The fluorometric assay of Kissane and Robins has been modified to monitor DNA in alkaline sucrose gradient fractions. Using this procedure the sedimentation analysis of DNA not only of liver, but also of brain, thymus, lung, pancreas, kidney, and skin was carried out. Like liver DNA, DNA released by the alkaline lysis of the above organs sedimented as heavy DNA (>1 x 109 daltons). A good correspondence was obtained for the sedimentation profiles of liver DNA whether DNA in the gradient fractions was determined by the fluorometric method or by measuring radioactivity.
Using the fluorometric assay, the strand breaks not only of liver DNA but also of brain and kidney DNA have been demonstrated following the intravenous administration of N-methylnitrosourea. Carcinogen-induced DNA damage and repair (as measured by sedimentation of DNA in alkaline sucrose gradients) in any organ including human biopsy specimens are potentially measurable by this procedure.
Many chemicals have been recognized as carcinogens for human beings for nearly two centuries (1,2). They are being added to our environment at a rate faster than their safety can be evaluated. Several short-term, predictive bioassays have been developed to measure the carcinogenic potential of chemicals (3-10). Carcinogens, either directly or after being metabolically activated can interact in vivo with DNA (11-14) and cause DNA damage (6,7,14-21). One of the ways of studying DNA damage and its subsequent repair is by sedimentation analysis of DNA in alkaline and or neutral sucrose gradients. Using this method it has been demonstrated that carcinogens induce DNA-strand damage in vivo (6,7,15-23). However, the sucrose density gradient method (24,25) is currently applicable only to those organs whose DNA can be radioactively prelabeled. This is because the method requires the loading of a relatively small amount of DNA (less than 1 p,g on a 5 ml sucrose gradient). To make this method applicable for studies in nonproliferating organs and human biopsy specimens, it becomes necessary to monitor DNA in the gradient fractions by methods other than by determining its radioactivity.
In this communication, we present the details for the estimation of DNA from alkaline sucrose gradient fractions by a modification of the