99mTc-Labeled HYNIC-DAPI Causes Plasmid DNA Damage with High Efficiency

Abstract

99mTc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, 99mTc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a 99mTc-labeled HYNIC-DAPI compound with that of 99mTc pertechnetate (99mTcO4 −). pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by 99mTc-HYNIC-DAPI (1.03) was twice that caused by 99mTcO4 − (0.51), and the number of DSBs increased fivefold in the 99mTc-HYNIC-DAPI-treated sample compared with the 99mTcO4 − treated sample (0.02 to 0.10). In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the 99mTcO4 – treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the 99mTc-HYNIC-DAPI-treated samples. These results indicated that 99mTc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the 99mTc-labeled compound with DNA. In contrast to these results, 99mTcO4 − induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of 99mTc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by approximately 10-fold in terms of inducing DSBs.

Subcellular Targeting of Theranostic Radionuclides

“…In cell-free studies, they showed a decrease in DNA damage when the distance between the intercalating moiety was increased from 0.3 to 1.5 nm, clearly demonstrating the need for close association with DNA molecules for AE-generated DNA lesion formation. Similarly, Kotzerke et al (2014) radiolabeled DAPI, a dual groove-binder, intercalator and commonly employed DNA dye, with 99m Tc. In related work, the anthracycline doxorubicin, a widely used topoisomerase II inhibitor and DNA intercalating chemotherapeutic, was conjugated to 99m Tc to enhance its potency and diagnostic potential ( Imstepf et al, 2015 ).…”

Re-assessing gallium-67 as a therapeutic radionuclide

“…When bound to DNA, damage induced by 125 I was not diminished by DMSO [37]. Also, non-plasmid-bound 99m TcO 4 − caused several fold lower induction of single strand breakage in the presence of DMSO [38,39]. Overall, direct incubation of radionuclides with plasmid DNA in a cell-free system is useful to understand DNA damage by radionuclide emissions and decay only.…”

Synthesis of a new HYNIC-DAPI derivative for labelling with 99mTechnetium and its in vitro evaluation in an FRTL5 cell line

“…Further investigations of our group confirm the thesis of a direct interaction with free DNA and prove that 99m Tc-HYNIC-DAPI is bound to plasmid DNA, introducing 4-fold more singlestrand breaks (SSB) and up to 10-fold more double-strand breaks (DSB) than free 99m Tc sodium pertechnetate. 13 As shown in section 2.4, the decomposition half-life of 99m Tc-HYNIC-DAPI is about 8 h in culture medium. Decomposition influences the internalisation into the cells and the distribution between the cell compartments, e.g.…”

“…First results of the effects of 99m Tc-HYNIC-DAPI on plasmid DNA have already been published. 13 3.3.2. The properties of 99m Tc-HYNIC-DAPI in cell culture.…”