Exp. Eye Res. (1989) 49, 685-698 Repair of H202-induced DNA Damage in Bovine Lens Epithelial Cell Cultures ABRAHAM SPECTOR*, NORMAN J. KLEIMANt, RUEY-RUEY C. HUANG$ A~D RE~-Ro~ WAnG Biochemistry and Molecular Biology Laboratory, Department of Ophthalmology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, U.S.A. (Received 13 March 1989 and accepted in revised form 25 May 1989) H202 concentrations only slightly higher than normal physiological levels found in the lens and aqueous fluid produce a significant number of DNA single-strand breaks in lens epithelial cell cultures. In this investigation, the repair of DNA damaged by short-term, H202-induced oxidation was examined in bovine tens epithelial cell cultures. Repair was rapidly initiated and was almost completed in 30 min. A drop in NAD concentration was associated with the DNA damage. 3-Aminobenzamide inhibition of poly(ADP-ribose) polymerase, an enzyme believed to be stimulated by DNA oxidation and involved in DNA repair, prevented the loss of NAD. In contrast, a similar drop in ATP concentration was only slightly lessened by the presence of this inhibitor. Inhibition of the polymerase by 3-aminobenzamide primarily affected only the early recovery period. Overall, recovery occurred almost as effectively in the presence of the inhibitor as in its absence. Preineubation of lens cultures with o-phenanthroline, an iron chelator, prevented the drop in NAD levels associated with DNA damage. Since a hydroxyl radical is produced from H~O 2 by a Fenton type reaction, this result supports the concept that the H~O~-induced oxidation of DNA is caused by hydroxyl radical. In contrast, peroxide-induced loss of activity of a cytosolic enzyme, glyceraldehyde-3-phosphate dehydrogenase, was unaffected by the presence of o-phenanthroline, suggesting direct H20 ~ oxidation of this enzyme. The results of these experiments suggest that lens epithelium contains enzymes that rapidly repair single-strand DNA breaks induced by H20 ~ insult. Key words : DNA damage; DNA repair; H202; lens epithelial cell cultures; alkaline elution; single-strand breaks; poly(ADP-ribose) polymerase; NAD; ATP; hydroxyl radical; 3-amino- benzamide ; o-phenanthroline. I. Introduction It is generally accepted that oxidative insult is an early or initiating event in the development of cataract (Spector and Roy, 1978 ; Augusteyn, 1981 ; Spector, 1984a). Oxidative damage to protein and possibly lipid during cataract development has been extensively reported (Augusteyn, 1981; Spector, 1984a, b; Bhuyan and Bhuyan, 1983). In contrast, there have been no reports linking lens oxidation to DNA damage and subsequent development of cataract. This is surprising in view of the vulnerability of the lens to oxidation. The lens contains a single layer of epithelial cells on its anterior surface overlaying an organized arrangement of elongated anuclear fiber cells (Harding and Crabbe, 1984). The cells in the central epithelium divide rarely, if at all, but cell division increases approaching the equatorial zone, the region of differentiation into fiber cells. In this region the epithelial cells begin to lose their nuclei, viable DNA and their * To whom correspondence should be addressed at Biochemistry and Molecular Biology Laboratory, Department of Ophthalmology, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032, U.S.A. t Recipient of a National Institutes of Health Individual National Research Service Award. :~ Current address: Department of Molecular Pharmacology, Merck, Sharp and Dohme Research Laboratories, Rahway, NJ 07065, U.S.A. 0014-4835/89/100685+ 14 $93.00/0 9 1989 Academic Press Limited