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DNA Damage in the Intracerebral Rat Gliosarcoma 9L Treated with 1-(2-Chloroethyl)-3-cyclohexyl-1-nitrosourea

Section of Neurosurgery, Baltimore Cancer Research Center, Division of Cancer Treatment, National Cancer Institute, NIH, Baltimore, Maryland 21201

Nitrosoureas are among the most effective agents for brain tumor chemotherapy, but their mode of action is uncertain. Damage to DNA in the intracerebral rat gliosarcoma 9L following in vivo exposure to 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) was determined by the alkaline sucrose gradient method. Following i.p. CCNU treatment, tumors were removed and mechanically dissociated. Tumor cells were lysed upon and sedimented through an alkaline sucrose gradient. DNA content of gradient fractions was assayed fluorometrically. DNA from tumor cells exposed to therapeutic, nontoxic CCNU doses sedimented more slowly than did untreated DNA, indicating single-strand breaks or alkali-labile strand damage. At this nontoxic dose and at short lysis times, anomalous sedimentation was observed, while conventional random sedimentation profiles were attained with the increased damage from higher doses. Damage to tumor DNA was also investigated by measuring the fraction of DNA remaining double stranded after exposure to alkali by determining its resistance to S₁ nuclease from Aspergillus oryzae. This fraction reflects its counterpart, the fraction of DNA that has completely unwound between single-strand breaks and separated irreversibly from the helix, and thus gives quantitative insight into the amount of damage present. Tumors from rats receiving a 3-hr treatment with CCNU (30 mg/kg) or methyl methanesulfonate (25 mg/kg) showed equivalent fractions of double-stranded DNA in alkali. The doublestranded DNA fraction for tumors treated with methyl methanesulfonate returned to control levels by 16 hr, demonstrating complete repair of the rat brain tumor DNA in vivo. CCNU-treated tumors showed no detectable ability to repair the damage by 16 hr.

¹ To whom requests for reprints should be addressed, at Department of Neurosurgery, University of California, San Francisco Medical Center, San Francisco, Calif. 94143.

Received 5/ 4/76. Accepted 7/15/77.