Characteristics of DNA damage induced by ultrasoft X-rays and upon photoionization.
AbstractThe initial indiscriminate damage of ionizing radiation on DNA in cells arises through direct energy deposition in DNA (non scavengeable component) or through water radicals (scavengeable component) together as clustered lesions. The non scavengeable component of damage leads to electron loss (and electron gain through electron migration) centres. In solution, it is usually difficult to separate the two components due to overwhelming water radicals. Evidence is presented (chapter 3), for clean production of mainly electron loss centres in DNA and base constituents at room temperature in solution, achieved by 193 nm pulsed high intensity laser light through a monophotonic ionization. Quantitative information on the role of the oxidative damage has been presented. Efficient migration of oxidative damage was found to occur over 2-3 bases from the generation site at adenine, thymine and cytosine to final localisation at guanine. The oxidative damage transfer appeared to be hindered when occurring between adenine and cytosine to guanine. Changes in light scattering intensities of polynucleotides and DNA indicated strand break formation upon a pulse of 193 nm laser light (chapter 4). Comparison of the transient optical absorption of the polynucleotides and DNA showed similar kinetics, indicating that strand breakage is initiated at the base radical cation and probably at guanine. It is proposed that the abundant cationic radical species in DNA is inefficient in causing strand break, but adds to the complexity of clustered damage by ionizing radiation through formation of base damage. Therefore strand break arise predominantly from OH radical attack and direct energy deposition on sugar-phosphate. The use of radiation which produces specific energy deposition events within DNA is imperative to extend our understanding of the effects of ionizing irradiation in biological systems. Characteristic aluminium K (A1K) (energy of 1.5 keV) and copper L (Cu L) (energy of ~0.95 keV) ultrasoft X-rays (USX) have been used as a probe for low energy electrons of track end structure for low LET ionizing radiation (chapter 5). Cellular inactivation, DNA dsb induction and their repair in Chinese hamster V79-4 cells as monolayer have been studied under aerobic and anaerobic conditions. Relative to 60Co y-rays, the relative biological effectiveness (r.b.e.) for cellular inactivation is 1.7 0.1 and 2.4 0.3 for A1 K and Cu L USX respectively. The oxygen enhancement ratio (OER) of 1.9 for A1 K USX is less than that for 60Co y-rays. DNA dsb induction by A1K and Cu L USX at 277 K, determined using the sensitive pulsed field gel electrophoresis technique is linear with dose, with r.b.e values of 2.6 0.2 and 2.8 0.3 for A1K and Cu L USX respectively. A reduced OER (of 2.1) was found for A1K USX. The repair kinetics for rejoining of DNA dsb following an irradiation dose of 15 Gy is similar for both USX and 60Co y-rays and has half-life of 22 5 min. The scavengeable component of damage ascribed to hydroxyl radicals for both 60Co y-rays and A1 K ultra-soft X-ray was investigated using 0.5 mol dm-3 ethylene glycol (EG) (chapter 6), showed a protection against cell inactivation to the same extent for both A1 K USX and 60Co y-rays. EG also reduced the number of DNA dsb measured by ~40%. It is confirmed that USX are more effective in both cell killing and the induction of DNA dsb than typical low-LET irradiation. From the findings with EG, dsb rejoining and the RBE values, it is inferred that the dsbs are similar in complexity for both radiation qualities. From the use of USX as a probe of track structure, it is inferred that cellular inactivation and DNA dsb break induction by 60Co y irradiation reflects clustered damage from predominantly track-end events which represents about 30% of the total dose. Although there are some minor differences, with 60Co y irradiation a distribution of severity of damage is expected including low energy electrons with energies greater than that of either Cu L- and A1 K-USX. USX are therefore an effective probe but not an exact model for the track-end clustered damage produced in low LET irradiations.