Abstract

The binding of metronidazole [1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole] to nucleic acids was quantitated and characterized in vitro. [¹⁴C]Metronidazole was incubated with nucleic acid under various conditions, the nucleic acid and associated ¹⁴C were separated from lower molecular weight compounds by molecular sieve chromatography, and nucleic acid concentration and radioactivity were measured in the eluate. Maximum binding occurred when metronidazole was reduced by sodium dithionite in the presence of calf thymus DNA (0.73 molecule of drug per 10³ nucleotides). Binding was significantly less if metronidazole was reduced prior to incubation with DNA, and did not occur with unreduced metronidazole. Binding of reductively activated metronidazole to bacterial and phage DNA and yeast tRNA was also demonstrated; it was 2 times greater to alkali-denatured than to native DNA, was less in the presence of MgCl₂ or NaH₂PO₄ and at higher pH, and did not affect the melting temperature or renaturation profile of calf thymus DNA. While 15-30% of bound ¹⁴C was released by dialysis or rechromatography, the majority of the label remained complexed to nucleic acid under a variety of conditions known to dissociate noncovalent complexes. Studies with synthetic polynucleotides suggested binding specificity of reductively activated metronidazole for guanine and cytosine. Both unreduced and dithionite-reduced metronidazole bound to bovine serum albumin, and binding of the reduced drug was approximately 35% of binding to nucleic acids. The results indicate that reduction of metronidazole in vitro yields a short-lived, activated compound(s) which, while not cross-linking, binds largely in a covalent fashion primarily to guanine and cytosine of mammalian, bacterial, and phage DNA. This binding may have implications for the antimicrobial, mutagenic, and radiosensitizing actions of this drug.