森 千里

自分のPCBs(Polychlorinated biphenyls:ポリ塩化ビフェニール)汚染状況に興味を持った参加者に採血を行い、汚染濃度データを郵送によって告知した。その後、自分の汚染濃度を知った後の心理状況と行動の変化に関するアンケート調査を実施したので結果を報告する。送付対象者144名(男性66名、女性78名)中、97名から回答があった(回収率67.4%)。内、有効回答は71(男性30名、女性41名)、有効回答率49.3%だった。参加動機として多かったものは「自分の汚染濃度を知りたかったから」が男性では70.0%、女性では80.5%、「環境問題に興味があるから」が男性で63.3%、女性で75.6%であった。調査結果を知った時の気持ちで、「最もよくあてはまる」と回答のあった項目は「生活習慣と濃度との関係を知りたい」と答えた人が男性で60.0%、女性で68.3%、「結果を知ってよかった」が男性で53.3%、女性で68.3%だった。調査後の行動については、本研究参加について、家族や友人など他者と「次世代への環境について話した」が男性では40.0%、女性では65.9%だった。本研究参加者は、延べ2000名以上の人にこの研究を話題として示しており、大きな社会的影響力を持つことがわかった。今回の対象者は、検査費を一部負担した上で調査に参加するという、元々環境問題に強い関心を持つ人々が多かったため、この結果をただちに一般化することはできない。化学物質の健康影響問題に関して、研究を通じての社会参加の意識や学習意欲について考察した場合、本研究は、若年層と比べて高齢の世代において、より積極性が高いことを指摘した。しかしながら環境汚染による次世代影響を考えると、より若い世代の男女が関心を持ち行動につなげることがより重要と推測され、今後の働きかけが重要であると考えられた。加えて、自分の汚染度を知ることによって、化学物質による健康影響問題についての関心を引き起こし、結果として生活習慣の見なおしや、周囲の人々に対する影響の波及の可能性が示唆された。(著者抄録)

Mammalian sperm require ATP for motility, and most of it is generated by glycolysis. The glycolytic enzymes segregate to the principal piece region of the flagellum, where some are bound tightly to a novel cytoskeletal structure defining this region, the fibrous sheath (FS), and others are easily extracted with detergents. One of the latter is the spermatogenic cell-specific variant isozyme of hexokinase type 1 (HK1S), characterized by an N-terminal 24-amino acid spermatogenic cell-specific region (SSR). Yeast two-hybrid screens carried out using the SSR as bait determined that HK1S is tethered to muscle-type phosphofructokinase (PFKM) in the principal piece region. This led to the identification of four testis-specific Pfkm splice variants, one that overlapped a variant reported previously (Pfkm-v1) and three that were novel (Pfkm-v2, Pfkm-v3, and Pfkm-v4). They differ from Pfkm transcripts found in somatic cells by encoding a novel 67-amino acid N-terminal extension, the testis-specific region (TSR), producing a spermatogenic cell-specific PFKM variant isozyme (PFKMS). An antiserum generated to the TSR demonstrated that PFKMS is present in the principal piece and is insoluble in 1% Triton X-100 detergent. In subsequent yeast two-hybrid screens, the TSR was found to interact with glutathione S-transferase mu class 5 (GSTM5), identified previously as a spermatogenic cell-specific component of the FS. These results demonstrated that HK1S is tethered in the principal piece region by PFKMS, which in turn is bound tightly to GSTM5 in the FS. © 2010 by the Society for the Study of Reproduction, Inc.

Polybrominated diphenyl ethers (PBDEs) are used as flame retardants to prevent combustion in consumer products, such as electronics, construction materials, and textiles and, therefore, have become important commercial substances. PBDEs were also detected in maternal blood, breast milk, umbilical cord blood, and cord tissue, thereby indicating that fetuses were also exposed to PBDEs. The purpose of this study is to identify the effect of PBDEs on human umbilical vein endothelial cells (HUVECs). Cultured HUVECs were exposed to a commercial mixture of penta-BDE (DE71), octa-BDE (DE79), and deca-BDE (DE83). Each gene expression that was altered in DNA microarray was confirmed by real-time reverse transcription-polymerase chain reaction and Western blotting analysis. The results indicated that gene expressions concerning antioxidant system, i.e., thioredoxin family, 24-dehydrocholesterol reductase (DHCR24), and tumor suppressor protein p53, were altered by PBDEs exposure in HUVECs. Moreover, it was demonstrated that thioredoxin-interacting protein (TXNIP) was a target gene in exposure to DE71 and DE79 in HUVECs, by drastically decreasing time-dependent TXNIP expression in HUVECs.

Perinatal exposure to diethylstilbestrol (DES) can have numerous adverse effects on the reproductive organs later in life, Such as vaginal clear-cell adenocarcinoma. Epigenetic processes including DNA methylation may be involved in the mechanisms. We subcutaneously injected DES to neonatal C57BL/6 mice. At days 5, 14, and 30, expressions of DNA methyltransferases (Dnmts) Dnmt1, Dnmt3a, and Dnmt3b, and transcription factors Sp1 and Sp3 were examined. We also performed restriction landmark genomic scanning (RLGS) to detect aberrant DNA methylation. Real-time RTPCR revealed that expressions of Dnmt1, Dnmt3b, and Sp3 were decreased at day 5 in DES-treated mice, and that those of Dnmt1, Dnmt3a, and Sp1 were also decreased at day 14. RLGS analysis revealed that 5 genomic loci were demethylated, and 5 other loci were methylated by DES treatment. Two loci were cloned, and differential DNA methylation was quantified. Our results indicated that DES altered the expression levels of Dnmts and DNA methylation.

At Chiba University, gross anatomy laboratory sessions (" laboratories") are required for physical therapy students. Though most physical therapy schools require their students to participate in laboratories so that they will better understand the structure of the human body, few data exist on the value of these laboratories specifically for physical therapy students. We administered questionnaires to physical therapy undergraduate students both before and after they participated in laboratories. Questionnaire items focused on student attitudes toward the laboratories and on human life and dignity. Data from 83 students were analyzed, with the following results: (1) 74.7% of students had a positive attitude about attending laboratories before doing so (2) with few exceptions, students' attitudes about upcoming laboratories grew more positive after experiencing the laboratory work (P &lt 0.001) (3) laboratories caused students to contemplate the topics of human life and dignity and (4) 83.1% of students hoped to participate in laboratories at least four times. These results indicate that laboratories reinforce physical therapy students' positive attitudes about laboratory learning and promote student reflection on human life and dignity. This study provides support for the implementation of multiple laboratory sessions using cadavers into a uniform curriculum for physical therapy students in Japan. © 2009 American Association of Anatomists.

Objectives: The aim of this study was to analyze epigenetic (specifically, DNA methylation) participation in the mechanisms of cleft palate only induced by maternal exposure to all-trans retinoic acid in mice. Design: Cleft palate only was induced in fetuses by maternal exposure to all-trans retinoic acid. Their secondary palates were excised for analysis. Cytosine extension assay and restriction landmark genomic scanning were performed to analyze DNA methylation status. The expression levels of the DNA methyltransferases were examined by real-time reverse transcriptase-polymerase chain reaction. Results: Using cytosine extension assay, on gestation day 14.5, the status of DNA methylation within CpG islands and in global DNA was decreased significantly in all-trans retinoic acid-treated groups compared with the controls (p < .01 and p < .05). In the controls, the status within CpG islands on gestation day 14.5 was significantly increased compared with gestation days 13.5 and 18.5 (p < .01). Using real-time reverse transcriptase-polymerase chain reaction, there was no significant change in the expression of DNA methyltransferases, except on gestation day 18.5. Using restriction landmark genomic scanning on gestation day 18.5, five spots (0.49%) in the controls and one spot (0.1%) in all-trans retinoic acid-treated groups were specifically detected. Conclusions: These results indicate that changes in DNA methylation may play an important role in the manifestation of cleft palate only caused by environmental factors such as maternal exposure to all-trans retinoic acid.

During epididymal transit, sperm acquire the ability to initiate rapid forward progressive motility on release into the female reproductive tract or physiological media. Glycolysis is the primary source of the ATP necessary for this motility in the mouse, and several novel glycolytic enzymes have been identified that are localized to the principal piece region of the flagellum. One of these is the spermatogenic cell-specific type 1 hexokinase isozyme (HK1S), the only member of the hexokinase enzyme family detected in sperm. Hexokinase activity was found to be lower in immotile sperm immediately after removal from the cauda epididymis (quiescent) than in sperm incubated in physiological medium for 5 min and showing rapid forward progressive motility (activated). However, incubating sperm in medium containing diamide, an inhibitor of disulfide bond reduction, resulted in lower motility and HK activity than in controls. HK1S was present in dimer and monomer forms in extracts of quiescent sperm but mainly as a monomer in motile sperm. A dimer-size band detected in quiescent sperm with phosphotyrosine antibody was not detected in activated sperm, and the monomer-size band was enhanced. In addition, the general protein oxido-reductase thioredoxin-1 was able to catalyze the in vitro conversion of HK1S dimers to the monomeric form. These results strongly suggest that cleavage of disulfide bonds in HK1S dimers contributes to the increases in HK activity and motility that occur when mouse sperm become activated.

Hexokinase is the first enzyme in the glycolytic pathway and utilizes ATP to convert glucose to glucose-6-phosphate (G6P). We previously identified three variant transcripts of Hk1 that are expressed specifically in spermatogenic cells, have different 5 ' untranslated regions, and encode a protein (HK1S, spermatogenic cell-specific type 1 hexokinase) in which the porin-binding domain (PBD) of HK1 is replaced by a novel N-terminal spermatogenic cell-specific region (SSR). However, the level of expression of the individual variant transcripts or of the other members of the hexokinase gene family (Hk2, Hk3, and Gck) in spermatogenic cells remains uncertain. We show that Hk1, Hk2, and Hk3 transcripts levels are quite low in spermatocytes and spermatids and Gck transcripts are relatively abundant in spermatids, but that glucokinase (GCK) is not detected in spermatozoa. Using real time RT-PCR (qPCR) with primers specific for each of the three variant forms and RNA from whole testis and isolated germ cells, we found that transcripts for Hk1_v2 and Hk1_v3, but not for Hk1_v1, are relatively high in spermatids. Similar results were seen using spermatogenic cells isolated by laser-capture microdissection (LCM). Immunoblotting studies found that HK1S is abundant in sperm, and immunostaining confirmed that HK1S is located mainly in the principal piece of the sperm flagellum, where other spermatogenic cell-specific glycolytic enzymes have been found. These results strongly suggest that HK1, HK2, HK3, and GCK are unlikely to have a role in glycolysis in sperm and that HK1S encoded by Hk1_v2 and Hk1_v3 serves this role.

Brominated flame retardants (BFRs) are used to prevent combustion in consumer products. Examples of BFRs are polybrominated diphenyl ethers (PBDEs), tetrabromobisphenol A (TBBPA), and tribromophenol (TBP). These compounds are reported to have adverse effects on human health and endocrine disrupting effects. The purpose of this study was to identify the Japanese perinatal exposure to PBDEs, hydroxylated PBDE metabolites (OH-PBDEs), TBBPA, and TBP compared with polychlorinated biphenyls (PCBs) and hydroxylated PCB metabolites (OH-PCBs). We investigated the concentrations of these compounds in maternal blood, maternal milk, cord blood, and umbilical cords from 16 Japanese mother-infant pairs by HRGC/HRMS. PBDEs were detected in all samples of maternal blood (mean ± SD median = 25 ± 23 pg/g 18 pg/g wet weight), maternal milk (140 ± 220 pg/g 59 pg/g wet weight), cord blood (4.8 ± 6.5 pg/g 1.6 pg/g wet weight), and umbilical cords (3.1 ± 3.1 pg/g 2.1 pg/g wet weight). The mothers were divided into two groups, a high-concentration group and a low-concentration group. The percentage of BDE-47 showed the greatest difference between the two groups. 6-OH-BDE-47, TBBPA, and TBP were detected in all umbilical cord samples (mean ± SD median = 8.4 ± 8.1 pg/g 8.0 pg/g, 16 ± 5.5 pg/g 15 pg/g, and 33 ± 8.2 pg/g 32 pg/g wet weight respectively), but not in all maternal blood or cord blood samples. These results indicate that OH-PBDEs, TBBPA, and TBP, in addition to PBDEs, PCBs, and OH-PCBs, pass through the blood-placenta barrier and are retained in the umbilical cord.

Polychlorinated biphenyls (PCBs) are a group of persistent pollutants that are detected in maternal serum and umbilical cord, suggesting that fetal exposure also needs to be considered. The effects of dioxin-like PCB congeners 3,3',4,4'-tetrachlorobiphenyl (PCB77) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) and a non-dioxin-like compound 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) on the expression of endothelial nitric oxide synthase (eNOS), known to maintain blood flow to the fetus, in human umbilical vein endothelial cells (HUVECs) were investigated. The mRNA levels of eNOS, aryl hydrocarbon receptor (AhR) and cytochrome P450 (CYP) 1A1 in cells treated with 5 mu M PCBs for 24 hours were analysed by real-time RT-PCR. Cells were also treated with alpha-naphthoflavone (alpha NF), an AhR antagonist or ICI 182780, an estrogen receptor (ER) antagonist, one hour prior to PCB exposure, to observe the effects of these receptors on eNOS modulation. Each PCB increased the eNOS mRNA level by 4.5-fold that was markedly inhibited by alpha NF. ERs were also suspected of altering eNOS levels because ICI 182780 treatment resulted in a decrease in the eNOS level. These results suggest that the eNOS mRNA expression increases due to the action of PCBs related to both AhR and ERs in HUVECs, and that maternal PCB exposure could influence fetal circulation.

In our previous study, the vagina] opening (VO) day of C57BL/6 mice was accelerated several days by chronic exposure to a 0.05% isoflavone (IF) fortified diet. The purpose of this study was to investigate whether the acceleration of VO by IF (1) has a critical window, (2) is modified by IF exposure combined with 17 beta-estradiol (E2), and (3) has any relation with gene expressions of estrogen-related receptors (ERRs). As a result, we determined that the critical window for the acceleration of VO was between 15 and 21 days postnatal. The combined effect of E2 and IF was thought to be additional in the acceleration of VO. The gene expression of ERR gamma was significantly decreased in vagina by IF. The reduction of ERR gamma may have two possible sequelae: disarrangement of vaginal development and high risk of vaginal cancer. In conclusion, IF exposure has a critical window for acceleration of VO and may have adverse effect on mouse vagina. (c) 2007 Elsevier Inc. All rights reserved.

The human gross anatomy is one of the important courses for a co-medical worker, such as a physical therapist, a sports trainer or a nurse. Similarly, the students at a co-medical school should learn the human gross anatomy course. To improve understanding of the human body, the educational human dissection view is important to translate the human structure into some optical information. In the present study, the survey was performed on the students in the school of physical therapy who took part in the educational human dissection tour at Chiba University. The total number of answers was 88 and the reply percentage was 77.2% (68 persons ; male=50, female=18). The 92.6% of students had interest in the anatomy and felt that their understanding of the structure of a human body was improved. Further, the students who had participated in the dissection for several times showed more understanding of the views. The results suggested that the multiple experience of the human anatomical views isuseful for the co-medical students to improve the quality of their understanding ofthe human body.

Previously, we reported that decreased epididymal expression of CD59 and decay accelerating factor (DAF) genes may affect sperm motility and the acrosome reaction in rats treated long-term (28 days) with sulfasalazine. To investigate the early effects of sulfasalazine on the male reproductive tract, we presently examined sperm motility, the acrosome reaction, and gene expression in the testes and epididymides of rats treated with sulfasalazine for 1, 7 or 14 days. Reduced sperm motility and acrosome reactions were noted on day 7, however, there were no remarkable changes in testicular gene expression. On the other hand, attenuated epididymal gene expression of CD59 and DAF was observed as early as day 1. As CD59 and DAF are secreted from the epididymis and play a role in sperm maturation, we hypothesize that sulfasalazine affects sperm maturation as an early effect and that CD59 and DAF genes are related to the negative effect. (c) 2006 Elsevier Inc. All rights reserved.

There is increasing number of people suffering from "Sick-building Syndrome" in Japan. Therefore, we started "Chemi-less Town Project", building a model town in which buildings are constructed with fewer chemicals. In this paper, we introduce the background, concept and current situation of the project. We made the key word, "Chemi-less", which means "fewer" chemicals. In the model town, there are houses, clinic, school, library and park. At the clinic, we will practice the environmental preventive medicine. While decreasing the harmful chemicals in the town, we will carry out to create better environment in order to prevent possible adverse health effects, and to increase the quality of life of future generations. In five years, we would like to suggest new standards of houses and buildings, so that people can choose materials and technique to make towns according to the target level. Using Chemi-less Town project, we will spread our idea world wide, about the necessity of sustainable health town for future generations from the view of the environmental preventive medicine.

Isoflavone (IF), a type of phytoestrogen, has multiple beneficial effects, but too much phytoestrogen can have adverse effects on offspring. To examine whether chronic exposure to high IF has adverse effects on reproductive development, mice offspring were exposed to IF through dietary administration to dams during pregnancy and lactation and to the offspring directly after weaning until sacrifice. In male offspring, there was no difference between the IF group and controls; however, in female offspring in the IF group, remarkably earlier puberty and induction of multioocyte follicles on postnatal day (PND) 21 were observed. Gene expression levels of estrogen receptor β decreased in the ovary and vagina on PND 21. These results suggest that chronic exposure to higher than normal levels of IF induces alterations in the reproductive development of female mice through an estrogenic effect.

Neonatal administration of diethylstilbestrol (DES) to rodents has adverse effects on spermatogenesis. However, not many studies have been conducted to determine which type of cell - germ or somatic - is the major target of DES. In order to clarify this, we tried reciprocal germ cell transplantation-transplantation of germ cells from DES-treated mice into intact mice and germ cells from normal mice into DES-treated mice. The donor germ cells were tagged with the green fluorescent protein (GFP) gene in order to distinguish the exogenous germ cells from the endogenous cells. Moreover, to obtain a large number of spermatogonia from the testes of adult mice, we performed fractionation by centrifugation with Percoll. Consequently, we found that the germ cells collected from DES-treated mice have differentiated into normal sperms in normal seminiferous tubules. However, in the case of the transplantation of normal germ cells into the seminiferous tubules of DES-treated mice, defective spermatogenesis was observed. In conclusion, DES has adverse effects on the somatic cells that are involved in spermatogenesis rather than the germ cells. (c) 2006 Elsevier Inc. All rights reserved.

In the gross anatomy laboratory, which is one of the compulsory subjects in most medical and dental schools, participants cannot avoid exposure to formaldehyde (FA), which is emitted from cadavers during dissection. FA has been recognized as a harmful chemical and we have previously reported that symptoms felt by participants in a gross anatomy laboratory are similar to those of allergic diseases. Although immunoglobulin E (IgE)-mediated sensitization to FA is a matter of controversy, it is possible that IgE production is evoked during a gross anatomy laboratory and is responsible for the reported symptoms. In order to test this hypothesis, we examined the relationships between the personal FA exposure levels and plasma IgE levels in a gross anatomy laboratory. In the laboratory, the personal FA exposure levels ranged from 0.33 to 1.47 ppm. Total blood IgE levels did not increase significantly and specific IgE to FA was negative during the laboratory sessions. Thus, from this study, we cannot support the hypothesis that the exposure to FA triggers an IgE-mediated reaction in this study. In conclusion, exposure to FA does not induce IgE production during gross anatomy laboratories at our school. <br>

Flutamide (FLUT) has potent anti-androgenic activity and is used in the medical field and in a screening test to detect endocrinologically active compounds. Our previous study demonstrated that FLUT induced histological deformation of spermatids and ultrastructural defects of the apical ectoplasmic specialization (ES) in the mouse testis. The apical ES is an actin-based junctional structure between the Sertoli cells and germ cells. Cortactin, an actin-binding protein, is found in the actin layer of ES. The protein level of cortactin was decreased in FLUT-treated testes as shown by Western blot analysis. The detailed analysis indicated that the protein level was drastically decreased in FLUT-treated seminiferous tubules of stages from VI to IX. Immunohistochemistry and immunoelectron microscopy showed that FLUT depressed cortactin expression in the apical ES. In addition, the effect of FLUT on cortactin localization appeared between 12 h and 8 days (about 180 h) after a one-day treatment. These results suggest that FLUT depressed the expression of cortactin in the apical ES with stage specificity. Therefore, the initial target of FLUT may be the cell-cell interactions between the Sertoli and germ cells. To our knowledge, this study is the first to document the decrease of cortactin expression in the abnormal apical ES following treatment with FLUT. (c) 2006 Elsevier Ltd. All rights reserved.

Macrophage migration inhibitory factor (MIF) is a multifunctional protein that acts as a pro-inflammatory cytokine, pituitary hormone, immunoregulator and mitogen. The MIF gene knockout (MIFKO) mouse has been used to study the MIF response in many tissues, such as with skin injury and spinal cord injury; however, there is little information about the MIFKO mouse testis. This study reports the levels of serum and intratesticular estradiol and testosterone, the ultrastructure of the testis, and preliminary findings from in vitro fertilization. Our results revealed a decrease in estradiol and testosterone levels and deformation in spermiogenesis, in the MIFKO mouse. These initial findings study may lead to a better understanding of the role that MIF plays in the mouse testis. (c) 2005 Elsevier Inc. All rights reserved.

Predictive biomarkers of testicular toxicity are needed for an efficient development of drugs. The purpose of the present study was to obtain further insight into the toxicity mechanisms of various male reproductive toxicants and to detect genomic biomarkers for rapid screening of testicular toxicity. Four reproductive toxicants, 2, 5-hexanedione (Sertoli cells toxicant), ethylene glycol monomethyl ether (EGME; spermatocytes toxicant), cyclophosphamide (spermatogonia toxicant) and sulfasalazine, were orally administered to male rats once. Six hours after the single dosing, gene expression in the testes was monitored by cDNA microarray and real-time RT-PCR and the testes were histopathologically examined. No histopathological abnormality was detected except for slight degeneration of spermatocytes in the EGME-treated testes. cDNA microarray analysis revealed differential gene expression profiles, and it was possible based on the profiles to characterize the action of the compounds in the testes. Interestingly, 3 spermatogenesis-related genes - heat shock protein 70-2, insulin growth factor binding protein 3 and glutathione S transferase pi - were affected by all the compounds. The above changes of gene expression were detectable within a short period after the dosing prior to the appearance of obvious pathological changes. These data suggest that cDNA microarray is a useful technique for evaluation of primary testicular toxicity. Furthermore, we propose the above 3 spermatogenesis-related genes as potential biomarkers of testicular toxicity.

Toxicogenomic analysis of human umbilical cords revealed that the hierarchical order of the umbilical cords clustered according to their gene expression profiles corresponded to their order according to their total concentrations of chemicals, with one exception. In the exceptional umbilical cord, the total concentration of chemicals was lowest, but its gene expression profile was most similar to that of the umbilical cord exhibiting the highest level of total chemical concentrations. These results suggest that gene expression in umbilical cords may reflect their exposure to some persistent chemicals. In addition, the expression profile may reflect contamination with a mixture of chemicals rather than that with each single compound. Furthermore an important suggestion from this study is that toxicogenomic analysis can detect potential high-risk groups, because genetically higher susceptibility as well as higher exposure of an individual to multiple chemicals can be regarded as a higher health risk to the individual.

Sulfasalazine (SASP) has been reported to depress the fertility in men and experimental male animals, but the fundamental mechanisms of infertility caused by SASP are still unknown. This study was designed to investigate the mechanisms of infertility in rats treated with SASP at a dose of 600 mg/kg for 28 days, including monitoring of sperm motility using computer associated sperm analysis system and acrosome reaction by FITC-concanavalin A lectin staining. The sperm motility and acrosome reaction, which are important for fertilization, were significantly reduced by SASP. Furthermore, to investigate the molecular mechanisms of infertility induced by SASP, mRNA expression analysis in the testes was performed using cDNA microarray as a first screening. It was revealed that CD59, which is located on the acrosomal membrane and is known to be important for the reproductive function of sperm, was affected in the testes; this was also confirmed by real-time PCR analysis, but the spermatogenesis-related genes examined in this study were not affected. Therefore, we focused on CD59 and two other acrosome membrane related-genes: MCP and DAF. CD59, MCP, and DAF in the epididymides of SASP-treated rats were significantly decreased as assessed by real-time RT-PCR analysis and additionally, the expression of CD59 protein was found to be decreased by Western blotting. These results allowed us to hypothesize that the suppression of epididymal acrosomal membrane proteins synthesis with their consequent reduced incorporation to the sperm membrane leads to a depressed sperm motility and acrosome reaction, and thereby leads to infertility in SASP treated male rats.