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DNA Damage in Rat 9L Cells Treated with Nitrogen Mustard and 1,3-Bis(2-chloroethyl)-1-nitrosourea Assayed by Viscoelastometry and S₁ Nuclease¹

Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California 94143

The techniques of viscoelastometry and S₁ nuclease digestion were applied to the analysis of DNA damage in rat 9L cells treated with nitrogen mustard and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Results of the S₁ nuclease assay permitted quantitation of the amount of single-strand (or alkali-labile) break formation as well as DNA interstrand cross-link formation. In the presence of 2% detergent, only cells treated with nitrogen mustard showed evidence of DNA cross-link formation as determined by this assay. Viscoelastic analysis of cell lysates under denaturing conditions (pH 12.15) showed that cells treated with nitrogen mustard led to substantial increases in both the viscoelastic retardation time and recoil, consistent with the presence of DNA cross-links, while treatment with BCNU led to decreases in these two properties, consistent with the induction of single-strand breaks. Viscoelastic analysis of cell lysates under nondenaturing conditions (pH 11.15) showed that nitrogen mustard produced an increase in retardation time, consistent with single-strand break induction, along with a fast recoiling component that eventually led to gel-like behavior, suggesting the possibility of drug-induced intermolecular DNA-DNA cross-links. BCNU treatment resulted in a decrease in retardation time. This decrease is consistent with induction of DNA interstrand cross-links by BCNU and shows that the single-strand breaks observed at denaturing conditions were due to the presence of alkali-labile sites rather than true strand breaks. While other methods using denaturing conditions have resulted in evidence for DNA cross-links following BCNU treatment, both viscoelastic and S₁ nuclease experiments showed negative results in this regard. Further work is needed to clarify this point.

¹ Work supported by Grant CA-19658 awarded by the National Cancer Institute, Department of Health, Education, and Welfare, and a grant from the Cancer Research Coordinating Committee, University of California. Partial support of this research from the donors of the Petroleum Research Fund administered by the American Chemical Society is also acknowledged. Analysis of data using nonlinear regression was performed at the Computer Graphics Laboratory, School of Pharmacy, University of California, San Francisco, supported by Grant RR1081 from the Division of Research Resources, NIH.

² To whom requests for reprints should be addressed.

³ Present address: Becton Dickinson, FACS System, 490-B Lakeside Drive, Sunnyvale, Calif. 94086.

Received 10/ 1/79. Accepted 5/15/80.

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