Gen Watanabe

Nanoparticle-rich diesel exhaust may disrupt testosterone biosynthesis and metabolism via growth hormone

We previously reported that exposure to low (22.5+/-0.2 nm in diameter, 15.4+/-1.0 microg/m(3) in mass weight, 2.27x10(5)/cm(3) in mean number concentration), and medium (26.1+/-0.5 nm, 36.4+/-1.2 microg/m(3), 5.11x10(5)/cm(3)) concentrations of nanoparticle-rich diesel exhaust (NR-DE) for 1 and 2 months (5 h/day, 5 days/week) significantly increased plasma testosterone in male Fischer 344 rats, whereas exposure to a high concentration (27.1+/-0.5 nm, 168.8+/-2.7 microg/m(3), 1.36x10(6)/cm(3)) did not. The present study attempts to clarify the mechanism of this elevation. Low and medium exposures to NR-DE for 1 and 2 months significantly increased steroidogenic acute regulatory protein (StAR)- and cytochrome P450 side-chain cleavage (P450scc)-mRNA and their protein expressions in the testis of rats, in which the elevation pattern was very similar to that of plasma testosterone levels. Interestingly, both exposure levels for 1 month significantly increased growth hormone (GH) receptor expression in the testis, and low exposure also increased testicular insulin-like growth factor I-mRNA levels and hepatic microsomal cytochrome P450 2C11-mRNA and their protein levels in rats. These two factors are thought to be related to growth hormone secretion. Disruption of testosterone biosynthesis by NR-DE exposure may be a mode of action for reproductive toxicity, which may, in part, be regulated by increasing StAR and P450scc expressions via GH signalling.

Expression of Nerve Growth Factor and Its Receptors NTRK1 and TNFRSF1B Is Regulated by Estrogen and Progesterone in the Uteri of Golden Hamsters

Abstract Experiments were conducted using female golden hamsters to identify the presence of nerve growth factor (NGF) and its receptors NTRK1 and TNFRSF1B in the uteri of female animals and regulation on their expression by estrogen and progesterone. NGF and its receptor NTRK1 were immunolocalized to luminal epithelial cells, glandular cells, and stromal cells. TNFRSF1B was immunolocalized in luminal epithelial and glandular cells, with no staining found in stromal cells of the uterine horns of normal cyclic golden hamsters. Strong immunostaining of NGF and its receptors NTRK1 and TNFRSF1B was observed in uteri on the day of proestrus as compared to the other stages of the estrous cycle. Results of immunoblot analysis of NGF revealed that there was a positive correlation between uterine NGF expression and plasma concentrations of estradiol-17β. To clarify the effects of estrogen and progesterone on NGF, NTRK1, and TNFRSF1B expression, adult female golden hamsters were ovariectomized and treated with estradiol-17β and/or progesterone. Immunoblot analysis and immunohistochemistry indicated that estradiol-17β stimulated expression of NGF and its two receptors in the uterus. Treatment with progesterone also increased NGF and NTRK1 expression in the uterus. However, no additive effect of these steroids on expression of NGF and its receptors was observed. Changes in uterine weights induced by estradiol-17β and/or progesterone showed the same profile with that of NGF, suggesting that a proliferative act of NGF may be involved in uterine growth. These results suggest that NGF may play important roles in action of steroids on uterine function.

Ovulation block by Pueraria mirifica: a study of its endocrinological effect in female monkeys.

Pueraria mirifica (PM), a Thai herb containing phytoestrogens, may act as estrogen and disturb reproduction. To investigate the effect of PM on the menstrual cycle length and related hormones, nine adult female monkeys (Macaca fascicularis) were separated into three groups. Each group (n=3) was fed with 10, 100, and 1000 mg/d of PM for three menstrual cycles. The menstrual cycle length increased significantly in monkeys treated with PM-10 and PM-100 and disappeared completely in monkeys treated with PM-1000. Serum follicle stimulating hormone, luteinizing hormone, estradiol, progesterone, and ir-inhibin were lower during the treatment period in a dose-dependent manner. Changes in menstrual cycle length and the hormonal levels recovered during the post-treatment period only in monkeys treated with PM-10 and PM-100. PM greatly influences menstrual cycles and may suppress ovulation by lowering serum levels of gonadotropins.

Alleviative Effects of Quercetin and Onion on Male Reproductive Toxicity Induced by Diesel Exhaust Particles

Diesel exhaust particles (DEPs) are particulate matter from diesel exhaust that contain many toxic compounds, such as polyaromatic hydrocarbons (PAHs). Some toxicities of PAH are thought to be expressed via aryl hydrocarbon receptors (AhRs). The male reproductive toxicity of DEPs might depend on AhR activation induced by PAHs. We hypothesized that AhR antagonists protect against the male reproductive toxicity of DEPs. Quercetin is a flavonoid and a well-known AhR antagonist, while onion contains many flavonoids, including quercetin. Hence, we examined whether quercetin and onion have alleviative effects against the male reproductive toxicity induced by DEPs. BALB/c male mice were fed quercetin- or onion-containing diets and received 10 injections of DEP suspension or vehicle into the dorsal subcutaneous layer over 5 weeks. The mice were euthanized at 2 weeks, after the last treatment, and their organs were collected. Daily sperm production and total incidence of sperm abnormalities were significantly affected in the DEP groups as compared with the vehicle group, but the total incidence of sperm abnormalities in the quercetin + DEP-treated mice was significantly reduced as compared with the DEP-treated mice. The numbers of Sertoli cells were significantly decreased in DEP-treated mice as compared with the vehicle-treated mice, but, the numbers of Sertoli cells were significantly increased in the quercetin and the onion + DEP-treated mice as compared with the DEP-treated mice. These results clearly indicate alleviative effects of quercetin and onion against the male reproductive toxicity induced by DEP.

Nitrophenols isolated from diesel exhaust particles regulate steroidogenic gene expression and steroid synthesis in the human H295R adrenocortical cell line

Studies of nitrophenols isolated from diesel exhaust particles (DEPs), 3-methyl-4-nitrophenol (PNMC) and 4-nitro-3-phenylphenol (PNMPP) have revealed that these chemicals possess estrogenic and anti-androgenic activity in vitro and in vivo and that PNMC accumulate in adrenal glands in vivo. However, the impacts of exposure to these compounds on adrenal endocrine disruption and steroidogenesis have not been investigated. To elucidate the non-receptor mediated effects of PNMC and PNMPP, we investigated the production of the steroid hormones progesterone, cortisol, testosterone, and estradiol-17beta and modulation of nine major enzyme genes involved in the synthesis of steroid hormones (CYP11A, CYP11B1, CYP17, CYP19, 17betaHSD1, 17betaHSD4, CYP21, 3betaHSD2, StAR) in human adrenal H295R cells supplied with cAMP. Exposure to 10(-7) to 10(-5) M PNMC and 1 mM 8-Br-cAMP for 48 h decreased testosterone, cortisol, and estradiol-17beta levels and increased progesterone secretion. At 10(-5) M, PNMC with 1 mM 8-Br-cAMP significantly stimulated expression of the 17betaHSD4 and significantly suppressed expression of 3betaHSD2. In comparison, 10(-7) to 2 x 10(-5) M PNMPP with 1 mM 8-Br-cAMP for 48 h decreased concentrations of estradiol-17beta, increased progesterone levels, but did not affect testosterone and cortisol secretion due to the significant suppression of CYP17 and the non-significant but obvious suppression of CYP19. Our results clarified steroidogenic enzymes as candidates responsible for the inhibition or stimulation for the production of steroid hormones in the steroidogenic pathway, thus providing the first experimental evidence for multiple mechanisms of disruption of endocrine pathways by these nitrophenols.

Effects of Pueraria mirifica, an herb containing phytoestrogens, on reproductive organs and fertility of adult male mice.

The effects of Pueraria mirifica (PM) on reproductive organs and fertility of adult male mice were investigated. Male mice were divided into four groups (10 mice/group). Groups 1–3 were orally treated with PM at doses of 0 (PM-0), 10 (PM-10), and 100 (PM-100) mg/kg BW/d in 0.2 mL distilled water, and group 4 was subcutaneously injected with 200 μg/kg BW/d of synthetic estrogen diesthylstibestol (DES). The treatment schedule was separated into two periods: treatment and posttreatment (8 wk for each period). The PM-10 and PM-100 treatments had no effect on testicular weight, sperm number, and serum LH, FSH, and testosterone levels. Only the PM-100 treatment reduced weights of epididymes and seminal vesicle and the sperm motility and viability. Histopathological examination demonstrated that testis, epididymis, and seminal vesicle were normal in all doses of PM treatment. PM-treated males showed no alterations in mating efficiency and on causing pregnancy of their female partners. DES injection impaired all those parameters. Offspring fathered by the PM-and DES-treated males exhibited neither malformations nor change of body weight gains, and the reproductive organ weights of 50-d old pups were in the normal range. The present data clearly demonstrate that a long-term treatment of PM at doses 10 and 100 mg/kg BW/d, via oral route, does not alter a male fertility and a hypothalamus pituitary-testis axis. Although PM-100 can cause some moderate impairment, no persistent effects were observed. Most of PM-treated mice increased the mating efficiency after stop treatment.

Styrene Trimer May Increase Thyroid Hormone Levels via Down-Regulation of the Aryl Hydrocarbon Receptor (AhR) Target Gene UDP-Glucuronosyltransferase

Background Styrene trimers (STs) are polystyrene-container–eluted materials that are sometimes detected in packaged foods. Although the possible endocrine-disrupting effects of STs, such as estrogenic activities, have been reported, their potential thyroid toxicity, such as that caused by the related endocrine disruptor 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has not been studied in detail. Objective Using wild-type and aryl hydrocarbon receptor (Ahr)–null mice, we investigated whether 2,4,6-triphenyl-1-hexene (ST-1), an isomer of STs, influences thyroxin (T4) levels in the same manner as TCDD, which induces UDP-glucuronosyltransferase (UGT) via the AhR, resulting in a decrease in T4 levels in the plasma of mice. Methods Both wild-type and Ahr-null mice (five mice per group) were treated for 4 days by gavage with ST-1 (0, 32, or 64 μmol/kg). Results High-dose (64 μmol/kg) ST-1 decreased the expression of AhR, cytochrome P450 (CYP) 1A1/2, UGT1A1/A6, and CYP2B10 mRNAs and the enzyme activity for CYP1A and UGT1A only in the wild-type mice. This dose decreased AhR DNA binding, but paradoxically increased AhR translocation to the nucleus. In contrast, a high dose of ST-1 increased T4 levels in the plasma in wild-type mice but did not influence T4 levels in AhR-null mice. Conclusions Although ST-1 treatment might cause an increase in AhR levels in the nucleus by inhibiting AhR export, this chemical down-regulated AhR mRNA, thus leading to down-regulation of AhR target genes and an increase in plasma T4 levels.

Quercetin attenuates oxidative damage induced by treatment of embryonic chicken spermatogonial cells with 4-nitro-3-phenylphenol in diesel exhaust particles.

Quercetin, an antioxidant flavonoid, is considered beneficial for human and animal health. In this study, the protective effect of quercetin on oxidative damage to testicular cells was studied in embryonic chickens after treatment with 4-nitro-3-phenylphenol (PNMPP) derived from diesel exhaust particles. Testicular cells were challenged with PNMPP (10−8–10−6 M) alone and in combination with quercetin for 48 h. The results showed that quercetin manifested no deleterious effect on spermatogonial cells up to 1.0 μg/ml. Exposure to PNMPP (10−6 M) induced condensed nuclei and vacuolated cytoplasm and reductions in testicular cell viability and spermatogonial cell numbers (p<0.05). It also induced lipid peroxidation by an elevation of thiobarbituric acid reactive substances and decreased glutathione peroxidase activity and superoxide dismutase activity (p<0.05). Simultaneous supplementation with quercetin restored these parameters to the same levels as in the control. These data indicate that quercetin protects spermatogonial cells from oxidative damage in embryonic chickens intoxicated with PNMPP.

Quercetin protects embryonic chicken spermatogonial cells from oxidative damage intoxicated with 3-methyl-4-nitrophenol in primary culture

Diesel exhaust particles (DEP) are considered to be one of the most important air pollutants. In this study, the protective effect of quercetin, an antioxidant flavonoid, on oxidative damage of testicular cells was studied by analysis of the intracellular antioxidant system of embryonic chickens after treatment with 3-methyl-4-nitrophenol (PNMC) derived from DEP. Testicular cells from 18-day-old embryos were cultured in serum-free McCoys5A medium and challenged with PNMC (10(-7) to 10(-5)M) alone or in combinations with quercetin (1.0 microg/ml) for 48h. Results showed that exposure to PNMC (10(-5)M) induced condensed nuclei and vacuolated cytoplasm, a decrease in testicular cell viability and spermatogonial cell number. Exposure to PNMC induced lipid peroxidation by an elevation of thiobarbituric acid reactive substances as well as decreasing glutathione peroxidation activity and superoxide dismutase activity. However, simultaneous supplementation with quercetin restored these parameters to the similar levels as the control. PNMC is therefore concluded to have induced the oxidative stress of the spermatogonial cells, which can be attenuated by combined quercetin treatment. Our results support the therapeutic use of quercetin in the prevention or treatment of the reproductive toxicity by environmental toxicant PNMC.

Schnurri-2 mutant mice are hypersensitive to stress and hyperactive

The bone morphogenetic protein (BMP)/transforming growth factor-beta (TGF-beta)/activin superfamily regulates development of the nervous system during embryogenesis and is also suggested to be involved in adult brain function. However, how BMP/TGF-beta/activin signals modulate neuronal function remains unknown. Schnurri is a transcription factor that contains two metal finger regions. Mammalian Shn-2 enters the nucleus from the cytoplasm in response to BMP-2 stimulation and plays an important role in BMP-dependent adipogenesis. To investigate whether mammalian Shn plays a role in adult brain function, we examined the behaviors of mutant mice lacking Shn-2 (Shn-2(-/-)). Shn-2(-/-) mice exhibited hypersensitivity to stress accompanied by anxiety-like behavior. Consistent with this, stress-induced corticosterone levels were significantly higher in Shn-2(-/-) mice compared to wild-type controls. Interestingly, Shn-2(-/-) mice were more active than wild-type mice in a familiar environment. The basal and stress-induced expression levels of the immediate early genes, including c-Fos, were decreased in Shn-2(-/-) mice compared to wild-type mice. Thus, Shn-2 plays a critical role in locomotion and anxiety-like behavior.