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Formation of Interstrand Cross-Links in Chloroacetaldehyde-treated DNA Demonstrated by Ethidium Bromide Fluorescence¹

Donner Laboratory, Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720

Chloroacetaldehyde, the stable metabolite of the human carcinogen vinyl chloride, forms interstrand cross-links in vitro in salmon sperm DNA and in the alternating copolymer, poly(deoxyadenylate-deoxythymidylate) [poly(dA-dT)]. Formation of the cross-link was a function of both time of reaction and concentration of chloroacetaldehyde. Cross-linking in chloroacetaldehyde-treated poly(dA-dT) was detected initially by changes in renaturation hysteresis [Singer et al., Carcinogenesis (Lond.), 5: 1165–1171, 1984]. This has been confirmed and quantitated using the relative fluorescence of ethidium bromide after denaturation and reannealing at 40°C. Three percent cross-linking was detected after 10 min reaction with 20 mM chloroacetaldehyde at 24°C. In DNA the relative fluorescence of ethidium bromide after denaturation and rapid cooling was used to estimate the number of cross-links formed. Three times as much cross-linking occurs in DNA compared to poly(dA-dT) under identical reaction conditions. The postulated structure for an interstrand cross-link in poly(dA-dT) is a hydroxyethyl bridge across the strands between the N⁶-amino groups of alternate adenine residues. In DNA, other amino groups in the proper configuration can be involved.

¹ This work was supported by grants, CA 42736 and CA47723 from the NIH, Bethesda, MD, which were administered by the Lawrence Berkeley Laboratory under DOE Contract DE-AC03-76SF00098.

² To whom requests for reprints should be addressed.

Received 2/26/88. Revised 5/25/88. Accepted 6/ 3/88.

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