Biological consequences of damage introduced in bacteriophage PM2 DNA by hydrogen peroxide-mediated free radical reactions

• Published in: Carcinogenesis (New York) 1996-01, Vol.17 (1), p.5-11
• Main Authors: Gille, JJP, Wientjes, N M, Lafleur, MVM, Joenje, H, Retel, J
• Format: Article
• Language: English
• Subjects: phage PM2
• ISSN: 0143-3334

In order to study the biological consequences of DNA damage induced by H sub(2)O sub(2)-mediated free radical reactions, DNA from bacteriophage PM2 was exposed to H sub(2)O sub(2), Fe super(3+)-citrate and ascorbate either alone or in combination. Induction of DNA lesions was determined as well as the biological activity of the phage DNA. Exposure to H sub(2)O sub(2) alone resulted in max. 0.2 single-strand breaks per molecule; in the presence of Fe super(3+)-citrate, the yield was similar to 4-fold higher. Under both conditions no double-strand breaks could be detected and the biological activity was not diminished. This indicates that low levels of single-strand breaks as generated by H sub(2)O sub(2)/Fe super(3+)-citrate do not inactivate PM2 DNA. Exposure to ascorbate in the presence Fe super(3+)-citrate resulted in extensive induction of single-strand breaks. However, at ascorbate concentration where similar to 3 single-strand breaks per molecule were induced, again no double-strand breaks could be detected and the biological activity of the DNA was not diminished. At 5 mM ascorbate, single-strand breaks were above the detection limit. Under these conditions, 0.02 double-strand breaks were induced and the biological activity was reduced to 50%. The contribution of double-strand breaks to biological inactivation was calculated to be similar to 3%. When PM2 DNA was exposed to H sub(2)O sub(2) in the presence of ascorbate/Fe super(3+)-citrate, a typical biphasic dose-effect relationship was observed both for the induction of double-strand breaks and biological inactivation, suggesting that one or more reactive species sensitive to H sub(2)O sub(2) play a critical role. The times OH scavenger t-butanol appeared to be relatively inefficient in protecting PM2 DNA, which may indicate that other reactive species than times OH are involved. Our data suggest that other reactive species than times OH, such as the ferryl ion, are involved in H sub(2)O sub(2)-mediated DNA damage induction and biological inactivation.