Incubation in the presence of increasing concentrations of nestorone, a synthetic progesterone that does not bind to AR and is used as a negative control, did not affect MENT induced reporter gene transcriptional activity. Similar results were also observed in AR-transfected PC3 prostate cell lines (data not shown) and in HEK-293 and T47D AR-positive cells for the effect of adding progesterone to HeLa cells on trestolone acetate induced AR-mediated trans activation (figures 5A and B, respectively).

Transactivation of the estrogen receptor by the aromatic metabolite 7α-methyl-E2

In vitro, trestolone acetate was aromatized to active estrogen-7α-methylE2 (LaMorte et al. 1994). Therefore, in this study, it was considered important to investigate the ability of this estrogen to activate transcription through two estrogen receptor (ER) subtypes. Figures 6 and 7 show the estrogen agonist activity of E2 and 7α-methyl E2 via transactivation of ESR1 or ESR2, respectively. It can be seen that the aromatised metabolites of trestolone acetate are similar by ED50 values (8.04×10−12 and 1.27×10−10 ESR1 and ESR2 are M, respectively) and E2 (7.69×10−12 and 1.84×10−10 are M for ESR1 and ESR2, respectively). Incubated in the presence of the ER antagonist ICI, the effects of 7α-methyl E2 on transcription were significantly inhibited by ESR1 (Figure 6B) and ESR2 (Figure 7B). Unlike control vectors, ICI, nestorone, or E2 alone did not have any effect on cells that were not transfected with ER subtypes.

The addition of 7α-methyl to 19-nortestosterone significantly enhanced its androgen potency and AR binding ability. However, this 7α-methyl-replaced steroid is not reduced by prostatic 5α-reductase (Liao et al. 1973). In fact, compared to testosterone, trestolone acetate has a weaker effect on male accessory gonads compared to its ability to suppress gonadotropins; trestolone acetate can be aromatized to 7α-methyl E2 (LaMorte et al. 1994). These observations suggest that trestolone acetate, as an ingredient of androgen replacement therapy or male birth control pills, has health-promoting effects, particularly by avoiding overstimulating effects on the prostate, including prostate-specific antigens and antigens from the aromatization of trestolone acetate.