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Endocrinological and Pathological Effects of Anabolic-Androgenic Steroid in Male Rats

Endocrinological and Pathological Effects of Anabolic-Androgenic Steroid in Male Rats

Endocrine Journal 2004, 51 (4), 425–434

Endocrinological and Pathological Effects of Anabolic-androgenic in Male Rats

MASATO TAKAHASHI, YUKITOSHI TATSUGI AND TOSHIHIKO KOHNO

International Budo University Faculty of Physical Education, Chiba 299-5295, Japan

Abstract. Many athletes use drugs, especially anabolic androgenic (AAS), but there are few reports on the endocrinological and pathological changes in AAS abusers. In this study we reported the results of endocrinological examinations in rats administered AAS and also physical changes. We separated 37 male Wistar rats (7 weeks old) into 3 groups: Group A was medicated with decanoate, , and dromostanolone; Group B with and saline; and Group C was given only saline. They were given subcutaneous injections of the medications or the control vehicle once a week for 6 weeks. Medications were stopped for 4 weeks, and then resumed for another 6 weeks. After that, rats were sacrificed. Serum level in Group A was significantly higher than that in Group C. Serum in Group A was significantly higher than that in both Groups B and C. Serum -17 levels in Groups A and B were significantly higher than that in Group C. In pathological evaluation, heart, testis, and adrenal gland were severely damaged. These findings indicate that there is a high degree of risk related to the use of AAS.

Key words: Anabolic androgenic steroid, Doping in sports, Endocrinology, Pathology (Endocrine Journal 51: 425–434, 2004)

BEN Johnson, a Canadian athlete who used the control efforts has naturally been fewer than those in anabolic-androgenic steroid (AAS) , was other countries [3, 4]. Because AAS abuse in athletics disqualified from the Seoul Olympics in an effort to is widespread the side effects elicited by AAS abuse control doping. After this notorious incident, reports have been extensively reported [5–10]. of such cases have been numerous. In Japan, body- Psychotic states and infertility have social implica- builders and the other athletes sometimes test positive tions [11–15] and are among the serious side effects for AAS use. Takahashi, who has investigated AAS elicited by AAS abuse. However, the pathophysiology abuse among Japanese athletes, reported two cases of and prognosis of AAS use remains unclear. Bashin et doping by Japanese athlete [1, 2]. Traditionally, it has al., Berman et al., Bross et al., and Tricker et al. been thought that Japanese athletes refrain from AAS attempted to quantify the side effects of testosterone use and so the public was shocked to hear of these in- administration in humans [16–19]. As is unethical to cidents. Greater anti-doping efforts and treatment for administer high doses of AAS in humans, in the the deleterious side effects of these drugs in Japanese current study we have used animals to determine the athletes are needed. However, since doping control in pathology and prognosis resulting from AAS use. We Japan has been minimal in the past, the number of previously reported the results of hematological and Japanese doctors and scientists participating in doping biochemical examinations following AAS administra- tion in rats, and included data reporting the changes in body and organ weights in these animals [20]. Based Received: April 2, 2003 Accepted: April 21, 2004 on our previous findings, we hypothesized that AAS Correspondence to: Dr. Masato TAKAHASHI, International Budo administration would result in significant deleterious University Faculty of Physical Education, 841, Shinkan, effects on organ tissue pathology and endocrine func- Katsuura, Chiba 299-5295, Japan tion. Pathological evaluation especially revealed 426 TAKAHASHI et al. severe damage to heart, testis, and adrenal gland. AAS nighttime hours. administration further resulted in a severe imbalance in the endocrine system. Endocrinological examinations

Endocrinological examinations included measure- Methods ment of adrenocorticotropic hormone (ACTH) and atrial natriuretic peptide (ANP) in plasma, and corti- Animals and experimental conditions costerone, testosterone, dihydrotestosterone (DHT), estradiol-17 (E2), and erythropoietin (EPO) in serum. We separated 37 male Wistar rats (7 weeks old) Furthermore, we measured prostate specific antigen into 3 groups: Group A (n = 12) was administered (PSA). Organs were fixed in 10% neutral buffered nandrolone decanoate (8.33 × 10–2 mg/g; Sankyo Co. formalin. Next the heart, liver, kidney, spleen, adrenal Ltd., Tokyo), (1.67 × 10–1 mg/g; gland, testis, prostate gland, and spleen were routinely Japan Schering Co., Ltd., Osaka), and dromostanolone processed for paraffin embedding. Organs were cut (8.33 × 10–2 mg/g; Shionogi Co., Ltd., Osaka); Group into 10 m thin sections and stained with hematoxylin B (n = 12) received nandrolone decanoate and saline; and eosin (H.E.). These tissue samples were then and Group C (n = 13) was given only saline. These examined under light microscope for quantification of drug dosages were converted from 100 times the gen- histopathological changes following AAS administra- eral dose for 60 kg body weight in humans, since this tion. dose has been previously found to be used by drug abusers [21]. Such doses were chosen based on the Statistical analysis fact that 100 times the normal dosing is necessary to pharmacologically investigate the pathological effects Significant differences were identified using one- of drug abuse. Each rat was housed in individual way ANOVA. If the ANOVA revealed significant cage (25 × 13 × 16 cm), and given access to rat chow main effects (p<0.05), Sheffe’s post-hoc method of one pellets (Oriental Yeast Co. Ltd., Tokyo) and water ad way factorial ANOVA and multiple comparison tests libitum. Temperature and moisture averaged 25 ± 2°C were then used. and 75 ± 10%, respectively. After a week of acclima- tion, animals were given subcutaneous injections of AAS or the control vehicle once a week for 6 weeks. Results Drug administration was stopped for 4 weeks, and then resumed for another 6 weeks. This dosing regimen Behavioral observations during experimental period was used to duplicate the “stacking” of AAS and the “steroid cycle” that is used by AAS abusers in athlet- After 3 weeks, some Group A and Group B rats ex- ics. These studies followed the ethical guidelines set hibited hair loss (Fig. 1). After 4 weeks, some Group by International Budo University. A (n = 5) and Group B rats (n = 9) exhibited such aggressive behavior that they could not be handled Sacrifice without gloves. After 5 weeks, some rats of Group B (n = 3) demonstrated extremely aggressive and hostile After two cycles of steroid administration, rats were behavior. After 1 cycle of AAS administration, all sacrificed by removing 12 ml of blood from the heart Group A and Group B animals exhibited inactive under ether anesthesia. Serum and plasma were pre- behavior, as evidenced by lack of locomotion upon served. We then extracted and recorded the wet stimulation. A very slow locomotor movement upon weight of skeletal muscles (M. extensor digitorum physical contact was further evident following this longus, M. gastrocnemius, M. plantaris, M. soleus, M. experiment. After 9 weeks, two rats of Group A and levator ani), liver, spleen, heart, kidney, adrenal gland, Group B caused self-inflicted injuries to their tails. and prostate gland. Collected blood and tissue sam- After 10 weeks, 6 of 12 rats in both Groups A and B ples were used to make a hormonal profile. In addi- were also observed to have self-inflicted bite wounds. tion, these experiments were performed during the After 2 cycles, all Group B and 4 rats of Group A EFFECTS OF ANABOLIC-ANDROGENIC STEROID 427

exhibited aggressive and hostile, behavior again. After 13 week, these rats became physically inactive (Fig. 2).

Endocrinological profile

The change the body weight is illustrated in Fig. 3 and a comparison of the organ weights is shown in Table 1. Results from endocrinological measurements are presented in Fig. 4. Data indicate that plasma ACTH in Groups A and B was significantly lower than in Group C (p<0.01). Changes in serum corticosterone exhibited no significant differences among the groups. Fig. 1. Rats experienced hair loss both systemically and at the Serum level of testosterone in Group A was signifi- site of subcutaneous AAS injections.

Fig. 2. Experimental Behavioral observations during in Group A and Group B rats. This figure shows the change of physical activity. *Loss of hair, **Self inflicted tail wounds

Fig. 3. Change of body weight in experimental period. Group A was administered 3 kinds of AAS. Group B was administered 1 kind of AAS and physiological saline. Control, Group C, was administered only physiological saline. 428 TAKAHASHI et al.

Table 1. Body weight (BW), muscle weight and internal organ weight in rats which were treated by AAS in saline. Group A, treated which 3 kinds of AAS; Group B, treated with 1 kind of AAS, and Group C, treated with only saline.

gastrocnemius gastrocnemius/ n BW (g) EDL (g) EDL/BW soleus (g) soleus/BW plantaris (g) plantaris/BW (g) BW A -group 12 506.5 ± 61.6 0.26 ± 0.04 0.51 ± 0.08 2.31 ± 0.31 4.57 ± 0.59 0.19 ± 0.05 0.37 ± 0.08 0.49 ± 0.06 0.98 ± 0.14 B -group 12 506 ± 17.3 0.26 ± 0.04 0.5 ± 0.07 2.45 ± 0.28 4.7 ± 0.53 0.18 ± 0.03 0.34 ± 0.07 0.53 ± 0.07 1.02 ± 0.15 C -group 13 642 ± 98.5 0.31 ± 0.04 0.48 ± 0.09 2.85 ± 0.23 4.47 ± 0.67 0.25 ± 0.06 0.39 ± 0.12 0.7 ± 0.2 1.08 ± 0.23 A-B A-B A-B A-B A-B A-B A-B A-B A-B A-C*** A-C** A-C A-C** A-C A-C* A-C A-C** A-C B-C*** B-C* B-C B-C** B-C B-C** B-C B-C** B-C

M.levator ani M.levator heart (g) heart/BW liver (g) liver/BW kidney (g) kidney/BW (g) ani/BW 3.59 ± 0.45 7.12 ± 1.01 1.93 ± 0.2 3.82 ± 0.36 17.36 ± 2.51 34.15 ± 2.67 3.03 ± 0.57 5.97 ± 1 3.5 ± 0.44 6.7 ± 0.78 2.25 ± 0.33 4.3 ± 0.62 17.01 ± 2.85 32.36 ± 3.43 3.02 ± 0.48 5.77 ± 0.89 3.83 ± 0.88 5.87 ± 0.62 2.1 ± 0.36 3.24 ± 0.25 20.83 ± 3.95 32 ± 2.4 2.04 ± 0.40 3.14 ± 0.27 A-B A-B A-B A-B* A-B A-B A-B A-B A-C A-C* A-C A-C** A-C* A-C A-C*** A-C*** B-C B-C* B-C B-C*** B-C* B-C B-C*** B-C***

adrenal (g) adrenal/BW testis (g) testis/BW prostate (g) prostate/BW spleen (g) spleen/BW 0.06 ± 0.04 0.11 ± 0.07 1.57 ± 0.15 3.14 ± 0.52 3.48 ± 0.5 6.93 ± 1.28 0.97 ± 0.17 1.92 ± 0.3 0.04 ± 0.02 0.08 ± 0.03 1.68 ± 0.11 3.22 ± 0.25 3.41 ± 0.53 6.54 ± 1.07 0.97 ± 0.16 1.86 ± 0.28 0.05 ± 0.02 0.07 ± 0.03 1.91 ± 0.2 2.98 ± 0.35 1.48 ± 0.63 2.22 ± 0.72 1.21 ± 0.25 1.86 ± 0.19 A-B A-B A-B A-B A-B A-B A-B A-B A-C A-C A-C*** A-C A-C*** A-C*** A-C* A-C B-C B-C B-C** B-C B-C*** B-C*** B-C* B-C Values are means ± SD. *(p<0.05); **(p<0.01); ***(p<0.001). BW; Body weight

cantly higher than in Group C (p<0.001), while di- sediments were also observed (Fig. 6). The prostate hydrotestosterone (DHT) in Group A was significantly gland of Group A was macroscopically hypertrophied. elevated as compared to Group B (p<0.001) and Group Adenomatous hypertrophy especially in the dorso- C (p<0.001). Estradiol-17 (E2) of Group A was lateral lobe, irregular epithelial proliferation and significantly higher than in Group C (p<0.001). Mea- starch-like sediments in the vacuoles of these cells sured erythropoietin (EPO) in Group A was signifi- were also observed (Fig. 7). In the testes of Group A, cantly lower than in Group B (p<0.01) and in Group C spermatozoa were scarred within the lumen of the (p<0.001). seminiferous tubules, and supporting Sertoli cells were reduced in numbers. Furthermore, few interstitial cells Pathological findings (Leydig cells) were visible (Fig. 8). Although the liver of Group A was not macroscopically altered, the cen- We examined the pathological changes in rats fol- tral veins of individual lobules were dilated, and partial lowing AAS administration by light microscopy. The congestion in interlobular tissue was observed. Be- myocardium and pericardium in the hearts of Group A cause liver cells were turbid and slightly swollen, sinu- rats exhibited partial fibrosis, and small round cells in- soidal capillaries were not clearly observable (Fig. 9). vaded the myocardium. Scarring of the pericardium Although the wet weight of the adrenal glands of was observed as well. Cardiac myofibrils were irregu- Group A was unchanged, vacuole degeneration could larly lined, and vacuolar degeneration was observed be observed within the zona reticularis. The compact (Fig. 5). In the kidney of Group A, tubular epithelial cells in the upper margin of the zona fasciculata tended cells were swollen and reduced in numbers. Distal tu- to be dominant (Fig. 10). bules were partially hemorrhaged, and PAS positive EFFECTS OF ANABOLIC-ANDROGENIC STEROID 429

Fig. 4. Endocrinological profile of plasma adrenocorticotropic hormone (ACTH). Significant difference between Groups A/B and C. Each column represents mean ± standard deviation. a. Dihydrotestosterone, Significant difference between Groups A and B, between Groups A and C. b. Estradiol-17, Significant difference between Groups A and C, between Groups B and C. c. Erythropoietin, Significant difference between Groups A and B, between Groups A and C. d. Testosterone, Significant difference between groups A and C.

Discussion gesting that AAS and might have some effect on the central nervous system which could in- In the current study we used supraphysiologic doses duce abnormal behavior. After a period of hostility, of AAS in rats to investigate the pathological and the animals became inactive evidenced by a lack of prognostic effects of AAS abuse. In general, 100 locomotor activity upon physical contact. Martinez et times the normal dosing is necessary to pharmacologi- al. reported that some testosterone treated male mice cally investigate pathological effects of drug abuse exhibited aggressive behaviors as well [25]. Taken [21]. It is well established that the psychological state together these data indicate that the neurological sys- of human subjects becomes unstable with AAS abuse tem becomes abnormal. [5, 22, 23]. Furthermore Pope et al. reported that Corticosterone level was not altered by AAS admin- supraphysiologic doses of testosterone administration istration, indicating that the rats had not experienced increased ratings of manic symptoms in a few normal any degree of significant stress in response to the dop- men, but most showed few psychological changes ing protocol. Levels of ACTH in Groups A and B [24]. In our study, we observed hostile, aggressive were lower than those in Group C. This finding indi- behaviors in rats following AAS administration, sug- cates that a negative feedback response to the increase 430 TAKAHASHI et al.

Fig. 5. Heart sample stained with hematoxylin and eosin (H.E.) from a representative animal from group A. a. myocar- dium (× 100) b. pericardium (× 200) Invasion of small Fig. 7. Prostate gland sample from a Group C (a) and a Group round cells (arrow), such as lymphatic cells, was ob- A (b) animal, respectively, stained with H.E. (× 40). served. Scaring in many areas was also observed. Adenomatous hypertrophy and irregular epithelial prolif- eration (arrow) as well as a starch-like sediment were observed (* ) in vacuoles of these cells.

Fig. 6. Kidney sample from a representative animal from group A (× 100 ) Fig. 8. Spermatozoa sample from a Group C (a) and a Group A a. Distal tubule stained with periodic acid-Schiff stain (b) animal stained with H.E.. In the testis of a Group A (PAS). Proliferative hypertrophy of epithelial cells was animal, few interstitial cells (* ) were seen and support- observed (arrow). Some PAS-positive sediment was ing cells (arrowhead) had dropped off from the basal seen which was indicative of denatured mucoid cells layer. Many cells, especially Leydig cells, were atrophic. (arrowhead). b. Distal tubule stained with hematoxyline eosine stain (H.E.). Epithelial cells were swollen and less numerous than normal. Deposition of fibrin was hypothesis. AAS administration in rats elicited a sig- observed in the tubules. nificant elevation in testosterone, dihydrotestosterone, and estradiol-17. We speculate that both endogenous in circulating androgens may have been initiated. In testosterone and exogenous testosterone or testosterone- this study, we hypothesized that plasma testosterone, like substances were measured. The differences in the dihydrotestosterone, and estradiol-17 levels of AAS- AAS metabolic system between rats and humans could administered rats would be lower as previously report- be responsible for these discrepant results. We were ed in humans [13]. However, the results from the cur- unable to replicate the same results in preliminary rent study were in complete opposition to our original studies. Bitran et al. reported that blood testosterone EFFECTS OF ANABOLIC-ANDROGENIC STEROID 431

erythropoietin levels [27]. We speculate that the dis- crepancy between these results and those in the present study is due to the difference in the dose of nandrolone utilized. Because we administered AAS at very high doses, most likely a negative feedback inhibition of EPO was elicited due to an elevation in hemoglobin levels in response to AAS administration [20]. As we originally hypothesized, pathological find- ings indicate that organs dependent on androgens were significantly damaged following AAS administration. The prostate gland was macroscopically and micro- scopically hypertrophied, although there was no evi- dence of carcinoma. Hypertrophy of the prostate to this degree most likely would have caused the drainage Fig. 9. Liver lobule sample stained with H.E. (× 40) in a Group of urine to be less likely. Prostate specific antigen was C (a) and a Group B (b) animal, respectively. The cen- within the normal range. Bitran et al. reported that tral veins of the lobules were dilated (* ) in the Group B the prostate gland weight of rats with 2 week of animal. Glisson’s capsule was partially congested methandrostenolone injected decreased after AAS (arrow). In the Group C animal, sinusoidal capillaries were clearly seen radiating in all directions. But in the discontinuation [26]. The true mechanism causing Group B animal, it was difficult to clearly observe the such discrepancy cannot be adequately explained. capillaries and sinusoidal capillaries because the liver In the testes, almost all Sertoli cells and Leydig cells cells were slightly swollen (× 200, upper right figure). were reduced in numbers, and the overall damage to the testes was tremendous. Due to negative feedback mechanisms, the secretion of endogenous testosterone was completely inhibited. There are a few reports that the testicular weights of male rats decreased through- out the AAS treatment period [28, 29]. The findings from the current study supports this fact. Although athletes and students readily use AAS, this finding is very shocking, and AAS abusers would most likely be quite alarmed if they knew of these pathological ef- fects on the testes [29]. When Leydig cells are de- stroyed, patients must be supplied with exogenous testosterone. We feel these findings are important, as we could find few reports regarding these pathological effects of AAS abuse. Fig. 10. Adrenal gland sample in a Group C (a) and Group A Our data indicate significant renal damage follow- (b) animal, stained with PAS. In the Group A animal, ing AAS administration as well. Some studies indicate a large degree of vacuole degeneration was observed in the presence of receptors in the kidney [30– the zona reticularis (arrowhead). 32], while another study suggests they are only present within the urogenital tract [33]. Data from the current levels were increased 7-fold by 1 week administration study suggest that AAS affected the epithelial cells of of testosterone propinate, and 10-fold by 2 weeks admin- urinary tubules via an androgen-dependent mechanism istration [26]. The elevations in dihydrotestosterone either directly or indirectly. However, biochemical ex- and estradiol-17 were also accompanied by an ele- amination indicated that renal function was normal vation in testosterone. [20]. Erythropoietin levels of Group A were significantly Although heart obstruction was not considered to be lower than those in Groups B and C. Saitoh et al. re- the cause of death in Olympic athlete Florence Joyner, ported that injection of nandrolone into mice enhanced data from our study indicates that cardiac damage 432 TAKAHASHI et al. results from AAS administration. Significant inflam- induced liver dysfunction. However, biochemical mation was observed within the myocardium and examination has yet to reveal liver dysfunction under pericardium. It is likely that this could be the cause these circumstances. Boada et al. reported that high of fatal arrhythmia in athletes who use AAS. Two doses of stanozolol treatment in male rats resulted in reports investigated the cardiac capillary bed of inflammation or degeneration in central lobular hepato- testosterone-propionate administered female rats [34, cytes and exert a proliferative effect on liver cells [43]. 35]. Although they observed the impairment of car- Furthermore, there are some reports indicating that diac capillaries by AAS, they did not report of any ob- levels of cytocrome P-450 in the liver are decreased structions in the myocardial or pericardial region. in male rats following AAS administration [44]. Woodiwiss et al. used nandrolone decanoate adminis- The androgen secreting zona reticularis of the adre- tration in male rats, and reported that AAS affected left nal gland would most likely suffer damage following ventricle remolding [36]. They also reported negative AAS abuse due to initiation of negative feedback mecha- findings with regard to AAS-induced cardiac damage, nisms. Furthermore, it was difficult to accurately although, the dose used was less than used in the measure the weight of each adrenal gland due to its current study. Regardless, the fact remains that many small size. We speculate that the adrenal gland weight athletes use large doses of AAS, thus significantly among the groups was not statistically differentiated increasing their risk for sustaining cardiac damage. because of this. Bentvelsen et al. reported the pres- Although sudden death of unknown origin is some- ence of androgen receptors in the adrenal gland [33]. times reported, it is possible that this may be due to The results from our study corroborate these findings AAS-induced cardiac damage. In light of our current as well. findings, and those in previous reports regarding the In conclusion, AAS administration has considerable serious deleterious effects of AAS on the myocardium, effects on various organs. We are unaware of any pre- this is a logical conclusion [37–40]. Furthermore viously published reports regarding the pathological Marsh et al. reported that androgen receptors were state of AAS abuse. Furthermore, the degree of tissue present in cardiac muscle [41]. Our data clearly indi- damage sustained by AAS administration was much cate that AAS-induces negative cardiac effects. Al- greater than we originally hypothesized. In addition, though experimental data obtained from animals our data indicated a great imbalance in the endocrine correlates well with data from human subjects, the system as a result of AAS administration. These re- pathophysiology of adverse cardiovascular effects of sults would strongly suggest that athletes should re- AAS use is still poorly understood [42]. frain from AAS use if they wish to avoid these serious Changes in hepatic tissue are typical following drug- deleterious effects.

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