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intense bodybuilder curlingby: Karl Hoffman

Anabolic Steroid Action In Vitro and In Vivo the traditional view of anabolic steroids is that their anabolic-androgenic potency is to a large extent dependent on the relative binding affinity of a given steroid to the androgen receptor. In an earlier edition of Mind and Muscle Magazine I reviewed some recent research suggesting that while binding affinity is still a factor controlling the action of anabolic steroids, the particular genes activated by any given steroid may be just as important or even more so.

There I made the comments that:

Surprisingly, despite the number of synthetic AAS that have been developed, their modes of action are poorly understood. This holds for the naturally occurring androgens as well. There is some evidence (which we will discuss below) that androgens are able to exert some of their actions independently of the androgen receptor (AR). Antagonism of the glucocorticoid receptor is one possible way androgens may exert an anabolic effect.
Binding affinity to the androgen receptor has also been invoked to explain the differences in potencies and effects of the natural and synthetic androgens. For example, dihydrotestosterone binds the androgen receptor much more strongly than does testosterone at the same concentration, yielding a higher degree of ligand-receptor stability. When the concentration of testosterone is increased however, the receptor stability increases to a level similar to that seen with dihydrotestosterone (1). This has led to the proposal that the weaker androgenic potency of testosterone compared to that of dihydrotestosterone in target tissues such as the prostate resides in testosterone’s weaker interaction with the androgen receptor. Yet it is well known that some steroids which are very potent anabolic agents, such as stanozolol or oxymetholone, bind the AR only very weakly (2). If we assume that AR binding affinity is the sole determinant of an agent’s ability to act via the AR to promote anabolic or androgenic actions, then we are forced into the conclusion that certain potent AAS that bind the AR with negligible affinity must be exerting their anabolic effects via some other routes that do not involve AR binding. Indeed, this has become to a large degree dogma in the bodybuilding literature.
Some interesting recent research has shed light on this problem by showing that AR binding affinity is only partly responsible for the androgen receptor mediated effects of both physiologic androgens and synthetic AAS. In the study I would like to discuss, the authors present evidence for the existence of distinct steroid specific target gene transcription profiles following AR activation (3). In other words, the structures of androgen responsive genes vary in such a way that some genes are more readily activated by certain androgens than by others. The set of genes readily switched on by a given androgen determines the net physiological effect of that androgen. This theory readily explains how an anabolic steroid like oxandrolone, whose AR binding affinity is quite low, can be so anabolic: it happens to preferentially turn on genes whose products promote skeletal muscle anabolism, while failing to activate genes which promote virilization.

In this paper I would like to discuss a different but not necessarily contradictory explanation of why some steroids that appear to bind only weakly to the AR still manage to exert potent anabolic and androgenic effects. The solution to the apparent paradox is a simple one: virtually all binding affinity studies to date have been carried out in vitro. Here we will look at the recently published research by Feldkoren and Andersson [1] who compared the in vivo and in vitro actions of the anabolic steroids stanozolol (Winstrol) and methanedienone (Dianabol). We will see that the interactions of these anabolic steroids with the AR are much different when measured in vitro when compared to measurements carried out in vivo.

In [1] the authors examined the interaction of the above mentioned steroids with the AR by using three different systems: (1) a recombinant AR ligand binding in vitro assay (the modern standard method of expressing binding affinities); (2) a cell based AR-dependent transactivation assay; and (3) an in vivoassay based on steroid induced cytosolic AR depletion in skeletal muscle. The logic behind system (3) is that normally the unbound AR resides in the cytosol of the cell. Upon ligand (steroid) binding the ligand-receptor complex translocates to the nucleus. So the degree of cytosolic depletion of the AR when exposed to a particular steroid serves as a measure of the degree of binding of the steroid to its receptor. The binding affinities of testosterone and 17-alpha methyltestosterone were examined as well.

System (1) measured the in vitro binding strengths of the given steroids by determining how effectively they displaced radiolabeled methyltrienolone (MT) from the recombinant AR. Methyltrienolone binds extremely strongly to the AR and to what degree it can be displaced from the AR serves as the standard assay for measuring the binding strength of any given androgen receptor ligand.

 

By observing the amount of displaced radiolabeled MT as a function of the concentration of the competing steroid, it’s possible to calculate the binding affinity of the competitor. The affinity is usually quantified as the equilibrium dissociation constant, Ki. The subscript i is used to indicate that the competitor inhibited radioligand binding. The Ki is the concentration of the competing ligand that will bind to half the binding sites at equilibrium, in the absence of radioligand or other competitors. If the Ki is low, the affinity of the receptor for the inhibitor is high, and vice versa.

Not surprisingly MT possessed the highest affinity with a Ki of 0.20 nM, the Ki for testosterone and 17alpha-methyltestosterone were 0.80 and 0.90 nM, respectively. Both stanozolol and methanedienone were the least effective competitors with a Ki for stanozolol of 4.5nM and methanedienone of 5.0 nM. The calculated in vitro binding affinities of the various agents are depicted below in Figure 1, excerpted from [1].


Fig. 1. Binding strength of various steroids to the recombinant rat AR. The data presented in Fig. 1 were analyzed to calculate the equilibrium dissociation constant Ki for each steroid mentioned above. T, testosterone; MT, methyltrienolone; 17alpha-MeT, 17alpha-methyltestosterone; S, stanozolol; MA, methanedienone.

So here we see the expected picture from all we have read about various anabolic steroids; namely, certain oral steroids like Winstrol and Dianabol only bind relatively weakly (compared to testosterone, for example) to the AR yet are well known to be quite potent.

System (2) employed a full length recombinant AR and a section of DNA consisting of the androgen response element (the portion of a gene to which the AR-ligand complex binds) spliced to a luciferase reporter gene. These are then inserted into a cell. Luciferase emits light when activated, and the idea here is that when the cell containing this artificial gene complex is exposed to a given steroid, the steroid will bind to the AR, attach to the artificial gene construct, and activate the luciferase. The amount of light emitted is a measure of the binding of the AR-ligand complex to the reporter gene, which in turn is a function of the strength of binding of the ligand to the AR.

Transcriptional activation is usually expressed in terms of so called EC50 of the ligand. EC50 is defined as the molar concentration of a ligand, which produces 50% of the maximum possible response for that ligand. Methyltrienolone was found to be the most effective transcriptional activator with an EC50 of 5 pM (picomoles), in agreement with its high affinity in vitro. The calculated EC50 values for the other steroids were 44pM for 17alpha-methyltestosterone, 52pM for stanozolol, and 79pM for methanedienone; all four steroids induced the same level of maximum transactivation. The researchers did not measure the EC50 of testosterone because it metabolized to the relatively inactive androgen androstenedione by the enzyme 17beta-hydroxysteroid dehydrogenase present in the type of cells used in the experiment. Hence, in intact cells, both stanozolol and methanedienone are potent activators of the AR, of the same order of magnitude as 17alpha-methyltestosterone.

Finally, to test the in vivo strength of the various steroids, rats were injected with each steroid at a dose 0.3 mg/kg of body weight. On hour after treatment, muscle cells were removed from the animals and the cytosolic depletion analysis of the AR was carried out. Methyltrienolone resulted in a 67% reduction of androgen binding sites in muscle cytosol. Stanozolol possessed almost as much activity (44% depletion) as testosterone (55%), followed by methanedienone, which caused a 33% reduction in binding sites. 17alpha-Methyltestosterone demonstrated the lowest degree of cytosolic AR depletion (11%) of all of the AS.

We see then a clear discrepancy between the typically published in vitro binding affinities of various anabolic steroids, and the ability of these steroids to evoke biological responses via classical androgen receptor mediated transcription in both cell based systems and in vivo.

It is interesting to compare the in vivo binding affinities obtained above with previously published binding data. Saartok et.al.[2] measured the binding affinities of a number of anabolic steroids relative to methyltrienolone (MT) using a system based on the binding of steroids to the AR in the cytosols obtained by grinding rat muscle and prostate tissue. Their relative binding affinities (RBA) were calculated as the ratio between the molar concentrations of unlabeled MT and of the competitor required to displace 50% of the radiolabeled MT from cytosolic binding sites (i.e. androgen receptors). If MT is arbitrarily given an RBA of 1, testosterone exhibited an RBA of 0.7; 17alpha-methyltestosterone’s was 0.10; stanozolol 0.03; and methanedienone 0.02.

We immediately see large differences in the results obtained in vivo in [1] compared to the in vitro data published in [2]. For example in [1] the binding affinities between MT and testosterone differed by only 18%. In [2] the difference was 30%. The disparity between studies for stanozolol and methanedienone is even greater. Looking at stanozolol, in [1] we see an affinity difference from MT of 34%. In [2] the difference is enormous, almost 2 orders of magnitude.

References

1. Feldkoren BI, Andersson S. Anabolic-androgenic steroid interaction with rat androgen receptor in vivo and in vitro: a comparative study. J Steroid Biochem Mol Biol. 2005 Apr;94(5):481-7.

2. Saartok T, Dahlberg E, Gustafsson JA. Relative binding affinity of anabolic-androgenic steroids: comparison of the binding to the androgen receptors in skeletal muscle and in prostate, as well as to sex hormone-binding globulin. Endocrinology. 1984 Jun;114 (6):2100-6.

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