Baiping Mao

Effects of Methoxychlor and Its Metabolite Hydroxychlor on Human Placental 3β-Hydroxysteroid Dehydrogenase 1 and Aromatase in JEG-3 Cells.

Progesterone and estradiol produced by the human placenta are critical for maintenance of pregnancy and fetal development. In the human placenta, 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) is responsible for the formation of progesterone from pregnenolone and aromatase (CYP19A1) for the production of estradiol from androgen. Insecticide methoxychlor (MXC) and its metabolite hydroxychlor (HPTE) may disrupt the activities of these 2 enzymes. In this study, we investigated the effects of MXC and HPTE on steroid production in human placental JEG-3 cells and on HSD3B1 and CYP19A1 activities. MXC and HPTE inhibited progesterone and estradiol production in JEG-3 cells. MXC and HPTE were potent HSD3B1 inhibitors with the half maximal inhibitory concentration (IC50) values of 2.339 ± 0.096 and 1.918 ± 0.078 μmol/l, respectively. MXC had no inhibition on CYP19A1 at 100 μmol/l, while HPTE was a weak inhibitor with IC50 of 97.16 ± 0.10 μmol/l. When pregnenolone was used to determine the inhibitory mode, MXC and HPTE were found to be competitive inhibitors of HSD3B1. When cofactor NAD+ was used, MXC and HPTE were the noncompetitive inhibitors of HSD3B1. When testosterone was used, HPTE was a mixed inhibitor of CYP19A1. In conclusion, MXC and HPTE are potent inhibitors of human HSD3B1, and HPTE is a weak CYP19A1 inhibitor.

Blood-Testis Barrier

In the mammalian testis, the blood-testis barrier (BTB) is an important ultrastructure in the epithelium of the seminiferous tubule—the functional unit of the testis that produces sperm—to support spermatogenesis. For instance, studies using multiple animal models have shown that a disruption of the BTB always leads to an impairment in spermatogenesis, including defects in meiosis, spermiogenesis, and spermatid cell differentiation and survival—cellular events that take place behind the BTB in the adluminal (apical) compartment of the seminiferous epithelium. In this article, besides summarizing the unique morphological features of the BTB, and many of its known functions including its role in supporting spermatogenesis, we focus on recent findings regarding the biology of a local autocrine-based functional axis known as the apical ectoplasmic specialization (apical ES)-blood-testis barrier/basal ES-the basement membrane axis in the testis, which is essential to maintain the homeostasis of the BTB. Specifically, we focus on the role of biologically active peptides generated at the apical ES and also at the basement membrane in the testis during the epithelial cycle of spermatogenesis, and their intriguing antagonistic effects on the BTB function. We also put forth a hypothetic model based on these data to serve as a framework for investigators in the field who seek to better understand the BTB biology.

Effects of Ziram on Rat and Human 11β-Hydroxysteroid Dehydrogenase Isoforms.

Ziram is a widely used fungicide for crops. Its endocrine disrupting action is largely unknown. 11β-Hydroxysteroid dehydrogenases, isoforms 1 (HSD11B1) and 2 (HSD11B2), have been demonstrated to be the regulators of the local levels of active glucocorticoids, which have broad physiological actions. In the present study, the potency of ziram was tested for its inhibition of rat and human HSD11B1 and HSD11B2. Ziram showed the inhibition of rat HSD11B1 reductase with IC50 of 87.07 μM but no inhibition of human enzyme at 100 μM. Ziram showed the inhibition of both rat and human HSD11B2 with IC50 of 90.26 and 34.93 μM, respectively. Ziram exerted competitive inhibition of rat HSD11B1 when 11-dehydrocorticosterone was used and mixed inhibition when NADPH was supplied. Ziram exerted a noncompetitive inhibition of both rat and human HSD11B2 when steroid substrates were used and an uncompetitive inhibition when NAD(+) was supplied. Increased DTT concentrations antagonized rat and human HSD11B2 activities, suggesting that the cysteine residues are associated with the inhibition of ziram. In conclusion, for humans, ziram is a selective inhibitor of HSD11B2, implying that this agent may cause excessive glucocorticoid action in local tissues such as the kidney, brain, and placenta.

The Effects of Fungicides on Human 3β-Hydroxysteroid Dehydrogenase 1 and Aromatase in Human Placental Cell Line JEG-3

Placenta secretes a large amount of progesterone and estradiol, which are critical for maintaining pregnancy. In human placenta, 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) catalyzes pregnenolone to form progesterone, and aromatase (CYP19A1) catalyzes testosterone into estradiol. Fungicides display antifungal activities and are widely used to prevent fungal infections in agricultural plants. These chemicals include azoles, such as tebuconazole (TEB), triadimefon (TRI), and vinclozolin (VCZ) or organotins, such as tributyltin (TBT) and tetrabutyltin (TTBT). Fungicides may disrupt the activities of these 2 enzymes. In the present study, we investigated the effects of these fungicides on steroid production in a human placental cell line JEG-3 and on HSD3B1 and CYP19A1 activities. Of all fungicides tested at 100 µmol/L, only TBT inhibited pregnenolone-mediated progesterone production in JEG-3 cells by over 50%. Except TTBT, all other 4 fungicides inhibited testosterone-mediated estradiol production by over 50%. TBT was a moderate HSD3B1 inhibitor with a half maximal inhibitory concentration (IC50) of 45.60 ± 0.12 µmol/L. When pregnenolone was used to determine the mode of inhibition, TBT was a competitive inhibitor of HSD3B1. The IC50 values of TEB, TRI, VCZ, and TBT for CYP19A1 were 56.84 ± 0.13, 58.73 ± 0.14, 57.42 ± 0.171, and 4.58 ± 0.048 µmol/L, respectively. TEB, TRI, and VCZ were noncompetitive inhibitors of CYP19A1, while TBT was a competitive inhibitor of this enzyme. Therefore, they are endocrine disruptors.

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

Abstract 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 μM) for 3 h in combination with 22R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone or dihydrotestosterone, and the concentrations of 5α-androstanediol and testosterone in the media were measured. Leydig cells were cultured with BHA (0.05–50 μM) 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 3β-hydroxysteroid dehydrogenase 1 (HSD3B1), 17α-hydroxylase (CYP17A1) and 17β-hydroxysteroid dehydrogenase 3 activities. In Leydig cells, BHA (50 μM) significantly inhibited LH- and 8Br-cAMP-mediated androgen production. BHA directly inhibited rat testis CYP17A1 and HSD3B1 activities. At 50 μM, 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.

Structure-activity relationships of phthalates in inhibition of human placental 3β-hydroxysteroid dehydrogenase 1 and aromatase

Phthalates are associated with preterm delivery. However, the mechanism is unclear. Progesterone formed by 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) and estradiol by aromatase (CYP19A1) in placenta are critical for maintaining pregnancy. In this study, we compared structure-activity relationships (SAR) of 14 phthalates varied in carbon atoms in alcohol moiety to inhibit human HSD3B1 in COS1 and CYP19A1 in JEG-3 cells. There were responses in that only diphthalates with 4-7 carbon atoms were competitive HSD3B1 inhibitors and diphthalates with 6 carbon atoms were CYP19A1 inhibitors. IC50s of dipentyl (DPP), bis(2-butoxyethyl) (BBOP), dicyclohexyl (DCHP), dibutyl (DBP), and diheptyl phthalate (DHP) were 50.12, 32.41, 31.42, 9.69, and 4.87μM for HSD3B1, respectively. DCHP and BBOP inhibited CYP19A1, with IC50s of 64.70 and 56.47μM. DPP, BBOP, DCHP, DBP, and DHP inhibited progesterone production in JEG-3 cells. In conclusion, our results indicate that there is clear SAR for phthalates in inhibition of HSD3B1 and CYP19A1.

Zearalenone Inhibits Rat and Human 11β-Hydroxysteroid Dehydrogenase Type 2

Zearalenone is a mycotoxin produced by Fusarium spp. 11β-Hydroxysteroid dehydrogenases, isoforms 1 (HSD11B1) and 2 (HSD11B2), have been demonstrated to be the regulators of the local level of active glucocorticoid, which has a broad range of physiological actions. In the present study, the potency of zearalenone was tested for the inhibition of HSD11B1 and HSD11B2 in rat and human tissues. Zearalenone showed potent inhibition of HSD11B2 with the half-maximal inhibitory concentration (IC50) calculated at 49.63 and 32.22 μM for the rat and human, respectively. Results showed that zearalenone competitively inhibited HSD11B2 when a steroid substrate was used. However, it served as an uncompetitive inhibitory factor when the cofactor NAD+ was used. In contrast, the potency of zearalenone to inhibit both rat and human HSD11B1 was diminished, with the concentration of 100 μM causing almost no inhibitory effect on the isoform. In conclusion, we observed that zearalenone is a selective inhibitor of HSD11B2, implying that this agent may cause excessive glucocorticoid action in local tissues such as kidney and placentas.

Gossypol enantiomers potently inhibit human placental 3β-hydroxysteroid dehydrogenase 1 and aromatase activities

Gossypol is a chemical isolated from cotton seeds. It exists as (+) or (-) enantiomer and has been tested for anticancer, abortion-inducing, and male contraception. Progesterone formed from pregnenolone by 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) and estradiol from androgen by aromatase (CYP19A1) are critical for the maintenance of pregnancy or associated with some cancers. In this study we compared the potencies of (+)- and (-)-gossypol enantiomers in the inhibition of HSD3B1 and aromatase activities as well as progesterone and estradiol production in human placental JEG-3 cells. (+) Gossypol showed potent inhibition on human placental HSD3B1 with IC50 value of 2.3 μM, while (-) gossypol weakly inhibited it with IC50 over 100 μM. In contrast, (-) gossypol moderately inhibited CYP19A1 activity with IC50 of 23 μM, while (+) gossypol had no inhibition when the highest concentration (100 μM) was tested. (+) Gossypol enantiomer competitively inhibited HSD3B1 against substrate pregnenolone and showed mixed mode against NAD(+). (-) Gossypol competitively inhibited CYP19A1 against substrate testosterone. Gossypol enantiomers showed different potency related to their inhibition on human HSD3B1 and CYP19A1. Whether gossypol enantiomer is used alone or in combination relies on its application and beneficial effects.

Determination of songorine in rat plasma by UPLC-MS/MS: Assay development and application to pharmacokinetic study.

An ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of songorine in rat plasma. Sample preparation was accomplished through a simple one-step deproteinization procedure with 0.2mL of acetonitrile to a 0.1mL plasma sample. Plasma samples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consisting of acetonitrile-0.1% formic acid in water with gradient elution. The total run time was 3.0min and the elution of songorine was at 1.68min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization (ESI) by multiple reaction monitoring (MRM) of the transitions at m/z 358.3→340.3 for songorine and m/z 237.2→194.3 for carbamazepine (internal standard). The calibration curve was linear over the range of 1-1000ng/mL with a lower limit of quantitation (LLOQ) of 1.0 ng/mL. Mean recovery of songorine in plasma was in the range of 75.2-87.5%. The intra- and inter-day precision (RSD) was between 3.1-8.5% and 4.3-9.6% and the intra- and inter-day accuracy (RE) ranged from -4.0 to 8.9% and -9.0 to 6.7%. This method was successfully applied in pharmacokinetic study after intravenous administration of 5.0mg/kg songorine in rats.

Environmental toxicants and cell polarity in the testis

During spermatogenesis, head-tail cell polarity, apico-basal cell polarity and planar cell polarity (PCP) are remarkably noted in the seminiferous epithelium in which the heads of developing haploid spermatids are pointed to the basement membrane, and with their tails to the tubule lumen. Furthermore, these polarized spermatids are laid unidirectionally across the plane of the seminiferous epithelium, mimicking PCP noted in hair cells of the inner ear. Treatment of rodents with environmental toxicants that lead to germ cell exfoliation, however, are associated with notable changes in spermatid polarity, and defects in spermatid polarity always precede spermatid loss from the epithelium. Studies have also shown that environmental toxicant-induced Sertoli cell or testis injury is mediated through changes in actin and/or microtubule (MT) cytoskeletons. Emerging evidence has illustrated that cell polarity and PCP also exert their regulatory effects through changes in cytoskeletal organization. Herein, we discuss and critically evaluate these recent findings, hoping that better efforts can be coordinated by investigators to address this rapidly developing field regarding the role of cell polarity and PCP proteins in toxicant-induced male reproductive dysfunction.