There is evidence for the induction by IR of thermally labile DNA lesions, which
contribute to DSB formation (tlDSBs), albeit in a delayed manner, even in cells
maintained under physiological temperatures (see Introduction). As a result of this
delayed formation, the total load of DSBs generated in an irradiated cell (tDBSs)
will be the sum of those induced promptly, i.e. those present immediately after
irradiation (prDBSs), and those generated within a non-DSB-CDS by the conversion of
a TLSL to a SSB (tlDBSs); thus, tDSBs = prDBSs + tlDBSs. It
is not known whether prDBSs and tlDBSs are detected and processed by the cell with
the same efficiency and, actually, arguments can be developed why this may not be
the case [17–19]. If cells detect and process differently prDBSs and tlDBSs, it is likely
that their biological consequences will also be different.
Experimentally, the yields of prDBSs can be determined by lysing cells immediately
after irradiation using low temperature (0 – 4°C) lysis protocols (LTL),
whereas the standard 50°C lysis allows determination of tDBSs. The difference
between tDBSs – prDBSs yields gives then estimates regarding the yields of
tlDBSs. There is evidence that IR induces a spectrum of TLSLs with different levels
of chemical and thermal stability . This raises the question how to determine the biologically relevant
subset of tlDBSs, i.e. the subset that also converts to a DSB in cells maintained
under physiological conditions. There are at present no established methods allowing
the reliable determination of the biologically relevant subset of tlDBSs. However,
as a first approximation, we assume that conversion of TLSLs to breaks is similar in
cells maintained at 37°C and cells analyzed by lysing at 50°C immediately
after exposure to IR .
Using the above outlined conceptual and experimental background we investigate here
how the yields of prDBSs and tlDBSs change in cells exposed to HI. The results
presented in the previous section extend trends previously reported for neutrons  and confirm a strong, inverse LET dependence of the yields of prDBSs and
tlDBSs. Specifically, while exposure to HI causes a strong reduction in the yields
of tlDBSs as compared to X-rays, it causes a strong increase in the yields of
prDBSs. These opposing effects partly compensate each other and as a result the
yield of tDSBs changes only modestly with increasing LET. This is in line with the
observation that RBEs close to 1 are frequently measured for the induction of DSBs . Notably, our results demonstrate that when prDBSs are specifically
detected by LTL protocols, much higher RBE values are measured that are approaching
those obtained for cell survival (Figure 6). This is a
potentially highly significant observation that warrants further investigations.
Notably, the RBE values for DSB-induction after exposure to high LET radiation, as
measured by γ-H2AX foci formation in diverse cell lines, is also very close to
one ( and references therein). This is significant as it shows, in line with
our earlier work , that the load of DSBs the cell ultimately “sees” is close to
that measured by HTL. If cells were only detecting prDBSs, as it is often assumed,
two to three times more DSBs (i.e. γ-H2AX foci) would have been expected after
exposure to high LET radiation than after low LET radiation. On the other hand, it
also demonstrates that the γ-H2AX marking of DSBs does not differentiate levels
of DSB complexity.
The increase in prDSBs observed with increasing LET can be explained by the expected
increase in the size of ionization clusters (more ionizations within the same
volume) that leads to the generation of higher complexity CDS, i.e. the presence of
a higher number of lesions at the site. As a result of this increase in lesion
number within a CDS it becomes more likely that prompt SSBs will combine to form a
prDSB. Even if TLSLs are present in these CDSs, their subsequent conversion to
breaks will remain inconsequential with reference to DSB formation. The chemical
reactions that convert a TLSL to a SSB remain uncharacterized, but may include
base-catalyzed hydrolysis or oxidation .
As noted above, TLSLs are not a uniform chemical entity but rather a spectrum of
lesions with different chemical and thermal sensitivities. The probability of their
formation from clusters of ionization events and radical attacks, as well as their
chemical evolution may be decisively determined by the chemical environment in their
immediate vicinity. In this respect, it is likely that the details of DNA
organization in chromatin within a CDS, including all participating histone and
non-histone proteins, will affect decisively not only the induction of TLSLs but
also their evolution to tlDBSs. This theoretically anticipated dependence provides
also a first explanation for the surprisingly large differences observed in the
yields of tlDBSs among different cell lines both after exposure to high as well as
to low LET radiation [17–19]. Furthermore, the large differences in LTL dose response curves among
different cell lines contrasts the surprisingly similar HTL dose response curves and
points to cell line specific variation in the chemical environment in the vicinity
of a clustered ionization hitting the DNA that alters the probability of generation
of a prDSB.
While the selection of cell lines used here reflects the intellectual evolution of
the TLSL problematic in our work during the past few years, future work will
certainly benefit from a hypothesis oriented selection of cell lines and an analysis
of TLSL production and evolution after treatments that alter chromatin organization.
It is also worth pointing out that the differences between cell lines persist even
after exposure of cells to high LET radiation  (see above).
The results discussed above demonstrate that the energy deposition pattern of
radiation is not the sole determinant of the yields of tlDSBs. Small differences in
DNA organization may cause changes in the induction and the subsequent chemical
processing of TLSLs and may strongly affect the form of DSBs induced – even
after exposure to high LET radiation. Worth noting is also that the differences
observed in lesion induction and evolution among cell lines exposed to HI are
largely eliminated if “naked” DNA is irradiated instead of cells.
Collectively, the results presented here, as well those published before [17–19], point to an unexplored dimension in the production of DNA damage by IR.
Temporal evolution of complex radiation damage to DSBs, and the suggested role of
DNA organization in this evolution go beyond current concepts of DNA damage
induction and repair  and indicate aspects of DDR that warrant further investigations.
It is tempting to speculate that transient, chemical stabilization of TLSLs, may
allow repair of SSBs and base damages within a non-DSB CDS, so that subsequent
conversion of the TLSL to a DNA break will not cause a DSB. Such agents may find
application in radiation protection on earth and in space, as well as in the
development of new strategies in radiation oncology [21, 36, 37].