Shinji Taneda

Prenatal exposure to bisphenol A up-regulates immune responses, including T helper 1 and T helper 2 responses, in mice

The effect of prenatal exposure to bisphenol A (BPA) on the immune system in mice was investigated. Virgin female mice were fed varying doses of BPA, on a daily basis, over a period of 18 days commencing on the day of pairing with stud males (day 0). On day 77, their male offspring of 8 weeks were immunized with hen egg lysozyme (HEL). Three weeks later, anti‐HEL immunoglobulin G (IgG) in sera, and proliferative responses of spleen cells to the antigen, were measured. Anti‐HEL IgG2a and interferon‐γ (IFN‐γ), secreted from splenic lymphocytes, were measured as indicators of T helper 1 (Th1) immune responses, while anti‐HEL IgG1 and interleukin‐4 (IL‐4) were measured as indicators of Th2 responses. The results showed that fetal exposure to BPA was followed by significant increases in anti‐HEL IgG as well as antigen‐specific cell proliferation. Both Th1 responses (including anti‐HEL IgG2a and IFN‐γ production) and Th2 responses (including anti‐HEL IgG1 and IL‐4 production) were augmented by prenatal exposure to BPA, although the augmentation of Th1 responses appeared to be greater than that of Th2 responses. Two‐colour flow cytometric analysis showed that mice exposed prenatally to BPA had 29% and 100% more splenic CD3+ CD4+ and CD3+ CD8+ cells, respectively, than control animals. Similar results were obtained from females whose mothers had consumed BPA during pregnancy. These results suggest that prenatal exposure to BPA may result in the up‐regulation of immune responses, especially Th1 responses, in adulthood.

Estrogenic Activities of Nitrophenols in Diesel Exhaust Particles

Abstract We recently isolated 3-methyl-4-nitrophenol (4-nitro-m-cresol; PNMC) and 4-nitro-3-phenylphenol (PNMPP) from diesel exhaust particles (DEP) and identified them as vasodilators. Because these compounds are alkylphenolic derivatives that might mimic hormones, we evaluated their estrogenic activity by using recombinant yeast screens, myometrial contractility assays, and in vivo uterotrophic assays. Recombinant yeast screen assays showed that both PNMC and PNMPP possess estrogenic activity. Furthermore, ovariectomized 25-day-old immature female rats injected with PNMC and PNMPP subcutaneously for 2 days showed significant increases in uterine weight among those receiving 100 mg/kg PNMC and 0.1 and 1.0 mg/kg PNMPP. To clarify further the estrogenic activity of PNMC and PNMPP, rat uterine horns were monitored in organ bath chambers for myometrial contractility in response to oxytocin (OT). Significant differences occurred in the initial and maximum contractilities to OT at 0.25 and 25 mIU/ml in uterine horns obtained from animals treated with 100 mg/kg PNMC and in the maximum contractilities to OT at 0.025, 0.25, and 25 mIU/ml in those from rats treated with 0.1 mg/kg PNMPP. These results clearly demonstrated that PNMC and PNMPP in DEP have estrogenic activity both in vitro and in vivo and might therefore be considered as endocrine-disrupting chemicals.

Effects of in utero exposure to nanoparticle-rich diesel exhaust on testicular function in immature male rats.

We investigated the effects of in utero exposure to nanoparticle-rich diesel exhaust (NR-DE) on reproductive function in male rats. Pregnant F344 rats were exposed to NR-DE (148.86 microg/m(3), 1.83 x 10(6)particles/cm(3), 3.40 ppm CO, 1.46 ppm NOx), filtered diesel exhaust (F-DE; 3.10 microg/m(3), 2.66 particles/cm(3), 3.30 ppm CO, 1.41 ppm NOx), or clean air (as a control) from gestation days 1 to 19 (gestation day 0=day of sperm-positivity). Male offspring were examined on postnatal day 28. The relative weights of the seminal vesicle and prostate to body weight were decreased after exposure to NR-DE or F-DE compared with controls. Serum concentrations of testosterone, progesterone, corticosterone, and follicle stimulating hormone and testicular concentrations of steroidogenic acute regulatory protein and 17beta-hydroxysteroid dehydrogenase mRNA were decreased after exposure to NR-DE or F-DE compared with control levels. In contrast, serum concentrations of immunoreative inhibin were increased after exposure to NR-DE or F-DE compared with control levels, whereas transcription of follicle stimulating hormone receptor mRNA was increased in the NR-DE exposure group only. These results suggest that prenatal exposure to NR-DE or F-DE leads to endocrine disruption after birth and suppresses testicular function in male rats. Because both the NR-DE and F-DE-exposed groups reacted to the same extent, the nanoparticles in DE did not contribute to the observed reproductive toxicity.

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.

Effects of inhaled nanoparticle-rich diesel exhaust on regulation of testicular function in adult male rats

We investigated the effects of nanoparticle-rich diesel exhaust (NR-DE) on reproductive function. Eight-week-old male F344 rats were divided into 12 experimental groups and exposed to either whole NR-DE at low (15.37 μg/m3, 2.27 × 105 particles/cm3), middle (36.35 μg/m3, 5.11 × 105 particles/cm3), or high (168.84 μg/m3, 1.36 × 106 particles/cm3) concentrations or clean air for 4, 8, or 12 weeks (5 hours/day, 5 days/week). NR-DE exposure for 4 or 8 weeks did not affect body weight; however, body weight was significantly decreased in rats exposed to low- or high- concentration NR-DE for 12 weeks compared to the control group. Relative weights of testes, epididymides, seminal vesicles, and prostate had increased non-significantly in all NR-DE-exposed rats at 4, 8, and 12 weeks. Adrenal gland relative weights were significantly increased at 4 weeks in rats exposed to low-concentration NR-DE. Plasma luteinizing hormone and follicle stimulating hormone concentrations did not change significantly. Plasma testosterone concentrations were significantly increased after exposure to low- or middle-concentration NR-DE for 4 or 8 weeks compared to controls. Plasma immunoreactive (ir-) inhibin concentrations were significantly increased after exposure to high-concentration NR-DE for 4 weeks or middle- or high-concentration NR-DE for 12 weeks compared to controls. Testicular testosterone concentrations were significantly increased at 4, 8, and 12 weeks after exposure to low-concentration NR-DE compared to controls. In contrast, with exposure to low- or high-concentration NR-DE, testicular ir-inhibin concentrations were significantly greater than in controls, but only at 4 weeks. These results suggest that NR-DE inhalation disrupts the endocrine activity of the male reproductive system.

Immunohistological study for estrogenic activities of nitrophenols in diesel exhaust particles

We recently isolated 3-methyl-4-nitrophenol (4-nitro-m-cresol; PNMC) and 4-nitro-3-phenylphenol (PNMPP) from diesel exhaust particles (DEP) and identified them as vasodilators and xenoestrogens. The estrogenic activity of PNMC and PNMPP was further examined by using immunohistochemical staining of proliferating cell nuclear antigen (PCNA) in uterine luminal epithelium of ovariectomized 25-d-old immature female rats injected with PNMC and PNMPP subcutaneously for 2 d. Significant increases were observed in uterine luminal epithelium in PCNA positive cells of animals receiving 10 and 100 mg/kg PNMC and 0.1 mg/kg PNMPP compared with controls. These results clearly show the estrogenic activity of PNMC and PNMPP by cell proliferation on the uterine luminal epithelium.

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.

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.

Effects of the phosphodiesterase IV inhibitor rolipram on Th1 and Th2 immune responses in mice

The present study was designed to investigate the effect of the phosphodiesterase IV inhibitor rolipram on Th1 and Th2 immune responses in mice. Mice were immunized subcutaneously at the base of the tail with ovalbumin (OVA) emulsified with complete Freunds adjuvant (day 0) and were treated daily with oral administration of various doses of rolipram from days 0 to 20. On day 21, production of anti‐OVA IgG and proliferative responses to the antigen were determined. Anti‐OVA IgG2a and interferon‐γ (IFN‐γ), as indicators of Th1 responses, and anti‐OVA IgG1 and interleukin‐10 (IL‐10), as indicators of Th2 responses, were also measured. The results showed that treatment with rolipram failed to affect the production of OVA‐specific IgG but decreased the proliferation of spleen cells to the antigen. Its inhibitory effect on these immune responses was correlated with a marked decrease in IFN‐γ but not IL‐10 production, although neither anti‐OVA IgG2a nor IgG1 production was affected by rolipram. These results suggest that rolipram may preferentially inhibit Th1 responses more effectively than Th2 responses. Administration of rolipram resulted in suppression of antigen (OVA)‐induced arthritis in mice. The suppression of joint inflammation by rolipram was associated with the inhibition of the OVA‐specific proliferative responses of spleen cells and IFN‐γ secretion. These results indicate that rolipram may be effective in regulating Th1‐mediated diseases such as rheumatoid arthritis.

Effect of nanoparticle-rich diesel exhaust on testicular and hippocampus steroidogenesis in male rats

Background: Nanoparticle-rich diesel exhaust (NR-DE) has potentially adverse effects on testicular steroidogenesis. However, it is unclear whether NR-DE influences steroidogenic systems in the brain. Objective: To investigate the effect of NR-DE on hippocampal steroidogenesis of adult male rats in comparison with its effect on the testis. Methods: F344 male rats (8-week-old) were randomly divided into four groups (n = 8 or 9 per group) and exposed to clean air with 4.6 ± 3.2 μg/m3 in mass concentration, NR-DE with 38 ± 3 μg/m3 (a level nearly equivalent to the environmental standard in Japan (low NR-DE)), NR-DE with 149 ± 8 μg/m3 (high NR-DE), or filtered diesel exhaust with 3.1 ± 1.9 μg/m3 (F-DE), for 5 hours/day, 5 days/week, for 1, 2 or 3 months. F-DE was prepared by removing only particulate matters from high NR-DE with an HEPA filter. Results: Exposures to the high NR-DE for 1 month, and low NR-DE for 2 months, significantly increased or tended to increase plasma and testicular testosterone levels compared to clean air exposure, which might have resulted from the increased expression of mRNA of steroidogenic acute regulatory protein and its protein in the testes of rats. In the hippocampus, high NR-DE exposure for 1 month significantly increased the androstendione level compared to the clean air exposure, while no significant difference was observed in the steroidogenesis between fresh air exposure and any exposure to NR-DE or F-DE. Conclusion: NR-DE may influence steroidogenic enzymes in the testis, but not those in the hippocampus.