DNA damage in the intracerebral rat gliosarcoma 9L treated with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea.

teins (2, 4) have been demonstrated after in vitro and in vivo exposure to these agents. The work of Schabel et a!. (19) showing that the hamster plasmacytoma is resistant to known alkylating agents and to 1,3-bis(2-chlonoethyl)-1-ni trosoureas hasgeneratedspeculation that the carcinostatic activity of the nitrosourea agents is principally related to their alkylating metabolites. Wheeler and Bowdon (23) of fered further support for this tenet when they showed that 1,3-bis(2-chloroethyl)-2-nitnosourea has metabolic effects similar to those of alkylating agents. Because the nitrosoureas have antitumor activity against malignant brain tumors, we chose the i.c.2 rat gliosancoma 9L for study of the in vivo alkylating activity of CCNU. When alkylated DNA is exposed to alkali, the strands begin to separate with single-strand breaks serving as points where unwinding can begin (21). Complete unwinding of regions between breaks results in the irreversible release of single-stranded fragments from the helix, which can be detected on alkaline sucrose gradients (14) on quantitated by their susceptibility to digestion by 5, nuclease, a single strand-specific exonuclease from Aspergi!!us oryzae (7, 13). Both the alkaline sucrose gradient technique and S1 nu clease determination have been used in the work presented here to demonstrate DNA damage in an experimental solid tumor treated with CCNU doses that have been shown to be nontoxic and therapeutic. Because it is difficult to effect radioactive prelabeling of the DNA of solid tissues in vivo, in both these techniques we depended upon the fluorescent assay for DNA developed by Kissane and Robbins (8). This assay has been previously applied by Wheeler and Lett (24) to a sucrose gradient analysis of radiation-induced DNA damage in a normal brain and by Zubroff and Sarma (26) to a similar analysis of carcinogen-induced DNAdamage in a variety of normal tissues.