Mass spectrometric assay and physiological-pharmacological activity of androgenic neurosteroids

Abstract

Steroid hormones play a key role in the pathophysiology of several brain disorders. Testosterone modulates neuronal excitability, but the underlying mechanisms are obscure. There is emerging evidence that testosterone-derived "androgenic neurosteroids", 3alpha-androstanediol and 17beta-estradiol, mediate the testosterone effects on neural excitability and seizure susceptibility. Testosterone undergoes metabolism to neurosteroids via two distinct pathways. Aromatization of the A-ring converts testosterone into 17beta-estradiol. Reduction of testosterone by 5alpha-reductase generates 5alpha-dihydrotestosterone, which is then converted to 3alpha-androstanediol, a powerful GABA(A) receptor-modulating neurosteroid with anticonvulsant properties. Although the 3alpha-androstanediol is an emerging neurosteroid in the brain, there is no specific and sensitive assay for determination of 3alpha-androstanediol in biological samples. This article describes the development and validation of mass spectrometric assay of 3alpha-androstanediol, and the molecular mechanisms underlying the testosterone modulation of seizure susceptibility. A liquid chromatography-tandem mass spectrometry assay to measure 3alpha-androstanediol is validated with excellent linearity, specificity, sensitivity, and reproducibility. Testosterone modulation of seizure susceptibility is demonstrated to occur through its conversion to neurosteroids with "anticonvulsant" and "proconvulsant" actions and hence the net effect of testosterone on neural excitability and seizure activity depends on the levels of distinct testosterone metabolites. The proconvulsant effect of testosterone is associated with increases in plasma 17beta-estradiol concentrations. The 5alpha-reduced metabolites of testosterone, 5alpha-dihydrotestosterone and 3alpha-androstanediol, had powerful anticonvulsant activity. Overall, the testosterone-derived neurosteroids 3alpha-androstanediol and 17beta-estradiol could contribute to the net cellular actions of testosterone in the brain. Because 3alpha-androstanediol is a potent positive allosteric modulator of GABA(A) receptors, it could serve as an endogenous neuromodulator of neuronal excitability in men. The 3alpha-androstanediol assay is an important tool in this area because of the growing interest in the potential to use adjuvant aromatase inhibitor therapy to improve treatment of epilepsy.

Fig. 1. Synthesis of the androgenic neurosteroid 3α-androstanediol and 17β-estradiol from testosterone

5α-Reductase converts testosterone into 5α-dihydrotestosterone, which is then reduced further to 3α-androstanediol by 3α-hydroxysteroid oxidoreductase. The 5α-reduction is irreversible and rate-limiting, while the 3α-reduction is reversible and occurs more readily. 17β-Estradiol is produced by the aromatase enzyme.

Fig. 2. Mass spectrometry assay of 3α-androstanediol

(A) Representative LC-MS-MS chromatogram of extracted blank rat plasma with 3α-androstanediol (100 ng/ml). The neurosteroid 3α-androstanediol was monitored from m/z 275 → m/z 257. (B) Plasma 5α-androstanediol concentrations following testosterone administration in rats. Pretreatment with finasteride (100 mg/kg, ip) completely blocked the metabolism of testosterone to 5α-androstanediol. * p<0.05 versus control (n = 6 per group).

Fig. 3. Potential mechanisms of testosterone modulation of seizure activity

Androstanediol, which is synthesized through androgen pathway, produces anticonvulsant effects that are most likely due to its ability to potentiate the GABA_A receptor-mediated inhibition. Estradiol, which is synthesized through estrogen pathway, facilitates seizure susceptibility by a complex mechanism, including increase in excitatory NMDA receptors because of its ability to enhance the dendritic spine density in the hippocampus.

Fig. 4. Antiseizure activity of androgenic neurosteroids

(A) Dose-dependent protection by 5α-dihydrotestosterone (DHT) and 3α-androstanediol against pentylenetetrazol (PTZ)-induced seizures. DHT and 3α-androstanediol were administered, respectively, 30 and 15 min before PTZ (85 mg/kg, sc) injection. Mice failing to show clonic spasms lasting longer than 5 seconds were scored as protected (n= 6–8 mice per group). (B) Time course for protection against PTZ-induced seizures by androgenic neurosteroids DHT (442 mg/kg) and 3α-androstanediol (100 mg/kg). Steroids were given at time 0 and seizure protection was assessed at different time points (n=6 mice per group). Adapted with permission from Reddy, 2004c.

Fig. 5. Androstanediol potentiation of GABA_A receptor function

Like allopregnanolone, 3α-androstanediol is believed to bind at GABA_A receptors and enhance GABA-mediated inhibitory neurotransmission in the brain. The GABA_A receptor is built from several subunits and composed of pentameric channel made of two α subunits, two β subunits and a γ subunit. GABA_A receptor are pluripotent drug targets mediating anxiolytic, sedative, anticonvulsant, and amnesic activities. Neurosteroids have a specific binding site at the GABA_A receptor and the subunit composition appears to have a great impact on neurosteroid modulation of receptor function. The binding site(s) for 3α-androstanediol is proposed to be distinct from that of the GABA, benzodiazepine and barbiturate sites. However, the exact location of 3α-androstanediol binding site is currently unknown. Thus, 3α-androstanediol, by allosteric potentiation of GABA_A receptor-mediated inhibitory synaptic currents, could promote enhanced inhibition and seizure protection.