Iron deprivation induces apoptosis and enhances ionizing radiation injury in endothelial cells

Bone marrow transplant conditioning regimens induce the displacement of iron into the vascular space which may contribute to regimen related toxicity through endothelial injury. Iron can exacerbate freeradical mediated injury but is necessary for DNA repair. We studied the effects of iron deprivation on radiation injury by exposing confluent monolayers of human umbilical vein endothelial cells (EC) to DFO (lOOuM) 24 hrs prior to 2.5 Gy irradiation, followed by plating in iron replete medium. There was no detectable toxicity immediately after 24 hrs DFO (trypan blue); however, clonogenic survival after DFO alone was reduced compared to untreated control (16.0% vs 26.1%). DFO followed by 2.5Gy resulted in 32% further reduction in survival compared to 2.5 Gy alone suggesting synergy for the combined treatments (p<0.01, ANOVA). Previous iron loading of EC by incubation with hemin 50uM for 24h had no effect on survival after 2.5 Gy. Because DFO may induce apoptosis in tumor cell lines, EC treated with DFO and 2.5 Gy were examined for apoptosis by morphologic criteria after passage in fresh iron-replete media. Few apoptotic cells were seen immediately after passage (1.9% control vs 1.8% DFO), but the number increased in the DFO group peaking after 24h in normal medium (0.79% control vs 6.4% DFO). The percentage of apoptotic cells decreased to baseline by 48h (0.79% control vs 1.12% DFO). The finding of apoptotsis was not abrogated by replacing iron replete media without passage. Radiation had no effect on the time course or peak number of apoptotic cells in the control or DFO group during the 48 hrs after passage. In contrast to apoptosis, the mitotic index over 48h did not differ significantly between control, DFO, or 2.5 Gy treated cells, but DFO & 2.5 Gy resulted in a 69% lower mitotic index compared to control after 48h in iron replete medium (p<0.01). DFO & 2.5 Gy significantly delayed entry into S and G2 phase by flow cytometry and increased the number of chromosome aberrations suggesting enhanced DNA damage or delayed repair. In conclusion, iron-deficiency reduces clonogenic survival in EC through induction of apoptosis, an effect which is independent of irradiation, but DFO enhances deleterious effects of radiation on clonogenic survival, cell proliferation, and entry into 5 and G2 phase. The synergy between tron-chelation and radiation is mediated perhaps through the enhancement of DNA damage or an impairment of DNA repair.