This study explores the histological features and Bmp4 expression patterns in the replaced tooth germ of Xenopus laevis . Tooth germ formation starts from the dental placode through epithelial-mesenchymal interactions, involving various signaling pathways such as Fgf, Shh, Bmp, and Wnt. In mice, Bmp4 expression in the dental placode inhibits Pax9 expression in the dental mesenchyme. Although absent in the presumptive dental lamina of birds and toothless mammals, Bmp4 remains conserved in reptiles and fish owing to gene duplication. However, its expression in amphibian tooth germs is poorly understood. Three-month-old X. laevis were employed in this study. Initially, samples underwent paraffin embedding and were sectioned into 5 or 12 μm ribbons for H&E staining and in situ hybridization, respectively. Results revealed teeth appearing in two maxillary rows: the labial side, with prefunctional and functional teeth, and the lingual side, with replaced tooth germs behind functional teeth. Enameloid was observed between the inner dental epithelium and dental mesenchyme at the cap or early bell stages, whereas enamel and dentin formed during the late bell or mineralization stages from the replaced tooth germ. Bmp4 expression was evident in the inner dental epithelium (ameloblasts), dental papilla (odontoblasts), stellate reticulum, and Hertwig’s epithelial root sheath. Overall, these findings highlight the conservation of Bmp4 expression in X. laevis tooth development.

Osteocalcin (OC) is the most abundant noncollagenous protein of extracellular matrix in the bone. In an OC deficient mouse, bone formation rates are increased in cancellous and cortical bones. OC is known as a negative regulator of mineral apposition. OC is also expressed in the tooth of the rat, bovine, and human. However, little is known about OC during tooth development in Xenopus. The purpose of this study is to compare the expression of OC with mineralization in the developing tooth of Xenopus, by using von Kossa staining and in situ hybridization. At stage 56, the developmental stage of tooth germ corresponds to the cap stage, and an acellular zone was apparent between the dental papilla and the enamel organ. From stage 57, calcium deposition was revealed by von Kossa staining prior to OC expression, and the differentiated odontoblasts forming predentin were located at adjoining predentin. At stage 58, OC transcripts were detected in the differentiated odontoblasts. At stage 66, OC mRNA was expressed in the odontoblasts, which was aligned in a single layer at the periphery of the pulp. These findings suggest that OC may play a role in mineralization and odontogenesis of tooth development in Xenopus.

환경호르몬(내분비계 교란물질)은 생체 외부에서 들어와 인간의 내분비기관에서 호르몬의 생리 작용을 교란시키는 화합물을 뜻한다. 환경호르몬은 생체 내 호르몬의 합성, 방출, 수송 등 다양한 과정에 관여해 각종 형태의 교란을 일으킴으로써 생태계 및 인간에게 영향을 주며, 생식 이상과 성장 억제 등을 초래하기도 한다. Alkylphenol ethoxylates의 일종인 4-tert-octylphenol(OP)은 호르몬과 유사한 작용을 하거나, 호르몬 작용을 방해할 수 있는 내분비계 장애물질로 알려져 있다. 비교적 약한 유사 에스트로겐 작용을 가지고 있지만, OP의 화학적 특성상 비이온성 지용성 물질로 인체에 축적된 다면 내분비 조절 기구에 영향을 미칠 수 있는 가능성을 갖고 있다. OP는 주로 가정용 및 공업용 세제 등의 계면활성제, 페인트, 살충제, 플라스틱, 합성 수지류의 산화방지제나 안정제 용도로 주로 사용되고 있다. 이러한 내분비계 장애물질인 OP는 수질오염 및 식습관에서 유발되는 환경적 요인에 의해 체내로 유입된다. 본 연구에서는 무당개구리(Bombina orientalis)와 아프리카 발톱개구리(Xenopus laevis) 의 배아를 이용하여 4-tert-octylphenol(OP)이 초기 발생에 미치는 영향을 온도와 노출 시간 그리고 종간의 차이로 분석하였다. OP 50μM이상 처리군에서 온도, 종에 차이 없이 생존율이 0%로 확인되었다. 23℃에서 embryo가 tadpole이 되기까지 Xenopus의 경우 96h, Bombina의 경우 144h이 소요되는데, 각각 OP 에 노출되는 시간의 차이가 있으므로 Xenopus는 23℃ 96h, 19℃ 144h , Bombina의 경우 26℃ 96h, 23℃ 144h으로 온도의 차이를 주어 노출시간을 같게 하여 초기 발생을 관찰 하였다. Bombina 배아에 OP 10μM 을 23℃, 144h 동안 노출시킬 경우 두부 연골의 기형을 갖는 올챙이로 성장한다. 하지만 동일한 온도조건에서 Xenopus 배아에 23℃ 96h동안 노출시킬 경우 두부 연골의 기형이 관찰되지 않고, 척추, 꼬리의 기형만이 관찰된다. 이러한 결과는 같은 온도 조건임에도 불구하고, OP이 종간에 미치는 영향이나 노출시간에 따라 영향이 다를 수 있음을 보여준다. 본 결과로서 수계에서 OP에 노출된 양서류의 종간 차이, 더 나아가 OP이 환경의 변화에 따라 양서류에 다른 영향을 줄 것으로 예상된다.

As alternatives of phthalate plasticizers harmful as endocrine disruptors, citrate esters have been considered for plasticizer in the production of cosmetics, PVC plastics, and pharmaceuticals. Though considered to be low toxic in mammals in vivo and in vitro toxicological information for citrate esters in aquatic lives remained poorly understood. In an effort to find alternative plasticizers we examined the developmental toxicity of tributyl O-acetylcitrate (ATBC), triethyl 2-acetylcitrate (ATEC) and trihexyl O-acetylcitrate (ATHC) together with dibutyl phthalate (DBP) as the positive control in Xenopus laevis embryos based on Frog Embryo Teratogenesis Assay Xenopus (FETAX). In X. laevis embryos LC50 and EC50 values of ATBC at 96 hours were calculated to be 12.7 ppm (13.3 mg/L) and 11.6 ppm (12.2 mg/L). The LC50 and EC50 values of ATEC at 96 hours were calculated to be 360.6 ppm (409.6 mg/L) and 364.3 ppm (413.8 mg/L), respectively. The LC50 values of ATHC at 96 hours were calculated to be 97.5 ppm (98.0 mg/L). The LC50 and EC50 values of dibutyl phthalate (DBP) at 96 hours were calculated to be 12.7 ppm (13.2 mg/L) and 7.1 ppm (7.4 mg/L), respectively. Developmental abnormality such as head malformation, gut malformation, bent trunk, ventral blister, abnormal tail and myotome were significantly increased by DBP at 8.9 ppm, and which was observed by citrate esters at much higher concentration (ATEC, 320 ppm; ATHC, > 75 ppm; ATBC, 15 ppm). In DBP treated embryos, overgrowth of nostrils was frequently observed and growth was inhibited at 6 ppm. ATEC and ATBC inhibited growth at 80 and 15 ppm, respectively. In ATHC treated embryos, the head and tail length were significantly increased at 14.8 ppm. Lipid peroxidation in tadpoles was significantly increased by DBP (10 ppm) but not by ATEC, ATBC, and ATHC. In tadpoles pro-apoptotic bad, bax and bak mRNA levels and DNA fragmentation were significantly increased by DBP (10 ppm) but not by citrate esters. Together, citrate esters could be considered as substitution for phthalate esters as plastic plasticizers.

We investigated the toxic effects of difenoconazole on the development in the African clawed frog, Xenopus laevis. To test the toxic effects, frog embryo teratogenesis assays using Xenopus were performed. Embryos were exposed to various concentrations of difenoconazole (0-30 μM). LC100 for difenoconazole was 30 μM, and the LC50 determined by probit analysis was 27.19 μM. Exposure to difenoconazole concentrations ≥5 μM resulted in 10 different types of severe external malformation. Histological examinations revealed dysplasia of the eye, heart, liver, somatic muscle, and swelling of the pronephric ducts. The tissue-specific toxic effects were investigated with an animal cap assay. Blood cells were normally induced at a high frequency by mSCF and activin A. However, the induction of blood cells was strongly inhibited by the addition of difenoconazole. Electron micrographs of tested embryos showed the degeneration of somatic muscle and the shrinkage of microvilli on pronephric duct. The gene expression of cultivated animal cap explants was investigated by reverse transcriptase-polymerase chain reaction (RT-PCR). It revealed that the expression of the blood-specific marker(β -globin Ⅱ) and muscle-specific marker (XMA) were more strongly inhibited than the neural-specific marker(XEn2) by the addition of difenoconazole.

We investigated the toxic effects of tebuconazole on development in the African clawed frog, Xenopus laevis. To test the toxic effects, frog embryo teratogenesis assays using Xenopus were performed. Embryos were exposed to various concentrations of tebuconazole(0-100 μM). LC100 for tebuconazole was 100 μM, and the LC50 determined by probit analysis was 82.35 μM. The exposure to tebuconazole concentrations ≥40 μM resulted in 11 different types of severe external malformations including gut dysplasia. Histological examinations revealed various dysplasia in the eye, heart, liver, intestine, somatic muscle, and in the pronephric ducts. The tissue-specific toxic effects were investigated with an animal cap assay. Blood cells are generally induced at a high frequency by the combination of mSCF and activin A, however, the induction of blood cells was strongly inhibited by the addition of tebuconazole. Electron micrographs of tested embryos showed many of multivesicular bodies and dysplasia of photo-receptive cell, however, the somatic muscle degeneration was not severe. The gene expression of cultivated animal cap explants was investigated by reverse transcriptase-polymerase chain reaction and revealed that expression of the blood-specific marker, β globin Ⅱ and muscle-specific marker, muscle actin were more strongly inhibited than the neural-specific marker, XEn2.

We investigated the toxic effects of carbaryl on early embryo development in the African clawed frog, Xenopus laevis. To test the toxic effects, frog embryo teratogenesis assays using Xenopus were performed. Embryos were exposed to various concentrations of carbaryl (5∼320 μM). LC100 for carbaryl was 320 μM, and the LC50 determined by probit analysis was the concentration of 235.68 μM. Exposure to 160 μM of carbaryl resulted in 10 different types of severe external malformations. Histological examination revealed dysplasia of the eyes, heart, guts, somatic muscle, dorsal, liver, blood vessel and swelling of the pronephric ducts. Malformation of neural tissue and brain was not severe even in the high dose of carbaryl. Benzidine blood stain showed distinct inhibition of inducing erythrocytes in embryos and animal cap explants. Electron micrographs of embryo revealed retinal detachment, loose photoreceptor lamella and the degeneration of sarcomeres in the carbaryl-treated group. The mitochondrial degeneration was also observed in the test group.