NITROBENZIMIDAZOLES AS SUBSTRATES FOR DT-DIAPHORASE AND REDOX CYCLINGCOMPOUNDS - THEIR ENZYMATIC-REACTIONS AND CYTOTOXICITY

We have synthesized a number of nitrobenzimidazoles containing nitro g roups in the benzene ring and found that they acted as relatively effi cient substrates for rat liver DT-diaphorase (EC 1.6.99.2), their reac tivity exceeding reactivities of nitrofurans and nitrobenzenes. Nitrob enzimidazoles were competitive with NADPH inhibitors of DT-diaphorase in menadione reductase reactions, their inhibition constant being unch anged in the presence of dicumarol and being increased in the presence of 2',5'-ADP. These data indicate that the poor reactivity of nitrobe nzimidazoles and other nitroaromatics in comparison to quinones could be determined by their binding in the adenosine-phosphate binding regi on of the NADPH-binding site, whereas quinones bind at the nicotinamid e-binding pocket at the vicinity of FAD of DT-diaphorase. The reductio n of 4,5,6-trinitrobenzimidazol-2-one by DT-diaphorase most probably i nvolves reduction of ii-nitro group to B-nitroso or B-hydroxylamine de rivative at the initial step. A certain parallelism existed between re activities of nitrobenzimidazoles toward DT-diaphorase and their react ivities in single-electron reduction by Anabaena ferredoxin:NADP(+) re ductase (EC 1.18.1.2) and Saccharomyces cerevisiae flavocyto-chrome bz (EC 1.1.2.3), the latter being determined by electronic factors. Howe ver, we suppose that the relatively high reactivity of polinitrobenzim idazoles toward DT-diaphorase was due not only to electronic effects, but also to a sterical crowding of nitrogroups by each other. The toxi city of nitrobenzimidazoles to bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) with a moderate amount of DT-diaphorase (260 U/mg protein) is partly prevented by dicumarol. That points out t o partial determination of nitrobenzimidazole cytotoxicity by their re duction by DT-diaphorase. Another important factor of nitrobenzimidazo le toxicity to this cell line was oxidative stress, catalyzed by singl e-electron transfering enzymes. (C) 1997 Academic Press.