Louisiana State University Physics & Astronomy

Ultraviolet light and ionizing radiation can kill and mutate cells, damage tissues and induce cancer in higher organisms. The critical radiation-induced damage is usually alternations in DNA. For biologically relevant doses, damage levels range from a few lesions per million DNA bases down to a few per billion. Measurement of such damages was challenging because of their low level, and, in many cases, the availability of only small quantities of DNA that could not be readily radiolabeled.

We developed methods that uses changes in the number-average-length of the DNA molecules in a sample resulting either from the action of the radiation, in the case of direct strand breaks, or from enzymatic or chemical treatments that convert damage sites into strand breaks. Our approach uses gel electrophoresis to separate DNA as a function of size combined with electronic imaging of fluorophore-labeled DNA to determine the relative mass distribution of the DNA in the gel. This information, combined with the dispersion function of the gel, determined from the distances of migration of DNA molecules of defined lengths, permits calculation of the number-average-length of a population of DNA molecules using a method-of-moments analysis.

For UV radiation, we measure pyrimidine dimers that are converted to single-strand breaks by treatment with a dimer-specific endonuclease. Applications have included measuring the action spectrum (cross-section as a function of wavelength) for dimer formation in intact plants and in human skin irradiated in situ.

A major cause of the biological damage induced by ionizing radiation (X-rays, g-rays, protons, and heavy ions – the latter two produced by the NASA Space Radiation Laboratory at BNL) was postulated to be bistranded clustered DNA damages – any combination of two or more closely opposed oxidized bases, abasic sites or single-strand breaks. However clustered damages other than double strand breaks had not actually been measured in isolated DNA or DNA from irradiated cells or tissues. We used the number average length approach to quantify both direct double-strand breaks and clustered damages that can be converted to double-strand breaks by lesion specific endonucleases, showing that approximately 4 times more enzyme-detected clusters are produced per radiation dose than frank double-strand breaks.

While damage induction is generally a linear function of dose, biological effects, such as clonogenic survival of cells, are not. Analytical methods were developed to combine an action spectrum for dimer induction with the irradiance of a polychromatic UV source, such as the sun, to predict biological effects such as the probability of cell survival as a function of dose. This work lead (indirectly) to a new stochastic model for radiation survival as a function of dose that has Euler gamma function solutions and is applicable to both UV and ionizing radiations. The number of lethal damages produced per unit dose, their potential reparability and the finite extent of repair per cell are treated explicitly in this model and can be determined from survival data using non-linear least-squared computational methods.