Growth hormone-responsive DT-diaphorase-mediated bioreduction of tetrazolium salts.
Microculture tetrazolium assays (MTAs) rely upon the bioreduction of tetrazolium salts to their intensely coloured formazans. Although these assays are being extensively used, the intracellular mechanisms responsible for the formazan production are not known. MTAs currently provide the basis for uniquely precise in vitro bioassays for human growth hormone (hGH) which use the Nb2 cells. We have compared two contrasting tetrazolium salts, namely 3-(4,5-dimethyl-thiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) and 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-++ +sulfophenyl) tetrazolium, inner salt (MTS), in this system. An intermediate electron acceptor (IEA) is obligatory for the MTS- but not the MTT-bioassay. We report that inhibitors of DT-diaphorase abolished MTS- but not MTT-formazan production. We conclude that substitution of MTT with MTS/menadione resulted in formazan production via a different electron transfer pathway which is exclusively mediated by DT-diaphorase.
Goodwin
C J
CJ
Department of Molecular Pathology, University College London, United Kingdom.
Holt
S J
SJ
Riley
P A
PA
Downes
S
S
Marshall
N J
NJ
D000818:Animals; D002460:Cell Line; D001728:Dicumarol / Q000494:pharmacology; D008058:Dihydrolipoamide Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005562:Formazans; D013006:Growth Hormone / Q000494:pharmacology*; D006801:Humans; D008223:Lymphoma; D010084:Oxidation-Reduction; D051381:Rats; D013379:Substrate Specificity; D013778:Tetrazolium Salts / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D005562:Formazans; D013778:Tetrazolium Salts; D001728:Dicumarol; D013006:Growth Hormone; D008058:Dihydrolipoamide Dehydrogenase