DNA damaging effects of benzotriazine-N-oxides

The aim of this study was to investigate the DNA damaging
abilities of SR 4233 (Tirapazamine) and a range of
congeners. These are novel benzotriazine-N-oxide compounds
of potential clinical importance in the treatment of
hypoxic tumours. The studies used a viral double
transfection assay which involved exposing purified $X174
DNA to the compounds under oxic, hypoxic and hypoxic
reductive conditions and subsequently transfecting the DNA
into E. coli AB1157 and E. coli C. Experiments under
reductive hypoxia were carried out between pH 4-7 to
establish if a reduction product required protonation for
the DNA damage process. The biologically relevant druginduced DNA damage was assessed by utilising a range of E. coli mutants deficient in specific DNA repair genes, the
products of which are involved in excision, recombination
and SOS repair. Viscometry was used to assess the effects
of various druginucleotide ratios and the ionic strength of
the buffer on the DNA damage caused.
DNA has been confirmed as a target for the action of the
benzotriazine-N-oxides. SR 4233, the lead compound, was
found to be the most active of all the compounds tested.
Damage was only induced upon reduction of the compounds and
there was no significant damage to DNA under oxic or
hypoxic conditions. SR 4233 exhibited increased DNA damage
at acid pH indicating that the radical anion responsible
for DNA damage has a requirement for protonation. The
damaging species is probably the 1-electron reduction
product of SR 4233 as SR 4317 and SR 4330, the 2-electron
and 4-electron reduction products respectively, caused
significantly less DNA damage under hypoxic reductive
conditions. There is also evidence that the
disproportionation reaction, which can lead to radical
production without DNA damage, may not be an important
reaction for SR 4233 bioactivation.
Studies with repair-deficient mutants of E. coli indicated
that SR 4233 is capable of inducing DNA damage which is
recognised and repaired by the ABC excinuclease complex.
However, the major damage caused is recognised and repaired
by the gene products of the xth and nth mutants. This
indicates that reduced SR 4233 induces primarily pyrimidine
based oxidative damage in DNA.

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