Junzo Yonemoto

Bisphenol-A Affects Spermatogenesis in the Adult Rat Even at a Low Dose.

Bisphenol‐A Affects Spermatogenesis in the Adult Rat Even at a Low Dose: Motoharu Sakaue,et al. Environmental Health Sciences Division, National Institute for Environmental Studies—Bisphenol‐A (BPA), a xenobiotic estrogenic compound widely used as a plastics monomer, has been suspected to have a so‐called low dose effect on the reproductive system when administered transplacentally. In the present study, we investigated possible low‐dose effects of BPA on spermatogenesis in adult rats. Male rats (13 weeks old; W13) were administrated a daily oral dose of BPA, ranging from 2 ng to 200 mg/kg, for 6 days and examined for testicular weight (TW) and daily sperm production (DSP) at W14 and W18. A BPA dose as low as 20 jug/kg tended to decrease TW and significantly reduced both DSP and the efficiency of spermatogenesis (DSP per gram testis) at W18, showing that BPA suppressed a normal increase in DSP and TW from W13 to W18. A single administration of 20 fig BPA/kg to W13 rats affected the intensity or mobility of several protein spots in the testicular cytosol fraction as shown by two‐dimensional gel electrophoresis analysis. The present study showed that BPA at a low dose affects spermatogenesis in the adult rat.

Screening and detection of the in vitro agonistic activity of xenobiotics on the retinoic acid receptor

The retinoic acid receptors (RARs) play key roles in various biological processes in response to endogenous retinoic acids. However, excessive embryonic exposure to specific ligands for each subtype of the RAR was reported to induce specific developmental abnormalities. We measured the RAR agonistic activity of 543 chemicals using an assay system adopting yeast cells transfected with the human RAR gamma and a coactivator. Eighty-five of the 543 chemicals, including 16 organochlorine pesticides, 14 styrene dimers, 9 monoalkylphenols and 6 parabens, exhibited RAR gamma agonistic effects in this assay. In particular, monoalkylphenols having a 6-9 carbon alkyl group para to the phenolic hydroxyl group possessed high affinity for the RAR gamma, and their activities were 1.363-0.446% of that of all-trans RA. para-Alkylphenols chlorinated at the ortho position also were about as active or more active than their unchlorinated analogs. In addition, all tested styrene dimers showed positive effects, and the activity of 1-phenyltetralin, the strongest in this category, was 1.169% that of all-trans RA. A number of chemicals having binding affinity for the RAR gamma were revealed in this study (both newly identified and confirmed), further comprehensive studies of in vitro and in vivo effects via the RARs are required for the reliable risk assessment of chemicals. In vitro receptor binding studies represent an important step in hazard identification and suggest a potential mechanism of action, which can be an important step in risk assessment and in particular for screening studies to identify potential toxicity and inform mechanistic studies.

Immunohistochemical localization of thyroid stimulating hormone induced by a low oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin in female Sprague-Dawley rats.

We have investigated how a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects thyroid hormone regulation, especially in relation to the localization of thyroid stimulating hormone (TSH) in the pituitary and that of thyroxin (T4) of the thyroid in the rat. Female Sprague-Dawley rats were given a single oral administration of TCDD ranging from 1.0 to 4.0 microg/kg body weight (bw), and then tissue specimens were removed on day 7 post-administration. Thyroid hormone concentrations were measured in serum, and the expression of the TCDD-responsive genes, UDP-glucuronosyltransferase-1 (UGT1) and cytochrome P4501A1 (CYP1A1) were examined in the liver. TCDD administration resulted in an increase in both immunostaining intensity and the number of TSH-positive cells in the anterior pituitary. T4 was found to localize only in the follicular lumen of the thyroid in vehicle-treated control rats, while TCDD administration caused a foamy change in the colloid of some follicles, an indication of accelerating the biosynthesis of T4 in the thyroid. By morphometrical analysis, the ratio of parenchymal/lumenal area of the thyroid was found to increase in response to TCDD. TCDD treatment as low as 2.0 microg TCDD/kg bw induced a significant decrease in both serum total T4 (TT4) and free T4 (FT4) concentrations in the rats, with a significant increase in serum TSH levels in the 4.0 microg TCDD/kg bw rats. Serum total triiodothyronine (TT3) level was unchanged in all groups. The UGT1 gene was significantly induced at a TCDD dose as low as 1.0 microg/kg bw in a dose-dependent manner. TCDD concentrations in the serum, liver and adipose tissues were detected in a dose-related fashion. The present immunohistochemical results clearly support the earlier biochemical findings on the perturbation of the thyroid-pituitary axis by TCDD and suggest that UGT1 is an immediate target of a low TCDD exposure that triggers the perturbation.

Induction of metallothionein in the livers of female Sprague-Dawley rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin

Metallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotective effects against metal toxicity and external stimuli including ionizing or ultraviolet B irradiation. Since 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to cause an exaggerated oxidative stress response in animals and in different organs, we have studied possible involvement of MT in the oxidative responses induced by TCDD. Female Sprague-Dawley (SD) rats (6-week old) were administered a single oral dose of TCDD that varied from 1.0 to 4.0 microg/kg body weight. The serum and tissues were collected 7 days after dosing. Indicators of oxidative damage were assessed. Significant increases in serum 8-hydroxydeoxyguanosine (8-OHdG) levels were observed in the rats dosed with 2.0 and 4.0 microg TCDD/kg bw. Only 4.0 microg TCDD/kg bw produced a decrease in reduced glutathione concentration in the liver. Immunohistochemical staining revealed a TCDD-induced increase in heme oxygenase-1 (HO-1) expression in the hepatic macrophages (Kupffer cells). Under these conditions, MT protein as well as the mRNAs of MT-I and MT-II, were dose-dependently induced in the liver by TCDD doses from 1.0 microg/kg bw. TCDD-induced MT was found to localize in the parenchymal cells of the liver. Serum concentrations of cytokines (TNF-alpha, IL-1beta and IL-6) were not affected by TCDD. The hepatic concentrations of Cu, Zn and Fe were all increased significantly by TCDD administration. Our results suggest that MT levels are increased in the liver upon exposure to TCDD, perhaps by TCDD-generated reactive oxygen species, and that it may play a protective role in TCDD-induced oxidative stress responses as an antioxidant.

Critical role of cyclooxygenase-2 activation in pathogenesis of hydronephrosis caused by lactational exposure of mice to dioxin.

Congenital hydronephrosis is a serious disease occurring among infants and children. Besides the intrinsic genetic factors, in utero exposure to a xenobiotic, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has been suggested to induce hydronephrosis in rodents owing to anatomical obstruction in the ureter. Here, we report that hydronephrosis induced in mouse pups exposed lactationally to TCDD is not associated with anatomical obstruction, but with abnormal alterations in the subepithelial mesenchyma of the ureter. In the kidneys of these pups, the expressions of a battery of inflammatory cytokines including monocyte chemoattractant protein (MCP)-1, tumor necrosis factor alpha (TNFalpha) and interleukin (IL)-1beta were up-regulated as early as postnatal day (PND) 7. The amounts of cyclooxygenase (COX)-2 mRNA and protein as well as prostaglandin E2 (PGE(2)) were conspicuously up-regulated in an arylhydrocarbon-receptor-dependent manner in the TCDD-induced hydronephrotic kidney, with a subsequent down-regulation of the gene expressions of Na+ and K+ transporters, NKCC2 and ROMK. Daily administration of a COX-2 selective inhibitor to newborns until PND 7 completely abrogated the TCDD-induced PGE(2) synthesis and gene expressions of inflammatory cytokines and electrolyte transporters, and eventually prevented the onset of hydronephrosis. These findings suggest an essential role of COX-2 in mediating the TCDD action of inducing hydronephrosis through the functional impairment rather than the anatomical blockade of the ureter.

The Japan Environment and Children's Study (JECS): A Preliminary Report on Selected Characteristics of Approximately 10 000 Pregnant Women Recruited During the First Year of the Study

BACKGROUND The Japan Environment and Childrens Study (JECS) is an ongoing nationwide birth cohort study launched in January 2011. In this progress report, we present data collected in the first year to summarize selected maternal and infant characteristics. METHODS In the 15 Regional Centers located throughout Japan, the expectant mothers were recruited in early pregnancy at obstetric facilities and/or at local government offices issuing pregnancy journals. Self-administered questionnaires were distributed to the women during their first trimester and then again during the second or third trimester to obtain information on demographic factors, physical and mental health, lifestyle, occupation, environmental exposure, dwelling conditions, and socioeconomic status. Information was obtained from medical records in the first trimester and after delivery on medical history, including gravidity and related complications, parity, maternal anthropometry, and infant physical examinations. RESULTS We collected data on a total of 9819 expectant mothers (mean age = 31.0 years) who gave birth during 2011. There were 9635 live births. The selected infant characteristics (singleton births, gestational age at birth, sex, birth weight) in the JECS population were similar to those in national survey data on the Japanese general population. CONCLUSIONS Our final birth data will eventually be used to evaluate the national representativeness of the JECS population. We hope the JECS will provide valuable information on the impact of the environment in which our children live on their health and development.Background The Japan Environment and Children’s Study (JECS) is an ongoing nationwide birth cohort study launched in January 2011. In this progress report, we present data collected in the first year to summarize selected maternal and infant characteristics. Methods In the 15 Regional Centers located throughout Japan, the expectant mothers were recruited in early pregnancy at obstetric facilities and/or at local government offices issuing pregnancy journals. Self-administered questionnaires were distributed to the women during their first trimester and then again during the second or third trimester to obtain information on demographic factors, physical and mental health, lifestyle, occupation, environmental exposure, dwelling conditions, and socioeconomic status. Information was obtained from medical records in the first trimester and after delivery on medical history, including gravidity and related complications, parity, maternal anthropometry, and infant physical examinations. Results We collected data on a total of 9819 expectant mothers (mean age = 31.0 years) who gave birth during 2011. There were 9635 live births. The selected infant characteristics (singleton births, gestational age at birth, sex, birth weight) in the JECS population were similar to those in national survey data on the Japanese general population. Conclusions Our final birth data will eventually be used to evaluate the national representativeness of the JECS population. We hope the JECS will provide valuable information on the impact of the environment in which our children live on their health and development.

The effects of perinatal exposure to low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin on immune organs in rats

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is revealed to exert diverse biological effects including immunotoxicity, mainly by inadvertently activating the transcription factor arylhydrocarbon receptor (AhR). In the present study, the developmental effects of perinatal exposure to low doses of TCDD on the major immune organs of offspring, thymus and spleen, were investigated focusing on weaning time (postnatal day (PND) 21), puberty (PND 49) and adulthood (PND 120) in male rats. Concurrently, TCDD contents in those organs were measured with a high-resolution gas chromatography-mass spectrometry (GC/MS). In the thymus and spleen, CYP1A1 mRNA induction, the sensitive reaction caused by activation of AhR, was also measured in order to examine whether perinatally administered TCDD can elicit gene expressions in these organs. When pregnant dams were administered a single oral dose of 12.5-800 ng TCDD/kg body weight on gestation day (GD) 15, the weights of the thymus and spleen of the offspring did not differ from those of control animals throughout the experiments. The thymus and spleen maternally exposed to 800 ng TCDD/kg contained 102.0 and 62.7 pg TCDD/g tissue on PND 21, respectively, and the amounts decreased thereafter. In the thymus, dose-dependent CYP1A1 mRNA induction was clearly observed by maternal exposure to 50-800 ng TCDD/kg on PND 5. The induction was gradually decreased on PND 21 and 49. On the other hand, CYP1A1 mRNA induction in the spleen was very weak. In these thymi, no reproducible change was observed by TCDD exposure in cell number and cellular population defined by CD4 and CD8 molecules at any time. In contrast, splenocyte number was shown to decrease by maternal exposure to 12.5-800 ng TCDD/kg in a dose-dependent manner on PND 49. The alteration in spleen cellularity by TCDD was not detected on PND 21 or 120. These results clarified that perinatal exposure to low doses of TCDD affects the immune organs, which is apparent in spleen around puberty and likely to be hardly relevant to AhR-dependent gene expressions.

Behavioral effects of trichloroethylene and tetrachloroethylene in mice.

This study was performed to clarify the toxicological profiles of trichloroethylene (TRCE) and tetrachloroethylene (TECE) when they are administered intraperitoneally in mice. The ED50 for loss of righting reflex were 2596 mg/kg in TRCE and 4209 mg/kg in TECE. TRCE and TECE impaired bridge test performance at 500 and 2000 mg/kg, respectively. An operant behavior performance was also inhibited by TRCE at 1000 mg/kg and by TECE at 2000 mg/kg. Both TRCE and TECE exhibited anticonflict effects in a Vogel-type task at 500 mg/kg. This effect was confirmed by the finding that TRCE exhibited anticonflict action in a Geller-type paradigm at 250 mg/kg and more, as did TRCE did at 1000 mg/kg. These results show that TRCE and TECE affect various behaviors in mice and suggest that conflict behaviors are one of the most sensitive behavioral indicators of the effects of these substances. The toxicological profiles of TRCE and TECE with respect to behavioral effects were very similar, and they can be classified in a single category.

Identification of Novel Low-Dose Bisphenol A Targets in Human Foreskin Fibroblast Cells Derived from Hypospadias Patients

Background/Purpose The effect of low-dose bisphenol A (BPA) exposure on human reproductive health is still controversial. To better understand the molecular basis of the effect of BPA on human reproductive health, a genome-wide screen was performed using human foreskin fibroblast cells (hFFCs) derived from child hypospadias (HS) patients to identify novel targets of low-dose BPA exposure. Methodology/Principal Findings Gene expression profiles of hFFCs were measured after exposure to 10 nM BPA, 0.01 nM 17β-estradiol (E2) or 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 24 h. Differentially expressed genes were identified using an unpaired Students t test with P value cut off at 0.05 and fold change of more than 1.2. These genes were selected for network generation and pathway analysis using Ingenuity Pathways Analysis, Pathway Express and KegArray. Seventy-one genes (42 downregulated and 29 upregulated) were identified as significantly differentially expressed in response to BPA, among which 43 genes were found to be affected exclusively by BPA compared with E2 and TCDD. Of particular interest, real-time PCR analysis revealed that the expression of matrix metallopeptidase 11 (MMP11), a well-known effector of development and normal physiology, was found to be inhibited by BPA (0.47-fold and 0.37-fold at 10 nM and 100 nM, respectively). Furthermore, study of hFFCs derived from HS and cryptorchidism (CO) patients (n = 23 and 11, respectively) indicated that MMP11 expression was significantly lower in the HS group than in the CO group (0.25-fold, P = 0.0027). Conclusions/Significance This present study suggests that an involvement of BPA in the etiology of HS might be associated with the downregulation of MMP11. Further study to elucidate the function of the novel target genes identified in this study during genital tubercle development might increase our knowledge of the effects of low-dose BPA exposure on human reproductive health.

Association of variants in genes involved in environmental chemical metabolism and risk of cryptorchidism and hypospadias

We hypothesized that single-nucleotide polymorphisms (SNPs) of genes involved in environmental endocrine disruptors (EEDs) metabolism might influence the risk of male genital malformations. In this study, we explored for association between 384 SNPs in 15 genes (AHR, AHRR, ARNT, ARNT2, NR1I2, RXRA, RXRB, RXRG, CYP1A1, CYP1A2, CYP1B1, CYP2B6, CYP3A4, CYP17A1 and CYP19A1) and risk of cryptorchidism (CO) and hypospadias (HS) in 334 Japanese (JPN) males (141 controls, 95 CO and 98 HS) and 187 Italian (ITA) males (129 controls and 58 CO). In the JPN study group, five SNPs from ARNT2 (rs2278705 and rs5000770), CYP1A2 (rs2069521), CYP17A1 (rs4919686) and NR1I2 (rs2472680) were significantly associated at both allelic and genotypic levels with risk of at least one genital malformation phenotype. In the ITA study group, two SNPs in AHR (rs3757824) and ARNT2 (rs1020397) were significantly associated with risk of CO. Interaction analysis of the positive SNPs using multifactor dimensionality reduction demonstrated that synergistic interaction between rs2472680, rs4919686 and rs5000770 had 62.81% prediction accuracy for CO (P=0.011) and that between rs2069521 and rs2278705 had 69.98% prediction accuracy for HS (P=0.001) in JPN population. In a combined analysis of JPN and ITA population, the most significant multi-locus association was observed between rs5000770 and rs3757824, which had 65.70% prediction accuracy for CO (P=0.055). Our findings indicate that genetic polymorphisms in genes involved in EED metabolism are associated with risk of CO and HS.