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Effect of some synthetic steroids on rat liver function
EFFECT OF SOME SYNTHETIC STEROIDS ON RAT LIVER FUNCTION Eric
Ho Yuen. Richard D. Goold and Beverley Faculty of Pharmacy, Rhodes University Grahamstown, 6140. Republic of South Africa :i 3i>i\ d.? 5-L.‘--"5 ABSTRACT
J. Wilson
Norethynodrel proved to have moderately potent effects on hepatic function in female rats. It causes (a) loss of body mass, (b) increase (c) rise in the protein content of liver, in the relative liver mass, (d) increase in the cytochrome P-450 leve1, (e) reduction in the length of pentobarbital-induced sleep, and (f) increase in the rate of In contrast, biotransformation of (i) aniline and (ii) aminopyrine. ethynyl estradiol was shown to have little or no effect as judged by the same criteria, but, like norethynodrel, progesterone and estradiol, caused loss of body mass. INTRODUCTION It their
has been well
synthetic
function
derivatives
does
administration there
steroids
not,
on the
been
however,
picture
of the diverse
progestational
mass, while
to bring
about
agents
some mixtures
have been associated
with
liver
inconsistencies
mass.
relative levels substrates
Similar
liver and
mass,
metabolic
activity
of the hepatic
January 1985
(3) prevail
as far
TDEOXDI
liver.
obtained
The
from
to
the
rats.
of mixtures
Nor
of
such
to cause no
and estrogens
certain
oral
contraceptives,
or with
no change, respect
(4, 5) in
cytochrome
(mfo)
liver
to body mass,
endogenous
oxidase
in
have
(l),
levels, as
on the
or no change
with
protein
mixed function
S
of the
and
function.
mass (l),
(2),
an increase,
effects
have been reported
agents,
mlcrosomal
that
effects
in liver
of these
hormones
pronounced
substances
of hepatic
a loss
gonadal
in the results
or estrogenic
or even to cause a loss, reported
the female
including
agree
common parameters
For example, change,
tissues,
of progestogens a clear
that
have more or less
of "non-target"
literature
is
established
and are
Volume
P-450 exogenous
concerned. 45, Number
1
Increases,
decreases,
administration
lack
have been reported
Because divergent
and
of
the evident
results,
of for
effect all
need for
we undertook
of these
further
a study
progesterone comparison
of certain
study
the
and estradiol
of
steroid
parameters. to
clarify
these
of norethynodrel
and ethynyl on
(4-pregnen-3,20-dione)
result
of the effects
(17~~ethinyl-17-hydroxy-5(lO)-estren-3-one) ethinyl-1,3,5(10)-estratriene-3,17-diol)
as a
estradiol
(17~
rat,
using
(estradiol-17e)
for
female
effects. EXPERIMENTAL
progesterone (Sigma Steroids. Norethynodrel (Searle Lab.) or estradiol (Sigma Chemical C ) ethynyl estradiol (Wyeth Lab j Fo.j was dissolved in ethyl oieite and individually Chemical administered intraperitoneally (i-p.) between 08h30 and 09hOO daily for Two dosage levels, 1 fig periods ranging from two to thirty days. of each steroid were used. Control animals received no and 20 mglkg drug but equivalent volumes of ethyl oleate. Animals. Female Wistar rats between 4 and 5 months old and These rats were acquired from weighing initially 195 + 25 g were used. the South African Institute for Medical Research and were housed in a room with temperature and humidity control, in stainless steel cages with raised bottom (4 animals per cage). The animals were allowed free access to food and water. Sampling of Animals and Dosing. Animals were initially sorted on the basis of the regularity of their estrus cycles, only those with regular 4-day cycles being selected. For each steroid and dosage level under study, thirty-six animals were sorted into groups of four, one group for each period of dosing. Determination of Sleeping Time. Sleeping time of the steroidtreated rats and control rats was determined for animals treated for 7. with the steroid. Thirty mg/kg of pentobarbitai l~di~~d(Sf~a~~?R) ) was administered i.p. Preparation of Microsomes. Animals were sacrificed by a blow on the head after the predetermined duration of steroid administration. Two animals from each treatment group and from the control group were sacrificed, one 2 h after the last administration and the other 24 h after. Livers were removed and all subsequent operations were performed at 0 to 4°C. After perfusion with 0.9% NaCl, the livers were homogenized in a Thomas tissue homogenizer in 3 volumes of cold 0.025 M sucrose solution. The animals in the control group were sampled as in the experimental, except that both livers were combined. Liver
microsomes
were prepared
by a calcium
precipitation
method
(6).
Protein concentration was Assay of Protein and Cytochrome P-450. determined by the method of Lowry -et ai. (/) as modified by Miller (8). The method used for the determination of cytochrome P-450 was that of Omura and Sato (9). Enzymatic Assays. Enzymatic activity was measured in the following DL-isocitric acid (10 pmoles), incubation mixture: NAOP (2 pmoles), MnC12 (0.01 pmoles), isocitric acid dehydrogenase MgC12 (25 pmoles), (0.4 Ill). Five pmoles of freshly distilled aniline, or of aminopyrine, served as substrate. Buffer of 0.2 M Tris.HCl, pH 7.8, was used to microsomes equivalent to 250 mg of bring the total mixture to 4 mL; wet liver was added to the reaction. The flasks were incubated in a metabolic shaker under rapid shaking for 20 min at 37°C. Estimation of Aniline Hydroxylase Activity. Aromatic hydroxylation of aniline was determined by measuring formation of p-aminophenol according to the method of Maze1 (10) and by using the aboTTe incubation mixture. Calorimetric determinations were performed with a Beckman DB spectrophotometer. Demethylation Estimation of Aminopyrine Demethylase Activity. of aminopyrine was determined by measuring formaldehyde formation according to the method of Maze1 (10) with a Beckman DB spectrophotometer. The procedure was modified to adjust the final volume to 6 mL and to use the same NADPH-generating system as for the aniline hydroxylase system. RESULTS AND DISCUSSION In this order
study,
to accelerate
levels
are
those
numerous authors. that
rats
than
do
maintain
to a
-reflex, ')I
adequate
and levels
20 mg/kg
treatment
might
of these
it
levels
rate
(11)
of metabolic
Both dose levels of
sleeping being
was clearly
evident
after
estradiol
most
in The
used
by
on the fact xenobiotics
high
enough
to
disappearance. of
time
regimen
ethynyl
of
agents.
been
in part,
doses be
dosage
with
and have
is based,
that
were employed
by these
of metabolism
in the face Time.
steroids
be induced
is essential
marked shortening
that
of the
recommended by Gunzel
on Sleeping
an effect
levels
changes which
Choice
humans,
the
Although
high
have a much higher
Effect rise
rather
as
norethynodrel based on
gave righting
the more effective only
(Figure
7 days of treatment.
at both dosage
levels
also
S
56
shortened treated the
sleeping animals
Student's
values
the small
times,
in the effect It'
obtained
days with
?F-EOXDI
test
after
either
difference
produced
7 days and those
steroid,
hence only
control
difference
values
was noted
determined
one set of results
and
according
was insignificant
Little
(P> 0.01).
between
to
between
after
14 or 21
is shown.
Figure 1. Effect on Sleeping Time. Female. Wistar rats were treated for seven days with norethynodrel (N), 1 mg/kg (1 N) or 20 mg/kg (20 N) or with ethynyl estradiol (E), 1 mg,$kg (1E) or 20 mg/kg (20 E). Sleeping following time was determined pentobarbital administration of Control rats were (30 mg/kg i-p.). injected daily for seven days with Each bar represents ethyl oleate. the average of 12 animals. mean f S.D.
It
has been reported
no effect
on narcosis
administration prolonged found
an increase
rats
after
mg/kg.
after
of certain narcosis
that
oral
contraceptive
On the other
in the --in vitro
a 3- or 21-day a agents
administration
of norethynodrel
30 days of treatment
(12).
Similarly,
progestational
(4)
regimen
mixtures hand,
metabolism of
(10 mg/kg/d)
of hexobarbital
norethynodrel
in sleeping
time
such
medroxyprogesterone
and
short
Juchau and
reduction as
for
has that
periods
Fouts
(13)
in male
at a level has been noted
of
50 with
(17-acetoxy-6a-
S
TDEOXDI
methyl-4-pregnene-3,20-dione) metabolism
with
(4)
quingestanol
acetate
(14).
Certain
3,5-pregnandien-20-one) been shown to decrease results
bear
out
females
below)
sex
in this
and
latter
an
differences species
oral
associated
(SER)
underlying
common phenomena.
Nonetheless,
on Mass. The animals
subjected
(20
mg/kg)
lost
the treatment
days
they
treated than
approximately
with
norethynodrel
did those
animals
days
they
weighed
with
approximately
comparison,
a group of animals
and
loss
their
ethynyl
estradiol
after
days than
they
did
receiving
estradiol.
initially.
in mass of control
treated
animals
weighed
(Table
1 and Figure
2).
steroids
decrease appears
in
smooth
result
approximately
to be dose related,
after
30 Those
dose,
after
so that
original
to
to
after
30
in the animals
in comparison
in the extreme
the higher
regimen
less
was noted
subjected
For
subjected
slightly
than
30
mass.
a progesterone
35% less
body mass of animals
regimen
much less
in mass must be viewed so that,
the
mass.
Those rats only
(see
mass, although
their
was subjected
well
endoplasmic
so that
original
lost
95% of
animals,
are
in
certainly
period
the higher
A similar
Any loss
the gain
The
also
dose weighed
those
to the norethynodrel
14 days was similar.
at either
in
drugs
almost
80% of their
(1 mg/kg) treated
the
are
Effect
weighed
cautious
enzyme induction
of
result)
mass throughout
Our
with
of
have
(4).
in the brain
in the metabolism
(unpublished
mixtures
in male rats
proliferation
reticulum
hexobarbital
but one must be
studies
(15).
of
contraceptive
findings
from
rate
(17-acetoxy-3-(cyclopentyloxy)-
of pentobarbital
obtained
since
recognized
levels
these
results
relating
and an enhanced
with
cases,
the
controls
to each
of
dose of the
the
the
steroid
S
58 Table
1.
Effect
Steroid
of stemld
adminlstratlon
Dosea %/kg
THEOXDI
on the mass of
Duration days
lnltlal
16
183.3
30 14 20
16
20 20
female
rats.
Mass
Final 9
Mass
f
8.5
179.3
f
7.3
97
179.8
2
7.6
170.0
f
6.8
95
205.5
f
2.3
201.1
*
0.4
98
182.2
f
6.2
168.8
*
8.0
08
30
186.0
*
8.3
154.8
f
9.3
80
14
218.5
t 13.5
200.6
f 15.2
92
16
177.8
f
9.3
175.q
t
9.7
98
30
179.0
* 12.4
175.3
t
12.7
98
14
219.5
i
8.0
213.8
f
10.8
97
9
Final/Initial Mean Mass x 100
norethynodrel progesterone
norethynodrel progesterone
ethynyl
estradiol
estradlol
ethynyl estradlol
ethyl
20
16
176.3 2
6.1
167.5
t
5.5
95
20
30
173.0 f
6.8
162.3
*
9.0
94
20
14
209.8
2.8
195.8
*
2.9
93
estradlol f
14
175.5
f
7.9
185.9
*
8.2
106
30
175.5
t
7.9
202.8
i
10.0
114
oleate
a Stemlds
were
administered
daily
in the doses
indicated
to 4 rats
Figure
2.
in each group.
Effect
of Treatment
of
Female Rats with Norethynodrel (0) Animals or Ethynyl Estradiol (m). were treated with 20 mglkgld of either steroid or with ethyl oleate (4) for the appropriate number of Each point represents the days. comparison of mass of 4 animals after treatment with the mass of the same animals at the beginning of the treatment period. (Animals treated with progesterone (0) and estradiol (er) 20 mg/kg. for comparison.)
S resulting
in
rats
a more marked decrease
in body mass accompanying
decrease (16)
and
loss of mass
59
W-EOXDI
in body mass in estradiol
been shown to
cases.
administration
male rats (17) has been reported
in castrated
all
A
to female
previously,
as
has
a
males (2, 17), while synthetic estrogens have
reduce the rate of gain of body mass in
males
(12).
Norethynodrel has not been shown previously to reduce the rate of gain of body mass. of mass
However,
in combination with estrogens,
at high doses (5) or a dose-dependent
gain of mass (3,
5,
12).
it causes a loss
reduction in the rate
of
Other oral contraceptive mixtures have also
been associated with a decreased gain in mass (12, Effect on Mass of Liver.
The mean relative
18).
liver mass
somatic index (LSI) expressed as g/lOOg body mass) in receiving norethynodrel (both dosages, Figure 3A) 7 A
(liver
animal groups increased,
markedly
B
,._,..._. ____..... z... 6 *l.l,f,;; ,,,~,,...,,...a............’ “““‘-‘“~~’ . -x----;-__----‘--kX 5 : %-4,.‘e 2*-Jf X St 4 i--x
Figure 3. Liver Somatic Index (LSI). LSI is the actual liver mass in g per 100 g body mass. A. Each point represents the values from a single animal to which norethynodrel (1 mg (x) or 20 mg (.)/kg in ethyl oleate) had been administered for the appropriate numberof days. Control animals were injected daily with ethyl oleate. The LSI of the 16 control animals on the final day was 3.85. B. Each point represents the value from a single animal to which ethynyl estradiol (1 mg (x) or 20 mg (m)/kg in ethyl oleate) had been administered for the appropriate numberof days. Control animals were injected daily with ethyl oleate. LSI of the 16 control animals on the final day was 3.7. an increase which appears to have begun early, until
the thirtieth
and which continued
day. Sixteen control animals, sacrificed
on the
S final was
a relative
had
day,
somewhat
receiving
higher
liver mass of 3.85.
than
that
norethynodrel.
progesterone
(20 mg/kg)
while
treated
those
TPEOXDI
of the controls
with
after
14 days,
1 mg/kg for
this
in both
groups
of
rats
animals were treated
For comparison, and,
liver mass itself
The
LSI of 5.07 f
had an period
with
0.03
had an LSI of 4.48
-+
0.04. Norethynodrel, while causing a significant increased the mass of the liver as did
body,
observation effect
to enzyme induction.
of progesterone
parameter
in
pointing
(4).
liver
(1)
has
or LSI (4))
An earlier
medroxyprogesterone
(4)
ethinyl-4-estrene)
(1)
also been reported and the same
or ethynodiol
was
another
progesterone, report
showed the hormone to be without
Norethynodrel
mass
in mass of the
decrease
effect
on
the
on
this
to cause no change
result
was noted Lhen
diacetate (3e,17l+diacetoxy-17-
administered.
On
the
other
hand,
chlormadinone (17-acetoxy-6-chloro-4,6-pregnadiene-3,2O-dione)
caused a
loss of liver mass (1).
however,
have
been
increase without
to
cause hypertrophy
in LSI (3,
5,
la),
Only one
while others
mixtures,
of the
liver
(3)
and
an
have been reported
to
be
(4).
significant
namely an
entirely,
oral contraceptive
reported
effect
occurred,
Some
change
attributable
to
ethynyl
estradiol
increase in LSI (Figure 3B), but it was largely, or
due to the fact that treated animals lost overall mass while
the mass of the liver remained unchanged(in partial
agreement with an
earlier
animals receiving
report
(12)).
A
similar
that
the
LSI
pattern
was noted
in
estradiol
in
treatment
with 20 mg/kg while that resulting from administration
mg/kg was 4.90 + 0.04.
was 5.34 + 0.08 following
Unlike the
animals treated
14 days
with
of
of
1
ethynyl
estradiol, liver
animals
receiving
Sixteen
control
animals
mass.
relative
liver
The
female
is rats
(16,
P-450. --
studied
and
levels
of
protein
the
less
twelfth
day of treatment. 22.4
cytochrome
Sixteen
mg protein/g
450/mg protein.
steroid
there
The values
P-450 were higher of
administration.
the
when either
(Figure
day when 20 mg/kg P-450 continued
up to the
of liver for
levels
of both
in animals
steroid Underlying
than
an increase
12th day day,
microsomal
with
off
by
the
on the 30th
protein
24 h after sacrificed in
of the
nmoles of cytochrome
sacrificed in those
while
particularly
sacrificed
and 0.54
4A)
per
and levelled rats
of protein
(Figure
48))
increase dosage of
in levels
period
to rise
control
male change
ensued an
of cytochrome
P-450 content,
noted
and Cvtochrome
1 mg/kg of norethynodrel marked
be
but
reported,
Protein
as the fourth
the 30-day
in
(17),
no significant
increases
Levels
and
(2).
in LSI,
continued
a dosage of
to
administration
studied
as early
throughout
should
a loss of mass was
an increase
was administered.
microsomal
It
of
(2)
or of a loss
of Microsomal
P-450 appeared
administration
21).
Notable
respect
cytochrome
20,
was administered.
appeared
day yielded
(17,
P-450 concentrations
At
effects
change,
and in one case in which
with
steadily
treatment.
in
day had a
in male rats
cytochrome
cytochrome
increase
with
findings
males were being
Concomitantly
of norethynodrel to
results
on the Concentration
norethynodrel and
on the final
mass associated
to earlier
other
castrated
in protein
liver
in the case in which
were being
Effect
showed an increase
sacrificed
19) of no significant
with
however that,
occurred,
in
in contrast
consistent
rats
estradiol
mass of 3.7.
increase
estradiol
is
however,
2
cytochrome
the h
Pand last
after P-450
levels
as noted
aminolevulinic steroids,
by us
of course)
It
acid.
including
is,
has
been demonstrated
progesterone,
in levels
an increase that
are good inducers
of
6-
a variety
of
of s-ALA synthetase
2.0
1.5
I.0 ,5
~~~~+Jw-----------~~,5-
i
0.5
L. ':
++ 5
10
15
OUNATlO"
OF
20
TREATWENT
25
0
5
10
15
OURATION
(0AYZ.l
OF
20
TNEATNENl
25
30
(OAYS)
and Cytochrome P-450 in Rats Treated Female Wistar rats were dosed with norethynodrel 20 mg/kg (A) or 1 mg/kg (B) for the number of days indicated. Protein content (mg/g liver) (0) was determined by the method of Lowry et al. (7) as modified by Miller (8). Cytochrome P-450 (nmo leslmg pEteYi$ (x) was determined by the method of Omura and Sato (9). Figure
4.
with Norethynodrel.
Microsomal Protein
Literature
levels of cytochrome
on
administration
prolonged
progestogen,
chlormadinone
(.l)
insignificant,
or
acetate,
a
following
wide range of
results.
to be increased (1,
decrease
of
(1) depending
levels
appeared
20 mg/kg and
microsomal protein
to
14) or
of
significantly
or of cytochrome
P-450.
to show slight,
specific
on the
Neither the
be
perhaps
steroid employed.
ethynyl estradiol
1 mg/kg respectively.
increase
in levels of the
levels have been seen to
5A and 58 show the effects
levels
while another
caused a reduction
Microsomal protein
P-450 (1).
unaffected
Figures
reveals
in the liver
was shown previously to be without effect,
Norethynodrel
cytochrome
of steroids
P-450
at dose
of these
concentrations
dose of
S The lack to
produce
the
of change
finding
in protein
a noticeable
shortening that
of
effect
the
the
TDEOXDI
level
is consistent
with
on the proliferation
lamellae
thereof
of the SER or
on
results).
The
(unpublished
SER is not markedly
increased
the failure
is in agreement
with
0 x
2.0
n
t.5
; 'I' g :: 2::
1 , 8
.
.
&.;...;
. . . . .R,
. . . . . . . "";
.. ... .. ... ..... ... ..
.
,..
.7P
.
I.0
zig SE -^
* [+(g+.-*x-x~--------~
,
o5
g f ;
:* 5
0
IO
o”Rlr*oN
15 OF
20
TREAWE”,
J 30
25 ,Ol”S,
Figure 5. Microsomal Protein and Cytochrome P-450 in Rats Treated with Ethynyl Estradiol. Female Wistar rats were dosed with ethynyl estradiol or 1 mg/kg (B) for the number of days 20 mg/kg (A) indicated. Protein content (mg/g liver) (0) was determined by the method of Lowry et al. (7) as modified by Miller (8). Cytochrome P-450 (nmoles/mg proteiii)(x) was determined by the method of Omura and Sato (9). previously report
reported
findings
but
is not
in agreement
with
another
(16). It
is not surprising
450 concentrations fact, a
(12),
it
marked castrated
of
in
levels
cytochrome
lowering males
of an increase
the change produced
by ethynyl
has been reported
reduction
types
that
that
P-450.
in our study was very
under
in males (24)
of cytochrome (2,
estradio
similar
conditions
and a specific
Estradiol
P-450 levels
in
(21,
25).
(23)
reduction
has been reported
P-450 levels
21) and in females
in cytochrome
in the cytochrome
males
small.
In
there
is
in certain to
(17,
cause 20,
Only one report
has been noted.
P-
Estradiol
a
20, (19) has
S
64 a
small
less
inductive
than
effects 450
capacity
have been reported of cytochrome
(5,
18)
report
an increase
administration
of cytochrome still
of
protein,
(3)
or no significant
on Enzyme Activities.
With
1 mg/kg of norethynodrel
activity
to 126%
aminophenol/mg mg/kg for
to 10.60 nmol/mg
in levels
estrogens,
of
some
cytochrome
P-450
others
found
ly altered
(14,
(3) _
that 18,
Similarly,
showed an increase change (5,
12,
with
(18))
slight
14) when oral
to
compared with protein
aminopyrine
in
has
for
12 (from
to 9.28 nmol).
hydroxylase days increased 7.39
nmol
p-_
of
20
At a level
the activity
been
of sleep
demethylase
to
activity,
12 days increased protein
these reported
P-450 system noted
duration
for
controls
increased
formaldehyde/mg
increase
administration
to aniline
administered
norethynodrel
administered
nmoles
respect
171% (6.20
protein).
respect
norethynodrel
cytochrome
as
microsomal
12 days,
With
(24)
were administered.
Effect activity,
b5
P-
(12).
and
a decrease
reports
in cytochrome
estradiol
of the steroids;
reported
to these
of cytochrome
P-450 were not significant
another
to microsomal
21, 24)
much
lack of change in reduction
progestogens
contraceptives
reduced
as has been the
but
In addition
level
of mixtures
prolonged
similar
(2,
case
and
(2.67
a fall
and a reduced
earlier,
to marked decrease
the
levels
(26).
the
levels
respect
and pregnenes
of ethynyl
fol lowing
26) ,
to &ALA synthetase
P-450 in the presence
workers
the
respect
P-450 itself,
c) reductase
In
with
have the pregnanes
on cytochrome (or
TDEOXD1
nmol/mg
caused
(27).
Induction studies
as increased
mg/kg
of
to
129%
protein).
A
the activity
activities
in these
as well
to 3.44
20
by of
progesterone the
would account enzymatic
hepatic for
activities
the in
vitro ._-
in
rats
These Fouts
18
results
(13)
substrates
subjected
of noted
to 48 hours
metabolic
end further an
ncrease
prior
as well
heightened
(I,
hepatic
norethynodrel
in the
14).
for
4 days
The increase brought A
about
dose
105%
in metabolic
(1,
to
estradiol
increased
(2.67
105%
inhibition effect,
hand,
rats
Enhanced
with
other
no indication
of
administration
diacetate
of (17~-
were
12).
or a st imulation
respect
was negligible
given
to the xenobiotics
and not
in aniline
to 7.14 nmol 1,
while
of demethy lation
Such minimal
of metabolism
with
significant.
hydroxylase
and a dose of 20 mg/kg resulted
the rate
as we1 1 (4,
days.
treatment
after
certain
or when norethisterone
in an increase
(6.80
nmol to 2.85).
workers
12)
activity
estradiol
7.42 nmol)
increase
of
and
(12).
by ethynyl
to
to
Juchau
to male
of 3
or ethynodiol
of 1 mg/kg resulted
(7.09
by
metabolism
a period
was noted
workers
report
On the other
ethinyl-17-hydroxy-4-estren-3-one) to male rats
of
related
mfo activity
by other
rate
or for
been 4,
to the
had been administered
to a drug, has
of norethynodrel.
support
when norethynodrel
activity
progestogens
to a regimen
20
of aminopyrine
changes have been noted
of endogenous of their
in a similar mg/kg
In the case of the estrogenic steroids
metabolism
and
have all
to
ethynyl to
by
107%
earlier
steroids,
an
xenobiotics,
no
been reported
(2,
16, 17, 19, 20, 21, 25, 28-35). In the of
effect
enhanced
case of oral reported,
the rates
4, 5,
14, 18, 26, 36),
(I,
reduced agents
contraceptive
cannot
(3,
4, readily
13,
18).
mixtures,
of metabolism while
Our results
be related,
there
has been a range
of some compounds
those with
however,
of other
compounds were
individual to the
being
steroidal effect
of
a
combination of Blackham
the
and
particular
Spencer
progestogen
5 to
This
1.
conflicting
especially in view of
agents,
(37) were able to and estrogen
phenomenon
nullify
report
the effects
by administering
might help,
the
however,
of both
a
in a ratio
them
in
that
explaining
of the
findings of various investigators.
Our results do show, however, that norethynodrel does influence metabolism in that it caused a loss of overall mass, an increase liver
mass (and
particularly
microsomal protein,
of
LSI),
increase
and
an increase In cytochrome
P-450
in
in
hepatic
levels
and
an
metabolism toward specific xenobiotics --in vitro and a reduction of xenobiotic effect in vivo, all of which indicate that the enhanced
rate
of
steroid is an inducer of hepatic activity. significant
effect
brought
about
On the other
by equivalent
quantities
estradiol was a loss of overall mass, resulting In contrast
in which the sacrifice
of
xenobiotics
the only
of
ethynyl
in an increase in LSI.
to any finding of enhancedmetabolism resulting
administration
the prolonged
hand,
from
of progestogens are the results of studies
agents have been administered a few hours prior animal,
the have
or
of
studies
in which steroids
to and
to microsomal mixtures -in the steroids inhibit metabolism of substances
been added simultaneously
vitro. In such cases, -serving as substrates for the hepatic enzymesystem in both males (13, 32, 38-40)
and
females (38, 39 ).
has been observed
in
38, 39), pregnanolone (3a-hydroxy-5B-
the case of progesterone
(13,
pregnan-20-one)
metabolites of progesterone (38,
and some
32,
Such inhibition
39, 41, 42),
norethynodrel (4, 5, 13, 38, 43), lynestrenol (17a-ethinyl-4-estren-1701) (38)
norethisterone
acetate
--in vivo as well as in vitro.
(38,
401,
and
ethynodiol diacetate (38)
S The differences a
prolonged
between
situation,
the
that
fact
xenobiotics,
where inhibition
estrogens,
are all
agents
spite
as
for
for
emerged from spectral
(2,
for
that
this
emerging
estrogens
reason,
be
from the
by suggesting endogenous
devoid
reports
that
29,
of
in the
of
therefore
unable
to affect
estrogen
receptors
(54).
which
are not so readily
effect
may be accounted
for
if
doses,
P-450
systems
steroid
dependence
(19,
21,
characteristics.
to differences 450 systems Such in
which
in which
(53).
the
may be induced
specificity endogenous
directly
39,
and,
in part
systems (19)
and
through
by are the
substances
some differences there
56-58),
in
are a number of different
each
differences
with
sexits
own
may be related
of those
cytochrome
P-
by steroids.
of induction steroids
liver
metabolized
possess
specificities
growth have
or when estrogenic
(55) which
has
discrepancies
are readily P-450
the
Studies
may be rationalized
Furthermore, substrate
to which
Hence,
are employed,
29,
systems
when
in the
one remembers that
cytochrome
many
from studies
46, 49-52).
metabolized
the hepatocytes
hepatic
inducibility
43,
cytochrome
metabolized
to
c larity
work,
and on rats
literature
At higher
P-450
obtained
metabolic
effect
appropriate
vitro
and
cons iderable
low doses of the steroids
levels
(44)
results
periods,
may be rapidly
--in
sites.
46-48),
(20,
noted with
is due largely
same cytochrome
--in vitro 20,
rule,
steroids
divergent
(45))
hormone has been administered revealed
the
prolonged
work rats
many
the binding
apparently
are administered
on hypophysectomized
for
sometimes
compounds and the is the
with
substrates
of the
activity
of estrogenic
and hence are competitors In
the enhanced
administration
metabolic
67
TIlEOXDl
act
is exemplified as
substrates
clearly for
in
studies
the
hepatic
S metabolizing
In castrated
system.
markedly reduces hydroxysteroid
3a- and
activity
the
dehydrogenase
17B-hydroxysteroid
levels of estradiol cytosolic
3~
enhanced
metabolism
of a
has
suggested
activity.
been
If
a
steroids (41), of
cytochrome
36 other
(20)
and
increased (28).
were
dehydrogenase
these
selectively
to
microsomal 3a-
a-reductase that
Low
with respect
and
and cortisone to the fact
to
while
(28))
feminization of activity
will metabolize substances
increase in
respect
corticosterone-4-hydrogenase
point
overall
administration
with
7a-hydroxylase
dehydrogenase
These results
450 systems
It
and
and 17e-hydroxysteroid
in such rats.
liver
dehydrogenase,
cause the
and 3$-hydroxysteroid
estradiol
males,
of the
specifically
activities,
enzyme
TBEOXDI
(28,
59)
cytochrome
and,, hence,
P-450 may not be
Pan
in the
reflected
specific xenobiotic.
role
that
gonadal
in regulation
hormones
is assigned
then results which reflect
regulate to
inhibitory
the
hepatic endogenous
metabolic effects
high levels while low levels are stimulatory may be evidence for
need to
inhibit
the metabolism of these steroids
a
themselves under
certain circumstances. all
Not
necessarily hepatic
of
the
effects
of estrogens
on
due to their direct effect on the
influence
hepatic liver,
has been seen to be due to their
hepatic organs as well.
(47),
(29), believed by other of GH reveal
that
workers
it exerts
the
effects
very
similar to those of estradiol
effect
on non-
60, 61) or some
to be GH (62).
effects
(2, 20, 43).
their
where they are known
to bring about the release of growth hormone (GH) (48, factor
are
however. Their
bring about some of
The estrogens
hepatic effects by affecting the pituitary
other
function
on the
Studies on
liver which are
The possibility a similar
that
nature
is evidenced
the animals
treated
with
the
action
of
known
further be
indicated
accounted
(electron
for
related
The
these
GH on carbohydrate
in part will
in
hepatic
result
of the progestational
result
of both
as is the case with
the result
Not similar,
however,
of progestational
activity
of
the progestogen,
and the activity
of cytochrome
In summary,
differences
the finally, liver
to and,
which
could
the
liver
in
paper),
another
of:
of more than P-450
or
norethynodrel,
in the
to the steroids
as
compared
Estrogens degree
their
in
metabolic
both
literature
the
levels
insufficient
with
sensitivities
induction
progestogens;
3)
enzymes (for
4) differences
substrate
between
specificity
of the pituitary
be
to cause
the
of specific
P-450); in
can largely
in
in inducibility
P-450 systems
employed.
of
differences
of cytochrome
in the
a
P-450.
estrogens
5) differences
or as
P-450.
induced
of
cytochrome
or as the
very moderate
2)
types
for
is the pronounced
cytochrome
effects;
particular
liver,
of estrogens
on hepatic
of results
sexes
accounted
the estrogens.
hepatic
the
be
on the hepatocyte,
low doses being
between
various
lipid
could
1) dosage,
characteristics
example,
divergence
in terms
noticeable
agent
the cytochrome
while
explained
of mass of
mobilization
of GH on the
to the effect agents
levels
levels
of the action
to cause an increase
the
fat
of
is of
may be related
in a subsequent
protein
direct
either
show loss
mass, an increase
by the deposition be published
agents
to GH.
increase
unable
and
in liver
as being
were
which
a loss which
agents,
either
effect
of the progestational
in our studies
by an increase
micrographs
function
the action
and and
the
ACKNOWLEDGEMENT The Medical Research Council support of B.J. Wilson.
of South Africa
is acknowledged
for
its
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Lloyd. H-M.. Meares. J.D.. Jacobi. J. and Thomas. F.J.. J. ENDOCRINOL. 51(3) 473 (1971) Mode. A.. Grstedt. G.. Eneroth. P. and Gustafsson. J.A. ENDOCRINOLOGY113(4) 1250 (1983)
Ho Yuen. Richard D. Goold and Beverley Faculty of Pharmacy, Rhodes University Grahamstown, 6140. Republic of South Africa :i 3i>i\ d.? 5-L.‘--"5 ABSTRACT
J. Wilson
Norethynodrel proved to have moderately potent effects on hepatic function in female rats. It causes (a) loss of body mass, (b) increase (c) rise in the protein content of liver, in the relative liver mass, (d) increase in the cytochrome P-450 leve1, (e) reduction in the length of pentobarbital-induced sleep, and (f) increase in the rate of In contrast, biotransformation of (i) aniline and (ii) aminopyrine. ethynyl estradiol was shown to have little or no effect as judged by the same criteria, but, like norethynodrel, progesterone and estradiol, caused loss of body mass. INTRODUCTION It their
has been well
synthetic
function
derivatives
does
administration there
steroids
not,
on the
been
however,
picture
of the diverse
progestational
mass, while
to bring
about
agents
some mixtures
have been associated
with
liver
inconsistencies
mass.
relative levels substrates
Similar
liver and
mass,
metabolic
activity
of the hepatic
January 1985
(3) prevail
as far
TDEOXDI
liver.
obtained
The
from
to
the
rats.
of mixtures
Nor
of
such
to cause no
and estrogens
certain
oral
contraceptives,
or with
no change, respect
(4, 5) in
cytochrome
(mfo)
liver
to body mass,
endogenous
oxidase
in
have
(l),
levels, as
on the
or no change
with
protein
mixed function
S
of the
and
function.
mass (l),
(2),
an increase,
effects
have been reported
agents,
mlcrosomal
that
effects
in liver
of these
hormones
pronounced
substances
of hepatic
a loss
gonadal
in the results
or estrogenic
or even to cause a loss, reported
the female
including
agree
common parameters
For example, change,
tissues,
of progestogens a clear
that
have more or less
of "non-target"
literature
is
established
and are
Volume
P-450 exogenous
concerned. 45, Number
1
Increases,
decreases,
administration
lack
have been reported
Because divergent
and
of
the evident
results,
of for
effect all
need for
we undertook
of these
further
a study
progesterone comparison
of certain
study
the
and estradiol
of
steroid
parameters. to
clarify
these
of norethynodrel
and ethynyl on
(4-pregnen-3,20-dione)
result
of the effects
(17~~ethinyl-17-hydroxy-5(lO)-estren-3-one) ethinyl-1,3,5(10)-estratriene-3,17-diol)
as a
estradiol
(17~
rat,
using
(estradiol-17e)
for
female
effects. EXPERIMENTAL
progesterone (Sigma Steroids. Norethynodrel (Searle Lab.) or estradiol (Sigma Chemical C ) ethynyl estradiol (Wyeth Lab j Fo.j was dissolved in ethyl oieite and individually Chemical administered intraperitoneally (i-p.) between 08h30 and 09hOO daily for Two dosage levels, 1 fig periods ranging from two to thirty days. of each steroid were used. Control animals received no and 20 mglkg drug but equivalent volumes of ethyl oleate. Animals. Female Wistar rats between 4 and 5 months old and These rats were acquired from weighing initially 195 + 25 g were used. the South African Institute for Medical Research and were housed in a room with temperature and humidity control, in stainless steel cages with raised bottom (4 animals per cage). The animals were allowed free access to food and water. Sampling of Animals and Dosing. Animals were initially sorted on the basis of the regularity of their estrus cycles, only those with regular 4-day cycles being selected. For each steroid and dosage level under study, thirty-six animals were sorted into groups of four, one group for each period of dosing. Determination of Sleeping Time. Sleeping time of the steroidtreated rats and control rats was determined for animals treated for 7. with the steroid. Thirty mg/kg of pentobarbitai l~di~~d(Sf~a~~?R) ) was administered i.p. Preparation of Microsomes. Animals were sacrificed by a blow on the head after the predetermined duration of steroid administration. Two animals from each treatment group and from the control group were sacrificed, one 2 h after the last administration and the other 24 h after. Livers were removed and all subsequent operations were performed at 0 to 4°C. After perfusion with 0.9% NaCl, the livers were homogenized in a Thomas tissue homogenizer in 3 volumes of cold 0.025 M sucrose solution. The animals in the control group were sampled as in the experimental, except that both livers were combined. Liver
microsomes
were prepared
by a calcium
precipitation
method
(6).
Protein concentration was Assay of Protein and Cytochrome P-450. determined by the method of Lowry -et ai. (/) as modified by Miller (8). The method used for the determination of cytochrome P-450 was that of Omura and Sato (9). Enzymatic Assays. Enzymatic activity was measured in the following DL-isocitric acid (10 pmoles), incubation mixture: NAOP (2 pmoles), MnC12 (0.01 pmoles), isocitric acid dehydrogenase MgC12 (25 pmoles), (0.4 Ill). Five pmoles of freshly distilled aniline, or of aminopyrine, served as substrate. Buffer of 0.2 M Tris.HCl, pH 7.8, was used to microsomes equivalent to 250 mg of bring the total mixture to 4 mL; wet liver was added to the reaction. The flasks were incubated in a metabolic shaker under rapid shaking for 20 min at 37°C. Estimation of Aniline Hydroxylase Activity. Aromatic hydroxylation of aniline was determined by measuring formation of p-aminophenol according to the method of Maze1 (10) and by using the aboTTe incubation mixture. Calorimetric determinations were performed with a Beckman DB spectrophotometer. Demethylation Estimation of Aminopyrine Demethylase Activity. of aminopyrine was determined by measuring formaldehyde formation according to the method of Maze1 (10) with a Beckman DB spectrophotometer. The procedure was modified to adjust the final volume to 6 mL and to use the same NADPH-generating system as for the aniline hydroxylase system. RESULTS AND DISCUSSION In this order
study,
to accelerate
levels
are
those
numerous authors. that
rats
than
do
maintain
to a
-reflex, ')I
adequate
and levels
20 mg/kg
treatment
might
of these
it
levels
rate
(11)
of metabolic
Both dose levels of
sleeping being
was clearly
evident
after
estradiol
most
in The
used
by
on the fact xenobiotics
high
enough
to
disappearance. of
time
regimen
ethynyl
of
agents.
been
in part,
doses be
dosage
with
and have
is based,
that
were employed
by these
of metabolism
in the face Time.
steroids
be induced
is essential
marked shortening
that
of the
recommended by Gunzel
on Sleeping
an effect
levels
changes which
Choice
humans,
the
Although
high
have a much higher
Effect rise
rather
as
norethynodrel based on
gave righting
the more effective only
(Figure
7 days of treatment.
at both dosage
levels
also
S
56
shortened treated the
sleeping animals
Student's
values
the small
times,
in the effect It'
obtained
days with
?F-EOXDI
test
after
either
difference
produced
7 days and those
steroid,
hence only
control
difference
values
was noted
determined
one set of results
and
according
was insignificant
Little
(P> 0.01).
between
to
between
after
14 or 21
is shown.
Figure 1. Effect on Sleeping Time. Female. Wistar rats were treated for seven days with norethynodrel (N), 1 mg/kg (1 N) or 20 mg/kg (20 N) or with ethynyl estradiol (E), 1 mg,$kg (1E) or 20 mg/kg (20 E). Sleeping following time was determined pentobarbital administration of Control rats were (30 mg/kg i-p.). injected daily for seven days with Each bar represents ethyl oleate. the average of 12 animals. mean f S.D.
It
has been reported
no effect
on narcosis
administration prolonged found
an increase
rats
after
mg/kg.
after
of certain narcosis
that
oral
contraceptive
On the other
in the --in vitro
a 3- or 21-day a agents
administration
of norethynodrel
30 days of treatment
(12).
Similarly,
progestational
(4)
regimen
mixtures hand,
metabolism of
(10 mg/kg/d)
of hexobarbital
norethynodrel
in sleeping
time
such
medroxyprogesterone
and
short
Juchau and
reduction as
for
has that
periods
Fouts
(13)
in male
at a level has been noted
of
50 with
(17-acetoxy-6a-
S
TDEOXDI
methyl-4-pregnene-3,20-dione) metabolism
with
(4)
quingestanol
acetate
(14).
Certain
3,5-pregnandien-20-one) been shown to decrease results
bear
out
females
below)
sex
in this
and
latter
an
differences species
oral
associated
(SER)
underlying
common phenomena.
Nonetheless,
on Mass. The animals
subjected
(20
mg/kg)
lost
the treatment
days
they
treated than
approximately
with
norethynodrel
did those
animals
days
they
weighed
with
approximately
comparison,
a group of animals
and
loss
their
ethynyl
estradiol
after
days than
they
did
receiving
estradiol.
initially.
in mass of control
treated
animals
weighed
(Table
1 and Figure
2).
steroids
decrease appears
in
smooth
result
approximately
to be dose related,
after
30 Those
dose,
after
so that
original
to
to
after
30
in the animals
in comparison
in the extreme
the higher
regimen
less
was noted
subjected
For
subjected
slightly
than
30
mass.
a progesterone
35% less
body mass of animals
regimen
much less
in mass must be viewed so that,
the
mass.
Those rats only
(see
mass, although
their
was subjected
well
endoplasmic
so that
original
lost
95% of
animals,
are
in
certainly
period
the higher
A similar
Any loss
the gain
The
also
dose weighed
those
to the norethynodrel
14 days was similar.
at either
in
drugs
almost
80% of their
(1 mg/kg) treated
the
are
Effect
weighed
cautious
enzyme induction
of
result)
mass throughout
Our
with
of
have
(4).
in the brain
in the metabolism
(unpublished
mixtures
in male rats
proliferation
reticulum
hexobarbital
but one must be
studies
(15).
of
contraceptive
findings
from
rate
(17-acetoxy-3-(cyclopentyloxy)-
of pentobarbital
obtained
since
recognized
levels
these
results
relating
and an enhanced
with
cases,
the
controls
to each
of
dose of the
the
the
steroid
S
58 Table
1.
Effect
Steroid
of stemld
adminlstratlon
Dosea %/kg
THEOXDI
on the mass of
Duration days
lnltlal
16
183.3
30 14 20
16
20 20
female
rats.
Mass
Final 9
Mass
f
8.5
179.3
f
7.3
97
179.8
2
7.6
170.0
f
6.8
95
205.5
f
2.3
201.1
*
0.4
98
182.2
f
6.2
168.8
*
8.0
08
30
186.0
*
8.3
154.8
f
9.3
80
14
218.5
t 13.5
200.6
f 15.2
92
16
177.8
f
9.3
175.q
t
9.7
98
30
179.0
* 12.4
175.3
t
12.7
98
14
219.5
i
8.0
213.8
f
10.8
97
9
Final/Initial Mean Mass x 100
norethynodrel progesterone
norethynodrel progesterone
ethynyl
estradiol
estradlol
ethynyl estradlol
ethyl
20
16
176.3 2
6.1
167.5
t
5.5
95
20
30
173.0 f
6.8
162.3
*
9.0
94
20
14
209.8
2.8
195.8
*
2.9
93
estradlol f
14
175.5
f
7.9
185.9
*
8.2
106
30
175.5
t
7.9
202.8
i
10.0
114
oleate
a Stemlds
were
administered
daily
in the doses
indicated
to 4 rats
Figure
2.
in each group.
Effect
of Treatment
of
Female Rats with Norethynodrel (0) Animals or Ethynyl Estradiol (m). were treated with 20 mglkgld of either steroid or with ethyl oleate (4) for the appropriate number of Each point represents the days. comparison of mass of 4 animals after treatment with the mass of the same animals at the beginning of the treatment period. (Animals treated with progesterone (0) and estradiol (er) 20 mg/kg. for comparison.)
S resulting
in
rats
a more marked decrease
in body mass accompanying
decrease (16)
and
loss of mass
59
W-EOXDI
in body mass in estradiol
been shown to
cases.
administration
male rats (17) has been reported
in castrated
all
A
to female
previously,
as
has
a
males (2, 17), while synthetic estrogens have
reduce the rate of gain of body mass in
males
(12).
Norethynodrel has not been shown previously to reduce the rate of gain of body mass. of mass
However,
in combination with estrogens,
at high doses (5) or a dose-dependent
gain of mass (3,
5,
12).
it causes a loss
reduction in the rate
of
Other oral contraceptive mixtures have also
been associated with a decreased gain in mass (12, Effect on Mass of Liver.
The mean relative
18).
liver mass
somatic index (LSI) expressed as g/lOOg body mass) in receiving norethynodrel (both dosages, Figure 3A) 7 A
(liver
animal groups increased,
markedly
B
,._,..._. ____..... z... 6 *l.l,f,;; ,,,~,,...,,...a............’ “““‘-‘“~~’ . -x----;-__----‘--kX 5 : %-4,.‘e 2*-Jf X St 4 i--x
Figure 3. Liver Somatic Index (LSI). LSI is the actual liver mass in g per 100 g body mass. A. Each point represents the values from a single animal to which norethynodrel (1 mg (x) or 20 mg (.)/kg in ethyl oleate) had been administered for the appropriate numberof days. Control animals were injected daily with ethyl oleate. The LSI of the 16 control animals on the final day was 3.85. B. Each point represents the value from a single animal to which ethynyl estradiol (1 mg (x) or 20 mg (m)/kg in ethyl oleate) had been administered for the appropriate numberof days. Control animals were injected daily with ethyl oleate. LSI of the 16 control animals on the final day was 3.7. an increase which appears to have begun early, until
the thirtieth
and which continued
day. Sixteen control animals, sacrificed
on the
S final was
a relative
had
day,
somewhat
receiving
higher
liver mass of 3.85.
than
that
norethynodrel.
progesterone
(20 mg/kg)
while
treated
those
TPEOXDI
of the controls
with
after
14 days,
1 mg/kg for
this
in both
groups
of
rats
animals were treated
For comparison, and,
liver mass itself
The
LSI of 5.07 f
had an period
with
0.03
had an LSI of 4.48
-+
0.04. Norethynodrel, while causing a significant increased the mass of the liver as did
body,
observation effect
to enzyme induction.
of progesterone
parameter
in
pointing
(4).
liver
(1)
has
or LSI (4))
An earlier
medroxyprogesterone
(4)
ethinyl-4-estrene)
(1)
also been reported and the same
or ethynodiol
was
another
progesterone, report
showed the hormone to be without
Norethynodrel
mass
in mass of the
decrease
effect
on
the
on
this
to cause no change
result
was noted Lhen
diacetate (3e,17l+diacetoxy-17-
administered.
On
the
other
hand,
chlormadinone (17-acetoxy-6-chloro-4,6-pregnadiene-3,2O-dione)
caused a
loss of liver mass (1).
however,
have
been
increase without
to
cause hypertrophy
in LSI (3,
5,
la),
Only one
while others
mixtures,
of the
liver
(3)
and
an
have been reported
to
be
(4).
significant
namely an
entirely,
oral contraceptive
reported
effect
occurred,
Some
change
attributable
to
ethynyl
estradiol
increase in LSI (Figure 3B), but it was largely, or
due to the fact that treated animals lost overall mass while
the mass of the liver remained unchanged(in partial
agreement with an
earlier
animals receiving
report
(12)).
A
similar
that
the
LSI
pattern
was noted
in
estradiol
in
treatment
with 20 mg/kg while that resulting from administration
mg/kg was 4.90 + 0.04.
was 5.34 + 0.08 following
Unlike the
animals treated
14 days
with
of
of
1
ethynyl
estradiol, liver
animals
receiving
Sixteen
control
animals
mass.
relative
liver
The
female
is rats
(16,
P-450. --
studied
and
levels
of
protein
the
less
twelfth
day of treatment. 22.4
cytochrome
Sixteen
mg protein/g
450/mg protein.
steroid
there
The values
P-450 were higher of
administration.
the
when either
(Figure
day when 20 mg/kg P-450 continued
up to the
of liver for
levels
of both
in animals
steroid Underlying
than
an increase
12th day day,
microsomal
with
off
by
the
on the 30th
protein
24 h after sacrificed in
of the
nmoles of cytochrome
sacrificed in those
while
particularly
sacrificed
and 0.54
4A)
per
and levelled rats
of protein
(Figure
48))
increase dosage of
in levels
period
to rise
control
male change
ensued an
of cytochrome
P-450 content,
noted
and Cvtochrome
1 mg/kg of norethynodrel marked
be
but
reported,
Protein
as the fourth
the 30-day
in
(17),
no significant
increases
Levels
and
(2).
in LSI,
continued
a dosage of
to
administration
studied
as early
throughout
should
a loss of mass was
an increase
was administered.
microsomal
It
of
(2)
or of a loss
of Microsomal
P-450 appeared
administration
21).
Notable
respect
cytochrome
20,
was administered.
appeared
day yielded
(17,
P-450 concentrations
At
effects
change,
and in one case in which
with
steadily
treatment.
in
day had a
in male rats
cytochrome
cytochrome
increase
with
findings
males were being
Concomitantly
of norethynodrel to
results
on the Concentration
norethynodrel and
on the final
mass associated
to earlier
other
castrated
in protein
liver
in the case in which
were being
Effect
showed an increase
sacrificed
19) of no significant
with
however that,
occurred,
in
in contrast
consistent
rats
estradiol
mass of 3.7.
increase
estradiol
is
however,
2
cytochrome
the h
Pand last
after P-450
levels
as noted
aminolevulinic steroids,
by us
of course)
It
acid.
including
is,
has
been demonstrated
progesterone,
in levels
an increase that
are good inducers
of
6-
a variety
of
of s-ALA synthetase
2.0
1.5
I.0 ,5
~~~~+Jw-----------~~,5-
i
0.5
L. ':
++ 5
10
15
OUNATlO"
OF
20
TREATWENT
25
0
5
10
15
OURATION
(0AYZ.l
OF
20
TNEATNENl
25
30
(OAYS)
and Cytochrome P-450 in Rats Treated Female Wistar rats were dosed with norethynodrel 20 mg/kg (A) or 1 mg/kg (B) for the number of days indicated. Protein content (mg/g liver) (0) was determined by the method of Lowry et al. (7) as modified by Miller (8). Cytochrome P-450 (nmo leslmg pEteYi$ (x) was determined by the method of Omura and Sato (9). Figure
4.
with Norethynodrel.
Microsomal Protein
Literature
levels of cytochrome
on
administration
prolonged
progestogen,
chlormadinone
(.l)
insignificant,
or
acetate,
a
following
wide range of
results.
to be increased (1,
decrease
of
(1) depending
levels
appeared
20 mg/kg and
microsomal protein
to
14) or
of
significantly
or of cytochrome
P-450.
to show slight,
specific
on the
Neither the
be
perhaps
steroid employed.
ethynyl estradiol
1 mg/kg respectively.
increase
in levels of the
levels have been seen to
5A and 58 show the effects
levels
while another
caused a reduction
Microsomal protein
P-450 (1).
unaffected
Figures
reveals
in the liver
was shown previously to be without effect,
Norethynodrel
cytochrome
of steroids
P-450
at dose
of these
concentrations
dose of
S The lack to
produce
the
of change
finding
in protein
a noticeable
shortening that
of
effect
the
the
TDEOXDI
level
is consistent
with
on the proliferation
lamellae
thereof
of the SER or
on
results).
The
(unpublished
SER is not markedly
increased
the failure
is in agreement
with
0 x
2.0
n
t.5
; 'I' g :: 2::
1 , 8
.
.
&.;...;
. . . . .R,
. . . . . . . "";
.. ... .. ... ..... ... ..
.
,..
.7P
.
I.0
zig SE -^
* [+(g+.-*x-x~--------~
,
o5
g f ;
:* 5
0
IO
o”Rlr*oN
15 OF
20
TREAWE”,
J 30
25 ,Ol”S,
Figure 5. Microsomal Protein and Cytochrome P-450 in Rats Treated with Ethynyl Estradiol. Female Wistar rats were dosed with ethynyl estradiol or 1 mg/kg (B) for the number of days 20 mg/kg (A) indicated. Protein content (mg/g liver) (0) was determined by the method of Lowry et al. (7) as modified by Miller (8). Cytochrome P-450 (nmoles/mg proteiii)(x) was determined by the method of Omura and Sato (9). previously report
reported
findings
but
is not
in agreement
with
another
(16). It
is not surprising
450 concentrations fact, a
(12),
it
marked castrated
of
in
levels
cytochrome
lowering males
of an increase
the change produced
by ethynyl
has been reported
reduction
types
that
that
P-450.
in our study was very
under
in males (24)
of cytochrome (2,
estradio
similar
conditions
and a specific
Estradiol
P-450 levels
in
(21,
25).
(23)
reduction
has been reported
P-450 levels
21) and in females
in cytochrome
in the cytochrome
males
small.
In
there
is
in certain to
(17,
cause 20,
Only one report
has been noted.
P-
Estradiol
a
20, (19) has
S
64 a
small
less
inductive
than
effects 450
capacity
have been reported of cytochrome
(5,
18)
report
an increase
administration
of cytochrome still
of
protein,
(3)
or no significant
on Enzyme Activities.
With
1 mg/kg of norethynodrel
activity
to 126%
aminophenol/mg mg/kg for
to 10.60 nmol/mg
in levels
estrogens,
of
some
cytochrome
P-450
others
found
ly altered
(14,
(3) _
that 18,
Similarly,
showed an increase change (5,
12,
with
(18))
slight
14) when oral
to
compared with protein
aminopyrine
in
has
for
12 (from
to 9.28 nmol).
hydroxylase days increased 7.39
nmol
p-_
of
20
At a level
the activity
been
of sleep
demethylase
to
activity,
12 days increased protein
these reported
P-450 system noted
duration
for
controls
increased
formaldehyde/mg
increase
administration
to aniline
administered
norethynodrel
administered
nmoles
respect
171% (6.20
protein).
respect
norethynodrel
cytochrome
as
microsomal
12 days,
With
(24)
were administered.
Effect activity,
b5
P-
(12).
and
a decrease
reports
in cytochrome
estradiol
of the steroids;
reported
to these
of cytochrome
P-450 were not significant
another
to microsomal
21, 24)
much
lack of change in reduction
progestogens
contraceptives
reduced
as has been the
but
In addition
level
of mixtures
prolonged
similar
(2,
case
and
(2.67
a fall
and a reduced
earlier,
to marked decrease
the
levels
(26).
the
levels
respect
and pregnenes
of ethynyl
fol lowing
26) ,
to &ALA synthetase
P-450 in the presence
workers
the
respect
P-450 itself,
c) reductase
In
with
have the pregnanes
on cytochrome (or
TDEOXD1
nmol/mg
caused
(27).
Induction studies
as increased
mg/kg
of
to
129%
protein).
A
the activity
activities
in these
as well
to 3.44
20
by of
progesterone the
would account enzymatic
hepatic for
activities
the in
vitro ._-
in
rats
These Fouts
18
results
(13)
substrates
subjected
of noted
to 48 hours
metabolic
end further an
ncrease
prior
as well
heightened
(I,
hepatic
norethynodrel
in the
14).
for
4 days
The increase brought A
about
dose
105%
in metabolic
(1,
to
estradiol
increased
(2.67
105%
inhibition effect,
hand,
rats
Enhanced
with
other
no indication
of
administration
diacetate
of (17~-
were
12).
or a st imulation
respect
was negligible
given
to the xenobiotics
and not
in aniline
to 7.14 nmol 1,
while
of demethy lation
Such minimal
of metabolism
with
significant.
hydroxylase
and a dose of 20 mg/kg resulted
the rate
as we1 1 (4,
days.
treatment
after
certain
or when norethisterone
in an increase
(6.80
nmol to 2.85).
workers
12)
activity
estradiol
7.42 nmol)
increase
of
and
(12).
by ethynyl
to
to
Juchau
to male
of 3
or ethynodiol
of 1 mg/kg resulted
(7.09
by
metabolism
a period
was noted
workers
report
On the other
ethinyl-17-hydroxy-4-estren-3-one) to male rats
of
related
mfo activity
by other
rate
or for
been 4,
to the
had been administered
to a drug, has
of norethynodrel.
support
when norethynodrel
activity
progestogens
to a regimen
20
of aminopyrine
changes have been noted
of endogenous of their
in a similar mg/kg
In the case of the estrogenic steroids
metabolism
and
have all
to
ethynyl to
by
107%
earlier
steroids,
an
xenobiotics,
no
been reported
(2,
16, 17, 19, 20, 21, 25, 28-35). In the of
effect
enhanced
case of oral reported,
the rates
4, 5,
14, 18, 26, 36),
(I,
reduced agents
contraceptive
cannot
(3,
4, readily
13,
18).
mixtures,
of metabolism while
Our results
be related,
there
has been a range
of some compounds
those with
however,
of other
compounds were
individual to the
being
steroidal effect
of
a
combination of Blackham
the
and
particular
Spencer
progestogen
5 to
This
1.
conflicting
especially in view of
agents,
(37) were able to and estrogen
phenomenon
nullify
report
the effects
by administering
might help,
the
however,
of both
a
in a ratio
them
in
that
explaining
of the
findings of various investigators.
Our results do show, however, that norethynodrel does influence metabolism in that it caused a loss of overall mass, an increase liver
mass (and
particularly
microsomal protein,
of
LSI),
increase
and
an increase In cytochrome
P-450
in
in
hepatic
levels
and
an
metabolism toward specific xenobiotics --in vitro and a reduction of xenobiotic effect in vivo, all of which indicate that the enhanced
rate
of
steroid is an inducer of hepatic activity. significant
effect
brought
about
On the other
by equivalent
quantities
estradiol was a loss of overall mass, resulting In contrast
in which the sacrifice
of
xenobiotics
the only
of
ethynyl
in an increase in LSI.
to any finding of enhancedmetabolism resulting
administration
the prolonged
hand,
from
of progestogens are the results of studies
agents have been administered a few hours prior animal,
the have
or
of
studies
in which steroids
to and
to microsomal mixtures -in the steroids inhibit metabolism of substances
been added simultaneously
vitro. In such cases, -serving as substrates for the hepatic enzymesystem in both males (13, 32, 38-40)
and
females (38, 39 ).
has been observed
in
38, 39), pregnanolone (3a-hydroxy-5B-
the case of progesterone
(13,
pregnan-20-one)
metabolites of progesterone (38,
and some
32,
Such inhibition
39, 41, 42),
norethynodrel (4, 5, 13, 38, 43), lynestrenol (17a-ethinyl-4-estren-1701) (38)
norethisterone
acetate
--in vivo as well as in vitro.
(38,
401,
and
ethynodiol diacetate (38)
S The differences a
prolonged
between
situation,
the
that
fact
xenobiotics,
where inhibition
estrogens,
are all
agents
spite
as
for
for
emerged from spectral
(2,
for
that
this
emerging
estrogens
reason,
be
from the
by suggesting endogenous
devoid
reports
that
29,
of
in the
of
therefore
unable
to affect
estrogen
receptors
(54).
which
are not so readily
effect
may be accounted
for
if
doses,
P-450
systems
steroid
dependence
(19,
21,
characteristics.
to differences 450 systems Such in
which
in which
(53).
the
may be induced
specificity endogenous
directly
39,
and,
in part
systems (19)
and
through
by are the
substances
some differences there
56-58),
in
are a number of different
each
differences
with
sexits
own
may be related
of those
cytochrome
P-
by steroids.
of induction steroids
liver
metabolized
possess
specificities
growth have
or when estrogenic
(55) which
has
discrepancies
are readily P-450
the
Studies
may be rationalized
Furthermore, substrate
to which
Hence,
are employed,
29,
systems
when
in the
one remembers that
cytochrome
many
from studies
46, 49-52).
metabolized
the hepatocytes
hepatic
inducibility
43,
cytochrome
metabolized
to
c larity
work,
and on rats
literature
At higher
P-450
obtained
metabolic
effect
appropriate
vitro
and
cons iderable
low doses of the steroids
levels
(44)
results
periods,
may be rapidly
--in
sites.
46-48),
(20,
noted with
is due largely
same cytochrome
--in vitro 20,
rule,
steroids
divergent
(45))
hormone has been administered revealed
the
prolonged
work rats
many
the binding
apparently
are administered
on hypophysectomized
for
sometimes
compounds and the is the
with
substrates
of the
activity
of estrogenic
and hence are competitors In
the enhanced
administration
metabolic
67
TIlEOXDl
act
is exemplified as
substrates
clearly for
in
studies
the
hepatic
S metabolizing
In castrated
system.
markedly reduces hydroxysteroid
3a- and
activity
the
dehydrogenase
17B-hydroxysteroid
levels of estradiol cytosolic
3~
enhanced
metabolism
of a
has
suggested
activity.
been
If
a
steroids (41), of
cytochrome
36 other
(20)
and
increased (28).
were
dehydrogenase
these
selectively
to
microsomal 3a-
a-reductase that
Low
with respect
and
and cortisone to the fact
to
while
(28))
feminization of activity
will metabolize substances
increase in
respect
corticosterone-4-hydrogenase
point
overall
administration
with
7a-hydroxylase
dehydrogenase
These results
450 systems
It
and
and 17e-hydroxysteroid
in such rats.
liver
dehydrogenase,
cause the
and 3$-hydroxysteroid
estradiol
males,
of the
specifically
activities,
enzyme
TBEOXDI
(28,
59)
cytochrome
and,, hence,
P-450 may not be
Pan
in the
reflected
specific xenobiotic.
role
that
gonadal
in regulation
hormones
is assigned
then results which reflect
regulate to
inhibitory
the
hepatic endogenous
metabolic effects
high levels while low levels are stimulatory may be evidence for
need to
inhibit
the metabolism of these steroids
a
themselves under
certain circumstances. all
Not
necessarily hepatic
of
the
effects
of estrogens
on
due to their direct effect on the
influence
hepatic liver,
has been seen to be due to their
hepatic organs as well.
(47),
(29), believed by other of GH reveal
that
workers
it exerts
the
effects
very
similar to those of estradiol
effect
on non-
60, 61) or some
to be GH (62).
effects
(2, 20, 43).
their
where they are known
to bring about the release of growth hormone (GH) (48, factor
are
however. Their
bring about some of
The estrogens
hepatic effects by affecting the pituitary
other
function
on the
Studies on
liver which are
The possibility a similar
that
nature
is evidenced
the animals
treated
with
the
action
of
known
further be
indicated
accounted
(electron
for
related
The
these
GH on carbohydrate
in part will
in
hepatic
result
of the progestational
result
of both
as is the case with
the result
Not similar,
however,
of progestational
activity
of
the progestogen,
and the activity
of cytochrome
In summary,
differences
the finally, liver
to and,
which
could
the
liver
in
paper),
another
of:
of more than P-450
or
norethynodrel,
in the
to the steroids
as
compared
Estrogens degree
their
in
metabolic
both
literature
the
levels
insufficient
with
sensitivities
induction
progestogens;
3)
enzymes (for
4) differences
substrate
between
specificity
of the pituitary
be
to cause
the
of specific
P-450); in
can largely
in
in inducibility
P-450 systems
employed.
of
differences
of cytochrome
in the
a
P-450.
estrogens
5) differences
or as
P-450.
induced
of
cytochrome
or as the
very moderate
2)
types
for
is the pronounced
cytochrome
effects;
particular
liver,
of estrogens
on hepatic
of results
sexes
accounted
the estrogens.
hepatic
the
be
on the hepatocyte,
low doses being
between
various
lipid
could
1) dosage,
characteristics
example,
divergence
in terms
noticeable
agent
the cytochrome
while
explained
of mass of
mobilization
of GH on the
to the effect agents
levels
levels
of the action
to cause an increase
the
fat
of
is of
may be related
in a subsequent
protein
direct
either
show loss
mass, an increase
by the deposition be published
agents
to GH.
increase
unable
and
in liver
as being
were
which
a loss which
agents,
either
effect
of the progestational
in our studies
by an increase
micrographs
function
the action
and and
the
ACKNOWLEDGEMENT The Medical Research Council support of B.J. Wilson.
of South Africa
is acknowledged
for
its
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