Absence of reactive intermediates in the formation of catechol estrogens by rat liver microsomes

Release of 3H2O from regiospecifically labeled estradiol was measured during 2-hydroxylation of this estrogen by rat liver microsomes. The amount of tritium remaining in the isolated catechol estrogen was also determined. Virtually all the tritium was removed from C-2 during the reaction confirming the absence of an NIH shift. About 20% of the tritium at C-1 was also lost without any such change occurring at C-4 or C-6,7 of the steroid molecule. These findings provide evidence for the formation of an arene oxide or o-semiquinone intermediate during the conversion of estradiol to 2-hydroxyestradiol. No indication of adduct formation at either C-1 or C-4 during this biotransformation was obtained although the 2-hydroxylated product was able to react with a nucleophile such as glutathione. The different regiospecificity of tritium loss in the generation of catechol estrogens and in their subsequent reaction leads to the important conclusion that the reactive intermediates in the two processes must be different. The possible role of catechol estrogens in neoplastic transformation is discussed.     

Morphological effects of the catechol estrogens on rat epididymal epithelia

The 2-hydroxy and 4-hydroxyestradiols (2-/4-OHE₂) caused marked cytotoxic effects, including vacuolation and nuclear changes, in rat epididymal epithelia, after exposure to very low levels (40 ng/rat/week) for 20 weeks. The effects of the 2-/4-OHE₂ metabolites were more pronounced than that of estradiol-17β(E₂).     

Active nonaromatic intermediates in the conversion of steroidal estrogens into catechol estrogens

A mechanism is proposed for mixed-function oxidase-catalyzed formation of the catechol estrogens 2-hydroxy- and 4-hydroxyestradiol from estradiol. This mechanism involves nonaromatic epoxyenones as intermediates. The isomeric 1 alpha,2 alpha-epoxy-17 beta-hydroxyestr-4-en-3-one and 1 beta,2 beta-epoxy-17 beta-hydroxyestr-4-en-3-one (the latter as its 17-acetate) were synthesized from 17 beta-hydroxy-5 alpha-estran-3-one. The isomeric 4 alpha,5 alpha-epoxy-17 beta-hydroxyestr-1-en-3-one and 4 beta,5 beta-epoxy-17 beta-hydroxyestr-1-en-3-one were prepared from 19-nortestosterone. From incubations of [6,7-3H]estradiol with microsomes from MCF-7 human breast cancer cells, which principally catalyze the formation of 2-hydroxyestradiol from estradiol, we were able to isolate a 3H-labeled product with the chromatographic properties of 1 beta, 2 beta-epoxy-17 beta-hydroxyestr-4-en-3-one (as its 17-acetate). The soluble protein fraction of homogenates of rat liver, which is devoid of estrogen 2-/4-hydroxylase activity, has been shown to catalyze the formation of 2- and 4-hydroxyestradiol from the 1 alpha,2 alpha-epoxide and from the 4 alpha,5 alpha- and 4 beta,5 beta-epoxides, respectively. We suggest that these results taken together strongly support a role for epoxyenones as intermediates in the formation of catechol estrogens.     

Androgens inhibit the formation of catechol estrogens by estrogen 2-hydroxylase present in rat liver microsomal preparations

The inhibition of estrogen 2-hydroxylase by androgens was demonstrated in screening assays and has been further investigated under initial velocity conditions. The ability of testosterone, 5 alpha-dihydrotestosterone, and dehydroepiandrosterone to block the conversion of estradiol to 2-hydroxyestradiol by male rat liver microsomal preparations was determined using two radiotracer methods--the conversion of [4-14C]estradiol to [4-14C]2-hydroxyestradiol and the release of 3H2O from [2-3H]estradiol. The apparent Ki's for the androgens ranged from 12.0 to 14.0 microM, with the apparent Km for the substrate estradiol in these assays of 2.08 microM. Multiple inhibition studies with the androgens and 2-bromoestradiol, an effective estrogen inhibitor, in male rat liver microsomes resulted in Dixon plots consisting of a series of nonparallel, intersecting lines. Thus, the androgens and 2-bromoestradiol are non-exclusive inhibitors, i.e. the binding of one compound to the enzyme does not interfere with the binding of the other. These interactions of androgens suggest that the steroid hormonal environment be considered in the examination of the physiological role(s) of the estrogen 2-hydroxylase and the catechol estrogen products.     

Carcinogenicity of catechol estrogens in Syrian hamsters

Estradiol and other estrogens induce renal carcinoma in male Syrian hamsters. The mechanism of carcinogenesis still remains unclear. Activation of estrogens to catechol metabolites has in the past been postulated to play a role in estrogen-induced carcinogenesis. Therefore, the carcinogenic activity of catechol estrogens was investigated. After 175 days of treatment, 4-hydroxyestradiol was found to be as carcinogenic as estradiol in male Syrian hamsters (4/5 and 4/5 animals with kidney tumors, respectively). Animals treated with 2-hydroxyestradiol (0/5) or 2-methoxyestradiol (0/6) did not develop renal carcinoma. The catechol estrogens failed to be mutagenic in the Ames test (reversions of his- S. typhimurium to histidine prototrophy in the TA 100 strain). The lack of carcinogenic activity of 2-hydroxyestradiol was not due to a failure to stimulate estrogen-dependent tumor growth. Growth of H-301 cells, an estrogen-dependent hamster kidney tumor cell line, was supported in vivo by estrogens in the following order: estradiol greater than 4-hydroxyestradiol greater than 2-hydroxyestradiol. Stimulation of tumor growth by 2-methoxyestradiol was not detected. It was concluded that the carcinogenic activity of 4-hydroxyestradiol was consistent with a role of catechol metabolites in estrogen-induced carcinogenesis. However, the intrinsic carcinogenic or hormonal activity of 2-hydroxyestradiol probably can not be assessed accurately in vivo because of its rapid methylation and metabolic clearance.     

Direct in vitro stimulation of pituitary LH release by alpha melanocyte stimulating hormone

The present $\text{in vitro}$ superfusion study demonstrates that synthetic α-MSH acts at the pituitary level, independent of the hypothalamus, to increase the release of LH in male but not in female rats.     

Adduction of catechol estrogens to nucleosides

We report the formation, detection, quantitation and structural characterization of products resulting from the adduction of deoxynucleosides (deoxyadenosine, deoxyguanosine, deoxycytidine and 5-methyldeoxycytidine) to the catechol estrogens (CE) of estrone, estradiol-17beta and estradiol-17 alpha. The crude products are obtained in a one-pot synthesis through oxidation of catechols to quinones and subsequent Michael-type reaction with the deoxynucleosides in acidic medium.In all experiments, adducts are detected by electrospray ionization mass spectrometry analysis after HPLC separation (LC/ESI/MS(n)). The two pyrimidines deoxycytidine and 5-methyldeoxycytidine yield only CE adducts to deoxynucleosides, which correspond to stable adducts on DNA. For purines, the results depend on the CE (2,3- or 3,4-catechols) used, the function and configuration on carbon 17 (ketone for estrone, alcohol for alpha and beta isomers of estradiol), and on the purine itself (deoxyadenosine or deoxyguanosine). Both stable adducts and deglycosylated adducts are formed, and therefore formation of stable adducts on DNA as well as the loss of purines from the DNA strands could be possible. MS(2) and MS(3) experiments prove to be relevant for further structural determinations, enabling in some cases the elucidation of the regiochemistry of adduction on the A and B rings of the steroid moiety.     

Direct inhibition of tyrosine hydroxylase activity by catechol estrogens.

Catechol estrogens, the 2-hydroxylated metabolites of estrogens, recently shown to be formed in brain, inhibit tyrosine hydroxylase, the enzyme that catalyzes the pivotal step in the biosynthesis of the neurotransmitters dopamine and norepinephrine. The nature of the inhibition is by competition with the pterin cofactor and thus resembles feedback inhibition of the enzyme by catecholamines.     

Mechanism of LH release in cultured rat pituitary cells

To investigate the mechanisms of the synthesis and the release of gonadotropin, rat anterior pituitary cells were stimulated in vitro with luteinizing hormone releasing hormone (LH-RH), [D-Ser(tBu)]6 des-Gly-NH2(10) ethylamide (Buserelin) and 12-0-tetradecanoyl phorbol-13-acetate (TPA), and then the LH and LH-beta subunit released into the medium were determined by radioimmunoassay. Buserelin showed its biological activity at a much lower concentration than LH-RH, but both of them caused the release of LH and LH-beta subunit in a dose-dependent manner. Furthermore, intracellular LH synthesis from LH-beta subunit by stimulation with LH-RH or Buserelin was also found. After inducing various degrees of desensitization by stimulation with LH-RH or Buserelin in a dose-dependent manner (the first stimulation), pituitary cells were stimulated with a fixed dose of TPA (the second stimulation) and the released LH was assayed. LH was released almost constantly by the second stimulation, regardless of the dose used for the first stimulation. These results suggest that the C-kinase pathway was unaffected by the desensitization induced with LH-RH or Buserelin.     

Convenient large scale preparation of catechol estrogens

2-Hydroxyestrone, 2-hydroxyestradiol-17β, 2-hydroxy-17α-ethyny1estradiol, 2-hydroxyestriol, 4-hydroxyestrone, 4-hydroxyestradiol-17β, 4-hydroxy-17α-ethynylestradiol and 4-hydroxyestriol are prepared on a preparative scale from the corresponding aminophenols using a new inverse oxidation procedure. By the synthesis described both the 2and 4-hydroxylated estrogens are available in high yields.     

In vitro metabolic conjugation of catechol estrogens

In vitro metabolic conjugation of the catechol estrogens, 2-hydroxyestrone and 4-hydroxyestrone, has been investigated by means of HPLC with electrochemical detection. Sulfation of 2-hydroxyestrone and 4-hydroxyestrone with the rat liver 105 000 g supernatant fortified with 3'-phosphoadenosine-5'-phosphosulfate provided the 2- and 4-monosulfates, respectively. Glucuronidation of the two catechols with the rat and human liver 1500 g supernatant in the presence of uridine-5'- phosphoglucuronic acid gave the 2- and 4-glucuronides, respectively. In contrast, incubation with the guinea pig liver 1500 g supernatant yielded both isomeric monoglucuronides . When 2'-hydroxyestrone was incubated with rat liver 1500 g supernatant and S-adenosyl-L-methionine, the 2- and 3-monomethyl ethers were formed in an equal amount, while 4-hydroxyestrone was transformed into the 4-methyl ether in 12 times greater yield than the 3-methyl ether. The participation of sulfation and glucuronidation in the formation of guaiacol estrogens is discussed.     

Norepinephrine-like effects of neuropeptide Y on LH release in the rat

Neuropeptide Y, a recently isolated neuropeptide exhibited norepinephrine-like effects on LH release after intracerebroventricular administration at doses from 0.5 to 10 micrograms. While it promptly suppressed LH release in ovariectomized rats, there was a dose-related stimulation of LH secretion in ovarian steroid primed-ovariectomized rats. In view of the evidence that neuropeptide Y may coexist with adrenergic neurotransmitters, these findings suggest that it may play a role in regulation of LH release in the rat, either independently or in concert with catecholamines.     

Formation of catechol estrogens by intestinal bacterial demethylation of 2-methoxyestrone

The intestinal bacterial metabolism of 2-methoxyestrone was studied by incubation in the isolated coecum from rats. Following isolation of estrogens by a combination of ion-exchange and ligand-exchange chromatography, the metabolites were identified by gas chromatography-mass spectrometry. The two main reactions were oxidoreduction at C-17 and extensive demethylation at C-2. Thus, the demethylation of 2-methoxyestrogens known to occur in vivo may be due to the action of microbial enzymes. The study also shows that the intestinal microflora is capable of converting biologically inactive into active steroid hormones.     

Nitrendipine-induced stimulation of renin release by the isolated perfused rat kidney

The direct effects of the organic calcium antagonist nitrendipine upon renin release were assessed using the isolated rat kidney perfused at constant pressure. This model circumvents the indirect actions of vasodilating agents by artificially maintaining perfusion pressure constant, thereby avoiding the hypotensive effects associated with the systemic administration of such agents. Renin release as assessed by radioimmunoassay was stimulated 2.6-fold upon the administration of 10(-6) M nitrendipine. Since this stimulation of renin release occurred in the absence of any alteration in perfusion pressure, we conclude that it represents a direct action of nitrendipine. This finding is in support of the current hypothesis concerning the inverse relationship between cytosolic Ca2+ and renin secretory rate, and suggests that Ca entry into the juxtaglomerular cells of the juxtaglomerular apparatus is sensitive to blockade by organic calcium antagonists such as nitrendipine.     

2-Hydroxylation of estrogens in the brain participates in the initiation of the preovulatory LH surge in the rat

Estradiol-2-hydroxylase, the enzyme responsible for the conversion of estrogens to catechol estrogens was measured in the brain of female rats at specific stages of the estrus cycle. Radiometric measurements of the enzyme activity in microsomal, mitochondrial, and synaptosomal fractions of the brain revealed a sharp increased in activity at proestrus just prior to the preovulatory LH surge. The enzyme activity declined to lower levels at diestrus and metestrus. No comparable fluctuations were noted in the liver enzyme. These changes in brain enzyme activity in conjunction with demonstrated positive feedback of exogenous catechol estrogens on pituitary LH release, suggest that a rise in endogenous catechol estrogen formation in the brain may be responsible for the physiological induction of the preovulatory LH surge.     

A simple chemical method for the synthesis of catechol estrogens

The preparation of 2-hydroxyestrone, 2-hydroxyestradiol-17, 4-hydroxyestrone and 4-hydroxyestradiol-17β by a simple one-step chemical reaction, treatment with potassium nitrosodisulfonate, is described. The structures of the products were established by nmr, ultraviolet, infrared and mass spectra as well as from their chemical and chromatographic properties.