Nitrobenzimidazoles as substrates for DT-diaphorase and redox cycling compounds: their enzymatic reactions and cytotoxicity.
We have synthesized a number of nitrobenzimidazoles containing nitro groups in the benzene ring and found that they acted as relatively efficient substrates for rat liver DT-diaphorase (EC 1.6.99.2), their reactivity exceeding reactivities of nitrofurans and nitrobenzenes. Nitrobenzimidazoles were competitive with NADPH inhibitors of DT-diaphorase in menadione reductase reactions, their inhibition constant being unchanged in the presence of dicumarol and being increased in the presence of 2',5'-ADP. These data indicate that the poor reactivity of nitrobenzimidazoles and other nitroaromatics in comparison to quinones could be determined by their binding in the adenosine-phosphate binding region of the NADPH-binding site, whereas quinones bind at the nicotinamide-binding pocket at the vicinity of FAD of DT-diaphorase. The reduction of 4,5,6-trinitrobenzimidazol-2-one by DT-diaphorase most probably involves reduction of 5-nitro group to 5-nitroso or 5-hydroxylamine derivative at the initial step. A certain parallelism existed between reactivities of nitrobenzimidazoles toward DT-diaphorase and their reactivities in single-electron reduction by Anabaena ferredoxin:NADP+ reductase (EC 1.18.1.2) and Saccharomyces cerevisiae flavocytochrome b2 (EC 1.1.2.3), the latter being determined by electronic factors. However, we suppose that the relatively high reactivity of polinitrobenzimidazoles toward DT-diaphorase was due not only to electronic effects, but also to a sterical crowding of nitrogroups by each other. The toxicity 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 to partial determination of nitrobenzimidazole cytotoxicity by their reduction by DT-diaphorase. Another important factor of nitrobenzimidazole toxicity to this cell line was oxidative stress, catalyzed by single-electron transferring enzymes.
Sarlauskas
J
J
Institute of Biochemistry, Vilnius, Lithuania.
Dickancaite
E
E
Nemeikaite
A
A
Anusevicius
Z
Z
Nivinskas
H
H
Segura-Aguilar
J
J
Cenas
N
N
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001562:Benzimidazoles / Q000138:chemical synthesis / Q000378:metabolism* / Q000633:toxicity*; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects; D003676:Deferoxamine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D007668:Kidney; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D015394:Molecular Structure; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D009574:Nitro Compounds / Q000378:metabolism / Q000633:toxicity; D010655:Phenylenediamines / Q000494:pharmacology; D051381:Rats; D012756:Sheep; D013053:Spectrophotometry; D013379:Substrate Specificity
D000975:Antioxidants; D001562:Benzimidazoles; D004791:Enzyme Inhibitors; D009574:Nitro Compounds; D010655:Phenylenediamines; D009249:NADP; C004845:N,N'-diphenyl-4-phenylenediamine; D016660:NAD(P)H Dehydrogenase (Quinone); D003676:Deferoxamine