Quantitative analysis of agarose gel electrophoresis for determination of single-strand breaks and double-strand breaks by plotting open circular (OC) and linear (L) plasmid fractions in absence of 0.2 M DMSO.

<p>Incubation of pUC 19 plasmid DNA with 3–15 MBq of [^99mTc]Ia, [^99mTc]Ib, [^99mTc]II, [^99mTc]III, and ^99mTcO₄^– reference for 24 h at room temperature.</p

Direct and Auger Electron-Induced, Single- and Double-Strand Breaks on Plasmid DNA Caused by ^99mTc-Labeled Pyrene Derivatives and the Effect of Bonding Distance

<div><p>It is evident that ^99mTc causes radical-mediated DNA damage due to Auger electrons, which were emitted simultaneously with the known γ-emission of ^99mTc. We have synthesized a series of new ^99mTc-labeled pyrene derivatives with varied distances between the pyrene moiety and the radionuclide. The pyrene motif is a common DNA intercalator and allowed us to test the influence of the radionuclide distance on damages of the DNA helix. In general, pUC 19 plasmid DNA enables the investigation of the unprotected interactions between the radiotracers and DNA that results in single-strand breaks (SSB) or double-strand breaks (DSB). The resulting DNA fragments were separated by gel electrophoresis and quantified by fluorescent staining. Direct DNA damage and radical-induced indirect DNA damage by radiolysis products of water were evaluated in the presence or absence of the radical scavenger DMSO. We demonstrated that Auger electrons directly induced both SSB and DSB in high efficiency when ^99mTc was tightly bound to the plasmid DNA and this damage could not be completely prevented by DMSO, a free radical scavenger. For the first time, we were able to minimize this effect by increasing the carbon chain lengths between the pyrene moiety and the ^99mTc nuclide. However, a critical distance between the ^99mTc atom and the DNA helix could not be determined due to the significantly lowered DSB generation resulting from the interaction which is dependent on the type of the ^99mTc binding motif. The effect of variable DNA damage caused by the different chain length between the pyrene residue and the Tc-core as well as the possible conformations of the applied Tc-complexes was supplemented with molecular dynamics (MD) calculations. The effectiveness of the DNA-binding ^99mTc-labeled pyrene derivatives was demonstrated by comparison to non-DNA-binding ^99mTcO₄^–, since nearly all DNA damage caused by ^99mTcO₄^– was prevented by incubating with DMSO.</p></div

Quantitative analysis of agarose gel electrophoresis for quantification of single-strand breaks and double-strand brekas by plotting open circular (OC) and linear (L) plasmid fractions in presence of 0.2 M DMSO.

<p>PUC 19 plasmid DNA was incubated with [^99mTc]Ia, [^99mTc]Ib, [^99mTc]II, [^99mTc]III, and 3–15 MBq ^99mTcO₄^– as reference.</p

Overview of the synthesized unlabeled compounds, radiolabeling procedures and the nomenclature of the ^99mTc-radiolabeled pyrene derivatives (M = ^99mTc/⁹⁹Tc).

<p>Overview of the synthesized unlabeled compounds, radiolabeling procedures and the nomenclature of the ^99mTc-radiolabeled pyrene derivatives (M = ^99mTc/⁹⁹Tc).</p

Representative radio-HPLC chromatogram of [^99mTc]Ib after radiolabeling and purification by HPLC; radioactive peak is shown at <i>t</i>_R = 11.3 min with RCP = 97% (HPLC-gradient: 5/95% A/B to 95/5% A/B within 11 min; A: H₂O (0.05% TFA) B: acetonitrile (0.05% TFA).

<p>Representative radio-HPLC chromatogram of [^99mTc]Ib after radiolabeling and purification by HPLC; radioactive peak is shown at <i>t</i>_R = 11.3 min with RCP = 97% (HPLC-gradient: 5/95% A/B to 95/5% A/B within 11 min; A: H₂O (0.05% TFA) B: acetonitrile (0.05% TFA).</p

Summary of quantitative analysis of the fractions of OC (SSB) and L (DSB) caused by incubation of 15 MBq [^99mTc]Ia, [^99mTc]Ib, [^99mTc]II, [^99mTc]III, and ^99mTcO4– in absence (–) and presence (+) of 0.2 M DMSO.

<p>Summary of quantitative analysis of the fractions of OC (SSB) and L (DSB) caused by incubation of 15 MBq [^99mTc]Ia, [^99mTc]Ib, [^99mTc]II, [^99mTc]III, and ^99mTcO4– in absence (–) and presence (+) of 0.2 M DMSO.</p

Overview of the detected OC (SSB) and L (DSB) plasmid fractions regarding to the radical scavenging qualities of the ⁹⁹Tc-labeled pyrene derivatives [⁹⁹Tc]Ia, [⁹⁹Tc]Ib, [⁹⁹Tc]II, and [⁹⁹Tc]III in comparison to 0.2 M DMSO.

<p>Incubation of pUC 19 plasmid DNA with 3 MBq ^99mTcO₄^– and 3 MBq equivalent amount of the respective ⁹⁹Tc-labeled pyrene derivative as well as 15 MBq ^99mTcO₄^– and 15 MBq equivalent amount of the respective ⁹⁹Tc-labeled pyrene derivative.</p

Representative agarose gel of supercoiled (SC) pUC 19 plasmid DNA incubated with various activities of [^99mTc]Ib and [^99mTc]III in presence of DMSO (0.2 M).

<p>Lane C is the control pUC 19 plasmid DNA (SC) and lane L is the linear control pUC 19 plasmid DNA (L). OC represent open circular conformation of pUC 19 plasmid DNA. Lanes 1–5 show pUC 19 plasmid DNA incubated with [^99mTc]Ib (3–15 MBq). Lanes 6–10 show pUC 19 plasmid DNA incubated with [^99mTc]III (3–15 MBq).</p

Summary of HPLC retention times (<i>t</i>_R) of chelators 4, 8a, 8b, 9 and the radiolabeled pyrene derivatives [^99mTc]Ia, [^99mTc]Ib, [^99mTc]II, [^99mTc]III in comparison with [^99mTc(CO)₃(H₂O)₃]⁺ and ^99mTcO4– as references.

<p>Summary of HPLC retention times (<i>t</i>_R) of chelators 4, 8a, 8b, 9 and the radiolabeled pyrene derivatives [^99mTc]Ia, [^99mTc]Ib, [^99mTc]II, [^99mTc]III in comparison with [^99mTc(CO)₃(H₂O)₃]⁺ and ^99mTcO4– as references.</p