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We compared the influence of thyroid hormone-disrupting chemicals (heptafluorobutanoic acid, PFBA and tris[1,3-dichloro-2-propyl] phosphate, TDCPP) and thyroid hormone (3,3',5-triiodo-L-thyronine, T3) on swim bladder inflation and thyroid hormone-related gene expression in Japanese medaka and zebrafish. The swim bladder of most larvae had inflated at 4 h post hatching (hph) in Japanese medaka and at 48 hph in zebrafish in controls. In both fish species, the swim bladder inflation was inhibited in larvae exposed to PFBA (lowest observed effect concentration [LOEC] in medaka: 40 mg/L; in zebrafish: 80 mg/L), TDCPP (LOEC in medaka: 1 mg/L; in zebrafish: 0.5 mg/L), and T3 (no inhibition in Japanese medaka; LOEC in zebrafish: 7.5 μg/L). We also examined the influence of PFBA, TDCPP, and T3 on the expression of thyroid stimulating hormone subunit beta (tshβ) or thyroid hormone receptor alpha (trα) and beta (trβ). No changes were observed in the expression of genes after PFBA and TDCPP exposure; however, T3 exposure upregulated trα and trβ expression in both fish species. When the results were compared between Japanese medaka and zebrafish, swim bladder inflation in both species was found to be inhibited by exposure to thyroid hormone-disrupting chemicals. Our results show that inhibition of the swim bladder inflation at 4 hph in Japanese medaka and 48 hph in zebrafish is a potential indicator of thyroid hormone-disturbing activity of chemicals.

Chemicals with androgenic or estrogenic activity induce the sex reversal and/or intersex condition in various teleost fish species. Previously, we reported that exposure to 17α-methyltestosterone, bisphenol A, or 4-nonylphenol induces changes in expression of the gonadal soma-derived factor (gsdf) gene accompanied by disruption of gonadal differentiation in Japanese medaka (Oryzias latipes). These findings suggest that gsdf expression might be a useful biomarker for predicting the potential effect of chemicals on gonadal differentiation. We examined the gsdf expression in Japanese medaka exposed to chemicals with estrogenic or androgenic activity. Exposure to the androgenic steroid 17β-trenbolone at 0.5-22.1 μg/L induced the development of ovotestis (presence of ovarian tissue with testicular tissue) and female-to-male sex reversal in XX embryos, and exposure at 6.32 and 22.1 μg/L significantly increased gsdf expression in XX embryos compared with controls at developmental stage 38 (1 day before hatching). In the present study, no statistically significant difference in gsdf mRNA expression was observed after exposure to 17β-estradiol, 17α-ethinylestradiol, and 4-t-octylphenol, which have estrogenic activity. In addition, antiandrogenic chemicals or chemicals without endocrine-disrupting activity did not induce changes in gsdf expression in XX or XY embryos. Thus, an increase in gsdf expression after androgen exposure was observed in XX embryos. Together, these findings indicate that gsdf expression might be useful for predicting the adverse effect of chemicals on gonadal differentiation. Environ Toxicol Chem 2022;41:1875-1884. © 2022 SETAC.

We investigated the lethal toxicity of Shibukuro and Tama river water near the inflow of Tamagawa hot-spring water in Akita Prefecture, Japan. We first measured heavy metal concentrations in both rivers. We detected iron, arsenic, and aluminum; the concentrations of each tended to decrease from upstream to downstream. We next examined the influence of river water on zebrafish Danio rerio and water flea Daphnia magna. We observed lethal effects in both species, with Daphnia magna more sensitive to toxicity than Danio rerio. For both species, the toxic effects of river water decreased with increasing distance downstream from the inflow of hot-spring water. Our results show that the heavy metals discharged from Tamagawa hot spring have a negative effect on aquatic organisms.

To determine whether the marine medaka Oryzias melastigma is a suitable model organism for evaluating the effects of environmental chemicals on marine teleosts, we examined the effect of salinity on physiological development and zinc toxicity. Growth as measured by total body length was significantly lower in fresh water compared to brackish water. Reproductive success was also significantly reduced in fresh water, although we observed cells in the pituitary producing gonadotropins such as Gpa (common glycoprotein hormone α), Fshb (follicle stimulating hormone β), and Lhb (luteinizing hormone β) at all salinities. These results indicate that O. melastigma is adaptable to various salinities from fresh to seawater, and brackish water is best for physiological processes including growth performance and reproduction. When zinc was dissolved in saltwater, a white precipitate formed immediately, and the dissolved concentration decreased in the supernatant and increased at precipitate. We performed zinc toxicity tests on early life stage and adult stage in fresh water, brackish water, and seawater. Among adults, the lowest observed effect concentration for mortality in freshwater (15.3 mg/L) was lower than in brackish water (>48 mg/L) or seawater (>48 mg/L). Similarly, among embryos and larvae, the lowest observed effect concentration for mortality in freshwater (4.8 mg/L) was lower than in brackish water (48 mg/L) or seawater (48 mg/L). These results highlight the importance of using marine organisms to evaluate the ecological effects of marine pollutants.

To determine whether the marine medaka Oryzias melastigma is a suitable model organism for in vivo acute toxicity bioassay in seawater, we first determined whether there were differences in the concentrations of chemicals that were toxic to marine medaka (O. melastigma) and freshwater medaka (O. latipes). We performed in vivo acute toxicity bioassay with 3-chloroaniline, triclosan, 3,4-dichloroaniline, fenitrothion, and pyriproxyfen on larvae of both species. Although the concentrations of 3-chloroaniline and fenitrothion that were lethal to the larvae were identical for both species, the toxic concentrations of triclosan, 3,4-dichloroaniline, and pyriproxyfen were lower for O. melastigma than for O. latipes. We then used an in vivo acute toxicity bioassay to monitor the quality of coastal seawater in Akita, Japan. No lethal effects were observed in the harbor and canal in 2019. O. melastigma could be used to monitor the quality of seawater with salinities in the range 2-25. Our findings suggest that O. melastigma can be used as the test fish for in vivo acute toxicity bioassay intended for water quality monitoring.

In the present study, we assessed the negative effects of triphenyltin (TPT) on zebrafish (Danio rerio) by exposing embryos and early-stage larvae to various concentrations of TPT from 2 h after fertilization (haf) until 30 days after hatching (dah). Whether test groups were fed or fasted during ecotoxicity studies using fish models has varied historically, and whether this experimental condition influences test results is unknown. Here, we confirmed that the lethal concentration of TPT to embryo and early-stage larvae (i.e., 3 dah or younger) showed in fed (lowest observed effect concentration (LOEC); 6.34 μg/L) and fasted (LOEC; 6.84 μg/L) groups. In addition, 84% and 100% of the larvae in the 2.95 and 6.64 μg/L exposure groups, respectively, had uninflated swim bladders; all affected larvae died within 9 dah. This finding suggests that morphologic abnormalities in early larval zebrafish are useful as endpoints for predicting the lethality of chemical substances after hatching. We then assessed the expression of several genes in the thyroid hormone pathway, which regulates swim bladder development in many fish species, including zebrafish. Larvae exposed to 6.64 μg/L TPT showed significant increases in the mRNA expression levels of thyroid hormone receptor α (trα) and trβ but not of thyroid stimulating hormone β subunit. These findings suggest that TPT disrupts the thyroid system in zebrafish.

The branched isomer mixture 4-nonylphenol (4-NP) has been used worldwide as a surfactant, and can have endocrine-disrupting effects on aquatic organisms. For instance, 4-NP induces the formation of testis-ova (i.e., testicular and ovarian tissue in the same gonad) or male to female sex reversal of various teleost fishes. Recently, our group revealed that altered gsdf gene expression is associated with disruption of gonadal differentiation in Japanese medaka (Oryzias latipes) embryos exposed to methyltestosterone or bisphenol A, suggesting that gsdf might be useful as a biomarker for predicting the impact of endocrine-disrupting chemicals (EDCs) on gonadal differentiation. Here, we used 4-NP to examine further whether gsdf expression at the embryo stage is useful for predicting EDC impact on gonadal sex differentiation. When fertilized medaka eggs were exposed to 32 or 100 μg/L 4-NP, testis-ova in genetic males and sex reversal from genetic male to phenotypic female were observed. At stage 38 (just before hatching), 4-NP exposure at 1-100 μg/L did not affect gsdf expression in XX embryos compared with the nontreated control; however, in XY embryos, the gsdf expression in the 100 μg/L-exposed group was significantly lower than that in the controls. The 4-NP concentration at which gsdf expression was suppressed was equal to that at which testis-ova and sex reversal were induced. These results indicate that expression of the gsdf gene at the embryonic stage in medaka is a useful biomarker for predicting the impact of EDCs on sexual differentiation.

To determine whether the marine medaka Oryzias melastigma is a suitable model organism for in vivo acute toxicity bioassay in seawater, we first determined whether there were differences in the concentrations of chemicals that were toxic to marine medaka (O. melastigma) and freshwater medaka (O. latipes). We performed in vivo acute toxicity bioassay with 3-chloroaniline, triclosan, 3,4-dichloroaniline, fenitrothion, and pyriproxyfen on larvae of both species. Although the concentrations of 3-chloroaniline and fenitrothion that were lethal to the larvae were identical for both species, the toxic concentrations of triclosan, 3,4-dichloroaniline, and pyriproxyfen were lower for O. melastigma than for O. latipes. We then used an in vivo acute toxicity bioassay to monitor the quality of coastal seawater in Akita, Japan. No lethal effects were observed in the harbor and canal in 2019. O. melastigma could be used to monitor the quality of seawater with salinities in the range 2-25. Our findings suggest that O. melastigma can be used as the test fish for in vivo acute toxicity bioassay intended for water quality monitoring.

Bisphenol A (BPA), a very important raw material in the plastics industry, is an endocrine-disrupting chemical in teleost fish. Although BPA induces testis-ova and sex reversal in teleost fish species, the molecular mechanism remains unclear. We evaluated the effects of BPA (measured concentrations: 45, 92, 326, 1030 and 3406 μg/L) on Japanese medaka (Oryzias latipes) using OECD TG234 (2011, Fish Sexual Development Test, OECD Guidelines for the Testing of Chemicals, Section 2). BPA at 1030 and 3406 μg/L induced testis-ova and sex reversal with female-type secondary sexual characteristics in XY males at 30 and 60 days posthatching (dph). Then we examined the BPA effect on the expression of sex differentiation genes related to the testis-ova and sex reversal in XY medaka. BPA exposure (1030 and 3406 μg/L) suppressed gsdf mRNA expression and increased cyp19a1a mRNA expression in XY individuals at stage 38 and 30 dph, although foxl2 mRNA expression showed no change. Interestingly, the concentration of BPA that suppressed gsdf mRNA expression at the larval stage was consistent with that needed to induce testis-ova and sex reversal. These results suggest that the gsdf gene at the embryonic stage can be used as a useful biomarker for predicting the impact of estrogenic endocrine-disrupting chemicals on sexual differentiation in Japanese medaka.

Although zinc is an essential element for organisms, excess zinc exposure is harmful. We assessed the possible negative influence of zinc (Zn) on the freshwater fish Danio rerio during its early life stage by using Organization for Economic Cooperation and Development test guideline no. 210. Lethality of Zn after hatching occurred in a concentration dependent manner. The LC50 and lowest observed effect concentration of mortality values in the present toxicity assay were 2.31 mg/L (95% confidence limit: 1.81–3.05) and 1.5 mg/L, respectively. These values were close to the reported concentration recorded in aquatic environments. Growth inhibition was observed at 15 and 30 days post-hatching with Zn exposure of 1.5 mg/L. In general, the growth hormone (Gh)/insulin-like growth factor-I (Igf-1) axis is important for growth in fishes, and Zn exposure induced a significant reduction of igf-1 expression at the concentration that caused growth inhibition. These findings suggest that the observed growth inhibition was induced by the suppression of igf-1 expression. In addition, these results suggest that by examining gene expression on the Gh/Igf-1 axis, it may be possible to predict growth suppression by chemical exposure.

We report a case of liposarcoma of the hypopharynx in a 76 year-old male that recurred repeatedly and was associated with histological changes over a period of 10 years. The initial histological diagnosis was a tumor of the well-differentiated type, and the patient underwent simple extirpation via lateral pharyngotomy. Eight years after the original surgery, lateral pharyngotomy was repeated for resection of a recurrent tumor, which proved to be of the dedifferentiated type. Two years after the second surgery, the tumor recurred again, growing more rapidly, and the patient displayed dyspnea and dysphagia. CT scans and MRI findings revealed a large hypopharyngeal tumor expanding from the mesopharynx to the cervical esophagus. The patient subsequently underwent pharyngolaryngoesophagectomy combined with free jejunum transplantation. The resected tumor was histologically diagnosed as myxoid in type.Most liposarcomas of the hypopharynx are histologically low-grade malignancies, patients with this disease often survive for years. However, in cases of recurrence, the histological features of the tumor occasionally change to higher-grade malignant types. In view of this possibility, complete surgical excision is very important in the initial treatment of liposarcoma, and physicians should not hesitate to perform wide excision in cases of recurrence.

Two new pterocarpans, sophoracarpans A and B, were isolated from Sophora tomentosa L. (Leguminosae) in addition to three known isoflavonoids, wighteone (erythrinin B), sophoraisoflavanone A and l-maackiain. The structures of sophoracarpans A and B were elucidated as 6β,9-dimethoxy-3-hydroxypterocarpan and 3-hydroxy-6β-methoxy-8,9-methylenedioxypterocarpan, respectively, on the spectroscopic basis. The configurations at C-6, C-6a, and C-11a of both compounds were determined by a series of nuclear magnetic resonance irradiation experiments. The isoflavone wighteone, known as an antifungal phytoalexin in several genera of leguminous plants, was also found to occur in the genus Sophora

Two new pterocarpans named sophoracarpans A and B were isolated from Sophora tomentosa L. (Leguminosae), and their respective structures have been elucidated as 6β, 9-dimethoxy-3-hydroxyperocarpan (I) and 3-hydroxy-6β-methoxy-8, 9-methylenedioxypterocarpan (III).

The present study attempted to examine whether clonal cell lines of the oral epithelium can differentiate into ameloblasts and regenerate tooth when combined with dental germ mesenchyme. Clonal cell lines with a distinct morphology were established from the oral epithelium of p53-deficient fetal mice at embryonic day 18 (E18). The strain of mouse is shown to be a useful source for establishing clonal and immortalized cell lines from various tissues and at various stages of development. Tooth morphogenesis is almost completed and the oral epithelium is segregated from the dental epithelium at E18. In RT-PCR analysis of cell lines, mucosal epithelial markers (cytokeratin 14) were detected, but ameloblast markers such as amelogenin and ameloblastin were not detected when cells were cultured on plastic dish. They formed stratified epithelia and expressed a specific differentiation marker (CK13) in the upper layer when cultured on feeder layer or on collagen gel for 1–3 wk, demonstrating that they are of oral mucosa origin. Next, bioengineered tooth germs were prepared with cell lines and fetal molar mesenchymal tissues and implanted under kidney capsule for 2–3 wk. Five among six cell lines regenerated calcified structures as seen in natural tooth. Our results indicate that some oral epithelial cells at E18 possess the capability to differentiate into ameloblasts. Furthermore, cell lines established in the present study are useful models to study processes in tooth organogenesis and tooth regeneration.