Molecular mechanism for the activation of the anti-tuberculosis drug isoniazid by Mn(III): First detection and unequivocal identification of the critical N-centered isoniazidyl radical and its exact location.

Isoniazid (INH), the most-widely used anti-tuberculosis drug, has been shown to be activated by Mn(III) to produce the reactive carbon-centered isonicotinic acyl radical, which was considered to be responsible for its anti-tuberculosis activity. However, it is still not clear whether the previously-proposed N-centered isoniazidyl radical intermediate can be initially produced or not; and if so, what is its exact location on the hydrazine group, distal- or proximal-nitrogen? Through complementary applications of ESR spin-trapping and HPLC/MS methods, here we show that the characteristic and transient N-centered isoniazidyl radical intermediate can be detected and identified from INH activation uniquely by Mn(III)Acetate not by Mn(III) pyrophosphate. The exact location of the radical was found to be at the distal-nitrogen of the hydrazine group by 15N-isotope-labeling techniques via using 15N-labeled INH. Diisonicotinyl hydrazine was identified as a new reaction product from INH/Mn(III). Analogous results were observed with other hydrazides. This study represents the first detection and unequivocal identification of the initial N-centered isoniazidyl radical and its exact location. These findings should provide a new perspective on the molecular mechanism of INH activation, which may have broad biomedical and toxicological significance for future research for more efficient hydrazide anti-tuberculosis drugs.

Mechanism of synergistic DNA damage induced by the hydroquinone metabolite of brominated phenolic environmental pollutants and Cu(II): Formation of DNA-Cu complex and site-specific production of hydroxyl radicals.

2,6-Dibromohydroquinone (2,6-DBrHQ) has been identified as an reactive metabolite of many brominated phenolic environmental pollutants such as tetrabromobisphenol-A (TBBPA), bromoxynil and 2,4,6-tribromophenol, and was also found as one of disinfection byproducts in drinking water. In this study, we found that the combination of 2,6-DBrHQ and Cu(II) together could induce synergistic DNA damage as measured by double strand breakage in plasmid DNA and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, while either of them alone has no effect. 2,6-DBrHQ/Cu(II)-induced DNA damage could be inhibited by the Cu(I)-specific chelating agent bathocuproine disulfonate and catalase, but not by superoxide dismutase, nor by the typical hydroxyl radical (•OH) scavengers such as DMSO and mannitol. Interestingly, we found that Cu(II)/Cu(I) could be combined with DNA to form DNA-Cu(II)/Cu(I) complex by complementary application of low temperature direct ESR, circular dichroism, cyclic voltammetry and oxygen consumption methods; and the highly reactive •OH were produced synergistically by DNA-bound-Cu(I) with H2O2 produced by the redox reactions between 2,6-DBrHQ and Cu(II), which then immediately attack DNA in a site-specific manner as demonstrated by both fluorescent method and by ESR spin-trapping studies. Further DNA sequencing investigations provided more direct evidence that 2,6-DBrHQ/Cu(II) caused preferential cleavage at guanine, thymine and cytosine residues. Based on these data, we proposed that the synergistic DNA damage induced by 2,6-DBrHQ/Cu(II) might be due to the synergistic and site-specific production of •OH near the binding site of copper and DNA. Our findings may have broad biological and environmental implications for future research on the carcinogenic polyhalogenated phenolic compounds.