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Betel nut is the second largest economic food product in Taiwan. In Southeast Asia, the habit of chewing betel nut seems to be highly correlated with oral submucous fibrosis and oral squamous cell carcinoma. Oral submucous fibrosis is characterized by abnormal accumulation of oral submucous collagen fibers and limitation of mouth opening. Although the mechanism responsible for tissue damage is still unknown, prolonged irritation caused by betel nut and tobacco is considered to be a major factor contributing to the pathogenesis of oral submucous fibrosis. The effect of betel nut chewing on immune system remains unknown. Present study aims to investigate the change of plasma hormones including cortisol, testosterone, and inflammatory cytokine concentrations in male chewing betel nut compared with normal subjects. Heparinized blood was obtained from control group (normal young+mid-aged individuals), betel nut-chewing, and oral cancer male subjects. The study was approved by the Ethics Committee of the Chang-Gung Memorial Hospital. Written informed consent was granted by the patients. Plasma cortisol and testosterone concentrations were detected by commercial enzyme-linked immunosorbent assay (ELISA). The inflammatory cytokines, including interleukin-1β (IL-1β), IL-15, tumor necrosis factor-α (TNF-α), were analyzed by ELISA with commercial monoclonal capture antibodies and polyclonal detection antibodies. The median concentrations of plasma IL-1β, IL-15, and TNF-α were 3.14pg/ml, 3.14pg/ml, and 6.85pg/ml, respectively, in patients with oral cancer, compared with median plasma IL-1β, IL-15, and TNF-α concentration of 2.64pg/ml, 5.86pg/ml, and 5.38pg/ml, respectively, in patients with betel nut-chewing habit. In contrast, the median concentrations of plasma IL-1β, IL-15, and TNF-α in mid-aged males (aged 30-50) were 7.00pg/ml, 10.64pg/ml, and 31.73pg/ml, respectively, compared with median plasma concentration of IL-1β, IL-15, and TNF-α of 4.48pg/ml, 33.36pg/ml, and 97.77pg/ml in young males (aged 20-22), respectively. Also, significantly elevated plasma cortisol concentration was noted in betel nut-chewing (median 727.2ng/ml) and oral cancer patients (561.9ng/ml) compared to the mid-aged (176.8ng/ml) and young males (173.4ng/ml), respectively. In addition, lower plasma testosterone concentrations were found in betel nut-chewing subjects compared with young males (2.6±3.3ng/ml vs 6.2±2.9ng/ml). To summarize, the inflammatory cytokines and steroid hormones may reflect the degree of inflammation in betel nut-chewing males and the oral cancer subjects. The above findings suggest that betel nut-chewing or oral cancer inhibits plasma cytokines and regulates steroid hormones concentrations compared to mid-aged or young normal subjects. It is also indicated that betel nut-chewing causes decreased inflammatory cytokines as the same levels as in oral cancer subjects.

Effects of calcitonin peptides, including human calcitonin (hCT), salmon calcitonin (sCT), and calcitonin gene-related peptide (CGRP), on the secretion of testosterone and luteinizing hormone (LH) in male rats were studied. Male rats were injected intravenously with human chorionic gonadotropin (hCG), calcitonin peptides, or hCG plus calcitonin peptides. Blood samples were collected at several intervals following hormone challenge. In an in vitro experiment, testis blocks were incubated with hCG (0, 0.05, 0.5, or 5 IU/ml) or hCG (0.5 IU/ml) plus calcitonin peptides (0-10(-9) or 10(-6) M) at 34 degrees C for 30 minutes. Both medium and plasma samples were extracted by ether and analyzed for testosterone by radioimmunoassay (RIA). The concentration of calcium in each plasma sample was measured by an automatic calcium analyzer. The anterior pituitary gland (AP) was incubated with or without calcitonin peptides (0-10 nM) at 37 degrees C for 30 minutes. They were then incubated with gonadotropin releasing hormone (GnRH, 10 nM) for a further 30 minutes. The concentration of LH in AP medium was measured by RIA. The accumulation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in both testicular tissues and APs were measured by RIA. A single intravenous injection of calcitonin peptides decreased the basal and hCG-stimulated levels of plasma testosterone gradually from 60 to 180 or 360 minutes after challenge. The plasma calcium was not altered by the injection of calcitonin peptides and/or hCG. Administration of calcitonin peptides in vitro resulted in a dose-dependent inhibition of both basal and hCG-stimulated release of testosterone.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypoxia has been shown to stimulate the expression of vascular endothelial growth factor (VEGF), which is a major mediator for angiogenesis and vasculogenesis. During hypoxia, VEGF promotes angiogenesis in the testis. However, the effect of VEGF on the steroidogenesis of testosterone and the cell proliferation in Leydig cells is unclear. To assess the effects and the action mechanisms of hypoxia, a mouse TM3 Leydig cell line was employed in the present study. The Leydig cells were incubated in an incubator chamber (95% N2-5% CO2) for 1–24 h. The cultured media were collected and assayed by testosterone RIA and VEGF enzyme immunoassay. 3-(4,50-Dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide assay was used to detect the proliferation of Leydig cells. The present results showed that the proliferation of Leydig cells was enhanced significantly by hypoxia. The basal VEGF release was increased, and the response of VEGF production to human chorionic gonadotropin (hCG) was also enhanced in hypoxic condition. During hypoxia, administration of hCG or VEGF stimulated proliferation of Leydig cells, but the stimulatory effect was abolished by the administration of anti-VEGF antibody. Higher doses of VEGF stimulated testosterone release in a dose-dependent manner. Administration of anti-VEGF antibody abolished the stimulatory effect of VEGF on testosterone release. These data suggest that hypoxia stimulates cell proliferation and testosterone release in Leydig cells via an increase of VEGF production.

The aim of this study was to explore the effect and action mechanisms of intermittent hypoxia on the production of testosterone both in vivo and in vitro. Male rats were housed in a hypoxic chamber (12% O2+ 88% N2, 1.5 l/ml) 8 h/day for 4 days. Normoxic rats were used as control. In an in vivo experiment, hypoxic and normoxic rats were euthanized and the blood samples collected. In the in vitro experiment, the enzymatically dispersed rat Leydig cells were prepared and challenged with forskolin (an adenylyl cyclase activator, 10−4M), 8-Br-cAMP (a membrane-permeable analog of cAMP, 10−4M), hCG (0.05 IU), the precursors of the biosynthesis testosterone, including 25-OH-C (10−5M), pregnenolone (10−7M), progesterone (10−7M), 17-OH-progesterone (10−7M), and androstendione (10−7-10−5M), nifedipine (L-type Ca2+channel blocker, 10−6-10−4M), nimodipine (L-type Ca2+channel blocker, 10−5M), tetrandrine (L-type Ca2+channel blocker, 10−5M), and NAADP (calcium-signaling messenger causing release of calcium from intracellular stores, 10−6-10−4M). The concentrations of testosterone in plasma and medium were measured by radioimmunoassay. The level of plasma testosterone in hypoxic rats was higher than that in normoxic rats. Enhanced testosterone production was observed in rat Leydig cells treated with hCG, 8-Br-cAMP, or forskolin in both normoxic and hypoxic conditions. Intermittent hypoxia resulted in a further increase of testosterone production in response to the testosterone precursors. The activity of 17β-hydroxysteroid dehydrogenase was stimulated by the treatment of intermittent hypoxia in vitro. The intermittent hypoxia-induced higher production of testosterone was accompanied with the influx of calcium via L-type calcium channel and the increase of intracellular calcium via the mechanism of calcium mobilization. These results suggested that the intermittent hypoxia stimulated the secretion of testosterone at least in part via stimulatory actions on the activities of adenylyl cyclase, cAMP, L-type calcium channel, and steroidogenic enzymes.

Nonylphenol (NP), a final metabolite of nonylphenol polyethoxylate, has been reported to interfere with male reproduction. However, its mechanisms are not fully understood. In the present study, we examined the effects of NP on steroidogenesis of testosterone in rat Leydig cells. The testosterone concentrations in rat plasma were examined after intravenous injection of NP (100 microg/kg) at different time intervals. In addition, rat Leydig cells were challenged with different concentrations of NP (4.25-127.5 microM) to evaluate its influences on testosterone steroidogenesis. Administration of NP showed a decrease of hCG-induced plasma testosterone. Moreover, in vitro experiments revealed that NP (127.5 microM) alone stimulated testosterone release through increase of both protein levels and activities of the StAR and P450(SCC). In contrast, NP inhibited hCG-induced testosterone release in rat Leydig cells. The inhibitory effect was also observed after incubation of the Leydig cells in the presence of different precursors. These results suggested that NP had differential effects on testosterone synthesis.

Arecoline is one of the major components of betel nuts, which have been consumed as chewing gum in Southeast Asia. In this study, the effects of arecoline on testosterone (T) secretion were explored. Male rats were injected with human chorionic gonadotropin (hCG, 5 IU/kg) or arecoline (1 μg/kg) plus hCG via a jugular catheter. Blood samples were collected at several time intervals subsequent to the challenge. Rat anterior pituitary was treated with gonadotropin-releasing hormone in vitro with or without arecoline, and then the concentration s of luteinizing hormone (LH) in the medium were measured. Rat Leydig cells were purified by Percoll density gradient centrifugation and incubated with arecoline, hCG, forskolin, 8-bromo-cAMP (8-Br-cAMP), nifedipine, nimodipine, or tetrandrine at 34°C for 1 h. A single intravenous injection of arecoline resulted in an increase of the hCG-induced level of plasma T. Administration of arecoline (10−8 to 10−6 M) in vitro increased T production in Leydig cells. The stimulatory effect of arecoline on T release in vitro was enhanced by hCG (0.001 IU/ml), forskolin (10−6 M), or 8-Br-cAMP (10−5 M). By contrast, nifedipine, nimodipine, or tetrandrine inhibited the increased T concentrations induced by arecoline. Western blot showed that arecoline increases steroidogenic acute regulatory (StAR) protein expression compared with vehicle. These results suggested that arecoline stimulates testosterone production by acting directly on Leydig cells via mechanisms involving an activation of L-type calcium channels, increasing the activity of 17β-hydroxysteroid dehydrogenase and enhancing the expression of StAR.