Localized irradiations, Evaluation through ''comet assay''

During the last 50 years various radiation accidents involving localized irradiations occurred, resulting mainly from improper handling of sealed sources Co60, Cs137 or Ir192 at workplaces for industrial gammagraphy. Severe skin reaction may develop at the contact sites. Such inhomogeneous irradiations lead to a differential exposure of lymphocytes in lymphatic tissues or other organs that may recirculate into the peripheral blood producing a mixed irradiated and unirradiated population of lymphocytes. Applying the mathematical models ''Contaminated Poisson'' of Dolphin and Qdr method of Sasaki, a mean dose in the irradiated body area and its size can be estimated from unstable chromosome aberration scoring. This give an indication of the proportion of haemopoietic stem cell compartment involved in the overexposure. There are also different biophysical techniques that can give responses in biological dosimetry. The ''Comet Assay'' (single cell gel electrophoresis) is a sensitive and rapid method for DNA strand break detection in individual cells. The advantages of the technique include: collection of data at the level of individual cell; the need for small numbers of cells per sample; its sensitivity for detecting DNA damage and that virtually any eukaryote cell population is amenable to analysis. The objective of this work is to apply ''Comet Assay'' method to evaluate the effect of radiation on skin and subcutaneous tissues, differentiating irradiated from unirradiated body areas. It could provide a useful tool to estimate the extension and the dose in the irradiated region, contributing with the current techniques. In this first study, we evaluate the alkaline comet assay as a method for detection of DNA radiation induced damage in keratinocytes from primary culture obtained from full thickness skin biopsies of patients requiring grafts. Skin and, particularly, keratinocytes were selected as an appropriate cellular system due to: Skin, the first barrier against radiation exposure and therefore the first target. Basal cells, from which keratinocytes derive, represent approximately 5% of all epidermal cell. It is the basal cell that responds to damage and initiates migration and repopulates the affected areas. As differentiating keratinocytes undergo an apoptotic via and it is not possible to distinguish apoptotic cells from strongly irradiated cells, we consider that keratinocytes from primary cultures are adequate. We evaluate the response of unirradiated and irradiated keratinocytes, using Co60 gamma rays, in order to get information on the extension of the body area for an early assessment of irradiation consequences in the exposed individuals. (author)