Prior to the banning of over-the-counter sales, androstenedione was used as a dietary supplement by individuals who likely believed it would increase muscle mass during training. Due to concern for adverse health effects associated with its chronic use, the NTP conducted genetic, subchronic, and chronic toxicity studies of androstenedione in male and female rats and mice. The health effects of androstenedione will vary since androstenedione is an androgenic hormone that can be metabolized to a more potent androgen (e.g., testosterone) or to an estrogen, and different hormonal effects may occur depending on the metabolism and presence of steroid receptors within a specific tissue, which may vary with the age and sex of the animal.
In the 14-week studies, androstenedione did not demonstrate any dose-limiting toxicity effects in male and female rats and mice. Female rat body weights increased compared to the vehicle controls in both the 3-month and 2-year studies. A similar result occurred with oxymetholone, an androgenic anabolic steroid, which increased female rat body weights in subchronic and chronic studies (NTP, 1999
). The increase in female rat body weights by androstenedione may be due to increased muscle mass, but neither muscle mass nor adipose mass were evaluated. The increased incidences of adrenal gland X-zone atrophy and X-zone cytoplasmic vacuolization seen in female mice after a 14-week exposure indicate that androstenedione, or a metabolite, had an androgenic effect. Regression of the adrenal X-zone in female mice normally occurs rapidly during the first pregnancy. Regression of the X-zone in female mice can be stimulated by administration of androgens and can be delayed in males via castration (Holmes and Dickson, 1971
; Tomooka and Yasui, 1978
The reduction in sperm concentration in the rat cauda epididymis that occurred in the 3-month study, in the absence of effects on spermatid numbers, suggests androstenedione may be interfering with sperm maturation. This effect is different from previous studies which observed a decrease in spermatid and spermatozoa concentrations. Administration of testosterone or estrogen via implants decreases spermatid concentrations within the testes and results in reduced fertility (Robaire et al., 1979
; Robaire et al., 1984
) and the anabolic steroid oxymetholone, reduced sperm concentrations in the epididymides and spermatid numbers (NTP, 1999
). Sperm motility in male mice was significantly decreased at the top dose, and similarly, oxymetholone reduced male mouse sperm motility (NTP, 1999
). Since sperm motility is achieved within the epididymis, androstenedione administration may also have affected sperm maturation in male mice. These effects indicate a potential for exogenous androstenedione to produce adverse effe fertility and reproductive performance. The genetic toxicity studies in bacteria and cts on male rodents reported here were negative, with the exception of an equivocal response in the peripheral blood micronucleus test in the high dose female mice exposed to androstenedione for 14 weeks. These results are in agreement with other published genetic toxicity test results (McKillop et al., 1983
In the chronic study, there were statistically significant increased incidences of MCL, a common neoplasm in female F344/N rats (Haseman et al., 1998
). Due to the low incidence in vehicle controls (10%) compared to the historical range for all routes (8% to 40%; mean, 22%) the relationship of this increased incidence to androstenedione administration was uncertain. Androstenedione also significantly increased the incidence of alveolar/bronchiolar adenoma or carcinoma (combined) in mid-dose male rats. Since these lung neoplasms were only marginally increased in the mid-dose (20 mg/kg) group and there was no reduction in survival or body weight in the 50 mg/kg group, it is unclear if the marginally increased incidence in the mid-dose group was compound related because of the lack of a dose response.
Interestingly, there were significant decreases in the incidences of mammary gland hyperplasia, mammary gland cysts, and mammary gland adenomas in female rats in the chronic study, which suggests that androstenedione administration ameliorated these lesions’ endocrine mechanism(s). The decrease in the incidences of testicular interstitial cell adenoma in F344/N male rats, a neoplasm common to this strain (Haseman et al., 1998
), also suggests that androstenedione administration had a similar effect in this 2-year study. The origination of testicular interstitial cell adenoma is thought to be through endocrine-mediated mechanisms (Cook et al., 1999
), which androstenedione administration may have alleviated. Oxymetholone administration had a similar effect on testicular adenomas in rats (NTP, 1999
). A previous study that administered testosterone, estradiol, or a luteinizing hormone (LH) receptor agonist to Fisher 344 rats demonstrated that these hormones reduced the high levels of circulating LH in older animals and inhibited formation of Leydig cell adenomas (Chatani et al., 1990
). Androstenedione, or an androstenedione metabolite, likely had a similar effect on circulating LH levels, which is believed to be a stimulating factor for interstitial cell adenoma formation in the Fisher 344/N rat testis.
In the chronic mouse study, androstenedione was carcinogenic in male and female mouse liver. Although B6C3F1 mice have a high background rate of liver tumors, males more so than females, the increase in adenomas, multiple adenomas and carcinomas, and marginal increase in hepatoblastomas indicates carcinogenic activity in the male mouse liver. In female mice, androstenedione increased the incidence of adenomas and carcinomas and multiple carcinomas. In contrast, male and female rats displayed no evidence of androstenedione-induced hepatocellular neoplasms, which may be due to differences in metabolism between the two species and/or genetic susceptibility. Androstenedione carcinogenicity within the liver is consistent with other androgens, which are known hepatocellular carcinogens (IARC, 1987
). The incidence of these neoplasms in mice might have declined if androstenedione administration had been stopped. Stopping treatment with oxymetholone, a human liver carcinogen, results in regression of hepatic tumors (Fremond et al., 1987
; Montgomery et al., 1980
; Obeid et al., 1980
; Velazquez and Alter, 2004
). The androgen receptor also contributes to tumor promotion in the liver. Male mice lacking a functional androgen receptor in the liver have a considerably lower prevalence of hepatocellular tumors after a N,N
-diethylnitrosamine challenge compared to wildtype mice (Kemp et al., 1989
The increase of pancreatic islet cell adenomas, a rare neoplasm, was considered related to androstenedione administration due to the incidence in the 50 mg/kg group exceeding concurrent and historical control rates from all routes of administration. Male mice had a similar increase at the high dose, which also was considered related to androstenedione administration since it exceeded the historical control range from all routes of exposure. In addition, the day of first incidence decreased with increasing dose in male mice, which is supportive of an effect in the pancreas. It is not clear by what mechanism androstenedione administration would induce these neoplasms, but the mouse pancreatic islet β cells express the androgen receptor, which may play a role in β-cell proliferation (Li et al., 2008
). CYP 17, the steroidogenic enzyme responsible for converting progestins to androgens, has been identified in rat pancreatic islets (Ogishima et al., 2008
), but has yet to be identified in mouse islet cells. The relation of androgens, pancreatic islet cells, and insulin is not well understood, but it is of interest due to polycystic ovary syndrome, in which individuals have increased levels of circulating androstenedione, and decreased insulin sensitivity (Schuring et al., 2008
The decrease in the incidence of malignant lymphoma in female mice, a common type of neoplasm for this strain (Haseman et al., 1998
), may be due to endocrine action since the immune system is sensitive to steroids (Beagley and Gockel, 2003
; Bouman et al., 2005
). Male B6C3F1 mice have a considerably lower background rate of malignant lymphoma compared to the female mice (average: 3% versus 21% for all routes), and the androgen action of androstenedione could be responsible for reducing malignant lymphoma incidence in female mice.
Androstenedione administration resulted in histological changes, indicating masculinization, in the kidney (), submandibular salivary gland (), and clitoral gland of female mice. The submandibular salivary gland is a sexually dimorphic and androgen-sensitive tissue. The withdrawal of androgens in male mice results in demasculinization within this gland and administration of androgens to females induces masculinization (Chretien, 1977
; Kronman and Spinale, 1965
; Sawada and Noumura, 1991
). In addition to the submandibular gland changes, androstenedione administration resulted in glomerulus metaplasia in female mice, an appearance similar to that of the male mouse. This effect within the female mouse glomerulus was also noted after oxymetholone administration (NTP, 1999
). The hormonal responsiveness of the clitoral gland to androgens is not well understood (Traish et al., 2002
), but the increased incidence of clitoral gland hyperplasia in female mice may be related to the androgenic effects of androstenedione.
There were similar findings between oxymetholone and androstenedione in the chronic exposure rat gavage studies (), but there were some noted differences. Oxymetholone is a potent anabolic steroid, but it displays poor androgen receptor binding, while androstenedione binds the androgen receptor, albeit less potently than dihydrotestosterone, and displays limited evidence of an anabolic effect (Jasuja et al., 2005
; Kicman, 2008
; Saartok et al., 1984
). In the chronic study, oxymetholone increased neoplasm incidences in the liver and skin of female rats, while androstenedione did not. The differences in liver tumor incidence may be related to differences in metabolism. The 17′ methyl group within oxymetholone decreases the rate of liver metabolism and 17α-alkylated androgens have side effects of hepatotoxicity (Snyder, 2001
). However, androstenedione, as an endogenous non-17′ methylated hormone, likely has a faster metabolism. The skin neoplasms observed with oxymetholone-treated female rats may be related to oxymethalone’s anabolic activity, as anabolic steroids induce skin lesions, and oxymetholone accumulates within this site (NTP, 1999
). In contrast there is limited evidence of anabolic activity by androstenedione (Jasuja et al., 2005
). The effects of diet, NIH-07 for oxymetholone versus NTP-2000 for androstenedione, on outcome are not known, but dietary differences may also be a contributing factor in differences between the two studies.
Comparison of chronic exposure results between oxymetholone (F344/N rats) and androstenedione (F344/N rats and B6C3F1 mice)
In summary, there was clear evidence of androstenedione carcinogenic activity in the mouse liver, which is generally consistent with other androgens. The pancreatic islet adenomas in male and female mice were considered to be related to androstenedione administration. The incidences, both increases and decreases, of neoplasia in rodents administered androstenedione were generally similar to oxymetholone, an anabolic androgen. The exceptions may be due to differences in androgen receptor binding, metabolism, and anabolic activity. In male and female rats, there was equivocal evidence of carcinogenic activity based on lung neoplasms and mononuclear cell leukemia, respectively. Androstenedione administration reduced the incidence of neoplasms in several tissues that are well known endocrine targets, suggesting an ameliorative effect within these tissues, possibly due to compensation for adverse endocrine-mediated mechanisms that arise during the aging process.