Tomoko Mitsuhashi

Fetal and neonatal exposure to three typical environmental chemicals with different mechanisms of action: Mixed exposure to phenol, phthalate, and dioxin cancels the effects of sole exposure on mouse midbrain dopaminergic nuclei

A major question is whether exposure to mixtures of low-dose endocrine disruptors (EDs) having different action mechanisms affects neurodevelopment differently than exposure to EDs individually. We therefore investigated the effects of fetal and neonatal exposure to three typical EDs - bisphenol A (BPA), di-(2-ethylhexyl)-phthalate (DEHP), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) - on the midbrain dopaminergic system associated with functions - including motor activity, emotion, and cognition - affected by neuropsychiatric diseases such as attention-deficit/hyperactivity disorder. ICR mouse dams and their pups were orally treated with BPA (5mg/(kg day)), DEHP (1mg/(kg day)), or TCDD (8ng/kg) individually, or with mixtures thereof, to compare the effects between sole and mixed administration. We analyzed tyrosine hydroxylase (TH)- and Fos-immunoreactive (ir) neurons as markers of dopamine and neuronal activation, respectively. The numbers of TH- and/or Fos-ir neurons and the intensity of TH-immunoreactivity within midbrain dopaminergic nuclei (A9, A10, and A8) of each sole administration group significantly differed from controls at 2, 4, and 6 weeks of age. In contrast, no significant differences were detected in the mixture groups, suggesting counteractions among those chemicals. These results indicate that ED mixtures as pollution have unique and elusive effects. Thyroid hormones and/or aryl hydrocarbon receptor-related mechanisms may be responsible for this counteraction.

When does the sex ratio of offspring of the paternal 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure decrease: In the spermatozoa stage or at fertilization?

Recent animal experiments confirmed that paternal 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure decreases the sex ratio of offspring at birth without altering litter size. However, the timing of this decrease remained unclear. Male mice were administered TCDD at 7-12 weeks of age and mated with non-treated females. The Y-bearing/X-bearing sperm ratio was examined by real-time PCR and FISH methods, and the sex ratio of the 2-cell embryos collected from non-treated females that had been mated with TCDD-exposed males were investigated by nested PCR. The Y-bearing/X-bearing sperm ratio was not significantly decreased in the TCDD group. However, the sex ratio of the 2-cell embryos of the TCDD group was significantly lower than that of the control group. These results may have resulted from a decrease in fertility of Y-bearing sperm. Thus, the results of this study suggested that the sex ratio of the offspring was decreased at fertilization and not during the spermatozoa stage.

Direct effects of diethylstilbestrol on the gene expression of the cholesterol side-chain cleavage enzyme (P450scc) in testicular Leydig cells.

AIMS To investigate the precise mechanisms underlying the action of estrogenic endocrine disruptors, we evaluated the direct effects of synthetic estrogen diethylstilbestrol (DES) on steroidogenesis in Leydig cells, with particular emphasis on the expression of the cholesterol side-chain cleavage enzyme P450scc. Furthermore, the mechanism underlying the action of DES was compared with that of endogenous estrogen 17beta-estradiol (E2), which has a potency equivalent to that of DES. MAIN METHODS TTE1 Leydig cells were treated with 5 x 10(-)(8) microM to 5 microM DES or E2 for 24h, and P450scc gene expression and the histone modifications underlying their transcriptional activation were examined using reverse transcription-polymerase chain reaction (RT-PCR) and chromatin immunoprecipitation (ChIP), respectively. KEY FINDINGS P450scc mRNA expression in the DES-treated and E2-treated cells reduced in inverse proportion to the dose of DES and E2, respectively; however, cAMP stimulation induced a recovery in the expression to a level approximately equal to those in the controls. In the DES-treated cells, ChIP assay revealed histone deacetylation in the P450scc promoter region. Interestingly, E2 did not cause histone deacetylation. SIGNIFICANCE In the early stages of steroidogenesis, DES and E2 directly induced a reduction in P450scc mRNA expression in inverse proportion to their doses, and treatment with cAMP restored the decreased P450scc mRNA expression. Furthermore, DES can induce alterations in the histone modification of the P450scc gene, and natural estrogen and synthetic estrogenic compounds such as DES may induce reproductive disorders through different molecular mechanisms.

Epigenetic abnormality of SRY gene in the adult XY female with pericentric inversion of the Y chromosome

In normal ontogenetic development, the expression of the sex‐determining region of the Y chromosome (SRY) gene, involved in the first step of male sex differentiation, is spatiotemporally regulated in an elaborate fashion. SRY is expressed in germ cells and Sertoli cells in adult testes. However, only few reports have focused on the expressions of SRY and the other sex‐determining genes in both the classical organ developing through these genes (gonad) and the peripheral tissue (skin) of adult XY females. In this study, we examined the gonadal tissue and fibroblasts of a 17‐year‐old woman suspected of having disorders of sexual differentiation by cytogenetic, histological, and molecular analyses. The patient was found to have the 46,X,inv(Y)(p11.2q11.2) karyotype and streak gonads with abnormally prolonged SRY expression. The sex‐determining gene expressions in the patient‐derived fibroblasts were significantly changed relative to those from a normal male. Further, the acetylated histone H3 levels in the SRY region were significantly high relative to those of the normal male. As SRY is epistatic in the sex‐determination pathway, the prolonged SRY expression possibly induced a destabilizing effect on the expressions of the downstream sex‐determining genes. Collectively, alterations in the sex‐determining gene expressions persisted in association with disorders of sexual differentiation not only in the streak gonads but also in the skin of the patient. The findings suggest that correct regulation of SRY expression is crucial for normal male sex differentiation, even if SRY is translated normally.

In vitro evaluation of gene expression changes for gonadotropin-releasing hormone 1, brain-derived neurotrophic factor and neurotrophic tyrosine kinase, receptor, type 2, in response to bisphenol A treatment

We evaluated the effects of bisphenol A (BPA) on embryonic mouse hypothalamic cells. Real‐time reverse transcription polymerase chain reaction (RT‐PCR) indicated that gonadotropin‐releasing hormone 1 (Gnrh1) expression in 0.02–20 μM BPA‐treated cells did not differ from that in control cells but decreased significantly in 200 μM BPA‐treated cells. The mRNA level for brain‐derived neurotrophic factor (Bdnf), which participates in GNRH1 secretory system development, decreased significantly in 200 μM BPA‐treated cells, but that for neurotrophic tyrosine kinase, receptor, type 2 (Ntrk2), did not change. This indicates that Gnrh1 gene expression in mice fetuses is not affected by exposure to <20 μM BPA and that the adverse effects of BPA on the BDNF‐NTRK2 neurotrophin system are induced by decrease in the mRNA level of the ligand, not of its receptor.

Global gene profiling and comprehensive bioinformatics analysis of a 46,XY female with pericentric inversion of the Y chromosome

XY females are rare individuals who carry a Y chromosome but are phenotypically female. In approximately 80–90% of these cases, there are no mutations in the SRY gene, a testis‐determining gene on the short arm of the Y chromosome, and the pathophysiology of XY females without SRY mutation remains unclear. In the present study, we used a molecular data mining technique to analyze the pathophysiology of an XY female with functional SRY and pericentric inversion of the Y chromosome, and compared the results with those of a normal male. Interestingly, upregulations of numerous genes included in the development category of the Biological Process ontology, including genes associated with sex determination and organ morphogenesis, were seen in the patient. Additionally, the transforming growth factor‐β (TGF‐β) signaling pathway and Wnt signaling pathway, in which most cell–cell interactions during embryonic development are involved, were altered. Alterations in the expression of numerous genes at the developmental stage, including alterations at both the gene and pathway levels, may persist as a vestige of anomalies of sex differentiation that presumably began in the fetal period. The present study indicates that a data mining technique using bioinformatics contributes to identification of not only genes responsible for birth defects, but also disorders of sex development (DSD)‐specific pathways, and that this kind of analysis is an important tool for clarifying the pathophysiology of human idiopathic XY gonadal dysgenesis. Our findings could serve as one of the basic datasets which will be used for future follow‐up investigations.

A Unique Pattern of Bisphenol A Effects on Nerve Growth Factor Gene Expression in Embryonic Mouse Hypothalamic Cell Line N-44

Abstract We investigated the toxicity of bisphenol A (BPA) by determining the gene expression of nerve growth factor (Ngf in the embryonic mouse cell line mHypoE-N44 derived from the hypothalamus exposed to BPA dose range between 0.02 and 200 μmol L-1 for 3 h. Ngf mRNA levels decreased in a dose-dependent manner, with significant reductions observed in the 2 to 50 μmol L-1 BPA treatment groups compared to controls. However, at 100 to 200 μmol L-1 the NgfmRNA gradually increased and was significantly higher than control, while the expression of the apoptosis-related genes Caspase 3 and transformation-related protein 73 decreased significantly. These results suggest that in an embryonic hypothalamic cell line the higher doses of BPA induce a unique pattern of Ngf gene expression and that BPA has the potential to suppress apoptosis essential for early-stage brain development.

Immunohistochemical analysis of steroidogenic acute regulatory protein (StAR) and StAR-binding protein (SBP) expressions in the testes of mice during fetal development

The expression patterns of steroidogenic acute regulatory protein (StAR) and StAR-binding protein (SBP) in fetal Leydig cells were compared by using immunohistochemistry. While StAR immunoreactivity was detected during the first steps of testis differentiation, SBP expression was detected slightly later. The timing of SBP expression closely correlated with that of the testosterone surge, an event which is known to induce masculinization. Our results suggest that SBP plays an important role in male sexual development via interactions with StAR.