Quantitative GFP fluorescence as an indicator of arsenite developmental toxicity in mosaic heat shock protein 70 transgenic zebrafish.
In transgenic zebrafish (Danio rerio), green fluorescent protein (GFP) is a promising marker for environmental pollutants. In using GFP, one of the obstacles which we faced was how to compare toxicity among different toxicants or among a specific toxicant in different model species with the intensity of GFP expression. Using a fluorescence detection method, we first validated our method for estimating the amount of GFP fluorescence present in transgenic fish, which we used as an indicator of developmental toxicity caused by the well-known toxicant, arsenite. To this end, we developed mosaic transgenic zebrafish with the human heat shock response element (HSE) fused to the enhanced GFP (EGFP) reporter gene to indicate exposure to arsenite. We confirmed that EGFP expression sites correlate with gross morphological disruption caused by arsenite exposure. Arsenite (300.0 microM) caused stronger EGFP fluorescence intensity and quantity than 50.0 microM and 10.0 microM arsenite in our transgenic zebrafish. Furthermore, arsenite-induced apoptosis was demonstrated by TUNEL assay. Apoptosis was inhibited by the antioxidant, N-acetyl-cystein (NAC) in this transgenic zebrafish. The distribution of TUNEL-positive cells in embryonic tissues was correlated with the sites of arsenite toxicity and EGFP expression. The EGFP values quantified using the standard curve equation from the known GFP quantity were consistent with the arsenite-induced EGFP expression pattern and arsenite concentration, indicating that this technique can be a reliable and applicable measurement. In conclusion, we propose that fluorescence-based EGFP quantification in transgenic fish containing the hsp70 promoter-EGFP reporter-gene construct is a useful indicator of development toxicity caused by arsenite.
Seok
Seung-Hyeok
SH
Department of Laboratory Animal Medicine, College of Veterinary Medicine and KRF Zoonotic Disease Priority Research Institute, Seoul National University, Seoul 151-742, Republic of Korea.
Baek
Min-Won
MW
Lee
Hui-Young
HY
Kim
Dong-Jae
DJ
Na
Yi-Rang
YR
Noh
Kyoung-Jin
KJ
Park
Sung-Hoon
SH
Lee
Hyun-Kyoung
HK
Lee
Byoung-Hee
BH
Ryu
Doug-Young
DY
Park
Jae-Hak
JH
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D030801:Animals, Genetically Modified; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D018053:Arsenites / Q000008:administration & dosage / Q000633:toxicity*; D015415:Biomarkers; D004305:Dose-Response Relationship, Drug; D004625:Embryo, Nonmammalian / Q000187:drug effects / Q000378:metabolism; D004785:Environmental Pollutants / Q000008:administration & dosage / Q000633:toxicity*; D005456:Fluorescent Dyes / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D017930:Genes, Reporter / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D009030:Mosaicism; D015027:Zebrafish / Q000235:genetics
D000975:Antioxidants; D018053:Arsenites; D015415:Biomarkers; D004785:Environmental Pollutants; D005456:Fluorescent Dyes; D018840:HSP70 Heat-Shock Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C015001:arsenite; D000111:Acetylcysteine