0021-972X/86/6305-1193$O2.00/0
`Journal of Clinical Endocrinology and Metabolism
`Copyright © 1986 by The Endocrine Society
`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Vol. 63, No. 5
`Printed in U. S.A.
`
`Mechanism of Inhibition of Human Testicular
`Steroidogenesis by Oral Ketoconazole*
`
`€<
`JACOB RAJFER, SURESH C. SIKKA, FABIO RIVERA, AND
`DAVID J. HANDELSMANT
`
`Division of Urology, Department of Surgery, UCLA School of Medicine, Harbor- UCLA Medical Center,
`Torrance, California 90509
`
`ABSTRACT. To determine the antisteroidogenic effect of ke-
`toconazole (KTZ) in the human testis, we measured the plasma
`A5-pregnenolone, A5-17oz—hydroxypregnenolone, dehydroepian-
`drosterone (DHEA), progesterone, 17a-hydroxyprogesterone,
`androstenedione (A), and testosterone (T) concentrations in
`three men with previously untreated metastatic prostate cancer
`at various time intervals for 24 h before and 48 h after the
`administration of 200 mg oral KTZ every 8 h. The adrenal
`glands of these three patients were suppressed (as measured by
`the plasma cortisol levels) by the administration of 1.0 mg
`dexamethasone daily for 7 days before and during’ the study.
`After six doses of KTZ, bilateral orchiectomy was performed,
`and the intratesticular concentration of the aforementioned
`seven steroids and the intratesticular activities of the 1701-
`hydroxylase, 17,20-desmolase, and 17,3-hydroxysteroid dehydro-
`genase enzymes in the A‘-steroidogenic pathway were deter-
`mined. These seven intratesticular steroids and three intrates-
`ticular enzyme activities were compared to those in five men
`
`with previously untreated prostate cancer who underwent or-
`chiectomy as primary treatment for their disease. Plasma A,
`DHEA, and T all significantly decreased during KTZ therapy.
`There was no significant change in the other four steroids in the
`plasma. In the testis, A5-pregnenolone, A5-17a-l1ydroxypregnen-
`clone, and A‘-17a-hydroxyprogesterone were all significantly
`elevated, whereas intratesticular DHEA, A, and T were signifi-
`cantly decreased in the three KTZ-treated patients compared to
`levels in the five non-KTZ-treated patients. Measurement of the
`enzyme activities demonstrated a significant reduction in both
`17oz-hydroxylase and 17,20-desmolase, but no change in 17,8-
`hydroxysteroid dehydrogenase,
`in the KTZ-treated patients
`compared to the levels in the non-KTZ.-treated patients. We
`conclude that oral KTZ decreases testicular T production by
`inhibiting the 17,20-desmolase and also the 17a-hydroxylase
`steps in both the A“- and A5—T biosynthetic pathways. (J Clin
`Endogrinol Metal) 63; 1193, 1986)
`
`ETOCONAZOLE (KTZ) is a synthetic, orally ac-
`—
`tive imidazole dioxolone derivative introduced ini-
`tially as a broad spectrum antifungal agent (.1). More
`recently, KTZ was found to be a potent inhibitor of
`human gonadal (2) and adrenal (3, 4) steroidogenesis. A
`common mechanism of action of KTZ has been proposed,
`since inhibition of a wide variety of cytochrome P-450-
`dependent enzymes by KTZ has been described in fungal
`membranes (5) and adrenal steroid production (4). Ste-
`roidogenesis in the testis, however, is much more sensi-
`tive to inhibition by KTZ compared to that in the adre-
`nal, since production of testicular C-19 steroids is pro-
`foundly inhibited by KTZ at doses that do not inhibit
`basal production of cortisol and other C-21 adrenal ste-
`roids (2, 3). Thus, it may be hypothesized that a major
`
`.
`
`Received March 10, 1986.
`Address requests for reprints to: Jacob Rajfer, M.D., Harbor-UCLA
`Medical Center, 1000 West Carson Street, Box 5, Torrance, California
`90509.
`* This work was supported in part by a grant from the NIH (GCRC
`RR-00425).
`“T National Health Medical Research Council Overseas Fellow (Aus-
`tralia){ Current address: Department of Medicine, University of Syd-
`ney, Sydney, 2006, New South Wales, Australia.
`
`site of steroidogenic blockade by KTZ in the testis is
`17,20-desmolase, a cytochrome P-450-dependent enzyme
`that catalyzes conversion of C-21 steroidal precursors to
`C-19 sex steroids.
`
`The inaccessibility of human steroidogenic tissues has
`limited the ability to test this hypothesis directly in men
`treated with KTZ. Previous studies attempted to infer
`the site of action of KTZ by examining the profiles of
`circulating steroid precursors (2, 3, 6). This indirect
`approach has several difficulties, including the dual ad-
`renal and gonadal sources of such steroid precursors,
`alterations in steroid clearance rates, and the introduc-
`tion of additional intermediate variables, such as possible
`drug effects on circulating steroid—binding protein levels
`or binding affinity. An alternative approach of studying
`the effects of KTZ on steroidogenesis in uitro (7) entails
`some difficulties in interpretation, since the possible
`effects of active metabolites of KTZ cannot be adequately
`addressed. In this study we examined the site(s) whereby
`KTZ inhibits testicular testosterone (T) production in
`man by direct measurement of testicular steroidogenic
`enzyme activities after in viva treatment. In addition, to
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`1194
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`RAJFER ET AL.
`
`JOE & M- 1986
`V0163 -No 5
`
`further define the relationship between the site of ste-
`roidogenic inhibition and circulating steroid precursors,
`we simultaneously measured both testicular and serum
`steroid levels in KTZ—treated men who underwent con-
`
`current adrenal suppression. Our results indicate that
`KTZ inhibits testicular steroidogenesis primarily by in-
`hibiting 17,20-desmolase activity.
`
`Materials and Methods
`
`Patients
`
`Three unselected men (mean age, 69 yr; range, 63-76 yr)
`with newly diagnosed metastatic (stage D) prostate cancer who
`were about to undergo bilateral ‘oi-chiectomy as the conven-
`tional primary treatment for their disease were enrolled into
`this study. Before entry into the study, each man gave written
`informed consent to undergo blood sampling and dexametha-
`sone (DEX) and KTZ treatment before orchiectomy. All pro-
`tocols and procedures were approved by the Human Subjects
`Ethical Review Committee of the Harbor—UCLA Medical Cen-
`ter. Testes excised from five other unselected men (mean age,
`69 yr; range, 63-82 yr) with (stage D) metastatic prostate cancer
`who had received no prior drug treatment were used as controls
`for measurement of testicular steroidogenic activity. None of
`the patients in the control or KTZ-treated groups had received
`any prior hormonal therapy for their disease.
`
`Protocol
`
`The men commenced taking oral DEX (0.5 mg daily) at least
`7 days before admission to the Clinical Study Center, Harbor-
`UCLA Medical Center, and DEX was continued until surgery
`3 days after admission. After admission, a venous cannula was
`inserted into a forearm vein, and aliquots of blood were with-
`drawn at various time intervals for the next '72 h until orchiec-
`
`tomy. During the control day (first 24 h after admission), blood
`was sampled at -24, -20, —16, —8, +4, -2, -1, and 0 h. At the
`start of the second day and for the following 48 h until surgery,
`200 mg KTZ (Janssen Pharmaceutica, Beerse, Belgium) were
`administered orally every 8 h in addition to DEX, and blood
`was sampled at 1, 2, 4, 8, 12, 16, 20, 24, 32, 40, and 48h. The
`blood was allowed to clot, and the serum was separated and
`stored at -20 C until performance of steroid assays.
`At surgery, both testes were removed, snap-frozen in liquid
`nitrogen, and stored at -70 C until processing for measurement
`of testicular steroidogenic enzymes and steroid levels.
`
`Measurement of steroid levels and enzyme activities
`
`The testes from both the control (untreated) and KTZ-
`treated groups were quickly thawed, decapsulated, and homog-
`enized at 4 C using a motor-driven glass homogenizer by 15-20
`strokes at about 2000 rpm in ice-cold 0.1 M phosphate buffer,
`pH 7.4 (1:5, wt/vol), containing 0.25 M sucrose and 1 mM
`dithiothreitol. The homogenate was centrifuged at 800 X g for
`20 min, and the supernatant was either used immediately for
`the enzyme assays or kept at -70 C until used for the deter-
`mination of steroid concentrations and enzyme activities.
`
`The steroid concentrations in testicular homogenates and
`‘serum samples were measured using RIA procedures, as de-
`scribed by Abraham et al. (8). An aliquot (1.0 ml) of homogenate
`(equivalent to ~200 mg tissue) or 1.0 ml serum was used for
`extraction with diethyl ether after trace amounts (~1000 cpm)
`of various steroids were added to determine their recoveries.
`Steroids were separated in batches by Celite column chroma-
`tography techniques, as described by Manlimos and Abraham
`(9). RIA procedures were carried out using purified tritiated
`tracers, authentic standards, and specific antibody preparations
`(RSL, Inc., Carson, CA). These specific antibodies were raised
`employing steroid—carboxyether-BSA or steroid-hemisuccin-,
`ate-human serum albumin conjugates. The assays were per-
`formed after making appropriate dilutions of the recovered
`steroid samples. Dextran-coated charcoal was used to separate
`the free from the bound radiolabeled. steroids. The specificity
`and accuracy of the assays were determined by assaying differ-
`ent vqlumes of the extracts as well as by monitoring the
`recovery of the added steroids. The recovery of the added
`steroids vzaried between 90-110%, and no systematic deviation
`of results from linearity was found. The cross-reactivity data
`showed that only dihydrotestosterone cross-reacted (~20%) in
`the T assay; for theghother steroid assays, the "cross-reactivities
`of major steroids were less than 1%. KTZ, in amounts up to 40
`Mg, did not cross-react with the T antibody. The sensitivity of
`the assay procedures was 20 pg for A5-pregnenolone (PG),1 15
`pg for progesterone (P), 10 pg for 17a—hydroxyprogeste'rone
`(17-P), 30 pg forwandrostenedione (A), and 10 pg for T. The
`intraassay variations for these steroids were 9% for PG, 7% for
`P, 8% for 17—P, 10% for A, and 7% for T,‘_while the interassay
`coefficient of variation varied from 10418%. Serum cortisol
`levels were determined, as previously described (10),_employing
`[i‘2"’I]cortisol and anticortisol antibody (RSL, Inc.). All samples
`from an individual patient were analyzed at the same time.
`Measurements of 17:1-hydroxylase, 17,20-desmolase, and
`17,8-hydroxysteroid dehydrogenase activities in the A“-steroid-
`ogenic pathway were carried out as previously described (11).
`
`Statistical analysis
`
`To‘ optimize analytical design of this intensive study (19
`samples/subject) of a small number of subjects (n = 3 subjects)
`and to account for baseline differences between subjects, serial
`measurements of serum steroids within individual patients were
`analyzed by repeated measures analysis of variance which
`considered each subject as his own control. Where appropriate,
`log transformations of hormone levels were used before analysis
`to ensure homogeneity of variance. The effects of KTZ during
`specified time periods were analyied with F tests on suitable 1
`degree of freedom linear contrasts. Analyses of variance were
`performed using the BMDP program 4V implemented on a
`VAX 11/750 computer (12). Intratesticular steroids and ste-
`roidogenic enzyme activities were compared between KTZ-
`
`‘The following trivial names are used. A5-pregnenolone (PG), 5-
`pregnen-3/3-ol-one; 17a-hydroxyprogestei-one (17-P), 4-pregnen-17oz-
`ol-3,20-dione; 17a-hydroxypregnenolone (17-PG), 5-pregnen-3;3—17oz-
`diol-20-one; dehydroepiandrosterone (DHEA), 5-androsten-36-ol-1%
`one; androstenedione (A), 4-androsten-3,17-dione.
`
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`

`
`KTZ AND HUMAN TESTIS
`
`1195
`
`treated and control men by unpaired t test. Statistical signifi-
`cance was set at a 2—tailed 0: level of 0.05.
`
`17-P (F = 7.6; P = 0.11), or P (F = 4.0; P = 0.184; Table
`1 and Fig. 1).
`
`Results
`
`Intratesticular steroids
`
`The KTZ-treated and control groups were similar in
`age and stage of prostate cancer. No side—effects (hepa-
`totoxicity) occurred during KTZ treatment and up to 1
`month after orchiectomy.
`
`Serum steroids
`
`Serum cortisol levels in the DEX—treated men were
`suppressed (Table 1), indicating virtually complete sup-
`pression of glucocorticoid output during the entire study.
`This degree of adrenal suppression suggested that further
`changes in serum steroid levels could be attributed to
`changes in testicular, not adrenal, steroidogenesisl. The
`low levels of serum cortisol before the initiation of KTZ
`therapy suggests compliance with DEX therapy.
`By comparison with the 24 h of treatment with DEX
`alone, the addition of KTZ (600 mg/day) produced sig-
`nificant suppression (Table 1) of serum T (F = 101.2; P
`= 0.01), A (F = 229.4; P = 0.004), and dehyd_roepian-
`drosterone (DHEA; F = 69.3; P = 0.014), a rnarginhl
`increase in 17-P (F = 4.9; P = 0.16), but no change in
`PG (F = 0; P = 0.96), 17-PG (F = 0.01; P = 0.95), or P
`(F = 0.01; P = 0.93). The decreases in serum T and
`DHEA were maximal by the end of the first treatment
`day, whereas A levels decreased further on the second
`treatment day (F = 41.9; P = 0.023). Serum 17-P in-
`creased significantly on the first day only (F = 20.4; P _=
`0.041), Diurnal effects during the 3,days of study were
`evident in serum T (F = 22.8; P = 0.041), DHEA (F =
`81.5; P = 0.012), A (F = 20.4; P = 0.046), and 17—PG (F
`= 332.8; P = 0.003), but not PG (F = 10.3; P‘-’= 0.085),
`
`There was a significant (P < 0.025) decrease (75%
`lower) in intratesticular T levels, from 661 : 155 (:
`SEM) ng/g testis in control patients to 163 : 101 in KTZ-
`treated patients (Fig. 2). Intratesticular A levels de-
`creased 75% (P < 0.05), from 139 : 52 ng/g testis in
`control patients to 33 : 12 in KTZ-treated patients.
`DHEA levels declined 43%, from 410 : 46 ng/g testis in
`control patients to 232 : 72 in KTZ-treated patients (P
`< 0.05). Intratesticular 17—P increased 1383%, from 12
`: 2 n.%/g testis in control patients to 178 : 82 in KTZ-
`treated patients (P < 0.05), while PG and 17—PG both
`increased 248% and 400%, respectively, in the KTZ-
`treated groups compared to levels in control patients.
`There was no difference in the intratesticular P content
`in the control and KTZ-treated patients. These results
`indicate that KTZ blocks primarily the 17,20—desmolase
`enzyme of both the A4- and A5—pathways of steroidogen-
`esis.
`
`Testicular steroidogenic enzyme activities
`
`Testicular 17,20—desmolase activity; (picomoles per
`min/mg protein) decreased 56%, from 590 : 105 (: SEM)
`in control patients to 260 : 20 in KTZ-treated patients
`(P < 0.025), while 17a:-hydroxylase activity decreased
`29%, from 1040 : 135 in control patients to 740 : 62 in
`KTZ-treated patients (P < 0.05). 1746-Hydroxysteroid
`dehydrogenase activity increased by 73% (P < 0.05) in
`the testes of KTZ-treated men (956 : 292) compared to
`that in control men (554 : 122; Fig. 2). These results are
`consistent with the inferences from product/precursor
`
`TABLE 1. Effect of KTZ on serum steroid levels in DEX-trealted prostate cancer patients
`Basal
`
`Steroids
`
`‘
`—-24h —20h —16h 8h —4h —2h —lh
`
`0“
`
`+1 h +2 h +4 h +8 h" +12 h +1611" +20 11' +24 11° +32 h“ +40 hf‘ +48 11"
`
`KTZ“
`
`PG (ng/dl)
`.
`17—PG (ng/dl)
`
`DHEA (ng/dl)
`
`P (ng/dl)
`
`17~P (ng/dl)
`
`A (ng/dl)
`
`T (ng/dl)
`'
`Cortisol (ng/d.l)"
`
`210
`193
`214
`213
`185
`177
`176
`168
`148
`112
`137
`165
`182
`:43
`:32
`:62
`:19
`:32
`:79
`:52
`:14
`:28
`:18
`:18
`:23
`:14
`35
`4
`42
`39
`47
`46
`38
`28
`33
`41
`18"
`27
`42
`:13
`:7
`:12
`:10
`:4 :18
`:8
`:8
`:5
`:14
`:3
`:7
`:17
`19
`23
`21
`24
`46 >
`42
`47
`33
`3
`34
`23
`30
`50
`:4
`:2
`:5
`:3
`:7
`:7
`:6 :12
`:5
`:10
`:3
`:2
`:8
`15
`53
`132
`151
`150
`161
`142
`164
`115
`139
`121
`122
`132
`:48
`:50
`:34
`:42
`:36
`:33
`:26
`:32
`:17
`:25
`:14
`:14
`:26
`95
`83
`71
`66
`49
`57
`61
`66
`76
`62
`45
`44
`66
`:18
`:21
`:10
`:21
`:17
`:10
`:22
`:21
`:27
`:18
`:17
`:15
`:14
`35
`42
`44
`49
`54
`' 57
`49
`57
`46
`48
`50
`52
`69
`:7
`:6
`:5
`:8
`:11
`:8
`:8 :14
`:10
`:9
`:6
`:4
`:10
`279
`338
`232
`408
`586
`549
`668
`E44
`610
`631
`503
`476
`708
`:90 :132 :119
`:148 :219 :231 :242 :252 :217 :176 :156 :176
`:351
`393
`270 ND
`450 ND
`ND
`240
`226 ND
`ND
`496 ND
`290
`:19O
`:43
`:26
`:29
`:90 :190
`:70
`
`150
`:6
`33
`:16
`15
`:2
`153
`:53
`82
`:14
`35
`:9
`295
`:134
`270
`:70
`
`166
`:14
`
`:19
`15
`:1
`144
`:49
`85
`:35
`34
`:13
`155
`:65
`ND
`
`250
`:16
`44
`:28
`11
`:3
`142
`:41
`84
`:28
`23
`:7
`219
`:62
`<200
`
`140
`:41
`3
`:22
`10
`:4
`139
`:38
`104
`:34
`19
`:5
`134
`:21
`<200
`
`154
`:20
`41
`:28
`16
`:1
`130
`:41
`127
`:50
`30
`:11
`265
`:40
`<200
`
`180
`:12
`37
`:25
`11
`:5
`133
`:35
`77
`:17
`23
`:5
`203
`:60
`<200.
`
`ND, not done. Values are the mean : SEM.
`,
`“ 200 mg KTZ, orally every 8 h.
`°Normal 0800 h values, 10,000—25,000 ng/dl; all men (11 = 3) received DEX (0.5 mg daily) throughout the study, starting 3 days before initiation of KTZ
`administration.
`I
`'
`
`WCK1038
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`

`
`RAJFER ET AL.
`
`JOE & M- 1986
`V0163-N05
`
`ANDROSTENEDIONE
`
`-s~4To*<i
`
`£3
`
`‘
`
`is
`
`TESTOSTERONE
`
`8
`I6.
`24
`32
`40
`TREATMENT PERIOD (Hrs)
`
`48
`
`-24
`
`-16
`
`-8-4 0 4 8
`
`I6
`
`24
`
`32
`
`40
`
`48
`
`Lmiuyn
`
`.i.j_
`
`80
`
`so
`
`40
`
`20
`
`O
`-24
`
`|
`-l6
`
`an
`>
`-E-4 0 4
`
`TREATMENT PERIOD (Hrs)
`FIG. 1. Mean (isEM) serum steroid levels (nanograms per dl) in three patients who received 3 days of KTZ therapy (200 mg, three times daily)
`and DEX (1.0 mg/day) during the 3 days of KTZ therapy and for 7 days befo‘re KTZ therapy.
`
`ratios of steroids in either serum or testis, and again
`suggest that the principal site wherein KTZ inhibits T
`biosynthesis is the l7,20—desmolase enzyme.
`
`Discussion
`
`These results demonstrate clearly that KTZ inhibits
`testicular steroidogenesis by inhibiting 17,20—desmolase
`activity, a cytochrome P—450-dependent enzyme. This
`conclusion is supported by the concordance of the results
`of direct assays of testicular steroidogenic enzyme activ-
`ities from KTZ—treated men and the contents of steroid
`precursors in the testis. For example, 17,20-desmolase
`activity was lower by nearly 60% in testes from KTZ-
`treated men compared to a 29% decrease in 17a-hydrox-
`ylase activity and a 73% increase in 176-hydroxysteroid
`dehydrogenase activity. Similarly, testicular concentra-
`tions of the postdesmolase products (T, A, and DHEA)
`were all decreased, whereas the predesmolase precursors
`(PG, 17-PG, and 17—P) were increased. Whereas the
`pattern of precursor accumulation and product deficit
`was clearly evident in the testicular steroid concentra-
`tions, the steroid precursor patterns in serum were less
`
`clear-cut, despite the effective concurrent suppression of
`adrenal steroidogenesis. These results highlight the dif-
`ficulty in making inferences about steroidogenic enzyme
`activities from concentrations of circulating steroids that
`have dual glandular origins and differing MCRs. The
`direct confirmation of KTZ inhibition of the 17,20-des-
`molase step in vivo effectively minimizes the importance
`of other potential KTZ—in_duced effects on circulating
`steroid-binding proteins or metabolic disposition of ste-
`roids.
`This predominant site of KTZ inhibition may explain
`the greater sensitivity of testicular compared with adre-
`nal steroidogenesis to KTZ (2, 3). The basal secretion of
`T is profoundly reduced by doses of KTZ that have no
`effect on basal cortisol secretion (2, 3). Theoretically,
`pure inhibition of the 17,20-desmolase step would inter-
`fere only with the production of C-19 steroids,
`thus
`altering secretion of sex steroids (C-19 androgens and
`estrogens), but not adrenal (C-21) glucocorticoids and
`mineralocorticoids. This is most closely exemplified by
`congenital 17,20-desmolase deficiency, which is associ-
`ated with genital ambiguity due to abnormal C-19 steroid
`production whereas adrenal function is normal (13).
`
`WCK1038
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`WCK1038
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`

`
`KTZ AND HUMAN TESTIS
`
`
`
`A5—STEROIDSng/qtestis)
`
`450
`400
`350
`300
`250
`200
`I50
`I00
`
`30
`20
`IO
`
`Pregnenolone
`
`l7u—OH-Pregnenolone
`
`450
`400
`350
`300
`250
`200
`I50
`I00
`
`30
`20 ‘
`I0
`
`300
`250
`200
`I50
`100
`so
`4o
`
`so
`2o
`
`'0
`
`Control Patients (n =5)
`
`KTZ Treated Patients (n=3)
`mean i SEM
`
`{mu
`
`T
`
`.50
`40
`
`so
`20
`
`'0
`
`Androstenedione
`
`Testosterone
`
`t\\
`ITB-HSD
`
`_
`
`300
`2 5 O
`200
`I50
`I 00
`so
`40
`
`30
`20
`
`'0
`
`|7u-OH- Progesterone
`t
`
`_m__.
`
`unoo
`I000
`900
`e00
`700
`600
`500
`400
`300
`200
`I00
`
`I|OO
`I000
`900
`800
`700
`600
`500
`400
`300
`200
`I00
`
`I7a—Hydroxy|nse
`
`I7, 20-Desmolose
`
`_
`'5'’C.
`U‘
`E\
`
`EE\T
`
`:ED
`
`.
`
`>-
`t:
`ZI-o
`
`<L
`
`u2)
`
`-N2L
`
`UI
`C‘
`<1
`
`
`
`
`
`A4-STEROIDS(Hg/gtestis)
`
`300
`2 50
`200
`I50
`|OO
`50
`4o
`
`30
`20
`
`'0
`
`P'°9€5'e'0“€
`
`FIG. 2. Comparison between mean (iSEM) A‘ and A5 intratesticular steroid concentrations (nanograms per g testis) and A‘ enzyme activities
`(picomoles per min/mg protein) in three patients who received KTZ and in five control patients. Note that enzyme activities are aligned to reflect
`l
`their appropriate relationship to their respective substrates and products. See Materials and Methods for details.
`
`The possibility of a second site of inhibition of KTZ
`is difficult to exclude from the data presented; Indeed,
`the general effects of KTZ on cytochrome P-450 enzymes
`(4) and their involvement in multiple steps in the ste-
`roidogenic pathways (13, 14)
`indicate that additional
`sites of action might be expected, albeit at higher doses
`reflecting the relative selectivity of KTZ on the 17,20-
`desmolase step. The lesser but significant inhibition of
`17oz-hydroxylase is consistent with a second site of action
`for KTZ, although recent evidence (15) confirmed the
`earlier hypothesis of Nakajin et al. (16) of the coexistence
`of 17ouhydroxylase and 17,20—desmolase in a single mul-
`tifunctional enzyme complex in pig testis. Thus, inhibi-
`tion of both enzymes in the human testis by KTZ might
`be attributable to a single site of action of KTZ on the
`multienzyme complex. The ability of KTZ to inhibit
`other adrenal cytochrome P—450 enzymes in vitro (4) and
`ACTH-stimulated adrenal reserve (3) in vivo suggests
`that at a sufficiently high dose, KTZ can inhibit other
`
`including the cholesterol
`cytochrome P—450 enzymes,
`side—chain cleavage (20,22—desmolase) and 11oz-hydrox-
`ylase enzymes.
`The pattern of enzyme inhibition demonstrated in this
`study is most directly applicable to the A4—pathWay, since
`the in vitro enzyme assays used A4-substrates. Neverthe-
`less, the consequences of 17,20—desmolase inhibition by
`KTZ were also clearly apparent in the accumulation in
`the testis of its A5—substrate, 17-PG, and in reduction of
`its product, DHEA, consistent with the suggestion that
`a single or very similar enzymes catalyze each of the
`equivalent steps in the A4— and A5-pathways (12-16). The
`lack of a uniform increase in steroid precursors may be
`due to secondary sites of action of KTZ, differences in
`the sensitivity of various enzymes for KTZ effects, or a
`combination of these effects with the removal of feedback
`inhibition of the enzymes. The failure of both A“- and
`A5-precursors to accumulate to an equivalent degree in
`serum compared to their accumulation in the testis may
`
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`
`1198
`
`RAJFER ET AL.
`
`JCE & M’ 1986
`Vol 63°No 5
`
`have several explanations. The small number of subjects
`as well as KTZ—related effects on steroid release, dispo-
`sition, or binding protein avidity are possible explana-
`tions. This does, however, illustrate the difficulties of
`making reliable inferences about the mechanism of ac-
`tion of drugs on steroidogenic enzymes from circulating
`steroid levels.
`
`Reservations about the interpretation of these studies
`need to be considered. Although the number of KTZ-
`treated subjects was small (11 = 3), the intensive sampling
`regimen considerably enhanced the power of these inves-
`tigations. Another question of validity arises in relation
`to the use of concurrent DEX therapy to effect adrenal
`suppression before and during KTZ treatment. This had
`the ethical advantage of protecting the subjects against
`hypersensitivity to the adrenal-blocking effects of KTZ.
`It also facilitated the interpretation of changes in circu-
`lating steroid levels, since in the presence of adrenal
`suppression, these could be attributed exclusively to go-
`nadal effects. It is unlikely that the effects of the dose of
`DEX used are important in the interpretation of changes
`in testicular steroid enzyme activities or steroid concen-
`trations. Although there. is evidence that excessive or
`pharmacological doses of glucocorticoids can inhibit tes-
`ticular function and steroidogenesis (17-20), it is unlikely
`to be important in the dose and time used in these
`studies. Furthermore, the comparison of peripheral ste-
`roid levels on the control day (DEX alone) with those on
`subsequent treatment days (KTZ plus DEX) indicated
`clear effects temporally related to KTZ treatment. It is,
`nevertheless, hard to entirely rule out small or very
`delayed effects of DEX.
`A final caveat is the possibility that some of the effects
`might be attributable in part to the effects of aging or
`chronic illness on testicular function. Since human tes-
`
`ticular tissue is only available from elderly men with
`prostate cancer undergoing therapeutic orchiectomy, this
`discussion must remain largely speculative. The principal
`effects of aging on Leydig cell function include loss of
`Leydig cell numbers, decrease in overall steroidogenic
`activity, and evidence of hypothalamic-pituitary com-
`pensation for impaired androgenic supply (21, 22). Nev-
`ertheless, there is no evidence that the actual steroido-
`
`genic mechanisms are qualitatively altered despite the
`quantitative changes associated with advancing age and/
`or chronic illness (13, 14). Thus, most likely, it is legiti-
`mate to extrapolate the effects in this study to the
`mechanism of KTZ-induced reductions in circulating
`testicular steroid levels in younger men (2, 6).
`In conclusion, this study demonstrates directly for the
`first time that KTZ inhibits testicular steroidogenesis
`primarily by inhibiting the activity of the cytochrome P-
`450-dependent 17,20—desmolase enzyme.
`
`Acknowledgments
`
`We thank Ms. Katherine Zozobrado for her technical assistance and
`J. Brasel, M.D., for measurement of the serum cortisol levels.
`
`References
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