RESUMO
T-2 toxin is one of the mycotoxins, a group of type A trichothecenes produced by several fungal genera including Fusarium species, which may lead to the decrease of testosterone secretion in primary Leydig cells derived from mouse testis. The previous study demonstrated T-2 toxin decrease the testosterone biosynthesis in the primary Leydig cells derived from the mouse testis directly. In this study, we further examined the direct biological effects of T-2 toxin on the process of steroidogenesis, primarily in Leydig cells of mice. Leydig cells of mature mouse were purified by Percoll gradient centrifugation and the cell purity was determined by 3ß-hydroxysteroid dehydrogenase (3ß-HSD) staining. To examine the decrease in T-2 toxin-induced testosterone secretion, we measured the transcription level of three key steroidogenic enzymes including 3ß-HSD-1, cytochrome P450 side-chain cleavage (P450scc) enzyme, and steroidogenic acute regulatory (StAR) protein in T-2 toxin/human chorionic gonadotropin (hCG) co-treated cells. Our previous study showed that T-2 toxin (10(-7), 10(-8), and 10(-9) M) significantly suppressed hCG (10 ng/ml)-induced testosterone secretion. The studies demonstrated that the suppressive effect is correlated with a decrease in the level of transcription of 3ß-HSD-1, P450scc, and StAR (p < 0.05).
Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Toxina T-2/toxicidade , 17-Hidroxiesteroide Desidrogenases/análise , 17-Hidroxiesteroide Desidrogenases/genética , Animais , Células Cultivadas , Centrifugação , Células Intersticiais do Testículo/enzimologia , Masculino , Camundongos , Fosfoproteínas/análise , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
A novel serine protease, with a molecular mass of 19 kDa and the N-terminal sequence of ARTPEAPAEV, was isolated from dried fruiting bodies of the mushroom Pholiota nameko. The purification protocol comprised ion exchange chromatography on DEAE-cellulose, Q-Sepharose and SP-Sepharose, and gel filtration on Superdex 75. It was unadsorbed on DEAE-cellulose and Q-Sepharose but adsorbed on SP-Sepharose. It exhibited an optimum temperature at 50°C, an optimum pH at pH 8.8, a Km of 5.64 mg/mL and a Vmax of 0.98 µmol/min/mL against substrate casein. A number of metal ions inhibited the enzyme including Pb(2+), Mn(2+), Ca(2+), Hg(2+), Zn(2+), Cu(2+), Co(2+), Fe(3+) and Al(3+), with the inhibition of the last two cations being the most potent. K(+) and Mg(2+) slightly enhanced, while Li(+) moderately potentiated the activity of the protease. The protease was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting that it is a serine protease.
