VOL. 1, No. 1, January 2004

A. J. Lee, et al. [2004] Med Hypotheses Res 1: 53-65.

Characterization of Novel Nonpolar 17β-Estradiol
Metabolites Formed by Human Cytochrome P450

Anthony J. Lee, Perry J. Pellechia, Michael D. Walla, and
Bao Ting Zhu*

Department of Basic Pharmaceutical Sciences, College of Pharmacy (A.J.L., B.T.Z.), and
Department of Chemistry and Biochemistry, College of Science and Mathematics (P.J.P.,
M.D.W.), University of South Carolina, Columbia, SC 29208, USA

Abstract.  We report here our findings on the NADPH-dependent formation of a novel
class of nonpolar estrogen metabolites by fifteen human cytochrome P450 (CYP) isoforms
(CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, 3A7, and
4A11). A total of some 20 nonpolar radioactive metabolite peaks (designated as M1
through M20) were detected following incubations of [3H]17β-estradiol with human CYP
isoforms and NADPH. Some of the nonpolar metabolites were formed at varying rates
with each of the 20 human CYP isoforms tested, but M15 and M16 were selectively
formed only with a few CYP isoforms. CYP3A4 and 3A5 had the highest catalytic activity
for the formation of M15 and M16, but CYP1A1, 2C8 and 2C9 had weak but detectable
catalytic activity for their formation. Kinetic analyses showed that the apparent Km
values for CYP3A4 and CYP3A5-dependent formation of M15 and M16 ranged from 46–
119 uM, and their apparent Vmax values ranged from 206–276 pmol/nmol of CYP/min.
Using mass and NMR spectrometric analyses, we unequivocally identified the structures of
M15 and M16 to be the dimers of 17β-estradiol, which were connected together through a
diaryl ether bond between a phenolic oxygen atom of one 17β-estradiol molecule and the
2- or 4-position aromatic carbon of the other 17β-estradiol. Further studies are needed to
determine any biological functions that may be associated with these novel nonpolar
estrogen metabolites.

*Address all correspondence to: Dr. Bao Ting Zhu, Department of Basic Pharmaceutical
Sciences, College of Pharmacy, University of South Carolina, Room 617 of Coker Life
Sciences Building, 700 Sumter Street, Columbia, SC 29208 (USA).

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