The use of rate constants of flavoenzyme-catalyzed single-electron reduction of nitroaromatics in the analysis of their cytotoxicity mechanisms

Enzymatic formation and redox cycling of free radicals is an intrinsic property of nitroaromatic compounds (ArNO₂). If the main mode of their action is redox cycling and subsequent oxidative stress, then the cytotoxicity can be described by regression log (cytotoxic concentration) = a - b E¹₇ – c log D, where E¹₇ is the single-electron reduction potential of compound, and log D is its octanol/water distribution coefficient at pH 7.0 ([1-3], and references therein). The deviation from the limits predicted by the redox cycling activity may point to additional mechanisms of cytotoxicity or therapeutic action of ArNO₂. However, the direct determination of E¹₇ values of ArNO₂ by pulse-radiolysis is complicated. On the other hand, E¹₇ may be calculated from the geometric average of their bimolecular reduction rate constants by single-electron transferring flavoenzymes such as NADPH:cytochrome P-450 reductase, ferredoxin:NADP⁺ oxidoreductase, or NADPH:adrenodoxin reductase/adrenodoxin complex [1-3]. Using this approach, we obtained the previously unavailable E¹₇ values of nitroaromatic antiandrogens nilutamide (-0.408 V), flutamide (-0.399 V), 2- hydroxyflutamide (-0.394 V) and (3-amino-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl) phenyl) propanamide (-0.377 V), and antiviral agents nitazoxanide (-0.380 V) and niclosamide (- 0.339 V). With an exception of more cytotoxic niclosamide, the cytotoxicity of these compounds in mouse hepatoma MH22a cells closely followed the regression previously obtained for the model nitroaromatic compounds (n = 15) with available E¹₇ values [3]. Taken together with the protection by antioxidants, it points to the prevailing oxidative stress-type cytotoxicity mechanism. On the other hand, the cytotoxicity of examined compounds in human colon carcinoma HCT-116 cells was almost by one order of magnitude higher than expected. This points to the prevalent other modes of action of above nitroaromatic antiandrogens and antiviral agents, whose mechanisms warrant further investigation, and to a definite potential of repurposing of these drugs. (author)