Parathyroid Hormone Reduces Persistent Proton Induced DNA Double-Strand Breaks in Osteocytes

Abstract

<h3>Purpose/Objective(s)</h3> Healthy bone is commonly irradiated during radiotherapy for many cancers, and bone fractures can be a late complication. A recent study revealed a higher incidence of rib fractures after breast irradiation with protons (7%) compared to photons (1%) at the same dose, but the underlying biological mechanisms are unknown. Here we used <i>in vitro</i> experiments, supported by Monte Carlo track structure simulations, to investigate differences in bone cell responses to proton and photon radiation, and to identify potential therapeutic interventions. <h3>Materials/Methods</h3> We used TOPAS-nBio, a radiobiology simulation toolkit, to simulate a segment of bone using geometric models of bone cells (osteoblasts, osteoclasts, and osteocytes). Photon and proton tracks were simulated through bone matrix to investigate energy distributions received by each cell. For <i>in vitro</i> experiments, we utilized the murine osteocytic cell line Ocy454. Cells were irradiated with different doses of protons or photons. Surviving fraction as a function of dose was determined using colony formation assays. DNA double-strand breaks were assessed by 53BP1 foci assay. For PTH treatments, cells were treated with 10 nM human PTH(1-34) for 24 hours prior to irradiation. All experiments were repeated independently three or more times. Statistical analysis was performed by unpaired t-test with Bonferroni correction. <h3>Results</h3> Monte Carlo track simulations showed osteocytes are the bone cell most likely to receive radiation damage, particularly with protons. In the case of large multi-nucleated osteoclasts, both photons and protons spared some of the nuclei, suggesting functional sparing of osteoclasts from radiation damage. We therefore focused our <i>in vitro</i> experiments on Ocy454 osteocytes. Interestingly, the surviving fraction of osteocytes was significantly lower after protons compared to photons for all investigated doses of 1 to 8 Gy. The dose for 10% surviving fraction was 5.38 Gy for protons and 7.46 Gy for photons (Relative Biological Effect 1.39). Although protons and photons induced similar numbers of 53BP1 foci at 1 hour (79% vs. 72% cells with foci), a significantly higher number of foci was observed at 24 hours after protons compared to photons (22% vs. 6.5%, p=0.0078), suggestive of persistent DNA damage after protons. Remarkably, we found that pre-treatment with parathyroid hormone (PTH) decreased 53BP1 foci at 24 hours in proton-irradiated cells to levels comparable to that of non-irradiated cells (22% without PTH vs. 6.3% with PTH, p=0.014). <h3>Conclusion</h3> Surviving fraction of osteocytes was lower after proton compared to photon irradiation, suggesting a greater sensitivity of osteocytes to protons. Protons result in greater DNA damage in osteocytes, but this damage may be mitigated with the addition of PTH. Further study is needed to investigate the potential of PTH to reduce bone damage induced by proton irradiation.