Volume 20, Issue 3

5α-reductases, a unique family of enzymes with a wide host of substrates and tissue distributions, play a key role in the metabolism of androgens, progestins, mineralocorticoids and glucocorticoids. These enzymes are the rate-limiting step in the synthesis of a host of neurosteroids, which are critical for central nervous system function. Androgens and glucocorticoids modulate mitochondrial function, carbohydrate, protein and lipid metabolism and energy balance. Thus, the inhibition of these regulatory enzymes results in an imbalance in steroid metabolism and clearance rates, which leads to altered physiological processes. In this report, we advance the hypothesis that inhibition of 5α-reductases by finasteride and dutasteride alters not only steroid metabolism but also interferes with the downstream actions and signaling of these hormones. We suggest that finasteride and dutasteride inhibit 5α-reductase activities and reduce the clearance of glucocorticoids and mineralocorticoids, potentiating insulin resistance, diabetes and vascular disease.

Drug-drug interactions (DDI) with oral contraceptives containing 17α-ethinylestradiol (EE2) have been well characterized with regard to interactions with phase I drug metaolizing enzymes; however, DDI with EE2 and phase II enzymes have not been as thoroughly addressed. Our laboratory recently reported that in vitro EE2 potently inhibits human cytosolic sulfotransferase (SULT) 1A1 while EE2 was not sulfated until micromolar concentrations. Molecular docking studies demonstrated that Tyr169 and isoleucine 89 (Ile89) may play a role in the inhibitory and/or catalytic positioning of EE2 within the active site of SULT1A1. Therefore, the current study focused on determining the role of Ile89 in the inhibition of SULT1A1 utilizing site-directed mutagenesis. Ile89 was mutated to an alanine and the effect of the mutation was characterized using kinetic and binding assays. SULT1A1-Ile89Ala was found to have a Km for EE2 that was 11-fold greater than wild-type enzyme. A decreased affinity (Kd) of EE2 for SULT1A1-Ile89Ala was apparently responsible for the increase in Km, and also resulted in the loss of the potent inhibition. Molecular modeling was used in an attempt to determine the atomic level changes in binding of EE2 to SULT1A1-Ile89Ala. However, analysis of the effect of the single Ile89 mutation on both the open and closed homology models was not consistent with the docking and kinetic results. Overall, the mechanism of inhibition of EE2 for SULT1A1 is apparently the result of interactions of Ile89 with EE2 holding it in a potent inhibitory conformation, and mutation of the Ile89 significantly decreases the inhibition.

Background: Calculated free testosterone (cFT) is determined from the values of total testosterone (TT), sex hormone binding globulin (SHBG), and albumin (Alb) using mathematical formulae. We evaluated any potential cFT variance when determined with fixed Alb (4.3 g/dL) compared to measured Alb, and the point at which low SHBG and Alb combinations produced significant cFT variance. Materials: We analyzed 2050 data points in 1222 women. cFT values with fixed vs. the actual measured Alb values were evaluated and contrasted. cFT levels were determined theoretically for all possible combinations of TT, SHBG, and Alb. Methods: Agreement between the two measures was assessed with Lin’s concordance coefficient. Statistical analyses were performed using R software version 2.12.1. Results: Mean Alb was 4.05±0.30 g/dL. Mean SHBG 73.0±53.3 nmol/L. A fixed Alb of 4.3 g/dL produced no significant variance for most evaluations of cFT. The accuracy decreased with Alb ≤3.5 g/dL in combination with SHBG ≤30 nmol/L and exists in 1.0% of the samples. Conclusions: A fixed Alb of 4.3 g/dL is acceptable for most clinical evaluations. If Alb is ≤3.5 g/dL, along with SHBG ≤30 nmol/L, the variance increases and a free testosterone (FT) measurement by equilibrium dialysis is warranted for better accuracy.

Flavin-containing mono-oxygenases (FMOs) are a family of microsomal chemical- and drug-metabolizing enzymes. FMO3 is a major FMO form in adult mouse and human liver. FMO3 mutations have been associated with the incidence and severity of trimethylaminuria (TMAU), a metabolic disorder characterized by the inability of the affected individual to metabolize the odorous trimethylamine to its non-odorous N-oxide. In addition to this primary genetic form, there are other forms of TMAU that support the hypothesis that FMO3 activity may be modulated by steroid hormones. To understand the molecular mechanism involved in the regulation of Fmo3 gene expression by steroid hormones, we performed this study in an in vitro cellular system, mouse liver cells, and on the human FMO3 gene. Dexamethasone, 5α-dihydrotestosterone, thyroid hormone, and progesterone had no effect on the accumulation of Fmo3 mRNA. The use of increased concentration of theophylline inhibited estrogen receptor α (ERα)-mediated transcription of Fmo3 mRNA. 17β-Estradiol inhibited Fmo3 mRNA accumulation. The use of ICI 164,384 abolished the inhibitory effect induced by estrogen. Gel-shift analyses showed a binding in the 5′ region of the Fmo3 gene. This binding was abrogated by an excess of a cDNA containing an estrogen-responsive element. An estrogen-binding site was also present in the first intron of the human gene, as demonstrated by the gel-shift assay. Supershift experiments confirmed the binding of ERα in both mouse and human samples. Furthermore, chromatin immunoprecipitation assay confirmed the binding of ERα in the promoter region of mouse Fmo3 and in the first intron of the human FMO3 gene. Thus, 17β-estradiol plays a fundamental role in the regulation of Fmo3 gene transcription.