Quantitative aspects of estrogen metabolism in mammalian female and male reproductive systems

How significant is local estrogen metabolism? In a biological response in vivo, the primary agent 17β-estradiol (E2) is delivered by the blood supply to the tissues where it acts mainly through binding to the estrogen receptors (ERs). However, active enzymes within the cells may intervene to regulate, in part, the levels of E2 available for ER binding. This can be illustrated clearly with radioactive estrogens as substrates in tissue incubations.

Earlier work on estrogen metabolism focused on the identification of products formed by microsomal preparations and highly purified enzymes – involved mainly in oxidative metabolism. An extensive catalysis of E2 and estrone (E1) by various cytochrome P450 isoforms present in the liver and in extrahepatic estrogen target organs resulted in the formation of various hydroxylated or keto metabolites (Zhu & Lee 2005). The impressive list of such metabolic products led to a hypothesis that the response to estrogen exposure was not solely from E2 but also from the results of the combined actions of specific metabolic enzymes present in, and characteristic of, the target tissues (Zhu & Conney 1998).

Although metabolism (e.g. oxidation, reduction, hydroxylation, sulfation and desulfation) is most impressive in the liver, the target tissues themselves express some of these enzymes to varying degrees and tissue specificities. A prime example is the interconversions between E1 and E2 by the 17-hydroxysteroid-dehydogenases (17β-HSDs); higher amounts of E2 result when reductive 17β-HSDs prevail. Competing actions of sulfatases and sulfotransferases could also be critical for estrogen exposure in target tissues (Mueller et al. 2015). A related feature is the presence of active mechanisms for the transport of E1S across the cell membrane by organic anion transporters; it is uncertain how quantitatively significant this might be.

Two further examples of the complexities of local estrogen metabolism are worth inclusion at this point. First, the formation of products such as the catechol estrogens, 2-OH and 4-OH, is of particular interest in cancer (Ziegler et al. 2015). Both may be oxidized further to semi-quinones and quinones that are active carcinogens, through mechanisms first revealed for polyaromatic hydrocarbons. Detoxification and removal of catechol estrogen quinones occur through the conjugation with glutathione. In contrast to the formation of quinones, the conversion to monomethyl ethers by catechol-o-methyltransferase (Roy et al. 1990) can be beneficial insofar as 2-methoxy-E2 has been shown to have antiproliferative activity.

Secondly, a surprising product of enzyme activity is the esterification of E2 with a variety of long-chain fatty acids (e.g. palmitic) specifically at carbon-17 on the D ring (Hochberg 1998). These lipophilic estrogens, found in the highest concentrations in ovarian follicular fluids, provide a large potential reservoir of E2 as a hydrophobic prohormone from various sites of adipose tissue. They are associated with lipoproteins for transport in blood, with some claims for antioxidative properties.

At present, quantitative data on the full extent of local metabolism of E2 are still unavailable, or incomplete, despite considerable advances in technology (Rister & Dodds 2020). The low levels of estrogens and their products, usually encountered in most target tissues, present challenges for separation, identification and quantification. Thus, difficulties remain in meeting the objective of gaining a full account of metabolic activity when biological samples of interest are limited in size. In this regard, studies with radiolabeled estrogens provide preliminary answers and afford a guide. A rewarding feature in the use of radiolabeled E1 and E2 is assurance that the products formed are indeed derived from the radioactive substrate.

After tissue incubation, the separation of steroids into unconjugated and conjugated fractions provides important information on the distribution of radioactivity as a first step. The amount of radioactivity in the conjugate fraction is a significant measure of inactivation of the estrogen substrate and its possible removal. Three components comprise the conjugate fraction – sulfates, glucuronidates and non-hydrolyzable products (e.g. glutathiones) – with some variation in total amounts and distribution. For example, in studies with boar tissues, the total conjugates were over 50% for the epididymis and 60% for vas deferens. Sulfate fractions for both tissues were about 80% of total conjugates, almost entirely present as E1S – suggesting that rapid sulfotransferase activity had limited further metabolism (Raeside et al. 1999). Marked differences, however, were observed with accessory sex glands where conjugation was 15 and 50% for prostate and seminal vesicles, respectively – with remarkably high proportions as glucuronidates (67%) by seminal vesicles. Recovery of ‘free’ steroids from glucuronidates is negligible in contrast to high levels of deconjugation of estrogen sulfates on entering cells. Thus, the extent and type of conjugation can have a marked bearing on the outcome of exposure to estrogens. Although direct action of some steroid conjugates can be cited, such as DHEAS and E1S, the major biological response is attributed to the unconjugated form, almost universally. For this reason, focus on unconjugated steroids has remained paramount in studies of estrogen metabolism.

Examples of chromatographic profiles for unconjugated fractions from diverse mammalian tissues were chosen to illustrate the significance of local metabolism of estrogens as reflected in quantitative terms (Fig. 1). They show only some of the variety in distribution patterns encountered in many other similar experiments. Although identification of some products was tentative and others were unknown, the primary information was (i) on substrate retention and (ii) on the extent of estrogen metabolism. Indeed, the salient feature in all instances was the presence of estrogen metabolites in greater amounts than for the substrate. Note that sulfated estrogens and other conjugated products had been removed before profiles of unconjugated fractions were examined.

HPLC profiles of unconjugated estrogen metabolites from tissue incubations with [³H]-estradiol and [³H]-estrone. Tissues were excised from animals euthanized under accepted University and National protocols, or at clinical surgery. They were then minced, rinsed and transferred to vials containing a medium with radioactive substrate and incubated for 2 h. Steroids were recovered from media by solid-phase extraction for examination by HPLC. Mouse mammary gland (A and B); canine skeletal muscle (C and D); stallion epididymis and vas deferens (E and F). Peaks: 1. 6-oxygenated-estrogens; 2. 5α,6α-epoxy-E2; 3. 5α,6α-epoxy-E1; 4. 2-methoxy-E2; 5. 2-methoxy-E1; 6. E2/E1-ethers. Radiolabeled products (solid lines); nonradioactive estrogen reference standards (dotted lines), 17β-estradiol (E2) and estrone (E1). Some variability was inevitable in the conditions of HPLC chromatography so that peak retention times are based on reference to those of internal nonradioactive standards.

Citation: Reproduction 164, 2; 10.1530/REP-22-0188

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HPLC profiles of unconjugated estrogen metabolites from tissue incubations with [³H]-estradiol and [³H]-estrone. Tissues were excised from animals euthanized under accepted University and National protocols, or at clinical surgery. They were then minced, rinsed and transferred to vials containing a medium with radioactive substrate and incubated for 2 h. Steroids were recovered from media by solid-phase extraction for examination by HPLC. Mouse mammary gland (A and B); canine skeletal muscle (C and D); stallion epididymis and vas deferens (E and F). Peaks: 1. 6-oxygenated-estrogens; 2. 5α,6α-epoxy-E2; 3. 5α,6α-epoxy-E1; 4. 2-methoxy-E2; 5. 2-methoxy-E1; 6. E2/E1-ethers. Radiolabeled products (solid lines); nonradioactive estrogen reference standards (dotted lines), 17β-estradiol (E2) and estrone (E1). Some variability was inevitable in the conditions of HPLC chromatography so that peak retention times are based on reference to those of internal nonradioactive standards.

Citation: Reproduction 164, 2; 10.1530/REP-22-0188

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HPLC profiles of unconjugated estrogen metabolites from tissue incubations with [³H]-estradiol and [³H]-estrone. Tissues were excised from animals euthanized under accepted University and National protocols, or at clinical surgery. They were then minced, rinsed and transferred to vials containing a medium with radioactive substrate and incubated for 2 h. Steroids were recovered from media by solid-phase extraction for examination by HPLC. Mouse mammary gland (A and B); canine skeletal muscle (C and D); stallion epididymis and vas deferens (E and F). Peaks: 1. 6-oxygenated-estrogens; 2. 5α,6α-epoxy-E2; 3. 5α,6α-epoxy-E1; 4. 2-methoxy-E2; 5. 2-methoxy-E1; 6. E2/E1-ethers. Radiolabeled products (solid lines); nonradioactive estrogen reference standards (dotted lines), 17β-estradiol (E2) and estrone (E1). Some variability was inevitable in the conditions of HPLC chromatography so that peak retention times are based on reference to those of internal nonradioactive standards.

Citation: Reproduction 164, 2; 10.1530/REP-22-0188

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The following discussion of the HPLC profiles relates to the fate of each substrate incubated, separately, in different types of mammalian tissues (Fig. 1). A complete view of the distribution of radioactivity is revealed in each case. Moreover, it allows the selection of peaks of possible interest for further examination. In this light, the results from the three tissue types are discussed with comments on the numbered peaks (no. 1–6) where present in the profiles. The peaks were examined later with further chromatography (data not shown).

With tissues from mouse mammary glands (Fig. 1A and B), about 50% of total radioactivity in extracts of incubation media was found in the unconjugated fractions. In the chromatographic profiles, it was noted that E1 was formed from E2 but no reduction of E1 to E2. Both substrates yielded large amounts of very polar metabolites (peak no. 1), possibly 6α/β-OH-E2 and 6α/β-OH-E1, respectively. Peak no. 2 tentatively identified as an E2 epoxide, is a major product, whereas the lesser peak no. 3 was possibly E1 epoxide (Raeside & Christie 2017). Interestingly, the nonpolar peak no. 5 was subsequently co-eluted with 2-methoxy-E1. The very nonpolar peak no. 6 was shown to be an unidentified estrogen ether of E2 and not an E2 fatty acid ester. This was also true for the E1 metabolite─note E1 esterification with long-chain fatty acids does not occur (Hochberg 1998). The formation of estrogen dimers (Zhu & Lee 2005) was considered (Raeside & Christie 2010) but rejected when an authentic standard became available (unpublished data).

In canine skeletal muscle (Fig. 1C and D), the unconjugated profiles of radioactivity (45% of total) for estrogen metabolism showed little, if any, interconversion between E2 and E1. Both substrates revealed only modest amounts of radiolabeled products in peak no. 1, suggestive of the presence of 6α-OH-E2 and 6α-OH-E1, respectively. Very nonpolar metabolites were the dominant feature (peak no. 6) with two components present in the peak in each profile. Perhaps, most noteworthy because of possible interest in cancer was the evidence for catechol-O-methyltransferase activity in the formation of 2-methoxy-E2 (peak no. 4) and 2 methoxy-E1 (peak no. 5).

Stallion tissues from the reproductive tract─epididymis and vas deferens, likewise, displayed distinctive profiles of estrogen metabolites in media extracts (Fig. 1E and F); however, only about 20% of total radioactivity was unconjugated. No interconversions between E2 and E1 occurred. Relatively small amounts of radioactivity were seen as several lesser peaks of polar products. The estrogen epoxide of each substrate was present as a significant peak in their respective profiles (no. 2 and no. 3). Their tentative identity was made (Raeside & Christie 2017). While some 2-MeOH-E2 was detected (peak no. 4), there was no clear evidence for 2-MeOH-E1. A marked difference was observed for the very nonpolar metabolites (peak no. 6), which was not typical for this type of tissue.

In conclusion, regardless of the definitive identification of the radiolabeled products, these studies reveal that the local metabolism of estrogens is quantitatively significant. Remarkedly in most instances, the metabolites were either very polar or very nonpolar products; furthermore, they were either ‘weak’ estrogens or of unknown activity resulting in a reduced availability of E2. These findings are limited, however, as a reflection of conditions in vivo. Granted data of a more dynamic nature are needed. One such means might be perfusion of organoids made from target tissues – using nonradioactive-labeled E2 (²H; ¹³C) in apparatus now available – to generate the profile and identification of estrogen metabolites with liquid chromatography–mass spectrometry. Similarly, information could also be obtained on factors that affect the activities of enzymes involved in estrogen metabolism and thus the extent of estrogen exposure in target tissues.