**DNA Double Strand Breaks as a Biomarker of Cancer Risk in the Rat Skin
Model**

Fredric J. Burns, PhD, Moon-shong (Eric) Tang,PhD, Krystyna Frenkel,
PhD,

Feng Wu and Ronghe Zhang, PhD

New York University School of Medicine

Department of Environmental Medicine

DNA double strand breaks (DSBs), a result of the tendency of ionizations
to cluster, are thought to be relevant to carcinogenesis, because of the
probability that such breaks could lead to genomic instability \[1-4\].
Clusters are capable of breaking both strands of the DNA backbone
simultaneously; a process that also breaks the chromosome and is
probably unique to ionizing radiation as a carcinogen \[5\]. DSBs are
also associated with the cytotoxic action of radiation based on
experiments with cells *in vitro* and on carcinogenic activity based on
speculations about carcinogenesis *in vivo* \[6\].

A model of radiation carcinogenesis based on 2 interacting DNA double
strand breaks (DSBs) has shown excellent consistency with malignant
tumor yields in rat skin exposed to acute doses of different types of
radiation. The expected cancer yield in cancers/animal is: Y (D,L) =
CLD + BD^2^, where D is acute dose, L is average LET and C and B are
coefficients to be evaluated empirically. The above equation is based on
the assumption that cancers arise from either of 2 sources; 1) 2 DSBs in
the same track (the CLD term) or 2) 2 DSBs in different tracks (the
BD^2^ term). In reality the tumors from these different sources are
indistinguishable by current technology. But by evaluating C and B based
on cancer induction, the 2 terms can be evaluated separately. Based on
these evaluations a dose, D~equal~, can be established where the 2 terms
contribute equally to total tumor yield, i.e. D~equal~ = CL/B. The
latter is a very useful quantity for risk assessment purposes. At doses
below D~equal~ cancer yield is predominantly linear with dose, while
above D~equal~ it is predominantly dose squared. Nearly all tumor
induction data in support of this model were derived from exposure of
rat skin to radiation in the LET range between 26 and 150 keV/µ or at
the LET =\~ 0.34 keV/µ (x-rays and electrons). Within these LET limits
the cancer yield (squamous, basal cell and other types of carcinomas)
conform very well to the equation above as dose and LET vary.

If double strand breaks are really the starting point of carcinogenesis,
an evaluation of their dose and LET dependence might provide a useful
biodosimetric marker of cancer risk. These breaks have the advantage of
being very frequent, at least in the first few hours after irradiation,
relative to the cancers that occur much later. A first step in this
evaluation is to show that DSBs follow the same dose and LET dependence
as observed for cancer induction, albeit at very different absolute
magnitudes. Following carcinogenic radiation doses in rat skin virtually
every epithelial cell is expected to exhibit numerous DSBs, while the
cancer probability is on the order of one per million cells exposed.
Nevertheless the question remains whether dose and LET dependencies
remain parallel for the 2 endpoints, particularly whether respective
D~equal~ values would be comparable.

Samples of rat skin keratinocytes were irradiated with either ^56^Fe
ions or x-rays and then were stained for DSBs by means of the γ-H2AX
antibody technique. The resulting patterns were analyzed based on the
track structure of the radiation, i.e. high-energy, heavy charged ions,
such as ^56^Fe, pass through the nucleus of cells essentially in
straight tracks that consist of a dense, LET-dependent core of
ionizations surrounded by a penumbra of ionization associated with delta
electrons that are ejected from the track at different energies. The
results showed H2AX-positive foci aligned along straight tracks in the
beam direction at time 0 (\~10 min after exposure). At later times the
straight tracks generally remained parallel to one another in the same
nucleus, but each nucleus showed a plus and minus rotations relative to
the zero time orientation. Foci within tracks were counted as a fraction
of total foci for 2 doses of ^56^Fe ions and 2 equicarcinogenic doses
x-rays. When plotted in the same manner and on the same scale, these DSB
fractions were quantitatively consistent the cancer yields derived from
fitting rat skin cancer data as described above. Even the extrapolated
D~equal~ for DSBs was within about 30% of the D~equal~ for
carcinogenesis. Upon further verification, particularly in human tissue,
DSBs within a few hours after irradiation might prove to be a useful
biodosimetric tool for estimating cancer risks for different types and
combinations of space radiation for which no human cancer data is ever
likely to be available.

References

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