The ultracytochemical acridine orange (AO) method has been employed to demonstrate DNA template activity within experimental brain tumors induced by transplacental administration of ethylnitrosourea (ENU) in Sprague‐Dawley rats. Ultrastructural examination revealed that acridine orange binds to DNA exclusively within the active extended euchromatin portion of the cell nucleus of brain tumor and of foci of atypical cell proliferation. No AO reaction products were visible in the non‐neoplastlc cell nuclei adjacent to the brain tumors. The percentages of AO positive cells in brain tumor ranged from 25.9 to 69.6, whereas average labeling indices of brain tumor cells in rats given an intraperitoneal injection of3H‐thymidine 1 hr before sacrifice were between 2.4 and 8.0. The average number of AO chromatin reaction products per single section of a cell nucleus varied between 7 and 21. The ratios of areas of euchromatin to heterochromatin were found to be slightly larger in AO positive nuclei than in AO negative nuclei in each of the tumors. The present results suggested that rat brain tumors induced by transplacental administration of ENU exhibit de‐repression of DNA template normally repressed in the adult state. The usefulness of this method for study on the development of experimental brain tumors is briefly di
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