Direct influence of S9 liver homogenate on fluorescence signals: impact on practical applications in a bacterial genotoxicity assay.
Assays based on the bacterial SOS-response offer the possibility of automatization of genotoxicity testing for screening of large compound libraries. While existing assays use colorimetric detection or luminescence read-out, we describe here the use of a fluorescence-based system to achieve high sensitivity of detection required for assay miniaturization. Three commonly used fluorophores--fluorescein, DDAO and resorufin--are evaluated. Experimental evidence is given that S9 liver homogenate contains a heat-labile, reversible fluorophore-binding activity and therefore, significantly reduces fluorescence intensities. We have worked out simple solutions to overcome the S9 related interference in order to be able to establish a robust bacterial genotoxicity assay.
Dreier
Jürg
J
Department of Biological Technologies, F. Hoffmann-La Roche Ltd., Pharma Division, CH-4002 Basel, Switzerland. juerg.dreier@basilapharma.com
Breitmaier
Eva B
EB
Gocke
Elmar
E
Apfel
Christian M
CM
Page
Malcolm G P
MG
D001419:Bacteria / Q000187:drug effects* / Q000235:genetics; D001483:Base Sequence; D005453:Fluorescence; D005966:Glucuronidase / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D009152:Mutagenicity Tests; D011401:Promoter Regions, Genetic; D013014:SOS Response, Genetics
D005966:Glucuronidase