Peripheral Serum Corticosteroid Concentrations in
Relation to the Rat Adrenal Cortical Circadian
Rhythm in Androgen-Induced Hypertension
CAROL S. FINK, B.A.,
SAMUEL GALLANT, P H . D . , A N D ALEXANDER C. BROWNIE, P H . D .
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SUMMARY Adrenal function was assessed in control rats and in rats treated for 2 and 4 weeks with 17amethylandrosteriediol (MAD; 17a-methyl-5-androstene-30,170-diol), a synthetic androgen known.to produce
hypertensive cardiovascular disease. In both groups and at both time periods, a circadian rhythm of blood corticosteroid concentrations was observed. The high point for serum corticosterone (B), 18-hydroxy-lldeoxycordcosterone (18-hydroxy-DOC), and 11-deoxycorticosterone (DOC) concentrations occurred at the
beginning of the dark period (1800 hours), and the low point occurred at the onset of the light period (0600
hours). Serum concentrations of DOC were always found to be higher in MAD-treated rats as compared with
controls. The serum concentrations of B and 18-hydroxy-DOC were lower than control values at 1800 hours
but higher than control concentrations at 0600 hours. The in vitro 11/3- and 18-hydroxylation of DOC was
markedly reduced with MAD treatment. In contrast, cholesterol side-chain cleavage activity was higher in
animals treated with MAD. These in vitro findings correlated with spectral studies that showed a decreased
binding of DOC to cytochrome P45O1U and increased binding of cholesterol to cytochrome P450^x. These
studies suggest that MAD treatment selectively decreases 11/9- and 18-hydroxylatlon in adrenal mitochondria,
and this results in an increased serum concentration of DOC, a hypertensinogenic steroid. This effect of MAD
on peripheral serum DOC concentration is most readily observed in quiescent animals at the high point of the
circadian rhythm. (Hypertension 2: 617-622, 1980)
KEY WORDS • androgen-induced hypertension
17a-methylandrostenediol • circadian rhythm
• adrenal cortex
S
KELTON1 found that hypertensive cardiovascular disease results from chronic treatment
of rats with the synthetic androgen, 17amethyl-androstenediol (MAD; 17arrnethyl-5-androstene-3/3,17/3-diol). An important observation that
allows one to propose a mechanism for this form of
experimental hypertension is that of Salgado and
Selye2 who demonstrated that the adrenal gland had
to be present for the development of hypertension. In
addition, Brownie and Skelton* found that steroid
11/8-hydroxylase activity was low in adrenal homogenates and mitochondria from MAD-treated rats,
leading to an accumulation of 11-deoxycorticosterone (DOC). MAD is converted to 17a-methyltestosterone (MT; 17a-methyl-4-androstene-17/3-ol-3-one)
in the adrenal4 and MT is similar to testosterone'
• serum corticosteroids
in being an inhibitor of the 11/3-hydroxylation of
DOC." This has led to general agreement that increased secretidn of the hypertensinogenic steroid
DOC7 is involved in the pathogenesis of MADinduced hypertension. Indeed, there is evidence*'9 for
increased secretion of DOC in the adrenal venous
effluent of MAD-treated rats at the same time as there
is decreased secretion of corticosterohe (B)8 and 18hydroxy-11-deoxycorticosterone (18-hydroxy-DOC).'
These studies in which adrenal venous blood was
collected, however, of necessity involve rats subjected
to the stress of anesthesia. Furthermore, effects of
MAD upon other enzymes of the corticosteroid
biosynthetic pathway have not been described. Accordingly, the present studies were designed to measure concentrations of DOC, 18-hydroxy-DOC, and B
in peripheral blood of control and MAD-treated rats,
with blood being collected undef quiescent conditions
and at 6-hour intervals during a 24-hour period1 in
order to take into account the circadian rhythm of
adrenal cortical activity.10-" In addition, adrenal
mitochondria were isolated for the determination of
cholesterol side-chain cleavage activity as this enzyme
catalyzes what is recognized as the rate-limiting step
in corticosteroidogenesis.11- "
From the Departments of Pathology and Biochemistry, State
University of New York at Buffalo, Buffalo, New York.
Supported by U.S. Public Health Service Grant HL-06975.
Address for reprints: Alexander C. Brownie, Ph.D., Department
of Biochemistry, State University of New York at Buffalo, 102
Cary Hall, Buffalo, New York 14214.
Received August 30, 1979; revision accepted February 4, 1980.
617
618
HYPERTENSION
Methods
Animals and Treatment
Male Sprague-Dawley rats weighing approximately
200 g (Holtzman Co.; Madison, Wisconsin) were
singly caged in three rooms with controlled
temperature (22 ± 1°C) and lighting (fluorescent illumination from 0600-1800 hours). All rats were uninephrectomized, given 1% NaCl as drinking solution,
and fed Charles River Rat Chow that contained 0.40%
sodium, ad libitum. The experimental groups received
10 mg MAD (Sigma), suspended in 0.2 ml corn oil, by
subcutaneous injection 6 days per week. Control
groups received the vehicle alone.
Blood Pressures
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Systolic blood pressures were measured indirectly
under light ether anesthesia14 with a Physiograph Four
(E and M Instrument Co., Inc., Houston, Texas).
Collection of Rat Serum and Serum Steroid Assays
After 2 or 4 weeks of treatment, seven control and
eight MAD-treated rats were killed by decapitation at
each of the following times: 0600, 1200, 1800, and
2400 hours. Exposure to stress was minimized by
handling the rats twice daily during the treatment
period and by having five people enter each room in
turn and begin blood collection within 12 seconds of
entry and removal of the rat from its cage. Blood was
individually collected from the trunk and allowed to
clot on ice. The serum was separated by centrifugation
and stored in the frozen state until assayed.
Corticosterone was measured by the direct radioimmunoassay (RIA) procedure of Gomez-Sanchez et
al.1B following methylene dichloride extraction of a 0.1
ml aliquot of serum. Serum 18-hydroxy-DOC was
measured by direct RIA according to the method of
Gallant." Serum DOC concentrations were determined by RIA. 17 The serum samples were extracted
and then chromatographed in a solvent system of
cyclohexane:methanol:water (100:50:25 v/v) by a
similar method previously described. 18 In this
chromatography system, DOC had an Rf value of
0.29 and was well separated from MT, MAD, B, 18hydroxy-DOC, and progesterone, which had Rf values
of 0.46, 0.11, 0.01, 0.001 and 0.81 respectively. The
cross-reactivities of these steroids with the DOC antibody were: MT (0.2%), MAD (0.1%), B (2%), 18hydroxy-DOC (0.01%), and progesterone (95%).
Collection of Adrenal Tissue and Enzyme Assays
Adrenal zona fasciculata-reticularis tissue was
obtained by adrenal enucleation in situ. 1 ' Adrenal
tissue was homogenized in cold buffered 0.25 M
sucrose, and mitochondria were obtained by differential centrifugation.20 The concentration of adrenal
mitochondrial protein used for each assay ranged
between 1.5-2.5 mg/ml and was determined by the
method of Lowry et al.21 Cytochrome P450 concentrations in the adrenal mitochondrial preparations
VOL 2, No 5, SEPTEMBER-OCTOBER
1980
were determined by using an Aminco DW2 Spectrophotometer by the method of Omura and Sato."
Cholesterol side-chain cleavage (CSCC) activity
was determined by measuring the pregnenolone
formed from endogenous cholesterol in adrenal
mitochondria incubated at 37°C under air in the
presence of cyanoketone, an inhibitor of sterol 3/S-oldehydrogenase, and isocitrate as a source of reducing
equivalents.2' The reaction medium contained 50 mM
sucrose, 50 mM NaCl, 5 mM MgCl 2 , 5 mM KC1, and
100 mM MOPS (Morpholinopropane Sulphonic
Acid, pH = 7.4; Calbiochem). The pregnenolone
formed was measured by RIA.2*
Mitochondrial 11/3- and 18-hydroxylase assays were
performed in the same reaction medium described
above; 60 ^M DOC substrate as well as a source of
reducing equivalents were added. The method of assay
employed high-pressure liquid chromatography for
simultaneous assay of 11/3- and 18-hydroxylase activity.2*
Spectral Analyses
Adrenal cortical mitochondrial cytochrome P450
systems, consisting of P450 llfl and P450KC, were examined by light absorption spectrophotometry.28 The
extent of cholesterol association with P450KC was
determined by the heat-generated Type I absorbance
change (HGI) which develops as the mitochondria are
warmed from 1°C to 37°C.27 An indirect measure of
cholesterol association with P450.cc, the pregnenolone-induced Type II (reverse Type I) absorbance
change (PII), was determined by adding saturating
concentrations of pregnenolone to warmed adrenal
mitochondrial samples.28 The extent of DOC binding
to P45O1W was measured by adding saturating
amounts of DOC to warmed adrenal mitochondria
and measuring the observed Type I absorbance
change (DOCI). 29
Results
Body Weights and Blood Pressures
Figure 1 shows the weekly mean systolic blood
pressures and body weights of representative control
and MAD-treated rats. Body weight increased more
rapidly in the control rats. By 4 weeks, the blood
pressures of the MAD-treated group were significantly greater than those of the controls (p < 0.05).
Eight of 14 (57%) MAD-treated rats and only one of
13 (7%) controls became hypertensive by 7 weeks.
Rats were considered hypertensive when systolic
blood pressures were in excess of 150 mm Hg.
Peripheral Serum Steroid Determinations
Figure 2 shows the peripheral serum concentrations
of DOC, 18-hydroxy-DOC, and B from quiescent control and MAD-treated male rats which were killed at
0600, 1200, 1800 or 2400 hours after 2 or 4 weeks of
treatment. A circadian rhythm for B, 18-hydroxyDOC, and DOC in the control rats at both 2 and 4
SERUM CORTICOSTEROIDS IN ANDROGEN-INDUCED HYPERTENSION/FwJt et al.
weeks is evident when the concentrations of these
steroids at 1800 hours (lights off) are compared with
those at 0600 hours (lights on). The low basal concentrations of all three steroids at 0600 hours in these
control rats indicate that they were quiescent. The apparent increase of serum B concentrations after 4
weeks of treatment, above that observed after 2 weeks
in both control and MAD-treated rats, may be the
result of an age-related maturation of the circadian
rhythm of plasma corticosterone concentrations.30
The MAD-treated rats also showed a circadian
rhythm of B, 18-hydroxy-DOC, and DOC similar to
that seen in the controls, with a high point at 1800
hours and a low point at 0600 hours. However, the absolute concentrations determined in the MAD-treated
rats varied from those achieved by the controls. At
0600 hours, concentrations of DOC, B, and 18hydroxy-DOC were higher in the MAD-treated rats
after both 2 and 4 weeks. However at 1800 hours, B
and 18-hydroxy-DOC concentrations were lower in
the MAD-treated animals, but DOC concentrations
remained higher than those of the control group. Even
when the effects of stress are minimized, as they have
been during these experiments, DOC concentrations
were higher in the MAD-treated rats at all times
studied, and were maximal at 1800 hours. The data indicate that these differences in serum steroid concentrations were magnified at 1800 hours.
CMTML
•II
171
111
IS!
141
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131
121
Adrenal Mitochondria! Incubations
Mitochondrial 11/3- and 18-hydroxylase and CSCC
activities were compared in the quiescent control and
MAD-treated rats which were killed at 0600, 1200,
1800 and 2400 hours after either 2 or 4 weeks (table 1).
These data indicate that MAD treatment decreases by
approximately the same proportion both 11/3 and 18hydroxylase activities. When the CSCC activities were
examined, a circadian rhythm was evident in both control and MAD-treated rats. Activities measured at
1800 hours were higher than those measured at 0600
hours in both groups at 2 and 4 weeks. Moreover, the
MAD-treated rats showed a greatly increased rate of
CSCC activity above control activity at 0600 and 1200
hours after 2 and 4 weeks. Although these data are expressed per nmole of cytochrome P450, similar
differences between control and MAD groups are
observed if CSCC activity is expressed as
pregnenolone formed per mg protein.
HI
111
411
441
. . - • *
. . * •
* • • • • •
411
, - * • • • "
3M
321
211
241
211
7
I
1. Mean body weights and blood pressures of
MA D-treated and control rats measured at weekly intervals
after the initiation of treatment. Vertical lines indicate ±
standard error of the mean (SE).
FIGURE
Spectral Data
Cholesterol and DOC association with cytochrome
P450 of adrenal mitochondria isolated from the
various groups is shown in table 2. Cholesterol
TABLE 1. Effect of MAD Treatment on Adrenal Mitochondrial Up- and 18-Hydroxylase and Cholederol SideChain Cleavage Activities
Rat
Activity*
ll^hydroxylase
18-hydroxylase
Cholesterol side chain
cleavage
group
control
MAD
control
MAD
control
MAD
619
After 2 wks of treatment
1800
1200
0600
2400
After 4 wks of treatment
1800
0600
1200
2400
hrs
hrs
hrs
hrs
hrs
hrs
hrs
hrs
6.83
2.90
2.66
1.58
0.08
0.28
9.00
3.22
5.03
1.65
0.18
0.48
6.08
3.74
2.86
2.02
0.36
0.43
8.63
2.70
3.98
1.48
0.35
0.34
7.77
3.59
4.62
1.35
0.16
0.34
12.32
4.19
4.84
1.90
0.12
0.39
8.20
2.99
4.69
1.57
0.38
0.49
8.20
3.11
4.73
1.64
0.33
0.58
*Enzyme activities are expressed in nmoles of product formed/minute/nmole P450. Each value represents
the'mean of duplicate assays; taken at 6-hour intervals during a 24-hour period.
HYPERTENSION
620
OMOfc
1
CONTROL
120011
1800 h
2, No 5, SEPTEMBER-OCTOBER
FOUR WEEKS TREATMENT
TWO WEEKS TREATMENT
60
VOL
2400 1
1200 h
1800 k
2400 k
•
MAD
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2.5
I
2I
1-5
S
i.i
1.5
FIGURE 2. Mean peripheral serum steroid concentrations of MAD-treated and control rats quiescently
killed at the hours indicated either 2 or 4 weeks after the initiation of treatment. Data are expressed in ng/dl
or ng/ml. Vertical lines indicate ± s£ Significance was determined by Student's t test. Asterisk (*) indicates
p < 0.05; dagger ft) indicates p = 0.067.
TABLE 2. Cholesterol and DOC Association vrith Cytochrome P45O of Adrenal Mitochondria Isolated from
Quiescent Control and MA D-treaied Ram a* Various Times of the Day*
Time of Day (hrs)
HG I
Control MAD
Pn
Control MAD
DOC I
Control MAD
Cytochrome
P450
(nmoles/mg
protein)
Control MAD
After 2 wks of treatment
0600
1200
1800
2400
19.7
21.2
26.6
36.2
36.0
48.1
44.7
55.3
10.3
13.7
22.3
29.8
30.0
41.2
38.2
50.0
50.9
41.6
42.8
44.7
22.7
20.6
17.5
27.6
1.84
1.75
2.19
2.01
1.24
1.16
1.36
1.05
After 4 wks of treatment
0600
1200
1800
2400
24.0
21.6
30.3
3Q.5
47.9
34.9
48.0
51.6
15.3
10.8.
22.7
22.6
42.7
21.3
46.0
43.6
50.0
47.8
45.5
42.5
24.0
27.4
24.0
16.4
2.06
1.55
1.83
1.86
0.98
1.05
0.97
1.13
•HGI ant! DOCI are expressed as AA 390-420/nmole P450 X 10,. PII is expressed as AA 420-390/nmole
P450 X 10,.
1980.
SERUM CORTICOSTEROIDS IN ANDROGEN-INDUCED HYPERTENSION/F/nfc el al.
association with P450.cc, measured by HGI and PII
assays on adrenal mitochondria, was increased in the
MAD-treated groups above control values at all time
periods studied. DOC binding to P450nS, measured by
DOCI assays on adrenal mitochondria, on the other
hand, was decreased in the MAD-treated groups at all
time periods. Total adrenal mitochondrial P450 concentrations were reduced by about 40% after treatment with MAD.
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Discussion
Previous studies suggest that following the injection
into a rat of MAD, a A6-3/9-ol-steroid, the corresponding A4-3-ketone, MT, is produced in sufficient
quantities to inhibit steroid lljS-hydroxylation. This
results in an increased secretion of DOC and, eventually, hypertensive cardiovascular disease. Another
effect of chronic MAD treatment is a significant
decrease in total adrenal mitochondrial cytochrome
P450.'1'3S This decrease has been related to the
reduced activity of steroid 1 10-hydroxylation.
However, cytochrome P450KC which is involved in the
rate-limiting step of corticosteroidogenesis, CSCC, is
also localized to the mitochondria of the adrenal cortical cell." Our studies show that, in contrast to
steroid 11/3- and 18-hydroxylation, the activity of
CSCC is not reduced by chronic treatment with
MAD. Thus, we can now propose that the androgen
treatment selectively decreases 11/3- and 18hydroxylation in adrenal cortical mitochondria and
leads to an increase in the secretion of DOC.
In the present study we have shown for the first time
that, compared to controls, MAD-treated rats have
elevated plasma DOC concentrations. Furthermore,
we have shown the advantage of carrying out
measurements at the high point of the circadian
rhythm when the concentrations of DOC achieved are
much higher than those seen in quiescent rats killed in
the morning at the start of the light period. At the high
point of the circadian rhythm one can readily see the
reduced ability of MAD-treated rats to maintain normal concentrations of corticosterone and 18-hydroxyDOC whereas there are increased concentrations of
DOC. These studies also show that despite alterations
in corticosteroidogenesis brought about by treatment
with MAD there is a circadian rhythm in DOC, 18hydroxy-DOC, and B, with the highest values occurring near the start of the dark period. Apparently,
these three corticosteroids are under similar control,
with their presumed source being the zona fasciculatareticularis.
There is some evidence that treatment of rats with
androgen reduces ACTH secretion." However,
despite that, our studies show that a circadian rhythm
of corticosteroids is retained in androgen-treated rats.
In control rats there is an apparent circadian rhythm
in CSCC activity with a maximum around 1800 hours
and a minimum around 0600 hours. These results are
similar to those in another report from our
laboratory.11 There is a less well-defined circadian
621
rhythm in CSCC activity in the MAD-treated groups.
This is related to a higher activity at 0600 and 1200
hours compared to controls. This higher rate of
cholesterol side-chain cleavage in MAD-treated rats
at 0600 and 1200 hours correlates with higher plasma
B and 18-hydroxy-DOC concentrations in MADtrcated groups. At these times, when control animals
are showing very low B and 18-hydroxy-DOC concentrations, DOC/B ratios arc not significantly higher in
MAD-treated rats at 2 weeks. However, when the
1800-hour blood samples are analyzed, it is clear that
the DOC/B ratio is much higher in the MAD-treated
rats than in controls. The advantage of measuring corticosteroid concentrations on blood samples
withdrawn at or near the high point of the circadian
rhythm has been shown by us for adrenal regeneration
hypertension18 and now this is also applicable to
androgen-induced hypertension.
Acknowledgments
The authors thank Luther Joseph, Marlene Telech, and Mary
Russo for their expert technical assistance, Drs. Robert Kramer and
Josephine Alfano for their help in preparing the manuscript, and
Berta Cole for her secretarial assistance.
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C S Fink, S Gallant and A C Brownie
Hypertension. 1980;2:617-622
doi: 10.1161/01.HYP.2.5.617
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