Nariaki Fujimoto

Thyroid hormonal activity of the flame retardants tetrabromobisphenol A and tetrachlorobisphenol A

The thyroid hormonal-disrupting activity of the flame retardants tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA) was examined and compared with that of bisphenol A, a typical estrogenic xenobiotic. TBBPA and TCBPA, halogenated derivatives of bisphenol A, markedly inhibited the binding of triiodothyronine (T(3); 1 x 10(-10) M) to thyroid hormone receptor in the concentration range of 1 x 10(-6) to 1 x 10(-4) M, but bisphenol A did not. The thyroid hormonal activity of TBBPA and TCBPA was also examined using rat pituitary cell line GH3 cells, which grow and release growth hormone (GH) depending on thyroid hormone. TBBPA and TCBPA enhanced the proliferation of GH3 cells and stimulated their production of GH in the concentration range of 1 x 10(-6) to 1 x 10(-4) M, while bisphenol A was inactive. TBBPA, TCBPA, and bisphenol A did not show antagonistic action, i.e., these compounds did not inhibit the hormonal activity of T(3) to induce growth and GH production of GH3 cells. TBBPA and TCBPA, as well as bisphenol A, enhanced the proliferation of MtT/E-2 cells, whose growth is estrogen-dependent. These results suggest that TBBPA and TCBPA act as thyroid hormone agonists, as well as estrogens.

Sex Hormone Dependency of Diethylnitrosamine‐induced Liver Tumors in Mice and Chemoprevention by Leuprorelin

The prevalence of liver tumors throughout the world makes it imperative to seek chemopreventive agents. This tumor appears to be hormone‐responsive and hormonal manipulations may therefore be beneficial. On this basis, both sexes of 12‐day‐old B6C3FJ mice were injected i.p. with diethylnitrosamine (DEN) at the dose of 2.5 μg/g body weight and observed for 32 weeks (males) or 36 weeks (females). In 100% of male mice, liver tumors were observed with an average diameter of 2.72 mm and multiplicity of 60.8. Orchidectomy at 6 weeks of age in these mice inhibited the incidence, multiplicity and size to 63%, 5.6 and 1.54 mm, respectively. By further implantation with an E2 pellet at monthly intervals, these parameters were reduced to 26%, 0.6 and 0.61 mm, respectively. Administration of a gonadotropin‐blocking chemical, leuprorelin, to DEN‐treated male mice significantly reduced the multiplicity and size of tumors to 18.3 and 2.54 mm (P<0.01 compared to those of DEN only). In female mice, the incidence of liver tumor was significantly smaller than that of males. However, ovariectomy and/or testosterone supplement significantly increased the occurrence of liver tumor. An anti‐estrogen, toremifene, caused a marked further decrease of liver tumors. Mitotic indices with bromodeoxyuridine in tumor tissues paralleled the occurrence of liver tumors. Serum testosterone levels were significantly reduced by orchidectomy or by leuprorelin administration. These results further confirm that liver tumor is testosterone‐responsive and hormonal manipulation by surgical orchidectomy or by chemical orchidectomy i.e. by leuprorelin, could substantially prevent the appearance of liver tumors.

Suppression by estrogen receptor β of AP-1 mediated transactivation through estrogen receptor α

Abstract The estrogen receptor (ER) is known to mediate gene transcription from AP-1 enhancer elements as well as the well-documented estrogen responsive elements (EREs). Investigations of AP-1 mediated transactivation through ER have been performed with rather complex promoters such as insulin like growth factor 1 (IGF-1) and collagenase promoters. In the present study, we investigated AP-1 mediated transactivation through ERα and ERβ with a less complicated reporter consisting of only consensus AP-1 motifs. NIH 3T3 cells were transiently transfected with human ERα and ERβ expression plasmids and AP-1-luc and ERE-luc reporters. 17β-Estradiol failed to activate ERβ-AP-1 responses while activating ERα-AP-1, ERα-ERE, ERβ-ERE mediated transcription. On the other hand, antiestrogens such as tamoxifen enhanced AP-1 mediated transactivation through both ERα and ERβ. An ERα positive human breast cancel cell line, MCF-7, also showed the same manner of AP-1 mediated transactivation through ERα. When NIH 3T3 with ERα and MCF-7 were co-transfected with ERβ, E2 dependent AP-1 responses decreased in both cell lines depending on the amount of the ERβ expression plasmid. These results suggest that ERα and ERβ may function in opposition with ERβ actually suppressing the function of ERα in AP-1 mediated transactivation.

Induction of Thyroid Tumours in(C57BL/6N×C3H/N)F1 Mice by Oral Administration of Kojic Acid

The tumorigenicity of kojic acid (KA), which is widely used for food and cosmetics in Japan, was examined in B6C3F1 mice. Female and male animals were divided into three groups and given 0, 1.5 and 3.0% KA containing food from the age of 6 weeks. At sacrifice after 20 months, thyroid weights were significantly increased in both sexes of mice receiving KA, especially in the male groups. The enlarged thyroid glands histologically featured diffuse hyperplasia and follicular adenomas, the incidences of the latter being 65% and 87%, respectively in 1.5% and 3.0% KA-treated males, significantly higher than the control value of 2%. In the females, the figures were 2%, 8% and 80% in the 0%, 1.5% and 3.0% KA groups, respectively. The serum free T3 levels in the 3.0% KA animals of both sexes at month 6 were significantly lower than in the controls. On the other hand, their serum TSH levels were higher, although the differences disappeared at later time points. In conclusion, continuous administration of high dose of KA induces thyroid adenomas in male and female B6C3F, mice, presumably by a mechanism involving decrease in serum free T3 levels and increased TSH.

PSPC1, NONO, and SFPQ Are Expressed in Mouse Sertoli Cells and May Function as Coregulators of Androgen Receptor-Mediated Transcription

Abstract In Sertoli cells of testis, androgen receptor-regulated gene transcription plays an indispensable role in maintaining spermatogenesis. Androgen receptor activity is modulated by a number of coregulators which are associated with the androgen receptor. Non-POU-domain-containing, octamer binding protein (NONO), a member of the DBHS-containing proteins, complexes with androgen receptor and functions as a coactivator for the receptor. Paraspeckle protein 1 alpha isoform (PSPC1, previously known as PSP1) and Splicing factor, proline- and glutamine-rich (SFPQ, previously known as PSF), other members of the DBHS-containing proteins, are also found in androgen receptor complexes, suggesting that these DBHS-containing proteins may cooperatively regulate androgen receptor-mediated gene transcription. We demonstrated that PSPC1, NONO, and SFPQ are coexpressed in Sertoli cell line TTE3 and interact reciprocally. The effect of the DBHS-containing proteins on the transcriptional activity was assessed using the construct containing androgen-responsive elements followed by a luciferase gene. The results showed that all the DBHS-containing proteins activate androgen receptor-mediated transcription, and PSPC1 is the most effective coactivator among them. Furthermore, we confirmed the presence of PSPC1, NONO, and SFPQ proteins in Sertoli cells of adult mouse testis sections. These observations suggest that PSPC1, NONO, and SFPQ form complexes with each other in Sertoli cells and may regulate androgen receptor-mediated transcriptional activity.

Identification of estrogen-responsive genes in the GH3 cell line by cDNA microarray analysis

To identify estrogen-responsive genes in somatolactotrophic cells of the pituitary gland, a rat pituitary cell line GH3 was subjected to cDNA microarray analysis. GH3 cells respond to estrogen by growth as well as prolactin synthesis. RNAs extracted from GH3 cells treated with 17beta-estradiol (E2) at 10(-9) M for 24 h were compared with the control samples. The effect of an antiestrogen ICI182780 was also examined. The array analysis indicated 26 genes to be up-regulated and only seven genes down-regulated by E2. Fourteen genes were further examined by real-time RT-PCR quantification and 10 were confirmed to be regulated by the hormone in a dose-dependent manner. Expression and regulation of these genes were then examined in the anterior pituitary glands of female F344 rats ovariectomized and/or treated with E2 and 8 out of 10 were again found to be up-regulated. Interestingly, two of the most estrogen-responsive genes in GH3 cells were strongly dependent on E2 in vivo. #1 was identified as calbindin-D9k mRNA, with 80- and 118-fold induction over the ovariectomized controls at 3 and 24 h, respectively, after E2 administration. #2 was found to be parvalbumin mRNA, with 30-fold increase at 24 h. Third was c-myc mRNA, with 4.5 times induction at 24 h. The levels were maintained after one month of chronic E2 treatment. Identification of these estrogen-responsive genes should contribute to understating of estrogen actions in the pituitary gland.

Estrogen enhancement of androgen-responsive gene expression in hormone-induced hyperplasia in the ventral prostate of F344 rats

It has been postulated that, in addition to the crucial role of androgens, estrogens may be involved in development of prostate hyperplasia and cancer. In rats, combined administration of estrogen and androgen synergistically increases ventral prostate weight, and continued treatment results in the development of glandular hyperplasia. Prostate adenocarcinoma can be induced by chemical carcinogens in rats, and estrogen given together with an androgen generally shortens the latent period or increases the incidence and/or multiplicity of carcinomas. However, the mechanisms responsible for these synergistic effects of estrogen and androgen are poorly understood. In the present study, we examined the combined effects of 17β‐estradiol (E2) and testosterone (T) on gene expression in an early stage of prostate hyperplasia in an F344 rat model. ERα expression, which has been suggested to contribute to development of prostatic hyperplasia, was increased by the combined treatment with T and E2, while it was suppressed by T alone. Expression levels of two androgen‐responsive genes, probasin and kallikrein S3, were increased in the ventral prostate of rats treated with T plus E2 for 4 weeks in a dose‐dependent manner, while short‐term treatment did not alter the expression. These results suggested that enhancing effects of E2 on transcription of androgen‐responsive genes, as well as an increased level of ERα may play roles in the synergistic effects of E2 on T‐induced prostate hyperplasia.

Strain Difference in Regulation of Pituitary Tumor Transforming Gene (PTTG) in Estrogen–induced Pituitary Tumorigenesis in Rats

Recently a novel oncogene, PTTG (pituitary tumor transforming gene) was isolated from a rat pituitary tumor cell line whose expression is apparently correlated with pituitary tumorigenesis. In the rat, estradiol (E2) is known to induce anterior pituitary hyperplasia. The effects of E2, however, vary greatly among rat strains. Therefore we examined the expression of PTTG and its regulation by E2 in F344, Wistar, Brown–Norway and Donryu rats. Four–week–old females were ovariecto–mized and a pellet containing 10 mg of E2 was given s.c. Total RNA was isolated from the pituitary gland and PTTG mRNA was measured with a competitive RT–PCR technique. The F344 strain was the most susceptible to E2 induction of pituitary tumorigenesis, followed by Wistar and Brown–Norway, while no increase in pituitary weight was noted in Donryu rats. PTTG mRNA in the gland was induced by E2 within 48–72 h in F344 and Wistar, but not in Brown–Norway or Donryu strains. These data suggest that PTTG expression may at least in part be responsible for strain differences in E2–induced pituitary tumorigenesis

Up-regulation of the estrogen receptor by triiodothyronine in rat pituitary cell lines

The effects of thyroid hormones on estrogen receptor (ER) levels in four different rat pituitary clonal cell lines designated as MtT/Se, SM, S and E, were investigated. When T3 was added at 10(-7) M, a significant increase in ER was evident after 9 h with maximum levels reached after 24-48 h in MtT/Se (270% of control), MtT/SM (160%) and MtT/S (140%). No significant increase was noted in MtT/E cells in which the T3 receptor (T3R) level was only one-tenth of that in the other cells, suggesting a direct link between ER expression and T3R binding. ER levels began to increase at 10(-10) M and reached maxima at 10(-8) M in MtT/Se, SM and S cells. Up to 10(-5) M of retinoic acid (RA) did not change ER levels in any of the cell lines. When Se cells were treated with cycloheximide before T3 administration, the increase in ER was completely blocked. Northern blot analysis of total RNA isolated from T3-treated MtT/Se cells revealed a limited 1.4-fold increase in ER mRNA at 10(-7) M. In conclusion, thyroid hormones (but not RA) increase ER levels in pituitary cells by a process requiring protein synthesis, and which is accompanied by an increase in the mRNA.

Estrogen Receptor Levels and Tumor Growth in a Series of Pituitary Clonal Cell Lines in Rats

Four kinds of in vitro clonal pituitary tumor cell lines named MtT/Se, MtT/SM, MtT/S and MtT/ E, each of which shows different sensitivity to estrogen on proliferation, were inoculated into fat pad of ovariectomized rats and estrogen‐loaded ovariectomized rats at 105 and 106 cells/site. They formed tumor with average latency ranging from 30 to 71 in ovariectomized rats and 13 to 63 days in estrogenized rats inoculated with 106 cells. MtT/Se was highly sensitive to estrogen for growth, and MtT/SM also grew well in estrogenized rats. With MtT/S and MtT/E, there was no significant shortening of average tumor latency in estrogenized rats. In vivo, the cytosolic estrogen receptor (ER) levels of MtT/Se, SM, S and E were measured to be 452 ±66, 370 ±115, 260 ±16 and 83 ±8 fmol/ mg protein, respectively. In vitro, however, the lowest ER level was noted in MtT/Se. Histologically, all four tumors grown in rats were composed of homogeneous round cells, and MtT/Se contained particularly large nucleated cells. In MtT/E, the cells appeared to be changing into ftbromatous cells. Three cell lines except MtT/E maintained the function of hormonal secretion in vivo as well as in vitro. Serum GH level was increased in rats with MtT/Se and MtT/S. Increased levels of both prolactin and growth hormone were measured in sera of rats with MtT/SM. Increases of hormones as well as tumor sizes were promoted by the estrogen.