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Biology of Reproduction 59, 248-254 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Imidazoles Suppress Rat Testosterone Secretion and Testicular Interstitial Fluid Formation In Vivo1

Michael L. Adams2,a, Edward R. Meyera, , and Theodore J. Ciceroa

a Department of Psychiatry, Washington University School of Medicine, St. Louis, Missouri 63110–1093


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of these studies was to examine the effects of imidazoles on testosterone secretion and testicular interstitial fluid (TIF) formation through measurement of serum LH, serum testosterone, TIF testosterone, and TIF volumes. Imidazole, 1-methylimidazole, 4-methylimidazole (4-MI), and ketoconazole, an oral imidazole antifungal agent, caused dose-dependent decreases in testosterone secretion and TIF formation. Imidazole, 2-methylimidazole, and 4-MI decreased LH secretion. 4-MI decreased testosterone secretion 1–6 h after injection, increased testosterone at 8–16 h, decreased LH secretion at 4 h, decreased TIF volumes at 1–8 h, and slightly increased TIF volumes at 24 h. 4-MI blocked the stimulatory effects of hCG on testosterone secretion and prevented an expected increase in LH secretion after the 4-MI-induced decrease in testosterone secretion. 4-MI also reversed the effects of three other stimulants of testosterone secretion that presumably act through three different testicular regulatory systems: N-methyl-D,L-aspartate, an excitatory amino acid; NG-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor; and naltrexone, an opioid antagonist. These results support the hypothesis that imidazoles inhibit testicular function and male reproductive function through inhibition of testosterone secretion, TIF formation, and LH secretion regulatory systems.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several drugs, illnesses, and injuries disrupt male fertility through direct or indirect suppression of testicular function [1, 2], but further study is required to characterize the mechanisms and sites of action involved. Among testicular suppressants we have found that alcohol [3–6], morphine [7, 8], and nitric oxide [8–11] inhibit testosterone secretion and testicular interstitial fluid (TIF) formation, two fundamental testicular regulatory functions in vivo in rats [12, 13]. These testicular effects are expressed as profound decreases in serum and TIF testosterone concentrations and TIF volumes.

Imidazole antifungals such as ketoconazole are clinically effective because they suppress fungal ergosterol production through a biosynthetic pathway analogous to the gonadal and adrenal steroidogenic pathways in humans [14]. In fact, ketoconazole and related antifungal imidazoles inhibit testosterone secretion [15–18] and corticosteroid secretion [19, 20]. These antisteroidogenic effects are related to the inhibition of cytochrome P450 enzyme systems [14, 21], which imidazoles inhibit through binding to heme iron [22–24]. Both imidazoles [25–28] and nitric oxide [29] inhibit nitric oxide synthase, a hemoprotein enzyme. This evidence suggests that imidazoles may affect testicular steroidogenesis and TIF formation like another testicular suppressant, nitric oxide [8, 10, 11].

Testicular steroidogenesis has been measured by serum and TIF testosterone concentrations, which parallel serum testosterone concentrations [4]. TIF formation, like testosterone secretion, is a major regulatory aspect of testicular function and is affected by testicular suppressants such as alcohol, morphine, and isosorbide dinitrate [5–8, 10–12]. TIF testosterone levels reflect testosterone secretion inside the testes, where it exerts paracrine and autocrine effects on steroidogenesis and spermatogenesis [30, 31]. TIF volumes have been measured to assess TIF formation and vascular perfusion inside the testis [13, 32, 33] that control access of important substances to testicular cells and structures [32, 34–36]. Disruption of TIF paracrine control systems is thought to be a major cause of idiopathic infertility in men [30], so these TIF measures reflect important aspects of gonadal function. Serum LH also has been measured because it is a major physiological stimulus of testosterone secretion and a major factor in the regulation of TIF formation [30–32].

In the present studies we examined the effects of low molecular weight imidazole and methylimidazoles, as well as the higher molecular weight imidazole antifungal medication used in humans, ketoconazole, on LH and testosterone secretion and on TIF formation in rats in vivo. The structures of the imidazoles used in these studies are shown in Figure 1. A prototypical imidazole, 4-methylimidazole (4-MI), was then tested alone and in combination with stimulants of testosterone secretion to start examining the mechanisms involved in 4-MI-induced suppression of testicular function. The effects of small imidazoles and methylimidazoles on the hypothalamic-pituitary-gonadal axis of rodents and humans have not been examined previously. The antisteroidogenic effects of the larger imidazole, ketoconazole, have not been compared to those of the small imidazole and methylimidazole. In addition, the effects of imidazoles on TIF formation have not been studied previously.



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  		FIG. 1. Chemical structures of the imidazoles used in these studies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials and Animals

Adult (60 days old) or adolescent (42 days old) male Sprague-Dawley-derived rats were used in all experiments. They were originally derived from Harlan Sprague-Dawley, Inc. (Indianapolis, IN) rats. Imidazole, 1-methyl imidazole, 2-methyl imidazole, 4-MI, NG-nitro-L-arginine methyl ester (NAME), naltrexone, N-methyl-D,L-aspartate (NMA), and hCG were obtained from Sigma Chemical Company (St. Louis, MO). Ketoconazole 200-mg tablets were obtained through a pharmacy at this institution. The testosterone antiserum (R-15P) was obtained from Radioimmunoassay Systems Laboratories, Inc. (Carson, CA) and had approximately 18% cross-reactivity with 5-alpha-dihydrotestosterone and less than 1% cross-reactivity with all other steroids. The National Hormone and Pituitary Program (Dr. Salvatore Raiti, NIDDK, Bethesda, MD) supplied the rat double-antibody RIA kits used for all serum LH measurements.

Treatments

Increasing doses (10–300 mg/kg or 122–4407 µmol/kg) of imidazole, 1-methylimidazole, 2-methylimidazole, and 4-MI or saline were injected in adult rats; samples of serum and TIF were collected 2 h later. Since 4-MI was the most potent agent among imidazole and the methylimidazoles in suppressing testosterone secretion and TIF formation at this time point, 50 mg/kg (609 µmol/kg) 4-MI or saline was injected in adult rats; then serum and TIF samples were collected 0.5, 1, 2, 4, 6, 8, 16, and 24 h after injection. All injections of imidazole and the methylimidazoles were subcutaneous, and 10 rats per group were injected as were saline control groups at each time point.

In addition, increasing doses (10–300 mg/kg or 57–565 µmol/kg) of ketoconazole (Fig. 1) were given by oral gavage to adult rats, and samples were collected 4 h later. The ketoconazole tablets (200 mg) were allowed to disintegrate in water; then the ketoconazole tablet suspensions were constantly stirred and given by oral gavage in 10 ml/kg volumes. Oral gavage with water was used as a control, since nonactive tablet ingredients like lactose and cellulose given by oral gavage did not alter testosterone secretion or TIF volumes in preliminary studies.

4-MI was injected in combination with injections of the testicular stimulants hCG (20 IU/kg), NMA (70 mg/kg), NAME (100 mg/kg), or naltrexone (5 mg/kg) with saline controls. NAME, naltrexone, and hCG were injected 2 h before serum and TIF collection; NMA was injected 1 h before sample collection at the times of peak effects on testosterone secretion. In all of these experiments, 4-MI was injected 4 h before sample collection at the time of its peak effects on testicular function; 2 h before NAME, naltrexone, and hCG; and 3 h before NMA. These doses and time intervals were chosen to exert maximal effects on testosterone secretion. Adult rats, 60 days old, were used in all except the NMA experiments. Younger, adolescent rats, 42 days old, were used in the NMA experiments because adult rats are not as sensitive as younger rats to the reproductive endocrine effects of NMA [37]. All injections were subcutaneous, and 10 rats per group were injected along with saline controls.

TIF Collection

TIF was collected as previously described [3, 4]. Immediately after serum collection from trunk blood, both testes were removed, small holes were cut in the caudal end of each testis, and each testis was then suspended in a tube to allow TIF drainage overnight. TIF volumes were then measured with a pipette and TIF testosterone was measured by RIA.

Testosterone and LH RIAs

Testosterone was extracted as previously described [7, 38] from serum with ethanol. The dried aliquots were reconstituted in RIA buffer and then assayed by RIA as previously described [3, 4]. TIF aliquots were diluted with RIA buffer and then subjected to testosterone RIA without extraction. LH was measured by RIA in unextracted serum aliquots as previously described, utilizing instructions and materials from the National Hormone and Pituitary Program from the NIDDK [3]. NIDDK rat LH reference protein-3 was used as a standard. The assay had a sensitivity of 0.02–0.03 ng/tube or 0.1–0.15 ng/ml serum. Intra- and interassay variations were less than 5% in all experiments [3]. Imidazoles did not cross-react with the testosterone or LH RIAs.

Statistics

ANOVA followed by post hoc analysis with Fisher's protected least-significant difference tests was used to determine significant differences between groups as described previously [4]. The level of significance was set at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Imidazole Dose Responses

Imidazole, 1-methylimidazole, 4-MI, and ketoconazole had decreased serum testosterone in a dose-dependent manner at 2 h after treatment (Fig. 2A). Doses are expressed in micromoles per kilogram of body weight to compare potencies of the various imidazoles on a molar basis. Intraperitoneally administered 4-MI can cause substantial toxicity at doses above 150 mg/kg (1827 µmol/kg) in mice. Toxic reactions include convulsions, hyperactivity, tremor, opisthotonos (tetanic spasm in which the spine is fixed in an extended position), and Straub tail (tail held off the floor pointing upward) [39]. Our studies were done with 50–100 mg/kg (609–1218 µmol/kg) subcutaneous doses of 4-MI that did not cause these signs of toxicity. Testosterone concentrations after control oral water gavage in the ketoconazole experiments were much lower than after control saline injections in the other experiments (see legends for Fig. 2, A-C). Although the cause of this is unknown, it may be related to the effects of injection versus gavage, the interval between treatment and sample collection, and/or the time of day samples were collected (the rats were gavaged instead of being injected at the same time of day and the samples were collected 4 h after ketoconazole as compared to 2 h in the other experiments).



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  		FIG. 2. Dose-response curves of the effects of imidazoles on serum testosterone (A), TIF testosterone (B), TIF volumes (C), and serum LH (D). Doses ranging from 10 to 300 mg/kg are expressed in micromoles per kilogram. A) Concentrations are shown as percentages of saline control levels: 6.8 ± 0.8 ng/ml for imidazole, 2-methylimidazole, and 4-MI; 4.8 ± 0.8 ng/ml for 1-methylimidazole; and 3.4 ± 0.4 ng/ml for ketoconazole. B) Concentrations are shown as percentages of saline control levels: 318 ± 37 ng/ml for imidazole, 2-methylimidazole, and 4-MI; 264 ± 46 ng/ml for 1-methylimidazole; and 131 ± 20 ng/ml for ketoconazole. C) Volumes are shown as percentages of saline control levels: 111 ± 6 µl/testis for imidazole, 2-methylimidazole, and 4-MI; 120 ± 4 µl/testis for 1-methylimidazole; and 116 ± 4 µl/testis for ketoconazole. D) Concentrations are shown as percentages of saline control levels: 0.39 ± 0.04 ng/ml for imidazole, 2-methylimidazole, and 4-MI; 0.30 ± 0.04 ng/ml for 1-methylimidazole; and 0.21 ± 0.04 ng/ml for ketoconazole. Values are means ± SEM of 10 animals per group. *Significantly (p < 0.05, ANOVA) different from the respective saline control groups.

All imidazoles dose-dependently decreased TIF testosterone concentrations (Fig. 2B), and all imidazoles except 2-methylimidazole decreased TIF volumes by 2 h after treatment (Fig. 2C). We found that the effects of 4-MI in suppressing testosterone secretion and TIF volumes were very consistent among all experiments.

4-MI and 2-methylimidazole decreased LH secretion by the 2-h time point (Fig. 2D). Ketoconazole increased serum LH concentrations at 4 h after oral gavage; but 4-MI, imidazole, and 2-methylimidazole decreased LH by 2 h after injection.

4-MI Time-Response

4-MI at 50 mg/kg significantly decreased serum testosterone levels by 2–6 h after injection, TIF testosterone levels at 2–4 h after injection, and TIF volumes at 1–8 h after injection (Fig. 3, A–C). Following these decreases, significant "rebound" increases in serum testosterone and TIF testosterone occurred 8–16 h after injection, and a slight increase in TIF volumes occurred 24 h after injection.



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  		FIG. 3. Time courses of the effects of saline or 4-MI (50 mg/kg; 609 µmol/kg) on serum testosterone (A), TIF testosterone (B), and TIF volume (C). Values are the means ± SEM of 10 animals per time point. *Significantly (p < 0.05, ANOVA) different from the saline control values.

4-MI did not significantly alter serum LH levels at any tested time point except 4 h after injection in this experiment (Fig. 4); this effect was similar to its suppression of LH secretion in the 4-MI dose-response experiment (Fig. 2D) at 2 h after injection.



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  		FIG. 4. Time courses of the effects of saline or 4-MI (50 mg/kg; 609 µmol/kg) on serum LH. Values are the means ± SEM of 10 animals per time point. *Significantly (p < 0.05, ANOVA) different from the saline control values.

Localization of 4-MI Effects

To more directly examine whether the testicular effects of imidazoles were dependent on changes in LH, 4-MI at 50 mg/kg was injected in combination with hCG, a gonadotropin with testicular-stimulatory actions like those of LH. Human CG at 20 IU/kg stimulated testosterone secretion without affecting TIF volumes (Fig. 5A). 4-MI completely blocked hCG-induced increases in testosterone secretion. These data indicate that the suppression of testosterone secretion by 4-MI is due to a direct effect on the testes rather than to other indirect effects associated with other changes in LH.



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  		FIG. 5. Effects of saline or 4-MI (50 mg/kg; 609 µmol/kg) pretreatment followed by saline or A) hCG (20 IU/kg), B) NMA (70 mg/kg; using 42-day-old rats), C) NAME (100 mg/kg), or D) naltrexone (5 mg/kg) treatment on serum LH (B-D only), serum testosterone (Serum T), TIF testosterone (TIF T), and TIF volume (TIF Vol) levels. Values are means ± SEM of 10 animals per group expressed as percentages of the control (saline pretreatment + saline treatment) values. A) Serum T, 5.3 ± 0.8 ng/ml; TIF T, 228 ± 45 ng/ml; and TIF Vol, 124 ± 4 µl/testis. B) LH, 0.25 ± 0.03; serum T, 1.0 ± 0.1 ng/ml; TIF T, 24 ± 4 ng/ml; and TIF Vol, 62 ± 3 µl/testis. C) LH, 0.34 ± 0.03; serum T, 6.1 ± 0.5 ng/ml; TIF T, 263 ± 28 ng/ml; and TIF Vol, 127 ± 4 µl/testis. D) LH, 0.38 ± 0.05; serum T, 5.8 ± 0.6 ng/ml; TIF T, 254 ± 42 ng/ml; and TIF Vol, 131 ± 5 µl/testis. *Significantly (p < 0.05, ANOVA) different from the control (saline injections only) values.

4-MI and NMA

4-MI was injected in combination with NMA in 42-day-old rats in order to examine its effect on excitatory amino acid-induced testicular stimulation. NMA stimulated LH and testosterone secretion and decreased TIF volumes (Fig. 5B). 4-MI inhibited NMA-induced stimulation of testosterone secretion and potentiated the effect of NMA on TIF formation, but it did not significantly affect NMA-stimulated LH secretion. These results indicate that 4-MI blocks the testosterone secretory effect of excitatory amino acids.

4-MI and NAME

The nitric oxide synthase inhibitor NAME, as expected [10], increased testosterone secretion without altering TIF formation or LH secretion (Fig. 5C). 4-MI suppressed LH secretion in this experiment (in contrast to others; see Fig. 5, B and D) and completely reversed NAME-induced stimulation of testosterone secretion. In addition, NAME appeared to potentiate a 4-MI-induced suppression of TIF volumes and reversed the suppressant effect of 4-MI on LH levels.

4-MI and Naltrexone

The opioid antagonist, naltrexone, stimulated testosterone secretion without affecting TIF formation (Fig. 5D) as expected from preliminary results. 4-MI completely blocked the effects of naltrexone on both LH and testosterone secretion.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
These results confirm an adverse effect of imidazoles on male reproductive function [15–18] and support the hypothesis that imidazoles inhibit male fertility through suppression of two important aspects of testicular function: testosterone secretion and TIF formation [12, 13, 32–36]. The results also suggest that imidazoles can disrupt pituitary LH secretion regulatory mechanisms. Imidazole, 1-methylimidazole, 2-methylimidazole, 4-MI, and ketoconazole (Fig. 1) inhibited testosterone secretion, and all imidazoles except 2-methylimidazole inhibited TIF formation (Fig. 2, A-C). 4-MI, 2-methylimidazole, and imidazole decreased LH secretion under some conditions (Figs. 2D, 4, and 5C). 4-MI was used as a prototypical imidazole in subsequent experiments and caused profound decreases in serum and TIF testosterone (Fig. 2D) that were qualitatively similar to the testicular effects of other testicular suppressants such as nitric oxide [8, 10, 11, 38], alcohol [3–5, 38], and morphine [7, 8].

Although 4-MI caused profound decreases in testosterone secretion 2 to 6 h after s.c. injection, those decreases were followed by substantial rebound increases in testosterone secretion 8–16 h after injection, with levels returning to normal by 24 h (Fig. 2D). These rebound increases are similar to those seen 8 h after s.c. morphine injections in rats [7], but we have found that another testicular suppressant, alcohol, does not result in any rebound increases in testosterone secretion after acute i.p. injection (unpublished results). In addition, no significant increases in serum LH were found before or during these rebound increases in testosterone secretion, indicating that the increases in testicular function were not due to prior increases in gonadotropin secretion. A compensatory increase in LH secretion would be expected to follow the large decrease in testosterone secretion, but no such increase occurred. These results also suggest that 4-MI suppresses negative feedback mechanisms in the pituitary, suppressing regulatory changes in LH secretion. In relation to the small imidazoles like 4-MI, it is not known whether the rebound effect is related to the elimination time of the imidazole and subsequent overaction of processes counteracting the initial testicular suppressant effect, or whether counteracting processes are independent of 4-MI levels at its sites of action. A slight rebound increase in TIF formation also occurred at 24 h after injection, and both rebound effects were independent of increases in LH secretion.

The testicular-suppressant actions of 4-MI were further characterized by experiments in which 4-MI blocked the stimulation of testosterone secretion by hCG, NMA, NAME, and naltrexone (Fig. 5, A-D). In these experiments, 4-MI suppressed testosterone secretion and TIF formation like other testicular suppressants that we have studied previously: nitric oxide [11], alcohol [4], and morphine [7]. At 2 or 4 h after injection, imidazole, 2-methylimidazole, and 4-MI had decreased LH (Figs. 2D and 4). The slight suppressant effects of 4-MI on LH secretion, and the suppressive effects of 4-MI on hCG-stimulated testosterone secretion (similar to those of nitric oxide, alcohol, and morphine), indicate that 4-MI-induced decreases in testosterone secretion were not due completely to prior inhibition of gonadotropin (LH) secretion. NMA is also known to stimulate LHRH/LH secretion [40], so the blockade of NMA-stimulated testosterone secretion by 4-MI without any effect on LH secretion (Fig. 5B) also suggests that prior changes in LH secretion are not involved. 4-MI blocked the stimulatory effect of a nitric oxide synthase inhibitor (NAME) on testosterone secretion (Fig. 5C), like nitric oxide [11], morphine [8], or alcohol [38]; and 4-MI also blocked both testosterone and LH secretion stimulated by the opioid antagonist naltrexone (Fig. 5D), like the opioid agonist morphine [7]. Together, the results suggest a major suppressive effect of imidazoles on testicular testosterone secretion whether it is basal or is stimulated by several different mechanisms: endogenous gonadotropin (LH), exogenous gonadotropin treatment (hCG), excitatory amino acid treatment (NMA), decreases in endogenous nitric oxide (NAME), or antagonism of endogenous opioids (naltrexone). However, beyond these very preliminary results, it is not known whether 4-MI interacts directly with these gonadotropin, excitatory amino acid, nitric oxide, or opioid testicular regulatory systems.

Although the smaller imidazoles suppress pituitary LH secretion mechanisms (Figs. 2D, 4, and 5, C and D), they also exert LH-independent effects on basal and stimulated testosterone secretion through diverse mechanisms, indicating a very important site of action in the testes. The exact mechanisms involved in the suppressive effects of imidazoles on testicular steroidogenesis are unknown; direct inhibitory effects on testicular steroidogenic enzymes are possible. Antifungal imidazoles like ketoconazole inhibit heme-containing, cytochrome P450-related steroidogenic enzyme systems [14, 21–24] and exert clinically undesirable endocrine side effects, inhibiting testosterone [15–18] and corticosteroid [19, 20] secretion. In relation to these effects, nitric oxide synthase is a heme-containing cytochrome P450-like enzyme [41, 42] that is inhibited by imidazoles [25–28] and nitric oxide [29], and nitric oxide regulates testosterone secretion [8, 10–12, 38]. It is also interesting to note that in the present studies, 2-methylimidazole was the least effective or least potent in suppressing testicular function (Figs. 1 and 2, A–C) in comparison to the other low molecular weight imidazoles and ketoconazole. 2-Methylimidazole was also less active than 1-methylimidazole in binding to cytochrome P450 and its inhibition of microsomal enzymes [23]. This suggests a possible correlation between the testicular-suppressant activities of imidazoles and their binding-affinity steroidogenic enzyme systems.

However, imidazoles may also exert important indirect actions on testicular steroidogenesis through effects on testicular blood flow. Other studies have suggested that testosterone secretion is regulated by testicular blood flow [43, 44]. Vasodilators without obvious effects on nitric oxide synthase systems inhibited testosterone secretion by themselves and blocked testosterone secretion that was stimulated by a vasoconstrictive nitric oxide synthase inhibitor [11]. This suggested that the vasodilating properties of the testicular-suppressant agents might be important factors in suppressing testicular steroidogenesis. Some evidence suggests that imidazoles may also have vasorelaxant properties that could contribute to their testicular-suppressant effects. Imidazoles were found to decrease blood pressure in mice [39] and to act through cytochrome P450-related mechanisms to inhibit calcium influx into cells [45, 46] and relax at least one type of vascular smooth muscle [47]. In addition, in the present studies the testosterone secretion stimulant effect of the vasoconstrictor NAME [8–12, 38] was completely blocked by pretreatment with 4-MI (Fig. 5C). Thus, at least part of the effect of imidazoles on testosterone secretion may be explained by indirect vascular actions.

Possible vascular effects of imidazoles may also be involved in TIF formation effects. Altered blood flow appears to be related to TIF formation [32, 48–50], but it is not clear how the possible effects of imidazoles on the testicular vasculature and the larger vascular beds of other organs interact to alter TIF formation [3]. As discussed above, imidazoles may exert important vascular effects [39, 45–47] that could alter TIF volumes because TIF volumes reflect testis vascular perfusion and TIF formation [13, 32, 33].

The rationale for the use of TIF measures is based on our own [12] studies and those of others [30–36]. The imidazoles were found to cause significant decreases in TIF volumes, which have been validated as a measure of TIF formation. Decreases in TIF volumes are indicators of decreases in testicular vasopermeability and testicular blood flow and of limited access of blood cells, hormones, and nutrients to their sites of action within the testes. TIF formation plays a major role in male fertility because problems with testicular paracrine control systems, based on TIF as a medium of transport, may account for a large portion of idiopathic infertility in men [30]. Just as the TIF-suppressant effects of ethanol, morphine, and nitric oxide suggest important testicular effects of those agents, the TIF-suppressant effects of imidazoles suggest another mechanism for potential adverse effects of imidazoles on male reproductive function and fertility, indicating that the TIF volume parameter is not a superfluous parameter in studies focusing on the testicular effects of imidazoles. TIF testosterone levels directly reflect testosterone secretion because testosterone is secreted into TIF before it is transported to the blood stream [13]. Both TIF yield and TIF output are a result of vasopermeability—movement of fluid across blood vessel walls—due to blood pressure in the blood vessels. Our own studies and those of others have shown that testosterone is not metabolized overnight during the TIF collection period. If anything, postmortem TIF testosterone measures seem to overestimate actual TIF testosterone concentrations at the time of testes collection [4].

Our results indicate that the smaller methylimidazoles and imidazole used in these studies exert antisteroidogenic effects in the testes, like the larger antifungal imidazoles such as ketoconazole that suppress steroidogenesis as a side effect of treatment of fungal infections. The imidazole antifungals inhibit human testosterone secretion [15–18] and corticosteroid secretion [19, 20]. This is such a prominent effect that ketoconazole has been studied for use as a testosterone-suppression test agent to help determine whether athletes have used androgenic steroids [18]. These studies indicate that the imidazole moiety of ketoconazole and possibly other imidazole-containing drugs may be responsible for potential suppressive effects on testosterone secretion, TIF formation, and male fertility in vivo.

An important consideration in these in vivo studies in male rats is whether the pharmacological doses used here in rats relate to doses used in humans. That problem was one reason for the inclusion of ketoconazole, a drug used in humans. It is known that ketoconazole at usual doses in clinical practice can significantly decrease testosterone levels [15, 17, 18]. It can be difficult to extrapolate effective human doses to rats and effective doses in rats to humans; but in this case, on a molar basis, ketoconazole was more potent than the other imidazoles in decreasing testosterone secretion in rats. Since ketoconazole has a higher molecular weight than the other imidazoles tested, that means that on a weight basis, smaller doses of the small imidazoles were more effective in rats than ketoconazole. The results apply to human use because, in rats, the effects of ketoconazole and the other imidazoles occur at similar doses.

In summary, our results indicate that imidazoles suppress the two major regulatory aspects of testicular function (testosterone secretion and TIF formation) and can suppress LH secretion regulatory systems in the pituitary in rats, supporting the hypothesis that imidazoles can suppress male reproductive function and fertility.


   ACKNOWLEDGMENTS
 
We thank Myrtle Barrett, Jingling Chen, and William D. Johnson for their technical assistance.


   FOOTNOTES
 
1 This work was supported by grants AA-09222, AA-07144, and AA-07466 from the National Institute on Alcohol Abuse and Alcoholism and DA-03833 from the National Institute on Drug Abuse. T.J.C. is the recipient of a Research Scientist Award (DA-00095) from the National Institute on Drug Abuse. Back

2 Correspondence: Michael L. Adams, Department of Psychiatry, Box 8134, Washington University School of Medicine, 4940 Children's Place, St. Louis, MO 63110–1093. FAX: (314) 747–2163; mike-a{at}dcm.wustl.edu Back

Accepted: March 11, 1998.

Received: September 9, 1996.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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