Effects of butylated hydroxyanisole on the steroidogenesis of rat immature Leydig cells

Butylated hydroxyanisole (BHA) is a synthetic antioxidant used for food preservation. Whether BHA affects testosterone biosynthesis is still unclear. The effects of BHA on the steroidogenesis in rat immature Leydig cells were investigated. Rat immature Leydig cells were isolated from 35-old-day rats and cultured with BHA (50 mu M) for 3 h in combination with 22R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone or dihydrotestosterone, and the concentrations of 5 alpha-androstanediol and testosterone in the media were measured. Leydig cells were cultured with BHA (0.05-50 mu M) for 3 h. Q-PCR was used to measure the mRNA levels of following genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1 and Akr1c14. The testis microsomes were prepared to detect the direct action of BHA on 3b-hydroxysteroid dehydrogenase 1 (HSD3B1), 17 alpha-hydroxylase (CYP17A1) and 17 beta-hydroxysteroid dehydrogenase 3 activities. In Leydig cells, BHA (50 mu M) significantly inhibited LH- and 8Br-cAMP-mediated androgen production. BHA directly inhibited rat testis CYP17A1 and HSD3B1 activities. At 50 mu M, it also reduced the expression levels of Hsd17b3 and Srd5a1 and their protein levels. In conclusion, BHA directly inhibits the activities of CYP17A1 and HSD3B1, and the expression levels of Hsd17b3 and Srd5a1, leading to the lower production of androgen in Leydig cells.