Kathryn D. Held1, Yvonne L. McCarey1, Laurence Tartier1,
Elena V. Rusyn1, Giuseppe Schettino2, Melvyn Folkard2,
Kevin M. Prise2, and Barry D. Michael2
1Department of Radiation Oncology, Massachusetts General Hospital/Harvard
Medical School, Boston, MA 02114; 2Gray Laboratory Cancer Research
Trust, Mount Vernon Hospital, Northwood, HA6 2JR, UK
Accurate evaluation of the risks associated with exposure to low doses of ionizing radiation (IR) is a major challenge for environmental sciences. Studies on the mechanisms of the actions of low doses of IR are needed to help understand possible risks. IR exerts its effects on cells through production of reactive oxidizing species (ROS) such as ·OH, H2O2 and O2·-. Cells also produce low levels of ROS during normal metabolic processes. Therefore, it has been suggested that cell adaptation to low endogenous levels of ROS may mean they are less affected by low level IR exposure. As part of this project we are comparing the effects of ROS generated through chemical means in cells, mimicking endogenous production of ROS, with the effects of IR. Endpoints include initiation of apoptosis, micronuclei production and loss of clonogenicity.
In one set of studies, we have used HL-60 human promyelocytic leukemia cells to compare the apoptotic pathways induced by four different treatments: bolus H2O2, an oxidizing thiol compound dithiothreitol (DTT) which we have shown acts as a "slow release" source of H2O2 in cells (Tartier et al., 2000), high dose IR and low dose IR. Addition of H2O2 to cell medium causes rapid apoptosis, evident by DNA fragmentation or appearance of a sub-G1 fraction in flow cytometry of propidium iodide-stained cells, starting within 1-2 h, depending on the concentration of H2O2. The H2O2-induced apoptosis occurs in a fashion that depends on both mitochondria and caspase 3 activation. In contrast, the "slow release ROS" agent, DTT, leads to apoptosis on a somewhat slower scale and in a fashion that requires caspase 3 activation as a relatively early event, but is independent of mitochondrial involvement and caspase 8 or 9. Different patterns still are seen by low (e.g., 3 Gy) and high (20 Gy) dose IR, with the most striking differences being the apparent lack of caspase 3 involvement in the relatively rapid apoptosis caused by high dose IR and the very long time to apoptosis after the low dose IR. The data are summarized in the table, and clearly show that not all ROS are created equal.
| ROS generation | Time to maximal apoptosis | Mitochondrial involvement | Caspase 3 activation | |
|
H2O2 |
<5-30 min | 1-4 h | Yes | 0.5-2 h |
| DTT | 5-60 min | 5 h | No | 2-4 h |
| High dose IR | Immediate | 4-5 h | 2-4 h | None |
| Low dose IR | Immediate | 72-96 h | 48-72 h | 48-72 h |
In studies where apoptosis is not a major pathway for expression of cell damage, primary human fibroblasts (AGO1522) were used to assess the responses of cells to low doses of IR using clonogenicity and micronuclei formation as endpoints. Both conventional X-rays and the focused soft X-ray microprobe were used. (See companion abstract by Prise et al. for description of the X-ray microprobe.) With conventional X-rays, hypersensitivity was seen at £ 0.2 Gy using both clonogenicity and micronuclei formation as end points. When low doses of focused X-rays were targeted to the nucleus, the cell sensitivity (clonogenicity) was greater than that seen using conventional X-rays. The low dose hypersensitivity to conventional X-rays was increased by pre-treating cells with the thiol depletor buthionine sulfoximine (BSO). These data suggest that ROS are involved in the increased response of cells to low dose X-rays.
References:
Tartier L, McCarey YL, Biaglow JE, Kochevar IE, Held KD. Apoptosis induced by dithiothreitol in HL-60 cells shows early activation of caspase 3 and is independent of mitochondria. Cell Death and Differentiation 7:1002-1010 (2000).
This work is funded by the DOE (DE-FG07-99ER62874). EVR is supported by NIH CA09078.