incubations of the vas deferens and epididymis showed the presence of several polar peaks but also a notable peak between E2 and E1 (Fig. 1) which became the focus of attention. Unexpectedly, when nonradioactive E1 was used as substrate no comparable peak was detected with UV absorption at 280 nm (Fig. 2). Thus it seemed that the characteristic phenolic structure of ring A was no longer present. With estrogen standards for reference, including ring B unsaturated estrogens (equilin, equilenin), efforts were made to learn more about the “unknown” metabolite by comparing Rts with that of the peak from epididymal tissue incubations of [3H]-E1; the only ‘match’ was with 17a-OH-E2 (data not shown). This possibility was dismissed because of its UV absorption at 280 nm, which is lacking in the “unknown” metabolite. All estrogens with an additional hydroxyl group (e.g. 2-, 4-, 6- and 16-OH-E1) are even more polar than E2, whereas their methylation products (2-, 3- and 4methoxy-E1) are less polar than E1. The failure of the “unknown” compound to align with the 9,11-dehydro-E1 reference standard, and because of the very non-polar nature of 16-dehydro-steroids, it is unlikely that the E1 metabolite had an additional site of unsaturation.
Fig. 1. Representative HPLC profiles of unconjugated steroids from incubations of [3H]-estrone with tissues from the reproductive tract of mature stallions. Note the presence of a prominent peak of radioactivity for an unidentified metabolite (U) with a retention time between E2 and E1. Larger amounts of radioactivity are seen as polar products— presumably 6-oxygenated-estrogens. Reference standards of 17b-estradiol (E2) and estrone (E1) were detected by UV absorbance at 280 nm (dotted line). (Solvent system 1).
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Fig. 2. Representative HPLC profiles for tissues from mature stallions when incubated with non-radioactive estrone. No peak was detected between the internal standard of E2 and the E1substrate. Small peaks at 4 and 6 min were seen with Rts similar to 6-oxygenated estrogens (Solvent system 2).
3.2. [3H]-E1 metabolism in the porcine accessory sex glands and reproductive tract With the boar, estrogen sulfates comprised 80–90% of the metabolites formed by regions of the epididymis (caput, corpus and cauda) and the vas deferens [22]. These fractions were not studied further, as was the case for the stallion work (see
Section 3.1). All HPLC profiles for the unconjugated products in incubations from the epididymis and sex glands revealed a radiolabeled metabolite between E1 and E2 (Fig. 3). It was the principal, or a major, peak in all chromatograms and was accompanied by a polar product tentatively identified by Rt as 6a-OH-E1. No evidence of the “unknown” compound was seen (UV 280 nm) when non-radioactive E1 was the substrate (data not
Fig. 3. Representative HPLC profiles of unconjugated steroids from incubations of [3H]-estrone with tissues from the reproductive tract of mature boars. A peak of radioactivity for an unidentified metabolite (U) was seen in all instances between reference standards of E2 and E1 (dotted line). Other metabolism was noted mainly as radioactive products with the retention time (5 min) of 6a-OH-E1 (Solvent system 1).
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shown). Subsequent efforts for identification of the “unknown” metabolite were made mainly with extracts from replicate incubations of [3H]-E1 with porcine tissues. 3.3. Chemical reactions with the “unknown” metabolite of [3H]-E1 3.3.1. Reduction with potassium borohydride The reduction with KBH4 resulted in a product from the radiolabeled metabolite that was more polar than E2 (Fig. 4A). Its position on HPLC reflected a moderate increase in polarity with the same relative shift as obtained regularly with reduction of E1 to E2. We concluded there was reduction of a keto group at C17. 3.3.2. Acetylation This was done to determine whether the hydroxyl group at C-3 of the E1 substrate remained intact. After the reaction it was clear that acetylation had occurred (Fig. 4B), and that a single acetylated product had been formed. Thus the presence of a single hydroxyl group seemed established for the metabolite. 3.3.3. Exposure to acid Acid treatment yielded only two products; the major one was coincident with 6a-OH-E1and the lesser eluted with the estrone reference standard (Fig. 4C). It was coincident with the Rt of the 6a-OH-E1 reference standard which suggested the presence of a third atom of oxygen in the molecule. This additional oxygen could
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not have been be present as a second carbonyl (see above, 3.3.1), or as a second hydroxyl group (3.3.2). In the context of exposure to acid, it should be noted that investigation of the sulfoconjugated estrogen metabolites has not yet been made because a solvolysis step, with its lower pH, would have resulted directly in the formation of 6a-OH-E1and/or E1. Similarly, the use of ascorbic acid to protect catechol estrogens from oxidation during our investigations (including HPLC) was omitted; as a consequence, the metabolism to 2-OH- and 4-OH-E1/ E2 was not examined. 3.3.4. Mass spectroscopy An attempt was made to establish the existence of an additional oxygen atom in the “unknown” compound by mass spectroscopy (LC/MS/MS). A composite sample collected from several tissue incubations of equine vas deferens showed no absorption at 280 nm but a small peak was detected at 200 nm, between E2 and E1 (data not shown). This material was submitted for examination; E1 and 6a-OH-E1 reference standards were included for comparison. LC-ESI/MS/MS revealed an Rt (7.2 min) which was slightly less than that for E1 (Rt, 7.6) but much greater than for 6a-OH-E1 (Rt, 4.4). The results in positive ion mode for the 6a-OH-E1 standard were clear with a mass ion [M+H]+ = 287.1638, and [M+HH2O]+ = 269.1530, indicating loss of the hydroxyl as water. However, due to the low abundance of ions in the MS spectrum for the “unknown” sample it was not possible to reliably propose a formula; the presence of a positive ion species of 269.1568 suggested [M+H-H2O]+ from a formula of C18H22O3. 3.4. Possible formation of a stable epoxide of E1
Fig. 4. Representative HPLC profiles of the “unknown” compound (U) after chemical reactions. A. Borohydride reduction resulted in a shift to a product more polar than E2 (Solvent system 1). B. Acetylation had a marked effect on polarity (U before, solid line; U after, dashed line). Solvent system 3. C. Acid hydrolysis yielded a peak co-eluting with 6a-OH-E1and a small amount of E1. Reference standards (dotted line). Solvent system 4.
The prospect of a stable epoxide involving the aromatic ring of E1 was entertained as the structural form of the “unknown” metabolite. Epoxides of estrogens as possible agents in breast cancer were hypothesized to be labile, reactive intermediates in the oxidative metabolism to catechol estrogens, and even mutagenic themselves [5]. Labile, non-aromatic ring A, dienol epoxides—thought to be an intermediate step in forming the 2-OH and 4-OH estrogens—can be stabilized as their enone epoxide tautomers [9]. In the case of our “unknown” metabolite, its existence as an epoxy-enone was ruled out by the absence of UV absorption at 254 nm. Because the ring A dienol epoxides (1,2- and 4,5-epoxides) are unstable it is most likely that a 5,10- epoxide would also be a highly labile structure (Scheme 1). In analogy with epoxide hydrolase (or non-enzymatic) action on 1,2- and 4,5epoxides, leading to 2-OH and 4-OH estrogens, it is suggested that similar action on a 5,10-epoxide of E1 might give rise to a hydroxyl at C-10 (10-hydroxy-3,17-diketo-estra-1,4-diene). Indeed, on investigating the inactivation of estrone in 1940, Westerfeld [23] concluded: “The introduction into the oestrone molecule of another hydroxyl group in the o- or p-position could theoretically produce three compounds; o-substitution would give rise to either 2:3- or 3:4-dihydroxy-17-keto-oestratriene; p-substitution would yield 10-hydroxy-3:17-diketo-oestradiene (1:4). These derivatives of oestrone are unknown at present”. The first catechol estrogen to be identified was 2-hydroxyestrone; in 1960, Fishman et al. reported its finding as a “new” metabolite of estradiol [24]. Since then, the catechol estrogens (2OH and 4-OH-E1/E2) have become well established as major products of estrogen metabolism, with well recognized biological significance from cancer [7] to neuroendocrine regulation [16]. Whereas 2,3- and 4,5-epoxides have been considered as intermediates in the formation of the catechol estrogens, we have been unable to find any report of an oxirane ring formed with C-5 and C10 of an estrogen. Although p-hydroxylation of a potential 5,10-
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Scheme 1. Oxidative metabolism of estrone. Intermediates in the formation of the catechol estrogens and 6a-hydroxyestrone are suggested; only metabolites with the a-configuration are illustrated. The stable “unknown compound” is proposed to be 5a, 6a-epoxy-E1. UV absorption: *280 nm; **254 nm; ***-ve for both.
epoxide might be considered, this is unlikely to be the case because the 10-hydroxy-product would differ by having an ab-unsaturated ketone and thus UV absorption at 254 nm. However, the possibility of enzymatic, or non-enzymatic, hydrolysis of a putative 5,10epoxide to form a stable 5,6-epoxide was considered as an option for the “unknown” metabolite. Then it was noted that the major peak on HPLC following acid exposure of the radiolabeled “unknown” compound was coincident with a 6a-OH-E1 reference standard. These observations reflect a severing of the oxygen bond to C 5 by nucleophilic attack at the ‘more substituted’ position of a 5a,6a-epoxide. No inversion of the stereochemistry was seen at C 6 since the 6a-configuration was retained in the 6a-OH-E1 formed by exposure to aqueous acid. Furthermore, a lesser peak of radioactivity with the Rt of the E1 reference standard points to an attack on the C O bond at C 6 to restore the aromatic ring with the product being a return to E1 (Fig. 4). In conclusion, the “unknown” compound which possesses remarkable stability in handling and storage is strongly favored to be 5a,6a-epoxyestrone. 3.5. Possible biological significance of a stable epoxide of E1 The potential significance of an estrogen epoxide in normal and malignant breast and other tissues was discussed briefly in a recent hypothesis [12]. It was considered in relation to an earlier hypothesis [5] that oxidative metabolism by cytochrome P-450 enzymes might produce a 4a,5a-epoxide, for example, as an intermediate step in the formation of 4-OH-E2. This labile
intermediate was found to be active as a neoplastic forming agent—presumably present for a limited time as the more stable 4a,5a-epoxy-enone [10]. Furthermore, the position of 4-OH-E2/E1 in the initiation of breast cancer through further oxidation to E2/ E1-quinones seems well established [8]. The existence of a stable 5a,6a-epoxy-E1 would represent an alternative and competitive pathway in estrogen metabolism that could reduce the intrinsic risk of catechol estrogen production. Formation of 6a-OH-E1 on exposure to aqueous acid suggests possible hydrolysis of 5a,6aepoxy-E1 to a 3,5a,6a-triol, as a transient intermediate, with subsequent dehydration at C5 to give 6a-OH-E1. An endogenous epoxide hydrolase might also be responsible for some of the 6aOH-E1 seen, in addition to the “unknown” compound, at the initial chromatography of most media extracts. It remains to be determined what biological actions, if any, might be demonstrated for the “unknown” compound when sufficient quantities can be made by chemical synthesis. Stable 5,6-epoxides can be formed by oxidative metabolism of steroids with a double bond between C-5 and C-6, such as cholesterol, pregnenolone and DHEA [1,2,25]; to date, biological implications have been considered only for cholesterol. Recent studies on cholesterol in relation to breast cancer have demonstrated that 5,6-epoxides of cholesterol are exceptionally stable compounds and surprisingly unreactive toward nucleophiles, thus ruling out a direct mutagenic and carcinogenic potency in mammals [26,27]. However, metabolism of 5a,6a-cholesterol by epoxide hydrolase produced 3b,5a,6b-trihydroxy-cholesterol which may be slightly carcinogenic; on further oxidation to 6oxo-cholestan-3b,5a-diol (OCDO) an oncogenic metabolite of cholesterol was revealed [28]. In subsequent studies, a new pathway in cholesterol metabolism was discovered that involves the action of an epoxide hydrolase on 5a,6a-epoxy-cholesterol to form a conjugation product with histamine (Dendrogenin A, a steroidal alkaloid) through a b-linkage at C-6 [29]. This unusual compound is now the focus of attention because it has been shown to have anti-tumor, as well as neuro-stimulating, properties [30]. It remains to be seen whether the relationship between the oxidative metabolism of E1 and cholesterol—to form stable 5,6epoxides—extends beyond the formation of 6-OH compounds on hydrolysis. Nevertheless, the occurrence of the “unknown” compound as a metabolite from incubations with E1 in nonhepatic tissues of mammalian domestic and laboratory species indicates a possible extensive, physiological significance. HPLC profiles of estrogen metabolites in media extracts from a series of experiments illustrate the broad range of its formation in potential target tissues. 3.6. Examples of the wide range in sites for production of a stable 5a,6a-epoxide of E1 The following examples extend the evidence beyond that from reproductive tissues of the stallion and domestic boar (Sus scrofa). 3.6.1. Early equine conceptus Components of the equine conceptus (embryo proper, allantois, bilaminar and trilaminar trophoblast) all revealed the presence of the ‘unknown’ metabolite in the HPLC profiles for E1 metabolites (Fig. 5). All possessed peaks with alignment for the “unknown” metabolite but with much greater amounts of radiolabeled material tentatively identified by Rts as 6-oxygenated products such as 6a-OH-E1 and 6-oxo-E1. The trophoblastic tissues were more active and showed some conversion of the substrate to E2, especially by the bilaminar layer (Fig. 5C). On exposure to aqueous acid the [3H]-labeled “unknown” compound co-eluted with the 6a-OH-E1 reference standard (data not shown).
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Fig. 5. Representative HPLC profiles of unconjugated steroids from incubations of [3H]-estrone with tissues from the components of early equine conceptuses (days 24–26). A, embryo proper; B, allantois; C, bilaminar trophoblast; D, trilaminar trophoblast. In all tissues a peak of radioactivity for an unidentified metabolite (U) was observed between the E2 and E1 standards. A low level of sulfoconjugation in the embryo proper was reflected in greater amounts of free radioactive material at HPLC. Metabolism to polar products was prominent in the profiles but E2 was also formed by the trophoblast, especially the bilaminar layer. (Solvent system 1).
3.6.2. Mouse tissues Results of metabolism of [3H]-E1 by several non-hepatic tissues and liver are given as representative profiles of similar findings in replicates of these tissues from other animals (n = 2–10). HPLC profiles (Fig. 6) show marked variation; a peak between E2 and E1,
having the Rt for the “unknown” metabolite, was seen in all tissues except for the liver. In liver incubations the metabolism of the substrate was almost complete; most of the radioactivity appeared as very polar products and none between E2 and E1 (Fig. 6D). The uterus had
Fig. 6. Representative HPLC profiles of unconjugated steroids from incubations of [3H]-estrone with several types of tissues from mice (A, mammary gland; B, ovary; C, uterus; D, liver; E, muscle; F, brain). With the exception of liver (Fig. 6D) all tissues showed the presence, to some degree, of an unidentified metabolite (U) between E2 and E1 standards. Metabolism was low in the uterus (Fig. 6C); all other profiles revealed significant amounts of polar products and the ovary alone (Fig. 5B) had a pronounced peak for E2. (Solvent system 2, A–D; Solvent system 1, E and F).
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the least metabolic activity, with the “unknown” metabolite and E2 being the principal products (Fig. 6C). While evidence for the formation of the “unknown” metabolite was clear in both ovary and mammary incubations, much of the radioactivity was seen as polar products. In the case of the ovary, a prominent peak for E2 was not unexpected (Fig. 6B). The HPLC profile of products for the mammary gland revealed more radioactivity as polar metabolites (Fig. 6A). Skeletal muscle provided clear evidence for the metabolite but with about equal amounts of radioactivity in the polar region (Fig. 6E). In brain tissue the metabolism of substrate was most marked, predominantly to polar products; however, the presence of the “unknown” metabolite was still detected (Fig. 6F). On the basis of Rts in the initial chromatography the presence of 6a-OH-E1, and lesser amounts of 6-oxo-E1, was strongly suspected in most experiments. A reciprocal, quantitative relationship between them and the “unknown” compound as products of metabolism is of interest but has not yet been established. If such were the case, it would suggest an even greater contribution of enzyme activities leading to the “unknown” compound and to 6oxygenated metabolites was present as a significant alternative to the formation of catechol estrogens. 3.7. Possible formation of a stable epoxide of E2 Lastly, some preliminary evidence for the production of a similar epoxide metabolite was provided from more limited investigations with E2 as substrate (Fig. 7). The “unknown” peak at 19 min in Fig. 7A, on acid hydrolysis, yielded not only E2 but also 6a-OH-E2 and 6b-OH-E2 (Fig. 7B). This again implied the presence of a third oxygen atom in the estrogen metabolite as seen with E1 as substrate.
4. Summary and conclusions Evidence is presented for the production of a stable 5a,6aepoxide of E1 in several non-hepatic tissues from the mouse and domestic animals. Attempts to identify a radiolabeled metabolite by HPLC (Rt, >E2 and
Fig. 7. Representative HPLC profiles of unconjugated steroids from incubations of [3H]-estradiol with tissues from the reproductive tract of mature stallions. A. Most of the radioactivity in all cases was seen as very polar metabolites (6hydroxylated?); lesser amounts were noted (U) as material slightly more polar than the E2 substrate (Solvent system 2). B. Acid hydrolysis of U resulted in two polar peaks co-eluting with 6a- and 6b-OH-E2 and a similar amount seen as E2. Solvent system 4).
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Journal of Steroid Biochemistry & Molecular Biology xxx (2016) xxx–xxx
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A stable epoxide of estrone: Evidence for formation of a ‘new’ estrogen metabolite James I. Raeside* , Heather L. Christie Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, Canada
A R T I C L E I N F O
Article history: Received 18 August 2016 Received in revised form 12 October 2016 Accepted 24 October 2016 Available online xxx Keywords: Estrone Epoxide Stable metabolite Radioactive steroids Tissue incubations 6a-Hydroxy-estrone
A B S T R A C T
Oxidative metabolism of estrogens is an important feature in liver and some non-hepatic tissues. In initial studies on estrogen metabolism in tissues from the reproductive tract of the stallion, where testicular estrogen secretion is remarkably high, a prominent radiolabeled product from [3H]-estrone (E1) was noted on chromatography; it had a retention time (Rt) between 17b-estradiol (E2) and E1. Unexpectedly, when non-radiolabeled E1 was the substrate no UV absorption at 280 nm was seen at the Rt for the [3H]labeled product—suggesting a non-aromatic ring A. The following efforts were made to reveal more about the nature of the “unknown” compound. Reduction and acetylation showed, separately, the presence of a single keto and hydroxyl group. Exposure to acid gave a single radiolabeled peak with Rt of 6a-hydroxyE1—suggesting the presence of a third molecule of oxygen. Mass spectrometry with limited material was inconclusive but supportive for a formula of C18H22O3. Thus, an epoxide involving the aromatic ring of E1 is suggested as a labile intermediate in the formation of the “unknown” metabolite. Estrogen epoxides as labile, reactive intermediates have been considered as potential precursors of the 2- and 4-hydroxy catechol estrogens with implications in breast cancer [Soloway, 2007]. Because of the association of the “unknown” metabolite with 6a-hydroxy-E1, the structural form proposed for the stable epoxide is that for 5a,6a-epoxy-estrone. This represents an alternative to the production of the 2- and 4-hydroxycatechol estrogens. The broad range in normal tissues where the “unknown” compound was shown to be a persistent metabolite (e.g. mouse mammary glands, ovary, uterus, brain, muscle, equine conceptus, stallion and domestic boar reproductive tracts) suggests more general biological implications. ã 2016 Elsevier Ltd. All rights reserved.
1. Introduction Steroid epoxides in biological systems were the subject of an early review [1]. They have been postulated as being participants in, or products of, selective biological oxidations. Attention was first drawn to them as possible intermediates in the biosynthesis of steroids when squalene epoxide was shown to undergo cyclization leading to the ultimate formation of cholesterol. Their biosynthesis and hydrolysis can occur by enzymatic or non-enzymatic processes—for example, cholesterol-5a,6a-epoxide is formed by ultraviolet light irradiation of skin. Although this epoxide of cholesterol was shown to be a stable compound it is generally conceded that steroid epoxides as oxidative metabolites in nature are quite labile. As a consequence, few have been detected and isolated under relatively physiological conditions. The formation of
* Corresponding author at: Department of Biomedical Sciences, University of Guelph, N1G 2W1, Guelph, ON, Canada. E-mail address: [email protected] (J.I. Raeside).
stable 5,6-epoxides of pregnenolone by liver microsomal preparations is at least one other exception [2]. The position of epoxides in the metabolism of estrogens has generated interest largely in relation to cancer. In analogy with the need for metabolic activation of some polycyclic aromatic hydrocarbons (PAHs) to become carcinogenic [3], a cytochrome P-450mediated oxidative metabolism of the primary estrogens, 17bestradiol (E2) and estrone (E1), seems necessary for carcinogenicity [4]. Some uncertainty remains as to the actual nature of the oxidized estrogen products that account for the initiation of tumor formation and support. It was proposed that epoxidation of E2 and E1 may be the underlying mechanism for inducing the carcinogenicity of these estrogens [5]. In addition, it has been shown that after reacting with dimethyldioxirane (DMDO), a versatile epoxide-forming oxidant, E2 was able to bind DNA forming DNA adducts [6]. However, an alternative pathway to DNA adducts has been the subject of extensive reviews, namely, the role of catechol estrogen quinones as initiators of breast and other human cancers [7,8]. After the initial oxidation to the catechol estrogens (CEs, 2hydroxy-E2/E1 and 4-hydroxy-E2/E1) by cytochrome P450
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enzymes, further oxidation gave rise to the semiquinones/ quinones that were the proximate carcinogens. The oxidative steps in the hydroxylation of E2 and E1 leading to the CEs themselves may well include epoxide formation as the corresponding epoxy-enone intermediates [9]. In fact, the facile conversion of the nonaromatic 1,2- and the 4,5-epoxyenones of E2/E1 to the catechol estrogens, 2- and 4-OH-E2/E1, respectively, was later demonstrated [10]. Although the chemical synthesis of these epoxy-enones was reported over twenty years ago [11], it remains to be determined whether any of the epoxy-estrogens are found in mammalian systems especially in the context of breast and ovarian cancers [12]. In studies on estrogen metabolism in domestic animals we have consistently encountered a metabolite of E1 which remained unidentified. One feature stood out, namely, it appeared as a metabolite of radiolabeled E1 ([3H]-E1) but was not detected (UV 280 nm) when unlabeled E1 was the substrate in incubations with a number of non-hepatic tissues. Its presence varied from being a relatively prominent to a barely detectable, radioactive peak on chromatography. From the literature the occurrence of an epoxide seemed unlikely; however, as the investigation of its nature progressed a point was reached where we now propose that the metabolite is the 5a,6a-epoxide of E1. The following is a brief account of the work leading to this conclusion. A few examples are also given to illustrate the relatively widespread formation of this metabolite and, thereby, to draw further attention to its possible biological significance. Similar results have also been obtained in more limited studies with [3H]-E2. 2. Materials and methods 2.1. Chemicals and reagents The radioactive steroids (NEN) obtained from Perkin–Elmer (Shelton, CT, USA) were [2,4,6,7-3H]-17b-estradiol (71.0 Ci/mmol) and [2,4,6,7-3H]-estrone (74.1 Ci/mmol). Nonradioactive steroids were purchased from Steraloids Inc (Newport, RI, USA). 6aHydroxy-estrone (6a-OH-E1) was made by Dalton Chemical Laboratories Inc., Toronto, ON, Canada. Solvents from Caledon Laboratories, Ltd (Georgetown, ON, Canada) were glass-distilled and reagent grade; acetonitrile (HPLC grade 190) was used for HPLC. Sep-Pak C18 cartridges were purchased from Waters Scientific (Mississauga, ON, Canada). Medium 199 and supplements were supplied by Sigma. All other chemicals were analytical grade from Fisher Scientific (Toronto, ON, Canada). 2.2. Collection and preparation of tissues 2.2.1. Stallion At castration of 8 stallions (3–17 years-old) the testes were placed on ice and taken to the laboratory where the epididymis and vas deferens were removed. Portions of the vas and mid regions of the caput, corpus and cauda epididymides were stripped of adventitious tissues, minced separately and transferred to a test tube containing physiological buffered saline (PBS). Three washes removed spermatozoa by suspension and gravity sedimentation. In each experiment replicates (2–4) of each tissue (250 mg) were dispensed for incubation within 3 h of the surgery. 2.2.2. Domestic boar (Sus scrofa) Reproductive tracts (n >10) were removed at slaughter from pubertal Yorkshire pigs (6 month-old) and at euthanasia, by intravenous injection of pentobarbital, from older breeding boars. Vas and epididymal tissues were prepared as above (2.2.1.). Prostate and seminal vesicles were also recovered from the older animals (n = 6) and dissected free of extraneous tissues before
mincing finely with scissors. Equal amounts of tissue (100–300 mg, wet weight) from each source were dispensed for incubation. 2.2.3. Early equine conceptus Mares from the research herd at the University of Guelph were used to recover conceptuses (Days 24–26), under conditions approved by the University Animal Care Committee. Conceptuses (n = 10) were collected by transcervical uterine lavage, as described previously [13]. The embryo proper (15–30 mg, wet weight) in PBS was dissected free of its extraembryonic membranes (yolk sac wall, allantois and amnion) under a dissecting microscope, using ophthalmic scissors and forceps. The trophoblast was divided into bilaminar (10–30 mg) and trilaminar (40–70 mg) components by dissection; these were incubated separately, without mincing, as was the case for the allantois (15–40 mg). 2.2.4. Mouse tissues Mammary glands were dissected from mature nulliparous female mice, 4–6 months-old (n = 20). In all experiments, the tissues were pooled for each animal, minced with scissors and washed by decanting in PBS. About 85–300 mg wet weight of tissue were dispensed for incubation. Additional tissues were collected from two mice (uterus, ovaries, brain, skeletal muscle and liver), washed and minced as above for the mammary gland. The amounts of tissues taken for incubation were (approximate wet weight, mg): uterus, 45–100; ovary, 10–40; brain, 100; muscle, 85–180; liver, 100. 2.2.5. Tissue incubation Tissues were incubated for 2–4 h in culture medium (TC-199, 5 ml for >300 mg; 2 ml for <300 mg) in small glass flasks in a shaking water-bath at 37 C under 5% CO2 in air, after either [3H]E1/E2 (1 106 cpm; or 2 106 cpm for >300 mg) or non-radioactive E1 (50 or 100 mg) were added as substrates. Within 2 h from time of collection, incubations were done in duplicate for radiolabeled substrates and 2–6 replicates for non-radiolabeled E1. After incubation and removal of the media, the tissues were rinsed twice with 200 ml of fresh medium which was then added to the previously collected media. When the larger amounts of tissues were involved, the contents of the flasks were transferred to tubes for centrifugation to recover the media; in which case the tissues were washed twice with centrifugation in 1 ml TC-199 and the washings pooled with the spent media. Media and tissues were stored in glass vials at 20 C until processed. 2.2.6. Analytical procedures Steroids were recovered from the medium by solid-phase extraction (SPE, Waters C18 Sep-Pak column) as described previously [14]. Briefly, the incubation medium was diluted with water to a volume of 5 ml, applied to a primed column and the unconjugated and conjugated steroids were eluted with 5 ml of diethyl ether and 5 ml of methanol, successively. The amount of radioactive material in each SPE fraction was determined by liquid scintillation counting (LSC) of an aliquot in 4 ml of Ecolite scintillation cocktail (MP Biochemicals, Solon, OH). The eluates were taken to dryness under nitrogen at <45 C. The dried residue was dissolved in 100 ml of acetonitrile:water (1:3) for injection on HPLC. Chromatographic profiles of steroids from SPE were generated on a Waters HPLC System using a Nova-Pak C18 column with UV absorbance monitored at 280, 254 and 210 nm. Four solvent systems were used: (1) a binary solvent gradient (Waters # 8) of acetonitrile: water (28:72 to 90:10); (2) an isocratic solvent system of 30% acetonitrile in water; (3) an isocratic solvent system of 40% acetonitrile in water; and (4) a binary solvent gradient (Waters # 8) of acetonitrile: water (20:80 to 90:10). All systems were run for
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40 min at a flow-rate of 700 ml/min. Fraction collections (LKB RediFrac, Pharmacia) were set for 0.5 or 1 min intervals for all systems with an aliquot (usually one-tenth) from each fraction taken for LSC. Identification of a radiolabeled metabolite was based initially on coincidence in elution with an authentic reference steroid run as internal standard. An intrinsic lag-time in fractional collection of the radiolabeled material resulted in a slight delay in recording the retention time (Rt) from that observed for the UV absorption of the associated reference standard.
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activities of the resultant metabolites that could activate their own unique receptors or effectors [18]. Our investigations were directed initially to estrogen metabolism in the reproductive tract of the stallion where the enigma of remarkably high production of estrogens by the testes was first noted by Zondek in 1934 [19]. The estrogen chosen was E1 because of its presence in much higher concentrations than E2 in peripheral blood, albeit as sulfates [20], where E1S is about a hundred times higher than testosterone [21]. 3.1. E1 metabolism in the equine reproductive tract
2.2.7. Chemical reactions Reduction: A modification of the method of Diczfalusy and Anne-Marie v. Münstermann, 1959, was used [15]. To dried residues from HPLC fractions in 1 ml of 90% aqueous MeOH, 5 mg of KBH4 was added and left overnight at room temperature. Water (2 ml) was added, tube shaken and left for 1 h before extraction of steroids with 3 2 ml diethylether, which was washed with 2 2 ml water and taken to dryness at <45 C under nitrogen. Acetylation: Dried residue in stoppered glass test tube was dissolved in pyridine (0.2 ml), an equal volume of acetic anhydride was added and left overnight at room temperature. Reagents were then removed under nitrogen. Water (5 ml) was added to the residue for SPE. Acid treatment: After exposure of the dried residue to 2N H2SO4 (1 ml) at 50 C for 2 h, water (1 ml) was added before neutralzing with 1N NaOH. Extraction was done with 2 5 ml of diethylether, which was washed with 2 2 ml water and the contents taken to dryness under nitrogen. 3. Results and discussion Oxidative metabolism of estrogens takes place mainly in the liver, but many examples of extra-hepatic metabolism have been reported since the early demonstration of 2-hydroxylation of E2 by the rat brain [16,17]. Importantly, a functional role for estrogen metabolism in target tissues may be seen to reside in the biological
Although estrogen sulfates comprise about 40–90% of the metabolites formed they have not yet been examined in this study because the use of solvolysis, as a preliminary step, would be detrimental (see Section 3.3.3). The HPLC profiles of [3H]-E1 metabolites in the unconjugated fraction from incubations of the vas deferens and epididymis showed the presence of several polar peaks but also a notable peak between E2 and E1 (Fig. 1) which became the focus of attention. Unexpectedly, when nonradioactive E1 was used as substrate no comparable peak was detected with UV absorption at 280 nm (Fig. 2). Thus it seemed that the characteristic phenolic structure of ring A was no longer present. With estrogen standards for reference, including ring B unsaturated estrogens (equilin, equilenin), efforts were made to learn more about the “unknown” metabolite by comparing Rts with that of the peak from epididymal tissue incubations of [3H]-E1; the only ‘match’ was with 17a-OH-E2 (data not shown). This possibility was dismissed because of its UV absorption at 280 nm, which is lacking in the “unknown” metabolite. All estrogens with an additional hydroxyl group (e.g. 2-, 4-, 6- and 16-OH-E1) are even more polar than E2, whereas their methylation products (2-, 3- and 4methoxy-E1) are less polar than E1. The failure of the “unknown” compound to align with the 9,11-dehydro-E1 reference standard, and because of the very non-polar nature of 16-dehydro-steroids, it is unlikely that the E1 metabolite had an additional site of unsaturation.
Fig. 1. Representative HPLC profiles of unconjugated steroids from incubations of [3H]-estrone with tissues from the reproductive tract of mature stallions. Note the presence of a prominent peak of radioactivity for an unidentified metabolite (U) with a retention time between E2 and E1. Larger amounts of radioactivity are seen as polar products— presumably 6-oxygenated-estrogens. Reference standards of 17b-estradiol (E2) and estrone (E1) were detected by UV absorbance at 280 nm (dotted line). (Solvent system 1).
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Fig. 2. Representative HPLC profiles for tissues from mature stallions when incubated with non-radioactive estrone. No peak was detected between the internal standard of E2 and the E1substrate. Small peaks at 4 and 6 min were seen with Rts similar to 6-oxygenated estrogens (Solvent system 2).
3.2. [3H]-E1 metabolism in the porcine accessory sex glands and reproductive tract With the boar, estrogen sulfates comprised 80–90% of the metabolites formed by regions of the epididymis (caput, corpus and cauda) and the vas deferens [22]. These fractions were not studied further, as was the case for the stallion work (see
Section 3.1). All HPLC profiles for the unconjugated products in incubations from the epididymis and sex glands revealed a radiolabeled metabolite between E1 and E2 (Fig. 3). It was the principal, or a major, peak in all chromatograms and was accompanied by a polar product tentatively identified by Rt as 6a-OH-E1. No evidence of the “unknown” compound was seen (UV 280 nm) when non-radioactive E1 was the substrate (data not
Fig. 3. Representative HPLC profiles of unconjugated steroids from incubations of [3H]-estrone with tissues from the reproductive tract of mature boars. A peak of radioactivity for an unidentified metabolite (U) was seen in all instances between reference standards of E2 and E1 (dotted line). Other metabolism was noted mainly as radioactive products with the retention time (5 min) of 6a-OH-E1 (Solvent system 1).
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shown). Subsequent efforts for identification of the “unknown” metabolite were made mainly with extracts from replicate incubations of [3H]-E1 with porcine tissues. 3.3. Chemical reactions with the “unknown” metabolite of [3H]-E1 3.3.1. Reduction with potassium borohydride The reduction with KBH4 resulted in a product from the radiolabeled metabolite that was more polar than E2 (Fig. 4A). Its position on HPLC reflected a moderate increase in polarity with the same relative shift as obtained regularly with reduction of E1 to E2. We concluded there was reduction of a keto group at C17. 3.3.2. Acetylation This was done to determine whether the hydroxyl group at C-3 of the E1 substrate remained intact. After the reaction it was clear that acetylation had occurred (Fig. 4B), and that a single acetylated product had been formed. Thus the presence of a single hydroxyl group seemed established for the metabolite. 3.3.3. Exposure to acid Acid treatment yielded only two products; the major one was coincident with 6a-OH-E1and the lesser eluted with the estrone reference standard (Fig. 4C). It was coincident with the Rt of the 6a-OH-E1 reference standard which suggested the presence of a third atom of oxygen in the molecule. This additional oxygen could
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not have been be present as a second carbonyl (see above, 3.3.1), or as a second hydroxyl group (3.3.2). In the context of exposure to acid, it should be noted that investigation of the sulfoconjugated estrogen metabolites has not yet been made because a solvolysis step, with its lower pH, would have resulted directly in the formation of 6a-OH-E1and/or E1. Similarly, the use of ascorbic acid to protect catechol estrogens from oxidation during our investigations (including HPLC) was omitted; as a consequence, the metabolism to 2-OH- and 4-OH-E1/ E2 was not examined. 3.3.4. Mass spectroscopy An attempt was made to establish the existence of an additional oxygen atom in the “unknown” compound by mass spectroscopy (LC/MS/MS). A composite sample collected from several tissue incubations of equine vas deferens showed no absorption at 280 nm but a small peak was detected at 200 nm, between E2 and E1 (data not shown). This material was submitted for examination; E1 and 6a-OH-E1 reference standards were included for comparison. LC-ESI/MS/MS revealed an Rt (7.2 min) which was slightly less than that for E1 (Rt, 7.6) but much greater than for 6a-OH-E1 (Rt, 4.4). The results in positive ion mode for the 6a-OH-E1 standard were clear with a mass ion [M+H]+ = 287.1638, and [M+HH2O]+ = 269.1530, indicating loss of the hydroxyl as water. However, due to the low abundance of ions in the MS spectrum for the “unknown” sample it was not possible to reliably propose a formula; the presence of a positive ion species of 269.1568 suggested [M+H-H2O]+ from a formula of C18H22O3. 3.4. Possible formation of a stable epoxide of E1
Fig. 4. Representative HPLC profiles of the “unknown” compound (U) after chemical reactions. A. Borohydride reduction resulted in a shift to a product more polar than E2 (Solvent system 1). B. Acetylation had a marked effect on polarity (U before, solid line; U after, dashed line). Solvent system 3. C. Acid hydrolysis yielded a peak co-eluting with 6a-OH-E1and a small amount of E1. Reference standards (dotted line). Solvent system 4.
The prospect of a stable epoxide involving the aromatic ring of E1 was entertained as the structural form of the “unknown” metabolite. Epoxides of estrogens as possible agents in breast cancer were hypothesized to be labile, reactive intermediates in the oxidative metabolism to catechol estrogens, and even mutagenic themselves [5]. Labile, non-aromatic ring A, dienol epoxides—thought to be an intermediate step in forming the 2-OH and 4-OH estrogens—can be stabilized as their enone epoxide tautomers [9]. In the case of our “unknown” metabolite, its existence as an epoxy-enone was ruled out by the absence of UV absorption at 254 nm. Because the ring A dienol epoxides (1,2- and 4,5-epoxides) are unstable it is most likely that a 5,10- epoxide would also be a highly labile structure (Scheme 1). In analogy with epoxide hydrolase (or non-enzymatic) action on 1,2- and 4,5epoxides, leading to 2-OH and 4-OH estrogens, it is suggested that similar action on a 5,10-epoxide of E1 might give rise to a hydroxyl at C-10 (10-hydroxy-3,17-diketo-estra-1,4-diene). Indeed, on investigating the inactivation of estrone in 1940, Westerfeld [23] concluded: “The introduction into the oestrone molecule of another hydroxyl group in the o- or p-position could theoretically produce three compounds; o-substitution would give rise to either 2:3- or 3:4-dihydroxy-17-keto-oestratriene; p-substitution would yield 10-hydroxy-3:17-diketo-oestradiene (1:4). These derivatives of oestrone are unknown at present”. The first catechol estrogen to be identified was 2-hydroxyestrone; in 1960, Fishman et al. reported its finding as a “new” metabolite of estradiol [24]. Since then, the catechol estrogens (2OH and 4-OH-E1/E2) have become well established as major products of estrogen metabolism, with well recognized biological significance from cancer [7] to neuroendocrine regulation [16]. Whereas 2,3- and 4,5-epoxides have been considered as intermediates in the formation of the catechol estrogens, we have been unable to find any report of an oxirane ring formed with C-5 and C10 of an estrogen. Although p-hydroxylation of a potential 5,10-
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Scheme 1. Oxidative metabolism of estrone. Intermediates in the formation of the catechol estrogens and 6a-hydroxyestrone are suggested; only metabolites with the a-configuration are illustrated. The stable “unknown compound” is proposed to be 5a, 6a-epoxy-E1. UV absorption: *280 nm; **254 nm; ***-ve for both.
epoxide might be considered, this is unlikely to be the case because the 10-hydroxy-product would differ by having an ab-unsaturated ketone and thus UV absorption at 254 nm. However, the possibility of enzymatic, or non-enzymatic, hydrolysis of a putative 5,10epoxide to form a stable 5,6-epoxide was considered as an option for the “unknown” metabolite. Then it was noted that the major peak on HPLC following acid exposure of the radiolabeled “unknown” compound was coincident with a 6a-OH-E1 reference standard. These observations reflect a severing of the oxygen bond to C 5 by nucleophilic attack at the ‘more substituted’ position of a 5a,6a-epoxide. No inversion of the stereochemistry was seen at C 6 since the 6a-configuration was retained in the 6a-OH-E1 formed by exposure to aqueous acid. Furthermore, a lesser peak of radioactivity with the Rt of the E1 reference standard points to an attack on the C O bond at C 6 to restore the aromatic ring with the product being a return to E1 (Fig. 4). In conclusion, the “unknown” compound which possesses remarkable stability in handling and storage is strongly favored to be 5a,6a-epoxyestrone. 3.5. Possible biological significance of a stable epoxide of E1 The potential significance of an estrogen epoxide in normal and malignant breast and other tissues was discussed briefly in a recent hypothesis [12]. It was considered in relation to an earlier hypothesis [5] that oxidative metabolism by cytochrome P-450 enzymes might produce a 4a,5a-epoxide, for example, as an intermediate step in the formation of 4-OH-E2. This labile
intermediate was found to be active as a neoplastic forming agent—presumably present for a limited time as the more stable 4a,5a-epoxy-enone [10]. Furthermore, the position of 4-OH-E2/E1 in the initiation of breast cancer through further oxidation to E2/ E1-quinones seems well established [8]. The existence of a stable 5a,6a-epoxy-E1 would represent an alternative and competitive pathway in estrogen metabolism that could reduce the intrinsic risk of catechol estrogen production. Formation of 6a-OH-E1 on exposure to aqueous acid suggests possible hydrolysis of 5a,6aepoxy-E1 to a 3,5a,6a-triol, as a transient intermediate, with subsequent dehydration at C5 to give 6a-OH-E1. An endogenous epoxide hydrolase might also be responsible for some of the 6aOH-E1 seen, in addition to the “unknown” compound, at the initial chromatography of most media extracts. It remains to be determined what biological actions, if any, might be demonstrated for the “unknown” compound when sufficient quantities can be made by chemical synthesis. Stable 5,6-epoxides can be formed by oxidative metabolism of steroids with a double bond between C-5 and C-6, such as cholesterol, pregnenolone and DHEA [1,2,25]; to date, biological implications have been considered only for cholesterol. Recent studies on cholesterol in relation to breast cancer have demonstrated that 5,6-epoxides of cholesterol are exceptionally stable compounds and surprisingly unreactive toward nucleophiles, thus ruling out a direct mutagenic and carcinogenic potency in mammals [26,27]. However, metabolism of 5a,6a-cholesterol by epoxide hydrolase produced 3b,5a,6b-trihydroxy-cholesterol which may be slightly carcinogenic; on further oxidation to 6oxo-cholestan-3b,5a-diol (OCDO) an oncogenic metabolite of cholesterol was revealed [28]. In subsequent studies, a new pathway in cholesterol metabolism was discovered that involves the action of an epoxide hydrolase on 5a,6a-epoxy-cholesterol to form a conjugation product with histamine (Dendrogenin A, a steroidal alkaloid) through a b-linkage at C-6 [29]. This unusual compound is now the focus of attention because it has been shown to have anti-tumor, as well as neuro-stimulating, properties [30]. It remains to be seen whether the relationship between the oxidative metabolism of E1 and cholesterol—to form stable 5,6epoxides—extends beyond the formation of 6-OH compounds on hydrolysis. Nevertheless, the occurrence of the “unknown” compound as a metabolite from incubations with E1 in nonhepatic tissues of mammalian domestic and laboratory species indicates a possible extensive, physiological significance. HPLC profiles of estrogen metabolites in media extracts from a series of experiments illustrate the broad range of its formation in potential target tissues. 3.6. Examples of the wide range in sites for production of a stable 5a,6a-epoxide of E1 The following examples extend the evidence beyond that from reproductive tissues of the stallion and domestic boar (Sus scrofa). 3.6.1. Early equine conceptus Components of the equine conceptus (embryo proper, allantois, bilaminar and trilaminar trophoblast) all revealed the presence of the ‘unknown’ metabolite in the HPLC profiles for E1 metabolites (Fig. 5). All possessed peaks with alignment for the “unknown” metabolite but with much greater amounts of radiolabeled material tentatively identified by Rts as 6-oxygenated products such as 6a-OH-E1 and 6-oxo-E1. The trophoblastic tissues were more active and showed some conversion of the substrate to E2, especially by the bilaminar layer (Fig. 5C). On exposure to aqueous acid the [3H]-labeled “unknown” compound co-eluted with the 6a-OH-E1 reference standard (data not shown).
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Fig. 5. Representative HPLC profiles of unconjugated steroids from incubations of [3H]-estrone with tissues from the components of early equine conceptuses (days 24–26). A, embryo proper; B, allantois; C, bilaminar trophoblast; D, trilaminar trophoblast. In all tissues a peak of radioactivity for an unidentified metabolite (U) was observed between the E2 and E1 standards. A low level of sulfoconjugation in the embryo proper was reflected in greater amounts of free radioactive material at HPLC. Metabolism to polar products was prominent in the profiles but E2 was also formed by the trophoblast, especially the bilaminar layer. (Solvent system 1).
3.6.2. Mouse tissues Results of metabolism of [3H]-E1 by several non-hepatic tissues and liver are given as representative profiles of similar findings in replicates of these tissues from other animals (n = 2–10). HPLC profiles (Fig. 6) show marked variation; a peak between E2 and E1,
having the Rt for the “unknown” metabolite, was seen in all tissues except for the liver. In liver incubations the metabolism of the substrate was almost complete; most of the radioactivity appeared as very polar products and none between E2 and E1 (Fig. 6D). The uterus had
Fig. 6. Representative HPLC profiles of unconjugated steroids from incubations of [3H]-estrone with several types of tissues from mice (A, mammary gland; B, ovary; C, uterus; D, liver; E, muscle; F, brain). With the exception of liver (Fig. 6D) all tissues showed the presence, to some degree, of an unidentified metabolite (U) between E2 and E1 standards. Metabolism was low in the uterus (Fig. 6C); all other profiles revealed significant amounts of polar products and the ovary alone (Fig. 5B) had a pronounced peak for E2. (Solvent system 2, A–D; Solvent system 1, E and F).
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the least metabolic activity, with the “unknown” metabolite and E2 being the principal products (Fig. 6C). While evidence for the formation of the “unknown” metabolite was clear in both ovary and mammary incubations, much of the radioactivity was seen as polar products. In the case of the ovary, a prominent peak for E2 was not unexpected (Fig. 6B). The HPLC profile of products for the mammary gland revealed more radioactivity as polar metabolites (Fig. 6A). Skeletal muscle provided clear evidence for the metabolite but with about equal amounts of radioactivity in the polar region (Fig. 6E). In brain tissue the metabolism of substrate was most marked, predominantly to polar products; however, the presence of the “unknown” metabolite was still detected (Fig. 6F). On the basis of Rts in the initial chromatography the presence of 6a-OH-E1, and lesser amounts of 6-oxo-E1, was strongly suspected in most experiments. A reciprocal, quantitative relationship between them and the “unknown” compound as products of metabolism is of interest but has not yet been established. If such were the case, it would suggest an even greater contribution of enzyme activities leading to the “unknown” compound and to 6oxygenated metabolites was present as a significant alternative to the formation of catechol estrogens. 3.7. Possible formation of a stable epoxide of E2 Lastly, some preliminary evidence for the production of a similar epoxide metabolite was provided from more limited investigations with E2 as substrate (Fig. 7). The “unknown” peak at 19 min in Fig. 7A, on acid hydrolysis, yielded not only E2 but also 6a-OH-E2 and 6b-OH-E2 (Fig. 7B). This again implied the presence of a third oxygen atom in the estrogen metabolite as seen with E1 as substrate.
4. Summary and conclusions Evidence is presented for the production of a stable 5a,6aepoxide of E1 in several non-hepatic tissues from the mouse and domestic animals. Attempts to identify a radiolabeled metabolite by HPLC (Rt, >E2 and
Fig. 7. Representative HPLC profiles of unconjugated steroids from incubations of [3H]-estradiol with tissues from the reproductive tract of mature stallions. A. Most of the radioactivity in all cases was seen as very polar metabolites (6hydroxylated?); lesser amounts were noted (U) as material slightly more polar than the E2 substrate (Solvent system 2). B. Acid hydrolysis of U resulted in two polar peaks co-eluting with 6a- and 6b-OH-E2 and a similar amount seen as E2. Solvent system 4).
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Please cite this article in press as: J.I. Raeside, H.L. Christie, A stable epoxide of estrone: Evidence for formation of a ‘new’ estrogen metabolite, J. Steroid Biochem. Mol. Biol. (2016), http://dx.doi.org/10.1016/j.jsbmb.2016.10.007