Beyond Vida's Book, Part 2/2: Androgen, Progesterone, Estrogen & Corticosteroid Receptor Activities of ALLmost All Anabolic Steroids - Metabolits, Progestins & More

Image 1: Tetrahydrogestrinone aka THG,
aka "The Clear" and the bone of contention
in the "BALCO Scandal" could probably
have been identified years before, if the
WADA detectives already had the new
 mammalian androgen responsive reporter
gene assays at their disposal.

As mentioned in part 1 of this post (cf. Beyond Vida's Book, Part 1/2), Houtman and the other scientists from BioDetection Systems B.V. and the Institute of Public Health and the Environment in the Netherlands, did not exactly want to provide juicers with data on which steroids may cause unexpected side-effects, when they conducted their study in 2008. The original idea was to demonstrate that their technology would be able to identify even those androgens, which have hitherto not been classified as illegal substances - so called "designer steroids", which would not turn up in the chemical–analytical approaches combining gas chromatography (GC) or liquid chromatography (LC) separations with mass spectrometry (MS) or tandem mass spectrometry (MS/MS) simply because their molecular structure is hitherto unknown (you cannot find what you ain't looking for ;-).

This is the second part of a two part series, click here to read more about nandrolone, trenbolone, testosterone and all the androgens on the list of prohibited substances of the World Anti Doping Agency.

If this method had been available back in the day, when Patrick Arnold developed THG (tetrahydrogestrinone aka "The Clear") for BALCO, the detectives from the World Anti Doping Agency (WADA) would probably have been able to identify Marion Jones and other athletes who used this highly performance-enhancing drug, even before baseball star Berry Bonds blew the whistle in 2001.

Figure 1: Relative potency at the androgen (compared to testosterone), progesterone (compared to progesterone) and estrogen alpha and beta receptors (compared to estrogen) of "The Clear" (THG), trenbolone and nandrolone
(calculated based on data from Houtman. 2008)

Now, more than 10 years later, THG is on the list of WADA prohibited compounds and we know that "The Clear" is 0.25x as androgenic as dihydrotestosterone and 1.15x more androgenic than testosterone. We also know that THG is a powerful progestin, with 91% of the receptor activity of "real" progesterone (trenbolone has 35%), but hardly any translational activity at the level of the estrogen (0.0017% for estrogen-beta) and corticosteroid receptor.

Figure 2: Relative potency (new reference:
progesterone!) of steroids not on the
WADA list at the progesterone receptor
(data calculated base on Houtman. 2008)

A few notes on the graphs: Houtman et al. did not measure the relative potency at the progesterone receptor for all of the "other exogenous androgens". Instead of meshing androgen and progesterone activity into a single graph, I thus decided to seperate the two, because otherwise it would have been difficult to identify which of the steroids actually have "zero" transcriptional activity at the progesterone receptor and which just have not been analyzed by the scientists. Additionally I recalculated the references using testosterone and progesterone instead of DHT and the over-potent progestin ORG2085.

Among the better known other (I am sticking to Houtman et al.'s nomenclature, here) exegenous androgens, the Dutch scientists analyzed, trestolone, or 7a-methyl-19-nor-T (MENT), is certainly the "meanest bitch". It's androgenic activity is almost 10x higher than that testosterone (and thus 3x more potant than nandrolone!) and its potency as a transcriptional activator at the level of the progesterone receptor is still 29% of that of progesterone and thus more than 9x higher than that of nandrolone aka "Deca".

Figure 2: Relative potency at of steroids
not on the WADA list at the androgen
receptor; note that I re-calculated the
values relative to testosterone as a new
reference, this means that you have to
divide the values by 4.68 to get DHT as
a reference, as in Part 1, fig. 2 (data
calculated base on Houtman. 2008)

The major offenders, as far as chances of immediate (cf. red box in part 1 of this article) progesterone-related side effects are concerned, are yet the norethisterone derivatives (relative potencies compared to progesteron)

1. 11b-ethynyl-NET - 880%
2. delta-15-NET - 290%
3. 11b-ethenyl-NET - 262%
4. 6a-methyl-NET - 253%
5. 11b-I37ethyl-NET - 139%

I guess, you probably already suspect that these are agents in progesterone-only or combined contraceptive pills. So, in essence nothing athletes will be using.

The 7-alpha-methylated derivate of the progestin nethisterone, 7a-methyl-NET, however, has an androgen/progesterone activity ratio of 427/8. Now, although the respective values for trenbolone and nandrolone are only 442/35 and 327/7 this does not necessarily go to tell you that it would make a powerful mass builder, especially in view of the results of previous studies using CHO-AR reporter gene assays, according to which nandrolone (I do not have data on trenbolone, here) has a 1.6x higher androgen receptor binding affinity than the aforementioned nethisterone derivate.

Image 2: The dissection of the
prostate (image by Gray. 2005) is
an unsavory, yet obligatory part
of the Hershberger assay, an in-
vivo method to evaluate the
androgenic activity of steroids
that was developed by
T.V. Hershberger in the 1960s.

Different tests, different results: Most of the data you will currently find on message-boards and certain webpages is based on the "old" CHO-AR reporter gene assays, the results of which often contradict relative activity levels measured by AR CALUX. According to the CHO-AR gene assay, for example the nandrolone would be the more potent androgen receptor agonist (2.65x more potent, Sonneveld. 2005). According to the Chemical Activated Luciferase gene eXpression test, on the other hand, its almost the other way around. Lastly, according to the good old Hershberger-test, which was developed in the 1960 and '70s as an in-vivo procedure to evaluate the androgenic activity of steroids in rats, 7a-methyl-NET has 6.25x the androgenic activity of nandrolone. While it is likely that the CALUX assay is the most reliable method, when it comes to the exact cellular mechanisms, the good old Hershberger-tests, Julius Vida (the "Vida" from "Vida's Book") used, as well, still have their merit, as they provide some insight into what may happen when the compound is administered to an actual living organism.

Similar to the WADA-prohibited compounds, where with the exception of 4-chloro-19-nor-T, 19-norclostebol (4%) and 19-nor-androstenediol (1%) most of the compounds that have been tested exhibited <1% of the receptor activity of estradiol at both the estrogen beta and alpha receptors, compared to estradiol none of the tested (estrogen receptor transcriptional activity was measured for only 10 out of 36 androgens) compounds had significant estrogenic activity. 7a-methyl-NET, the progestin discussed in the previous paragraph, for example has a relative potency of 0.038%, which is more than 4x higher than the 0.009% potency of nandrolone, yet still less than half of the activity of DHEA (0.081%).

Figure 3: Relative potency of steroid metabolits at the androgen receptor, note that I re-calculated the values relative to testosterone as a new reference, this means that you have to divide the values by 4.68 to get DHT as a reference, as in Part 1, fig. 2 (data calculated base on Houtman. 2008)

Similarly, out of the less-androgenic (cf. figure 3) metabolites and isomers Houtman et al. actually tested, only 5a-androstane-3b,17b-diol (0.543%) and 4-androstenediol (4-AD) (0.125%) presented significant interactions with the estrogen beta receptor (0.039% and 0.012% at the alpha receptor).

If I may remind you of the the high transcriptional activity of nandrolone and trenbolone, I've discussed in the first part of this article, which corresponds well with the progesterone related side-effects many athletes complain about, the relatively high activity of 4-AD at the estrogen receptor (25x higher than testosterone) in the CALUX assay is yet another sign for the real-world relevance of the luciferase assay data (cf. red box "Different tests, different results"). After all, androst-4-ene-3b,17b-diol, which happens to be one of the pro-hormones that has lately re-appeared on the "supplement" market, is well-known to be one of the "wetter" compounds.

Figure 4: Relative potency of several natural and synthetic steroids at the corticosteroid receptor with cortisol as a reference (data calculated based on Houtman. 2008)

Finally, we will have a brief look at those steroids which mess with the glucocorticoid receptor. Other than the usual suspects in figure 4, only fluoxymesterone (0.27%), 5a-hydrogen-11b-methyl-NET (0.52%), 11b-methyl-19-nor-T (1.97%), 7a-methyl-19-nor-T aka "Trestolone" or "MENT" (1.56%) and progesterone (0.65%) exhibit any notable corticosteroid activity (expressed relative to the potency of cortisol). However, nandrolone, trenbolone and other anabolics with progestational activity appear to exert indirect effects on the mammalian corticosteroid metabolism (Moor. 1971) and may thus induce downstream effects the CALUX assay obviously cannot detect (suggested read: red box on DHEA and aromatization in part 1 of this article).

With the issue of literal "side-effects", i.e. effects not related to direct transcriptional activity at the receptor sites, I want to conclude this two part series on the immediate effects of a broad range of androgens on androgen, progesterone, estrogen and corticosteroid receptors by reminding you of the fact that despite an ever-increasing accuracy and the constant development of even more sophisticated analytical methods, there still is (and probably never will be) a machine, where you insert a certain molecule, press a few buttons and get a print out that says: "Person A; age: 25, sex: male; training: 5x a week; nutrition: [...] will gain Xlbs of lean mass and shed Ylbs of fat on a Z-week cycle of XYZmg of compound ALPHA".

Beyond Vida's Book, Part 1/2: Androgen, Progesterone, Estrogen & Corticosteroid Receptor Activities of ALLmost All Anabolic Steroids - WADA Prohibited Compounds.

Image 1: Data on the interaction of androgens
with the progesterone receptor is scarce,
this study has it!

This is only blogpost #499, yet at the same time, its a premiere! It's the first time that I am aware of that something you read at the SuppVersity has been covered by the "competition" before. Kudos to my dutch friends from ergogenics.org, who dug up a 2008 paper by Corine J. Houtman et al. (Houtman. 2008) with extensive CALUX(R) bioassay data on the androgen, progesterone, estrogen and corticosteroid receptor response to allmost all popular steroids - it would probable be the "A", as in 4-androstenediol (the "good old 4-AD"), to "Z", if there actually was a common androgen with a "z" as its first character.

Note! Due to the fact that the sheer amount of data from this study exceeds the time I can spend on analyzing and compiling it for you, today, this is going to be a two part series, with the second part on what the authors describe as "potential" AAS (among these are such illustrious names as 7a-methyl-19-nor-T aka Trestolone or MENT) will follow tomorrow. And don't get mad at me for that, at least it does have the advantage that you can comment / pose questions today and have them answered in detail, by tomorrow ;-)

Update: click here for part 2!

The original intention of the study obviously was to demonstrate that by the means of the mammalian androgen receptor responsive reporter gene assay (AR CALUX® bioassay), the anti-doping agency would be able to identify hitherto unknown "designer steroids", which would not show up in the usual tests, where the chemical structure of the substance you are looking for must be known beforehand. The CALUX bioassay, on the other hand, directly measures the transcriptional activity of a specific steroid receptor when it is exposed to a given substance and is thus a very reliable measure of the biological effect a certain anabolic will have on the cellular level.

Figure 1: Enzymes, their cellular location,
substrates and products in human
steroidogenesis; DHEA is the first
compound in the left androgen column
(figure by Slashme and Mikael Häggström)

While it is of course grandiose to be able to measure receptor activity directly, a non-negligible weakness of these bioassays is that they do not provide any insight into the effects of downstream-metabolits of the tested androgen. Let's take dehydroepiteandrosterone (DHEA) as an example. It is well known that after several enzymatic reactions DHEA can eventually be converted to estrogen (cf. figure 1), if however you measure the transcriptional activity of DHEA at the estrogen receptor it turns out to be 0.0813% of 17β-Estradiol (E2). This example clearly shows that, at least in the case of aromatizeable steroids such as DHEA, CALUX provides only part of the overall picture. Keep that in mind, when you are trying to interpret the data!

Other than "Vida" in his famous rat studies, these assays obviously do not provide any information about the "anabolic" value of the respective compounds. Oxandrolone, for example, is known as a highly anabolic steroid, which has about 6x the anabolic activity of testosterone. Nevertheless, its activity at the androgen receptor is only 1% of that of dihydro-testosterone (DHT) and thus no more than 1/20 of the androgen receptor activity of testosterone (cf. figure 2). Accordingly, the following data will not really tell you how much muscle an athlete will be able to accrue whilst taking a certain steroid, but rather which androgen, progesterone, and estrogen related side-effects he or she may experience in the course of that cycle.

Figure 2: Relative potency at the
androgen (reference: DHT) and
progesterone (reference: ORG2085)
receptor of androgens that are
officially prohibited by WADA
(based on Houtman. 2008)

If you have a look at the androgens from the WADA's list of prohibited compounds in figure 2, you will notice that compared to the synthetic progestin 16a-ethyl-21-hydroxy-19nor-4-pregnene-3,20-dione (ORG2085) only a handful of compounds exhibits a significant transcriptional activity at the progesterone receptor:

• 17a-ethyl-19-nor-T (norethandrolone) - 24%
• norbolethone - 21%
• tetrahydrogestrinone (THG) - 7%
• gestrinone - 5%
• 17b-trenbolone - 3%

If the occurrence of trenbolone as the last item on the list of androgens with a high activity at the level of the progesterone receptor puzzled you, you obviously have not heard of the dreaded "progestin-gyno" this powerful steroid is supposed to induce!? While 3% of the activity of a synthetic progestin does not sound much, trenbolone is a 6423x more potent activator of the progesterone receptor than testosterone and it is still 525x more active than nandrolone aka "deca", another of the commonly used mass and strength agents the use of which is rumored to have induced gynecomastia in a non-negligible number of drug using athletes.

Only 4-chloro-19-nor-T, 19-norclostebol (4%), 19-nor-androstenediol and methyl-androstenediol (0.1%) exhibit transcriptional activity >0.1% of that elicited by estradiol at the estrogen alpha and beta receptors and none of the tested androgens from the WADA list has an activity >0.002% (this is fluoxymesterone) of that of dexamethasone at the corticosteroid receptor. In order not to overcomplicate things, I have decided to exclude this additional data from figure 2.

This is only part 1! Don't forget to check back tomorrow for more information on metabolites and isomers, such as 4AD & Co., other exogenous androgens that are not yet on the WADA anti-doping list, such as 7a-methyl-19-nor-T (Trestolone,  MENT) and other steroids!

Update: click here for part 2!