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Targeted and enantioselective delivery of chiral diagnostic-probes and therapeutics into specific compartments inside cells is of utmost importance in the improvement of disease detection and treatment. The classical DNA ‘light-switch’ ruthenium(II)-polypyridyl complex, [Ru(DIP)2(dppz)]Cl2 (DIP = 4,7-diphenyl-1,10-phenanthroline, dppz = dipyridophenazine) has been shown to be accumulated only in the cytoplasm and membrane, but excluded from its intended nuclear DNA target. In this study, the cationic [Ru(DIP)2(dppz)]2+ is found to be redirected into live-cell nucleus in the presence of lipophilic 3,5-dichlorophenolate or flufenamate counter-anions via ion-pairing mechanism, while maintaining its original DNA recognition characteristics. Interestingly and unexpectedly, further studies show that only the Δ-enantiomer is selectively translocated into nucleus while the Λ-enantiomer remains trapped in cytoplasm, which is found to be mainly due to their differential enantioselective binding affinities with cytoplasmic proteins and nuclear DNA. More importantly, only the nucleus-relocalized Δ-enantiomer can induce obvious DNA damage and cell apoptosis upon prolonged visible-light irradiation. Thus, the use of Δ-enantiomer can significantly reduce the dosage needed for maximal treatment effect. This represents the first report of enantioselective targeting and photosensitization of classical Ru(II) complex via simple ion-pairing with suitable weak acid counter-anions, which opens new opportunities for more effective enantioselective cancer treatment.

We have found recently that nuclear uptake of the cell-impermeable DNA light-switching Ru(II)-polypyridyl cationic complexes such as [Ru(bpy)2(dppz)]Cl2 was remarkably enhanced by pentachlorophenol (PCP), by forming ion-pairing complexes via a passive diffusion mechanism. However, it is not clear whether the enhanced nuclear uptake of [Ru(bpy)2(dppz)]2+ is only limited to PCP, or it is a general phenomenon for other highly chlorinated phenols (HCPs); and if so, what are the major physicochemical factors in determining nuclear uptake? Here, we found that the nuclear uptake of [Ru(bpy)2(dppz)]2+ can also be facilitated by other two groups of HCPs including three tetrachlorophenol (TeCP) and six trichlorophenol (TCP) isomers. Interestingly and unexpectedly, 2,3,4,5-TeCP was found to be the most effective one for nuclear delivery of [Ru(bpy)2(dppz)]2+, which is even better than the most-highly chlorinated PCP, and much better than its two other TeCP isomers. Further studies showed that the nuclear uptake of [Ru(bpy)2(dppz)]2+ was positively correlated with the binding stability, but to our surprise, inversely correlated with the lipophilicity of the ion-pairing complexes formed between [Ru(bpy)2(dppz)]Cl2 and HCPs. These findings should provide new perspectives for future investigations on using ion-pairing as an effective method for delivering other bio-active metal complexes into their intended cellular targets.

The carcinogenicity of N-hydroxy-2-acetamidofluorene (N-OHAAF), the major genotoxic metabolite of the classic model aromatic amine (AA) carcinogen 2-acetylaminofluorene, has been attributed mainly to the formation of DNA adducts via arylnitrenium upon enzymatic activation. Here, we show, unexpectedly, that exposure of N-OHAAF to UV or sunlight irradiation can not only induce the formation of the well-known covalent DNA adducts, but, more interestingly, simultaneous generation of oxidative DNA damage was also observed as measured by the formation of DNA single-/double-strand breaks (SSBs/DSBs) and 8-oxo-2'-deoxyguanosine (8-oxodG), which were partly inhibited by the typical hydroxyl radical (•OH) scavengers. Electron spin resonance spin-trapping and fluorescent studies unequivocally confirmed that the highly reactive •OH was generated from photolysis of N-OHAAF. Further DNA sequencing investigations suggest that photoactivation of N-OHAAF caused preferential cleavage at guanine, thymine and cytosine sites. More importantly, the formation of 8-oxodG and DSBs were also observed when fibroblast Balb/c-3T3 cells were co-exposed to N-OHAAF/UV irradiation as measured by double immunofluorescence staining. Taken together, we propose that both •OH and amidyl radicals can be readily produced via N-OH homolysis in N-OHAAF by photoirradiation, which can induce both oxidative and covalent DNA damage. This represents the first report of •OH production and site-specific DNA damage via photoactivation of the genotoxic hydroxamic acid intermediate, which provides a new free radical perspective to better understand the molecular mechanism for the carcinogenicity of AAs.

DNA methylation has been proven to be a critical epigenetic mark important for various cellular processes. Here, we report that redox-active quinones, a ubiquitous class of chemicals found in natural products, cancer therapeutics and environment, stimulate the conversion of 5 mC to 5 hmC in vivo, and increase 5 hmC in 5751 genes in cells. 5 hmC increase is associated with significantly altered gene expression of 3414 genes. Interestingly, in quinone-treated cells, labile iron-sensitive protein ferritin light chain showed a significant increase at both mRNA and protein levels indicating a role of iron regulation in stimulating Tet-mediated 5 mC oxidation. Consistently, the deprivation of cellular labile iron using specific chelator blocked the 5 hmC increase, and a delivery of labile iron increased the 5 hmC level. Moreover, both Tet1/Tet2 knockout and dimethyloxalylglycine-induced Tet inhibition diminished the 5 hmC increase. These results suggest an iron-regulated Tet-dependent DNA demethylation mechanism mediated by redox-active biomolecules.