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Original Studies |
Biomedicum Helsinki, Department of Physiology (T.R., J.J.P., O.A.J.), Institute of Biomedicine, University of Helsinki, FIN-00014 Helsinki; Hospital for Children and Adolescents (L.D., S.W.), University of Helsinki, FIN-00029 Huch; Institute of Biotechnology (J.J.P.), University of Helsinki, FIN-00014 Helsinki; and Department of Clinical Chemistry (O.A.J.), University of Helsinki, FIN-00014 Helsinki, Finland
Address all correspondence and requests for reprints to: Taneli Raivio, M.D., Ph.D., Biomedicum Helsinki, Department of Physiology, Institute of Biomedicine, University of Helsinki, P. O. Box 63 (Siltavuorenpenger 20 J), FIN-00014 Helsinki, Finland. taneli. raivio{at}helsinki.fi
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-dihydrotestosterone,
dehydroepiandrosterone, or androstenedione levels. We conclude that our
novel bioassay enables quantitation of mammalian cell response to
bioactive androgens in human serum, even in pediatric patients with
relatively low androgen levels. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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AR-interacting protein 3 (ARIP3), a 572-amino acid nuclear protein expressed primarily in the testis, represents a potential coregulator of AR-dependent transcription (6). Although the exact physiological role of ARIP3 is not yet known, we have observed that it can facilitate considerably the androgen-dependent interaction between the AR LBD and N-terminal region (6). Herein, we describe the development of a bioassay that is based on ARIP3-facilitated interaction between the LBD and N-terminal region of AR. This assay seemed useful for the quantitation of circulating androgen bioactivity in pediatric patients. We expect that the assay will have important applications in clinical endocrinology.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All plasmid constructs have been reported previously (4, 6, 7), and only a short description of each is given here. The LBD of human AR (containing amino acids 657–919; Ref. 7) and the N-terminal region of rat AR (5–538; Ref. 4) were created by PCR, and the products were cloned into the pM and VP16 vectors (CLONTECH Laboratories, Inc., Palo Alto, CA), respectively. The luciferase reporter pG5-LUC contains five Gal4-binding sites in front of the minimal TATA box. pFLAG-ARIP3 has been described (6).
Cell culture and transfection
COS-1 cells (American Type Culture Collection, Manassas, VA) were maintained in phenol red-free DMEM (Life Technologies, Inc., Santa Clara, CA) containing penicillin (25 U/mL), streptomycin (25 U/mL), and 10% (vol/vol) FCS (Life Technologies, Inc., Paisley, UK). Twenty-four hours before transfection, the cells were divided onto a 96-well plate (NUNC, Roskilde, Denmark) at a density of 5000 cells/well. The plates were incubated overnight at 37 C in a humidified atmosphere of 5% CO2-95% air. Three hours before transfection, the cell culture medium was replaced by DMEM containing 8% charcoal-stripped FCS. The cells were transfected using FuGene reagent (Roche Molecular Biochemicals, Mannheim, Germany) according to the instructions provided by the manufacturer. Each well received a total of 69 ng DNA [pG5-LUC, 25 ng; pM-hAR (657–919), 13 ng; pFLAG-ARIP3, 13 ng; pVP16-rAR (5–538), 13 ng; and pCMVß, 5 ng] delivered as a single mastermix of plasmids.
Twenty-four hours after transfection, medium in each well was replaced by 90 µL phenol red-free DMEM without FCS, and 10 µL testosterone-containing FCS in triplicate (standard) or 10 µL human serum (unknown sample) in duplicate was added. After an overnight incubation at 37 C humidified atmosphere of 5% CO2-95% air, the wells were aspirated empty, the cells were lysed in 30 µL diluted reporter lysis buffer (Promega Corp., Madison, WI), and 10 µL of cell lysates were transferred to 96-well plates for measurements of ß-galactosidase (8) and luciferase (9) activities.
Sex steroids and nonsteroidal antiandrogens
Dehydroepiandrosterone (DHEA;
3ß-hydroxy-5-androsten-17-one), androstenedione (4-androstene-3,
17-dione), and 5-dihydrotestosterone (DHT;
17ß-hydroxy-5-androstan-3-one) were obtained from Steraloids Inc.
(Wilton, NH) and were dissolved and serially diluted in ethanol and
added to charcoal-stripped FCS to yield the following concentrations:
6.13 nmol/L, 25 nmol/L, and 100 nmol/L. DHT was diluted further in FCS
to result in a serum concentration of 0.78 nmol/L. These steroids were
tested in the bioassay in parallel with testosterone standard curve. To
investigate the transactivation potential of estradiol, 17ß-estradiol
was dissolved in charcoal-stripped FCS to result in a serum
concentration of 500 nmol/L. The nonsteroidal antiandrogens Casodex
[(2RS)-4'-cyano-3-(4-fluorophenylsulfony)-2-hydroxy-2-methyl-3'(trifluoromethylpropionanilide)],
and hydroxyflutamide
(4-hydroxy-,,-trifluoro-2-methyl-4'-nitro-m-propionotoluidide)
were obtained from Zeneca Pharmaceuticals (Macclesfield,
UK) and Schering AG Corp. (Broomfield, NJ), respectively.
Antiandrogens were serially dissolved in ethanol and added to
charcoal-stripped FCS containing 10 nmol/L testosterone. The highest
antiandrogen concentrations in the resulting FCS were 1 µmol/L
hydroxyflutamide and 10 µmol/L Casodex; the measurements were carried
out in quadruplicate in one transfection.
Subjects
Thirty-two boys, 1.0–16.8 yr old, were investigated. Twenty-three boys, 13.9–16.8 yr old, had constitutional delay of puberty (CDP). Clinical data, together with serum hormone levels in 19 boys have been published previously (10). The boys were in early puberty (18 boys were at Tanner stage G2, and 5 were at stage G3) and had no underlying diseases that could have accounted for the delay in puberty. Sixty-five per cent had a history of pubertal delay in the family. The boys were clinically examined, puberty was staged according to Tanner (11), the testes were measured with a ruler, testicular volume was calculated from the formula: length x width2 x 0.52 (12), and a single blood sample was drawn. Twenty-two boys have been followed up for 12 months or longer; puberty has progressed in each subject. Another study group consisted of 9 boys, 1.0–6.4 yr old, with cryptorchidism (clinical data and serum hormone levels have been published previously; 13). These boys were treated with human CG (hCG; 1500–5000 IU im, 3 times, with 1-week interval). Blood samples were drawn immediately before the treatment and on the fourth day after the last hCG injection. The blood samples were allowed to clot, serum was separated by centrifugation, and the sera were stored at -70 C until required. The study protocol was accepted by the ethical committee of the Hospital for Children and Adolescents, University of Helsinki. Informed consent was obtained from the guardian and, in addition, from the boys with CDP.
Preparation of standards and patient sera for the bioassay
Testosterone (Steraloids Inc.) was dissolved, serially diluted in ethanol, added to charcoal-stripped FCS (HyClone Laboratories, Inc., Logan, UT), and divided in aliquots, which were stored at -70 C for future use as standards in the bioassay. Sixty microliters of serum from each boy (see below) was centrifuged briefly, filtered through a 0.22-µm Spin-X centrifuge filter unit (Corning Costar Corporation, New York, NY), and stored at -70 C until used.
Diethyl ether extraction
Testosterone was added to pooled sera of 10 prepubertal (age range, 1.0–8.0 yr) boys with cryptorchidism (13). The serum pool was divided in 300-µL aliquots in glass tubes, followed by 300 µL diethyl ether (Merck KGaA, Darmstadt, Germany). The tubes were vortexed briefly, centrifuged for 10 min at 4 C, and placed in a dry ice-ethanol (-70 C) bath to freeze the water phase, after which the organic phase was transferred to a new glass tube. Freezing was repeated once, diethyl ether was evaporated, and the samples were reconstituted in 300 µL charcoal-stripped FCS (5 tubes). The tubes were shaken gently overnight at 4 C and filtered through a 0.22-µm Spin-X centrifuge filter unit; 30–60 µL serum from each tube was taken for testosterone RIA (see below). Serum sex hormone-binding globulin (SHBG) level in the pooled sera of the boys (and in charcoal-stripped FCS) was measured using the assay described below.
Immunoassays
Serum testosterone concentrations were measured using a commercially available RIA kit [Orion Diagnostica, Espoo, Finland; (10, 13)]. According to the manufacturer, the assay has a 4.5% cross-reactivity with DHT, and minimal cross-reactions to other steroid hormones. Serum DHT, androstenedione, and DHEA concentrations were measured in boys with CDP, after separation of steroid fractions on Lipidx-5000 microcolumn (Packard-Becker, Groningen, The Netherlands), as previously described (14). Serum SHBG concentrations in 22 boys with CDP were measured using time-resolved fluoroimmunoassay (Perkin-Elmer Life Sciences, Wallac Finland Oy, Turku, Finland). According to the manufacturer, the sensitivity of the SHBG assay is better than 0.5 nmol/L; inter- and intraassay coefficients of variation (CVs) are both less than 5%.
Data analysis
Relative luciferase activities were calculated by dividing luciferase activities by ß-galactosidase activities to correct for differences in transfection efficiency. Standard curves for the bioassay were fitted with a 4-parameter weighted equation using the AssayZap program (Biosoft, Cambridge, UK); the results are expressed in nmol/L testosterone equivalents. Pearson’s correlation coefficient was calculated between paired variables to investigate their relationship. Mean values of different parameters were tested by paired and unpaired t tests, when appropriate. All mean values are expressed ± SD. Statistical significance was accepted for P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Androgens from the serum enter COS-1
cells and induce the interaction between the LBD and the N-terminal
region of AR. This interaction is enhanced by ARIP3 (6).
The complexes bind to Gal4-binding sites, located in the pG5-LUC
reporter construct, and the VP16 transcriptional activation domains are
tethered to the luciferase gene promoter, leading to activation of
luciferase gene transcription. Luciferase activity in cell lysates
corresponds to androgen bioactivity in human serum.
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Different amounts of testosterone were added to charcoal-stripped
FCS, and the resulting dose-response curves are shown in Fig. 2. Values are presented as units of
relative luciferase activity (luciferase activities divided by
ß-galactosidase activities obtained for each well). The steepest
increase in relative luciferase activity occurred at testosterone
concentrations in FCS below 10 nmol/L. The median of maximal fold
induction (calculated as the ratio of relative luciferase activity
induced by 100 nmol/L testosterone to activity induced by
charcoal-stripped FCS without added testosterone) from five different
assay runs was 745.
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To investigate the biopotencies of different naturally occurring androgens, DHT, androstenedione, and DHEA were added to charcoal-stripped FCS. Dihydrotestosterone was the most active androgen; FCS containing 0.78 nmol/L DHT induced relative luciferase activity equal to 10 nmol/L testosterone equivalents. Only the highest concentration of androstenedione (100 nmol/L in FCS) induced a signal that corresponded to 1.3 nmol/L testosterone equivalents. DHEA did not activate luciferase gene expression at any the doses examined. The relative luciferase activity induced by FCS containing a high concentration of estradiol (500 nmol/L) was less than 0.1% of that achieved with FCS containing a saturating testosterone concentration (100 nmol/L).
Inhibition by nonsteroidal antiandrogens
The effect of antiandrogens on the interaction between the N- and C-terminal domains of AR was investigated by first adding testosterone at a subsaturating concentration (10 nmol/L) to charcoal-stripped FCS. Then, increasing amounts of hydroxyflutamide and Casodex were added to aliquots of the testosterone-containing FCS, and 10 µL of each dilution was subjected to the bioassay. FCS containing 100 (hydroxyflutamide) or 1000 (Casodex) times the molar amount of testosterone suppressed relative luciferase activities to levels of approximately 5% of the activity achieved by testosterone-containing FCS without added antiandrogens.
Sensitivity and precision
The sensitivity of the bioassay was defined as mean +2 SD of multiple luciferase activities induced by charcoal-stripped FCS without added testosterone; it was below the signal induced by FCS containing 1.0 nmol/L testosterone (cell culture medium containing 0.1 nmol/L testosterone). Intraassay CV was defined as repeated measurement of the same human serum sample. At 4.9 nmol/L testosterone equivalents, the intraassay CV was 8.3%. Interassay CV was 21%, as determined from 11 consecutive assay runs.
Patient data
Serum androgen bioactivity levels were above the assay
sensitivity in 15 boys with CDP, and in all 9 prepubertal boys with
cryptorchidism during treatment with hCG (androgen bioactivity levels
before the hCG treatment were below the detection limit of the assay).
The mean androgen bioactivity level in samples containing measurable
activity (n = 24) was 4.3 ± 3.2 nmol/L (range, 1.0–14.5
nmol/L testosterone equivalents). These values were on the rising part
of the dose-response curve, and they correlated strongly with serum
testosterone levels measured by RIA (r = 0.93, P
< 0.0001, n = 22; Fig. 3). When
expressed as a function of serum total testosterone, the average serum
androgen bioactivity levels were 26 ± 3.7% and 33 ± 9% in
boys with CDP or cryptorchidism, respectively (P <
0.05). In boys with CDP, this percentage and testis volume correlated
positively (r = 0.49, n = 13, P = 0.09).
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Diethyl ether extraction of human serum
To investigate the relationship between serum androgen
bioactivity and testosterone levels further, testosterone was added to
pooled sera of 10 prepubertal boys with cryptorchidism, to yield the
serum concentration of 21 nmol/L. In this pool, serum testosterone
concentration, as measured with RIA, was 20.6 ± 0.9 nmol/L,
whereas the androgen bioactivity level determined by the current assay
was 5.7 ± 0.6 nmol/L testosterone equivalents (Fig. 4). Concentration of SHBG in this serum
pool was 135 nmol/L. We next extracted the pooled sera with diethyl
ether, which releases steroid hormones from their binding proteins,
after which the steroid-containing phase was reconstituted in
charcoal-stripped FCS (without measurable SHBG). This should render
free plus initially protein-bound androgens in human serum available to
the cells of the bioassay. Indeed, after the procedure, serum androgen
bioactivity in the pool increased from 5.7 ± 0.6 nmol/L to
13.0 ± 1.6 nmol/L testosterone equivalents (Fig. 4;
P < 0.005). The actual rise in serum androgen
bioactivity is likely to be even higher, because not all testosterone
was extracted from serum by diethyl ether; the mean testosterone level
measured by RIA after the reconstitution was 14.1 ± 1.3
nmol/L (mean recovery 69%; Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The sensitivity of the assay was better than the signal induced by 1.0 nmol/L testosterone in the 10-µL FCS aliquot, corresponding to 0.1 nmol/L testosterone in cell culture medium. This is of the same order of magnitude as the dissociation constant of the receptor for testosterone (17, 18), which casts some doubt on the possibility to enhance significantly the assay sensitivity without additional manipulation of the sample. Although diethyl ether extraction and subsequent concentration of the sample in FCS increased the relative sensitivity of the bioassay, the assay without any extraction has the benefit of being able to measure directly the androgen bioactivity in human serum.
In boys, serum androgen bioactivity and serum testosterone concentrations correlated strongly. Testosterone in serum is bound with high affinity to SHBG and with low affinity to albumin; only approximately 2–3% of total testosterone seems to be free (18, 19, 20). The amount of biologically active testosterone has been suggested to be a function of free testosterone (21). The androgen bioactivity levels that we measured were obviously too high to represent merely the free testosterone fraction. Indeed, in the course of the assay, sera were diluted with cell culture medium in a ratio of 1 to 10, which should result in the dissociation of the weakest protein-steroid complexes. Thereafter, both the free plus initially albumin-bound testosterone fractions in serum, which together are often referred to as the bioavailable testosterone (22, 23, 24, 25), should be available for the cells in the bioassay.
Because of the high androgenic potential of DHT, one would expect to
find a positive correlation between serum androgen bioactivity and DHT
levels in boys with CDP. Lack of relationship between these variables
may, however, reflect the fact that the affinity of DHT for SHBG is
three times higher than that of testosterone (26), which
may render the low amounts of circulating DHT in boys biologically
inert. On the other hand, biologically active DHT is produced in
androgen target cells expressing the 5-reductase enzymes. Thus, the
local intracellular androgen milieu may differ from that measurable in
the peripheral circulation. Addition of testosterone to SHBG-containing
serum pool of prepubertal boys yielded androgen bioactivity values that
were approximately one-fourth of the testosterone levels measured by
RIA. Likewise, extraction of human serum with diethyl ether and
subsequent reconstitution of the sample into charcoal-stripped FCS
increased bioactivity levels. Taken together, although we cannot
exclude the existence of yet-unidentified factor(s) in human serum that
inhibit sex steroid entry into or action within the target cells, these
findings suggest that the SHBG-bound steroids are not available to
COS-1 cells in the bioassay.
Serum androgen bioactivity levels and testis volumes correlated strongly, which probably reflects an increased testicular testosterone production toward adulthood. The ratio of serum androgen bioactivity to testosterone also tended to increase as a function of puberty, which is to be expected because of the reciprocal changes in serum testosterone and SHBG levels during adolescence (23, 27, 28). On the other hand, this ratio was higher in prepubertal boys with cryptorchidism during hCG treatment than in the older boys with CDP. This was not anticipated, because prepubertal subjects have higher serum SHBG levels than boys in early puberty (23, 27).
The interaction between the N-terminal domain and the LBD of AR is dependent on the presence of androgen and is influenced only minimally by estradiol (3, 5). This was also observed in the present study. Moreover, nonsteroidal antiandrogens Casodex and hydroxyflutamide suppressed reporter gene activity, suggesting that the current assay is also applicable to screening for compounds with either androgenic or antiandrogenic activity. However, caution is required in the interpretation of these results because, in the assays employing fragments of AR or full-length AR, some synthetic compounds may act somewhat differently (5). Nevertheless, our unpublished results indicate that androgen bioactivity levels in boys measured with the current assay correlate strongly with those measured by a bioassay based on the use of full-length AR.
From the clinical point of view, bioassays such as ours should be, in several instances, superior to the conventional assays based on immunological detection of steroid hormones. For example, the current bioassay measures supranormal androgen bioactivity values in males after a prolonged administration of anabolic steroids, despite the low levels of physiological androgens measured by RIA in serum of these subjects (our unpublished observation). Likewise, it is conceivable that exposure to a number of medicinal and/or environmental compounds will alter the levels of immunoassayable androgens and androgen bioactivity to a differential degree, and sometimes in opposite directions.
In conclusion, we have developed a bioassay that is based on the ARIP3-facilitated interaction between the N-terminal domain and LBD of AR. The assay has a wide dynamic range and is minimally influenced by estradiol. In boys, serum androgen bioactivity levels and testosterone concentrations measured by RIA correlate strongly, but the relative bioactivity levels are lower, which probably reflects the binding of androgens to SHBG.
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Footnotes |
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Received August 3, 2000.
Revised November 17, 2000.
Accepted December 5, 2000.
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References |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
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, Boys with CDP
(n = 13); •, prepubertal boys with cryptorchidism (n = 9)
during hCG treatment.
) and androgen bioactivity levels (mammalian cell bioassay, 
)
were measured. To release testosterone from the binding proteins, serum
was extracted with diethyl ether, the steroid-containing phase was
reconstituted in charcoal-stripped FCS, and the measurements were
repeated. Asterisk, Different from the mean androgen
bioactivity level before extraction-reconstitution procedure
(P < 0.005). Each bar represents
the mean + SD of five measurements.