Loss of Cellular Transformation Efficiency Induced by DNA Irradiation with Low-Energy (10 eV) Electrons

Abstract

Low energy electrons (LEEs) of energies less than 20 eV are generated in large quantities by ionizing radiation in biological matter. While LEEs are known to induce single (SSBs) and double strand breaks (DSBs) in DNA, their ability to inactivate cells by inducing nonreparable lethal damage has not yet been demonstrated. Here we observe the effect of LEEs on the functionality of DNA, by measuring the efficiency of transforming Escherichia coli with a [pGEM-3Zf (-)] plasmid irradiated with 10 eV electrons. Highly ordered DNA films were prepared on pyrolitic graphite by molecular self-assembly using 1,3-diaminopropane ions (Dap 2+ ). The uniformity of these films permits the inactivation of approximately 50% of the plasmids compared to <10% using previous methods, which is sufficient for the subsequent determination of their functionality. Upon LEE irradiation, the fraction of functional plasmids decreased exponentially with increasing electron fluence, while LEE-induced isolated base damage, frank DSB, and non DSB-cluster damage increased linearly with fluence. While DSBs can be toxic, their levels were too low to explain the loss of plasmid functionality observed upon LEE irradiation. Similarly, non-DSB cluster damage, revealed by transforming cluster damage into DSBs by digestion with repair enzymes, also occurred relatively infrequently. The exact nature of the lethal damage remains unknown, but it is probably a form of compact cluster damage in which the lesions are too close to be revealed by purified repair enzymes. In addition, this damage is either not repaired or is misrepaired by E. coli, since it results in plasmid inactivation, when they contain an average of three lesions. Comparison with previous results from a similar experiment performed with γ-irradiated plasmids indicates that the type of clustered DNA lesions, created directly on cellular DNA by LEEs, may be more difficult to repair than those produced by other species from radiolysis.

Clustered DNA Damages induced by 0.5 to 30 eV Electrons

“…As a means of quantifying the damage to plasmid DNA, it is also possible to directly measure the transformation of cells whose survival depends on intact DNA [89]. In this case, LEE-irradiated plasmid DNA is transferred into E. Coli bacteria to increase their resistance against an antibiotic.…”

“…In this case, LEE-irradiated plasmid DNA is transferred into E. Coli bacteria to increase their resistance against an antibiotic. For example, E. Coli bacteria can be incubated in an ampicillin-rich environment that would normally kill them [89]. However, the cells can survive by the injection of undamaged plasmids that encode an enzyme that is capable of inactivating the antibiotic.…”

“…In this manner, the results of curve 2B serve to relate LEE-induced damages to cell death. A detailed analysis of DNA damages induced by 10-eV electrons has also showed that non-DSB clustered lesions are the most likely to be responsible for inactivating the cells [89]. The energy correlation between all the peaks in the curves of Figure 1 and Figure 2A provides further evidence that the LEE-induced clustered lesions that are formed by TA decay into destructive channels, which can be lethal to cells (i.e., they can decrease plasmid DNA functionality, which increases cells death).…”

Transient Anions in Radiobiology and Radiotherapy: From Gaseous Biomolecules to Condensed Organic and Biomolecular Solids

“…Figure 4 schematically shows that how utilizing of X-ray interactions with gold nanoclusters within living cells, which subsequently results in releasing both Auger and ICD electrons, has been suggested as a potential strategy for targeted cancer treatment [148]. After such extensive studies on LEE-induced damage under "near"-cellular conditions, it was only very recently that the lethal effects of LEEs in cells have been demonstrated by Sahbani et al [154], who investigated the biological functionality of DNA, via a simple model system comprising E. coli bacteria and plasmid DNA bombarded by LEEs. In these experiments, highly ordered DNA films were arranged on pyrolytic graphite surface by molecular self-assembly technique using 1,3-diaminopropane ions to bind together the plasmid DNAs [155].…”

Reactivity of prehydrated electrons toward nucleobases and nucleotides in aqueous solution

“…Sanche and coworkers ( 8 ) provided the first experimental observation of DNA strand cleavage induced by LEEs. This work led to numerous mechanistic studies on LEE-induced damage in naked DNA models including DNA building blocks in gas phase or condensed states, single- and double-stranded oligomers of defined sequences, plasmid DNA, and cellular DNA ( 9 , 10 ). These studies established that, initially, LEEs lead to the formation of resonant transient negative ions (TNIs) in DNA models.…”