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Influence of Various Estrogens on BiotransformationAffinity to Cytochrome P-450 Structure Activity Relationships and Scavenger Function.

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Influence of Estrogens on Biotransformation
Influence of Various Estrogens on Biotransformation: Affhity to
Cytochrome P-450, Structure Activity Relationships, and Scavenger
Sabine Gemhardt a), Elke Karge
Bruno Schonecker b), and Wolfgang Klinger*a)
Institut fiir Pharmakologie und Toxikologie der Friedrich-Schiller-Universitat Jena, Loebdertstr. 1, D-07743 Jena, Germany
Institut fiir Organische Chernie und Makromolekulare Chemie der Friedrich-Schiller-UniversitiitJena
Key Words: Estrogens; biotransformution; scavengerfunction; enantiomer; structure activity relationship
Nine natural and synthetic estrogens, all derived from endogenous
17P-estradio1, were tested for their affinity to cytochrome P-450
(P450).Binding spectra of the estrogens with rat liver microsomal
P450 and inhibition kinetics with characteristic monooxygenase
model reactions (ethylmorphine N-demethylation, EN, and
ethoxycoumarin 0-deethylation, EO) were determined. In addition, uncoupling effects and/or free radical scavenger functions
were analysed by NADPH/Fe++stimulated microsomal luminoland lucigenin-amplified chemiluminescense (CL). 17P-Estradio1,
17a-ethynylestradiol,and D-estradiol 3-methyl ether inhibited
both monooxygenase reactions of cytochrome P-450,whereas
L-estradiol3-methyl ether inhibited EO only. 17P-Estradio1, 17aethynylestradiol, and D-estradiol3-methyl ether seem to act as free
radical scavengers. From the results both structure activity relationships could be established and data on possible interferences
with drug metabolism obtained. The enantiomers D- and L-estradiol3-methyl ether differ in their effects on these systems.
Oxidation catalysed by the cytochrome P450-enzyme-complex (P450) belongs to the most important biotransformation
reactions. Both endogenous (e.g. steroids) and exogenous
compounds (xenobiotics) can be oxidised. Therefore binding
to and hydroxylation by P450 of steroids may compete with
the biotransformation of drugs and practical therapeutic consequences must be considered. According to our present
knowledge P450 comprises 74 families with over 48 1 different P450 genes and 22 pseudogenes [11 and this number is
steadily increasing. All these enzymes differ in their substrate
specificity and inducibility.
The most important P450 enzymes for drug biotransformation are found in four main families (1 to 4). According to
their inducibility the P450-forms are divided into two main
groups: inducers of the barbiturate type and inducers of the
3-methylcholanthrene type. The 3-methylcholanthrene type
induces two P450 forms, 1Al and 1A2, phenobarbital induces the P450 families 2 and 3 (2B1, 2B2, 3A2 to 3A4).
These enzymes are responsible both for the biotransformation
of many drugs and the hydroxylation of steroids. Other specific inducers, e.g. ethanol for 2E1, are of minor importance
Arch. Phann. Pharm. Med. Chem.
Substrates with high affinity can act as inhibitors. One
inhibitor type is characterised by its reversible inhibition of
P450-dependent hydroxylation of substances. These inhibitors (e.g. macrolide-antibiotics) bind reversibly to P450 and
produce stable complexes. Therefore, P450 remains in an
inactive state [3941.However, so-called suicide inhibitors (e.g.
ally1 isopropyl acetamide) produce an irreversible modification of the apoprotein [51.
Spectral changes occurring on binding to P450 result in
characteristic binding curves and are a parameter of binding.
Likewise the inhibition of typical monooxygenase reactions
ethylmorphine N-demethylation (EN) and ethoxycoumarin
0-deethylation (EO) give information about the interference
at the binding site of different P450 forms; these reactions are
catalysed only by a few P450 species and that is why they
also represent sensitive test methods for the determination of
inducers and inhibitors.
In the P450 reaction cycle dislinking reactions permanently
occur with releasing of reactive oxygen species (ROS). ROS
are the superoxide anion radical (02”), the superoxide radical
(HOO’), the hydroxyl radical (OH’) and hydrogen peroxide
(H202) which can cause cell and tissue damages. Furthermore, toxic metabolites could be generated during the reaction of substrates at P450. These and the above mentioned
highly reactive ROS are controlled by a protection system,
the antioxidative system. If the antioxidative system (with
ascorbic acid, GSH, and vitamin E) decompensates, either
toxic metabolites can bind to liver cells or ROS are formed.
This intracellular toxic stress leads to cell death or to other
damage such as inflammation, mutation, or cancer.
The investigation of luminol- (Lm) or lucigenin- (Lc)amplified chemiluminescence (CL) gives information about
possible dislink functions of the substances to be tested. An
inhibition of CL indicates an inhibition of oxidative functions
of P450 as a shunt reaction or rather for scavenger functions.
Therapeutic relevance could be deduced from this.
Chemical Structures of Tested Estrogens
All tested estrogens are derived from natural l7p-estradiol
(Rl): cf. Fig. 1. In addition, the 3-methyl ethers attached to
them were examined.
0 VCH Verlagsgesellschaft rnbH, D-6945 1 Weinheirn, 1997
0365-6233/97/0505-0135$17.50 +.50/0
Gemhardt, Karge, Schonecker, and Klinger
Fig. 1: 17P-Estradiol and derived estrogens (3-methyl ethers).
All of the other estrogens, except the enantiomers D- and
L-estradiol3-methyl ether were without any influence on the
monooxygenase system.
With the Lineweaver-Burk diagram a competitive inhibition type of 17P-estradiol for EN was established (Fig. 3). It
may be concluded that a common substrate binding site
exists. All of other tested estrogens showed non-competitive
17a--ethynylestradiol3-methyl ether (mestranol)
3 10.4
inhibition types. Accordingly binding to P450 takes place at
an allosteric center, and here also interactions with other
estriol 3-methyl ether
drugs are to be expected.
Possible uncoupling effects can be demonstrated by an
increase of ROS formation, measured as Lm- and Lc-CL. As
For l7P-estradiol a spectral change of type I (hexobarbital) Lm-CL is based on two univalent oxidation steps, one univawas recognized. The difference spectra of 17a-ethynylestra- lent reduction of lucigenin is followed by oxidation [71,Thus
diol, mestranol, estriol, and estriol 3-methyl ether evidently Lm- and Lc-CL may be influenced differently.
17j3-Estradioland 17a-ethynylestradiolsignificantly inhibindicated reverse t e I according to the structure of steroids
without nitrogen
Estrone and estrone 3-methyl ether did ited Lm-CL. A significant inhibition of Lc-CL was observed
not show any characteristic extinction differences or curves. for 17a-ethynylestradiol, which indicates an inhibited genAs the determination of spectral changes only allows state- eration of H 2 0 2 or hydroxy radicals [81(Fig. 4).
ments about the affinity of substrates to P450 but not about
Additionally the binding of the two enantiomersof estradiol
the monooxygenase and/or oxidase function of P450s with 3-methyl ether and their influence on P450-dependent
the given substrate, the next step was to test the influence of biotransformation were investigated. The D-form did not
estrogens on monooxygenase reactions.
show any defined binding curves, however for the L-form an
For 17&estradiol an inhibition of EN at concentrations
maximum in a similar position like aniline was
M was found. 17a-ethynylestradiol showed a similar
inhibitory effect at the same concentration. Both substances
For D-estradiol3-methylether an inhibition of EN ( 104M)
also proved to be inhibitors of EO (each at concentration
M) (Fig. 2). These results indicate binding and possi- was found, while L-estradiol3-methyl ether did not reveal a
ble hydroxylation of l7P-estradiol and 17a-ethynylestradiol clear influence. But both enantiomers inhibited EO at concentrations > lo4 M.
at P450s.
D-estradiol3-methyl ether
L-estradiol 3-methyl ether
estrone 3-methyl ether
Arch. Pharm.P h a m Med. Chem 330,135-140 (1997)
Influence of Estrogens on Biotransformation
FA (pmollg pfot x mln.]
FA @mol/g prot x mln.]
7-OH-C bmoi I Q pmt x mind
7-OH4 bmol I g prot. x min.]
° L
6o l
Fig. 2: Influence of different estrogens on ethylmorphine (EM) N-demethylation (EN) and ethoxycoumarin (EC) 0-deethylation (EO). The metabolites
formaldehyde (FA) and 7-OH-coumarin (7-OH-C) were determined. K: controls without any addition to the standard incubation mixture, K(ac): controls
after addition and evaporation of the solvent for the estrogens. Structures of the estrogens: RI = 17P-estradio1, R2 = D-estradiol 3-methyl ether, R6 =
17a-ethynylestradiol.Concentrations of the estrogens: E-6 = lo4 M (exponent -6) etc.
Summary of Results
X E-4
m 2 0
1,333 2,888
11s [rnllprnol]
Fig. 3: Influence of R1 on EN, Lineweaver-Burk-plotof the data presented
in Fig.2. Demonstrated are control values and the data obtained with the
highest concentration used (E-4).
For the D-enantiomer a binding at P450 was suspected, as
both EN and EO were inhibited. For the L-form the results of
inhibition of EO and the spectral changes correlate.
In the CL experiments the D-form significantly inhibited
Lc-CL, but not the L-enantiomer (Fig. 4). Lm-CL was not
affected by D- or L-estradiol3-methyl ether.
Arch Phamr P h a m Med Chem. 330,135-140 (1997)
10-4M 1 0 4 M
Scheme 1: Influence of all estrogens tested (abbr. cf. Chemical structures)
on binding type to microsomal cytochrome P-450(spectral changes), on
monooxygenase reactions (EN and EO) and on oxidase functions (Lm-CL
and LC-CL). Lm = luminol, Lc = lucigenin. Given are the minimum
concentrations at which significant inhibitions could be observed.
The lack of characteristic extinction differences or binding
curves in the determination of spectral changes for the estrogens D-estradiol3-methyl ether, estrone and estrone-3methyl
ether can be discussed as following: The difference spectra
arise by a shift of spin equilibrium to high spin (type I) or low
Gemhardt, Karge, Schonecker, and Klinger
'1B 'II t m
RW [E+6 CPM I mg pr0t.l
RW p+5 CPM I mg prot.]
I: 1111
:i Im
Fig. 4 Influence of different estrogens on luminol (upper panel) and lucigenin amplified chemiluminescence. Abbreviations cf. caption of Fig. 2. RLU =
relative luminescence units, CPM =counts per minute. Statistically significant different values to control values (K and K(ac), cf. caption of Fig.2) are marked
by asterisks (student's r-test, p 50.05).
spin (reverse type I). That means, that different affinities to
high or low spin of P450 induce the shift. The same affinity
of one substrate to both conformations exclude formation of
a difference spectrum [91. So with this method a binding or
transformation of D-estradiol 3-methyl ether, estrone and
estrone-3-methyl ether at P450 was not proved conclusively.
For all other estrogens a classification into type 1 or reverse
type I was shown by characteristic spectra.
For 17P-estradiol a binding curve of type I and a competitive inhibition of EN and EO was shown. The proved inhibition of EN by 17p-estradiol and also by 17a-ethynylestradiol
confirms earlier tests [lo]. Thus it can be assumed, that 17pestradiol binds, at least in particular, at forms of IA, 2B and
3A families of P450s.
From steroids which contain an ethynyl grou at C 17 (like
EE) it is known, that they are direct inhibitors by covalent
binding to the specific P450s [12].
It was found, that 17a-eth nylestradiol reacts among others
with the P450 form 3A4 [13rwhich correlates with the finding
that EN is a special indicator reaction for 3A4 [I4].
Also for the estrogens mestranol and estriol the lack of
influence on the monoxygenase system confirms earlier test
results with simiIar methods [I5]. The lack of influence of
estrone, estrone 3-methyl ether, mestranol, estriol, and estriol
3-methyl ether on EN and EO can be explained by the
structure of these estrogens. For the estrogens estrone and
estrone 3-methyl ether the double bond of the carbonyl oxygen at C17 could explain the lack of affinity to P450s and the
lack of reactions. Although mestranol with an ethynyl group
at C17 is considered to be an inhibitor of P450s this effect
could not been proved by determination of EN and EO. Thus
mestranol seems to bind and react at other P450 forms. The
special quality of structure of estriol and and estriol3-methyl
ether is the OH group at C 16. In liver microsomes of male
rats special P450 forms like 2C11 and 2D exist to which C16
hydroxylated estrogens bind [16]. So it could be explained,
that an affinity to and reaction of these estrogens with P450
could not be demonstrated with the indicator reactions EN
and EO.
Furthermore it is possible that these estrogens bind at distinct P450s and are transformed, but this is not proven with
specific model reactions like EN and EO, which are regarded
as indicator reactions only for special families like lA, 2B;
2C and 3A.
In these experiments inhibitory concentrations are in the
range of 10-5-104M. These concentrationscannot be correlated with in vivo concentrations in plasma: the estrogens
were dissolved in acetone, and the solvent was evaporated.
During the 10 min incubation period with shaking only small
parts are dissolved in the phosphate buffer, the amount is
Arch. Pharm.P h a m Med. Chem 330,135-140 ( I 997)
Influence of Estrogens on Biotransformation
considered to be proportional to the amount added to the test
tubes. The concentration found in the lipid phase near the
P450 active center is unknown, the same holds true for the in
vivo concentrationsof estrogens in the target areas, the estrogen receptors. The amounts binding to P450 in vivo are
dependent on both the dissociation constants (for steroids in
the FM range) and the turnover number. Therefore direct
conclusions as to inhibiting plasma concentrations after administration of therapeutic doses cannot be drawn.
The decreases of CL by 17p-estradiol and 17a-ethynylestradiol indicate an inhibition of oxidative functions of P450
as a shunt reaction, but also the interpretation that these
estrogens act as scavengers is possible.
In particular estriol and 17 -estradiol are known as naturally existing antioxidants [ l I. At present estro ens can be
considered as optimal free radical scavengers [18 . Considering all these facts we conclude, that the inhibition of CL by
l7p-estradiol and 17a-ethynylestradiol is due to a scavenger
function. Possible uncoupling effects of these estrogens can
be excluded, because CL is not increased.
With regard to the results for the two enantiomers on
determination of the monooxygenase system it is assumed
that D-estriol3-methyl ether is predominantly bound to and
catalysed by 2B and 3A enzyme families, while the L-enantiomer is transformed by 1A and 2B species. Evidently the
chirality has an influence on the affinity to special P450forms.
Until now there are no publicationsabout effects of estrogen
enantiomers on biotransformation.Only for the non-steroidal
estrogen diethylstilbestrolwas it demonstrated that estrogen
receptors show chiral preferences and thus different interactions with enantiomers
These results point to an inhibition of oxidative effects of
P450, but could also be interpreted as a scavenger function of
the D-enantiomer.
Ethylmorphine N-demefhylation(EN): The estrogens dissolved in acetone
lo4 M in the final volume) were pipetted into test tubes and the
solvent was evaporated.
A 1 ml portion of incubation mixture of contained 0.5 m10.5 M sodium
phosphate buffer (pH 7.4) 0.05 ml NADP solution (0.3 pmo1/0.05 ml);
0.05 ml glucose-6-phosphate ( 5 pmo110.05 ml); 0.05 ml MgClz
(5 pmoV0.05 ml); 0.05 ml semicarbazide (10 pmoV0.05 ml); 0.2 ml9000 g
liver residue; 0.1 ml ethylmorphine (6 pmoVO.l d).The reaction was
started by adding 9OOO g liver supernatant. After 10 min incubation at 37 "C
the reaction was stopped with 0.1 ml cold TCA (75%). The samples were
centrifuged at 6000-9000 rpm. To 0.5 ml of clear residue was added 0.5 ml
of NASH agent. After incubation at 37 "C for 40 min and cooling the
extinction was measured (Specol 11 of Carl Zeiss Jena) at a wavelength of
412 nm (Klinger and Miiller [241).
Efhoxycoumarin 0-deerhylution (EO): Estrogens dissolved in acetone
( lo4,
lo4 M in the final volume) were pipetted into test tubes and the
solvent was evaporated.
The incubation mixture of 0.5 ml contained: 0.1 ml 7-ethoxycoumarin
(0.2 pmol/O.l ml), 0.1 ml NADPH (0.25 pmol/O.l ml); 0.1 ml glucose-6phosphate (2.5 pmoYO.1 ml); 0.1 ml MgClz (lOpmoVO.l ml); 0.1 ml sodium phosphate buffer (pH 7.4); 0.1 mi 9000 g liver residue. The reaction
was started by adding 9000 g liver supernatant. After 10 rnin incubation at
37 "C the reaction was stopped with 0.5 ml cold TCA (75%). After addition
of 4.0 ml NaOH solution 7-hydoxycoumarin was measured fluonmetrically
at 454 nm (excitation at 375 nm; Hitachi fluorimeter F 2000). (Aitio [251).
Luminol- and lucigenin amplified chemiluminescence (Lm-CL; Lc-CL):
0.05 ml of the estrogens (lo4, l o 5 , 104M) were pipetted into special test
tubes for CL measurement and the solvent was evaporated.
The incubation mixture of 2.0 ml for Lm-CL contained 1.2 ml 0.1 M
sodium phosphate buffer (pH 7.4); 0.2 ml Few solution (0.05 mmolll); 0.2
ml luminol (0.3 mmolA); 0.2 ml microsome suspension (1 mg proteid2.0 ml); 0.2 ml NADPH solution (0.1 mmol/l).
The incubation mixture of Lc-CL contained 1.2 mlO.1 M sodium phosphate buffer (pH 7.4); 0.2 ml Fe++solution (12.5 pmolll); 0.2 ml lucigenin
(5 mmo1A); 0.2 ml microsome suspension (0.1 mg proteid2.0 ml); 0.2 ml
NADPH solution (0.1 mmol/l). The reaction started by injecting 203.7 pmol
NADPH-solution in a luminometer (BIOLUMAT LB 953; programme K11). The counts are given for a measurement period of 3.5 min (Klinger et
al. [*I).
( lo4,
Experimental Part
Animal and liverpreparation: 50 to 70 day-old male untreated rats, raised
and housed in the Institute of Pharmacology and Toxicology of the Friedrich
Schiller University Jena, were used. They were kept under controlled conventional conditions (athmospheric humidity > 50 %, temperature 22-24 "C,
natural light-dark rhythm). The animals were nourished with Altromin pellets
and tap water ad libitum.
The rats were anaesthetised with ether and decapitated. The livers were
prepared and weighed. For the preparation of the 9000 g supernatant liver
was homogenised with two parts 0.1 M sodium phosphate buffer (pH 7.4).
Then the homogenate was centrifuged for 20 min at 9000 g and 0 "C.
Microsomes were prepanted according to Schenkman [2'1: 25 mM MgClz
solution was added 1 + 2.5 to the 9000 g supernatant followed by a second
turn of centrifugation at 9000 g and 0 "C for 20 min.
Protein determination: Protein was determined with the Biuret method of
Gornall 1221.
0.25 ml 5% sodium desoxycholate solution was added to 9000 g supernatant or microsome suspension. After 5 rnin the substances were mixed with
4.5 ml "Biuret" solution. 4.5 ml NaOH were added to the blanks. After
30 min the extinction was measured (spectral photometer Specol 11 from
Carl Zeiss Jena).
Spectral changes at binding at P450: The mixture contained the following
substances: 0.1 ml 0.1 M sodium phosphate buffer (pH 7.4) or 0.1 d
1.1 mM hexobarbital(1.1 pmol/O.1 mi) or 0.1 ml 1.O mM aniline hydrcchlorid(l.l prnol/O.l ml)or0.1 mlestrogenR1 t o R 9 ( c = 104M)and 1.Oml
microsome suspension (5.5 mg/ml), following Schenkman [231and measured
with programme 13 of Specord M 40,Carl Zeiss Jena.
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Received February 24, 1997 [Fp194]
Arch P h a m P h a n Med. Chem 330,135-140 (1997)
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estrogen, structure, cytochrome, activity, scavenger, 450, function, relationships, influence, biotransformationaffinity, various
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