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.