Simple radioactive assay for the estimation of DNA breaks

Abstract

The intactness of DNA is an important part of the normal cellular structure. Any change to the DNA in the form of breaks leads to a change in the integrity, which in turn leads to abnormality in the cellular activity. Many discrepancies have been reported among the various methods of detecting DNA damage. Here, a simple, sensitive and reproducible method has been developed for the detection of DNA breaks by radioactive labelling of 5' broken ends. The method was evaluated by studying chemically induced DNA damage by using both organochloride (2,4-dichlorophenoxyacetic acid and lindane) and organophosphorus (sevin and phosphamidon) compounds at different concentrations. Phosphamidon, one of the organophosphorus compounds studied, showed complete degradation of the DNA after treatment. Radioactive analysis of phosphamidon showed higher counts at the lowest concentration (20 microg) of the chemical when compared with the control (2752 scintillation counts per minute, scm). Studies on the chemically induced DNA breaks by radiolabelling revealed that the cumulative effect of the organophosphorus and organochloride compounds showed maximum counts in all the samples (the highest being 2904 scm) when compared with the organophosphorus and organochloride compounds studied separately (the highest being 1881 and 2260 scm, respectively). Radiolabelling studies on the blood samples of 23 pesticide workers by the newly developed assay showed a significant positive correlation (0.893) between the number of years of exposure and the scintillation counts. A maximum of 11,702 scm (for 18 years of exposure) and a minimum of 1682 scm (for 4 years of exposure) were recorded compared with 1253 scm for the negative control. This method can be used effectively for estimation of the DNA breaks, irrespective of its nature.