Analysis of anabolic steroids in hair: Time courses in guinea pigs

Sensitive, specific, and reproducible methods for the quantitative determination of eight anabolic steroids in guinea pig hair have been developed using LC/MS/MS and GC/MS/MS. Methyltestosterone, stanozolol, methandienone, nandrolone, trenbolone, boldenone, methenolone and DHEA were administered intraperitoneally in guinea pigs. After the first injection, black hair segments were collected on shaved areas of skin. The analysis of these segments revealed the distribution of anabolic steroids in the guinea pig hair. The major components in hair are the parent anabolic steroids. The time courses of the concentrations of the steroids in hair (except methenolone, which does not deposit in hair) demonstrated that the peak concentrations were reached on days 2-4, except stanozolol, which peaked on day 10 after administration. The concentrations in hair appeared to be related to the physicochemical properties of the drug compound and to the dosage. These studies on the distribution of drugs in the hair shaft and on the time course of their concentration changes provide information relevant to the optimal time and method of collecting hair samples. Such studies also provide basic data that will be useful in the application of hair analysis in the control of doping and in the interpretation of results.     

Gas chromatographic and mass spectrometric analysis of conjugated steroids in urine

This study was carried out qualitatively and quantitatively to investigate the presence and the concentrations of anabolic steroids in urine collected from orally administered humans. Microanalysis of conjugated steroids by gas chromatography and mass spectrometry (GC/MS) has been carried out. Following oral administration three major metabolites of anabolic steroid drugs have been detected and partially characterized. The six steroids can be analysed at the same time in 17 min. The lower detection limit was 10 ng/ml in 5 ml of urine. The conjugated steroids from urine were centrifuged to 2,430g for 10 min, the supernatant solution passed through Amberlite XAD-2 column and the steroids eluted fraction esterified by using MSTFA and TMSI. The rate of metabolism and urinary excretion seem to be reasonably fast.     

Reference ranges for urinary concentrations and ratios of endogenous steroids, which can be used as markers for steroid misuse, in a Caucasian population of athletes

The detection of misuse with naturally occuring steroids is a great challenge for doping control laboratories. Intake of natural anabolic steroids alters the steroid profile. Thus, screening for exogenous use of these steroids can be established by monitoring a range of endogenous steroids, which constitute the steroid profile, and evaluate their concentrations and ratios against reference ranges. Elevated values of the steroid profile constitute an atypical finding after which a confirmatory IRMS procedure is needed to unequivocally establish the exogenous origin of a natural steroid. However, the large inter-individual differences in urinary steroid concentrations and the recent availability of a whole range of natural steroids (e.g. dehydroepiandrosterone and androstenedione) which each exert a different effect on the monitored parameters in doping control complicate the interpretation of the current steroid profile.The screening of an extended steroid profile can provide additional parameters to support the atypical findings and can give specific information upon the steroids which have been administered.The natural concentrations of 29 endogenous steroids and 11 ratios in a predominantly Caucasian population of athletes were determined. The upper reference values at 97.5%, 99% and 99.9% levels were assessed for male (n = 2027) and female (n = 1004) populations. Monitoring minor metabolites and evaluation of concentration ratios with respect to their natural abundances could improve the interpretation of the steroid profile in doping analysis.     

Speeding up the screening of steroids in urine: Development of a user-friendly library

This work presents a novel database search engine – MLibrary – designed to assist the user in the detection and identification of androgenic anabolic steroids (AAS) and its metabolites by matrix assisted laser desorption/ionization (MALDI) and mass spectrometry-based strategies. The detection of the AAS in the samples was accomplished by searching (i) the mass spectrometric (MS) spectra against the library developed to identify possible positives and (ii) by comparison of the tandem mass spectrometric (MS/MS) spectra produced after fragmentation of the possible positives with a complete set of spectra that have previously been assigned to the software. The urinary screening for anabolic agents plays a major role in anti-doping laboratories as they represent the most abused drug class in sports. With the help of the MLibrary software application, the use of MALDI techniques for doping control is simplified and the time for evaluation and interpretation of the results is reduced. To do so, the search engine takes as input several MALDI-TOF–MS and MALDI-TOF–MS/MS spectra. It aids the researcher in an automatic mode by identifying possible positives in a single MS analysis and then confirming their presence in tandem MS analysis by comparing the experimental tandem mass spectrometric data with the database. Furthermore, the search engine can, potentially, be further expanded to other compounds in addition to AASs. The applicability of the MLibrary tool is shown through the analysis of spiked urine samples.     

Detection of new exemestane metabolites by liquid chromatography interfaced to electrospray-tandem mass spectrometry

Exemestane is an irreversible aromatase inhibitor used for anticancer therapy. Unfortunately, this drug is also misused in sports to avoid some adverse effects caused by steroids administration. For this reason exemestane has been included in World Anti-Doping Agency prohibited list. Usually, doping control laboratories monitor prohibited substances through their metabolites, because parent compounds are readily metabolized. Thus metabolism studies of these substances are very important. Metabolism of exemestane in humans is not clearly reported and this drug is detected indirectly through analysis of its only known metabolite: 17β-hydroxyexemestane using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and gas chromatography coupled to mass spectrometry (GC-MS). This drug is extensively metabolized to several unknown oxidized metabolites. For this purpose LC-MS/MS has been used to propose new urinary exemestane metabolites, mainly oxidized in C6-exomethylene and simultaneously reduced in 17-keto group. Urine samples from four volunteers obtained after administration of a 25mg dose of exemestane were analyzed separately by LC-MS/MS. Urine samples of each volunteer were hydrolyzed followed by liquid-liquid extraction and injected into a LC-MS/MS system. Three unreported metabolites were detected in all urine samples by LC-MS/MS. The postulated structures of the detected metabolites were based on molecular formulae composition obtained through high accuracy mass determination by liquid chromatography coupled to hybrid quadrupole-time of flight mass spectrometry (LC-QTOF MS) (all mass errors below 2ppm), electrospray (ESI) product ion spectra and chromatographic behavior.     

Anabolic steroids: a review of their effects on the muscles, of their possible mechanisms of action and of their use in athletics.

Anabolic steroids are synthetic molecules developed in the hope of obtaining a complete separation of the androgenic and myotrophic (anabolic) actions of testosterone. Such a goal has never been fully achieved. However, some synthetic steroids present a partial dissociation between these two activities. Since a single hormonal receptor apparently mediates the androgenic as well as the anabolic actions of testosterone, differences in patterns of androgen metabolization in the muscles and the sex accessory organs have been proposed as a possible cause of this phenomenon. Undoubtedly, androgens are able to exert a trophic effect on skeletal and cardiac muscle fibres in subjects with low circulating levels of testosterone such as prepubertal or hypogonadal males and females; however, the widespread use of anabolic steroids in male athletes to increase their physical performances poses the question of whether these compounds are active in the presence of normal circulating levels of testosterone. Most experimental animal studies indicate that anabolic steroids are ineffective in this situation. Since the results of the experiments performed in humans are largely contradictory, it is still not clear whether anabolic steroids are able to improve athletic performances. These and other relevant issues are reviewed.     

Athletic aids: fact or fiction?

A major problem facing physicians involved in the daily care of amateur athletes in international competition is that of so-called doping. While there are many substances used that might be considered ineffectual or physiologic, the two main categories of substances considered as doping agents are stimulants and anabolic steroids. No substance is as yet known that will improve an athlete''s performance. Often such substances are taken in error rather than in a misguided attempt to improve one''s ability. These substances can be dangerous and, because of this, doping rules were established basically to protect the athlete. Anabolic steroids are particularly dangerous; they usually are taken by those engaged in lifting or throwing sports in an attempt to improve performance by increasing muscle bulk. There is as yet no scientific proof that performance is improved. Dope testing for stimulants should be carried out in a quantitative rather than qualitative manner so that the athlete who has taken a small amount of a so-called stimulant (such as an asthma or common cold preparation) would not be excluded from competition. Rigid testing for anabolic steroids should be continued.     

Gas chromatographic–tandem mass spectrometric determination of anabolic steroids and their esters in hair: Application in doping control and meat quality control

We have developed a powerful and simple sensitive method for testing hair for anabolic steroids and their esters. A 100-mg amount of powdered hair was treated with methanol in an ultrasonic bath for extraction of esters, then alkaline digested with 1 M NaOH for an optimum recovery of other drugs. The two liquid preparations were subsequently extracted with ethyl acetate, pooled, then finally highly purified using a twin solid-phase extraction on amino and silica cartridges. The residue was derivatized with N-methyl-N(trimethylsilyl)-trifluoracetamide (MSTFA) prior to injection. Analysis was conducted by gas chromatography coupled to a triple quadrupole mass spectrometer. The generally chosen parent ion was the molecular ion while two daughter ions were selected for each compound with collision energies ranging from −16 to −21 eV. Internal standards were nandrolone d₃ for non-esterified drugs and testosterone phenyl propionate for esters. The limits of detection calculated from an analysis of the blanks (n=30) were 0.08 pg/mg for nandrolone, 6.20 pg/mg for boldenone, 0.07 pg/mg for methyl testosterone, 0.15 pg/mg for ethinyl estradiol, 2.10 pg/mg for metandienone, 0.86 pg/mg for testosterone propionate, 0.95 pg/mg for testosterone cypionate, 1.90 pg/mg for nandrolone decanoate, 3.10 pg/mg for testosterone decanoate and 4.80 pg/mg for testosterone undecanoate. Application to doping control has been demonstrated. In a series of 18 sportsmen, two tested positive for anabolic steroids in hair whereas urinalysis was negative for both of them. The first positive case was nandrolone and the second case concerned the identification of testosterone undecanoate. Measured in 10 white males aged between 22 and 31 years, the testosterone concentration was in the range 1.7–9.2 pg/mg (mean=5.0 pg/mg). The method was also applied in meat quality control. Of the 187 analyses realized based upon hair and urine sampling in slaughter houses, 23 were positive for anabolic steroids in hair: one case for boldenone, one case for metandienone, two cases for testosterone propionate, three cases for nandrolone, five cases for testosterone decanoate and 11 cases for methyl testosterone. In the meantime, urinalysis was always negative for these drugs or their metabolites.     

The impact of nandrolone decanoate and growth hormone on biosynthesis of steroids in rats

Growth hormone (GH) and anabolic androgenic steroids (AAS) are commonly used in sports communities. Several studies have suggested an association between GH and AAS. We have investigated the impact of GH in rats treated with nandrolone decanoate (ND). Male Wistar rats received ND (15 mg/kg) every third day during three weeks and were subsequently treated with recombinant human GH (1.0 IU/kg) for ten consecutive days. Plasma samples were collected and peripheral organs (i.e. heart, liver, testis and thymus) were dissected and weighed. Concentration of thirteen endogenous steroids was measured in the rat plasma samples using high specificity LC–MS/MS methods. Seven steroids were detected and quantified, and concentrations of estrone, testosterone, and androstenedione were significantly different among the groups, while concentrations of pregnenolone, DHEA, 17-hydroxyprogesterone and corticosterone were not altered. Administration of rhGH alone altered the plasma steroid distribution, and the results demonstrated significantly increased concentrations of plasma estrone as well as decreased concentrations of testosterone and androstenedione in the ND-treated rats. Administration of rhGH to ND-pretreated rats did not reverse the alteration of the steroid distribution induced by ND. Administration of ND decreased the weight of the thymus, and addition of rhGH did not reverse this reduction. However, rhGH administration induced an enlargement of thymus. Taken together, the plasma steroid profile differed in the four groups, i.e. control, AAS, rhGH and the combination of AAS and rhGH treatment.     

Ecdysteroids: A novel class of anabolic agents?

Increasing numbers of dietary supplements with ecdysteroids are marketed as “natural anabolic agents”. Results of recent studies suggested that their anabolic effect is mediated by estrogen receptor (ER) binding. Within this study the anabolic potency of ecdysterone was compared to well characterized anabolic substances. Effects on the fiber sizes of the soleus muscle in rats as well the diameter of C2C12 derived myotubes were used as biological readouts. Ecdysterone exhibited a strong hypertrophic effect on the fiber size of rat soleus muscle that was found even stronger compared to the test compounds metandienone (dianabol), estradienedione (trenbolox), and SARM S 1, all administered in the same dose (5 mg/kg body weight, for 21 days). In C2C12 myotubes ecdysterone (1 µM) induced a significant increase of the diameter comparable to dihydrotestosterone (1 µM) and IGF 1 (1.3 nM). Molecular docking experiments supported the ERβ mediated action of ecdysterone. To clarify its status in sports, ecdysterone should be considered to be included in the class “S1.2 Other Anabolic Agents” of the list of prohibited substances of the World Anti-Doping Agency.     

Lipid profile of body builders with and without self-administration of anabolic steroids

Twenty-four top-level body builders [13 anabolic steroid users (A); 11 non-users (N)] and 11 performance-matched controls (C) were examined to determine the effect on lipids, lipoproteins and apolipoproteins of many years of body building with and without simultaneous intake of anabolic steroids and testosterone. After an overnight fast, triglycerides (TG), total cholesterol (TOTC), high density lipoprotein cholesterol (HDLC), low density lipoprotein cholesterol (LDLC), the HDLC subfractions HDL2C and HDL3C, as well as apolipoprotein A-I (Apo A-I), apolipoprotein A-II (Apo A-II) and apolipoprotein B (Apo B) were determined. Both A and N, compared to C, showed significantly lower HDLC and higher LDLC concentrations, with the differences between A and C clearly pronounced. In a subgroup of 6 body builders taking anabolic steroids at the time of the study, HDLC, HDL2C, HDL3C, Apo A-I and Apo A-II were all significantly lower and LDLC was significantly higher than in a second subgroup of 7 body builders who had discontinued their intake of anabolic steroids at least 4 weeks prior to the study. In some single cases HDLC was barely detectable (2-7 mg.dl-1). The TG and TOTC remained unchanged. The present findings suggest that many years of body building among top-level athletes have no beneficial effect on lipoproteins and apolipoproteins. Simultaneous use of anabolic steroids results in part in extreme alterations in lipoproteins and apolipoproteins, representing an atherogenic profile. After discontinuing the use of anabolic steroids, the changes in lipid metabolism appear to be reversible.     

Anabolic steroids abuse and male infertility

For several decades, testosterone and its synthetic derivatives have been used with anabolic and androgenic purposes. These substances were first restricted to professional bodybuilders, but become more and more popular among recreational athletes. Up to date, 3,000,000 anabolic-androgenic steroids (AAS) users have been reported in the United States with an increasing prevalence, making AAS consumption a major public health growing concern. Infertility is defined by the WHO as the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse and a male factor is present in up to 50 % of all infertile couples. Several conditions may be related to male infertility.Substance abuse, including AAS, is commonly associated to transient or persistent impairment on male reproductive function, through different pathways. Herein, a brief overview on AAS is offered. Steroids biochemistry, patterns of use, physiological and clinical issues are enlightened. A further review about fertility outcomes among male AAS abusers is also presented, including the classic reports on transient anabolic steroid-induced hypogonadism (ASIH), and the more recent experimental reports on structural and genetic sperm damage.     

Effects of steroids on the brain opioid system

The experiments reported here add further evidence in support of the view that sex steroids may influence the binding characteristics of brain opioid receptors. In particular, it has been shown that: (a) the number of μ-opioid receptors varies in the hypothalamus of regularly cycling female rats according to the different phases of the estrous cycle, which are characterized by fluctuations of circulating levels of sex steroids; (b) the number of μ-opioid receptors decreases in the hypothalamus and in the corpus striatum when ovariectomized rats are submitted to treatments with estradiol and progesterone able to induce a “positive” feedback effect on LH release. A treatment with estrogen alone able to induce a “negative” feedback effect on LH release brings about an increase of the number of μ-opioid receptors in the thalamus and in the hippocampus; (c) in addition to the μ-receptors, receptors of the delta type may also be involved in the control of gonadotropin secretion; recent results here presented indicate that a line of immortalized hypothalamic cells (GT1 cells), which synthesize and secrete LHRH, present δ opioid receptors on their membranes; these are apparently involved in the control of LHRH release from these cells.     

Telmisartan as metabolic modulator: a new perspective in sports doping?

The World Antidoping Agency (WADA) has introduced some changes in the 2012 prohibited list. Among the leading innovations to the rules are that both 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (peroxisome proliferator-activated receptor-δ [PPAR-δ]-5' adenosine monophosphate-activated protein kinase [AMPK] agonist) and GW1516 (PPAR-δ-agonist) are no longer categorized as gene doping substances in the new 2012 prohibited list but as metabolic modulators in the class "Hormone and metabolic modulators." This may also be valid for the angotensin II receptor blocker telmisartan. It has recently been shown that telmisartan might induce similar biochemical, biological, and metabolic changes (e.g., mitochondrial biogenesis and changes in skeletal muscle fiber type) as those reported for the former call of substances. We suspect that metabolic modulators abuse such as telmisartan might become a tangible threat in sports and should be thereby targeted as an important antidoping issue. The 2012 WADA prohibited list does not provide telmisartan for a potential doping drug, but arguments supporting the consideration to include them among "metabolic modulators" are at hand.     

Detection of anabolic steroid abuse using a yeast transactivation system

The classical analytical method for detection of anabolic steroid abuse is gas chromatography followed by mass spectrometry (GC/MS). However, even molecules with a chemical structure typical for this class of substances, are sometimes not identified in routine screening by GC/MS when their precise chemical structure is still unknown. A supplementary approach to identify anabolic steroid abuse could be a structure-independent identification of anabolic steroids based on their biological activity. To test the suitability of such a system, we have analyzed the yeast androgen receptor (AR) reporter gene system to identify anabolic steroids in human urine samples. Analysis of different anabolic steroids dissolved in buffer demonstrated that the yeast reporter gene system is able to detect a variety of different anabolic steroids and their metabolites with high specificity, including the so-called 'designer steroid' tetrahydrogestrinone. In contrast, other non-androgenic steroids, like glucocordicoids, progestins, mineralocordicoids and estrogens had a low potency to stimulate transactivation. To test whether the system would also allow the detection of androgens in urine, experiments with spiked urine samples were performed. The androgen reporter gene in yeast responds very sensitive to 5alpha-dihydrotestosterone (DHT), even at high urine concentrations. To examine whether the test system would also be able to detect anabolic steroids in the urine of anabolic steroid abusers, anonymous urine samples previously characterized by GCMS were analyzed with the reporter gene assay. Even when the concentration of the anabolic metabolites was comparatively low in some positive samples it was possible to identify the majority of positive samples by their biological activity. In conclusion, our results demonstrate that the yeast reporter gene system detects anabolic steroids and corresponding metabolites with high sensitivity even in urine of anabolic steroid abusing athletes. Therefore we believe that this system can be developed towards a powerful (pre) screening tool for the established doping tests. The system is easy to handle, robust, cost-efficient and needs no high-tech equipment. But most importantly, a biological test system does not require knowledge of the chemical structure of androgenic substances and therefore suitable to detect previously unidentified substances, especially those of the class of so-called designer steroids.     

Structural specificity of steroids in stimulating DNA synthesis and protooncogene expression in primary rat hepatocyte cultures

Among the chemical compounds of varied structure which possess liver tumour-promoting are steroids, such as estrogens, pregnenolone derivatives and anabolic steroids. Although the mechanism(s) of tumour promotion in liver by these xenobiotics is not well understood, it is clear that growth stimulation is one important element in their action. As a basis for better defining whether steroids stimulate growth by a common mechanism or fall into sub-groups with differing actions, the effects of 46 steroids on DNA synthesis and the expression of protooncogenes c-fos and c-myc were examined in primary cultures of normal rat hepatocytes. Tentative groupings of steroids have been identified based on apparent structural requirements for stimulation of DNA synthesis, and effects of auxiliary factors in modulating this growth stimulus. For a "progestin" group, insulin appeared to be permissive for stimulation of DNA synthesis, and presence of an ester or hydroxyl group at 17alpha-position in combination with a non-polar group at C(6) appeared to be required for stimulation. For the pregnenes, dexamethasone was stimulatory. Structural requirements include a non-polar substitution at 16alpha-position and presence of a 6alpha-methyl group. Androgens were weak or ineffective stimulators of DNA synthesis. Anabolic steroids were weak to strong stimulators and alteration to A ring structure in combination with non-polar substitution at 17alpha-position appeared to be required for the activity. With the exception of the anabolic steroid, dianabol, there do not appear to be strong correlation between ability to stimulate DNA synthesis and ability to induce protooncogene expression among the steroids. This study provides a starting point for future more detailed examination of growth-stimulatory mechanism(s) of action of steroids in the liver.     

Anabolic steroid usage in athletics: facts, fiction, and public relations

Anecdotal evidence suggests the widespread usage of anabolic steroids among athletes (20-90%), particularly at the professional and elite amateur levels. In contrast, scientific studies indicate that usage is rare and no higher than 6%. Conclusions from scientific studies suggest that anabolic steroid usage declines progressively from high school to college and beyond; however, anecdotal evidence claims the opposite trend. In this clash between "hard" scientific data vs. "soft" anecdotal information, it is natural that professionals would gravitate toward scientifically based conclusions. However, in the case of anabolic steroids (a stigmatized and illegal substance), should word-of-mouth testimony from individuals closest to the issues--those who have participated in and coached sports, those who have served as drug-testing overseers, and journalists who relentlessly track leads and verify sources--be set aside as irrelevant? Not if a complete picture is to emerge. In this review, hard scientific evidence is placed on the table side-by-side with soft anecdotal evidence, without weighting or bias. The purpose is to allow the opportunity for each to illuminate the other and, in so doing, potentially bring us a step closer to determining the true extent of anabolic steroid usage in athletics.     

Detection, quantification and confirmation of anabolic steroids in equine plasma by liquid chromatography and tandem mass spectrometry

Anabolic androgenic steroids are related to the male sex hormones and are abused in equine sports. In an effort to deter the abuse of anabolic steroids, a sensitive LC-MS/MS method was developed for detection, quantification and confirmation of eight major anabolic steroids (testosterone, normethandrolone, nandrolone, boldenone, methandrostenolone, tetrahydrogestrinone (THG), trenbolone, and stanozolol) in equine plasma. Formation of solvent adduct ions of the analytes was observed under electrospray ionization (ESI) conditions, and desolvation of the solvent adduct ions by source collision-induced decomposition (CID) increased the abundance of the [M+H]+ ions as well as the multiple-reaction monitoring (MRM) signals. ESI (+) and APCI (+) were compared with respect to sensitivity for the analytes and the former provided better sensitivity. The matrix effect on ion suppression or enhancement was evaluated, and was negligible. Confirmation of the analytes was performed using criteria of three ion transitions and LC retention time of each analyte. The limit of detection (LOD) and quantification (LOQ) was 25 pg/mL. The limit of confirmation (LOC) was 25 pg/mL for boldenone; 50 pg/mL for normethandrolone, nandrolone, and methandrostenolone; and 100 pg/mL for testosterone, THG, trenbolone, and stanozolol. The analytes were evaluated for stability and found to be stable in plasma for 24h at room temperature, 13 days at 4 degrees C, and 34 days at -20 and -70 degrees C. The method was successfully applied to analyses of equine plasma samples for pharmacokinetics study. This method is sensitive and useful for detection, quantification and confirmation of these anabolic steroids in equine plasma.     

Vasopressin differentially modulates aggression and anxiety in adolescent hamsters administered anabolic steroids

Adolescent Syrian hamsters (Mesocricetus auratus) treated with anabolic/androgenic steroids display increased offensive aggression and decreased anxiety correlated with an increase in vasopressin afferent development, synthesis, and neural signaling within the anterior hypothalamus. Upon withdrawal from anabolic/androgenic steroids, this neurobehavioral relationship shifts as hamsters display decreased offensive aggression and increased anxiety correlated with a decrease in anterior hypothalamic vasopressin. This study investigated the hypothesis that alterations in anterior hypothalamic vasopressin neural signaling modulate behavioral shifting between adolescent anabolic/androgenic steroid-induced offensive aggression and anxiety. To test this, adolescent male hamsters were administered anabolic/androgenic steroids and tested for offensive aggression or anxiety following direct pharmacological manipulation of vasopressin V1A receptor signaling within the anterior hypothalamus. Blockade of anterior hypothalamic vasopressin V1A receptor signaling suppressed offensive aggression and enhanced general and social anxiety in hamsters administered anabolic/androgenic steroids during adolescence, effectively reversing the pattern of behavioral response pattern normally observed during the adolescent exposure period. Conversely, activation of anterior hypothalamic vasopressin V1A receptor signaling enhanced offensive aggression in hamsters exposed to anabolic/androgenic steroids during adolescence. Together, these findings suggest that the state of vasopressin neural development and signaling in the anterior hypothalamus plays an important role in behavioral shifting between aggression and anxiety following adolescent exposure to anabolic/androgenic steroids.