The administration of anabolic-androgenic steroids (AAS) to achieve a body composition of low fat and high muscle mass and to improve athletic performance has increased notably during the past three decades (29). In the past, AAS use was restricted to elite athletes and weightlifters, but today other populations (e.g., bodybuilders and recreational athletes) also use these drugs. Previous studies have estimated that approximately 1-3 million men and women in the United States use AAS (3,8). These people use the drugs in concentrations 10-60 times above the physiologic dose, by administration of a single type or a mixture of several AAS (2,10).
Many side effects of AAS use have been described in androgen-dependent organs, depending on the dose and the duration of administration (24). Bronson et al. (10) showed that supraphysiologic doses of AAS shorten life span in male rats: 52% of treated animals had died after 1 yr of treatment, compared with 12% of control animals. The main causes of these deaths were kidney tumors, glomerulonephropathy, and peliosis hepatitis. The major documented effects of AAS abuse are those on liver, serum lipids, and the reproductive system. In the liver of AAS abusers are seen structural and functional alterations, such as cholestatic jaundice, peliosis hepatitis, hepatocellular hyperplasia, and hepatocellular adenoma. An increased cardiovascular risk factor caused by the administration of this sex steroid might be secondary to changes in lipoprotein fraction, increased triglyceride and clotting factors levels, hypertension, and changes in the myocardium itself (24). In the reproductive system, endogenous testosterone levels, gonadotrophic hormones, and sex hormone-binding globulin (SHBG) are reduced, as are testis weight, sperm count, and sperm motility. Other affected systems include behavior, glucose tolerance, hyperinsulinemia, cerebrovascular events, and changes in prostate and immune functions (24).
Thyroid cells express androgen receptors and sex steroids might directly influence thyroid function. Banu et al. (4) showed that testosterone stimulates thyrocyte proliferation in culture, an effect independent of TSH. In rats, testosterone might increase TSH synthesis, secretion, and the response of TSH to TRH, because castrated male rats have lower serum and pituitary TSH concentrations (7). It is well known that the type 1 iodothyronine deiodinase (D1) is positively modulated by androgens. Castrated male rats have decreased D1 activity, which is restored after androgen replacement (22). In humans, thyroid hormone-binding globulin (TBG), which binds thyroxine (T4) and 3', 3, 5 - triiodothyronine (T3) in the circulation, is also affected by androgens. In a clinical study, Deyssig and Weissel (15) showed that total T4, total T3, and TBG were significantly decreased, and the response of pituitary after TRH stimulation was increased in five bodybuilders who used high concentrations of AAS compared with eight control subjects. Thus, the abuse of AAS may cause a mild impairment of thyroid function in humans (15). In a similar study, Daly et al. (14) found elevated serum TSH and free T4, but decreased total T4, total T3, and TBG in AAS users. These authors proposed that the elevated TSH and free T4 could be caused by an increased sensitivity of the pituitary to TRH or to a decreased sensitivity of the thyrotroph to hormonal feedback (14).
The previous studies on the possible interference of AAS on thyroid function have been conducted in humans, who usually take other drugs (e.g., GH, insulin, and thyroid hormones) together with AAS. Hence, the aim of the present study was to elucidate the effects of AAS administration on thyroid function using experimental animals in order to exclude the influence of other variables.
We postulate that high doses of DECA administered to male rats could interfere with thyroid gland function and thyroid hormone metabolism.
MATERIALS AND METHODS
Adult male Wistar rats weighing 200-250 g were maintained in an animal room with controlled lighting (12-h light-dark cycle) and temperature (23-24°C). The institutional committee for evaluation of animal use in research (CAUAP) approved the study, according to the international guiding principles for biomedical research involving animals (Geneva, Switzerland). The animals were divided into two groups: normal control rats (submitted to vehicle injection; peanut oil with 10% of benzoic alcohol) and rats treated with nandrolone decanoate (Deca Durabolin (50 mg·mL−1 Organon)) 1 mg·100 g−1 b.w. Steroid and vehicle were administered by a single intramuscular injection in the hind limb once a week for 8 wk. The dose used was 60 times higher than the dose usually recommended for hypogonadism in humans, but corresponds to the dose generally used by anabolic steroids abusers. After the experimental period, the animals were decapitated and blood was collected for hormone concentration analyses. Serum was obtained after centrifugation of the blood at 3000 rpm for 15 min, and stored at −20°C. The rat tissues were dissected out, weighed, and stored in liquid nitrogen until processing for enzymatic measurements.
Radioimmunoassay for total T3 and T4, free T4, and TSH.
Serum TSH levels were measured using a specific radioimmunoassay (RIA) for rat TSH obtained from the National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK, Bethesda, MD), and expressed in terms of the reference preparation 2 (RP-2). Intra- and interassay coefficients of variation were 7.7 and 6.5%, respectively. The sensitivity was 0.63 ng·mL−1.
The serum total T3 and T4 and free T4 concentrations were measured using commercial RIA kits using 125-I as tracer (T3: DLS, 3100 Active, sensitivity of 4.3 ng·dL−1, inter- and intraassay coefficients of variation varied from'4.2 to 6.0% and from 5 to 6.5%, respectively; T4: DLS, 3200 Active, sensitivity of 0.4 μg·dL−1, inter- and intraassay coefficients of variation varied from 7.1 to 7.4% and 2.9 to 5.1%, respectively; free T4: DSL, 40100 Active, sensitivity of 1 pg·mL−1), based on the presence of specific antibodies adhered to the internal surface of propylene tubes. Rat hormone-free serum was used in the standard curves for total T3 and T4 and TSH. All the procedures were carried out following the recommendations of the kit.
Thyroperoxidase (TPO) iodide oxidation activity.
For extraction of TPO, each rat thyroid gland was homogenized in 50 mM Tris HCl buffer, pH 7.2, and centrifuged at 200.000 × g, 4°C for 35 min. The pellet was suspended in digitonin (1%, w/v; Sigma) and stored for 24-48 h at 4°C. After this incubation period, a new centrifugation was done in the same conditions, and the supernatant containing solubilized TPO was stored at −20°C (11,16,23).
The TPO iodide-oxidation assay (11,23) was performed as follows: 1.0 mL 50 mM sodium phosphate buffer, pH 7.4, containing 24 mM KI and 11 mM glucose was placed in a 2-mL cuvette; the volume of solubilized TPO (from 10 to 100 μL) was added, and the final volume adjusted to 2.0 mL with 50 mM sodium phosphate buffer, pH 7.4. The assay was started by the addition of 10 μL of 0.1% (w/v) glucose oxidase (Boehringer Grade I). The increase in absorbance at 353 nm (ΔA353 nm·min−1, triiodide formation) was followed for 2 min on a Hitachi spectrophotometer (U-3000). The ΔA353 nm·min−1 was determined from the linear portion of the reaction curve and corrected for a blank determined in the absence of TPO. One unit of iodide oxidation activity is defined as ΔA353 nm·min−1 = 1.0. The unit of activity was related to protein concentration in the enzyme preparation. Protein was measured by the method of Bradford (9).
Type 1 iodothyronine deiodinase activity.
Each thyroid, a pool of two pituitary glands, and 25 mg of liver or kidney were homogenized in 1 mL 0.1 M sodium phosphate buffer containing 1 mM EDTA, 0.25 M sucrose, and 10 mM dithiothreitol, pH 6.9. Homogenates (150 μg of protein for pituitary samples and 30 μg of protein for liver, thyroid, or kidney) were incubated in triplicate for 1 h at 37°C with 1 μM [125I] rT3 (Perkin Elmer Life and Analytical Sciences), and 10 mM dithiothreitol (USB) in 100 mM potassium phosphate buffer containing 1 mM EDTA, pH 6.9, in a reaction volume of 300 μL, as previously described (5). Blank incubations were carried out in the absence of protein. The reaction was stopped at 4°C by the addition of 100 μL fetal bovine serum (Cultilab, Brazil) and 200 μL trichloroacetic acid (50%, v/v) followed by vigorous agitation. The samples were centrifuged at 10,000 rpm for 3 min, and the supernatant was collected for measurement of 125I liberated during the deiodination reaction. Although type 2 deiodinase (D2) is also present in the thyroid and pituitary glands, our assay conditions only measured D1 activity because a specific D1 inhibitor (100 mM PTU) completely blocked deiodinase activity.
The D1 activity was related to the protein concentration in the homogenates. Protein was measured by the method of Bradford (9), after incubation of homogenates with NaOH (2.5N).
The results are expressed as the mean ± SEM. Data from total T3, total T4, free T4, and deiodinase activities were analyzed by unpaired t-test. The results of serum TSH and TPO activities were analyzed by Mann-Whitney U-test. Statistical analyses were done using the software Graphpad Prism (version 4, Graphpad Software, Inc., San Diego, CA). A value of P ≤ 0.05 was considered statistically significant.
Body and tissue weights.
Body weight and retroperitoneal adipose tissue were not significantly altered by the use of DECA, although retroperitoneal adipose tissue was slightly decreased (P < 0.07) when expressed in relation to body weight (Table 1).
The animals that received supraphysiologic doses of DECA had significant decreased testis weight, and increased heart and kidney weights (Table 2, P < 0.05), when compared with the control group. The relative and absolute thyroid weights were also significantly increased by DECA administration (Table 3, P < 0.05). The relative weights of liver and lungs were not statistically different between groups.
Serum total T3 and T4, free T4, and TSH concentrations.
Serum TSH levels were significantly decreased (P < 0.05) in the DECA-treated group (Fig. 1A). Serum total T3 was significantly decreased in DECA-treated rats (P < 0.001) in comparison with control animals (Fig. 1B). Serum total T4 was not significantly changed in the DECA-treated group (Fig. 1C), but the concentrations of serum-free T4 were significantly diminished (Fig. 1D, P < 0.01).
Thyroperoxidase (TPO) activity.
Thyroperoxidase is a key enzyme in the biosynthesis of thyroid hormones, and an impaired TPO activity could account for decreased T4 and T3 biosynthesis. The use of DECA in high concentrations, however, did not modify the activity of this enzyme (DECA = 2.43 ± 0.34 U·mg−1 protein, N = 11; C = 2.21 U·mg−1 protein ± 0.28, N = 11).
In rats, a large proportion of T4 is deiodinated in the thyroid, generating approximately 40-50% of circulating T3 (6), thus decreased thyroidal D1 activity could lead to lower serum T3 levels. Serum T3 also originates from the outer ring deiodination of T4 by renal and hepatic D1.
Hepatic D1 activity was significantly increased in the treated group, as well as the renal D1 enzyme activity (Fig. 2A and B, P < 0.05). Thyroidal and pituitary D1 activities, however, were unchanged in the DECA-treated (Fig. 2C and D).
The indiscriminate use of AAS, both among athletes and outside the sports scenario, has become a sociogovernmental concern (13). Approximately 1-3 million men and women of diverse ages and socioeconomic condition use anabolic steroids in the United States, with the objective to improve performance, or simply for aesthetic ends (8). These individuals are likely to develop a lot of diseases; however, the effects of high dosages of AAS on thyroid function are still controversial and poorly addressed in the literature (8,24). In the present study, we used rats as the animal model to elucidate the effects of AAS super dosage on thyroid gland function to avoid the interference of other drugs.
The final body weight and the rate of growth of the treated animals were not significantly different from those of the control group. These findings are in accordance with the results found by Woodiwiss et al. (28) and Saborido et al. (25) but disagree from those of Joumaa and Léoty (18) and Takahashi et al. (26), who observed a reduction in the corporal weight of animals treated with AAS. This discordance probably results from differences in the ages of the animals used and the protocols used for drug treatment.
Despite the previous description that testosterone can promote fat loss in humans (27), both the absolute and relative retroperitoneal fat weights were unchanged in the DECA-treated group. Mauras et al. (21), using healthy men with androgen deficiency, have demonstrated an increase in the adipose tissue mass, however, Woodhouse et al. (27) showed that the effect of testosterone on adipose tissue depends on the dose administered and also on the different regions of the body. We only analyzed rat retroperitoneal tissue, and our results are in accordance with the previous findings of unchanged intraabdominal adipose tissue in healthy young men receiving high doses of testosterone (27).
Administration of DECA caused significant changes in the relative weight of different organs. The significant reduction of testis weight found in our study can be caused by atrophy, probably because of the inhibition of gonadotropin secretion caused by DECA (2,14,24). The increase in the relative weight of the kidneys in the treated animals agrees with the findings of Takahashi et al. (26), who also found hemorrhage of distal renal tubules and cellular hypertrophy probably caused by a direct action of the anabolic steroid on the organ (24,26). Increased heart weight is a common finding reported in the literature, probably reflecting cardiac hypertrophy secondary to AAS use (26).
Thyrotropin is a trophic hormone to the thyroid gland that acts by stimulating both thyroid hormone biosynthesis and thyroid cell proliferation. Because serum TSH is significantly decreased in DECA-treated animals, we could expect decreased thyroid weights. Thus, the increase in the absolute and relative weights of the thyroid found should be attributed to a direct stimulation of cellular proliferation produced by the steroid. Reinforcing the possibility of a direct action of androgens on thyrocytes, Banu et al. (4) demonstrated that testosterone induces thyroid cell proliferation in cultures.
The DECA-treated animals had a significant reduction in serum total T3, free T4, and TSH, without a significant change in serum total T4. Thus, the reduction in total T3 and free T4 in the treated group could be explained by a decrease in thyroid hormone production caused by decreased TSH and a concomitant increase in serum binding proteins, because serum total T4 is unchanged. Further studies are needed to better understand the mechanisms involved in the maintenance of thyroid T4 production despite decreased serum TSH levels. Another possibility is that T4 production by the thyroid is maintained regardless of decreased serum TSH and that the decreased serum total T3 and free T4 could be explained by changes in the peripheral metabolism of T4 by deiodinases. In fact, hepatic and renal D1 activities were stimulated by DECA in the present study; however the outer ring deiodination, which is responsible for T3 generation from T4, may not be occurring adequately. Sulfated iodothyronines are preferential substrates for inner ring deiodination by D1. Thus, T4 sulfatation increases its inner ring deiodination, generating rT3 sulfate and not T3 (20). Moreover, sulfated T4 is degraded 100 times faster than T4, which could explain the decreased free T4 concentrations (20). Sulfotransferases are the enzymes responsible for T4 sulfatation and these enzymes could also be induced by DECA administration, explaining the decreased outer ring T4 deiodination and, therefore, the decreased serum T3 levels. The evaluation of sulfotransferase activity in DECA-treated animals, however, has yet to be done.
The reports of serum TSH changes found in AAS abusers are controversial. A previous study in athletes who used AAS also found a reduction in serum TSH levels (1). Increased serum TSH, however, has been described in individuals submitted to a short-term treatment with AAS (12), whereas in weightlifters who used AAS no significant difference in TSH levels were found (15). Some reports have shown that serum TSH concentrations are higher in male rats than in female rats (12,17), and gonadectomy of males results in decreased TSH levels that are restored by testosterone replacement (4). Whether the decreased production of TSH in DECA-treated rats is caused by a direct effect of the hypothalamus-pituitary axis or to an indirect effect caused by testicular hypofunction must be evaluated by further studies.
Thyroperoxidase activity was unaltered in DECA-treated animals. A higher TPO gene expression in young male rats than in female rats has been described. A reduction in the expression of this gene occurs with aging, suggesting that testosterone can be a positive regulator of TPO gene expression (12).
The D1 enzyme, which is responsible for the deiodination of the outer or the inner ring of T4, is modulated by DECA in a tissue-specific manner. The results of increased (liver and kidney) or normal (thyroid) D1 activity together with the findings of significantly decreased T3 indicate that the D1 outer ring deiodination of T4 might be decreased in animals treated with DECA. Furthermore, thyroid D1 activity is positively regulated by TSH, which is decreased in DECA-treated animals, so a direct stimulatory action of DECA on D1 could explain the normal activity found in the thyroid. Thyroid hormones are the main positive regulators of hepatic and renal D1 (30). Thus, the increased D1 activity in DECA-treated animals, with low serum T3, also suggests a direct stimulatory action of DECA on D1 activity. These results are in accordance with previous reports that demonstrated an increase of hepatic D1 mRNA and activity in male rats when compared with female rats and a reduction in these levels with castration (19,22). Moreover, a stimulatory effect of testosterone replacement in castrated male rats (19,22) and a direct action of this steroid in hepatocyte cell cultures have been described (22).
The results found in this study suggest a probable direct action of DECA on the thyroid gland and on liver and kidney D1 activities. The reductions of serum total T3 and free T4 in the treated group are probably related to a change in the hepatic metabolism of T4. The reduction of serum TSH seems to occur because of a direct central action of DECA; however, the mechanisms remain to be determined. In conclusion, the use of DECA alters thyroid function and thyroid hormone economy, and could lead to clinical or subclinical thyroid dysfunction in users of these drugs.
This work was supported by grants from Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Programa de Nu`cleos de Excelência (PRONEX), and Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq). We are grateful for the technical assistance of Norma Lima de Arau`jo Faria, Advaldo Nunes Bezerra, and Wagner Nunes Bezerra. Rodrigo S. Fortunato and Norma L. A. Faria were recipients of fellowships from CNPq during the present study.
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Keywords:©2006The American College of Sports Medicine
THYROPEROXIDASE; TYPE 1 IODOTHYRONINE DEIODINASE; THYROID HORMONES; THYROTROPIN; NANDROLONE DECANOATE