Investigation of the in vitro metabolism of 17b-estradiol by LC-MS/MS using ESI and APC

Abstract

The steroid hormone estradiol is mainly metabolised (i) by oxidation of the hydroxyl function at the C17 position via steroid dehydrogenase, leading to estrone, and (ii) by hydroxylation via cytochrome P450 enzymes, occurring on the preferred positions C16α, C2 and C4 of the steroid (Fig. 1). When the hydroxylation takes place on the aromatic A ring of the steroid (positions 2 and 4), it leads to the formation of catechol-estrogens [1]. These compounds undergo several enzymatic processes to be either converted into inactive O-methoxylated metabolites or oxidised into quinone forms. Due to their highly electrophilic properties, estrogen quinones are extremely reactive species. Although they can be inactivated by conjugation with glutathione [2], they are also likely to react with DNA bases to form covalent adducts. This process can induce mutagenicity phenomenons [3], and is known to constitute a critical first step in several tumor initiation processes.The aim of this work was to study the in vitro metabolism of 17β-estradiol using hepatocytes in order to assess the formation of catechol estrogens at the cellular level. Thus, the detection of the catechol estrogens and their subsequent biotransformation products, i.e. glucuronide or glutathione conjugates and O-methoxylated derivatives, has been particularly emphazised. The extracts obtained from rat hepatocyte incubations have been directly analysed by LC-MS/MS using ESI and APCI ionisation techniques, which proved to be very complementary for the determination of polar as well as non-polar metabolites. [4,5,6,7]. HPLC grade solvents from Scharlau (Scharlau, Barcelona, Spain) and water from a Milli-Q system (Millipore, Saint-Quentin-en-Yveline, France) were used for LC-MS analyses. Material and methods Chemicals Cell preparationHepatocytes were prepared from Wistar rat livers according to the method described by Seglen * Corresponding authorLiquid chromatography coupled to tandem mass spectrometry with electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI) were used for the investigation of the in vitro metabolism of 17β-estradiol on rat isolated hepatocytes. The catechol estrogens corresponding to 17β-estradiol and estrone were identified by ESI, together with their glutathione and glucuronide conjugates, whereas more apolar compounds such as methoxylated catechol estrogens were characterised using APCI.

Schistosoma haematobium: Identification of new estrogenic molecules with estradiol antagonistic activity and ability to inactivate estrogen receptor in mammalian cells

“…It is known from the literature that estradiol is mainly metabolised by (i) oxidation of the hydroxyl function at the C17 position (via steroid dehydrogenase) to yield estrone, and (ii) hydroxylation via cytochrome P450 enzymes, occurring preferentially on C2, C4 and C16 (Bolton et al, 1998;Rathahao et al, 2000). When hydroxylation takes place on the steroid aromatic ring A, catechol-estrogens are produced (Iverson et al, 1996).…”

The role of estradiol metabolism in urogenital schistosomiasis-induced bladder cancer

“…When hydroxylation takes place on the steroid aromatic ring A, catechol estrogens are produced. [50][51][52] CYP1A1, CYP1A2, and CYP3A4 participate in this process; they exhibit catalytic activity dominantly for the 2-hydroxylation in estrogen 38 to liberate 2-hydroxyestrone/estradiol (2-OHE 1 (E 2 )). Thereafter, further oxidation to semiquinones (E 1 E 2 -2,3-SQ) proceeds, and to quinones (E 1 E 2 -2,3-Q) catalyzed by P450 enzymes.…”

Targeting molecular signaling pathways of Schistosoma haemotobium infection in bladder cancer

“…It is known from the literature that estradiol is mainly metabolised by (1) oxidation of the hydroxyl function at the C17 position (via steroid dehydrogenase) to yield estrone and (2) hydroxylation via cytochrome P450 enzymes, occurring preferentially on C2, C4 and C16. 81,82 When hydroxylation takes place on the steroid aromatic ring A, catechol-estrogens are produced. 83 Furthermore, the proposed molecular structure of these new estrogenic molecules explains the cellular effects of Sh demonstrated previously in reference 26.…”