Hidenobu Miyaso

CADMIUM EXPOSURE INCREASES SUSCEPTIBILITY TO TESTICULAR AUTOIMMUNITY IN MICE

Cadmium, one of various environmental toxicants, is known to suppress systemic immunity and to injure the testicular capillary endothelia with resultant necrosis of testicular tissues in mice and rats treated with high doses. Recently, it also became evident that cadmium can affect the integrity of the blood–testis barrier (BTB), the endocrine function of Leydig cells, apoptosis of germ cells and systemic immunity, even on treatment with a low dose that does not induce spermatogenic disturbance. Experimental autoimmune orchitis (EAO), i.e., an organ‐specific autoimmunity of the testis, can be induced by repeated immunization with testicular antigens, and its pathology is characterized by lymphocytic inflammation and spermatogenic disturbance. In the present study, we investigated the morphological and functional changes of testes in mice treated with a low dose of cadmium chloride (CdCl2) and also examined its toxicity as to susceptibility to EAO. The results showed that exposure to 3 mg CdCl2 kg−1 body weight did not affect the spermatogenic state. However, the BTB at the tubuli recti and the rete testis, but not the seminiferous tubules, was slightly weakened, and intra‐testicular mRNA expression of interleukin (IL)‐6, tumor necrosis factor‐α and IL‐1β was significantly increased by the CdCl2 treatment. Furthermore, immunization with testicular antigens after the CdCl2 exposure significantly augmented the EAO severity. Therefore, exposure to a low dose of CdCl2 induces no significant disturbance of spermatogenesis, however, it does change the immunological microcircumstances in the testis, resulting in increased susceptibility to testicular autoimmunity. Copyright

Effects on the local immunity in the testis by exposure to di-(2-ethylhexyl) phthalate (DEHP) in mice.

Exposure to di-(2-ethylhexyl) phthalate (DEHP) has been reported to induce spermatogenic disturbance through oxidant stress and affect the immune system as an adjuvant. However, the effect of DEHP on the testicular immune microenvironment has not yet been investigated. In the present study, we examined the testicular immune microenvironment after exposure to doses of DEHP, previously identified as no-observed-adverse-effect levels. Adult male mice were administered food containing 0%, 0.01% or 0.1% DEHP and then testes were analyzed. The results showed that a slight but significant spermatogenic disturbance appeared in the 0.1% DEHP group but not in the 0.01% DEHP group at 8 weeks. It was also demonstrated that lymphocytes and F4/80- and MHC class II- positive cells were significantly increased with the elevation of IL-10 and IFN-γ mRNA expressions in the testes of not only the 0.1% DEHP group but also the 0.01% DEHP group at 8 weeks. Histochemical analyses involving horseradish peroxidase (HRP) as a tracer showed that a little blood-borne HRP had infiltrated into the lumen of a few seminiferous tubules beyond the blood-testis-barrier in both the 0.1% and 0.01% DEHP groups at 8 weeks. This indicates that a dose of DEHP that has little effects on spermatogenesis can change the testicular immune microenvironment with functional damage of the blood-testis barrier.

Chiba study of Mother and Children's Health (C-MACH): cohort study with omics analyses

Purpose Recent epidemiological studies have shown that environmental factors during the fetal period to early childhood might affect the risk of non-communicable diseases in adulthood. This is referred to as the developmental origins of health and disease (DOHaD) concept. The Chiba study of Mother and Childrens Health (C-MACH) is a birth cohort study based on the DOHaD hypothesis and involves multiomics analysis. This study aims to explore the effects of genetic and environmental factors—particularly the fetal environment and postbirth living environment—on childrens health, and to identify potential biomarkers for these effects. Participants The C-MACH consists of three hospital-based cohorts. The study participants are pregnant women at <13 weeks gestation. Women who underwent an examination in one of the three hospitals received an explanation of the study. The participants consented to completing questionnaire surveys and the collection and storage of biological and house/environmental samples. Participants were provided unique study numbers. All of the data and biological specimens will be stored in the Chiba University Center for Preventive Medical Sciences and Chiba University Center for Preventive Medical Sciences BioBank, respectively. Findings to date Consent to participate was obtained from 433 women. Of these women, 376 women completed questionnaires in the early gestational period. The mean age was 32.5 (4.4) years. The mean body mass index (BMI) was 21.1 (3.0) kg/m2. Before pregnancy, 72.3% of the women had a BMI of 18.5–24.9 kg/m2. During early pregnancy, 5.0% of the participants smoked. Future plans Primary outcomes are allergy, obesity, endocrine and metabolic disorders, and developmental disorders. Genome-level, metabolome-level, umbilical cord DNA methylation (epigenome), gut microbiota and environmental chemical exposure variables will be evaluated. We will analyse the relationships between the outcomes and analytical variables.

Pilot study of a dissection table for gross anatomy laboratory equipped with a photocatalytic device that decomposes formaldehyde.

Recently, there has been concern about the effect of formaldehyde (FA) on the human body. The International Agency for Research on Cancer (IARC) has reported that exposure to FA can induce cancer in humans . FA is also classified by the Japan Society for Occupational Health as “a substance that is considered to cause cancer in human beings and there is evidence that it does cause cancer”. FA is also a potent contact sensitizer and can elicit contact dermatitis and respiratory symptoms, probably through irritant mechanisms . Gross anatomy laboratory is a compulsory subject in most medical and dental schools and is used for studying the normal structure of the human body. Cadavers for the gross anatomy laboratory are generally injected with embalming fluid, which contains FA as a principal component. Since long periods in the gross anatomy laboratory are required to learn human body structures in detail, it is necessary for the cadavers to be fixed and preserved. Antisepsis using FA is used for this purpose because no better method exists at present. To reduce exposure to FA, the Japanese Ministry of Education, Culture, Sports, Science and Technology has released “The improvement plan for the dissection course in medical and dental schools” 4) which requires a reduction of FA concentrations in gross anatomy laboratories. To prevent significant sensory irritation in the general population, the World Health Organization (WHO) recommends an air quality guideline value of 0.1 mg/m 3

Early postnatal exposure to a low dose of decabromodiphenyl ether affects expression of androgen and thyroid hormone receptor-alpha and its splicing variants in mouse Sertoli cells.

Decabromodiphenyl ether (decaBDE) adversely affects reproduction and development. Our previous study showed that postnatal exposure to a low dose of decaBDE (0.025 mg/kg body weight/day) by subcutaneous injection on postnatal days (PNDs) 1 through 5 leads to reductions in testicular size and number of Sertoli cells and sperm, while higher dose of decaBDE (2.5 mg/kg body weight/day) had no significant differences about these. In the present study, we examined the molecular mechanism of these effects on mouse testes following postnatal exposure to a low decaBDE dose. We hypothesized that postnatal exposure to decaBDE may alter levels of serum thyroid hormones (THs) and testosterone, or the level of TH receptor alpha (Thra) transcripts and its splicing variants and androgen receptor (Ar) in Sertoli cells, adversely affecting spermatogenesis. To test this hypothesis, we examined serum TH and testosterone levels and the levels of transcripts of the Ar, Thra and its splicing variants, and Thra splicing factors (Hnrnpa1, Srsf1, and Hnrnph1) with qPCR in isolated mouse Sertoli cells exposed postnatally to decaBDE (0.025, 0.25, and 2.5 mg/kg). Levels of serum testosterone and transcripts encoding Ar, Thra, and its variant, Thra1, declined significantly in Sertoli cells of mice exposed to 0.025 mg decaBDE/kg. No significant differences in serum TH level or Thra2, Hnrnph1, or Srsf1 transcript levels were observed between control and decaBDE-exposed mice. However, the Thra1:Thra2 and Hnrnpa1:Srsf1 ratios were altered in Sertoli cells of mice exposed to 0.025 mg decaBDE/kg but not in cells exposed to 0.25 or 2.5 mg decaBDE/kg. These results indicate that postnatal exposure to a low dose of decaBDE on PNDs 1 through 5 lowers the testosterone level and the levels of Ar and Thra transcripts in Sertoli cells, accompanied by an imbalance in the ratios of Thra splicing variants, resulting in smaller testicular size and impaired spermatogenesis.

The effects of early postnatal exposure to a low dose of decabromodiphenyl ether (BDE-209) on serum metabolites in male mice

The toxicity of decabromodiphenyl ether (BDE-209) has been reported in several studies. However, there is not much known about the toxicological biomarkers that characterize BDE-209 exposure. In this study, we subcutaneously exposed mice to 0.025 mg/kg/day BDE-209 on postnatal days 1‑5 and sacrificed the animals at 12 weeks of age (day 84). Flow injection analysis and hydrophilic interaction chromatography-triple quadrupole mass spectrometry were used to determine the serum metabolomes of these mice in order to characterize the effects of BDE-209 exposure. Data analysis showed a good separation between control and exposed mice (R(2) = 0.953, Q(2) = 0.728, and ANOVA of the cross‑validated residuals (CV‑ANOVA): P‑value = 0.0317) and 54 metabolites were identified as altered in the exposed animals. These were selected using variable importance (VIP) and loadings scaled by a correlation coefficient criteria and orthogonal partial least squares discriminant analysis (OPLS‑DA). BDE‑209‑exposed mice showed lower levels of long-chain acylcarnitines and citrate cycle-related metabolites, and higher levels of some amino acids, long-chain phospholipids, and short-chain acylcarnitines. The disruption of fatty acid, carbohydrate, and amino acid metabolism observed in the serum metabolome might be related to the previously observed impaired spermatogenesis in mice with early postnatal exposure to a low dose of BDE-209.

The Changes of Cortactin p80/85 Isoform Profiles and Tyrosine Phosphorylation Status During Spermatogenesis in the Mouse Testis

Cortactin--an F-actin-binding protein--regulates actin polymerization and plays an important role in cytoskeleton actin dynamics. Cortactin functions are reportedly regulated by changes in its isoform (p80/85) profiles and phosphorylation status. Although essential for spermatogenesis, the exact role of cortactin in the testis has not been well elucidated. Herein, we examined its dynamics in isoform profiles and tyrosine phosphorylation levels during spermatogenesis in mice. Western blot analysis showed that the amount of the p85 isoform of cortactin particularly decreased during stages VI-VIII. In the p80 isoform, cortactin phosphorylations at both tyrosine 421 and 466 were detected during stages XII-V and VI-VIII. For the p85 isoform, tyrosine 466 phosphorylation of cortactin, not 421, was detected during stages XII-V and VI-VIII, and this phosphorylation particularly increased during stages VI-VIII. The tyrosine 466 phosphorylation level of the p85 isoform of cortactin relative to the total amount of the p85 isoform notably increased during stages VI-VIII. Additionally, immunohistochemical analysis showed localization of the tyrosine 466-phosphorylated cortactin around the heads of elongating and elongated spermatids. Finally, we examined the effect of flutamide--an antiandrogen--on cortactin isoform profiles and phosphorylation status. The amount of tyrosine 466-phosphorylated p85 isoform of cortactin relative to the total amount of the p85 isoform of cortactin decreased after flutamide injection, whereas no change was detected with regard to the total amount of cortactin p85 isoform. These data suggest the possibility that localization of the more tyrosine 466-phosphorylated p85 isoform of cortactin around the heads of elongated spermatids is important for stages VII-VIII processes and that its phosphorylation site of the p85 isoform might be a target for creating a novel contraceptive and treating infertility in the future.

The methylation levels of the H19 differentially methylated region in human umbilical cords reflect newborn parameters and changes by maternal environmental factors during early pregnancy

Abstract H19 is a tumor‐suppressor gene, and changes in the methylation of the H19‐differential methylation region (H19‐DMR) are related to human health. However, little is known about the factors that regulate the methylation levels of H19‐DMR. Several recent studies have shown that maternal environmental factors during pregnancy, such as smoking, drinking, chemical exposure, and nutrient intake, can alter the methylation levels of several genes in fetal tissues. In this study, we examined the effects of maternal factors on changes in the methylation levels of H19‐DMR in the human umbilical cord (UC), an extra‐embryonic tissue. Participants from the Chiba study of Mother and Childrens Health (C‐MACH) were enrolled in this study. Genomic DNA was extracted from UC samples, and the methylation level of H19‐DMR was evaluated by methylation‐sensitive high resolution melting analysis. Individual maternal and paternal factors and clinical information for newborns at birth were examined using questionnaires prepared in the C‐MACH study, a brief‐type self‐administered diet history questionnaire (BDHQ) during early pregnancy (gestational age of 12 weeks), and medical records. Univariate and multivariate logistic regression analyses indicated that reduced H19‐DMR methylation (<50% methylation) in UC tissues was positively related to decreased head circumference in newborns [odds ratio (OR) =2.82; 95% confidence intervals (CI): 1.21–6.87; p=0.0183 and OR =2.51; 95% CI: 1.02–6.46; p=0.0499, respectively]. Moreover, multiple comparison test showed that H19‐DMR methylation in UC tissues was significantly reduced in the low calorie group (intake of less than 1,000 kcal/day; methylation level: 40.98%; 95% CI: 33.86–48.11) compared with that in the middle (1,000–1,999 kcal/day; methylation level: 51.28%; 95% CI: 48.28–54.27) and high (≥2,000 kcal/day; methylation level: 52.16%; 95% CI: 44.81–59.51) calorie groups (p=0.0054 and 0.047, respectively). In the subpopulations with low to moderate calorie intake (<2,000 kcal/day), reduced H19‐DMR methylation in UC tissues was significantly related to serum homocysteine concentration (OR =0.520; 95% CI: 0.285–0.875; p=0.019), maternal age (OR =1.22; 95% CI: 1.01–1.52; p=0.049), and serum folate levels (OR =0.917; 95% CI: 0.838–0.990; p=0.040). These data indicated that H19‐DMR methylation levels in human UC tissues could be modulated by maternal factors during early pregnancy and may affect fetal and newborn growth. HighlightsWe examined the effect of maternal factors on H19‐DMR methylation in umbilical cords (UC).Reduced H19‐DMR methylation in UCs was related to decreased head circumference.H19‐DMR methylation in UCs was reduced in the low calorie group.Decreased serum folate levels were associated with lower H19‐DMR methylation in UCs.H19‐DMR methylation in UCs could be modulated by maternal factors during pregnancy.

Associations between changes in the maternal gut microbiome and differentially methylated regions of diabetes-associated genes in fetuses: A pilot study from a birth cohort study

Several intrauterine environmental factors can increase the future risk of type 2 diabetes. The microbiome can influence the balance between health and disease. However, the influence of the maternal gut microbiome on the future risk of diabetes in the fetus is unknown. The present study investigated the associations between maternal gut microbiome and differentially methylated regions of diabetes‐associated genes in umbilical cord samples. The present study included 10 pregnant participants from a birth cohort study. 16S ribosomal ribonucleic acid metagenome analysis of maternal stool samples and deoxyribonucleic acid methylation assays of umbilical cord samples were carried out. The present study found that changes in the UBE2E2 and KCNQ1 methylation rates in umbilical cord samples were associated with the proportion of Firmicutes in the maternal gut, albeit with marginal correlations after adjustment for age and body mass index. These findings suggest a link between the methylation of diabetes‐associated genes in fetuses and maternal microbiota components during pregnancy.

Levels of formaldehyde vapor released from embalmed cadavers in each dissection stage

Formaldehyde (FA) is an aldehyde used in antiseptics and adhesives. The World Health Organization (WHO) and other institutes have linked FA to sick building syndrome and allergic diseases. Recent studies have reported that cadavers embalmed using formalin and ethanol-based preservative solutions release FA vapor during dissection and that FA vapor may adversely affect students and lecturers in gross anatomy laboratories. However, few details have been reported correlating dissection stage with increased FA vapor release. In this study, we evaluated the vapor level of FA released in each dissection stage. Six cadavers for which consent was given for use in anatomy research and education were examined in this study. Using an active sampling method, FA vapor was collected above the thoracoabdominal region of each dissected cadaver. FA was eluted from each sampler using acetonitrile and analyzed by high-performance liquid chromatography. Our data show that FA levels significantly increase after skin incision and that the vapor level of FA released differs between male and female cadavers. We also found that subcutaneous adipose tissues of the thoracoabdominal-region release FA vapor and that female cadavers release significantly higher levels of FA per kilogram of subcutaneous adipose tissue than do male cadavers. Based on these data, we propose the methods be developed to prevent exposure to FA vapors released from cadavers.