Effect of Diazepam on Serum Testosterone and the Ventral Prostate Gland in Male Rats
Syst Biol Reprod Med Downloaded from informahealthcare.com by University of Calgary on 09/06/13 For personal use only.
P. S. COOK,M. NOTELOVITZ, P. S. KALRA,and S. P. KALRA A study was initiated to investigate the effect of diazepam on serum testosterone and testosterone-sensitive tissues in male rats. Diazepam was administered in a dose of 50 m a g body weight daily for 10 days. When compared with an equally matched group of control male rats, this dose of diazepam was associated with a significant reduction in both the weight of the ventral prostate of treated rats and the serum testosterone levels. These changes were not associated with alterations in serum LH and FSH and the hypothalamic luteinizing hormonereleasing hormone content. Since diazepam does not interfere with the radioimmunoassay of testosterone, it is suggested that the above observations were biologically induced via direct suppression of the interstitial cells of the testis. Key Words: Diazepam (Valium);Testosterone; Prostate gland.
INTRODUCTION
A study was initiated to investigate the effect of diazepam (Valium) on serum testosterone (T) in rats, following the clinical observation that its administration to surgically castrated young women was associated with a significant reduction in plasma T values (Notelovitz, unpublished data, 1977). The objectives of the present investigation were threefold: (a) to determine whether this observed effect was due to interference of the drug in the radioimmunoassay (RIA)of T or its reduced product, 5adihydrotestosterone (DHT);(b) to study the effect of diazepam on the testis and ventral prostate in vivo; and (c) to determine if the effect of diazepam on T production (if any) was mediated via pituitary gonadotropin secretion or hypothalamic luteinizing hormone-releasing hormone (LH-RH). MATERIALS AND METHODS Cross-reactivity of diazepam in radioimmunoassay. T and DHT were analyzed by RIA in accordance with the procedure of Coyotupa et al. [2]. The two androgens were isolated on Sephadex LH-20 columns following extraction of serum with diethyl ether. Antiserum to testosterone-3-oxine conjugated to human serum albumin was obtained from Dr. G. E. Abraham. The cross-reactivity of diazepam in the T and DHT RIA was examined in three separate experiments.
Received February 28, 1979. From the Department of Obstetrics and Gynecology, University of Florida College of Medicine, Gainesville, Florida. Address reprint requests to: M. Notelovitz, Department of Obstetrics and Gynecology, Box J-294 JHMHC, University of Florida College of Medicine, Gainesville, Florida 32610.
31 0 Elsevier North Holland, Inc., ARCHIVES OF ANDROLOGY. 3, 31-35 (1979)
0148-5016/79/01003 1-05$02.25
Syst Biol Reprod Med Downloaded from informahealthcare.com by University of Calgary on 09/06/13 For personal use only.
32
P. S. Cook et el.
1. Varying doses of pure diazepam were dissolved in buffer and processed in the RIA as unknown samples without chromatography. Binding of the 3H-T to antiserum was higher at all dose levels of diazepam compared with the binding in “zero” tubes (tubes containing no T or diazepam). This indicated the presence of “negative” blanks, possibly leading to an underestimation of unknowns when diazepam was present in the assay tubes. 2. Diazepam (0.5 mg) was added to a set of assay tubes containing known amounts of T. Binding was again higher in the tubes containing diazepam, indicating the possibility of an underestimation of unknowns. 3. Diazepam (0.5 mg/ml) was added to human blood samples containing known amounts of T and DHT. Replicate samples (with and without added diazepam) were processed as unknowns for the determination of T and DHT following extraction with diethyl ether and isolation by column chromatography (Sephadex LH-20).
In vivo assessment. Twenty-two Charles River CD adult male rats were maintained (two in a cage) in a room with a 14-hr light-10-hr dark cycle. The animals were supplied with Purina chow and water ad libitum. They were weighed at the start of the study and then every other day until the completion of the study. The rats were divided into four groups: Group I rats were treated daily for 10 days with 50 mg diazepamkg body weight. The diazepam was administered intraperitoneally as a 0. l-ml suspension (12.5 mg/ml; 40% propylene glyco1:absolute alcohol mixture, 9:l) per 25 g body weight. Group I1 rats were treated with diazepam as in group I, but were allowed an ll-day recovery period on completion of the treatment cycle. Group 111 rats served as a control for group I and were treated daily with a comparable volume of the vehicle for 10 days. Group IV served as control for group I1 and were treated with vehicle only for 10 days, after which they were allowed an 11-day recovery period. At the end of the 10-day treatment period, the rats in groups I and I11 were sacrificed by decapitation; groups I1 and IV were sacrificed in a similar fashion 11 days later. Trunk blood was centrifuged for 20 min at 3000 rpm and the serum was stored at -20°C until assayed for androgens and gonadotropins. The testes and the ventral prostate were dissected out and weighed. The brain from each animal was removed from the cranium and the neural tissue encompassing the medial basal hypothalamus was dissected out and frozen in 0.1 N HCl for LH-RH analysis. The RIA for estimation of T , DHT, LH, FSH, and LH-RH have been described [4]. For statistical analysis, Student’s t test was used to compare group means. RESULTS Cross-Reactivity of Diazepam in Radioimmunoassay (Table 1)
There was no significant difference in the T levels of samples with and without diazepam. DHT levels were significantly lower in samples containing diazepam. Thus, diazepam appears either to interfere with binding to antiserum or to prevent the adequate separation of the free-labeled antigen by the dextran-coated charcoal technique. However, when samples were extracted with diethyl ether and T and DHT were purified by column chromatography, the estimations of T were not altered by the presence of diazepam. Diazepam appears to elute along with DHT in our system and, if present in sufficient quantities, may result in an underestimation of DHT values. Body Weight
At the end of 10 days of treatment, there was no significant change in the body weights of both the control (316.2 2 14.8 g) and the diazepam-treated rats (310.6 k 9.9 g) when
Diazepam on Testosterone and Ventral Prostate
33
TABLE 1 Cross-Reactivity of Diazepam (V) in the Radioimmunoassay of Testosterone and Dihydrotestosterone
DHT
TESTOSTERONE (NG/ML)
SUBJECTS
26.9 t 2.9" (3)* 567.2 t 28.3 (4)
Females Males
Syst Biol Reprod Med Downloaded from informahealthcare.com by University of Calgary on 09/06/13 For personal use only.
' Mean
No V
?
0.5
MG V / M L
25.7 t 1.8 (5) 638.1 ? 20.8 (3)
No V 10.1 ? 0.6 (3) 19.7 2 1.7 (4)
(NG/ML)
0.5
MG
V/ML
3.3 t 0.3 (5)r 5.9 t 0.7 (5)r
SEM.
* Number of experiments. e p =
Syst Biol Reprod Med Downloaded from informahealthcare.com by University of Calgary on 09/06/13 For personal use only.
P. S. COOK,M. NOTELOVITZ, P. S. KALRA,and S. P. KALRA A study was initiated to investigate the effect of diazepam on serum testosterone and testosterone-sensitive tissues in male rats. Diazepam was administered in a dose of 50 m a g body weight daily for 10 days. When compared with an equally matched group of control male rats, this dose of diazepam was associated with a significant reduction in both the weight of the ventral prostate of treated rats and the serum testosterone levels. These changes were not associated with alterations in serum LH and FSH and the hypothalamic luteinizing hormonereleasing hormone content. Since diazepam does not interfere with the radioimmunoassay of testosterone, it is suggested that the above observations were biologically induced via direct suppression of the interstitial cells of the testis. Key Words: Diazepam (Valium);Testosterone; Prostate gland.
INTRODUCTION
A study was initiated to investigate the effect of diazepam (Valium) on serum testosterone (T) in rats, following the clinical observation that its administration to surgically castrated young women was associated with a significant reduction in plasma T values (Notelovitz, unpublished data, 1977). The objectives of the present investigation were threefold: (a) to determine whether this observed effect was due to interference of the drug in the radioimmunoassay (RIA)of T or its reduced product, 5adihydrotestosterone (DHT);(b) to study the effect of diazepam on the testis and ventral prostate in vivo; and (c) to determine if the effect of diazepam on T production (if any) was mediated via pituitary gonadotropin secretion or hypothalamic luteinizing hormone-releasing hormone (LH-RH). MATERIALS AND METHODS Cross-reactivity of diazepam in radioimmunoassay. T and DHT were analyzed by RIA in accordance with the procedure of Coyotupa et al. [2]. The two androgens were isolated on Sephadex LH-20 columns following extraction of serum with diethyl ether. Antiserum to testosterone-3-oxine conjugated to human serum albumin was obtained from Dr. G. E. Abraham. The cross-reactivity of diazepam in the T and DHT RIA was examined in three separate experiments.
Received February 28, 1979. From the Department of Obstetrics and Gynecology, University of Florida College of Medicine, Gainesville, Florida. Address reprint requests to: M. Notelovitz, Department of Obstetrics and Gynecology, Box J-294 JHMHC, University of Florida College of Medicine, Gainesville, Florida 32610.
31 0 Elsevier North Holland, Inc., ARCHIVES OF ANDROLOGY. 3, 31-35 (1979)
0148-5016/79/01003 1-05$02.25
Syst Biol Reprod Med Downloaded from informahealthcare.com by University of Calgary on 09/06/13 For personal use only.
32
P. S. Cook et el.
1. Varying doses of pure diazepam were dissolved in buffer and processed in the RIA as unknown samples without chromatography. Binding of the 3H-T to antiserum was higher at all dose levels of diazepam compared with the binding in “zero” tubes (tubes containing no T or diazepam). This indicated the presence of “negative” blanks, possibly leading to an underestimation of unknowns when diazepam was present in the assay tubes. 2. Diazepam (0.5 mg) was added to a set of assay tubes containing known amounts of T. Binding was again higher in the tubes containing diazepam, indicating the possibility of an underestimation of unknowns. 3. Diazepam (0.5 mg/ml) was added to human blood samples containing known amounts of T and DHT. Replicate samples (with and without added diazepam) were processed as unknowns for the determination of T and DHT following extraction with diethyl ether and isolation by column chromatography (Sephadex LH-20).
In vivo assessment. Twenty-two Charles River CD adult male rats were maintained (two in a cage) in a room with a 14-hr light-10-hr dark cycle. The animals were supplied with Purina chow and water ad libitum. They were weighed at the start of the study and then every other day until the completion of the study. The rats were divided into four groups: Group I rats were treated daily for 10 days with 50 mg diazepamkg body weight. The diazepam was administered intraperitoneally as a 0. l-ml suspension (12.5 mg/ml; 40% propylene glyco1:absolute alcohol mixture, 9:l) per 25 g body weight. Group I1 rats were treated with diazepam as in group I, but were allowed an ll-day recovery period on completion of the treatment cycle. Group 111 rats served as a control for group I and were treated daily with a comparable volume of the vehicle for 10 days. Group IV served as control for group I1 and were treated with vehicle only for 10 days, after which they were allowed an 11-day recovery period. At the end of the 10-day treatment period, the rats in groups I and I11 were sacrificed by decapitation; groups I1 and IV were sacrificed in a similar fashion 11 days later. Trunk blood was centrifuged for 20 min at 3000 rpm and the serum was stored at -20°C until assayed for androgens and gonadotropins. The testes and the ventral prostate were dissected out and weighed. The brain from each animal was removed from the cranium and the neural tissue encompassing the medial basal hypothalamus was dissected out and frozen in 0.1 N HCl for LH-RH analysis. The RIA for estimation of T , DHT, LH, FSH, and LH-RH have been described [4]. For statistical analysis, Student’s t test was used to compare group means. RESULTS Cross-Reactivity of Diazepam in Radioimmunoassay (Table 1)
There was no significant difference in the T levels of samples with and without diazepam. DHT levels were significantly lower in samples containing diazepam. Thus, diazepam appears either to interfere with binding to antiserum or to prevent the adequate separation of the free-labeled antigen by the dextran-coated charcoal technique. However, when samples were extracted with diethyl ether and T and DHT were purified by column chromatography, the estimations of T were not altered by the presence of diazepam. Diazepam appears to elute along with DHT in our system and, if present in sufficient quantities, may result in an underestimation of DHT values. Body Weight
At the end of 10 days of treatment, there was no significant change in the body weights of both the control (316.2 2 14.8 g) and the diazepam-treated rats (310.6 k 9.9 g) when
Diazepam on Testosterone and Ventral Prostate
33
TABLE 1 Cross-Reactivity of Diazepam (V) in the Radioimmunoassay of Testosterone and Dihydrotestosterone
DHT
TESTOSTERONE (NG/ML)
SUBJECTS
26.9 t 2.9" (3)* 567.2 t 28.3 (4)
Females Males
Syst Biol Reprod Med Downloaded from informahealthcare.com by University of Calgary on 09/06/13 For personal use only.
' Mean
No V
?
0.5
MG V / M L
25.7 t 1.8 (5) 638.1 ? 20.8 (3)
No V 10.1 ? 0.6 (3) 19.7 2 1.7 (4)
(NG/ML)
0.5
MG
V/ML
3.3 t 0.3 (5)r 5.9 t 0.7 (5)r
SEM.
* Number of experiments. e p =
