Endocrine Journal 2004, 51 (4), 425–434 Endocrinological and Pathological Effects of Anabolic-androgenic Steroid 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 steroids (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 nandrolone decanoate, metenolone acetate, and dromostanolone; Group B with nandrolone decanoate 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 testosterone level in Group A was significantly higher than that in Group C. Serum dihydrotestosterone in Group A was significantly higher than that in both Groups B and C. Serum estradiol-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) stanozolol, 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), metenolone acetate (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