Relative importance of oxidation and Ca(2+) in DNA strand breakage by H(2)O(2) in the 2sFou cell line.
The rat hepatoma cell line (2sFou) was used to assess the mechanism of DNA strand breakage using the model oxidant H(2)O(2). Exposure of the cells to H(2)O(2) (100 mum) at 37 degrees C significantly reduced the amount of double-stranded DNA remaining after alkaline unwinding at 15 degrees C (control = 75.0 +/- 7.3%, n = 9, 100 mum H(2)O(2) = 46.5 +/- 8.0, n = 8, P < 0.02). The catalase inhibitor 3-amino-1,2,4-triazole halved the concentration of H(2)O(2) needed to give a significant level of strand breakage (control = 58.6 +/- 3.9%, n = 8, 50 mum H(2)O(2) = 36.5 +/- 2.1, n = 3, P < 0.02). The calcium chelator Quin-2 and the endonuclease inhibitor aurintricarboxylic acid both inhibited DNA strand breakage following treatment with 100 mum H(2)O(2) by 92% and 94%, respectively, whereas the lipid peroxidation inhibitor N, N-diphenyl-1,2,4-phenylene-diamine had no effect on H(2)O(2)-induced strand breakage. Analysis of the 2sFou cellular DNA for the 8-oxodG adduct following H(2)O(2) exposure provided no evidence for direct attack of the DNA mediated by hydroxyl radicals. Ca(2+) appears to be the major mediator of H(2)O(2)-induced DNA strand breakage.
Davies
J
J
School of Biochemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Chipman
J K
JK