![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
 |
Abstract |
|---|
 Top Abstract Experimental ProceduresResults and DiscussionReferences |
|---|
),
although their metabolic formation has only been reported very recently
(Ohe et al., 2000). Estrone
(E11)and estradiol
(E2)-derived quinols [10ß-hydroxy-1,4-estradiene-3,7-dione (E1-quinol)
and 10ß,17ß-dihydroxy-1,4-estradiene-3-one (E2-quinol)] were
detected from the respective estrogens during metabolic oxidation catalyzed by
several cytochrome P450 isoenzymes in rat liver microsomal systems. Contrary
to the well known catechol metabolites of estrogens
(Zhu and Conney, 1998),
quinols possess no aromatic A-ring; thus, their (bio)chemistry should be
substantially different from that of catechols. Although Ohe et al.
(2000) suggested quinol
formation as a potential contributor to the carcinogenecity of estrogens,
actual studies have shown no significant toxicity by estrogen-derived quinols
(Solaja et al., 1996;
Milic et al., 2001).
Incidentally, we recently found that the cytochrome P450 mimic,
5,10,15,20-tetrakis(pentafluorophenyl)-21H,23H-porphine
Fe(III)/3-chloroperbenzoic acid system
(Higuchi and Hirobe, 1996),
yielded E1-quinol as a principal product from E1, instead of the expected
catechols (L. Prokai, K. Prokai-Tatrai, and P. Perjesi, unpublished results).
Quinols are obtained chemically from the corresponding
para-substituted phenols mostly via metal-catalyzed oxidative
dearomatization (Yamada et al.,
1974); however, it is possible to convert the quinols back to the
parent compounds through reduction by, e.g., Zn/acetic acid
(Gold and Schwenk, 1958).
Therefore, we hypothesized that such a reaction for estrogen-derived quinols
may also be possible with endogenous reducing agent(s), and enzymes in the
liver may further catalyze this process. This proposed pathway would, then, be
unique, because it would allow for the regeneration of phenolic A-ring
estrogens from the metabolites. A facile regeneration would also explain why
quinols might be unable to accumulate as major products upon metabolism by
liver microsomes, yet the corresponding route may represent a significant part
in the overall estrogen-metabolizing process in the system. In this study, we
tested the hypothesis for the existence of reductive conversion in enzyme-free
and rat liver microsomal incubations using a representative substrate
(E1-quinol), as shown in Fig.
1.
|
|
Experimental Procedures |
|---|
|
TopAbstractExperimental ProceduresResults and DiscussionReferences |
|---|
). Cytochrome P450
reductase (EC 1.6.2.4
[EC]
) and all other chemicals were obtained from
Sigma-Aldrich Inc. (St. Louis, MO). Male rat liver microsomes (Sprague-Dawley
strain, untreated) were purchased from XenoTech LLC (Kansas City, KS). Incubation of E1-Quinol with Endogenous-Reducing Agents. E1-quinol (0.1 mM) and 1.0 mM NADH, NADPH, sodium ascorbate, or GSH in 0.1 M sodium phosphate buffer (1 ml of final volume, pH 7.5) was incubated at 37°C. At incremental time points, 100-µl aliquots were removed into ice-cooled centrifuge tubes, and 50 µl of glacial acetic acid was added. After immediate extraction with ethyl acetate (2 x 500 µl), the organic layer was evaporated under nitrogen stream at room temperature. Reconstitution of the samples with the LC mobile phase was followed by LC/MS analyses. For the control experiment, no reducing agent was used.
Microsomal Incubations of E1-Quinol. The incubation mixture (final
volume 1 ml) containing 0.4 mM of NADP+, 60 mM of KCl, 4 mM of
MgCl2, 4 mM of G-6-P, 5 U of G-6-P dehydrogenase, and 0.1 mM
E1-quinol in sodium phosphate buffer (pH 7.5) was preincubated for 2 min at
37°C before microsomes (1 mg/ml protein) and 0.4 U of cytochrome P450
reductase (Roy and Liehr, 1988)
were added. After 2.5 min of incubation, aliquots (100 µl) were taken, and
glacial acetic acid (50 µl) was added to stop the reaction. To the mixture,
ethyl acetate (3 x 500 µl, vortexed for 1 min, centrifuged at 10,000
rpm for 5 min) was added. The organic layers were removed and combined
followed by evaporation under nitrogen stream. The residue was dissolved in
the mobile phase for LC/MS analysis.
Qualitative Analysis. LC separation was done using a 5 cm x 2.1 mm i.d. Discovery HS C18 reversed-phase column (Supelco, Bellefonte, PA) with 0.25 ml/min water/methanol/2-propanol/acetic acid/dichloromethane (53:35:5:5:2, v/v) as a mobile phase. The sample residues were dissolved in 40 µl of mobile phase, respectively, and 5 µl of the solution was injected for analysis. Mass spectra were recorded on a quadrupole ion-trap instrument (LCQ; ThermoFinnigan, San Jose, CA) using positive-ion APCI as the method of ionization. MS/MS and MS3 product-ion scans were obtained after collision-induced dissociation with helium as the target gas. Comparison with authentic reference compound (retention time, tR, and mass spectra) was used for unambiguous identification of E1.
Quantitative Analysis. As an internal standard,
1,3,5(10)-estratrien-17
-ethynyl-17ß-ol (0.3 µM) was added
before each sample extraction. E1 and E1-quinol levels were determined by
LC/APCI-MS/MS and calibration with solutions of known concentrations of E1
(0.02 to 11 µM) and E1-quinol (0.2 to 125 µM) extracted for analyses.
The chromatographic peak areas for E1, E1-quinol, and the internal standard
were obtained from the m/z 271 
253, m/z 287
269, and m/z 279 133 + 159 MS/MS transitions, respectively.
Kinetic analysis of the microsomal metabolism was done by nonlinear
curve-fitting (Scientist for Windows; MicroMath, Inc., Salt Lake City, UT)
presuming consecutive first-order processes in the cascade of E1-quinol
E1 E1-metabolites.
|
Results and Discussion |
|---|
|
TopAbstractExperimental ProceduresResults and DiscussionReferences |
|---|
). The
quinol was meticulously purified by column chromatography (silica gel,
dichloromethane, and dichloromethane/ethyl acetate, 8.5:1.5) to ensure that no
contamination from E1 was present. Although Ohe et al.
(2000) used gas
chromatography/MS system for the detection of quinol in rat liver microsomes,
we developed an LC/MS assay, because gas chromatography/MS gave us unreliable
results due to artifacts detected during the analysis of the underivatized and
also of the trimethylsilylated standard. Among the possible ionization
techniques compatible with routine LC/MS, APCI is the method of choice for the
ionization of nonpolar analytes such as steroids
(Ma and Kim, 1997). The
selection of E1 as a parent compound for quinol preparation was beneficial for
the LC/MS, because positive-ion APCI-MS properties of E1 were more
advantageous than those of E2, which showed extensive water loss (-18 u) from
the protonated molecule (MH+). With the LC/MS method developed, we
have also shown the formation of E1-quinol upon incubation of estrone with rat
liver microsomes under the conditions employed by Ohe et al.
(2000). To probe the hypothesis for the in vitro reductive regeneration of estrogens from quinols, we incubated E1-quinol in phosphate buffer (pH 7.5) supplemented with various endogenous reducing agents, such as ascorbic acid, GSH, NADH, or NADPH and in a rat liver microsomal system. When E1-quinol (100 µM) was treated with a 10-fold molar excess GSH, we failed to detect E1—similarly to the control experiment where no potential reducing agent was used. Only trace amount of E1 was obtained even after 12 h of incubation upon the addition of ascorbic acid. However, formation of E1 was clearly detectable even after a short period of time, when the incubation was carried out in the presence of NADH and, especially, NADPH (Fig. 2). The presence of E1 in the incubation sample was unequivocally determined by matching retention time and mass spectra (MS/MS and MS3 product-ion spectra) to authentic E1, as shown in Fig. 2. Although the exact mechanism of the reaction is yet to be elucidated, our results clearly show that enzymes are not ultimately necessary for the reductive aromatization of estrogen-derived quinol metabolites.
|
When rat liver microsomal incubation of E1-quinol was done, the
quinol-to-phenol transformation progressed very rapidly. The initial rate of
estrone formation at 37°C was 6.5 ± 1.5 nmol ·
min-1 · (mg of protein)-1, whereas the
initial rate of the second-order reaction for the reduction of E1-quinol to
estrone by a 10-fold excess of NADPH in a microsome-free buffer solution and
under identical incubation conditions was 0.62 ± 0.03 nmol ·
min-1. Consequently, while reduction of E1-quinol to E1
takes place in the mere presence of NAD(P)H, enzymes available in the liver
apparently catalyze this reaction. The involvement of flavoprotein reductases
distinct from the cytochrome P450 hemoprotein has been suggested in the
metabolism of structurally similar steroids in mammalian tissue
(Vaz et al., 1995); however,
further studies are needed to address the nature of the catalyst present in
the microsomal system.
In summary, our results imply a novel metabolic cycle for phenolic A-ring
estrogens, which proceeds through quinols that are then regenerated to the
parent estrogen consumed during preceding metabolic process by
enzyme-catalyzed NAD(P)H-dependent reduction. Additionally, while redox
cycling between catechol estrogen and their quinones produces reactive oxygen
species (Roy and Liehr, 1988;
Liehr and Roy, 1990;
Nutter et al., 1994;
Bolton et al., 2000), it is
highly unlikely that steroidal quinols show similar properties to catechol
estrogens in this regard. The cyclic dienone structure would preclude the
formation of "semiquinol" radicals crucial to the prooxidant
effect. Further studies are needed, however, to associate biological functions
with the newly identified and apparently very efficient metabolic regeneration
of estrogens via quinols.
Laszlo Prokai
Katalin Prokai-Tatrai
Pal Perjesi
Alevtina D. Zharikova
James W. Simpkins
Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, Florida (L.P., P.P., A.D.Z.); Center for Neurobiology of Aging, College of Medicine, University of Florida, Gainesville, Florida (K.P.-T.); Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas (J.W.S.)
|
Footnotes |
|---|
1 Abbreviations used are: E1, estrone; E2, estradiol; E1-quinol,
10ß-hydroxy-1,4-estradiene-3,7-dione; E2-quinol,
10ß,17ß-dihydroxy-1,4-estradiene-3-one; LC, liquid chromotography;
APCI, atmospheric-pressure chemical ionization; MS, mass spectrometry; GSH,
glutathione; G-6-P, glucose-6-phosphate. 
Address correspondence to: Dr. Laszlo Prokai, Department of Medicinal Chemistry, College of Pharmacy, 1600 SW Archer Road, University of Florida, Gainesville, Florida 32610-0485. E-mail: lprokai{at}grove.ufl.edu
|
References |
|---|
|
TopAbstractExperimental ProceduresResults and DiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  J. M. Flynn, D. A. Lannigan, D. E. Clark, M. H. Garner, and P. R. Cammarata RNA suppression of ERK2 leads to collapse of mitochondrial membrane potential with acute oxidative stress in human lens epithelial cells Am J Physiol Endocrinol Metab, March 1, 2008; 294(3): E589 - E599. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Singh, J. A. Dykens, and J. W. Simpkins Novel Mechanisms for Estrogen-Induced Neuroprotection. Experimental Biology and Medicine, May 1, 2006; 231(5): 514 - 521. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
