Assessment of the genotoxicity of S9-generated metabolites using the GreenScreen HC GADD45a-GFP assay.
Genotoxicity can be assessed by monitoring expression of a GADD45a-GFP reporter in the human lymphoblastoid cell line TK6. A flow cytometric method has been developed to effectively distinguish GFP fluorescence from coloured and fluorescent test samples as well from the S9 liver extracts used to generate metabolites from pro-genotoxins. The method includes the use of propidium iodide exclusion for the determination of cellular viability. This paper describes the method development, the derivation of decision thresholds for the identification of genotoxins using the method, and presents data from a 56-compound validation study of the method. The results illustrate that the method permitted the detection of the majority of pro-genotoxins tested and, importantly, the high specificity of the GADD45a-GFP assay was maintained.
Jagger
Christopher
C
Gentronix Ltd, Manchester, UK.
Tate
Matthew
M
Cahill
Paul A
PA
Hughes
Chris
C
Knight
Andrew W
AW
Billinton
Nicholas
N
Walmsley
Richard M
RM
D000818:Animals; D002273:Carcinogens / Q000032:analysis / Q000633:toxicity*; D018797:Cell Cycle Proteins / Q000096:biosynthesis* / Q000235:genetics; D002457:Cell Extracts / Q000737:chemistry / Q000633:toxicity; D045744:Cell Line, Tumor; D002470:Cell Survival; D004249:DNA Damage; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D008099:Liver / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D008297:Male; D009153:Mutagens / Q000032:analysis / Q000633:toxicity*; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000235:genetics; D011419:Propidium / Q000633:toxicity; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D000097642:GADD45 Proteins
D002273:Carcinogens; D018797:Cell Cycle Proteins; D002457:Cell Extracts; D049452:Green Fluorescent Proteins; D009153:Mutagens; D009687:Nuclear Proteins; D011419:Propidium; C439070:GADD45A protein, human; D000097642:GADD45 Proteins