Effects of Etomidate on the Steroidogenesis of Rat Immature Leydig Cells.

Hua-Cheng Liu,Hongguo Guan,Yuanyuan Hu,Qing-Quan Lian,Cong Hu,Chan Wang,Danyan Zhu,Ren-Shan Ge,Han Lin,Senlin Li,Zhichuan Chen,Daqing Ma

doi:10.1371/journal.pone.0139311

Abstract

BackgroundEtomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of etomidate is the reduced plasma concentration of corticosteroids, leading to the abnormal reaction of adrenals. Cortisol and testosterone biosynthesis has similar biosynthetic pathway, and shares several common steroidogenic enzymes, such as P450 side chain cleavage enzyme (CYP11A1) and 3β-hydroxysteroid dehydrogenase 1 (HSD3B1). The effect of etomidate on Leydig cell steroidogenesis during the cell maturation process is not well established.MethodologyImmature Leydig cells isolated from 35 day-old rats were cultured with 30 μM etomidate for 3 hours in combination with LH, 8Br-cAMP, 25R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone and dihydrotestosterone, respectively. The concentrations of 5α-androstanediol and testosterone in the media were measured by radioimmunoassay. Leydig cells were cultured with various concentrations of etomidate (0.3–30 μM) for 3 hours, and total RNAs were extracted. Q-PCR was used to measure the mRNA levels of following genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14. The testis mitochondria and microsomes from 35-day-old rat testes were prepared and used to detect the direct action of etomidate on CYP11A1 and HSD3B1 activity.Results and ConclusionsIn intact Leydig cells, 30 μM etomidate significantly inhibited androgen synthesis. Further studies showed that etomidate also inhibited the LH- stimulated androgen production. On purified testicular mitochondria and ER fractions, etomidate competitively inhibited both CYP11A1 and HSD3B1 activities, with the half maximal inhibitory concentration (IC50) values of 12.62 and 2.75 μM, respectively. In addition, etomidate inhibited steroidogenesis-related gene expression. At about 0.3 μM, etomidate significantly inhibited the expression of Akr1C14. At the higher concentration (30 μM), it also reduced the expression levels of Cyp11a1, Hsd17b3 and Srd5a1. In conclusion, etomidate directly inhibits the activities of CYP11A1 and HSD3B1, and the expression levels of Cyp11a1 and Hsd17b3, leading to the lower production of androgen by Leydig cells.

Highlights

Etomidate is a carboxylated imidazole first synthesized in 1964 and reported in 1965 by Janssen Pharmaceuticals [1]
Because the inhibitions were comparable between luteinizing hormone (LH) and 8Br-cAMP stimulations, it suggests that the inhibition site(s) may be beyond the LH signaling cascade
To further elucidate the specific cascades by which etomidate may affect androgen productions, we tested all the enzymatic cascades by providing the cells with different substrates that start the pathway reactions from different points

Summary

Introduction

Etomidate is a carboxylated imidazole first synthesized in 1964 and reported in 1965 by Janssen Pharmaceuticals [1]. Developed as the anti-fungal agent, its potent hypnotic activity was found during animal testing. Etomidate is a short acting intravenous anaesthetic agent for general anaesthesia and has an elimination half-life of 3–5 hours [4]. Its anaesthetic effects in the central nerve system are thought to be mediated by type A γ-aminobutyric acid receptors [5]. Etomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of etomidate is the reduced plasma concentration of corticosteroids, leading to the abnormal reaction of adrenals. The effect of etomidate on Leydig cell steroidogenesis during the cell maturation process is not well established

Methods

Results

Conclusion