We are grateful to Ms. Kaori Shimizu and Mr. Masaki Takasu of the Graduate School of Life Sciences, Toyo University, for their technical support. This project was supported by research grants from the Special Research Foundation and Bio-Nano Electronics Research Centre of Toyo University (to SK); by the Science Research Promotion Fund of the Promotion and Mutual Aid Corporation for Private Schools of Japan (to SK); by the New Project Fund for Risk Assessments, from the Ministry of Economy, Trade and Industry (to SK); by a Grant-in-Aid for Challenging Exploratory Research (award 23651028 to SK); by a Grant-in-Aid for Scientific Research (B) and (C) (award 23310026 and 26340030 to SK); and by a Grant-in-Aid for Strategic Research Base Project for Private Universities (award S1411016 to SK) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

본 연구에 사용 된 일본 송사리는 고통과 불편의 완화에 대한 상황을 고려하여, 인도적 토요 대학의 제도적 지침에 따라 처리 하였다. 1. 실버 Nanocolloids (SNCs) <ol><li> 정제 SNCs 구매 (20 ㎎ / ℓ -1, 99.99 % 순도, 입자 증류수에 현탁 28.4 ± 8.5 nm의에 대한 평균 직경). </li><li> 유도 결합 플라즈마 질량 분석에 의해 은의 순도와 농도의 유효성을 검사 (ICP-MS) 취급 설명서 (8)에 따라 분석한다. ICP-MS의 전처리 방법은, (7)에서 설명하는 분석한다. </li></ol> SNC 솔루션 다른 염분도와 (실버 콜로이드와의 Ag +의 혼합물) 2. 준비 <ol><li> 60 g의 NaCl, 1.8 g의 KCl, 2.4 g의 염화칼슘 2 · 2H 2 O, 및 9.78 g 황산 4 · 7H 2 O ULT의 L 1로 구성된 60 ×의 ERM 준비rapure 물; 초순수에서의 NaHCO3 1.25 %와 7.0 pH를 조정합니다. </li><li> 하룻밤 25 ° C에서 ERM 솔루션을 저어. </li><li> 희석 ERM과 SNCs을 섞는다. 각 SNC-ERM 혼합 용액을 40 ml의를 준비합니다. 최종 농도는 ERM의 서로 다른 농도의 SNCs 10 ㎎ / L -1 (1 배, 5 배, 10 배, 15 배, 20 배, 또는 30 배). </li><li> 초순수에서 0.625 %와 7.0에 SNC-ERM 혼합 용액의 NaHCO3의 pH를 조정합니다. 의 Ag + 방출은 산성 조건 (9)에 의해 촉진되기 때문에 pH 조정은 SNC 용액을 제조하는데 매우 중요하다. </li><li> SNCs에 대한 참조 화합물로서 AGNO 3을 사용합니다. <ol><li> 희석 ERM과 AGNO 3 섞는다. 15.7 ㎎ / ℓ의 AGNO 3 농도 AGNO 3 -ERM 혼합 용액을 40 ml의 준비 -1 ERM의 서로 다른 농도 (10 ㎎ / ℓ -1 실버) (1 배, 5 배, 10 배, 15 배, 20 배, 또는 30 배) . 참고 : AGNO 3 실버 콜로이드 독성을 검사하려면수용성 실버의 소스 솔루션은, 실버 콜로이드 및 수용성 실버의 혼합물이다 SNCs에 대한 기준 화합물로 사용됩니다. </li></ol></li></ol> 3. 메다카 문화와 계란 수확 <ol><li> 메다카 (O.의 latipes) (오렌지 - 붉은 주) (60 명의 남성과 60 명의 여성)을 얻었다. </li><li> 그룹으로 배양 메다카 (남성 및 20 일 그룹으로 20 명의 여성)를 메다카 관류 배양 시스템을 사용하여 3 L의 탱크 1 배 ERM있다. <ol><li> 다음과 같은 조건에서 문화 : 배지의 pH 범위 6.2 6.5까지 빛 : 어두운주기 : 16 : 8 시간 문화 매체의 온도 : 24 ± 0.5 ° C 배양 배지의 삼투압 : 257 mOsm </li></ol></li><li> (하루에 다섯 번 () 하루에 한 번) 10시 아테 살리나 노 플리 우스에 송사리를 공급 09:00 11:00 13:00 15:00, 17:00에서 인공 건조 물고기 다이어트를 피드. <ol><li> A.를 얻 살리나 노 플리 우스. </li> &lt;리&gt; 플라스틱 비커에 3.0 % 염 용액의 5 L를 준비합니다. </li><li> 비커에서 소금 용액에 소금물 새우 달걀 30g을 추가합니다. </li><li> 폭기 펌프를 사용하여 (4 L / 분 -1)를 버블 링 48 시간 동안 25 ° C에서 알을 부화. </li><li> 48 시간 후, 버블 링을 중지합니다. </li><li> 5 ~ 10 분은 부화 A를 분리하는 솔루션이 방치 해칭 달걀과 달걀 껍질에서 살리나 노 플리 우스 (용액의 아래 부분) (용액의 상부). </li><li> 데칸 테이션에 의한 용액의 상층을 제거한다. </li><li> 283 μm의의 구멍을 가진 체를 통해 솔루션의 하부 필터, 198 μm의의 구멍과 그물에 통과 노 플리 우스를 수집합니다. </li><li> 6 시간 이내에 송사리에 노 플리 우스를 공급. </li></ol></li><li> 암형 메다카가 산란 한 후 암컷 &#39;본체로부터 부드럽게 외부 달걀 클러스터를 제거 또는 SM을 사용하여 수조의 바닥에서 계란을 수집모든 네트 (그물 크기 5cm × 5 cm의 구멍 크기 0.2 mm X 0.2 mm). </li><li> 5 초 동안 수돗물 흐르는 계란 클러스터를 씻어. </li><li> 30 배 ERM 솔루션에 세척 계란 모든 클러스터를 추가합니다. </li><li> 1 분 후 솔루션에서 클러스터를 제거하고 부드럽게 마른 종이 타올과 롤 사이의 계란 클러스터를 배치합니다. </li><li> 30 배 ERM에 다시 계란을 넣습니다. </li><li> 해부 현미경 수정란을 선택합니다. </li><li> 집게를 사용하여 여섯 잘 플라스틱 접시에 1 배의 ERM에 810 알을 선택한 놓습니다. </li><li> (21) 발달 단계까지 인큐베이터에서 25 ± 0.1 ° C에서 알을 품어 (메다카 배아의 발달 단계가 Iwamatsu (10)의 작업에서 정의되었다.) </li><li> 해부 현미경 발달 단계 (21)에서 배양 계란을 선택합니다. </li><li> 1 배의 ERM로 선택한 계란을 씻어. </li><li> 노출 실험 (4 절)로 세척 계란을 가하지. </li></ol> 4. 독성 SNCs의 시험 또는 AGNO 3 </sub&gt; 다른 ERM의 염분도에서 <ol><li> 송사리 계란을 씻어 (단계 21) 테스트 솔루션 [SNCs (10 ㎎ / ℓ -1) 또는 AGNO 3 3 회 ERM의 각 농도 (1 배, 5 배에서 (15.7 ㎎ / ℓ -1 10 ㎎ / ℓ -1 실버 등) , 10 배, pH가 7에서 15 배, 20 배, 또는 30 배)]. 컨트롤로, pH가 7에서 30 ×의 ERM 1 × 계란을 사용합니다. </li><li> 여섯 웰 플라스틱 플레이트에서 각 시험 용액 5 ml의 15 세척 계란을 추가합니다. (SNC의 노출 실험을 세 번 수행하거나 AGNO 3 독성 각 시험 용액을 사용하여 시험). </li><li> 알루미늄 호일 접시를 감싸. </li><li> 부화 할 때까지 14 일 동안 어둠 속에서 25 ° C에서 랩 번호판을 품어. </li><li> 생물학적 변화와 죽은 달걀 노출 된 계란마다 24 시간을 관찰한다 (그림 1, 2). </li><li> 테스트 솔루션마다 24 시간을 교환한다. </li><li> 다음과 같은 관측을 수행합니다. <ol><li> 노출의 날 6 일, 오 (15 초당) 심박수를 계산스톱워치 (도 3a)를 사용하여 해부 현미경 F 메다카 배아. </li><li> 노광 6 일째, 마이크로 미터 (도 3b)를 사용하여 해부 현미경 메다카 배아의 눈의 크기 (직경)를 측정한다. </li><li> 해칭 날, 마이크로 미터 (도 3C)를 사용하여 해부 현미경 유충의 몸 전체의 길이를 측정한다. </li><li> 십사일 (도 3d) 위에 부화 노출 알의 총 수를 계산. </li></ol></li></ol> SNC 솔루션의 가용성 실버, 실버 분석 5. 분리 <ol><li> 14,000 ×에서 3 kDa의 멤브레인 필터로 여과하여 각각 SNC 수용액 (콜로이드 실버와 실버 수용성의 혼합물)으로부터 가용성은 격리 g 및 10 분 동안 4 ° C. 1 배 ERM의 응집 SNCs의보고 된 평균 직경이 67.8 내지 11이므로 SNCs에서 수용성은을 분리하는 3 kDa의 멤브레인 필터를 사용하여차의 Ag +의는 0.162 내지 12; 3 kDa의 막은 2 내지 13 이상의 직경의 입자를 제외한다. </li><li> ICP-MS 운영 매뉴얼 8 항에 ICP-MS 분석 (그림 3E)에 의해 여과 된 용액 (= 가용성은 농도)의 50 μL의 실버 농도를 측정한다. ICP-MS의 전처리 방법은, (7)에서 설명하는 분석한다. </li></ol> 메다카 배아에서 실버 생물 농축 6. 측정 <ol><li> 4 절에 설명 된대로 메다카 SNCs에 계란 (단계 21) 또는 AGNO 3 노출. </li><li> 노출의 날 (6)에서 메다카 도서 (14)에 기술 된 프로토콜에 따라 효소를 부화 송사리를 사용하여 계란 (즉, dechorion)에서 융모막를 제거합니다. </li><li> ICP-MS 운영 매뉴얼 8 (그림 3 층)에 따라 ICP-MS 분석에 의해 dechorioned 계란의 실버 농도를 측정한다. 전처리ICP-MS에 대한 방법은 7 장에서 설명하는 분석. </li></ol> ICP-MS 분석에 의해 실버 농도 7. 측정 <ol><li> (; 섹션 1은 농도의 검증을위한) 샘플 [실버 용액 50 μl를 추가; 세 dechorionated 배아 (섹션 5); 또는 50 ㎖ 테플론 비커에 여과 된 용액 (섹션 5)]의 50 μL. </li><li> 50 ML의 비커에 초순수 질산 2.0 ML을 추가합니다. </li><li> 그것은 (약 3 시간)을 밖으로 건조 직전까지 110 ° C에서 핫 플레이트의 혼합물을 가열한다. </li><li> 완전 유기물을 용해 초순수 2.0 질산 ㎖의 비이커에 과산화수소 0.5ml를 추가한다. </li><li> 그것은 (약 3 시간)을 밖으로 건조 직전까지 핫 플레이트에 다시 혼합물을 가열한다. </li><li> 초순수 1.0 % 질산 수용액 4 ㎖에 잔류 녹인다. </li><li> 전송 원심 튜브 용액 4 mL를. </li><li> (3 회 총)을 두 번 7.7-7.6를 반복합니다. 최종 부피는 12.0이고ml의. </li><li> 취급 설명서 8 항에 ICP-MS 분석을 이용하여 (1.0 %의 고순도 질산에 용해) 시료의 실버 농도를 측정한다. <ol><li> 내부와 외부 표준 용액을 사용하여 실버 농도를 정량화 (자료 목록을 참조하십시오). 내부 및 외부 표준 용액은 실험실 인증을위한 미국 협회 (A2LA)의 인증을 받았으며. 실버의 검출 한계는 0.0018 ng를 / ㎖ -1 (솔루션)와 0.016 NG mg의 무게 -1 (배아 체)이었다. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

송사리는 해수에 매우 관대있는 민물 고기이다 잘 물고기의 원래의 자연 서식지는 일본 해안 6 떨어져 바닷물 것을 알 수 없습니다. 따라서, 송사리는 염화 세포 (7) 잘 개발했습니다. 이 독특한 특성은 물고기의 단일 종을 사용하여 (담수 해수) 염분의 함수로서 환경에서 화학 물질의 독성을 시험하는 새로운 방법을 제공 과학자. 단계 21에서 송사리 계란을 얻...

1979 년 시험 화학 물질에 대한 경제 협력 개발기구 (OECD) 테스트 가이드 라인을위한기구의 설립 이후, 38 테스트 가이드 라인은 가이드 라인, 바이오 틱 시스템 1에 미치는 영향의 제 2 항에 발표되었다. 담수 서식지, 즉 담수 공장에서왔다 테스트 수생 생물의 모든; 조류; 이러한 물벼룩 류와 chironomids 같은 무척추 동물; 이러한 송사리, 제브라 피쉬, 무지개 송어 등 물고기. 바닷물 환경에 비해 담수 환경은 더 직접적으로 인간의 경제 및 산업 활동에 의해 영향을받습니다. 그들은 오염 위험이 높은 있기 때문에 따라서, 담수 환경은 테스트를 위해 우선 순위가되었습니다. 하구를 포함한 해안 지역에서, 염분도는 소금기있는 물과 해수 조건 사이에서 변화하고,이 지역은 종종 산업 활동 (2)에 의해 오염된다. 해안 지역 및 관련 습지는 시간을 특징으로한다고등학교의 생태 학적 생물 다양성과 생산성. 해안 생태계 따라서 화학 오염으로부터 보호해야합니다. 그러나, 기수 및 해수 서식지의 생태 독성 학적 연구가 제한되어있다. Sakaizumi 3 일본 메다카 계란 메틸 수은 염분의 독성 작용을 연구 및 시험 용액의 삼투압을 높이면 메틸 수은의 독성 향상된 것으로 나타났다. . Sumitani 등 4 매립지 침출수의 독성을 조사하기 위해 송사리 계란을 사용; 그들은 계란에 침출수의 삼투압 동등성이 배 발생 과정의 이상을 유도하는 열쇠 것을 발견했다. 또한, Kashiwada 5 플라스틱 나노 입자 (직경 39.4 nm의) 쉽게 기수 조건에서 송사리 계란 융모막 (15 배 배아 양육 매체 (ERM))를 통해 투과 보도했다. 전형적인 작은 물고기 모델, 일본 송사리 (오리 지 아스 쿠르 latipes </em은&gt;) 및 기초 생물학 독물학 (6)에 사용되어왔다. 일본어 송사리는 해수 담수에서 그들의 고도로 발달 된 염화 세포 (7)에 이르기까지 환경에서 살 수 있습니다. 그들은 염분도의 광범위한 조건에서 테스트에 유용하기 때문에 가능성이 높습니다.

<table border="0" cellpadding="0" cellspacing="0" width="785">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Silver nanocolloids</td>
			<td style="width:188px;">Utopia Silver Supplements</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">NaCl</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">31319-45</td>
			<td>For making ERM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">KCl</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">28513-85</td>
			<td>For making ERM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2&#183;2H2O</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">06730-15</td>
			<td>For making ERM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4&#183;7H2O</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">21002-85</td>
			<td>For making ERM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaHCO3&#160;</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">31212-25</td>
			<td>For making ERM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AgNO3</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">31018-72</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pH meter</td>
			<td>HORIBA, Ltd.</td>
			<td style="width:119px;">F-51S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Balance</td>
			<td>Mettler-Toledo International Inc.</td>
			<td style="width:119px;">MS204S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">medaka (Oryzias latipes) orange-red strain&#160;</td>
			<td colspan="2">National Institute for Environmental Studies</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">medaka flow-through culturing system</td>
			<td>Meito Suien Co.</td>
			<td>MEITOsystem</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Artemia salina nauplii eggs</td>
			<td>Japan pet design Co. Ltd</td>
			<td>4975677033759</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">aeration pomp</td>
			<td>Japan pet design Co. Ltd</td>
			<td>non-noise w300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Otohime larval &#946;-1</td>
			<td style="width:188px;">Marubeni Nissin Feed Co. Ltd</td>
			<td style="width:119px;">Otohime larval &#946;-1</td>
			<td>Artificial dry fish diet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dissecting microscope</td>
			<td style="width:188px;">Leica microsystems</td>
			<td style="width:119px;">M165FC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micrometer</td>
			<td>Fujikogaku, Ltd.</td>
			<td>10450023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">incubator</td>
			<td>Nksystem</td>
			<td>TG-180-5LB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">shaker</td>
			<td style="width:188px;">ELMI Ltd.</td>
			<td style="width:119px;">Aizkraukles 21-136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well plastic plates</td>
			<td>Greiner CELLSTAR</td>
			<td style="width:119px;">M8562-100EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">aluminum foil</td>
			<td>AS ONE Co.</td>
			<td style="width:119px;">6-713-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">stopwatch</td>
			<td>DRETEC Co. Ltd.</td>
			<td style="width:119px;">SW-111YE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-kDa membrane filter</td>
			<td>EMD Millipore Corporation</td>
			<td colspan="2">0.5-mL centrifugal-type filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50-mL Teflon beaker</td>
			<td>AS ONE Co.</td>
			<td style="width:119px;">33431097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Custom claritas standard</td>
			<td style="width:188px;">SPEXertificate</td>
			<td style="width:119px;">ZSTC-538</td>
			<td>For internal standard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Custom claritas standard</td>
			<td style="width:188px;">SPEXertificate</td>
			<td style="width:119px;">ZSTC-622</td>
			<td>For external standard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">ultrapure nitric acid</td>
			<td style="width:188px;">Kanto Chemical Co.</td>
			<td style="width:119px;">28163-5B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">hydrogen peroxide&#160;</td>
			<td style="width:188px;">Kanto Chemical Co.</td>
			<td style="width:119px;">18084-1B</td>
			<td>for atomic absorption spectrometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">ICP-MS</td>
			<td>Thermo Scientific</td>
			<td colspan="2">Thermo Scientific X Series 2&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hot plate</td>
			<td>Tiger Co.</td>
			<td>CRC-A300</td>
			<td></td>
		</tr>
	</tbody>
</table>

salinity-dependent toxicity assay of silver nanocolloids using medaka eggs

염분은 수생 환경의 중요한 특성이다. 수생 생물의 경우는 담수, 기수 및 해수의 서식지를 정의합니다. 화학 물질 및 수생 생물에 그들의 생태 학적 위험 평가의 독성 시험은 자주 담수에서 수행되지만, 수생 생물에 화학 물질의 독성의 pH, 온도, 염도에 따라 달라집니다. 있는 방법은 수생 생물에 독성의 염분 의존성을 테스트하기 위해, 그러나,이 없습니다. 그들은 담수, 기수 및 해수에 적응 할 수 있기 때문에 여기서 우리는 송사리 (오리 지 아스 쿠르 latipes)을 사용했다. 배아 양육 매체 (ERM) (1 배, 5 배, 10 배, 15 배, 20 배 및 30 배)의 계란 (1 배의 ERM과 30 배 ERM을 송사리하는 실버 nanocolloidal 입자 (SNCs)의 독성을 테스트하기 위해 사용 된이 삼투 압력 상당의 다른 농도 각각 담수와 해수에). 여섯 아니라 플라스틱 판에, 세중 15 송사리 계란 10 밀리그램 / L &amp;에 SNCs에 노출# 8722; 1 어둠 속에서 pH가 7, 25 ° C에서 ERM의 서로 다른 농도이다. 우리는 하루 6 일 (섹션 4) 부화의 유충의 몸 전체 길이에 15 초와 눈 직경에 따라 심장 박동을 측정하기 위해 해부 현미경 마이크로 미터를 사용했다. 배아 또는 부화는 14 일째까지 관찰되었다; 우리는 그 다음 십사일 (4 절)에 대해 매일 부화 속도를 계산. 배아에 실버 축적을 확인하려면, 우리는 유도 실버 테스트 솔루션의 농도 (섹션 5) dechorionated 배아 (6 절) 분명히 염분 증가와 함께 증가 메다카 배아에 SNCs의 국지적 인 독성을 측정하는 플라즈마 질량 분석을 결합 사용합니다. 이 새로운 방법은 우리가 다른 염분도에서 화학 물질의 독성을 테스트 할 수 있습니다.

SNC 독성에 염분의 효과는 매우 분명했다 : 기형 또는 사망의 유도가 염분 따라 (그림 1, 2)이었다. 우리는 (10 ㎎ / ℓ -1) -exposed 배아 SNC에서 표현형 바이오 마커 (심장 박동, 눈의 크기, 몸 전체 길이, 부화 속도)를 측정 하였다. 이러한 표현형 바이오 마커는 염분에 의존 SNC 독성을 밝혔다. 심장 ?...

Watch this Scientific Journal Video about Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs at JoVE.com

Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

Embryonic stages are the most susceptible to xenobiotics. Although chemical toxicity depends on salinity, no method exists to test the salinity dependence of toxicity to aquatic organisms. Here, we describe a new and high-throughput method for determining the salinity dependence of toxicity to aquatic embryos.

메다카 계란을 사용하여 실버 Nanocolloids의 염도에 의존하는 독성 분석

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and ...

salinity-dependent-toxicity-assay-silver-nanocolloids-using-medaka

Research

JoVE Journal

Developmental Biology

7.9K 조회수.  Toyo University. Embryonic stages are the most susceptible to xenobiotics. Although chemical toxicity depends on salinity, no method exists to test the salinity dependence of toxicity to aquatic organisms. Here, we describe a new and high-throughput method for determining the salinity dependence of toxicity to aquatic embryos.

비디오: Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

화학 물질 안전 검사 및 시스템 생물학 데이터 생성을 위해 발생 독성 시험 법을 바탕으로 인간 다 능성 줄기 세포

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

연구

발생학

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

선충을 사용하여 생식선 화학 독성의 종합 평가<em&gt; 예쁜 꼬마 선충</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

Method of Studying Palatal Fusion using Static Organ Culture

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that adheres to form the midline epithelial seam (MES). The theories suggest that MES cells follow an epithelial to mesenchymal transition (EMT), apoptosis and migration, making a fused palate 1. Complete disintegration of the MES is the final essential phase of palatal confluence with surrounding mesenchymal cells. We provide a method for palate organ culture. The developed in vitro protocol allows the study of the biological and molecular processes during fusion. The applications of this technique are numerous, including evaluating responses to exogenous chemical agents, effects of regulatory and growth factors and specific proteins. Palatal organ culture has a number of advantages including manipulation at different stages of development that is not possible using in vivo studies.

Studies of palate development are motivated by the incidence of cleft palate, a birth defect that imposes a tremendous health burden and can leave lasting disfigurement. We demonstrate here a technique to culture palatal shelves that can be used to study different signaling pathways involved in palatal development and fusion.

정적 장기 문화를 사용하여 구개 퓨전 공부의 방법

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that ...

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

심 외막 파생물 문화 분석 및<em&gt; 전의 VIVO</em&gt; 심 외막 유래 세포 마이그레이션 평가

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

약물 치료와<em&gt; 생체</em&gt;은 메다카 물고기 골다공증 모델에서 조골 파골 세포 상호 작용의 영상

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including embryogenesis and development, wound healing, tumor growth and metastasis, and ocular and cardiovascular diseases. Effective in vitro models that recapitulate the biology of angiogenesis are needed to appropriately study this process and identify mechanisms of regulation that can be ultimately targeted for novel therapeutic strategies. The bead angiogenesis assay has been previously demonstrated to recapitulate the multiple stages of endothelial sprouting in vitro. However, a limitation of this assay is a lack of endothelial &#8211; mural cell interactions, which are key to the molecular and phenotypic regulation of endothelial cell function in vivo. The protocol given here presents a methodology for the incorporation of mural cells into the bead angiogenesis assay and demonstrates a tight association of endothelial cells and pericytes during sprouting in vitro. The protocol also details a methodology for effective silencing of target genes using siRNA in endothelial cells for mechanistic studies. Altogether, this protocol provides an in vitro assay that more appropriately models the diverse cell types involved in sprouting angiogenesis, and provides a more physiologically-relevant platform for therapeutic assessment and novel discovery of mechanisms of angiogenesis regulation.

This protocol presents a novel in vitro bead assay that more appropriately models the process of in vivo sprouting angiogenesis by incorporating pericytes. This modification enables the bead assay to more faithfully recapitulate the heterotypic cellular interactions between endothelial cells and mural cells that are critical for angiogenesis.

분석 결과 돋 아 내 피 세포 구슬 Pericytes 통합

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including ...

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the formation of new blood vessels and involve several signal transduction pathways. Here we show that the tube formation assay is a simple in vitro method to evaluate the impact of natural products on angiogenesis and to investigate the molecular mechanisms involved. In particular, in the presence of the water extract of Ruta graveolens (RGWE), endothelial cells are no longer able to form a cell-cell network and that the RGWE effects on human umbilical vein endothelial cell (HUVEC) tube formation is abolished by the constitutive activation of MEK.

Here, we evaluate the effects of the water extract of Ruta graveolens on vessel network formation by using a tube formation assay on a gelled basement matrix.

HUVEC 튜브 형성 분석은 혈관 신생에 천연 물의 영향을 평가하기 위해

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the ...

Injecting Gryllus bimaculatus Eggs

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been developed in traditional model systems, it is difficult to manipulate genes or messenger RNA (mRNA) in most other organisms. At the same time, evolutionary and comparative approaches rely on an exploration of gene function in many different species, necessitating the development and adaptation of techniques for manipulating expression outside currently genetically tractable species. This protocol describes a method for injecting reagents into cricket eggs to assay the effects of a given manipulation on embryonic or larval development. Instructions for how to collect and inject eggs with beveled needles are described. This relatively straightforward technique is flexible and potentially adaptable to other insects. One can gather and inject dozens of eggs in a single experiment, and survival rates for buffer-only injections improve with practice and can be as high as 80%. This technique will support several types of experimental approaches including injection of pharmacological agents, in vitro capped mRNA to express genes of interest, double-stranded RNA (dsRNA) to achieve RNA interference, use of clustered regularly interspaced short palindromic repeats (CRISPR) in concert with CRISPR-associated protein 9 (Cas9) reagents for genomic modification, and transposable elements to generate transient or stable transgenic lines.

Injecting Gryllus bimaculatus Eggs

Here we present a protocol to inject cricket eggs, a technique which serves as a foundational method in many experiments in the cricket, including, but not limited to, RNA interference and genomic manipulation.

그릴루스 바이마쿨라투스 계란 주입

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

내피 콜로니 형성 세포및 중간엽 줄기 세포에 의해 형성된 공동 배양 스페로이드를 이용한 3차원 혈관신생 분석 시스템

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

근접 결찰 분석술을 사용하여 C. 예쁜꼬마선에서 리보뉴클레오 단백질 복합어셈블리의 시투 검출

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...