• Environmental Analysis Health and Toxicology
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• v.15 no.4
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• pp.131-137
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• 2000

The release of hazardous waste materials into the environment poses serious risks in humans and ecosystems. The risk assessment of environmental pollutants including hazardous chemicals requires a comprehensive measurement of hazard and exposure of the chemicals that can be achieved by toxicity evaluation using a biological system such as biomarkers. In this report we have tried to develop a biomarker used to elucidate a molecular basis of, and to monitor abnormal behaviors caused by diazinon in Japanese medaka (Oryzias latipes) as a model organism. First, an attempt was made to clone tyrosine hydroxylase gene from Japanese medaka that would be a candidate for a biomarker for neuronal modulations and behaviors. For monitoring experiments at behavioral and molecular biological levels, the fish were treated under different sublethal conditions of diazinon and their behavioral responses were observed . In this study we have successfully cloned a partial TH gene from the medaka fish through PCR screening of an ovary cDNA library. DNA sequencing analysis revealed that the amplified fragment was 327 bp encoding 109 amino acids. Comparing the DNA sequence of medaka TH with other species, TH gene revealed the DNA sequence was completely identical to that of rat TH. In the RT-PCR, 330 Up of mRNA was consistently amplified in all the treated samples including control There were no significant differences in the TH expression level regardless of treating concentrations (1∼5,000 ppb) and time (0∼48 hr) The reason appeared to be that RT-PCR was not performed using through a quantitative analysis normalized against an actin gene expression. Organ or tissue - specific detection of TH activity and mRNA as biomarkers will be a useful monitoring tool for neurobehavioral changes in fish influenced by toxic chemicals. Furthermore, quantitative analysis of locomotive patterns and its correlation with the neurochemical and molecular data would be highly useful in measuring toxicity and hazard ofvarious environmental pollutants.

• Proceedings of the Korean Environmental Health Society Conference
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• 2005.06a
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• pp.279-285
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• 2005

Endocrine disrupting effects of four pharmaceutical products were evaluated with fish. The test pharmaceuticals, i.e., sulfamethoxazole, sulfamethazine, oxytetracycline and tetracycline have been often detected in aquatic environment, but their ecological hazard on receptors of various trophic levels has seldom been evaluated. In the present study, we conducted acute toxicity assays with a fish, Japanese medake (Oryziα lαtipes). The vitellogenin induced in female fish normally, but a endocrine disrupting chemical could give effects even male fish. We have tried 4 pharmaceutic chemicals to find out the endocrine disrupting effects. Sulfamethoxazole 1, 0.5 ppm induced vitellogenin even at male Japanese medaka. Sulfamethazine 10, 5, 1 ppm could induced vitellogenin at male fish. Oxytetracycline 10, 5, 1ppm could induced vitellogenin With the fish. Tetracycline 10, 5 ppm could induced vitellogenin at male fish. Some pharmaceuticals such as sulfamethoxazole, sulfamethazine, oxytetracycline and tetracycline could give effects to male Oryzias latipes. They could induced vitellogenin under exposure range 0.5 ${\sim}$ 10 ppm of chemicals at male Oryzias latipes.

• The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
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• v.16 no.1
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• pp.39-51
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• 2011

Acute toxicity test and behavioral change analysis of seawater acclimated Japanese medaka were conducted to identify the brine effects on fish by seawater desalination. 7 day acute toxicity test of brine revealed linear concentration-response relationship from 40.0~80.0 psu treatment groups. There was no significant brine effect for 30-40 psu groups and mass mortality was observed from >50 psu exposure (7-day $LC_{50}$=51.4 psu). Images from the real time camera system were analyzed to observe the changes in behavioral patterns of medaka exposed to various salinity. 40.0 and 50.0 psu exposed groups were stabilized in behavioral patterns after 3.1 and 4.6 hours, respectively and 60.0 psu group showed sharp increase in activity during first 12 hours and 50% mortality thereafter. Similar patterns were observed to 70 and 80 psu groups and both experimental groups showed 100% mortality within 12 hours. Acute toxicity test and behavioral patterns showed very similar toxicity results which revealed the increases in mortality and behavioral activities from 50.0 psu. This critical salinity for fish impacts must be implemented to brine discharge strategy by seawater desalination into the coastal area. Also, we recommend that real time camera monitoring system must be a useful tool for early warning of fish toxicity for other applications. This research was funded by Ministry of Land, Transport and Maritime Affairs, Korea.

• Environmental Analysis Health and Toxicology
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• v.23 no.3
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• pp.221-230
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• 2008

Chlorpyrifos (CPF) is a widely used organophosphate insecticide. Japanese medaka, Oryzias latipes was selected to investigate the effects of chlorpyrifos on survival rate, hatching rate, reproductivity and histopathological changes. Adult fish were exposed to CPF at concentrations of 20, 40, 80 and $160{\mu}g/L$, and the test duration was 4 weeks. An experiment with eggs was also performed under CPF exposure continued for 7 days covering from the fecundation to end-hatching stages. Survival rate of the adult medaka decreased in CPF $80{\mu}g/L$ and $160{\mu}g/L$ treatment groups compared with control, solvent control, $20{\mu}g/L$ or $40{\mu}g/L$L treatment group. Spontaneous start of feeding significantly decreased compared with the control (p < 0.01) when exposed to CPF at above 40 $\mu g/L$. Total spawning decreased by the 20 or $40{\mu}g/L$ CPF treatment compared with the control or solvent control in the test with eggs obtained from untreated adult medaka, and abnormal eggs increased in those CPF-treated groups. There were certain adverse effects at above $40{\mu}g/L$ determined from liver and gonad histological examinations. The results indicate that CPF has insignificant toxic effects in Japanese medaka at less than $20{\mu}g/L$ in long term exposure.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.343-348
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• 2018

Although the efforts to establish fish embryonic stem cells (ESCs) have been made for a long time, derivation of authentic ESCs that possess pluripotency is still difficult suggesting a need for the stepwise optimization of the methods to establish fish ESCs. Primary culture of the blastomeres from the embryos at blastula stage is a critical step for establishing continuous ESC lines. Here, we evaluated the effects of temperatures and basal media on primary culture of blastula embryo-derived blastomeres in marine medaka (Oryzias dancena). The blastomeres were isolated from the blastula embryos and cultured in various conditions designed by the combination of 4 temperatures including $28^{\circ}C$, $31^{\circ}C$, $34^{\circ}C$, and $37^{\circ}C$ and 2 basal media including Dulbecco's modified eagle's medium (DMEM) and Leibovitz's L-15 medium (L15). With the exception of a case cultured in L15 at $31^{\circ}C$, the rate of primary cell adherence reached 100% when the blastomeres were cultured over $31^{\circ}C$. The period for primary adherence was significantly shorter in the groups cultured in $34^{\circ}C$ and $37^{\circ}C$ than in the ones in $28^{\circ}C$ and $31^{\circ}C$. The proportion of subculture was significantly high in the group cultured in DMEM at $31^{\circ}C$ compared to the other groups. Collectively, we demonstrated that the culture in DMEM at $31^{\circ}C$ was effective to primary culture of the blastomeres derived from blastula embryos.

• Proceedings of the Korea Society of Environmental Toocicology Conference
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• 2002.10a
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• pp.156-156
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• 2002

The goal of this study is to develop a biomarker used in monitoring abnormal behaviors of Japanese medaka (Oryzias latipes) as a model organism caused by hazardous chemicals that are toxic and persistent in the ecosystem. A widely used insecticide, diazinon (O, O-diethyl O- (2-isopropyl-4-methyl-6-pyrimidinyl) phosphorothioate), is highly neurotoxic to fish, and it is also well known that it causes vertebral malformation and behavioral changes of fish at relatively low concentrations. The fish behaviors were observed on a real time basis using an image processing and automatic data acquisition system. The genes potentially involved in the abnormal behaviors were cloned using suppression subtractive hybridization (SSH) technique. The untreated individuals showed common behavioral characteristics. When the test fish was affected by diazinon at a concentration of 0.1 and 1 ppm, some specific patterns were observed in its behavioral activity and locomotive tracks. The typical patterns were enhanced surfacing activity, opercular movement, erratic movement, tremors and convulsions as reported previously. The number of genes up-regulated tty diazinon treatment were 97 which includes 27 of unknown genes. The number of down-regulated genes were 99 including 60 of unknown genes. These gene expression patterns will be analyzed by the artificial neural networks such as self organization map (SOM) and multilayer perceptron (MLP), revealing the role of genes responsible for the behaviors. These results may provide molecular biological and neurobehavioral bases of a biomonitoring system for diazinon using a model organism such as fish.

• The Korean Journal of Pesticide Science
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• v.8 no.4
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• pp.309-318
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• 2004

Acute toxicity test and chronic toxicity test were conducted with killifish (Oryzias latipes, Medaka) to evaluate toxicity effect of pesticides. Acute toxicity test was investigated mortality in 48 hours and 96 hours after treatment, chronic toxicity test was examined with the early life stage of 30 days after hatching be started embryos of Medaka. The test substances were two pesticides, Carbosulfan and Phenthoate, applied to the paddy rice plant and well-known to the high fish toxicity. As the result of acute toxicity test, median concentration $(LC_{50})$ at 96 hours in Medata was Carbosulfan 0.102 mg/L and Phenthoate 0.167 mg/L, and Fish early life stage toxicity test was conducted on basis of the result of acute toxicity test and concluded from the investigation of hatching success, period of hatching, survival post hatching, length and weight of surviving fishes and abnormal fish. The results of early life stage toxicity test were represented by no observed effect concentration (NOEC), lowest observed effect concentration (LOEC) and maximum acceptable toxicant concentration (MATC). NOEC was Carbosulfan 0.0067ppm and Phenthoate 0.011ppm, LOEC of PCP-Na, Carbosulfan and Phenthoate were 0.017ppm and 0.029ppm, MATC of Carbosulfan and Phenthoate were 0.011ppm and 0.018ppm. These studies will be expected to supply more varied chronic toxicity effects at lower concentration than acute toxicity test. Therefore, evaluation data will be more realistic and the risk assessment of pesticide will be leveled up.

• Environmental Analysis Health and Toxicology
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• v.22 no.4
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• pp.305-312
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• 2007

환경오염을 신속하게 모니터링하기 위한 생물지표의 개발은 증가하고 있는 오염의 심각성에 비추어 매우 중요한 과제로 여겨지고 있다. 본 연구에서는 독성물질처리에 의하여 선택적으로 발현이 조절되는 단백질의 동정을 통하여 독성물질에 대한 단백질 생물지표를 발굴하고자 시도하였다. 즉, 송사리(Oryzias latipes)를 유기인계 살충제인 다이아지논(diazinon)에 0, 0.1, 1, 5 mg/L 농도로 24시간 노출시킨 후, 머리와 몸통부분으로 나누어 단백질 발현패턴을 분석하였다. 본 시스템에서 다이아지논 처리에 의하여 유의적으로 발현이 증가된 단백질로서 alpha-tubulin, ribonuclease pancreatic precursor, protein hfq 등을 동정하였으며, 이 가운데 alpha-tubulin과 $hsp90{\beta}$의 발현이 다이아지논 농도에 의존적으로 증가하는 것을 semi-quantitative RT-PCR방법으로 확인하였다. 이와 같이 다이아지논 처리에 특이적으로 발현이 증가된 송사리 단백질들은 노출평가를 위한 생물지표로서 개발에 응용될 수 있을 것으로 평가된다.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.50 no.2
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• pp.160-168
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• 2017

Optimizing the conditions for stem cell culture is an essential prerequisite for the efficient utilization of stem cells. In the culture of fish embryonic stem cells (ESCs) or ESC-like cells, embryo extracts are important for stable growth, but there is no rule for determining the developmental stage of the embryos used to obtain extracts. Therefore, this study investigated the effects of the developmental stage of extract donor embryos on the culture of Oryzias dancena ESC-like cells. O. dancena ESC-like cells were cultured in different media containing each of four types of embryo extract depending on the developmental stage of the extract donor embryos. Growth, morphology, colony-forming ability, alkaline phosphatase (AP) activity, and embryoid body (EB) formation of the cells were investigated. While the developmental stage of the extract donor embryos did not influence the growth, morphology, AP activity, or EB formation of ESC-like cells, colony-forming ability was affected and the pattern of the effects differed completely between the two ESC-like cells investigated. These results suggest that the developmental stage of extract donor embryos should be selected carefully for the culture of ESC-like cells, according to the research purpose and type of cell line.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.57 no.3
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• pp.239-252
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• 2024

Recombination activating genes (RAGs) play a crucial role in initiating V(D)J recombination, which is essential for developing adaptive immunity in vertebrates. In this study, we cloned and characterized RAG1/2 cDNA from the marine medaka Oryzias dancena (OdRAG1/2) and investigated their mRNA expression patterns during ontogenetic developmental stages. The OdRAG1 and OdRAG2 cDNA contained open reading frames (ORFs) encoding proteins containing 1,078 and 531 amino acids, respectively. Multiple sequence alignment and phylogenetic analysis revealed that OdRAG1 and OdRAG2 are highly conserved with their corresponding orthologs, featuring distinct core and non-core regions. Notably, expression analysis showed that, in contrast to other fish RAGs studied, OdRAG1/2 expression peaked at 0 days post-hatching (DPH). Additionally, for the expression of T and B cell differentiation markers, CD3γ and CD20, also peaked at 0 DPH. Collectively, adaptive immunity in O. dancena potentially begins during embryonic development, which is critical for V(D)J recombination and essential immune component development, suggesting the early ontogenetic stage interactions between innate and adaptive immunity.