Zebrafish 동물모델에서 human HtrA2의 expression system 정립에 관한 연구

조성원,박효진,김구영,남민경,김호영,고인호,김철희,임향숙,Cho, Sung-Won,Park, Hyo-Jin,Kim, Goo-Young,Nam, Min-Kyung,Kim, Ho-Young,Ko, In-Ho,Kim, Cheol-Hee,Rhim, Hyang-Shuk 한국생명과학회 2006 생명과학회지 Vol.16 No.4

Mitochondrial serine protease로 알려진 human HtrA2 (hHtrA2)는 apoptosis 유도 과정에서 중요한 역할을 담당하고 있을 뿐만 아니라 hHtrA2가 motor neuron degeneration과 관련이 있다는 최근 연구 결과가 있으나, hHtrA2의 생리적 기능은 아직 명확하게 밝혀져 있지 않다. 이와 같이 생체내에서 필수적인 업무를 담당하는 hHtrA2의 기능을 심도 있게 연구하기 위해서는 적절한 동물모델 시스템이 필요하나 이에 대한 연구도 미흡한 실정이다. 따라서 본 연구에서는 hHtrA2의 기능 분석을 위한 기본적인 실험으로 zebrafish라는 동물모델을 선택하여 hHtrA2의 발현 시스템을 정립하였다. 먼저 zebrafish에 hHtrA2를 발현시키기 위하여 zebrafish에서 일반적으로 사용되는 발현 시스템인 pCS2+ vector에 hHtrA2와 GFP를 cloning하고 plasmid를 HEK293 cell에 transfection한 후, hHtrA2-GFP fusion 단백질의 발현을 immunoblot과 immunofluorescence staining assay로 확인한 바 약 64 kDa의 hHtrA2 단백질의 발현을 확인할 수 있었다. Zebrafish에서 hHtrA2-GFP fusion 단백질의 발현양상은 immunofluorescence microscope으로 확인하였다. hHtrA2-GFP DNA와 mRNA를 zebrafish embyro에 microinjection하여 두 가지 component의 발현을 비교 분석한 결과, DNA는 dot 형태로 mRNA는 몸 전체에 퍼져보이는 형태로 발현 양상의 차이는 있었으나 둘 다 zebrafish embryo에서 잘 발현되는 것을 알 수 있다. 다음 DNA를 주 component로 microinjection하여 zebrafish embryo에서 발현을 확인한 결과 hHtrA2는 72 hpf 까지 발현이 지속되는 것을 확인하였다. 본 연구에서 정립한 hHtrA2의 zebrafish 발현 조건은 앞으로 zebrafish에서 hHtrA2의 생리적 기능을 심도있고 정확하게 연구하는 데 있어 기본적인 자료로 활용 할 수 있을 것이다. HtrA2/Omi, a mitochondrial trypsin-like serine protease, is pivotal in regulating apoptotic cell death. Several lines of recent evidence suggest that HtrA2 is associated with the pathogenesis of neurodegenerative disorders; however, the physiological function of HtrA2 still remains elusive. For studying physiological function of HtrA2 in depth, it is necessary to develop a suitable expression system in the model animal. We therefore utilized the zebrafish as a model animal to establish expression of human HtrA2 (hHtrA2) in vivo. For expression of mature HtrA2 as GFP fusion in zebrafish embryos, the HtrA2 (WT) or (S306A) cDNAs with the C-terminal GFP tag were inserted into the pCS2+ plasmid. Expression patterns of HtrA2 in HEK293 cells were first monitored by immunofluorescence staining and immunoblot assays, showing approximately 64 kDa of the HtrA2-GFP fusion proteins. Subsequently, the hHtrA2 plasmid DNA or in vitro transcribed mRNA was microinjected into zebrafish embryos. The expression patterns of HtrA2 in Zebrafish embryos were monitored by GFP fluorescence in 24 hours-post-fertilization (hpf). Although expression patterns of HtrA2-GFP in developing embryos were different between the injected DNA and mRNA, both nucleic acids revealed good expression levels to further study the physiological role of HtrA2 in vivo. This study provides a suitable condition for expressing hHtrA2 in the zebrafish embryos as well as a method for generating useful system to investigate physiological properties of the specific human genes.

Evaluation of cytotoxicity, genotoxicity, and zebrafish embryo toxicity of mixtures containing Hyssopus officinalis, Morus alba, Engraulis japonicus, and 27 other extracts for cosmetic safety assessment

하유나,김영진,Choi Jeongyeon,황인철,Ko Ju-Young,전희경,김연정 대한독성 유전단백체 학회 2021 Molecular & cellular toxicology Vol.17 No.2

Background Although various natural ingredients and formulated reagents are used as ingredients in cosmetics, it is important to have alternatives to animal tests, such as in vitro tests of toxicity, as animal testing of cosmetics is prohibited in many countries. Objectives Here, cytotoxicity, genotoxicity, and embryotoxicity on zebrafish embryos were evaluated to confirm the safety of ingredients formulated with 30 extracts from natural resources used as cosmetic ingredients. The blended ingredients were tested by dividing them into mixtures of 25 natural products (NMA), 30 natural products (NMB), and concentrated NMB (NMC), depending on the number of blended natural components and their application. Results First, in vitro, HaCaT cells were treated with concentrations of 0 to 20% for 24 h to measure cell viability. DNA damage within the IC20 value was measured by comet assay. In the comet assay, 10 mM H2O2 was used as a positive control. Additionally, zebrafish embryos were used as a substitute for conventional animal testing to observe natural mixture toxicity. The olive tail moment (OTM) were not affected by NMA, NMB, or NMC in comparison with the positive control versus three representatives within the IC20 value concentration of each mixture. Zebrafish embryo results were analysed in a manner similar to the cytotoxicity test results. NMA mixtures showed no cytotoxicity, genotoxicity, or toxicity on zebrafish embryos. Other mixtures showed non-toxicity within the representatively concentrated range. Conclusion Therefore, the 3 mixtures containing Hyssopus officinalis, Morus alba, Engraulis japonicus, and 27 other extracts were considered to be not significantly toxic in HaCaT cell and zebrafish, so it is suggested that they could be used as cosmetic ingredients. Background Although various natural ingredients and formulated reagents are used as ingredients in cosmetics, it is important to have alternatives to animal tests, such as in vitro tests of toxicity, as animal testing of cosmetics is prohibited in many countries. Objectives Here, cytotoxicity, genotoxicity, and embryotoxicity on zebrafish embryos were evaluated to confirm the safety of ingredients formulated with 30 extracts from natural resources used as cosmetic ingredients. The blended ingredients were tested by dividing them into mixtures of 25 natural products (NMA), 30 natural products (NMB), and concentrated NMB (NMC), depending on the number of blended natural components and their application. Results First, in vitro, HaCaT cells were treated with concentrations of 0 to 20% for 24 h to measure cell viability. DNA damage within the IC20 value was measured by comet assay. In the comet assay, 10 mM H2O2 was used as a positive control. Additionally, zebrafish embryos were used as a substitute for conventional animal testing to observe natural mixture toxicity. The olive tail moment (OTM) were not affected by NMA, NMB, or NMC in comparison with the positive control versus three representatives within the IC20 value concentration of each mixture. Zebrafish embryo results were analysed in a manner similar to the cytotoxicity test results. NMA mixtures showed no cytotoxicity, genotoxicity, or toxicity on zebrafish embryos. Other mixtures showed non-toxicity within the representatively concentrated range. Conclusion Therefore, the 3 mixtures containing Hyssopus officinalis, Morus alba, Engraulis japonicus, and 27 other extracts were considered to be not significantly toxic in HaCaT cell and zebrafish, so it is suggested that they could be used as cosmetic ingredients.

The roles of NF-κB and ROS in regulation of pro-inflammatory mediators of inflammation induction in LPS-stimulated zebrafish embryos

Ko, Eun-Yi,Cho, Su-Hyeon,Kwon, Seung-Hae,Eom, Chi-Yong,Jeong, Myeong Seon,Lee, WonWoo,Kim, Seo-Young,Heo, Soo-Jin,Ahn, Ginnae,Lee, Kang Pa,Jeon, You-Jin,Kim, Kil-Nam Elsevier 2017 FISH AND SHELLFISH IMMUNOLOGY Vol.68 No.-

<P><B>Abstract</B></P> <P>In this study, the roles of reactive oxygen species (ROS) and NF-κB on inflammation induction in lipopolysaccharide (LPS)-stimulated zebrafish embryos were evaluated using N-acetyl-<SMALL>L</SMALL>-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), specific inhibitors of ROS and NF-κB, respectively. LPS-stimulated zebrafish embryos showed increasing production of NO and ROS and expression of iNOS and COX-2 protein, compared to a control group without LPS. However, NAC significantly inhibited production of NO and ROS and markedly suppressed expression of iNOS and COX-2 protein in LPS-stimulated zebrafish embryos. The mRNA expressions of NF-κB such as p65NF-κB and IκB-A were significantly increased after LPS stimulation, whereas PDTC attenuated mRNA expression of NF-κB. PDTC also inhibited production of NO and reduced expression of iNOS and COX-2 protein in LPS-stimulated zebrafish embryos. Taken together, these results indicated that LPS increases pro-inflammatory mediators in zebrafish embryos through ROS and NF-κB regulation.</P> <P><B>Highlights</B></P> <P> <UL> <LI> We confirmed the roles of ROS and NF-κB in LPS-stimulated zebrafish embryos. </LI> <LI> We employed NAC and PDTC, which are specific inhibitors of ROS and NF-kB. </LI> <LI> NAC inhibited LPS-induced NO and ROS production and iNOS and COX-2 expression. </LI> <LI> PDTC also inhibited LPS-induced NO production and iNOS and COX-2 expression. </LI> <LI> PDTC significantly inhibited LPS-induced increases in the mRNA expression of NF-κB. </LI> </UL> </P>

Symphoricarpos albus의 항산화능과 Zebrafish 배아 독성 및재생 효능 평가

이찬우,허혜연,김면수,장영표,김보애 한국응용과학기술학회 2024 한국응용과학기술학회지 Vol.41 No.2

: 본 연구는 Symphoricarpos albus(S. albus) 추출물과 발효 추출물을 대상으로 zebrafish 배아를이용하여 천연 화장품 소재로서의 유효성 평가 및 꼬리지느러미 재생력을 비교 평가하였다. 이를 위한 S. albus 추출물의 항산화 활성은 10-200μg/mL 농도에서 DPPH radical scavenging, FRAP activity, ABTS+ radical scavenging을 진행하였으며, 모두 농도 의존적인 radical 소거 활성을 보이고, S. albus 잎 추출물에서 가장 높을 항산화 활성을 나타냈다. Zebrafish는 현재 각광받고 있는 실험 대체 동물로서 수많은 화장품연구에 활용되고 있으며, 본 연구는 zebrafish 배아를 채취하여 응고율, 부화율, 심장 독성을 평가하였다. 그 결과 발효 추출물의 경우 100μg/mL 이상의 농도에서는 독성을 나타내는 것을 확인하였다. 재생 효능을 평가하기 위해 zebrafish 꼬리지느러미를 절단하였고, 3일 동안 상처 회복력을 관찰하였다. 그 결과 72 시간 부터 S. albus 잎 추출물 200μg/mL에서 대조군 대비 17%의 재생 효과를 나타내었다. 이러한 결과는 S. albus가 피부 개선용 항산화 및 재생을 위한 천연소재로서 활용 가능한 것으로 사료된다 This study compared and evaluated the antioxidant activities of Symphoricarpos albus(S. albus) extract and fermented extract. Antioxidant activity was measured by DPPH radical scavenging, FRAP, and ABTS. Concentrations were measured at 200, 100, 50, and 10 μg/mL, and antioxidant activity increased in a concentration-dependent manner. S. albus leaves fermented extracts had the highest antioxidant activity. And this study evaluated the safety and tail regeneration of S. albus extract using zebrafish model embryos. Zebrafish are in the spotlight as an alternative animal and can be used for cosmetic research. Zebrafish embryos were collected and evaluated for coagulation rate, hatching rate, and cardiotoxicity. As a result, it was toxic at concentrations above 100μg/ml. The tail was cut and the regenerative effect was observed for 3 days. As a result, from 72 hours, S. albus 200ug/ml leaf extract showed a 17% regenerative effect compared to the control group. These results suggest that S. albus can be used as a natural material for antioxidant and regeneration for skin improvement.

Sequential assessment via daphnia and zebrafish for systematic toxicity screening of heterogeneous substances

Jang, Gun Hyuk,Park, Chang-Beom,Kang, Benedict J.,Kim, Young Jun,Lee, Kwan Hyi Elsevier 2016 Environmental pollution Vol.216 No.-

<P><B>Abstract</B></P> <P>Environment and organisms are persistently exposed by a mixture of various substances. However, the current evaluation method is mostly based on an individual substance’s toxicity. A systematic toxicity evaluation of heterogeneous substances needs to be established. To demonstrate toxicity assessment of mixture, we chose a group of three typical ingredients in cosmetic sunscreen products that frequently enters ecosystems: benzophenone-3 (BP-3), ethylhexyl methoxycinnamate (EHMC), and titanium dioxide nanoparticle (TiO<SUB>2</SUB> NP). We first determined a range of nominal toxic concentration of each ingredient or substance using Daphnia magna, and then for the subsequent organismal level phenotypic assessment, chose the wild-type zebrafish embryos. Any phenotype change, such as body deformation, led to further examinations on the specific organs of transgenic zebrafish embryos. Based on the systematic toxicity assessments of the heterogeneous substances, we offer a sequential environmental toxicity assessment protocol that starts off by utilizing Daphnia magna to determine a nominal concentration range of each substance and finishes by utilizing the zebrafish embryos to detect defects on the embryos caused by the heterogeneous substances. The protocol showed additive toxic effects of the mixtures. We propose a sequential environmental toxicity assessment protocol for the systematic toxicity screening of heterogeneous substances from Daphnia magna to zebrafish embryo in-vivo models.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Contemporary ecological evaluation protocols are mostly based on an individual substance’s toxicity. </LI> <LI> This study proposes a systematic toxicity screening method for heterogeneous substances by demonstrating a proof-of-concept. </LI> <LI> The systematic environmental toxicity assessment protocol used two in-vivo models, Daphnia magna and zebrafish embryo. </LI> <LI> The combined toxicity effects of the mixtures were additive. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

살균소독제의 zebrafish embryos 발달 독성 및 갑상선호르몬 합성에 미치는 영향

지성근(Seonggeun Zee),박준우(June-Woo Park),박창범(Chang-Beom Park) 환경독성보건학회 2021 한국독성학회 심포지움 및 학술발표회 Vol.2021 No.5

COVID-19 바이러스 전염 사태의 장기화에 따른 살균소독제의 생산과 사용증가와 함께 직·간접적으로 인체 및 환경에 미치는 악영향에 대한 우려에 대응하기 위해, 본 연구에서는 zebrafish 배아를 이용한 독성평가 및 관련 바이오마커를 검증하였다. COVID-19 바이러스 전염 방지용 손소독제(에어로졸 타입, A; 젤 타입, B)와 손소독제 주요 구성성분인 benzalkonium chloride(BAC)를 대상으로, 각 제품 내 화학물질 및 BAC 함유량을 기준으로 노출 농도를 설정하여, zebrafish embryo 발달 단계에서의 독성값(Effective concentraions, ECx)을 산출하였다. 서로 다른 타입의 손소독제와 BAC의 독성값(50% Effective concentraions, EC50)은 각 0.05 mg/L(A 손소독제), 119 mg/L(B 손소독제), 0.59 mg/L(BAC)으로, 독성 강도는 A 손소독제> BAC> B 손소독제 순이었다. zebrafish embryos의 발달 독성 관련 바이오마커 발굴 및 검증 연구를 수행하기 위해, 각 손소독제와 BAC의 LOEC (Lowest observed effect concentration)(즉, EC25 and EC10)의 노출에 따른 thyroids 합성 경로에 관여하는 유전체(thyroid stimulating hormone, TSH; deiodinase enzyme, Dio; thyroid hormone receptor, THR) 발현량 변화를 관찰하였다. 대조군에서의 유전체 발현과 비교했을 때, zebrafish embryo의 발달에 관여하는 갑상선 호르몬(Thyroid) 관련 유전체(THR, Dio, THR) mRNA 발현량들이 모두 감소하였다. 따라서 낮은 독성의 살균소독제 노출은 zebrafish embryo 단계에서 갑상선호르몬 합성 저해를 유발할 가능성이 있고, 이 연구 결과는 살균소독제 사용에 있어 인체 및 환경생물에 대한 안전성 검증이 필요함을 시사한다.

Sub-lethal pharmaceutical hazard tracking in adult zebrafish using untargeted LC–MS environmental metabolomics

Sotto, Ryan B. De,Medriano, Carl D.,Cho, Yunchul,Kim, Hyuk,Chung, In-Young,Seok, Kwang-Seol,Song, Kyung Guen,Hong, Seok Won,Park, Youngja,Kim, Sungpyo Elsevier 2017 Journal of hazardous materials Vol.339 No.-

<P><B>Abstract</B></P> <P>Antibiotics in the aquatic environment are dispersed through anthropogenic activities at low concentrations. Despite their sub lethal concentration, these biologically active compounds may still have adverse effects to non-target species. This study examined the response of adult zebrafish to 0.1mg/L concentration of clarithromycin, florfenicol, sulfamethazine, and their mixture using environmental metabolomics. Embryo and larvae of the fish were also used to assess fish embryo acute toxicity and behavior tests respectively. The fish embryo toxicity test did not show any inhibition of growth and development of the embryos after 96h of exposure to the antibiotics. Changes in swimming activity were seen in 5-dpf larvae which is believed to be correlated with the length of exposure to the compounds. Meanwhile, environmental metabolomics revealed diverse metabolites and pathways that were affected after 72h of exposure of the adult fish to sub-lethal concentration of the compounds. We found that even at low concentration of the antibiotics, behavioral and metabolic effects were still observed despite the lack of visible morphological changes. Further studies involving other aquatic organisms and bioactive compounds are encouraged to strengthen the findings presented in this novel research.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Individual and mixture of antibiotics at 0.1mg/L did not impair growth of embryos. </LI> <LI> Zebrafish larvae preferred blue over yellow regardless of antibiotic’s presence. </LI> <LI> Swimming behavior of 5-dpf larvae significantly changed relative to exposure time. </LI> <LI> Metabolites choline, guanosine, and ADP were regulated in the exposed zebrafish. </LI> <LI> Antibiotics’ mechanism of action seems to play a role in zebrafish’s metabolism. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

3‑Bromo‑5‑(ethoxymethyl)‑1,2‑benzenediol inhibits LPS-induced pro-inflammatory responses by preventing ROS production and downregulating NF-κB <i>in vitro</i> and in a zebrafish model

Ko, Eun-Yi,Heo, Soo-Jin,Cho, Su-Hyeon,Lee, WonWoo,Kim, Seo-Young,Yang, Hye-Won,Ahn, Ginnae,Cha, Seon-Heui,Kwon, Seung-Hae,Jeong, Myeong Seon,Lee, Kang Pa,Jeon, You-Jin,Kim, Kil-Nam Elsevier 2019 INTERNATIONAL IMMUNOPHARMACOLOGY Vol.67 No.-

<P><B>Abstract</B></P> <P>The anti-inflammatory effects of 3‑bromo‑5‑(ethoxymethyl)‑1,2‑benzenediol (BEMB) from <I>Polysiphonia morrowii</I> were evaluated in lipopolysaccharide (LPS)-induced RAW264.7 cells and zebrafish embryo. BEMB showed anti-inflammatory effects by inhibiting the production of nitric oxide (NO) and reactive oxygen species (ROS), and the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in the LPS-activated RAW264.7 cells and zebrafish embryo without cytotoxicity. Moreover, BEMB suppressed the protein and mRNA expression levels of nuclear factor (NF)-κB (p65 and inhibitor of NF-κB [IκB]-A) in RAW264.7 cells and zebrafish embryo, respectively. Collectively, the results of this study indicate that BEMB suppressed the production of pro-inflammatory mediators such as NO, iNOS, and COX-2 as well as their regulation in LPS-induced RAW264.7 cells and zebrafish embryos by inhibiting ROS production and NF-κB expression. Therefore, this study suggests that BEMB could be a potential anti-inflammatory agent for the treatment of inflammatory diseases.</P> <P><B>Highlights</B></P> <P> <UL> <LI> 3‑Bromo‑5‑(ethoxymethyl)‑1,2‑benzenediol (BEMB) was isolated from <I>Polysiphonia morrowii</I>. </LI> <LI> BEMB inhibited LPS-induced pro-inflammatory mediators production in RAW264.7 cells. </LI> <LI> BEMB also inhibited pro-inflammatory mediators production in zebrafish embryos. </LI> <LI> Anti-inflammatory effect of BEMB by regulating ROS and NF-κB <I>in vitro</I> and <I>in vivo</I>. </LI> </UL> </P>

Inhibition of Plk1 induces mitotic infidelity and embryonic growth defects in developing zebrafish embryos

Jeong, K.,Jeong, J.Y.,Lee, H.O.,Choi, E.,Lee, H. Academic Press 2010 Developmental Biology Vol.345 No.1

Polo-like kinase 1 (Plk1) is central to cell division. Here, we report that Plk1 is critical for mitosis in the embryonic development of zebrafish. Using a combination of several cell biology tools, including single-cell live imaging applied to whole embryos, we show that Plk1 is essential for progression into mitosis during embryonic development. Plk1 morphant cells displayed mitotic infidelity, such as abnormal centrosomes, irregular spindle assembly, hypercondensed chromosomes, and a failure of chromosome arm separation. Consequently, depletion of Plk1 resulted in mitotic arrest and finally death by 6days post-fertilization. In comparison, Plk2 or Plk3 morphant embryos did not display any significant abnormalities. Treatment of embryos with the Plk1 inhibitor, BI 2536, caused a block in mitosis, which was more severe when used to treat plk1 morphants. Finally, using an assay to rescue the Plk1 morphant phenotype, we found that the kinase domain and PBD domains are both necessary for Plk1 function in zebrafish development. Our studies demonstrate that Plk1 is required for embryonic proliferation because its activity is crucial for mitotic integrity. Furthermore, our study suggests that zebrafish will be an efficient and economical in vivo system for the validation of anti-mitotic drugs.

A zebrafish model for the rapid evaluation of pro-oxidative and inflammatory death by lipopolysaccharide, oxidized low-density lipoproteins, and glycated high-density lipoproteins

Park, K.H.,Cho, K.H. Academic Press 2011 Fish & shellfish immunology Vol.31 No.6

Oxidation and inflammation are leading causes of nearly all chronic metabolic disorders, and play major roles in cardiovascular disease, cancer, and chronic age-dependent disease. High-density lipoprotein (HDL) and apolipoprotein (apo) A-I have strong antioxidant and anti-inflammatory properties in the plasma. Fructose-induced non-enzymatic glycation of apoA-I can lead to the production of dysfunctional apoA-I and HDL. To compare the physiologic effects of dysfunctional apoA-I and HDL, reconstituted HDL containing native apoA-I (nA-I) or glycated apoA-I (gA-I) was injected into zebrafish embryos in the presence of inflammatory molecules. Co-injection of reconstituted HDL containing VLDL and LDL gA-I (gA-I-rHDL) and lipopolysaccaride (LPS) resulted in acute embryo deaths, while rHDL containing nA-I (nA-I-rHDL) and LPS resulted in significantly enhanced survival. Co-injection of oxidized LDL (oxLDL) and nA-I-rHDL improved embryo survival, while co-injection of oxLDL and gA-I-rHDL aggravated inflammatory deaths. Furthermore, co-injection of oxLDL and HDL<SUB>2</SUB> (5 ng of protein) or HDL<SUB>3</SUB> (15 ng of protein) from the young group (22 +/- 2 years old) showed significantly increased embryo survival compared with the same co-injection of HDL from the elderly group (71 +/- 4 years old). In conclusion, our assay system provides a rapid and economic method to screen antioxidant and anti-inflammatory agents using zebrafish embryos.