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

Abstract

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.

Subcellular Targeting of Theranostic Radionuclides

“…These studies provided valuable knowledge that illuminated the inverse relationship between AE-emitter distance to the DNA and DNA damage and cell kill ( Kortylewicz et al, 2012 ). Since then, several small molecule DNA intercalators have been proposed as nuclear-targeting agents for AE-emitting radionuclides, such as radiolabeled derivatives of acridine orange ( Pereira et al, 2017 ), pyrene ( Häfliger et al, 2005 ; Reissig et al, 2016 ), doxorubicin ( Imstepf et al, 2015 ), and daunorubicin ( Fondell et al, 2011 ). Acridine orange is a cell-permeable molecule with anticancer drug and radiosensitizing properties.…”

“…The Alberto group explored the effect of 99m Tc-labeled pyrene-NLS (SV40 NLS peptide) conjugates on cell survival and found that these compounds exhibited a DNA damaging effect, leading to mitotic catastrophe ( Haefliger et al, 2005 ). Reissig et al (2016) synthesized related pyrene constructs with 99m Tc-labeled alkyne groups of variable length. 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.…”

Radio- und Photosensitivierung von Plasmid-DNA durch den DNA-bindenden Liganden Propidiumiodid: Untersuchungen zur Auger-Elektronen-Induktion und zum Nachweis von Cherenkov-Strahlung

“…Es konnte weiterhin nachgewiesen werden, dass Tc-99m-markiertes DAPI an die DNA bindet und dosisabhängig ESB und DSB induziert, welche durch den Radikalfänger DMSO nicht verhindert werden können und somit auf eine direkte Wechselwirkung und nicht auf eine indirekte, Radikal-vermittelte Wirkung zurückgehen [8]. In einer Folgeuntersuchung wurde der Abstand des Radioisotops zur Bindungsstelle variiert, so dass bei zunehmender Entfernung der Anteil an DSB rückläufig war und die DNA-Schäden durch DMSO effektiv verhindert werden konnten [9]. Außerdem konnte gezeigt werden, dass der Alphastrahler Ra223 im Vergleich zu Re-188 und Tc-99m ebenfalls primär Radikal-vermittelte schädigende Wirkung hat [15].…”

“…7 Darstellung der relativen Anteile der Fluoreszenzintensität der OC-Konformation (ESB) und der L-Konformation (DSB) bei Bestrahlung mit 200 Gy Röntgenstrahlung in Abhängigkeit von der PI-Konzentration (MW aus 3 Versuchen ± SEM). mehr zeigen können [9]. Es lässt sich auch damit begründen, dass PI eine ionische Verbindung ist und zumindest ein großer Teil des Iodids in der wässrigen Lösung frei vorliegt.…”

“…B. durch Einlagerung in die kleine Furche der DNA-Helix). Für die DNA-bindenden Substanzen Tc-99m-DAPI und Tc-99m-Pyren konnte an Plasmid-DNA gezeigt werden, dass die Auger-Elektronen des Tc99m DSB induzieren [8,9], dass demnach eine Anlagerung einer DNA-bindenden Substanz in der kleinen Furche hinreichend nah zu den vulnerablen Strukturen ist, so dass selbst Auger-Elektronen geringer Reichweite ein hohes Schädigungspotential aufweisen.…”