Nucleic Acids Research, Vol 24, Issue 9 1625-1631, Copyright © 1996 by Oxford University Press
B Epe, D Ballmaier, W Adam, GN Grimm and CR Saha-Moller
N-Hydroxypyridine-2-thione (2-HPT), known to release hydroxyl radicals on
irradiation with visible light, and two related compounds, viz. N-
hydroxypyridine-4-thione (4-HPT) and N-hydroxyacridine-9-thione (HAT), were
tested for their potency to induce DNA damage in L1210 mouse leukemia cells
and in isolated DNA from bacteriophage PM2. DNA single- strand breaks and
modifications sensitive to various repair endonucleases (Fpg protein,
endonuclease III, exonuclease III, T4 endonuclease V) were quantified.
Illumination of cell-free DNA in the presence of 2-HPT and 4-HPT gave rise
to damage profiles characteristic for hydroxyl radicals, i.e. single-strand
breaks and the various endonuclease-sensitive modifications were formed in
the same ratios as after exposure to established hydroxyl radical sources.
In contrast, HAT plus light gave rise to a completely different DNA damage
profile, namely that characteristic for singlet oxygen. Experiments with
various scavengers (t-butanol, catalase, superoxide dismutase) and in D2O
as solvent confirmed that hydroxyl radicals are directly responsible for
the DNA damage caused by photoexcited 2-HPT and 4-HPT, while the damage by
HAT plus light is mediated by singlet oxygen and type I reactions. The type
of DNA damage characteristic of hydroxyl radicals was also observed in
L1210 mouse leukemia cells when treated with 2-HPT plus light or with H2O2
at 0 degrees C. t-Butanol (2%) inhibited the cellular DNA damage by
approximately 50%. A dose of 2-HPT plus light that generated single-strand
breaks at a frequency of 5 x 10(-7)/bp was associated with 50% cell
survival. No DNA damage and cytotoxicity was observed after treatment with
2-HPT in the dark. We propose that 2-HTP and 4-HTP may serve as new agents
to study the consequences of DNA damage induced by hydroxyl radicals in
cells. In addition, the data provide direct evidence that hydroxyl radicals
are ultimately responsible for the genotoxic effects caused by H2O2 in the
dark.
ARTICLES
Photolysis of N-hydroxpyridinethiones: a new source of hydroxyl radicals for the direct damage of cell-free and cellular DNA
Institute of Pharmacy, University of Mainz, Germany.
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