Progressive metastatic pheochromocytoma induced by multiple endocrine neoplasia type 2A with a lethal outcome.

Introduction: Patients with multiple endocrine neoplasia type 2A (MEN2A) harboring a pathological variant in the RET gene are characterized by medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism. Although pheochromocytoma is currently defined as a malignant tumor, MEN2A-associated pheochromocytoma is known to have a small risk of metastasis. Case presentation: The case was a 62-year-old Japanese male with bilateral pheochromocytoma, multiple metastases in the liver and bones, and a cardiac thrombus. Genetic testing revealed a pathological variant at codon 634 of the RET gene, thereby leading a diagnosis of MTC. We considered that the multiple metastases were due to MTC; however, a liver biopsy revealed metastasis of pheochromocytoma. Conclusion: When pheochromocytoma precedes MTC, the diagnosis of MEN2A may be difficult.

Molecular cloning and characterization of ligand- and species-specificity of amphibian estrogen receptors.

Estrogens are essential for normal reproductive activity in both males and females as well as for ovarian differentiation during a critical developmental stage in most vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in amphibians, we isolated cDNAs encoding the estrogen receptors (ERalpha and ERbeta) from the Japanese firebelly newt (Cynops pyrrhogaster), Tokyo salamander (Hynobius tokyoensis), axolotl (Ambystoma mexicanum), and Raucous toad (Bufo rangeri). Full-length amphibian ER cDNAs were obtained using 5' and 3' rapid amplification of cDNA ends. The predicted amino acid sequences of these amphibian ERs showed a high degree of amino acid sequence identity (over 70%) to each other. We analyzed the relationships of these amphibian ER sequences to other vertebrate ER sequences by constructing a phylogenetic tree. We verified that these were bona fide estrogen receptors using receptor dependent reporter gene assays. We analyzed the effects of natural estrogens, ethinylestradiol, and DDT and its metabolites on the transactivation of the four amphibian species listed above, and Xenopus tropicalis ERs and found that there were species-specific differences in the sensitivity of these ERs to hormones and environmental chemicals. These findings will expand our knowledge of endocrine-disrupting events in amphibians.

A case in which bladder cancer invaded the ureteral orifice and was resected via photodynamic diagnosis-assisted transurethral resection involving orally administered 5-aminolevulinic acid.

INTRODUCTION: Transurethral resection of bladder tumor is widely used in combination with photodynamic diagnosis to treat non-muscle invasive bladder cancer. We experienced an intriguing case, in which bladder cancer infiltrated into the right ureteral orifice and was resected via photodynamic diagnosis-assisted transurethral resection involving the oral administration of 5-aminolevulinic acid. CASE PRESENTATION: This case was a 71-year-old Japanese man. He was diagnosed with bladder carcinoma, which had infiltrated into the right ureter (clinical classification: T1, N0, M0). He underwent transurethral resection involving the oral administration of 5-aminolevulinic acid. We successfully resected the tumor in the ureteral orifice, which was accomplished by resecting the ureteral orifice until the non-luminescent lumen was exposed. After the surgery, to prevent recurrence, Bacillus Calmette-Guérin was administered intravesically after right ureteral stent placement. CONCLUSION: Photodynamic diagnosis-assisted transurethral resection involving the oral administration of 5-aminolevulinic acid has the potential to treat ureteral tumors derived from bladder tumors.

Effect of atrazine on metamorphosis and sexual differentiation in Xenopus laevis.

There is a growing international concern that commonly used environmental contaminants have the potential to disrupt the development and functioning of the reproductive system in amphibians. One such chemical of interests is the herbicide atrazine. Effects of atrazine on sex differentiation were studied using wild-type Xenopus laevis tadpoles and all-ZZ male cohorts of X. laevis tadpoles, produced by mating wild-type ZZ male to sex-reversed ZZ male (female phenotype). Stage 49 tadpoles were exposed to 0.1-100 ppb atrazine or 0.27 ppb (1 nM) 17beta-estradiol (E(2)) until all larvae completed metamorphosis (stage 66). Metamorphosis, gonadal morphology and histology, CYP19 (P450 aromatase) mRNA induction, and hepatic vitellogenin (VTG) induction were investigated. Effects of atrazine on VTG-induction were also assessed in vitro in primary-cultured X. laevis hepatocytes. Atrazine had no effect on metamorphosis of developing wild-type or all-male X. laevis larvae. Statistical increase in female ratios was observed in 10 and 100 ppb atrazine groups in comparison with control group. While no hermaphroditic froglet was observed in all atrazine groups. In ZZ males, sex reversal was induced by 0.27 ppb E(2), but not by atrazine at concentrations of 0.1 and 1.0 ppb. In addition, neither P450 aromatase mRNA in the gonad nor hepatic VTG were induced by atrazine. Furthermore, VTG was not induced by 1000 ppb atrazine in primary-cultured hepatocytes. Our results indicate that female ratios in developing X. laevis tadpoles were increased by 10 and 100 ppb atrazine under the present experimental conditions. While the other endpoints showed no effect in the range of 0.1-100 ppb atrazine. These results suggest that effect of atrazine on sexual differentiation was not caused by estrogenic action and has no induction ability of P450 aromatase gene in gonad.

Simple and high-sensitivity detection of dioxin using dioxin-binding pentapeptide.

The purposes of this study are to construct a simple dioxin detection system using peptides that bind to dioxin, and to test the system on real environmental samples. In this method, dioxin and N-NBD-3-(3',4'-dichlorophenoxy)-1-propylamine (NBD-DCPPA) are competitively bound to the peptides synthesized on beads. The fluorescence intensity of the bead decreases with increasing dioxin concentration. The concentration of dioxin is determined by measuring the fluorescence intensity using a fluorescence microscope equipped with a CCD camera. The fluorescence microscope system was equipped with a motor-driven stage and could be used with 96-well microplates and analytical software that automatically measured the fluorescence intensity of the bead images in the wells. Dioxin detection conditions, reaction temperature, number of beads and concentration of the organic solvent were optimized. About 0.5 nM (150 pg mL(-1)) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TeCDD) could be detected under the optimized conditions. Environmental soil samples were subjected to the detection system using the peptide beads. Although the results obtained correlated poorly with the toxicity equivalency quantity (TEQ) concentration obtained by a GC/MS method, our method is robust enough as a prescreening method to detect at least 250 pg-TEQ g(-1), the survey level for soil as stipulated in the law concerning special measures against dioxins in Japan.

Development of biomarkers of endocrine disrupting activity in emerging amphibian model, Silurana (Xenopus) tropicalis.

Because amphibians show peculiar ecological features and interesting responses to some hormones, it is conceivable that amphibians are very useful animals for assessing the toxic effects of environmental contaminants, including endocrine disrupters. To develop methods of detecting endocrine toxicity of environmental chemicals in amphibians, we have started to assemble a biomarker tool kit for an emerging amphibian model, Silurana (Xenopus) tropicalis. We isolated full-length cDNAs encoding estrogen receptor alpha (ERalpha), ERbeta, thyroid hormone receptor alpha (TRalpha), and TRbeta of S. (X.) tropicalis to develop a reporter gene assay system, as an estimation tool for environmental chemicals. The amino acid sequences inferred from the four full-length cDNAs were highly homologous to those of ERalpha, TRalpha and TRbeta of X. laevis, and ERbeta of the Japanese quail. In particular, the S. (X.) tropicalis ERalpha shared a higher similarity of amino acid sequence with X. laevis ERalpha than the previously reported S. (X.) tropicalis ERalpha, as determined by Wu et al. RT-PCR analysis showed that the two ERalpha and ERbeta transcripts were expressed relatively abundantly in the brain, liver, and gonad/kidney complex of the S. (X.) tropicalis tadpole after gonadal sex differentiation occurring at developmental stages 54-59, suggesting that they are susceptible to estrogenic substances. A similar result was obtained in the two TR transcripts, although their expression levels were lower in the gonad/kidney complex than in the other tissues. Moreover, we identified vitellogenin A (Vtg A) and Vtg B as estrogen-responsive genes expressed in the female S. (X.) tropicalis liver using macroarray analysis and RT-PCR. In addition, Rana japonica Vtg was purified from serum using anion-exchange chromatography to produce anti-Vtg antibody as a protein marker. In the future, we are going to construct reporter gene assay systems using the full-length ER and TR cDNAs, analyze histologically the differentiation of gonads and thyroid glands in the S. (X.) tropicalis tadpole exposed to estrogenic chemicals, and produce sex-reversed male S. (X.) tropicalis to obtain all-male tadpoles. Using these tools, we hope to be able to identify endocrine disrupting compounds in laboratory experiments for hazard assessment purposes, and also detect endocrine toxicity in environmental samples as part of an integrated approach to assessing the impact of environmental contaminants on wild amphibian populations in Japan and the UK.

Investigating potential for effects of environmental endocrine disrupters on wild populations of amphibians in UK and Japan: status of historical databases and review of methods.

Concern over global declines among amphibians has resulted in increased interest in the effects of environmental contaminants on amphibian populations, and more recently, this has stimulated research on the potential adverse effects of environmental endocrine disrupters in amphibians. Laboratory studies of the effects of single chemicals on endocrine-relevant endpoints in amphibian, mainly anuran, models are valuable in characterizing sensitivity at the individual level and may yield useful bioassays for screening chemicals for endocrine toxicity (for example, thyroid disrupting activity). Nevertheless, in the UK and Japan as in many other countries, it has yet to be demonstrated unequivocally that the exposure of native amphibians to endocrine disrupting environmental contaminants results in adverse effects at the population level. Assessing the potential of such effects is likely to require an ecoepidemiological approach to investigate associations between predicted or actual exposure of amphibians to (endocrine disrupting) environmental contaminants and biologically meaningful responses at the population level. In turn, this demands recent but relatively long-term population trend data. We review two potential sources of such data for widespread UK anurans that could be used in such investigations: records for common frogs and common toads in several databases maintained by the Biological Records Centre (UK Government Centre for Ecology and Hydrology), and adult toad count data from 'Toads on Roads' schemes registered with the UK wildlife charity 'Froglife'. There were little abundance data in the BRC databases that could be used for this purpose, while count data from the Toads on Roads schemes is potentially confounded by the effects of local topology on the detection probabilities and operation of nonchemical anthropogenic stressors. For Japan, local and regional surveys of amphibians and national ecological censuses gathering amphibian data were reviewed to compile survey methodologies and these were compared with methods used in the UK and other countries. Substantial consensus exists in amphibian survey methodologies and this should be exploited in the initiation of coordinated monitoring programs for widespread and common anuran amphibians in Japan and the UK to generate long-term robust and standardized population trend data. Such data would support comparative ecoepidemiological assessments of the impact of environmental endocrine disrupters in these two cooperating countries.

Medaka (Oryzias latipes) for use in evaluating developmental effects of endocrine active chemicals with special reference to gonadal intersex (testis-ova).

Japanese medaka (Oryzias latipes) has been widely used for the evaluation of the toxicity of endocrine active chemicals (EACs) and other chemicals as well as for monitoring the adverse effects of effluent discharges in relation to sexual development and function. It is useful for these evaluations for many reasons including the following: 1) it has a short life cycle facilitating studies extending over long phases of development and over multigenerations, 2) it is easy to rear, 3) male and female phenotypes can easily be distinguished on the basis of secondary sex characteristics, and 4) a genetic marker (DMY) is available for identifying the true genotypic sex. Several biomarkers have been found to be useful for identifying the effects of exposure to estrogenic and androgenic chemicals in medaka and they include increased levels of hepatic vitellogenin (VTG) and testis-ova induction in males for exposure to estrogenic chemicals, and decreased levels of hepatic VTG in females and an altered morphology of dorsal and anal fins and formation of papillae for androgenic chemicals. In this paper, we present a critical analysis of the use of medaka as a test species for studies of endocrine disruption and report on the use of sex-related genetic markers and alterations in gonadal development, including the induction of testis-ova formation, for assessing the disruptive effects of EACs. In this paper, we focus on some of the more recent studies and findings.

Molecular cloning of estrogen receptor alpha (ERalpha; ESR1) of the Japanese giant salamander, Andrias japonicus.

Estrogens are essential for normal reproductive activity in females and males and for ovarian differentiation during a critical developmental stage in many vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in the Japanese giant salamander (Andrias japonicus), we isolated cDNA encoding the estrogen receptor (ER) from the liver. A full-length Japanese giant salamander ER cDNA (jgsER) was obtained using 5' and 3' rapid amplification cDNA ends (RACE). The deduced amino acid sequence of the jgsER showed high identity to the Xenopus ERalpha (ESR1) (77.7%). We have applied both the conventional ERE-luciferase reporter assay system and the GAL4-transactivation system to characterize this receptor. In two different transient transfection assay systems using mammalian cells, the jgsER protein displayed estrogen-dependent activation of transcription. The GAL4-transactivation system showed about 10-fold greater activity of the estrogen receptor by hormone when compared to the conventional ERE-luciferase reporter assay system. Tissue distribution of ERalpha mRNA was examined and kidney, ovary and liver exhibited expression. This is the first isolation of an estrogen receptor from a salamander and also is the first functional cDNA obtained from the Japanese giant salamander, an endangered species considered a special natural monument of Japan.

Low-dose dioxins alter gene expression related to cholesterol biosynthesis, lipogenesis, and glucose metabolism through the aryl hydrocarbon receptor-mediated pathway in mouse liver.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a common environmental contaminant. TCDD binds and activates the transcription factor aryl hydrocarbon receptor (AHR), leading to adverse biological responses via the alteration of the expression of various AHR target genes. Although small amounts of TCDD are consumed via contaminated daily foodstuffs and environmental exposures, the effects of low-dose TCDD on gene expression in animal tissues have not been clarified, while a number of genes affected by high-dose TCDD were reported. In this study, we comprehensively analyzed gene expression profiles in livers of C57BL/6N mice that were orally administered relatively low doses of TCDD (5, 50, or 500 ng/kg body weight (bw) day(-1)) for 18 days. The hepatic TCDD concentrations, measured by gas chromatography-mass spectrometry, were 1.2, 17, and 1063 pg toxicity equivalent quantity (TEQ)/g, respectively. The mRNA level of the cytochrome P450 CYP1A1 was significantly increased by treatment with only TCDD 500 ng/kg bw day(-1). DNA microarray and quantitative RT-PCR analyses revealed changes in the expression of genes involved in the circadian rhythm, cholesterol biosynthesis, fatty acid synthesis, and glucose metabolism in the liver with at all doses of TCDD employed. However, repression of expression of genes involved in energy metabolism was not observed in the livers of Ahr-null mice that were administered the same dose of TCDD. These results indicate that changes in gene expression by TCDD are mediated by AHR and that exposure to low-dose TCDD could affect energy metabolism via alterations of gene expression.

Development of metamorphosis assay using Silurana tropicalis for the detection of thyroid system-disrupting chemicals.

The West African clawed frog (Silurana tropicalis), which resembles the South African clawed frog (Xenopus laevis), but is somewhat smaller, has a diploid genome and a shorter generation time. Therefore, S. tropicalis has the potential for use as a new model in ecotoxicology. We demonstrated a S. tropicalis metamorphosis assay based on Xenopus Metamorphosis Assay (XEMA) using 1 microg/L thyroxine (T4) and 75 mg/L propylthiouracil (PTU). Tadpoles at developmental stages 48-50 were exposed to chemicals for 28 days and total body length, developmental stage, and hind limb length were recorded every 7 days. Significant differences in developmental stage and total body length were found for both T4 and PTU after 7-day exposure, which were similar to the results of the XEMA ring-test using the same chemicals. Moreover, in the present study, we measured hind limb length as a new endpoint of thyroid axis. Significant differences in the hind limb length were encountered in both T4 and PTU treatments after 7 days of exposure. These results suggest that S. tropicalis can be used in a XEMA-like protocol to detect agonist and antagonist effects of chemicals on the thyroid system. Hind limb length is also a suitable endpoint in such protocols. A new test protocol detecting both thyroid disruption and reproductive effects of chemicals using S. tropicalis should be established in the near future.

All ZZ male Xenopus laevis provides a clear sex-reversal test for feminizing endocrine disruptors.

We investigated the possibility of using all ZZ male Xenopus laevis tadpoles produced by mating normal ZZ males with feminized ZZ males to detect estrogenic chemical activity. We examined the effects of 17beta-estradiol (E2) on sex differentiation by treating NF stage 49/50 to stage 57 tadpoles with 0.1, 1, 10, and 20 nM E2 for 4 weeks. Following this, the tadpoles were allowed to develop in clean water until the animals reached stage 66. Increased developmental abnormalities and mortality were not observed in all E2-exposed groups during metamorphosis. Feminization of gonads was detected at all E2 concentrations, whereas nonexposed controls developed testes. Morphological and histological analyses showed that feminized gonads were ovaries. Five and one hermaphroditic frogs were found in the 0.1 and 1 nM E2 groups, respectively, showing testicular as well as ovarian regions within one gonad. These results indicate that phenotypically normal females can be produced from genetic males and demonstrate the utility of a sex-reversal test based on all ZZ males for examining in vivo effects of chemicals with estrogenic activity. The testing of all ZZ male tadpoles might be a useful tool for assessment of feminizing compounds not only estrogenic substance.

Dioxin-binding pentapeptide for use in a high-sensitivity on-bead detection assay.

The purpose of this study is to develop a dioxin detection method using a short peptide alternative to an immunoantibody. A full peptide library consisting of 2.5 million possible amino acid combinations was constructed by a solid-phase split synthesis approach using 19 natural amino acids. The peptide beads were subjected to a competitive binding assay between 2,3,7-trichlorodibenzo-p-dioxin and N-NBD-3-(3',4'-dichlorophenoxy)-1-propylamine (NBD-DCPPA) in a buffer containing 20% 1,4-dioxane. Two almost identical pentapeptides, FLDQI and FLDQV, that could bind dioxin were screened from the combinatorial library. NBD-DCPPA and the peptide synthesized on resin beads could be utilized to determine dioxin concentrations. The fluorescence intensity of the beads was measured using fluorescence microscopy to make a calibration curve for the dioxin concentrations. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TeCDD) could also detected in the presence of 30% 1,4-dioxane. To optimize the peptide sequence, a one-amino acid-substituted library was prepared using amino acids including nonnatural amino acids. The internal amino acids, LDQ, could not be substituted by any other amino acids. This result indicates that these three side chains are essential to recognize dioxins. The peptide C terminus substituted by phenylglycine showed a 10 times lower detection limit of 2,3,7,8-TeCDD of 150 pM (50 pg/mL) than the original sequence FLDQV. The cross reactivity of the dioxin binding peptides including the secondary derivatives was investigated. Some polycyclic aromatic hydrocarbons bound to the peptide beads, but nonchlorinated dibenzo-p-dioxin and PCB did not. From these results, we demonstrate the potential of short peptides as a practical sensor material targeting low molecular weight compounds such as dioxin.